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 bet a-HSD) of Leydig cells protects against glucocorticoid-induced inhibit ion of testosterone (T) production. However, Leydig cells express type I 11 beta-HSD, which has been shown to be reductive in liver parenchy mal cells. Because reduction would have the opposite effect of activat ing glucocorticoid, the present study was designed to determine: 1) wh ether Leydig cell 11 beta-HSD is primarily; oxidative or reductive; an d 2) whether oxidative and reductive activities are separately modifie d by known regulators of Leydig cell steroidogenic function. Leydig ce lls and liver parenchymal cells were purified from mature male Sprague -Dawley rats (250 g BW), and 11 beta-HSD oxidative and reductive activ ities were measured using radiolabeled substrates and TLC of triplicat e media samples from l-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), de xamethasone (DEX, 100 nM), T (50 nM), or epidermal growth factor (EGF, 50 ng/ml). In confirmation of previous reports, the reductive activit y of 11 beta-HSD was predominant over oxidation in Liver parenchymal c ells. In contrast, 11 beta-HSD oxidative activity prevailed over reduc tion in Leg-dig 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 en dogenous glucocorticoid levels were pressed by adrenalectomy (ADX). Ox idative activity declined in Leydig cells after ADX (22.53 +/- 1.12 pm ol/h . 10(6) cells, mean +/- SEM vs. 31.47 +/- 1.48 pmol/h . 10(6) cel ls in sham-operated controls, P < 0.05), whereas there was no change i n reductive activity. This indicated that physiologically active corti costerone is involved in maintaining the predominance of 11 beta-HSD o xidation. When enzyme activities were analyzed in Leydig cells after 3 days of hormonal treatment in vitro, oxidation and reduction acre obs erved to change in opposing directions. Culture of Leydig cells Dom sh am-operated control rats with either LH, T, or EGF resulted in decline s 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), fespeeti vely. However, EGF stimulated 11 beta-HSD reductive activity in cultur ed 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 i ncreased oxidative activity and simultaneously decreased reductive act ivity in Leydig cells from ADX rats. This increase accentuated the pre dominance of oxidative activity in Leydig cells, with a ratio of oxida tive to reductive activity of 4:1 after DEX treatment, compared with 2 :1 in controls that were untreated. We conclude that 11 beta-HSD activ ity in Legdig cells is primarily oxidative. Moreover, oxidation and re duction are regulated separately by hormones.