Hormonal regulation of oxidative and reductive activities of 11 beta-hydroxysteroid dehydrogenase in rat Leydig cells.

We have proposed that the 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) of Leydig cells protects against glucocorticoid-induced inhibition of testosterone (T) production. However, Leydig cells express type I 11 beta-HSD, which has been shown to be reductive in liver parenchymal cells. Because reduction would have the opposite effect of activating glucocorticoid, the present study was designed to determine: 1) whether Leydig cell 11 beta-HSD is primarily oxidative or reductive; and 2) whether oxidative and reductive activities are separately modified by known regulators of Leydig cell steroidogenic function. Leydig cells and liver parenchymal cells were purified from mature male Sprague-Dawley rats (250 g BW), and 11 beta-HSD oxidative and reductive activities were measured using radiolabeled substrates and TLC of triplicate media samples from 1-h incubations immediately after cell isolation. Enzyme activities also were examined in purified Leydig cells at the end of 3 days of culture in vitro in the presence of LH (10 ng/ml), dexamethasone (DEX, 100 nM), T (50 nM), or epidermal growth factor (EGF, 50 ng/ml). In confirmation of previous reports, the reductive activity of 11 beta-HSD was predominant over oxidation in liver parenchymal cells. In contrast, 11 beta-HSD oxidative activity prevailed over reduction in Leydig cells by a ratio of 2:1. The activities of 11 beta-HSD also were analyzed in Leydig cells that were purified 7 days after endogenous glucocorticoid levels were suppressed by adrenalectomy (ADX). Oxidative activity declined in Leydig cells after ADX (22.53 +/- 1.12 pmol/h.10(6) cells, mean +/- SEM vs. 31.47 +/- 1.48 pmol/.10(6) cells in sham-operated controls, P < 0.05), whereas there was no change in reductive activity. This indicated that physiologically active corticosterone is involved in maintaining the predominance of 11 beta-HSD oxidation. When enzyme activities were analyzed in Leydig cells after 3 days of hormonal treatment in vitro, oxidation and reduction were observed to change in opposing directions. Culture of Leydig cells from sham-operated control rats with either LH, T, or EGF resulted in declines in oxidative activity from 33.35 +/- 0.77 to 28.24 +/- 1.93, 27.30 +/- 0.96, and 24.13 +/- 1.02 pmol/ h.10(6) cells (x +/- SE), respectively. However, EGF stimulated 11 beta-HSD reductive activity in cultured Leydig cells from both control (from 18.97 +/- 1.10 to 27.16 +/- 0.71 pmol/h.10(6) cells and ADX rats (from 16.51 +/- 0.75 to 23.56 +/- 0.84 pmol/h.10(6) cells). Among the hormonal treatments, only DEX increased oxidative activity and simultaneously decreased reductive activity in Leydig cells from ADX rats. This increase accentuated the predominance of oxidative activity in Leydig cells, with a ratio of oxidative to reductive activity of 4:1 after DEX treatment, compared with 2:1 in controls that were untreated. We conclude that 11 beta-HSD activity in Leydig cells is primarily oxidative. Moreover, oxidation and reduction are regulated separately by hormones.