REGULATION BY DEXAMETHASONE OF THE 3-BETA-HYDROXYSTEROID DEHYDROGENASE-ACTIVITY IN ADULT-RAT LEYDIG-CELLS

The effect of long-term in vitro treatment with dexamethasone, insulin and/or LH on the 3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity and the testosterone level was examined in cultures of Leydig cells from adult rats. A rapid and simple method for measuring the 3beta-HSD activity has been developed, in which the NADH, generated by 3beta-HSD, reduced nitroblue tetrazolium to a product with absorption maximum at 560 nm. K(m) for the reaction was 8. 1 mu M and V(max) was 12.7 nmol/min x mg protein. Addition of 0. 1 or 1 muM dexamethasone for 44 h decreased the 3beta -HSD activity to 83% and the basal testosterone level to 64% of control value after 22 and 44 h of culture. Addition of 1 nM insulin inhibited the 3beta-HSD activity to 90% after 44 h of culture, whereas the testosterone level was increased after 3 h. Addition of 0.1 ng/ml LH did not affect the 3beta-HSD activity in Leydig cells from adult rats. Concomitant treatment of the cells with dexamethasone and insulin inhibited the 3beta-HSD activity to 74%, indicating an additive effect, whereas no additive effect on the testosterone level was observed. The results demonstrate that the 3beta-HSD activity can be measured in a rapid and reliable way by measuring the reduction of nitroblue tetrazolium. Furthermore, the results suggest that dexamethasone acts on 3beta-HSD through a mechanism different from that of insulin, as an additive effect was observed.