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(Hypertension. 1998;31:303.)
© 1998 American Heart Association, Inc.

Scientific Contributions

Effect of Selective Inhibition of Soluble Guanylyl Cyclase on the K_Ca Channel Activity in Coronary Artery Smooth Muscle

Pin-Lan Li; Man-Wen Jin; William B. Campbell

From the Departments of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wis.

Correspondence to Pin-Lan Li, MD, PhD, Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226. pli{at}post.its.mcw.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Activation of a soluble guanylyl cyclase plays an important role in nitric oxide (NO)-induced vasodilation. Recently, we have reported that NO increases the calcium-activated potassium (K_Ca) channel activity in vascular smooth muscle cells from coronary arteries. The present study examined the role of the soluble guanylyl cyclase in the control of basal activity of the K_Ca channels and in mediating NO-induced activation of the K_Ca channels in vascular smooth muscle cells, using a selective inhibitor of this enzyme, 1H-[1,2,4]oxadiazolo[4,2-]quinoxalin-1-one (ODQ). In the cell-attached patch-clamp mode, addition of ODQ into the bath solution (10 µmol/L) decreased the K_Ca channel activity by 59% and attenuated activation of the channels induced by the NO donor, deta nonoate, by 70%. ODQ had no effect on 8-bromo-cGMP-induced activation of the K_Ca channels. Deta nonoate produced a concentration-dependent relaxation of precontracted coronary arteries. When ODQ was added to the bath, the deta nonoate-induced relaxations were inhibited. The IC₅₀ for deta nonoate was decreased by about 25-fold and the maximal effect of deta nonoate was reduced by about 60%. A specific K_Ca channel inhibitor, iberiotoxin, decreased deta nonoate-induced vasodilation but to a lesser extent than ODQ. However, ODQ was without effect on the vasodilation induced by a prostacyclin analog, iloprost, and by adenosine. These results indicate that a soluble guanylyl cyclase and cGMP play an important role in the control of the K_Ca channel activity in coronary arterial smooth muscle cells. K_Ca channel activation participates in the NO-induced vasodilation in coronary circulation.

Key Words: potassium channels • nitric oxide • endothelium • guanylyl cyclase • vasodilation • coronary artery

Abbreviations: K_Ca = calcium-activated potassium • NO = nitric oxide • NOS = nitric oxide synthase • ODQ = 1H-1,2,4-oxadiazolo[4,2-]quinoxalin-1-one • PKG = protein kinase G • VSM = vascular smooth muscle

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
NO plays an important role in mediating the biological activity of endothelium-dependent agonists.¹ Recent studies indicated that activation of the K⁺ channels participates in endothelium-dependent vasodilation and NO-mediated vascular effects.^2–5 More recently, we have reported that NO markedly increased the activity of two different K⁺ channels in small coronary arterial smooth muscle cells.⁶ However, the mechanism by which NO activates these K⁺ channels remains unknown.

Previous studies have demonstrated that NO stimulates cGMP production and that increased cGMP production correlates with the NO-mediated vasodilation.^7–10 Guanylyl cyclase inhibitors such as methylene blue or LY83583 lower tissue or cellular cGMP concentrations and block the vascular relaxation induced by stimulators of NOS or NO donors.^11,12 It was concluded that activation of guanylyl cyclase mediates the vasodilator effect of NO. This conclusion, however, has been challenged by several recent studies. These studies demonstrated that NO-induced relaxation of VSM may be dissociated from increases in cGMP production,^2,9,13 and classical inhibitors of guanylyl cyclase such as methylene blue and LY83583 also inhibit the NOS activity and lower NO concentrations.^14–19 Inhibition of endogenous NO production by these compounds may result in vasoconstriction and consequently counteract the vasodilator effect of the NOS stimulators or NO donors. A decrease in the concentration of NO due to its oxidization by these compounds may block the vascular effect of NO before it acts on VSM cells. Therefore, the role of cGMP in mediating the vasodilator effect of NO remained to be further confirmed.

More recently, a novel soluble guanylyl cyclase inhibitor, ODQ, was reported to block the vasodilator effect of NO.²⁰ This compound inhibits the deta nonoate-stimulated increase in cGMP production in rat aortic VSM, but it had no effect on the activity of other forms of guanylyl cyclase such as membrane-associated guanylyl cyclase A, B, and C. In contrast to methylene blue and LY83583, ODQ neither inhibits the NOS activity and nor oxidizes NO.²⁰ Therefore, ODQ is a selective soluble guanylyl cyclase inhibitor that can be used to further determine the role of cGMP in mediating activation of the K_Ca channel and the vasodilation induced by NO.

The present study examined the effect of ODQ on NO-induced K⁺ channel activation in coronary arterial smooth muscle cells and relaxation of coronary arteries. The purpose of this study was to clarify the contribution of cGMP-mediated K_Ca channel activation to the vasodilation induced by NO.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Patch-Clamp Studies
Patch-clamp studies were performed as described in our recent reports.^6,21 Briefly, VSM cells were enzymatically dissociated from bovine small coronary arteries, and single channel K⁺ currents were recorded using the cell-attached mode. Smooth muscle cells were placed in a 1-mL perfusion chamber mounted on the stage of a Nikon inverted microscope. After the tip of the pipette was positioned on a cell, a high-resistance seal (5 to 15 G) was formed between the pipette tip and the cell membrane by applying a light suction. Then, the activity of the K⁺ channels in the membrane spanning the pipette tip was recorded.

A List EPC-7 patch-clamp amplifier (List Biological Laboratories, Inc) was used to record single-channel currents. The amplifier output signals were filtered at 1 kHz with an eight-pole Bessel filter (Frequency Devices Inc). Currents were digitized at a sampling rate of 3 kHz and stored on the hard disk of a Gateway 486 DS66 computer for off-line analysis. Data acquisition and analysis were performed with pClamp software (version 6.03, Axon Instruments). Total open state probability values in patches were determined from recordings of several minutes by

where N is the maximal number of channels observed in the condition of high levels of P_o. P_o is the open state probability, T is the duration of the recording, and j is the time with j=1,2,...N channels open.

In these experiments, the VSM cells were bathed with a solution containing (in mmol/L): KCl 145, CaCl₂ 1.8, MgCl₂ 1.1, glucose 10, and HEPES 5 (pH 7.4). The pipette solution contained (in mmol/L): KCI 145, CaCl₂ 1.8, MgCl₂ 1.1, and HEPES 5 (pH 7.4). To determine the effect of ODQ on the activity of K⁺ channels, a 3-minute channel current recording at a membrane potential of +40 mV was obtained before and 10 minutes after addition of ODQ at a concentration of 10 µmol/L (n=7). In an additional group of cells, a NO donor, deta nonoate, at a concentration of 10 µmol/L was added into the cell bath solution in the absence or presence of ODQ (10 µmol/L) and after 3 minutes of incubation, the K⁺ channel activity was recorded.

Vascular Reactivity Studies
Vascular reactivity in bovine coronary arteries was determined as previously described by our laboratory.^22,23 Briefly, the epicardial left anterior descending coronary artery was dissected, cleaned of adhering fat and connective tissue, and placed in a Krebs-bicarbonate solution containing (in mmol/L) NaCl 119, KCl 5, NaHCO₃ 24, KH₂PO₄ 1.2, MgSO₄ 1.2, glucose 11, EDTA 0.02, and CaCl₂ 3.2. The rings were prepared and suspended in a 6-mL water-jacketed organ chamber at 37°C. The contractile responses were monitored using a Grass polygraph. After an equilibration period of 1.5 hours, the vessels were activated by addition of KCl (40 mmol/L) until reproducible contractions were obtained. Then, one ring of each pair received vehicle (0.01% ethanol) and other ring received ODQ (10 µmol/L) (n=8) or iberiotoxin (100 nmol/L) (n=7) for 10 minutes before the addition of the thromboxane-mimetic agent U46619 (20 nmol/L). U46619 was selected as the precontracting agent, because it produced reproducible, sustained contractions in coronary arteries. After a sustained contraction by U46619 was obtained, cumulative additions of deta nonoate (10^-9 to 10^-5 mol/L), iloprost (10^-9 to 10^-5 mol/L), and adenosine (10^-8 to 10^-4 mol/L) were made every 4 minutes or until a plateau response was reached. Results were expressed as percent relaxation relative to the U46619 contraction with 100% relaxation reaching the basal tension before U46619 contraction.

Statistics
Data are presented as mean±SEM; n indicates number of bovine hearts. The significance of the differences in mean values between and within multiple groups was examined using an analysis of variance for repeated measures followed by a Duncan’s multiple range test. A Student’s t test was used to evaluate statistical significance of differences between two paired observations. A value of P<.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of ODQ on the Increase in the K_Ca Channel Activity Induced by Deta Nonoate
The results of these experiments are presented in Fig 1. Fig 1A shows representative recordings of single channel K_Ca currents depicting the effects of deta nonoate in the absence or presence of ODQ. Deta nonoate markedly increased the activity of the K_Ca channels, and ODQ decreased the activity of these channels. In the presence of ODQ, the effects of deta nonoate were significantly attenuated. Fig 1B summarizes the effect of ODQ on basal K_Ca channel activity and deta nonoate-induced alterations of the NP_O of the K_Ca channels. The NP_O of the K_Ca channels was increased from 0.047±0.004 to 0.216±0.009 when deta nonoate was added to the bath at concentrations of 100 µmol/L. ODQ at a concentration of 10 µmol/L decreased basal NP_O of the K_Ca channels by 59% and reduced deta nonoate-induced increases in the NP_O of the K_Ca channels by 70%. Deta nonoate and ODQ did not alter the current amplitude of these channels (Fig 1C).

View larger version (31K): [in this window] [in a new window]   Figure 1. Effect of deta nonoate, a NO donor, on the KCa channel activity in cell-attached patches of coronary arterial smooth muscle cells isolated in the absence and presence of the selective inhibitor of soluble guanylyl cyclase inhibitor, ODQ. A, Representative recordings of the KCa channels under control conditions and after addition of 100 µmol/L deta nonoate to the bath at a membrane potential of +40 mV in the absence and presence of ODQ (10 µmol/L). B and C, Effect of deta nonoate on the NPO and current amplitude of the KCa channel in smooth muscle cells, respectively. *indicates significant difference from control (P<.05). indicates significant difference from values obtained after ODQ was added alone (P<.05).

Effect of ODQ on the Increase in the K_Ca Channel Activity Induced by 8-Bromo-cGMP
Representative recordings of single channel K_Ca currents under control conditions and after administration of a cell-permeable analog of cGMP, 8-bromo-cGMP, and ODQ are presented in Fig 2A. 8-Bromo-cGMP significantly increased the activity of the K_Ca channels when added into the bath solution. ODQ had no effect on 8-bromo-cGMP-induced activation of the K_Ca channels. Fig 2B summarizes the effect of 8-bromo-cGMP on the NP_O of the K_Ca channels in the absence and presence of ODQ. 8-Bromo-cGMP at a concentration of 10 µmol/L produced a 18-fold increase in the NP_O of the K_Ca channels. The effect of 8-bromo-cGMP was not altered by pretreatment of cells with ODQ.

View larger version (33K): [in this window] [in a new window]   Figure 2. Effect of a cell-permeable analog of cGMP, 8-bromo-cGMP on the KCa channel activity in cell-attached patches of coronary arterial smooth muscle cells in the absence and presence of the selective inhibitor of soluble guanylyl cyclase inhibitor, ODQ. A, Representative recordings of the KCa channels under control conditions and after addition of 10 µmol/L 8-bromo-cGMP to the bath at a membrane potential of +40 mV in the absence and presence of ODQ (10 µmol/L). B and C, Effect of 8-bromo-cGMP on the NPO and current amplitude of the KCa channel in smooth muscle cells, respectively. * indicates significant difference from control (P<.05). indicates significant difference from values obtained after ODQ was added alone (P<.05).

Effect of ODQ on the Relaxation of Coronary Arteries Induced by Different Vasodilators
The result of these experiments are presented in Fig 3. Deta nonoate produced a concentration-dependent relaxation in U46619-precontracted coronary arterial rings. Complete relaxation to deta nonoate occurred at 10^-5 mol/L with an IC₅₀ of 2.5x10^-7 mol/L. In the presence of ODQ, deta nonoate-induced relaxation was significantly attenuated, and the concentration-response curve was shifted to the right (Fig 3A). The IC₅₀ for the deta nonoate effect was increased by 25-fold, and maximal relaxation was decreased by 60% by ODQ. In contrast to the effect on deta nonoate-induced relaxation, ODQ had no effect on the relaxation of coronary arteries induced by iloprost (10^-9 to 10^-5 mol/L) and adenosine (10^-8 to 10^-4 mol/L) (Fig 1B and 1C).

View larger version (17K): [in this window] [in a new window]   Figure 3. Effect of ODQ (10 µmol/L) on the relaxation of coronary arteries induced by deta nonoate, iloprost, and adenosine. A, Effect of ODQ on the deta nonoate-induced relaxation. B, Effect of ODQ on the iloprost-induced relaxation. C, Effect of ODQ on the adenosine-induced relaxation. *indicates significant difference from control (P<.05).

Effect of Iberiotoxin on the Relaxation of Coronary Arteries Induced by Deta Nonoate
These experiments were designed to determine the contribution of the K_Ca channel activity to NO-induced vasodilation in the coronary circulation (Fig 4). In the presence of a selective inhibitor of the K_Ca channel activity, iberiotoxin, deta nonoate-induced relaxation was attenuated, and the concentration-response curve also shifted to the right. However, iberiotoxin inhibited deta nonoate-induced relaxation to a much lesser extent than ODQ. The treatment of arterial rings with iberiotoxin did not alter the inhibitory effects of ODQ on deta nonoate-induced relaxation (data not shown).

View larger version (15K): [in this window] [in a new window]   Figure 4. Effect of iberiotoxin (100 nmol/L) on the relaxation of coronary arteries induced by deta nonoate. * indicates significant difference from control (P<.05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study demonstrated that a selective inhibitor of soluble guanylyl cyclase, ODQ, markedly decreased the activity of the K_Ca channels, suggesting that cGMP may play an important role in gating the K_Ca channels under physiological conditions. To determine the role of a cGMP-mediated signaling mechanism in NO-induced activation of the K_Ca channels, we examined the effect of a NO donor, deta nonoate, on the K_Ca channel activity in the absence and presence of ODQ. We found that deta nonoate significantly increased the K_Ca channel activity. When the VSM cells were pretreated with ODQ, the effect of deta nonoate on the K_Ca channel activity was attenuated by 70%. In contrast to the effect on NO, ODQ had no effect on 8-bromo-cGMP-induced activation of the K_Ca channels. These results strongly support the view that NO stimulates a soluble guanylyl cyclase and activates the K_Ca channels in VSM cells.

Previous studies have demonstrated that methylene blue or LY83583 also blocked the effect of NO on the K_Ca channel activity in smooth muscle cells, indicating that activation of soluble guanylyl cyclase mediates the effect of NO on the K_Ca channel activity.^24–27 However, since these compounds have been reported to inactivate reactive NO and inhibit the NOS activity in addition to inhibiting the guanylyl cyclase activity,¹⁵ the conclusion based on their inhibitory effects on NO response is questionable. The results of the present study with ODQ yield a clearer conclusion than previous studies, since ODQ does not inhibit the NOS activity, does not autooxidize NO, and does not have cross-inhibitory effects on other guanylyl cyclases.²⁰

In the present study, ODQ did not fully block the effect of deta nonoate on the K_Ca channel activity at a concentration that completely abolished the NO-induced cGMP production.²⁰ It appears that a cGMP-independent effect may also contribute to activation of the K_Ca channels by NO. The present study did not attempt to address this issue. However, a recent study demonstrated this cGMP-independent effect of NO on the K_Ca channel activity.² With cell-free membrane patches from rabbit aortic smooth muscle cells, both exogenous and native NO directly activates single K_Ca channels. In this excised membrane patch-clamp recording mode, cGMP could not be formed or its action was excluded.² Taken together, these findings indicate that activation of the K_Ca channels may be associated with both cGMP-dependent and cGMP-independent mechanisms. The present study indicates that a cGMP-dependent mechanism may contribute to 70% of the effects of NO on the K_Ca channel activity.

We also examined the effect of ODQ on the NO-induced vasodilation in coronary arteries. ODQ significantly attenuated deta nonoate-induced relaxation in coronary arterial rings with a 25-fold increase in the IC₅₀ and a 60% decrease in maximal relaxation. It had no effect on iloprost- and adenosine-induced relaxation, which is associated with activation of the cAMP-protein kinase A pathway or an increase in the activity of delayed rectifier K⁺ channels.⁶ These results are consistent with previous reports demonstrating that ODQ increased the IC₅₀ for deta nonoate by 30-fold and reduced maximal relaxation to deta nonoate by 75% in rat aortic rings precontracted by phenylephrine,²⁰ which indicates that activation of a soluble guanylyl cyclase plays an important role in mediating the NO-induced vasodilation in coronary arteries. However, ODQ did not fully block the NO-induced vasodilation in coronary arteries. It seems that a cGMP-independent mechanism may account for the vasodilator effect of deta nonoate at high concentrations.

Recent studies have indicated that alteration of the K_Ca channel activity plays an important role in mediating vasoconstrictor or vasodilator responses to different vasoactive agonists.³ Inhibition of the K_Ca channel activity contributes to the depolarization and vasoconstriction induced by angiotensin II, norepinephrine, endothelin, and serotonin in VSM from different vascular beds.^28,29 Activation of the K_Ca channels participates in the vasodilation in cerebral and coronary arteries induced by ß-adrenergic stimulation, NO, and an endothelium-derived hyperpolarization factor, epoxyeicosatrienoic acids.^4–6,30,31 In the present study, we demonstrated that deta nonoate activated the K_Ca channels and produced vasodilation in coronary arteries. It is possible that activation of the K_Ca channels hyperpolarizes coronary smooth muscle and subsequently decreases Ca⁺⁺ influx, leading to vasodilation. However, activation of the K_Ca channels is not the only mechanism mediating NO-induced vasodilation of coronary arteries. We demonstrated that a selective K_Ca channel inhibitor, iberiotoxin, at a concentration that completely blocks the activity of K_Ca channels⁶ did not fully abolish the NO-induced relaxation in coronary arteries. Compared with ODQ, iberiotoxin attenuated the NO-induced vasodilation to a much lesser extent. These results indicate that activation of the K_Ca channels may represent only part of the cGMP-mediated effect in NO stimulation. The present study did not explore other mechanisms for cGMP pathways in NO-induced vasodilation. Because activation of PKG and the resulting protein phosphorylation are fundamental mechanisms of the cGMP-mediated effect, it is possible that the phosphorylation of other contractile or regulatory proteins in VSM cells contributes to the K_Ca channel-independent effect of cGMP in NO-induced vasodilation.³ Moreover, the combination of iberiotoxin and ODQ did not block the vasodilator effect of deta nonoate at high concentrations, suggesting that cGMP-independent vasodilator effects of NO are not associated with activation of the K_Ca channels.

In summary, a selective soluble guanylyl cyclase inhibitor, ODQ, reduces the K_Ca channel activity and inhibits the NO-induced activation of these K channels, suggesting that NO may activate the K_Ca channels through cGMP/PKG signaling pathway. This cGMP/PKG-mediated activation of the K_Ca channels contributes in part to the vasodilator effect of NO in coronary circulation.

	Acknowledgments

 
This work was supported by grants from the National Heart, Lung and Blood Institute (HL-51055 and HL-57244) and American Heart Association, Wisconsin Affiliate (95-GB-52). The authors thank Gretchen Barg for her secretarial assistance.

Received September 17, 1997; first decision October 13, 1997; accepted October 20, 1997.

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