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1. We tested the hypothesis that agonist-stimulated Ca2+ entry, and thus formation of endothelium-derived nitric oxide (EDNO) in vascular endothelial cells, is related to activation of microsomal P450 mono-oxygenase (P450 MO) and the biosynthesis of 5,6-epoxyeicosatrienoic acid (5,6-EET). 2. Several P450 inhibitors diminished the sustained [Ca2+]i plateau response to agonist or intracellular Ca2+ store depletion with ATPase inhibitors by 31-69% (fura-2 technique). Mn2+ influx stimulated by agonists or ATPase inhibitors was prevented by P450 inhibitors. 3. Histamine- or ATPase inhibitor-stimulated formation of EDNO was strongly attenuated (50-83%) by P450 inhibitors, without any effect on EDNO formation by the Ca2+ ionophore A23187, indicating that decreased EDNO synthesis is due specifically to the inhibition of Ca2+ entry by these compounds. 4. Induction of P450 MO by beta-naphthoflavone potentiated agonist-induced Ca2+ and Mn2+ influx by 60 and 53%, respectively. Intracellular Ca2+ release remained unchanged. 5. The P450 MO product, 5,6-EET (< 156 nmol l-1), activated Ca2+/Mn2+ entry without any depletion of intracellular Ca2+ stores. The 5,6-EET-stimulated Ca2+/Mn2+ entry was not affected by P450 inhibitors. 6. As with the bradykinin-stimulated Ca2+ entry pathway, the 5,6-EET-activated Ca2+ entry pathway was permeable to Mn2+ and Ba2+, sensitive to Ni2+, La3+ and membrane depolarization, and insensitive to the removal of extracellular Na+ or the organic Ca2+ antagonist, nitrendipine. 7. In the presence of 5,6-EET, stimulation with bradykinin only transiently increased [Ca2+]i. Vice versa, 5,6-EET failed to increase [Ca2+]i further in bradykinin-stimulated cells. The sustained [Ca2+]i plateau phase induced by a co-stimulation with bradykinin and 5,6-EET was identical to that observed with bradykinin or 5,6-EET alone. 8. These results demonstrate that Ca2+ entry induced by the P450 MO product, 5,6-EET, is indistinguishable to that observed by stimulation with bradykinin. 9. All data support our hypothesis that depletion of endothelial Ca2+ stores activates microsomal P450 MO which in turn synthesizes 5,6-EET. We propose that the arachidonic acid metabolite 5,6-EET or one of its metabolites is a second messenger for activation of endothelial Ca2+ entry.