Activation of microsomal cytochrome P450 mono-oxygenase by Ca2+ store depletion and its contribution to Ca2+ entry in porcine aortic endothelial cells.
1. We investigated how microsomal cytochrome P450 mono-oxygenase (Cyp450 MO) is regulated in cultured porcine aortic endothelial cells. The hypothesis that a Cyp450 MO-derived metabolite links Ca2+ store depletion and Ca2+ entry was studied further. 2. Microsomal Cyp450 MO was monitored fluorometrically by dealkylation of 1-ethoxypyrene-3,6,8-tris-(dimethyl-sulphonamide; EPSA) in saponin permeabilized cells or in subcellular compartments. Endothelial Ca2+ signalling was measured by a standard fura-2 technique, membrane potential was determined with the potential-sensitive fluorescence dye, bis-(1,3-dibutylbarbituric acid) pentamethine oxonol (DiBAC4(5)) and tyrosine kinase was quantified by measuring the phosphorylation of a immobilized substrate with a horseradish peroxidase labelled phosphotyrosine specific antibody. 3. Depletion of cellular Ca2+ pools with inositol 1,4,5-trisphosphate (IP3), thapsigargin or cyclopiazonic acid activated microsomal Cyp450 MO. Similar to direct Ca2+ store depletion, chelating of intramicrosomal Ca2+ with oxalate stimulated Cyp450 MO activity, while changing cytosolic free Ca2+ failed to influence Cyp450 MO activity. These data indicate that microsomal Cyp450 MO is activated by depletion of IP3-sensitive stores. 4. Besides the common cytochrome P450 inhibitors, econazole, proadifen and miconazole, thiopentone sodium and methohexitone inhibited Cyp450 MO in a concentration-dependent manner. The physiological substrate of Cyp450 MO, arachidonic acid, inhibited EPSA dealkylation. In contrast to most other cytochrome P450 inhibitors used in this study, thiopentone sodium did not directly interfere with Ca2+ entry pathways, membrane hyperpolarization due to K+ channel activation or tyrosine kinase activity. 5. Inhibition of Cyp450 MO by thiopentone sodium diminished Ca2+/Mn2+ entry to Ca2+ store depletion by 43%, while it did not interfere with intracellular Ca2+ release by IP3 or thapsigargin. 6. Cyp450 MO inhibition with thiopentone sodium diminished autacoid-induced membrane hyperpolarization. 7. Induction of Cyp450 MO with dexamethasone/clofibrate for 72 h yielded increases in thapsigargin-induced Cyp450 MO activity (by 35%), Ca2+/Mn2+ entry (by 105%) and membrane hyperpolarization (by 40%). 8. The Cyp450 MO-derived compounds, 11,12 and 5,6-epoxyeicosatrienoic acids (EETs) yielded membrane hyperpolarization, insensitive to thiopentone sodium. 9. These data demonstrate that endothelial Cyp450 MO is activated by Ca2+ store depletion and Cyp450 MO produced compounds that hyperpolarize endothelial cells. 10. The data presented and our previous findings indicate that Cyp450 MO plays a crucial role in the regulation of store-operated Ca2+ influx. We propose that Cyp450 MO-derived EETs constitute a signal for Ca2+ entry activation and increase the driving force for Ca2+ entry by membrane hyperpolarization in porcine aortic endothelial cells.
Hoebel
B G
BG
Department of Medical Biochemistry, University of Graz, Austria.
Kostner
G M
GM
Graier
W F
WF
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001011:Aorta / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002994:Clofibrate / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000502:physiology*; D003907:Dexamethasone / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000378:metabolism*; D004789:Enzyme Activation; D008345:Manganese / Q000494:pharmacology; D008564:Membrane Potentials; D008861:Microsomes / Q000201:enzymology; D013552:Swine
D008345:Manganese; C046783:11,12-epoxy-5,8,14-eicosatrienoic acid; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System; D015126:8,11,14-Eicosatrienoic Acid; D002994:Clofibrate; D002118:Calcium