11,12-Epoxyeicosatrienoic Acid Stimulates Endogenous Mono-ADP-Ribosylation in Bovine Coronary Arterial Smooth Muscle

Abstract

Abstract —The role of endogenous ADP-ribosylation in mediating the activation of the Ca 2+ -activated K + channels was determined in bovine coronary arteries. Endogenous ADP-ribosylation was examined by incubating coronary arterial homogenates or lysates of cultured coronary arterial smooth muscle cells with [adenylate- 32 P]NAD. Four 32 P-labeled proteins were observed at 51, 52, 80, and 124 kDa in the homogenates and lysates. This reaction was enhanced by the addition of 11,12-epoxyeicosatrienoic acid (11,12-EET), a cytochrome P450–derived eicosanoid, and GTP to the incubation. By Western blot analysis, 42- and 70-kDa proteins were recognized by specific antibodies against ADP-ribosyltransferase in the coronary arterial homogenates and smooth muscle cell lysate but not in the lysate of endothelial cells. The 52-kDa acceptor protein of endogenous ADP-ribosylation comigrated with a protein ADP-ribosylated by cholera toxin and was recognized and immunoprecipitated by an anti–G S α antibody. These results suggest that G S α is one of several acceptors of the ADP-ribose moiety. As shown by the patch-clamp technique, 11,12-EET stimulated the activation of the K + channels in the smooth muscle cells, and this activation was completely blocked by novobiocin, vitamin K 1 , 3-aminobenzamide, and m -iodobenzylguanidine, inhibitors of endogenous mono-ADP-ribosyltransferases. We conclude that endogenous mono-ADP-ribosyltransferases are present in smooth muscle from bovine coronary arteries. These enzymes transfer ADP-ribose to the cellular proteins such as G S α and may mediate intracellular signal transduction in coronary vascular smooth muscle. In the coronary circulation, the ADP-ribosylation signaling pathway may play an important role in mediating the activation of the K + channels induced by 11,12-EET.