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)

Cultured inner medullary collecting duct (mIMCD-3) cells express Na+/H+ exchanger isoforms NHE-2 and NHE-1 (Soleimani et al. (1994) J. Biol. Chem. 269, 27973-27978). In the present studies we examined the effect of lethal acid stress on Na+/H+ exchanger activity and isoform expression in mIMCD-3 cells. mIMCD-3 cells were incubated for 10 min with 20 mM ammonium, and exposed to an ammonium-free acidic solution (pH 6.0) for 120 min. Thereafter, cells were recovered and grown in normal culture media. The surviving clones were isolated and subjected to two additional cycles of acid stress. A mutant clone was isolated and characterized for Na+/H+ exchange activity and isoform expression. The mutant mIMCD-3 clone demonstrated significant over-expression of Na+/H+ exchange activity as assessed by acid-stimulated 22Na influx (11.56 nmol/mg protein in mutant vs. 4.06 nmol/mg in parent cells, P < 0.001, n = 4) and sodium-dependent pHi recovery from an acid load (0.55 pH/min in mutant vs. 0.28 pH/min in parent cells, P < 0.01, n = 6). A dose-response inhibition of the exchanger showed that the mutant cells were very sensitive to dimethylamiloride (IC50 158 nM in mutant vs. 889 nM in parent mIMCD-3 cells, P < 0.001). To compare the Na+/H+ exchanger isoforms in mutant and parent mIMCD-3 cells, poly(A)+ RNA was isolated from each group and probed with radiolabeled NHE-1 or NHE-2 cDNA. The expression of NHE-1 mRNA was increased by approximately 100% in mutant cells. The NHE-2 mRNA, on the other hand, was found to be absent in mutant mIMCD-3 cells. Examination of the regulatory mechanisms of the Na+/H+ exchanger isoforms in parent mIMCD-3 cells, which express NHE-2 and NHE-1, and mutant mIMCD-3 cells, which only express NHE-1, would be helpful in elucidating the roles of NHE-2 and NHE-1 in inner medullary collecting duct cells.
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PMID:The effect of lethal acid stress on Na+/H+ exchanger isoforms in cultured inner medullary collecting duct cells: deletion of NHE-2 and over expression of NHE-1. 754 47

Recent cloning experiments have identified the existance of four distinct Na+/H+ exchanger isoforms designated as NHE-1, NHE-2, NHE-3, and NHE-4. The cellular distribution, subcellular localization, and regulation of one of these isoforms, NHE-2, in the kidney remains unknown. Northern hybridization showed that NHE-2, along with NHE-1, is expressed in cultured renal medullary collecting duct (mIMCD-3) cells. Acid-stimulated, dimethyl amiloride-sensitive 22Na+ uptake and sodium-dependent pHi recovery occurred only from the basolateral surface of the cells, indicating localization of Na+/H+ exchanger to the basolateral membrane domain. Incubation of IMCD cells in high osmolality media (510 mosm/liter) for 72 h stimulated the Na+/H+ exchanger activity by 59% (p < 0.001). NHE-1 mRNA abundance decreased, whereas NHE-2 mRNA increased in high osmolality media. Incubation of IMCD cells in acid media (pH 7.1) for 48 h did not affect the Na+/H+ exchanger activity compared with control (pH 7.4) (p > 0.05). Northern hybridization, however, indicated that NHE-1 mRNA increased, whereas NHE-2 mRNA decreased in acid media. In conclusion, mIMCD-3 cells express NHE-1 and NHE-2 mRNAs. The cell functional studies in mIMCD-3 cells strongly suggest that NHE-2, along with NHE-1, is expressed in the basolateral membrane domain. They further demonstrate differential regulation of NHE-1 and NHE-2 mRNAs in response to acidosis and high osmolality and suggest that NHE-2 may be involved in volume regulation of IMCD cells.
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PMID:Na+/H+ exchanger isoforms NHE-2 and NHE-1 in inner medullary collecting duct cells. Expression, functional localization, and differential regulation. 796 30

Ischemic renal injury is associated with changes in the expression of a number of genes. Although pH regulation is undoubtedly important during the recovery from ischemia, the expression of acid-base transporters during acute ischemic renal failure has not been studied. In the present study, levels of mRNA encoding the colonic H+-K+-ATPase and four isoforms of the Na+/H+ exchanger (NHE-1, NHE-2, NHE-3 and NHE-4) were measured by quantitative Northern analysis in rat renal cortex and medulla following ischemia-reperfusion injury. Rats were subjected to 30 minutes of renal artery occlusion and then sacrificed either 12 or 24 hours after the occlusion was released. The most striking changes followed 30 minutes of occlusion and 12 hours of reperfusion and involved the mRNA for NHE-3 (involved in HCO3- reabsorption in proximal tubule and thick limb) and colonic H+-K+-ATPase (involved in HCO3- reabsorption in collecting duct). These changes were: (1) a approximately 75% decrease in NHE-3 mRNA in both cortex and medulla; and (2) an approximately 8-fold increase in colonic H+-K+-ATPase mRNA in the cortex. At 12 hours of reperfusion, there was a 66% reduction in the Na+/H+ exchanger (NHE-3) activity as assayed by acid-stimulated 22Na+ influx into brush border membrane vesicles (P < 0.01). After 24 hours of reperfusion, NHE-3 mRNA remained suppressed while cortical colonic H+-K+-ATPase mRNA declined to only twice the control level. Medullary colonic H+-K+-ATPase mRNA did not change significantly. Gastric H+-K+-ATPase mRNA in cortex or medulla remained the same at 0, 12, and 24 hours after reperfusion. Cortical NHE-1 increased mildly at 12 and 24 hours of reperfusion whereas a moderate decrease in NHE-2 and NHE-4 mRNAs was observed in cortex and medulla after both 12 and 24 hours of reperfusion. We suggest that overexpression of colonic H+-K+-ATPase in the early phase of renal reperfusion injury may be responsible for compensatory reabsorption of increased HCO3- load resulting from suppression of NHE-3. This was supported by a fourfold increase in colonic H+-K+-ATPase mRNA in rats treated with acetazolamide, which causes renal HCO3-wasting. Rapid decline in colonic H+-K+-ATPase expression at 24 hours after reperfusion is likely due to reduced HCO3- delivery to distal tubules resulting from decreased GFR. Overexpression of H+-K+-ATPase may be vital to acid-base homeostasis in the early phase of acute ischemic renal failure.
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PMID:Ischemic-reperfusion injury in the kidney: overexpression of colonic H+-K+-ATPase and suppression of NHE-3. 908 76

Four members of the Na+/H+ (NHE) exchange family are present in the renal tubule. NHE1, ubiquitously expressed, amiloride-sensitive and growth factor-activatable, is present in the basolateral membrane of most segments of the renal tubule. NHE2 and NHE3 are present in the apical membrane of renal tubule segments, except the collecting duct. NHE4 is probably a basolateral isoform. NHEs are involved in multiple functions, cell pH and volume regulation, and transepithelial transport of NaCl, NaHCO3, and NH4+. The present review deals with the recent developments in the functions of NHEs, the effects of hormones, osmolality, acid base status, and potassium stores of Na+/H+ exchange at the cellular and molecular levels. Finally, the alterations of NHE activities in some renal disease processes are briefly discussed.
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PMID:Na+/H+ exchanger subtypes in the renal tubule: function and regulation in physiology and disease. 925 81

Na+/H+ exchangers (NHE) play a critical role in many cellular and transport processes in the inner medullary collecting duct (IMCD). Morphologically, the IMCD is divided into the outer (IMCD1), middle (IMCD2), and inner (IMCD3) segments. The inner, IMCD3 segment contains only one cell type, the IMCD cell, which is distinct in ultrastructure and in function from the principal and intercalated cells that are present in other portions of the IMCD. NHEs constitute a gene family containing several isoforms (NHE1, NHE2, NHE3, NHE4 and NHE5) which possess distinct characteristics and serve specialized functions. To understand the molecular basis of NHE-related processes in the IMCD, it is critical to know the molecular identity of the NHEs in this tubule segment. The purpose of the present study was to identify the NHE isoforms present and their polar distribution in IMCD3. Applying the reverse transcription-polymerase chain reaction (RT-PCR) technique to IMCD3 (obtained from distal 50% of inner medulla) of mouse and rat kidneys, we found that NHE1, NHE2 and NHE4, but not NHE3 were expressed in both species. The polar localization of NHE in IMCD3 was examined in tubules isolated from rats and perfused in vitro with HEPES-buffered solutions under isotonic conditions. pHi was measured by BCECF fluorescence. Na+-dependent, amiloride-inhibitable pHi recovery from cell acidification (consistent with NHE) was detected in the basolateral, but not the apical, membrane of IMCD3. We conclude that NHE1, NHE2 and NHE4, but not NHE3, are present in both the mouse and rat IMCD3. Functionally, NHE is limited to the basolateral membrane. Additional studies are needed to determine the physiological roles and regulation of basolateral NHE isoforms in this tubule segment.
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PMID:Expression of Na+/H+ exchanger isoforms in inner segment of inner medullary collecting duct. 969 Nov 22

Four Na+/H+ exchangers (NHE1 to NHE4) have been detected in the kidney. Renal NHE2 expression sites have not been fully established. We have raised rabbit antisera against an oligopeptide related to the amino acids 652 to 661 of rat NHE2. Western blot analysis of plasma membrane fractions isolated from rat renal cortex showed that affinity-purified anti-NHE2 antibody detected an 85-kDa protein in apical but not in basolateral membranes. The labeling of this 85-kDa protein was specifically blocked by preincubation of the antibody with its monomeric peptide, indicating specific recognition. Indirect immunolabeling was performed on sections of paraformaldehyde-fixed rat kidney embedded in paraffin. Strong staining was seen in the apical membrane of cortical thick ascending limbs, distal convoluted tubules, and connecting tubules. Much weaker apical staining was found in medullary thick ascending limbs of Henle. In the inner medulla, some thin limbs were intensively labeled by the anti-NHE2 antibody. No staining could be detected in any segments of the proximal tubule and collecting duct.
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PMID:Immunolocalization of the Na+/H+ exchanger isoform NHE2 in rat kidney. 972 10

Na(+)/H(+) exchanger (NHE) proteins perform a variety of functions in the kidney and are differentially distributed among nephron segments. The purpose of this study was to identify NHE isoforms in murine M-1 cells as a model of cortical collecting duct principal cells. It was found that mRNAs corresponding to NHE1, NHE2, and NHE4 are expressed in M-1 cells. NHE-dependent regulation of intracellular pH (pH(i)) was investigated in the absence of extracellular HCO. Application of a 20 mM NH(4)Cl pulse resulted in a reversible intracellular acidification from which recovery was partially inhibited by application of 1 mM amiloride to either the apical or the basolateral membranes and was abolished when amiloride was applied to both sides of the monolayers, which suggests that NHEs are expressed in both the apical and the basolateral cell membranes of M-1 cells. The purinergic agonists ATP and benzoylbenzoyl-ATP caused a reduction of pH(i) when applied to the apical membrane, which suggests pH(i) may be influenced by extracellular nucleotides in the luminal fluid of the cortical collecting duct.
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PMID:Expression of isoforms of the Na(+)/H(+) exchanger in M-1 mouse cortical collecting duct cells. 1188 Mar 26

The Na+/H+ exchanger (NHE) constitutes a gene family containing several isoforms that display different membrane localization and are involved in specialized functions. Although basolateral NHE-1 activity was described in the cortical collecting duct (CCD), the localization and function of other NHE isoforms is not yet clear, This study examines the expression, localization, and regulation of NHE isoforms in a rat cortical collecting duct cell line (RCCD1) that has previously been shown to be a good model of CCD cells. Present studies demonstrate the presence of NHE-1 and NHE-2 isoforms, but not NHE-3 and NHE-4, in RCCD1 cells. Cell monolayers, grown on permeable filters, were placed on special holders allowing independent access to apical and basolateral compartments. Intracellular pH (pHi) regulation was spectrofluorometrically studied in basal conditions and after stimulation by NH4Cl acid load or by a hyperosmotic shock. In order to differentiate the roles of NHE-1 and NHE-2, we have used HOE-694, an inhibitor more selective for NHE-1 than for NHE-2. The results obtained strongly suggest that NHE-1 and NHE-2 are expressed in the basolateral membrane but that they have different roles: NHE-1 is responsible for pHi recovery after an acid load and NHE-2 is mainly involved in steady-state pHi and cell volume regulation.
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PMID:Differential role of Na+/H+ exchange isoforms NHE-1 and NHE-2 in a rat cortical collecting duct cell line. 1247 76

The sodium/proton exchanger type 1 (NHE-1) plays an important role in the proliferation of vascular smooth muscle cells (VSMC). We have examined the regulation of NHE-1 by two potent mitogens, serotonin (5-HT, 5-hydroxytryptamine) and angiotensin II (Ang II), in cultured VSMC derived from rat aorta. 5-HT and Ang II rapidly activated NHE-1 via their G protein-coupled receptors (5-HT(2A) and AT(1)) as assessed by proton microphysiometry of quiescent cells and by measurements of intracellular pH on a FLIPR (fluorometric imaging plate reader). Activation of NHE-1 was blocked by inhibitors of phospholipase C, CaM, and Jak2 but not by pertussis toxin or inhibitors of protein kinase C. Immunoprecipitation/immunoblot studies showed that 5-HT and Ang II induce phosphorylation of Jak2 and induce the formation of signal transduction complexes that included Jak2, CaM, and NHE-1. The cell-permeable Ca(2+) chelator BAPTA-AM blocked activation of Jak2, complex formation between Jak2 and CaM, and tyrosine phosphorylation of CaM, demonstrating that elevated intracellular Ca(2+) is essential for those events. Thus, mitogen-induced activation of NHE-1 in VSMC is dependent upon elevated intracellular Ca(2+) and is mediated by the Jak2-dependent tyrosine phosphorylation of CaM and subsequent increased binding of CaM to NHE-1, similar to the pathway previously described for the bradykinin B(2) receptor in inner medullary collecting duct cells of the kidney [Mukhin, Y. V., et al. (2001) J. Biol. Chem. 276, 17339-17346]. We propose that this pathway represents a fundamental mechanism for the rapid regulation of NHE-1 by G(q/11) protein-coupled receptors in multiple cell types.
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PMID:Mitogen-induced activation of Na+/H+ exchange in vascular smooth muscle cells involves janus kinase 2 and Ca2+/calmodulin. 1279 14

Large-scale phosphoproteomic analysis employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) often requires a significant amount of manual manipulation of phosphopeptide datasets in the post-acquisition phase. To assist in this process, we have created software, PhosphoPIC (PhosphoPeptide Identification and Compilation), which can perform a variety of useful functions including automated selection and compilation of phosphopeptide identifications from multiple MS levels, estimation of dataset false discovery rate, and application of appropriate cross-correlation (XCorr) filters. In addition, the output files generated by this program are compatible with downstream phosphorylation site assignment using the Ascore algorithm, as well as phosphopeptide quantification via QUOIL. In this report, we utilized this software to analyze phosphoproteins from short-term vasopressin-treated rat kidney inner medullary collecting duct (IMCD). A total of 925 phosphopeptides representing 173 unique proteins were identified from membrane-enriched fractions of IMCD with a false discovery rate of 1.5%. Of these proteins, 106 were found only in the membrane-enriched fraction of IMCD cells and not in whole IMCD cell lysates. These identifications included a number of well-studied ion and solute transporters including ClC-1, LAT4, MCT2, NBC3, and NHE1, all of which contained novel phosphorylation sites. Using a label-free quantification approach, we identified phosphoproteins that changed in abundance with vasopressin exposure including aquaporin-2 (AQP2), Hnrpa3, IP3 receptor 3, and pur-beta.
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PMID:An automated platform for analysis of phosphoproteomic datasets: application to kidney collecting duct phosphoproteins. 1768 30


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