Real-time fluorescence analysis on molecular mechanisms for regulation of cytochrome P450scc activity upon steroidogenic stimulation in adrenocortical cells

• Published in: Journal of inorganic biochemistry 2000-11, Vol.82 (1), p.171-180
• Main Authors: Homma, Ryota, Kimoto, Tetsuya, Niimura, Yoshihito, Krivosheev, Alexander, Hara, Takayuki, Ohta, Yoshihiro, Kawato, Suguru
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Adrenal cortex; Adrenal Cortex - cytology; Adrenal Cortex - metabolism; Adrenocorticotropic Hormone - metabolism; Animals; Calcium - metabolism; Calcium signal; Calcium Signaling - physiology; Cattle; Cholesterol - metabolism; Cholesterol Side-Chain Cleavage Enzyme - metabolism; Fluorescence analysis; Fluorescent cholesterol; Mitochondria - metabolism; NADP - metabolism; Oxazines - metabolism; P450scc; Proteolipids - metabolism
• ISSN: 0162-0134; 1873-3344
• DOI: 10.1016/S0162-0134(00)00160-4

Real-time fluorescence analysis revealed that the activity of cytochrome P450scc was related to Ca 2+ signals arising from extracellular NADPH, ACTH and ATP stimulation in adrenocortical fasciculata cells. The side-chain cleavage reaction by cytochrome P450scc was measured with 3β-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 μM NADPH. The extracellular NADPH stimulation was prevented by the addition of thapsigargin and EGTA which abolished Ca 2+ oscillations induced by NADPH. Suramin, a specific antagonist of the P 2y type ATP receptor, also completely abolished the NADPH-induced cholesterol–resorufin conversion. These results imply that extracellular NADPH (membrane impermeable) produced Ca 2+ oscillations through its binding to ATP receptor thereby stimulating the activity of P450scc. The application of 45–500 μM extracellular ATP to cells did not, however, significantly increase the resorufin production. These three stimulators produced very different types of Ca 2+ signals. ACTH induced mainly a series of Ca 2+ spikes superimposed on a long-lasting basal Ca 2+ elevation. The Ca 2+ signals induced by NADPH showed predominantly a series of Ca 2+ spikes without elevation of the basal Ca 2+ concentration. Only long-lasting Ca 2+ elevation was induced by extracellular ATP. The stimulation of cytochrome P450scc may thus be correlated with the different patterns of Ca 2+ signals.