(Stroke. 2000;31:2500.)
© 2000 American Heart Association, Inc.
Original Contributions |
Extracellular pH, Ca2+ Influx, and Response of Vascular Smooth Muscle Cells to 5-Hydroxytryptamine
From the Departments of Pediatrics (V.N., J.A-D., M.A.) and Cellular and Molecular Physiology (M.A.), Yale University School of Medicine, New Haven, Conn.
Correspondence to Michael Apkon, MD, PhD, Department of Pediatrics, Yale University, PO Box 208064, New Haven, CT 06520-8064. E-mail michael.apkon{at}yale.edu
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Background and Purpose—Cerebral vascular smooth muscle cells (VSMCs) contract on extracellular pH (pHo) increases and relax on pHo decreases. These changes in tone are believed to result from changes in [Ca2+]i, although the responsible mechanisms are not fully understood. VSMCs also contract in response to 5-hydroxytryptamine (5-HT), which increases [Ca2+]i via both Ca2+ release and influx. We hypothesized that examining effects of pHo decreases on 5-HT–induced [Ca2+]i changes would allow us to identify mechanisms whereby pHo influences tone. Accordingly, we compared [Ca2+]i increases in cerebral VSMCs, evoked by 5-HT, with increases evoked by increased pHo and examined 5-HT–dependent [Ca2+]i increases at normal and decreased pHo.
Methods—We monitored [Ca2+]i,, using the Ca2+-sensitive dye fura 2, in cultured rat cerebral VSMCs obtained by enzymatic digestion of middle cerebral arteries and their branches (passages 1 to 3) grown on glass coverslips and superfused with physiological saline.
Results—Increasing pHo from 7.3 to 7.8 increased [Ca2+]i, and these increases were prevented in Ca2+-free solutions. Decreasing pHo from 7.3 to 6.9 did not alter [Ca2+]i unless [Ca2+]i was first raised by treatment with 5-HT (10 µmol/L). 5-HT resulted in biphasic [Ca2+]i increases characterized by transient peaks blocked by the Ca2+-ATPase inhibitor thapsigargin (10 nmol/L) and prolonged plateaus blocked by the Ca2+ channel blocker Ni2+ (1 mmol/L). Acidification did not alter the transient peaks but significantly reduced 5-HT–induced Ca2+ influx.
Conclusions—We conclude that increasing pHo induces Ca2+ influx in rat cerebral VSMCs and decreasing pHo inhibits 5-HT–stimulated Ca2+ entry but not intracellular Ca2+ release.
Key Words: calcium • calcium channels • muscle, smooth • pH
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Introduction |
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Arterioles integrate a multitude of vasoactive inputs arising from neurohumoral signals, environmental cues such as pH, and mechanical factors such as flow and pressure. Each of these inputs may act directly on the vascular smooth muscle cell (VSMC) or may modulate the release of vasoactive substances from endothelial cells. Agents acting directly on the VSMC may alter tone by 3 mechanisms: altering intracellular Ca2+ concentrations ([Ca2+]i), altering the sensitivity of the contractile regulatory apparatus to [Ca2+]i, or modulating the sensitivity to other vasoactive inputs. Examining the mechanisms responsible for modulation of the response to one agent by another may yield important clues as to how each stimulus results in changes in vascular tone.
Systemic VSMCs contract on extracellular alkalinization and relax on extracellular acidification. Changes in vascular tone during extracellular pH (pHo) changes are believed to occur as a result of changes in [Ca2+]i, although the mechanisms responsible for the changes in [Ca2+]i are not fully understood. Systemic VSMCs also contract to a number of other neurohumoral stimuli in a Ca2+-dependent manner. For example, cerebral VSMCs contract on exposure to the vaso-constrictor serotonin (5-hydroxytryptamine [5-HT]), which evokes a biphasic increase in [Ca2+]i: an initial rapid but transient increase in [Ca2+]i followed by a more slowly decaying component that produces an apparent "plateau."1 2 3 4 Inasmuch as the [Ca2+]i increase caused by 5-HT is thought to reflect both Ca2+ release and Ca2+ influx, we hypothesized that examining the effect of pHo decreases on the response to 5-HT would allow us to determine which pathways are pH sensitive and might contribute to the decrease in VSMC tone during acidification.
To determine whether the Ca2+ release mechanisms or Ca2+ influx pathways stimulated by 5-HT were pHo sensitive, we measured [Ca2+]i in single, cultured VSMCs from rat middle cerebral arteries (MCAs) and their penetrating branches. We compared the increases in [Ca2+]i evoked by pHo increases with those evoked by 5-HT, and we examined the 5-HT–dependent [Ca2+]i increases at normal and decreased pHo.
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Materials and Methods |
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Preparation of Cerebral VSMC
Cerebral VSMCs were isolated from adult rats by enzymatic dissociation of the intact MCA and its branches according to a previously published protocol.5 Briefly, adult male Sprague-Dawley rats (weight, 250 to 300 g) were anesthetized with methoxyflurane (Metafane, PitmanMoore) and decapitated according to institutional guidelines. The skull was opened, the dura mater was stripped away, and the brain was transferred to an ice-cold Pucks solution (GIBCO BRL) and then to the refrigerated stage (4°C) of a dissecting microscope. A section of cortex underlying the MCA was isolated, and the overlying pial membranes were gently removed, preserving the penetrating arterioles arising from the primary branches of the MCA. The MCAs and their first- and second-degree pial branches were dissected free of connective tissue and incubated in an enzyme solution containing collagenase, elastase, and deoxyribonuclease. After incubation, tissue culture medium (M-199, GIBCO BRL) supplemented with 10% fetal calf serum was added to terminate enzymatic digestion, and the cells were dissociated by gentle trituration. The cells were then centrifuged, resuspended, and plated onto 35-mm plastic tissue culture dishes or onto glass coverslips. The tissue culture medium (M-199) was supplemented with 10% fetal calf serum, insulin (5 µg/mL), selenium (5 ng/mL; ITS Premix, Collaborative Biomedical Products), penicillin (50 U/mL), and streptomycin (50 µg/mL; Pen-Strep, GIBCO BRL). After growing to confluence, the cells on plastic were harvested with trypsin (0.25%) and EDTA (1 mmol/L) in Ca-free PBS and passed onto glass coverslips or plastic culture dishes. The cells were studied in primary culture or at passages 1 to 3. Cells isolated and cultured in this manner maintain a contractile phenotype.5
Measurement of Intracellular Ca2+
We measured
[Ca2+]i of cells grown on
glass coverslips using ratiometric video fluorescence
microscopy and the Ca2+-sensitive dye fura
2.6 The coverslips were transferred from plastic tissue
cultural dishes into the bottom of an experimental chamber and placed
against an oil-immersion objective on the inverted microscope. Cells
were loaded with the cell-permeant acetoxymethyl ester fura 2-AM
(Molecular Probes) at room temperature for 2 hours7 and
then superfused with physiological saline solution
for 30 minutes at 37°C to allow intracellular cleavage of the ester
group before experiments were started. The fura 2-AM stock (1
mmol/L) was combined with Pluronic (20% wt/vol in dimethyl sulfoxide)
before dilution in tissue culture media to a final concentration of
10 µmol/L fura 2, 0.025% Pluronic. Fura 2 was excited at 340
and 380 nm, with emission measured at 510 nm.
Our microscopy apparatus consisted of a Nikon Diaphot inverted microscope and a x40 oil-immersion objective. The excitation source consisted of a light from a xenon arc lamp passing through a high-speed filter wheel (Ludl Electronics). The fluorescent images were detected by a variable gain intensified charge-coupled device camera (ICCD-350F, VideoScope), digitized by a video frame grabber (Occulus F64DSP, Coreco), and stored on the hard disk of a personal computer. Filter changing, specimen illumination, camera gain, image capture, and processing were all controlled by custom software developed with the use of Optimas (Optimas Corp).
Our software system incorporated camera gain control to ensure that the detected image intensities remained in the center of the camera’s linear range. Identical gains were used for the 2 images of each pair (1 data point), which allowed reliable intensity ratios to be calculated over a wide range of specimen intensities. The software also incorporated frame averaging, image thresholding, and dark current subtraction. For each area of interest, the pixel intensities of the paired images were averaged, and pixels with mean intensity values below a specified threshold did not contribute to the calculated average ratio. The average pixel intensities were calculated for the remaining pixels for each image, and the ratio of the average intensities was used to represent the value for the area of interest at that time point. Areas of interest were delineated by hand at the start of the experiment. We identified areas of interest encompassing the perinuclear regions of each cell in the microscope field. Typically, between 1 and 3 cells were examined in each experiment, and the results from each experiment were averaged together to establish the response for that particular experiment.
Given the difficulties in measuring reliable in vivo calibrations of fura 2, we inferred changes in [Ca2+]i from changes in the ratio of emitted light intensity excited at each of the 2 wavelengths (I340/I380). We monitored I340/I380 at 60-second intervals during the 30-minute period of superfusion before starting the experiment and at 10-second intervals during the experiments. Percent changes in I340/I380 were used to compare the experimental results.
Measurement of Intracellular pH
Intracellular pH (pHi) was measured
fluorometrically by a dual-excitation (440 versus 490 nm),
single-emission (530 nm) ratiometric technique8 with the
pH-sensitive fluorophore
2,7-bis-carboxyethyl-5(6)-carboxyfluorescein (BCECF;
Molecular Probes Inc).9 Cells were loaded at 37°C for 30
minutes with BCECF (10 µmol/L), followed by a period of
superfusion for 30 minutes with bath solution heated to 37°C. In each
experiment, the fluorescence signals were calibrated using a
variation of the high-[K+]/nigericin
technique,10 in which we calibrated the dye at the single
pHi of 7.00.8 The ratio of light
intensity excited at 490 nm to that excited at 440 nm measured at each
data point is normalized to the ratio in each cell measured at
pHi 7.00. pHi is then
calculated with the use of calibration curves spanning a
pHi range from 6.5 to 7.8 generated in a
population of cells. This approach allows a single point calibration
and compensates for differences in the intensity of the excitation
light across the microscope field and from experiment to experiment.
Solutions
The composition of the standard HEPES-buffered bathing solution
was (in mmol/L) 145.8 Na, 3 K, 1 Ca, 1.2 Mg, 130 Cl, 1.2
SO4, 2 PO4, 32 HEPES, and
10.5 glucose. Ca2+-free solutions were identical
except that CaCl2 was omitted. The pH of all
solutions was measured and titrated to the required pH at 37°C with
an Orion pH meter (model 811, Orion Research Inc). Solutions were
delivered at 37°C by warming them in an in-line heater immediately
before entering the experimental chamber.
Ketanserin (ICN Biomedicals) solutions were prepared daily by serially diluting a freshly prepared 20 mmol/L stock solution (in water) in the bathing solution to the final concentrations indicated. Nickel chloride was added to the bathing solution to the final concentration indicated. Thapsigargin (Sigma Chemical) solutions were prepared as a 5 mmol/L stock solution in water and diluted to a final concentration of 10 nmol/L in bath solution on the day of the experiments. 5-HT (Sigma Chemical) solutions were also prepared fresh daily.
Statistical Analysis
All results are expressed as mean±SD. Student’s paired
t tests were used for statistical comparisons. The
Bonferroni adjustment was applied for multiple comparisons.
P<0.05 was considered statistically significant.
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Changes in pHo Cause Minimal Changes in pHi
In intact cerebral arterioles, changes in pHo caused much smaller changes in pHi (
0.1 pH unit change in
pHi per pH unit change in
pHo).11 To determine whether
cultured cerebral VSMCs exhibit similar properties, we measured
pHi in cultured VSMCs during alkalinization of
the bath solution from pH 7.3 to 7.8. pHi
increased from 7.14±0.06 to 7.19±0.06 (n=8), although the mean
pHi values measured at pHo
7.3 and 7.8 were not significantly different. The average change in
pHi was 0.05±0.006 pH units.
Changes in pHo Cause Changes in
[Ca2+]i
To examine the direct effects of pHo changes
on [Ca2+]i, we measured
[Ca2+]i during increases
and decreases in pHo. In each experiment we
compared the [Ca2+]i
increase of the VSMC during alkalinization with the
[Ca2+]i increase during
5-HT application. As illustrated in Figure 1
, increasing pHo
from pH 7.3 to 7.8 caused an increase in
I340/I380 that developed
slowly, was maintained, and reversed when pHo was
restored to 7.3. This cell also exhibited the typical response to 5-HT:
a rapid rise to a transient peak, followed by a more slowly decaying
component that produces an apparent plateau. Because the rates of this
slower decay varied among experiments, we measured plateau values at
specific times after the onset of 5-HT exposure. Figure 2 illustrates the mean (±SD) percent
change in I340/I380 for the
response to alkalinization as well as the peak and plateau (150 seconds
after 5-HT exposure).
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The alkalinization-induced increase in
[Ca2+]i required
extracellular Ca2+. As shown in Figure 3A, pHo raised in a
nominally Ca2+-free bathing solution caused no
increase in I340/I380. When
Ca2+ was restored to the bathing solution while
pHo remained elevated,
I340/I380 promptly began to
rise. The steady state percent change in
I340/I380 under each
condition is shown in Figure 3B, along with the 5-HT–induced
peak and plateau values.
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Whereas increases in pHo caused increases in [Ca2+]i, decreases in pHo infrequently caused decreases in [Ca2+]i. On average, the acidification-induced change in [Ca2+]i was no different than the small increase that was seen on average during sustained superfusion at constant pHo. We reasoned that the failure to observe a decrease in [Ca2+]i with acidification could be caused by the following: (1) acidification relying on a mechanism independent of [Ca2+]i reductions for relaxing VSMCs, (2) the acidification-inhibitable mechanism being already quiescent under resting conditions, or (3) basal [Ca2+]i being sufficiently low that it is not possible to reliably measure a decrease with fura 2. We next examined the effects of pHo decreases under conditions in which [Ca2+]i was already increased by application of 5-HT.
Responses to 5-HT
Activation of 5-HT2 Receptor Increases
[Ca2+]i
As described above, application of 5-HT (10 µmol/L) caused
a rapid transient increase in
[Ca2+]i followed by a
slower decay. The slower decay leads to an apparent plateau phase, with
[Ca2+]i remaining above
its initial value for the duration of 5-HT application. This is similar
to the temporal pattern reported for 5-HT2 12 and other
agonists7 13 in other VSMCs. Both the peak and plateau
responses to 5-HT were inhibited by the 5-HT2
receptor antagonist ketanserin14 at low
concentrations. We observed nearly complete inhibition of the 5-HT
response at 10 nmol/L and 40% inhibition at 1 nmol/L.
5-HT Activates Both Ca2+ Release and
Ca2+ Influx Pathways
To verify that the rapid transient phase of
[Ca2+]i increases during
5-HT application reflects Ca2+ release from
intracellular pools, we examined the effect of the
sarcoplasmic-endoplasmic reticulum Ca2+-ATPase
(SERCA) inhibitor thapsigargin on 5-HT–induced
[Ca2+]i transients.
Thapsigargin has been previously shown to effectively deplete the
norepinephrine and caffeine releasable
Ca2+ stores within isolated rat
VSMCs.15 In cerebral VSMCs exhibiting typical
5-HT–induced [Ca2+]i
responses before application of thapsigargin, application of the SERCA
inhibitor (10 nmol/L) caused a marked increase in
[Ca2+]i that slowly
decayed over a period of 20 to 30 minutes of exposure toward the values
measured before thapsigargin application (Figure 4A). After thapsigargin application, 5-HT
failed to activate the rapid and transient peak in the
[Ca2+]i waveform,
although slower increases in
[Ca2+]i were observed in
some cells. These increases were considerably smaller than those
observed during the plateaus of the responses before thapsigargin
exposure (Figure 4B). On average, 5-HT applied after exposure to
thapsigargin did not raise
I340/I380 above the values
measured immediately before the onset of that 5-HT application.
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To test whether the plateau phases of the 5-HT–induced
[Ca2+]i responses reflect
Ca2+ influx, we blocked
Ca2+ entry using the Ca2+
channel inhibitor Ni2+. When
Ni2+ (1 mmol/L) was added to the
5-HT–containing solution, the transient peak was still observed, but
the plateau phase was inhibited. This did not simply reflect
desensitization or tachyphylaxis to 5-HT because the effect was
reversible: a third application of 5-HT after washing out
Ni2+ from the bath resulted in a
[Ca2+]i increase similar
to that observed during the first application (Figure 5A). Comparing the responses to the
second 5-HT applications in the presence of Ni2+
with the response to 5-HT after washing out the
Ni2+, we found that Ni2+
significantly inhibited only the plateau response (Figure 5B).
Ni2+ alone had no effect on
I340/I380.
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pHo Decreases Inhibit 5-HT–Induced Ca2+
Influx But Not Ca2+ Release
We tested the effect of decreasing pHo on
[Ca2+]i after first
elevating [Ca2+]i with
5-HT. We found that decreasing pHo from 7.3 to
6.9 in the continued presence of 5-HT resulted in a reversible decrease
in [Ca2+]i during the
plateau phase of the 5-HT response (Figure 6A). The magnitude of the decrease can be
illustrated by comparing
[Ca2+]i after sustained
acidification with those values measured immediately before
acidification and on restoring pHo to 7.3 (Figure 6B). The I340/I380
measured at pHo 6.9 was significantly lower than
the values measured before acidification or after restoring the
pHo to 7.3. However, the slow decay in
[Ca2+]i during the
plateau complicates calculating the precise degree of inhibition. To
explore the effects of acidification on the different modes of
[Ca2+]i increase (ie,
Ca2+ release versus Ca2+
influx), we examined the effect of acidification on the overall
response to 5-HT. We first exposed cells to 5-HT while decreasing the
bath pHo to 6.9. After a sufficient period for
washout of 5-HT and restoration of pHo to 7.3, we
reexposed cells to 5-HT without altering pHo
(Figure 7A). We measured peak and plateau
(defined as 150 seconds after onset of exposure)
[Ca2+]i increases and
calculated the ratio of the values observed during the first exposure
to those observed during the second exposure (Figure 7B). We
found that acidification significantly inhibited the plateau increase
in [Ca2+]i with no effect
on the peak increase. This did not represent merely a
sensitization to the second 5-HT administration by prior exposure
because paired 5-HT exposures, each at pHo 7.3,
caused nearly identical peak and plateau
[Ca2+]i increases (Figure 7C).
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pHo Dependence of [Ca2+]i
Vascular tone may be altered via changes in [Ca2+]i or via changes in the sensitivity of the contractile/regulatory apparatus to [Ca2+]i. We have found that for cerebral VSMCs, increased pHo increases [Ca2+]i. This pHo dependence of [Ca2+]i is also observed in mesenteric arterial smooth muscle cells,16 and the concurrent increase in tone in those cells reflects the increase in [Ca2+]i rather than an increase in Ca2+ sensitivity. It is possible, however, that the 2 cell types may transduce the pHo changes through different mechanisms. In mesenteric smooth muscle, the increases in tone during pHo increases reportedly result from increases in pHi rather than from direct effects of the pHo increases.17 In cerebral VSMCs within intact arterioles, pHi changes relatively little when pHo is altered,11 and it is the pHo change rather than this pHi change that underlies the motor responses of cerebral arterioles and cultured cerebral VSMCs.5 11 The cultured VSMCs studied here behave similarly to those in arterioles in that they exhibit a nearly identical change in pHi with pHo changes (0.1 pH unit per 1 pH unit change in pHo).
Whereas pHo increases led to [Ca2+]i increases, pHo decreases, on average, failed to alter [Ca2+]i. This is similar to the results of Dietrich and coworkers,18 who found that pHo decreases from 7.3 to 6.8 caused dilation of penetrating arterioles without significant decreases in [Ca2+]i. We may have failed to detect significant decreases in [Ca2+]i for several reasons. First, it is possible that the resting [Ca2+]i is already sufficiently low that I340/I380 is near Rmin, the ratio observed in Ca2+-free solutions. We think this unlikely given that the I340/I380 ratios we measured in vivo were higher than the ratios observed with our apparatus in vitro for Ca2+-free solutions containing fura.
A second possibility is that, under resting conditions, the pH-responsive Ca2+ influx mechanisms are inactive. Hence, pHo decreases would only reduce [Ca2+]i if the cells were first activated by agents that activated these mechanisms. Indeed, when [Ca2+]i was first elevated by applying 5-HT, pHo decreases led to [Ca2+]i decreases. If the pHo-responsive Ca2+ influx process were inactive at rest, then one would expect that pHo decreases would not alter the resting tone in the cultured VSMCs. However, pHo decreases do relax cerebral VSMCs when they are grown on a flexible silicone substratum (dimethylpolysiloxane) on which they develop spontaneous tone.5 This apparent paradox may result from influences on the substratum on cell phenotype or may result from phenotypic variation among the cultured VSMCs. This latter possibility is intriguing when we consider that as many as 25% of the VSMCs grown on dimethylpolysiloxane do not undergo detectable relaxation on acidification, and decreases in [Ca2+]i were observed in some VSMCs grown on glass coverslips during pHo decreases. We have observed significant ultraviolet absorption of the dimethylpolysiloxane, which precludes simultaneous measurements of tone and [Ca2+]i in single cells with fura 2.
It is possible that the increase in I340/I380 during alkalinization reflects alterations in the binding affinity of the fura 2 for Ca2+ as a result of changes in pHi during the pHo change.19 This is unlikely because fura 2 is relatively insensitive to pH near the resting pH (approximately pH 7.15), and the increase in pHi measured during a pHo increase from pH 7.3 to 7.8 is only 0.05 pH units.
Mechanisms Underlying Alkalinization-Induced
[Ca2+]i Increases
[Ca2+]i
represents a balance point at which release from intracellular
stores and influx from the extracellular space balance reuptake into
the stores and extrusion from the cytoplasm, and each process is served
by multiple mechanisms. Alkalinization-induced
Ca2+ increases require extracellular
Ca2+. This suggests that
pHo modulates the Ca2+
influx process rather than Ca2+ release from
intracellular stores. This is corroborated by the effects of
pHo on 5-HT–induced
[Ca2+]i increases. We
cannot distinguish from these experiments whether
pHo affects Ca2+ influx
directly via activating voltage-activated
Ca2+ channels or indirectly by depolarizing the
VSMC or stimulating some other Ca2+ influx
process.
Mechanisms Underlying 5-HT–Induced
[Ca2+]i Increases
We chose to examine the response to 5-HT because this amine causes
a biphasic increase in Ca2+ and because we
believed it possible to separate 2 distinct processes leading to these
Ca2+ increases.20 We applied
relatively high doses of 5-HT to activate maximally the
receptor-mediated transduction pathways. Our intention was to enable
the dissection of the pathways leading to
[Ca2+]i increases and to
allow us to examine the effects of extracellular acidification on these
pathways. We found that, in cerebral VSMCs, 5-HT induced a biphasic
increase in [Ca2+]i that
was blocked by the 5-HT2A antagonist
ketanserin. Inhibiting Ca2+ channels with the
nonselective antagonist Ni2+eliminates the plateau phase without altering the transient
peak. Moreover, discharging the intracellular
Ca2+ stores with thapsigargin, an endoplasmic
reticulum Ca2+ pump inhibitor,
eliminates the transient peak in
[Ca2+]i. The failure to
observe the peak responses after thapsigargin are not due to the fact
that [Ca2+]i is elevated
to a maximal level because thapsigargin does not cause elevations in
[Ca2+]i itself as great
as the peak responses to 5-HT observed before thapsigargin exposure. It
is difficult to quantitatively compare the 5-HT–dependent increases in
[Ca2+]i before and after
thapsigargin treatment because
[Ca2+]i levels often
decay slowly after the thapsigargin is washed out of the bath solution.
We conclude that 5-HT activates Ca2+
release followed by an increase in Ca2+
permeability of the cell.
Our differentiation of 2 modes for [Ca2+]i increases is consistent with the findings of other investigators. Transient 5-HT–induced, ketanserin-sensitive [Ca2+]i increases were observed in cultured rat cerebral VSMCs by Wang et al.2 These investigators also concluded that the transient increase resulted from Ca2+ release because Ca2+ channel antagonists such as Co2+, La3+, or nifedipine did not inhibit it. Interestingly, the [Ca2+]i increase observed by these investigators lasted only approximately 1 minute, and there was no sustained increase in [Ca2+]i. Ca2+ release has also been observed on 5-HT2 receptor activation in rat aortic VSMCs and VSMC cell lines,1 21 and Ca2+ release likely reflects generation of inositol(1,4,5)-trisphosphate.21
The later, sustained [Ca2+]i increase results from Ca2+ influx that, in cerebral VSMCs, is inhibited by Ni2+. The influx pathway is not known but could be either via store-operated Ca2+ channels22 or voltage-operated Ca2+ channels. In rat aortic VSMCs, 5-HT2 receptor activation potentiates L-type Ca2+ currents via protein kinase C activation.23 5-HT also increases the open probability of the L-type Ca2+ current in rabbit cerebral arterioles.24 This is consistent with the finding that the Ca2+ channel antagonist verapamil reduces 5-HT–induced contraction of rat aortic strips and tail arteries.25 Others have found inconsistent antagonism of 5-HT2–induced plateau [Ca2+]i increases with L-type Ca2+ channel antagonists and have suggested that 2 of these antagonists, verapamil and D600, interfere with binding of 5-HT to its receptor.3 26 Taken together, it seems likely that both store-operated and voltage-operated Ca2+ channels contribute to the plateau phase of the 5-HT2 response.
Acidification reduces the plateau Ca2+ increase but not the transient peak. The specificity of pHo decreases for inhibiting the plateau, as opposed to the peak, suggests that the pHo decrease does not interfere with 5-HT binding to its receptor or the generation of the intracellular second messengers leading to Ca2+ release. Rather, acidification specifically inhibits the Ca2+ influx process. This may reflect decreased L-type channel activity at low pHo.27 28 29 It is also possible that low pHo decreases Ca2+ influx through voltage-operated channels indirectly via change in membrane potential resulting from activation of K+ conductances.30 The [Ca2+]i decrease might also result from direct inhibition of the store-operated Ca2+ entry pathways, as observed in A7r5 rat aortic smooth muscle cells.31 The decrease in Ca2+ influx at acid pH may well underlie the decrease in maximal 5-HT–mediated contractions at acid pH in rabbit basilar artery.32
It is important to consider whether the responses to pHo or 5-HT are general properties of cerebral arterioles or of all systemic VSMC. The cerebral VSMCs studied here derive from arterioles of varying size, from MCA to penetrating arterioles as small as 70 µm in diameter. These cells have a contractile response to 5-HT.5 Although the responses reported here were homogeneous, the response to locally applied 5-HT on pial vessels has been shown to vary with vessel size,33 with contraction of large vessels and dilation of small vessels. It is certainly possible that 5-HT causes different responses in vessels of different sizes or origins. This may relate to differences in the phenotypes of the vascular smooth muscle within the walls of the vessels or to differences in effects on smooth muscle compared with endothelium in different vessels. The importance of our results, however, is that pHo modulates the Ca2+ influx pathway activated by 5-HT. This is particularly important since this Ca2+ influx pathway is likely activated by multiple vasoconstrictor substances. Moreover, this mechanism of [Ca2+]i regulation by pH is distinct from effects of pHi on the intracellular release process.34
pHo modulates vascular tone by altering Ca2+ influx, and the ability of pHo to modulate [Ca2+]i depends on the resting activity of the various Ca2+ influx processes. Moreover, pHo may modulate entry through store-operated Ca2+ channels as well as through voltage-operated Ca2+ channels. This underlies the importance of considering interaction between transduction processes when examining the influence of pHo on vasomotor control.
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Acknowledgments |
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This study was supported by grants from the Charles H. Hood Foundation, the Connecticut chapter of the American Heart Association, and National Institutes of Health grant P30HD27757 to Joseph B. Warshaw. Dr Apkon was an established investigator of the Society for Critical Care Medicine.
Received May 31, 2000; revision received July 26, 2000; accepted July 26, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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Editorial Comment
Department of Neurosurgery Washington University School of Medicine St Louis, Missouri
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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The strong sensitivity of cerebral vascular tone to changes in extracellular pH is one of the features that differentiates cerebrovascular blood flow regulation from other vascular networks. As such, understanding the cellular mechanisms by which metabolic need of the cerebral tissue, as reflected by tissue pH, causes alkalotic smooth muscle constriction or acidotic dilation is an important issue. To this end, Nazarov et al observed the intracellular calcium activity in cultured cerebral smooth muscle cells in response to extracellular pH changes. They find that extracellular alkalization causes external calcium influx, possibly via calcium channel activation, which would result in vessel constriction. Extracellular acidification, however, had no appreciable effect on the calcium activity. In addition, agonist (serotonin)-induced intracellular calcium release is reduced by acidosis. Physiologically, this would attenuate an agonist-induced vasoconstriction and may be a safeguard mechanism to allow for higher blood flow in situation of local metabolic need.
Several questions regarding the mechanism of pH-induced vasomotor responses still remain. A previous study of this groupR1 showed that cerebral smooth muscle cells in culture relax to acidic pH. Since the present study found no decrease in calcium activity, another calcium-independent mechanism needs to be postulated to explain the acidic relaxation. Further, the nature of the observed alkalotic calcium channel activation is still unexplained. As stated by the authors, this study does not discern whether alkalosis directly opens calcium channels and, if so, which calcium channel type may be involved. Alternatively, because increased pH depolarizes cerebral macrovessels and microvessels,R2 R3 alkalosis-induced calcium influx could be secondary to membrane depolarization. As such, the primary effect of increased extracellular pH on the smooth muscle needs to be elucidated further.
Received May 31, 2000; revision received July 26, 2000; accepted July 26, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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1. Apkon M, Weed RA, Boron WF. Motor responses of cultured rat cerebral vascular smooth muscle cells to intra- and extracellular pH changes. Am J Physiol. 1997;273(Heart Circ Physiol):H434–H445.
2. Smeda JS, Lombard JH, Madden JA, Harder DR. The effect of alkaline pH and transmural pressure on arterial constriction and membrane potential of hypertensive cerebral arteries. Pflugers Arch.. 1987;408:239–242.[Medline] [Order article via Infotrieve]
3. Dietrich HH, Dacey RG Jr. Effects of extravascular acidification and extravascular alkalinization on constriction and depolarization in rat cerebral arterioles in vitro. J Neurosurg.. 1994;81:437–442.[Medline] [Order article via Infotrieve]
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