Submitochondrial distribution of three key steroidogenic proteins (steroidogenic acute regulatory protein and cytochrome P450 sub(scc) and 3 beta -hydroxysteroid dehydrogenase isomerase enzymes) upon stimulation by intracellular calcium in adrenal glomerulosa cells

• Published in: The Journal of biological chemistry 1997-03, Vol.272 (12), p.7899-7907
• Main Authors: Cherradi, N, Rossier, M F, Vallotton, M B, Timberg, R, Friedberg, I, Orly, J, Wang, Xing Jia, Stocco, D M, Capponi, A M
• Format: Article
• Language: English
• ISSN: 0021-9258
• DOI: 10.1074/jbc.272.12.7899

In adrenal glomerulosa cells, angiotensin II (Ang II) and potassium stimulate aldosterone synthesis through activation of the calcium messenger system. The rate-limiting step in steroidogenesis is the transfer of cholesterol to the inner mitochondrial membrane. This transfer is believed to depend upon the presence of the steroidogenic acute regulatory (StAR) protein. The aim of this study was 1) to examine the effect of changes in cytosolic free calcium concentration and of Ang II on intramitochondrial cholesterol and 2) to study the distribution of StAR protein in submitochondrial fractions during activation by Ca super(2+) and Ang II. To this end, freshly prepared bovine zona glomerulosa cells were submitted to a high cytosolic Ca super(2+) clamp (600 nM) or stimulated with Ang II (10 nM) for 2 h. Mitochondria were isolated and subfractionated into outer membranes, inner membranes (IM), and contact sites (CS). Stimulation of intact cells with Ca super(2+) or Ang II led to a marked, cycloheximide-sensitive increase in cholesterol in CS (to 143 plus or minus 3.2 and 151.1 plus or minus 18.1% of controls, respectively) and in IM (to 119 plus or minus 5.1 and 124.5 plus or minus 6.5% of controls, respectively). Western blot analysis revealed a cycloheximide-sensitive increase in StAR protein in mitochondrial extracts of Ca super(2+)-clamped glomerulosa cells (to 159 plus or minus 23% of controls). In submitochondrial fractions, there was a selective accumulation of StAR protein in IM following stimulation with Ca super(2+) (228 plus or minus 50%). Similarly, Ang II increased StAR protein in IM, and this effect was prevented by cycloheximide. In contrast, neither Ca super(2+) nor Ang II had any effect on the submitochondrial distribution of cytochrome P450 sub(scc) and 3 beta -hydroxysteroid dehydrogenase isomerase. The intramitochondrial presence of the latter enzyme was further confirmed by immunogold staining in rat adrenal fasciculata cells and by immunoblot analysis in MA-10 mouse testicular Leydig cells. These findings demonstrate that under acute stimulation with Ca super(2+)-mobilizing agents, newly synthesized StAR protein accumulates in IM after transmiting through CS. Moreover, our results suggest that the import of StAR protein into IM may be associated with cholesterol transfer, thus promoting precursor supply to the two first enzymes of the steroidogenic cascade within the mitochondria and thereby activating mineralocorticoid synthesis.