The action of some vascular smooth muscle relaxants depends on the presence of the endothelium. We have recently shown that relaxation may be mediated through the formation of cyclic GMP. The nitrovasodilators are another class of relaxants which exert their effects through the formation of cyclic GMP, although their relaxation is independent of the presence of the endothelium. Their relaxant properties seem to depend on free radical formation--specifically, the formation of nitric oxide. The NO-induced smooth muscle relaxation is proposed to occur through activation of guanylate cyclase and the formation of cyclic GMP. Protein phosphorylation is thought to be a common event in the pathway for many biological phenomena. Moreover, sodium nitroprusside and 8-bromo cyclic GMP induce similar patterns of protein phosphorylation in intact rat thoracic aorta. Here we report that the patterns of protein phosphorylation induced by the endothelium-dependent vasodilators and nitrovasodilators were identical. Incorporation of 32P into myosin light chain was decreased by both classes of agents. Removal of the endothelium abolished the changes in phosphorylation with the endothelium-dependent vasodilator (acetylcholine), but not those with the nitrovasodilator (sodium nitroprusside). These results suggest that endothelium-dependent vasodilators and nitrovasodilators induce relaxation through cyclic GMP-dependent protein phosphorylation and dephosphorylation of myosin light chain.

The effects of sodium nitroprusside, 8-bromo cyclic GMP, 8-bromoguanosine 5'-monophosphate, 8-bromo cyclic AMP, dibutyryl cyclic AMP, and isoproterenol on incorporation of 32P into proteins in intact rat thoracic aorta were studied. Aortas were incubated in [32P]orthophosphate in order to label endogenous adenosine triphosphate. Agents were then added for various times and the tissues were homogenized and fractionated (100,000 x g for 60 min) into soluble and particulate fractions. Soluble and particulate fractions were subjected to isoelectric focusing followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiographs were made. Nitroprusside induced a concentration-dependent increase in incorporation of 32p into nine proteins and a decrease in 32P incorporation into two proteins. Some of these proteins appeared in both the soluble and particulate fractions of homogenates; others appeared only in the soluble fraction. The pattern of 32p incorporation was identical after 2-or 15-min exposure to nitroprusside and was mimicked by exposure to 50-500 p.M 8-bromo cyclic GMP. 8-Bromoguanosine 5'-monophosphate did not alter 32p incorporation. Dibutyryl cyclic AMP at 50 jaM had no effect upon 32p incorporation whereas a higher concentration (0.5 mM) caused increased or decreased 32P incorporation into some, but not all, of the same proteins. 8-Bromo cyclic AMP (5 mM) produced only small changes in 32P incorporation. The pattern of 32P incorporation induced by a relatively high concentration of isoproterenol 0.1 mM was similar but not identical to that seen with 0.5 mM dibutyryl cyclic AMP. The present study indicates that the incorporation of 32p into endogenous proteins of intact rat aorta can be regulated by nitroprusside. These effects can be mimicked by cyclic GMP analogues and only partially by cyclic AMP analogues or isoproterenol. Presumably, these effects of nitroprusside are mediated through a cyclic GMP-dependent process (protein kinase or phosphatase) which may play a role in the relaxant properties of nitroprusside and cyclic GMP.The present study investigates the hypothesis that smooth muscle relaxation induced by sodium nitroprusside may be mediated by cyclic GMP-dependent phosphorylation of endogenous proteins. Increased levels of cyclic GMP have been observed in response to sodium nitroprusside in various tissues including smooth muscle preparations (1-4). Cyclic GMP analogues have also been shown to cause relaxation or decreased contraction in smooth muscle (1, 5-7). Furthermore, agents that inhibit nitroprusside activation of guanylate cyclase in broken cell preparations have been shown to inhibit relaxation as well as nitroprusside-induced increased levels of cyclic GMP (6-9).However, the molecular mechanism by which nitroprusside and cyclic GMP induce relaxation is not known. Protein phosphorylation is thought to be a final common pathway for many biological processes (10). The addition of cyclic GMP to crude particulate fractions ofvarious smooth muscle preparations s...

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