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The peripheral-type benzodiazepine receptor is functionally linked to Leydig cell steroidogenesis
V Papadopoulos1, A G Mukhin, E Costa
1Department of Anatomy and Cell Biology, Georgetown University School of Medicine, Washington, D.C. 20007.
Abstract:
Testicular mitochondria were previously shown to contain an abundance of peripheral-type benzodiazepine recognition site(s)/receptor(s) (PBR). We have previously purified, cloned, and expressed an Mr 18,000 PBR protein (Antkiewicz-Michaluk, Mukhin, A. G., Guidotti, A., and Krueger, K. E. (1988) J. Biol. Chem. 263, 17317-17321; (Sprengel, R., Werner, P., Seeburg, P. H., Mukhin, A. G., Santi, M. R., Grayson, D. R., Guidotti, A., and Krueger, K. E. (1989) J. Biol. Chem. 264, 20415-20421); and in this report, we present evidence that PBR are functionally linked to Leydig cell steroid biosynthesis. A spectrum of nine different ligands covering a range of over 4 orders of magnitude in their affinities for PBR were tested for their potencies to modulate steroidogenesis in the MA-10 mouse Leydig tumor cell line. The Ki for inhibition of [3H]1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide binding and the EC50 for steroid biosynthesis for this series of compounds showed a correlation coefficient of r = 0.95. The most potent ligands stimulated steroid production by approximately 4-fold in these cells. This stimulation was not inhibited by cycloheximide, unlike human chorionic gonadotropin- or cyclic AMP-activated steroidogenesis. The action of PBR ligands was not additive to stimulation by human chorionic gonadotropin or cyclic AMP, but was additive to that of epidermal growth factor, another regulator of MA-10 Leydig cell steroidogenesis. Moreover, PBR ligands stimulated, in a dose-dependent manner, pregnenolone biosynthesis by isolated mitochondria when supplied with exogenous cholesterol. This effect was not observed with mitoplasts (mitochondria devoid of the outer membrane). Cytochrome P-450 side chain cleavage activity, as measured by metabolism of (22R)-hydroxycholesterol, was not affected by PBR ligands in intact cells. Similar results were also obtained with purified rat Leydig cells. In conclusion, PBR are implicated in the acute stimulation of Leydig cell steroidogenesis possibly by mediating the entry, distribution, and/or availability of cholesterol within mitochondria.
Insights
Peripheral-type benzodiazepine receptors (PBR) are functionally linked to Leydig cell steroid biosynthesis. PBR ligands stimulate steroid production by regulating cholesterol availability within mitochondria.
Area of Science:
- Endocrinology
- Mitochondrial Biology
- Steroidogenesis
Background:
- Testicular mitochondria possess peripheral-type benzodiazepine receptors (PBR).
- Previous studies purified, cloned, and expressed an 18,000 Mr PBR protein.
Purpose of the Study:
- To investigate the functional link between PBR and Leydig cell steroid biosynthesis.
- To determine the role of PBR ligands in modulating steroidogenesis.
Main Methods:
- Tested nine different PBR ligands for their potency to modulate steroidogenesis in MA-10 mouse Leydig tumor cells.
- Assessed the effect of PBR ligands on pregnenolone biosynthesis in isolated mitochondria and mitoplasts.
- Measured cytochrome P-450 side chain cleavage activity in intact cells and purified rat Leydig cells.
Main Results:
- A strong correlation (r=0.95) was observed between PBR ligand binding affinity and steroid biosynthesis potency.
- PBR ligands stimulated steroid production up to 4-fold, independent of cycloheximide.
- PBR ligands stimulated pregnenolone biosynthesis in isolated mitochondria, suggesting a role in cholesterol transport.
Conclusions:
- Peripheral-type benzodiazepine receptors are implicated in the acute stimulation of Leydig cell steroidogenesis.
- PBR may mediate cholesterol entry, distribution, and/or availability within mitochondria.
- These findings highlight a novel regulatory pathway in steroid hormone production.