In all steroidogenic cells, the side-chain cleavage of cholesterol by cytochrome P450scc determines the rate of total steroid production and is hormonally regulated. In adrenal fasciculata and glomerulosa cells, this process is controlled by distinct signalling pathways (respectively, ACTH/cAMP and angiotensin/Ca++). Cholesterol moves to cytochrome P450scc through hormonal stimulation of both movement of cholesterol to the mitochondria and then cholesterol transfer between mitochondrial membranes. An increase in free cytosolic cholesterol is followed by movement through the cytoskeleton, possibly facilitated by sterol carrier protein-2. Other factors (steroidogenesis activator peptide (SAP), endozepine, 5-HPETE] have been implicated in intramitochondrial cholesterol transfer. This may require specialized contact sites formed by outer membrane peripheral benzodiazepine receptors (activated by endozepine) and an inner membrane, hormonally stimulated 30 kDa phosphoprotein. We will use a recently developed permeabilized adrenal cell preparation to address the mechanism of activated cholesterol transfer. We will also purify the 30 kDa mitochondrial protein (p30) in order to further define its role in adrenal mitochondria. In both fasciculata and glomerulosa cells, ACTH and angiotensin, respectively, stimulate synthesis of the same multiple forms of p30, suggesting a similar activation process. Rat adrenal fasciculata cells which have been permeabilized (>90% trypan blue uptake) with streptolysin O, show double the activity of intact cells, retain the 50-fold stimulation generated by ACTH prior to permeabilization, and are stimulated after permeabilization by cAMP and Ca++. We will optimize and then characterize these preparations in relation to intact adrenal cells. They will be used to analyze the activation of cholesterol metabolism in fasciculata and glomerulosa cells in terms of cholesterol transfer. Ultrastructural changes associated with both permeabilization and hormonal activation will be examined, with particular reference to cytoskeleton and cholesterol distribution. We will determine to what extent the processes of intact cells are retained by the permeabilized cells; notably, activation of cholesterol esterases, synthesis, and phosphorylation of p30 and cholesterol metabolism. Permeabilized cells will be used to test the participation of specific proteins and mediators, either by direct uptake or by addition of specific antibodies. Mitochondrial activation by potential mediators (Ca++, endozepine, 5-HPETE) in permeabilized cells will be,compared to effects on isolated mitochondria: We will continue efforts to analyze the cellular synthesis of the multiple forms of p30.
We aim to purify bovine adrenal p30 (or use sequences established elsewhere) in order to generate specific antibodies that will be used to localize p30 in the mitochondria, and to determine associated proteins. This work will develop a new experimental approach for analysis of steroidogenesis in cells from all steroidogenic organs.
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