EVIDENCE AGAINST A ROLE OF CYTOCHROME P450-DERIVED ARACHIDONIC-ACID METABOLITES IN ENDOTHELIUM-DEPENDENT HYPERPOLARIZATION BY ACETYLCHOLINEIN RAT ISOLATED MESENTERIC-ARTERY

1 In rat mesenteric artery, acetylcholine (ACh) causes endothelium-dep endent hyperpolarization by releasing endothelium-derived hyerpolarizi ng factor (EDHF). Recent evidence suggests that EDHF may be a cytochro me P450-derived arachidonic acid metabolite. The aim of the present st udy was to investigate whether such a metabolite is indeed contributin g to ACh-induced hyperpolarization observed in rat mesenteric artery. 2 The phospholipase A(2) inhibitor quinacrine (30 mu M) nearly complet ely eliminated ACh-induced hyperpolarization. However, the hyperpolari zing effect of pinacidil was also abolished in the presence of quinacr ine. 3 The imidazole antimycotic agents ketoconazole (50 mu M), clotri mazole (30 mu M) and miconazole (10 mu M), which bind to the heme moie ty of cytochrome P450, eliminated not only ACh-induced hyperpolarizati ons but also those induced by pinacidil. SKF525A (30 mu M), a prototyp e inhibitor of the enzyme, also abolished the hyperpolarizing response s to both agents. In contrast, neither 17-octadecynoic acid (10 mu M), a mechanism-based inhibitor of cytochrome P450 metabolism of fatty ac ids, nor eicosatetraynoic acid (20 mu M), an inhibitor of all arachido nic acid metabolic pathways, altered ACh-induced hyperpolarization. Fu rthermore, the hyperpolarization was unaffected by the preferential in hibitors of specific cytochrome P450 isozymes, alpha-naphtoflavone (1 mu M), diedthyldithiocarbamate (50 mu M), metyrapone (20 mu M) and tro leandomycin (10 mu M). 4 Pretreatment of rats with lipopolysaccharide (2 mg kg(-1)) and exposure to nitroprusside (10 mu M), both of which a re expected to inhibit cytochrome P450 activity due to nitric oxide ov erproduction, were without effect on ACh-induced hyperpolarization. Pr etreatment of rats for 3 days with pentobarbitone (80 mg kg(-1) day(-1 )), a cytochrome P450 inducer, also did not affect the hyperpolarizing response to ACh. 5 Arachidonic acid in concentrations up to 100 mu M had no detectable effect on smooth muscle membrane potential. 11,12-Ep oxyeicosatrienoic acid (EET, 10 mu M), one of cytochrome P450-derived epoxygenase metabolites of arachidonic acid, elicited a small endothel ium-independent membrane hyperpolarization. The hyperpolarizing respon se to EET was blocked by glibenclamide (30 mu M), in contrast to the r esponse to ACh. 6 These results suggest that the contribution of a cyt ochrome P450-derived metabolite of arachidonic acid to ACh-induced hyp erpolarization via EDHF release is minimal or absent in rat mesenteric artery.