Files
Abstract
Evidence suggests that the vasodilator actions of S-nitrosothiols more closely resemble those of endothelium-dependent-relaxing factor (EDRF) than do those of nitric oxide (NO). S-nitrosothiols such as L-S-nitrosocysteine are thought to be stored within cytoplasmic vesicles in endothelial cells. Stimulation of endothelial cells with agonists or shear stress is thought to mobilize these vesicles by exocytotic mechanisms. We have shown that cytoplasmic vesicles exists in the endothelium of rat cerebral and basilar arteries, and in small rat pulmonary, mesenteric and femoral arteries. The presence of these vesicles suggests that a ubiquitous process, namely, vesicular exocytosis, may be involved in the ability of the endothelium to regulate vascular tone. This study demonstrates the presence of NADPH diaphorase positive vesicles, which may be a histochemical marker for S-nitrosothiols, rather than for NO synthase. We have demonstrated that endothelial cells contain fusion proteins necessary for vesicular exocytosis at the light level and ultrastructurally. Ultrastructurally, vesicle-associated membrane protein (VAMP) was shown to be closely associated with the vesicle membrane, while synaptosomal-associated protein of 25KDa (SNAP-25) and syntaxin were located in the endothelial cell membrane. The fusion proteins are therefore ideally located for core complex formation, which precedes exocytosis. We found that the endothelium-dependent agonist, acetylcholine, elicited a pronounced relaxation of small pulmonary, mesenteric and femoral arteries. This observation was paired with a significant increase in the number of fused vesicles at the endothelial cell membrane. In systemic arteries, hypoxia elicited relaxation and an increase in the numbers of fused vesicles at the endothelial cell membrane. In the small pulmonary artery, hypoxia elicited vasoconstriction and an increase in the number of fused vesicles. These results support the possibility that endothelium-dependent relaxation in response to stimuli such as shear stress and hypoxia is achieved by (1) mobilization of S-nitrosothiol-containing vesicles to the endothelial plasma membrane, (2) fusion of the vesicle to the plasma membrane and then exocytosis (release), and (3) S-nitrosothiol-mediated relaxation of vascular smooth muscle. Keywords: Endothelial cell, Endothelium-dependent-relaxing factor, Exocytosis, S-nitrosothiols,