23 January 1997, Volume 120, Issue 3, Pages 439 – 446

Article

Evidence against a role of cytochrome P450-derived arachidonic acid metabolites in endothelium-dependent hyperpolarization by acetylcholine in rat isolated mesenteric artery Mitsuhiro Fukao1,3, Yuichi Hattori1, Morio Kanno1, Ichiro Sakuma2 & Akira Kitabatake2 1Department of Pharmacology, Hokkaido University School of Medicine, Sapporo 060, Japan     2Department of Cardiovascular Medicine, Hokkaido University School of Medicine, Sapporo 060, Japan     3Author for correspondence

Keywords

Endothelium-derived hyperpolarizing factor;   acetylcholine;   hyperpolarization;   arachidonic acid;   cytochrome P450;   epoxyeicosatrienoic acid;   rat mesenteric artery

Abstract

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 A2 inhibitor quinacrine (30 µM) 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 µM), clotrimazole (30 µM) and miconazole (10 µM), which bind to the heme moiety of cytochrome P450, eliminated not only ACh-induced hyperpolarizations but also those induced by pinacidil. SKF525A (30 µM), a prototype inhibitor of the enzyme, also abolished the hyperpolarizing responses to both agents. In contrast, neither 17-octadecynoic acid (10 µM), a mechanism-based inhibitor of cytochrome P450 metabolism of fatty acids, nor eicosatetraynoic acid (20 µM), 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, -naphtoflavone (1 µM), diedthyldithiocarbamate (50 µM), metyrapone (20 µM) and troleandomycin (10 µM). 4   Pretreatment of rats with lipopolysaccharide (2 mg kg-1) and exposure to nitroprusside (10 µM), 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 µM had no detectable effect on smooth muscle membrane potential. 11,12-Epoxyeicosatrienoic acid (EET, 10 µM), 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 µM), 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.

Received 16 July, 96; Revised 7 October, 96; Accepted 29 October, 96

^© Macmillan Publishers Ltd 1997