Nitric oxide and cGMP cause vasorelaxation by activation of a charybdotoxin-sensitive K channel by cGMP-dependent protein kinase.

Abstract

Nitric oxide (NO)-induced relaxation is assodated with increased levels ofcGMP in vascular smooth muscle cells. However, the mechanism by which cGMP causes relaxation is unknown. This study tested the hypothesis that activation of Ca-sensitive K (Kc.) chanels, mediated by a cGMPdependent protein kinase, is responsible for the relaxation occurring in response to cGMP. In rat pulmonary artery rings, cGMP-dependent, but not cGMP-independent, relaxation was inhibited by tetraethylammonium, a classical K-channel blocker, and charybdotoxin, an inhibitor of Kca channels. Increasing extrcellular K concentration also inhibited cGMPdependent relaxation, without reducing vascular smooth muscle cGMP levels. In whole-cell patch-clamp experiments, NO and cGMP increased whole-cell K current by activating Kc. channels. This effect was mimicked by intracellular administration of (Sp)-guanosine cyclic 3',5'-phosphorothioate, a preferential cGMP-dependent protein kinase activator. Okadaic acid, a phosphatase inhibitor, enhanced whole-cell K current, consistent with an important role for channel phosphorylation in the activation of NO-responsive Kc channels. Thus NO and cGMP relax vascular smooth muscle by a cGMP-dependent protein kinase-dependent activation of K channels. This suggests that the final common pathway shared by NO and the nitrovasodilators is cGMP-dependent K-channel activation.Nitric oxide (NO) and nitrovasodilators cause vasodilatation by activating guanylate cyclase and increasing cGMP in vascular smooth muscle (VSM) (1). The mechanism by which cGMP reduces vascular tone has been uncertain. Several experiments suggest that cGMP-mediated vasodilation is associated with changes in membrane potential. (i) KCl, which depolarizes VSM cells, inhibits endothelium-dependent vasodilatation (2). (ii) NO itself hyperpolarizes VSM in many (3-5), but not all (6, 7), studies. Finally, agents that increase cGMP can activate K channels (8-10). K-channel activity is the main determinant of membrane potential, and K efflux resulting from K-channel opening causes hyperpolarization, inhibits voltage-gated Ca channels, and promotes relaxation (Fig. 1).The current investigation evaluated two hypotheses: (i) K-channel activation is essential for cGMP-induced VSM relaxation and (ii) increases in cGMP activate K channels by stimulating cGMP-dependent protein kinase (cGK).To precisely characterize the role of NO/cGMP-activated K channels in vascular relaxation, it is necessary to combine studies of vascular tone [isolated pulmonary artery (PA) rings] and electrophysiology (whole-cell patch-clamp studies of PA VSM). These studies prove that NO and agents that increase cGMP cause relaxation in large part by a cGKmediated activation of Ca-sensitive K (Kca) channels. MATERIALS AND METHODSDrugs. Drugs and reagents were from Sigma and were dissolved in normal saline unless otherwise stated. Bath concentrations of solvents were <0.1% and all vehicles were tested to exclude nonspecific effects. Saturated NO solutions (2-3 mM) were prepared...

Hypoxia enhances a cGMP-independent nitric oxide relaxing mechanism in pulmonary arteries

“…Because TEA blocks most K ϩ channels under the conditions employed, it appears that the NO donor SNAP does not induce a cGMP-independent relaxation through the activation of K ϩ channels in BPA. The role for NO in opening K ϩ channels via a cGMP mechanism may be an important part of smooth muscle relaxation in some systems (1), and evidence for the functional significance of this mechanism was detected in the present study when vascular segments were contracted with U-46619 under 21% O 2 . An increase in the level of membrane depolarization with 30 mM K ϩ and/or hypoxia (20) may have prevented the detection of cGMP-mediated relaxation in BPA through the opening of K ϩ channels under these conditions.…”

“…There is evidence that the opening of K ϩ channels in vascular smooth muscle is an important process involved in cGMP-dependent and cGMP-independent NO-mediated relaxation mechanisms (1,3). Thus the opening of K ϩ channels and the production of relaxation through hyperpolarization could potentially contribute to the mechanisms involved in NO-mediated relaxation under hypoxia.…”

“…Vasodilation resulting from hyperpolarization caused by the opening of K ϩ channels is a potential mechanism through which NO can promote relaxation (1,3). Therefore, a nonspecific K ϩ channel blocker that inhibits almost all vascular K ϩ channels, TEA (10 mM), was used to examine the role of the K ϩ channel in the mechanism of relaxation elicited by NO (Fig.…”

“…Previous studies (7) have shown that the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) causes a cGMP-dependent relaxation in bovine pulmonary arteries (BPA) and that 1H- [1,2,4]oxadiazolo- [4,3-a]quinoxalin-1-one (ODQ) inhibits this response under 21% O 2 (8,10). Although the mechanism of sGC inhibition by ODQ is still under investigation, the original concept that ODQ is oxidizing the iron group of the sGC heme from Fe 2ϩ 3 Fe 3ϩ , preventing the binding of NO to sGC (23), appears to best explain its mechanism of action (8).…”

Echinacoside induces rat pulmonary artery vasorelaxation by opening the NO-cGMP-PKG-BKCa channels and reducing intracellular Ca2+ levels

“…We also found that the ECH-induced relaxation was significantly weaker at the highest concentration of ECH in rings after treatment with TEA (1 mmol/L) compared with the control. NO-induced vasodilation is due in part to a cGMP-dependent, protein kinase G-mediated activation of BK Ca channels [24] . Therefore, we assumed that ECH induced rat pulmonary artery vasorelaxation via the NO-cGMP-PKG-BK Ca channels, which led to a decrease in intracellular Ca 2+ levels and the relaxation of PASMCs.…”