11,12-Epoxyeicosatrienoic acid stimulates endogenous mono-ADP- ribosylation in bovine coronary arterial smooth muscle

Abstract

The role of endogenous ADP-ribosylation in mediating the activation of the Ca2+-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-32P]NAD. Four 32P-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 K1, 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.