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Circ Res. Author manuscript; available in PMC 2013 January 20.
Published in final edited form as:
Circ Res. 2012 January 20; 110(2): 285–294. doi:10.1161/CIRCRESAHA.111.258145.

Acidosis dilates brain parenchymal arterioles by conversion of

calcium waves to sparks to activate BK channels
Fabrice Dabertrand, Ph.D.*, Mark T. Nelson, Ph.D.*, and Joseph E. Brayden, Ph.D.*
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*Department of Pharmacology, University of Vermont, College of Medicine, Burlington, VT 05405,

USA

Abstract
Rationale—Acidosis is a powerful vasodilator signal in the brain circulation. However, the
mechanisms by which this response occurs are not well understood, particularly in the cerebral
microcirculation. One important mechanism to dilate cerebral (pial) arteries is by activation of
large-conductance, calcium-sensitive potassium (BKCa) channels by local Ca2+ signals (Ca2+
sparks) through ryanodine receptors (RyRs). However, the role of this pathway in the brain
microcirculation is not known.
Objective—The objectives of this study were to determine the mechanism by which acidosis
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dilates brain parenchymal arterioles (PAs) and to elucidate the roles of RyRs and BKCa channels
in this response.
Methods and Results—Internal diameter and vascular smooth muscle cell (VSMC) Ca2+
signals were measured in isolated pressurized murine PAs, using imaging techniques. In
physiological pH (7.4), VSMCs exhibited primarily RyR-dependent Ca2+ waves. Reducing
external pH from 7.4 to 7.0 in both normocapnic and hypercapnic conditions decreased Ca2+ wave
activity, and dramatically increased Ca2+ spark activity. Acidic pH caused a dilation of PAs which
was inhibited by about 60% by BKCa channel or RyR blockers, in a non-additive manner.
Similarly, dilator responses to acidosis were reduced by nearly 60% in arterioles from BKCa
channel knockout mice. Dilations induced by acidic pH were unaltered by inhibitors of KATP
channels or nitric oxide synthase.
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Conclusions—These results support the novel concept that acidification, by converting Ca2+
waves to sparks, leads to the activation of BKCa channels to induce dilation of cerebral
parenchymal arterioles.

Keywords
brain parenchymal arteriole; acidosis; Ca2+ sparks; ryanodine receptor; potassium channel

Introduction
Brain parenchymal arterioles (PAs) constitute a unique vascular bed. In contrast to pial
arteries on the surface of the brain, PAs do not receive extrinsic innervation and are
enveloped by astrocytic processes called endfeet over nearly their entire basolateral
surface 1, 2. PAs account for approximately 40% of total cerebral vascular resistance 3. Thus

Corresponding author: Joseph E. Brayden, University of Vermont College of Medicine, Department of pharmacology, 89 Beaumont
Avenue, B-303 Given building, Burlington, VT 05405-0068, USA. Fax: 802-656-4523, Phone: 802-656-1985,
Joe.Brayden@uvm.edu.
Disclosure
None
 Dabertrand et al. Page 2

regulation of their diameter is a determinant process for appropriate perfusion of brain

tissue 2 and for neurovascular coupling 4. Compared with pial arteries, PAs depolarize and
constrict to lower levels of intravascular pressure, a physiologically appropriate response
given that PAs experience lower pressure levels in situ 5, 6, 7, 8. One plausible explanation is
that negative feedback elements which limit depolarization and constriction to pressure in
pial arteries are lacking, or less active in parenchymal arterioles. In large diameter pial
arteries, activation of smooth muscle large-conductance potassium channels (BKCa) by local
Ca2+-release events (Ca2+ sparks) through ryanodine receptors (RyRs) opposes pressure-
induced depolarization and constriction 9, 10. In contrast, BKCa blockers have little effect on
the diameter of cerebral arterioles 11, 12 even though functional BKCa channels are present 7.
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Regulation of cerebral blood flow (CBF) by changes in pH has been recognized as a critical
homeostatic mechanism for more than a century 13. It is well established that acidic pH
increases CBF by relaxing vascular smooth muscle cells (VSMC) 14, 15, 16. Protons are
thought to act through multiple mechanisms including the inhibition of Ca2+ entry through
voltage-dependent Ca2+ channels (VDCCs) 17, and activation of KATP channels 18 and NO
pathways 19. In addition, protons can decrease the open probability (P0) of RyRs 20, which
will modify the Ca2+-release profile of the sarcoplasmic reticulum (SR) and influence
contractile function in VSMCs. The goals of the present study were to determine the
mechanism by which normocapnic and hypercapnic acidosis (pH 7.0) causes arteriolar
dilation and the roles of Ca2+ signaling and BKCa channels in this process.

Methods
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Pressurized parenchymal arterioles (PAs)

Animal procedures used in this study were in accordance with institutional guidelines and
approved by the Institutional Animal Care and Use committee of the University of Vermont.
Male mice were euthanized by intraperitoneal injection of sodium pentobarbital (100 mg/kg)
followed by rapid decapitation. The brain was removed and placed into 4°C MOPS-buffered
saline. PAs (10–40 μm in diameter) were dissected from the middle cerebral artery territory.
Pre-capillary arteriolar segments were then mounted and pressurized in an arteriograph
system (Living Systems Instrumentation, Inc.) containing artificial CSF solution, and inner
diameter or Ca2+ release events were measured as described in the supplementary methods.

Solutions
The composition of artificial cerebrospinal fluid (aCSF) was (in mmol/L): 125 NaCl, 3 KCl,
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26 NaHCO3, 1.25 NaH2PO4, 1 MgCl2, 4 D-glucose, 2 CaCl2; pH was 7.4 when aerated with
5% CO2 and 7.0 when aerated with 15% CO2 (hypercapnia). In normocapnic conditions,
acidic pH was obtained by decreasing NaHCO3 concentration (in mmol/L): 23 for pH 7.3,
19 for pH 7.2, 15 for pH 7.1, 11 for pH 7.0, 8 for pH 6.9. NaCl concentration was increased
in order to keep [Na+]o constant at 152.25 mmol/L.

Drugs
Paxilline was purchased from A.G. Scientific, Inc. Ryanodine and U46619 were purchased
from Enzo Life Sciences. NS11021 was kindly donated by Dr. Søren-Peter Olesen,
Neurosearch A/S, Ballerup, Denmark. Iberiotoxin (IBTX) was purchased from Peptides
International. All other chemicals were purchased from Sigma-Aldrich.

Data Analysis and Statistics

Changes in arterial diameter were calculated as % change from baseline: [(change in
diameter)/initial diameter] or calculated as % of maximal dilation: (change in diameter)/
(maximal diameter − initial diameter). Ca2+ image experiments were analyzed with custom

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software (SparkAn) created by Dr Adrian D. Bonev in our laboratory. Fractional

fluorescence (F/Fo) was determined by dividing the fluorescence intensity (F) within a
region of interest by a mean 2+ fluorescence value (Fo) determined from 10 images before
stimulation and without any Ca events. Data are expressed as means ± s.e.m. Differences
between two means were determined using Student t test. Statistical significance was tested
at 95% (P<0.05) confidence level.

Results
Intact parenchymal arterioles display little functional BKCa or Ca2+ spark activity at normal
pH
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Elevation of intravascular pressure to 40 mm Hg constricted mouse parenchymal arterioles

by 40%, which is similar to the high level of myogenic tone previously observed in rat
parenchymal arterioles (PAs) 21, 8, 22, 7. The mean diameter after development of myogenic
tone was 19.1 ± 1.5 μm and the mean maximal diameter was 31.9 ± 2.1 μm (n = 31 PAs).
Inhibition of BKCa channels by paxilline (1 μmol/L) or RyRs by ryanodine (10 μm/L)
caused very small constrictions (3.3 ± 0.1%, n=6 and 3.9 ± 1.4%, n=11 respectively) (Figure
1A, 1B, and 1D). Responses to the thromboxane analog U46619 attested to the robust
constrictor capability of the arterioles (Figure 1A). Smooth muscle cells in myocytes from
PAs have a BKCa channel current density comparable to pial arteries 7. To test the
functionality of the BKCa channel we used the agonist NS11021 (3 μmol/L) 23, which
induced a robust, iberiotoxin-sensitive dilation of intact PAs (Figure 1C and 1D). Caffeine
was used to test for the presence of functional RyRs. Caffeine, by opening simultaneously
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all RyRs, typically triggers a large increase in cytosolic Ca2+, leading to vasoconstriction 24.
Caffeine, in a concentration-dependent manner, caused vasoconstriction, which was
prevented by the RyR inhibitor ryanodine, supporting the presence of functional RyRs
(Figure 1E and 1F). These results indicate that BKCa and RyRs in parenchymal arteriolar
smooth muscle cells have little influence on vessel diameter in the absence of exogenous
activators.

Intracellular Ca2+ signaling in VSMCs has a central role, particularly in regard of the
activation of BKCa channels. To examine spontaneous Ca2+ signals, intact pressurized PAs
were loaded with a fluorescent Ca2+ dye (Fluo-4). We found that 78% of the VSMCs
exhibited asynchronous Ca2+ waves propagating through the cytoplasm over a duration of
about 10 s, and very few localized Ca2+ events (Figure 2A, 2B and 2C). Figure 2A
illustrates the difference in spontaneous Ca2+ signaling between pial arteries and
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parenchymal arterioles, with similar levels of tone. In pial arteries (proximal middle cerebral
artery) about 80% of the cells exhibited typical brief (<0.5 s) and localized (<20 μm2) Ca2+
events (Ca2+ sparks), whereas less than 5% of the cells displayed propagated Ca2+ waves
(Figure 2C). In PAs only 12% of cells exhibited Ca2+ sparks.

Depletion of Ca2+ stores by application of the sarcoplasmic reticulum Ca2+- ATPase

inhibitor cyclopiazonic acid (30 μmol/L) abolished spontaneous Ca2+ waves in all smooth
muscle cells of PAs after 3 minutes (n=13 cells). Treatment with ryanodine (10 μmol/L) for
30 minutes, had a similar effect (n=22 cells). To further investigate the role of the RyRs in
the generation and propagation of the Ca2+ waves, we have tested the effect of the RyR
inhibitor tetracaine. In pressurized PAs, perfusion with tetracaine (100 μM) blocked Ca2+
waves in 99% of the cells (n=20 cells). Ca2+ sparks were not observed in PAs exposed to
ryanodine or tetracaine. These results indicate that RyRs mediate both Ca2+ waves and
sparks in PAs.

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Acidic pH converts Ca2+ waves to Ca2+ sparks

The preceding data indicate that Ca2+-spark activity and BKCa channel function in PA
myocytes are minimal under normal conditions. We next tested the hypothesis that acidosis,
a powerful physiological vasodilator signal, relaxes VSMCs in cerebral parenchymal
arterioles by enhancing Ca2+ spark activity. To test this hypothesis, pH was decreased from
7.4 to 7.0 by decreasing the bicarbonate concentration in the aCSF. This intervention led to a
sizeable decrease in Ca2+ wave activity (only 9% of cells exhibited waves at pH 7.0, data
not shown), accompanied by a robust increase in Ca2+ spark activity (Figure 3). Acidic pH
induced an increase in both spark frequency and the percentage of cells with Ca2+ sparks
(Figure 3B). Since this increase in Ca2+ spark activity should induce a parallel increase in
BKCa channel activity 9, we tested the hypothesis that acidosis induces an acute dilator
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response of intracerebral arterioles through Ca2+ spark-induced activation of BKCa channels.

BKCa or RyR blockers inhibit acidosis-induced dilation

PAs responded to both normocapnic and hypercapnic extraluminal acidosis with a fast,
stable, and reproducible vasodilation of about 70% (Figure 4A, 4C and 4D). Ryanodine (10
μmol/L) inhibited 65.5% of the acidic pH-induced dilation in normocapnic conditions and
60.1% in hypercapnic conditions (Figure 4A, 4C and 4D). Paxilline 1 μmol/L induced a
similar decrease (60.2%) in the normocapnic acidosis-induced dilation (Figure 4B and 4D).
In hypercapnic conditions, ryanodine and paxilline together did not inhibit the dilations
beyond that observed during paxilline alone (not shown) or ryanodine alone (Figure 4B and
4D). These results indicate that the Ca2+ sparks observed at pH 7.0 indeed activate BKCa
channels and contribute to the vasodilator response. They also suggest that BKCa channels
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are not directly activated by protons or CO2 because RyR and BKCa blockers have non-
additive effects.

Acidosis-induced dilation depends on BKCa channel expression

To further examine the role of BKCa channels in acidosis-induced dilation, we used a
genetic knockout approach with mice lacking the pore-forming α subunit of the BKCa
channel (Slo−/−/Kcnma1−/−) 25. In the absence of the functional channel, the dilation of
intact PAs induced by the agonist NS11021 (1 μmol/L) was reduced to the one observed in
wild-type mice in presence of paxilline (1 μm/L) (Figure 5A and 5B). The BKCa channel
blocker paxilline did not affect tone, or the residual dilations to pH or NS11021 of arterioles
from Kcnma1−/− mice. Moreover the dilations to reduced pH, in both normocapnia and
hypercapnia conditions, were substantially attenuated in arterioles from the Kcnma1−/− mice
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(Figure 5A and 5B). Indeed, the percent reduction in the response to acidosis in BKCa
knockout versus wild-type arteries was very close (57.5 % in hypercapnia and 55.2 % in
normocapnia) to that observed in normal arteries treated with ryanodine or paxilline versus
control. These results support the important role of BKCa channels in acidic pH-induced
vasodilation.

Acidosis-induced dilation is not affected by KATP channel inhibition in mouse PAs

KATP channels are directly activated by protons in VSMCs 18. However, in normocapnic
conditions, the selective KATP channel inhibitor glibenclamide (1 μmol/L) failed to inhibit
pH 7.0-induced dilation (n=6, Figure 6A). Higher glibenclamide concentrations (3 μmol/L
or 5 μmol/L) also did not alter the vasodilation (n=4 and n=3 respectively, data not shown).
Similarly, hypercapnia dilated PAs by 69.4 ± 2.9% and by 68.2 ± 2.0% in the absence and
presence of glibenclamide (1 μmol/L) respectively (n=6). To test for the presence of
functional KATP channels, cumulative additions of the KATP channel opener cromakalim
were applied to PAs and to third order mesenteric arteries from the same animal (Figure 6B,
6C, and 6D). Cromakalim (3 μmol/L) relaxed mesenteric arteries up to 65.1 ± 9.8% and this

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action was fully reversed by glibenclamide (1 μmol/L) (Figure 6C and 6D). In contrast,
cromakalim had no effect on PA diameter, suggesting these arterioles lack functional KATP
channels.

Inhibition of endothelial nitric oxide synthase (eNOS) does not significantly affect acidosis
induced-dilation
The eNOS inhibitor L-NAME (100 μmol/L) induced a modest constriction of PAs under
normal conditions of pH, temperature, and pressure, which is not surprising given the
prominent tonic influence of NO on PA tone 7, 21. Nevertheless, the dilations induced by
normocapnic or hypercapnic acidosis were not different in the absence and presence of L-
NAME (Figure 7).
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Decreasing extracellular pH inhibits calcium channel activation by Bay K 8644

In the presence of paxilline 1 μM, we measured the vasoconstriction induced by increasing
concentrations of the L-type channel agonist Bay K 8644, at pH 7.4 and pH 7.0. Decreasing
extracellular pH significantly increased the Bay K 8644 EC50 (p = 0.0005) from 16.3 nmol/
L (95% CI 9.1 nmol/L to 29.1 nmol/L) at pH 7.4, to 65.4 nmol/L (95% CI 47.3 nmol/L to
90.4 nmol/L) at pH 7 (Online Figure I). This result suggests an inhibition of the gating
propriety of the channel by protons.

Discussion
Acidosis activates Ca2+ sparks and BKCa channels
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Acidification of brain tissue can occur with reduced respiratory rate, during CO2 inhalation,
or during cerebral lactic acidosis related to ischemia 26 and hypoxia 27. Under those
conditions, a decrease in extracellular pH of 0.4 units occurs rapidly, e.g. within seconds
during global ischemia. This tissue acidification leads to cerebral vasodilation. We found
that acidification from pH 7.4 to 7.0 rapidly dilated parenchymal arterioles (Figure 4 and 5)
and that this dilator response is correlated with a dramatic increase in Ca2+ spark activity
and BKCa channel function (figure 3). Each Ca2+ spark delivers micromolar Ca2+ to the
adjacent BKCa channels to increase its open probability ~105- to 106-fold 28, causing up to a
20 mV hyperpolarization 29. Our results are consistent with the idea that acidification
increases Ca2+ spark activity, which in turn activates BKCa channels to hyperpolarize and
dilate PAs. The present report is the first investigation of the effects of acidosis on Ca2+
release from the SR in vascular smooth muscle and it implies a prominent role for Ca2+
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sparks as a mediator of cerebral vasodilation associated with acidosis.

Reshaping of RyRs-dependent Ca2+ release by protons

At physiological pH, Ca2+ spark activity in PA myocytes is low, and Ca2+ wave activity
predominates. The slight vasoconstriction observed in the presence of 1 μmol/L paxilline or
100 nmol/L IBTX in the present study suggests that RyR-mediated Ca2+ release in the form
of waves does not cause sufficient BKCa channel activation to modulate membrane
potential, and hence diameter. The simplest interpretation of this result is that the necessary
micromolar Ca2+ concentrations are not reached at the BKCa channel, likely because the
waves disperse the released Ca2+ ions whereas the sparks concentrate Ca2+ in spatially
limited areas. Acidosis causes a fundamental shift of this Ca2+ signaling dynamic from
waves to sparks. While we have not directly assessed the mechanism by which this
reshaping of the Ca2+ signal events occurs, we propose the following as a possible
explanation. The kinetic properties of Ca2+ waves and their inhibition by both ryanodine and
tetracaine strongly suggest that Ca2+ waves travel through the VSMCs by a RyR-dependent
Ca2+-induced Ca2+ release mechanism (CICR) 30. By this mechanism, a local increase in

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Ca2+ activates neighboring RyRs because the resting open probability (P0) of the RyRs is
high 31. However, if the resting P0 is low, RyR excitability is not sufficient to induce the
regenerating process described above and Ca2+ release remains limited to activation of
single RyR (Ca2+ quark) or clusters of RyRs (Ca2+ sparks) 31. From these observations and
because PAs show more Ca2+ waves than the pial arteries, we propose that the resting P0 of
RyRs in PAs is higher than in large diameter pial arteries, possibly due to altered activity of
one or more RyR modulators including [Ca2+]i, the FK506-binding protein, cyclic ADP-
ribose, the RyR phosphorylation state 32 or perhaps intracellular pH. Recent studies have
suggested that spontaneous Ca2+ sparks in smooth muscle are likely encoded by the RyR2
isoform 33, 34, which we have found to be expressed in PA myocytes, along with RyR1 and
RyR3 (data not shown). At physiological pH, the lack of Ca2+ sparks, and thereby
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diminished contribution of BKCa channels to control of membrane potential could explain

the enhanced myogenic tone observed in parenchymal arterioles. Physiologically, enhanced
myogenic tone may allow a higher “vasodilator reserve” during functional hyperemia but
also a greater tendency towards vasospasm associated with pathologies.

The P0 of RyRs is modulated by intracellular pH. For instance in cardiac sarcoplasmic

reticulum vesicles 35 and in planar lipid bilayers 36, 37 an increase in proton concentration
decreases P0. Further, acidic pH decreases the efficiency of CICR in skeletal muscle 38 and
reduces Ca2+ spark frequency in rat ventricular myocytes 39. Conversely alkalinization
increases the Ca2+ sensitivity of RyRs 40. We previously demonstrated that increases in pH
to 7.6 and beyond changed the primary Ca2+ signaling modality from Ca2+ sparks to
ryanodine-sensitive Ca2+ waves in large diameter pial artery smooth muscle 41. However,
although our previous study demonstrated the apparent pH sensitivity of RyRs in vascular
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smooth muscle cells, the functional link between altered Ca2+ signaling dynamics and
vascular tone appeared minimal since the constriction induced by alkalosis was mediated
mostly (82%) by direct enhancement of voltage-dependent Ca2+ channel activity. The
mechanism described in the present study relies on the same regulation of RyR P0 by
protons. However, here, we provide genetic and pharmacological evidence that the
reversible activation of Ca2+ sparks and BKCa channels is a major mechanism by which
acidosis, over a narrow and physiologically relevant pH range, induces cerebral
vasodilation.

Role of potassium channels in acidic-pH induced dilation

Our results indicate that substantial portion (60%) of acid pH-induced dilation depends on
RyR-mediated Ca2+ spark activation of BKCa channels. A number of studies have also
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suggested involvement of BKCa channels in acidic pH-induced vasodilation 42. On the

contrary, in rat penetrating arterioles, vasodilation to acid was not reduced by the BKCa
blocker TEA 12. However, species and methodological differences between this previous
study and ours, particularly in the perfusion solution composition and the drugs used, make
direct comparisons difficult.

Many studies support a role for KATP channels in the response to acidosis in mesenteric and
cerebral arteries, including brain penetrating arterioles 12, 18, 42. In light of this previous
work, the lack of effect of glibenclamide on both normocapnic and hypercapnic acidosis-
induced vasodilation shown here was not expected. However the absence of dilation in
response to the KATP agonist cromakalim, observed in the present study, strongly suggests a
lack of functional KATP channels in mouse PAs. Interestingly, rat arterioles, lacking
perivascular nerves, also are 43, 44 (for review see 45). Another study resistant to the
activation of KATP channels by cromakalim found that glibenclamide attenuates the
response of rabbit pial arterioles in vivo to low level of hypercapnia but it is ineffective with
more intense hypercapnia 46. This is consistent with the suggestion that KATP channels are
activated only by mild hypercapnia 47. However, other studies have found that vasodilation

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induced by direct, strong acidification is inhibited by glibenclamide 1242. It appears that

additional studies will be needed to clarify the specific role of KATP channels in the
vasodilator response to reduced pH.

The RyR- and BKCa-independent component of dilation induced by acidosis

Multiple in vivo studies report a role for NOS in the increase of cerebral blood flow elicited
by hypercapnia 48, 46. Interestingly, using an in vivo endothelial injury model, Wang and
coworkers reported, that the origin of NO in hypercapnic cerebrovascular dilation was
nonendothelial, and likely due to neuronal NOS activation 49. Their conclusion is supported
by the absence of effect of a NOS inhibitor on the CO2-induced increased blood flow in
mice lacking neuronal NOS 50. In vitro, relaxation of rat mesenteric arteries induced by
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hypercapnia is not altered by endothelial removal 15. A more recent study using isolated pial
arteries implies that the regulation of the arteriolar diameter by pH involves NO released
from perivascular nerves and not from endothelial cells 19. Since we used isolated brain
parenchymal arterioles, which lack extrinsic innervation, our observations support the
concept that the NOS-dependent component of increased cerebral blood flow elicited by
hypercapnia in vivo, results from neuronal NOS activation. In situ, this neuronal source of
NO is likely derived from neurons that encompass the “neurovascular unit” along with
arterioles and astrocytes.

In the present study, vasodilation induced by normocapnic or hypercapnic acidosis was not
significantly different when eNOS or KATP channels were inhibited. However, a residual
dilation, about 40% of the initial response, remained when RyR or BKCa channel blockers
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were applied during acidic pH induced-dilation. Vascular L-type Ca2+ channels are inhibited
by protons both internally and externally 17, 51 and this mechanism has been proposed as a
contributor to the decrease in [Ca2+]i and tone observed when pH is reduced 16. Therefore
the BKCa- and RyR-independent dilation observed at pH 7.0 (figure 4 and 5), is likely
induced by the inhibition of L-type channels by protons. This hypothesis is supported by the
observed decrease in the vasoconstriction induced by L-type channel agonist Bay K 8644
during acidosis.

Summary
Inhibition of RyRs or BKCa channels has little effect on the diameter of pressurized
parenchymal arterioles at physiological pH. Normocapnic or hypercapnic acidosis converts
smooth muscle Ca2+ waves to sparks, and causes profound vasodilation, which is
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significantly reduced by inhibitors of RyRs or BKCa channels (Figure 8). Our results support
the novel concept that pH modulates local Ca2+ signaling (“Ca2+ sparks”) to regulate BKCa
channel activity, and thereby dramatically influence arteriolar tone.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Acknowledgments
The authors would like to thank Andrea L. Meredith, PhD, and Richard W. Aldrich, PhD, for providing the
Kcnma1−/− mouse, Thomas J. Heppner, PhD, for insightful comments on pH regulation of RyRs, Adrian D.
Bonev, PhD, for design of custom software used for the analysis of dynamic Ca2+ events and.

Sources of funding

This work was supported by NIH grants RO1 HL44455, RO1 HL58231 and PO1 HL095488, Totman Trust for
Medical Research, and a postdoctoral fellowship from the American Heart Association 09POST2290090 to F.D.

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 Dabertrand et al. Page 8

Non-standard abbreviations

PAs parenchymal arterioles

BKCa large conductance potassium channel
VSMC vascular smooth muscle cell
RyR ryanodine receptor
CBF cerebral blood flow
VDCC voltage-dependent Ca2+ channel
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SR sarcoplasmic reticulum
aCSF artificial cerebrospinal fluid
IBTX iberiotoxin
CICR Ca2+-induced Ca2+ release
Po open probability

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Novelty and Significance

What Is Known?
• Cerebral blood flow (CBF) is highly regulated by pH, with acidification causing
profound vasodilation.
• In arteries on the surface of the brain (pial arteries), activation of large
conductance potassium calcium-activated channels (BKCa) channels by local
Ca2+ signals (Ca2+ sparks) through ryanodine receptors opposes
vasoconstriction.
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• Unlike pial arteries, inhibition of BKCa channels has little effect on the diameter
of (parenchymal) arterioles within the brain, even though their smooth muscle
cells have a significant BKCa channel density.
What New Information Does This Article Contribute?
• Ca2+ waves are the predominant spontaneous Ca2+ signals in parenchymal
arteriolar smooth muscles cells.
• Acidosis reshapes spontaneous Ca2+ waves into Ca2+ sparks which activate
BKCa channels.
• This mechanism accounts for 60% of acidosis-induced dilation of parenchymal
arterioles.
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Summary
Regulation of CBF by changes in pH has been recognized as a critical homeostatic
mechanism for more than a century, yet little is known about this effect in parenchymal
arterioles. Here, we provide genetic and pharmacological evidences that the reversible
activation of Ca2+ sparks and BKCa channels is a prominent mechanism by which
acidosis, over a narrow and physiologically relevant pH range, induces cerebral
vasodilation. Our study supports the novel concept that protons can induce the
conversion of smooth muscle Ca2+ waves to Ca2+ sparks, which triggers activation of
BKCa channels, causing dilation. When pH is shifted from 7.4 to 7.0, this mechanism
accounts for about 60% of a substantial and maintained vasodilator response. A change
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of 0.4 units in pH can occur within seconds in brain tissue during reduced respiratory
rate, CO2 inhalation, or cerebral lactic acidosis related to ischemia and hypoxia. The
mechanism presented here likely is relevant to these pathological situations. Moreover,
the parenchymal arterioles, along with neurons and astrocytes, constitute a functional
neurovascular unit that coordinates blood flow and neuronal activity. The present study
leads us to propose that protons, through modulation of Ca2+ signaling, may mediate or
modulate neurovascular coupling under normal and pathophysiological conditions.
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Figure 1.
Mouse parenchymal arterioles express functional BKCa channels and RyRs but the
myogenic response is not counterbalanced by Ca2+ spark driven BKCa currents under basal
conditions. Typical recordings of the internal diameter of pressurized parenchymal arterioles
(40 mm Hg) during the perfusion of the BKCa blocker paxilline 1 μmol/L (A), RyR blocker
ryanodine 10 μmol/L (B), or BKCa channel agonist NS11021 3 μmol/L (C). (D) Summary
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data showing change in luminal diameter expressed as percentage change in baseline

diameter with the number of experiment indicated in parentheses. *P < 0.05 (E) typical
recordings of the internal diameter showing the vasoconstriction induced by noncumulative
addition of caffeine in papaverine 100μmol/L in absence (left) or presence (right) of
ryanodine 10 μmol/L. (F) summary data showing change in luminal diameter expressed as
percentage of baseline diameter. (n = 5).

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Figure 2.
Spontaneous Ca2+ signals in intact cerebral arteries. (A) Representative recordings showing
Ca2+ sparks in pressurized (80 mm Hg) mouse pial artery (middle cerebral artery, ~100 μm
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diameter) and Ca2+ waves in pressurized (40 mm Hg) mouse parenchymal arterioles (~ 15
μm diameter). Squares on the photographs are regions of interest, where F/F0 is measured.
Spontaneous Ca2+ signals (traces below photographs) were detected as F/F0 from regions of
interest marked as colored squares in the top panels. (B) Progression of a Ca2+ wave from
one end of the cell to the other (bracketed cell from a parenchymal arteriole). Images are
color coded as indicated by the color bar. Images were acquired every 35.9 ms (for display,
every twentieth image is shown). (C) Summary data of the percent of cells exhibiting Ca2+
sparks and Ca2+ waves in pial arteries and parenchymal arterioles.

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Figure 3.
Acidic pH reshapes the intracellular Ca2+ dynamic from Ca2+ waves to Ca2+ sparks. (A)
Spontaneous Ca2+ signals recorded from the same regions of interest of a pressurized
parenchymal arteriole at pH = 7. 4 and pH = 7.0. (B) Compiled data showing Ca2+ spark
frequency (sparks/cell/s, black bars) and the percentage of cells presenting sparks (% of cells
with spark, grey bars) at different pHs. At pH 7.2, both the percentage of cells with Ca2+
sparks and spark frequency became significantly different from values observed at pH 7.4 (P
< 0.05).

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Figure 4.
Normocapnic and hypercapnic acidosis dilate pressurized parenchymal arterioles by
activating Ca2+ spark-driven BKCa channel activity. (A) Typical recording of the internal
diameter of a pressurized parenchymal arteriole (40 mm Hg) during the perfusion of acidic
aCSF under normocapnic conditions, without and with the RyR blocker ryanodine (10
μmol/L). (B) Effect of BKCa channel blocker paxilline (1 μmol/L) on acidic pH-induced
dilation under normocapnic conditions. (C) Effect of RyR blocker ryanodine (10 μmol/L)
and BKCa channel blocker paxilline (1 μmol/L) on acidic pH-induced dilation under
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hypercapnic conditions. (D) Summary data showing vasodilator responses related to initial
level of myogenic tone (0%) and fully relaxed diameter (100%; 0 Ca2+, papaverine 100
μmol/L), with the number of experiments indicated in parentheses. *P < 0.05
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Figure 5.
Hypercapnic-, normocapnic- and NS11021-induced dilations are decreased in Slo-KO
mouse. (A) Typical recordings of the internal diameter of pressurized parenchymal arterioles
(40 mm Hg) from a wild type mouse (Kcnma1+/+, upper trace) or a mouse lacking the α
subunit of the BKCa channel (Kcnma1−/−, lower trace) during the perfusion of acidic aCSF
under hypercapnic or normocapnic conditions, or in presence of the BKCa channel agonist
NS11021, in the absence or presence of the BKCa channel blocker paxilline. (B) Summary
data showing vasodilator responses related to initial level of myogenic tone (0%) and fully
relaxed diameter (100%; 0 Ca2+, papaverine 100 μmol/L), with the number of experiments
indicated in parentheses. *P < 0.001

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Figure 6.
Acidic pH-induced dilation of PAs is not affected by glibenclamide. (A) Typical recording
of the internal diameter of a pressurized (40 mm Hg) parenchymal arteriole (PA) during the
perfusion of acidic aCSF under normocapnic conditions with and without the KATP blocker
glibenclamide (1 μmol/L). (B) Absence of effect of KATP channel agonist cromakalim on a
parenchymal arteriole. (C) Vasodilation of a pressurized (60 mm Hg) third order mesenteric
artery (Mesenteric A) induced by cumulative addition of Cromakalim (10−8 to 3.10−8 mol/
L). Glibenclamide (1 μmol/L) completely blocked the cromakalim-induced dilation of
mesenteric artery. (D) Cromakalim concentration response relationships in parenchymal
arterioles (n = 5) and mesenteric arteries (n = 5). Dilation is related to initial level of
myogenic tone (0%) and fully relaxed diameter (100%).
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Figure 7.
NOS-inhibition does not alter dilations induced by acidic pHo in normocapnic and
hypercapnic conditions. (A) Typical recording of the internal diameter of a pressurized
parenchymal arteriole (40 mm Hg) during the perfusion of acidic aCSF induced by reduced
bicarbonate concentration (normocapnia) with and without the eNOS inhibitor L-NAME
(100 μmol/L). (B) Summary data (n = 4). (C) Typical recording of the internal diameter of a
pressurized parenchymal arteriole (40 mm Hg) during the perfusion of acidic aCSF induced
by increased pCO2 (hypercapnia) with and without the eNOS inhibitor L-NAME (100 μmol/
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L). (D) Summary data (n = 6). Dilation is expressed as percent of maximum dilation (0
Ca2+, papaverine 100 μmol/L).

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Figure 8.
Proposed mechanism for RyRs- and BKCa-dependent, acidosis-induced dilation of brain
parenchymal arterioles. By decreasing the P0 of RyRs in VSMCs, protons reshape
intracellular Ca2+ signaling from waves to sparks which activate BKCa channels and then
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cause dilation.
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