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)

We have obtained a polyclonal antiserum by immunizing fawn Burgundy rabbits with the mineralocorticoid receptor (MCR) purified biochemically from rat kidneys. High titers of anti-MCR activity were obtained in radioimmunoassays within 3 weeks and increased with a booster shot. In Western blot analysis, the antibody revealed a major band of 94-98 kDa in renal cytosol from rat and beef kidneys. We also developed a fluorographic procedure where the MCR linked covalently to tritiated R-5020, following ultraviolet irradiation, gave imprints superimposable on the Western blot profile. The fluorographic pattern was specific since it was largely abolished in the presence of cold RU 26752 that is specific to MCR, or mineralocortin. The immune IgG precipitated rat renal MCR(-)[3H]RU 26752 complexes in a dose-dependent manner and also recognized MCR bound to the natural hormone aldosterone. During gel permeation chromatography on Sephacryl, the elution profile of [3H]RU 26752 shifted to high-molecular-weight regions in the presence of immune IgG. The receptor protein could be immunolocalized primarily to the principal cells of the collecting duct in rat kidney but the intercalated cells and glomeruli were not labeled, contrary to beef kidney where a uniform pattern of immunostaining was evident. These should permit large-scale purification of the MCR for detailed physicochemical studies and for screening of the MCR-positive tissues during various pathophysiological syndromes.
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PMID:Immunophotochemical analysis of mineralocortin by polyclonal antibodies against the native receptor from rat kidney. 132 65

Eleven-beta-hydroxysteroid dehydrogenase (11 beta OHSD) protects the aldosterone receptor (MR) against its occupancy by glucocorticoid hormones. We examined the intrarenal distribution of 11 beta OHSD, as compared to that of MR. MR were localized in histological sections from rabbit kidney, using immunohistochemical methods with an anti-MR monoclonal antibody. 11 beta OHSD activity was measured in isolated tubular segments from rabbit, rat and mouse kidneys. Tubules were incubated in the presence of tritiated corticosterone (3H-B:2 x 10(-8)M). Then the rate of degradation of 3H-B into 3H-11-dehydrocorticosterone (3H-A) was determined by HPLC. MR was immunodetected in the distal tubule and the collecting duct. No positive staining was present in the proximal tubule. The conversion rate of 3H-B into 3H-A was high (approximately 80%) in the distal and collecting tubule. It was low in the proximal tubule (less than 15%) except in the rat (approximately 50%). These results indicate that MR and 11OHSD are colocalized along the mammalian nephron. This colocalization constitutes a strong argument in favor of the MR-protective role of 11 beta OHSD, and of a role of a defect of this enzyme in the genesis of some types of arterial hypertension.
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PMID:[A new regulatory mechanism of the action of corticosteroid hormones: cellular 11beta-hydroxycorticosteroid dehydrogenase]. 150 75

Aldosterone selectivity in mineralocorticoid target tissues has been suggested to be due to 11 beta-hydroxysteroid dehydrogenase (11-OHSD), which, by inactivating the endogenous glucocorticoids cortisol and corticosterone (CS), would allow aldosterone to bind to the mineralocorticoid receptor that has equal affinity for aldosterone and natural glucocorticoids. However, a recent immunohistochemical study failed to colocalize 11-OHSD and mineralocorticoid receptors in the kidney. The goal of this study was to determine 1) whether metabolism of CS occurs in the renal target cells of aldosterone, i.e. in cortical collecting duct cells, and 2) if it does so, whether this activity is sufficient to reduce intracellular CS levels to allow binding of aldosterone to the mineralocorticoid receptor. Cortical collecting duct cells were isolated by solid phase immunoadsorption, with a cell purity of approximately 98%. Metabolism of CS was studied in both freshly isolated cells and primary cultures grown as monolayers on permeable supports. Freshly isolated cells rapidly converted CS to 11-dehydro-CS, which was the only major metabolite detected. In intact collecting duct cells 11-OHSD had an apparent Km for CS of approximately 60 nM, a value more than 100-fold lower than the Km of 11-OHSD in the rat liver, and a maximum velocity of approximately 1.7 x 10(-14) mol/min.1000 cells. In cultured cells, when [3H]CS was applied to one side of the monolayer, almost all radioactivity on the opposite side was 11-dehydro-CS. The cells were able to almost completely metabolize CS passing through them for up to a concentration of 2.5 x 10(-7) M. Carbenoxolone, an inhibitor of 11-OHSD, reduced CS degradation by 88%. Neither freshly isolated nor cultured collecting duct cells converted [3H]11-dehydro-CS back to CS in a significant amount (less than 1%). These data provide functional evidence for 11-OHSD activity in renal aldosterone target cells and indicate that this enzyme might be a collecting duct-specific isoform of 11-OHSD which can sufficiently reduce intracellular CS concentrations to contribute to the apparent mineralocorticoid selectivity of the collecting duct.
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PMID:11 beta-Hydroxysteroid dehydrogenase activity in the renal target cells of aldosterone. 205 80

To investigate the direct epithelial effects of corticosteroids on renal ion transport, we studied the influence of the pure glucocorticoid agonist RU 28362 and aldosterone on Na+ and K+ transport in primary cultures of immunodissected rabbit cortical collecting duct (CCD) cells. When grown on permeable supports in a steroid-free medium, CCD monolayers exhibited a lumen-negative transepithelial potential difference (PD) of 5.2 +/- 1.07 mV and a short-circuit current (SCC) of 8.54 +/- 2.2 microA/cm2. Transepithelial resistance averaged 660 +/- 49 omega/cm2. The cultures actively reabsorbed Na+ and secreted K+. Both aldosterone and RU 28362 significantly increased PD and SCC; the effects were time and dose dependent. The effect of RU 28362 was completely prevented by the glucocorticoid receptor antagonist RU 486, whereas ZK 91587, a specific mineralocorticoid receptor antagonist, did not block its effect. Both aldosterone and RU 28362 increased the bath-to-lumen concentration ratio of Na+ while lowering that of K+, indicating an increased Na+ reabsorption and K+ secretion. The number of Na(+)-K(+)-ATPase units was significantly enhanced (approximately 2-fold) by both RU 28362 and aldosterone. These results demonstrate that, in cultured CCD cells, not only aldosterone but also a pure glucocorticoid is able to exert mineralocorticoid-like effects, and this latter effect is mediated by glucocorticoid receptors. Because all parameters studied responded similarly to aldosterone and RU 28362, we speculate that in CCD cells glucocorticoids and mineralocorticoids might act by regulating the same gene(s).
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PMID:Glucocorticoid receptors mediate mineralocorticoid-like effects in cultured collecting duct cells. 222 Nov 5

We have synthesized three peptides corresponding to putative antigenic regions in the immunogenic domain, hinge region, and carboxy-terminus of the protein. A rabbit immunized with a peptide derived from the hinge region of the receptor produced an antiserum which showed 50% displacement with 20 pg peptide at a final serum dilution of 1:35,000. When the antiserum was immunopurified and applied to sections of intact rat and human kidney it stained cells lining segments corresponding to distal tubule, connecting piece, and initial cortical collecting duct, consistent with the known sites of mineralocorticoid action. In both human (formaldehyde-fixed) and rat (Bouin's solution) there was ample evidence for both nuclear and cytoplasmic staining. The thymus, in which previously we have found [3H]aldosterone binding to be below detection limits, showed little or no staining. Western blot analyses demonstrated that the polyclonal antibody recognized an epitope of the expected molecular size. The availability of antibodies to the mineralocorticoid receptor should, thus, facilitate investigation of the steroid specificity-conferring mechanism which allows mineralocorticoids, but not glucocorticoids, access to the nonselective receptor in the kidney.
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PMID:Immunolocalization of renal mineralocorticoid receptors with an antiserum against a peptide deduced from the complementary deoxyribonucleic acid sequence. 247 68

Using a polyclonal antiserum against the hinge region of the recently cloned human mineralocorticoid receptor (MR) and indirect peroxidase immunohistochemistry, we have shown MR-like immunoreactivity (LI) in superficial nephron segments, including distal convoluted tubule, connecting piece and initial cortical collecting duct. The absence of staining in cells tentatively identified as intercalated cells on light microscopy was confirmed by pre-embedding electron microscopy. Though the intracellular distribution of immunostaining varied with the fixative used, the cellular distribution of MR-LI is in good general agreement with earlier micropuncture and autoradiographic studies.
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PMID:Immunocytochemical demonstration of mineralocorticoid receptors in rat and human kidney. 248 90

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

The purpose of this paper it to briefly review recent work from our laboratory dealing with the form of 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) present in renal aldosterone target cells. It is well established that aldosterone is the physiological mineralocorticoid hormone. The observation that mineralocorticoid receptors have equal affinity for aldosterone and endogenous glucocorticoids, coupled with the fact that circulating levels of glucocorticoids are much higher than those of aldosterone, raises the question of how aldosterone can fulfill its function. To explain this paradox, it was hypothesized that in mineralocorticoid target tissues 11 beta-OHSD rapidly inactivates glucocorticoids (but not aldosterone), thereby decreasing intracellular glucocorticoid levels, so that aldosterone can exert specific regulation via the mineralocorticoid receptor. However, the only form of this enzyme which has been cloned thus far might not be the enzyme which is able to confer aldosterone selectivity on the mineralocorticoid receptor. On the other hand, a new form of 11 beta-OHSD (11 beta-OHSD/CD) that we have discovered in renal collecting duct cells possesses all the properties necessary for protecting the mineralocorticoid receptor: very high affinity for endogenous glucocorticoids, high abundance in target cells, and irreversible dehydrogenase activity. Our hypothesis is that 11 beta-OHSD/CD is the enzyme that ensures aldosterone selectivity in mineralocorticoid target cells, and that is the product of a gene different from the one previously cloned.
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PMID:11 beta-Hydroxysteroid dehydrogenase in renal collecting duct cells. 819 37

In the present study, a competitive polymerase chain reaction (PCR) technique was used to quantitate the relative levels of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) mRNA in microdissected nephron segments from the rat kidney and of MR mRNA from isolated principal and intercalated collecting duct cells from rabbit. RNA was isolated from cells and isolated tubules, cDNA was synthesized, and receptor cDNA was coamplified by PCR with a competitive control template. beta-Actin PCR products were also obtained from each nephron segment studied, to assess variations in RNA extraction and cDNA synthesis. MR mRNA, as determined by this competitive PCR technique, was 10-fold more abundant in cortical collecting duct (CCD), outer medullary collecting duct, and inner medullary collecting duct segments than in the proximal tubule and thick ascending limb segments (P < 0.05). Both principal and beta-intercalated cells of the CCD contained detectable levels of MR mRNA, although the levels in the principal cells were threefold higher (P < 0.01). GR mRNA was twofold more abundant in glomeruli, proximal tubule, and thick ascending limb segments than in the collecting duct segments (P < 0.05). In general, the distribution pattern of MR and GR mRNA is consistent with the distribution of adrenal corticosteroid function along the nephron.
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PMID:Distribution of mineralocorticoid and glucocorticoid receptor mRNA along the nephron. 838 51

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


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