Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P41181 (collecting duct)
 5,183 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

It is widely accepted that in vivo the function of the papilla of the mammalian kidney is supported primarily by anaerobic metabolism. As a result, the major source of energy for support of function in the papilla is considered to be derived from glycolysis. This orientation originates from two concepts: 1) that in vivo the gaseous environment of the papilla has such a low PO2 that O2 availability limits O2 consumption, and 2) that papillary tissue has a high rate of glycolysis when compared with either cortical tissue or extrarenal tissues. It has also been tacitly assumed that papillary tissue has a "low" O2 uptake. Review of the measurements of PO2 of papillary tissue and of urine PO2 indicates that the PO2 of papillary tissue should not limit its aerobic mitochondrial oxidative metabolism. While the rate of aerobic glycolysis in papillary tissue is high, simultaneously papillary tissue has a rate of O2 uptake similar to that of liver and higher than that of muscle. The major (two-thirds) source of energy for papillary tissue in vitro is from O2 uptake. That papillary tissue is not exclusively dependent on glucose for its energy requirements is indicated by the greater stimulation of papillary tissue QO2 by succinate than by glucose. Thus, papillary tissue has both a high aerobic mitochondrial oxidative metabolism and a high aerobic glycolytic metabolism. It is suggested that the mechanism for the high rate of aerobic glycolysis in the presence of an adequate O2 supply is due to the relatively small mass of mitochondria in papillary tissue in relation to the amount of work done by the tissue. As a result of the limited rate of ATP production by the mitochondrial electron transport chain, the phosphorylation state ([ATP]/[ADP][Pi]) is reduced and the cytoplasmic redox state ([NAD+]/[NADH]) of the papillary collecting duct cells also becomes more reduced; changes in both ratios enhance the rate of glycolysis. This limited metabolic capacity of the collecting duct cells may permit an excess volume of solute and water to be excreted during volume expansion diuresis. The metabolic characteristics of the papilla, when compared to cortex, also provide a basis for the observed differences in substrate selectivity of cortex and medulla with respect to utilization of glucose and lactate. The experimental approaches that may provide information bearing on the suggested mechanisms for regulation of papillary metabolism in relation to tubular work functions are indicated.
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PMID:Is the function of the renal papilla coupled exclusively to an anaerobic pattern of metabolism? 22 Aug 81

Enzymatic properties of the enzyme 11 beta-hydroxysteroid dehydrogenase (11-HSD), which confers mineralocorticoid selectivity, have been explored in the aldosterone-sensitive collecting duct (CCD) and the aldosterone-insensitive Pars Recta (PR) of the rat kidney. After incubation of freshly isolated tubular segments with [3H]corticosterone (3H-B) or [3H]dehydrocorticosterone (3H-A), the rate of transformation of 3H-B into 3H-A (dehydrogenase activity), or the reverse reaction (reductase activity) were measured by HPLC, Vmax for dehydrogenase activity was found to be 8- to 10-fold higher in CCD than PR. The enzyme functions over a very wide range (0.1-5000 nM) of corticosterone concentration. In CCD, enzyme kinetics suggest either the presence of two 11-HSD forms, differing by their affinity for corticosterone, or complex kinetics. Addition of NAD or NADP to permeabilized tubules revealed that dehydrogenase activity is NAD-dependent in CCD and NADP-dependent in PR. Cofactor addition was ineffective in intact tubules. CCD exhibited an exclusive dehydrogenase activity, whereas in PR dehydrogenase and reductase activity were found. No regulation of dehydrogenase activity could be evidenced in adrenalectomized rats receiving or not aldosterone, corticosterone or dexamethasone, for 2 h, 3 days or 4 days. We conclude that 11-HSD in the CCD and PR differs by its Vmax and cofactor dependence. Corticosteroid hormones do not influence 11-HSD activity.
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PMID:Characteristics and regulation of 11 beta-hydroxysteroid dehydrogenase of proximal and distal nephron. 772 21

11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) is thought to confer aldosterone specificity to mineralocorticoid target cells by protecting the mineralocorticoid receptor from occupancy by endogenous glucocorticoids. We have recently described a novel isoform of 11-OHSD in the renal aldosterone target cells (11 beta-OHSD/CD) that differs from the previously characterized isoform (11 beta-OHSD-1). Unlike 11-OHSD-1, the collecting duct enzyme catalyzes irreversible dehydrogenation, has a very high affinity for its substrate, and is tissue-specific. We report here the isolation, sequence, and characterization of a complementary DNA (cDNA) encoding the rabbit collecting duct 11 beta-OHSD/CD or 11 beta-OHSD type 2. The cDNA, isolated using expression screening in Xenopus oocytes, is 1.9 kilobases in length and encodes a protein of 406 amino acids with a predicted molecular mass of 44,130 daltons. The cloned enzyme has a Michaelis constant (Km) for corticosterone of 6.6 +/- 3 nM, catalyzes exclusively dehydrogenation, and uses only NAD as cofactor. The cloned enzyme shows 85% and 75% amino acid identity to the recently cloned human type 2 11 beta-OHSD and sheep kidney 11 beta-OHSD, respectively, whereas the overall homology to rat liver 11 beta-OHSD-1 is less than 20% The messenger RNA for this 11 beta-OHSD is expressed at very high levels in the renal collecting duct and at much lower levels in the colon. The intrarenal distribution was determined by reverse-transcription polymerase chain reaction in isolated nephron segments or cell types. The messenger RNA is present only in aldosterone target cells within the kidney, at highest levels in principal cells, at lower levels in intercalated cells, and in inner medullary cells. These data suggest that the 11 beta-OHSD cDNA from rabbit collecting duct cells encodes the enzyme that confers aldosterone selectivity to mineralocorticoid target cells.
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PMID:Expression cloning of the aldosterone target cell-specific 11 beta-hydroxysteroid dehydrogenase from rabbit collecting duct cells. 775 Apr 80

11 beta-Hydroxysteroid dehydrogenase (11-OHSD) plays a critical role in conferring aldosterone specificity to mineralocorticoid target cells. We have recently described a novel isoform of 11-OHSD in the collecting duct (11-OHSD/CD), which differs from the previously characterized isoform (11-OHSD-1) in kinetic parameters, cofactor dependency, and reversibility of the reaction. Unlike 11-OHSD-1, the collecting duct enzyme catalyzes irreversible dehydrogenation of endogenous glucocorticoids, has a very high affinity for its substrate, and is located in mineralocorticoid target cells; it thus appears well suited to "protect" the mineralocorticoid receptors from occupancy by glucocorticoids. As a first step in attempting to isolate the cDNA for the 11-OHSD/CD isoform, we isolated mRNA from immunodissected cortical collecting duct (CCD) cells and characterized the 11-OHSD in oocytes injected with this mRNA. Water-injected oocytes had no measurable 11-OHSD activity. In contrast, oocytes injected with as little as 1 ng CCD mRNA expressed detectable 11-OHSD activity. Expression of 11-OHSD activity was dependent on the amount of mRNA injected and was maximal with 30 ng mRNA. Similar to the findings in CCD cells, the expressed enzyme preferred NAD over NADP (activity was 0.46 +/- 0.04 and 0.011 +/- 0.01 fmol.min-1.oocyte-1 with 0.1 mM NAD and NADP, respectively). The Michaelis constant (Km) for corticosterone was 11.5 +/- 3.7 nM. Similar to the findings in CCD cells, the expressed enzyme worked predominantly in the oxidative direction, as back-conversion of [3H]dehydrocorticosterone to corticosterone was negligible. (ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Expression and characterization of a new species of 11 beta-hydroxysteroid dehydrogenase in Xenopus oocytes. 828 21

Aldosterone exerts complex effects on the cortical collecting duct (CCD): it increases Na+ and K+ transport, and it also influences H+ and HCO3 transport. Whether these latter effects represent direct action of aldosterone on intercalated cells (ICC) or are secondary to changes in the transport of other electrolytes is unclear. Because the presence of specific receptors is the prerequisite of a direct steroid action, and mineralocorticoid receptors (MR) have not yet been demonstrated in ICC, in this study we determined the density of MR directly in isolated principal cells (PC) and beta-ICC. Purified populations of these two cell types were obtained from rabbit renal cortex by immunodissection and fluorescence-activated cell sorting. We found that both PC and beta-ICC contained a significant number of MR, although receptor density was higher in PC than in beta-ICC (6,704 +/- 912 vs. 2,181 +/- 388 MR sites/cell; P < 0.001). 11 beta-Hydroxysteroid dehydrogenase (11 beta-OHSD), an enzyme that is present predominantly in mineralocorticoid target cells, exhibited a distribution similar to that of MR in the two cell types. 11 beta-OHSD activity, determined by measuring the rate of conversion of [3H]corticosterone to 11-dehydrocorticosterone, was 1.08 +/- 0.14 and 0.34 +/- 0.08 fmol.min-1 x 1,000 cells-1 (P < 0.001) in intact PC and beta-ICC, respectively. 11 beta-OHSD in both cell types utilized NAD as cofactor. These results suggest that beta-ICC are potential direct targets of aldosterone and that MR in both PC and beta-ICC are protected by 11 beta-OHSD.
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PMID:Minealocorticoid receptors and 11 beta-steroid dehydrogenase activity in renal principal and intercalated cells. 830 86

11 beta-Hydroxysteroid dehydrogenase has been proposed to play an important role in aldosterone target cells by degrading endogenous glucocorticoids, thus allowing aldosterone to bind to the relatively nonselective mineralocorticoid receptor. The physiologically important species of this enzyme in renal aldosterone target cells appears to be kinetically and antigenically distinct from the previously characterized liver enzyme. Here we show that 11 beta-steroid dehydrogenase in the microsomal fraction of isolated renal collecting duct cells has a Km for corticosterone of 25.9 +/- 2.4 nM, about 100 times lower than the rat liver enzyme. Surprisingly, the collecting duct enzyme utilizes almost exclusively NAD as cofactor versus NADP used by the liver form. Conversion of corticosterone to 11-dehydrocorticosterone is 2.6 +/- 0.5 and 0.07 +/- 0.01 fmol/min/mg protein with 100 microM of NAD and NADP, respectively, demonstrating a 37.4 +/- 3.5-fold preference for NAD versus NADP. There is practically no conversion of 11-dehydrocorticosterone to corticosterone either with NADH or NADPH, indicating that in collecting duct cells the enzyme operates only in the direction of oxygenation. In addition, 11 beta-steroid dehydrogenase activity is dose dependently inhibited by the end product 11-dehydrocorticosterone while the liver enzyme does not show end product inhibition. We conclude that renal collecting duct cells, the major physiological targets of aldosterone, are protected from circulating glucocorticoids by a hitherto undescribed enzyme of the 11-dehydrogenase family, which differs from the known liver enzyme in having a significantly higher affinity for corticosterone and a different cofactor requirement.
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PMID:A new isoform of 11 beta-hydroxysteroid dehydrogenase in aldosterone target cells. 849 39

11 beta-Hydroxysteroid dehydrogenase (11 beta-OHSD) transforms circulating glucocorticoids to their "biologically inert" 11-dehydro derivatives. Isoforms of 11 beta-OHSD with different cofactor requirements and biochemical properties [Michaelis constant (Km) and maximal velocity (Vmax)] exist in the kidney. Since epithelial cells derived from the toad bladder also contain this enzyme, we wished to further characterize its properties in prepared cell homogenates. 11 beta-OHSD from toad bladder demonstrated a clear preference for NAD+ over NADP+ as a cofactor similar to that observed in renal cortical collecting duct (CCD) cells. Furthermore, 11 beta-OHSD had a rapid onset of action. The apparent Km for corticosterone was 16.3 x 10(-8) M, a value comparable to that observed for enzyme from CCD, and a Vmax of 4.8 x 10(-12) mol.mg protein-1.min-1. The end product, 11-dehydrocorticosterone (compound A), influenced enzyme activity; it increased 11 beta-OHSD activity at corticosterone concentrations below the apparent Km for the enzyme and inhibited 11 beta-OHSD activity at corticosterone concentrations above the Km for the enzyme. The inhibitory effects of compound A appeared noncompetitive with an apparent equilibrium constant (Ki) of 2.8 x 10(-7) M. Consistent with its inhibitory action on 11 beta-OHSD, compound A (10(-6) M) enhanced the short-circuit current response to corticosterone (10(-7) M) in the intact toad bladder (experimental 2.03 +/- 0.33 vs. control 1.40 +/- 0.17 times above baseline; n = 7, P < 0.01). Thus 11 beta-OHSD in toad bladder resembles the isoform found in CCD, and compound A may be biologically important as a regulator of 11 beta-OHSD.
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PMID:Activity of 11 beta-hydroxysteroid dehydrogenase in toad bladder: effects of 11-dehydrocorticosterone. 849 39

11Beta-hydroxysteroid dehydrogenase (11beta-HSD) is thought to confer aldosterone specificity to mineralocorticoid target cells by protecting the mineralocorticoid receptor (MR) from occupancy by endogenous glucocorticoids. In aldosterone target cells the type 2 11beta-HSD is present, which, in contrast to the type 1 11beta-HSD, has very high affinity for its substrate, is unidirectional and prefers NAD as cofactor. cDNAs encoding 11beta-HSD2 have been recently cloned from different species, and the cell-specific expression of its mRNA and protein were determined. 11Beta-HSD2 is expressed in every aldosterone target tissue. Northern analysis revealed that the rabbit 11beta-HSD2 is expressed at high levels in the renal collecting duct and at much lower levels in the colon. RT-PCR experiments demonstrated that 11beta-HSD2 mRNA is present only in aldosterone target cells within the kidney. We determined the subcellular localization of the rabbit 11beta-HSD2 using a chimera encoding 11beta-HSD2 and the green fluorescent protein (GFP). This construct was stably transfected into CHO and MDCK cells. The expressed 11beta-HSD2/GFP protein retained high enzymatic activity, and its characteristics were undistinguishable from those of the native enzyme. The intracellular localization of this protein was determined by fluorescence microscopy. 11Beta-HSD2-associated fluorescence was observed as a reticular network over the cytoplasm whereas the plasma membrane and the nucleus were negative, suggesting endoplasmic reticulum (ER) localization. Co-staining with markers for ER proteins, the Golgi membrane, mitochondria and nucleus confirmed that 11beta-HSD2 is localized exclusively to the ER. To determine what structural motifs are responsible for the ER localization, we generated deletion mutants missing the C-terminal 42 and 118 amino acids, and fused them to GFP. Similarly as with the intact 11beta-HSD2, these mutants localized exclusively to the ER. Both C-terminal deletion mutants completely lost dehydrogenase activity, independently whether activity was determined in intact cells or homogenates. These results indicate that 11beta-HSD2 has a novel ER retrieval signal which is not localized to the C-terminal region. In addition, the C-terminal 118 amino acids are essential for NAD-dependent 11beta-HSD activity.
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PMID:The role of 11beta-hydroxysteroid dehydrogenase in steroid hormone specificity. 969 85

Mineralocorticoid receptors in the inner medullary collecting duct (IMCD) are protected from glucocorticoid binding by an enzyme, 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2). To study the role of 11 beta-HSD2 in acid-base homeostasis, 11 beta-HSD2 activity was measured in rat IMCD-enriched cell suspensions. Homogenates of cell suspensions were incubated in buffers ranging in pH from 6.00 to 8.15 in the presence of 1 microCi of 3H-corticosterone (CS) and 400 microM NAD+. Enzyme activity was expressed as the amount of 3H-CS converted to 3H-11-dehydrocorticosterone (DHCS). IMCD 11 beta-HSD2 activity at pH 6.5 was 49% of activity at pH 7.5; 22.5 versus 11.0 fmol/microgram of protein per h. Experiments also were performed on intact cell suspensions at pH 7.5 and 6.5. There was a 42% inhibition in the IMCD cell suspension conversion rate of 3H-CS to 3H-11-DHCS at pH 6.5; 13.1 versus 7.6 fmol/microgram per h (P < 0.005). In cell suspensions at pH 7.5, 1-day acid loading caused a 26% inhibition in conversion rate, 13.2 versus 9.9 fmol/microgram per h (P < 0.05), when compared with controls. These results suggest that during acute metabolic acidosis, IMCD 11 beta-HSD2 is inhibited and may allow access to the mineralocorticoid receptors by glucocorticoids.
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PMID:Inhibition of IMCD 11 beta-hydroxysteroid dehydrogenase type 2 by low pH and acute acid loading. 1049 81

The kidney (11-HSD2 or 11-HSDK) isozyme of 11beta-hydroxysteroid dehydrogenase confers specificity for aldosterone on mineralocorticoid receptors in target tissues. In rodent kidney, this isozyme is expressed mainly in cortical collecting ducts and is undetectable in proximal tubules. Using mouse M-1 and rabbit RCD cortical collecting duct cells, we analyzed the 5'-flanking region of the human HSD11K gene encoding this enzyme in an attempt to identify transcriptional regulatory elements responsible for gene expression in the kidney. M-1 and RCD cells had high levels of NAD+ dependent 11-HSD activity with corticosterone as the substrate. Luciferase reporter constructs containing 1785 or 327 nucleotides (nt) upstream of the initiator ATG codon were expressed at similar levels in each cell line, but deletion to 167 nt almost completely abolished expression in both cell types. This region is GC-rich and contain Sp1 binding sites. Electrophoretic mobility shift assays of the region containing the putative Sp1 sites showed several DNA-protein complexes in both the cell types. Mutations of the Sp1 sites decreased transcriptional activity in M-1 cells; however, these mutations had a marginal effect in the RCD cells. These results suggest that elements controlling renal cell type expression are located in the proximal 327 nucleotides of the 5' flanking region of HSD11K.
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PMID:Expression of HSD11K (NAD+ dependent 11beta-hydroxysteroid dehydrogenase) promoter constructs in renal cell lines. 1092 54


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