Evidence against a role of cytochrome P450‐derived arachidonic acid metabolites in endothelium‐dependent hyperpolarization by acetylcholine in rat isolated 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 A 2 inhibitor quinacrine (30 mM) nearly completely eliminated ACh-induced hyperpolarization. However, the hyperpolarizing eect of pinacidil was also abolished in the presence of quinacrine. 3 The imidazole antimycotic agents ketoconazole (50 mM), clotrimazole (30 mM) and miconazole (10 mM), which bind to the heme moiety of cytochrome P450, eliminated not only ACh-induced hyperpolarizations but also those induced by pinacidil. SKF525A (30 mM), a prototype inhibitor of the enzyme, also abolished the hyperpolarizing responses to both agents. In contrast, neither 17-octadecynoic acid (10 mM), a mechanism-based inhibitor of cytochrome P450 metabolism of fatty acids, nor eicosatetraynoic acid (20 mM), an inhibitor of all arachidonic acid metabolic pathways, altered AChinduced hyperpolarization. Furthermore, the hyperpolarization was unaected by the preferential inhibitors of speci®c cytochrome P450 isozymes, a-naphto¯avone (1 mM), diedthyldithiocarbamate (50 mM), metyrapone (20 mM) and troleandomycin (10 mM). 4 Pretreatment of rats with lipopolysaccharide (2 mg kg 71 ) and exposure to nitroprusside (10 mM), both of which are expected to inhibit cytochrome P450 activity due to nitric oxide overproduction, were without eect on ACh-induced hyperpolarization. Pretreatment of rats for 3 days with pentobarbitone (80 mg kg 71 day 71), a cytochrome P450 inducer, also did not aect the hyperpolarizing response to ACh. 5 Arachidonic acid in concentrations up to 100 mM had no detectable eect on smooth muscle membrane potential. 11,12-Epoxyeicosatrienoic acid (EET, 10 mM), 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 mM), 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.

EDHF-mediated vasodilation involves different mechanisms in normotensive and hypertensive rat lungs

“…Various CYP450 inhibitors have been found to reduce EDHF-mediated relaxation of rat (34) and dog (31) pulmonary arteries. However, CYP450 inhibitors have not inhibited EDHFmediated vasodilation in other studies or have done so nonspecifically (14,18,23,24,33,36,42,58). In our study, 7-ethoxyresorufin and sulfaphenazole alone reduced, and the combination of inhibitors abolished, EDHF-mediated vasodilation in normotensive lungs.…”

“…For example, sulfaphenazole, an inhibitor of cytochrome 2C isozymes, inhibits EDHFmediated vasorelaxation in the porcine coronary artery (21) and 7-ethoxyresorufin, an inhibitor of CYP450 1A isozyme, inhibits the vasodilation in perfused rat mesenteric (1) and renal (2) vascular beds and the guinea pig cerebral artery (14). However, other studies in various arteries show that these and other CYP450 inhibitors fail to inhibit EDHF-mediated vasorelaxation, or do so nonspecifically, and do not support the involvement of CYP450 in the EDHF mechanism (18,23,24,32,36,42,58).…”

14,15-Epoxyeicosa-5(Z)-Enoic-mSI

“…For example, relaxation to potassium channel activators such as cromakalim or pinacidil was inhibited by some cytochrome P450 inhibitors. [13][14][15] Additionally, the P450 inhibitor clotrimazole directly inhibits Ca 2ϩ -activated K ϩ channels. 16 -18 Therefore, to establish the role of endogenous EETs in vascular relaxation, pharmacological tools are required that selectively inhibit only the action or synthesis of EETs without other nonspecific effects.…”

“…In some studies, inhibitors of cytochrome P450 blocked the relaxation to bradykinin and acetylcholine, whereas in other studies, these inhibitors were without effect. 1,2,4,5,[11][12][13][14][15] In this regard, cytochrome P450 inhibitors may have other nonspecific vascular effects. For example, relaxation to potassium channel activators such as cromakalim or pinacidil was inhibited by some cytochrome P450 inhibitors.…”

Role of Cytoplasmic Phospholipase A2 in Endothelium-Derived Hyperpolarizing Factor Dilations of Rat Middle Cerebral Arteries

“…The above studies implicating PLA 2 are supported by inhibition of the EDHF response with quinacrine, a less specific PLA 2 inhibitor (Bauersachs et al, 1994;Fukao et al, 1997). Interestingly, Dong et al (2000) reported that 1 mol/L AACOCF 3 had no effect on guinea pig mesenteric arteries but inhibited the EDHF response in guinea pig MCAs by 48%.…”