Evidence against a role of cytochrome P450-derived arachidonic acid metabolites in endothelium-dependent hyperpolarization by acetylcholine in rat isolated mesenteric artery

1 In rat mesenteric artery, acetylcholine (ACh) causes endothelium-dependent hyperpolarization by releasing endothelium-derived hyerpolarizing factor (EDHF). Recent evidence suggests that EDHF may be a cytochrome P450-derived arachidonic acid metabolite. The aim of the present study was to investigate whether such a metabolite is indeed contributing to ACh-induced hyperpolarization observed in rat mesenteric artery. 2 The phospholipase A(2) inhibitor quinacrine (30 mu M) nearly completely eliminated ACh-induced hyperpolarization. However, the hyperpolarizing effect of pinacidil was also abolished in the presence of quinacrine. 3 The imidazole antimycotic agents ketoconazole (50 mu M), clotrimazole (30 mu M) and miconazole (10 mu M), which bind to the heme moiety of cytochrome P450, eliminated not only ACh-induced hyperpolarizations but also those induced by pinacidil. SKF525A (30 mu M), a prototype inhibitor of the enzyme, also abolished the hyperpolarizing responses to both agents. In contrast, neither 17-octadecynoic acid (10 mu M), a mechanism-based inhibitor of cytochrome P450 metabolism of fatty acids, nor eicosatetraynoic acid (20 mu M), an inhibitor of all arachidonic acid metabolic pathways, altered ACh-induced hyperpolarization. Furthermore, the hyperpolarization was unaffected by the preferential inhibitors of specific cytochrome P450 isozymes, alpha-naphtoflavone (1 mu M), diedthyldithiocarbamate (50 mu M), metyrapone (20 mu M) and troleandomycin (10 mu M). 4 Pretreatment of rats with lipopolysaccharide (2 mg kg(-1)) and exposure to nitroprusside (10 mu M), both of which are expected to inhibit cytochrome P450 activity due to nitric oxide overproduction, were without effect on ACh-induced hyperpolarization. Pretreatment 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-Epoxyeicosatrienoic acid (EET, 10 mu M), one of cytochrome P450-derived epoxygenase metabolites of arachidonic acid, elicited a small endothelium-independent membrane hyperpolarization. The hyperpolarizing response to EET was blocked by glibenclamide (30 mu M), in contrast to the response to ACh. 6 These results suggest that the contribution of a cytochrome P450-derived metabolite of arachidonic acid to ACh-induced hyperpolarization via EDHF release is minimal or absent in rat mesenteric artery.