Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.1.1.3 (HSD)
 3,464 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Renal tissue of several species has been shown to express considerable 11 beta-hydroxysteroid dehydrogenase (11-HSD, EC 1.1.1.146) activity. However, it is uncertain as to which renal cell types exhibit 11-HSD activity. In the present study, we investigated corticosterone metabolism in BSC-1 cells, a continuous renal epithelial cell line derived from the African green monkey (Cercopithecus aethiops). In incubation experiments using 3H-labelled corticosterone and HPLC, we have demonstrated oxidative 11-HSD activity in intact monolayers of BSC-1 cells as well as in BSC-1 cell homogenates. 11-HSD activity in cell homogenates could be stimulated 7-9-fold by the addition of exogenous NADP+ (1 mM). In contrast, no reductive 11-HSD could be detected either in intact cells or in cell homogenates under various experimental conditions which were designed to favor reductive 11-HSD activity. Pilot experiments were performed in cell homogenates from two other renal epithelial cell lines derived from canine (MDCK) and porcine (LLC-PK1) kidney. They also revealed oxidative but no reductive 11-HSD activity. The data provide evidence for an epithelial localization of renal oxidative 11-HSD activity.
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PMID:Renal epithelial cell lines (BSC-1, MDCK, LLC-PK1) express 11 beta-hydroxysteroid dehydrogenase activity. 292 Jan 78

Mineralocorticoid receptors (MRs) are nonselective in vitro, binding corticosterone, cortisol, and aldosterone with similar affinity. In the distal nephron in vivo, MRs are selectively activated by aldosterone despite much higher glucocorticoid levels. This has been suggested to reflect the action of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), which catalyzes rapid inactivation of corticosterone to 11-dehydrocorticosterone (cortisol to cortisone). However, cellular models of this effect have not been reported, and a recent study suggested that properties intrinsic to MR contribute to aldosterone selectivity. We have screened clonal mammalian cell lines for 11 beta-HSD activity. Pig kidney epithelial LLC-PK1 cells expressed by far the greatest 11 beta-HSD activity. In cell homogenates, this was NAD-dependent, with Km for corticosterone of 34.4 nM and cortisol of 89.7 nM. Intact LLC-PK1 cells showed similar apparent Km for corticosterone (13.9 nM) and cortisol (79.4 nM); only 11 beta-dehydrogenation was detected. These biochemical data indicate the expression of the type 2 isoform, 11 beta-HSD2. Using primers to conserved regions of 11 beta-HSD2, a reverse transcriptase-polymerase chain reaction product was obtained from LLC-PK1 cell RNA. Sequence analysis revealed close homology to previously cloned 11 beta-HSD2 cDNAs from several species. LLC-PK1 cell 11 beta-HSD activity was inhibited by carbenoxolone (IC50 approximately 10(-8) M) and high concentrations of estradiol or progesterone (10(-7) and 10(-6) M), but was induced at lower estradiol concentrations (10(-8) and 10(-9) M). To examine whether the 11 beta-HSD2 activity in LLC-PK1 cells regulates corticosterone access to MR, cells were transfected with the corticosteroid-inducible mouse mammary tumor virus long terminal repeat-luciferase reporter construct. Cell transfection by a lipofection method did not alter 11 beta-HSD activity in LLC-PK1 cells. LLC-PK1 cells expressed low levels of MR (13.9 fmol/mg protein, dissociation constant (Kd) 0.3 x 10(-9) M for aldosterone) and glucocorticoid receptors (GR; 18.5 fmol/mg protein, Kd 0.3 x 10(-9) M for dexamethasone). Transfection with mouse mammary tumor virus long terminal repeat-luciferase reporter construct alone suggested that the endogenous levels of MR and GR were insufficient to affect transcription. However, cotransfection of LLC-PK1 cells with pRShMR, an MR expression plasmid, allowed at least 50-fold induction of luciferase with 10(-8) M aldosterone; the ED50 0.3 x 10(-9) M closely reflects the in vitro affinity of MR for aldosterone. Corticosterone only weakly induced luciferase (maximum of 6-fold induction).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:LLC-PK1 cells model 11 beta-hydroxysteroid dehydrogenase type 2 regulation of glucocorticoid access to renal mineralocorticoid receptors. 758 9

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.
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PMID:Characterization of 11beta-hydroxysteroid dehydrogenase activities in the renal cell line LLC-PK1: evidence for a third isoform? 1078 27