It is controversial whether the endothelial cell release of nitric oxide (NO) or a different factor(s) accounts for endothelium-dependent hyperpolarization, because in many arteries endothelium-dependent relaxation and hyperpolarization resists inhibitors of NO synthase. The contribution of NO to acetylcholine-induced endotheliumdependent hyperpolarization and relaxation of the rabbit carotid artery was determined by measuring NO with electrochemical and chemiluminescence techniques. In the presence of phenylephrine to depolarize and contract the smooth muscle cells, acetylcholine caused concentration-dependent hyperpolarization and relaxation which were closely correlated to the release of NO. N -nitro-L-arginine methyl ester (30 M) partially reduced the release of NO and caused a similar reduction in smooth muscle cell relaxation and hyperpolarization. To determine if the residual responses were mediated by another endothelium-derived mediator or NO released despite treatment with N -nitro-L-arginine methyl ester, N -nitro-L-arginine (300 M) was added. The combined inhibitors further reduced, but did not eliminate, NO release, smooth muscle relaxation, and hyperpolarization. Hyperpolarization and relaxation to acetylcholine remained closely correlated with the release of NO in the presence of the inhibitors. In addition, the NO donor, SIN-1, caused hyperpolarization and relaxation which correlated with the concentrations of NO that it released. These studies indicate that (i) the release of NO by acetylcholine is only partially inhibited by these inhibitors of NO synthase when used even at high concentrations, and (ii) NO rather than another factor accounts fully for endothelium-dependent responses of the rabbit carotid artery.Nitric oxide (NO) is now accepted to be the major mediator of endothelium-dependent, arterial smooth muscle relaxation (1). However, whether or not NO accounts for endotheliumdependent hyperpolarization is controversial (2, 3). Authentic NO and NO donors hyperpolarize vascular smooth muscle cells (4); however, in some studies, concentrations of NO that were too high to be considered physiological were required, or the hyperpolarization observed was smaller than that caused by an endothelial cell agonist (2, 3). In addition, in many in vitro and in vivo studies of human and animal arteries, endotheliumdependent relaxation, vasodilatation, and hyperpolarization persist in the presence of L-arginine analogues that are inhibitors of NO synthase (NOS) (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). This has led to the supposition that factors other than NO are important mediators of endothelium-dependent responses including hyperpolarization (2, 3). Cytochrome P450 metabolites of arachidonic acid (21,22), and carbon monoxide (23) have been proposed as alternative mediators. Neither the release from the endothelium of sufficient quantities of these mediators to account for the responses has been measured, nor has NO release been measured directly to exclude its contributio...
