NAD-dependent 11 beta-hydroxysteroid dehydrogenase in cultured human colonic epithelial cells.
The inactivation of physiological glucocorticoids by 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) confers mineralocorticoid specificity to certain aldosterone target tissues. However, 11 beta-HSD activity in a human mineralocorticoid-responsive tissue has never been characterized. The present studies describe the features of 11 beta-HSD in the cultured human colonic epithelial cell line, T84. The 11 beta-HSD activity of T84 cells resided in the microsomal fraction and showed a marked preference for NAD rather than NADP as cofactor. NAD or NADP (200 microM) increased the conversion of corticosterone to 11-dehydrocorticosterone by 24.1 +/- 2.1 and 0.5 +/- 0.7 pmol.mg protein-1.20 min-1, respectively, indicating a > 40-fold preference for NAD vs. NADP. The Michaelis constant values for corticosterone and cortisol were 11.3 +/- 1.5 and 79.8 +/- 10 nM, respectively. The T84 11 beta-HSD was inhibited by 11-dehydrocorticosterone in a noncompetitive fashion [inhibition constant (Ki) = 180 +/- 9.6 nM] and by carbenoxolone in a competitive fashion (Ki = 17.4 +/- 1.3 nM). The expression of mineralocorticoid receptors in these cells was demonstrated by reverse transcriptase-polymerase chain reaction of mRNA isolated from T84 cells and by [3H]aldosterone binding studies. The coexpression of this NAD-dependent isoform of 11 beta-HSD and mineralocorticoid receptors is consistent with the view that the NAD-dependent isoform is responsible for the specificity of mineralocorticoid responses.
Reeves
W B
WB
Division of Nephrology, University of Arkansas College of Medicine, Little Rock 72205, USA.
D042842:11-beta-Hydroxysteroid Dehydrogenases; D000230:Adenocarcinoma; D000450:Aldosterone / Q000378:metabolism; D002229:Carbenoxolone / Q000494:pharmacology; D002460:Cell Line; D003110:Colonic Neoplasms; D003345:Corticosterone / Q000031:analogs & derivatives / Q000494:pharmacology; D004848:Epithelium / Q000201:enzymology; D015870:Gene Expression; D006801:Humans; D006913:Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D018161:Receptors, Mineralocorticoid / Q000096:biosynthesis; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D018161:Receptors, Mineralocorticoid; D009243:NAD; D000450:Aldosterone; D009249:NADP; D006913:Hydroxysteroid Dehydrogenases; D042842:11-beta-Hydroxysteroid Dehydrogenases; C003552:11-dehydrocorticosterone; D002229:Carbenoxolone; D003345:Corticosterone