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(Stroke. 1996;27:1603-1608.)
© 1996 American Heart Association, Inc.

Articles

Role of Ca²⁺-Dependent K⁺ Channels in Cerebral Vasodilatation Induced by Increases in Cyclic GMP and Cyclic AMP in the Rat

Roberto Paterno, MD; Frank M. Faraci, PhD Donald D. Heistad, MD

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, University of Iowa College of Medicine, Iowa City, IA 52242-1081.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
Background and Purpose The mechanisms by which cAMP and cGMP produce vasorelaxation are not entirely clear. In this study we examined the hypothesis that relaxation of cerebral arterioles in response to receptor-mediated activation of adenylate cyclase (increase in cAMP) is mediated through Ca²⁺-dependent K⁺ channels.

Methods We measured the diameter of cerebral arterioles (basal diameter, 47±1 µm) using an open cranial window in anesthetized rats. Agonists and antagonists were applied locally in the cranial window.

Results Topical application of adenosine (0.1 and 1 mmol/L), a receptor-mediated activator of adenylate cyclase, and dibutyryl cAMP (60 and 200 µmol/L), a cell-permeable analogue of cAMP, dilated cerebral arterioles. Iberiotoxin (50 nmol/L), a selective inhibitor of Ca²⁺-dependent K⁺ channels, reduced vasodilatation in response to 0.1 and 1 mmol/L adenosine by 66% and 28%, respectively. Tetraethylammonium (TEA) (1 mmol/L), another inhibitor of Ca²⁺-dependent K⁺ channels, reduced vasodilatation to 0.1 and 1 mmol/L adenosine by 58% and 42%, respectively, and reduced vasodilatation in response to 60 and 200 µmol/L dibutyryl cAMP by 75% and 66%, respectively. Topical application of sodium nitroprusside (0.1 and 1 µmol/L), a direct activator of guanylate cyclase, and 8-bromo cGMP (200 and 600 µmol/L), a cell-permeable analogue, produced dilatation of cerebral arterioles that was inhibited by iberiotoxin (50 nmol/L) and TEA (1 and 3 mmol/L). In contrast, dilatation of cerebral arterioles in response to papaverine (which produces vasodilatation in large part by inhibition of Ca²⁺ channels) and aprikalim (which produces vasodilatation by activation of ATP-sensitive K⁺ channels) was not inhibited by iberiotoxin or TEA.

Conclusions These findings suggest that dilatation of cerebral arterioles by receptor-mediated activation of adenylate cyclase and by direct activation of guanylate cyclase in the rat is mediated in large part by activation of Ca²⁺-dependent K⁺ channels.

Key Words: adenosine • arterioles • cyclic AMP • cyclic GMP • nitric oxide • potassium channels

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
Two biochemical pathways that produce relaxation of vascular muscle are activation of adenylate cyclase with accumulation of cAMP^{1 2} and activation of guanylate cyclase with accumulation of cGMP.³ Adenosine produces receptor-mediated activation of adenylate cyclase² and dilatation of cerebral arteries in vitro^{4 5} and in pial arterioles in vivo.^{6 7} Application of dibutyryl cAMP, a cell-permeable analogue of cAMP, also produces dilatation of cerebral vessels.⁸ Nitric oxide and sodium nitroprusside, a nitric oxide donor, activate guanylate cyclase and increase concentration of cGMP, producing relaxation of cerebral vessels. 8-Bromo cGMP (a cell-permeable analogue of cGMP) also produces dilatation of cerebral arteries in vitro^{9 10} and in pial arterioles in vivo.^{11 12}

Activation of K⁺ channels in vascular muscle produces hyperpolarization, which is a major mechanism of vasorelaxation.^{1 13} Patch-clamp experiments suggest that levels of cAMP and/or cGMP are important determinants of activity of Ca²⁺-dependent K⁺ channels in vascular muscle.^{14 15 16} Thus, these studies suggest that the activity of Ca²⁺-dependent K⁺ channels may be a common pathway by which elevated levels of both cAMP and cGMP produce vasodilatation.

A recent study suggested that dilatation of cerebral arterioles in response to forskolin, a direct activator of adenylate cyclase, is mediated by activation of Ca²⁺-dependent K⁺ channels.⁸ To further examine this hypothesis, we determined whether cerebral vasodilatation in response to receptor-mediated activation of adenylate cyclase (using adenosine) was dependent on activation of Ca²⁺-dependent K⁺ channels. Iberiotoxin^{1 17} and tetraethylammonium (TEA) ions^{18 19 20} were used as inhibitors of K⁺ channels. In initial studies, sodium nitroprusside was used as an internal control. Because we found that vasodilatation in response to nitroprusside was also inhibited by TEA and iberiotoxin in the rat, additional experiments were performed with dibutyryl cAMP and 8-bromo cGMP. Papaverine, which relaxes vascular muscle in large part by inhibition of Ca²⁺ channels,²¹ and aprikalim, which relaxes vascular muscle by activation of K_ATP, were used as internal controls.¹³

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
Animal Preparation
Sprague-Dawley rats (n=59; mean±SEM weight, 360±7 g) were anesthetized with sodium pentobarbital (50 mg/kg IP). Pentobarbital was supplemented at approximately 25 mg/kg IV per hour. We evaluated depth of anesthesia by applying pressure to a paw and observing changes in heart rate or blood pressure. When such changes occurred, additional anesthetic was administered. The trachea was cannulated, and the animals were ventilated mechanically with air and supplemental oxygen. A catheter was placed in the femoral artery to measure systemic arterial pressure and to obtain arterial blood samples. A femoral vein was cannulated for infusion of drugs. Gallamine triethiodide (13 to 18 mg/kg IV) was used for skeletal muscle paralysis. Arterial blood gases were monitored and maintained within normal limits throughout the experiments (pH 7.42±0.001, PCO₂=38±0.1 mmHg, PO₂=147±2 mmHg).

A cranial window was prepared over the left parietal cortex and suffused with artificial cerebrospinal fluid (CSF) bubbled continuously with a gas mixture. In artificial CSF (temperature approximately 37.5°C), pH was 7.41±0.01, PCO₂ was 47±0.01 mmHg, and PO₂ was 79±1 mmHg. The diameter of pial arterioles (baseline=47±1 µm) (fourth order of the middle cerebral artery) was measured with a microscope equipped with a television camera coupled to a video monitor. Images were recorded on videotape, and vessel diameters were measured with an image shearing device.

Experimental Protocol
Four groups of animals were studied. In each group of rats, cerebral vessels were superfused with artificial CSF for 60 minutes before application of agonists.

In group 1 (time controls), arteriolar diameter was measured before application of agonists and during maximal vasodilatation in response to topical suffusion of adenosine (0.1 and 1 mmol/L) (n=5), dibutyryl cAMP (60 and 200 µmol/L) (n=5), sodium nitroprusside (0.1 and 1 µmol/L) (n=5), 8-bromo cGMP (0.2 and 0.6 mmol/L) (n=5), papaverine (0.1 and 0.3 mmol/L) (n=4), and aprikalim (10 and 100 µmol/L) (n=5). In each experiment, only two of these agonists were tested. Drugs and order of application were altered, and concentrations of agonists were applied in a cumulative manner. After a 40-minute recovery period to allow diameters of arterioles to return to baseline, the application of each agonist was repeated (recovery period). This group of animals functioned as a time control to establish whether responses to adenosine, dibutyryl cAMP, sodium nitroprusside, 8-bromo cGMP, and papaverine were reproducible.

In group 2 (iberiotoxin), arteriolar diameter was measured before application of agonists and during maximal vasodilatation in response to topical suffusion of adenosine (0.1 and 1 mmol/L) and sodium nitroprusside (0.1 and 1 µmol/L). After a 40-minute recovery period, application of agonists was repeated in the presence of iberiotoxin 5 nmol/L (n=5) or 50 nmol/L (n=5). The cranial window was treated with iberiotoxin for 10 minutes before application of agonists. The purpose of these experiments was to determine whether iberiotoxin inhibits dilatation of cerebral arterioles in response to adenosine and sodium nitroprusside.

In group 3 (TEA), arteriolar diameter was measured before the application of agonists and during the maximal vasodilatation in response to topical suffusion of adenosine (0.1 and 1 mmol/L), dibutyryl cAMP (60 and 200 µmol/L), sodium nitroprusside (0.1 and 1 µmol/L), and 8-bromo cGMP (200 and 600 µmol/L). After a 40-minute recovery period, application of agonists was repeated in the presence of TEA 1 mmol/L (n=6) or 3 mmol/L (n=4).

In group 4 (papaverine), arteriolar diameter was measured before the application of agonist and during maximal vasodilatation in response to topical suffusion of papaverine (0.1 and 0.3 mmol/L). After a 40-minute recovery period, application of agonist was repeated in the presence of iberiotoxin (n=5) (50 nmol/L). In other experiments, application of agonist was repeated in the presence of TEA (1 mmol/L) (n=6). The cranial window was treated with iberiotoxin or TEA for 10 minutes before application of agonist. Papaverine produces vasodilatation in large part by inhibition of Ca²⁺ channels²¹ and was used to determine whether TEA and iberiotoxin produce nonspecific inhibition of vasodilatation.

In group 5 (aprikalim), arteriolar diameter was measured before the application of agonist and during maximal vasodilatation in response to topical suffusion of aprikalim (10 and 100 µmol/L). After a 1-hour recovery period, application of agonist was repeated in the presence of TEA (n=6) (1 mmol/L). The cranial window was treated with TEA for 10 minutes before application of agonist. Aprikalim produces vasodilatation by activation of ATP-sensitive K⁺ channels and was used to determine whether TEA produces nonspecific inhibition of vasodilatation.

Drugs
Adenosine, dibutyryl cAMP, sodium nitroprusside, 8-bromo cGMP, papaverine, and TEA were purchased from Sigma Chemical Co. Aprikalim was a gift from Rhone-Poulenc Rorer, Vitry-Alfortville, France. Iberiotoxin was purchased from Research Biochemicals International.

Statistical Analysis
Values are percent change in diameter of cerebral arterioles and are expressed as mean±SEM. A paired t test was used for comparison of percent change in the absence and presence of inhibitors. Values of P<.05 were considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
Adenosine (0.1 and 1 mmol/L), dibutyryl cAMP (60 and 200 µmol/L), sodium nitroprusside (0.1 and 1 µmol/L), 8-bromo cGMP (200 and 600 µmol/L), papaverine (0.1 and 0.3 mmol/L), and aprikalim (10 and 100 µmol/L) produced reproducible, concentration-related dilatation of cerebral arterioles.

Iberiotoxin and TEA had no significant effect on resting diameter of cerebral arterioles; the change in diameter of cerebral arterioles was 2±1%, 0±1%, and 0±1% in response to iberiotoxin (50 nmol/L), 1 mmol/L TEA, and 3 mmol/L TEA, respectively.

Iberiotoxin (50 nmol/L) inhibited dilatation of cerebral arterioles in response to adenosine (Fig 1) and sodium nitroprusside (Fig 2). Inhibition by iberiotoxin was 66% and 28% in response to low and high concentrations, respectively, of adenosine. Inhibition was 40% and 44% in response to low and high concentrations of sodium nitroprusside. A low concentration of iberiotoxin (5 nmol/L) had no effect on vasodilatation in response to adenosine or sodium nitroprusside.

View larger version (14K): [in this window] [in a new window]   Figure 1. Change in diameter of cerebral arterioles in response to adenosine in the absence and presence of iberiotoxin (50 nmol/L). Values are percent change in diameter (mean±SEM) in six animals. *P<.05 vs control response.

View larger version (14K): [in this window] [in a new window]   Figure 2. Change in diameter of cerebral arterioles in response to sodium nitroprusside in the absence and presence of iberiotoxin (50 nmol/L). Values are percent change in diameter (mean±SEM) in five animals. *P<.05 vs control response.

TEA (1 mmol/L) produced significant inhibition of dilatation of cerebral arterioles in response to adenosine, dibutyryl cAMP, sodium nitroprusside, and 8-bromo cGMP. Inhibition by TEA, in response to low and high concentrations of these agonists, was 58% and 42%, respectively, with adenosine (Fig 3); 75% and 66% with dibutyryl cAMP (Fig 4); 44% and 32% (TEA 1 mmol/L) and 64% and 45% (TEA 3 mmol/L) with sodium nitroprusside (Fig 5); and 53% and 48% with 8-bromo cGMP (Fig 6). Iberiotoxin (50 nmol/L) and TEA (1 mmol/L) had no effect on vasodilatation in response to papaverine (Fig 7). TEA (1 mmol/L) had no effect on vasodilatation in response to aprikalim (Fig 8).

View larger version (13K): [in this window] [in a new window]   Figure 3. Change in diameter of cerebral arterioles in response to adenosine in the absence and presence of tetraethylammonium (TEA) (1 mmol/L). Values are percent change in diameter (mean±SEM) in six animals. *P<.05 vs control response.

View larger version (15K): [in this window] [in a new window]   Figure 4. Change in diameter of cerebral arterioles in response to dibutyryl cAMP in the absence and presence of tetraethylammonium (TEA) (1 mmol/L). Values are percent change in diameter (mean±SEM) in five animals. *P<.05 vs control response.

View larger version (17K): [in this window] [in a new window]   Figure 5. Change in diameter of cerebral arterioles in response to sodium nitroprusside in the absence and presence of tetraethylammonium (TEA) (1 mmol/L and 3 mmol/L). Values are percent change in diameter (mean±SEM) in six and four animals, respectively. *P<.05 vs control response.

View larger version (15K): [in this window] [in a new window]   Figure 6. Change in diameter of cerebral arterioles in response to 8-bromo cGMP in the absence and presence of tetraethylammonium (TEA) (1 mmol/L). Values are percent change in diameter (mean±SEM) in five animals. *P<.05 vs control response.

View larger version (18K): [in this window] [in a new window]   Figure 7. Change in diameter of cerebral arterioles in response to papaverine in the absence and presence of iberiotoxin (50 nmol/L) and tetraethylammonium (TEA) (1 mmol/L). Values are percent change in diameter (mean±SEM) in five and six animals, respectively. *P<.05 vs control response.

View larger version (11K): [in this window] [in a new window]   Figure 8. Change in diameter of cerebral arterioles in response to aprikalim in the absence and presence of tetraethylammonium (TEA) (1 mmol/L). Values are percent change in diameter (mean±SEM) in six animals. *P<.05 vs control response.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
The major finding in the present study is that dilatation of cerebral arterioles induced by adenosine and dibutyryl cAMP, as well as sodium nitroprusside and 8-bromo cGMP, is inhibited by iberiotoxin and TEA in the rat. These results suggest that dilatation of cerebral arterioles induced by both receptor-mediated activation of adenylate cyclase and direct activation of guanylate cyclase is mediated in part by activation of Ca²⁺-dependent K⁺ channels.

Large-conductance Ca²⁺-dependent K⁺ channels, which are activated by membrane depolarization and increases in intracellular calcium, have been described in a variety of types of smooth muscle.¹ Studies in vitro in which patch-clamp techniques were used suggest that these channels are present in cerebral vessels and that they participate in regulation of vascular tone.¹⁸

Patch-clamp studies also suggest that activation of a cAMP-dependent protein kinase increases the open probability of Ca²⁺-dependent K⁺ channels in vascular muscle.^{16 22} Dilatation of cerebral arterioles in the rabbit in response to direct activation of adenylate cyclase, with the use of forskolin, is mediated in large part by activation of Ca²⁺-dependent K⁺ channels.⁸

In the present study adenosine produced dilatation of cerebral arterioles that was inhibited by iberiotoxin and TEA. These findings suggest that cerebral vasodilatation during receptor-mediated activation of adenylate cyclase is dependent on activation of Ca²⁺-dependent K⁺ channels. To further study the adenylate cyclase/cAMP pathway, we used dibutyryl cAMP (a cell-permeable analogue of cAMP). Dibutyryl cAMP produced dilatation of cerebral arterioles that was also inhibited by TEA (1 mmol/L). Thus, dilatation of cerebral arterioles in response to receptor-mediated activation of adenylate cyclase and cAMP is mediated in part by activation of Ca²⁺-dependent K⁺ channels.

Patch-clamp and in vitro studies suggest that activation of cGMP-dependent protein kinase increases the open probability of Ca²⁺-dependent K⁺ channels in vascular muscle from the pulmonary and basilar arteries.^{14 23} Activation of Ca²⁺-dependent K⁺ channels in the aorta appears to play an important role in mediating responses to activation of guanylate cyclase.²⁴ In contrast, relaxation of some blood vessels in response to activation of guanylate cyclase is not dependent on activation of Ca²⁺-dependent K⁺ channels. Nitric oxide, which increases intracellular concentrations of cGMP, does not hyperpolarize vascular muscle in large cerebral arteries in vitro, suggesting that K⁺ channels are not activated in response to nitric oxide.^{25 26}

In rabbit cerebral arterioles, we found that vasodilatation in response to nitroprusside was not affected by iberiotoxin or charybdotoxin (another inhibitor of Ca²⁺-dependent K⁺ channels).⁸ In contrast, in this study nitroprusside produced dilatation of cerebral arterioles of the rat that was inhibited by iberiotoxin and TEA. Similar results were obtained with 8-bromo cGMP and TEA. These findings suggest that dilatation of cerebral arterioles in the rat in response to activation of guanylate cyclase and cGMP is dependent on activation of Ca²⁺-dependent K⁺ channels. Taken together, the present findings in rats and our previous study in rabbits suggest that there may be species differences in the role of Ca²⁺-dependent K⁺ channels in mediating relaxation of cerebral arterioles in response to activation of guanylate cyclase. There are examples in vascular smooth muscle²⁷ and other cell types^{28 29 30} in which specific ion channels are activated by both cGMP and cAMP.

We speculate that the remaining vasodilatation, after application of agonists and inhibitors of Ca²⁺-dependent K⁺ channels, may be mediated by other cAMP-dependent and cGMP-dependent mechanisms or perhaps by other K⁺ channels. Application of inhibitors of Ca²⁺-dependent K⁺ channels, in large cerebral vessels in vitro, produces depolarization of vascular muscle and vasoconstriction.³¹ These same inhibitors produce contraction of the basilar artery in vivo.^{32 33} In contrast, in other studies it was found that application of Ca²⁺-dependent K⁺ channel inhibitors has little effect on baseline diameters of cerebral microvessels in vivo, suggesting that the inhibitors did not induce significant depolarization of vascular muscle.^{8 34} We found that iberiotoxin and TEA had a very modest effect on resting diameter of cerebral arterioles. These findings suggest that Ca²⁺-dependent K⁺ channels may play a more important role in the regulation of basal tone of large cerebral arteries than of cerebral arterioles.

It is important to emphasize that the conclusions obtained from the experiments are dependent on selectivity of agonists and antagonists. Because iberiotoxin and TEA inhibited both cAMP- and cGMP-mediated responses, it was important to address the specificity of the inhibitors in these experiments. For that purpose, we used papaverine and aprikalim. Papaverine is not a direct activator of adenylate or guanylate cyclase and produces vasodilatation mainly by inhibition of voltage-dependent Ca²⁺ channels.²¹ Aprikalim is a direct activator of ATP-sensitive K⁺ channels.¹³ The finding that iberiotoxin and TEA did not inhibit vasodilatation in response to papaverine or aprikalim suggests that the blockers did not produce nonspecific inhibition of vasodilator responses.

In conclusion, dilatation of cerebral arterioles of the rat in response to receptor-mediated activation of adenylate cyclase and cAMP as well as direct activation of guanylate cyclase appears to be dependent on activation of Ca²⁺-dependent K⁺ channels.

	Acknowledgments

 
This study was supported by National Institutes of Health grants NS-24621, HL-16066, HL-14388, AG-10269, and HL-38901 and by a Grant-in-Aid from the American Heart Association. Dr Faraci is an Established Investigator of the American Heart Association.

Received October 30, 1995; revision received May 28, 1996; accepted June 5, 1996.

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Editorial Comment

Joseph E. Brayden, PhD, Guest Editor

Department of PharmacologySmooth Muscle Ion Channel GroupUniversity of Vermont Medical Research FacilityColchester, Vt

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
Using an open cranial window technique to monitor cerebral arteriolar diameter in vivo, Paterno et al have investigated the possible mechanisms by which cAMP and cGMP dilate cerebral arteries. Dilations in response to adenosine, sodium nitroprusside, or cyclic nucleotide analogues, which activate these second messenger signaling pathways, were reduced by approximately 50% in the presence of inhibitors of Ca²⁺-dependent K⁺ channels but not other K⁺ channel blockers. These findings imply an important role for Ca²⁺-dependent K⁺ channels in the regulation of cerebrovascular tone by endogenous vasodilators that generate either cAMP or cGMP. Activation of K⁺ channels will hyperpolarize the vascular smooth muscle membrane potential, which will close voltage-dependent calcium channels, decrease calcium entry, and reduce intracellular free calcium and vascular tone.

The results of the present study are consistent with work by other investigators who have used in vitro approaches (ion channel, membrane potential, and diameter measurements) to reveal a significant role for Ca²⁺-dependent K⁺ channels in regulation of vascular tone. The present study is significant because it is a clear example of the importance of this system in vivo under conditions in which multiple physiological inputs (pressure, flow, hormonal factors, neurotransmitters, endothelial factors) are simultaneously contributing to regulation of vascular tone and blood flow. An important direction for future experiments in which this approach is used will be to determine the significance of this dilator system under conditions in which cerebral blood flow is altered by physiological or pathophysiological stimuli.

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