Abstract-In the porcine coronary artery, a cytochrome P450 (CYP) isozyme homologous to CYP 2C8/9 has been identified as an endothelium-derived hyperpolarizing factor (EDHF) synthase. As some CYP enzymes are reported to generate reactive oxygen species (ROS), we hypothesized that the coronary EDHF synthase may modulate vascular homeostasis by the simultaneous production of ROS and epoxyeicosatrienoic acids. In bradykinin-stimulated coronary arteries, antisense oligonucleotides against CYP 2C almost abolished EDHF-mediated responses but potentiated nitric oxide (NO)-mediated relaxation. The selective CYP 2C9 inhibitor sulfaphenazole and the superoxide anion (O 2 Ϫ ) scavengers Tiron and nordihydroguaretic acid also induced a leftward shift in the NO-mediated concentration-relaxation curve to bradykinin. CYP activity and O 2 Ϫ production, determined in microsomes prepared from cells overexpressing CYP 2C9, were almost completely inhibited by sulfaphenazole. Sulfaphenazole did not alter the activity of either CYP 2C8, the leukocyte NADPH oxidase, or xanthine oxidase. ROS generation in coronary artery rings, visualized using either ethidium or dichlorofluorescein fluorescence, was detected under basal conditions. The endothelial signal was attenuated by CYP 2C antisense treatment as well as by sulfaphenazole. In isolated coronary endothelial cells, bradykinin elicited a sulfaphenazole-sensitive increase in ROS production. Although 11,12 epoxyeicosatrienoic acid attenuated the activity of nuclear factor-B in cultured human endothelial cells, nuclear factor-B activity was enhanced after the induction or overexpression of CYP 2C9, as was the expression of vascular cell adhesion molecule-1. These results suggest that a CYP isozyme homologous to CYP 2C9 is a physiologically relevant generator of ROS in coronary endothelial cells and modulates both vascular tone and homeostasis. Key Words: coronary artery Ⅲ cytochrome P450 Ⅲ endothelium-derived hyperpolarizing factor Ⅲ NADPH oxidase Ⅲ reactive oxygen species T he bioavailability of nitric oxide (NO) within the vascular wall and, as a consequence, NO-mediated relaxation is attenuated by an elevation in superoxide anion (O 2 Ϫ ) production. 1 Enzymes capable of generating reactive oxygen species (ROS) within the vasculature are, for example, NO synthases (NOS), cyclooxygenases, lipoxygenases, xanthine oxidase, and NADPH oxidase, all of which are reported to be functional in endothelial cells. 2 Diphenyleneiodonium has been used extensively to characterize ROS-generating enzymatic systems; however, this compound inactivates flavoproteins during electron-transfer reactions 3 and completely inhibits the activity of all isoforms of NOS, 3-6 xanthine oxidase, 3,7 and NADPH oxidase. 8 Thus, although pharmacological studies using isolated arteries have demonstrated that the production of ROS is markedly increased in animal models of hypertension, atherosclerosis, and heart failure, 9 the relative contribution of each of the potential O 2 Ϫ -producing enzymes to the overall ROS prod...

1 Cannabinoids are potent inhibitors of endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxations. We set out to study the mechanism underlying this eect and the possible role of cannabinoid-induced changes in intercellular gap junction communication. -THC prevented both the bradykinin-induced hyperpolarization and the nitric oxide and prostacyclin-independent relaxation of pre-contracted rings of porcine coronary artery. These eects were prevented by PD98059 as well as U0126. 5 In the absence of D 9 -THC, neither PD98059 nor U0126 aected the NO-mediated relaxation of coronary artery rings but both substances induced a leftward shift in the concentration ± relaxation curve to bradykinin when diclofenac and N o nitro-L-arginine were present. Moreover, PD98059 and U0126 prolonged the bradykinin-induced hyperpolarization of porcine coronary arteries, without aecting the magnitude of the response. 6 These results indicate that the cannabinoid-induced activation of ERK1/2, which leads to the phosphorylation of connexin 43 and inhibition of gap junctional communication, may partially account for the D 9-THC-induced inhibition of EDHF-mediated relaxation. Moreover, the activation of ERK1/2 by endothelial cell agonists such as bradykinin, appears to exert a negative feedback inhibition on EDHF-mediated responses.

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