RESUMO
To identify additional potential functions for the multi-PDZ domain containing protein Na+/H+ exchanger regulatory factor 2 (NHERF2), which is present in the apical domain of intestinal epithelial cells, proteomic studies of mouse jejunal villus epithelial cell brush border membrane vesicles compared wild-type to homozygous NHERF2 knockout FVB mice by a two-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS)-iTRAQ approach. Jejunal architecture appeared normal in NHERF2 null in terms of villus length and crypt depth, Paneth cell number, and microvillus structure by electron microscopy. There was also no change in proliferative activity based on BrdU labeling. Four brush border membrane vesicles (BBMV) preparations from wild-type mouse jejunum were compared with four preparations from NHERF2 knockout mice. LC-MS/MS identified 450 proteins in both matched wild-type and NHERF2 null BBMV; 13 proteins were changed in two or more separate BBMV preparations (9 increased and 4 decreased in NHERF2 null mice), while an additional 92 proteins were changed in a single BBMV preparation (68 increased and 24 decreased in NHERF2 null mice). These proteins were categorized as 1) transport proteins (one increased and two decreased in NHERF2 null); 2) signaling molecules (2 increased in NHERF2 null); 3) cytoskeleton/junctional proteins (4 upregulated and 1 downregulated in NHERF2 null); and 4) metabolic proteins/intrinsic BB proteins) (2 upregulated and 1 downregulated in NHERF2 null). Immunoblotting of BBMV was used to validate or extend the findings, demonstrating increase in BBMV of NHERF2 null of MCT1, coronin 3, and ezrin. The proteome of the NHERF2 null mouse small intestinal BB demonstrates up- and downregulation of multiple transport proteins, signaling molecules, cytoskeletal proteins, tight junctional and adherens junction proteins, and proteins involved in metabolism, suggesting involvement of NHERF2 in multiple apical regulatory processes and interactions with luminal contents.
Assuntos
Jejuno/metabolismo , Fosfoproteínas/genética , Proteoma/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Animais , Caderinas/metabolismo , Proliferação de Células , Cromatografia Líquida , Citoesqueleto/metabolismo , Regulação para Baixo , Imunofluorescência , Masculino , Camundongos , Camundongos Knockout , Microvilosidades/genética , Microvilosidades/metabolismo , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , beta Catenina/metabolismoRESUMO
Na/H exchanger regulatory factor 1 (NHERF1) is a scaffold protein made up of two PDZ domains and an ERM binding domain. It is in the brush border of multiple epithelial cells where it modulates 1) Na absorption by regulating NHE3 complexes and cytoskeletal association, 2) Cl secretion through trafficking of CFTR, and 3) Na-coupled phosphate absorption through membrane retention of NaPi2a. To further understand the role of NHERF1 in regulation of small intestinal Na absorptive cell function, with emphasis on apical membrane transport regulation, quantitative proteomic analysis was performed on brush border membrane vesicles (BBMV) prepared from wild-type (WT) and homozygous NHERF1 knockout mouse jejunal villus Na absorptive cells. Jejunal architecture appeared normal in NHERF1 null; however, there was increased proliferative activity, as indicated by increased crypt BrdU staining. LC-MS/MS analysis using iTRAQ to compare WT and NHERF1 null BBMV identified 463 proteins present in both WT and NHERF1 null BBMV of simultaneously prepared and studied samples. Seventeen proteins had an altered amount of expression between WT and NHERF1 null in two or more separate preparations, and 149 total proteins were altered in at least one BBMV preparation. The classes of the majority of proteins altered included transport proteins, signaling and trafficking proteins, and proteins involved in proliferation and cell division. Affected proteins also included tight junction and adherens junction proteins, cytoskeletal proteins, as well as metabolic and BB digestive enzymes. Changes in abundance of several proteins were confirmed by immunoblotting [increased CEACAM1, decreased ezrin (p-ezrin), NHERF3, PLCß3, E-cadherin, p120, ß-catenin]. The changes in the jejunal BBMV proteome of NHERF1 null mice are consistent with a more complex role of NHERF1 than just forming signaling complexes and anchoring proteins to the apical membrane and include at least alterations in proteins involved in transport, signaling, and proliferation.
Assuntos
Jejuno/metabolismo , Fosfoproteínas/genética , Proteoma/análise , Trocadores de Sódio-Hidrogênio/genética , Vesículas Transportadoras/metabolismo , Animais , Caderinas/análise , Cromatografia por Troca Iônica , Feminino , Immunoblotting , Imuno-Histoquímica , Jejuno/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Microvilosidades/metabolismo , Microvilosidades/ultraestrutura , Fosfoproteínas/metabolismo , Proteômica/métodos , Trocadores de Sódio-Hidrogênio/metabolismo , Espectrometria de Massas em Tandem , beta Catenina/análiseRESUMO
We investigated the role of the Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) on intestinal salt and water absorption, brush border membrane (BBM) morphology, and on the NHE3 mRNA expression, protein abundance, and transport activity in the murine intestine. NHERF1-deficient mice displayed reduced jejunal fluid absorption in vivo, as well as an attenuated in vitro Na(+) absorption in isolated jejunal and colonic, but not of ileal, mucosa. However, cAMP-mediated inhibition of both parameters remained intact. Acid-activated NHE3 transport rate was reduced in surface colonocytes, while its inhibition by cAMP and cGMP was normal. Immunodetection of NHE3 revealed normal NHE3 localization in the BBM of NHERF1 null mice, but NHE3 abundance, as measured by Western blot, was significantly reduced in isolated BBM from the small and large intestines. Furthermore, the microvilli in the proximal colon, but not in the small intestine, were significantly shorter in NHERF1 null mice. Additional knockout of PDZK1 (NHERF3), another member of the NHERF family of adaptor proteins, which binds to both NHE3 and NHERF1, further reduced basal NHE3 activity and caused complete loss of cAMP-mediated NHE3 inhibition. An activator of the exchange protein activated by cAMP (EPAC) had no effect on jejunal fluid absorption in vivo, but slightly inhibited NHE3 activity in surface colonocytes in vitro. In conclusion, NHERF1 has segment-specific effects on intestinal salt absorption, NHE3 transport rates, and NHE3 membrane abundance without affecting mRNA levels. However, unlike PDZK1, NHERF1 is not required for NHE3 regulation by cyclic nucleotides.
Assuntos
Colo/metabolismo , Absorção Intestinal/fisiologia , Jejuno/metabolismo , Fosfoproteínas/deficiência , Cloreto de Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Imuno-Histoquímica , Mucosa Intestinal/metabolismo , Camundongos , Microvilosidades/ultraestrutura , Trocador 3 de Sódio-HidrogênioRESUMO
The multi-PDZ domain containing protein Na(+)/H(+) Exchanger Regulatory Factor 1 (NHERF1) binds to Na(+)/H(+) exchanger 3 (NHE3) and is associated with the brush border (BB) membrane of murine kidney and small intestine. Although studies in BB isolated from kidney cortex of wild type and NHERF1(-/-) mice have shown that NHERF1 is necessary for cAMP inhibition of NHE3 activity, a role of NHERF1 in NHE3 regulation in small intestine and in intact kidney has not been established. Here a method using multi-photon microscopy with the pH-sensitive dye SNARF-4F (carboxyseminaphthorhodafluors-4F) to measure BB NHE3 activity in intact murine tissue and use it to examine the role of NHERF1 in regulation of NHE3 activity. NHE3 activity in wild type and NHERF1(-/-) ileum and wild type kidney cortex were inhibited by cAMP, whereas the cAMP effect was abolished in kidney cortex of NHERF1(-/-) mice. cAMP inhibition of NHE3 activity in these two tissues is mediated by different mechanisms. In ileum, a protein kinase A (PKA)-dependent mechanism accounts for all cAMP inhibition of NHE3 activity since the PKA antagonist H-89 abolished the inhibitory effect of cAMP. In kidney, both PKA-dependent and non-PKA-dependent mechanisms were involved, with the latter reproduced by the effect on an EPAC (exchange protein directly activated by cAMP) agonist (8-(4-chlorophenylthio)-2'O-Me-cAMP). In contrast, the EPAC agonist had no effect in proximal tubules in NHERF1(-/-) mice. These data suggest that in proximal tubule, NHERF1 is required for all cAMP inhibition of NHE3, which occurs through both EPAC-dependent and PKA-dependent mechanisms; in contrast, cAMP inhibits ileal NHE3 only by a PKA-dependent pathway, which is independent of NHERF1 and EPAC.
Assuntos
Íleo/metabolismo , Túbulos Renais/metabolismo , Fosfoproteínas/fisiologia , Trocadores de Sódio-Hidrogênio/fisiologia , Animais , AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Isoformas de Proteínas , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Distribuição TecidualRESUMO
Histidine residues in Na+/H+ exchangers are believed to participate in proton binding and influence the Na+/H+ exchanger activity. In the present study, the function of three highly conserved histidines in the juxtamembrane cytoplasmic domain of NHE3 was studied. His-479, His-485, and His-499 were mutated to Leu, Gln or Asp and expressed in an Na+/H+ exchanger null cell line and functional consequences on Na+/H+ exchange kinetics were characterized. None of the histidines were essential for NHE3 activity, with all mutated NHE3 resulting in functional exchangers. However, the mutation in His-475 and His-499 significantly lowered NHE3 transport activity, whereas the mutation in H485 showed no apparent effect. In addition, the pH profiles of the H479 and H499 mutants were shifted to a more acidic region, and lowered its set point, the intracellular pH value above which the Na+/H+ exchanger becomes inactive, by approximately 0.3-0.6 pH units. The changes in set point by the mutations were further shifted to more acidic values by ATP depletion, indicating that the mechanism by which the mutations on the histidine residues altered the NHE3 set point differs from that caused by ATP depletion. We suggest that His-479 and His-499 are part of the H+ sensor, which is involved in determining the sensitivity to the intracellular H+ concentration and Na+/H+ exchange rate.
Assuntos
Membrana Celular/metabolismo , Histidina/química , Histidina/metabolismo , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Membrana Celular/patologia , Fibroblastos , Histidina/genética , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/genética , Relação Estrutura-Atividade , Transfecção/métodosRESUMO
Cell biological approaches were used to examine the location and function of the brush border (BB) Na(+)/H(+) exchanger NHE3 in the opossum kidney (OK) polarized renal proximal tubule cell line. NHE3 epitope tagged with the vesicular stomatitis virus glycoprotein epitope (NHE3V) was stably expressed and called OK-E3V cells. On the basis of cell surface biotinylation studies, these cells had 10-15% of total NHE3 on the BB. Intracellular NHE3V largely colocalized with Rab11 and to a lesser extent with EEA1. The BB location of NHE3V was examined by confocal microscopy relative to the lectins wheat germ aggluttinin (WGA) and phytohemagluttin E (PHA-E), as well as the B subunit of cholera toxin (CTB). The cells were pyramidal, and NHE3 was located in microvilli in the center of the apical surface. In contrast, PHA-E, WGA, and CTB were diffusely distributed on the BB. Detergent extraction showed that total NHE3V was largely soluble in Triton X-100, whereas virtually all surface NHE3V was insoluble. Sucrose density gradient centrifugation demonstrated that total NHE3V migrated at the same size as approximately 400- and approximately 900-kDa standards, whereas surface NHE3V was enriched in the approximately 900-kDa form. Under basal conditions, NHE3 cycled between the cell surface and the recycling pathway through a phosphatidylinositol (PI) 3-kinase-dependent mechanism. Measurements of surface and intracellular pH were obtained by using FITC-WGA. Internalization of FITC-WGA occurred largely into the juxtanuclear compartment that contained Rab11 and NHE3V. pH values on the apical surface and in endosomes in the presence of the NHE3 blocker, S3226, were elevated, showing that NHE3 functioned to acidify both compartments. In conclusion, NHE3V in OK cells exists in distinct domains both in the center of the apical surface and in a juxtanuclear compartment. In the BB fraction, NHE3 is largely in the detergent-insoluble fraction in lipid rafts and/or in large heterogenous complexes ranging from approximately 400 to approximately 900 kDa.
Assuntos
Túbulos Renais Proximais/metabolismo , Glicoproteínas de Membrana , Trocadores de Sódio-Hidrogênio/metabolismo , Androstadienos/farmacologia , Animais , Compartimento Celular/fisiologia , Linhagem Celular , Membrana Celular/metabolismo , Eletroforese em Gel de Poliacrilamida , Endossomos/metabolismo , Imunofluorescência , Corantes Fluorescentes , Expressão Gênica , Concentração de Íons de Hidrogênio , Líquido Intracelular/metabolismo , Túbulos Renais Proximais/citologia , Proteínas Luminescentes/genética , Substâncias Macromoleculares , Microvilosidades/metabolismo , Gambás , Transporte Proteico/efeitos dos fármacos , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/efeitos dos fármacos , Trocadores de Sódio-Hidrogênio/genética , Transfecção , Proteínas do Envelope Viral/genética , WortmaninaRESUMO
Na+/H+ exchangers are integral membrane proteins that exchange Na+ and H+ across cell membranes. The Na+/H+ exchangers 2 and 3 are epithelial isoforms in mammals and contribute to acid-base homeostasis. The gills of fishes, including elasmobranchs, are also associated with acid/base balance, and are probably the primary acid/base regulatory organ. This study examines the presence of Na+/H+ exchangers 2 and 3 using immunohistochemistry and immunoblotting in the gills of four species of elasmobranchs, the banjo ray (Trygonorrhina fasciata), southern eagle ray (Myliobatis australis), the gummy shark (Mustelus antarcticus) and the Australian angel shark (Squatina australis) using heterologous antibodies. Na+/H+ exchanger 2-like immunoreactivity was observed in the gills of the banjo ray, eagle ray and angel shark. In the banjo and eagle rays, this Na+/H+ exchanger-like immunoreactivity co-localised with immunoreactivity to Na+ /K+ -ATPase, a marker for the mitochondrial-rich cells of fishes. Na+/H+ exchanger 3-like immunoreactivity was only observed in the gills of the angel and gummy sharks, some Na+/H+ exchanger 3-like cells also showed Na+ /K+ -ATPase immunoreactivity. However, immunoblotting of banjo and eagle ray gill membranes demonstrated Na+/H+ exchanger 3-like immunoreactivity, which was not consistent with the immunohistochemical results. These data demonstrate the presence of epithelial Na+/H+ exchangers 2 and 3 in the gills of elasmobranchs and a link with acid/base regulation is suggested.
Assuntos
Elasmobrânquios/metabolismo , Brânquias/química , Trocadores de Sódio-Hidrogênio/análise , Animais , Western Blotting , Reações Cruzadas , Imuno-Histoquímica , Especificidade de Órgãos , Isoformas de Proteínas/análise , Isoformas de Proteínas/imunologia , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/imunologiaRESUMO
Enterohemorrhagic Escherichia coli producing Shiga toxins 1 and/or 2 have become major foodborne pathogens. The specific binding of Shiga toxin 1 B-subunit to its receptor, a neutral glycolipid globotriaosylceramide Gb(3), on the apical surface of colonic epithelium followed by toxin entry into cells are the initial steps of the process, which can result in toxin transcytosis and systemic effects of infection including hemolytic uremic syndrome. Understanding the complex mechanisms of Shiga toxin 1 binding and internalization may help to develop new strategies directed at preventing toxin internalization. Fluorescence resonance energy transfer microscopy revealed the clustering of Shiga toxin receptors Gb(3) in lipid rafts with another glycosphingolipid G(M1) on the apical surface of highly polarized intestinal epithelial Caco-2 cells. Lipid rafts disruption significantly decreased internalization of Shiga toxin 1 B-subunit. Although disruption of lipid rafts by cholesterol depletion did not affect the amount of bound Shiga toxin 1 B-subunit, lipid rafts are necessary for toxin uptake across the apical membrane of Caco-2 cells.
Assuntos
Transporte Biológico/fisiologia , Membrana Celular/metabolismo , Microdomínios da Membrana/metabolismo , Toxina Shiga I/metabolismo , Triexosilceramidas/metabolismo , Células CACO-2 , Polaridade Celular , Colesterol/metabolismo , Transferência de Energia , Corantes Fluorescentes/metabolismo , Gangliosídeo G(M1)/metabolismo , Humanos , Microdomínios da Membrana/química , Microscopia de Fluorescência/métodos , Agregação de Receptores/fisiologia , Receptores de Superfície Celular/metabolismoRESUMO
1. Rabbit ileal Na+-absorbing cell Na+-H+ exchanger 3 (NHE3) was shown to exist in three pools in the brush border (BB), including a population in lipid rafts. Approximately 50% of BB NHE3 was associated with Triton X-100-soluble fractions and the other approximately 50% with Triton X-100-insoluble fractions; approximately 33% of the detergent-insoluble NHE3 was present in cholesterol-enriched lipid microdomains (rafts). 2. The raft pool of NHE3 was involved in the stimulation of BB NHE3 activity with epidermal growth factor (EGF). Both EGF and clonidine treatments were associated with a rapid increase in the total amount of BB NHE3. This EGF- and clonidine-induced increase of BB NHE3 was associated with an increase in the raft pool of NHE3 and to a smaller extent with an increase in the total detergent-insoluble fraction, but there was no change in the detergent-soluble pool. In agreement with the rapid increase in the amount of NHE3 in the BB, EGF also caused a rapid stimulation of BB Na+-H+ exchange activity. 3. Disrupting rafts by removal of cholesterol with methyl-beta-cyclodextrin (MbetaCD) or destabilizing the actin cytoskeleton with cytochalasin D decreased the amount of NHE3 in early endosomes isolated by OptiPrep gradient fractionation. Specifically, NHE3 was shown to associate with endosomal vesicles immunoisolated by anti-EEA1 (early endosomal autoantigen 1) antibody-coated magnetic beads and the endosome-associated NHE3 was decreased by cytochalasin D and MbetaCD treatment. 4. We conclude that: (i) a pool of ileal BB NHE3 exists in lipid rafts; (ii) EGF and clonidine increase the amount of BB NHE3; (iii) lipid rafts and to a lesser extent, the cytoskeleton, but not the detergent-soluble NHE3 pool, are involved in the EGF- and clonidine-induced acute increase in amount of BB NHE3; (iv) lipid rafts and the actin cytoskeleton play important roles in the basal endocytosis of BB NHE3.
Assuntos
Íleo/metabolismo , Metabolismo dos Lipídeos , Trocadores de Sódio-Hidrogênio/metabolismo , Actinas/fisiologia , Animais , Proteínas do Citoesqueleto/fisiologia , Citoesqueleto/metabolismo , Citoesqueleto/fisiologia , Detergentes , Endocitose/fisiologia , Técnicas In Vitro , Masculino , Proteínas de Membrana/metabolismo , Microvilosidades/metabolismo , Fosfoproteínas/fisiologia , Proteínas Qa-SNARE , Coelhos , Trocador 3 de Sódio-Hidrogênio , SolubilidadeRESUMO
The Na(+)/H(+) exchangers NHE2 and NHE3 are involved in epithelial Na(+) and HCO absorption. To increase insights into the functions of NHE2 vs. NHE3, we compared their cellular processing with each other and with the housekeeping isoform NHE1. Using biotinylated exchanger, we determined that the half-life of plasma membrane NHE2 was short (3 h) compared with that of NHE1 (24 h) and NHE3 (14 h) in both PS120 fibroblasts and Caco-2 cells. NHE2 transport and plasma membrane levels were reduced by 3 h of Brefeldin A treatment, whereas NHE1 was unaffected. NHE2 was degraded by the lysosomes but not proteosomes, as demonstrated by increasing levels of endocytosed NHE2 protein after inhibition of the lysosomes, but not with proteosome inhibition. Unlike that of NHE3, basal NHE2 transport activity was not affected by phosphatidylinositol 3-kinase inhibition and did not appear to be localized in the juxtanuclear recycling endosome. Therefore, for NHE2, protein degradation and/or protein synthesis probably play important roles in its basal and regulated states. These results suggest fundamental differences in the cellular processing and trafficking of NHE2 and NHE3. These differences may underlie the specialized roles that these exchangers play in epithelial cells.
Assuntos
Transporte Proteico/fisiologia , Trocadores de Sódio-Hidrogênio/metabolismo , Androstadienos/farmacologia , Animais , Biotinilação , Brefeldina A/farmacologia , Linhagem Celular , Cricetinae , Cisteína Endopeptidases/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Meia-Vida , Humanos , Concentração de Íons de Hidrogênio , Lisossomos/metabolismo , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/metabolismo , Complexo de Endopeptidases do Proteassoma , Isoformas de Proteínas/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Trocador 3 de Sódio-Hidrogênio , Espectrometria de Fluorescência , WortmaninaRESUMO
Mammalian phospholipase C-beta isozymes are activated by a heterotrimeric GTP-binding protein linked to various cell surface receptors. Recent reports suggest that PDZ domain proteins play a significant role of PDZ-containing proteins in the regulation of mammalian PLC-beta isozymes. PDZ-containing proteins mediate the clustering of receptors and signaling molecules and thereby regulate agonist-induced signal transduction in polarized cells such as neuronal and epithelial cells. NORPA, a Drosophila PLC-beta, is known to be a component of a signaling complex that includes TRP and rhodopsin through interaction with INAD, a PDZ-containing protein. Mammalian PLC-beta1 and -beta2 isoforms interact with a PDZ-containing protein NHERF which is coupled to Trp4, a Ca(2+) channel. In addition, PLC-beta3 specifically interacts with E3KARP, another protein closely related to NHERF, through its C-terminal PDZ-binding motif. E3KARP up-regulates the PLC-beta3 activation coupled to muscarinic receptor. In this review, the role of signaling complexes mediated by PDZ-containing proteins in the regulation of PLC-beta isoforms will be discussed.
Assuntos
Proteínas de Drosophila , Isoenzimas/fisiologia , Transdução de Sinais , Fosfolipases Tipo C/fisiologia , Animais , Canais de Cálcio/metabolismo , Proteínas do Citoesqueleto/fisiologia , Drosophila/fisiologia , Proteínas do Olho/metabolismo , Isoenzimas/química , Modelos Biológicos , Fosfolipase C beta , Fosfoproteínas/fisiologia , Estrutura Terciária de Proteína , Trocadores de Sódio-Hidrogênio , Canais de Cátion TRPC , Fosfolipases Tipo C/química , Visão OcularRESUMO
The proximal duodenal mucosa secretes HCO that serves to protect the epithelium from injury. In isolated human duodenal enterocytes in vitro, multiple luminal membrane proteins are involved in acid/base transport. We postulated that one or more isoforms of the Na(+)/H(+) exchanger (NHE) family is located on the apical surface of human duodenal mucosal epithelial cells and thereby contributes to duodenal mucosal HCO transport. Duodenal biopsies were obtained from human volunteers, and the presence of NHE2 and NHE3 was determined by using previously characterized polyclonal antibodies (Ab 597 for NHE2 and Ab 1381 for NHE3). In addition, proximal duodenal mucosal HCO(3)(-) transport was measured in humans in vivo in response to luminal perfusion of graded doses of amiloride; 10(-5)--10(-4) M amiloride was used to inhibit NHE2 and 10(-3) M amiloride to inhibit NHE3. Both NHE2 and NHE3 were localized principally to the brush border of duodenal villus cells. Sequential doses of amiloride resulted in significant, step-wise increases in net duodenal HCO(3)(-) output. Inhibition of NHE2 with 10(-5) M and 10(-4) M amiloride significantly increased net HCO(3)(-) output. Moreover, there was an additional, equivalent increase (P < 0.05) in duodenal HCO(3)(-) output with 10(-3) M amiloride, which inhibited NHE3. We conclude that 1) NHE2 and NHE3 are localized principally to the brush border of human duodenal villus epithelial cells; 2) sequential inhibition of NHE2 and NHE3 isoforms resulted in step-wise increases in net HCO(3)(-) output; 3) NHE2 and NHE3 participate in human duodenal villus cell HCO(3)(-) transport; and 4) the contribution of NHE-related transport events should be considered when studying duodenal HCO(3)(-) transport processes.
Assuntos
Bicarbonatos/metabolismo , Duodeno/metabolismo , Mucosa Intestinal/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Adulto , Amilorida/farmacologia , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Diuréticos/farmacologia , Duodeno/química , Duodeno/citologia , Imunofluorescência , Humanos , Técnicas In Vitro , Mucosa Intestinal/química , Mucosa Intestinal/citologia , Masculino , Microvilosidades/química , Microvilosidades/metabolismo , Pessoa de Meia-Idade , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/análiseRESUMO
Phosphatidylinositol 3-kinase (PI 3-kinase) is a cytoplasmic signaling molecule that is recruited to activated growth factor receptors and has been shown to be involved in regulation of stimulated exocytosis and endocytosis. One of the downstream signaling molecules activated by PI 3-kinase is the protein kinase Akt. Previous studies have indicated that PI 3-kinase is necessary for basal Na(+)/H(+) exchanger 3 (NHE3) transport and for fibroblast growth factor-stimulated NHE3 activity in PS120 fibroblasts. However, it is not known whether activation of PI 3-kinase is sufficient to stimulate NHE3 activity or whether Akt is involved in this PI 3-kinase effect. We used an adenoviral infection system to test the possibility that activation of PI 3-kinase or Akt alone is sufficient to stimulate NHE3 activity. This hypothesis was investigated in PS120 fibroblasts stably expressing NHE3 after somatic gene transfer using a replication-deficient recombinant adenovirus containing constitutively active catalytic subunit of PI 3-kinase or constitutively active Akt. The adenovirus construct used was engineered with an upstream ecdysone promoter to allow time-regulated expression. Adenoviral infection was nearly 100% at 48 h after infection. Forty-eight hours after infection (24 h after activation of the ecdysone promoter), PI 3-kinase and Akt amount and activity were increased. Increases in both PI 3-kinase activity and Akt activity stimulated NHE3 transport. In addition, a membrane-permeant synthetic 10-mer peptide that binds polyphosphoinositides and increases PI 3-kinase activity similarly enhanced NHE3 transport activity and also increased the percentage of NHE3 on the plasma membrane. The magnitudes of stimulation of NHE3 by constitutively active PI 3-kinase, PI 3-kinase peptide, and constitutively active Akt were similar to each other. These results demonstrate that activation of PI 3-kinase or Akt is sufficient to stimulate NHE3 transport activity in PS120/NHE3 cells.
Assuntos
Proteínas Musculares , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/fisiologia , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Transporte Biológico , Epitélio/metabolismo , Glucose/metabolismo , Transportador de Glucose Tipo 4 , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Proto-Oncogênicas c-akt , Coelhos , Trocador 3 de Sódio-HidrogênioRESUMO
Na(+)/H(+) exchanger regulatory factor (NHERF) and NHERF2 are PDZ motif proteins that mediate the inhibitory effect of cAMP on Na(+)/H(+) exchanger 3 (NHE3) by facilitating the formation of a multiprotein signaling complex. With the use of antibodies specific for NHERF and NHERF2, immunocytochemical analysis of rat kidney was undertaken to determine the nephron distribution of both proteins and their colocalization with other transporters and with ezrin. NHERF was most abundant in apical membrane of proximal tubule cells, where it colocalized with ezrin and NHE3. NHERF2 was detected in the glomerulus and in other renal vascular structures. In addition, NHERF2 was strongly expressed in collecting duct principal cells, where it colocalized with ROMK. These results indicate a striking difference in the nephron distribution of NHERF and NHERF2 and suggests NHERF is most likely to be the relevant biological regulator of NHE3 in the proximal tubule, while NHERF2 may interact with ROMK or other targets in the collecting duct. The finding that NHERF isoforms occur in different cell types suggests that NHERF and NHERF2 may subserve different functions in the kidney.
Assuntos
Néfrons/metabolismo , Fosfoproteínas/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Proteínas do Citoesqueleto , Glomérulos Renais/metabolismo , Glomérulos Renais/ultraestrutura , Túbulos Renais Coletores/metabolismo , Túbulos Renais Coletores/ultraestrutura , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/ultraestrutura , Masculino , Néfrons/ultraestrutura , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Trocador 3 de Sódio-HidrogênioRESUMO
The highly conserved and ubiquitously expressed 14-3-3 proteins regulate differentiation, cell cycle progression and apoptosis by binding intracellular phosphoproteins involved in signal transduction. By screening in vitro translated cDNA pools for the ability to bind 14-3-3, we identified a novel transcriptional co-activator, TAZ (transcriptional co-activator with PDZ-binding motif) as a 14-3-3-binding molecule. TAZ shares homology with Yes-associated protein (YAP), contains a WW domain and functions as a transcriptional co-activator by binding to the PPXY motif present on transcription factors. 14-3-3 binding requires TAZ phosphorylation on a single serine residue, resulting in the inhibition of TAZ transcriptional co-activation through 14-3-3-mediated nuclear export. The C-terminus of TAZ contains a highly conserved PDZ-binding motif that localizes TAZ into discrete nuclear foci and is essential for TAZ-stimulated gene transcription. TAZ uses this same motif to bind the PDZ domain-containing protein NHERF-2, a molecule that tethers plasma membrane ion channels and receptors to cytoskeletal actin. TAZ may link events at the plasma membrane and cytoskeleton to nuclear transcription in a manner that can be regulated by 14-3-3.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Proteínas 14-3-3 , Aciltransferases , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Linhagem Celular , Galinhas , Proteínas de Ligação a DNA/química , Regulação da Expressão Gênica , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Fosforilação , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Initiation of intestinal Na(+)-glucose cotransport results in transient cell swelling and sustained increases in tight junction permeability. Since Na(+)/H(+) exchange has been implicated in volume regulation after physiological cell swelling, we hypothesized that Na(+)/H(+) exchange might also be required for Na(+)-glucose cotransport-dependent tight junction regulation. In Caco-2 monolayers with active Na(+)-glucose cotransport, inhibition of Na(+)/H(+) exchange with 200 microM 5-(N,N-dimethyl)- amiloride induced 36 +/- 2% increases in transepithelial resistance (TER). Evaluation using multiple Na(+)/H(+) exchange inhibitors showed that inhibition of the Na(+)/H(+) exchanger 3 (NHE3) isoform was most closely related to TER increases. TER increases due to NHE3 inhibition were related to cytoplasmic acidification because cytoplasmic alkalinization with 5 mM NH(4)Cl prevented both cytoplasmic acidification and TER increases. However, NHE3 inhibition did not affect TER when Na(+)-glucose cotransport was inhibited. Myosin II regulatory light chain (MLC) phosphorylation decreased up to 43 +/- 5% after inhibition of Na(+)/H(+) exchange, similar to previous studies that associate decreased MLC phosphorylation with increased TER after inhibition of Na(+)-glucose cotransport. However, NHE3 inhibitors did not diminish Na(+)-glucose cotransport. These data demonstrate that inhibition of NHE3 results in decreased MLC phosphorylation and increased TER and suggest that NHE3 may participate in the signaling pathway of Na(+)-glucose cotransport-dependent tight junction regulation.
Assuntos
Mucosa Intestinal/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Junções Íntimas/metabolismo , Ácidos/metabolismo , Álcalis/metabolismo , Amilorida/farmacologia , Animais , Antiulcerosos/farmacologia , Anti-Hipertensivos/farmacologia , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Células CACO-2 , Cimetidina/farmacologia , Clonidina/farmacologia , Citoplasma/metabolismo , Diuréticos/farmacologia , Impedância Elétrica , Eletrofisiologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Glucose/metabolismo , Guanidinas/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Metacrilatos/farmacologia , Microvilosidades/metabolismo , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Quinase de Cadeia Leve de Miosina/metabolismo , Fosforilação , Sódio/metabolismo , Trocador 3 de Sódio-Hidrogênio , Sulfonas/farmacologiaRESUMO
We developed a confocal morphometric analysis to quantitate the relative plasma membrane (PM) expression of the Na/H exchanger NHE3 in living PS120 fibroblasts. NHE3 is a membrane transport protein that is acutely regulated by changes in the number of molecules expressed at the PM. To quantitate the PM expression of NHE3 under various experimental conditions, we stably expressed a chimera of rabbit NHE3 and green fluorescent protein (NHE3-GFP) in PS120 fibroblasts. A three-dimensional (3D) map of the intracellular distribution of NHE3-GFP was obtained by confocal laser scanning microscopy (CLSM) of cells superfused with a styryl dye, FM 4-64. This fluorophore rapidly and reversibly labeled the outer lipid layer of the PM, which allowed generation of a digital mask of the PM and calculation of the fraction of a total cellular NHE3-GFP expressed at the PM. This analysis was successfully used to quantitate the relative PM expression of NHE3-GFP in control cells (25%) and a decrease in the expression caused by subsequent exposure of cells to wortmannin (5.1%). Reliability of the method was confirmed by cell surface biotinylation, which yielded very similar results. Confocal morphometric analysis is fast and reproducible and could potentially be used for investigations on regulation of expression of other membrane proteins.
Assuntos
Fibroblastos/metabolismo , Proteínas Luminescentes/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Microscopia Confocal , Coelhos , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/genéticaRESUMO
Teleost fishes, living in fresh water, engage in active ion uptake to maintain ion homeostasis. Current models for NaCl uptake involve Na(+) uptake via an apical amiloride-sensitive epithelial Na(+) channel (ENaC), energized by an apical vacuolar-type proton pump (V-ATPase) or alternatively by an amiloride-sensitive Na(+)/H(+) exchange (NHE) protein, and apical Cl(-) uptake mediated by an electroneutral, SITS-sensitive Cl(-)/HCO(3-) anion-exchange protein. Using non-homologous antibodies, we have determined the cellular distributions of these ion-transport proteins to test the predicted models. Na(+)/K(+)-ATPase was used as a cellular marker for differentiating branchial epithelium mitochondria-rich (MR) cells from pavement cells. In both the freshwater tilapia (Oreochromis mossambicus) and rainbow trout (Oncorhynchus mykiss), V-ATPase and ENaC-like immunoreactivity co-localized to pavement cells, although apical labelling was also found in MR cells in the trout. In the freshwater tilapia, apical anion-exchanger-like immunoreactivity is found in the MR cells. Thus, a freshwater-type MR chloride cell exists in teleost fishes. The NHE-like immunoreactivity is associated with the accessory cell type and with a small population of pavement cells in tilapia.
Assuntos
Proteínas de Transporte/metabolismo , Oncorhynchus mykiss/metabolismo , Tilápia/metabolismo , ATPases Vacuolares Próton-Translocadoras , Animais , Antiporters/metabolismo , Antiportadores de Cloreto-Bicarbonato , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Brânquias/metabolismo , Brânquias/ultraestrutura , Imuno-Histoquímica , Transporte de Íons , Microscopia Imunoeletrônica , Modelos Biológicos , Oncorhynchus mykiss/anatomia & histologia , ATPases Translocadoras de Prótons/metabolismo , Canais de Sódio/metabolismo , Cloreto de Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Especificidade da Espécie , Tilápia/anatomia & histologiaRESUMO
The branchial epithelium of the mudskipper Periophthalmodon schlosseri is densely packed with mitochondria-rich (MR) cells. This species of mudskipper is also able to eliminate ammonia against large inward gradients and to tolerate extremely high environmental ammonia concentrations. To test whether these branchial MR cells are the sites of active ammonia elimination, we used an immunological approach to localize ion-transport proteins that have been shown pharmacologically to be involved in the elimination of NH(4)(+) (Na(+)/NH(4)(+) exchanger and Na(+)/NH(4)(+)-ATPase). We also investigated the role of carbonic anhydrase and boundary-layer pH effects in ammonia elimination by using the carbonic anhydrase inhibitor acetazolamide and by buffering the bath water with Hepes, respectively. In the branchial epithelium, Na(+)/H(+) exchangers (both NHE2- and NHE3-like isoforms), a cystic fibrosis transmembrane regulator (CFTR)-like anion channel, a vacuolar-type H(+)-ATPase (V-ATPase) and carbonic anhydrase immunoreactivity are associated with the apical crypt region of MR cells. Associated with the MR cell basolateral membrane and tubular system are the Na(+)/K(+)-ATPase and a Na(+)/K(+)/2Cl(-) cotransporter. A proportion of the ammonia eliminated by P. schlosseri involves carbonic anhydrase activity and is not dependent on boundary-layer pH effects. The apical CFTR-like anion channel may be serving as a HCO(3)(-) channel accounting for the acid-base neutral effects observed with net ammonia efflux inhibition.
Assuntos
Proteínas de Transporte/metabolismo , Brânquias/metabolismo , Perciformes/metabolismo , ATPases Vacuolares Próton-Translocadoras , Amônia/metabolismo , Animais , Anidrases Carbônicas/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Feminino , Brânquias/ultraestrutura , Imuno-Histoquímica , Transporte de Íons , Masculino , Microscopia Imunoeletrônica , Mitocôndrias/ultraestrutura , Modelos Biológicos , Perciformes/anatomia & histologia , ATPases Translocadoras de Prótons/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Simportadores de Cloreto de Sódio-Potássio , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
The transmembrane topology of Na(+)/H(+) exchanger NHE3 has been studied using in vitro transcription/translation of two types of fusion vectors designed to test membrane insertion properties of cDNA sequences encoding putative NHE3 membrane spanning domains (msds). These vectors encode N-terminal 101 (HKM0) or 139 (HKM1) amino acids of the H,K-ATPase alpha-subunit, a linker region and a reporter sequence containing five N-linked glycosylation consensus sites in the C-terminal 177 amino acids of the H,K-ATPase beta-subunit. The glycosylation status of the reporter sequence was used as a marker for the analysis of signal anchor and stop transfer properties of each putative msd in both the HKM0 and the HKM1 vectors. The linker region of the vectors was replaced by sequences that contain putative msds of NHE3 individually or in pairs. In vitro transcription/translation was performed using [(35)S]methionine in a reticulocyte lysate system +/- microsomes, and the translation products were identified by autoradiography following separation using SDS-PAGE. We propose a revised NHE3 topology model, which contains a cleaved signal peptide followed by 11 msds, including extracellular orientation of the N-terminus and intracellular orientation of the C-terminus. The presence of a cleavable signal peptide in NHE3 was demonstrated by its cleavage from NHE3 during translational processing of full-length and truncated NHE3 in the presence of microsomes. Of 11 putative msds, six (msds 1, 2, 4, 7, 10, and 11) acted as both signal anchor and stop transfer sequences, while five (msds 3, 5, 6, 8, and 9) had signal anchor activities when tested alone. Of the latter, 3, 5, 6, and 9 were shown to act as stop transfer sequences after C-terminal extension. The actual membrane orientation of each sequential transmembrane segment of NHE3 was deduced from the membrane location of the N- and C-termini of NHE3. The regions between putative msds 8 and 9 and between msds 10 and 11, which correspond to the fourth and fifth extracellular loops, did not act as msds when tested alone. However, the extension of the fifth extracellular loop with adjacent putative msds showed some membrane-associated properties suggesting that the fifth extracellular loop might be acting as a "P-loop"-like structure.