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Identification of a Kinetically Distinct Activity of 11ß-Hydroxysteroid Dehydrogenase in Rat Leydig Cells¹

The Population Council (R.-S.G., H.-B.G, G.L.G., M.P.H.) and Rockefeller University (M.P.H.), 1230 York Avenue, New York, New York 10021; and Department of Obstetrics and Gynecology, State University of New York Health Science Center-Brooklyn (V.L.N.), Brooklyn, New York 11203

Address all correspondence and requests for reprints to: Matthew P. Hardy, The Population Council, 1230 York Avenue, New York, New York 10021. E-mail: m-hardy{at}popcbr.rockefeller.edu

Leydig cells are susceptible to direct glucocorticoid-mediated inhibition of testosterone biosynthesis but can counteract the inhibition through 11ß-hydroxysteroid dehydrogenase (11ß-HSD), which oxidatively inactivates glucocorticoids. Of the two isoforms of 11ß-HSD that have been identified, type I is an NADP(H)-dependent oxidoreductase that is relatively insensitive to inhibition by end product and carbenoxolone (CBX). The type I form has been shown to be predominantly reductive in liver parenchymal cells and other tissues. In contrast, type II, which is postulated to confer specificity in mineralocorticoid receptor (MR)-mediated responses, acts as an NAD-dependent oxidase that is potently inhibited by both end product and CBX. The identity of the 11ß-HSD isoform in Leydig cells is uncertain, because the protein in this cell is recognized by an anti-type I 11ß-HSD antibody, but the activity is primarily oxidative, more closely resembling type II. The goal of the present study was to determine whether the kinetic properties of 11ß-HSD in Leydig cells are consistent with type I, type II, or neither. Leydig cells were purified from male Sprague-Dawley rats (250 g), and 11ß-HSD was evaluated in Leydig cells by measuring rates of oxidation and reduction, cofactor preference, and inhibition by end product and CBX. Leydig cells were assayed for type I and II 11ß-HSD and MR messenger RNAs (mRNAs), and for type I 11ß-HSD protein. Leydig cell 11ß-HSD had bidirectional catalytic activity that was NADP(H)-dependent. This is consistent with the hypothesis that type I 11ß-HSD is present in rat Leydig cells. However, unlike the type I 11ß-HSD in liver parenchymal cells, the Leydig cell 11ß-HSD was predominantly oxidative. Moreover, analysis of kinetics revealed two components, the first being low a Michaelis-Menten constant (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 · 10⁶ cells. The second component consisted of high K_m activities that were consistent with type I: NADP-dependent oxidative activity with K_m of 5.87 ± 0.46 µM and V_max of 419 ± 17 pmol · min · 10⁶ cells, and NADPH-dependent reductive activity with K_m of 0.892 ± 0.051 µM and V_max of 117 ± 6 pmol · min · 10⁶ cells. The results for end product and CBX inhibition were also inconsistent with a single kinetic activity in Leydig cells. Type I 11ß-HSD mRNA and protein were both present in Leydig cells, whereas type II mRNA was undetectable. We conclude that the low K_m NADP-dependent oxidative activity of 11ß-HSD in Leydig cells does not confirm to the established characteristics of type I and may reside in a new form of this protein. We also demonstrated the presence of the mRNA for MR in Leydig cells, and the low K_m component could allow for specificity in MR-mediated responses.

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