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(Stroke. 1997;28:1264-1271.)
© 1997 American Heart Association, Inc.

Articles

Role of Potassium Channels in Relaxations of Canine Middle Cerebral Arteries Induced by Nitric Oxide Donors

Hisashi Onoue, MD, PhD; Zvonimir S. Katusic, MD, PhD

From the Departments of Anesthesiology and Pharmacology, Mayo Clinic and Mayo Foundation, Rochester, Minn.

Correspondence to Zvonimir S. Katusic, MD, PhD, Departments of Anesthesiology and Pharmacology, Mayo Clinic, Rochester, MN 55905.

	Abstract

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Background and Purpose The mechanisms underlying smooth muscle relaxations of cerebral arteries in response to nitric oxide (NO) and cyclic GMP (cGMP) are still not completely understood. The present study was designed to determine the role of potassium channels in the relaxations to NO donors 3-morpholinosydnonimine (SIN-1) and sodium nitroprusside (SNP), as well as 8-bromo-3',5'-cGMP (a synthetic analogue of cGMP) and zaprinast (a selective cGMP phosphodiesterase inhibitor).

Methods Rings of canine middle cerebral arteries without endothelium were suspended in Krebs-Ringer bicarbonate solution for isometric tension recording. The levels of cGMP were measured by radioimmunoassay. Relaxations to NO donors 8-bromo-cGMP and zaprinast were studied in the presence and in the absence of K⁺ channel blockers charybdotoxin (large-conductance Ca²⁺-activated K⁺ channels), glyburide (ATP-sensitive K⁺ channels), 4-aminopyridine (delayed rectifier K⁺ channels), and BaCl₂ (multiple types of K⁺ channels).

Results Concentration-dependent relaxations caused by NO donors (SIN-1 and SNP) were significantly reduced in arteries treated with BaCl₂ (3x10^-4 mol/L) or charybdotoxin (3x10^-8 mol/L). Relaxations to 8-bromo-cGMP were not affected by the same concentrations of BaCl₂ and charybdotoxin; however, they were reduced by higher concentrations of BaCl₂ (3x10^-3 mol/L) and charybdotoxin (10^-7 mol/L). Zaprinast-induced relaxations were significantly reduced by BaCl₂ (3x10^-4 mol/L) or charybdotoxin (3x10^-8 mol/L). Glyburide (10^-5 mol/L) and 4-aminopyridine (10^-3 mol/L) did not alter the relaxations to SIN-1 or SNP. The production of cGMP stimulated by SIN-1 in the vascular smooth muscle was not affected by BaCl₂ (3x10^-3 mol/L) or charybdotoxin (10^-7 mol/L).

Conclusions These results indicate that in canine middle cerebral arteries, a significant portion of relaxations to NO liberated from nitrovasodilators is mediated by large-conductance Ca²⁺-activated K⁺ channels. Other K⁺ channels, sensitive to BaCl₂, may also be involved in the mechanism of relaxations induced by NO.

Key Words: cerebral arteries • cyclic GMP • nitric oxide • vasodilation • dogs

	Introduction

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Nitric oxide is a major mediator of endothelium-dependent relaxations in various vascular beds.^{1 2} It is generally accepted that NO and exogenous nitrovasodilators relax smooth muscle cells by activation of the soluble form of guanylate cyclase and subsequent production of cGMP.³ However, the precise mechanisms underlying vascular smooth muscle relaxations via NO and cGMP still remain to be defined. In recent years, electrophysiological and pharmacological studies demonstrated an important role of K⁺ channels in the hyperpolarization and relaxations of smooth muscle cells.^{4 5 6} Several studies have suggested that native endothelium-derived relaxing factor/NO and NO liberated from nitrovasodilators can activate K⁺ channels in blood vessels. Although NO reportedly activates large-conductance K_Ca through cGMP-dependent protein kinase,^{7 8 9} it has been also demonstrated that NO itself can directly (independently of cGMP) activate K_Ca in rabbit aortic smooth muscle cells.¹⁰

NO plays an essential role in regulation of the cerebral circulation,^{11 12} and impaired NO-mediated relaxations appear to be involved in the pathogenesis of cerebral vasospasm associated with subarachnoid hemorrhage.^{13 14 15} Recent evidence suggests that several types of K⁺ channels, including K_Ca, K_ATP, K_IR, and K_DR, are functional in cerebral blood vessels.^{16 17 18 19 20} The role of K⁺ channels in mediation of vasodilation to NO has not been studied in large cerebral arteries. Therefore, the present study was designed to determine whether activation of K⁺ channels may play a role in NO-induced relaxations of isolated canine middle cerebral arteries.

	Materials and Methods

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The experiments were performed on rings (4 mm long) of middle cerebral arteries taken from dogs (15 to 20 kg) anesthetized with 30 mg/kg IV pentobarbital sodium. All procedures were conducted in accordance with institutional guidelines. The arterial rings were placed in modified Krebs-Ringer bicarbonate solution (control solution) of the following millimolar composition: NaCl 118.3, KCl 4.7, CaCl₂ 2.5, MgSO₄ 1.2, KH₂PO₄ 1.2, NaHCO₃ 25.0, calcium EDTA 0.026, and glucose 11.1. In all rings, the endothelium was removed mechanically by gentle rubbing of the intimal surface with a stainless-steel wire (31-gauge diameter).²¹ Each ring was connected to an isometric force-displacement transducer (Grass FT03, Grass Instrument Co) and suspended in an organ chamber filled with 25 mL control solution (37°C, pH 7.4) aerated with 94% O₂-6% CO₂. Isometric tension was recorded continuously. The rings were allowed to stabilize at a resting tension of 0.2 to 0.4 g for 1 hour. Each ring was then gradually stretched to the optimal point of its length-tension curve (approximately 3.0 g) as determined by the contraction to 10^-5 mol/L UTP.²² The successful removal of endothelium was verified by the absence of relaxation induced by 10^-6 mol/L bradykinin.²³

Radioimmunoassay of cGMP
A radioimmunoassay technique was used to determine the levels of cGMP. Rings without endothelium were initially incubated in control solution bubbled with a 94% O₂-6% CO₂ gas mixture and maintained at 37°C. After 1 hour, the rings were incubated for an additional 30 minutes in a solution containing 10^-3 mol/L IBMX to inhibit the degradation of cGMP by phosphodiesterases. During the last 10 minutes of the incubation period, some arteries were treated with SIN-1 (10^-6 mol/L). To determine the effect of K⁺ channel blockers on production of cGMP, BaCl₂ (3x10^-3 mol/L) or charybdotoxin (10^-7 mol/L) was added to the solution 10 minutes before the addition of SIN-1. To determine the effect of zaprinast on cGMP levels, control rings were not treated with IBMX, whereas treated rings were incubated in a solution containing 10^-3 mol/L zaprinast for 30 minutes. After the incubation, the rings were immediately removed from the solution and frozen in liquid nitrogen. cGMP radioimmunoassay kits (Amersham) were used to perform the measurements. Protein assay was performed by DC Protein Assay Kit (Bio-Rad).

Drugs
The following pharmacological agents were used: UTP (Sigma Chemical Co), bradykinin (Sigma), SIN-1 (Molecular Probes), SNP (Sigma), 8-bromo-cGMP (Sigma), zaprinast (BIOMOL Research Laboratories, Inc), diltiazem hydrochloride (Sigma), BaCl₂ (Sigma), charybdotoxin (Sigma), glyburide (BIOMOL), 4-aminopyridine (Research Biochemicals International), and papaverine hydrochloride (Sigma). Drugs were dissolved in distilled water; volumes of <0.15 mL were added to the organ chambers. Stock solutions of zaprinast and glyburide were prepared in DMSO (Sigma). Concentrations of all drugs are expressed as final molar (moles per liter) concentration in the control solution. The rings were contracted with 10^-5 mol/L UTP 10 minutes before the addition of vasodilator agents. Concentration-response curves were obtained in a cumulative fashion. Several rings prepared from the same artery were studied in parallel, and a concentration-response curve was established by each preparation. The relaxations were expressed as a percentage of maximal relaxations induced by 3x10^-4 mol/L papaverine. The drugs used as K⁺ channel blockers were added 20 minutes before obtaining the concentration-response curve for each vasodilator agent. K⁺ channel blockers (except glyburide) caused contractions of quiescent middle cerebral arteries (Table 1). However, because UTP produced only small contractions in the rings already contracted by K⁺ channel blockers, absolute values of tension did not differ significantly between the control arteries and arteries treated with K⁺ channel blockers (see figure and table legends). In certain experiments, the EC₅₀ was calculated for each ring by linear interpolation between the two concentrations evoking responses just above and below 50% of the maximal response.

View this table: [in this window] [in a new window]   Table 1. Effect of BaCl2, Charybdotoxin, and 4-Aminopyridine on Resting Tension of Canine Middle Cerebral Artery Rings Without Endothelium

Statistical Analysis
The results are expressed as mean±SEM; n refers to the number of animals studied. Statistical evaluation of the data was performed by ANOVA, followed by Fisher's test. Statistical significance was accepted at the level of P<.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effects of K⁺ Channel Blockers on Relaxations to NO Donors SIN-1 and SNP
BaCl₂ (3x10^-5 to 3x10^-3 mol/L) caused concentration-dependent reduction of relaxations to SIN-1 (Fig 1). The effect was significant in the presence of 3x10^-4 and 3x10^-3 mol/L BaCl₂; these concentrations of BaCl₂ also significantly reduced maximal relaxations to SIN-1. In the presence of increasing concentrations of charybdotoxin (10^-8 to 10^-7 mol/L), relaxations to SIN-1 were also significantly reduced (Fig 2). Small but significant reduction of maximal relaxations to SIN-1 was detected in the presence of the highest concentration (10^-7 mol/L) of charybdotoxin. The inhibitory effect of 3x10^-3 mol/L BaCl₂ on relaxations to SIN-1 was significantly greater than that of 10^-7 mol/L charybdotoxin (Fig 3A); however, the effect of 3x10^-4 mol/L BaCl₂ was identical to inhibition observed in the presence of 10^-7 mol/L charybdotoxin (Fig 3A). Further significant reduction of relaxations to SIN-1 was obtained in arteries treated with charybdotoxin (10^-7 mol/L) plus BaCl₂ (3x10^-4 mol/L) compared with arteries treated with charybdotoxin alone (Fig 3B).

View larger version (22K): [in this window] [in a new window]   Figure 1. Concentration-response curves to SIN-1 in canine middle cerebral artery rings without endothelium in the absence (control) and presence of BaCl2 (3x10-5, 3x10-4, and 3x10-3 mol/L). Relaxations were obtained during contractions induced by 10-5 mol/L UTP. Data are shown as mean±SEM and are expressed as percent of maximal relaxations induced by 3x10-4 mol/L papaverine; 100%=3.85±0.41 g (n=12), 4.88±0.51 g (n=5), 4.21±0.45 g (n=9), and 5.34±0.73 g (n=5) for control rings and rings treated with 3x10-5, 3x10-4, and 3x10-3 mol/L BaCl2, respectively. *Significantly different from control; P<.05.

View larger version (21K): [in this window] [in a new window]   Figure 2. Concentration-response curves to SIN-1 in canine middle cerebral artery rings without endothelium in the absence (control) and presence of charybdotoxin (CTX; 10-8, 3x10-8, and 10-7 mol/L). Relaxations were obtained during contractions induced by 10-5 mol/L UTP. Data are shown as mean±SEM and are expressed as percent of maximal relaxations induced by 3x10-4 mol/L papaverine; 100%=4.43±0.39 g (n=10), 4.00±0.39 g (n=5), 5.44±0.92 g (n=5), and 5.00±0.81 g (n=5) for control rings and rings treated with 10-8, 3x10-8, and 10-7 mol/L CTX, respectively. *Significantly different from control; P<.05.

View larger version (16K): [in this window] [in a new window]   Figure 3. Concentration-response curves to SIN-1 in canine middle cerebral artery rings without endothelium in the absence (control) and presence of BaCl2 (3x10-4 and 3x10-3 mol/L) and charybdotoxin (CTX; 10-7 mol/L) (A) and in the absence (control) and presence of CTX (10-7 mol/L) alone and CTX (10-7 mol/L) plus BaCl2 (3x10-4 mol/L) (B). Relaxations were obtained during contractions induced by 10-5 mol/L UTP. Data are shown as mean±SEM and are expressed as percent of maximal relaxations induced by 3x10-4 mol/L papaverine; 100%=4.01±0.58 g (n=9), 4.21±0.45 g (n=9), 5.34±0.73 g (n=5), and 5.00±0.81 g (n=5) for control rings and rings treated with 3x10-4 and 3x10-3 mol/L BaCl2, and 10-7 mol/L CTX (A) and 4.18±0.47 g (n=6), 4.93±0.68 g (n=6), and 4.65±1.00 g (n=6) for control rings and rings treated with CTX and CTX+BaCl2 (B), respectively. *Significantly different from rings treated with 3x10-4 mol/L BaCl2 or 10-7 mol/L CTX (A) and from rings treated with 10-7 mol/L CTX alone (B); P<.05.

The relaxations to SNP were also inhibited by BaCl₂ and charybdotoxin in the same manner as those to SIN-1 (Figs 4 and 5).

View larger version (21K): [in this window] [in a new window]   Figure 4. Concentration-response curves to SNP in canine middle cerebral artery rings without endothelium in the absence (control) and presence of BaCl2 (3x10-5, 3x10-4, and 3x10-3 mol/L). Relaxations were obtained during contractions induced by 10-5 mol/L UTP. Data are shown as mean±SEM and are expressed as percent of maximal relaxations induced by 3x10-4 mol/L papaverine; 100%=3.98±0.32 g (n=12), 4.26±0.90 g (n=5), 4.26±0.59 g (n=9), and 5.26±0.68 g (n=5) for control rings and rings treated with 3x10-5, 3x10-4, and 3x10-3 mol/L BaCl2, respectively. *Significantly different from control; P<.05.

View larger version (20K): [in this window] [in a new window]   Figure 5. Concentration-response curves to SNP in canine middle cerebral artery rings without endothelium in the absence (control) and presence of charybdotoxin (CTX; 10-8, 3x10-8, and 10-7 mol/L). Relaxations were obtained during contractions induced by 10-5 mol/L UTP. Data are shown as mean±SEM and are expressed as percent of maximal relaxations induced by 3x10-4 mol/L papaverine; 100%=4.20±0.28 g (n=10), 4.80±0.24 g (n=5), 4.72±0.57 g (n=5), and 5.28±0.73 g (n=5) for control rings and rings treated with 10-8, 3x10-8, and 10-7 mol/L CTX, respectively. *Significantly different from control; P<.05.

Glyburide (10^-5 mol/L) and 4-aminopyridine (10^-3 mol/L) did not alter values of EC₅₀ and maximal relaxations induced by SIN-1 or SNP (Tables 2 and 3).

View this table: [in this window] [in a new window]   Table 2. Effect of Glyburide on EC50and Maximal Relaxations Obtained in Response to SIN-1 and SNP in Canine Middle Cerebral Arteries Without Endothelium

View this table: [in this window] [in a new window]   Table 3. Effect of 4-Aminopyridine on EC50 and Maximal Relaxations Obtained in Response to SIN-1 and SNP in Canine Middle Cerebral Arteries Without Endothelium

Effects of K⁺ Channel Blockers on Relaxations to 8-Bromo-cGMP
In contrast to NO donors, the relaxations to 8-bromo-cGMP were not affected by 3x10^-4 mol/L BaCl₂ and 3x10^-8 mol/L charybdotoxin (Fig 6A and 6B). However, BaCl₂ (3x10^-3 mol/L) and charybdotoxin (10^-7 mol/L) significantly suppressed the responses to 8-bromo-cGMP.

View larger version (15K): [in this window] [in a new window]   Figure 6. Concentration-response curves to 8-bromo-cGMP in canine middle cerebral artery rings without endothelium in the absence (control) and presence of BaCl2 (3x10-4 and 3x10-3 mol/L) (A) and in the absence (control) and presence of charybdotoxin (CTX; 3x10-8 and 10-7 mol/L) (B). Relaxations were obtained during contractions induced by 10-5 mol/L UTP. Data are shown as mean±SEM and are expressed as percent of maximal relaxations induced by 3x10-4 mol/L papaverine; 100%=3.93±0.33 g (n=9), 4.26±0.65 g (n=5), and 5.21±0.73 g (n=7) for control rings and rings treated with 3x10-4 and 3x10-3 mol/L BaCl2 (A) and 3.65±0.34 g (n=13), 3.68±0.41 g (n=5), and 4.08±0.50 g (n=8) for control rings and rings treated with 3x10-8 and 10-7 mol/L CTX (B), respectively. *Significantly different from control; P<.05.

Effects of K⁺ Channel Blockers on Relaxations to Zaprinast
The relaxations induced by zaprinast (10^-7 to 10^-4 mol/L) were significantly reduced by BaCl₂ (3x10^-4 mol/L) or charybdotoxin (3x10^-8 mol/L; Fig 7).

View larger version (15K): [in this window] [in a new window]   Figure 7. Concentration-response curves to zaprinast in canine middle cerebral artery rings without endothelium in the absence (control) and presence of BaCl2 (3x10-4 mol/L) and charybdotoxin (CTX; 3x10-8 mol/L). Relaxations were obtained during contractions induced by 10-5 mol/L UTP. Data are shown as mean±SEM and are expressed as percent of maximal relaxations induced by 3x10-4 mol/L papaverine; 100%=4.44±0.26 g (n=6), 4.50±0.39 g (n=6), and 4.86±0.48 g (n=6) for control rings and rings treated with BaCl2 and CTX, respectively. *Significantly different from control; P<.05.

Effects of K⁺ Channel Blockers on Relaxations to Diltiazem
Diltiazem-induced relaxations were not affected by BaCl₂ and charybdotoxin even in the presence of the highest concentrations of 3x10^-3 and 10^-7 mol/L, respectively. EC₅₀ (-log mol/L) and maximal relaxations to diltiazem detected in the absence or presence of BaCl₂ (3x10^-3 mol/L) were 6.30±0.08 and 87.8±2.0% (n=10) and 6.32±0.09 and 95.3±1.3% (n=5), respectively. Those in the presence of charybdotoxin (10^-7 mol/L) were 6.45±0.08 and 91.1±2.2% (n=5).

Effects of K⁺ Channel Blockers on Production of cGMP
In canine middle cerebral arteries without endothelium, SIN-1 (10^-6 mol/L) produced approximately a 10-fold increase in levels of cGMP. The increase in cGMP levels was not affected by the highest concentrations of BaCl₂ (3x10^-3 mol/L) and charybdotoxin (10^-7 mol/L; Fig 8).

View larger version (41K): [in this window] [in a new window]   Figure 8. Effect of SIN-1 (10-6 mol/L) on cGMP levels in canine middle cerebral arteries without endothelium and modification by BaCl2 (3x10-3 mol/L) and charybdotoxin (CTX; 10-7 mol/L) of the increased cGMP production. Values are expressed as mean±SEM (n=10).

Effects of Zaprinast on cGMP Levels
In canine middle cerebral arteries without endothelium, zaprinast (10^-3 mol/L) caused a significant increase in cGMP levels (Fig 9).

View larger version (15K): [in this window] [in a new window]   Figure 9. Effect of zaprinast (10-3 mol/L) on cGMP levels in canine middle cerebral arteries without endothelium. Values are expressed as mean±SEM (n=7). *Significantly different from control; P<.05.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The results of the present study demonstrate that K_Ca play an important role in mediation of cerebral arterial relaxations to NO. This conclusion is based on the experiments demonstrating that a selective large-conductance K_Ca inhibitor, charybdotoxin,^{5 24 25 26} reduced the relaxations induced by two chemically different NO donors, SIN-1 and SNP.³ Selectivity of charybdotoxin was confirmed by the fact that relaxations to a calcium channel antagonist, diltiazem, were not affected in the presence of the highest concentration of charybdotoxin (10^-7 mol/L). Furthermore, charybdotoxin did not affect production of cGMP stimulated by SIN-1, ruling out the possibility that the inhibitory effect of charybdotoxin is due to nonselective inactivation of guanylate cyclase. Our findings are in agreement with conclusions of the previous in vivo study performed in rat cerebral arterioles.²⁷ However, we extended previous observations to demonstrate that K_Ca mediate a significant portion of relaxations to NO in large canine cerebral arteries.

BaCl₂ also strongly reduced the relaxations to NO donors without having any effect on relaxations to diltiazem or production of cGMP. These results further supported the proposition that K⁺ channels play an important role in cerebral arterial relaxations to NO. However, previous studies demonstrated that even at the very high concentration of 10^-2 mol/L, BaCl₂ has little effect on K_Ca.^{5 28} The highest concentration of BaCl₂ used in our study was 3x10^-3 mol/L, suggesting that the effect of BaCl₂ is possibly mediated via K⁺ channels other than K_Ca. This conclusion is also supported by the fact that the inhibitory effect of BaCl₂ on relaxations to NO donors was significantly larger than the inhibitory effect of charybdotoxin. Furthermore, in arteries treated with a high concentration of charybdotoxin (10^-7 mol/L), a lower concentration of BaCl₂ (3x10^-4 mol/L) exerted an additional inhibitory effect on relaxations to SIN-1, suggesting that charybdotoxin and BaCl₂ may affect different populations of K⁺ channels. K_IR are most sensitive to extracellular barium ions and should be blocked by micromolar concentrations of BaCl₂^{5 29 30 31 32} ; however, the relaxations to SIN-1 and SNP were not reduced in the presence of 3x10^-5 mol/L BaCl₂. These observations minimize the possibility that K_IR activation is involved in the relaxant actions of nitrovasodilators. In addition, our results demonstrated that glyburide (10^-5 mol/L), a selective K_ATP inhibitor,^{5 17 33 34} and 4-aminopyridine (10^-3 mol/L), the most reliable K_DR inhibitor,^{5 18 35 36} did not affect the relaxations to SIN-1 or SNP, ruling out a contribution of these K⁺ channels to NO-induced relaxations. The results of our study do not allow any conclusion regarding the type of K⁺ channels responsible for a BaCl₂-sensitive component of relaxations to nitrovasodilators. Interestingly, in canine colonic smooth muscle cells, NO activates K⁺ channels that are resistant to known specific K⁺ channel blockers.³⁷ This finding is in agreement with our results, and it is very likely that in canine middle cerebral artery, NO may activate a population of K⁺ channels that cannot be characterized by the available pharmacological tools used in the present study.

In contrast to NO donors, the relaxations to a cGMP analogue, 8-bromo-cGMP, were not affected by 3x10^-4 mol/L BaCl₂ and 3x10^-8 mol/L charybdotoxin. Such selective inhibition of relaxations to NO donors by K⁺ channel blockers suggests that NO may directly (independently of cGMP) activate K⁺ channels. Although previous studies using patch-clamp techniques have documented that NO activates large-conductance K_Ca through cGMP-dependent protein kinase in calf thoracic aorta,⁷ rabbit basilar artery,⁸ and rat pulmonary artery,⁹ it has also been reported that NO itself can directly activate K_Ca in rabbit aortic smooth muscle cells.¹⁰ Therefore, it is possible that a similar direct effect of NO is involved in the mechanisms of relaxation in canine cerebral arteries. Another possibility is that SIN-1 and SNP may generate peroxynitrite and cyanide, respectively,^{38 39} and that these products may activate K⁺ channels independently of cGMP production. However, we obtained identical results with two different NO donors, suggesting that our findings are best explained by formation of NO rather than peroxynitrite or cyanide. More importantly, the relaxations to zaprinast, which selectively inhibits cGMP phosphodiesterase and thereby increases endogenous cGMP levels,^{40 41 42} were strongly reduced by 3x10^-4 mol/L BaCl₂ or 3x10^-8 mol/L charybdotoxin. Furthermore, the relaxations to 8-bromo-cGMP were reduced by higher concentrations of BaCl₂ (3x10^-3 mol/L) or charybdotoxin (10^-7 mol/L), suggesting that the relaxations caused by exogenous cGMP are also mediated partly by the activation of K⁺ channels. An exact reason for the lower sensitivity of 8-bromo-cGMP–induced relaxations to K⁺ channel blockers is not clear. One possible explanation is that exogenous cGMP may not have access to the same molecular targets as endogenous cGMP generated after guanylate cyclase activation or phosphodiesterase inhibition. Thus, our results suggest that the activation of K⁺ channels by NO donors is most likely mediated by increased production of cGMP. It remains to be determined whether cGMP-independent interaction between NO and K⁺ channels is an important mechanism of relaxations in cerebral arteries.

The results of the present study suggest that in canine cerebral arteries, large-conductance K_Ca on the smooth muscle play a role in mediation of relaxations to NO. The activation of K_Ca appears to be dependent on cGMP production. Other K⁺ channels, sensitive to BaCl₂, may also be involved in the mechanism of relaxations induced by NO. These findings provide a basis for further analysis of the physiological and pathological significance of NO in the regulation of the cerebral circulation.

	Selected Abbreviations and Acronyms

 

8-bromo-cGMP = 8-bromo-3',5'-cyclic GMP cGMP = cyclic GMP IBMX = 3-isobutyl-1-methylxanthine KATP = ATP-sensitive K+ channels KCa = Ca2+-activated K+ channels KDR = delayed rectifier K+ channels KIR = inward rectifier K+ channels NO = nitric oxide SIN-1 = 3-morpholinosydnonimine SNP = sodium nitroprusside

	Acknowledgments

 
This work was supported in part by National Heart, Lung, and Blood Institute grant HL-53524 and by the Mayo Foundation. Dr Onoue was supported by a scholarship from Uehara Memorial Foundation (Tokyo, Japan).

Received November 21, 1996; revision received March 5, 1997; accepted March 28, 1997.

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