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 hyperpolarizing 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 microM) 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 microM), clotrimazole (30 microM) and miconazole (10 microM), which bind to the heme moiety of cytochrome P450, eliminated not only ACh-induced hyperpolarizations but also those induced by pinacidil. SKF525A (30 microM), a prototype inhibitor of the enzyme, also abolished the hyperpolarizing responses to both agents. In contrast, neither 17-octadecynoic acid (10 microM), a mechanism-based inhibitor of cytochrome P450 metabolism of fatty acids, nor eicosatetraynoic acid (20 microM), 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 microM), diedthyldithiocarbamate (50 microM), metyrapone (20 microM) and troleandomycin (10 microM). 4. Pretreatment of rats with lipopolysaccharide (2 mg kg-1) and exposure to nitroprusside (10 microM), 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 microM had no detectable effect on smooth muscle membrane potential. 11, 12-Epoxyeicosatrienoic acid (EET, 10 microM), 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 microM), 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.
Fukao
M
M
Department of Pharmacology, Hokkaido University School of Medicine, Sapporo, Japan.
Hattori
Y
Y
Kanno
M
M
Sakuma
I
I
Kitabatake
A
A
D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology; D016718:Arachidonic Acid / Q000378:metabolism*; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000502:physiology*; D006146:Guanidines / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D066298:In Vitro Techniques; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D008638:Mesenteric Arteries / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000187:drug effects*; D010741:Phospholipases A / Q000037:antagonists & inhibitors; D054467:Phospholipases A2; D020110:Pinacidil; D051381:Rats; D017208:Rats, Wistar; D014665:Vasodilator Agents / Q000494:pharmacology
D000935:Antifungal Agents; D065607:Cytochrome P-450 Enzyme Inhibitors; D006146:Guanidines; D007093:Imidazoles; D008070:Lipopolysaccharides; D014665:Vasodilator Agents; D016718:Arachidonic Acid; D020110:Pinacidil; D003577:Cytochrome P-450 Enzyme System; D010741:Phospholipases A; D054467:Phospholipases A2; D000109:Acetylcholine