Real-time fluorescence analysis on molecular mechanisms for regulation of cytochrome P450scc activity upon steroidogenic stimulation in adrenocortical cells.
Real-time fluorescence analysis revealed that the activity of cytochrome P450scc was related to Ca2+ signals arising from extracellular NADPH, ACTH and ATP stimulation in adrenocortical fasciculata cells. The side-chain cleavage reaction by cytochrome P450scc was measured with 3beta-hydroxy-22,23-bisnor-5-cholenyl ether (cholesterol-resorufin) by observing the distinct increase in fluorescence upon conversion of cholesterol-resorufin to resorufin and pregnenolone. Adrenocorticotropic hormone (ACTH) induced a relatively small stimulation of the P450scc activity. A significant production of resorufin was revealed after stimulation of cell cultures with 100 pM, 1 nM of ACTH for 3 h. On the other hand, extracellular NADPH was found to rapidly and greatly stimulate the resorufin production in intact cells immediately after the addition of 50-500 microM NADPH. The extracellular NADPH stimulation was prevented by the addition of thapsigargin and EGTA which abolished Ca2+ oscillations induced by NADPH. Suramin, a specific antagonist of the P2y type ATP receptor, also completely abolished the NADPH-induced cholesterol-resorufin conversion. These results imply that extracellular NADPH (membrane impermeable) produced Ca2+ oscillations through its binding to ATP receptor thereby stimulating the activity of P450scc. The application of 45-500 microM extracellular ATP to cells did not, however, significantly increase the resorufin production. These three stimulators produced very different types of Ca2+ signals. ACTH induced mainly a series of Ca2+ spikes superimposed on a long-lasting basal Ca2+ elevation. The Ca2+ signals induced by NADPH showed predominantly a series of Ca2+ spikes without elevation of the basal Ca2+ concentration. Only long-lasting Ca2+ elevation was induced by extracellular ATP. The stimulation of cytochrome P450scc may thus be correlated with the different patterns of Ca2+ signals.
Homma
R
R
Department of Biophysics and Life Sciences, Graduate School of Arts and Sciences, University of Tokyo at Komaba, Japan.
Kimoto
T
T
Niimura
Y
Y
Krivosheev
A
A
Hara
T
T
Ohta
Y
Y
Kawato
S
S
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000302:Adrenal Cortex / Q000166:cytology / Q000378:metabolism*; D000324:Adrenocorticotropic Hormone / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology; D002417:Cattle; D002784:Cholesterol / Q000378:metabolism; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D009249:NADP / Q000378:metabolism*; D010078:Oxazines / Q000378:metabolism; D011510:Proteolipids / Q000378:metabolism
D010078:Oxazines; D011510:Proteolipids; C020485:proteoliposomes; D009249:NADP; C014180:resorufin; D000255:Adenosine Triphosphate; D000324:Adrenocorticotropic Hormone; D002784:Cholesterol; D002786:Cholesterol Side-Chain Cleavage Enzyme; D002118:Calcium