11-BETA-HYDROXYSTEROID DEHYDROGENASE TYPE-2 COMPLEMENTARY DEOXYRIBONUCLEIC-ACID STABLY TRANSFECTED INTO CHINESE-HAMSTER OVARY CELLS - SPECIFIC-INHIBITION BY 11-ALPHA-HYDROXYPROGESTERONE

The 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta HSD-2) enzyme is thought to confer aldosterone specificity upon mineralocorticoid t arget tissues by protecting the mineralocorticoid receptor from bindin g by the more abundant glucocorticoids, corticosterone and cortisol. W e have developed a Chinese hamster ovary cell line stably transfected with a plasmid containing the rat 11 beta HSD-2 complementary DNA. Thi s cell line has expressed the enzyme consistently for many generations . The 11 beta HSD-2 was located primarily in the microsomes, but signi ficant amounts also existed in the nuclei and mitochondria. The enzyma tic reaction was unidirectional, oxidative, and inhibited by the produ ct, 11-dehydrocorticosterone, with an IC50 of approximately 200 nM. Th e K-m for corticosterone was 9.6 +/- 3.1 nM, and that for NAD(+) was a pproximately 8 mu M. The enzyme did not convert dexamethasone to 11-de hydrodexamethasone. Tunicamycin, an N-glycosylation inhibitor, had not effect enzyme activity. 11 alpha-Hydroxyprogesterone (11 alpha OH-P) was an order of magnitude more potent a competitive inhibitor of the 1 1 beta HSD-2 than was glycyrrhetinic acid (GA) (approximate IC50 = 0.9 vs. 15 nM). 11 beta OH-P, progesterone, and GA were almost equipotent (IC50 = 10 and 6 nM, respectively), adn 5 alpha-pregnandione and 5 be ta-pregnandione was less potent (IC50 = 100 and 500 nM, respectively) inhibitors of the enzyme. When the inhibitory activities were examined with intact transfected cells, 11 alpha OH-P was more potent than GA (IC50 = 5 and 150 nM, respectively). 11 alpha OH-P was not metabolized by 11 beta HSD-2. We were unable to demonstrate the presence of 11 al pha OH-P in human urine. In conclusion, a cell line stably transfected with the rat 11 beta HSD-2 was created, and the enzyme kinetics, incl uding inhibition, were characterized. 11 alpha OH-P was found to be a potent relatively specific inhibitor of the 11 beta HSD-2 enzyme. Its potential importance is that it is the most specific inhibitor of the 11 beta HSD-2 so far encountered and would aid in the study of the phy siological importance of the isoenzyme.