sPECIAL CONTRIBUTION

alcohol, effect on cellular membranes;

cell membranes, effect of alcohol

Effect of A l c o h o l on C e l l u l a r M e m b r a n e s

Ethanol disrupts the physical structure of cell membranes. The most fluid Dora B Goldstein, MD
membranes, including those that are low in cholesterol, are the most easily Stanford, California
disordered by ethanol. Although the membrane-di'sordering effect is small,
there is pharmacological, temporal, and genetic evidence that it is impor- From the Department of Pharmacology,
tant. Animals that are resistant to ethanol intoxication because of their ge- Stanford University School of Medicine,
netic background or because of previous exposure to ethanol are found to Stanford, California.
have brain membranes that are not easily disordered in vitro. An exception
is the increased behavioral sensitivity in aging animals, which is rJot Received for publication February 13,
matched by changes i~ their membranes. When animals are treated chron- 1986. Revision received May 15, 1986.
Accepted for publication June 5, 1986.
ically with ethanol, their membranes become stiffer, a response that can be
regarded as adaptive. Ethanol m a y favor the uptake of cholesterol or saturat-
ed fatty acids into membranes, thus reducing its own effect. [Goldstein DB: Presented at the UAEM/IRIEM Research
Symposium on Toxicology in San
Effect of alcohol on cellular membranes. A n n Emerg Med September Francisco, California, February 1986.
1986;15:1013-1018.]

MEMBRANE DISORDER HYPOTHESIS OF INTOXICATION Address for reprints: Dora B Gotdstein,

MD, Department of Pharmacology,
Ethanol and other anesthetic drags can disorder the physical structure of Stanford University School of Medicine,
cell membranes, and it is worth asking whether this is the actual mecha- Stanford, California 94305.
nism of intoxication. All types of biomembranes and model membranes are
disordered by ethanol. The disruption can be shown by a concentration-re-
lated decrease in membrane order on addition of ethanol in vitro. The degree
of membrane disordering can be measured using physiochemical techniques,
including electron paramagnetic resonance (EPR),1 fluorescene polarization, 2
differential scanning calorimetry,3 and optical transmission. 4 Ethanol inter-
feres with the packing of molecules in the phospholipid bilayer of the cell
membrane, thus increasing membrane fluidity. The fimctional consequences
of this disruption are probably mediated by proteins embedded in the too-
fluid lipid environment. The disordering action of ethanol and other anes-
thetic drugs s can be seen in almost any membrane (although not to the same
extent, as will be discussed below). Plasma membranes of several cell types
(neurons, erythrocytes, and hepatocytes, for example) and the membranes of
intracellular organelles show this effect, as do liposomes made from lipids
extracted from these cells. Because pure phospholipid vesicles are also disor-
dered by ethanol, it is obvious that proteins are not required.
Many different anesthetic drugs have this disordering effect. Their poten-
cies in vivo correspond well with their ability to disorder lipids and with
their lipid solubilities. But one cannot conclude from such data that exces-
sive fluidity of membranes is the actual cause of intoxication or anesthesia,
and the question whether lipid or protein is the primary site of anesthetic
action is by no means settled. Our working hypothesis is that ethanol pri-
marily affects the lipids of membranes. However, it is obvious that ethanol-
induced changes in membrane lipids are small. Experimental evidence for a
direct effect of ethanol on protein molecules has been sparse until quite re-
cently, but Franks and Lieb6 have now elegantly shown that a water-soluble
enzyme, luciferase, can be inhibited (competitively with substrate) by a
number of nonspecific lipid-soluble anesthetic agents, including ethanol.
These compounds apparently occupy a hydrophobic pocket on the surface of
the soluble protein. Their relative potencies as inhibitors of luciferase nicely
match their relative anesthetic potencies. Interactions of lipid-soluble drugs
with membrane-bound proteins are more likely than with soluble proteins,

15:9 September 1986 Annals of Emergency Medicine 1013/43

ALCOHOL AND CELLULAR MEMBRANES
Goldstein
FIGURE 1. Spin labels in a bilayer
m e m b r a n e . The cartoon shows a
phospholipid/cholesteroI bilayer with
embedded proteins. The spin labels
are 5-doxylstearic acid and 12-dox-
ylstearic acid, which are I8-carbon
f a t t y acids w i t h an a t t a c h e d ox-
azolidine ring that contains a stable
free radical and serves as the signal
for EPR measurements. This "doxyl"
group can be placed at different posi-
tions along the acyl chain, resulting in
signals coming from the core of the
m e m b r a n e (12-doxylstearic acid) or
near the surface (5-doxylstearic acid).
but they are more difficult to demon-
strate because we lack methods for
rigorous analysis of enzyme kinetics
in the presence of membranes or de-
tergents.
Measuring Membrane Order
Spectroscopic techniques measure
the motion of probes embedded in the
lipid bilayer. It is assumed that the
probe is distributed evenly in the hy-
drophobic regions of the membrane.
When EPR methods are used for stud-
ies of membrane structure, the probe
is usually a stable free radical, ie, a ni-
troxide-containing analog of a mem-
brane c o m p o n e n t (Figure 1). Its un-
paired electron acts as a tiny magnet
that absorbs energy in the field of a
laboratory magnet. The energy absorp-
tion spectra can be interpreted in
terms of the average orientation of the
probe in the membrane, S ie, its free-
dom to move. When the probe is in a
fluid environment, it moves freely and
the energy absorption spectrum has
sharp, symmetrical peaks. In a more
rigid environment, the probe is con-
strained and the s p e c t r u m flattens
out. Measuring the distance between
the peaks and troughs of the spectrum
allows an order parameter calculation,
which describes the orderliness of the
lipid bilayer around the probe. The
order parameter by definition varies
from zero (in a liquid) to one (in a
crystal). When the probe is allowed to
increase its wobbling motion, the
order p a r a m e t e r decreases and the
membrane environment is said to be
disordered.
Fluorescence polarization data have
similar meaning.S Here the probe is a
fluorescent dye that can be incorporat-
ed into a m e m b r a n e and irradiated
with vertically polarized light. The in-
dividual dye molecules that are ver-
tically oriented at any given moment
44/1014
5-dox dstearic acid
,.)°
(
(
(
will absorb a photon and will then re-
main in the excited state for about 10
nanoseconds before e m i t t i n g light
along the same molecular axis as the
a b s o r p t i o n . Because the m o l e c u l e
m o v e s during this brief time, the
emitted light (fluorescence) will no
longer be vertically polarized and the
extent of motion can be measured by
the degree of depolarization of the
light. Thus the fluorescence polariza-
tion indicates the rigidity of the
probe's environment.
Nuclear magnetic resonance
(NMR), a versatile but insensitive ap-
proach, can also detect an increase in
molecular motion within membranes
on addition of an alcohol. For exam-
ple, the acyl chains of a phospholipid
can be selectively deuterated at specif-
ic positions and examined with deu-
terium NMR. Addition of benzyl alco-
hol (a local anesthetic) decreases the
quadrupole splitting, indicating in-
creased m o b i l i t y of the i n t r a m e m -
brane chain segments. 9
M o d u l a t i o n b y Lipids
The intrinsic order of a membrane
bilayer is determined primarily by its
lipid composition. Plasma nlembranes
are composed of lipid and protein in
about equal amounts by weight; the
lipid is primarily various phospho-
lipids and cholesterol. Many different
kinds of lipid are present in a single
membrane. Intracellular membranes
contain little or no cholesterol and are
thus quite different f r o m p l a s m a
membranes. Membranes are stiffened
by an increase in their cholesterol
Annals of Emergency Medicine
12- doxylstearic acid holest'erol
)o:
,.J-
,o
content or an increased degree of sat-
uration of the fatty acyl chains of
their phospholipids. Cholesterol, with
its rigid sterol nucleus, tends to stiffen
the acyl chains of neighboring phos-
pholipids. Saturated acyl chains of
phospholipids tend to be packed in a
tightly ordered way, in contrast to
chains containing double bonds. The
latter cause kinks in the chain, allow-
ing carbon atoms of adjacent chains to
slip in and out. In mammalian mem-
branes, cholesterol seems to be the
main determinant of order.
T h e c o m p o s i t i o n of m e m b r a n e
lipids can be modified experimentally,
sometimes by diet but more easily in
vitro; such changes affect the sen-
sitivity of the membrane to disorder-
ing by ethanol. A few years ago we
studied the order parameters and dis-
ordering by ethanol in vesicles made
of egg lecithin, a m i x t u r e of fluid
phospholipids, lo Addition of cholester-
ol up to about 40 mole percent in-
creased the order p a r a m e t e r pro-
gressively; further additions had little
ordering effect. When ethanol was
added, its d i s o r d e r i n g effect was
b l u n t e d by the c h o l e s t e r o l . This
seemed to us a significant finding be-
cause it indicated that ethanol may
act more strongly in certain regions of
m e m b r a n e s where the cholesterol
content is low. There is some evidence
in the literature that the "boundary
lipids" that i m m e d i a t e l y surround
some intrinsic proteins in the mem-
branes have a low cholesterol con-
tent. 11 Thus ethanol may act at the
protein surface, having greater effects
15:9 September 1986
 FIGURE 2. Disordering effect of alco-
hols in vitro. Mouse synaptosomal
ALCOHOL CONCENTRATION(mM) plasma membranes spin-labeled with
200 400 600 5-doxylstearic acid were incubated
I I I with alcohols. The concentration-re-
lated decrease in the order parameter
demonstrates the disordering effect of
the alcohols, an effect that increases
~,~ethanol with their chain length and thus with
their lipid solubility.
-0.01
the sterol.
This protective m e c h a n i s m does
not operate in vitro. Mouse synap-
t o s o m a l p l a s m a m e m b r a n e s could
easily be enriched with cholesterol by
-0.02 incubation with liposomes of high
p- cholesterol content./2 The order pa-
W panol rameters rose according to the choles-
<
n~ terol/phospholipid ratio. Again the
high-cholesterol membranes were rel-
n," atively resistant to the disordering ef-
fect of ethanol as measured by EPR.
or"
-0.03 The effect of cholesterol on the re-
0
sponse to ethanol may explain some
Z
n-butanol specificity of action of this ordinarily
w nonspecific drug. It is supposed that
r._9
Z
lipids are i n h o m o g e n e o u s l y dis-
tributed in biomembranes and are seg-
g -0.04 regated into microdomains with dif-
ferent lipid composition and physical
properties. Cholesterol may associate
p r e f e r e n t i a l l y w i t h c e r t a i n phos-
pholipid domains and thus decrease
the ability of e t h a n o l to disorder
-0.05 them. This might account for the dif-
ferential sensitivity of different mem-
brane functions to ethanol. Those
enzymes or transport proteins that
have little or no cholesterol in their
immediately surrounding lipids m a y
be the most ethanol-sensitive.
I I I
2 CORRELATIONS OF
MEMBRANE ORDER WITH
DRUG A C T I O N S / N V/VO
there than in the bulk lipid where our raised the serum cholesterol more Although the membrane-disorder-
probes are. It may also have unusually than six-fold. The cholesterol in red ing effect of ethanol is small, requiring
strong effects in low-cholesterol intra- cell membranes was also elevated sig- sensitive methods to detect it at sub-
cellular membranes. nificantly, with a resultant increase in lethal drag concentrations, it is inti-
Recently we have carried this no- order parameters. Consistent with our mately related to the pharmacological
tion further, to see how manipulation liposome data, we observed that the effect of the drug. We can show this
of the cholesterol content in a natural erythrocyte membranes of cholester- relation either by varying the drug or
biological m e m b r a n e affects its re- ol-treated birds were resistant to the by varying the animals.
Sponse to ethanol. We used dietary disordering effect of ethanol in vitro.
n~ethods to change the erythrocyte A most remarkable incidental finding Chain Length
membrane cholesterol in an avian was that the cholesterol/phospholipid To vary the drug, we changed its
model and used liposomes as choles- ratio of synaptosomal plasma mem- lipid solubility. Modern experiments
terol donors for synaptosomal mem- branes in these quail was absolutely recapitulate the dogma of Meyer and
branes in vitro. 12 unaffected by the cholesterol diet. Overton in that the most lipid-soluble
lapanese quail were given cholester- Even though the brain was perfused by drugs are the most potent, both in
ol in their diet for several weeks; con- the same high-cholesterol plasma that v/vol3,14 and in their ability to disor-
trol birds ate the same diet without had stiffened the erythrocytes, it was der membranesA 4 We have shown this
cholesterol. The high-cholesterol diet somehow protected from taking up with aliphatic alcohols of different
15:9September1986 Annals of EmergencyMedicine 1015/45
ALCOHOL AND CELLULAR MEMBRANES
Goldstein
chain lengths (Figure 2). Increasing the
chain length increases the oil/water or
membrane/buffer partition coefficient
by about a factor of 3 for each addi-
tional methylene group, and the disor-
dering potency of the alcohols in-
creases correspondingly. Furthermore,
the potency of the alcohols as hypnot-
ics for mice increases in the same
m a n n e r (up to a point, where the
drugs cannot be easily injected be-
cause they are no longer sufficiently
water-soluble). The same relationship
between chain length and disordering
potency also holds for a series of
short-chain fatty acids, relatives of
valproic acid, that disorder m e m -
branes in v i t r o and have anticonvul-
sant potencies to match. 15
Genetics
The above is not strong evidence
that disordering causes the acute ef-
fects of ethanol in vivo. No one would
be surprised to hear that disordering
potencies are related to the ability of
drugs to dissolve in m e m b r a n e s .
Much better evidence that drugs may
work i n v i v o by disordering mem-
branes is the observation that the sen-
sitivity of different animals to ethanol
intoxication is related to the ease with
which their membranes can be disor-
dered.
We have examined groups of mice
whose sensitivity to ethanol differs,
either genetically or because of ac-
quired tolerance. We used mice of the
lines designated as Long Sleep and
Short Sleep that had been selectively
bred for sensitivity and resistance, re-
spectively, to the hypnotic effect of
ethanol. 16 Isolated synaptosomal plas-
ma membranes from these mice dif-
fered in their sensitivity to ethanol i n
v i t r o . ~7 The n e u r o n a l m e m b r a n e s
from the ethanol-sensitive Long Sleep
mice were more easily disordered by
ethanol than were those of ethanol-re-
sistant Short Sleep mice. Furthermore,
the same differential sensitivity was
evident in the erythrocyte membranes
of these mice, giving some hope for
eventual studies of the red cells of
human subjects with different genetic
backgrounds. We followed up our Long
Sleep/Short Sleep study with a similar
experiment in which no selective
breeding was needed. We simply test-
ed individual mice of a heterogeneous
stock for their sensitivity to ethanol,
using a test of balance. The synap-
tosomal membranes from the most
sensitive and most resistant of these
46/1016
animals were isolated and tested for
their response to ethanol i n v i t r o ;
again, the ethanol-sensitive mice had
ethanol-sensitive membranesAZ
Tolerance
The same correlation can be shown
when mice are made tolerant to eth-
anol by a liquid diet 18 or inhalation 19
regimen. When the mice become tol-
erant, their brain m e m b r a n e s are
found to be resistant to ethanol-in-
duced disordering (Figure 3). The
change is reversible within a day or
two, like tolerance in vivo. Other in-
vestigators have confirmed our find-
ings about tolerance, using mem-
branes from liver as well as brain,
intracellular membranes as well as
plasma membranes, and protein-free
lipid extracts as well as intact mem-
branes. 2o-2~ This is evidence that the
tiny disruption of membrane structure
induced by ethanol must be directly
related to the i n t o x i c a t i n g effect.
S o m e t h i n g is different about the
m e m b r a n e lipids w h e n a n i m a l s
change their sensitivity to ethanol.
Changes with Age
Not every change in sensitivity of
the animals to ethanol, however,
is m i r r o r e d in their m e m b r a n e s .
Changes take place over the lifespan
of animals that do not correlate with
the v u l n e r a b i l i t y of their synap-
tosomal membranes. Studies of very
young animals and of aging animals
indicate that rats and mice become
more ethanol-sensitive as they age.
Hollstedt and Rydberg~3 observed that
very young rats are remarkably insen-
sitive to ethanol, with respect to both
the tilt-plane test (ataxia) and the
LDso. Similarly mice become more
sensitive to the sedative effects of eth-
anol as they approach the end of their
life span.~4 But this is not caused by
increased sensitivity of membranes to
alchol-induced disorder.
On the contra~ several studies in-
dicate that plasma membranes be-
come progressively more rigid with
age and their cholesterol c o n t e n t
steadily increases, a condition associ-
ated with decreased vulnerability to
ethanol as described above. For exam-
ple, Schwarz et al ~s showed that the
microvillus membrane of rabbit small
intestine increased its cholesterol/
phospholipid ratio progressively dur-
ing the period from 14 days to adult.
The microviscosity paralleled the cho-
lesterol/phospholipid ratio (despite the
Annals of Emergency Medicine
fact that the degree of saturation of
the membrane fatty acids decreased
over the same time span). In another
investigation, fatty acyl chains appar-
ently mediated an increase in mem-
brane order with age. Hubbard and
Garratt 26 reported that adipocyte
membranes from aged rats contained
more saturated fatty acids than did
the corresponding membranes from
young animals. Fluroescence polariza-
tion data showed increased order in
the membranes from older animals.
Synaptic membranes, of particular
interest in relation to ethanol, were
examined by Hitzemann and John-
son. 27 They reported that desmosterol
(the immediate biosynthetic precursor
of cholesterol) is a major sterol in
membranes in young rats. With matu-
rity the desmosterol is replaced by
c h o l e s t e r o l and the s t e r o l / p h o s -
pholipid ratio rises. Fluorescence po-
larization values increase. These mea-
surements were made in rats at ages 7
and 14 days and in adult rats, and the
increased order was evident in pro-
tein-free lipid extracts as well as in-
tact membranes. Armbrecht and co-
workers 28 did not detect a change in
order p a r a m e t e r in m o u s e synap-
tosomal membranes, brain micro-
somes, or erythrocyte membranes be-
y o n d t h e age of t h r e e m o n t h s .
Different probes (reporting from differ-
ent depths in the membrane) were
used in the two studies; this is some-
times a cause of discrepant results.
Thus several investigators report
that biomembranes tend to become
more ordered with the age of the ani-
mal and some have measured a higher
level of cholesterol in plasma mem-
branes as the animals grow olden Ac-
cording to data cited above in which
membrane cholesterol and order were
experimentally manipulated, a change
.in this direction should make mem-
branes of aging animals less sensitive
to ethanol. However, in both rats ~9
and mice (Rydberg and Goldstein, un-
published data), the brain membranes
of very young, developing animals
maintained a constant degree of disor-
dering on addition of ethanol, even
though they became stiffer and richer
in c h o l e s t e r o l w i t h age. Indeed,
Armbrecht et a128 have directly dem-
o n s t r a t e d t h a t the s y n a p t o s o m a l
membranes of aging mice are more
ethanol-resistant i n v i t r o than are
those of young mice, in accord with
data on lipid composition. Neverthe-
less the mice become more ethanol-
15:9 September 1986

I I
.590
6 .585
.580
w
0 presence of certain lipids or by the in-
.575 I I
0 200
ETHANOL, rnM
sensitive. Thus the correlations that
worked so nicely with respect to ge-
netic differences and tolerance fail us
when we deal with developmental
changes. We cannot rely on the sim-
plistic n o t i o n that m e m b r a n e sen-
sitivity determines sensitivity in vivo.
Not surprisingly, it is more compli-
cated than that. That animals become
more easily sedated as they age is not
unexpected, but this change resides
elsewhere in the brain than in the
membrane lipid composition.
CHRONIC A D M I N I S T R A T I O N
OF E T H A N O L
Membranes change their properties
after ethanol has been administered
chronically. It is a c o m m o n finding
that the membranes of chronically
ethanol-treated animals are more rigid
than controls, with appreciably higher
order p a r a m e t e r s of f l u o r e s c e n c e
anisotropy values.19,2¢ This can even
begin before birth. 3o Rat pups that
were treated with ethanol during ges-
tation and lactation had liver plasma
membranes that were abnormally rich
in cholesterol. The cholesterol/phos-
pholipid ratio of the membranes in-
creased with age (5 to 25 days) and
t5:9September1986
I anol in m e m b r a n e s from tolerant
D ethanol, compared to controls.
CONTROL" ~
I I
400 600
was higher in the ethanol-treated pups
than in the controls at each time
point. The difference persisted at least
a few days after the ethanol was with-
drawn at weaning. These observations
suggest that infants born of drinking
mothers may have membranes that
are abnormally ordered, presumably
because of a change in lipid synthesis
and membrane assembly.
Increased cholestero131, 3~ and in-
creased saturation of fatty acids 21,33
have been reported in animals chron-
ically treated with ethanol, but not
uniformly. Increased order and de-
creased responsiveness to ethanol in
vitro can occur in experiments in
which no change in cholesterol can be
measured. 19 One may speculate that
the organism is adapting to a physical
phenomenon, the disorder in its mem-
branes, and may use any of a number
of different chemical strategies to re-
spond under different conditions.
It is likely that the reduced sen-
sitivity to ethanol in membranes of
animals chronically treated with eth--
anol is mediated by a decreased parti-
tion coefficient of ethanol. For exam-
ple, ethanol does not enter the synap-
tosomal membranes of ethanol-toler-
Annals of Emergency Medicine
FIGURE 3. Loss of sensitivity to eth-
mice. S y n a p t o s o m a l p l a s m a m e m -
branes were prepared from mice that
had been treated w i t h ethanol for
three days by inhalation. The m e m -
branes were spin-labeled with 5-dox-
ylstearic acid and the order param-
eters were determined in the presence
of ethanol. These m e m b r a n e s were
relatively resistant to disordering by
ant rats as readily as it enters the
m e m b r a n e s of control animals, zo
Other lipophilic compounds, such as
halothane, are also differentially ex-
cluded from membranes of ethanol-
treated rats, but the anesthetic effect
at a given intramembrane concentra-
tion is unchanged. 34 It is not known
whether ethanol is excluded by the
creased order per se.
How does ethanol cause the mem-
branes to become more rigid and to
accumulate cholesterol or saturated
fatty acids? Some experiments in vitro
suggest that this is another purely
physical effect; perhaps rigid mole-
cules like cholesterol can diffuse more
easily into m e m b r a n e s w h e n the
bilayer has been disordered by eth-
anol. Cholesterol can easily be trans-
ferred from various donors (plasma
l i p o p r o t e i n s , l i p o s o m e s , etc) to
erythrocyte membranes in vitro. At 37
C, the process reaches equilibrium
with half-times of several hours, de-
pending on the nature of the donor.
Addition of ethanol (350 mM) to the
incubation mixture markedly stimu-
lates the rate of transfer of cholesterol,
apparently without affecting the equi-
librium. 35 In analogous experiments
with saturated and unsaturated fatty
acids ( G o l d s t e i n and C h i n , u n -
published data), we found that ethanol
stimulates the uptake of palmitate
more than that of oleate into erythro-
cyte membranes in vitro. Thus in the
presence of ethanol, the membranes
accumulate the stiffer saturated fatty
acid, at least temporarily. The relation
of these equilibrium experiments in
vitro to steady-state conditions that
obtain in vivo cannot be predicted,
but it seems possible that a physical
mechanism based on the ease of pas-
sive transfer of rigid membrane com-
ponents could account for the stiff-
ness of membranes that have been
chronically exposed to ethanol.
1017/47
ALCOHOL AND CELLULAR MEMBRANES
Goldstein
CONCLUSION
These are speculative ideas. There
is still m u c h experimental variability
in studies Of the actions of ethanol, es-
pecially in chronic administration,
and we have far to go before we really
u n d e r s t a n d the relation of intoxica-
tion, tolerance, and physical depen-
dence to the physical properties of
membranes. The observation that eth-
anol disorders membranes, that toler-
ance develops to this effect, and that
m e m b r a n e s may become stiffer after
c h r o n i c exposure to e t h a n o l fit to-
gether well as evidence of an adaptive
response to ethanol, but this idea re-
mains an unproven hypothesis.
The work in Dr Goldstein's laboratory is
supported by the USPHS (grant AA01066)
and by the Alcoholic Beverage Medical
Research Foundation.
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