Characterization of 11beta-hydroxysteroid dehydrogenase activities in the renal cell line LLC-PK1: evidence for a third isoform?
We studied 11beta-hydroxysteroid dehydrogenase activities in the renal cell line LLC-PK1 and the effects of different steroids on them. Cortisol was oxidized in the presence of NAD as well as NADP, reflecting the presence of two different 11beta-HSD forms. Enzyme kinetics for cortisol 11beta-oxidation were: Vmax = 5.9 pmol/(min x mg), Km = 0.2 microM with NAD, and Vmax = 4.5 pmol/(min x mg), Km = 1.0 microM with NADP. Interestingly, no reverse reaction was observed when using cortisone and NADPH as substrate and cosubstrate, respectively. Exposure of cells to a variety of steroids had different effects on cortisol 11beta-oxidation rates with NADP compared to those with NAD. Dexamethasone initially (3-60 min of exposure) decreased the NAD-dependent 11beta-HSD activity to about 60%, which was no longer evident after 2 h or longer. By contrast, the 11beta-oxidation of cortisol with NADP increased by dexamethasone treatment of the cells, after a lagtime of about 2 h, and this effect was still evident after 32 h. The increase of 11beta-HSD activity with NADP by dexamethasone was concentration dependent (estimated EC50:125 nM). The antiglucocorticoid RU486 did not antagonize dexamethasone induction. Exposure of cells for 19 h to 1 microM cortisol, cortisone, progesterone, and estradiol also increased NADP-dependent cortisol 11beta-oxidation, but had no effect on the NAD-dependent 11beta-HSD activity. Immunoblot and reverse transcriptase-polymerase chain reaction experiments failed to detect any 11beta-HSD 1 protein or mRNA in these cells. Our observations suggest that in LLC-PK1 cells, two forms of 11beta-HSD exist, which differ in cosubstrate dependency, kinetics for cortisol, and modulation by steroids. Whereas the NAD-dependent form seems identical to renal 11beta-HSD 2, the NADP-dependent 11beta-HSD possibly resembles an as yet unknown third isoform.
Möbus
E
E
Department of Pharmacology and Toxicology, Philipps-University, Marburg, Germany.
Hermans
J J
JJ
Maser
E
E
D042842:11-beta-Hydroxysteroid Dehydrogenases; D000818:Animals; D003348:Cortisone / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D004958:Estradiol / Q000494:pharmacology; D005938:Glucocorticoids / Q000494:pharmacology; D006854:Hydrocortisone / Q000378:metabolism / Q000494:pharmacology; D006913:Hydroxysteroid Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D018374:LLC-PK1 Cells; D015735:Mifepristone / Q000494:pharmacology; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D011374:Progesterone / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine
D005938:Glucocorticoids; D012333:RNA, Messenger; D009243:NAD; D015735:Mifepristone; D011374:Progesterone; D004958:Estradiol; D009249:NADP; D003907:Dexamethasone; D006913:Hydroxysteroid Dehydrogenases; D042842:11-beta-Hydroxysteroid Dehydrogenases; D003348:Cortisone; D006854:Hydrocortisone