Cytochrome P450 mono‐oxygenase‐regulated signalling of Ca2+ entry in human and bovine endothelial cells.

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.