SUBMITOCHONDRIAL DISTRIBUTION OF 3 KEY STEROIDOGENIC PROTEINS (STEROIDOGENIC ACUTE REGULATORY PROTEIN AND CYTOCHROME P450(SCC) AND 3-BETA-HYDROXYSTEROID DEHYDROGENASE ISOMERASE ENZYMES) UPON STIMULATION BY INTRACELLULAR CALCIUM IN ADRENAL GLOMERULOSA CELLS

In adrenal glomerulosa cells, angiotensin II (Ang II) and potassium st imulate aldosterone synthesis through activation of the calcium messen ger system, The rate-limiting step in steroidogenesis is the transfer of cholesterol to the inner mitochondrial membrane, This transfer is b elieved to depend upon the presence of the steroidogenic acute regulat ory (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 i ntramitochondrial cholesterol and 2) to study the distribution of StAR protein in submitochondrial fractions during activation by Ca2+ and A ng II, To this end, freshly prepared bovine zona glomerulosa cells wer e submitted to a high cytosolic Ca2+ clamp (600 nM) or stimulated with Ang II (10 nm) for 2 h, Mitochondria were isolated and subfractionate d into outer membranes, inner membranes (IM), and contact sites (CS), Stimulation of intact cells with Ca2+ or Ang II led to a marked, cyclo heximide-sensitive increase in cholesterol in CS (to 143 +/- 3.2 and 1 51.1 +/- 18.1% of controls, respectively) and in IM (to 119 +/- 5.1 an d 124.5 +/- 6.5% of controls, respectively), Western blot analysis rev ealed a cycloheximide-sensitive increase in StAR protein in mitochondr ial extracts of Ca2+-clamped glomerulosa cells (to 159 +/- 23% of cont rols), In submitochondrial fractions, there was a selective accumulati on of StAR protein in IM following stimulation with Ca2+ (228 +/- 50%) . Similarly, Ang II increased StAR protein in IM, and this effect was prevented by cycloheximide, In contrast, neither Ca2+ nor Ang II had a ny effect on the submitochondrial distribution of cytochrome P450(sec) and 3 beta-hydroxysteroid dehydrogenase isomerase, The intramitochond rial 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 tha t under acute stimulation with Ca2+ mobilizing agents, newly synthesiz ed StAR protein accumulates in IM after transiting through CS, Moreove r, 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 mitoc hondria and thereby activating mineralocorticoid synthesis.