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 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity and the testosterone level was examined in cultures of Leydig cells from adult rats. A rapid and simple method for measuring the 3 beta-HSD activity has been developed, in which the NADH, generated by 3 beta-HSD, reduced nitroblue tetrazolium to a product with absorption maximum at 560 nm. Km for the reaction was 8.1 microM and Vmax was 12.7 nmol/min x mg protein. Addition of 0.1 or 1 microM dexamethasone for 44 h decreased the 3 beta-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 3 beta-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 3 beta-HSD activity in Leydig cells from adult rats. Concomitant treatment of the cells with dexamethasone and insulin inhibited the 3 beta-HSD activity to 74%, indicating an additive effect, whereas no additive effect on the testosterone level was observed. The results demonstrate that the 3 beta-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 3 beta-HSD through a mechanism different from that of insulin, as an additive effect was observed.