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.
Graier
W F
WF
Department of Medical Biochemistry, University of Graz, Austria.
Simecek
S
S
Sturek
M
M
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000594:Amino Acid Oxidoreductases / Q000378:metabolism; D000818:Animals; D001464:Barium / Q000494:pharmacology; D001571:Benzoflavones / Q000494:pharmacology; D001920:Bradykinin / Q000378:metabolism; D002118:Calcium / Q000502:physiology*; D002417:Cattle; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000819:agonists / Q000378:metabolism*; D004464:Econazole / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D005260:Female; D006632:Histamine / Q000378:metabolism; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D019001:Nitric Oxide Synthase; D010105:Oxygenases / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015221:Potassium Channels / Q000187:drug effects; D015290:Second Messenger Systems / Q000187:drug effects*; D019324:beta-Naphthoflavone
D001571:Benzoflavones; D065607:Cytochrome P-450 Enzyme Inhibitors; D015221:Potassium Channels; D001464:Barium; D019324:beta-Naphthoflavone; D004464:Econazole; C040776:5,6-epoxy-8,11,14-eicosatrienoic acid; D006632:Histamine; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D000251:Adenosine Triphosphatases; D015126:8,11,14-Eicosatrienoic Acid; D008274:Magnesium; D001920:Bradykinin; D002118:Calcium