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Vol. 61, No. 2, April 1981

Printed in U.S.A.

Intracellular pH

Departments of Physiology and Biophysics and of Anesthesiology, Washington

University School of Medicine, St. Louis, Missouri; and Department of
Physiology, Yale University School of Medicine,
New Haven, Connecticut

I. Introduction ......................................................... 297

II. Techniques for Measuring Intracellular pH ............................. 298
A. Measurements on homogenates. .................................... 298
B. Distribution of weak acids or bases ................................. 300
C. Colorimetry and fluorometry ....................................... 317
D. Microelectrode methods ........................................... 321
E. 31P nuclear magnetic resonance spectroscopy ........................ 329
III. Summary of Normal Values of Intracellular pH ......................... 334
IV. Effects of Nonionic Permeation of Weak Acids and Bases ................ 334
A. Historical perspective ............................................. 334
B. Weakacids..........: ............................................ 335
C. Weakbases ....................................................... 346
V. Effects of Ionic Permeation of Weak Acids and Bases; H+ Permeability. ... 348
A. Experimental evidence ............................................ 348
B. Models of ionic permeation ......................................... 353
VI. Effect of Temperature on Intracellular pH .............................. 355
A. Experimental evidence ............................................ 355
B. Theoretical considerations ......................................... 356
C. Model buffer systems and imidazole ................................. 357
VII. Is H+ at Equilibrium Across the Cell Membrane? ....................... 359
A. Historical perspective ............................................. 359
B. Measurements with glazed microelectrodes .......................... 360
VIII. Regulation of Intracellular pH ......................................... 364
A. Historical perspective and introduction .............................. 364
B. Short-term intracellular pH homeostasis; metabolic contributions ...... 366
C. Response to intracellular acid loads: acid extrusion ................... 367
D. Methods of intracellular acid loading ................................ 369
E. Acid extrusion: coupled transport of Na+, HCO;, and Cl- ............. 372
F. Acid extrusion: Na-H exchange. .................................... 382
G. Acid extrusion in vertebrate cells ................................... 383
H. Response to intracellular alkaline loads ............................. 385
I. Factors influencing steady-state intracellular pH ..................... 385
IX. Intracellular Buffering ................................................ 389
A. Physicochemical buffering ......................................... 389
B. Mechanisms of cellular buffering. ................................... 392
C. Techniques for measuring intracellular buffering power ............... 395
D. Estimates of intracellular buffering power ........................... 398
X. IntemalpH oforganelles ............................................. 400
A. Mitochondria ..................................................... 400
B. Chloroplasts ...................................................... 403
C. Lysosomes ....................................................... 403


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D. Chromaffingranules ............................................... 405

E. Sarcoplasmic reticulum ............................................ 407
XI. Properties and Processes Affected by Intracellular pH ................... 408
A. Membrane permeability and conductance ............................ 408
B. Cell-cell coupling .................................................. 409
C. Epithelial secretion and reabsorption of acid ......................... 411
D. Mechanical properties of muscle .................................... 412
E. Metabolism ....................................................... 416
F. Fertilization ...................................................... 417
XII. Miscellaneous ........................................................ 418
A. Anoxia and metabolic inhibitors .................................... 418
B. Calcium and intracellular pH ....................................... 419
C. Carbonic anhydrase inhibitors ...................................... 420
D. Conceptual errors in using CO,-containing solutions .................. 421


Considerable progress has been made toward understanding the mecha-

nisms of intracellular pH (pHi) regulation since the last comprehensive reviews
appeared (96,364,454). This new understanding is principally the result of new
techniques that permit the measurement of transient changes in pHi. Perhaps
the most fruitful among these methods has been the application of pH-sensitive
microelectrodes. Nuclear magnetic resonance spectroscopy and spectrophotom-
etry have also contributed to the advance of our knowledge and hold even
greater promise for the futu .re. In addition progress has been made with older
techniques, particularly . the weak acid and base methods. Normal pHi values
have been established for a wide variety of preparations. New insights have
been gained into the effect on pHi of permeation by the neutral and ionized
forms of weak acids and bases. Studies of the temperature dependence of pHi
have been extended, and the controversy over the supposed equilibrium dis-
tribution of H+ has been laid to rest. Considerable attention has been paid to the
careful definition and measurement of intracellular buffering power. Studies on
the internal pH of organelles have intensified. Finally, the implications of pHi
regulation for phenomena ranging from muscle tension to epithelial acid secretion
are becoming more appreciated. The purpose of this review is to critically
exam .ine these recent adv ‘antes (with particular emphasis on results obtained
with microelectrodes), to analyze older work in the light of recent progress,
and to provide an historical perspective whenever appropriate.
Previous comprehensive reviews include Caldwell’s classic PaPer (74) and
the more recent efforts of Robson et al. (364), Waddell and Bates (454), and
Cohen and Iles (96). Walker and Brown’s review (459) on intracellular ion
activities contains a section on measurements of pHi with microelectrodes.
Short reviews of limited scope have been written by Siesjo (406), Sorokina
(419), Hinke and Menard (189), De Weer (116), Thomas (433), Roos (369),
Boron (38, 39), and Gillies and Deamer (145, 146).

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298 A. ROOS AND W. F. BORON Volume 61


A. Measurements on Homogenates

One of the oldest methods for estimating pHi, the measurement of the pH of
cell lysate or homogenate, was first employed in 1912 by Michaelis and Davidoff
(290) with red blood cells. They noted that although blood pH (measured with a
Pt/H, electrode) is practically unaffected by a fivefold dilution of the blood with
normal saline, it falls by about 0.10 when water is the diluent, which lead them to
conclude t hat the pHi of the erythrocytes must be less than the pH of the plasma.
Although lysis or homogenization would seem to provide a rather
straightforward method for estimating pHi, the technique’s potentially serious
problems were recognized early on. For example, lactic acid and CO, production
continue after cellular destruction and lead to a fall in pH (291). In addition the
mixing of intra- and extracellular fluids can lead to changes in pH because the
solutions are of dissimilar pH and because of the dilution of intra- and extra-
cellular buffers (23). Finally, the disruption of intracellular organelles makes it
impossible to calculate the pH of the bulk intracellular fluid. (96).
To prevent continued metabolism after cellular destruction, Michaelis and
Kramsztyk (291) boiled the tissues immediately after dissection, although they
realized that boiling causes the loss of CO, and hence leads to an erroneously
high pH. The authors found a pH of 6.4-6.7 in various mammalian tissues that
had not been boiled and a pH of about 7.0 (38°C) in the tissues after boiling. They
concluded that the true pHi is about 6.8. This value is 0.2-0.3 lower than the
best present estimates for pHi in mammalian muscle (see sect. III). In 1924 Vies
and his colleagues (452) avoided the problem of CO, loss in their experiments
with sea urchin eggs by freezing the cells at - 15°C and then measuring the pH of
a cell mash at about 0°C. The value obtained, about 5.1 for unfertilized eggs,
was 1.2-1.7 lower than the best present estimates (see sect. III). This
discrepancy is probably due both to the slowness of the freezing process (which
would allow acid metabolites to be produced) and to the use of the platinum-
hydrogen electrode, which may be affected by interfering substances
(see sect. IID). Both difficulties were overcome in 1927 by Furusawa and
Kerridge (140), who worked with cat skeletal, cardiac, and uterine muscle. The
muscles were plunged into liquid air immediately after dissection, minced, and
then brought to O”C, whereupon the pH was measured with a glass electrode.
The pH values obtained for skeletal and cardiac muscle at 0°C were 7.04 and
7.07, respectively. Applying the authors’ temperature correction, these values
extrapolate to 6.89 and 6.92 at 37”C, only 0.1-0.2 less than the best present
estimates (see sect. III).
Erythrocytes and sea urchin eggs are the only two preparations for which
there are sufficient data for a direct comparison of the lysate-homogenate
method with another technique. In 1961 Fitzsimons and Sendroy (132) com-
pared the pHi estimated from lysates of human red cells (hemolyzed with
saponin, apparently at 37°C) with that calculated by the CO, method and from

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the chloride distribution between serum and cells (assuming [H+]J[H+]i= [Cl-],/
[Cl+],). At pH,, 7.40 and 37°C the three methods yielded pHi values of 7.20, 7.22,
and 7.23, respectively. Four years later, Bromberg and colleagues (53) used lysis,
5,5-dimethyl-2,4-oxazolidine-dione-2- 14C(DMO), and Cl- distribution to estimate
pHi in human erythrocytes. Packed cells were lysed by freezing at - lO”C, and the
lysate’s pH was measured anaerobically after the temperature had been raised to
37°C. After correcting for plasma trapping, pHi (lysis) was 7.15 t 0.013 (n = 13),
close to the DMO-derived value of 7.11 -e 0.008 (n = 16). As in the study by
Fitzsimons and Sendroy (132), the Cl- distribution yielded a somewhat higher
value, 7.21 t 0.008 (n = 19). In 1966 Funder and Wieth (139) used the lysis and
Cl- distribution techniques to calculate the pHi of human erythrocytes over a
range of pH, and found good agreement between the two methods. At pH, 7.47,
lysis and the Cl- distribution yielded values (obtained from the equation of best
fit) of 7.24 and 7.27, respectively. These are very close to the values obtained by
Bone et al. (36) in 1976 on human erythrocytes (pH, = 7.47), 7.27 t 0.010
(n = 8), calculated from the DMO distribution, and 7.26 t 0.004 (n = 8),
determined by the NH,+ distribution. In addition Funder and Wieth’s pHi
values at pH,, 7.40, namely 7.19 by lysis and 7.22 from the Cl- distribution, are
reasonably close to those obtained in 1970 by Calvey (78) on rabbit erythrocytes
(PH = 7.40), namely 7.27 t 0.05 (n = 40) with the DMO method and 7.27 from
the “Cl- distribution. Thus the lysis technique apparently produces reliable
results in mammalian erythrocytes.
In their 1976 study on sea urchin eggs, Johnson and co-workers (220)
first washed the eggs (StrongyZocentrotus purpuratus) in unbuffered, HCO,-
free artificial seawater (ASW) and then made a 5% suspension in 0.5 M KCl. The
suspension was homogenized and its pH immediately measured. The authors
arrived at a value of 6.48 for unfertilized eggs and 6.78 for eggs that had been
fertilized 10 min earlier. These values are similar to the homogenate data ob-
tained on the same species a year later by Lopo and Vacquier (262): 6.34 and
6.76 in unfertilized and fertilized eggs, respectively. The results of these two
studies, however, conflict on three counts with data obtained by other methods.
First, in 1978 Shen and Steinhardt (397), using microelectrodes, arrived at
substantially higher pHi values for the eggs of Lytechinus pictus, another
species of sea urchin: 6.84 in unfertilized eggs and 7.26 10 min after fertilization.
Second, they found that contrary to the results of Johnson et al., the rise in
pHi following fertilization is not preceded by a transient fall. Finally, the DMO
and methylamine data of Gillies and Deamer (146) indicate that fertilization
causes a sustained increase of pHi, whereas Lopo and Vacquier’s data show
pHi eventually returning to its prefertilization value. Apart from the con-
tradictory data on the pHi time course, the homogenate-derived pHi values
still fall some 0.5 below those obtained with microelectrodes. Although species
differences may account for part of the discrepancy, most is probably due to
other causes. I ) Homogenization of a 5% egg suspension results in a ZO-fold
dilution of intracellullar fluid that can be expected to alter pHi even if the ex-
tracellular fluid is unbuffered. 2) Unlike mammalian erythrocytes, these eggs

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300 A. ROOS AND W. F. BORON Volume 61

are compartmentalized; thus regions inaccessible to microelectrodes contribute

to the homogenate’s pH. 3) Since recently fertilized eggs undergo striking
metabolic changes, they may be especially susceptable to metabolically induced
artifacts. It would have been desirable if the eggs had been lysed by a rapid-
freezing technique, as in the experiments of Furusawa and Kerridge (140).
Also, it should be emphasized that the discrepancy between lysate-homogenate
and microelectrode-derived pHi values need not remain constant under all
experimental conditions. For example, changes in the pH of intracellular
subcompartments, under conditions in which the pH of the bulk intracellular
fluid (i.e., that in contact with the cell membrane) remains unchanged, would
be reflected by a shift in the pH of the homogenate but not in that measured
by a microelectrode.
We conclude that the present data justify the use of the lysate-homogenate
approach for the measurement of pHi only in nonnucleated erythrocytes.

B. Distribution of Weak Acids or Bases

1. Historical perspective

The weak acid or base methods rest on the assumption that only the un-
charged forms of the se electrolytes are permeant and that after sufficient
length of exposure, their concentrations are the same in extracellular and
intracellular water. In 1871 De Vries (115) first showed what can now be inter-
preted as the entry of the weak base NH, into living red beet cells: beet slices
turn brown w hen ex posed to a solution of N H,. The color change reflects a rise in
the pH of the central vacuole, which in plants forms most of the cell’s content.
De Vries observed the same change when ammonia was added to a solution of
the coloring material. In 1897 Overton (324) demonstrated the entry of am-
monia, primary, secondary, and tertiary amines, and various alkaloids into
plant (Spirogyra) cells by observing the formation of intracellular pre-
cipitates due to combination of these bases with normally present tannin. He
found that no precipitation occurs when the concentration of the uncharged
partner is reduced by acidifying the solutions. Overton concluded that it is only
the free, uncharged form that can penetrate. Quaternary amines do not enter,
“just as solutions of KOH and NaOH are ineffective” in leading to tannin pre-
cipitation. Somewhat later Overton (325) studied the influx of weak acids such
as lactic and acetic acid into frog muscle, using osmotic swelling as an index of
acid entry. He observed that the swelling is much greater than in more acidic
solutions of strong “mineral” acids. Again he explained this by assuming
membrane permeability to only the undissociated molecule. Weak acids, in
contrast to mineral acids, are soluble in ether, a property that Overton ascribed
to the presence of their undissociated forms. Since ether solubility parallels
lipid solubility, these findings reinforced his postulate of membrane largely
made up of lipids.

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The work of Overton was first confirmed by Warburg (464) in the sea urchin
egg (Strongylocentrotus lividus ). He observed that eggs supravitally stained
with neutral red do not change color in strong bases, but rapidly turn yellow
(indicating alkalinity) in solutions of NH&I. Harvey (168) performed similar
experiments on many plant cells (Elodea canadensis, Spirogyra) and an.imal
cells (Paramecium, Vorticella, and marine eggs of Toxopneustes and
Hipponoe ), again staining them with neutral red and exposing them to ammonia
and various amines. The great permeability of the cell walls of the colored flower
petals of Symphytum peregrinum to molecular CO, was dramatically demon-
strated in 1920 by Jacobs (211). The originally blue, mature petals rapidly
turned red (i.e., became more acid) not only when immersed in water gassed
with 100% C02, but even in a HCO,-CO2 mixture of pH - 7.4. Conversely
the originally red petals of a rhododendron hybrid turned bright blue (i.e.,
alkaline) not only when immersed in an alkaline solution of NH,OH, but
even in an acid (pH 6.2) solution of (NH&SO, (212). These last results are indic-
ative of permeability to NH, rather than to NH,+. This great preference of the
cell membrane for the uncharged form extends to dyes as well. For instance,
in 1911 Harvey (168) found that plant and animal cells take up basic dyes (e.g.,
neutral red) when dissolved in alkaline solutions but not in acidic solutions.
The reverse is the case for acidic dyes. Irwin (207) subsequently showed
that over a wide range of external pH (pH,), both rate of accumulation
and final concentration of brilliant cresyl blue in the vacuole of Nitella are
roughly proportional to the external concentration of the undissociated
form of the dye.
Adding quantitative refinement to the previous observations, Osterhout et
al. (323) exposed the marine alga Vatonia macrophysa to solutions of H,S (322) and
CO, at various values of pH, and measured the steady-state concentrations of
the weak acids in the vacuole. He assumed that only the undissociated form
of these acids was present in the vacuole; at least in the case of HzS, this does
not introduce a large error (vacuolar pH = 5.8, pK, of H,S = 7.0, pK, of CO,
= 6.3). Osterhout found that the vacuolar concentrations are about the same
as those of the undissociated form in the external fluid and concluded that
both CO, and H,S gain entry predominantly in the undissociated form. Later
Cooper and Osterhout (101) directly measured the pH of the vacuolar fluid and
found that it becomes alkaline when the cells are exposed to NH&l.

2. Calculation of pHi

When only the uncharged partner of a weak acid or base is permeant, the
pHi can be derived from the steady-state distribution of the electrolyte if
pH, and the apparent pK, values in the external and internal fluids (pK, and
pKi) are known. For a weak acid (HA _ A- + H+), [HAli = [HA], = [HA]
in the steady state. Therefore
VW, - [HAI
PH, = PKI + 1% (0

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A. ROOS AND W. F. BORON Volume 61

pHi = iTAli - CHAI

PKi + log (2)
where [TA] = [A-] + [HA]. Combining Equations 1 and 2, we have

pHi = PKi + log -
(lOpHo-pKo + 1) - 1
1 (3)

Similarly for a weak base (HB+ _ H+ + B)

rTBli (lOpKo-pHo+ 1) - 1
pHi = pKi - log -
rm, 1 (4)

where [TB] = [B], + [HB+]. If we assume that the pK is the same on either
side of the membrane, we obtain by rearranging Equation 3

-[TAli = (5)
rm, l(-pHo--ph’

which was first derived by Jacobs (213). It indicates that when pHi < pH,,
weak acids are more concentrated in the external fluid than in cell water. A
similar rearrangement of Equation 4 yields

l(p-3 + 1
-LTBli = (6)
n-B10 lop”-PHO
which shows that under the same relative pH conditions, weak bases are more
concentrated in cell water than in extracellular fluid; this was previously ob-
served by Overton (324).
In practice [TA]i and [TB]i are not measured directly because the sample
cannot be freed completely of extracellular fluid. Rather [TA] or [TB] in the total
sample water (i.e., intra- and extracellular) is determined, and a correction is
applied based on the extracellular volume measured with an impermeant.
marker such as inulin or sorbitol. Waddell and Butler (456) have presented equa-
tions for calculating pHi that incorporate the extracellular space corrections.
These principles governing the distribution of weak acids were first applied
in 1920 by Fridericia (137), who derived the pHi of ox red blood cells from the
CO, distribution between cells and medium, obtaining a value of 7.30
(PH -- 7.35, Pcop = 39.1). Two years later, E. J. Warburg (463) used the same
method in a detailed study of horse red blood cells. He obtained a pHi of
7.29 at a pH, of 7.40 and confirmed the relation pHi < pH, over a wide range
(6.5-7.9) of pH, (varied by changing Pco,). In 1935 Fenn and Maurer (129)

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applied the CO, method to frog skeletal muscle in vivo, using Cl- as an extra-
cellular marker. At a plasma pH of 7.34, and assuming a CO, tension equivalent
to 5% CO, (by their own admission a somewhat high estimate), Fenn and
Maurer calculated a pHi of 6.90, a reasonable value even by modern standards.
Wallace and Hastings (460), using the same approach, derived a pHi of 6.89
for in vivo cat skeletal muscle (pH,, = 7.24, Pco,! = 50).
Although much useful information has been amassed with the CO, method
[see the review by Waddell and Bates (454)], it was a significant step forward
when in 1959 Waddell and Butler (456) introduced the weak acid DMO, the
product of metabolic N-demethylation of the antiepileptic drug trimethadione.
In contrast to CO*, this acid (pK = 6.1 at 37°C and ionic strength 0.16 M; for
pK under other conditions see 46,188,265) is nonvolatile at physiologic tempera-
tures and thus is much easier to work with. Other weak acids such as lactic acid
(pK = 3.7; 153, 155, 368), propionic acid (pK = 4.9; 393, 2,4-dinitrophenol
(PK = 4.9; 415), and phenobarbital (pK = 7.8; 54) have also been used as
pHi indicators.
The first weak base for measuring pHi was NH,, (pK = 9.2), used in 1929
by Netter (312) on cow red blood cells to show that the ratio of NH,’ across the
cell membrane equals that of H+. In later experiments (313) Netter found that
the muscles of frogs perfused with solutions containing NH&l have about 5
times as much total NH,, (NH,, plus NH,+) as the perfusate. He postulated that
a similar ratio must pertain to the H+ ions. Fenn et al. (128) used NH:, on in vitro
frog muscle. The total NH, ratio between intracellular and extracellular water
was found to approximately equal the H+ ratio previously obtained with the
CO, method (129). Weiss (469) first used nicotine (pK = 8.2) as a pHi indicator.
After correcting for bound nicotine, he obtained a pHi for frog sartorius muscle
that reasonably agreed with that obtained using DMO. Other weak bases used
for pHi measurement include procaine (pK = 9.0; 54, 201), lidocaine (pK = 7.9;
201), methylamine (pK = 11.8; 46, 148, 375), morpholine (pK = 8.5; 167), mor-
phine (pK = 9.9; 54), atropine (pK = 4.4; 54), and trimethylamine (pK = 9.7; 54).

3. Advantages ofweak acid and base methods

The main advantages of weak acid and base methods are the simplicity of
their execution and the ease with which they can be applied to the various
tissues of the intact animal, to isolated tissues, or to cells and organelles
too small to be impaled by microelectrodes. Thus measurements have been
made in mitochondria (DMO; 2), chromaffin granules (methylamine; 221>,
chloroplasts (methylamine; 375), sarcoplasmic reticulum vesicles (DMO; 317),
and lysosomes (methylamine; 357); see sect. X.

4. Limitations and sources of error

There are eight major disadvantages or pitfalls in estimating pHi from the
distribution of a weak acid or base.

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304 A. ROOS AND W. F. BORON tblum 61

I) The relative slowness of distribution of the indicators limits their use

to steady-state conditions or to the measurement of relatively slow pHi
transients. The equilibration time for DMO in the brain and skeletal muscle
of the intact dog or cat is about 1 h (365,366,456); in the isolated rat diaphragm
(370) or barnacle muscle fiber (46) it is about 0.5 h; in the isolated, perfused rat
heart it is about 3 min (310); and in leukocytes the equilibration time is at the
most 20 min (254). For suspensions of organelles the required time is much
shorter: for instance, in mitochondria it is less than 30 s (2).
2) The methods require destruction of the cells. Therefore, only one single
measurement can be made.
3) Errors are introduced because of binding or metabolic transformation
of the indicators. In the case of C02, the suggestion that intracellular COZ is
present in a form other than HCO; and dissolved CO, (100) has largely been
disproved (66, 403; see also sect. VIIA ). DMO is not bound to bovine serum
albumin (455), to human plasma (466), or to homogenates of dog skeletal muscle
(456); evidence to the contrary (80) is inconclusive. Also the transmembrane
distribution of DMO in mammalian muscle cells (7) and liver cells (205), beef
heart mitochondria (l), and red cells (78) remains unchanged over a wide range
of concentrations. Although DMO is not metabolized in dog skeletal muscle (456)
and rat sympathetic ganglia (55), metabolic transformation does occur in the
cells of Acer pseudoplatanus (sycamore maple; 244,245). For methylamines no
metabolic transformation is demonstrable in barnacle muscle (46); information
on its binding is not available. Hannan and Wiggins (167) could not detect a
breakdown of morpholine into amines or nonamines in frog muscle. Nicotine
may bind to or be metabolized by frog sartorius muscle, since the distribution
ratio of this compound fell by as much as 30% when the concentration in the
medium was raised from 0.08 mM to 1.25 mM (469). Binding of nicotine to rat
diaphragm can also not be excluded: although ultrafiltration of nicotine-
containing muscle homogenates removed all the nicotine (3), the membrane used
was permeable to molecules with molecular weights as high as 50,000. There is
no metabolic transformation of nicotine in rat sympathetic ganglia (56), but the
situation in rat diaphragm muscle is less clear (3). When homogenates of
diaphragm that had been incubated with labeled nicotine were shaken with a
heptane-chloroform mixture, 16.5% of the label went into the chloroform versus
only 1.5% of the label of the incubation medium treated similarly. Since,
according to Adler (3), nicotine is soluble in heptane, but its major metabolites
are soluble only in chloroform, this suggests some metabolic transformation of
nicotine and an inability of the metabolites to leave the cell. In a recent study by
Brown and Garthwaite (54), morphine, atropine, trimethylamine, or pheno-
barbital were found not to be metabolized by rat sympathetic ganglia, but
procaine was partially broken down, presumably to p-aminobenzoic acid.
4) Uncertainty is introdu .ced because of the need to estimate the vol ume
of extracellular fluid contained in the sample since the pH indicator is also
present in this fluid. Supposedly impermeant markers (e.g., SO;*, Cl-, sorbitol,
and inulin), used to estimate the extracellular space, give somewhat different

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results. Fortunately the derived pHi is often rather insensitive to extracellular

volume. For instance, in mammalian skeletal muscle at a pH, of 7.40, the pHi
derived from a measured DMO distribution ratio (concentration in tissue water
divided by that in extracellular water) of 0.70 is reduced by only 0.03 (from 7.17 to
7.14) when the fractional extracellular water increases from 0.2 to 0.3. At very
low distribution ratios (i.e., where pHi G pH, for weak acids, or pHi B pH, for
weak bases), however, the effect of variations in extracellular space can be
considerable. For instance, in barnacle muscle, at a pH, of 8.0 and a distribution
ratio of 0.35, a fractional extracellular water content of 0.2 yields at pHi of 7.25,
and a fractional extracellular water content of 0.3 gives a pHi of 6.78.
5) The dissociation constant of the indicator in intracellular water is un-
known although it is assumed to be the same as that in extracellular water.
Equations 3 and4 show that any error in intracellular pK produces an identical
error in derived pHi. Differences in pK may be due to differences in ionic
strength, ionic composition, or both. The finding that the pK of DMO in 160-
600 mM NaCl is .02- .07 lower than in KC1 of the same molarity (46) may suggest
sensitivity to the cationic species, whereas its reduction with increasing NaCl
(46) and KC1 concentrations (265) might be due to either effect.
6) Weak acid and base methods yield an average pHi. It is well recognized
that there are striking differences in the pH of various membrane-bound
organelles. Thus the pH of the mitochondrion may be several units greater
than that of the lysosome (see sect. X). In a pre vious review Caldwell (74), as-
suming uniform intracellular distribution of the uncharged partner, concluded
that the distribution of weak bases between the cell’s interior and external
fluid reflects the mean H+ concentration of the cell and that the distribution
of weak acids reflects the mean OH- concentration. According to Robson
et al. (364), the weak acid distribution reflects a form of the harmonic mean intra-
cellular H+ concentration. Although such conclusions are arithmetically correct,
statistical refinements of this kind are not permissible: the modern operational
definition of pH does not treat pH as the negative logarithm of H+ concentra-
tion or activity (see sect. 0). One can, however, define the pHi derived from
weak acid or base distributions in terms of the pH values (rather than the
H+ or OH- concentrations) of the cellular subcompartments. Let it be
assumed that all membranes are permeable only to the uncharged species
whose intracellular concentration is therefore uniform and equal to that in
extracellular water. The total concentration of a weak acid in the water of the
jth subcompartment, [TA]j, is, according to the Henderson-Hasselbalch
equation (see Eq. 2)

iTAl, = [HA] l ( 10”Hj-PK + 1) (V

where pHj is the pH of the subcompartment. Thus the amount of TA in the

water volume (vj) of the subcompartment is

TA j = Vj[HA] l ( 10PHj-P” + 1) (8)

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306 A. ROOS AND W. F. BORON Volume 61

The mean concentration of TA ([TA],) in the total cell water (v,> is therefore

[TAl,JEi i Vj*(lOPHj-PK + 1) (9)

vt j=l

where n is the number of compartments. The average pHi derived from this
concentration is

= pK + log -*
Substituting Equation 9 into Equation 10 and rearranging yields

PHacid = log i fj. 10PHj


where fj = vJvt, the fractional volume of the jth compartment. A similar cal-
culation applied to weak bases yields

= -log 2 fj* 10mpHj (121

We can conclude from Equations 11 and 12 that both pH,,id and $Ibase are
independent of pK, and that pH,,id > IZbase, as revealed by a comparison of
the two expressions. Thus whenever there is intracellular pH inhomogeneity,
acids always give a higher pHi than bases. This has been previously
stated with elaboration by Waddell and Bates (454).
It is interesting to compare these two measures of overall pHi with the
volume-weighted average pHi, defined as

pHi = i fj*pHj

A calculation shows that pHhase and pH,,id always bracket this average pHi,
that is, pH,,id > pHi > pHbase. A numerical example for a two-compartment
system is given by Siesjo and Ponten (409).
?‘J A crucial assumption in the derivation of Equations 3-6 is that
[HAli = [HA], for weak acids and that [B]i = [B], for weak bases. These
equalities are valid only if there is a steady state of indicator distribution
across the plasma membrane and if the membrane is impermeable to the charged
form. If the charged form can traverse the membrane, however, the calculation
of pHi from the distribution of a weak acid or base may be erroneous, as il-
lustrated in the following example. Let a weak acid HA be equilibrated across
a cell membrane that is impermeable to A-. Then [HAli = [HA],, and the pHi

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FIG. 1. Passive movements of charged and uncharged forms of weak acids and bases. A: weak
acids. Under normal conditions of V,, pHi, and pH,,, the electrochemical gradient of the anionic
partner (A-) of the conjugate weak acid (HA) favors net efflux of A-. As [A-Ii decreases, the
intracellular equilibrium HA s H+ + A- shifts to the right, thereby lowering [HAli and leading
to influx of HA. Net result is inward shuttling of protons. A steady state can be maintained if
H+ is extruded as rapidly as it is shuttled in; then the influx of HA exactly balances the efflux of
A-. If H+ is in equilibrium across the membrane, A- is also in equlibrium, and there is no net
flux of either A- or HA. B: weak bases. Under normal circumstances, the electrochemical gradient
of the cationic partner (HB+) of the conjugate weak base (B) favors net influx of HB+. As [HB+]i
increases, the intracellular equilibrium HB+ e H+ + B shifts to the right, leading to efflux of B.
Net effect is inward shuttling of protons. Steady-state and equilibrium considerations are analogous
to the case for weak acid. [From Boron and Roos (46).]

can be calculated from Equation 3. Now let the membrane also be made per-
meable to A-. Under conditions of normal pHi, pH,, and V,, A- leaves the
cell. As illustrated in Fig. lA, the efflux of A- moves the reaction HAi + Hr
+ Ai to the right, thereby reducing [HA]1 below [HA],. The greater the
discrepancy between these two concentrations, the greater will be the error in
calculating pHi from Equation 3. The discrepancy increases with increasing
membrane permeability to A- (P,) and with decreasing permeability to HA
(PHA). The efflux of A- and reduction of [HA]1 cause [TA]i/[TA]” to fall below
its ideal value. If this reduced ratio is unwittingly inserted into Equation 3,
an erroneously low value for pHi results. For a weak base, permeability to
BH+ leads to the entry of this ion (under normal conditions), which in turn drives
the reaction BH: L- Bi + Hr to the right (see Fig. 1B). Thus [B]i is raised above
[B], and [TB]i/[TB], is greater than the ideal value. Insertion of this higher
ratio into Equation 4 again results in an erroneously low calculated pHi*
Roos (365, 368) and Boron and Roos (46) have expressed these considera-
tions quantitatively, on the assumptions that the net passive flux of the uncharged
partner is governed by the Fick equation and the next flux of the charged partner
is given by the constant-field treatment (147, 191). (For the mathematical ex-
pressions of these assumptions, see sect. vB.) In the steady state, the sum of the
two fluxes equals zero. It can be shown that under these conditions the distri-
bution ratio for a weak acid is

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308 A. ROOS AND W. F. BORON Volume 61

[TAl i [H+]i + K R[H+], + aK

-wu, = [H+], + K l R[H+]i + PK
where R = Pf.JPA, a = FV,I{ RT [l - exp( -FV,IRT)] } , and p = (FVJRT)
exp( -FV,/R T )/[ 1 - exp( -F&JR T )].

For weak bases

VBli [H+]i + K R’K - r[H’l,

-[TBIO = [H+]i + K
R’K - S[H’li

where R’ = PB/PHB, y = FV,I{RT [l - exp(FV,/RT)]}, and 6 = (FVIRT)

exp(FV,/RT)/[l - exp(FVJU’)]; F is the Faraday constant, T is the absolute
temperature, V, is the membrane potential, and K is the apparent acid dissocia-
tion constant.
WhenV, = 0, thena! = p = y = d = 1, and Equation 14 reduces to that
of Irvine et al. (206), and Equation 15 to that of Orloff and Berliner (321)

kTAli [H]i + K+ R[H+], + K

-[wo = [H+], + K l R[H+]i + K
iTBli [H+]i + K R’K - [H+],
-= [H+],+K ’ R’K-[H+]i
One other special case might be mentioned, namely that in which H+ is in
equilibrium across the membrane. If only HA is permeant (i.e., [HAli = [HA],),
and K is the same on both sides of the membrane, then we have from the
mass-action law
[H+],[A-1, = K[HA], = K[HA]i = IH+Ii[A-Ii U8)

[H+]i/[H+]”= [A-],/[A-]i uw
It follows that the electrochemical gradient for A- is equal and opposite to
that for H+. Because H+ is in equilibrium, A- also must be in equilibrium.
In the theoretical case in which only A- is permeant, this ion will equilibrate
across the membrane. Because H+ is also in equilibrium, Equation 18a again
applies, and thus [HAli = [HA],. In summary whatever the permeabilities
are of HA and A-, both species will equilibrate when H+ is at equilibrium.
Similar reasoning can be applied to weak bases. It follows that for cells such
as nonnucleated erythrocytes, in which H+ is normally at equilibrium (132,
139, 193, 446; see sect. Im5a), the calculation of pHi from the distribution

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of a weak acid or base is unaffected by ionic permeability, and Equation 3

or 4 can be applied (365).
Even though permeability to the charged form of a weak acid or base is a
potentially serious source of error in estimating pHi from the weak electrolyte’s
distribution, the error in most cases is rather small. A permeability to the
charged form as high as 0.005 of that to the neutral form leads to an under-
estimation of the true pHi by only a few hundredths (46, 365), provided the
indicator’s pK is within l-2 units of the prevailing pHi and pH,. The com-
monly used weak acids (i.e., CO, and DMO) satisfy this requirement, but
weak bases often do not. For instance, in the case of methylamine (pK = 11.8),
the neutral molecule accounts for less than 0.01% of the total indicator at physio-
logic values of pH, that is, [HB+] B [B]. Here even a relatively low permeability
to HB+ leads to [B]i being substantially greater than [B], and thus to a sig-
nificant error in calculated pHi, when Equation 4 is applied (46).
Only limited information is available on the membrane permeability of weak
acids and bases. Keifer and Roos (227) recently found in barnacle muscle a
permeability of 1.9 x 10s4cm/s to the molecular form of DMO and a permeability
of 1.5 x low7 cm/s to the DMO ion, assuming the fiber to be a cylinder. Thus
the ratio of permeabilities ( PHAIPA) is about 1,000. This ratio leads to the
DMO-derived pHi being only about 0.01 lower than the true pHi (46). In rat
diaphragm muscle, the distribution ratio of D-lactate was found to be only 0.65
of that for DMO (368). If only the lactic acid molecule were permeant (and if
this indicator were subject to none of the other pitfalls listed in this section),
the two ratios should have been about equal (see Eq. 5; pHi = pH, % pK).
The observed discrepancy is consistent with a ratio, PAIPHA, for lactic acid of
about 3 x 10m4. Note that as with methylamine discussed above, the pK of
lactic acid (-3.7) is far removed from the physiologic pH range; thus the effects
of a given ionic permeability on the distribution ratio are greatly exaggerated.
Some degree of lactate ion permeability was also found in the electrical studies
by Woodbury and Miles (482) of frog sartorius muscle. Evidence for ionic
permeation of weak acids and bases has also been obtained in experiments
in which the slow fall in pHi caused by the influx of HB+ or the efflux of A- was
monitored using pH-sensitive microelectrodes or other techniques (see sect. V).
Data consistent with a significant permeability to HCO, have been gathered on
rat (7, 8, 232), mouse (lo), and frog (35, 209) skeletal muscle; on crab (9) and
barnacle (37, 44) muscle; and on sheep heart Purkinje fibers (122). In view
of the high permeability to molecular COP, however, it is doubtful that under
most conditions permeability to HCO,? vitiates the pHi derived from the total
CO, distribution. In the case of NH,+, evidence for permeation has been
obtained in the squid axon (42), snail neuron (429), barnacle muscle (37),
mouse soleus muscle (lo), and crayfish muscle (300). In barnacle muscle (46),
a slow decline in the DMO-derived pHi was observed during prolonged ex-
posures to 10 mM methylammonium, suggesting a substantial permeability
to the methylammonium ion. (An analysis of these pHi transients is given in
sect. v.) The increase of 0.3-0.4 of the nicotine-derived pHi observed by Brown

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310 A. ROOS AND W. F. BORON Vdume 61

and Halliwell (55) in the rat superior cervical ganglion upon depolarization
([K+], raised from 6 to 124 mM) is far too great to be accounted for by ionic
8) Weak acids and bases can directly affect pHi by two mechanisms. First,
the entry of the neutral form decreases pHi in the case of weak acids and
increases it in the case of weak bases. These effects, which are dependent
on concentration, pHi, and pH,, are considered in detail in section IV. Second,
as mentioned above, the efflux of the anionic form of weak acids or the entry
of the cationic form of weak bases leads to a slow fall of pHi. These effects,
which are dependent on concentration, membrane permeability, and V,, are
examined in section V.

5. Results obtained with weak acid and base methods

A considerable amount of data is available from experiments in which weak

acid or base methods were compared with other techniques of pHi measure-
ment. Studies on organelles are reviewed separately in section X.
a) Evaluation of weak acid and base methods in erythrocytes. In non-
nucleated red cells, the distribution of weak acids and weak bases should yield
the same value for pHi, and the H+ distribution should be the reciprocal of that
of Cl-, because these cells are homogeneous and because the permeant
ions Cl- and HCO; (and thus H+) are distributed according to a Donnan
equilibrium. The distribution of the Cl- and HCO; ions has been shown in electro-
lyte studies (167a, 446), including those in which the distribution of the
permeant anions was changed by raising the intracellular concentrations of
nonpermeant organic anions (119). The existence of a Donnan distribution was
confirmed in studies in which V, (measured with fluorescent probes) was com-
pared with Cl- distributions (193). In two studies (132, 139) on human red cells,
the pHi derived from the distributions of CO, (pH-CO,) and Cl- (pH-Cl) closely
agreed over a wide range of pH, values (6.4-8.6). Thus for oxygenated cells in
bicarbonate buffer (pH,, = X4), pH-CO, was 7.23 and pH-Cl was 7.22, whereas
pHi directly measured in the hemolysate (pH-hem) (see sect. IIA) was 7.20
(132). Sanslone and Muntwyler (387) found excellent agreement between pH-
DMO and pH-Cl for rat cells suspended in a bicarbonate buffer (at pH,
7.40, pH-Cl was 7.16 and pH-DMO was 7.18), and Calvey (78) made a similar
observation for rabbit red cells suspended in phosphate buffer. Nine com-
parisons (mostly in human erythrocytes) are available in which equilibrations
with CO, and/or DMO were carried out in vivo; the results were usually com-
pared with pH-hem. In four of these (58, 111, 338, 417), there was excellent
agreement between pH-hem and either pH-CO, or pH-Cl, and in another
(363) between pH-DMO and pH-Cl. In the remaining four studies (53, 330,344,
437), pH-hem differed by 0.05-0.16 from the other measurement (pH-CO,,
pH-Cl, or pH-DMO). In three in vitro studies on human red cells (pH, = 6.9-
8.0), in which pH-DMO was compared with the pHi derived from the distribu-

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tion of a weak base, ammonia (36), or nicotine (54, 466), the agreement was
good. Thus at pH, 7.47, pH-amm was 7.25 and pH-DMO was 7.29 (36), whereas
at pH, 7.34, pH-nit was 7.06 and pH-DMO was 7.10 (466), and at pH, 7.26, pH-
nit 7.08 and pH-DMO was 7.15 (54). We conclude that the weak acid and base
methods applied to nonnucleated red cells yield consistent and reasonable
results that agree with those derived from the chloride distribution.
The situation in nucleated cells is different. When pH-NH3 and pH-DMO
were simultaneously measured in avian (cockerel) erythrocytes in bicarbonate
buffer, pH-DMO was greater than pH-NH, over the entire pH, range studied
(6.9-7.8; 36). Thus at pH, 7.40, pH-DMO was 7.31 and pH-NH3 was 7.16. The
authors’ interpretation is based on the inhomogeneity of these cells. Both pH-
DMO and pH-NH, of isolated red cell nuclei suspended in phosphate buffer
(PH 0 = 7.10) were 6.98. If we assume, with the authors, a relative nuclear
volume of 0.5, then a cell with a uniform extranuclear pHi of 7.10 and a nuclear
pHi of 6.98 yields a pH-DMO of 7.040 and a pH-NH, of 7.036 (Eqs. 12 and
13). In actuality, at a $I-DMO of 7.04, the measured S-NH, was only about
6.9. Thus intracellular inhomogeneity, by itself, seems insufficient to explain
the difference between the two pHi values. The possibility of NH,+ permeabil-
ity of the plasma membrane was not considered. If the equilibrium potential for
H+(EH) is greater than V, (neither V, nor Cl- distribution was measured),
permeability to NH,+ may lead to erroneously low values for pH-NH3, which
could account for the remainder of the discrepancy.
b) Comparison of CO, and DMO methods in muscle and nerve tissue. In
skeletal muscle under normal conditions, the CO, method always gives higher
pH values than the DMO method. Thus in the isolated rat diaphragm (pH,
= 7.4, PC02 = 40), pH-CO, was 7.03 and pH-DMO was 6.89 (293). The dis-
crepancy increased when pH, was raised to 7.93 by reducing PcoB (pH-CO2
was 7.55 and pH-DMO was 7.10) but was reduced to zero when pH, was
lowered to 6.93 by raising Pco2 (both methods yielded a value of 6.72). Dis-
crepancies of 0.1 were also observed under normal acid-base conditions in
skeletal muscle (in vivo) of the rat (17) and dog (89). Reeves (350) found more
striking differences in sartorius and gastrocnemius muscles of bullfrogs ac-
climatized at temperatures between 5 and 28°C: pH-CO, was about 0.5 higher
than pH-DMO over the entire temperature range. Some possible explanations
for these discrepancies come to mind. I) The plasma membrane and/or the
membrane surrounding some organelle may be permeable to the DMO ion.
This would produce an erroneously low pH-DMO if EH were greater than
V,, which is generally the case for the plasma and mitochondrial (see sect. X)
membranes. 2) Cellular Pco2 may exceed the measured extracellular Pco~,
which would lead to an erroneously high pH-CO,. The effect of such a difference
is greatest at low Pco2. 3) The intracellular pK of DMO may exceed the extra-
cellular value; the converse might be the case for CO,. 4) There may have been
technical problems with the CO, method. It was pointed out by Ponten and
Siesjo (339) that unless special precautions are taken, CO, from the air con-
denses on frozen tissue specimens, thereby increasing their apparent pH-CO,.

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312 A. ROOS AND W. F. BORON Volume 61

Two closely similar in vivo studies on rat brain allow comparison between
the CO, (237) and the DMO (366) methods. The agreement was within 0.04 over
the pH, range studied (7.2-7.8).
c) Comparison of CO, and creatine phosphokinase methods. In a study on
rat brain, Siesjo et al. (407) compared pH-CO, (taking extracellular volume
as 15%, a reasonable value) with pHi derived from the creatine phosphokinase
equilibrium. Extracellular volume enters the latter method only in the pre-
liminary calculation of the equilibrium constant. The agreement between the
two methods was within 0.01 over the tissue Pco2 range of 45-266.
d) Evaluation of lactate and pyruvate methods. Granholm et al. (153, 155)
compared, in cat and rat brains, lactate and pyruvate as possible pHi indicators
with HCO;. On the assumption that only the undissociated forms were
permeant and that the measured concentrations in cerebrospinal fluid
were representative of those in the interstitial fluid, the transmembrane con-
centration ratios for the three ions should have been identical. At arterial
PCO~of 34, however, the distribution ratio in cat cortex was 1.51 for lactate,
3.43 for pyruvate, and 1.91 for HCO;. Similar differences were encountered
in the rat brain.
Roos (368) compared the distribution of DMO with that of D-lactate in the
rat diaphragm in vitro (pH, = 7.4, Pco2 = 40) and found a pH-DMO of 7.10
and a pH-lactate of about 6.94. He interpreted this difference as suggestive
of membrane permeability to the D-lactate ion.
e) Comparison of DMO and spectroscopic methods. Thomas and co-
workers (428) compared the pHi derived from DMO distribution with that
measured spectroscopically in Ehrlich ascites tumor cells after the cells were
exposed to the diacetate derivative of 6-carboxyfluorescein (CF). This
colorless molecule diffuses into the cell, after which the chromophore CF is
released. Its absorption spectrum is a function of pH. At pH, 7.4, the DMO-
derived pHi was 7.45 and the CF-derived pHi was 7.4. The agreement was
equally good at a pH, of 6.2 (7.15 and 7.1, respectively) and after rotenone,
valinomycin, or nigericin had been added to the medium.
f) Evaluation of methods using other weak bases. In experiments on rat
diaphragm muscle (pH, = 7.39, Pco2 = 30-38), Adler (3) obtained values of
7.17 t 0.06 for pH-DMO but only 6.69 t .07 for pHi derived from the distribu-
tion of nicotine (pH-nit). He ascribed this difference to intracellular com-
partmentalization, but intracellular binding or metabolic transformation of
nicotine cannot be ruled out (see sect. IB~c). The pHi difference persisted
under a variety of acid-base and osmotic conditions (5, 6). When the tempera-
ture was reduced from 38 to 25 or 4°C (4), the discrepancy diminished. This
effect may at least partly be explained by a reduction of the transmitochondrial
pH difference, which has been observed in rat skeletal muscle mitochondria
as a consequence of cooling (414). Yet damage to compartmental boundaries
cannot be ruled out, since the reversibility of the effect was not tested. A
puzzling feature in Adler’s (4) results was the constancy of pH-nit (at 38°C)
when PCO* was raised from 36 to 148, whereas pH-DMO fell as expected.
This insensitivity of pH-nit to Pco2 disappeared at reduced temperature.

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After correcting for bound nicotine, Weiss (469) found that pH-nit of frog
sartorius muscle equals pH-DMO. Adding as little as 0.08 mM nicotine to the
medium, however, raised pH-DMO by 0.2-0.3, and the addition of 2.5 mM
nicotine produced a further rise of 0.1. Calculations show that given a reason-
able intracellular buffering power, the action of nicotine as a weak base can
account for only a small fraction of this pHi increase. On the other hand, Hannan
and Wiggins (167), also working with drog sartorius muscle (pH, = 7.4, 2O”C),
obtained results comparable to those of Adler (3) in mammalian muscle: pH-
DMO was 7.10, pH-nit was 6.92, and pH-morpholine was 6.99.
In experiments in which isolated rat superior cervical ganglia were incu-
bated in HCO;-Ringer’s solution (pH, = 7.37) containing 30 PM nicotine,
Brown and Halliwell (55) obtained values of 7.18 and 6.54 for pH-DMO and
pH-nit, respectively. When 2.5 mM hexamethonium was added to block the
nicotine-induced depolarization, the two values increased to 7.29 and 6.63, re-
spectively. This pH-DMO closely resembled that obtained in the absence of
nicotine (7.33). At [K+], = 124, pH-DMO was not affected by nicotine, and
hexamethonium was without effect on pH-nit (the effect on pH-DMO was not
studied). These data suggest, in contrast to the authors’ conclusion, that the
reduction of pHi with nicotine is due to depolarization. The question of why
nicotine in frog muscle raises pH-DMO (469) but lowers pH-DMO in the ganglion
remains unresolved. Brown and Garthwaite (54) measured in rat ganglia a pH-
DMO of 7.31 t .003, whereas pH-nit (in the presence of 2.5 mM hex-
amethonium), pH-atropine, pH-morphine, pH-trimethylamine, and pH-pro-
Caine all ranged between 6.4 and 6.6. Raising the nicotine concentration from
0.3 to 210 PM had no effect on pH-nit. Uncouplers of oxidative phos-
phorylation [2,4-dinitrophenol (DNP) and carbonylcyanide m-chlorophenyl-
hydrazone (CCCP)] greatly reduced the difference between pH-DMO and
pH-nit, but respiratory inhibitors (antimycin A, rotenone, and cyanide) were
without effect. These data suggest that the difference between pH-DMO
and the weak base-derived pH values is due to compartmentalization. Un-
couplers, by increasing proton conductance of the membranes separating
the subcompartments, reduce regional pHi differences. In agreement with
these conclusions, pH-DMO and pH-nit of human red cells were nearly
the same, whereas the difference between the two in the brown fat of cold-
stressed rats (which is abundant in mitochondria) amounted to more than one
unit. The DNP reduced the difference to about 0.4. Discrepancies between
pH-DMO and pH-nit have also been described by Putney and Borzelleca (345)
in experiments on slices of rat submaxillary gland. At pH, values of 6.80,
7.40, and 8.00, pH-DMO was 6.72, 7.24, and 7.82, respectively, whereas
pH-nit was 6.23, 6.45, and 6.66.
g) Comparison of DlWO and microelectrode methods. The DMO and
microelectrode techniques have been directly compared in two studies. Boron
and Roos (46) used the two techniques to measure pHi in squid giant axons
and giant barnacle muscle fibers bathed in CO,-free media. For the axon
(PH = 7.7, 22”C), the pH-DMO was 7.36 t 0.025 (n = 6) and the pH-elec was
7.35& 0.006 (n = 126). Results of the barnacle experiments (pH, = 7.2, 7.8,

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314 A. ROOS AND W. F. BORON Volume 61

or 8.0) also showed that pH-DMO and pH-elec are very similar, although
the latter always exceeds the former by 0.05-0.06. For instance, at a pH, of 7.8,
pH-DMO was 7.29 t 0.009 (n = 27), and pH-elec was 7.34 t 0.025 (n = 6). The
distribution of methylamine was also used to compute pHi (pH-MA) of barnacle
fibers. The pH-MA values were on the average 0.09 lower than the pH-DMO
values; comparison of pH-MA with pH-elec confirmed the sequence pH-elec
> pH-DMO > pH-MA. The authors pointed out that the methylamine tech-
nique, in addition to yielding rather low estimates for pHi, requires a pro-
longed incubation time to ensure equilibration of the indicator.
Hinke and Menard (188) also compared the DMO and microelectrode tech-
niques in barnacle muscle. They found that the DMO distribution reaches
a stable value after about a l-h incubation but that the apparent extracellular
space, measured with [3H]inulin, continues to drift upward after its initial
rapid rise, the latter lasting about 45 min. This drift is probably an artifact due to
the uptake of low-molecular-weight, labeled inulin fragments (see 272). By back-
extrapolating to zero time, the authors properly obtained the correct extra-
cellular space, which, taken with the average of the DMO distribution values
obtained after 1 h, would have yielded the correct pH-DMO. The authors
calculated a number of pH-DMO values from the simultaneous DMO and
inulin measurements during, the 3 h of incubation, however. The supposedly
correct pH-DMO was then obtained either by back-extrapolating the pH-DMO-
regression line to zero time or by averaging all pH-DMO values after 45 min.
The first method should yield an erroneously high pH-DMO, since the negative
slope of the regression is almost certainly caused by the spurious increase of
the extracellular space. As for the second method, since the pH-DMO values
after 45 min were all derived on the basis of too high an extracellular space,
their mean must be too low. Indeed the first method yielded pH-DMO values
that were 0.1-0.2 higher than pH-elec, whereas the relationship was reversed
with the second method. The correct pH-DMO would therefore have agreed
rather well with pH-elec.
Hinke and Menard applied a similar analysis to studies in which they exposed
fibers to either 5% CO, (pH, = 6.2) or to 5 mM (NH&SO, (pH = 8.2). Both
treatments led to a progressive fall in the DMO distribution, beginning after
20-30 min of exposure. This most likely reflects a true decline of pHi, due to
HCO; efflux in the first instance and to NH,+ influx in the second (see
sects. v and VIII). The authors’ microelectrode records also showed these down-
ward drifts of pHi. Since the fibers were not in the steady state, application
of the DMO technique is of questionable value.

6. Novel applications of weak acid and base methods

a) Mean transit time of DMO. Effros and colleagues (120) calculated the
pHi of in situ dog myocardium from the mean transit times through the heart of
each of four isotopes that were injected in known amounts into the descending

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coronary artery and serially sampled from the great cardiac vein. The mixture
consisted of ‘251-albumin (indicator of plasma water volume), 51Cr-EDTA
(indicator of plasma water plus interstitial volume), 3H,0 (indicator of total
water volume), and [14C]DM0. From the dilution profiles, corrected for
recirculation, the four mean transit times were derived and from them the
steady-state distribution ratio of DMO could be calculated. This ratio, in
combination with the extracellular (plasma) pH obtained by direct measure-
ment, yielded the pHi in the usual way (Eq. 5). The technique permits repeated
measurements to be made. At an arterial pH of 7.39 and a PCO~of 33, pHi was
found to be 6.94 t 0.07 (n = ZO), a reasonable value. Studies were also per-
formed after periods of ischemia and after injection of HCl and Na,C03.
b) DMO washout. Poole-Wilson (340) derived pHi from the washout of
DMO from the isolated, arterially perfused and rhythmically stimulated rabbit
ventricular septum (at 26°C). He interpreted the two slowest of the four com-
ponents into which the washout curve could be resolved as representing wash-
out of DMO from the intracellular space and calculated a pH-DMO of 7.23 t 0.05
(n = 11). In separate experiments he also determined pH-DMO in the con-
ventional fashion, obtaining a value of 7.28 t 0.02 (n = 5).
c) Autoradiogv-aphy with DMO. Lyman and Waddell (265) used auto-
radiography followed by photometric densimetry to measure the [14C]DM0
concentrations in different regions of teeth of lo- to 11-day-old mice. Total water
content of the regions was similarly measured on a second group of animals
with N-[ 14C]acetyl-4-aminoantipyrine. Both compounds were injected intra-
peritoneally, and their densities in the teeth were compared with those in the
blood. On the assumption of a blood pH of 7.35, the pH of the soft tissue of the
teeth (such as the central pulp) was 7.0-7.2, whereas that of the calcified areas
(such as dentine, enamel, and bone) was 7.3-8.5.
d) Transient pHi measurements with DMO. Roos and Boron (370) moni-
tored changes in the pH-DMO of rat diaphragms at 30-min intervals. The
changes were produced by first acid-loading the tissues for 2 h in media contain-
ing either 5% CO, or 118 mM DMO and then returning them to the original
solutions while maintaining pH, at -7.4 throughout. Upon acid-loading, pH-
DMO fell and then partially recovered. When the diaphragms were returned
to the original solutions, pH-DMO overshot the control value. This pattern of
pH-DMO changes is the same as that of the pHi changes measured with micro-
electrodes in other types of cells (see sect. VIII).
e) External NH,+ electrode. Rottenberg and Grunwald (374) suspended
chloroplasts in a well-buffered medium containing very small amounts (2O-
40 PM) of NH: and followed the course of external NH,+ activity with an
NH,+-selective electrode. Since the electrode was also sensitive to Na+ and
K+, these ions were omitted from the medium. They found that on illumination,
[NH,+], falls and then levels off. From this final value, the magnitude of the fall,
and pH,, the authors calculated the pH of the chloroplasts (pH,) prevailing at
the end of the illumination period as follows. Both in the medium and in the
organelles, the equilibrium NH,+ L, NH, + H+ holds. Before illumination

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316 A. ROOS AND W. F. BORON Volume 61

[NH,], equals [NH,],, since the thylakoid membrane is permeable to NH, but
not to NH,+. With illumination the pH, falls (see sect. x); this leads to a
reduction of [NH,], and thus to an influx of NH3, which in turn reduces
[NH&. When a steady state has been reached, [NH,]i once more equals
[NH,], and thus [NH&/[NH,+], = [H+],/[H+],. If the osmotic volume of the
chloroplasts relative to the volume of the medium is known, [NH,+], in the new
steady state can be calculated from the reduction in [NH,+],, provided
[NH,+], during control conditions (i.e., in the dark) is negligible. Since this
was the case, [H+], could be obtained from the above equation. The method
has some limitations: 1) transient pH, changes cannot be measured, since only
in the steady state does the transmembrane [NH,+] ratio equal that of
[H+]; 2) the method requires a closed system, since the total ammonia
(NH,+ + NH,) must remain constant; 3) as used in this study, the method is
very insensitive due to the relatively small volume occupied by the chloroplasts
(0.1% of the total volume); and 4) the massive entry of NH, into the chloroplasts
and its subsequent protonation to NH,+ (about 35 mM NH: accumulated dur-
ing illumination) should produce a substantial increase in pH, by itself. There-
fore the transmembrane pH gradient (pH, - pH,) produced by illumination
may well have been greater than the value of 3.4 reported by the authors.
f) Fluorescence quenching. In 1972 Deamer et al. (110) used fluorescence
quenching to determine the pH gradient across the liposome membrane, and
Schuldiner et al. (392) used this approach to measure the fall of the pH of
chloroplasts when they were illuminated. The fluorescence of atebrin and 9-
aminoacridine, both weak bases (BH L, B + H+), is supposed to be quenched
100% when these probes are inside chloroplasts. Before illumination most of
the probes are outside, and the level of fluorescence is relatively high. Upon
illumination pHi falls, and inside the chloroplasts the reaction [BH+], L, [B],
+ [H+]i shifts to the left, lowering [B], and causing uncharged amine to enter
the chloroplast where its fluorescence is quenched. Net uptake of amine from
the medium and thus the distribution ratio from which pHi can be derived
(see sect. III?) is calculated from the decrease in fluorescence. The assump-
tion that internal amine is quenched 100% is probably valid, since the relative
fluorescence of the suspension drops to nearly zero when its concentration of
chloroplasts is sufficiently high. Other assumptions are that initial [BH+], - 0,
that the chloroplasts maintain constant volume, that pH, is constant, and that
the total amine in the system remains constant. Although initial [BH+], is
never zero, typical light-induced decreases of pHi are so large (e.g., 3 pH
units) that the change in [BH+], is far larger than the initial level. In general
fluorescence quenching has the disadvantage that the ability to detect transient
pHi changes is limited by the diffusion of the neutral form of the probe, since
pHi can be calculated only in the near-steady state ([B]i = [B],). Also the tech-
nique is rather insensitive, the smallest detectable pH gradient being about a
full pH unit, i.e., 0.2-l% quenching. If the initial pH gradient is very large
(e.g., pH, = 8.0, pHi = 5.0), however, then much smaller pHi changes can be
detected. It is evident that fluorescence quenching of weak bases is useful for

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measuring increases in pHi only if there is a significant initial organellar con-

centration of the charged form of the probe.

C. Calorimetry and Fluorometry

1. Introduction and historical perspective

De Vries (115) in 1871 was the first to observe color change of a naturally
occurring intracellular pigment, in this case a betacyanin, resulting from an
alteration of pHi. When he exposed tissue slices of red beets to a solution of
NH3, their red color turned to brown, then to yellow. In 1915 Willstatter
and Mallison (478) extracted and chemically identified the colored substances
in the petals of many flowers. These amphoteric anthocyanins are generally
red in acid solutions and blue in alkaline ones. The authors ascribed the color
variations of various flowers to differences in pHi rather than in chemical
structure of the pigments. Three years later, Crazier (105) observed the pH-
dependent color changes of pigments extracted from various sponges and from
the nudibranch Chromodoris zebra and estimated the pHi of the intact cells by
color. In 1920 (211) and 1922 (212), Jacobs observed the changes in color of flower
petals when he exposed them to alkaline or acid solutions. Since he was mainly
interested in using the pHi changes as the index of the membrane’s permeability
to acids and bases, he made no attempt to establish absolute pHi values (for
details see sect. II??).
The application of foreign dyes dates back to 1893 when Metchnikoff (286)
observed that particulate litmus ingested by protozoa changes in color from
blue to red. He postulated that this intracellular acidity is responsible for the
death of ingested organisms. Similar observations were made on other species
(for references see 261). Another approach was initiated in 1906 by Loeb (258),
who, stimulated by Ehrlich’s application of dyes to biological systems, supra-
vitally stained the eggs of the sea urchin (S. puripuratus) with neutral red and
then monitored pHi changes associated with fertilization. The same dye and
biological preparation were used 4 years later by Warburg (464) to show that
NHS, but not NaOH penetrates into the eggs, turning them from red to yellow.
Later Harvey (168) used neutral red to study the entry of a number of amines
into plant and animal cells, and Jacobs (212) used the dye to study the effect
of exposure to NaHCO, and NH&l on starfish eggs.
Since most dyes are impermeant, ways were studied of introducing
the indicators directly into the cell. In 1913 Kite (236) injected small drops of
solutions of azolitmin, alizarin Na-sulfonate, tropeolin ooo no. 1, and other
dyes into living Amoeba proteus and estimated that the organisms have a
“neutral to slightly alkaline reaction.” This seems to have been the first reason-
ably precise pHi measurement by any method. A more primitive method for
introducing the dyes was that used by Vies and his group (451). They partially
crushed the cell in drops of dye; this drew the indicator into the cell, which was

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318 A. ROOS AND W. F. BORON Volume 61

then rapidly washed and its color examined. In 1925 Needham and Needham
(309), using an approach similar to that of Kite, obtained a value of about 7.6 for
the pHi of Amoeba proteus. Schmidtmann (388, 389), at about the same time,
made extensive use of the injection method. She introduced microscopic dye
granules into various mammalian cells and found a pHi of 6.6-6.8 for
skeletal muscle and heart muscle fibers from a number of mammals. Sig-
nificantly she observed that skeletal muscle pHi remains unchanged when the
external pH is raised from 5.2 to 8.0 (phosphate buffers) and that on staining
with neutral red, a narrow red zone often appears around the nucleus while
the rest of the cytoplasm stains yellow (i.e., more alkaline). This difference in
staining is not due to damage to the nucleus, since it remains unstained unless
its membrane is punctured. No attempt was made to prevent loss of CO, from
her preparations. Other early work with dyes has been summarized by
Caldwell (74).
After the early 1930’s, interest in dyes as pHi indicators waned. This may
partially have been due to the increasing popularity of the CO, method, but
the realization must also have dawned that color indicator methods have serious
limitations. Metachromatic, protein, and salt errors complicate absolute de-
terminations of pHi; moreover, the very presence of a dye can alter pHi. Never-
theless the dye technique offers several advantages. It can be used with small
cells or even with organelles such as lysosomes (218) and mitochondria (85). Also
modern spectrophotometric methods have allowed greater sensitivity with
lower dye concentration. Finally, the nearly instantaneous response of dyes
permits the study of rapid changes in pHi, a feature that has been used in the
study of pHi transients during muscle contraction and gastric acid secretion.

2. Direct application of pH-sensitive dyes

In two careful studies, Piontek and Herbst (176, 337) photometrically

measured both the resting pHi of frog skeletal muscle and directional changes
in pHi occurring during isometric contraction. The muscle (semitendinosus)
was transilluminated in a direction parallel to its fibers. This longitudinal
illumination has the advantage that the muscle can be kept in its natural state,
exposed to the bathing solution, in contrast to the arrangement in the conven-
tional transverse illumination in which the muscle must be flattened between
optical surfaces. Moreover the total thickness of membrane crossed by the light
beam, and thus artifacts due to membrane-bound dye, are minimized. In
earlier work (for references see 337) no account was taken of the possible
presence of some of the dye in the extracellular space. To cancel the effect of
extracellular dye, the present authors “plugged” the extracellular spaces with
3 mM bromcresol purple (BCP). Preliminary work (337) had shown that the
application of this dye to the muscle produces an exponential fall in transmitted
light to about 2% of the initial value. Since a l-mm layer of 3 mM BCP absorbs
more than 99% of the incident light, the authors concluded that BCP had

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blocked the extracellular but not the intracellular light paths (otherwise
transmittance would have fallen to zero). The authors then compared the
transmittance in two groups of muscles. Both had been treated with BCP, but
in addition one had also been equilibrated with bromthymol blue (BTB). The
reduced transmittance in the latter group must have been due solely to the
intracellular presence of BTB. The (unknown) concentration effect of BTB
on transmitted light intensity was canceled by taking the difference between
the reduction of transmittance at two wavelengths. To increase accuracy,
measurements were made at two pairs of two wavelengths. The data were
compared with measurements on muscle protein-containing solutions of BTB
of different pH values (175, 176). The pHi of frog muscle in bicarbonate-
Ringer’s solution (pH, of 7.2, 2% COz, T = 4°C) was found to be 7.1 t 0.1 (9), a
reasonable value. The authors also carried out experiments during isometric
contraction (176). In these studies no BCP was used (after the muscles had been
stained with BTB, they were soaked in Ringer’s solution to remove extra-
cellular dye). Since in dyefree studies the reduction of transmitted light
(due to iricrease in scattering) with contraction was largely independent of
wavelength, the authors used, in the stained muscle, the difference in trans-
mitted light at two wavelengths as an index of directional changes in pHi; no
absolute calibration was attempted. Piontek and Herbst (176) found that dur-
ing the rise in tension, pHi always falls. At the start of relaxation, pHi began
to return to the resting value, but this was not always reached by the end of
relaxation. In successive twitches, there were small but systematic changes
in the time course of tension and of pHi. Treatment with iodoacetate caused
pHi to overshoot transiently the resting value early in relaxation.
More recently MacDonald and Jobsis (266) reported their spectrophoto-
metric observations on pH changes in frog sartorius muscle during isometric
contraction. The muscles, loaded either with BCP or with neutral red (NR),
were “held firmly in place” between glass plates and transversely trans-
illuminated. During contraction the authors observed optical changes with
both dyes in the direction of a fall in pH. Since the absorbance shifts of the dyes
with pH were in opposite directions, the findings were interpreted as a fall in
pHi: movement artifacts should have either increased or decreased the ab-
sorbance of both dyes. However, we must question the authors’ claim that
“since the pHi shifts exhibit the same general form, either dye may be used to
examine relative shifts in pHi.” By their own admission and on the basis of
prior evidence (337), BCP remains outside the fibers. This is in agreement
with their findings that the time course of return to resting absorbance was
different for the two dyes. Neutral red does enter the fiber, but at least in the
squid axon, it is also attached to the membrane, as shown by the voltage de-
pendence of its fluorescence (95). It is difficult to evaluate the efficacy of the
optical measures taken to cancel the effects of increased scattering during
contraction, since no control studies were performed in which these same meas-
ures were applied to unstained preparations. The authors used a technique of
averaging the signals from 20 successive twitches; thus systematic shifts in

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320 A. ROOS AND W. F. BORON Volume 61

optical behavior previously reported by Herbst and Piontek (176) would not be
apparent. The conclusions about the time course of pHi during a muscle
twitch (especially during the relaxation phase) and about the effect of metabolic
inhibitors must therefore be accepted with caution.
We should mention some dynamic dye studies, carried out by Hersey
on the pHi of the acid-producing cells of the stomach. He used either BCP
(178, 181) or BTB (179, 180) to estimate changes in the pHi of frog gastric
mucosa. The dyes were incorporated into the tissue during a 1- to 2-h
incubation (dye on serosal side) followed by a rinse. There is no assurance that
BCP can enter the cells any more than it can enter muscle fibers (see above),
but the author provided indirect evidence (179) that some of the BTB was
located intracellularly. He assumed that at least part of it was in the acid-
producing cells and found that intracellular binding of BTB resulted in a shift
of its apparent pK, from 6.95 to 8.00 (180). Both BCP (178) and BTB (180)
indicated an alkaline shift at the onset of acid secretion; the average pHi of
all mucosal cell types could exceed a value of 8 (180). The carbonic anhydrase
inhibitor acetazolamide (Diamox), added to the serosal side of the preparation,
enhanced this shift, which was followed in 5- 10 min by a diminution of acid
excretion (178, 181). Both thiocyanate and imidazole inhibited the theophyl-
line-stimulated acid secretion of the stomach, but only thiocyanate prevented
the rise in pHi monitored with BTB (179).

3. Fluorescent probes phagocytixed by cells

This technique was introduced by Ohkuma and Poole (319) in their 1978
study of intralysosomal pH and exploits the pH sensitivity of a fluorescent
marker, fluorescein isothiocyanate-labeled dextran. This compound was
excited at wavelengths of either 495 or 450 nm, whereas the fluorescence
intensity was measured at 519 nm. The ratio of intensities produced by the
two excitations was taken as an index of pH. This fluorescence-intensity ratio
is only slightly affected (corresponding to pH changes of ~0.1) by ionic
strength (O-100 mM NaCl), the presence of protein (200 mg/ml of bovine
serum albumin), or buffer composition [lo mM lactate, acetate, phosphate,
borate, or 2-amino-2-hydroxymethyl-1,3-propanediol (Tris)]. Although not
verified by the authors, the plot of the ratio versus pH seems to be a simple
pH titration curve with an apparent pK of about 5.8. This implies that the
fluorescence-intensity ratio is a measure of the fraction of probe molecules in the
form of the conjugate base. Ohkuma and Poole (319) exposed macrophages
to the fluorescein isothiocyanate-labeled dextran and observed that this
material is endocytosed and remains intact inside the lysosomes (see sect.
XC). The fluorescence-derived pH is 4.75 t 0.01 (n = 38) at 37°C and, as
expected, reversibly increases during exposures to weak bases such as NH,
and methylamine. Advantages of the fluorescence technique are that it is non-
invasive, nondestructive, and not extremely expensive. The authors followed
rapid changes in pH, though with some sacrifice in accuracy, by monitoring

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fluorescence while exciting only at 495 nm. (The ratio method requires about
2 min.) The fluorescence-ratio technique is reported to be accurate to within
0.1-0.2 pH units, although no results obtained from other methods (under
similar conditions) are available for comparison. At present this particular
fluorescence method can be used only to study intralysosomal pH in cells that
phagocytize dextran. For this purpose, however, it is of great value, since the
lysosomes being studied remain inside living macrophages.

4. Spectroscopic probes generated in situ

Thomas and colleagues (428) exposed Ehrlich ascites tumor cells to the
diacetyl derivatives of the chromophores fluorescein or CF. In contrast to the
deacetylated compounds, these derivatives, which are uncharged and color-
less, readily diffuse into the cells (pH, must be lowered to 6.2 for CF), where
the acetate groups are cleaved by native enzymes. Especially for fluorescein,
the efflux of the deacetylated probe is a difficulty. At 2O”C, 90% of this com-
pound was lost within 60 min at pHi 7.4; 90% was lost in only 10 min at pHi
6.2, suggesting that it is the protonated form that leaves the cell. On the other
hand, only about 20% of the CF (which carries an additional negative charge)
was lost in 60 min at pHi 7.4. A second advantage of CF over fluorescein
is that it is sensitive only to the bulk intracellular pH, whereas the latter also
senses mitochondrial pH.
Both the absorbance and fluorescence spectra of the two probes are pH
sensitive. In extracellular fluid the ratio of absorbances at 490 and 465 nm fol-
lowed a simple pH titration curve, pK - 6.5. Since intracellular binding was
suspected, intracellular calibration of CF was also carried out; nigericin and
high [PI, were used to collapse the pH gradient across the cell membrane.
The discrepancy between the intra- and extracellular calibration curves was
substantial at low pH. Under a variety of conditions, the values obtained by
the DMO and spectroscopic methods agreed very closely (usually within 0.05).
When cells were exposed to glucose or lactic acid, the CF-derived pHi re-
sponded as anticipated (i.e., it reflected a decrease in pHi).
A disadvantage of this technique is that because of backleak from the
cells, some lo-20% of the CF in a sample is extracellular. This can be corrected
for, however, by centrifuging cells and determining [CF] in the supernatant.
The method offers the advantages that rapid pHi transients can be readily
followed and that even pHi shifts of less than 0.01 can be detected.

D. Microelectrode Methods

I. Introduction

The microelectrode techniques requires that the cell be impaled by two

microelectrodes (or one double-barreled electrode), one reversible to H+ and the

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322 A. ROOS AND W. F. BORON Volume 61

other serving as an indifferent reference. The voltage difference between the

two electrodes (usually obtained electronically) is a linear function of PHi. The
voltage difference between the internal reference electrode and an extra-
cellular reference electrode is the membrane potential (V,), usually recorded
simultaneously with the PHi. Several varieties of pH-sensitive electrodes have
been used, including platinum-hydrogen, antimony, tungsten, pH-sensitive
glass, and pH-sensitive liquid ion-exchange resin. In commercial macroscopic
systems, the pH electrode is glass, whereas the reference is calomel contact-
ing the test solution by a saturated KC1 salt bridge. The pH of a solution x,
measured with any of these electrodes, is calculated according to the

pH(x) = pH(s) + (E, - E,)*FI(RT In 10) (19)

where pH(s) is the value assigned to a standard solution, E, is the voltage dif-
ference between the pH and reference electrodes in solution x, and E, is the
voltage difference in the standard solution; F is the Faraday constant, R is the
gas constant, and T is absolute temperature. In practice the electrode’s slope
(i.e., voltage change per unit change in pH) is determined empirically,
since it is often less than the ideal Nerstian value of FI(RT In 10). Note that
Equation 19 represents an operational definition of pH in which the scale of
acidity and akalinity is relative; it provides no precise information about H+
ion activity (aH). Such an operational scale is used because of the theoretical
impossibility of determining the activity coefficient of the Cl- in the pH standard
solutions. The U. S. National Bureau of Standards has provided a series of six
primary pH standards, each a well-defined, dilute solution of simple electro-
lytes. For these solutions, the activity coefficient for Cl- has been somewhat
arbitrarily defined by a convention based on the Debye- Huckel theory. Thus the
PH( s) of these solutions is defined as PaH. If the composition of a test solu-
tion matches the composition of one of the standards, then it is possible to
calculate a value for aH that has quasithermodynamic validity (i.e., the
measured pH approximates pa& For intracellular pH, such a maneuver is
probably never valid: the intracellular fluid differs greatly from the standards
and moreover is not a simple solution. In conclusion an intracellular measure-
ment of pH should be regarded only as a relative index of cellular acid-
base balance. For an authoritative discussion of the theory and measure-
ment of pH, the monograph by Bates (23) should be consulted.
As stated above, the measurement of PHi requires that both pH-sensitive
and reference microelectrodes be placed within the cell. Serious difficulties
arise when the pH-sensitive electrode is not entirely within the cell; these
are discussed in section VII. It is also important that no significant voltage
drop occurs between the pH -sensitive an.d reference el.ectrodes; that is , the two
should be placed as closely together as possible. For a multicellular prepara-
tion, for which it may be difficult to be certain that the two electrodes are located
in the same cell, Aickin and Thomas (10) have suggested a method to

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ensure proper electrode placement. One first introduces the pH electrode,

waits until its voltage is stable, and then introduces the reference electrode.
The second impalement usually results in a short-lived decay of V,. If both
electrodes are in the same cell (and therefore sensing thesame V,), the electron-
ically subtracted difference between their voltages should not change during
this slow drift in V,, since pHi remains constant.
With cells too small to accommodate both electrodes at the same time,
sequential impalements may have to be made or two cells may have to be
impaled, each by one electrode. This clearly is not ideal. Ellis and Thomas
(122, 123) impaled separate cells in mammalian Purkinje fibers (which are
electrically coupled) with pH and reference microelectrodes and found that
depolarization with KC1 (which should not alter pHi) alters the measured
pHi only very slightly, indicating that the V, changes by about equal amounts
in nearby cells. Simultaneous impalements of separate cells have also been made
successfully with isolated proximal tubules of the salamander kidney (40), where
the cells are probably also well coupled electrically. Note that this approach
does not require that the cells be tightly coupled, but only that they have
identical membrane potentials.
An alternative to impaling cells with two microelectrodes is to use a double-
barreled electrode. Three types have been described, one based on pH-sensi-
tive glass (113), another on antimony (2’78), and a third on a pH-sensitive liquid
ion exchanger (277). Disadvantages of a double-barreled electrode are that it
has a larger tip diameter than the corresponding single-barreled electrode
and that it is more difficult to make.

2. Metal pH-sensitive electrodes

a) Hydrogen. The hydrogen electrode, the first pH-sensitive electrode

used in biological work, is made by bubbling hydrogen gas over a metal
capable of catalyzing the reaction

H+ + e- e %Hz (g) (W

Platinum black is the most effective catalyst, although palladium black, bright
platinum, palladium, and iridium may also be used (23). Although the hydrogen
electrode is the ultimate pH standard, it has several disadvantages. I) The
reversibility of the reaction of Equation 20 is inhibited by cyanide, hydrogen
sulfide, arsenic compounds, and by cations of metals such as silver and mercury
(23). 2) The electrode is sensitive to several oxidizing agents (nitro-
phenols, benzoic acid, and other aromatics). 3) The developed potential is
sensitive to the partial pressure of hydrogen. Because of its slow response,
the electrode must be held in the test solution for a significant length of time, and
gases such as 0, or COZ, when present, may displace the hydrogen. In 1927
Taylor and Whitaker (427) succeeded in measuring the vacuolar pH of NiteIZa

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324 A. ROOS AND W. F. BORON Vohme 61

with a miniature platinum-hydrogen electrode. They obtained a value of 5.5-

6.6, about a unit higher than is now generally accepted.
b) Antimony. The basis for the antimony electrode’s usefulness as a pH
sensor is thought to be an oxidation-reduction reaction following a thin layer of
antimonous oxide on the electrode’s surface (23)

Sb,O,(s) + 6H+ + 6e- L, 2Sb(s) + 3H,O (21)

Unfortunately this reaction is not completely reversible. In addition the

electrode is sensitive to the condition of the metal surface, oxidizing and
reducing agents, various anions (23) including phosphate, and ionic strength
(156). The calibration slope is apparently pH sensitive (30), possibly because dif-
ferent buffers are used for different pH ranges. Therefore the electrodes must
be calibrated in solutions resembling the test solution as closely as possible (72).
The first to use an antimony electrode to measure pHi were Buytendijk and
Woerdeman (67), who in 1927 used it with fertilized and unfertilized eggs of
amphibians and chickens. In 1972 Bicher and Ohki (30) used antimony micro-
electrodes (consisting of a KCl-filled glass pipette coated with antimony and
insulated except at the extreme tip) to estimate pHi in squid giant axons. They
found a value of - 7.0, which is 0.3-0.4 lower than values obtained with pH-
sensitive glass electrodes. Finally Matsumura and colleagues (278) used a
double-barreled antimony microelectrode to measure pHi in frog sartorius
muscle and proximal tubule cells. The values obtained (7.12 and 7.49,
respectively) agree reasonably well with those arrived at by other methods. We
conclude that these electrodes may be useful for measuring pHi, provided that
they are meticulously calibrated and that experimental maneuvers directly
affecting electrode behavior are carefully avoided.
c) Tungsten. The pH sensitivity of tungsten is probably due to a surface
coating of the oxide (23), as is the case for antimony. A miniature version of this
electrode was used by Caldwell (73) in 1954 in his study of pHi in crab muscle
fibers. The electrodes often gave about the same value as glass electrodes, but
sometimes the results were bizarre. The electrodes had slopes of 40-50 mV/pH
unit between a pH of 4.0 and 9.2 and, as expected, were sensitive to oxidizing
and reducing agents. Contamination with copper caused the electrodes to
acquire some Cl- sensitivity. Thus Caldwell concluded that tungsten electrodes
were not suitable for pHi measurements.

3. Glass pH-sensitive microelectrodes

a) Introduction. The pH sensitivity of soft glass was first noted in 1906

by Cremer (103) and further characterized in the 1920’s by Hughes (202) and by
MacInnes and Dole (267). These authors demonstrated that the voltage across a
thin layer of soda-lime glass closely resembles that of the platinum-hydrogen
electrode. Somewhat later, MacInnes and Dole (268) found that of the glasses

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available, Corning’s 015 was optimal for pH measurements (low asymmetry

potential, relatively low resistance, low alkaline error). Since that time,
however, further refinements in glass composition have been made (see 23). In
1927 Furusawa and Kerridge (140) used a pH-sensitive glass electrode to
measure the pH of cell lysates as an estimate of muscle pHi* Five years later,
Voegtlin and Kahler (453) reduced the size of the glass electrode sufficiently
to permit pH measurements of the extracellular fluid of solid tumors.
In view of the difficulties inherent in the use of metal electrodes, it is under-
standable that pH-sensitive glass electrodes have been very popular. The vol-
tage across a thin membrane of Corning 015 glass responds to changes in pH
over a wide range, having a slope greater than 99% of the theoretical value
between pH values of 1 and 10 (23). The glass electrode is insensitive to oxidizing
and reducing agents, and although the liquid junction potential of the reference
cell is by its very nature sensitive to changes in solution composition, the
glass electrode itself is not sensitive to anions, dissolved gases, or changes in
buffer composition. It displays excellent stability, has a reasonably rapid re-
sponse time (which depends on electrode geometry), and is easily cared for.
The only drawbacks of glass microelectrodes are that at high pH they are sensi-
tive to the cations of alkali metals (especially Nat) and that their high
resistance requires the use of a high-impedance electrometer for the voltage
b) CaLdwelL’s electrode. It was not until 1954 that Caldwell (73) became the
first to use pH-sensitive glass electrodes intracellularly. The pH-sensitive
glass of his electrode was 50-80 pm in diameter and was exposed for
the terminal 0.5 cm; the remainder was covered with insulating glass and
shellac. The electrodes, which sometimes had a reference electrode attached
beside them, were inserted longitudinally into crab muscle fibers (diam - 600
pm) and yielded pHi values of - 6.9. Even in these large cells, the electrode
must have done some damage, however, since Caldwell observed opacities and
contractures in the muscle, and the membrane was very often depolarized beyond
-40 mV. The electrodes seem to have produced less injury in squid giant
axons (diam - 500 pm), in which Caldwell (75), in 1958, obtained values of V,
that were generally more negative than -50 mV. In 1960 Spyropoulos, using
a pH electrode of similar size, found an average V, of about -58 mV in
squid axons (420).
c) Hinke’s electrode. In 1967 Hinke (186) produced a pH-sensitive glass
microelectrode consisting of an insulating micropipette made of lead glass
(Corning 012) through which protruded a length of ion-sensitive glass (Corning
015) about 20 pm in diameter and 150 pm long (see Fig. 2A). He had previously
used a similar design for Na+- and K+-selective microelectrodes (185). This style
of electrode is rugged, responds within 1 s, and has been successfully used to
measure pHi in muscle fibers of the giant barnacle (diam - 500 pm) (37,44-46,
48, 188,227,280) and squid giant axons (42,43,379). Hinke and colleagues (188,
279b) took special care to minimize tissue damage in their experiments on barnacle
muscle fibers, cannulating the tendon of the vertically mounted fiber, introduc-

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326 A. ROOS AND W. F. BORON Volume 61


pH -sensitive
glass ----

FIG. 2. Exposed-tip (A) and recessed-tip (B) microelectrodes. With exposed-tip micro-
electrode, designed by Hinke (186), entire pH-sensitive surface must be introduced into the cell.
With recessed-tip microelectrode, designed by Thomas (429, 434), only extreme tip need be
within cell. [From Boron (39).]

ing the pH electrode through the cannula, and then advancing it for at least 1
cm. A conventional microelectrode was then introduced radially at the level of
the pH electrode tip. An alternative approach (42) is to mount barnacle muscle
fibers or squid axons horizontally and cannulate them at both ends. pH-sensitive
and open-tipped reference electrodes are introduced through cannulas and
advanced for about 2 cm so that their tips nearly meet at the center of the cell.
The cannulated ends of the fiber are isolated by grease seals from the super-
fused central region. Althou gh Hi nke- style electrodes cannot be used with small
cells, these electrodes remain the preferred method for squid axons and
barnacle muscle.
d) Kostyulc-Sorolcina electrode. In 1961 Kostyuk and Sorokina (241) de-
scribed a miniature version of the Hinke electrode, with a tip fine enough
to radially impale frog muscle fibers (diam - 100 pm). The pHi value they
obtained, -7.12, has been verified by other techniques. The exposed end of pH-
sensitive glass (like that shown in Fig. 2A ) was lo-20 pm long and “some tenths
of a micron” in diameter at its tip. The electrodes were made by inserting a
length of pH-sensitive glass tubing into soft, insulating glass tubing (composi-
tion not given), cementing one to the other, and then drawing them out together.

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Although the authors did not comment on their success rate in fabricating their
microelectrodes, in our laboratory this has been extremely low. This is probably
the major reason for this electrode’s failure to gain widespread favor.
e) LavaWe’s electrode. In 1964, Lavallee (248) proposed a microelectrode
system for measuring pHi, consisting of two sharp, open-tipped microelectrodes,
one made of borosilicate (Pyrex) glass and the other of uninsulated pH-sensitive
(Corning 015) glass. Both were filled with 0.5 M KCl, and had similar tip
diameters (-0.5 pm) and resistances (30-100 Ma). Each electrode was
sensitive to K+ (-36 mV/lO-fold change in K+ activity), but pH sensitivity was
confined to the 015 glass pipette. Thus when paired, the electrodes produced a
voltage that presumably was sensitive only to pH. Probably because of the pH
electrode’s K+ sensitivity, the slope was only about half the theoretical ‘value,
about 30 mV/unit change in pH. The response was linear between pH 2 and
8.5. Lavallee used this system in stimulated (2OO/min) rat atria1 muscle and ob-
tained a pHi of 6.92 t 0.015 (n = 15) at 30°C a reasonable value. The some-
what disturbing possibilities exist that the pH and Pyrex electrodes do not have
exactly the same sensitivity to K+ or that this sensitivity is altered on im-
palement. The system’s sensitivity to ions other than K+ is not known. Although
the precise basis of the pH sensitivity is unclear, breaking off the electrode’s
terminal 2-3 pm eliminates the pH response. Lavallee has suggested that the
specificity for H+ arises as a result of fixed charges on the inner surface of the
glass electrode.
f) ELectrode ofCarter et al. In 1967 Carter and colleagues (82) described an
electrode made by insulating pH-sensitive glass tubing with glaze and then
drawing the tubing out into micropipettes. The electrodes had very fine
tips (~1 pm) and, according to the authors, were insulated except for the
terminal 5-20 pm. The electrode’s behavior, however, as tested in mammalian
and crab muscle, was bizarre (82, 326), and it must be concluded that the
electrode was so poorly insulated that an appreciable fraction of the pH-sensing
surface remained outside the cell. This topic is considered in detail in section VII.
g) Thomas’ microelectrode. The introduction of the recessed-tip micro-
electrode by Thomas (429) in 1974 represents the most significant methodo-
logical advance in recent years in the intracellular pH field. The electrode,
illustrated schematically in Figure 2B, is constructed by inserting a closed-tip
pH-sensitive glass micropipette into an open-tipped micropipette made of
insulating glass (typically aluminosilicate with a very high melting point) until
the tips are close together and then forming a tight seal between the two by
applying high pressure to the inside of the pH pipette while applying local heat
with a microforge. Since it melts at a much lower temperature than the
aluminosilicate, the pH glass is blown out against the insulating glass. The result
is a pH electrode whose pH-sensitive tip is recessed with respect to its open-
tipped glass sheath. This design offers several advantages: I) only the open tip
of the insulating glass need enter the cell, 2) the tip is fine enough ((1 pm) to
permit the impalement of fairly small cells, and 3 ) the electrodes can be manu-
factured with relatively little waste. Also the electrode can be made in the

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328 A. ROOS AND W. F. BORON Volume 61

double-barreled configuration, as described by De Hemptinne and Marrannes

(113) for use with rat soleus muscle. A more complete description of this
electrode and its applicat,ions has recently appeared (llla). The major draw-
back is a rather long response time, which varies directly with the recessed
volume and inversely with the sq uare of the diameter of the insulati ng pipette’s
tip opening. Thus if the response is to be kept reasonably short [Aicken and Thomas
(10) recommend a 90% response within 1 min after a solution change], that is, the
recessed volume must be minimized, a requirement that becomes increasingly
important as the tip size is reduced. So far it has been possible to retain an
acceptable response time while reducing the tip diameter of single-barreled
electrodes sufficiently (~0.5 pm) to permit impalement of mouse soleus muscle
fibers, and cells of the salamander proximal tubule (40). Further miniaturization
may be hampered by the inability to further lessen the recessed volume or, if this
is possible, by the reduction of the surface area of pH-sensitive glass to such an
extent as to raise the electrode’s electrical resistance (already loll- 1012a) to
unacceptably high levels. The trade-off between response time and tip diameter is
especially acute for double-barreled electrodes. These difficulties might be
lessened by using a pH-sensitive glass with a lower resistivity (such as uranium
glass; see 23), by decreasing the thickness of the pH-sensitive glass mem-
brane, or by sharply beveling the tip and thereby increasing the area for diffusion.
The construction of recessed-type electrodes is described in detail in a recent
monograph (434).
h) Pucacco-Carter electrode. Introduced in 1976 (343), this electrode over-
comes the difficulty of inadequate insulation by confining the pH-sensitive
surface to the extreme tip. A very thin (-0. 1 pm) membrane of p H- sensitive
glass (Corning 015 or urani urn oxid .e) is form .ed and then softened bY heating.
A boro- or aluminosilicate micropipette, beveled to 2-200 pm at the tip, is then
advanced through the softened pH glass so that the latter completely covers its
tip. The electrode can easily be made in the double-barreled configuration. The
surface area of exposed PH glass is so small that the el.ectrical resistance,
response time, and noise can be expected to be high. The authors found that
electrodes with 4- to 8-pm tips required l-3 min to respond to solution changes,
whereas those with 2-pm tips required 5-35 min.
i) Yamaguchi-Stephens electrode. This is a recessed-tip microelectrode
(485) constructed much like the electrode of Kostyuk and Sorokina (241). A
length of pH-sensitive glass tubing is placed inside a piece of soda-lime glass
tubing, the two are glued together, and then drawn out together. According
to the authors, placing a few strands of fiberglass inside the pH tubing before
pulling causes the pH glass tip to seal spontaneously and also produces a glass-
glass seal with the outer glass (like that shown in Fig. 2B). Other workers have
been unable to successfully duplicate this process, despite significant effort.
j) Hollande r’s electrode. No details of design or construction are available
(194) for Hollander’s electrode except that the electrodes were fabricated from
Corning 015 glass and had tip diameters of 0.06 pm (!). In mammalian cardiac

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muscle they yielded an average pHi of 6.96 t 0.04 (n = ISO), a reason-

able value.

4. Liquid ion-exchange microelectrodes

a) HCO; exchanger. A liquid ion-exchange microelectrode consists of

a micropipette siliconized at its tip so that it can retain a small volume of
organic solvent in which the exchanger is dissolved. In 1974 Khuri and co-
workers (230, 231) used a 3:1:6 mixture of tri-n-octylpropylammonium chloride
to octanol to trifluoroacetyly-butylbenzene as an HCO;-sensitive exchanger.
The sensitivity of the electrode to HCO; was over 50 times greater than that
to Cl- or H,PO;, and the sensitivity to OH- reportedly was low enough not
to be a problem at pH values less than 8. This type of microelectrode is po-
tentially useful due to its small size (5 l-pm combined tip diam for a double-
barreled electrode, the second barrel being the reference), rapid response time
(~1 s), and ease of construction. The electrode is sensitive to Pco~, however,
so that the calibrating solution must have the same PCO~as the test solution.
Also pHi must be less than 8 to minimize the OH- error, and since [HCO,]i
varies with pHi at constant Pcop, the absolute magnitude of the Cl- error varies
with pH. Khuri and colleagues have used these electrodes to measure steady-
state [HCO,]i in skeletal muscle fibers of frog (231) and rat (231, 232), and
their estimates have corresponded to reasonable values of pHi.
b) H+ exchanger. Very recently Matsumura et al. (277) have described
a double-barreled, pH-sensitive microelectrode based on a nigericin-
containing liquid ion exchanger. In the pH range 5-9, the microelectrodes
have a linear response and an average slope of about 53 mV. The deviation at
extreme pH values and the electrode resistance were unspecified. Repro-
ducibility is t 1 mV, and the response time is a few seconds. Although the
electrode’s response to Na+, KS, Ca2+, and Mg*+ is only 2-3 mV/lO-fold change
in ion activity, the sensitivity to NH,+ is substantial: 14 mV/lO-fold change
above 10 mM. The values for pHi obtained with this electrode, 6.98 t 0.12 for
frog sartorius muscle and 7.36 t 0.12 for the renal proximal tubule of Nectums,
are reasonable. Because these electrodes should be easy to make and use, even
in the double-barreled configuration, they hold great promise for the future.

E. 31P Nuclear Magnetic Resonance Spectroscopy

In 1973 Moon and Richards (302) employed 31Pnuclear magnetic resonance

(NMR) spectroscopy to estimate the intracellular pH of intact erythrocytes.
Since that time several other investigators have extended NMR methods to
yeast (386), bacteria (308), various lymphoid and tumor cells (306,307), isolated
rat liver cells (98), whole skeletal muscle of rat (199) and frog (62, 108, log),
whole perfused rat heart (141, 214, 215), and other tissues. Since NMR

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330 A. ROOS AND W. F. BORON Volume 61

spectroscopy is inherently nondestructive, the time course of a changing pHi

can be followed as can the time course of intracellular concentrations of several
phosphorus-containing metabolites. The application of NMR spectroscopy to
intact cells has been the subject of several reviews (61, 63, 346, 401). As NMR
spectroscopy is unfamiliar to most physiologists, a brief introduction is in order.
The technique exploits a quantum mechanical property called spin,
possessed by several atomic nuclei, among them 31P. The spin generates a
magnetic moment along the axis of the spin, causing the nucleus to behave like
a tiny bar magnet. When such a nucleus is placed in a magnetic field, the
magnetic moment precesses like a top about the axis of the applied field. The
angle the precessing moment makes with the applied field is quanta1 and for
31P is designated +1/Z (moment aligned parallel to field; low-energy state) or
- l/2 (moment aligned antiparallel to field; high-energy state). The distribution
between the two energy states is of the Boltzmann type. The frequency of the
precessing moment is proportional to the magnetic field strength at the nucleus.
This local field is the vector sum of the applied field and the fields generated
by electrons near the nucleus. Thus there is a unique local magnetic field and
precession frequency for each chemically distinct 31Pnucleus. When a nucleus is
irradiated at a radiofrequency (RF) equal to its precession frequency, it ab-
sorbs energy or “resonates,” and the population’s distribution moves away
from a Boltzmann distribution as more nuclei are in the higher energy state.
In practice 31PNMR spectra are obtained by the pulsed Fourier transform
method. Here the sample is irradiated with an intense multifrequency pulse at
intervals of about 1 s. The pulse is constructed such that all 31Pnuclei are stim-
ulated simultaneously. After each pulse they undergo a relaxation process
in which a time-dependent signal, called the free induction decay, is emitted.
The average of several hundred of these decays is then converted by Fourier
transform to an NMR spectrum containing peaks for each chemically distinct
31P nucleus. The separation between these peaks is termed the chemical shift,
since it has as its origin the differences in electron configuration, or chemistry,
affecting the various nuclei. Chemical shift (6; given in ppm) is defined as
0-h - vref)/vo, where v, and vref are the frequencies, in Hz, of the sample and
reference peaks, respectively, and v, is the operating frequency, in MHz. The
total time required for a detailed spectrum can be as little as 1 min (442).
31P Nuclear magnetic resonance spectroscopy owes its usefulness for
estimating pHi to the pH sensitivity of the chemical shifts of several phos-
phorus-containing compounds. The most commonly used of these is inorganic
phosphate (Pi), which is generally present in a high enough concentration to
produce an easily detected peak and which has an apparent pK, in the physio-
logic range of pHi. In muscle the chemical shift (relative to that of phospo-
creatine, which is rather insensitive to pH changes in the physiologic range) of
the acidic form of Pi (H,PO,) is -3.35 ppm (-430 Hz for a 129-MHz RF
signal), whereas that of the basic form (HPO$-) is -5.6 ppm (-720 Hz) (108).
Since the exchange rate between individual H2P0; and HPOi- ions (log-

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lOlo s) is far greater than the frequency separating the peaks (-310 Hz),
the signals merge as a single peak whose chemical shift is the concentration-
weighted average of the chemical shifts of the two members of the conjugate
pair. Thus a typical titration curve is obained when the chemical shift of Pi is
plotted against pH

0 + 3.35
PH = 6.9 + log - (22)
56. -CT

where 6.9 is the p& for Pi, and u is the observed chemical shift (c < 0).
Assuming that such an in vitro standardization curve is applicable to
intracellular fluid, pHi can readily be obtained from the chemical shift of
intracellular phosphate.
Nuclear magnetic resonance spectroscopy offers several advantages for
measuring pHi. First, the technique is noninvasive and nondestructive, so that
the course of pHi over a period of time can be followed. Second, the concentra-
tions of various phosphorus-containing metabolites (i.e., ATP, ADP, phospho-
arginine, phosphocreatine, and sugar phosphates) can simultaneously be
followed. Third, the technique is relatively fast. Although a detailed spectrum
(necessary for determining metabolite levels) may require several minutes,
a pHi determination (for which only the position of the Pi peak is needed) can
be obtained in as little as 10 s (346), although 1 min is more representative.
Fourth, the sensitivity of NMR spectroscopy for detecting changes in pHi is
high. Modern spectrometers can resolve chemical shifts of 0.03 ppm (R. Shul-
man, personal communication), which corresponds to about 0.02 pH units at
values near the pK (the pH resolution, of course, decreases as pH deviates
from pK). Finally, the method can be applied to organelles and small cells and
may also provide some insight into cellular compartmentation (65, 394, 401),
as discussed in sect. 1123.
There are also limitations to the use of NMR spectroscopy for monitoring
pHi. The technique requires a great deal of instrumentation, and to avoid
technical pitfalls, the operator must be thoroughly versed in its theory and
application. Perhaps the most serious drawback is that the absolute amount of
intracellular Pi in the sample must be sufficient for the peak to be detected.
Since the intracellular concentration of Pi is in the mM range, the cells must
be tightly packed. As a result, addition of nutrients and removal of wastes
may be insufficient, and the experiment may be performed under unphysio-
logic conditions. Another potential difficulty is that the calibration curve ob-
tained in vitro may not be applicable intracellularly, an objection that can only
be dealt with by directly comparing the NMR-derived value with an established
technique (see below). Finally, on rare occasion the Pi peak may be
buried by that of another compound with a similar chemical shift. For example,
Colman and Gadian (99) found that in early toad embryos the Pi peak was

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332 A. ROOS AND W. F. BORON Volume 61

masked by resonances readily identified as being due to the dominant yolk

Values for pHi obtained in NMR studies are fairly reasonable, though
unfortunately NMR spectroscopy has not been directly compared with other
methods. Data are available for a few indirect comparisons. In rat leg muscle at
2O”C, Hoult et al. (199) found a pHi of 7.1, which is not too far below Aickin and
Thomas’ microelectrode value of 7.23 for mouse soleus muscle at 28OC(10). The
muscle in the NMR experiments, however, was packed into a closed NMR tube,
whereas that in the microelectrode studies was superfused with Ringer’s solu-
tion equilibrated with 5% CO,-95% 0,. Burt and colleagues (62) measured the
pHi of frog skeletal muscle by NMR, also confining the tissue to a closed tube. To
correct for tissue deterioration, they extrapolated pHi values back to zero
time, obtaining a value of 7.21. In contrast to the muscles in the previous
studies, the toad muscle used in the NMR experiments of Dawson et al. (108)
was superfused with Ringer’s solution equlibrated with 100% 0, (pH, = 7.7,
4°C). Their pHi value of 7.5 compares with the frog muscle value of 7.05, ob-
tained by Bolton and Vaughan-Jones (35) with microelectrodes at 25OC (pH,
= 7.2). The value obtained by Bolton and Vaughan-Jones would probably have
been about 0.4 higher at 4”C, assuming the temperature sensitivity of pHi
parallels that of the pH of neutral water (see sect. VIB) and thus is in reasonably
close agreement with the NMR-derived pHi. Dawson et al. (108, 109) observed
a rise in pHi of about 0.2 immediately after contraction had begun, possibly
due to phosphocreatine breakdown. This alkalinization was followed by
acidification; eventually pHi returned toward normal. Finally, Jacobus et al.
(214) used NMR to obtain a pHi of 7.18 in isolated, perfused rabbit heart at
37OC. In comparison Neely et al. (310) and Steenbergen et al. (423) obtained
values of 7.05 and 7.02, respectively, on isolated perfused rat heart with the
DMO method, while Ellis and Thomas (122) used microelectrodes in obtaining
a value of 7.14 for isolated rat and ferret ventricular muscle. In the perfused rat
heart, the NMR methods gave a somewhat higher value of 7.4 (215).
It is clear that if the reliability of the NMR method for pHi measurement
is to be firmly established, the biological preparations must be examined
under more physiologic conditions than in the past. Failure to observe a dif-
ference between pHi and pH, in some studies (306, 307) may have been due to
accumulation of metabolites in a closed NMR tube. Therefore continuous
superfusion, as done by Dawson et al. (108, log), is essential. The NMR method
should also be evaluated by examining pHi responses to various manipulations
(e.g., variations in the CO, and NH, concentration) whose effects on pHi have
already been studied by other methods. Even under similar conditions, com-
parisons between the NMR and other methods should be made with caution,
since the different techniques measure different parameters. Microelectrodes
measure the pH of the bulk intracellular fluid (in immediate contact with the
electrode), as presumably does NMR. On the other hand, DMO and other weak
acids and bases provide some average pH of the entire intracellular content.
Therefore apparent discrepancies may be reconcilable.

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TABLE 1. Neurons and nerve fibers

Tissue Met hod ture, “C PI-L Buffer pHi Comments Ref.

Squid axon (L. forbesi) el( Cw) 7.4-8.05 Unspecified (no CO,) 7.10 2 0.028 (29) 75
Squid axon (L. pealeii) eKS> 7.95 Seawater 7.25-7.45 (65) v, = -55 to -61 420
el( Sb) 7.8? Seawater 7.0 + 0.2 (7) No change with high [K+], 30
or pH, 5.0 or 9.0
e1U-U 23 7.6-7.8 5 mM Tris-HEPES 7.32 + 0.013 (22) V, = -56.7 t 0.7 42
el( H) 22 7.70 5 mM Tris-HEPES 7.35 + 0.006 (126) If,= -57 46
DMO 22 7.70 5 mM Tris-HEPES 7.36 _+ 0.025 (6) Sorbitol for extracellular
Snail neuron el(W 7.81 7.26 + 0.10 v, = -59.7 242, 418
(H. pomatia)

Snail neuron cl(T) 8.0 20 mM Tris-maleate 7.41 + 0.015 (32) v, = -44.2 xt 0.76 429
(H. aspersa)

Barnacle photoreceptor cl(T) 7.2 (8) Dark-adapted for 0.5 h; 57

(B. eburneus) after illumination, pH i
= 7.0-7.1
Rat superior cervical DMO 22-27 7.37 HCO,-5% CO, 7.33 + 0.018 (10) Mannitol 55
ganglion Nicotine 22-27 7.37 HCO,-5% CO, 6.54 (8) Mannitol
DMO 25 7.4 HCO,-5% CO, 7.31 + 0.003 (46) Medium contained 2.5 mM 54
Nicotine 25 7.4 HCO,-5% CO, 6.51 + 0.011(36) hexamethonium; pHi
measured with atro-

Copyright © 1981 American Physiological Society. All rights reserved.

pine, morphine, and
trimethylamine were
nearly identical

Numbers in parentheses are number of studies. el(Cw), Caldwell-style glass electrode; cl(S), Spyropoulos-style glass electrode; cl(H), Hinke-style
glass electrode; el( K), Kostyuk-style glass electrode; cl(T), Thomas-style glass electrode; el( Sb), antimony electrode.

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334 A. ROOS AND W. F. BORON Volume 61

TABLE 2. h ViVO bmk?

Method PKJ PI-6 Comments Ref.

DMO 7.36-7.42 7.09 + 0.007 (7) Cat; assuming 17% ECS and pH, 365
= pH of mean cerebral capillary
7.39-7.43 7.05 ?I 0.013 (14) Assuming 17% ECS and pH = pHCsF 366
7.42 6.94 2 0.03 (10) Assuming 12% ECS and pH, = pHivc 246
7.31 7.05 Dog; 21.9% ECS measured with 14
sulfate, pH, = pH,ist. pH, of
superficial cerebral cortex
7.45 7.10 Assuming 12% ECS and pH, = pHCsF 237
7.43 7.06 -+ 0.01 (14) Assuming 12% ECS and pH, = pHcsF 284
7.40 7.04 -+ 0.010 (9) Assuming 15% ECS and pH, = pHclst 407
7.41 7.04 + 0.006(13) Assuming 15% ECS and pH, = pHcist 269

Values are for rat brain unless otherwise indicated. Numbers in parentheses are number of
studies. ECS, extracellular space; CSF, cerebrospinal fluid; cist, cisterna magna; ivc, inferior
vena cava.


Tables 1 through 12 summarize the normal values for pHi. With few ex-
ceptions, these data have been derived from work published after the 1969
review of Waddell and Bates (454). Measurements on organelles, plant cells,
and microorganisms are omitted.


A. Historical Perspective

The development of the concept of nonionic permeation of weak acids and

bases has been traced in section IIB. A few words may be said here about some
early studies on the biological implications of this concept. In 1913 Loeb (259)
stated that the effects of acids and bases on egg development depend on the
penetration of the uncharged member of the conjugate pair. He found that weak
acids induce the development of a “fertilization membrane” in unfertilized
eggs of the sea urchin, whereas weak bases allow development to a few cell
divisions. Loeb, however, did not explicitly ascribe the effects to changes in pHi.
(For further details on the relationship between pHi and fertilization, see
sect. XII?) Jacobs (210) found in 1920 that tadpoles and protozoa in solutions
equilibrated with 100% CO2 (pH, = 3.8) are rapidly rendered motionless,
whereas HCl and organic acids at the same pH, are largely ineffective. Even CO,
solutions buffered with HCO;(pH, = 6.9) were effective. Jacobs ascribed this
to the great penetrating power of the CO, molecule, which results in internal

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TABLE 3. &?ati

Tissue Method ture, “C pH, PHI Comments Ref.

Rat ventricle (v) CO* 7.34 7.13 + 0.03 (24) Cl- for ECS 28
DMO 7.42 6.83 * 0.02 (10) Cl- for ECS 246

Rat heart (v) DMO 38 7.36 6.95 Inulin for ECS 381
DMO 7.41 6.91 + 0.01 (7) Inulin for ECS 92

Rat heart (p) DMO 30 7.39 7.06 2 0.02 (10) Inulin for ECS 360
DMO 37 7.25 7.02 + 0.03 (6-12) Sorbitol for ECS; working heart 310
DMO 37 7.33 7.05 t 0.02 (4-10) Sorbitol for ECS; working heart 423
31PNMR 30 7.4 215

Rat ventricle (i) elU’) 35 7.40 7.14 k 0.03 (9) V, = -76.7 k 1.6; in 100% 0, 122
(10 mM HEPES, same pH,J,
pH, = 7.20 + 0.03 (7); same
results in ferret ventricle

Dog ventricle (v) DMO 7.36 6.94 k 0.07 (20) Wr-EDTA for ECS; pH, derived 120
from mean transit times;
arterial pH = 7.39

Rabbit left DMO 36.5-39 7.44 6.80 + 0.02 (30) SICr-EDTA for ECS; same pH, 341
ventricle (v) for right ventricle and atria

Rabbit septum (p) DMO 26 7.38 7.28 t 0.02 (5) 5*Cr-EDTA for ECS; pH, from 340
washout of DMO was
7.23 2 0.05 (11)

Rabbit heart (p) DMO 37 7.28 7.18 Cl- for ECS 149
3’P NMR 37 7.18 + 0.07 (?) Perfusate was phosphate-free 214

Turtle ventricle (v) DMO 20 7.68 7.21 (13) Inulin for ECS 272

Turtle heart (p) DMO 23-25 7.60 7.67 Cl- for ECS 457

Sheep Purkinje (i) cl(T) 35 7.40 7.02 * 0.04 (14) V, = -78.1 2 2.5. In 100% 0, 123
(10 mM HEPES or Tris-
maleate, same pH,-J, pH,
= 7.20 t 0.05 (4)
cl(T) 36.5 7.40 7.09 2 0.02 (6) In absence of Cl-, pHi = 7.40 448,449
2 0.02 (3)

Perfusates and incubation fluids buffered with HCO.;-5% CO*, except for turtle heart, which was perfused with mixture
of acetate, borate, and Tris. Numbers in parentheses are number of studies. ECS, extracellular space; (v), in vivo; (p), perfused
heart; (i), isolated muscle preparation; cl(T), Thomas-style glass eiectrode.

acidification, and suggested that a similar mechanism operating in sensory taste

cells may be responsible for the sensation of acid taste. This hypothesis,
however, which was first proposed by Overton (325), has not been con-
firmed (see 25).

B. Weak Acids

I. CO,-induced acidification

More recent work has confirmed and expanded the earlier observation
that CO, produces a fall in pHi. In 1958 Caldwell (75), who was the first to

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336 A. ROOS AND W. F. BORON Volume 61

TABLE 4. In vitro rat and mouse skeletal muscle

Met hod PHO PHI Comments Ref.

DMO 7.39 6.89 2 0.14 (89) Inulin for ECS 7

DMO 7.40 7.17 t 0.06 (35) Inulin for ECS; 2 mM nicotine in baths 3
Nicotine 7.40 6.69 + 0.07 (35)
DMO 7.40 6.94 (18) Inulin for ECS, which was 44% (!) 170
of total water
7.38-7.45 7.10 iI 0.017 (35) Inulin for ECS 368
7.40 7.077 + 0.007 (23) Sorbitol for ECS 370
7.40 7.163 + 0.007 (63) 5 mM HEPES buffer; HCO;-free

31P NMR 7.1 Rat hindleg muscles, 20°C 199

el(W 7.34 Rat muscle 242

cl(C) 7.33 6.68 + 0.03 (7) Rat leg muscle, 36°C; V, = -75.9 326
+ 4.2 (36); deep penetration of

eKT) 7.40 7.07 k 0.007(65) Mouse soleus; at 28”C, pHi = 7.23 10

+ 0.01 (33); V, = -71.8 + 0.8
(37°C) and -64.3 + 1.0 (28°C)

Unless stated otherwise, rat diaphragm was used at 37”C, and buffer was HCO;-5% CO,.
Numbers in parentheses are number of studies. ECS, extracellular space; cl(T), Thomas-style
glass electrode; el( K), Kostyuk-style glass electrode; el( C), Carter-style glass electrode.

use pH-sensitive glass microelectrodes, observed that the pHi of the muscle
fibers of crabs (Carcinus maenas and Maja squinado) and of the giant axon
of the squid (Loligo forbesi) falls by more than 0.5 within a few minutes after
the cells are exposed to a solution equilibrated with 100% CO,. Saline acidified

TABLE 5. In vivo rat skeletal muscle

Met hod PHO PHI Comments Ref.

DMO 7.41-7.49 6.90 + 0.0094(14) Diaphragm, inulin for ECS; pH, = PH,,~ 367
7.45 6.97 + 0.03 (11) Gracilis; inulin for ECS; pH, = PH,,~ 476
v, = -91.9 AI 0.6 (72)
7.42 6.90 + 0.01 (10) Thigh muscle; Cl- for ECS; pH, = pHive 246
7.41 6.84 k 0.02 (10) Gracilis; inulin for ECS; pH, = pHi”c 92

cl(C) 7.41 6.68 2 0.08 (11) Thigh muscle; deep penetration of 326
electrode; V, = -80.5 + 4.5 (66)

el( HCO;) 7.03 Thigh muscle; calculated from [HCO;], 232

and arterial PCO~

Numbers in parentheses are number of studies. ECS, extracellular space; cl(C), Carter-style
glass electrode; el(HCO;), bicarbonate electrode; art, artery; ivc, inferior vena cava.

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TABLE 6. In viva dog, rabbit, human, and chicken skeletal muscle

Species Method PHO PK Comments Ref.

Dog DMO 7.42 6.84 IL 0.06 (33) Neck muscle; inulin for ECS; 60
PH0 = PHart
DMO 7.33 6.96 Hindleg muscle; sucrose for ECS; 150
pH, = pHf,, V + 0.02
DMO 7.35 6.92 Gracilis artificially perfused with 151
dog’s own blood; sucrose for
EC% PHO = PH,m v
co* 7.35 6.94 Quadriceps; Cl- for ECS; 152
PI% = Ph?ln v
Rabbit DMO 7.44 6.79 _+ 0.04 (30) Diaphragm and quadriceps; 341
51Cr-EDTA for ECS; pH,
= PH rt ventr

Human CO, 7.4 7.00 + 0.06 (13) Quadriceps; Cl- for ECS, assuming 385
v, = -88; pH, and PCO~ as in
fern v blood

Chicken DMO 7.31 (22) 6.93 (2) Pectoral muscle; inulin, Cl- or SCN- 411
for ECS; pH, = pHb, v + 0.02

Numbers in parentheses are number of studies. ECS, extracellular space; art, artery;
fern v, femoral vein; grac v, gracilis vein; rt ventr, right ventricle; br v, brachial vein.

with sodium hydrogen phthalate or sodium phosphate, or alkalinized with

sodium tetraborate, produced much slower drifts in pHi. Subsequent micro-
electrode studies have confirmed the striking effect of CO, in frog sartorius
muscle (35, 241), rat muscle (326), crab muscle (9, 326), barnacle muscle (37,
280), mammalian cardiac muscle and Purkinje fibers (122, 123), mouse soleus
muscle (lo), snail neuron (429), squid giant axon (42,420), crayfish neuron (301),
and the cells of the amphibian renal proximal tubule (40). A similar observation
has been made on rat diaphragm muscle in which the DMO method was used to
follow pHi transients (370). In a series of microelectrode experiments on snail
neurons, Thomas (429) confirmed Caldwell’s observation that non-CO, buffers
are much less effective in lowering pHi than CO,. He found that when the ex-
ternal solution is changed from one buffered with 20 mM Tris-maleate (pH, = 8.0)
to one equilibrated with 10% CO, (pH, = 6.5), the pHi falls abruptly (within 5
min) from 7.4 to 6.8 and rapidly recovers when the control solution is read-
mitted. On the other hand, an exposure to a solution buffered to pH 6.5 with
Tris-maleate produced only a slow, sustained fall in pHi (co.2 in 12 min). The pHi
recovery after return to the control solution was similarly sluggish.
The mechanism of intracellular acidification by CO, was clearly understood
by Jacobs (213) in 1940; it is the result of the intracellular hydration of the enter-
ing CO, molecule and the subsequent dissociation of H&O, to form H+ and
HCO,. Although at the start of exposure to CO, an inward electrochemical
gradient for HCO; also exists, the influx of this ion (which would blunt the CO,-

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338 A. ROOS AND W. F. BORON Volume 61

TABLE 7. Frog and turtle skeletal muscle

Met hod ture, “C PHO PHi Comments Ref.

Bromt hymol 7.2 7.1 2 0.1 (9) Semitendinosus; bicarbonate buffer; ECS; 176, 337
blue optically plugged with bromocresol

eW 7.36-7.40 7.12 + 0.07 (50) Sartorius; V, = -90.4 + 2.9; bicarbonate 241

buffer; same pH, in phosphate buffer
and in vivo

el[ HCO,] 25 7.0 Sartorius; bicarbonate buffer; pH, 231

calculated from [HCO.?] and PCO~
el(Sb) 7.66 2 0.06 (12) 7.10 + 0.05 (12) In vivo; V, = -55.8 + 8.6 (12); 278
pH, = pH of arterial blood

el(nig) 7.57 2 0.09 (7) 6.98 + 0.05 (7) In vivo; V, = -74.4 t 10.6 (7); 277
pH, = pH of arterial blood
:jlP NMR 28 7.21 2 0.06 (6) Gastrocnemius; anaerobic conditions 62
4 7.17 7.5 ‘-t 0.03 (5) Sartorius; ? buffer 108

DMO 23.5-26 7.35 7.16 + 0.01 Toe muscle; bicarbonate buffer 209

co2 20 7.77 7.49 In vivo sartorius and gastrocnemius; 350

inulin for ECS; pH, = pHplasma
DMO 20 7.77 6.96 (31) In vivo gracilis, gastrocnemius, and 272
sartorius; inulin for ECS;
PH, = pHp,asma
DMO 20 7.10 t 0.05 Sartorius; bicarbonate buffer; sulfate 167
Nicotine 20 6.92 + 0.03 for ECS; V, = -88 + 1.2 in
Morpholine 20 6.99 2 0.08 separate experiments

DMO 20 7.68 7.05 (3) In vivo pectoral muscle; pH, = pHplaqma 272

In vitro except as indicated. Numbers in parentheses are number of studies. ECS, extracellular space; cl(K), Kostyuk-style
glass electrode; el[HCO;], bicarbonate electrode; el(Sb), antimony electrode; el(nig), nigericin electrode.

induced fall in pHi) is probably very small due to its low permeability, the effect
of membrane potential, and the short interval during which the gradient is
inward. With a reduction in external Pco~, the loss of cellular CO, leads to
the association of H+ and HCO; and therefore to a rise in pHi. This mecha-
nism is illustrated by an experiment in which HCO; was iontophoretically
injected into a snail neuron (429). As the HCO;combines with H+ ions to form
H&O, (which after dehydration leaves the cell as CO,), pHi should rise; this was
indeed observed. Later Thomas (430) showed in the same preparation that the
immediate pHi changes in response to changes in PCO~are slowed with 10s5 M
acetazolamide, an inhibitor of carbonic anhydrase. This indicates that under
these conditions the hydration, respectively dehydration, of CO, becomes the
rate-limiting step. In mouse soleus muscle, Aickin and Thomas (10) found that
acetazolamide has no effect; in sheep Purkinje fibers, Ellis and Thomas (122)
observed only a marginal effect of the drug. For further details see section XIIC.
Two trends that have been observed in CO, experiments are consequences
of the mass-action law. First, the magnitude of the CO,-induced acidification
decreases as the initial pHi decreases. This was first observed by Thomas

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TABLE 8. Crab, crayfish, and barnacle muscle

Met hod ture, “C PHO Buffer PK Comments Ref.

Crab (C. maenas)

el( Cw) 7.53 2.6 mM HCO; 6.91 + 0.07 (7) pH, near fiber = 7.06; V, = -50 to -59 73

el( Cw) 7.8 2.6 mM HCO; 7.17 k 0.038(15) pH, near fiber = 7.2; V, = -21 to -54; 75
pHi similar to that of muscle of crab
M. squinado

eKC) 6.96 2.6 mM HCO, 6.96 2 0.10 (8) Deep penetration of electrode; 326
v, = -55.8 t 1.6 (8); also buffered
with Tris-maleate

cl(T) 20 7.5 20 mM Tris-maleate 7.27 + 0.02 (33) v, = -64.9 zt 0.6 9

Crayfish (P. clarkii)

W) 20-23 7.4 HEPES 7.17 + 0.12 (17) v, = -74.8 + 4.6 (17) 300

Barnacle (Z3. nubilus)

el(W 23-25 7.6 25 mM Tris 7.32 in 0.009 (4) v, = -71 280

eKW 15 7.8 5 mM Tris 7.34 + 0.025 (6) v, = -50.5 * 0.7; pH-el > pH-DMO 46
DMO 15 7.8 5 mM Tris 7.29 + 0.009 (27) Sorbitol for ECS

cl(H) 23.5 7.6 25 mM Tris -7.3 V, = -60 188

Copyright © 1981 American Physiological Society. All rights reserved.

DMO 23.5 7.6 25 mM Tris -7.25 Inulin for ECS

el(W 22 7.50 5 mM HEPES 7.31 * 0.01 (43) v, = -59.1 5 01 377

Numbers in parentheses are number of studies. ECS, extracellular space; el(Cw), Caldwell-style glass electrode; cl(C), Carter-style glass electrode;
cl(T), Thomas-style glass electrode; cl(H), Hinke-style glass electrode.

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TABLE 9. Blood cells in vitro

Species Met hod PHO Buffer PHi Comments Ref.

Rabbit DMO 7.40 Phosphate 7.27 + 0.05 (40) Agrees with pH derived from Cl distribution; 78
temperature not given

31P NMR 6.7 c&)6.86 + 0.05 Hemoglobin reduced with CO; cells suspended 302
b) 6.89 or 0.06 in plasma; a) from P peaks of diphospho-
glycerate; b) from P peak of Pi; temp.
not given

Human DMO 7.47 HCO,-5% CO, 7.27 Between 2 and 24% COz, pH-DMO remains 36
M-LMO, 7.47 HCO,-5% CO, 7.25 equal to pH-(NH&SO,

Cockerel DMO 7.40 HCO,-5% CO, 7.31 Between 2 and 24% COz, pH-DMO > pH- 36
(NHJW, 7.40 HCO,-5% CO, 7.16 (NH&%
Human DMO 7.26 HEPESZO mM 7.15 +_ 0.006 (4) Both measurements on same samples; 54
Nicotine 7.26 HEPES 20 mM 7.08 2 0.007 (4) difference not significant

DMO 7.34 Plasma 7.10 (20) Both measurements on same samples; 466
Nicotine 7.34 Plasma 7.06 (20) difference not significant

Leukocytes, DMO 7.40 HCO,- 5% CO, 7.11 2 0.01 (8) 254

Copyright © 1981 American Physiological Society. All rights reserved.


Temperature 37OC unless otherwise indicated. Numbers in parentheses are number of studies.

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TABLE 10. Mammalian and avian cells (other than nerve,

muscle, and blood cells)

Tissue PH0 PHI Comments Ref.

Rat liver 7.4 7.17 -e 0.09 (4) Inulin for ECS; liver was perfused at 35°C 79
7.45 7.20 + 0.03 (11) In vivo; inulin for ECS; V, = -41 2 0.3 (120) 476
7.37-7.40 7.17 2 0.023 (11) In vivo; inulin for ECS 205

Rat brown fat -7.4 7.39 + 0.010 (20) In vitro; HCO; buffer at 25°C; rats first 54
6.39 + 0.028 (9) exposed to 4°C for 24 h; second value
obtained with nicotine

Mice pancreatic 7.4 6.98 In vitro; obese, hyperglycemic mice; sucrose 172
islets for ECS; 3 mM glucose in medium;
HCO; buffer at 37°C

Baby mice teeth

Soft tissues 7.35 7.0-7.2 In vivo; soft tissue includes pulp, epithelium, 265
Calcified tissues 7.35 7.3-8.5 odontoblast, ameloblast; calcified tissue
includes predentine, dentine, enamel,
Dog stomach 7.38-7.45 7.28 2 0.02 (7) In vitro; inulin for ECS; mucosa first treated 104
parietal cells with pronase and collagenase; HCO,
buffer at 37°C

Chicken shell 7.29 7.02 (2) In vivo; inulin, Cl-, or SCN- for ECS; the 411
gland pH, falls to 6.53 2 .39 (10) between 2 and
10 h of calcification

Unless otherwise indicated, pH, values obtained with DMO. Numbers in parentheses are number of studies. ECS,
extracellular space.

(429) in the snail neuron: 10% CO, (pH, s 6.5) reduced pHi by 0.7, 0.5, and 0.3
when the initial pHi was 7.5, 7.3, and 6.9, respectively. Similar observations
have been made in crab muscle (9) and barnacle muscle (37). The basis of this
effect is that at a low initial pHi, newly formed H&O, undergoes relatively little
dissociation (the equilibrium H&O, * H+ + HCO; is shifted to the left). The
second trend is that for a given initial pHi, the C02-induced acidification in-
creases less than proportionally to the increase in Pco~. Thus when a barnacle
muscle is successively exposed to brief pulses of 1, 2, 5, and 10% CO,
([Hcmo = 10 mM), the reduction in pHi with 2% CO, is less than twice that
with 1% C02, and with 10% CO, it is less than twice that with 5% CO, (37). This
nonlinearity can largely be explained by the mass-action principle. For example,
a 2% pulse can be imagined to consist of two consecutive 1% pulses. The first
lowers pHi and thus the acidification produced by the second must be smaller,
as discussed above. The logarithmic relationship between [H+] and pH also
contributes to the nonlinearity.

2. Dependent of steady-state pHi on PCO~

Adler et al. (7) used the DMO method to study steady-state pHi (measured
after 4-6 h incubation) in the isolated, intact rat diaphragm muscle over a

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TABLE 11. Amphibian and reptilian cells (other than muscle cells)

Tissue Met hod ture, “C P&l Buffer PH, Comments Ref.

Nectumcs proximal [HCOc]i and 25 7.33 7.44 (46) In vivo 230

tubule PC02
Frog proximal tubule el( Sb) 7.66 + 0.06* (12) 7.49 2 0.03 (12) In vivo; pH of tubular fluid 278
7.60 2 0.07 (12)
el( nig) 7.57 2 0.09* (7) 7.36 2 0.05 (7) In vivo; V, = -77.3 + 4.8 (7) 277
Salamander proximal NJ3 7.5 13 mM HEPES 7.36 Isolated, perfused tubule
tubule (Amby-
stoma tigrinum) 3
Toad bladder DMO 7.37 (s) Phosphate + 5% CO, 7.30 * 0.05 (4) Inulin for ECS added to serosal 250 E
(Bufo marinus) 7.18 (m) side
Frog gastric mucosa DMO 6.9 HCO,-5% CO, 7.47 + 0.04 (20) Inulin for ECS; pHi increases 450 g
after histamine
Bromthymol 7.4 (s) HCO,-5% CO, 7.43 + 0.06 (6) Nonsecreting (metiamide inhib.); 180 .r
blue 4.2 (m) no buffer other pHi value: 7.13 + 0.07 w
(6) (SCN inhib.); pHi :
increases after histamine g
Frog oxyntic cells DMO 25 7.11 22 mM TES 7.18 2 0.05 (5) Inulin for ECS; cells detached 292
+ HCO,-5% CO, with pronase
Turtle liver (P. scripta) DMO 20 7.68 7.29 (11) In vivo; inulin for ECS 272
Turtle smooth muscle DMO 20 7.68 7.23 (13) In vivo; inulin for ECS 272

Copyright © 1981 American Physiological Society. All rights reserved.

(P. scripta)

In vitro unless otherwise indicated. Numbers in parentheses are number of studies. ECS, extracellular space; el(Sb), antimony electrode; el(nig), $
nigericin electrode; cl(T), Thomas-style glass electrode; s, serosal side; m, mucosal side. * Arterial blood. a

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TABLE 12. Embryos and tumor cells &
Tempera- ,Q
Cells Met hod ture, “C Buffer P&l PK Comments Ref.

Salamander eggs el( Sb) In gelatin 5.9 or 7.7 7.2 v, = l-5 mV (? polarity); 67
(T&on taeniatus) after fertilization,
pHi = 8.5
Sea urchin eggs homog 17 In seawater 8.0 6.48 + 0.01 (13) No correction for ECF; 220
(S. purpuratus) 10 min after fertiliza-
tion, pHi = 6.76
+ 0.02 (8)

Sea urchin eggs homog 15 In seawater 7.8 6.34 2 0.1 (41) No correction for ECF;
(L. pictus) 10 min after fertiliza-
tion, pHi = 6.76
+ 0.16 (5)
eU’) 17-19 In seawater 8.1 6.84 k 0.02 (44) v, = -10.5; 5-6 min after
fertilization, pHi
= 7.26 t 0.06 (8)

Frog embryo eU’) 2 mM Tris 7.5 7.7 v, = -8 to -40; 4-cell

(Xenopus) stage to midblastula
E hrlich ascites cells 31P NMR ZO? 6.36 6.82 Either from Pi or from 307

Copyright © 1981 American Physiological Society. All rights reserved.

Spectr 20 50 mM HEPES 7.4 7.4 428
DMO 20 50 mM HEPES 7.45 7.45

Numbers in parentheses are number of studies. ECF, extracellular fluid; el(Sb), antimony electrode; cl(T), Thomas style electrode; homog, pH
measured on homogenates; Spectr, spectrophotometric method with /3-carboxyfluorescein.

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344 A. ROOS AND W. F. BORON Volume 61

wide range of CO, concentrations; [HCOJ 0 was kept constant at about 22 mM.
He found that the pHi remains unchanged when PCO~is raised from 40 to about
70 mmHg (pH, = ‘7.4- 7. l), but falls linearly from 6.9 to 6.6 when Pcoz is raised
from 70 to 200 mmHg (pH, = 7.1-6.7). The pHi rises linearly from 6.9 to 7.45
when Pco2 is reduced from 40 to 90 mmHg (pH, = 7.4-8.0). Gonzalez and co-
workers, however, failed to observe a pHi plateau between a PCO~of 40 and 70.
In the first of their two studies (150), the pHi of the hindleg muscles of the
nephrectomized dog was measured after venous PCO* had been kept for 1.5-
2.0 h at 21-94. In the second study (151), the dog’s gracilis muscle was perfused
for 2 h with the animal’s own blood equilibrated with different CO,-O2 mixtures
(venous PCO~of 35- 135). It must be admitted that in these studies, [HCO,I,, did
not remain constant but was allowed to rise with Pco~.
It is now realized that the long-term changes in pHi are the net result of
passive movements of CO, and HCO;, metabolic acid production, and an
active pHiregulating mechanism. The results obtained by Adler et al. (7, 8),
Gonzalez et al. (150, 151), and others are reinterpreted in section VIIIZ in light of
this recent information.

3. Other weak acids

Sies et al. (402) observed a transient rise in the pH of the effluent of the
perfused rat liver when weak acids such as benzoic or propionic were added
to the perfusate. Removing the acids produced a transient fall. These changes
would be expected if the acids crossed the plasma membrane of the liver cells
mainly in the undissociated form and should be associated with pHi changes
in the opposite direction.
Roos (368) measured the DMO-derived pHi of rat diaphragm muscle ex-
posed for 2 - 5.5 h to solutions containing high levels of L- or D-lactate or of DMO;
pH, was kept at -7.4 (5% CO,). He observed that even very high external
concentrations of the acids produce only relatively small decreases in pHi* For
instance, in 2 h, 118 mM D-lactate reduced pHi from 7.06 t 0.01 (5) to 6.99
-+ 0.02 (6); 118 mM DMO reduced pHi from 7.07 t 0.01 (6) to 6.95 t 0.01 (5).
The measured transmembrane distributions of lactate and DMO indicate that
the acids mainly (though not exclusively) penetrate the cells in the undissociated
form (see sect. II&) and thus should produce a sizable fall in pHi. The author
interpreted the relative stability of pHi in terms of extrusion of H+ ions from the
fibers under the stimulus of the acid load (see sect. VIIIG). When acid extrusion
in barnacle muscle was minimized by reducing pH,, (see sect. VIIIE), addition
of 15 mM DMO to the medium produced a marked fall in pHi, followed by a nearly
flat plateau; the measurements were made with microelectrodes (227). Similar
studies on the effect of other weak acids (formic, acetic, propionic, butyric, iso-
butyric, cy- and P-hydroxybutyric, valeric, isovaleric, caproic, heptanoic,
crotonic, lactic, and salicylic) on barnacle muscle yielded comparable results
from which the permeability to the acids’ molecular forms could be calculated
(D. W. Keifer, G. Mayes, and A. ROOS,manuscript in preparation).

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A number of workers have observed a fall in pHi after exposing cells to

propionic acid. Such studies were made on sheep Purkinje fibers (276, 443), rat
soleus muscle (113), crab muscle (396), and barnacle muscle (226). The ac-
companying transient rise in pH, at the cell surface measured by Marannes et al.
(276) must have been because the rate of disappearance of propionic acid from
the unstirred layer into the cell exceeded the rate at which the acid was replaced
by diffusion from the bulk solution (for unstirred layers, see next sect.IV& ).
The work of Mainwood and Worsley-Brown (271) also emphasizes the im-
portance of nonionic diffusion. They examined the factors that influence the net
efflux of total lactate ([L-l + [HL]) from frog sartorius muscle during rhythmic
stimulation and found that this efflux is enhanced either by raising pH, (with
either Pco2 or [HCO;], constant, or in nonbicarbonate buffers) or by raising
Pco2 at constant pH,. The former procedure reduces [HL],, the latter raises
[HL],. Therefore both increase the outward-directed gradient for HL and thus
enhance net HL efflux. The authors also saw an increase of lactate efflux when
the external buffer concentration was raised. This may be explained by the
existence of an unstirred layer of external fluid next to the membrane. A high
buffer concentration prevents accumulation of HL in this layer by minimizing
the localized fall in pH that occurs as a consequence of HL efflux. Mainwood and
Worsley-Brown also found that at low, but not high, buffer concentration,
K+-induced depolarization further decreased the already sluggish lactate efflux.
Since a low buffer concentration reduces efflux of HL, the contribution of efflux
of L- to total efflux becomes significant. Thus its decrease, engendered by a
reduction of its outward-directed electrochemical gradient caused by depolariza-
tion, becomes noticeable. This effect of depolarization assumes, of course, that
L- efflux is electrogenic.

4. Unstirred layers

The role played by the unstirred layer in the transmembrane movement

of weak acids has recently been studied by Gutknecht and Tosteson (162). A lipid
bilayer was interposed between an Na salicylate solution at pH = 4.9 (either
poorly or well buffered) and a well-buffered salicylate-free solution at pH = 7.4.
Salicylic acid (pK 5 3.0) in its neutral form (HS) is highly lipid soluble and per-
meant; S- is impermeant. A trace of [14C]salicylate was added to the Na salic-
ylate solution, both solutions were continuously stirred, and the net trans-
membrane flux of HS was measured from the appearance of label in the salicy-
late-free solution. The authors found that the flux is 43 times greater when the
salicylate solution is well buffered than when it is poorly buffered and explained
this result as follows. The concentration of HS in the unstirred layer on the
salicylate side falls as HS diffuses into the salicylate-free side. This decrease
shifts the equilibrium HS L, H+ + S- to the left. In the absence of buffer, how-
ever, the limited supply of protons greatly restricts the conversion of S- into
HS, and [H+] is greatly reduced (Gutknecht and Tosteson calculated that the

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346 A. ROOS AND W. F. BORON Volume 61

steady-state pH in the film of unstirred layer closest to the membrane must have
been more than 2 units higher than in the bulk of the salicylate solution). As a
result [HS] in the unstirred layer of the salicylate side remains low, and thus its
transmembrane flux is small. On the other hand, a high buffer concentration in
the unstirred layer provides a large reservoir of protons and permits the rapid
transport of H+ in the form of protonated buffer from the bulk solution into the
unstirred layer. When HS diffuses into the salicylate-free side, which results
in a shift to the left of the above equilibrium, the formation of HS from S- and H+
is not limited by lack of H+. Consequently [HS] remains high in the unstirred
layer and the transmembrane flux of HS is large, whereas [S-l in the layer is
reduced. Gutknecht and Tosteson’s calculations showed that [S-l next to the
membrane is as low as 7.4 mM, whereas that in the bulk is 40 mM; in contrast,
pH is only 0.2 higher than in the bulk. It can be concluded that buffers, by
providing protons to the unstirred layer, greatly increase the transmembrane
flux of the uncharged species of weak acids. In comparable experiments, Gut-
knecht et al. (160) described the enhancing effect of buffers on the net flux of CO,
across lipid membranes. Because the rate of interconversion of HCO; and CO,
is slow due to the slowness of CO, hydration, carbonic anhydrase must be
present for the effect to be evident. In solutions well buffered to a pH of 7-8 and
containing carbonic anhydrase, the rate-limiting step for transmembrane CO,
movement is the diffusion of HCO; through the unstirred layer, whereas
at pH > 9 (at which [HCO,I is very great) it is the diffusion of CO, across the
membrane. Gutknecht and co-workers have published two additional studies
on this subject (161, 163).
Gros and co-workers (158, 159) have demonstrated a comparable en-
hancing effect of albumin and of phosphates on the diffusion of CO, through
liquid layers. They emphasized the significance of their observations for the
rapid equalization of [H+] and [CO,l in intracellular regions where free dif-
fusion of molecules and ions can occur. Engasser and Horvath (124) made a
theoretical analysis of this process. It is evident that the same approach also
holds for weak bases such as NH3, whose rate of membrane permeation from
solutions of its salts should be accelerated by the presence of buffers.

C. Weak Bases

I. NH,-induced alkalinixation

Thomas (429) was the first to use pH-sensitive microelectrodes to follow

the pHi transients induced by ammonium salts. He observed a rapid and revers-
ible rise in the pHi of snail neurons exposed to 5 mM (NH&SO,. The mechanism
of this rise was already well understood by Jacobs (213). In the external solution
there exists the equilibrium NH,+ e NH, + H+. The NH, rapidly diffuses
into the cell where most of it combines with protons, resulting in a rise of pHi*
This process continues until [NH,]i = [NH,],. (If the membrane is also per-
meable to NH,+, the alkalinization is somewhat blunted, since a small fraction

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of the entering NH,+ dissociates into NH, and H+; this effect is discussed in
detail in sect. v). The importance of [NH,], in determining the extent of the
alkalinization was demonstrated by Boron (37), who exposed a barnacle muscle
fiber first to 10 mM NH&I at pH, 7.7, and then (after washout and recovery)
to 5 mM NH&l at pH, 8.0. The [NH,], in the two solutions was the same, and
indeed pHi rose by nearly the same amount in the two pulses. Successive
equal increments of NH, should be progressively less effective in removing
intracellular H+ ions. This follows from considerations of mass action, since
each increment will encounter a lower [H+]i than the preceding one. Thomas’
experiments (429) point to the same phenomenon: pHi rose by about 0.1 on ex-
posure to 5 mM (NH&SO4 at a pH, of 7, whereas it rose by only about 10 times as
much when NH, was increased IOO-fold by raising the pH, to 9. Similarly, when
barnacle muscle fibers were exposed to pulses of increasing [NH&l], at the
same pH,, doubling [NH&l],, and thus [NH&,, produced less than a doubling
of the alkalinization (37). The alkalinizing effect of external NH: has also been
demonstrated in squid axons (42), mouse soleus muscle (lo), crayfish muscle
(300), crayfish neurons (301), and cells of the amphibian renal proximal
tubule (40).
In a study by Thomas (429), NH,+ was injected
. iontophoretically into snail
neurons. This resulted in a fall of pHi because some of the injected ions dis-
sociate into NH, and H+, the NH, diffusing out of the cell. The fall in pHi corre-
sponds to that observed on removal of external NH,‘, which is discussed in
section v.

2. Other weak bases

Weak bases other than NH, are also able to alkalinize the interior of cells
or organelles. The older observations of Overton (324) and others have already
been mentioned (see sect. IIB). More recently Bianchi and Bolton (29) observed
a rise in pHi (measured with DMO) in frog sartorius muscle exposed to either
lidocaine (pK, = 7.85) or procaine (pK, = 8.95). At constant external procaine
concentration (3.7 mM), raising pH, from 7.2 to 8.2 increased the alkalinizing
effect of the drug, though very much less than lo-fold (the factor by which
external nonionized concentration increased). This nonlinearity has been dis-
cussed above for NH,. The procaine effect was confirmed by Weiss (470). Lopo
and Vacquier (262) observed a reversible rise in pHi (estimated in cell homoge-
nates) from about 6.4 to 7.1 in sea urchin eggs (S. purpuratus) exposed to 10 mM
procaine at pH, 7.8. Boron and Roos (46), who exposed barnacle muscle to 10
mM methylamine at pH, 7.7, observed that after 2 h the pHi (measured with
DMO) was about 0.4 higher than without the base.

3. Effect of weak bases on organelles

NH:, also alkalinizes the internal milieu of lysosomes. Reijngoud et al.

(355, 367) found that pHi of rat liver lysosomes rises progressively from

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348 A. ROOS AND W. F. BORON Volume 61

about 6.0 to 6.8 when, at constant pH,, [NH&l], is increased from 0 to 20 mM.
Ohkuma and Poole (319), who used a fluorescence technique to estimate the
pH of lysosomes in living, cultured mouse peritoneal macrophages, observed
a concentration-dependent and reversible rise in lysosomal pH when the intact
cells were exposed to NH&l. Rottenberg and Grunwald (374) found that chloro-
plasts take up NH3 from the medium when exposed to light. The uptake is due
to the light-induced fall of internal pH, which leads to an increase in the inward
gradient for NH, by shifting the equilibrium NH,+ e NH, + H+ to the left.
Both lysosomes (355, 356) and chloroplasts (375) exhibit a progressive rise
in pHi when the concentration of methylamine (pK, = 11.8) in the medium
is raised. Johnson and Scarpa (222) used methylamine to measure the pHi of
chromaffin granules of the adrenal gland. The low value observed, about 5.5,
suggests that nonionic permeation may play a role in the accumulation and
storage of the weakly basic catecholamines. In a later study, Johnson et al.
(221) observed a dose-dependent increase of the pHi of isolated chromaffin
granules exposed to dopamine. Increasing pH, led to an influx of methylamine
and norepinephrine; after a new steady state had been reached, decreasing
pHi produced an efflux of methylamine, but not of norepinephrine, epinephrine,
or dopamine. These effects are compatible, and nonionic diffusion is the mode of
transport of these compounds only if it is assumed that intragranular binding
prevents the efflux of the catecholamines.



Since the early part of this century, it has been recognized that cell mem-
branes are generally far more permeable to neutral molecules than to ions.
For weak acids and bases, one would expect that permeation by the charged
partner plays, at best, a minor role in the transmembrane movement of the
conjugate pair. Yet there is now good evidence that the permeability of the
cell membrane to at least one charged species, NH:, is sufficiently great for
the passive movement of the ion to have significant second-order effects on pHi*
Permeability to the charged form of weak acids or bases, which we refer to as
ionic permeability, is also of interest because of its implications for calculating
pHi from the distribution of weak acids or bases; this is discussed in section IIB.
Here we examine the evidence for ionic permeation and discuss theoretical
analyses of this phenomenon.

A. Experimental Evidence

The first experimental evidence for ionic permeation of NH,+ was pre-
sented in 1974 by Thomas (429), who briefly exposed snail neurons to NH:
while monitoring pHi with a microelectrode. If plasma membranes were per-
meable only to NH3, then pHi would rise and level off during an NH: exposure

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o 7.3


,I5 mln,

FIG. 3. Effects of brief and of prolonged NH,+ exposures on intracellular pH (pH,) and mem-
brane potential (V,) of squid giant axon. During indicated periods, artificial seawater (pH,
= 7.70 throughout) contained 10 mM NH:. Brief, first exposure caused pH, to rise and nearly
level off; after return to an NH:-free solution, pHi undershot its initial value by -0.06. With
the second, more prolonged NH: exposure, pHi rose as before, but then slowly declined during
the plateau phase; removal of NH: caused an exaggerated undershoot. [From Boron and
De Weer (429.1

and then return to its initial level upon removal of external NH,+. Instead
Thomas found that pHi is always lower after removal of NH,+ than before
its application. A similar undershoot was also observed in 1976 by Boron and
De Weer (42) in experiments in which they briefly exposed squid giant axons
to NH,+ (Fig. 3A). In addition these authors noted that when axons are
exposed to NH,+ for more prolonged periods (Fig. 3B), the rapid, initial rise
in pHi is followed by a slower fall (this period of relatively slow pHi change is
termed the plateau phase) and that the pHi undershoot, upon return to an
NH&free solution, is greatly exaggerated. Thomas (429) had suggested
that undershoots are due to passive influx of NH,f during the NH,+ exposure.
Boron and De Weer pointed out that permeability to NH,+ could also account
for the slow plateau-phase acidifications observed during their longer experi-
ments and proposed the following scheme. During a brief exposure to NH, and
NH,+, the NH, rapidly enters and combines with H+ to form NH,+, thereby
raising the pHi. This alkalinization, however, is blunted as NH,+ also enters,
though more slowly, and dissociates to form NH, and H+. [The acidifying effect
of entering NH,f is illustrated by experiments in which snail neurons bathed in
normal Ringer’s solution were iontophoretically injected with NH,+ (429): pHi
fell as the injected NH,+ gave up its H+ and exited at NW,.] If the cell is now
returned to an NH,+-free solution, NH3 rapidly leaves the cell, thereby leading
to the dissociation of internal NH,+ into NH, and H+ and a fall in pHi. Some
NH,+ probably also leaves the cell, but since the force driving NH,f out of the
cell is less than that which had previously driven it in (due to the effect of V,>,
the fiber loses fewer of these proton carriers than it gains. The retained protons
yielded by this excess internal NH,+ are responsible for the observed under-

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350 A. ROOS AND W. F. BORON Volume 61

FIG. 4. Proposed mechanism for inward shuttling

of Hi by NH,-NH,+ couple during plateau phase of
Figure 3. Influx of NH,f down its steep electrochemi-
cal gradient shifts the intracellular equilibrium NH$ =
H+ + NH, to the right, thereby acidifying the cell and
leading to efflux of NH,. Although there is also an
electrochemical gradient for H+ favoring influx of this
ion, absolute flux of H+ per se is very small due to its
very low concentration. In some cells, a portion of the
NH,f influx may be mediated by the Na-K pump.
[From Boron and De Weer (42).]

shoot of pHi. On prolonged exposure to NH, and NH,+, the rate of NH, entry
eventually falls to zero as [NH,], approaches [NH&. Alkalinization then ceases,
and the subsequent course of pHi is determined by the entry of NH,‘. When
NH, is at equilibrium, and assuming the pKL of NH,+ to be identical on both
sides of the membrane, [IJ+]i/[NH,‘3i = KLI[NH,]i = KL/‘[NH,I, = [H+],/
[NH,+],. In other words, the equilibrium potential for NH,+ (ENH4) is the
same as that for H+ (E.), which can be calculated from pHi and pH,. In the
experiment of Fig. 3, E, during the plateau phase (pHi = 7.7, pH, = 7.7) comes
to 0 mV, substantially more positive than the prevailing V, of -59 mV. Thus
the positively charged ammonium ion is driven into the cell by a substantial
electrochemical gradient. The intracellular dissociation of this NH,+ not only
causes pHi to fall, but also raises [NH,]i above [NH& and thus leads to the
efflux of NH3. As illustrated in Figure 4, the effect of the simultaneous fluxes
of NH,’ and NH, is the inward shuttling of protons. If the exposure were
sufficiently long (and if pHi were not being independently regulated), this
shuttling would continue until H+, and therefore NH,+, had reached elec-
trochemical equilibrium (i.e., until, in the present example, pHi was -6.7). It
is clear from the above that the longer the exposure to NH,‘, the greater the
magnitude of both plateau-phase acidification and undershoot.
This model predicts that if the electrochemical gradient for NH,+ were re-
versed, pHi during the plateau phase of an NH,+ exposure would rise rather
than fall. This indeed was observed in experiments on barnacle muscle fibers
exposed to 230 nm K+ in addition to 7 mM NH&l (pH,, = 8.0) (37). At the start
of the plateau phase, pHi was about 7.65, so that &H4 (assumed equal to EH)
was about -20 mV, whereas V, was only about -5 mV. The electrochemical
gradient for NH,+ was thus directed outward. When the fiber was returned to
an NH&free solution, pHi fell to a value that was higher rather than lower
than the initial pHi.
It might be noted that the magnitude of the fall in pH during the
plateau phase in Figure 3 (-0.07) is much less than the magnitude of the under-
shoot (-0.27). One reason for this is that not all the NH,+ enters during the
plateau phase: some enters during the period of rising pHi, as evidenced bY
undershoots after even brief exposures to N H+4 . The major reason for the
discrepancy, however, is that the NH,-NH,’ pair contributes significantly

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to intracellular buffering, so that total buffering is far greater during the

NH,+ exposure than after it. This is discussed in more detail in section IX.
In addition to the aforementioned data from pH microelectrode experi-
ments, there is independent evidence that the plasma membrane is permeabable
to NH,+. For example, Binstock and Lecar (31) showed that NH,S partially
replaces Na+ or K+ during the action potential of the squid axon. Hille (183, 184)
made similar observations on myelinated frog nerve fibers. In addition it is
known that NH,+ depolarizes the plasma membrane in a number of prepara-
tions. From the degree of depolarization produced in squid axons by 480 mM
NW- (pH, = 7.8), Hagiwara et al. (164) estimated that the ratio of resting
NH,+ to K+ permeability is 0.2-0.3. Boron and De Weer (42) obtained a
value closer to unity when they compared depolarizations produced by in-
creasing either [K+], or [NH,+], by 10 mM (pH, = 7.7). It must be admitted
that especially the former of these estimates is complicated by NH,+-induced
pHi changes, which might independently influence V,.
In certain cases, NH,+ may enter cells by other than passive mechanisms.
Aickin and Thomas (11) found that removing external K+ from mouse soleus
muscle exposed to NH,+ causes the rate of the plateau-phase acidification to
greatly increase. This effect is blocked by ouabain, suggesting that in the
absence of K+, NH,+ may be transported inward by the Na+-K+ pump. The
effect on pHi is, of course, the same, regardless of the mode of NH: entry.
Removal of K+ has no influence on the plateau-phase acidification rate in
barnacle muscle, squid axons (W. F. Boron, unpublished observation), or cray-
fish neurons (W. Moody, unpublished observation).
The evidence for permeability to NH,+ has been obtained in several
preparations: the snail neuron (429), the squid giant axon (42), the barnacle
muscle fiber (37), mouse soleus muscle (lo), crayfish slow flexor muscle (300),
crayfish neuron (301), and cells of the salamander’s renal proximal tubule (40).
Less work has been done with ions of other weak acids and bases. Boron and
Roos (46) exposed barnacle muscle fibers to 10 mM methylamine and observed
a plateau-phase acidification in experiments in which they measured pHi with
DMO. No attempt was made, however, to observe an undershoot after methyl-
amine removal. In microelectrode experiments on barnacle muscle, Keifer and
Roos (227) found a plateau-phase acidification during exposures to DMO and a
pHi shortfall after removal of the weak acid. Both results indicate a permeability
to the DMO anion, which the authors estimate to be about 0.1% of the per-
meability to the neutral molecule.
Several groups of workers have obtained results consistent with membrane
permeability to HCO,, showing that pHi falls when cells are exposed to a low-
pH, low-[HCOJ solution (so that an outward-directed electrochemical gradi-
ent for HCO; was established). The studies include the DMO experiments by
Adler et al. (7,8) on rat diaphragm and by Izutsu (209) on frog muscle, as well as
microelectrode experiments on crab muscle (9), sheep Purkinje fibers (122),
rat skeletal muscle (232), barnacle muscle (37), mouse soleus muscle (lo), and
frog skeletal muscle (35). Also Gonzalez and Clancy (149) observed a fall in

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352 A. ROOS AND W. F. BORON Volume 61

pHi and a net loss of HCO; from cardiac muscle when hearts were perfused
with a CO,-HCO; solution of low pH. It must be admitted that in addition to the
passive efflux of HCO;, other factors may also have contributed to the decline
in pHi observed in these studies. For example, lowering pH, decreases the rate
of acid extrusion by the pHi regulating system (see sect. VIII), and this in itself
would produce a fall in pHi. Other data indicate that HCO; efflux does indeed
occur when pH, and [HCO;], are simultaneously lowered, but that this
HCO; efflux may be carrier mediated, rather than via a pore or channel. When
barnacle muscle fibers are exposed to acidic (pH = 6.5) CO,-HCO; solutions,
pHi, after its initial fall (due to the influx of CO,), continues to drift downward
(37). Although pHi rises upon withdrawal of the external CO,-HCO;, it al-
ways falls short of its initial value (37). Transport of HCO,? is implicated by
the observation that both the plateau-phase acidification and the shortfall
depend on [HCO;], independent of pH, and pHi (44). Although these results
are consistent with a passive egress of HCO;, the observation that both the
plateau-phase acidification and the shortfall are blocked by 4-acetamido-4’-
isothiocyanatostilbene-2,2’-disulfonate (SITS; 44), a potent inhibitor of anion
exchange in several preparations, suggests that carrier-mediated transport is
involved. Specifically data of Thomas for snail neurons (436) and of Keifer for
barnacle muscle (225) suggest that the aforementioned barnacle results can be
explained by reversed operation of the pHi regulating system (see sect. VIIIE).
Because of the unusually high mobility of the H ion in water (some 5 times
that of the K ion), a relatively high membrane permeability to H+ might be
expected. Two recent estimates are in agreement with this expectation. Izutsu
(209) exposed frog skeletal muscle to a relatively acid medium (pH, = 6.6,
CHw31, = 5 mM, 5% CO,). The pHi measured with DMO declined approxi-
mately exponentially (time constant 2.3 h) from 7.0 to 6.6. From the rate of fall,
the permeability to H+ was then computed on the assumption that the decline
was exclusively the consequence of passive H + in .flux. At a total intracellular
buffering power of 10 mM (a rather low estimate), Izutsu obtained an H+ per-
meability of 10m3cm/s, 3 orders of magnitude greater than that of K+. If the ob-
served pHi fall had been brought on exclusively by passive HCO, efflux, a mem-
brane permeability to HCO; of 3 x 10V8 cm/s would be required, 2 orders of
magnitude less than that to Cl-. The presence of active acid extrusion (H+
transport from the cell and/or HCO; transport into it), which would lead to an
und .erestimate of both permeabilities, is probably of little importance, since a
low pH, suppresses this process (see sect. VIII). At least during the early part
of the pHi fall, however, the production of acid by the fibers may have led to an
overestimate. Later in the pHi fall, lactic acid production was most likely re-
duced by the low pHi (see sect. XIE). If both H+ influx and HCO, efflux had
contributed to the acidification, both permeabilities would have been over-
Woodbury (481) has attempted, in frog muscle, to derive the ratios of H+
and HCO; permeabilities to that of Cl- from changes in membrane voltage.
Using media in which Cl- had been replaced by glutamate (an impermeant ion),

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he observed a depolarization when pH, was drastically reduced from 7.4 to 5.8
(solution nominally CO,-free) and a hyperpolarization when [HCO;], was
drastically raised from 1 to 20 mM (10% CO,). By comparing these changes
with the hyperpolarization produced by the addition of 2 mM Cl- to the medium,
Woodbury estimated the relative permeabilities to H+ and HCO;. He ascribed
the changes in V, (and in membrane resistance measured simultaneously),
observed in the pH and HCO; studies, exclusively to passive transmembrane
H+ and/or HCO; movements. This is valid only if the fluxes of the other per-
meant ions (mainly Na+ and K+) remain in the steady state, that is, if their
permeabilities and driving forces do not change. There is good reason, however,
to question this assumption of invariance of permeabilities with pH, and pHi
(see sect. XIA). Woodbury’s results (PHI& = 500, PHCO.&I = 0.1) must
therefore be viewed with considerable caution.

B. Models of Ionic Permeation

In 1940 Jacobs (213) described the transmembrane distribution of weak

acids and bases on the assumption that only the uncharged partner is permeant.
Sixteen years later, Orloff and Berliner (321) derived a steady-state model for
the distribution of weak bases (BH+ L- B + H+), which for the first time con-
sidered permeation by both members of the conjugate pair; V, was assumed to
be zero. The authors were aware that permeability to BH+ would lead to a
shuttling of protons, as depicted in Figure 4, and that for the steady state to be
maintained, acid would have to be added to one compartment and alkali to the
other. Irvine and colleagues (206) subsequently presented a similar equation for
weak acids. In 1965 Roos (365), taking into account V, and permeability to both
charged and uncharged forms, derived an equation for the steady-state dis-
tribution of a weak acid; a similar equation has also been derived by Boron and
Roos (46) for a weak base. For this, the net passive flux of B was assumed to be
governed by Fick’s law whereas the constant field treatment (147,191) was used
to describe the flux of BH+

JB = PB ([Blo - [BIJ (23)

J BH =
. [BHlo - [BHIiE (24)
RT E-1

where JB and JBH represent the net inward fluxes of B and BH+, respectively,
PB and PBH the permeabilities, and E = exp( VZIRT). The steady-state dis-
tribution was obtained by setting JB = -JBH. An analogous approach was used
for weak acids. The final equations, presented in section IIB, reduce to those
of Orloff and Berliner (321) and of Irvine et al. (206) when V, is set to zero. For
more physiologic values of V, (e.g., -90 mV), however, the Roos and Boron-
Roos models differ considerably from the older ones. For example, during the

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354 A. ROOS AND W. F. BORON Volume 61

plateau phase in the experiment of Figure 3, pHi 55 pH, (i.e., [NH,+]i r [NH,],).
The Orloff-Berliner model would predict that there would be no net flux of
NH,+ or NH, and that pHi would remain constant. The newer model, on the
other hand, predicts a substantial electrochemical gradient favoring the influx
of NH: and the observed fall in pHi.
Boron and De Weer (42), starting from Equations 23 and 24, presented
a model that describes the time-dependent pHi changes observed when a cell
is exposed to a weak base such as NH,. Their analysis, when modified to include
the refinement of Keifer and Roos (227), shows that for weak bases in general

dlITBli K
- = p h3(mo - [TBli)
dt rHli + K

[HB], - ‘H1i [TBlie

+ PHBKnF LHli + K
RT ’ E-l 1 (25)

-d[Hli = -In lO[H]ip K PHBVrnF

dt P [H]i + K + In lOK[TB]i[H]ilp([H]i + K) ’ RT ’

[HB], - ‘H1i [TBlie

[Hli + K - [HI .
E -1 [H]i + K + In IOK[Ti]i[H]i/fl([H]i + K) l

w[w” - K
IHli + K
where total internal concentration of base ([TB],) = [B]i + [BH+]i, p is the sur-
face/volume ratio, K is the apparent acid dissociation constant, t is time, and
p is the intrinsic intracellular buffering power. As expected, Equations 25 and
26, when limited to the steady state (d[H]ildt = 0, and thus [H]i = constant),
reduce to the equations of Boron and, Roos (46). Although these differential equa-
tions have no general analytical solution, they can be solved numerically, and
typical solutions have pHi versus time courses similar to those of Figure 3. The
equations predict that when PNHd= 0, an exposure to NH,+ produces a stable
alkalinization, and removal of NH,+ causes pHi to return to its initial value.
At values of P NH4greater than zero, the magnitude of the initial alkalinization
decreases, the plateau phase is marked by an acidification, and removal of the
external NH,+ produces an undershoot. The blunting of the initial alkaliniza-
tion, the rate of plateau-phase acidification, and the magnitude of the under-
shoot all increase as the value chosen for PNHd increases. When a reasonable
value (e.g., -10S7 cm/s) is used for PNHr, the solution of Equations 25 and 26
gives a good approximation of the actual data. Finally, the equations predict

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that reversing the electrochemical gradient for NH,+ should lead to a plateau-
phase alkalinization and a pHj shortfall after NH,+ removal, as has been actually
observed (37). Analogous equations have also been derived for a weak acid (42).


A. Experimental Evidence

The interest in the relation between the pH of biological fluids and tem-
perature dates back to the mid-1920’s, when Stadie and co-workers (421,
422) first examined the thermodynamic principles on which such a relation
must be founded. Their experiments on whole blood and true and separated
plasma showed that at constant “total C02” content (i.e., with these fluids in a
closed space without gas phase), pH falls as a temperature is raised. These
results have been confirmed many times since. Extensive literature has arisen
on the relation between acid-base balance and temperature, which has recently
been reviewed by Reeves (353). Here we shall largely confine ourselves to the
temperature effect on intracellular pH. All evidence indicates that in this case
also, there is an inverse relation between temperature and pH. Adler (4) showed
that when the temperature is reduced from 37 to 25OC and then to 4”C, at
constant pH,, both the pH-DMO and pH-nit of isolated rat diaphragm muscle
increase; this occurs over the pH, range of 6.8-7.8. The medium was buffered
with HCO;-CO,, and [HCO;], was always kept at 25 mM. Hinke and
McLaughlin (187) measured PHi of giant barnacle muscle fibers with micro-
electrodes and found that from 5 to 4O”C, PHi falls linearly with increased tem-
perature (at 5”C, PHi was 7.6; at 3O”C, it was 7.4). Above 3O”C, the
fall in PHi is steeper (at 4O”C, PHi was 6.8), and eventually the fibers contract.
Other workers, using the same preparation and technique, found PHi values of
7.35 t 0.016 (n = 15) at 15°C (46) and 7.31 t 0.01 (n = 43) at 22OC (37). In
Aickin and Thomas’ (10) microelectrode experiments, the PHi of mouse soleus
muscle in HCO;-Ringer’s solution (5% COZ, pH, kept at 7.40) fell from 7.23
t O.Ol(33) at 28OCto 7.07 t 0.007 (65) at 37OC.These observations were confirmed
in a second paper (ll), in which the rate of PHi recovery from an acid load at
28°C was found to be only 35% of that at 37OC. In contrast Ellis and Thomas
(122) observed no effect of temperature on PHi of rat ventricle: at 2O”C, PHi
was 7.17 t O.O6(n = 7), and at 35”C, it was 7.20 t 0.03 (n = 7). Under in vivo
conditions, Saborowski et al. (381) found pH-DMO of rat ventricle at 37°C to be
about 6.95, and at 21°C, pH-DMO was 7.2. Arterial pH at both temperatures
was 7.40.
In an extensive study, Malan et al. (272) acclimatized bullfrogs (Rana
catesbeiana) and turtles (Pseudemys sctipta) for 3-8 days at tempera-
tures ranging from 5 to 32OC. The in vivo pH-DMO of various tissues was then
determined using purified [3H]inulin as the extracellular marker. They meas-
ured, at 2O”C, a PHi of frog skeletal muscle of 6.96 (n = 31, no SE given) and a

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356 A.ROOSANDW.F.BORON Vobume 61

pHi of turtle muscle of 7.05 (n = 13). According to the linear regressions, dpHi/
dt was -O.O15/“C for pH-DMO of frog muscle, and -0.019, -0.012, -0.023,
and -0.014 for turtle muscle, ventricle, liver, and esophageal smooth muscle,
respectively. For comparison, the value for both frog and turtle blood plasma
was -0.021. In contrast to the behavior of the pHi, the in vivo PCO* of frog
blood is known to rise with temperature: it is 6 mmHg at lO”C, 11 mmHg
at 2O”C, and 18 mmHg at 30°C (200).

B. Theoretical Considerations

The relation between temperature and the pH of biological systems has

both physicochemical and physiologic aspects. In the simplest case of a solu-
tion containing only one buffer, the change of pH with temperature parallels
the change of pK of the buffer; that is, the degree of dissociation of the buffer
remains practically unchanged. The temperature dependence of the pK is given
by the integrated form of the van’t Hoff equation

PKl (27)

where A H is the heat of ionization (Cal), T, and T, are absolute temperatures,

and 4.576 is In 10 times the gas constant, R (cal deg-l mol-l). It is evident that
l l

the relation between pK and T is nonlinear. When more than one buffer is
present, the pH change of the solution with temperature equals the sum of
the individual pK changes, each weighted by the buffer’s relative contribution
to total buffering (64). This principle (for derivation see 351) can be applied to
biological fluids such as blood plasma, provided these fluids are in a closed space
without gas phase (i.e., the amount of total buffer, including CO, and HCO;,
must remain unchanged). It might also be applicable, though caution must
be used, to the pH of cells maintained in a solution of constant pH and Pco~,
as were the mouse soleus fibers in Aickin and Thomas’ study (10) referred to
above. Under these conditions the observed changes in pHi may largely be
determined by the temperature dependence of the pK values of the intracellular
buffers. Yet other factors, such as temperature-dependent changes in passive
ion fluxes (i.e., H+, OH-, or HCO;), acid-extrusion rate, and metabolism,
could also contribute. For example, if a decrease in temperature produced a
proportionally greater fall in the acid-extrusion rate than in the rate of
passive HCO; efflux, the steady-state pHi would be lower than one would
otherwise predict. Another complication in interpreting temperature-induced
pHi shifts is introduced when PCO* is kept constant. If the model of Reeves
and Malan (354), which is discussed below, is applicable to mammalian cells,
then reducing temperature at constant Pco2 would produce a smaller increase

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in pHi of the mouse muscle fibers than if the system were closed (in which case
PCO* would fall).

C. Model Buffer Systems and Zmidaxole

Reeves and Malan (354) have attempted to model the temperature-

dependent changes of muscle pHi that were observed by Malan et al. (272)
in intact, acclimatized frogs (discussed above). The initial conditions, taken
from the experiments, were pHi = 6.89, PCO~ = 15, T = 25OC. The model was
endowed with three buffer systems in physiologic concentrations: HCO;-CO,,
imidazole (50 mM), and phosphate (15 mM). The variations of PCO~ with
temperature imposed on the model were those observed in the blood of
the acclimatized frogs (200). Since the pK and the temperature dependence
(i.e., A H) of the three buffers are known, the temperature dependence of the
model’s pHi can be calculated. Reeves and Malan (354) found that this depend-
ence closely resembles that actually observed in the muscle and concluded that
the conditions imposed on the model must be similar to those in vivo. Since the
plasma membrane of the model was assumed to be impermeable to all sub-
stances other than C02, the agreement implies that during the in vivo tempera-
ture transitions, all transmembrane traffic affecting pHi (i.e., movements of
H+, OH-, and HCO;) remained in the steady state. This may not be as
unreasonable as it appears at first glance, since in frog muscle the traffic seems
to be very light (for further comment on this point, see sect. VIII@. Also
there is no recovery of the pHi when frog muscle fibers, at 2O”C, are exposed
to 5% CO, (pH, = 7.2; 35), whereas in every other cell studied, pHi returns
toward normal after an intracellular acid load has been imposed (see sect. VIII,
especially sect. I). It must be admitted that the proposed muscle model
is quite insensitive to even large changes in selected buffer composition. For
instance, ‘at 5”C, variations in phosphate concentration of lo-15 mM change
pHi by only 0.05; at 30°C the model’s pHi response is practically inde-
pendent of its phosphate content. Taking into account the standard error of
the experimentally observed (272) temperature dependence, ApHi/AT (-0.0152
- 0. 0032) the data could adequately be explained by any combination of 5-30
mM phosihate and 40-75 mM imidazole.
When the temperature of dog plasma in a closed system was raised from 7
to 37.5”C, the course of the pH was such that the degree of dissociation of the
imidazole groups (pK values -7) increased by less than 4% (351). These near-
constant values indicate (see sect. VIB) that these groups, which are part of the
histidine residues of the (plasma) proteins, play the dominant buffering role.
This is not surprising since carbonic acid, the other plasma buffer present in
significant amounts, has a pK, that is more than a unit less than the pH and thus
contributes little to buffering (see sect. ~4). Reeves found that the tempera-
ture dependence of the plasma pH is hardly affected by the presence of red
cells (351, 352). Thus the temperature changes did not induce net trans-

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358 A. ROOS AND W. F. BORON Volume 61

membrane movements of H+, OH-, or HCO;. Also both the Cl- distribution
between red cells and plasma and the cell volume remained practically un-
changed, in contrast to the situation where, at constant temperature (3%5”C),
the CO, tension was varied (352). Therefore [Cl-]i/[Cl-1, and [H+]J[H+]i were
unaffected by temperature. The membrane voltage should also have remained
nearly unchanged; this point has not yet been tested.
The constancy of the H+ distribution means that a temperature change
induced identical pH changes in both red cells and plasma. This would be self-
evident if imidazole were the system’s only buffer, since the pH shifts would
then equal imidazole’s shift in pK (see sect. VIES). Especially in the intracellular
milieu, however, other buffers besides hemoglobin (mostly phosphates,
prominent among them diphosphoglycerate) are present whose pK values have
different sensitivities to temperature. Fortu itously these substances are pres-
ent in concentrations such that they induce a temperature response of cell pH
that closely matches that of plasma pH.
It is of considerable interest that cooling blood in a closed air-free
space produced nearly the same increase in plasma pH (and decrease in Pco,) as
acclimatizing whole conscious animals to the same lower temperatures (272).
Reeves (349, 351) and Malan et al. (272) proposed that maintaining the degree
of dissociation of the imidazole group is an overriding aim of in vivo acid-base
regulation. Indeed the ionized fraction of this group in the plasma of acclimatized
frogs and turtles hardly changed when the temperature was reduced from 30
to 10°C (272). In the intracellular milieu, the constancy was not quite so striking
(272): in frog skeletal muscle, the ionized imidazole fraction fell by about 13%
(from 0.60 to 0.52), in turtle muscle the decrease was less than half as much, and
in turtle heart it was about 20%. In turtle liver the fraction increased
by about 11%.
The heat of ionization ( A H) of imidazole (about 7 kcal/mol) is approxi-
mately half that of water, and thus ApKim = 0.5 ApK, for the same tempera-
ture change (see Eq. 27). Since temperature-induced changes in plasma pH of
the acclimatized animals paralleled those in pKim (i.e., A pH, = A pKi,),
A pH, must about equal 0.5 ApK,. Therefore since ApK, = ApH,, + ApOH,,,
A pH, must about equal A pOH,. In other words the difference between pH, and
pOH,, or the equivalent ratio [OH-],/[H+],, remains approximately unchanged
when the temperature is altered. It has recently been proposed (200) that
constancy of [OH-l&H+], is the prime aim of acid-base regulation in the blood,
a rival concept to that of constancy of imidazole ionization and one that was
vaguely considered many years ago(16) . There is neither thermodynamic
nor physiologic justification for su.ch an assu mption ?however. The rate of many
physical, chemical, and physiologic processes is affected by temperature and
by [H+] or, possibly in some cases, by [OH-], but not by the ratio of the two con-
centrations. Moreover the constancy of the ratio is only approximate. Even
under the carefully controlled conditions of a closed, air-free environment (351),
[OH-]/[H+] of in vitro blood ([total CO,] = 25 mM, pH of plasma = 7.4 at
37.5”C) increased from 12.6 to 16.4 when the temperature was raised from

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0 to 45”C, whereas the degree of imidazole dissociation increased only from

0.79 to 0.82.


A. Historical Perspective

Although it is now generally accepted that H+ is not in equilibrium across

the plasma membrane of animal cells other than erythrocytes, this dictum was
challenged as recently as the late 1960’s. In this section we offer a short his-
torical perspective of the subject and a detailed analysis of the recent work.
Donnan’s theory of ion equilibria (see 118) so dominated the thinking of the
1920’s and early 1930’s that nearly all contemporary investigators postulated
that the distribution of the hydrogen ion across the plasma membrane of any
animal cell must obey a Donnan equilibrium. This assumption is well illustrated
by the work of Netter (311) and of Mond and Netter (299). Netter (311)
predicted that in muscle [H+]i/[H+], must equal [K+]i/[K+]o, since he considered
both ion species to be permeant. The pHi estimated from the pH of “pressed
juice” of frog muscle, however, was always higher than the value predicted by
the K+ ratio. Rather than question the validity of applying Donnan’s theory to
the distribution of H+, Netter concluded that the discrepancy must have been
due either to compartmentalization or to CO, loss. Somewhat later Mond and
Netter (299) were puzzled that net K+ efflux from frog muscle was not reduced by
repetitive stimulation. They assumed that stimulation produced a decrease in
pHi and therefore expected it to reduce [K+], and/or raise [K+]i, maintaining
the equality of the H+ and K+ distributions as required by Donnan theory.
Since they could find no evidence for changes in K+ concentrations, these
authors postulated that pH, must have declined in parallel with pHi.
The first direct evidence that H+ is not in equilibrium across the cell
membrane of muscle was provided by Fenn and colleagues in the mid-1930’s.
Using the CO, method (though not correcting for extracellular space) to estimate
the pHi of frog skeletal muscle incubated at pH, 7.0, Fenn and Cobb (127) found a
pHi of 7.0, much higher than the value of 5.7 predicted from the K+ distribu-
tion ratio. Later Fenn and Maurer (129) obtained an in vivo value of 6.9 after
correcting for extracellular (chloride) space and assuming a pH, of 7.34.
After these experiments the nonequilibrium distribution of H+ became
generally accepted. There have been two attempts to resuscitate the old
equilibrium concept, however. The first is by Conway and Fearon (100) and the
second by Carter et al. (82). The argument in both cases concerned the validity
of methods for measuring pHi. Conway and Fearon questioned the CO, tech-
nique. As discussed earlier (see sect. II??), this method requires that intra-
cellular CO, be present only in the form of free HCO, and of dissolved CO,.
In experiments on mammalian skeletal muscle, these authors found that the
total (acid-labile) COZ, measured as the CO, released upon addition of strong

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360 A. ROOS AND W. F. BORON Volume 61

acid, is more than twice the amount that can be precipitated with barium. In
their view barium-precipitable CO, is identical to dissolved CO, plus free HCO;
(and CO:-), whereas acid-labile CO, also includes CO, derived from carbamino
compounds and other stores. Therefore use of acid-labile CO, would lead to an
overestimate of [HCOy]i and thus an overestimate of CO,-derived pHi (pH-
CO,). On the other hand, the barium-precipitated CO, leads to a value for
pH-CO, of about 6.0, close to that expected if H+ were in equilibrium.
This conclusion was effectively disputed by Hill (182), who pointed out
that at such a low pHi, lactate would cease to be formed, the critical pHi being
about 6.3. Yet he found that even at an external pH as low as 4.5 (phosphate
buffer), muscle cells still produce lactate, even though the pHi required by the
Donnan theory would be about 3.0. Hill reasoned that the pHi of muscle is
normally near 7, whereas it is reduced below 6.3 by exposure to 100% C02,
which is known to stop lactate production. A second rebuttal of Conway and
Fearon’s proposal was made by Butler and colleagues (66). They confirmed that
barium precipitates only a fraction of the acid-labile C02, but found that even
added HCO; cannot be quantitatively recovered by barium precipitation
from muscle extracts previously stripped of their CO,. They noted a similar
discrepancy after adding HCO; to solutions of l-4% albumin. Although these
experiments admittedly leave open the possibility that some of the added
HCO, was transformed and hence not in free solution, they can most simply
be explained by the assumption that for some reason barium fails to precipitate
all the total CO, dissolved in protein solutions.

B. Measurements With Glazed Microelectrodes

The second major attempt to demonstrate a passive distribution of H+

was made by Carter and colleagues (82) in 1967. These authors attempted to
measure the pHi of rat skeletal muscle with a pH-sensitive glass pipette coated
with an insulating glaze, except at the terminal 5-20 pm. Double-barreled
electrodes permitted the simultaneous measurement of V,, whereas those
equipped with a third barrel allowed current to be passed as well. In rat skeletal
muscle in vivo, with V, spontaneously ranging from -30 to -90 mV, the ap-
parent pHi (“pHi”) always equaled the equilibrium value

“pHi” = pH, +
RT lnm10

At a normal Vm of -89 mV and a pH, of 7.41, “pHi” was 5.99 t 0.02 (n = 38).
When Vm was clamped at +15 to -233 mV, “pHi” always assumed the
equilibrium value within lo-15 s, the response time of the recording system.
Five years later, Carter (81) radially inserted the same style of electrode
into muscle fibers (diam - 1 mm) of the giant barnacle. When Vm was -59

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-+ 0.9 mV and pH, was 7.05 t 0.02, “pHi” was 6.12 t 0.03 (n = 253), again
close to the equilibrium pHi. The pHi measured with larger, Caldwell-style
electrodes, introduced longitudinally, was 7.0, the same as that obtained with
the glaze-insulated electrode subsequently introduced into the hole made by
the larger one. The pHi measured with DMO was 6.68 t 0.02 (n = 13). The
author postulated that the smaller electrode sensed the pH of a small (3% of total
cell volume), acidic compartment beneath the membrane, whereas the larger
electrode reflected the pH of the main, alkaline compartment, and DMO meas-
ured the volume-weighted average of the two. The disruption produced by the
large electrode would cause the small electrode, when subsequently introduced,
to sense the same high pH.
More recently Hannan and Wiggins (167) used electrodes similar to those
of Carter to measure pHi in frog sartorius muscle, obtaining a value of 5.92
to.09 (n = 14), not significantly different from the equilibrium value (5.89
t 0.02). The pHi derived from the distributions of DMO, nicotine, and morpho-
line ranged between 6.9 and 7.0. The authors concluded from the microelectrode
experiments that H+ is indeed in equilibrium; however, the weak acid-base
experiments seemed to indicate that the true pHi was greater than 6. Yet this
true pHi, they maintained, cannot be determined by any method, due to the
supposedly unique structure of intracellular water. As a result the standard
chemical potential of intracellular water (p&J would differ (by an unknown
amount) from that of extracellular water (473) and would obviate the use of
microelectrodes (since p&Z0 determines electrode voltage) and weak acids and
bases (since JLC~~~influences the apparent dissociation constant). This ad hoc
assumption is supported by no evidence.
There are at least three a priori reasons for doubting the results obtained
by Carter et al. First, other methods consistently have produced values for
pHi considerably higher than would be expected if H+ were in equilibrium.
Carter’s attempt to reconcile these discrepancies by resorting to com-
partmentalization requires several unlikely ad hoc assumptions, such as an
alkaline subcompartment occupying 95% of cell volume. Also there is no reason
why his electrodes should do less damage than larger pipettes, given the
relatively small size of most intracellular structures compared to either device.
The second reason concerns the instability of pHi, which would be the
consequence of its being regulated by both V, and pH,. There are numerous
cellular processes sensitive to changes in pH, and it is therefore to the cell’s
advantage to maintain pHi independently of parameters such as pH, and
V,, which are sensitive to many influences originating in the cell and its environ-
ment. A third argument against the findings of Carter et al. concerns the voltage
clamping experiments. As pointed out by Roos (367), changes in V, could not
lead to the very rapid alterations of pHi supposedly observed unless the
plasma membrane’s permeability to H+ or HCO; was preposterously high.
The required H+ permeability would have to be more than 300 cm/s, a value
that exceeds the H+ permeability of a 50-A layer of pure water. An HCO;
permeability of about low3 cm/s would be required if an HCO; flux would

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362 A. ROOS AND W. F. BORON Volume 61

have been responsible for the rapid pHi change; this value is 3 orders of mag-
nitude greater than the Cl- permeability.
In addition to these a priori arguments, there is direct experimental
evidence that the glazed electrodes used in these experiments were inade-
quately insulated. In 1972 Paillard (326) reported experiments on rat and crab
muscle fibers in which he used glaze-insulated electrodes of Carter’s design.
For in vitro experiments in which the electrodes were introduced into super-
ficial skeletal muscle fibers of rat, they registered a pHi of 6.12 t .02 (n = 6),
a value very close to the calculated equilibrium pHi (pH, = 7.33, V, = -76
mV). When the electrodes were advanced farther into the muscle, they
registered a much higher pHi, 6.68 t 0.03 (n = 7), although V, remained un-
changed. Depolarization by raising [K+], to 20 mM caused the superficially
measured pHi to change rapidly so as to remain consistent with a passive H+
distribution, whereas the deep pHi remained higher. Similarly, imposed varia-
tions in pH, caused the superficial pHi to rapidly shift to values predicted
by the Nernst equation (equilibrium distribution), whereas similar treatment
produced only slow changes in deep pHi values, which remained considerably
higher than the superficial ones. The experiments with crab muscle fibers
yielded similar results. Most importantly, when the fibers were impaled with
completely uninsulated electrodes, the pHi values were not different from those
obtained by impaling superficial fibers with glaze-insulated electrodes.
Paillard’s results, especially this last finding, strongly suggest that the
glazed microelectrodes were inadequately insulated. The results obtained by
Carter et al. (81, 82) can thus be explained as follows. Imagine that a cell is
impaled with a Ling-Gerard microelectrode that measures membrane voltage
and that a glazed, pH-sensitive microelectrode is placed in the bathing solu-
tion. The voltage difference between the Ling-Gerard electrode and an indif-
ferent electrode in the bath is V,, whereas that between the glazed and indif-
ferent electrodes (V,) is exclusively a measure of pH,. To measure pHi, the pH
electrode’s entire pH-sensitive surface must be with the cell so that the voltage
it measures (Vi), minus V, (i.e., Vi - V,), is exclusively a measure of pHi* If,
however, the unwitting experimenter believes that the pH-sensitive surface of
the glazed electrode is completely intracellular when, in fact, it is partly extra-
cellular, a predictable error will be made. In the extreme case, the supposedly
intracellular electrode is actually entirely outside the cell, and the voltage meas-
ured by the glazed electrode is V,. Since the experimenter believes the
electrode to be inside, however, he subtracts V, and obtains V, - V, as the
measure of pHi, instead of the desired Vi - V,. The effect of this error on the
calculated pHi can be appreciated from the following. The pH of any solu-
tion can be calculated from the voltage of the pH electrode (V) by the
PH = V/S + K (29 >
where S (the electrode’s pH sensitivity, given in volts/pH) and K are
empirically determined constants. Therefore

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PH i= w i - v,)/S + K (30)

When (V, - V,) is mistakenly used in Equation 30, the erroneous PHi
(pH,) is given by

PH X = w 0 - v,)lS + K (31)

From Equation 29, however, it follows that pH, = V&S + K, or V,

= S(pHo - K). When this expression for V, is substituted into Equation 31,
we have

PH X = m?Ho - K) - V&S + K = pH, - v,/s (32)

Now the equilibrium PHi is given by the Nernst equation

pHfq = pH, + V,l(2.3RT/F) (33)

The ideal sensitivity of the electrode is also described by the Nernst equation:
S = -(2.3RT)lF. Therefore Equations 32 and 33 are identical, and thus pH,
= pHfq. That is, a pH-electrode so poorly insulated as to be effectively extra-
cellular will erroneously indicate a value for PHi, namely pH,, which is the same
as the equilibrium PHie As a consequence, when pH, or V, change, pH, instan-
taneously changes with them, so that it remains equal to pHfq.
If part of the electrode is inside the cell and part of it is outside the cell, the
equation of Wiggins (474) describes the dependence of pH, on the fraction f of
the electrode’s pH-sensitive surface that is intracellular

PH X = f*pHi + (1 - f)[(pH, + V,l(2.3RT/F)] (34)

Equivalent circuit analysis (W. F. Boron, unpublished observations) shows

this equation to be valid if the resistance of the plasma membrane is negligible
compared to that of the pH-sensitive membrane. When the electrode is entirely
intracellular (f = l), pH, = pHi. As the electrode is withdrawn (f -+ 0), pH,
approaches pH fq. The equation indicates that if H+ were indeed in equilibrium
across the membrane, it would be immaterial how much of the pH-sensitive
surface was intracellular, since pH, would always equal pHf~.
The results of Carter et al., obtained with glazed electrodes, clustered
around 6.0. According to Equation 34, this would require either that H+ be in
equilibrium across the membrane or that in all experiments f be approximately
zero. Wiggins (474) claimed that if in these experiments the electrodes were
partly inside the cell and partly outside, f should have ranged between 0 and 1,
and the measured PHi between 6.0 and 7.0. It is quite possible, however, that
consistently only a small fraction of the total pH-sensitive surface area had
entered the cell. Paillard (326) estimated, on the basis of the depth to which
the glazed electrode had to be advanced into the muscle to obtain a maximal

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364 A. ROOS AND W. F. BORON Volume 61

pHi value, that the length of the exposed pH-sensitive tip is 90-100 pm. If this
is so, then for a rat muscle fiber 30 pm in diam., a maximum of about 30% of the
tip’s length, or only about 10% of the tip’s area (assuming the tip to be a cone),
could have been intracellular. For f = 0.1, pHi = 7.1, pH, = 7.4, and V,
= -0.090 V, Equation 34 predicts a pH, of 6.02, not far from the values obtained
by Carter et al. (82). In a delicate impalement, very much less than 30 pm of
the glazed electrode’s tip would have entered. In fact, even if the length of
exposed pH-sensitive glass were only about 20 pm, as Carter et al. claim, an
impalement with 5 pm of tip length would have resulted in only about 6% of the
tip’s surface area being intracellular. Thus contrary to Wiggins’ claim, it is
quite reasonable for the erroneous pHi obtained with glaze-insulated
electrodes always to be near the equilibrium pHi.
The case against an equilibrium distribution for H+ is reinforced by the
results of experiments with recessed-tip, pH-sensitive microelectrodes (see
sect. Ml). The open tip of the insulating glass sheath has a diam. of 1 pm or
less and thus is nearly as fine as that of the glaze-insulated electrodes.
There is no question, however, that the electrodes are adequately insulated.
Results obtained with these electrodes on crab muscle (average pHi = 7.27;
9) agree with those of Caldwell (75) on the same preparation (pHi = 7.0) and
with those of Boron and Roos (46) and of Boron (37) on barnacle muscle fibers
(PH. = 7.3). Boron and Roos (37,46) used relatively large electrodes that Carter
(81)’ claimed would disrupt the supposedly alkaline subcompartment. Also
results obtained with recessed-tip electrodes on mammalian muscle (10, 11, 122,
123) and frog skeletal muscle (35) agree with those obtained using DMO (209,
368), NMR (62, 108, 199), and other methods outlined in section II.
These arguments lead us to conclude that H+ is not in equilibrium across
the plasma membrane of any cell s so far exam .ined (with the exception of
erythrocytes) and that the results of Carter and colleagues can be attributed
to the use of inadequately insulated electrodes.


A. Historical Perspective and Introduction

In the 1920’s and early 1930’s, it was generally assumed that small ions
such as H+ and K+ were distributed across the plasma membrane according to
a Donnan equilibrium. The regulation of pHi would merely involve the re-
distribution of ions according to the relation: [H+]i/[H’], = [K+]i/[K+]o = exp-
(-V,F/RT). Contrary evidence was not obtained until 1934 with the work of
Fenn and Cobb (127). Measuring both pHi (by the CO, method) and [K+]i in
frog sartorius muscle, they found that [H+]i/[H+], is substantially less than
[K+]i/[K+], (i.e., pHi is far higher than predicted from the K+ distribution).
Although Fenn and co-workers (127, 129) considered other explanations for
this discrepancy, they judged it most likely that K+ is indeed in equilibrium

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but that “some independent mechanism within the muscle . . . [regulates]

. . . the pH to approximate neutrality in spite of the demands of membrane
equilibrium. ” They also recognized that “some continuous supply of energy
would obviously be necessary for this purpose.” These conclusions were
eventually supported by the pioneering microelectrode studies of Caldwell
(75), Spyropoulos (420), and Kostyuk and Sorokina (241). For example, the last
authors, also working with frog sartorius muscle, showed that the equilibrium
pHi (calculated from the measured pH, of 7.4 and V, of -90 mV) is about 5.9,
far lower than the observed, steady-state pHi of 7.1. In the absence of some
compensatory process, the predicted passive influx of H+ (or efflux of OH-)
would eventually reduce pHi to its equilibrium value. Fenn and Maurer (129)
believed this compensatory process to involve phosphocreatine and lactic
acid metabolism. The possibility of ion transport was not entertained until
1955, when Hill (182) proposed an H+-extruding device analogous to the
Na+ pump.
The compensatory process envisaged by the early workers in the field
would not only have to neutralize H+ passively entering the cell, but also the
acid contributed by at least two other sources: metabolism and fluxes of the
ionized forms of weak acids and bases. The acidifying effects of metabolism
are well known. Extreme examples include the fall in pHi produced by anoxia
(429), metabolic inhibitors (42, 241, 429), and, in muscle cells, prolonged con-
tractile activity (108, 140, 177). Fluxes of the ionized forms of weak acids and
weak bases can also acidify the cell interior (see sect. v). When the neutral
form (e.g., NH:, or CO,) is equilibrated across the cell membrane and if
pKi = pK,, a gradient is established for the corresponding ion (e.g., NH,+ or
HCO;). For cations, this gradient is the same as for H+; for anions, it is the
same as for OH-. Thus cations such as NH,+ (if permeant) would normally
enter the cell, and anions such as HCOZ would leave it, both fluxes tending
to lower pHi. Significantly, because weak acid and base ions are generally
present in far higher concentrations than H+ or OH-, their fluxes (assuming
reasonable permeability) are expected to have far greater acidifying effects
than those of H+ or OH-. This has been confirmed in an in vitro system by
Gros et al. (159), who found that the addition of phosphate buffer increases
the effective proton flux by about 4 orders of magnitude.
The preceding discussion points out the central problem of pHi regulation:
the neutralization of intracellular acid derived from a variety of sources.
In the short term, considerable acid can be neutralized by several reversible
and rapidly responding mechanisms that serve to “buffer” acid loads; these
are discussed in section VII@?. In the long term, however, pHi homeostasis
reli es on the cell .‘s ability to extrude H+ and/or accumulate HCO; or OH-.
These so-called acid-extrusion mechanisms were first identified by their ability
to return pHi toward normal after acute intracellular acid loads, as discussed
in section VIIIC. Several methods of intracellular acid loading, discussed in
section VIIID, have since been used to examine the ionic mechanism of acid
extrusion, and three patterns have emerged. In squid axons, snail neurons,

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366 A. ROOS AND W. F. BORON Volume 61

and barnacle muscle, acid extrusion is achieved by coupling the entry of Na+
and HCO,? to the exit of Cl- and possibly H+ (sect. VIIIE). In crayfish neurons
and amphibian proximal tubular cells, Na-H exchange (sect. VII@) is the main
mechanism for acid extrusion, whereas in mammalian muscle (sect. VIIIG),
Na-H and Cl-HCO,, exchangers operate in parallel. The response of cells to
intracellular alkaline loads is presented in section VIIIH, followed by an examina-
tion of the determinants of steady-state pHi in section VIIIL

B. Short-Term Intracellular pH Homeostasis; Metabolic Contributions

The long-term stability of pHi simply requires that acid be removed as

rapidly as it appears. The short-term regulation of pHi is more complex. When
responding to an acute internal acid load, the cell first recruits several rela-
tively rapid mechanisms that consume H+, thereby minimizing the magnitude
of the pHi decrease. Later pHi slowly returns toward normal as acid is ex-
truded from the cell. The rapidly responding mechanisms include I) physico-
chemical buffering, 2) cellular consumption of nonvolatile acids, and 3) the
transfer of acid or alkali between the cytosol and organelles. Mechanisms
I and z were considered by Siesjo and Messeter (408) in their analysis of
short-term pHi homeostasis of brain cells. In the broadest sense, all three are
buffering mechanisms, since they reversibly consume H+ (for further justifica-
tion of this designation, see sect. IXB). Together they neutralize more than
99.99% of the acid of alkali introduced into a cell. (For example, the addition
of lo-” mol of H+ to a liter of cell content might lower pHi from 7.1 to 7.0, repre-
senting an increase of only about 2 x lO+ M in free [H+].)
Physicochemical buffering, probably the single most important buffer sys-
tem, is considered in section IXA. The transfer of acid or alkali across organellar
membranes is considered in section X. The contribution of biochemical reactions
to pHi homeostasis has been studied extensively in mammalian brain by Siesjo
and colleagues (133-135, 153, 155, 269, 284, 285, 406-408) and has been re-
viewed by Cohen and Iles (96). To summarize briefly, the conversion of a weak
acid (e.g., lactic acid) to a neutral product (e.g., glucose) or to one that can readily
leave the cell (e.g., CO,> results in the loss of intracellular H+. Internal pH
can also be influenced by other reactions, such as the hydrolysis of ATP (which
releases H+) or phosphocreatine (which consumes H+). In rat brain,
Folbergrova et al. (134) showed that intracellular acid loading (accomplished
by increasing Pco,) leads to a reduction in the levels of several acidic metabolic
intermediates (pyruvate, lactate, citrate, cr-ketoglutarate, maleate, glutamate,
and aspartate) and to an elevation of glucose and glucose 6-phosphate levels.
This pattern suggests that reducing pHi inhibits a step (possibly the phos-
phofructokinase reaction) in the glycolytic pathway. Indeed Trivedi and Dan-
forth (439) have shown that phosphofructokinase isolated from mouse muscle
is markedly pH sensitive: lowering the pH from 7.2 to 7.1 inhibits the reaction
by more than 90%. Thus the net cellular consumption of pyruvic and other

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acids in the experiments of Folbergrova et al. (134), if it resulted in the pro-

duction of neutral or readily diffusible molecules, represents a partial
neutralization of the imposed acid load. The magnitude of the contribution
can, in theory at least, be calculated from the change in the steady-state
concentration of each metabolite. Such an analysis (data taken from 408)
predicts that the maximum amount of H+ that can be neutralized by the
consumption of these acidic intermediates is about 50% of that taken up by
physicochemical buffers. Other evidence for metabolic consumption of acid
comes from Cohen et al. (97), who showed that increased lactate uptake by
the isolated, perfused rat liver is associated with a rise in pHi, as would be
expected if lactate ions entered the cell and were converted to neutral prod-
ucts. Intracellular alkalosis (produced by decreasing Pco,) leads to increased
levels of pyruvate, lactate, and other acidic metabolic intermediates in rat brain
(269), and these metabolic changes thereby partially neutralize an alkaline load.
It must be recognized that physicochemical, biochemical, and organellar
buffering mechanisms offer only partial and short-term solutions to acid load-
ing. They can only minimize decreases in pHi and are of limited capacity.
The restoration of a normal pHi after an acute acid load requires the extrusion
of all added acid. As this extrusion proceeds, buffers yield H+ previously
consumed and are thereby restored to their initial state. Thus the central
role in maintaining pHi is played by those transport mechanisms that remove
acid from the cell.

C. Response to Intracellular Acid Loads: Acid Extrusion

As discussed in section VII& the existence of a pHiregulating mecha-

nism can be proved on purely thermodynamic grounds: in the steady state, the
intracellular accumulation of H+ must be balanced by the energy-requiring
extrusion of acid. More direct evidence is obtained by studying the cell’s re-
sponse to acute intracellular acid loading, as first done in 1971 by Messeter
and Siesjo (284). They followed the overall pHi of rat brain (measured with
the CO, method) after acutely raising PCO~ and found that although pHi
fell from its initial value of 7.06 to about 6.93 after 15 min, it subsequently
recovered to about 6.97 after 45 min and reached 7.03 after 3 h. The authors
reasoned that the initial, CO,-induced fall in pHi is minimized by physiochem-
ical and biochemical buffering; but two observations led them to conclude
that the pHi recovery must be due to the active transport of H+ or HCO, across
the cell membrane. I) Other studies (285) indicated no further biochemical
buffering after 45 min of hypercapnia, and 2) the required H+ extrusion or
HCO: uptake would have occurred against a concentration gradient. Although
their analysis neglected the membrane potential, inclusion of a negative V,
would only have strengthened the case for active transport. A similar recovery
of pHi from a hypercapnia-induced acid load was demonstrated 5 years later
in dog brain by Arieff et al. (14) with the DMO technique. Evidence for acid

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368 A. ROOS AND W. F. BORON Volume 61

7.01 A f-y 20 mln

80 1 5%

FIG. 5. Recovery of pH, of snail neurons from a Con-induced acid load. A: brief CO, exposure.
During indicated period, a neuron was exposed to Ringer’s solution equilibrated with 5% CO, (pH,
7.0), causing pHi to fall and then to recover slightly. Removal of CO, led to an overshoot of pHi.
B: prolonged CO, exposure. During indicated periods, a neuron was exposed to Ringer’s solution
equilibrated with 0.9, 2.2, and 4.4% CO,; pH, was 7.5 throughout. Application of CO, produced
an abrupt fall in pHi, followed by an exponential recovery toward the initial value; CO, removal
led to a substantial pHi overshoot. (Note that pHi tracings are upside down, possibly reflecting
the aberrant automobile traffic pattern in the author’s native country.) [A and B from Thomas (429
and 430, respectively).]

extrusion was also provided by Roos (368), who used the DMO method to
measure the steady-state pHi of rat diaphragm muscle. Incubating the
muscles 2-3 h in HCO;-Ringer’s solution (pH, = 7.4) containing 118 mM
lactate or DMO caused pHi to fall by only 0.06-0.12. Since the influx and
dissociation of these neutral weak acids should have caused a much larger
intracellular acidification, the author attributed the relative stability of pHi
to a proton-extruding mechanism and proposed that its rate was responsive
to intracellular acid loads.
The first direct microe1ectrod.e evidence for acid extrusion was obtained
by Thomas (429) in 1974. Exposing a snail neuron to 5% C02--Ringer’s solution
at pH, 7.0 (Fig. 5A), he found that pHi initially fell (due to the influx of CO,) and
then recovered somewhat, whereas removal of the CO, caused pHi to return to
a value greater than the initial one (overshoot). During the pHi recovery of
this CO, exposure, V, was approximately -55 mV, so that the calculated
equilibrium pHi was about 6.05. Since the actual pHi was near 7.0, passive move-
ments of H+, OH-, or of HCO; could only have led to a further fall in pHi,
rather than the observed rise. Thomas therefore ascribed the pHi recovery to
the active extrusion of H+, or an equivalent mechanism. Additional evidence
for acid extrusion was obtained in the microelectrode experiments of Boron
and De Weer on squid giant axons (42). When CO, exposures were very brief,
no pHi recovery occurred and the overshoots after CO, removal were small.

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After prolonged CO, exposures, however, pHi recovery was plainly evident
and overshoots were greatly exaggerated. They noted that the magnitude of
the overshoot is directly related to the amount of acid extruded during the
period of acid loading. Examples of the effects of prolonged CO, exposures
on the pHi of snail neurons are given in Figure 5B (430). A similar time course
of pHi after CO, exposure has been observed in crab muscle (9), sheep Purkinje
fibers (122, 123), barnacle muscle fibers (37), mouse soleus muscle (lo), rat
diaphragm muscle (370), and Xenopus embryos (441). In addition recoveries
from propionic acid-induced acid loads have been demonstrated in rat soleus
muscle (113) and sheep Purkinje fibers (443). We point out two early papers
that, in retrospect, may have provided evidence of acid extrusion. In 1920
Jacobs (21 l), monitoring the vacuolar pH of flowers of Symphytum peregrinum,
found that exposure to 100% CO, at a pH, of 3.8 causes a rapid and sustained
fall in pH,. When the CO, solution contained 500 mM HCO; (pH, = 7.4),
however, the initial fall in pH, is followed by a slower recovery. In 1945 Brandt
(50) measured pHi in a suspension of heat-fixed yeast cells both before and after
exposure to 50% CO,. Although he found no difference in the two pHi values
when the cells were incubated anaerobically, the pHi after CO, removal was
substantially higher than before exposure when the cells were incubated
in 50% 0,.

D. Methods of Intracellular Acid Loading

Since the recovery of pHi from acid loads is the basis for studying the ionic
mechanism of pHi regulation, this section is devoted to the methods available
for reducing pHi*

1. Exposure to weak acids

As described in section IV, pHi can be lowered by exposing the cell to weak
acids such as CO*, DMO, or lactic, acetic, or propionic acids. The technique
is technically simple, but has disadvantages: 1) the weak acid must continually
be present for the acid load to be maintained and 2) since many weak acids are
cellular products, they may influence metabolism.

2. Intracellular injection of acid

In 1974 Thomas (429) iontophoretically injected NH,+ into snail neurons

by passing current between two microelectrodes, one filled with KC1 and the
other with NH&l. Since the NH: dissociates into NH, (which leaves the cell)
and H+, this is equivalent to injecting H+ directly. Thomas (430) and others
(373) have injected H+ by passing current between KCl- and HCl-filled micro-
electrodes (for an example see Fig. 7); the amount of H+ injected is determined

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370 A. ROOS AND W. F. BORON Volume 61

from the amount of current passed and the transport number of H+ (430). Acid
can also be pressure injected as done by Meech and Thomas (283) on snail
neurons. They determined the amount of H+ injected from the rise in internal
Cl- activity, which they monitored with a Cl- sensitive microelectrode. With
these techniques the size of the acid load can be precisely controlled, several
successive injections can be made in the same cell, and the composition of the
external fluid is left unchanged. Unfortunately these techniques can only be
used in spherical cells (in which no significant concentration gradients are pro-
duced), which are resilient enough to withstand multiple electrode impalements.

3. Transient exposure to NH:

As discussed in section V, pHi can also be lowered by pretreatment with

NH,+ (42, 43). During an exposure to NH,+, pHi first rises rapidly (due to the
influx of NH,) and then slowly declines (due to the passive influx of NH,+). When
the external NH,+ is removed, pHi falls to a level less than the initial one, this
undershoot reflecting the previous net entry of NH& This method (for an
example see Fig. 6) has been used in squid giant axons (431), mouse soleus
muscle (lo), barnacle muscle fibers (37), crayfish muscle (300), crayfish neurons
(301), and cells of the amphibian renal proximal tubule (40). Its advantages are
that 1) it can be applied to very large or elongated cells, 2) no NH: remains at
the completion of acid loading, and3 ) the degree of acidification can be controlled
by judicious choice of [NH,+], and length of exposure. On the other hand,
some cell swelling may occur during the exposure, membrane potential is often
somewhat lower after NH,+ exposure than before its application, and NH,
may alter metabolism. In mouse soleus muscle, removal of external K+ en-
hances the acidification, apparently by causing the Na+ jump to transport
NH,+ inward instead of K+ (11).

4. Internal dialysis

Internal dialysis was introduced by Brinley and Mu1 ins (52) to control
intracellular levels of low-molecular-weight (i.e., < 1,000) solutes. The tech-
nique requires that a horizontally mounted cell be threaded by a length of porous
dialysis tubing. Since the composition of the fluid driven through the tubing
can be varied, the intracellular environment can be controlled to a consider-
able extent. Russell and Boron (379) imposed acid loads on squid giant axons
by dialyzing them with solutions of low pH and, after halting dialysis, monitor-
ing the subsequent recovery of pHi with microelectrodes (see Fig. 9). In
another approach (48, 225), barnacle muscle fibers were continuously dialyzed
with a low-pH solution so that the steady-state pHi reflected the balance
between acid extrusion and acid loading. Assuming th e acid-loading rate to
remain constant, a change in pH ‘i can be attributed to a change in the acid-
extrusion rate. The advantages of the dialysis method are 1) pHi can be lowered

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pH 7.7 PH 8.0

Q 76
7.4- *

30 min

FIG. 6. Dependence of acid extrusion on HCO; in squid giant axon. Axon was acid-
loaded by exposing it to 50 mM NH:; removal of NH,’ caused pHi to undershoot its initial level
(see Fig. 3). At pH,, 8.0, and in the nominal absence of HCO;, pHI recovered only very slowly (seg-
ment AB). Introduction of 10 mM HCO,-0.4% CO, produced a rapid fall in pHi (BC), followed
by a fairly rapid recovery (CD). Removal of CO,-containing solution produced a pHi overshoot.
[From Boron and De Weer (43).]

rather quickly, 2) the composition of the external fluid is not affected, 3) internal
levels of Na+, Cl-, ATP, and other small solutes can be altered, and 4) drugs
can be directly introduced into the cells.

5. Internal perfusion

With this approach perfusate is introduced directly into the cell rather
than through dialysis tubing. Although it has yet to be used to acid-load cells,
internal perfusion has been employed in electrophysiologic studies of squid
axons (18, .320), snail neurons (240, 251), myocardial cells (252), myoballs (198),
and barnacle muscle (229).

6. Reduction of pH,

In several preparations (9, 10,41, 122,229), reducing pH, has been shown
to cause a fall in pHi, probably due to increased HCO; efflux in combination
with a reduced acid-extrusion rate. When pH, is raised to its initial value, pHi

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372 A. ROOS AND W. F. BORON Volume 61

returns to normal 122). This technique is of limited use because the rate and
magnitude of the decrease are both low.

E. Acid Extrusion: Coupled Transport of Na+, HCO;, and Cl-

I. Introduction and summary

The regulation of pHi has been studied most extensively in three inverte-
brate preparations that apparently have similar mechanisms of acid extrusion.
The squid giant axon, snail neuron, and giant barnacle muscle fiber possess
an ion-transport mechanism that responds to decreases in pHi by extruding
acid. The rate of acid extrusion is low at normal values of pHi and increases
progressively as pHi is lowered. At a given pHi, the acid-extrusion rate in-
creases with increasing pH, and, at least in barnacle muscle, with increasing
[cyclic AMP]i. Although acid extrusion requires external HCO; and internal
Cl- and is sensitive to inhibitors of anion transport, suggesting a simple Cl-
HCO, anion exchange, external Na+ is also required. Experiments with ion-
specific microelectrodes and with isotopes confirm that acid extrusion is ac-
companied by the influx of Na+ and the efflux of Cl-. Apparently the stoichiom-
etry is two equivalents of intracellular acid neutralized for each equivalent
of Na+ entering and of Cl- leaving the cell. These data are consistent with
several models for acid extrusion: 1) Na+ and HCO; enter in exchange for
H+ and Cl-, 2) Na+ and two HCO; enter in exchange for Cl-, and 3) the
NaCO; ion pair exchanges for internal Cl-. The ion-transport mechanism
responsible for acid extrusion also seems capable of mediating Cl-Cl exchange
(at least in barnacle muscle) and of operating in reverse. The energy required
for acid extrusion is probably provided by the coupled entry of Na+ down
its electrochemical gradient, although an ATP requirement has been described
for squid axons.
Brief mention should be made of an observation by Thomas (435) showing
that after complete blockade of the normal pHiregulating system of the snail
neuron, acid extrusion can be initiated by raising [K+], to about 100 mM. This
may represent a separate K-H exchange mechanism that is nonfunctional at
normal [KS],.

2. Response to acid loads

As discussed above (sect. VIIIC), when a cell is exposed to an increased

level of Co*, pHi declines and then slowly recovers. The recovery of pHi occurs
regardless of the means of acid loading, as would be expected if acid extrusion
were stimulated by a decrease in pHi. Thus recovery of pHi has been observed
in squid axons (43) and barnacle muscle (37) after acid-loading by pretreatment
with NH,+, in snail neurons after iontophoretic (430) or pressure (283) injection

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of H+, and in squid axons (379) and barnacle muscle (48) after dialysis with
acidic fluid.

3. Requirement for external HCO;

As originally shown in squid axons (43) and snail neurons (430), the rate
of acid extrusion is greatly enhanced by HCO;. This is illustrated by the
experiment in Figure 6, in which a squid axon was acid-loaded by pretreating
with NH,+. Comparable results have been found barnacle muscle (37). The
dependence of acid extrusion on [HCO;), was first quantified by Thomas (432),
who found that regardless of pHi, the acid-extrusion rate of snail neurons
increases with [HCO,],, at constant pH, (7.5). For instance, when pHi was 0.25
below the control value, the rate was about 0.2 mmolliter-l=min-l at 0 mM
HCO;, 1.3 at 4.5 mM, and 2.3 at 21 mM. In the barnacle muscle (45) and the
squid axon (380), the dependence of acid-extrusion rate on [HCO;], appears
to follow simple Michaelis-Menten kinetics. In barnacle muscle (pHi = 6.8,
[Na+], = 440 mM), the apparent K, for HCO; is 4.2 at pH, 8.0 and 10.3 mM
at pH, 7.4; lowering pH, has no effect on apparent V,,,. For squid axons
(PH i = 6.7, [Na+], = 425 mM), the apparent K, for HCO; is 2.3 mM at pH,
8.0. These data strongly suggest that external HCO; is a substrate of the
pHi-regulating mechanism. The demonstrable (although low) rate of acid ex-
trusion in the nominal absence of HCO.7 might be due to the presence of
traces of HCO, derived from metabolic CO,.

4. Sensitivity to stil bene derivatives and furosemide

The ability of the disulfonic stilbene derivatives 4,4l-diisothiocyanostilbene-

2,2’-disulfonic acid (DIDS) and 4-acetamido-4’-isothiocyanostilbene-2,2’-disul-
fonic acid (SITS) to inhibit the pHi-regulating mechanism was first demon-
strated in experiments on snail neurons (431). As illustrated in Figure 7, the
iontophoretic injection of H+ elicited a rapid recovery of pHi under control con-
ditions and in the presence of 100 PM ouabain but not with 10 PM DIDS. Similar
results have been obtained with SITS in squid axons (379) and barnacle muscle
(37). Pyridoxal5’-phosphate and p-isothiocyanatobenzenesulfonate (PIBS) also
inhibit acid extrusion in barnacle muscle (37).
These compounds (for structures see Fig. 8) were first found to specifically
inhibit anion fluxes in erythrocytes (for a review see ref. 69); SITS decreases
the efflux of SOi- without affecting K+ efflux (238). Both SITS and DIDS are
confined to the erythrocyte’s exterior (69, 270) and bind to a fixed number of
sites (166, 253, 400). The linear relationship between binding and inhibition of
SO:- efflux (71, 253, 400) points to a rather specific functional activity of these
compounds. In agreement with this, over 95% of labeled derivative is asso-
ciated with a band III protein of -95,000 mol wt (70, 400). The related com-
pounds PIBS (190) and pyridoxal5’-phosphate (68) similarly inhibit erythrocyte

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374 A. ROOS AND W. F. BORON Volume 61

2‘ ot
,4 35 HCl iniehion current
r 20 min ’

Ouabain ‘DIDS
100pM 10pM

FIG. 7. Inhibition of acid extrusion by 4,4’-diisothiocyanostilbene-2,2’-disulfonic acid (DIDS) in

snail neuron. Neuron was iontophoretically injected with HCl at the 4 indicated times. pHi rapidly
recovered from the 1st 3 acid loads, even though ouabain (100 PM) was present in the 3rd case.
Presence of 10 PM DIDS, however, produced a substantial inhibition of pHI recovery after 4th
injection. [From Thomas (431).]

anion transport; PIBS seems to bind to a band III glycoprotein (190), whereas
pyridoxal Y-phosphate is associated both with a band III protein and, to a lesser
extent, with three glycoproteins (68). It is likely that the band III protein with
which these inhibitors interact is identical with at least a portion of the trans-
port protein.


R,-NCS + H2N -R2 F R-NH-C-NH-R2

Edmon Reaction

CH2-H@04 Cl NHCH2 L-J0


Pyridoxal Phosphate Furosemide

FIG. 8. Structural formulas of inhibitors of Na-coupled Cl-HCO, exchange. Edman reaction,

by which isothiocyanate derivatives react with free amines to form NJ-disubstituted thiourea
derivatives, is also shown. Exact mechanism by which furosemide reacts with the cell is unknown.
Pyridoxal 5’-phosphate reacts with free amines to form a Schiff base.

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Internal Fl- -free, 3SOmM Cl-

FIG.9. Dependence of acid
extrusion on internal Cl- in squid
fluid pH 7,3pH 6.1 -.1
giant axon. Axon was internally External HCO: HCOi
t t
dialyzed with a Cl-free, low-pH fluid
solution. After dialysis was halted
pH1 did not recover (segment ab), 7.4-
even in the presence of 10 mM c
HCO; (bc). After the axon
was dialyzed with a solution con- 7.2-
taining 350 mM Cl-, however, ap-
plication of HCO; caused a re- PHi +

covery of pHi (fs) that was blocked 7.0 c

(gh) by 0.5 mM 4-acetamido-4’-
isothiocyanostilbene - 2,2’ - disul- c
fonic acid (SITS). pHi recovery
could also be supported by 150 mM 6X.-
internal Cl- (normal level) . [From
Russell and Boron (379).] 30 min
6.6 t - d

Pyridoxal 5’-phosphate, SITS, DIDS, and PIBS can all react covalently
with free amines (see Fig. 8). Pyridoxal phosphate inhibition of SO;- efflux
in erythrocytes (68) and acid extrusion in barnacle muscle (37) is reversible,
whereas SITS and PIBS inhibition of acid extrusion in barnacle muscle (37) is
irreversible. Those stilbene derivatives incapable of a covalent interaction, how-
ever, can still inhibit anion fluxes in erythrocytes (70). Apparently the elec-
trostatic attraction between the sulfonate groups and a positively charged
area on the cell surface, which may represent the initial interaction of all
stilbenes with the transport protein, is sufficient to block transport. Thus the
inhibition of acid extrusion in squid axons by the noncovalently reacting 4,4’-
dinitrostilbene-2,2’-disulfonic acid (DNDS) is fully reversible (380).
Acid extrusion in barnacle muscle (48) and squid axons (380) is reversibly
inhibited by the diuretic furosemide (0.6-1.0 mM). The drug also inhibits
active Cl- reabsorption from the thick ascending loop of Henle in the rabbit
(59) and Cl- self-exchange in human erythrocytes (51). Furosemide does not
specifically affect anion transport, however, as evidenced by Sachs’ observation
(384) that the drug inhibits both ouabain-sensitive and -insensitive Na+ fluxes
in human erythrocytes.

5. Dependence on internal Cl-

The dependence of acid extrusion on HCO; and its sensitivity to various

inhibitors of anion transport suggested the involvement of Cl-. This was first
confirmed in experiments on dialyzed squid axons (379), which showed that the
ability to recover from an acid load requires internal Cl- and is blocked by SITS
(see Fig. 9). Parallel experiments demonstrated that a component of Cl’ efflux

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376 A. ROOS AND W. F. BORON Volume 61

(similar in magnitude to the calculated HCO; uptake) requires HCO;,

is stimulated by acid loading, and is blocked by SITS. A linkage between Cl-
efflux and acid extrusion has also been established in snail neurons and barnacle
muscle and implicated in A.@ysia neurons. Thomas (432) showed that internal
Cl- is required for acid extrusion in snail neurons and that intracellular Cl-
activity falls during acid extrusion. In dialyzed barnacle muscle (48), both acid
extrusion and Cl- efflux are stimulated by acid loads and cyclic AMP (see sect.
VIIIZD) and inhibited by SITS and furosemide. Although the earlier data of
Di Polo (117), which show that Cl- efflux from barnacle muscle rises as pHi is
reduced or as pH, is increased, were interpreted in terms of a fixed-charge
model, it is likely that at least a portion of these fluxes represent activity of the
pHiregulating system. Finally, Russell (377) has found that acid-loading
ApZysia neurons results in an HCO c-stimulated, SITS-inhibited fall of intra-
cellular Cl- activity.

6. Dependence on external Na+

The involvement of Na+ in regulating pHi was first demonstrated by

Thomas (432), who showed that replacing external Na+ with bis(2-hydroxy-
ethyl)dimethylammonium produced a reversible and nearly complete blockade
of pHi recovery in acid-loaded snail neurons. Removal of Na+ has no effect on
pHi in neurons pretreated with SITS. The author also demonstrated that acid
extrusion is accompanied by a transient rise of intracellular Na+ activity, in-
dicating a net Na+ uptake. The Na+ dependence of acid extrusion has also been
demonstrated in barnacle muscle (45) and squid axons (380). In both cases the
dependence on [Na+], seems to follow simple Michaelis-Menten kinetics. For
barnacle muscle, the apparent K, for external Na+ in seawater containing 10
mM HCO; (pH, = 8.0) is 59 mM. Reducing [HCO;] to 2.5 mM at the same
pH, substantially reduces V,,, but does not significantly alter K,. Conversely,
reducing pH, from 8.0 to 7.4 at constant [HCO;], substantially increases K,
but does not significantly affect V,,,. In squid axons the apparent K, for Na+ is
77 mM (pH, = 8.0, [HCO;], = 12 mM). Radioisotopic studies have revealed
that acid extrusion is accompanied by the net HCO;-dependent uptake
of Na+ (380).

7. Dependence on pHi and pH,

The first demonstration that the recovery rate of pHi from acid loads is ap-
proximately exponential was made on snail neurons (see Figs. 5 and 7; 430).
Thus assuming the total intracellular buffering power to be invariant of pHi (see
sect. IX), the rate of acid extrusion per unit cell volume should vary linearly with
pHi (432), this has been confirmed in barnacle muscle (see Fig. 10; 44). Figure
10 also shows that at any pHi, the acid-extrusion rate increases as pH, and
[HCO;], are simultaneously raised (constant Pco,), a trend first noted in squid

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0.4% co2


r,,,_,o w-I&, 6.0

67. 68. 69. 70

. 71
. 72
. 73
. 74
FIG. 10. Dependence of acid-extrusion rate in giant barnacle muscle fibers on pH, at constant
Pco*. Curves summarize data from 63 fibers that were acid-loaded by the NH: technique and
then allowed to recover from the acid load while bathed in HCO;-containing artificial seawaters
equilibrated with 0.4% CO,. Measurements were made at 4 different values of pH,, (8.6, 8.0, 7.4,
and 6.8) produced by adding the appropriate amounts of HCO; (40, 10, 2.5, and 0.6 mM, respec-
tively). Acid-extrusion rates were obtained by multiplying rate of pHi recovery (dpHi/dt) by
measured intracellular buffering power. Data for pH, values 8.6,8.0, and 7.4 were normalized to pHi
at which dpHi/dt = 0. Each point represents mean of 2-7 fibers at pH, 6.8, 3-11 fibers at pH 7.4,
7-20 fibers at pH 8.0, and 7-19 fibers at pH 8.6. Vertical bars indicate standard errors; lines
represent linear regressions. [From Boron et al. (44).]

axons (43) and later confirmed in barnacle muscle (37) and snail neurons (432).
Other studies on barnacle muscle (44) have shown that the acid-extrusion rate
increases with pH, even at constant [HCO;],, although the increase is only
about 35% of that observed when the two parameters are raised concurrently.
This is to be expected, since changes in [HCO;], alone influence acid extrusion.
Regarding possible mechanisms of this pH, sensitivity, recall that in barnacle
muscle (45), lowering the pH, increases the apparent K, (i.e., it reduces the
apparent affinity) for both external HCO; and Na+ (see sect. VIIIE~ and VIIIEG).

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378 A. ROOS AND W. F. BORON Volume 61

6. ZnJluence of cycZic AMP

When barnacle muscle fibers are acid-loaded by dialyzing with a low pH

solution, and dialysis is subsequently halted to permit pHi to recover, the
addition of 20 PM cyclic AMP to the dialysis fluid causes the rate of the pHi
recovery to increase by about threefold, an effect blocked by SITS (48). Sub-
stantially lower levels of cyclic AMP are effective when dialysis with the low-pH
solution is continuous. Under these conditions, pHi falls to a steady-state level
that is higher than the pH of the dialysate, the pH difference reflecting acid
extrusion. Addition of 1 PM cyclic AMP to the dialysate causes the steady-state
pHi to rise by about 0.1. As expected, furosemide reverses this effect, causing
pHi to fall toward the pH of the dialysate. Other experiments showed that
cyclic AMP has no effect at pHi 7.30, where the acid-extrusion rate is
normally zero.
As anticipated if acid extrusion in barnacle muscle involves HCO; and
Cl- transport, cyclic AMP stimulates Cl- efflux, an effect blocked by SITS and
furosemide. Interestingly Cl- influx was also stimulated by cyclic AMP and
inhibited by SITS and furosemide, suggesting that both Cl-Cl and HCO,-Cl
exchanges avail themselves of the same mechanism (see sect. VIIIEH).
Bittar and colleagues (32-34), also using barnacle muscle, have described
a ouabain-insensitive Na+ efflux that is stimulated by cyclic AMP when pH, is
reduced to 5.8. Such a low pH,, used with an HCO; ASW, is known to cause a
fall in pHi that is likely to be mediated by reversed operation of the pHi-regu-
lating system. Since, as shown above, cyclic AMP stimulates the system, and
since its reverse operation may well involve Na efflux (see sect. VIIIEII),
Bittar’s data can be accounted for without recourse to a new Na+-transport

9. ATP dependence and potential sources of energy

Acid extrusion in intact squid axons is blocked by either cyanide or DNP

(43), agents known to reduce intracellular ATP levels (76). Furthermore work
with dialyzed axons (379) has shown that addition of ATP to the dialysate re-
stores acid extrusion, even while axons are poisoned with cyanide, and that the
linked efflux of Cl- is similarly affected. It must be emphasized that these results
do not prove that acid extrusion is linked to ATP hydrolysis. The known Na+
dependence of acid extrusion and the associated influx of Na+ down its electro-
chemical gradient (380) suggest that Na+ entry may provide the energy neces-
sary for acid extrusion. Although it is possible that both ATP hydrolysis and
the NA+ gradient energize this process, it is more likely that one fills only a
catalytic role. Interestingly Russell has observed that the coupled influx of
Na+ and Cl- into squid axons also requires ATP, though again the Na+ gradient
could provide sufficient energy (376, 378). These dependencies on ATP may
reflect the axon’s attempt to minimize dissipative processes in the face of
diminished energy sources.

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There seems to be no requirement for ATP in snail neurons (431), where

carbonyl cyanide m-chlorophenylhydrazone - although reducing ATP levels
sufficiently to inhibit the Na+ pump- has no effect on acid extrusion. Of course,
a very high affinity of the acid-extruding mechanism for ATP cannot be ruled
out. The dependence on ATP of acid extrusion has not been examined in
barnacle muscle, where a high glycogen content makes substantial reductions in
[ATP]i very difficult. Given the Na+ requirement in all three preparations and
the demonstration of net Na+ uptake in the snail neuron (432) and squid axon
(380), the most likely energy source for acid extrusion in these cells is the Na+
gradient. A simple calculation shows this gradient to be more than adequate
to drive the ion fluxes discussed below.

IO. Stoichiometry and models of acid extrusion

Because V, changes by no more than a few millivolts during acid extrusion

in the snail neuron (431) and since no significant change in V, occurs in barnacle
muscle after acid extrusion is blocked with SITS (37), acid extrusion in these
cells is probably electroneutral. Thomas has proposed a model for the snail
neuron (see Fig. 11, top), in which Na-H and HCO,-Cl exchanges are tightly


7-k Cl-+ H+

FIG. 11. Models of Na-coupled, Cl-

HCOZ exchange. The 1st model (top),
proposed by Thomas (432), suggests that
external Na+ and HCO; exchange for
internal H+ and Cl-. The 2nd model
(middle), also by Thomas (432), is like
lst, but H+ efflux is replaced by influx of
Na++ ZHCO; -
an extra HCO;. In 3rd model (bottoms),
proposed by Becker and Duhm (24), the
NaCO, ion pair exchanges with internal
Cl-. All models are electroneutral and
have a stoichiometry of 1 Na+ taken up
for each Cl- lost and for each pair of
protons neutralized intracellularly.

No++ co; s Na CO;


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380 A. ROOS AND W. F. BORON Vohme 61

coupled and driven by the energy of the Na+ electrochemical gradient (432,
436). This model is indistinguishable from one in which H+ efflux is replaced
by the influx of either OH- or an additional HCO; (Fig. 11, middle); again
the process would be electroneutral. The stoichiometry of acid extrusion has
been examined in only a few studies. In three experiments, Thomas (432) ob-
tained an acid extrusion/Na+ uptake ratio of about 4, rather than the expected 2.
In a single experiment he injected equal amounts of Na+ and H+ into the same
cell, finding that the Na+ injection caused intracellular [Na+]i to rise by 8.5 mM,
whereas acid extrusion after the H+ injection produced an increase in [Na+]i
of 4.6 mM. This ratio is close to the expected 2. Thomas pointed out that the
discrepancy between this and the previous three experiments would be resolved
if only half as much Na+ were distributed as H+. Intracellular binding of Na+
would also account for the data. In seven similar experiments Thomas measured
intracellular Cl- depletion during pHi recovery, obtaining a mean ratio of acid
extrusion/Cl- loss of about 4. This value could also be reconciled with the models
of Figure 11 if internal Cl- behaved similarly to Na+ regarding binding and/or
distribution. In squid axons, Russell and Boron (379) have used “Vl to estimate
the Cl- efflux accompanying acid extrusion and obtained a value of 3.9 pmol
cm-2 l s-1
12) at 15°C at pHi = 6.7, pH, = 8.0, and [HCO,], = 10 mM. The
(n =

estimated acid-extrusion rate in three experiments was about 5 pmol crnm2 s-l. l l

More recently a larger series of experiments, under similar conditions and

using a better estimate for intracellular buffering power, has yielded an acid-
extrusion rate of 7.5 pm01 cm-* s-l (n = 15) (W. F. Boron and J. M. Russell,
l l

unpublished observations). Also measured were Na+ influx, which amounted to

about 3 pmol~cm-2s-1 (n = 6), and Cl- influx and Na+ efflux, which were
negligible (380). Taken with the previous estimate for Cl- efflux, these re-
sults are consistent with a stoichiometry of two equivalents of intracellular acid
neutralized for each equivalent of Na+ taken up and Cl- released.
A different model of acid extrusion, based on the existence of the ion pair
NaCo;, was proposed by Becker and Duhm (24) in 1978 (Fig. 11, bottom).
Thirteen years earlier, Wieth and Funder (472) had observed a threefold in-
crease of Na+ influx into ouabain-treated, nonnucleated red cells when ex-
ternal Cl- was replaced by HCO;. Wieth (471) suggested that Na+ (and also
Li+) enters these cells after combining with CO:- to form the ion pair NaCO;
(or LiCO;), the existence of which had previously been demonstrated by
Garrels and colleagues (142). Later Callahan and Goldstein (77) showed that the
HCO;-stimulated Na+ influx is reduced by SITS and furosemide, inhibitors
of anion exchange. Also Funder et al. (138) demonstrated that the inhibition
by DIDS of both Cl-Cl exchange and HCO;-induced Li+ uptake is linearly re-
lated to DIDS binding to band III proteins derived from the cell membrane.
These findings suggest that the erythrocyte’s anion transport system can ex-
change NaCO; for Cl- and provide the basis of Becker and Duhm’s model of
acid extrusion. Since CO:-, after having entered the cell, can neutralize two
protons, the stoichiometry of this model is the same as Thomas’ model. Re-
cently one prediction of the ion-pair model, namely that reducing [HCO;], at

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constant pH, should raise the apparent K, for Na+ without affecting V,,,, was
contradicted in acid-extrusion experiments on barnacle muscle (45): K, was
not significantly changed, whereas V,,, was reduced (see sect. VW!%).

11. Cl-Cl exchange and reversal of acid extrusion

The rate of acid extrusion is greatest at low pHi values, and in the barnacle
muscle, progressively falls to zero as pHi approaches the threshold value of
-7.35. Although at this threshold, no net acid extrusion occurs, there is evi-
dence that the pHi-regulating mechanism slowly exchanges internal Cl- for ex-
ternal Cl-. 1) At pH 7.3, small and approximately equal Cl- fluxes in both
directions have been demonstrated (48); 2) both fluxes are inhibited by SITS and
by furosemide (48); and 3) internal Cl- is required for the Cl- influx (J. M.
Russell and M. S. Brodwick, unpublished observations). As pHi decreases
below the threshold, two changes in the ion-transport system apparently occur:
1) the system partially shifts away from the Cl-Cl mode to permit acid ex-
trusion and 2) the total rate of ion exchange increases. Supporting the second
point is that both Cl- influx and efflux (48) and the acid-extrusion rate pro-
gressively increase as pHi is lowered. Furthermore evidence for competition
between HCO, and Cl- suggests that Cl-Cl exchange and acid extrusion are
mediated by the same carrier: at pHi values below the threshold, increasing
[HCO& causes the acid-extrusion rate to rise and Cl- influx to fall. At the
threshold, however, increasing [HCO;], has no effect on either the acid-ex-
trusion rate (which remains at zero) or Cl- influx. Finally, cyclic AMP appears
to enhance the anion-exchange mechanism in both the Cl-Cl and acid-extrusion
modes. Ashley et al. (15) also provided evidence for Cl-Cl exchange in barnacle
muscle. They showed that Cl- efflux is reversibly inhibited by furosemide and
by removal of external Cl-. Their failure to detect an HCO; dependence of
Cl- efflux is to be expected, inasmuch as pHi was probably normal.
Under certain conditions, the pHi-regulating systems of barnacle muscle
and snail neurons seem to operate in “reverse,” mediating a fall in pHi. When
barnacle fibers are exposed to an acidic, bicarbonate-containing solution (pH,
- 6.5), the initial fall in pHi due to influx of CO, is followed by a further decay
of pHi (37). This decay is dependent on HCO; and SITS, suggesting that it
involves the pHiregulating mechanism (44). The work of Keifer (225) on acid-
loaded barnacle muscle confirms the participation of Na+: either removing
external Na+ or raising [Na+]i by dialysis causes a SITS-sensitive fall in pHi.
Similar results have been obtained in snail neurons (436), where lowering pH,
from 7.5 to 6.5 at constant PCO~leads to a SITS-sensitive fall in pHi. In addition
the decline in pHi is accompanied by a SITS-sensitive rise in intracellular Cl-
activity and is blocked by removal of external Cl-. As expected, raising internal
Na+ accelerates the fall in pHi, suggesting the involvement of Na+.
The above observations on Cl-Cl exchange and reversal of the pHi-
regulating system are not inconsistent with any of the models of Figure 11.

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382 A. ROOS AND W. F. BORON Vobume 61

all could be expanded to permit several modes of ion exchange (e.g., Cl-Cl ex-
change as well as forward and reversed acid extrusion), with net transport
determined by the balance among the various modes. The first two models
question whether Cl-Cl exchange would require Na+. If so, Cl-Cl exchange
might be accompanied by Na-Na exchange. Apparent Na-Na exchange might
also occur as the result of simultaneous forward and reversed acid extrusion.
According to the third model, Cl-Cl exchange would have no Na+ requirement
and thus would not be accompanied by Na-Na exchange. Apparent Na-Na ex-
change could be produced, however, by (NaCO&(NaCO& exchange or by
simultaneous (NaCO,)i-Cl, and Cli-(NaCO,), exchanges. All three models
predict that net acid extrusion should increase with increasing [Na+], and
[HCO& and with decreasing [Cl-], as well as with the opposite changes

F. Acid Extrusion: Na-H Exchange

Initial evidence for exchange of external Na+ for internal H+ was obtained
from sea urchin eggs (220) and membrane vesicles from the brush borders of rat
small intestine and kidney (305). In sea urchins, fertilization induces an
amiloride-sensitive rise in pHi and fall in pH,, events accompanied by an
amiloride-sensitive uptake of Na+. In rat brush-border membrane vesicles,
Na+ uptake is stimulated by lowering pHi, and this is attended by a fall in pH,.
Subsequent studies with pH-sensitive microelectrodes have confirmed and
extended these observations in other preparations. In mouse soleus muscle
(ll), crayfish neurons (301), and proximal tubule cells (41), the recovery of
pHi from an intracellular acid load is accompanied by an increase in intra-
cellular Na+ activity, it is substantially inhibited by removal of external Na+,
and it is insensitive to stilbene derivatives. Also amiloride reversibly inhibits
both the pHi recovery and the attendant rise in intracellular Na+ activity in
muscle. Although amiloride produced a 70% blockade of acid extrusion in mouse
muscle at 0.1 mM, it was only minimally effective in the proximal tubule
at that concentration. With the latter preparation, however, 2 mM amiloride
produced nearly a 100% blockade at an external Na+ concentration of 10 mM
and a 75% blockade at 100 mM. This suggests competition between amiloride
and Na+ and may provide an explanation for the drug’s apparent failure (at
0.1 mM) to inhibit acid extrusion in crayfish neurons bathed in 195 mM Na+.
The Na+ gradient probably provides the energy for Na-H exchange: in crayfish
neurons the recovery of pHi from an acid load, which normally proceeds until
pHi returns to the normal value of -7.15, halts prematurely when pH, is lowered.
In an experiment at a pH, of 5.9, pHi leveled off at 6.74, representing an
[H+]J[H+]i of 6.92. This is not far from the measured [Na+]J[Na+]i of 6.96 and
suggests that the Na+ gradient was no longer adequate to fuel H+ extrusion.
It should not be conclu .ded that pH, and the Na+ gradient norm .ally determine
the level at which pHi stabilizes after recovery from an acid load. Under

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physiologic conditions, pHi stabilizes at about 7.1 in crayfish neurons, even

though the energy of the Na+ gradient is sufficient to drive PHi more than a full
pH unit higher. Apparently at pHi values above some threshold, acid extrusion
is turned off irrespective of the abundance of the energy supply.
Evidence for Na-H exchange has also been obtained in two other prepara-
tions of renal origin, brush-border membrane vesicles isolated from rabbit
renal cortex and a cell line derived from dog kidney. Kinsella and Aronson (233)
showed that the brush-border membrane vesicles, derive primarily from the
proximal tubule’s luminal membrane and extrude H+ in the presence of inward-
directed gradients of Na+ and Li+ but not K+. Conversely, Na+ uptake into
vesicles (K, = 5.6 mM) is stimulated by acid loads and inhibited by amiloride
(Ki = 15.6 PM) and harmaline (234); SITS, furosemide, and acetazolamide
are without effect (233). Kinsella and Aronson also showed that Na+ uptake
is competitively inhibited by Li+ but not by K+, Rb+, Cs+, and choline (233).
Interestingly Na+ efflux is stimulated by external Na+ (235). If this trans effect
proves to be amiloride sensitive, it would indicate an Na-H exchanger that also
engages in Na-Na exchange. In the second study, Rindler et al. (361) showed
that a ouabain-insensitive component of Na+ influx into cultured dog kidney cells
is apparently electroneutral, sensitive to amiloride (Ki = 16 PM), competitively
inhibited by external Li+, insensitive to SITS and to Cl- replacement, and
stimulated by CO, and acetic acid. These last results suggest that the Na+ up-
take is sensitive to intracellular acid loads. Under steady-state conditions,
much of the uptake depended on internal Na+; this suggests once again that the
carrier responsible for Na-H exchange may also mediate Na-Na exchange.

G. Acid Extrusion in Vertebrate Cells

Although not as extensively studied, the vertebrate pHi-regulating mech-

anism shares a number of properties with its invertebrate counterpart.
Mammalian skeletal and cardiac muscle respond to acid loads by returning pHi
toward normal; this recovery is not affected by inhibitors of the Na+ pump
(11, 122, 368). In rat cardiac muscle, acid extrusion is enhanced by cyclic AMP
(130). A SITS-sensitive Cl-HCO, exchanger has been identified in mouse
soleus muscle. In addition these cells possess an independent, amiloride-
sensitive Na+-H+ exchanger (11). In contrast to the situation in mammalian
muscle, pHi of frog skeletal muscle does not recover after modest acid loading
(pHi reduced by O.l-0.2), even in an HCO;-Ringer’s solution of pH, 7.4 (35).
Nevertheless an acid-extruding mechanism is indicated because the steady-state
pHi values are substantially higher than if H+ were passively distributed.

I. Ouabain

If H+ were transported out of the cell by the Na+ pump, as suggested by

Woodbury (480), acid extrusion would be blocked by ouabain. The failure of

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384 A. ROOS AND W. F. BORON Volume 61

Adler (5) to observe a fall in pHi when rat diaphragm muscle was exposed to
ouabain, even though [Na+]i increased sixfold, might have been due to the fibers
being insufficiently acid loaded. Roos (368) later showed, however, that even
after diaphragm muscle is acid-loaded by exposure to 46 mM DMO or 118 mM
lactate, ouabain does not significantly reduce steady-state pHi, although it does
raise [Na+]i. Similarly no effect of ouabain on pHi after acid loading was ob-
served by Ellis and Thomas (122) in sheep Purkinje fibers or by Aickin and
Thomas (11) in mouse soleus muscle. Inhibition of the Na+ pump by removal
of external K+ equally fails to inhibit acid extrusion (11). Thus it can be con-
cluded that acid extrusion is not mediated by the Na+ pump.

2. Cyclic AMP

Riegle and Clancy (360), using DMO to measure pHi of perfused rat cardiac
muscle in vivo, found that the ability to resist a CO,-induced decrease in
pHi is enhanced by norepinephrine and diminished by P-adrenergic blockade.
Later Fenton et al. (130) found that either dibutyryl cyclic AMP or glucagon,
when added to the coronary perfusate, exerts a similar effect. Since dibutyryl
cyclic AMP can directly enter the cell, whereas norepinephrine and glucagon
both stimulate cyclic AMP formation, these data strongly suggest that cyclic
AMP enhances myocardial acid extrusion. Experiments on whole rats (92), in
which P-adrenergic blockade reduced the ability of skeletal muscle to resist a
respiratory or metabolic acidosis-induced decrease in pHi, suggest that the
same may be true in skeletal muscle. These results are thus in harmony with
the previously discussed studies on barnacle muscle (48).

3. Evidence for Na-H exchange

This has been examined in detail in section VIIIF. Experiments on mouse

soleus muscle indicate that the amiloride-sensitive exchange of external Na+ for
internal H+ accounts for about 70% of this tissue’s total acid-extruding capacity
(11). The remainder is presumably due to HCO,-Cl exchange.

4. Evidence for HCO,-Cl exchange

In addition to Na-H exchange, pHi regulation in mouse soleus muscle may

involve HCO,-Cl exchange (11). This is suggested by the findings that 1) re-
moval of HCO; or addition of SITS reduces the rate of pHi recovery from an
acid load by about 30%; 2) the effect of SITS is additive to that of amiloride ap-
plication or Na+ removal; and 3) the &lo of the pHi recovery is 2.6 before but
6.9 during inhibition by amiloride, suggesting that amiloride blocks the normally
dominant, low-Qlo Na-H exchanger and leaves intact a low-capacity, high-Q10
HCO,-Cl exchanger. Implicit in the conclusion that two independent systems

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exist is the assumed specificity of action of amiloride and SITS. Furthermore

if HCO,-Cl exchange requires Na+ (as in several invertebrate cells; see above),
it would be difficult to distinguish between Na-H and Na+-dependent HCO,-Cl
It is possible that the HCO,-Cl exchange system primarily serves to
regulate [Cl-Ii. It is of interest that an HCO,-Cl exchange has been demon-
strated in sheep Purkinje fibers (449). Removal of external Cl- causes intra-
cellular Cl- activity to fall slowly and pHi to rise. Restoration of Cl- to the
external medium causes a rapid, HCO;-dependent recovery of intracellular
Cl- activity and a simultaneous fall in pHi; both transients are sensitive to SITS.
A study of the pHi dependence of this Cl- recovery might elucidate the role
played by HCO,-Cl exchange in regulating intracellular Cl- activity and pHi.

H. Response to Intracellular Alkaline Loads

There is no evidence to suggest that cells possess a specialized transport

mechanism to accumulate acid in response to alkaline loads. Surely cells can
recover from alkaline loads. For example, after a post-CO, overshoot, pHi
eventually returns toward normal (10, 37, 430). On the other hand, when
fresh barnacle muscle is alkaline-loaded by exposure to NH, (37) or by internal
dialysis with an alkaline solution (39), the rate of pHi recovery can be negligibly
small. The mechanism of recovery from alkaline load has not been carefully
studied. Aickin and Thomas (10) have suggested that the recovery is due to
passive fluxes of ions. Boron et al. (44) have pointed out that metabolically
produced acid may also contribute to the recovery. After barnacle muscle fibers
have been mounted in a chamber for an appreciable period (but especially after
recovery from an acid load) blockade of acid extrusion produces a progressive
fall in pHi, wheres such blockade is without effect in fresh fibers (44). This sug-
gests that an alteration in metabolism is responsible for the acidification. Al-
though there is evidence that the ion-transport mechanism responsible for acid
extrusion in snail neurons (436) and barnacle muscle (44) can operate in reverse
at a very low pH, (see sect. VIIIE~ I ), it is not clear how significant this reversal
is during alkaline loading at normal pH,. The most likely explanation for the
pHi recovery from alkaline loads is that the increase in pHi inhibits acid ex-
trusion (see sect. VIIIE7), thereby leaving unchecked the acidifying effects of
passive ion fluxes and metabolism.

I. Factors Influencing Steady-State Intracellular pH

1. Theoretical considerations

By definition pHi is in a steady state when the rate of acid loading of the
cell (JJ equals the rate of acid extrusion (&). Thus changes in steady-state
pHi can result from alterations in JL and/or &. As pointed out in section VIII A, J,

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A. ROOS AND W. F. BORON Volume 61

FIG. 12. Schematic representation of

dependence of acid-extrusion rate on pH1.
Lines A, B, and C reflect differences in
rate constant of acid extrusion under dif-
ferent conditions of pH, or substrate con-
centrations. pHi is the threshold pHi,
above which acid-extrusion rate falls to 0
regardless of pHi.

is determined by the rate of metabolic acid production and by the fluxes of ions
across the plasma membrane. As discussed in sections VIIIE and VIIIF, JE is
largely determined by pHi, pH,, and substrate (e.g., Na+) concentrations.
Figure 12 schematically illustrates the dependence of J, on steady-state pHi for
three conditions of pH, or substrate concentration (curves A, B, and C).
At the threshold pHi (PHI), J, is zero; at lower values of pHi, J, linearly in-
creases (44, 48,432). The slope of the relationship between JE and pHi is deter-
mined by pH, and substrate concentration: the greater their composite positive
effect on JE, the steeper the slope. Imagine that curve B represents the de-
pendence of JE on pHi under control conditions and that J, has the value J,. In
the steady state, this requires that J, = J, = JZ, and thus pHi = pHb. Now let
JL remain unchanged while pH, and/or substrate levels are altered such that the
JEwpHi relationship is represented by curve A. This change, per se, would lead
to a reduction of J, from J, to J1, so that J, > J,. This discrepancy results in a
reduction of the pHi, until JE and JL are once more in balance. That is, pHi falls
from pHb to pH,. Conversely, had the conditions been altered so that the JE-
pHi relationship would now be represented by curve C, then a new balance
between JE and J, would be achieved at pHi = pH,. The diagram also illustrates
the effect on pHi of changes in J1,. For example, let curate C represent the
JEwpHi relationship and let the initial J, have the value Jp, corresponding
to an initial steady-state pHi of pH,. If JL is now increased to a value represented
by Js, pHi falls ( JL > JE) until once more J, J = JE. That is, the new steady-state
pHi is pHb.
This elementary analysis may be useful for gaining insight into the mech-
anisms by which pHi changes to a new steady-state value. It must be admitted
that not infrequently the situation is more complicated than assumed above.
Thus the new imposed condition (e.g., a change in Pco,) might bring about
changes both in pH, and pHi. Since the pHi change might affect metabolism
and ion fluxes, both J1, and JE would be changed simultaneously.

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2. Application to some experimental findings

We can now interpret some of the older steady-state pHi data in the light
of our present knowledge through an approach comparable to the one above.
We have selected as a prototype the two 1965 papers by Adler et al. (7, 8) on
mammalian skeletal muscle. (For similar, though less complete, recent work on
this tissue, both in vivo and in vitro, see 60,150- 152,170,232,246,326,341,367.
The earlier work has been reviewed by Waddell and Bates in 454.) Adler et al.
measured the pHi with DMO in excised, intact rat diaphragms exposed for
4-6 h to various solutions. The internal acid load the muscle cells experienced
under normal conditions (pH, = 7.39, PCO~= 39 mmHg, [HC0,3, = 23 mM,
PH i N 6.89, [HCOy]i z 7 mM) has two components, the H+ generated by
cellular metabolism and the HCO; passively leaving the cells, which is driven
by an outward-directed electrochemical gradient for HCO; of approximately
60 mV, assuming a membrane voltage of -90 mV. [Although H+ ions are driven
into the cell by an electrochemical gradient of equal magnitude and their
permeability should be much greater than that of HCO; (see sect. v), their
net flux is probably very small because prevailing H+ concentrations are 4-6
orders of magnitude smaller than those of HCO;.] As stated above, this acid
load is balanced by an acid-extrusion mechanism that in mammalian muscle
probably consists mainly of an exchange of internal H+ for external Na+ (see
sect. VIIIG; 11).
The data of Adler et al. (7, 8) show that proportionally raising Pco2 and
[HCO& so that pH, remains unchanged generally causes the steady-state pHi
to fall. Thus at pH, 7.5, pHi was 7.24, 7.04, and 6.94 at CO, tensions of
12, 28, and 37 mmHg, respectively. This fall in pHi can be explained as follows.
As Pcop is raised, the influx of CO, produces a rapid fall in pHi and an increase in
[HCOi]i. Whereas the decrease in pHi stimulates acid extrusion and
possibly suppresses metabolic acid production (228), both of which tend to
increase pHi, the attendant rise in [HCOT]i leads to an increased HCO;
efflux, which results in a further fall in pHi. [Note that when Pcop and [HCO;],
are proportionally increased, the increase in [HCO;], is generally greater
than the increase in [HCOc]i. Yet since the latter’s influence on net HCO;
efflux, as given by the constant field equation (147, 191), is weighted by the
factor exp(-V, FIRT), which is about 29 at a V, of -90 mV, the result is an
increase in HCO; efflux.] Whether pHi recovers from the initial, CO*-induced
acid load or continues to fall depends largely on the increment in HCO; efflux
and thus on HCO; permeability and V,. If HCO; efflux predominates,
pH, continues to fall until J, = J,. The greater the Pco~, the greater [HCOc]i
and thus the lower steady-state pHi will be. If acid extrusion prevails, the re-
covery of pHi will be increasingly inhibited by HCO; efflux at higher CO,
levels. Thus regardless of whether pHi continues to fall or recovers, the new
steady-state pHi is expected to decrease with increasing Pco*. The partial,
though incomplete, recovery of pHi has actually been observed with micro-
electrode studies on sheep heart Purkinje fibers (122): the proportional increase

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388 A. ROOS AND W. F. BORON Vohme 61

in PCO* and [HCOJ, causes pHi to fall, recover, and level off at a somewhat
lower value than the control. In some of the studies by Adler et al., the acidifying
and alkalinizing effects of raising PCO~at constant pH, were apparently about
equal, since pHi remained unchanged. Thus pHi remained at 6.88 when, at pH,
7.42, Pco2 was raised from 38 to 119 mmHg (8).
Of interest is the time course of pHi observed with microelectrodes in
mouse soleus muscle (10) and sheep heart Purkinje fibers (122) when, at constant
pH,, Pco2 is reduced below its normal value. After its initial rise due to CO,
efflux, pHi slowly drifts down to a new value, which is still higher than the
control. The cause of this drift is not known; it may be due to metabolic adjust-
ments of the cell (see sect. VIIIH). The mechanisms that determine the increase
in steady-state pHi are otherwise the same as those discussed above.
We can make two predictions from this model of pHi regulation. 1) When
Pcoz is raised while [HCO;],, rather than pH,, is maintained unchanged, the
initial, rapid fall in pHi should be the same under the two conditions (i.e.,
constant [HCO&, and constant pH,). Since pH, falls, however, acid ex-
trusion is inhibited. Also since [HCO,], is constant, the increase in [HCOT]i
dictates an even greater increment in HCO; efflux (assuming constant V,
and membrane permeability to HCO;). Thus the recovery of pHi is inhibited
and the further decay is enhanced, with the result that the new steady-state
pHi is lower than when pH, is held constant. Indeed Adler et al. found that
raising PCO*from 38 to 119 at 23 mM [HCO;], (pH, 7.42 and 6.87, respectively)
causes the steady-state pHi to fall from 6.89 to 6.73 (7); the same PCO~change at a
constant pH, of 7.42 has no effect on pHi (8). 2) The model also predicts that at
constant PC02, steady-state pHi should rise or fall with pH, (i.e., with [HCOJ,).
The effect of lowering pH, in this way is a reduction in the rate of acid extrusion,
whereas the accompanying reduction of [HCO;], enhances HCO; efflux. Each
of these changes tends to reduce the value to which pHi must fall until JE and
J, are once more in balance. Conversely raising pH, at fixed Pcoz enhances
acid extrusion, whereas the accompanying increase in [HCO,I, reduces
HCO; efflux. Thus the steady-state pHi must rise. In general the data of Adler
et al. (7,8) confirm these predictions. However, these workers (7) and Heisler (170)
claimed that at a PCO~of about 38, pHi remains unchanged as pH, is varied
between 7.4 and about 7.0. Microelectrode studies on mouse soleus muscle
have failed to confirm such a pHi plateau (10).
A number of workers have carried out experiments similar to the above
on intact mammalian heart (120, 149, 246, 247, 341, 425, 426; for a review of
earlier studies see 454). The observed changes in steady-state pHi are quali-
tatively similar to those in skeletal muscle. Lai et al. (246, 247) and Poole-
Wilson and Cameron (341) have tentatively ascribed the smaller fall in myo-
cardial pHi than in skeletal muscle pHi in response to the same increase in PCO~
to a higher rate of acid extrusion in heart muscle.
Izutsu (209) has examined the effect of various combinations of [HCO,I,
and PCO~on the steady-state pHi of bullfrog toe muscles, measured with DMO
after 7 h of incubation. Many of his findings are similar to those obtained on

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mammalian muscle. Thus altering Pco2 at constant [HCO;], causes reciprocal

changes in pHi. Also reducing [HCO;], from 24 to 5 mM at constant PCO~
leads to a reduction in pHi. The author made three interesting observations. 1)
At high [HCO;], (24-50 mM), a new steady state is achieved in only about 15
min; pHi then remains unchanged for as long as 10 h. In contrast when [HCO,],
is lowered, it takes about 5 h to reach a steady state. 2) Increasing [HCO&
from 24 to 40 or 50 mM at constant PCO~(7-42 mmHg) has no effect on pHi.
3) The relationship between calculated steady-state [HCOc]i and pHi (obtained
in experiments in which PcoB was varied between 7 and 90 mmHg while
[HCO,], was kept constant at 5, 24, or 40-50 mM) could be represented
by three different straight lines, one for each value of [HCO;],. The line for
40-50 mM [HCO,I, was steepest and had a slope of 38 mM/pH unit. This slope
was experimentally indistinguishable from the theoretical titration slope cal-
culated by Woodbury (480) by summing the buffering power of the muscle
constituents. This theoretical slope, about -41 mM/pH unit, represents the
negative of the intrinsic buffering power of the myoplasm (see sect. IX) and
agrees rather well with the value of 35 mM obtained in a microelectrode study on
frog muscle (35). The similarity of the theoretically derived intrinsic buffering
power with the observed negative slope of the [HCOg]i-pHi relationship at
high [HCO;], would indicate the absence of significant net ionic fluxes (both
active and passive) in response to changes in Pco*. That is, at 40-50 mM
[HCO;I, frog muscle seems to behave as a closed system except regarding
CO,. This hypothesis would also explain the first two of Izutsu’s observations: a
new steady state is reached as soon as CO, has equilibrated, and raising
[HCOJ-, has no effect on pHi as long as PCO* is kept unchanged. These
phenomena would require a low membrane permeability to HCO; and a
sluggish response of the acid-extrusion mechanism to a fall in pHi* Interest-
ingly microelectrode studies on frog muscle (35) have failed to show evidence
of acid extrusion for at least 20 min after the abrupt fall in pHi accomplished by
increasing Pco2 at constant pH, (7.2). These findings are in striking contrast
to those on numerous other preparations, where a similar acid load is followed
by a rapid recovery of pHi (see sect. VIIIC).


A clear understanding of intracellular buffering and of the parameters af-

fecting its magnitude is crucial for gaining quantitative insight into pHi regula-
tion and other transport processes involving H+ or equivalent ions. Here we
develop a framework for interpreting buffering power data.

A. Physiochemical Buffering

Physicochemical buffering is a property of weak acids and bases whereby

these compounds minimize shifts in pH by reacting with exogenous H+ accord-
ing to the equation

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390 A. ROOS AND W. F. BORON Volume 61

Mn + H+ % MHn+’ (35)

where Mn is a weak base of valence n and MHn+l is a weak acid of valence

n + 1. The subject of buffering was of considerable interest at the turn of the
century (for references see 93), but no progress was made in quantifying the
effect until 1908 when Henderson (173) and Washburn (467) pointed out that a
weak acid exerts maximum buffering when its dissociation constant equals
[H+]. It must be added that the two authors proved this point only for acids
whose dissociation constant equals the [H+] at neutrality, that is, whose
pK ‘v 7. Six years later Koppel and Spiro (239) introduced the first general
measure of “buffer action,” P

P = as - s,> (36)

where dS is the amount of strong acid (M) required to produce a small pH

shift (dpH) in the buffered solution, and dS, is the amount required to produce
the same pH shift in an unbuffered solution of the same pH. Since the addition
of acid lowers pH, P is negative. The authors showed that for a monobasic weak
acid (HA L, H+ + A-) of apparent dissociation constant K

P - -203K[H+l ,TA]
(K + [H+])2

where [TA] = [HA] + [A-]. Furthermore by setting dP/dpH = 0, they demon-

strated that P achieves its maximal value of -0.58[TA] when [H+] = K. Thus
they were the first to rigorously derive the general conditions of maximal buf-
fering. A consequence of this analysis, noted by the authors, is that the maximal
buffer action of all monovalent weak acids at equivalent total concentration
is the same. They also demonstrated that the total buffer action of a mixture of
buffers is the sum of the individual buffering actions, each calculated from
Equation 37. An equation analogous to Equation 37 was also derived for a weak
base, and the buffering powers of dibasic weak acids and ampholytes
were given.
This innovative work failed to draw the attention of Clark (93), but was
hailed in 1922 by Michaelis in his monograph (288). Michaelis modified the
definition of buffering power so that


where dB represents the amount of added strong base. Also in 1922 Van
Slyke (444), apparently unaware of Koppel and Spiro’s work (239) until after his
paper was in press, repeated part of their analysis but defined buffer value as in

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Equation 38. The differences between the definitions given in Equations 36

and 38 are that p and P are of opposite sign, and that p includes the so-called
self-buffering of water. This effect, subtracted in the definition of Koppel and
Spiro, becomes apparent at extreme pH values but is negligible in the
physiologic pH range. The definition of buffering power as given by Michaelis
and Van Slyke (Eq. 38) is now generally accepted. It is unfortunate that
the pioneering contribution of Koppel and Spiro (see 371 for a translation and
historical details) has been ignored to the extent that Woodbury (480) has
proposed naming the unit of buffering power after Van Slyke.
Equation 37 is valid when the buffer concentration is constant but not
when one of the buffer partners can exchange with the surroundings. This
was recognized by Van Slyke (444) in his analysis of the contribution of the
CO,-HCO; pair to the buffering of blood. In a closed system, where the CO,
buffer concentration ([CO,] + [ HCO,I) is constant, the buffer value of the
CO,-HCO; pair, pco2 (given by Eq. 37), is rather small, due to the large dif-
ference between pK; (-6.1) and the pH of the blood. Thus pcoz contributes
only about 10% to total buffering of the blood in a closed system (444). On
the other hand, CO,-HCO; may make a significant contribution in an open
system in which [COJ, rather than [CO,] + [HCO;], is constant. When acid
is added to such an open system, some of it combines with HCO; to form
H&O, that leaves in the form of COz. The amount of acid neutralized equals
the fall in [HCO;]. The opposite occurs when alkali is added. Thus in an open
system, pco2 is given by ( .[HCO;]/apH),ro,, which is evaluated by dif-
ferentiating the Henderson-Hasselbalch equation at constant Pco2

pco2 = (a’;;;“‘),,02 = 2.3sPco,10~~-p~ = 23[HCO,] (39)

where s is the solubility of CO,. When pH is sufficiently high, the equilibrium

HCO, = CO;- + H+ cannot be ignored, and PcOais given by the more gen-
eral expression (64)

PCO;! = 23[HCO,] + 4.6[CO;-I (40)

The contribution of CO,-HCO; to overall buffering in an open system can be

substantial. In blood, for example, pcoz is more than twice as great as the
buffering provided by all other buffers combined.
This analysis is of considerable importance in obtaining a figure for the total
buffering power &) of intracellular fluid. The plasma membrane is highly
permeable to CO, and because the extracellular fluid approximates an infinite
reservoir for C02, the cell can be considered an open system with respect to CO,.
In mammalian muscle (pHi = 7.1 and PCO~ = 37 mmHg), in which the non-CO,
or “intrinsic” (37) buffering power, p,, may amount to about 40 mM,
PCO2 is approximately 29 mM (lo), that is, about 40% of the total buffering

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392 A. ROOS AND W. F. BORON Volume 61

power. The cell may also behave as an open system for other weak acids
(HA L, H+ + A-) or bases (B + H+ G BH+), provided the plasma membrane
is sufficiently permeable to their uncharged form only, so that in the steady
state, [HAli = [HA],or [B]i = [B],. A striking example of the contribution made
by a weak base to PT in an open system is seen in experiments with the
NH,/NH,+ pair. Recall from section v that exposing a squid axon to 10 mM
NH,+ at pH, 7.7 causes a rapid rise in pHi that is followed by a plateau phase
during which pHi falls at a much slower rate (see Fig. 3B). During this plateau
phase, [NH,]i remains nearly equal to [NH& (i.e., the system is open to NH,),
so that the contribution of NH,/NH,+ to PT is given by (a[NH,‘]i/
apHi)fNH,j, or 2*3[NH,+]i* Under the conditions of the experiment, PNH3comes
to 23 mM. Since PI during the plateau phase (pHi = 7.70) is about 9 mM (meas-
ured as described in sect. IxCZ), PT = 9 mM + 23 mM = 32 mM. A con-
sequence of the large contribution made by NH,-NH,+ to PT in this open
system is that the fall in pHi during the plateau phase is much smaller than
the pHi undershoot observed when the cell is returned to an NH,+-free solution
(see Fig. 3B). Since both the plateau-phase acidification and the undershoot
are manifestations of the same NH,+ influx, the ratio of the two should be
inversely related to the ratio of PT values prevailing during the plateau phase
and after NH, removal. The inverse PT ratio in Figure 3 was 32 mM/9 mM or
3.6, in good agreement with the observed ApHi ratio of 0.27/0.07 = 3.9.

B. Mechanisms of Cellular Buffering

The nature and relative significance of the mechanisms that contribute to

intracellular buffering have been subjects of considerable debate (410). We
limit ourselves here to the buffering of the bulk intracellular fluid (i.e., the
fluid in direct contact with the cell and organellar membranes). Intracellular
buffering is evaluated by observing the response of pHi to acute acid or alkaline
loads of known magnitude and therefore reflects the contributions of several
homeostatic mechanisms (outlined in sect. VIII@. Thus in response to acid loads,
protons are removed both by physicochemical buffers and by metabolic
processes and also may be sequestered by certain organelles. These events
are rapid and usually reversible and serve to minimize the initial fall in pHi. In
addition a slower mechanism comes into play, acid extrusion. During acute
alkaline loads, the slower mechanism probably consists of a decrease in acid
extrusion and an increase in passive acid influx and alkali efflux.

I. Mechanisms included in the definition “intracellular buffering”

It would be ideal if the individual contributions of these various homeostatic

mechanisms could be clearly distinguished. One basis for such distinction is their
time course (see discussions of Brown and Woodbury in 410). The reactions
involved in physicochemical buffering are ionic (with the exception of the

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hydration of CO,) and thus reach completion in a fraction of a second. The

metabolic responses are somewhat slower but probably are still largely com-
pleted within several seconds. The large surface/volume ratio of organelles
is also conducive to a rapid response. In contrast ion transport across the cell
membrane is a relatively slow process. For instance, it takes about 15 min
for complete recovery of pHi from an acid load in mouse soleus muscle (11) and
considerably longer in cells as large as barnacle muscle fibers (44). Thus
monitoring the course of pHi (in these two studi .es, with microelectrodes)
allows acid extrusion to be distinguished from the other processes. A second
approach for separating the mechanisms is to inhibit one or more of them.
Thus in several cells, acid extrusion can be blocked either by applying SITS
(37, 379, 431) or by sufficiently lowering pH, (37, 43, 44, 432). When acid
extrusion in the snail neuron is fully inhibited, acid-loading with CO, causes
pHi to fall to a new, stable value within 2 min, whereas about 15 min are re-
quired for a complete pHi recovery without the inhibition (432).
Woodbury’s proposal (see 410) that “the term ‘buffer’ . . . be restricted to
chemical buffering’ has little practical merit, since physicochemical, metabolic,
and organellar buffering occur nearly simultaneously. We recommend that
the term include the combined effect of all three processes, (see sect. IXC) but
exclude transmembrane ion transport for three reasons: r> it can easily be
distinguished on the basis of time course and response to inhibitors; 2) its
inclusion would make “intracellular buffering power” time dependent; and 3)
it has the special distinction of being sensitive to extracellular factors such as
PH,, [HCwo, and wa+1,.

2. Quantification of cellular buffering

Three practical definitions have been suggested.

a) Michaelis-Van Slyke modi,fication of Koppel and Spiro definition.
p = dB/dpH, where dB is the amount of strong base that would have to be
added to the intracellular fluid to raise pHi by dpHi. Although the definition of
p was originally confined to physicochemical buffering power for in vitro sys-
tems, when applied to cells it should be expanded to include biochemical and
organellar buffering. This definition is applicable to each of three fundamental
methods of applying an intracellular acid or alkaline load.
dB/dpH = & In the absence of C02, PT is simply p1 (which is understood to
include non-CO,-HCO; physicochemical buffering as well as metabolic and
organellar buffering); in the presence of CO, (Pco, constant), & is p1 + Pact.
Note that in the latter case, pco2 can be directly obtained as -a[HCOy]i/
dpHi = -2.3[HCOT]i (see Eq. 39), since for every proton supplied by the
CO,-HCO; buffer system, one HCO; is formed.
II) ALTERATION OF Pco,. Here an acid load is provided by the intracellular
dissociation of H&O, into H+- and HCO;; an alkaline load is provided by the

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394 A. ROOS AND W. F. BORON Volume 61

reverse reaction. Thus the equivalent addition of strong base is -d[HCOT]i

and as pointed out by Peters and Van Slyke (334), -d[HCOs]i/dpHi is the
buffering power of non-CO* or intrinsic buffers, pi.
(B +H+L Here the equivalent addition of strong base is -d[A-]i
BH+). or
d[BH+]i. By analogy with the previous case, it is clear that -d[A-]i/dpHi is
the buffering power of all non-HA-A- buffers, whereas d[BH+]i/dpHi is the
buffering power of all non-B-BH+ buffers. In the absence of CO*, these
ratios are simply PI; in the presence of CO, (Pco, constant), they are pi + pcoz.
As with addition of a strong acid on base, PcOn= -a[HCOT]i/apHi.
b) % pH regulation. Siesjo (405) found that in the rat brain, the fall in
pHi produced by acute acid loads (high Pco,) is followed by a gradual recovery,
which he ascribed to metabolic adjustments and ion transport. Application
of Equation 38 would thus yield a time-dependent buffer value. For this reason,
Siesjo proposed a measure of pHi regulation that combines the contributions
of all homeostatic mechanisms

% pH regulation = APKnax - ApH,bs ’ 100 (41)


where ApH,,, is the pH shift that would have been produced by an acute acid
or alkaline load in the absence of any homeostatic mechanism (including physico-
chemical buffering), and ApHob, is the pH shift actually observed. The
difference (ApH,,, - ApH,& was termed ApH,,,, reflecting the contribution
of all regulatory mechanisms. If the acid or alkaline load is applied by
altering Pco~, then

= A log [HCW
% pH regulation
. 1oo
A Pco2

where A is the difference between initial and prevailing conditions (405).

The Siesjo definition provides a purely descriptive yet unambiguous
measure of pHi homeostasis. It was particularly useful before the advent of
microelectrodes (which allowed the measurement of pHi transients) and the
discovery of inhibitors of acid extrusion. These new techniques permit one to
distinguish the contribution of ion transport to overall pHi homeostasis
and thus provide information that is obscured when all homeostatic mecha-
nisms are pooled.
c) SZope of Astmp plot. d log PCO,/dpHi, where d log PCO, refers to the
applied change in Pco,, and dpHi is the resultant shift in pHi. This formulation
is necessarily limited to the case in which an acid or alkaline load is imposed by
altering Pco~. As shown by Woodbury (410) and Karman and Held (223), the
derivative h = -d log Pco,/dpH is related to the intrinsic (non-CO&
buffering power (pi) as follows

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A=--. d log PC02 = 1 + P

dpHi In lO[HCO;1
Thus h is a function both of [HCO,]i and of pi. We see no advantage of this
involved expression over p = dB/dpH.

C. Techniques for Measuring Zntracellular Buffering Power

I. Titration of cell homogenates

Titration of cellular homogenates, the earliest method of measuring intra-

cellular buffering power, was first used by Furusawa and Kerridge (140) in
1927 in experiments on cat skeletal, cardiac, and smooth muscle. The frozen
and minced muscles, mixed with ice-cold normal saline, were titrated under
oil at 0°C with KOH or lactic acid. Dilution did not substantially alter the pH.
This is one of the few studies in which buffering power has been determined
over a wide pHi range (6.4-7.4; p, in all three muscle types, rose as pH fell
below 7). No correction was made for the presence of extracellular fluid and
PCO, was not measured. The results are in reasonable harmony with those of
modern studies carried out at 37°C (see Table 13).
In recent years several workers have used the in vitro titration approach.
Spyropoulos (420) extruded cytoplasm from squid giant axons; this approach
makes contamination of the axoplasm by extracellular water unlikely. However,
the estimate for p (about 20 mM) that can be derived from his titration curve
is considerably higher than that obtained by other methods (42, 380),
possibly due either to failure to stop metabolism or to evaporative water loss from
the sample. Siesjo and Messeter (408) measured the buffering power of whole
rat brains that were frozen in situ with liquid nitrogen, homogenized in an
F- solution, and equilibrated anaerobically with CO, (40 mmHg) at 37°C (403).
The homogenates were then titrated with NaOH to pH 7.1, which was taken
as the normal pHi at PCO~= 40, after which variations in PCO~were imposed
to obtain A (-d log Pcoz/dpH). The studies were done at several degrees of
dilution and the results extrapolated to zero dilution, yielding a value for A of
brain tissue of 1.8 at Pco2 = 40 and pH 7.1 (corresponding to p = 22 mM).
This figure necessarily includes the contributions of the extracellular fluid
and of any metabolism not blocked by F- and the absence of oxygen. A third
recent application of in vitro titration is the study by Johnson et al. (221).
They first used strong acid or base to titrate a suspension of intact chromaffin
granules and then a suspension of lysed granules, calculating internal buffering
by difference. The implicit assumption that the acid or alkali used for the
titration does not cross the intact granule m.embrane is supported by the
authors’ observation that the weak base method for calculating the granular
buffering power (see sect. 1xC2) yields similar results. Determinations of cell
buffering by titrations of cell homogenates of rat, chicken, and pigeon skeletal

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396 A. ROOS AND W. F. BORON Volume 61

and heart muscle have also been made by Lai et al. (247), Heisler and Piiper
(Nl), and Lykkeboe and Johansen (264).

2. Weak acid and base methods

Intracellular buffering power can also be determined by titrating the intra-

cellular fluid in situ, either by injecting acid or alkali (see sect. 1xC3) or by
exposing the cell to a weak acid or base. For the latter, originally outlined by
Van Slyke (444), raising Pcoz produces an influx of C02, which after hydration
yields H+ and HCO;. Nearly all the released H+ is consumed by non-CO,-
HCO; buffers; the remaining small quantity is responsible for the fall in pHi*
Since one HCO; is formed for each H+ released, the amount of acid added to
the cell is given by -A[HCOT]i. Thus

AB -A[HCOn]i
PI =apH,= (44 >
A pHi
where Pr is the mean non-CO, or intrinsic buffering power, a value that
necessarily includes biochemical and organellar buffering. In Equation 44
ApHi is directly measured, whereas A[HCO,]i is calculated from the initial
and final values of pHi and PCO~

LHCO.TIi = spco,lOpHi-pK (45)

where s is the solubility of CO, in cell water. Measurements are available of

the solubility and the apparent first pK of CO, in homogenates of red cells
(445, 446), mammalian skeletal muscle (107), and mammalian brain (403, 404).
Even if the extracellular Pcoz is precisely known (as for in vitro studies),
however, some doubt remains about cellular Pco~, especially in rapidly
metabolizing cells. An underestimation of Pcop would lead to an overestima-
tion of pi, the error becoming increasingly significant at low Pco~.
The process of acid extrusion from the cell, though relatively slow, begins
as soon as an acid load is imposed. Three methods have been used to cleanse
PI of this contamination. In the first (10, 430), the course of pHi recovery
during exposure to CO, is back-extrapolated to some early time when pre-
sumably acid extrusion has not yet begun, though CO, is in equilibrium across
the membrane. It is, of course, not certain how much extrapolation is needed
to satisfy these requirements. A second approach is to block acid extrusion
(44) by exposing the cell to a stilbene derivative (in the case of coupled Na+,
HCO;, Cl- transport) or amiloride (to block Na-H exchange) or by removing
external Na+ or lowering pH, (see sect. VIII). Finally, it is possible to calculate
PI from the pHi change observed when PCO~is decreased (37). Although the
resulting abrupt increase in pHi may be followed by a recovery, the recovery
will be substantially slower than the recovery after an acid load.

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Intracellular buffering power can also be calculated from the pHi changes
produced by exposing a cell to weak acids other than CO, or to weak bases.
The most commonly used base is NH,. As discussed in section IV, exposing a
cell to a solution containing-NH,-NH,+ leads to the rapid entry of NH3, which
then combines with H+ to yield NH,+. Since for each NH,+ formed one intra-
cellular H+ is consumed

P- (46)

where p is the mean buffering power of all non-NH,-NH,+ intracellular

buffers. In the absence of CO, and other exogenous weak acids and bases,
Equation 46 gives the cell’s Pi. The value obtained should be the same as that
measured by the CO, method at the same pHi. In the presence of CO, (Pco,
constant), Equation 46 gives PI + pcoz. A[NH,+]i in Equation 46 is calculated

[NH,+], = [NH,], 10pKaWpHi (47)

and thus depends on accurate knowledge of [NH,]i, pK,, and pHi. [NHs]i is
assumed equal to [NH,],, which is obtained from pH,, pK, and ([NH,],
+ [NH,+],,). As discussed in section V, the initial rise in pHi is followed by a
slower downward drift due to entry of NH 4+.Three methods are available to
subtract the effect of NH,+ entry from the pHi change. 1) The pHi course of
the slow downward drift can be back-extrapolated to a time early in the NH,
exposure. 2) The NH,+ entry during the NH, exposure can be eliminated by
abolishing the NH,+ electrochemical gradient. This can be achieved by
judicious choice of [NH,+], and pH,. 3) The value for p can be calculated from
the pHi fall observed upon removal of external NH,. Although NH, removal
causes pHi to undershoot its initial value, thereby stimulating acid extrusion,
the acid extrusion can be blocked by one of the procedures previously outlined.
Importantly a single exposure to CO, or to NH, yields the mean buffering
power only over the pHi interval examined. Although in crab muscle (9) PI
appears constant over a wide pHi range, this does not seem to be generally true.
Thus the buffering power of homogenates of cat skeletal, cardiac, and smooth
muscle increases as pH is reduced (140). More recently a similar trend was
found in intact barnacle muscle, using the CO, and NH, methods (37, 44). The
data from a number of NH3 exposures, at different values of pHi, were com-
bined to obtain the relationship between PI and pHi over the pHi range 6.7-
7.8. The dependence of PI on pHi was given by PI = A + B(pHJ, where A = 155.6
t 11.8 and B = -17.4 t 1.6 (44). Note that in the presence of CO, (Pco, con-
stant), PT = PI + PcOn, and since pco2 = 2.3[HCOc]i, PT is by definition a
function of pHi.

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398 A. ROOS AND W. F. BORON Vohme 61

3. Microinjection methods

Intracellular fluid can also be titrated in situ by the direct intracellular

injection of acid or alkali. This approach, which has been applied to snail
neurons by Thomas (430) and by Meech and Thomas (283), requires knowledge
of the amount of acid (-AB) or alkali (AB) injected per unit volume of intra-
cellular fluid and the resultant shift in pHi. In his study, in which ions were
injected iontophoretically, Thomas (430) impaled the cell with two microelec-
trodes for measuring pHi and two more for passing current. From the cur-
rent’s magnitude and duration, the total injected charge was calculated and
then converted to equivalents of injected HCO; or H+ by means of the transfer
number of these ions (obtained in separate in vitro experiments). From the
estimated cell volume (the cells are nearly spherical), AB and therefore AB/
ApHi = p could be obtained. In COz-free Ringer’s solution, the HCO, injec-
tions yielded a value for p of 10.7 t 0.8 mM (n = 16), whereas the H+
injections gave a value of 10.9 t 0.7 mM (n = 18). Note that this technique
measures total buffering, which in the absence of CO, is identical to the intrinsic
value. As expected, Thomas found that adding CO, to the system increased
the measured PT. Two limitations are inherent in this technique: 1) the true
intracellular volume of distribution for the injected ions is difficult to determine
and 2) the pHi shift may be underestimated due to pHi recovery after the
Meech and Thomas (283) pressure-injected HCl into snail neurons and
estimated the amount of HCl injected by monitoring intracellular Cl- activity
with a Cl-sensitive microelectrode. This procedure obviates the first limita-
tion stated above, since the intracellular Cl- space is presumably identical
to the intracellular H+ space. In nominally CO,-free Ringer’s solution, the
authors obtained a value for /3 of 10.3 (mean of two values), which is very
close to the iontophoresis-derived estimate. The calculation of p by the pres-
sure-injection method requires an accurate value for the Cl- activity coefficient.
Neither of these microinjection methods can be used in cells such as muscle
fibers because of diffusional delays.
A serious discrepancy apparently exists between the buffering power of
the snail neuron obtained by microinjection and that obtained by the CO,
technique. The former value is about 11 mM, the latter 25-30 mM. It was
originally suggested by Thomas (430) that this was due to the injected ions
being distributed in only about one-third of the intracellular volume; thus 30
mM was presumed to be the correct value. The results of the more recent
HCl pressure-injection experiments from the same laboratory (283) seem to
indicate that the CO,-derived estimate was erroneously high.

D. Estimates of Intracellular Buffering Power

Table 13 summarizes the available data on intracellular buffering power.

The values obtained by titrating cell homogenates ideally reflect physico-

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TABLE 13. htracehdar buffering power

mmol . pH-’ * Tempera-
Preparation liter-’ Method PH, ture, “C Comments Ref.

Squid giant axon -20 Homog -7 21 Estimated from titration curve 420
9 NH, 7.3-7.8 22 42
11 NH:, 6.5 22 $
Snail neuron 25 co* 6.8-7.4 RT 429
29.5 CO2 7.0-7.5 RT pH, range estimated from figures 430
11 Inj 7.0-7.5 RT Microiontophoresis of HCO; 430
11 Inj 7.0-7.5 RT Microiontophoresis of H+ 430
10 Inj -7.3 18-20 Pressure injection of HCl 283

Crayfish neuron 25 7.0-7.2 21 301

Barnacle photoreceptor 15 -7.3 20 57a

Rat brain, whole 18.5 Homog 7.1 37 Calculated from d log PcoJdpH 408

Crab muscle 47 co, 7.0-7.3 20 9

Barnacle muscle 27.7 NH, 7.3 22 Least-squares /3 vs. pH, fit 37

28.5 co* 6.9-7.5 22 37
37.3 NH, 6.8 22 Least-squares p vs. pH, fit 44

Frog sartorius muscle 35 co* 6.85-7.05 25-26 35

Cat uterine muscle 36 Homog 6.8 0 140

Cat gastrocnemius muscle 58 Homog 6.8 0 140

Rat diaphragm muscle 67 Homog 6.3-7.1 37 171

Rat skeletal muscle 68 Homog ? 37.5 247

Mouse soleus muscle 43 6.8-7.2? 37 10

Pigeon breast muscle 77 Homog 6.3-7.3? 40 264

Pigeon leg muscle 65 Homog 6.3-7.3? 40 264

Chicken breast muscle 118 Homog 6.3-7.3? 40 264

Chicken leg muscle 55 Homog 6.3-7.3? 40 264

Cat cardiac muscle 23 Homog 6.8 0 140

Rat cardiac muscle 51 Homog ? 37.5 247

Sheep Purkinje fibers 35 co2 6.8-7.2? 35 122

Ferret ventricular muscle 69 co* 6.8-7.2? 35 122

Rat ventricular muscle 77 co, 6.8-7.2? 35 122

Pigeon cardiac muscle 64 Homog 6.3-7.3? 40 264

Chicken cardiac muscle 58 Homog 6.3-7.3? 40 264

Salamander proximal tubule 36 6.7-7.4 22 40

Xenopus embryo 18 co* 6.3-7.7 18-22 441

* Assume intracellular water 64% of tissue wet wt. t Assume intracellular water 85% of tissue water. $ Russell
and Boron, unpublished data. Homog, homogenate; inj, microinjection; RT, room temperature.

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400 A. ROOS AND W. F. BORON Volume 61

chemical buffering but may also reflect a degree of metabolic and organellar
buffering and some contamination from metabolic breakdown of the sample. In
some instances, extracellular contributions were not taken into account. The
remainder of the values were obtained by titrating intracellular fluid in situ,
either by exposing cells to weak acids and bases or by microinjection tech-
niques. The ApHi measurements for these in situ data were all obtained with
microelectrodes. In some cases acid extrusion occurring after acute intracellular
acid loads may not have been totally compensated for. Experiments were
usually performed at an approximately normal pH,, so that passive fluxes of
acid or base are unlikely to have had a substantial effect.
A considerable number of estimates of buffering power, many from in
vivo mammals, are omitted from Table 13 because they are likely to have been
heavily contaminated by active or passive transmembrane fluxes of acid or
base. The value of p has long been estimated by changing PCO~ and then
measuring the resultant fall of pHi several hours later. In their studies of
mammalian skeletal and cardiac muscle, Clancy and Brown (89) in 1966 and
Lai et al. (247) in 1973 clearly recognized that such measurements are con-
taminated by transmembrane fluxes of ions. This explains the time dependence
of p (see sect. IxB), which was demonstrated by Messeter and Siesjo (284) in
1971 and by Lai et al. (246) in 1973. Estimates of p may also be contaminated
by passive ion fluxes. For example, in the presence of COZ, a low pH, could
induce passive HCO, efflux, which might lead to a fall in pHi; the opposite
would be the case for high pH,. Thus estimates of p that are contaminated by
ion fluxes lack definition and have meaning only when the experimental con-
ditions are carefully defined.


A. Mitochondria

Perhaps the most crucial transmembrane proton gradient in a living

animal cell is that across the inner mitochondrial membrane. There is strong
evidence that this electrochemical gradient constitutes a second “point of
intersection of biological transformation pathways” (414), the first being ATP
and related chemical substances.
It is beyond the scope of this review to discuss in detail the concept of
mitochondrial energy transformations originally proposed by Mitchell in 1961
(294) and its expansion and substantiation by his group and by subsequent
workers. The reader is referred to the original literature and to reviews by
Mitchell (295-297) and others (49, 131, 413, 414). In brief the only role of the
respiratory oxidation-reduction chain is considered to be the translocation of
protons from the inside of the mitochondrion, across its inner membrane, and
into the cytoplasm. During respiration, electrons derived from substrate H
atoms are transferred, step by step, along the chain positioned in the inner mem-

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brane. The changes in free energy for most of the electron transfers are modest,
but at three points the energy release is large. At each of these points, called
coupling sites, the bond energy released is used for the extrusion of two protons
per pair of electrons transferred. (There is some difference of opinion about
the exact H+-electron stoichiometry.) At the end of the oxidation-reduction
chain, two electrons are donated to an oxygen atom and two protons are supplied
by the interior medium to form a water molecule. Thus a total of six protons
are extruded for every oxygen atom that is reduced to water. The energy ex-
pended in extruding the protons is stored in a proton gradient having an electrical
component, V, (the voltage across the inner membrane) and a chemical
component, l,OOO(RTIF) In ([H+],it/[H+],,,) or O.ZT(PH,~~ - pH,it), where cyt
and mit refer to cytoplasm and mitochondrial matrix, and the values are given
in mV. Thus the total electrochemical proton gradient, A$, is given by V,
+ 0*2UpH,,t - pH,it). The relative contributions of the two components to
A+ depend on the properties of the inner mitochondrial membrane. This mem-
brane has a very low permeability to ions in general and to protons in par-
ticular; its H+ conductance is around 0.5 &S/cm2 (296). Therefore in the ab-
sence of mechanisms for outward translocation of anions to accompany the
protons being extruded or for inward translocation of cations in exchange
for the protons, electrostatic repulsion forces would prevent the establishment
of a chemical proton gradient, and the energy of proton extrusion would be
stored solely in the form of a voltage difference (inside negative). Actually
the membrane contains carrier systems for the passive transport of anions
(e.g., phosphate) and cations (e.g., Na+ and Ca’+). By maintaining electro-
neutrality during transport, the carrier systems influence the activities of
H+ and other ions in both cytoplasm and mitochondrial matrix and thereby
control the relative contributions of membrane voltage and pH difference to A+.
The main function of the proton gradient is to provide the free energy for
ATP formation. This formation is mediated by a membrane-bound ATPase.
The external protons pass back into the mitochondrion, not through passive
leakage pathways but through this enzyme. The reentering protons yield their
excess energy, which is required for the phosphorylation of ADP at a
stoichiometry of two protons per molecule of ATP formed. Under suitable
energetic conditions, the synthesis of ATP can be reversed; its hydrolysis
then establishes an electrochemical proton gradient of the same polarity as
that produced by the oxidative chain.
Mitchell and Moyle (298) were the first to make an estimate of both the
membrane voltage and the pH difference across the inner membrane of rat liver
mitochondria, suspended in an artificial medium. The mitochondria were oxidiz-
ing P-hydroxybutyrate in the absence of phosphate acceptor (ADP) and thus of
ATP synthesis. The authors obtained an electrochemical H+ gradient of 230
mV, composed of a membrane voltage of 200 mV (inside negative) and a pH
difference of 0.5 (pH,i, = 7.5; pH, = 7.0). Without ATP synthesis, the H+
gradient is not dissipated, and the redox reaction and the H+ gradient are
poised at equilibrium, that is, there is neither net substrate utilization nor

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402 A. ROOS AND W. F. BORON Volume 61

further proton extrusion. When phosphate acceptor (ADP) was now added,
ATP synthesis was initiated; simultaneously the proton gradient fell to about
200 mV. Mitchell and Moyle estimated from the prevailing substrate (ADP,
ATP, Pi) concentrations that a A+ of 250 mV is required to provide two ex-
ternal protons with enough energy to form one ATP molecule as demanded
by the stoichiometry. The close agreement between this theoretical value and
that actually observed lends evidence to the concept that ATP synthesis is
fueled by the proton electrochemical gradient.
These findings of Mitchell and Moyle are in agreement with those of many
other workers. Skulachev (414) derived V, under various conditions from the
(supposedly passive) distribution of lipid-soluble synthetic anions such as
tetraphenylborate. Addanki et al. (2) used the DMO method to measure pH,it,
obtaining a value of 7.4-7.5 in state 4 (succinate supported) beef heart
mitochondria (pH, about 7.0). They observed that the addition of 0.5 mM
Ca2+ to the medium raises pH,it to 8.3-8.6 and simultaneously lowers the
pH, of the weakly buffered medium by 0.4-0.5, indicating stimulation of H+
extrusion; this was previously shown by others to accompany Ca2+ accumula-
tion. The authors also found that the DMO distribution, and thus pH,it, re-
mains unchanged at 7.7 when, at a pH, of 7.5, [DMO], was raised from 9 to
290 mM (1). This increase in [DMO], must have led to the net influx and sub-
sequent dissociation of this weak acid and therefore must have produced a
sizable intramitochondrial acid load. Since even a considerable buffering power
does not explain the observed constancy of pH,it, the results indicate that H+
extrusion is stimulated by the large acid load. More recently the technique
of 31P NMR has provided the first direct measurements of a pH gradient be-
tween the cytoplasm and mitochondria of intact rat liver cells under a variety
of conditions (401).
The electrochemical proton gradient across the inner mitochondrial
membrane can be reduced or abolished by diverse chemicals such as DNP.
All these “uncouplers” are weak acids of which both the protonated and de-
protonated forms are soluble in the mitochondrial membrane. For a recent
review on the transport of protons by weak acids, see McLaughlin and
Dilger (279a). The two partners provide a shuttling system for the movement
of protons down their electrochemical gradient. The mode of action of these
chemicals is thus comparable to that discussed in sect. v for the HCO;-CO,
and NH,-NH,f pairs in the plasma membrane, except that uncouplers may
go back and forth many times across the membrane without leaving it. The
erosion of the proton electrochemical gradient reduces and eventually
abolishes the energy source for ATP synthesis, which therefore stops,
whereas the oxidative process, released from the “braking’ effect of the proton
gradient, proceeds at an accelerated rate. Thus phosphorylation is uncoupled
from oxidation; the oxidative energy is dissipated as heat.
As might be expected, mitochondrial proton transport affects not only
pH,i, but also pH,,t. In the snail neuron, Meech and Thomas (282, 283) found
that intracellular injection of Ca2+ leads to a transient decrease in pHcYt. This

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may be expected if some of the injected Ca*+ is taken up by mitochondria

in exchange for H+, as referred to above. The authors pointed out that some
of the physiologic effects seen upon Ca*+ injection or cytoplasmic accumulation
may actually be the result of increased cell acidity.

B. Chloroplasts

Electrochemical gradients of H+ have also been demonstrated across the

plasma membrane of bacteria (for references see 347) and across the thylakoid
membrane of chloroplasts, both of which have a very low permeability to
H+ (296). The origin and use of these gradients are quite comparable to those
in the mitochondrion. In the chloroplast, protons energized by light are
pumped from the external medium into the organelle, thereby generating a
transmembrane voltage (inside positive) and reducing the pH of the interior
(pH,). Once established, the H+ gradient can be used as an energy source for
ATP formation, even in the dark (217). In 1964 Neumann and Jagendorf (314)
first inferred the light-induced internal acidification from the alkaline shift of
the unbuffered medium; uncouplers prevented the shift (216). Rottenberg et al.
(375) determined the pH, from methylamine distribution and upon illumina-
tion observed a fall from 7.5 to 6.0 (pH, = 8.0). Electron transport cofactors
reduced the pH,, whereas uncouplers increased it. Schuldiner et al. (392), using
fluorescent amines, found the pH, attained upon illumination (4.5-5.0) to be
nearly independent of pH, values in the range of 6.3-8.0. At higher pH, values,
pH, did rise. Similar conclusions were drawn by Rottenberg and Grunwald
(374), who calculated pH, resulting from illumination by measuring the fall of
[NH,+], with an ammonium-selective electrode in the medium.

C. Lysosomes

Lysosomes are vesicular organelles that contain hydrolytic enzymes with

acid pH optima. Their membranes are in a dynamic state: pinocytotic vesicles
filled with water and solute s or particulate matter constantly fuse with the
lysosomes, and the empty mem branous fragments somehow return to the
plasma membrane. More than 80 years ago, Metchnikoff (286) observed that
blue litmus particles turn red after having been ingested by protozoa. This
was the first demonstration of the acidity of lysosomal content. [For more
recent work with dyes see Jensen and Bainton (218).] Goldman and Rottenberg
(148) used the methylamine distribution to calculate the internal pH of lysosomes
(pH,,,) isolated from liver cells. At pH, 7.0 (the medium was buffered with 1
mM HEPES and contained 250 mM sucrose) and O”C, pH1,, was 5.4. Addition
of 0.3- 100 mM KC1 progressively raised pH1,, from 5.3 to 6.7. At all values of
[K+],, this ion’s distribution ratio remained equal to that of H+ and neither
ratio was affected by valinomycin (which increases K+ permeability). This
suggests that both K+ and H+ are at equilibrium and that V, is negative but

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404 A. ROOS AND W. F. BORON Volume 61

becomes progressively less so as [K+], is increased. Reijngoud and co-

workers (355-358) examined some of the factors affecting the pHi of isolated
Triton WR-1339-filled rat liver lysosomes. They confirmed that at O”C, pHI,,
rises with [K+],. At %“C, however, pH1,, remained nearly unchanged at about
6.3 even in the presence of 130 mM KC1 (pH, = 7.5). Valinomycin did not
affect pHI,, at any [K+],, but the addition of nigericin (which promotes elec-
troneutral K+-H+ exchange) raised pHI,, as [K+], was increased (358). It is
curious that the authors do not mention osmotic swelling or lysis of the
organelles, which should be the consequences of nigericin application. The
inability of valinomycin to raise pHI,, at high [K+], invalidates the authors’
conclusion that the pH1,, stability at 25OC was due to impermeability to K+,
even though this permeability may indeed have been low. Rather it indicates
a low membrane permeability to H+ that could be overcome by nigericin.
(Actually in a later paper [356], the authors’ data showed that substituting
130 mM KC1 for 250 mM mannitol raises pHI,, by 0.2-0.5.) Even though the
membrane of these detergent-filled organelles contains an ATPase (390),
their transmembrane pH difference could not have been maintained by an active
inward transport of protons [a distinct possibility for in situ lysosomes (see
above)], since the medium did not contain ATP. Instead acidic proteins in the
organelle, combined with the low membrane permeability to H+, are most
likely responsible for the low and stable pHIYs.
Reijngoud and Tager (357) observed a rise in pHI,, from 5.0 to 7.0 when
pH, was raised from 5.0 to 8.5; they ascribed this to an H+ efflux. The medium
contained 150 mM Tris, however, and the rise in pH1,, may have been caused
by the influx and subsequent protonation of Tris molecules, whose external
concentration increases with pH,. In a later paper (355), the same group of
workers found that methylamine accumulation in lysosomes was “inhibited”
by raising the concentration of either methylamine or NH&l in the medium.
These effects can be explained from simple mass-action considerations: a
progressively smaller fraction of entering base is protonated as pH1,, increases
and thus the base’s entry is progressively slowed (see sect. IvC). A review
by these workers of the permeability properties of lysosomes has ap-
peared (359).
When interpreting the above experiments, remember that the properties
of detergent-filled organelles suspended in a synthetic medium may be quite
different from those of native lysosomes. Recently Okhuma and Poole (319)
succeeded in studying lysosomal behavior under more physiologic conditions.
They measured pH1,, in intact, living mouse macrophages with a fluorescent
probe, obtaining a value of 4.75 (SEM = 0.01, n = 38) at a pH, of 7.4. The
authors offered evidence that this low pH1,, is maintained by an inward-directed,
energy-requiring transport of protons: when 2-deoxyglucose (which inhibits
glycolysis) and either azide or cyanide (which inhibit oxidation) are simul-
taneously applied, pH1,, reversibly rises by more than 0.5 in the course of about
7 min. On exposing the macrophages to 0. l- 10 mM NH&l, the authors saw
a reversible and concentration-dependent rise in pH1,,. With 0.1 mM NH&l,

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the initial alkalinization was followed by a slow drift in acid direction; on re-
moval of the salt, pHI,, fell to below the control value. If the existence of an
inward-directed proton pump can be accepted, then the initial rise in pHI,, re-
sulting from NH, influx could have stimulated its activity. This would explain
both the slow fall in pH1,, and the pHI,, undershoot after NH&l removal. (Note
that this is analogous to the pHi transients observed when intact cells are
acid-loaded with CO,; see sect. VIIIC.) The failure of these pHI,, transients
to occur with higher NH&l concentrations might indicate a balance between
the acidifying effect of the proton translocation and the alkalinizing effect of
NH,f efflux. Provided that the membrane is permeant to NH,+, this ion
should leave the lysosome since its electrochemical gradient is the same as
that for H+ and, on the basis of the above experiments, should be
directed outward.
A possible pharmacological application of lysosomal acidity was mentioned
by Homewood et al. (197). They found that when red cells containing
chloroquine-sensitive malarial parasites (P. berghei) are exposed to chloro-
quine, the cells accumulate 100 times as much of the drug as normal red cells and
also much more than cells containing chloroquine-insensitive parasites. The
authors postulated that the drug enters the digestive vacuole (lysosome) of the
sensitive parasite in the monoprotonated form (pK, = 10.2, pK, = 8.1), raises
the originally low PH lYS?and thereby interferes with proteolysis of hemoglobin
with resulting suppression of the parasite’s development.

D. Chromaffin Granules

The organelles of adrenal medullary cells are intimately involved in the

synthesis, storage, and secretion of catecholamines. They contain about 0.5 M
catecholamines, more than 0.1 M ATP, various cations such as K+, and signifi-
cant amounts of acidic proteins. There is general agreement that the interior
of the granule is acid: granular pH (pH,) values of 5.3-5.7 have been derived
from methylamine distribution (83, 195, 196, 221, 222) and with the 31P NMR
technique (83). The pH, is relatively insensitive to pH, and other changes in the
external medium (222). The membrane seems to have a low permeability to Na+
and K+ and to anions such as phosphate, SO:-, and HCO,; Cl- is relatively
permeant (83, 196, 222, 335).
Recently Holz (196) derived from the distribution of the triphenylmethyl-
phosphonium cation a membrane voltage of the chromaffin granule (V,) of about
-70 mV (inside negative). The medium (pH = 6.9) contained no ATP, and
sulfate and phosphate were the only anions. The V, was nearly the same as
EH (calculated from the methylamine distribution); the equality was maintained
when pH, was changed. Holz concluded that under these conditions the mem-
brane behaves like an H+ electrode. When ATP and Mg*+ were added to the
medium, pH, remained about 5.7, but V,, derived from the SCN- distribution
(195>, reversed its polarity to about +50 mV. Holz observed that subsequent

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406 A. ROOS AND W. F. BORON Volume 61

application of the uncoupler carbonylcyanide p-trifluoromethoxyphenyl-

hydrazine (FCCP), which increases membrane conductance to H+, restores
V, to nearly the same value prevailing before addition of ATP (about -70 mV),
again without changing pH, (195, 196). Casey et al. (83), who also examined
the effect of ATP, did observe a reduction in pH, by 0.2-0.5 when Cl-, a
permeant anion, was present. This effect was blocked by the uncoupler S13.
Though these workers did not measure V,, Holz (196) found that in the presence
of Cl- (without ATP), the membrane depolarizes and pH, rises. The work of
Casey et al. and of Holz indicates the existence of an inward-directed, electro-
genie, ATP-dependent mechanism for the translocation of protons. The failure
of either ATP or FCCP to affect pH, in the experiments of Holz was correctly
ascribed by the author to the lack of an anion to accompany the permeating H+
ion. Other data are in agreement with this concept. Johnson et al. (221) showed
that the rise in pH, due to catecholamine accumulation can be reversed with
ATP; this effect is inhibited by FCCP. Phillips and Allison (336), working with
sucrose- and buffer-filled “ghosts” of chromaffin granules in a Cl-containing
medium, found that ATP produces a prompt increase in V, (from 0 to +60 mV),
whereas pH, falls in 15 min from about 7.1 to 5.3; FCCP greatly reduces
both effects.
A question arises about the roles played by V, and pH in the accumulation
of catecholamines by the chromaffin granule. The low pH, could have at least
two functions: it could slow breakdown of the amines that rapidly oxidize at
alkaline pH, and it could promote uptake. Johnson et al. (221) showed that the
accumulation of epinephrine increases both as pH, is lowered and as pH, is in-
creased. Raising pH, increases the external concentration of uncharged cate-
cholamine, whereas lowering pH, prolongs the existence of an inward gradient
for this form. The authors therefore proposed that nonionic movement plays
an important role in the uptake of catecholamines. The failure of internal alka-
linization to produce catecholamine release was ascribed to binding. Nichols
and Deamer (315) found a similar sensitivity of catecholamine uptake to the
transmembrane pH difference in liposomes, artificial vesicles with a phos-
phatidylcholine membrane. Here the uptake, which was reversible, is most
likely mediated by diffusion of the uncharged species, whereas in the chromaffin
granule a carrier-mediated transport cannot be ruled out (see below).
In studies on granules that were lysed, reconstituted, and filled with buffer
([K+li = 10 mM), Schuldiner et al. (391) found that ATP produces a 20-fold
increase in the uptake of epinephrine; Cl- was present in the medium. This finding is
in agreement with the concepts discussed above, since under these conditions
ATP reduces pH, (83, 221, 336). Schuldiner et al. also observed that nigericin,
at a pH,, of 8.5 and a [K+], of 15 mM, completely blocks this stimulation; valinomycin
has no effect. The authors reasoned that nigericin, which facilitates electro-
neutral K+-H+ exchange, should have left the membrane voltage intact but
raised pH,, whereas valinomycin, which only increases K+ permeability, should
have depolarized the membrane without changing pH,. They therefore con-
cluded that it is primarily the transmembrane pH difference, rather than V,,

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that promotes catecholamine uptake. Holz (195), however, has provided direct
evidence that V, does play a role. Under his experimental conditions (absence
of significantly permeant anions), ATP plus Mg*+ stimulated catecholamine
transport four- to sixfold. Yet as mentioned earlier, this treatment has no ef-
fect on pH, (5. ‘7), but reverses the polarity of V, from inside negative to positive.
Holz found that FCCP, which prevents the reversal, completely blocks the
stimulation by ATP. He suggested that a negatively charged catecholamine
complex may be involved in the uptake; other possibilities include an electro-
genie exchange of two protons for one catecholamine ion or one proton for an
uncharged catecholamine molecule.
We should briefly mention an interesting but poorly understood feature
in catecholamine uptake: its dose-dependent inhibition by reserpine (22 1). This
drug does not affect pH,. It has been suggested that catecholamine transport
is mediated by a carrier and that reserpine blocks uptake by binding to this
The data thus suggest that there are at least two ways in which catechol-
ami nes can gain entrance into the chromaffin granule: 1) by diffusional or carrier-
mediated transport of the uncharged form, dependent on a transmembrane
pH difference (inside acid) maintained or enhanced by the ATP-dependent
inward translocation of protons; and 2) by some form of electrogenic transport,
possibly of the charged form of the amine, dependent on the reversal of V,
(inside negative to positive) resulting from the proton translocation energized
by ATP. The relevance of these mechanisms to the accumulation of catechol-
amine by in situ chromaffin granules remains to be investigated. A review of
bioenergetic processes in chromaffin granules has recently appeared (316).

E. Sarcoplasmic Reticulum

Nomura and Nakamaru (317) used DMO to calculate the internal pH, pH,,,
of vesicles derived from the sarcoplasmic reticulum of rabbit skeletal muscle.
They observed that as pH, is increased from 6.5 to 8.5 in a solution containing
105 mM KS and 40 mM Tris-maleate at 4”C, pH,, increases from 6.48 to 8.33.
It is possible that the entry of Tris molecules (whose external concentration
would rise with pH,,) was at least partially responsible for the observed rise
in pH,,. The transmembrane pH difference increased considerably in K+-free
medium: at pH,, 7.63, pH,, is only 7.07. The authors also transferred vesicles,
prepared in the absence of K+, to either a K+-free or a K+-contain .ing solution
(the anion in the latter, methanesulfate, is imperme ant) while monitoring
pH,. They found that pH,, decreases much more in the K+-containing solution
than in the K+-free one. Nomura and Nakamaru are justified in saying that
these data provide evidence for a KS-H+ exchange, but they are not justified in
concluding from the responsiveness of pHsr to pH,, that a Donnan distribution
of H+ obtains. Moreover if this were the case, the necessity of K+ to also be in
equilibrium would jeopardize the osmotic stability of the vesicle.

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408 A. ROOS AND W. F. BORON Volume 61


A. Membrane Permeability and Conductance

The selectivity of membrane permeability has often been ascribed to the

presence of fixed charges that attract and provide passage to oppositely charged
ions. As a corollary, Michaelis (289) proposed in 1926 that ionic permeability
should vary with pH when the structural groups are weakly ionized. An increase
of the H+ concentration at the membrane’s surface(s) would accordingly lead
to an increase in anionic permeability. A large increase in Cl- conductance
(gcl) was indeed observed in 1968 by Hagiwara et al. (165) in barnacle muscle
when, at constant pHi, pH, was reduced below 5. [Studies yielding similar
results in other cells have been reviewed by Hutter and Warner (203).] A com-
parable reduction of pHi below about 6 (at constant pH,) also steeply increased
g,,. On the other hand, K conductance (gK) decreased with pH, over the entire
pH range (from 10 to 4.5). The gradual fall in the g,lg,, ratio that occurs when
pHi is reduced from 10 to about 6 is probably also due to a fall in gK. Also in agree-
ment with Michaelis’ hypothesis are the observations made in 1971 and 1972 by
Fishman (121 and unpublished studies summarized in 22) that Na+ conductance
of squid axons perfused under voltage clamp steeply falls (with concomitant
loss of excitability) when pHi is reduced from 7.75 to 6.0. The relation between
Na+ conductance and pHi was sigmoidal, with an inflection at about 6.6 that
may correspond to the pK, of the ionizable group(s) on the membrane respon-
sible for the conductance changes. In comparable, more recent studies, Wanke
et al. (462a) concluded that the Na+ channels in the squid axon are controlled by
two independent groups, one on the external side of the channel with a pK, of
4.6, and one on the internal side with a pK, of 5.8. The same authors (462)
found that reduction of pHi in squid axons from 10 to 5.2 also reduces
K conductance. Again the K conductance-pHi relationship was sigmoidal
with an inflection at pHi = 6.9. The authors quoted previous work indicating
that changes in pH, (4.7- 10) do not appreciably affect K conductance and pro-
posed that the ionizable group (pK, = 6.9) is located on the intracellular side of
the membrane.
The response of membrane conductance to changes in pH, is by no means
the same for all cells. To explain their findings on frog muscle, Hutter and
Warner (203,204) resorted to a more complicated model than that of Michaelis.
A reduction of pH, led to a decrease in membrane conductance, mainly due to
a fall in g,,; gK was only slightly affected. A fall in membrane conductance
with acidosis was observed in 1958 by Meves and Volkner (287), who found
about the same effect whether the extracellular acidification was accomplished
by CO, or phosphate. Since CO, would also reduce pHi, the latter would seem
to have no additional effect on conductance.
In an extensive study, Woodbury and Miles (482) expanded the work of
Hutter and Warner and examined the conductance of frog muscle as affected by
a large number of Cl- substitutes in the external solution. They found that in

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the presence of ions such as Br- and NO;, conductance decreases as pH, de-
creases (from 9.2 to 5.4), just as with Cl-. Conductance had the opposite pH,
sensitivity, however, when benzoate or the ions of saturated, unbranched fatty
acids with one to five carbon atoms were used as substitutes. As pH, is reduced,
the external concentration of the undissociated forms of these acids (pK 3.8-4.9)
rises, and therefore so does their rate of entry into the muscle. This probably
causes pHi to fall progressively as pH, is reduced and may play a role in the
anomalous pH, sensitivity of the membrane conductance, a possibility not
examined by the authors.
An interesting but not yet completely explained finding by Krnjevic and
Lisiewicz (243) and by Meech (281) in nerve cells is the increase in K+ per-
meability resulting in hyperpolarization when the intracellular Ca2+ concen-
tration is raised. Simultaneously the pHi falls (see sect. XIIB). The relative roles
of the elevated [Ca’+]i and [H+]i in this phenomenon remain to be analyzed.
Walker and Brown (458) provide an example of a cell type in which gel is
affected by pH, but not by pHi* These workers found that lowering pH, from
8.0 to 6.5 causes the hyperpolarization of some cells in the abdominal ganglion
of Aplysia and the depolarization of others. Both effects can be explained by
an increase in gc1, since in the two kinds of cells, V, is respectively more and
less positive than the Cl- equilibrium potential. Because identical effects were
elicited in responsive cells whether the ASW was acidified with 5% CO, or with
strong acid and because ASW held at pH 8 with either 5% or 50% CO, was with-
out effect, neither pHi nor external CO,-HCO, seems to play a role in the
observed changes.
In striking contrast are the results of a study by Nonner et al. (318), who
observed that lowering pH not only exerted a marked effect from the inside,
but was nearly ineffective from the outside. They found that reduction of pHi,
but not of pH,, markedly slows inactivation gating in the Na+ channels of frog
skeletal muscle. In common with other effective chemical agents (some active
on the outside, some on the inside), activation gating was hardly affected.
although all treatments except low pHi reduced the peak Na+ current.
Moody (300) recently observed that reducing the pHi of crayfish slow
muscle fibers from 7.2 to 6.4-6.5 enables the fibers to generate all-or-none Ca
action potentials. (At normal pHi, only small graded responses are observed.)
The low pHi partially blocks the voltage-sensitive outward K current, which
normally shunts the inward Ca current and thereby prevents the generation
of action potentials.

B. Cell-Cell Coupling

Electrical coupling between certain adjacent cells is believed to be medi-

ated by gap junctions. In 1967 Loewenstein and colleagues (260) demonstrated
that coupling between the salivary gland cells of Chironomus larvae could be
abolished by the intracellular injection of Ca2+. Later Rose and Loewenstein
(372) showed that this uncoupling effect is produced only when the injected Ca2+

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410 A. ROOS AND W. F. BORON Volume 61

reaches a cell-cell junction. The authors localized the intracellular Ca2+from the
glow emitted by the previously injected Ca2+-sensitive photoprotein aequorin
and found that uncoupling occurs at the moment when the glow reaches the
junction. They also produced uncoupling by exposing cells to cyanide, DNP,
or Ca2+ ionophores, procedures that raise [Cil’+]i. Although these data impli-
cate a rise in Ca2+level as the cause of electrical uncoupling, other evidence sug-
gests that the Ca2+ effect might be mediated by a fall in pHi. It had previously
been shown that treatment with cyanide (42) or DNP (42,429) lowers pHi, and in
1977 Meech and Thomas (282) demonstrated that Ca2+injections also transiently
lower pHi, probably due to Ca-H exchange by mitochondria. Later Turin and
colleagues on Ambystoma blastomeres (27) and Fundulus embryos (26) and by
by acidifying the Ringer’s solution with 100% CO, (pH, 6.3); this caused pHi to
fall from 7.7 to about 6.3. A similar reduction in pH,, but in a CO,-free medium,
produced neither uncoupling nor a fall in pHi. Comparable observations of the
effect of 100% CO, on cell coupling were subsequently made by Bennett and
colleagues on Am&stoma blastomeres (27) and Fundulus embryos (26) and by
Iwatsuki and Petersen (208) on mouse pancreatic acinar cells. Most recently
De Mello (114) has shown that intracellular injections of H+ uncouple cardiac
Purkinje fibers.
Several questions have been the subjects of much debate. Is Ca”+-induced
electrical uncoupling mediated by a fall in pHi? Is low pHi-induced uncoupling
secondary to a rise in free Ca2+ levels? Can a reduced pHi and an elevated
[Ca”+]i each directly produce uncoupling? Rose and Rick (373), using Chirono-
mus larvae salivary glands, showed that although cyanide and DNP indeed
produce a fall in pHi, these pHi shifts precede uncoupling by several minutes
and are too small to account by themselves for the uncoupling. In other experi-
ments in which pHi was monitored with microelectrodes, these authors demon-
strated that uncoupling could occur without a fall in pHi and, even with intra-
cellular alkalosis, provided Ca2+ levels rise. Thus in this preparation, intra-
cellular acidification is not required for uncoupling. Rose and Rick ascribed
the uncoupling they observed after injecting H+ or exposing the cells to 100%
CO, to the direct effect of the elevated [Ca2+]i, which they demonstrated by
the aequorin glow. They emphasized that after H+ injection, pHi usually re-
covered before coupling was reestablished. They also stressed that in the CO,
experiments, the pHi at which coupling was abolished upon CO, application
(6.65 t 0.05, n = 10) was usually lower than that at which coupling was
reestablished after CO, removal (6.80 t 0.05, n = 12). These observations
would be expected, however, if both acidification and increased [Ca2+]i directly
induce uncoupling, since the H+ injection may elicit a rise in [Ca2+]i that outlasts
the acidification. In another study Rink and colleagues (362) used Ca*+-sensitive
microelectrodes to quantitatively monitor Ca’+ activities in Xenopus embryos.
They confirmed Rose and Rick’s observation (373) that 100% CO, produces a rise
in free Ca’+ but found that this elevation (from 0.20 PM to 0.35 PM) is too small
to account by itself for uncoupling (which requires free Ca2+ in the range of 50
pm in this preparation). They concluded that CO,-induced uncoupling is not
mediated by a rise in free Ca2?

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The present data seem most consistent either with species differences or
with the hypothesis that both an increase in free Ca*+ and a fall in pHi can
uncouple. This is supported by the work of Peracchia and Peracchia (332, 333),
which shows that either an increase in free Ca*+ or a fall in pHi can independ-
ently influence the morphology of gap junctions.

C. Epithelial Secretion and Reabsorption of Acid

The net secretion or reabsorption of acid or base that occurs across several
epithelia, including renal tubules, stomach, choroid plexus, and urinary bladder
has undergone considerable investigation (for reviews see 12, 144, 273-275,
348, 465). Regardless of the epithelium, the transport is a two-step process:
acid or alkali must be independently transported across each of the two barriers
of the epithelial cell. Invariably net movement of acid into or alkali out of the
cell across one barrier is thermodynamically downhill (although this may be
carrier mediated), whereas removal of acid from the cell across the opposite
membrane requires active transport. Experiments with vesicles isolated from
mammalian brush-border membranes (233, 305) and research on intact am-
phibian cells with pH-sensitive microelectrodes (41) indicate that the active step
in the renal proximal tubule is Na-H exchange (see sect. VIIIF). In the stomach
an ATP-dependent K-H exchange has been identified (382, 383). In light of the
preceding discussion on acid extrusion by symmetrical cells, it is expected that
pHi regulation plays a crucial role in the genesis and regulation of epithelial
acid transport.
Recently Boron and Boulpaep (41) have proposed that the cells of the proxi-
mal tubule, in secreting acid, take advantage of a pHi-regulating mechanism
that these cells have in common with symmetrical cells. Their data show that
the tubule cells regulate pHi quite efficiently: when acid loaded, the cells rapidly
extrude acid and return pHi toward normal. The mechanism of acid extrusion
is apparently an amiloride-sensitive Na-H exchange taking place across the
apical and basolateral membranes. The asymmetry of the cells is conferred by an
HCO; pathway(s), confined to the basolateral membrane, that permits the net
passive efflux of HCO.7. This efflux represents a continuous intracellular acid
load and thereby stimulates the pHiregulating mechanisms on both mem-
branes. Net acid secretion proceeds so that H+ is extruded across the apical
membrane and is therefore envisaged as a consequence of the cell’s proclivity
to regulate its pHi. [The suggestion that pHi regulation may play a role in acid
secretion had previously been made by Rector (348).] Accordingly acid secretion
would be enhanced by any process that stimulates Na-H exchange. Thus the
increased acid secretion that occurs after raising Pco2 at constant pH, can be
explained as the consequence of the fall in pHi produced both by the influx of
CO, and by the increased HCO, efflux secondary to the rise in [HCOT]i.
There may be a comparable mechanism in the choroid plexus that secretes
HCO.7 into the CSF. Wright (483, 484) has suggested that the apical mem-
brane (facing CSF) is permeable to HCO;, whereas the basolateral membrane
contains an Na-H exchanger (note that this orientation is opposite to that of the

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412 A.ROOSANDW.F.BORON Volume 61

proximal tubule). According to his view, CO, (or other lipid-soluble buffers)
diffuses into the cells across the basolateral membrane and dissociates, thereby
lowering pHi and stimulating Na-H exchange. Presumably this would in turn
favor an HCO; efflux across the apical membrane. Note that in this case, the
cause-and-effect relation is the reverse of that in the proximal-tubule model
(discussed above), where an HCO; efflux stimulates Na-H exchange. When
applied to CSF, the renal model predicts that the observed regulation of pHcsF
is actually a direct consequence of pHi regulation. For example, lowering
[HCO&SF at constant Pcoz would enhance net HCO, efflux, thereby lower-
ing pHi and stimulating Na-H exchange. The result would be an increased net
flux of acid from CSF to blood, returning pHcsv toward normal.
The transepithelial secretion of base into the avian oviduct (uterus) can
also be understood in terms of the pHi-regulation model. The avian egg remains
for about 18 h in the shell-gland region of the oviduct while some 5 g of CaCO,
are deposited on it. This precipitation requires an alkaline environment.
Nothing is known about the mechanism by which Ca ions are secreted by the
shell gland nor about the stimulus that sets off the secretion. Simkiss (411)
has shown that the pHi (measured with DMO) of the domestic chicken’s shell
gland falls from -7.0 to -6.5 between the 2nd and 10th h of the calcification
process and slowly returns to the original value in the subsequent 6 h, during
which shell formation presumably continues. The initial fall in pHi can be
ascribed to the passive efflux of base (although the author proposed OH-,
HCO; is more likely) from .the cells to the lumen of the gland. This efflux
represents an acid load on the cell that results in stimulation of the acid-extru-
sion mechanism, probably located at the basolateral membrane of the epithelial
cells. Thus acid is secreted into the blood. Indeed deposition of the shell is
accompanied by a marked metabolic acidosis (blood pH falls from 7.5 to 7.4, and
[HCO;] falls from 32 to 21; for references see 94, 412) to which the chicken re-
sponds by hyperventilating. Simkiss (412) was able to prevent the fall in pHi of
the shell gland by feeding the animal acetazolamide, an inhibitor of the carbonic
anhydrase. The shell gland contains this enzyme (see 411). By slowing the
hydration of CO*, the drug probably reduces the rate of intracellular formation
of HCO; and thereby minimizes the efflux of this ion and the resultant intra-
cellular acid load.
It would thus seem that the primary concern of acid-transporting
epithelial cells is the homeostasis of pHi. When coupled with an asymmetric
pathway for HCO; transport, however, the pHiregulating mechanism can be
used for net acid secretion or reabsorption and thus for the regul ation of the pH
of a body compartment or cavity (e.g., bl ood, CSF, oviduct).

D. Mechanical Properties of Muscle

1. Heart muscle

According to the Journal Books of the Royal Society, Isaac Newton ob-
served that when he put a drop of vinegar on pieces of eel heart, the rhythmic

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contractions stopped. This might be interpreted as the end result of the negative
inotropic effect of the acid on the heart. In 1880 Gaskell(143) demonstrated that
the frog ventricle, quiescent and in diastole during perfusion with 0.75% NaCl,
beats again for a short time and then stops in systole when the perfusate is
changed to a dilute NaOH solution. Dilute lactic acid, sent through a beating
ventricle, stops it in diastole. Thirty years later Jerusalem and Starling (219)
observed in the cat heart-lung preparation that raising PCO~in the coronary
perfusate increases both systolic and diastolic volumes. Later Gremels and
Starling (157), using the dog heart-lung preparation, and Smith (416), using the
turtle heart, found that acidosis has a greater depressant effect when produced
by CO, than by fixed acid. These studies suggest that lowering pHi has a nega-
tive inotropic effect: respiratory acidosis depresses pHi more than metabolic
acidosis (see sect. VIIIZ). The depressant effect of a reduced pHi was explicitly
considered in 1969 by Katz and Hecht (224) in their analysis of the mechanism of
the decrease in contractility during myocardial ischemia. [Neely et al. (310) and
Jacobus et al. (215) have since demonstrated that ischemia reduces pHi.1
These early findings have been abundantly confirmed. More recent workers
have examined the effect on cardiac performance of injecting solutions of
varying Pco~, bicarbonate, and/or fixing acid concentrations into the
peripheral vessels (13, 475) or coronary arteries (461) of dog, into the coronary
circulation of the isolated heart or heart-lung preparation of dog (87, 90), or
into the coronary perfusate of the isolated heart of guinea pig (279), rat
(423, 477), or rabbit (149,214). Acidosis reversibility reduces contractility (meas-
ured by a strain gauge attached to the ventricular surface) and maximal rate of
rise of left ventricular pressure and increases left ventricular end-diastolic
pressure and circumference. The effects are much more striking when Pco2 is
raised than when [HCO,], is reduced, and they parallel the reduction in
pHi (423). Conversely alkalosis has a reversible positive inotropic effect, more
pronounced when Pcoz is lowered than when [HCO;], is raised.
A few details in these studies are worth mentioning. In work on dog heart,
both Anderson et al. (13) and Clancy et al. (90) observed that raising [HCO,I,
results in a transient negative inotropic effect followed by recovery. The nega-
tive transient can be ascribed to the initial fall in pHi due to influx of CO, derived
from the added bicarbonate, but it is only of short duration because acid ex-
trusion is stimulated both by the reduced pHi and by the elevated pH,, (see sect.
VIII). In the carefully controlled study by Clancy et al. (90), return from relative
alkalosis of the coronary perfusate ([HCO& = 51, pH, = 7.33, Pcop = 100)
to control conditions ([HCO;], = 20 mM, pH,, = 7.22, PCO~= 50) resulted in an
increase in maximal rate of rise of ventricular pressure and a reduction of
diastolic pressure and circumference beyond the original values. This inotropic
overshoot upon lowering PCO~can be explained by the expected overshoot in
pHi beyond the control value, which should be the consequence of the preceding
acid extrusion (see sect. VIIIC).
Note that in the intact animal, reduction of blood pH by raising Pcoz (but
not so much by fixed acid addition) stimulates release of catecholamines (255),
which have a positive inotropic effect on the heart, most likely because they

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414 A. ROOS AND W. F. BORON Volume 61

reduce the fall in pHi produced by CO, (see sect. VIIIG). It is interesting that
acidosis reduces the myocardial response to extraneous catecholamines (475).
Studies on isolated cat papillary muscle (86, 88, 91, 112, 327, 328, 395) and
on ventricular strips of rat heart (88) and frog heart (263) have confirmed the
reversible inotropic effect of changes in pH,,, especially those accomplished
by changes in Pcoz. Both De Hemptinne et al. (112) and Serur et al. (395) ob-
served that the initial depression of isometric tension and rate of tension devel-
opment by respiratory acidosis is followed by a slow recovery and, on return
to control conditions, by an overshoot in these properties. These transients can
be interpreted in terms of corresponding changes in pHi (see sect. VIIIC).

2. Skeletal muscle

The inotropic effects of acid-base changes on skeletal muscle are quali-

tatively similar to those on heart muscle but much less striking. These effects
are nicely illustrated by the 1950 study by Creese (102) on the isolated, repeti-
tively stimulated rat diaphragm preparation. When at constant pH,, 7.5
the external HCO;-5% CO, buffer was replaced by 1.2 mM phosphate,
a rapid transient rise in contraction height (isotonic contraction) was followed
by a slow reduction over the next 1.5 h. When the original medium was re-
admitted, a transient fall in contraction height preceded a slow return to the
original value. The twitch tension in isometric contractions followed a similar
course. The two transients can be ascribed to the prompt pHi changes resulting
from removal and readmission of CO,. The reversible slow fall in contraction
height or tension should coincide with a slow fall in pHi caused by accumulation
of lactic acid generated during the contractions, since I) the (nominal) absence
of intracellular bicarbonate must have about halved the total intracellular
buffering power (see sect. IxA), and 2) the efflux of lactic acid must have been
impaired owing to the unstirred layer effect accentuated by the very low ex-
ternal buffer concentration (see sect. 1vB4). The modest reduction in acid ex-
trusion due to the absence of HCO,-Cl exchange (see sect. VIIIG) may have
played a minor role. When, on the other hand, Creese raised pH, to 8.8
([HCO,y], unchanged, nominally 100% 0,), contraction height immediately
increased and then continued to drift upward. This slow rise might be ascribed
to passive HCO,? influx, which would raise pHi; in phosphate buffer at the
same pH,, contraction height diminished. Creese suggested that “the effect
of bicarbonate may perhaps be attributed not to the external pH but to the
. l internal PH.”

Foulks and Perry (136) found that twitch tension of frog muscle is affected
very little by pH, changes between 5 and 9 (Pco, constant). According to Main-
wood and Worsely-Brown (271), stimulating frog muscle to fatigue (which re-
duces pHi) reduced twitch tension to about 20% of control. As lactic acid left
the muscle, tension recovered along a time course roughly similar to that of
lactate efflux. Increasing this efflux (and thus raising pHi) by raising pH, or

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external buffer concentration (see sect. IVB) led to nearly 100% recovery of
tension. In experiments in which they monitored pHi with 31P NMR, Dawson
et al. (109) found that repetitively stimulating frog muscle under anaerobic con-
ditions at 4°C reduces pHi and twitch tension. In other experiments on frog
muscle, Washio and Mashima (468) substituted 20% of the external Na+ with
NH,+ and found that twitch tension first increases and then slightly falls but al-
ways remains greater than the control value. More recently Heald (169) made
similar observations and in addition noted an immediate and progressive fall
in tension to below control value upon removal of the NH,+. Since the time
course of twitch tension in these two studies strikingly resembles the course
of pHi of other cells during and after exposure to NH,+ (see sect. VIIID),
changes in pHi may be responsible for the observed mechanical changes.
The fall in maximal developed tension, observed by Peiper et al. (331) in vascular
smooth muscle when [HCO;], was reduced, might also be explained by a re-
duction in pHi.

3. Analysis of negative inotropic effect of low pHi

In 1978 Fabiato and Fabiato (126), using segments of single cells of frog
skeletal muscle and rat ventricle from which the sarcolemma had been removed
(“skinned” fibers), analyzed some of the intracellular events responsible for the
fall in tension with intracellular acidosis. Their technique has the advantage
that the intracellular environment can be directly altered. They made the inter-
esting observation that when pH is reduced from 7.40 to 6.20 ([Ca”+] being kept
constant by a high [EGTA]), the [Ca2+] required for development of 50% of
maximum tension increases by a factor of 5.5 in cardiac muscle and 3.5 in skele-
tal muscle. The maximum tension (at saturating [Ca2+]) was reversibly de-
pressed by low pHi in both tissues. Since increasing [Ca”+] did not overcome the
depressing effect of acidosis, the authors concluded that this effect was not
caused by direct competition between Ca2+ and H+ for binding sites on troponin.
It is interesting that according to their quoted data, myofibrillar ATPase ac-
tivity is also depressed at low pH.
Fabiato and Fabiato (126) also studied Ca2+ loading and release from the
sarcoplasmic reticulum and the dependence of these processes on pH (6.2-7.4).
They found that the pH optimum for Ca2+-loading the sarcoplasmic reticulum
is relatively low (i.e., probably lower than the normal pHi) in skeletal muscle,
but relatively high (i.e., probably higher than the normal pHi) in cardiac muscle.
Thus modest reductions in pHi should have opposite effects on Ca2+ release in
the two kinds of muscle. The authors argue that under physiologic conditions a
small decrease in pHi should have little effect in skeletal muscle, since the pH
effect on the sarcoplasmic reticulum (i.e., increased Ca2+ release) may com-
pensate for that on the myofilaments (i.e., decreased sensitivity to Ca2+). They
suggest that this might explain the finding of Pannier et al. (329) that in rat
soleus muscle an increase in Pco2 actually enhances twitch tension. In cardiac

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416 A. ROOS AND W. F. BORON Volume 61

muscle, on the other hand, a fall in pHi should always have a pronounced negative
inotropic effect, since acidosis depresses both the sensitivity of the myofila-
ments to Ca2+ and its release from the sarcoplasmic reticulum

E. Metabolism

There are numerous interactions between intracellular pH and metabolic

transformations. The active sites on enzymes often contain ionizable groups
whose state of ionization may affect the enzyme’s conformation and thus its
substrate-binding and catalytic properties. Also the degree of ionization of
groups on a substrate or cofactor may affect its ability to bind to the enzyme.
On the other hand, the metabolic transformations themselves quite frequently
produce pH changes due to the direct uptake or release of either H+ or CO,.
At least two loci on the glycolytic pathway are pH sensitive. One is the
conversion of (inactive) phosphorylase b into (active) phosphorylase a. This
enzyme, which is present in tissues such as liver and muscle, catalyzes glycogen
breakdown or synthesis. In 1965 Danforth (106) showed that in intact frog
muscle, the lag period before the appearance of phosphorylase a in response to
stimulation of the muscle is prolonged as the CO, concentration i s rai sed. At 60%
C02, the maximal amount of phosphorylase a attained is also decreased. In a
purified in vitro system, reduction of pH similarly depresses phosphorylase con-
version by phosphorylase b kinase, confirming that the ambient pH plays the
crucial role in such effects (for references see 106).
The discovery of a second pH-sensitive glycolytic locus has a long history.
In 1933 Kerly and Ronzoni (228) showed that resting isolated frog muscle
exposed to Ringer’s solution equilibrated with 100% CO, ([HCO& = 20, pH,
= 6.0) produces practically no lactic acid during the first 3 h, whereas with 100%
N2 (PH, = 9.0), large amounts of the acid are generated. Hill (182) computed
that the pHi of these muscles at 100% CO, would be 6.1. Trivedi and Danforth
(439) in 1966 proposed that the effect may be due to the marked pH sensitivity
of phosphofructokinase, the enzyme that phosphorylates fructose 6-phosphate.
They observed in an in vitro system (phosphofructokinase was derived from
either frog or mouse skeletal muscle) a lo- to 20-fold reduction in the enzyme’s
activity when pH was reduced by as little as 0.1. The actual pH range in which
this striking effect occurs depended on the fructose 6-phosphate concentration;
at 0.4 mM, the pH range was 6.6-6.7 for the frog enzyme.
If the inhibition of enzyme activity results in changes in concentration of
acidic intermediates, it can itself affect the pHi. In fact, Trivedi and Danforth
suggested that “control of glycolysis by pH may also contribute to the regulation
of pHi.” Siesjo and co-workers (406,408; for details see sect. VIIIB) have shown
in the rat brain that the levels of pyruvate and many other acidic compounds fall
when the PCO~is raised. This reduces the fall in pHi resulting from the acid load.
Phosphofructokinase inhibition may be involved in the changes, but the activity
of other enzymes might also have been affected. The findings by Arieff et al.
(14) in the dog brain can be explained in a similar way.

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A few other studies point to a relationship between PHi and metabolism.

Cohen et al. (97) observed that the PHi of the isolated, perfused rat liver in-
creases when lactate is added to the perfusate. They ascribed this to the con-
version of lactate to glucose or to its oxidation to CO, and concluded that some
of the lactate must have entered as the ion. Thus lactate metabolism, in their
opinion, plays a role in regulating liver PHi. In two later studies from the same
laboratory, Lloyd et al. (256, 257) have elaborated on this hypothesis. The
studies of Sies et al. (402) suggest, on the other hand, that lactate and several
other m on.ocarboxylates cross the li ver cell membrane mainly in the Pro tonated
form. The pH, of the effluent of the perfused rat liver rose transiently when
these ions were added to the perfusate; removal had the opposite effect. When
the PHi was reduced (elevated Pco,), these acids transiently accumulated in
the effluent. Thus changes in PHi may affect the intracellular levels of metabo-
lites when one member of the conjugate pair of these substances is permeant.
In turn this would affect the PHi.
Moore et al. (303, 304) showed that insulin increases PHi (measured with
DMO) in frog skeletal muscle. The effect was blocked by amiloride or drastic
reduction of [Na+lO, but not by ouabain. These inhibitory procedures, in the
presence of HCO;-CO,, also inhibited insulin’s ability to stimulate glycolysis
(304). The authors suggested a causal connection between the two effects. The
PHi-glycolysis correlation, whose possible enzymatic basis was examined above,
is also illustrated by the work of Tomoda et al. (438). Glycolysis in human red
cells was stimulated when the cells were transferred from Ringer’s solution to
isotonic sucrose of the same pH. This transfer was accompanied by a rise in
red cell pHi (measured with DMO), which at pH, 7.0, amounted to about 0.8.
The rise may be ascribed* to the exchange of cellular Cl- with external HCO, (for
references see 438).

F. Fertilization

The observation that fertilization is accompanied by an increase in PHi was

first made in 1927 by Buytendijk and Woerdeman (67), who measured PHi in
frog and axolotl eggs with antimony microelectrodes and found that the PHI of
axolotl eggs increased from 7.2 to 8.3 upon fertilization. In 1976 Johnson et al.
(220) measured the pH of sea urchi n (S. purpuratus) egg homogenates and
demonstrated a PHI increase of about 0.3w i.thin the first 4 min after fertilization.
This intracellular alkalinization was accompanied by a fall in pH,, and the latter
was blocked by amiloride and also by removal of external Na+. In addition the
efflux of acid was balanced by the amiloride-sensitive influx of Na+, as measured
by 22Na uptake. The authors suggested that fertilization activates an Na-H ex-
change that raises PHi and thereby initiates development. The crucial role
played by the increase in PHi is emphasized by the observation that the fertiliza-
tion-induced increases in both K conductance (399, 424) and biosynthesis (125)
are mimicked by exposing unfertilized eggs to the weak base NH3 (see sect. IV),
thereby raising PHi directly (47, 479).

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418 A. ROOS AND W. F. BORON Volume 61

The fertilization-induced increase in pHi has also been observed in S. per-

purutus by Lopo and Vacquier (262), using homogenization, and by Gillies and
Deamer (146), using DMO and methylamine. Shen and Steinhardt (397, 398)
have made a similar observation on another species of sea urchin (L. pictus) with
pH-sensitive microelectrodes. Lopo and Vacquier have claimed that after its
initial rise,pHi falls back to its initial level within 60 min. This is refuted by
the observations of Gillies and Deamer, however, who used more reliable
methods and found that the elevated pHi remained stable for at least 100 min.
The Na-H exchange hypothesis of Johnson et al. has been called into
question by Shen and Steinhardt (398) based on their observations of the Na
dependence and amiloride sensitivity of the fertilization-induced increase in
pHi. They found that [Na+10 had to be reduced below 5 mM to demonstrably
inhibit the pHi increase. Also, in the presence of 25 mM Na+, 0.1 mM amiloride
had no effect on either the rise in pHi or subsequent cell division. Some in-
hibition of the pHi increase was observed ([Na+], = 25 mM) at 0.25-0.5 mM
amiloride, and the increase was completely blocked (though irreversibly) by
1 mM amiloride. They concluded that the relative insensitivity to amiloride
and to large decreases in [Na+], ruled out Na-H exchange and proposed instead
that Na+ merely plays an allosteric role. This conclusion is not necessarily
correct, however, since in other systems Na+ and amiloride compete with one
another. With cells of the salamander proximal tubule (41), for example, the
inhibition of Na-H exchange by 0.1 mM amiloride is almost negligible at 100 mM
[Na+lO, whereas the inhibition by 2 mM amiloride is about 75% and fully rever-
sible. At 20 mM [Na+],, the inhibition by 1 mM amiloride is nearly complete.
These results are in excellent agreement with those of Kinsella and Aronson
(234) on Na-H exchange in vesicles isolated from renal brush-border mem-
branes. They found a K, for external Na+ of about 5 mM, and a KI for amiloride
of about 15 PM. Thus Shen and Steinhardt’s data (398) are consistent with a high
affinity of an Na-H exchanger for Na and a relatively low affinity for amiloride.


A. Anoxia and Metabolic Inhibitors

Experiments with pH-sensitive microelectrodes have shown that removal

of oxygen or exposure to metabolic inhibitors produces a fall of pHi in frog
muscle, snail neurons, and squid axons. When frog sartorius muscle (241) is
exposed to 0.1 mM DNP at pH 6.4, pHi falls by -0.5 in 60 min. In snail neurons
(429) anoxia produces a slow, progressive fall in pHi that is fully reversible and
amounts to 0.2 after 10 min. With azide (5 mM, pH, 8.0) and DNP (0.2 mM,
pH, 8.0), pHi falls by about 0.2 in the first few minutes, and then continues fall-
ing at a lower rate. Squid axons subjected to anoxia (W. F. Boron and P. De
Weer, unpublished observations) undergo an intracellular acidification similar
to that observed in snail neurons. Cyanide (2 mM, pH, 7.7) has a similar effect,

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causing pHi to level off about 0.3 lower than the control after 1 h (42). Azide
(3 mM, pH, 7.7) and DNP (1 mM, pH, 7.7) both produce a two-stage fall at
pHi (42). This is most striking with azide: pHi abruptly falls by 0.1 and then
levels off within 2 min; after a delay of 5 min, further acidification continues
at a slower rate. The initial, rapid fall of pHi with azide and DNP is probably
due to the entry of the protonated form of the weak acid (see sect. IV). The later
and slower acidification with these drugs, and also with anoxia and cyanide,
might be ascribed to the intracellular accumulation of metabolic acids.

B. Calcium and Zntracellular pH

1. Ca-H exchange

Since isolated mitochondria can take up divalent cations in exchange for

H+ (21,84), the injection of Ca2+ into intact cells could be expected to produce a
fall in extramitochondrial pH (i.e., in pHi). Such an acidification has actually
been observed by Meech and Thomas (282,283) in experiments on snail neurons:
pressure injection of CaCl, causes an abrupt fall in pHi followed by a slower
recovery due to acid extrusion. In addition the transient rise in [Ca2+]i produces
a hyperpolarization due to increased K permeability (243,281). Neither the pHi
nor the V, effects are seen after injection of Mg2+ (282). Additional support
for the Ca-H exchange hypothesis comes from experiments in which neurons
were poisoned with carbonylcyanide-m-chlorophenylhydrazone, an uncoupler
of mitochondrial oxidative phosphorylation. When Ca2+ is injected in the pres-
ence of this compound, the resultant hyperpolarization is much larger and more
prolonged than normal, which would be expected if diminished mitochondrial
Ca2uptake permitted an exaggerated rise in [Ca2+]i. Ruthenium red, which also
inhibits mitochondrial Ca2+ uptake (447), similarly enhances the hyperpolari-
zation produced by Ca2+ injection. As anticipated, ruthenium red also alters
the pHi transient observed after Ca2+ injection: there was a very small, abrupt
decrease in pHi, presumably due to residual mitochondrial Ca-H exchange,
followed by a much slower fall. Since the total magnitude of the acidification
was about the same as in the absence of ruthenium red, the slower fall in pHi
probably reflects a rather sluggish, nonmitochondrial uptake of the injected
Ca2+in exchange for H+ (perhaps by endoplasmic reticulum or other organelles).
The authors concluded that under normal conditions most injected Ca2+ is
sequestered by mitochondria in exchange for H+ and estimated the stoichi-
ometry to be 1:1. This value was arrived at by calculating the amount of CaC1,
injected from the resultant change in intracellular Cl- activity and by deter-
mining the amount of H+ released with the observed change in pHi and the
measured intracellular buffering power. These results suggest that physiologic
events not be ascribed solely to changes in [Ca2+]i unless such events have been
shown to be independent of the anticipated changes in pHi.

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420 A.ROOSANDW.F.BORON Volume 61

The opposite transport process, H+ uptake in exchange for Ca*+ by mito-

chondria and/or other organelles, may also be of physiologic importance. The
rise in [Ca*+]i produced by injection of H+ (373) or exposure to 100% CO, (362,
373) has been clearly documented (see sect. XII?). When barnacle muscles are
bathed in Na-free media (preventing extrusion of Ca*+ in exchange for extra-
cellular Na+), abrupt decreases in pHi often result in contraction (45), pre-
sumably due to release of Ca*+ in exchange for H+.

2. Effect of pHi 0% Ca*+ transport

Changes in pHi may also affect Ca*+ transport across the cell membrane.
In experiments on squid axons, Baker and colleagues found that reducing pHi,
either by injecting acid or by exposing the cell to CO*, reduces both Ca-de-
pendent Na+ efflux (20) and Na-dependent Ca*+ influx (19). Conversely, raising
pHi by exposing the cell to NH,+ stimulates both fluxes (19). By itself, the
reduction in Ca2+ influx due to intracellular acidosis should reduce the level of
intracellular Ca*+. Such a reduction has actually been observed in experiments
with the Ca-sensitive photoprotein aequorin (19). In contrast to the squid axon,
barnacle muscle undergoes an increase in [ Ca*+]i during intracellular acidosis, as
demonstrated by Lea and Ashley (249), who also used aequorin. Since it was not
blocked by La”+, the effect was probably not due to a change in Ca*+ fluxes
across the plasma membrane. Instead the authors suggest that Ca*+ is released
from an intracellular store not present in squid axons: the fall in pHi may I)
inhibit Ca*+ uptake by the sarcoplasmic reticulum, as observed in frog skeletal
and rat cardiac muscle (126); 2) cause a Ca-dependent release of Ca*+ from the
sarcoplasmic reticulum; or 3) cause a displacement of Ca*+ from another
organelle or from a calcium-binding protein. A Ca-H exchange by mitochondria
may also contribute to the Ca*+ release (see above).

C. Carbonic Anhydrase Inhibitors

The fall in pHi produced by raising PCO* is the result of the release of H+
from the acid H2CO3, which is in turn formed in the cell by the hydration of CO*,
the permeant species. Thomas (430) suggested that the speed of the fall in pHi
when snail neurons are exposed to CO, indicates that the hydration, normally
a slow reaction, is catalyzed by carbonic anhydrase. Indeed 10B5M acetazol-
amide, an inhibitor of the enzyme, considerably slowed the fall in pHi upon
CO, application and also slowed its rise upon CO, withdrawal. In addition he
observed that acetazolamide, in the presence of 2.2% CO*, increases the tran-
sient fall in pHi induced by intracellular H+ injection. This is expected because
less of the injected H+ is neutralized by HCO; due to the greatly slowed de-
hydration of H,CO,. Had steps been taken to block the subsequent pHi recovery
due to acid extrusion, however, pHi should eventually have fallen to the same
value whether or not acetazolamide had been present. It is therefore incorrect to

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ascribe to the drug a generalized ability to reduce the cell’s buffering power
(see sect. IX). Rather the apparent reduction in buffering power is directly
related to the net effect of the rates of acid loading and acid extrusion, which
are very large for H+ injection.
In contrast to its effect in the snail neuron, acetazolamide (4 x 10s6 M) in
sheep Purkinje fibers had only a marginal effect (122); in mouse soleus muscle,
it (10B3 M) changed neither the speed nor size of the pHi response to changes
in PCO~(10). This is compatible with the near absence of carbonic anhydrase
in skeletal muscle (for references on this, see 10). Comparison of the correspond-
ing records obtained from the two preparations suggests that the immediate
pHi changes in snail neuron occur faster than in mouse muscle. The effect of
acetazolamide on the pHi of the avian shell gland was discussed in section XIC.

D. Conceptual Errors in Using CO,-Containing Solutions

Two recent publications (174, 192) have convinced us of the need to

emphasize that the relationship among Pco~, pH, and [HCO,] in a solution
is given by an ironclad expression, based on the law of mass action. Should un-
certainty arise about the relative importance of [HCO;], and PCO~for some
some physiologic effect, it is purposeless to attempt to produce a solution
with the same Pco2 and pH as the original one, but without HCO;. If an
initially HCO;-free solution is equilibrated with the same gas mixture as the
original one and then titrated to the same pH with strong base, the two solu-
tions, if properly prepared, will be identical. If they have different effects,
this must be because the addition of base to the second solution only transiently
raised the local pH to the desired value. As a result neither [HCO,I nor
Pcoz will be the same as in the original. If the second solution is used in this form,
its pH will drift downward as gassing is continued and Pcoz increases. It is
equally erroneous to label as “CO2-free, HCO; containing’ a solution that
initially contains the same [HCO;] as the original one, but that is then titrated
with strong acid to the same pH while being gassed with a CO,-free mixture
(192). Such a solution will have a finite Pcoz and an [HCO;] that is less than
the initial value. Its pH will steadily fall with continued gassing as HCO; is
converted to CO, that is blown off. Only when all HCO; has evolved will
be Pco2 be zero.

We thank Janice J. Wuelling and Susan J. Eads (Washington University) for their out-
standing and consistently cheerful secretarial assistance, without which this work would not have
been possible. We also thank Jeffrey B. Hughes (Yale University) for his help with the references.
Professor Ervin Y. Rodin (Washington University) proved not unequal to the task of proving the
inequalities in section IIB; we thank him for his help. We also thank Drs. Robert J. Gillies
and Jeffrey Alger (Yale University) for their critical comments on section 1123.
This work was supported by Public Health Service Grants HL-00082 to A. Roos and GM-
06499 (National Research Service Award) to W. F. Boron. A. Roos is the recipient of National
Institutes of Health Research Career Award HR-19608.

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422 A. ROOS AND W. F. BORON Volume 61


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