Multi time-resolved spectroscopic analysis on molecular mechanisms of steroid hormone synthesis in adrenal cortex.

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ACTH,ATP or angiotensin II was used to stimulate the steroidogenesis. In a single adrenocortical fasciculata cell, calcium signal was observed for all stmuli examined. In all cases, we found the co-exsitence of Ca-signaling cells and non-Ca-signaling cells. Multiple imaging of calcium signal and enzyme distribution within a single cell revealed that a Ca signaling cell did not contain 3beta-hydroxysteroid dehydrogenase and cytochrome P450scc and P450c21, implying that a Ca-signaling cell does not synthesize steroid hormones. On the other hand, non-Ca-signaling cell contained all these enzymes, indicating that the non-Ca-signaling cell is a steroidogenic cell. With a confocal microscope, the increase of calcium concentration was also observed in nucleus.We succeeded to label adrenodoxin (ADX) and adrenodoxin reductase (ADR) with phosphorescent probes without decreasing the electron transfer activity. Protein-protein interactions were investigated between ADX and P450scc and between ADR and P450scc by measuring time-resolved phosphorescence anisotropy. The anistropy measurements revealed the formation of ternery association consisting of P450scc, ADX and ADR.