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Department of Pharmacology, College of Medicine, The University of Vermont, Burlington, Vermont 05405
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The role of Ca2+ mobilization from
intracellular stores and Ca2+-activated
Cl
channels in caffeine- and histamine-induced
depolarization and contraction of the rabbit middle cerebral artery has
been studied by recording membrane potential and isometric force.
Caffeine induced a transient contraction and a transient followed by
sustained depolarization. The transient depolarization was abolished by ryanodine, DIDS, and niflumic acid, suggesting involvement of Ca2+-activated Cl channels.
Histamine-evoked transient contraction in Ca2+-free
solution was abolished by ryanodine or by caffeine-induced depletion of
Ca2+ stores. Ryanodine slowed the development of
depolarization induced by histamine in Ca2+-containing
solution but did not affect its magnitude. In arteries treated with 1 mM Co2+, histamine elicited a transient depolarization and
contraction, which was abolished by ryanodine. DIDS and niflumic acid
reduced histamine-evoked depolarization and contraction. Histamine
caused a sustained depolarization and contraction in
low-Cl solution. These results suggest that
Ca2+ mobilization from ryanodine-sensitive stores is
involved in histamine-induced initial, but not sustained,
depolarization and contraction. Ca2+-activated
Cl channels contribute mainly to histamine-induced
initial depolarization and less importantly to sustained
depolarization, which is most likely dependent on activation of
nonselective cation channels.
intracellular calcium stores; ryanodine; calcium-activated chloride channels; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; niflumic acid; nonselective cation channels
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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MOBILIZATION of Ca2+ from intracellular stores, mainly sarcoplasmic reticulum, plays an important role in excitation-contraction coupling in vascular smooth muscle cells (SMCs) (1, 5, 16a, 20, 23, 27, 29, 40-42). There is increasing evidence that Ca2+ ions released from internal stores not only initiate smooth muscle contraction but also modulate the ion conductances of the plasma membrane. The latter modifies the membrane potential of SMCs, thereby regulating the influx of Ca2+ through voltage-dependent Ca2+ channels (5, 16a, 20, 23, 27, 29). Depletion of the intracellular Ca2+ pool can also stimulate Ca2+ entry into SMCs through specific store-operated Ca2+ channels (6, 7, 34).
The importance of Ca2+ mobilization in the regulation of
cell excitability, membrane potential, and Ca2+ influx
substantially varies among different types of smooth muscle. The
spontaneous or vasoconstrictor-stimulated Ca2+ release from
sarcoplasmic reticulum can result in stimulation of inward currents,
outward currents, or both, depending on the level of membrane potential
and type of channels being activated. Three classes of channels,
Ca2+-activated K+ and Cl
channels and nonselective cation channels, are known to be regulated by
cytosolic Ca2+ and, therefore, might underlie the membrane
depolarization or hyperpolarization induced by
Ca2+-mobilizing vasoconstrictors (5, 9, 15-18, 20, 22,
24, 27, 29, 41).
A number of vasoconstrictors and also caffeine can induce contraction
of cerebral arteries in Ca2+-free media, demonstrating
functional importance of Ca2+ mobilization from the
sarcoplasmic reticulum in the initiation of contraction (1, 26, 37).
The role of Ca2+ released from internal stores in the
modulation of cerebrovascular membrane potential is less understood.
The spontaneous release of Ca2+ from intracellular stores
(Ca2+ sparks) has resulted in hyperpolarization of rat
cerebrovascular SMCs as a result of the opening of
Ca2+-activated K+ channels (29). Outward
K+ currents induced by photorelease of the caged
Ca2+ have been described in rat myocytes (36). However,
Ca2+-activated inward currents have not been found in these
studies (29, 36). In contrast, in rabbit cerebrovascular myocytes, inward (most likely Cl) and outward K+
currents in response to histamine- or caffeine-induced Ca2+
mobilization from intracellular stores have been described (16, 16a).
In our previous study (8) we found that histamine induced a transient
contraction of the rabbit middle cerebral artery (MCA) when the influx
of Ca2+ through voltage-dependent Ca2+ channels
was prevented by nifedipine or Co2+. In arteries treated
with Co2+, the transient contraction was associated with a
transient depolarization, suggesting the role of Ca2+
released from intracellular stores in the initiation of contraction and
depolarization. The present study was undertaken 1) to examine the contribution of Ca2+ mobilized from internal stores in
histamine-induced depolarization and contraction and 2) to
evaluate the functional role of Ca2+-activated
Cl channels in this response. Inasmuch as caffeine
is frequently used to release Ca2+ from sarcoplasmic
reticulum, we compared contractile and electrophysiological responses
caused by caffeine and histamine.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Adult male New Zealand White rabbits (2-3 kg) were anesthetized with pentobarbital sodium (39 mg/kg), heparinized (1,000 U/kg), and killed by rapid exsanguination. The brain was removed and placed in physiological salt solution (PSS) at room temperature. Ring arterial segments were prepared from the proximal part of the MCA and mounted in a resistance artery myograph for simultaneous recordings of membrane potential and isometric contractile force. After an equilibration period of 15-20 min and heating to 37°C, each arterial segment was stretched to a resting tension of 100 mg. One hour later the arterial segment was constricted by the combination of histamine (3 µM) and serotonin (1 µM). ACh (3 µM) was then added to the superfusate to verify the functional integrity of vascular endothelium. The dissection procedure, recording of isometric force, and electrophysiological method have been described in detail in the companion article (8).
For measurement of membrane potential, we used glass microelectrodes
that were filled with 0.5 M KCl and had tip resistances of 110-150
M
. An Ag-AgCl pellet was used as an indifferent electrode. For
recording of membrane potential in low-Cl solution,
an Ag-AgCl electrode was connected to the bath solution through an agar
bridge. Microelectrode intracellular impalements of SMCs were made from
the adventitial surface of arterial segments. The changes in the
membrane potential and isometric force were displayed and recorded on a
desktop computer with use of the Axotape 2.0 (Axon Instruments) data
acquisition program and on a chart recorder.
In experiments with Ca2+-free solution, histamine or caffeine was applied 10-15 min after removal of Ca2+ from the bathing solution. In a separate set of experiments, when arterial segments were treated with ryanodine, responses to histamine were tested after a brief (2-min) exposure of arteries to caffeine to accelerate Ca2+ depletion from internal stores.
Solutions and drugs.
We used PSS of the following composition (in mM): 130.0 NaCl, 4.7 KCl,
1.18 KH2PO4, 1.17 MgSO4, 14.9 NaHCO3, 0.026 EDTA, 11.1 glucose, and 1.6 CaCl2
(pH 7.4). To prepare K+-rich solutions, equimolar amounts
of NaCl were replaced with KCl. Ca2+-free PSS contained no
Ca2+ and 0.5 mM EGTA. Low-Cl solution
was made by replacing 130 mM NaCl in the PSS with 130 mM sodium
glutamate. Superfusion solutions were equilibrated with 95%
O2-5% CO2. To study the effects of
Co2+, we used HEPES-PSS solution of the following
composition (in mM): 135.0 NaCl, 5.6 KCl, 1.0 MgCl2, 10.0 HEPES, 10.0 glucose, and 1.8 CaCl2; pH was adjusted to 7.4 with 10.0 N NaOH. All solutions contained cimetidine (3 µM), an
inhibitor of H2-histamine receptors.
Data analysis and statistics. The data recorded were imported as ASCII files into SigmaPlot for graphical representation and statistical analysis. Values are means ± SE of n arterial segments. A Student's t-test was used to determine the significance of differences between sets of data. Differences were considered significant when P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Effects of histamine and caffeine in
Ca2+-free solution.
In Ca2+-containing PSS, histamine in submaximal
concentration (3 µM) induced a rapidly developing contraction of
rabbit MCA. After reaching maximum, this contraction declined to a
slightly lower steady-state level (95.4 ± 2.4% of maximum) that was
usually maintained throughout the histamine application (10-30
min; Fig. 1A). In
Ca2+-free PSS, histamine produced a transient contraction
(41.3 ± 2.8% of control) followed by a very small sustained
contraction (6.1 ± 0.7% of control, n = 20; Fig. 1,
A and B).
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Effects of ryanodine on caffeine- and histamine-induced depolarization and contraction. Because of very marked depolarization of SMCs induced by Ca2+ withdrawal from the bathing solution, it was difficult to compare the depolarizing responses to histamine in regular PSS with those in Ca2+-free solution. Our data indicate that histamine and caffeine contract rabbit MCA by releasing Ca2+ from the same intracellular pool. In smooth muscle, caffeine mobilizes Ca2+ by activating the ryanodine receptors located in the membrane of sarcoplasmic reticulum (20, 35). Therefore, ryanodine can be used as a tool in studying the role of Ca2+ mobilized from internal stores in the histamine-induced responses of rabbit MCA. In our next experiments we first characterized the caffeine-induced changes in membrane potential and contractile force and then studied the effects of caffeine and histamine before and after functional removal of intracellular stores with ryanodine.
Figure 2 demonstrates a typical effect of caffeine on membrane potential and force in Ca2+-containing PSS. Caffeine (10 mM) caused a transient contraction (18.6 ± 1.8% of maximal K+-induced response) and membrane depolarization consisting of transient and sustained components. The onset of transient depolarization of 10.6 ± 0.8 mV (n = 28) coincided in time with the onset of contraction. The transient depolarization was followed by a slowly developing sustained depolarization of 6.9 ± 0.9 mV (n = 28), which was maintained until the washout of caffeine. When resting membrane potential of SMCs in the arterial wall was less negative than
60 mV, caffeine
application resulted in more complex changes in membrane potential
consisting of a transient initial hyperpolarization of 3.6 ± 0.7 mV
followed by transient and then sustained depolarization (see Fig.
5A).
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67.6 ± 0.9 mV before and 67.8 ± 1.0 mV 20-30 min after
application of ryanodine (n = 13). In the presence of
ryanodine, the first application of caffeine resulted in depolarization
and contraction of variable amplitude usually smaller than in control.
The second caffeine application failed to evoke any significant
transient depolarization (0.5 ± 0.3 mV, n = 4) and
contraction and resulted in a slowly developing and weak sustained
depolarization of 3.0 ± 1.0 mV (n = 4; Fig. 2A). The
effect of ryanodine on caffeine-induced changes in membrane potential
and force was not different in arteries with and without endothelium
(Fig. 2, B and C). These experiments demonstrate that
caffeine-induced transient depolarization and contraction are due to
Ca2+ release from ryanodine-sensitive intracellular stores
of SMCs.
If caffeine and histamine release Ca2+ from the same
intracellular store, ryanodine should effectively inhibit the
histamine-induced contraction in Ca2+-free solution.
Indeed, histamine-evoked contraction in Ca2+-free solution
was reduced by 10 µM ryanodine from 41.3 ± 2.8 to 6.1 ± 0.7%
(n = 9) of maximal response in Ca2+-containing PSS
(n = 9; data not shown). Similar results were obtained from
endothelium-denuded arteries (n = 3; data not shown). Thus
histamine contracts the rabbit MCA in Ca2+-free solution as
a result of Ca2+ mobilization from ryanodine-sensitive
intracellular stores of SMCs.
To evaluate the physiological significance of Ca2+ release
from intracellular stores in the overall histamine-induced electrical and contractile responses, we studied the effects of histamine before
and after treatment of arteries with ryanodine (10 µM). In
Ca2+-containing PSS, histamine induced sustained
depolarization and contraction (Fig.
3A). In ryanodine-treated artery,
exposure to histamine initially resulted in a slowly developing
depolarization but without contraction. The generation of action
potentials (APs) on reaching threshold depolarization was followed by
acceleration in the development of the depolarization and initiation of
the contraction (Fig. 3B). The histamine-induced depolarization
(32.2 ± 1.0 mV, n = 12) was sustained and was not different
from the control values (31.9 ± 0.8 mV). Time to reach half-maximal
depolarization was longer in ryanodine-treated arteries (238 ± 38 s,
n = 10, P < 0.05) than in control (74 ± 13 s, n = 10). In the presence of ryanodine, histamine-induced
contraction was significantly reduced to 89.5 ± 6.7% of control
(n = 12).
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70 mV. When SMCs were depolarized by
BaCl2 (30-50 µM) from 72.3 ± 0.6 to
64.0 ± 1.1 mV, a subsequent application of histamine resulted
in a strong additional depolarization to 37.8 ± 1.0 mV and
development of contraction. These findings indicate that the level of
resting membrane potential is an important determinant of
histamine-induced sustained depolarization of rabbit MCA.
Our experiments with ryanodine demonstrate that Ca2+
mobilization from intracellular stores is not obligatory for
development of histamine-induced sustained depolarization of rabbit MCA
but might contribute to the early initial component of the
depolarization. In our previous study (8), we reported that histamine
induced a transient depolarization and contraction in arteries treated with 1 mM Co2+. This concentration of Co2+ is
effective for inhibition of nonselective cation channels and voltage-dependent Ca2+ channels. A similar pattern of the
response was observed in Ca2+-free
Co2+-containing PSS. This suggests that the
histamine-induced transient depolarization is due to Ca2+
mobilization from intracellular stores. To verify this hypothesis, we
studied the effect of histamine in arteries treated with 1 mM
Co2+ and 10 µM ryanodine (Fig.
4). Under this condition, histamine depolarized SMCs by only 4.3 ± 0.9 mV and induced no contraction (n = 6). In the continuous presence of ryanodine and histamine, removal of Co2+ from the bath solution was followed by a
slowly developing depolarization, generation of APs, and development of
contraction (Fig. 4, A and B). The effect of ryanodine
on histamine-induced depolarization in Co2+-treated
arteries is summarized in Fig. 4C.
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Effect of Cl channel inhibitors on caffeine- and
histamine-induced depolarization and contraction.
The above data demonstrate a contribution of Ca2+ release
from the ryanodine-sensitive pool to the depolarization induced by histamine and caffeine in rabbit MCA. One of the possible mechanisms of
this depolarization might be an activation of
Ca2+-dependent Cl channels (5, 20, 23).
To evaluate this possibility, we studied the effect of
Cl channel inhibitors, DIDS and niflumic acid, on
caffeine- and histamine-induced responses. Both inhibitors were applied
for 15-30 min before testing of caffeine or histamine.
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channels
(16a, 19, 23, 41). Niflumic acid (100 µM) caused hyperpolarization of
5.0 ± 0.7 mV (n = 11). Similar to DIDS, niflumic acid
substantially decreased the transient component of caffeine-induced
depolarization from 12.1 ± 2.1 to 3.6 ± 0.7 mV (n = 4) with
no significant changes in the sustained component of the depolarization
(9.3 ± 0.3 mV in control vs. 7.7 ± 0.7 mV in the presence of
niflumic acid, n = 4). Caffeine-induced contraction was almost
unchanged (94.8 ± 8.5% of control). Thus DIDS and niflumic acid
attenuated the transient component of caffeine-induced depolarization,
suggesting involvement of Ca2+-dependent
Cl channels.
The role of Ca2+-activated Cl channels
in histamine-induced depolarization was then studied in arteries
treated with niflumic acid or DIDS in concentrations abolishing the
caffeine-induced transient depolarization (100 and 200 µM,
respectively). In arteries treated with niflumic acid, histamine caused
sustained depolarization from 60.1 ± 1.4 to 44.2 ± 0.8 mV (n = 6), which developed more slowly than in control
(Fig. 6A). Initial (18.5 ± 2.0 mV, n = 7) and sustained (14.5 ± 1.7 mV, n = 6)
depolarizations were significantly reduced compared with those in
untreated arteries (31.5 ± 1.3 and 30.5 ± 1.3 mV, respectively,
n = 13). We also observed a potent inhibition of the sustained
contraction (22.1 ± 7.4% of maximal contraction in control,
n = 7) with less effect on the initial contraction (75.8 ± 8.1%, n = 4). Washout of niflumic acid in the presence of
histamine was followed by additional depolarization and restoration of
the contraction (data not shown).
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62.0 ± 2.7 to 37.8 mV. Initial and sustained
depolarizations were significantly smaller (23.4 ± 3.2 and 24.2 ± 3.2 mV, respectively, n = 5) than in untreated arteries (Fig.
7A). Maximal histamine-induced contraction was not different from control, but the sustained component
was reduced by DIDS to 72.6 ± 10.3% of control (Fig. 7B).
Thus both Cl channel inhibitors decreased
histamine-induced depolarization and contraction, niflumic acid being
more potent than DIDS.
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Effect of histamine in low-Cl solution.
Our experiments demonstrate that histamine can substantially (by
15-20 mV) depolarize SMCs in the presence of DIDS or niflumic acid. This indicates that mechanisms other than activation of Ca2+-dependent Cl channels are
responsible for the major part of histamine-induced sustained
depolarization. To confirm this idea, we studied the effects of
histamine in low-Cl solution. Arteries were
superfused with low-Cl solution for 15-30 min.
This resulted in a slowly developing depolarization from 60.6 ± 3.2 to 46.4 ± 0.9 mV (n = 4; Fig. 8). Exposure to histamine caused an
additional sustained depolarization to the same level (32.3 ± 1.3 mV) as in regular PSS (34.5 ± 1.1 mV). Initial and
sustained components of histamine-induced contraction were not
different from those in regular PSS (Fig. 8, A and C).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The main finding of the present study is that, in the rabbit MCA,
transient mobilization of Ca2+ from the ryanodine-sensitive
intracellular pool contributes to caffeine- and histamine-induced
depolarization of SMCs through stimulation of
Ca2+-activated Cl channels.
Histamine and caffeine release Ca2+ from ryanodine-sensitive internal stores. Intracellular Ca2+ stores importantly contribute to physiological responses of smooth muscle induced by vasoconstrictors (1, 5, 20, 21, 23, 27). Inositol trisphosphate (IP3)- and ryanodine-sensitive Ca2+-release channels have been described in SMCs, and they can be partially or completely codistributed in the same intracellular pool or localized in two different pools (20, 35, 40). Activation of the H1-histamine receptor in vascular SMCs results in inositol phospholipid breakdown with subsequent formation of diacylglycerol and IP3, the latter causing Ca2+ mobilization from the sarcoplasmic reticulum (12). In our experiments with Ca2+-free PSS, ryanodine abolished the contractile response to histamine, and caffeine failed to cause any significant contraction after application of histamine. These results indicate that, in SMCs of the rabbit MCA, histamine releases Ca2+ from ryanodine-sensitive internal stores. Our findings correlate well with a recent observation on the same tissue that ryanodine also abolished the transient increase in cytoplasmic Ca2+ induced by histamine in Ca2+-free solution (37).
The simplest interpretation of these data is that, in cerebral arteries, IP3 and ryanodine receptors are structurally coupled to the same intracellular Ca2+ pool. On the basis of the results of sequential application of caffeine and excitatory agonists, an overlapping of ryanodine and IP3-sensitive intracellular pools has been suggested in a number of vascular SMCs (18, 20, 23, 35, 41). However, the possibility cannot be ruled out that IP3 releases only a small amount of Ca2+ from a separate IP3-sensitive pool, which in turn causes a massive Ca2+ mobilization from the ryanodine-sensitive pool through a Ca2+-induced Ca2+-release mechanism (35). In any case, ryanodine-sensitive internal stores are a major source of Ca2+ mobilized by histamine in rabbit MCA.Ca2+-activated
Cl channels contribute to caffeine-induced
depolarization of rabbit MCA.
In our experiments, application of caffeine initially resulted in a
transient contraction and depolarization. Two features of the
caffeine-induced response, coincidence in the onset of initial
depolarization and contraction and their similar transient time course,
suggest that both events are closely related. Depletion of internal
Ca2+ stores with ryanodine completely abolished
caffeine-induced contraction and transient depolarization in rabbit MCA
(Fig. 2). It follows that Ca2+ released from the
ryanodine-sensitive intracellular pool is functionally involved in the
modulation of the ionic permeability of cerebrovascular smooth muscle.
channels by Ca2+ ions
mobilized from internal stores has been described in a number of
vascular tissues (5, 9, 16-18, 20, 23, 27, 36, 41) and can
potentially contribute to the caffeine-induced depolarization in rabbit
MCA. DIDS and niflumic acid, relatively potent inhibitors of
Ca2+-activated Cl channels, have been
widely used to explore the functional role of these channels in
electrophysiological responses of smooth muscle evoked by caffeine and
excitatory agonists (2, 16a, 19, 23). In our experiments, DIDS and
niflumic acid effectively abolished the caffeine-induced transient
depolarization. We have also found that caffeine-induced transient
depolarization (9.8 ± 0.8 mV, n = 3) was unaffected by 200 µM Co2+ (9.3 ± 0.5 mV, n = 3), indicating that
nonselective cation channels are not involved (unpublished
observation). These findings support the hypothesis that
Ca2+-activated Cl channels are the major
contributors to caffeine-induced transient depolarization in rabbit MCA.
It has been demonstrated that single SMCs from the rat posterior
cerebral artery show spontaneous outward (K+), but not
inward, currents (29). Photorelease of caged Ca2+ in single
myocytes from rat basilar artery evoked no inward currents, indicating
an absence of Ca2+-activated Cl channels
in this tissue (36). On the other hand, Ca2+-activated
Cl channels in response to Ca2+
mobilization by caffeine or histamine have been demonstrated in the
rabbit basilar artery (16, 16a). These findings suggest that expression
of Ca2+-activated Cl channels and their
role in the modulation of membrane potential of cerebrovascular
myocytes may be species dependent.
Role of Ca2+-activated
Cl channels in histamine-induced depolarization.
Because histamine can release Ca2+ from intracellular
stores, we suggest that, similar to caffeine, this Ca2+
mobilization might contribute to histamine-induced depolarization. In
arteries treated with 1 mM Co2+ to prevent Ca2+
influx through voltage-dependent Ca2+ channels and
nonselective cation channels, histamine induced a transient contraction
accompanied by a transient depolarization (8). The transient
depolarization and contraction were abolished by combined treatment
with 1 mM Co2+ and ryanodine. These experiments provide
direct evidence for a role of Ca2+ mobilization from
internal stores in histamine-induced depolarization. In our
experiments, after functional depletion of internal stores with
ryanodine, histamine-induced depolarization developed more slowly than
in control, although the level of sustained depolarization achieved was
the same (Fig. 3). This suggests that Ca2+ mobilization
contributes mainly to the initial component of histamine-induced depolarization.
solution or by niflumic acid, implicating a role of
Ca2+-activated Cl channels. It has been
demonstrated that Ca2+-activated Cl
channels contribute to histamine-induced inward current in vascular SMCs from rabbit pulmonary artery (41). Using relatively specific inhibitors of these channels, we evaluated the functional significance of Ca2+-activated Cl channels in
histamine-induced depolarization. The treatment of arteries with DIDS
and niflumic acid slowed the development of histamine-induced
depolarization and significantly diminished the initial and, to a
lesser extent, the sustained component of this depolarization. These
findings favor the contribution of Ca2+-activated
Cl channels in histamine-induced depolarization.
Niflumic acid was a more effective inhibitor of the histamine-induced
depolarization than DIDS. It may be that part of the inhibitory effect
of niflumic acid was related to the opening of
Ca2+-activated K+ channels (32, 39). The fact
that in our study niflumic acid can cause a hyperpolarization
strengthens this hypothesis. However, this issue remains unclear, since
in myocytes from the basilar artery, niflumic acid at the same
concentration produced no effect on Ca2+-activated
K+ current induced by application of histamine (16a) and,
in another study, did not dilate pressurized small cerebral arteries,
suggesting no effect on Ca2+-activated K+
channels (30).
Evidence for the role of nonselective cation channels in
histamine-induced sustained depolarization.
In the presence of Cl channel inhibitors or in
low-Cl solution, histamine still produced a strong
(15- to 20-mV) sustained depolarization. These findings clearly
indicate that a mechanism other than an activation of
Ca2+-sensitive Cl channels is involved.
Taking into consideration that the sustained depolarization caused by
histamine can be inhibited by Co2+ and by reduction in
extracellular Na+, we suggest that nonselective cation
channels play an important role. To our knowledge, this is the first
(although indirect) evidence for the potential role of nonselective
cation channels in histamine-induced depolarization of cerebrovascular
SMCs. Additional support for this hypothesis came from our experiment
demonstrating that, in arteries with a resting membrane potential more
negative than 70 mV, histamine failed to cause any
depolarization or contraction after functional removal of intracellular
stores with ryanodine (Fig. 3C). A slight depolarization of the
membrane with BaCl2 to 65 mV resulted in the
restoration of histamine-induced depolarization. A similar observation
has been reported in rabbit vertebral artery, where
glibenclamide-induced depolarization potently augmented the
depolarizing effect of histamine (28). These data suggest that
histamine-induced depolarization in cerebrovascular SMCs might be
regulated by the level of resting membrane potential. In the guinea pig
ileum, a slight electrical membrane hyperpolarization abolished the
ACh-induced depolarization due to a strong voltage dependence of
ACh-activated nonselective cation channels (13). A modulation of
histamine-induced depolarization by the level of resting membrane
potential in our study most likely reflects a voltage dependence of
nonselective cation channels, a property described in a number of
smooth muscle preparations (5, 15, 21, 22).
channels by Ca2+ released from internal stores might
accelerate the activation of nonselective cation channels. Therefore,
an additional physiological significance of Ca2+
mobilization might be regulation of the responsiveness of rabbit MCA to
histamine and possibly also to some other Ca2+-mobilizing vasoconstrictors.
It has been shown that nonselective cation channels in smooth muscle
can be facilitated by cytosolic Ca2+ because of its
mobilization from internal stores or as a result of entry through
voltage-dependent Ca2+ channels, thus providing a
positive-feedback regulating mechanism (5, 14, 15, 21, 24, 27, 33). We
found that histamine can induce a sustained depolarization in cerebral
arteries treated with ryanodine or a combination of ryanodine and
nifedipine or in Ca2+-free solution, suggesting that an
elevation of cytosolic Ca2+ is not obligatory for
activation of nonselective cation channels in this tissue. In cerebral
arterioles, endothelin-induced depolarization was also observed after
depletion of internal stores with thapsigargin, indicating less
importance of Ca2+ in initiation of this depolarization
(11). Elevation of cytosolic Ca2+ is not essential for
initiation of the depolarization of the mesenteric artery in response
to norepinephrine (31). We cannot, however, exclude some facilitator
role of Ca2+ in the activation of nonselective cation
channels in our experiments. For example, in arteries treated with
ryanodine, generation of APs greatly accelerated the development of
histamine-induced depolarization, which can be explained by both
voltage- and Ca2+-dependent activation of nonselective
cation channels.
In SMCs from the rabbit basilar artery, histamine- induced transient
inward current is most likely carried through
Ca2+-activated Cl channels. There was no
evidence for the contribution of nonselective cation channels (16, 25).
It is possible that, in rabbit basilar artery,
Ca2+-activated Cl channels might be the
major contributors to histamine-induced depolarization. Histamine at a
maximal concentration (10 µM) depolarized SMCs of rabbit basilar
artery from the resting potential 62 mV by only 2-3 mV and
induced a continuous generation of large-amplitude APs (38). In
contrast, maximal histamine-induced depolarization in our study was
~30 mV. A contribution of nonselective cation channels in addition to
Ca2+-activated Cl channels might account
for a much stronger depolarizing effect of histamine in the MCA than in
the basilar artery.
Nonselective cation channels of cerebrovascular SMCs might be a target
not only for histamine, but also for some other vasoconstrictors. We
have observed that Co2+ (200 µM) completely reversed
serotonin-induced depolarization (14.2 ± 0.9 mV, n = 3;
unpublished observations). Activation of the same nonselective cation
channels by a threshold concentration of different vasoconstrictors
might underlie the phenomenon of potentiation described in cerebral
arteries (4). In our experiments the exposure of rabbit MCA to
subthreshold concentrations of serotonin or histamine (0.1 and 1 µM,
respectively) produced no effect. However, combined application of
these vasoconstrictors resulted in a strong depolarization of rabbit
MCA from 67.0 ± 2.0 to 34.3 ± 1.9 mV and contraction
(n = 4; unpublished observation).
Caffeine-induced sustained depolarization. One of the unexpected and interesting observations of our study is that caffeine can induce a sustained depolarization of rabbit MCA. We suggest that caffeine-induced sustained depolarization resulted from opening of cation channels, since Co2+ (100-200 µM) can reversibly abolish this depolarization (unpublished observation). Nonselective cation channels activated by caffeine have been identified in single SMCs from portal vein (24) and toad stomach (10). In terms of their Ca2+ and voltage dependence as well as permeability to Ca2+, these channels are clearly a divergent group. In our experiments, sustained caffeine-induced depolarization, in contrast to that evoked by histamine, was greatly attenuated by ryanodine, suggesting that the underlying mechanisms might be different. Caffeine can induce a transient followed by a sustained elevation of cytosolic Ca2+ in cerebral arterioles (11). After treatment with thapsigargin, the sustained elevation in Ca2+ was abolished, suggesting a role of capacitative Ca2+ influx. The same mechanism might also operate in rabbit MCA.
In our experiments, caffeine-induced sustained depolarization, although it occasionally reached the threshold for activation of contraction (40 mV), was not in fact associated with sustained contraction.
It has been shown that caffeine also has a potent inhibitory effect on
smooth muscle contraction possibly mediated by stimulation of cAMP
production (42). This additional effect of caffeine is most likely
responsible for the absence of contraction during caffeine-induced
sustained depolarization in our experiments.
In conclusion, our experiments demonstrate that, in rabbit MCA,
Cl channels activated by Ca2+
mobilization from the ryanodine-sensitive intracellular stores importantly contribute to the transient caffeine-induced depolarization and the initial and, to a lesser extent, the sustained depolarization caused by histamine. We present evidence that nonselective cation channels appear to be important contributors to histamine-evoked sustained depolarization. This depolarization can be controlled by the
level of the membrane potential, and the initial depolarization due to
Ca2+-induced activation of Cl channels
might provide an important functional link between Ca2+
mobilization and activation of nonselective cation channels in rabbit MCA.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. J. E. Brayden (University of Vermont) for helpful discussions and advice.
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FOOTNOTES |
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This study was supported by National Heart, Lung, and Blood Institute Grant HL-32985.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: N. I. Gokina, Dept. of Obstetrics and Gynecology, College of Medicine, The University of Vermont, Burlington, VT 05405 (E-mail: gokina{at}salus.med.uvm.edu).
Received 29 January 1999; accepted in final form 14 December 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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