Received for publication April 14, 2003.
Revised May 9, 2003.
Accepted for publication August 8, 2003.

Upregulation of endothelial nitric oxide synthase by endothelium-derived hyperpolarizing factor involves MAPK and PKC signaling pathways

Abstract

Cytochrome P450-dependent metabolites of arachidonic acid, the epoxyeicosatrienoic acids (EETs), are proposed to be endothelium-derived hyperpolarizing factors (EDHF) that affect vascular tone; however, the effects of EDHF on endothelial-derived nitric oxide biosynthesis remain unknown. We examined the regulation of endothelial nitric oxide synthase (eNOS) by EDHF and investigated the relevant signaling pathways involved. The P450 epoxygenases CYP102 F87V mutant, CYP2C11-CYPOR and CYP2J2 were transfected into cultured bovine aortic endothelial cells and the effects of endogenously formed or exogenously applied EETs on eNOS expression and activity were assessed. Transfection with the P450 epoxygenases led to increased eNOS protein expression, an effect that was attenuated by co-treatment with the P450 inhibitor 17- ODYA. Northern analysis demonstrated that P450 transfection led to increased eNOS mRNA levels consistent with an effect at the pretranslational level. P450 epoxygenase transfection resulted in increased eNOS activity as measured by the conversion of L-arginine to L- citrulline. Addition of synthetic EETs (50-200nM) to the culture media also increased eNOS expression and activity. Treatment with MAPK, MAPK kinase, and protein kinase C inhibitors apigenin, PD98059 and H-7, respectively, significantly inhibited the effects of P450 transfection on eNOS expression. Overexpression of P450 epoxygenases or addition of synthetic EETs increased Thr495 phosphorylation of eNOS, an effect that was inhibited by both apigenin and PD98059. Overexpression of P450 epoxygenases in rats resulted in increased aortic eNOS expression providing direct evidence that EDHF can influence vascular eNOS levels in vivo. Based on this data, we conclude that EDHF upregulates eNOS via activation of MAPK and PKC signaling pathways.

Key words: arachidonic acid, cytochrome P450, endothelium, epoxyeicosatrienoic acid, epoxygenase, nitric oxide

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