IDENTIFICATION OF A KINETICALLY DISTINCT ACTIVITY OF 11-BETA-HYDROXYSTEROID DEHYDROGENASE IN RAT LEYDIG-CELLS

Leydig cells are susceptible to direct glucocorticoid-mediated inhibit ion of testosterone biosynthesis but can counteract the inhibition thr ough 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), which oxidati vely inactivates glucocorticoids. Of the two isoforms of 11 beta-HSD t hat have been identified, type I is an NADP(H)-dependent oxidoreductas e that is relatively insensitive to inhibition by end product and carb enoxolone (CBX). The type I form has been shown to be predominantly re ductive in liver parenchymal cells and other tissues. In contrast, typ e II, which is postulated to confer specificity in mineralocorticoid r eceptor (MR)-mediated responses, acts as an NAD-dependent oxidase that is potently inhibited by both end product and CBX. The identity of th e 11 beta-HSD isoform in Leydig cells is uncertain, because the protei n in this cell is recognized by an anti-type I 11 beta-HSD antibody, b ut the activity is primarily oxidative, more closely resembling type I I. The goal of the present study was to determine whether the kinetic properties of 11 beta-HSD in Leydig cells are consistent with type I, type II, or neither. Leydig cells were purified from male Sprague-Dawl ey rats (250 g), and 11 beta-HSD was evaluated in Leydig cells by meas uring rates of oxidation and reduction, cofactor preference, and inhib ition by end product and CBX. Leydig cells were assayed for type I and II 11 beta-HSD and MR messenger RNAs (mRNAs), and for type I 11 beta- HSD protein. Leydig cell 11 beta-HSD bad bidirectional catalytic activ ity that was NADP(H)-dependent. This is consistent with the hypothesis that type I 11 beta-HSD is present in rat Leydig cells. However, unli ke the type I 11 beta-HSD in liver parenchymal cells, the Leydig cell 11 beta-HSD was predominantly oxidative. Moreover, analysis of kinetic s revealed two components, the first being low a Michaelis-Menten cons tant (K-m) NADP-dependent oxidative activity with a K-m of 41.5 +/- 9. 3 nM and maximum velocity (V-max) of 7.1 +/- 1.2 pmol.min.10(6) cells. The second component consisted of high K-m activities that were consi stent with type I: NADP-dependent oxidative activity with K-m of 5.87 +/- 0.46 mu M and V-max of 419 +/- 17 pmol.min.10(6) cells, and NADPH- dependent reductive activity with K-m of 0.892 +/- 0.051 mu M and V-ma x of 117 +/- 6 pmol.min.10(6) cells. The results for end product and C BX inhibition were also inconsistent with a single kinetic activity in Leydig cells. Type I 11 beta-HSD mRNA and protein were both present i n Leydig cells, whereas type II mRNA was undetectable. We conclude tha t the low K-m NADP-dependent oxidative activity of 11 beta-HSD in Leyd ig cells does not confirm to the established characteristics of type I and may reside in a new form of this protein. We also demonstrated th e presence of the mRNA for MR in Leydig cells, and the low K-m compone nt could allow for specificity in MR-mediated responses.