title: 11 beta-Hydroxysteroid dehydrogenase type 2 complementary deoxyribonucleic acid stably transfected into Chinese hamster ovary cells: specific inhibition by 11 alpha-hydroxyprogesterone.
abstract: The 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta HSD-2) enzyme is thought to confer aldosterone specificity upon mineralocorticoid target tissues by protecting the mineralocorticoid receptor from binding by the more abundant glucocorticoids, corticosterone and cortisol. We have developed a Chinese hamster ovary cell line stably transfected with a plasmid containing the rat 11 beta HSD-2 complementary DNA. This cell line has expressed the enzyme consistently for many generations. The 11 beta HSD-2 was located primarily in the microsomes, but significant amounts also existed in the nuclei and mitochondria. The enzymatic reaction was unidirectional, oxidative, and inhibited by the product, 11-dehydrocorticosterone, with an IC50 of approximately 200 nM. The K(m) for corticosterone was 9.6 +/- 3.1 nM, and that for NAD+ was approximately 8 microM. The enzyme did not convert dexamethasone to 11-dehydrodexamethasone. Tunicamycin, an N-glycosylation inhibitor, had no effect on enzyme activity. 11 alpha-Hydroxyprogesterone (11 alpha OH-P) was an order of magnitude more potent a competitive inhibitor of the 11 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), and 5 alpha-pregnandione and 5 beta-pregnandione were 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 alpha 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, including 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 physiological importance of the isoenzyme.
authors.affiliation: Department of Internal Medicine, University of Missouri, Columbia 65212, USA.
authors.forename: H
authors.initials: H
authors.lastname: Morita
authors.forename: M
authors.initials: M
authors.lastname: Zhou
authors.forename: M F
authors.initials: MF
authors.lastname: Foecking
authors.forename: E P
authors.initials: EP
authors.lastname: Gomez-Sanchez
authors.forename: E N
authors.initials: EN
authors.lastname: Cozza
authors.forename: C E
authors.initials: CE
authors.lastname: Gomez-Sanchez
mesh_terms: D042842:11-beta-Hydroxysteroid Dehydrogenases; D000818:Animals; D016466:CHO Cells / Q000201:enzymology*; D003345:Corticosterone / Q000031:analogs & derivatives / Q000378:metabolism; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics*; D003907:Dexamethasone / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D006801:Humans; D006908:Hydroxyprogesterones / Q000494:pharmacology* / Q000652:urine; D006913:Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D007668:Kidney / Q000648:ultrastructure; D008297:Male; D008861:Microsomes / Q000201:enzymology; D009243:NAD / Q000494:pharmacology; D011247:Pregnancy; D051381:Rats; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D006908:Hydroxyprogesterones; D009243:NAD; C030228:11-hydroxyprogesterone; D003907:Dexamethasone; D006913:Hydroxysteroid Dehydrogenases; D042842:11-beta-Hydroxysteroid Dehydrogenases; C003552:11-dehydrocorticosterone; D003345:Corticosterone