EFFECTS OF CYTOCHROME-P450 INHIBITORS ON EDHF-MEDIATED RELAXATION IN THE RAT HEPATIC-ARTERY

1 The possibility that the endothelium-derived hyperpolarising factor (EDHF) in the rat hepatic artery is a cytochrome P450 mono-oxygenase m etabolite of arachidonic acid was examined in the present study. In th is preparation, acetylcholine elicits EDHF-mediated relaxations in the presence of the nitric oxide (NO) synthase and cyclo-oxygenase inhibi tors N-omega-nitro-L-arginine (L-NOARG) and indomethacin, respectively . 2 17-Octadecynoic acid (17-ODYA, 50 mu M), a suicide-substrate inhib itor of the cytochrome P450 mono-oxygenases responsible for the produc tion of 5,6-, 8,9-, 11,12- and 14,15-epoxyeicosatrienoic acids (EETs), had no effect on acetylcholine-induced relaxations in the presence of L-NOARG (0.3 mM) plus indomethacin (10 mu M). Furthermore, 5,6-, 8,9- , 11,12- and 14,15- EETs failed to relax arteries without endothelium in the presence of L-NOARG plus indomethacin. 3 Proadifen and clotrima zole, which are inhibitors of several isoforms of cytochrome P450 mono -oxygenases, inhibited acetylcholine-induced relaxations in the presen ce of L-NOARG plus indomethacin. The concentration of acetylcholine wh ich caused half-maximal relaxation was about 3 and 30 times higher in the presence than in the absence of clotrimazole (3 mu M) and proadife n (10 mu M), respectively. The maximal relaxation was reduced by proad ifen but not by clotrimazole. Proadifen (10 mu M) also inhibited acety lcholine-induced hyperpolarization in the presence of L-NOARG plus ind omethacin. 4 In the presence of 30 mM K+ plus indomethacin (10 mu M), acetylcholine induced an L-NOARG-sensitive relaxation mediated via rel ease of NO. Under these conditions, proadifen (10 mu M) shifted the ac etylcholine concentration-response curve 6 fold to the right without a ffecting the maximal relaxation. Clotrimazole (3 mu M) was without eff ect on these responses. The relaxant actions of the NO donor, 3-morpho lino-sydnonimine, were unaffected by proadifen (10 mu M). 5 The relaxa nt effects of the opener of ATP-sensitive potassium channels, levcroma kalim, were abolished by proadifen (10 mu M) and strongly attenuated b y clotrimazole (3 mu M). Proadifen (10 mu M) also abolished the hyperp olarization induced by levcromakalim (1 mu M). 6 The lack of effect of 17-ODYA on relaxations mediated by EDHF, together with the failure of extracellularly-applied EETs to produce relaxation, collectively sugg est that EDHF is not an EET in the rat hepatic artery. It seems likely that inhibition of ion channels in the smooth muscle rather than redu ced EDHF formation in the endothelium offers a better explanation for the actions of the cytochrome P450 inhibitors proadifen and clotrimazo le.