Hydroxylamine-induced relaxation inhibited by K⁺ channel blockers in rat aortic rings

Abstract

Hydroxylamine, a putative endogenous nitric oxide donor, relaxed rat aorta in a concentration-dependent manner (0.01–30 μM). Removal of endothelium or pretreatment of aortic tissue with N^G-nitro-l-arginine (L-NOARG, 100 μM) did not affect the relaxant effect of hydroxylamine but L-NOARG at 100 μM abolished the acetylcholine-induced relaxation. Methylene blue (10 μM) significantly reduced the relaxant effect of hydroxylamine in endothelium-denuded arteries. Tetrapentylammonium ions (0.3–3 μM), tetraethylammonium ions (1–3 mM) and charybdotoxin (100 nM) reduced the relaxant effect of hydroxylamine in the endothelium-denuded arteries while glibenclamide (3 μM) had no effect. Neither tetrapentylammonium nor tetraethylammonium ions affected relaxations induced by forskolin and verapamil. The effects of tetrapentylammonium ions (3 μM) and charybdotoxin (100 nM) were additive. Tetrapentylammonium ions (3 μM), tetraethylammonium ions (3 mM) and charybdotoxin (100 nM) decreased the relaxation induced by sodium nitroprusside in the endothelium-denuded arteries while glibenclamide (3 μM) had no effect. The concentration–relaxation curve for the relaxant effect of hydroxylamine was shifted to the right in the presence of high extracellular K⁺ (15–60 mM). Neither tetrapentylammonium ions (3 μM) nor charybdotoxin (100 nM) affected hydroxylamine-induced relaxation of the endothelium-denuded aorta precontracted with 60 mM K⁺. These results indicate that hydroxylamine relaxes the rat aorta partially through activation of tetrapentylammonium-, tetraethylammonium- and charybdotoxin-sensitive K⁺ channels and its action is comparable with that of sodium nitroprusside, an exogenous nitric oxide donor. The endothelium is not involved in the aortic response to hydroxylamine.

Introduction

The movement of K⁺ ions across the plasma membrane of arterial smooth muscle is an important determinant of the membrane potential, which in turn regulates the influx of Ca²⁺ through voltage-sensitive Ca²⁺ channels and thus muscle contractility. Activation of K⁺ channels causes membrane hyperpolarization, which closes voltage-sensitive Ca²⁺ channels and relaxes vascular smooth muscle (Nelson et al., 1990b). Both Ca²⁺-activated K⁺ (K_Ca) channels and ATP-sensitive K⁺ (K_ATP) channels with distinct pharmacological properties have been extensively studied in arterial smooth muscle (Brayden and Nelson, 1992; Langton et al., 1991; Nelson et al., 1990b; Standen et al., 1989).

In response to hormonal stimuli which trigger Ca²⁺ influx (Himmel et al., 1993), the endothelial cells in intact arteries regulate the contractility of the underlying arterial smooth muscle by releasing vasoactive factors, which cause relaxation via a variety of mechanisms. Nitric oxide, formed enzymatically from the terminal guanidine nitrogen atoms of l-arginine in the endothelium (Moncada et al., 1991), is the best known endothelium-derived relaxing factor. Nitric oxide, apart from stimulating guanylate cyclase in smooth muscle, may modulate the activity of ionic channels on the plasma membrane. For example, nitric oxide hyperpolarizes rabbit mesenteric arteries through activation of K_ATP channels (Murphy and Brayden, 1995) and activates K_Ca channels indirectly by increasing the activity of cyclic GMP-dependent protein kinase in cerebral and coronary arteries (Archer et al., 1994; Robertson et al., 1993; Taniguchi et al., 1993). Hydroxylamine is suggested to be an intermediate product of the pathway for the oxidative conversion of l-arginine to nitric oxide and thus may be an endogenous nitric oxide donor (DeMaster et al., 1989; Thomas and Ramwell, 1989). This mechanism may account for its relaxant effect on rabbit and rat aorta (Kruszyna et al., 1982; Rapoport and Murad, 1984). Hydroxylamine requires a catalase-dependent reaction to give rise to nitric oxide (Craven et al., 1979; DeMaster et al., 1989), whilst sodium nitroprusside releases nitric oxide through a non-enzymatic process. More recently, hydroxylamine was shown to activate K_ATP channels in pancreatic β-cells (Antoine et al., 1996). However, the exact mechanisms underlying the relaxant effect of hydroxylamine are still unclear.

The present investigation was intended to determine whether activation of K_ATP or K_Ca channels contributes toward the vasorelaxant responses induced by hydroxylamine and the exogenous nitric oxide donor sodium nitroprusside in rat isolated aortic rings. Specifically, the relaxant responses to each nitric oxide donor were compared in the absence and presence of the blockers of K_ATP and K_Ca channels.