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(Circulation Research. 1995;76:1057-1062.)
© 1995 American Heart Association, Inc.

Articles

Dilatation of Cerebral Arterioles in Response to Activation of Adenylate Cyclase Is Dependent on Activation of Ca²⁺-Dependent K⁺ Channels

Hisao Taguchi, Donald D. Heistad, Takanari Kitazono, Frank M. Faraci

From the Departments of Internal Medicine and Pharmacology, Cardiovascular Center and Center on Aging, University of Iowa College of Medicine, Iowa City.

Correspondence to Frank M. Faraci, PhD, Department of Internal Medicine, Cardiovascular Center, University of Iowa College of Medicine, Iowa City, IA 52242.

	Abstract

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Abstract The role of Ca²⁺-dependent potassium channels in mediating vascular responses to activation of adenylate cyclase in vivo is not known. The goal of this study was to examine the hypothesis that dilatation of cerebral arterioles in response to activation of adenylate cyclase is mediated by activation of Ca²⁺-dependent potassium channels. Diameters of cerebral arterioles were measured in vivo in anesthetized rabbits. Topical application of forskolin (1 and 10 µmol/L), a direct activator of adenylate cyclase, dilated cerebral arterioles by 40±8% (mean±SEM) and 71±9%, respectively, from a control diameter of 85±4 µm. Iberiotoxin (50 and 100 nmol/L), a selective inhibitor of Ca²⁺-dependent potassium channels, inhibited dilatation in response to both concentrations of forskolin by 45% to 60%. We obtained similar results by using charybdotoxin (50 nmol/L), another inhibitor of Ca²⁺-dependent potassium channels. Vasodilatation in response to dibutyryl cAMP (a cell-permeable cAMP analogue) was also inhibited by iberiotoxin. In contrast, dilatation of cerebral arterioles in response to sodium nitroprusside and acetylcholine (activators of guanylate cyclase) and aprikalim (activator of ATP-sensitive potassium channels) was not inhibited by iberiotoxin. These findings suggest that dilatation of cerebral arterioles in response to forskolin and increases in intracellular concentrations of cAMP are mediated by activation of Ca²⁺-dependent potassium channels. Thus, activation of Ca²⁺-dependent potassium channels may be a major mechanism of cerebral vasodilatation in response to activation of adenylate cyclase in vivo.

Key Words: cerebral arterioles • forskolin • sodium nitroprusside • aprikalim • iberiotoxin

	Introduction

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Activation of adenylate cyclase and accumulation of cAMP is a major mechanism that produces relaxation of vascular muscle.^{1 2} Forskolin, a direct activator of adenylate cyclase, produces increases in the intracellular concentration of cAMP^{3 4} and relaxation of cerebral arteries in vitro.^{3 5 6 7} In vivo, forskolin produces dilatation of cerebral arterioles^{7 8 9} and the basilar artery¹⁰ and increases cerebral blood flow.¹¹ Mechanisms by which forskolin, or increases in intracellular cAMP, produce vasodilatation are not clear.

Activation of potassium channels in vascular muscle produces hyperpolarization and relaxation.² Patch-clamp experiments suggest that increases in cAMP increase activity of Ca²⁺-dependent potassium channels in cultured coronary vascular muscle and airway smooth muscle.^{12 13} Thus, based on patch-clamp approaches, it seemed likely that vasodilation in response to increases in intracellular cAMP may be due in part to activation of Ca²⁺-dependent potassium channels. The role of Ca²⁺-dependent potassium channels in mediating relaxation of blood vessels in response to activation of adenylate cyclase has not been examined in vivo in any vascular bed.

The goal of the present study was to test the hypothesis that dilatation of cerebral arterioles to forskolin and a cAMP analogue (dibutyryl cAMP) is mediated by activation of Ca²⁺-dependent potassium channels. We tested whether iberiotoxin and charybdotoxin, which are selective inhibitors of Ca²⁺-dependent potassium channels,^{2 14} inhibit cerebral vasodilatation in response to forskolin and dibutyryl cAMP in vivo.

	Materials and Methods

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Animal Preparation
Experiments were performed on 46 New Zealand White rabbits (2.5 to 3.5 kg) that were anesthetized with pentobarbital sodium (35 mg · kg^-1 IV). Pentobarbital was supplemented regularly at 10 mg · kg^-1 · h^-1. The trachea was cannulated, and the animals were ventilated mechanically with air and supplemental oxygen. Arterial blood gases were monitored throughout the experiment (PCO₂, 36±1 [mean±SEM] mm Hg; PO₂, 160±6 mm Hg; and pH, 7.39±0.01). A femoral artery was cannulated for measurement of systemic pressure and to sample arterial blood. A femoral vein was cannulated for infusion of drugs. Gallamine triethiodide (5 mg/kg) was administered intravenously to produce paralysis of skeletal muscle. Depth of anesthesia was evaluated by applying pressure to a paw or the tail and observing changes in heart rate or blood pressure. When such changes occurred, additional anesthetic was administered. Arterial pressure was similar in the different groups of animals and averaged 85±2 mm Hg.

Rabbits were placed in a head holder, and a closed cranial window was placed over the parietal cortex as described previously.¹⁵ The cranial window was filled with artificial cerebrospinal fluid (CSF) warmed to 37°C. Values for pH, PCO₂, and PO₂ of the artificial CSF were 7.41±0.01, 37±1, and 40±3 mm Hg, respectively. Diameters of pial arterioles were measured by using a microscope equipped with a TV camera coupled to a video monitor. Diameters of blood vessels were measured by using a microscope equipped with a TV camera coupled to a video monitor and an image-shearing device.

Experimental Protocol
Seven groups of animals were studied. In group 1 (time controls), arteriolar diameter was measured under control conditions and 1 to 2 minutes after filling the window with CSF containing forskolin (1 and 10 µmol/L) (n=8) or sodium nitroprusside (1 and 10 µmol/L) (n=10). The diameter of cerebral arterioles was stable during this time; thus, all reported values represent steady state conditions. The order of application of agonists was altered, and concentrations of agonists were applied in a cumulative manner. The addition of agonists does not affect the pH of artificial CSF used to fill the cranial window. A 30- to 60-minute recovery period was included after each agonist to allow diameters of arterioles to return to baseline. Application of the agonists was then repeated after a 60-minute recovery period. This group of animals served as a time control to establish whether responses to forskolin and sodium nitroprusside were reproducible.

In group 2 (low concentration of iberiotoxin), arteriolar diameter was measured under control conditions and 1 to 2 minutes after filling the window with CSF containing forskolin (1 and 10 µmol/L) (n=7) or sodium nitroprusside (1 and 10 µmol/L) (n=7). After a 60-minute recovery period, application of forskolin and sodium nitroprusside was repeated in the presence of iberiotoxin (50 nmol/L, Research Biochemicals International). The cranial window was treated with iberiotoxin for 5 to 15 minutes before application of forskolin or sodium nitroprusside. The purpose of these experiments was to determine if iberiotoxin inhibits dilatation of cerebral arterioles in response to forskolin or sodium nitroprusside.

In group 3 (high concentration of iberiotoxin), arteriolar diameter was measured under control conditions and 1 to 2 minutes after filling the window with CSF containing forskolin (1 and 10 µmol/L) (n=4) or sodium nitroprusside (1 and 10 µmol/L) (n=4). After a 60-minute recovery period, application of forskolin and sodium nitroprusside was repeated in the presence of iberiotoxin (100 nmol/L). The cranial window was treated with iberiotoxin for 15 minutes before the application of forskolin or sodium nitroprusside. The purpose of these experiments was to determine if a higher concentration of iberiotoxin produced greater inhibition of dilator responses of cerebral arterioles to forskolin or sodium nitroprusside.

In group 4 (charybdotoxin), arteriolar diameter was measured under control conditions and 1 to 2 minutes after filling the window with CSF containing forskolin (1 and 10 µmol/L) (n=8) or sodium nitroprusside (1 and 10 µmol/L) (n=11). After a 60-minute recovery period, application of forskolin and sodium nitroprusside was repeated in the presence of charybdotoxin (50 nmol/L, Calbiochem). The cranial window was treated with charybdotoxin for 5 minutes before the application of agonists. The purpose of these experiments was to determine if charybdotoxin inhibits dilatation of cerebral arterioles in response to forskolin or sodium nitroprusside.

In group 5, arteriolar diameter was measured under control conditions and 1 to 2 minutes after filling the window with CSF containing acetylcholine (1 and 10 µmol/L) or aprikalim (0.1, 1, and 10 µmol/L), a direct activator of ATP-sensitive potassium channels (n=4). After a 60-minute recovery period, application of acetylcholine and aprikalim was repeated in the presence of iberiotoxin (100 nmol/L). The cranial window was treated with iberiotoxin for 15 minutes before the application of acetylcholine or aprikalim. The purpose of these experiments was to determine if iberiotoxin produced inhibition of vasodilator responses to acetylcholine (which produces dilatation of cerebral arterioles by release of endothelium-derived relaxing factor, nitric oxide) and aprikalim. This group addressed the specificity of iberiotoxin.

In group 6, arteriolar diameter was measured under control conditions and 15 minutes after filling the window with CSF containing dibutyryl cAMP (100 and 300 µmol/L), a cell-permeable analogue of cAMP (n=3). After a 60-minute recovery period, during which time diameters of cerebral arterioles returned to baseline, application of dibutyryl cAMP was repeated. This group of animals served as a time control to establish whether the response to dibutyryl cAMP was reproducible.

In group 7, arteriolar diameter was measured under control conditions and 15 minutes after filling the window with CSF containing dibutyryl cAMP (100 and 300 µmol/L) (n=7). After a 60-minute recovery period, application of dibutyryl cAMP was repeated in the presence of iberiotoxin (100 nmol/L). The cranial window was treated with iberiotoxin for 15 minutes before the application of dibutyryl cAMP. The purpose of these experiments was to determine if iberiotoxin produced inhibition of dilatation of cerebral arterioles in response to dibutyryl cAMP. In three of these animals, we also examined the effects of 1,9-dideoxyforskolin (1 and 10 µmol/L) on the diameters of cerebral arterioles. 1,9-Dideoxyforskolin is a structural analogue of forskolin that does not activate adenylate cyclase and thus serves as a control for possible nonspecific effects of forskolin independent of activation of adenylate cyclase.^{16 17}

Statistics
To examine the effects of antagonists on baseline vessel diameter, paired t tests were used on absolute values (not percent change). For comparison of percent change data in the absence and presence of inhibitors, statistical analysis was performed with Wilcoxon's test. All values are expressed as mean±SEM. A value of P<.05 was considered significant.

	Results

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Under control conditions, diameters of cerebral arterioles were similar in the different groups and averaged 85±4 µm. Forskolin (1 and 10 µmol/L) and sodium nitroprusside (1 and 10 µmol/L) produced reproducible concentration-related dilatation of cerebral arterioles (Fig 1).

View larger version (15K): [in this window] [in a new window]   Figure 1. Bar graphs showing reproducibility of changes in diameters of cerebral arterioles in response to two applications of forskolin and sodium nitroprusside. Values are mean±SEM (n=8 for forskolin and n=10 for nitroprusside).

In contrast to forskolin, 1,9-dideoxyforskolin had minimal effect on diameters of cerebral arterioles. The low concentration of 1,9-dideoxyforskolin (1 µmol/L) did not alter vessel diameter (change in diameter of 2±1%). The high concentration of 1,9-dideoxyforskolin (10 µmol/L) produced modest vasodilatation, but the increase in vessel diameter (14±3%) was much less than that produced by 1 or 10 µmol/L concentrations of forskolin (40% and 77%, respectively). Thus, 1,9-dideoxyforskolin is relatively inactive compared with forskolin, which suggests that dilatation of cerebral arterioles in response to forskolin is due in very large part to activation of adenylate cyclase and not to nonspecific effects.

Baseline diameter of cerebral arterioles tended to increase in the presence of the low concentration of iberiotoxin (50 nmol/L), but the change in diameter was very modest (86±7 versus 89±8 µm). The low concentration of iberiotoxin (50 nmol/L) produced marked inhibition of dilatation of cerebral arterioles in response to forskolin (Fig 2). In contrast, iberiotoxin had no significant effect on vasodilatation in response to sodium nitroprusside (Fig 2).

View larger version (16K): [in this window] [in a new window]   Figure 2. Bar graphs showing changes in diameters of cerebral arterioles in response to forskolin and sodium nitroprusside in the absence and presence of iberiotoxin (50 nmol/L). Baseline diameter of cerebral arterioles was 86±7 µm in the absence of iberiotoxin and 89±8 µm in the presence of iberiotoxin. Values are mean±SEM (n=7). *P<.05 vs control response.

The high concentration of iberiotoxin (100 nmol/L) produced very modest, but significant, constriction of cerebral arterioles. The baseline diameter of cerebral arterioles was decreased from 81±5 to 79±6 µm (a reduction of 3±1%, P<.05, n=15). Forskolin increased the diameter of cerebral arterioles by 38±10% and 74±8% in the absence and 18±7% and 31±6% in the presence of 100 nmol/L iberiotoxin (P<.05 versus control). Dilatation of cerebral arterioles in response to nitroprusside was not affected by 100 nmol/L iberiotoxin: the increase in diameter was 20±3% and 44±7% in response to 1 and 10 µmol/L nitroprusside in the absence of iberiotoxin versus 20±3% and 45±8% in the presence of iberiotoxin. These findings suggest that dilatation of cerebral arterioles in response to forskolin is dependent in large part on activity of Ca²⁺-dependent potassium channels. Vasodilatation in response to sodium nitroprusside is not dependent on the activity of iberiotoxin-sensitive potassium channels.

Baseline diameters of cerebral arterioles tended to increase in the presence of charybdotoxin (50 nmol/L), but the change in diameter was very modest (77±5 versus 80±5 µm). Charybdotoxin produced marked inhibition of dilatation of cerebral arterioles in response to forskolin (Fig 3) without affecting vasodilatation in response to sodium nitroprusside (Fig 3). The findings with charybdotoxin support those with iberiotoxin and suggest that dilatation of cerebral arterioles in response to forskolin is dependent on the activity of Ca²⁺-dependent potassium channels.

View larger version (16K): [in this window] [in a new window]   Figure 3. Bar graphs showing changes in diameters of cerebral arterioles in response to forskolin and sodium nitroprusside in the absence and presence of charybdotoxin (50 nmol/L). Baseline diameter of cerebral arterioles was 77±5 µm in the absence of charybdotoxin and 80±5 µm in the presence of iberiotoxin. Values are mean±SEM (n=8 for forskolin and n=11 for nitroprusside). *P<.05 vs control response.

Dibutyryl cAMP produced concentration-dependent vasodilatation. In response to dibutyryl cAMP (100 and 300 µmol/L), diameters of cerebral arterioles increased by 16±3% and 42±9% and by 18±1% and 48±6% during the first and second applications, respectively. These findings indicate that dilatation of cerebral arterioles in response to dibutyryl cAMP are reproducible.

Iberiotoxin (100 nmol/L) inhibited vasodilatation in response to dibutyryl cAMP (Fig 4). The increase in diameter of cerebral arterioles in response to the low and high concentrations of dibutyryl cAMP was inhibited by 35% and 28%, respectively. These findings suggest that cerebral vasodilatation in response to increases in intracellular concentrations of cAMP is mediated, in part, by activation of Ca²⁺-dependent potassium channels.

View larger version (16K): [in this window] [in a new window]   Figure 4. Bar graph showing changes in diameters of cerebral arterioles in response to dibutyryl cAMP in the absence and presence of iberiotoxin (100 nmol/L). Baseline diameter of cerebral arterioles was 75±12 µm in the absence of iberiotoxin and 73±11 µm in the presence of iberiotoxin. Values are mean±SEM (n=7). *P<.05 vs control response.

To further examine the specificity of iberiotoxin, we examined effects of iberiotoxin on vasodilatation in response to acetylcholine and aprikalim. Dilatation of cerebral arterioles in response to acetylcholine (Fig 5) and aprikalim (Fig 6) was not affected by iberiotoxin. These findings suggest that iberiotoxin does not inhibit (1) dilatation in response to acetylcholine, which is mediated by endothelium-derived relaxing factor (nitric oxide) and activation of guanylate cyclase in vascular muscle, or (2) cerebral vasodilatation in response to activation of ATP-sensitive potassium channels.

View larger version (15K): [in this window] [in a new window]   Figure 5. Bar graph showing changes in diameters of cerebral arterioles in response to acetylcholine in the absence and presence of iberiotoxin (100 nmol/L). Baseline diameter of cerebral arterioles was 76±11 µm in the absence of iberiotoxin and 72±12 µm in the presence of iberiotoxin. Values are mean±SEM (n=4).

View larger version (13K): [in this window] [in a new window]   Figure 6. Bar graph showing changes in diameters of cerebral arterioles in response to aprikalim in the absence and presence of iberiotoxin (100 nmol/L). Baseline diameter of cerebral arterioles was 75±12 µm in the absence of iberiotoxin and 73±11 µm in the presence of iberiotoxin. Values are mean±SEM (n=4).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our results suggest that dilatation of cerebral arterioles in response to forskolin and dibutyryl cAMP in vivo is mediated, in part, by activation of iberiotoxin- and charybdotoxin-sensitive potassium channels. This finding suggests that activation of adenylate cyclase stimulates Ca²⁺-dependent potassium channels in cerebral arterioles. The finding that acetylcholine-induced and sodium nitroprusside–induced dilatation of cerebral arterioles is not inhibited by charybdotoxin or iberiotoxin suggests that activation of guanylate cyclase does not stimulate Ca²⁺-dependent potassium channels in cerebral arterioles. To our knowledge, this is the first study to examine the role of Ca²⁺-dependent potassium channels in any vascular bed in vivo.

Large-conductance Ca²⁺-dependent potassium channels appear to be present in most types of vascular muscle.² Patch-clamp studies suggest that activation of a cAMP-dependent protein kinase increases the open probability of Ca²⁺-dependent potassium channels in vascular muscle.^{12 18} Thus, we anticipated that dilator responses of cerebral arterioles to stimuli that increase intracellular cAMP may be mediated by activation of Ca²⁺-dependent potassium channels. To test this hypothesis, we examined the effects of charybdotoxin, an inhibitor of Ca²⁺-dependent potassium channels,^{2 19} on the dilatation of cerebral arterioles in response to forskolin (a direct activator of adenylate cyclase).²⁰ Forskolin produced a marked dilatation of cerebral arteries that was inhibited by charybdotoxin. Because charybdotoxin may block other types of potassium channels under some conditions,^{2 21} we also tested the effects of iberiotoxin (a very selective inhibitor of Ca²⁺-dependent potassium channels^{2 21} ) on forskolin-induced dilatation of cerebral arterioles. Iberiotoxin, like charybdotoxin, produced marked inhibition of forskolin-induced cerebral vasodilatation. These findings suggest that dilatation of cerebral arterioles in response to forskolin is mediated, in large part, by activation of Ca²⁺-dependent potassium channels in vivo.

Although it is well documented that forskolin increases intracellular levels of cAMP (including in cerebral blood vessels^{3 4} ), we considered the possibility that forskolin may also exhibit nonspecific effects, such as activation of potassium channels independent of the activation of adenylate cyclase.¹⁶ To address this possibility, we examined the effects of 1,9-dideoxyforskolin on the diameters of cerebral arterioles. Although this structural analogue of forskolin has been used as a control for nonspecific effects of forskolin,^{16 17 22} we are not aware of any studies that examined the effects of this compound on the relaxation of blood vessels in vitro or in vivo. Nonspecific effects of forskolin on potassium channels (independent of activation of adenylate cyclase) have been found to be mimicked by 1,9-dideoxyforskolin.¹⁶ Thus, if forskolin were activating potassium channels in the present study via a mechanism that does not involve adenylate cyclase, one would expect forskolin and 1,9-dideoxyforskolin to exhibit similar effects and potency. We found that 1,9-dideoxyforskolin is relatively inactive compared with forskolin, which suggests that dilatation of cerebral arterioles in response to forskolin is due in very large part to the activation of adenylate cyclase and not to nonspecific effects. It is unclear why the high concentration of 1,9-dideoxyforskolin produced modest dilatation of cerebral arterioles, although the response may be due to the activation of potassium channels independent of activation of adenylate cyclase.¹⁶

If the iberiotoxin-sensitive component of forskolin-induced dilatation of cerebral arterioles is due to the activation of adenylate cyclase and the endogenous accumulation of cAMP, we anticipated that vasodilatation in response to exogenous cAMP would also be inhibited by iberiotoxin. In support of this hypothesis, we found that vasodilatation in response to dibutyryl cAMP (a cell-permeable analogue of cAMP) was inhibited by iberiotoxin.

A cGMP-dependent protein kinase increases the open probability of Ca²⁺-dependent potassium channels in dispersed cerebral vascular muscle.²³ Thus, we considered the possibility that a cGMP-dependent mechanism might modulate Ca²⁺-dependent potassium channels in cerebral arterioles. We found that neither charybdotoxin nor iberiotoxin altered dilator responses of cerebral arterioles to sodium nitroprusside (which activates guanylate cyclase and causes accumulation of cGMP). In addition, iberiotoxin had no effect on cerebral vasodilatation in response to acetylcholine. Dilatation of cerebral arterioles in response to acetylcholine is mediated by the release of endothelium-derived relaxing factor (nitric oxide) and the activation of guanylate cyclase. Thus, dilatation of cerebral arterioles produced by the activation of guanylate cyclase does not appear to be mediated by the activation of Ca²⁺-dependent potassium channels in vivo.

Our findings with acetylcholine and nitroprusside agree with previous studies of isolated vessels in vitro, in which relaxation in response to acetylcholine and nitroglycerin (which also increases cGMP concentrations in vascular muscle) was not affected by charybdotoxin.^{24 25} Tetraethylammonium ion, which also inhibits Ca²⁺-dependent potassium channels,² had little effect on the relaxation of coronary arteries in response to acetylcholine and nitroprusside in vitro.²⁶ Direct measurements of membrane potential also suggest that increases in cGMP levels do not open potassium channels in cerebral vessels.^{27 28} Nitric oxide (which increases cGMP concentrations and produces relaxation) does not hyperpolarize cerebral vascular muscle in intact vascular segments.^{27 28} In some noncerebral blood vessels, increases in cGMP may influence the activity of Ca²⁺-dependent potassium channels.²⁹

It is unlikely that the inhibitory effects of charybdotoxin and iberiotoxin on the dilatation of cerebral arterioles in response to forskolin were nonspecific. These toxins, particularly iberiotoxin, appear to be highly selective in their effects.² Both iberiotoxin and charybdotoxin produced an inhibition of responses to forskolin without affecting vasodilatation in response to sodium nitroprusside. Iberiotoxin also did not inhibit vasodilatation in response to acetylcholine. We have shown that the dilatation of cerebral arterioles in response to aprikalim, a direct activator of ATP-sensitive potassium channels,³⁰ is not affected by charybdotoxin,³¹ and in the present study, vasodilatation in response to aprikalim was not inhibited by iberiotoxin. Thus, there is considerable evidence demonstrating that the effects of iberiotoxin and charybdotoxin on cerebral arterioles are specific.

In large cerebral arteries in vitro, inhibitors of Ca²⁺-dependent potassium channels produce depolarization of smooth muscle and vasoconstriction.^{14 32} In the present study, the application of charybdotoxin and iberiotoxin had little effect on the baseline diameters of cerebral arterioles. The higher concentration of iberiotoxin (100 nmol/L) constricted cerebral arterioles, but the effect was very small. These findings suggest that although Ca²⁺-dependent potassium channels may play an important role in agonist-induced vasodilatation, these channels appear to have a more minor role in the regulation of basal tone of cerebral arterioles (cerebral microvessels) in vivo. The difference in magnitude of influence of these channels on basal tone in the present study and in previous studies^{14 32} may relate to conditions of the experiment (in vitro versus in vivo) but more likely represents segmental differences (large versus small vessels). In another study,³³ we observed constriction of the basilar artery in response to tetraethylammonium ion in vivo. Constriction of the basilar artery in response to tetraethylammonium in vivo³³ may reflect an inhibition of activity of Ca²⁺-dependent potassium channels in large cerebral vessels. It is also possible that constrictor effects of charybdotoxin and iberiotoxin on cerebral arterioles are attenuated by other compensatory mechanisms, so that basal vascular tone in vivo increases minimally.

It is important to recognize that in addition to effects on Ca²⁺-dependent potassium channels, recent evidence suggests that cAMP may also activate ATP-sensitive potassium channels in some blood vessels.^{2 17 22} Dilatation of the basilar artery in response to forskolin is inhibited modestly by glibenclamide, an inhibitor of ATP-sensitive potassium channels.³⁴ Inhibition of the cerebrovascular response to forskolin by glibenclamide,³⁴ however, was much less than we observed in the present study using charybdotoxin and iberiotoxin. Thus, dilatation of cerebral vessels in response to forskolin appears to be mediated in large part by activation of Ca²⁺-dependent potassium channels. However, because the highest concentration of iberiotoxin did not completely inhibit the dilatation of cerebral arterioles in response to forskolin or dibutyryl cAMP, activation of ATP-sensitive potassium channels or mechanisms that are independent of potassium channels also may contribute to the vascular response.³⁵

In conclusion, dilatation of cerebral arterioles in response to forskolin (which activates adenylate cyclase) is mediated, in large part, by activation of Ca²⁺-dependent potassium channels in vivo. Vasodilatation in response to increases in intracellular concentrations of cAMP was also mediated, in part, by activation of Ca²⁺-dependent potassium channels. The implication of these findings is that the activity of Ca²⁺-dependent potassium channels may have a major influence on cerebral vascular tone in response to vasoactive stimuli that increase intracellular cAMP levels. Such stimuli include adenosine, prostacyclin, vasoactive intestinal peptide, and some types of seizures.^{3 36 37 38} Vasodilatation in response to nitroprusside (which activates guanylate cyclase) and acetylcholine (which causes release of endothelium-derived relaxing factor, which activates guanylate cyclase)³⁹ does not appear to be dependent on the activation of Ca²⁺-dependent potassium channels in cerebral arterioles.

	Acknowledgments

 
This study was supported by National Institutes of Health grants HL-38901, NS-24621, AG-10269, HL-16066, and HL- 14355 and a Grant-in-Aid from the American Heart Association (92015170). Dr Faraci is an Established Investigator of the American Heart Association. The technical assistance of Cynthia Lynch is gratefully acknowledged.

Received July 25, 1994; accepted February 14, 1995.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Olsson RA, Pearson JD. Cardiovascular purinoceptors. Physiol Rev. 1990;70:761-845. [Free Full Text]

2. Nelson MT. Ca²⁺-activated potassium channels and ATP-sensitive potassium channels as modulators of vascular tone. Trends Cardiovasc Med. 1993;3:54-60.

3. Edvinsson L, Fredholm BB, Hamel E, Jansen I. Perivascular peptides relax cerebral arteries concomitant with stimulation of cyclic adenosine monophosphate accumulation or release of an endothelium-derived relaxing factor in the cat. Neurosci Lett. 1985;58:213-217. [Medline] [Order article via Infotrieve]

4. Suzuki Y, Huang M, Lederis K, Rorstad OP. The role of adenylate cyclase in relaxation of brain arteries: studies with forskolin. Brain Res. 1988;457:241-245. [Medline] [Order article via Infotrieve]

5. Ayajiki K, Toda N. Regional difference in the response mediated by ß1-adrenoceptor in bovine cerebral arteries. J Cereb Blood Flow Metab. 1992;12:507-513. [Medline] [Order article via Infotrieve]

6. Okumura R, Hatake K, Wakabayashi I, Suehiro A, Hishida S, Kakishita E. Vasorelaxant effect of trapidil on human basilar artery. J Pharm Pharmacol. 1992;44:425-428. [Medline] [Order article via Infotrieve]

7. Horsburgh K, Jansen I, Edvinsson L, McCulloch J. Second messenger systems: functional role in cerebrovascular smooth muscle regulation. Eur J Pharmacol. 1990;191:205-211. [Medline] [Order article via Infotrieve]

8. Busija DW, Chen J. Reversal by increased CSF [H⁺] and [K⁺] of phorbol ester-induced arteriolar constriction in piglets. Am J Physiol. 1992;263:H1455-H1459. [Abstract/Free Full Text]

9. Rosenblum WI. In vivo evidence that an adenylate cyclase-cAMP system dilates cerebral arterioles in mice. Stroke. 1988;19:888-891. [Abstract/Free Full Text]

10. Kitazono T, Heistad DD, Faraci FM. ATP-sensitive potassium channels in the basilar artery during chronic hypertension. Hypertension. 1993;22:677-681. [Abstract/Free Full Text]

11. Wysham DG, Brotherton AF, Heistad DD. Effects of forskolin on cerebral blood flow: implications for a role of adenylate cyclase. Stroke. 1986;17:1299-1303. [Abstract/Free Full Text]

12. Minami K, Fukuzawa K, Nakaya Y, Zeng X, Inoue I. Mechanism of activation of the Ca²⁺-activated K⁺ channel by cyclic AMP in the cultured porcine coronary artery smooth muscle cells. Life Sci. 1993;53:1129-1135. [Medline] [Order article via Infotrieve]

13. Kume H, Hall IP, Washabau RJ, Takagi K, Kotlikoff MI. ß-Adrenergic agonists regulate K_CA channels in airway smooth muscles by cAMP-dependent and -independent mechanisms. J Clin Invest. 1994;93:371-379.

14. Brayden JE, Nelson MT. Regulation of arterial tone by activation of calcium-dependent potassium channels. Science. 1992;256:532-535. [Abstract/Free Full Text]

15. Mayhan WG, Amundsen SM, Faraci FM, Heistad DD. Response of cerebral arteries after ischemia and reperfusion in cats. Am J Physiol. 1988;255:H879-H884. [Abstract/Free Full Text]

16. Hoshi T, Garber SS, Aldrich RW. Effect of forskolin on voltage-gated K⁺ channels is independent of adenylate cyclase activation. Science. 1988;240:1652-1655. [Abstract/Free Full Text]

17. Quayle JM, Bonev AD, Brayden JE, Nelson MT. Calcitonin gene-related peptide activated ATP-sensitive K⁺ currents in rabbit arterial smooth muscle via protein kinase A. J Physiol (Lond). 1994;475:9-13. [Abstract/Free Full Text]

18. Sadoshima J, Akaike N, Kanaide H, Nakamura M. Cyclic AMP modulates Ca⁺⁺-activated K channel in cultured smooth muscle cells of rat aortas. Am J Physiol. 1988;255:H754-H759. [Abstract/Free Full Text]

19. Miller CE, Moczydlowski E, Latorre R, Philips M. Charybdotoxin, a protein inhibitor of single Ca²⁺-activated K⁺ channels from mammalian skeletal muscle. Nature. 1985;313:316-318. [Medline] [Order article via Infotrieve]

20. Seamom KB, Daly JW. Forskolin: a unique diterpene activator of cyclic-AMP generating systems. J Cyclic Nucleotide Res. 1981;7:201-224. [Medline] [Order article via Infotrieve]

21. Galvez A, Gimenez-Gallego G, Reuben JP, Roy-Contancin L, Feigenbaum P, Kaczorowski GJ, Garcia ML. Purification and characterization of a unique, potent, peptidyl probe for the high conductance calcium-activated potassium channel from venom of the scorpion Buthus tamulus. J Biol Chem. 1990;265:11083-11090. [Abstract/Free Full Text]

22. Nakashima M, Vanhoutte PM. Isoproterenol causes hyperpolarization through opening of ATP-sensitive potassium channels in vascular smooth muscle of the canine saphenous vein. J Pharmacol Exp Ther. 1995;272:379-384. [Abstract/Free Full Text]

23. Robertson BE, Schubert R, Hescheler J, Nelson MT. cGMP dependent protein kinase activates Ca-activated K channels in cerebral artery smooth muscle cells. Am J Physiol. 1993;265:C299-C303. [Abstract/Free Full Text]

24. Najibi S, Cowan CL, Palacino JJ, Cohen RA. Enhanced role of potassium channels in relaxations to acetylcholine in hypercholesterolemic rabbit carotid artery. Am J Physiol. 1994;266:H2061-H2067. [Abstract/Free Full Text]

25. Hamaguchi M, Ishibashi T, Imai S. Involvement of charybdotoxin-sensitive K⁺ channel in the relaxation of bovine tracheal smooth muscle by glyceryl trinitrate and sodium nitroprusside. J Pharmacol Exp Ther. 1991;262:263-270. [Abstract/Free Full Text]

26. Cabell F, Weiss DS, Price JM. Inhibition of adenosine-induced coronary vasodilation by block of large-conductance Ca²⁺-activated K⁺ channels. Am J Physiol. 1994;267:H1455-H1460. [Abstract/Free Full Text]

27. Brayden JE. Membrane hyperpolarization is a mechanism of endothelium-dependent cerebral vasodilation. Am J Physiol. 1990;259:H668-H673. [Abstract/Free Full Text]

28. Plane F, Garland CJ. Differential effects of acetylcholine, nitric oxide and levcromakalim on smooth muscle membrane potential and tone in the rabbit basilar artery. Br J Pharmacol. 1993;110:651-656. [Medline] [Order article via Infotrieve]

29. Khan SA, Mathews WR, Meisheri KD. Role of calcium activated K⁺ channels in vasodilatation induced by nitroglycerine, acetylcholine and nitric oxide. J Pharmacol Exp Ther. 1993;267:1327-1335. [Abstract/Free Full Text]

30. Aloup JC, Farge D, James C, Mondot S, Cavero I. 2-(3-Pyridyl)-tetrahydrothiopyran-2-carbothiamide derivatives and analogues: a novel family of potent potassium channel openers. Drugs Future. 1990;15:1097-1108.

31. Mayhan WG, Faraci FM. Responses of cerebral arterioles in diabetic rats to activation of ATP-sensitive potassium channels. Am J Physiol. 1993;265:H152-H157. [Abstract/Free Full Text]

32. Asano M, Masuzawa-Ito K, Matsuda T, Suzuki Y, Oyama H, Shibuya M, Sugita K. Functional role of charybdotoxin-sensitive K⁺ channels in the resting state of cerebral, coronary and mesenteric arteries of the dog. J Pharmacol Exp Ther. 1993;267:1277-1285. [Abstract/Free Full Text]

33. Fujii K, Heistad DD, Faraci FM. Flow-mediated dilatation of the basilar artery in vivo. Circ Res. 1991;69:697-705. [Abstract/Free Full Text]

34. Kitazono T, Faraci FM, Heistad DD. Effect of norepinephrine on rat basilar artery in vivo. Am J Physiol. 1993;264:H178-H182. [Abstract/Free Full Text]

35. Torphy TJ. ß-Adrenoceptors, cAMP and airway smooth muscle relaxation: challenges to the dogma. Trends Pharmacol Sci. 1994;15:370-374. [Medline] [Order article via Infotrieve]

36. Edvinsson L, Fredholm BB. Characterization of adenosine receptors in isolated cerebral arteries of cat. Br J Pharmacol. 1983;80:631-637.[Medline] [Order article via Infotrieve]

37. Ferrendelli JA, Blank AC, Gross RA. Relationships between seizure activity and cyclic nucleotide levels in brain. Brain Res. 1980;200:93-103. [Medline] [Order article via Infotrieve]

38. Huang M, Rorstad OP. Cerebral vascular adenylate cyclase: evidence for coupling to receptors for vasoactive intestinal peptide and parathyroid hormone. J Neurochem. 1984;43:849-856. [Medline] [Order article via Infotrieve]

39. Faraci FM, Brian JE. Nitric oxide and the cerebral circulation. Stroke. 1994;25:692-703.[Abstract]

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