Up-regulation of endothelial nitric oxide synthase by cytochrome P450 arachidonic acid epoxygenase BM3F87V

Cytochrome P450 (CYP)-dependent metabolites of arachidonic acid, epoxyeicosatrienoic acids (EETs), have been suggested to be an endothelium-derived hyperpolarizing factor (EDHF). However, the interaction or relation between EDHF and endothelial nitric oxide synthase (eNOS) is still to be elucidated. In the present study, the regulation of eNOS by endogenous EDHF is examined. The cytochrome P450 epoxygenase BM(3)F87V is cloned into the mammalian expression vector pCB(6). Cultured bovine aortic endothelial cells (BAECs) less than 4 passages are used and transfected with BM(3)F87V. The effects of endogenous EETs result from BM(3)F87V transfection on eNOS are assessed in the endothelial cells by Western blot and Northern blot, and eNOS activity is also measured by the conversion of L-arginine to L-citrulline. Compared to transfection with the empty pCB(6) vector, transfection of BAECs with BM(3)F87V significantly elevates the levels of eNOS protein expression, which is markedly inhibited by treatment with CYP inhibitor 17-ODYA. BM(3)F87V transfection also elevates the eNOS mRNA level and increases the eNOS activity. This study suggests that EDHF up-regutates eNOS gene expression.