title: Characterization of 11β-hydroxysteroid dehydrogenase activities in the renal cell line LLC-PK(1) : Evidence for a third isoform?
abstract: We studied 11β-hydroxysteroid dehydrogenase activities in the renal cell line LLC-PK(1) 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 11β-HSD forms. Enzyme kinetics for cortisol 11β-oxidation were: V (max)=5.9 pmol/(min×mg), K (m)=0.2 μM with NAD, and V (max)=4.5 pmol/(min×mg), K (m)=1.0 μM 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 11β-oxidation rates with NADP compared to those with NAD. Dexamethasone initially (3-60 min of exposure) decreased the NAD-dependent 11β-HSD activity to about 60%, which was no longer evident after 2 h or longer. By contrast, the 11β-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 11β-HSD activity with NADP by dexamethasone was concentration dependent (estimated EC(50): 125 nM). The antiglucocorticoid RU 486 did not antagonize dexamethasone induction. Exposure of cells for 19 h to 1 μM cortisol, cortisone, progesterone, and estradiol also increased NADP-dependent cortisol 11β-oxidation, but had no effect on the NAD-dependent 11β-HSD activity. Immunoblot and reverse transcriptase-polymerase chain reaction experiments failed to detect any 11β-HSD 1 protein or mRNA in these cells. Our observations suggest that in LLC-PK(1) cells, two forms of 11β-HSD exist, which differ in cosubstrate dependency, kinetics for cortisol, and modulation by steroids. Whereas the NAD-dependent form seems identical to renal 11β-HSD 2, the NADP-dependent 11β-HSD possibly resembles an as yet unknown third isoform.
authors.affiliation: Department of Pharmacology and Toxicology, Philipps-University, School of Medicine, Karl-von-Frisch-Strasse 1, 35033, Marburg, Germany.
authors.forename: E
authors.initials: E
authors.lastname: Möbus
authors.forename: J J
authors.initials: JJ
authors.lastname: Rob Hermans
authors.forename: E
authors.initials: E
authors.lastname: Maser