An Inducible Functional Peripheral Benzodiazepine Receptor in Mitochondria of Steroidogenic Granulosa Cells*

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Abstract
Granulosa cell lines, transformed by SV40 T-antigen and Ha-ras oncogene, have recently been established that can produce progesterone at levels comparable to those of highly differentiated cultures of primary granulosa cells (1-4). Here, the hypothesis that these cells contain a mitochondrial benzodiazepine receptor, and that stimulation of the receptor can trigger progesterone production in these cells, was tested. The agonist of the peripheral benzodiazepine receptor, Ro5-4864, produced a 3- to 5-fold stimulation (P less than 0.005) of progesterone production both in differentiated granulosa primary cultures and in the oncogene-transformed cell lines. Ro5-2807 (diazepam, Valium) exerts a similar effect on granulosa cell steroidogenesis while the specific agonist of central benzodiazepine receptor Ro15-4513 was without effect. The effects of Ro5-4864 or Ro5-2807 were not additive to those of gonadotropins and cAMP. Intact isolated mitochondria possessed high-affinity binding sites to [3H]-Ro5-4864 (Kd = 3.03 +/- 0.70 nM), which were enriched by 1 order of magnitude in these organelles compared to total cell homogenate. Bound Ro5-4864 could be competitively displaced with 1 microM unlabeled Ro5-4864 and Ro5-2807, but not with specific ligands of central benzodiazepine receptors Ro15-4513 and Ro15-1788. Prolonged elevation of cAMP in these cells caused a 30% (P less than 0.01) rise in the number of receptors. Mitochondria of NIH 3T3 cells contained only 30-40% (P less than 0.001) of the Ro5-4864 binding sites of mitochondria from steroidogenic cells, whereas yeast mitochondria lacked them completely. The existence of functional peripheral benzodiazepine receptors in mitochondria suggests that they may have a physiological role in the mobilization of cholesterol into mitochondria, and in elevating progesterone production in ovarian cells. The modulation of the interaction between benzodiazepine compounds and the gamma-aminobutyric acid receptor by progesterone metabolites suggests new interrelationships between peripheral and central nervous system receptors sensitive to benzodiazepines.

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