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.
Highlights
Effect of peripheral-type benzodiazepine recognition site(s)/receptor(s) (PBR) Ligands on MA-10 Leydig Cell Steroid Biosynthesis-To investigate whether the different ligands affected Leydig cell steroidogenesis, increasing concentrations of these compounds were incubated for 4 h with MA-10 cells, and their effects on progesterone production were measured (Fig. 3)
Regulation of Steroidogenesis by PBR Liands in Leydig Cells Purified from Rat Testis-To exclude the possibility that the involvement of PBR in steroidogenesis may be specific to the MA-10 cell line rather than representative of normal Leydig cells, we examined the action of PBR ligands on purified rat Leydig cell steroidogenesis
A number of preliminary reports [18,19,20, 29, 45] show that some PBR ligands can increase steroidogenesis; a detailed pharmacological analysis was lacking to establish whether the mediator of this action was PBR
Summary
DBI displaces PBR ligands from rat adrenal gland cortex [25, 26], which raises the possibility that DBI may interact physiologically with PBR and elicit an as yet undefined biological activity Another laboratory [27, 28] has reported the purification of an 8.2-kDa polypeptide from bovine adrenal fasciculata which exhibited the ability to stimulate pregnenolone synthesis in mitochondrial preparations of adrenal glands. The localization of PBR on the mitochondrial compartment, the above-mentioned preliminary studies, and the finding that DBI is localized in Leydig cells raised the possibility that PBR may participate in the regulation of testicular steroidogenesis and prompted us to examine whether PBR ligands could alter Leydig cell function For this purpose, two cell models were used: the mouse Leydig cell line MA-10 and purified rat Leydig cells
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