Role of Ca2+-Activated K+ Channels in the Regulation of Membrane Potential and Tone of Smooth Muscle in Human Pial Arteries
Smooth muscle cells (SMCs) in 58% of human pial arteries obtained during surgery showed no spontaneous contractions and displayed a stable resting membrane potential (MP) of −54.7±1.5 mV. Those that exhibited periodic spontaneous contractions associated with periodic depolarization and generation of spontaneous action potentials (APs) had a less negative MP of −43.1±0.5 mV (42%). Inhibition of calcium-activated potassium (KCa) channels in the silent arteries by charybdotoxin (CTX) and tetraethylammonium ions (TEA) induced dose-dependent depolarization, AP generation, and contraction. TEA and CTX enhanced the spontaneous depolarization and force in arteries that exhibited spontaneous activity. They also prolonged the spontaneous APs up to several times and increased their upstroke amplitude. Both TEA and CTX failed to produce significant depolarization in arteries treated with nifedipine. It is concluded that KCa channels are important regulators of human pial artery SMC resting MP and tone. They are also involved in the control of AP amplitude and duration and the associated contractions. These data suggest that alterations in the activity of SMC KCa channels could be responsible for the appearance of spontaneous activity in human pial arteries in vitro and that impaired function of these channels might be related to vasospastic phenomena in human cerebral circulation.
- human cerebral arteries
- membrane potential
- action potentials
- calcium-activated potassium channels
The tone of the smooth muscle in small cerebral arteries is regulated by a number of mechanisms, including the electrical events at the plasma membrane. Various types of K+ channels have been implicated in the control of the MP and excitability of SMCs. Large-conductance KCa channels are found in the majority of vascular SMCs; however, their physiological role remains unclear.1 2 3 4 5 6 7 8 9 KCa channels are activated both by membrane depolarization and elevation of cytoplasmic Ca2+ and thus are potentially important in the regulation of resting MP and tone and also in the repolarization phase of APs in spike-generating SMCs.1 2 3 4 5 6 7 8 9 10
The functional role of KCa channels can be investigated using specific pharmacological blocking agents. TEA (0.1 to 1 mmol/L) preferentially blocks the KCa channels in a variety of vascular tissues.4 6 7 CTX and iberiotoxin, small peptides isolated from scorpion venom, are probably their most selective blockers.4 5 6 7 Although KCa channels have been identified in cerebral arteries of a number of species,5 7 10 11 12 13 14 15 16 they have not been studied in humans. We have found that SMCs in the majority of human pial arteries in vitro displayed a stable resting MP between −50 and −70 mV, but some arteries were more depolarized and exhibited periodic spontaneous contractions associated with membrane depolarization and generation of APs. After a period of spontaneous activity, the SMCs transiently repolarized and relaxed.17 Ca2+ entering the cells during AP generation would be expected to enhance the activity of KCa channels. The present study was undertaken (1) to evaluate the involvement of large-conductance KCa channels in the control of the resting MP in human pial arteries, (2) to determine the contribution of KCa channels to the repolarization phase of APs, and (3) to examine whether these channels are responsible for the transient repolarization that occurs after long-lasting spontaneous depolarization.
Materials and Methods
Human pial arteries were obtained from 57 patients (14 to 73 years of age) during neurosurgical resection of brain tumors. Sixteen of the patients were hypertensive and 3 were diabetic. Almost all patients (50 of 57) received dexamethasone during the presurgery period. Only normal arteries (those not feeding a tumor) were used. They were transported to the laboratory in cold PSS and either studied on the day of surgery or refrigerated and used on the second day. Usually only one arterial segment from a patient was studied. Segments 1 to 1.5 mm long were mounted in resistance artery myograph as previously described.17 They were continuously perfused with fresh, warm (37°C) PSS.
For measurements of MP, we used microelectrodes filled with 0.5 mol/L KCl and with tip resistances of 110 to 150 MΩ. SMCs were impaled from the adventitial surface of the arterial segment. The changes in the MP and isometric force were displayed and recorded on a desktop computer. Further methodological details and the criteria for acceptance of MP recordings have been described.17
The endothelium was removed by air injected through the lumen of the small arteries (diameter 100 to 300 μm) for 10 minutes or mechanically by a human hair (for larger arteries). The effectiveness of endothelium removal was confirmed by the absence of relaxation to acetylcholine in arteries precontracted with 5-hydroxytryptamine.
The PSS was of the following composition (mmol/L): 130 NaCl, 4.7 KCl, 1.18 KH2PO4, 1.17 MgSO4, 14.9 NaHCO3, 0.026 EDTA, 11.1 glucose, and 1.6 CaCl2. To prepare K+-rich solutions, equimolar amounts of NaCl were replaced with KCl. Superfusion solutions were equilibrated with a mixture of 95% O2 and 5% CO2, and the pH in the experimental chamber was 7.4. The drugs used were: CTX (Peptides International), 5-hydroxytryptamine creatinine sulfate, acetylcholine chloride, tetraethylammonium chloride, and nifedipine hydrochloride (Sigma).
In our preliminary experiments, we used TEA in concentrations of 0.1 to 0.5 mmol/L. TEA in these concentrations produced depolarization and contraction; however, the effects developed slowly and were weak. Therefore, in this study we used a higher dose of TEA (1 mmol/L) to get more rapid and substantial responses.
Results are expressed as mean±SEM. Student's t test was used to determine the significance of differences between sets of data, which was considered to be a value of P<.05.
Intracellular recordings of MP were obtained from 64 of the 75 arterial segments from 57 patients. The stable resting MP of −54.7±1.5 mV was observed in 58% of the arteries (146 cells from 37 segments). The balance of the segments exhibited slow, long-lasting waves of depolarization, with bursts of APs separated by silent periods of up to several minutes. The MP of 78 cells from 27 arteries, measured during these silent periods and referred to below as resting MP, was −43.1±0.5 mV. The incidence of spontaneous activity was higher in arteries from hypertensive (82.4%) than normotensive (31.0%) patients.
Effect of TEA and CTX on Quiescent Human Pial Arteries
In 13 of 17 arteries, 1 mmol/L TEA induced contractions that were 30.8±6.4% of the maximum K+-induced contraction (66 mmol/L). Application of 5 mmol/L TEA resulted in 57.6±9.4% contraction in 9 of 11 arteries. CTX (20, 40, and 100 nmol/L) induced contractions that were 33.7±5.5% (n=6), 57.9±9.0% (n=7), and 80.9±15.3% (n=3) of the maximum K+-induced contraction, respectively. In 5 of 15 arteries, CTX (20 to 40 nmol/L) was without effect. In most arteries, TEA or CTX caused periodic contractions (7 to 10 minutes' duration) separated by periods of quiescence, although some responded with tonic contractions.
TEA (1 mmol/L) depolarized the SMCs and induced fast oscillations in the MP of five arteries (Fig 1A⇓). TEA-evoked depolarization was interrupted by transient membrane repolarization and disappearance of membrane oscillations. In the experiment shown in Fig 1A⇓, shortly after starting the next wave of depolarization, the concentration of TEA was increased to 5 mmol/L to test whether it would eliminate the transient MP repolarizations. Higher concentrations of TEA (5 mmol/L) caused further depolarization and enhancement of membrane oscillations. Depolarization subsequently switched to transient repolarization and hyperpolarization in spite of the presence of the relatively high dose of the drug. TEA in concentrations of 1 and 5 mmol/L depolarized the SMCs by 6.5±1.6 mV (n=5) and 12.7±2.9 mV (n=3), respectively (Fig 2⇓). The period of depolarization with oscillations in MP was associated with a contraction composed of partially fused phasic components.
Exposing the same artery to 20 nmol/L CTX depolarized SMCs and evoked oscillations in MP (Fig 1B⇑). An increase in concentration of CTX to 40 nmol/L resulted in additional depolarization and an increase in the amplitude of the oscillations. CTX-induced contraction was composed of partially fused phasic contractions and displayed periodicity. CTX (20, 40, and 100 nmol/L) depolarized the SMCs by 6.0±1.0 mV (n=3), 14.2±2.8 mV (n=6), and 14.3±2.3 mV (n=3), respectively (Fig 2⇑). Within 15 to 20 minutes of the washout of TEA or CTX, complete restoration of resting MP occurred.
Resting MPs of the SMCs that contracted to TEA or CTX were in the range of −45 to −55 mV. TEA (1 to 5 mmol/L) in 4 of 17 and CTX (40 to 100 mmol/L) in 5 of 15 arteries failed to induce contraction. The mean resting MP of SMCs in these arteries was −62.8±1.9 mV (n=7). TEA (1 mmol/L) and CTX (40 nmol/L) depolarized the SMCs by only 2.3±0.3 mV and 3.8±2.2 mV, respectively, values significantly less than those obtained in arteries having resting MPs of −45 to −55 mV. To test whether these differences in the response to CTX and TEA were related to variation in the levels of MP, we studied the effect of CTX (or TEA) before and after moderate depolarization of SMCs by Ba2+. Ba2+ (10 to 100 μmol/L) induced dose-dependent depolarization of SMCs in human pial arteries,18 presumably due to block of inward rectifier K+ channels.19 Fig 3⇓ shows responses induced by CTX (40 nmol/L) at different levels of the resting MP. CTX depolarized the SMCs from −52 to −28 mV (24 mV) and caused MP oscillations and force development (Fig 3A⇓). One hour after a washout, the SMCs had spontaneously hyperpolarized from −52 to −58 mV. A second application of CTX induced a smaller depolarization (7 mV) and no contraction (Fig 3B⇓). After the washout, SMCs were depolarized by Ba2+ (25 μmol/L), and then a third application of CTX led to additional depolarization, generation of membrane oscillations, and contraction (Fig 3C⇓), a response similar to that observed after the first application of CTX (Fig 3A⇓). In the presence of Ba2+, CTX depolarized SMCs by 15.0±5.0 mV (compared with 3.8±2.2 mV in the control) and evoked force generation (n=4). Similar results were obtained when TEA was tested in the presence of Ba2+. TEA depolarized SMCs by 18.3±4.4 mV (compared with 2.3±0.3 mV in the control) and induced contraction (n=3). Thus, the level of the resting MP is an important determinant of TEA- and CTX-induced depolarization and contraction in human pial arteries.
Effects of TEA and CTX on Spontaneous Electrical and Contractile Activity
TEA (1 mmol/L) caused an increase in the amplitude and duration of the contractile events to 184.0±18.3% (n=14) and 170.8±49.2% (n=6) of control, respectively. The amplitude and duration of periodic spontaneous depolarization were also increased (Fig 4A⇓). After a period of enhanced spontaneous depolarization, SMCs repolarized in spite of the presence of TEA. The repolarization resulted in cessation of AP generation and relaxation. The effect was transient, and the next wave of spontaneous depolarization initiated AP generation and contraction. In 12 segments, TEA increased spontaneous depolarization by 11.0±1.9 mV (Fig 5⇓).
As with TEA, CTX (20 and 40 nmol/L) increased the amplitude of periodic contractions to 135.8±8.2% (n=6) and 153.3±8.8% (n=8) and their duration to 124.0±12.1% (n=3) and 163.9±19.1% (n=5) of control, respectively (Fig 4B⇑).This activity was associated with an increase in spontaneous depolarization of 6.8±0.9 mV (20 nmol/L; n=6) and 9.9±1.0 mV (40 nmol/L; n=7) (Fig 5⇑). CTX did not attenuate the periodic repolarization of SMCs associated with arterial relaxation. In two arteries, 100 nmol/L CTX enhanced the spontaneous contractions without affecting their periodicity.
TEA and CTX not only depolarized SMCs but also modified the spontaneous APs. TEA (1 mmol/L) increased the AP duration to 385.5±71.7% (n=5) and slowed repolarization. Additional spikes or membrane oscillations during the prolonged plateau were frequently observed (Fig 6A⇓). Similar results were obtained with CTX (20 and 40 nmol/L): AP duration was increased to 249.9±75.5% (n=4) and 256.2±72.1% (n=4) of control, respectively (Fig 6B⇓). TEA (1 mmol/L) and CTX (20 and 40 nmol/L) also increased the amplitude of the AP upstroke to 128.4±8.1%, 115.6±11.5%, and 124.4±7.4% of control, respectively. The AP plateau phase was slightly enhanced by TEA but not by CTX (Fig 7⇓).
Effect of Nifedipine on TEA-Induced and CTX-Induced Depolarization
Lowering the concentration of cytoplasmic Ca2+ dramatically decreases the open-state probability of KCa channels.4 5 7 12 14 If the effects of CTX and TEA on human pial arteries are primarily due to a specific block of KCa channels, then CTX- or TEA-induced depolarization should be diminished after a blockade of Ca2+ entry into the SMCs. Exposure of spontaneously active arteries to CTX (40 nmol/L) resulted in depolarization (8.3±1.2 mV; n=4) and contraction. Nifedipine (5 μmol/L) either did not change the MP or caused only weak (2 to 3 mV) hyperpolarization and completely inhibited the spontaneous APs and contractions. Fifteen to 20 minutes after exposure to nifedipine, CTX induced a weak depolarization of 1.8±0.9 mV (n=4). TEA (1 mmol/L) failed to produce significant depolarization in two arteries treated with nifedipine.
Effect of TEA and CTX on Endothelium-Denuded Human Pial Arteries
There is evidence in the literature for the presence of KCa channels in vascular endothelial cells.20 Inhibition of endothelial KCa channels will alter the production and secretion of vasoactive factors that would influence the underlying smooth muscle. For this reason, TEA and CTX were tested in arterial segments in which the endothelium was removed mechanically. After mechanical disruption, acetylcholine-induced (3 μmol/L) relaxation was reduced from 86.5±3.7% to 5.8±1.2% of 5-hydroxytryptamine–induced contraction (n=5). In denuded arteries, CTX (40 to 100 nmol/L, n=4) and TEA (1 to 5 mmol/L, n=3) induced tonic or periodic contractions.
We present the first evidence for a functional role of KCa channels in the regulation of tone of human pial arteries. Our experiments suggest that KCa channels are an important determinant of SMC resting MP, excitability, and tone in these vessels. Both CTX and TEA induced dose-dependent depolarization and contraction of SMCs and had a similar effect on arterial tone after endothelium removal. These findings are consistent with a direct action of CTX and TEA on the SMCs. In vascular SMCs from a number of species, 100 nmol/L CTX almost completely blocked large-conductance KCa channels without exerting a significant effect on Kv channels.5 6 7 12 In our preliminary study, blockade of Kv channels by 4-aminopyridine caused sustained depolarization of SMCs, in contrast to periodic CTX- or TEA-induced depolarization, and a blocker of KATP channels, glibenclamide, did not affect the MP.18 These data suggest that the observed effects of TEA and CTX are not related to blockade of Kv or KATP channels. CTX in a relatively low concentration (20 nmol/L) depolarized and contracted SMCs in human pial arteries, favoring a specific effect on KCa channels. TEA had effects similar to those induced by CTX (see Fig 1⇑). Neither CTX nor TEA induced significant depolarization after blockade of Ca2+ entry by nifedipine. This observation further supports the idea that both CTX and TEA produce depolarization of SMCs in human pial arteries mainly due to blockade of KCa channels.
The depolarization induced by CTX and TEA was critically dependent on the level of the resting MP. KCa channel activity is enhanced by membrane depolarization and elevation in the concentration of intracellular Ca2+.4 5 7 10 Dependence of KCa channel open-state probability on the concentration of cytoplasmic Ca2+ seems to be more important, since CTX and TEA failed to produce significant depolarization in the presence of nifedipine in spontaneously depolarized SMCs. Nifedipine or Ca2+-free solution did not depolarize the SMCs, which might be expected as a result of a decrease in KCa channel activity due to lowering of cytoplasmic Ca2+ concentration. It seems that some other Ca2+-regulated channels (or mechanisms) might be involved in the control of MP in human pial arteries and that the final level of MP represents the balance between them.
Accumulation of cytoplasmic Ca2+ during spontaneous activity might be expected to enhance the activity of KCa channels that contribute to transient repolarization of SMCs after long-lasting spontaneous depolarization. However, TEA and CTX did not attenuate the transient repolarization and relaxation of human pial arteries. On the contrary, both agents can induce periodic spontaneous activity in quiescent pial arteries. A similar ability of CTX (or iberiotoxin) and TEA to evoke or enhance periodic spontaneous contractions has been shown in other tissues.15 21 As this behavior is seen in human pial arteries (present study) and in the dog basilar artery15 after mechanical removal of the endothelium, the periodicity is probably due to some intrinsic property of SMCs and does not involve the activity of KCa channels.
Our experiments showed that SMCs in human pial arteries can generate APs (or oscillations in MP) associated with the phasic contractions that occur when they depolarize spontaneously or with high K+.17 Recently, spontaneous oscillations in wall tension of the same artery have been described by others.22 In spite of the demonstration of KCa channels in the majority of AP-generating SMCs in vascular and nonvascular tissues, their involvement in the repolarization phase of the AP varies with tissue type and source. Exposure to CTX or iberiotoxin did not modify the duration of spontaneous APs in the circular muscle of the canine colon23 nor the guinea pig mesotubarium24 but prolonged their duration in Bay K 8644–treated circular smooth muscle from the canine colon.23 Since in our experiments, in the presence of both TEA and CTX, the falling phase of APs occurred with substantial delay, resulting in their prolongation, it is suggested that KCa channel activation can contribute to the repolarization phase of the APs in human pial arteries.
We found that TEA or CTX also increased the upstroke component of spontaneous APs. A similar effect of KCa channel blockers has been described in some other smooth muscle preparations. CTX induced or enhanced the spikes in the longitudinal muscle of the canine colon23 and the guinea pig stomach25 and caused an increase in the amplitude of spontaneous slow waves and their conversion into spikelike APs in guinea pig trachealis.26 These effects can be explained by the involvement of KCa channels in the production of an early outward current during AP generation. Normally this current overlaps with the inward Ca2+ current, so that the net inward current seems insufficient to elicit regenerative all-or-nothing APs. Instead, SMCs generate small, graded APs or oscillations in MP during depolarization. Suppression of this outward current by TEA and CTX would enhance net inward current and increase the amplitude of AP upstroke or even facilitate AP generation in those SMCs that normally do not generate APs. These data suggest that KCa channels can play an important role in the regulation of AP upstroke and excitability of SMCs in human pial arteries.
SMCs of human pial artery segments displayed different levels of resting MP. A low level of MP might be one of the reasons spontaneous electrical and contractile activity occurs in some of these vessels. Although the incidence of spontaneous activity was higher in arteries from hypertensive (82.4%) than normotensive (31.0%) patients, the number of our observations is not sufficient to make definitive conclusions. The present study strongly suggests that KCa channels participate in the regulation of MP of SMCs in human pial arteries and that altered function of these channels might be responsible for the less negative MP and consequent generation of spontaneous APs. As CTX and TEA were unable to induce substantial depolarization in SMCs having very negative resting MP, some additional mechanisms must be responsible for the depolarized state of SMCs showing spontaneous activity in vitro.
Cerebral arteries in vivo possess myogenic tone. This tone has been demonstrated in isolated pressurized cerebral arteries of humans as well as many other species and has been associated with SMC depolarization.5 27 28 29 30 It has been proposed that this depolarization and subsequent elevation of cytoplasmic Ca2+ due to influx through voltage-dependent Ca2+ channels lead to activation of KCa channels and so counteract the pressure-induced depolarization and contraction. Thus, activation of KCa could represent an important negative feedback control mechanism in the regulation of membrane depolarization and tone caused by pressure or other factors.5 7 MPs of arterial SMCs in vivo are in the range of −40 mV to −55 mV.7 Our findings suggest that KCa channels might be partially activated under these conditions and might thereby participate in the regulation of MP and tone of human pial arteries in vivo.
In conclusion, our data demonstrate that blockade of KCa channels by CTX and TEA resulted in membrane depolarization, an increase of the amplitude and duration of APs, and marked contraction of SMCs in human pial arteries. These findings suggest that KCa channels are probably importantly involved in the regulation of cerebrovascular tone and their impaired function might contribute to pathophysiological conditions such as cerebral vasospasm.
Selected Abbreviations and Acronyms
|KATP channel||=||ATP-sensitive K+ channel|
|KCa channel||=||calcium-activated potassium channel|
|Kv channel||=||delayed-rectifier K+ channel|
|PSS||=||physiological salt solution|
|SMC||=||smooth muscle cell|
This study was supported by the Totman Medical Research Fund.
Previously presented in part in abstract form (FASEB J. 1996;10:A301).
- Received January 22, 1996.
- Accepted July 24, 1996.
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