In the aorta of the spontaneously hypertensive rat (SHR), endothelium-dependent contractions are enhanced by inhibitors of NO synthase and scavengers of NO, but not by methylene blue, an inhibitor of guanylyl cyclase, suggesting that the endotheliumderived contracting factor (EDCF) interacts chemically with NO and is inactivated by the latter. However, in view of the relative lack of specificity of methylene blue this hypothesis was re-examined. Acetylcholine-induced endothelium-dependent contractions of isolated rings of SHR aorta were significantly and similarly potentiated by two NOS inhibitors, by two structurally different NO scavengers, by two inhibitors of guanylate cyclase ODQ and NS2028, but to a lesser extent by methylene blue. The contraction of the isolated rat trachea in response to methacholine and the contraction of the rat aorta in response to both 8-isoprostane and KCl were inhibited significantly by methylene blue. Methylene blue binds to the M3 muscarinic receptor subtype but not to the TP receptor. Therefore, methylene blue is an antagonist of the M3 muscarinic receptor subtype, involved in the release of EDCF, and a non-specific inhibitor of TP receptormediated contractions, the receptor involved in the action of EDCF. These inhibitory effects of methylene blue are likely to counteract the effect of the inhibition of soluble guanylate cyclase. These results rule out the hypothesis according to which NO would chemically inactivate EDCF.

We have investigated the effects of insulin on the phosphorylation of glycogen phosphorylase in skeletal muscle. Rat epitrochlearis muscles were incubated in vitro with 32Ptio label cellular phosphoproteins, before being treated withho rmones. Phosphorylase, phosphorylase kinase, and glycogen synthase were immunoprecipitated under conditions that prevented changes in their phosphorylation states. Based on measurements of the activity ratio( -AMP/+AMP) and the 32P content of phosphorylase, 4-8% of the phosphorylase in untreated muscles appeared to be phosphorylated. Epinephrine promoted increases of approximately 4-fold in the32P content and activity ratio. Neither these effects nor the epinephrine-stimulated increases in phosphorylation of glycogen synthase andp hosphorylase kinase were attenuated by insulin. However, insulin at physiological concentrations rapidly decreased the 32P content of phosphorylase in muscles incubated without epinephrine. Results from peptide mapping experiments indicate that phosphorylase was phosphorylated at a single site in both control anhdo rmonetreated muscles. The maximum effect of insulin on phosphorylase represented a decrease in32Po f approximately 50%. By comparison, the 32P content of glycogen synthase and the

1 Experiments were designed to investigate whether or not oxygen-derived free radicals mediate endothelium-dependent contractions to acetylcholine in the aorta of spontaneously hypertensive rat (SHR). 2 Isometric tension was measured in aortic rings taken from adult male SHR and Wistar-Kyoto rat (WKY) in the presence of NG-nitro-L-arginine. 3 Endothelium-dependent contractions to acetylcholine were signi

The present study was designed to determine whether or not an increase in endothelial intracellular concentration of calcium ([Ca2+]i) evokes endotheliumdependent contractions in the aorta from normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Acetylcholine, adenosine triphosphate (ATP) and the calcium ionophore, A 23187, produced endothelium-dependent relaxations in isolated aortic rings of both WKY and SHR. These relaxations in response to the three agonists were significantly smaller in the SHR when compared with the WKY. Endothelium-dependent contractions to acetylcholine, ATP and A 23187 were observed only in the aorta isolated from the SHR. In the presence of NG-nitro-Larginine, an NO synthase inhibitor, the endothelium-dependent contractions in response to acetylcholine, ATP and A 23187 were potentiated significantly in the aorta SHR and were unmasked in that of WKY. However, the contractions were still significantly greater in SHR than in WKY. These contractions were abolished by indomethacin and valeryl salicylate (two cyclo-oxygenase inhibitors) as well as by S 18886 (a TP-receptor antagonist), indicating that the endothelium-dependent contraction produced by the three agonists share the same characteristics. The results of the present study indicate that the release/generation of endotheliumderived contracting factor, requires an increase in endothelial [Ca2+]i.