RESUMO
Carbonic anhydrase XIV (Car14) is highly expressed in the hepatocyte, with predominance in the canalicular membrane and its active site in the extracellular milieu. The aim of this study is to determine the physiological relevance of Car14 for biliary fluid and acid/base output, as well as its role in the maintenance of hepatocellular and cholangiocyte integrity. The common bile duct of anesthetized car14-/- and car14+/+ mice was cannulated and hepatic HCO3- output was measured by microtitration and bile flow gravimetrically before and during stimulation with intravenously applied tauroursodeoxycholic acid (TUDCA). Morphological alterations and hepatic damage were assessed histologically and immunohistochemically in liver tissue from 3- to 52-week-old car14-/- and car14+/+ mice, and gene and/or protein expression was measured for pro-inflammatory cytokines, fibrosis, and cholangiocyte markers. Biliary basal and more so TUDCA-stimulated HCO3- output were significantly reduced in car14-/- mice of all age groups, whereas bile flow and hepatic and ductular morphology were normal at young age. Car14-/- mice developed fibrotic and proliferative changes in the small bile ducts at advanced age, which was accompanied by a reduction in bile flow, and an upregulation of hepatic cytokeratin 19 mRNA and protein expression. Membrane-bound Car14 is essential for biliary HCO3- output, and its loss results in gradual development of small bile duct disease and hepatic fibrosis. Bile flow is not compromised in young adulthood, suggesting that Car14-deficient mice may be a model to study the protective role of biliary canalicular HCO3- against luminal noxi to the cholangiocyte.
Assuntos
Bicarbonatos , Ductos Biliares , Animais , Bicarbonatos/metabolismo , Ductos Biliares/metabolismo , Anidrases Carbônicas , Proliferação de Células , Fígado/metabolismo , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , CamundongosRESUMO
Members of the solute carrier 26 (SLC26) family have emerged as important players in mediating anions fluxes across the plasma membrane of epithelial cells, in cooperation with the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Among them, SLC26A3 acts as a chloride/bicarbonate exchanger, highly expressed in the gastrointestinal, pancreatic and renal tissues. In humans, mutations in the SLC26A3 gene were shown to induce congenital chloride-losing diarrhea (CLD), a rare autosomal recessive disorder characterized by life-long secretory diarrhea. In view of some reports indicating subfertility in some male CLD patients together with SLC26-A3 and -A6 expression in the male genital tract and sperm cells, we analyzed the male reproductive parameters and functions of SLC26A3 deficient mice, which were previously reported to display CLD gastro-intestinal features. We show that in contrast to Slc26a6, deletion of Slc26a3 is associated with severe lesions and abnormal cytoarchitecture of the epididymis, together with sperm quantitative, morphological and functional defects, which altogether compromised male fertility. Overall, our work provides new insight into the pathophysiological mechanisms that may alter the reproductive functions and lead to male subfertility in CLD patients, with a phenotype reminiscent of that induced by CFTR deficiency in the male genital tract.
Assuntos
Antiporters/metabolismo , Epididimo/metabolismo , Epididimo/fisiopatologia , Fertilização , Infertilidade Masculina/metabolismo , Capacitação Espermática , Transportadores de Sulfato/metabolismo , Animais , Antiporters/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Diarreia/congênito , Diarreia/etiologia , Masculino , Erros Inatos do Metabolismo/etiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Fenótipo , Contagem de Espermatozoides , Motilidade dos Espermatozoides , Espermatozoides/patologia , Transportadores de Sulfato/genética , Testículo/fisiopatologiaRESUMO
Following superficial injury, neighbouring gastric epithelial cells close the wound by rapid cell migration, a process called epithelial restitution. Na+ /H+ exchange (NHE) inhibitors interfere with restitution, but the role of the different NHE isoforms expressed in gastric pit cells has remained elusive. The role of the basolaterally expressed NHE1 (Slc9a1) and the presumably apically expressed NHE2 (Slc9a2) in epithelial restitution was investigated in the nontransformed rat gastric surface cell line RGM1. Migration velocity was assessed by loading the cells with the fluorescent dye DiR and following closure of an experimental wound over time. Since RGM1 cells expressed very low NHE2 mRNA and have low transport activity, NHE2 was introduced by lentiviral gene transfer. In medium with pH 7.4, RGM1 cells displayed slow wound healing even in the absence of growth factors and independently of NHE activity. Growth factors accelerated wound healing in a partly NHE1-dependent fashion. Preincubation with acidic pH 7.1 stimulated restitution in a NHE1-dependent fashion. When pH 7.1 was maintained during the restitution period, migratory speed was reduced to â¼10% of the speed at pH 7,4, and the residual restitution was further inhibited by NHE1 inhibition. Lentiviral NHE2 expression increased the steady-state pHi and reduced the restitution velocity after low pH preincubation, which was reversible by pharmacological NHE2 inhibition. The results demonstrate that in RGM1 cells, migratory velocity is increased by NHE1 activation, while NHE2 activity inhibit this process. A differential activation of NHE1 and NHE2 may therefore, play a role in the initiation and completion of the epithelial restitution process.
Assuntos
Movimento Celular , Mucosa Gástrica/citologia , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Linhagem Celular , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Lentivirus/metabolismo , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/genética , CicatrizaçãoRESUMO
A detrimental perceptive consequence of damaged auditory sensory hair cells consists in a pronounced masking effect exerted by low-frequency sounds, thought to occur when auditory threshold elevation substantially exceeds 40 dB. Here, we identified the submembrane scaffold protein Nherf1 as a hair-bundle component of the differentiating outer hair cells (OHCs). Nherf1(-/-) mice displayed OHC hair-bundle shape anomalies in the mid and basal cochlea, normally tuned to mid- and high-frequency tones, and mild (22-35 dB) hearing-threshold elevations restricted to midhigh sound frequencies. This mild decrease in hearing sensitivity was, however, discordant with almost nonresponding OHCs at the cochlear base as assessed by distortion-product otoacoustic emissions and cochlear microphonic potentials. Moreover, unlike wild-type mice, responses of Nherf1(-/-) mice to high-frequency (20-40 kHz) test tones were not masked by tones of neighboring frequencies. Instead, efficient maskers were characterized by their frequencies up to two octaves below the probe-tone frequency, unusually low intensities up to 25 dB below probe-tone level, and growth-of-masker slope (2.2 dB/dB) reflecting their compressive amplification. Together, these properties do not fit the current acknowledged features of a hypersensitivity of the basal cochlea to lower frequencies, but rather suggest a previously unidentified mechanism. Low-frequency maskers, we propose, may interact within the unaffected cochlear apical region with midhigh frequency sounds propagated there via a mode possibly using the persistent contact of misshaped OHC hair bundles with the tectorial membrane. Our findings thus reveal a source of misleading interpretations of hearing thresholds and of hypervulnerability to low-frequency sound interference.
Assuntos
Percepção Auditiva/fisiologia , Células Ciliadas Auditivas Externas/metabolismo , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Som , Animais , Células Ciliadas Auditivas Externas/citologia , Camundongos , Camundongos Knockout , Fosfoproteínas/genética , Trocadores de Sódio-Hidrogênio/genéticaRESUMO
The electrogenic Na(+)HCO3 (-) cotransporter NBCe1 (Slc4a4) is strongly expressed in the basolateral enterocyte membrane in a villous/surface predominant fashion. In order to better understand its physiological function in the intestine, isolated mucosae in miniaturized Ussing chambers and microdissected intestinal villi or crypts loaded with the fluorescent pH-indicator BCECF were studied from the duodenum, jejunum, and colon of 14- to 17-days-old slc4a4-deficient (KO) and WT mice. NBCe1 was active in the basal state in all intestinal segments under study, most likely to compensate for acid loads imposed upon the enterocytes. Upregulation of other basolateral base uptake mechanism occurs, but in a segment-specific fashion. Loss of NBCe1 resulted in severely impaired Cl(-) and fluid secretory response, but not HCO3 (-) secretory response to agonist stimulation. In addition, NBCe1 was found to be active during transport processes that load the surface enterocytes with acid, such as Slc26a3 (DRA)-mediated luminal Cl(-)/HCO3 (-) exchange or PEPT1-mediated H(+)/dipeptide uptake. Possibly because of the high energy demand for hyperventilation in conjunction with the fluid secretory and nutrient absorptive defects and the relative scarcity of compensatory mechanisms, NBCe1-deficient mice developed progressive jejunal failure, worsening of metabolic acidosis, and death in the third week of life. Our data suggest that the electrogenic influx of base via NBCe1 maintains enterocyte anion homeostasis and pHi control. Its loss impairs small intestinal Cl(-) and fluid secretion as well as the neutralization of acid loads imposed on the enterocytes during nutrient and electrolyte absorption.
Assuntos
Ânions/metabolismo , Dipeptídeos/metabolismo , Absorção Intestinal/fisiologia , Intestino Delgado/metabolismo , Simportadores de Sódio-Bicarbonato/deficiência , Simportadores de Sódio-Bicarbonato/metabolismo , Acidose/metabolismo , Animais , Bicarbonatos/metabolismo , Transporte Biológico/fisiologia , Cloretos/metabolismo , Eletrólitos/metabolismo , Enterócitos/metabolismo , Concentração de Íons de Hidrogênio , Mucosa Intestinal/metabolismo , CamundongosRESUMO
Slc26a9 is an anion transporter that is strongly expressed in the stomach and lung. Slc26a9 variants were recently found associated with a higher incidence of meconium ileus in cystic fibrosis (CF) infants, raising the question whether Slc26a9 is expressed in the intestine and what its functional role is. Slc26a9 messenger RNA (mRNA) was found highly expressed in the mucosae of the murine and human upper gastrointestinal tract, with an abrupt decrease in expression levels beyond the duodenum. Absence of SLC26a9 expression strongly increased the intestinally related mortality in cystic fibrosis transmembrane conductance regulator (CFTR)-deficient mice. Proximal duodenal JHCO3(-) and fluid secretion were reduced in the absence of Slc26a9 expression. In the proximal duodenum of young Slc26a9 KO mice, the glands and villi/crypts were elongated and proliferation was enhanced. This difference was lost with ageing, as were the alterations in fluid movement, whereas the reduction in JHCO3(-) remained. Laser dissection followed by qPCR suggested Slc26a9 expression to be crypt-predominant in the duodenum. In summary, deletion of Slc26a9 caused bicarbonate secretory and fluid absorptive changes in the proximal duodenal mucosa and increased the postweaning death rates in CFTR-deficient mice. Functional alterations in the duodenum were most prominent at young ages. We assume that the association of meconium ileus and Slc26a9 variants may be related to maldigestion and impaired downstream signaling caused by loss of upper GI tract digestive functions, aggravating the situation of lack of secretion and sticky mucus at the site of obstruction in CF intestine.
Assuntos
Antiporters/genética , Bicarbonatos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/metabolismo , Duodeno/metabolismo , Absorção Intestinal , Animais , Antiporters/metabolismo , Proliferação de Células , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Duodeno/crescimento & desenvolvimento , Duodeno/patologia , Humanos , Mucosa Intestinal/metabolismo , Transporte de Íons , Camundongos , Camundongos Endogâmicos C57BL , Transportadores de SulfatoRESUMO
A dysfunction of the Na(+)/H(+) exchanger isoform 3 (NHE3) significantly contributes to the reduced salt absorptive capacity of the inflamed intestine. We previously reported a strong decrease in the NHERF family member PDZK1 (NHERF3), which binds to NHE3 and regulates its function in a mouse model of colitis. The present study investigates whether a causal relationship exists between the decreased PDZK1 expression and the NHE3 dysfunction in human and murine intestinal inflammation. Biopsies from the colon of patients with ulcerative colitis, murine inflamed ileal and colonic mucosa, NHE3-transfected Caco-2BBe colonic cells with short hairpin RNA (shRNA) knockdown of PDZK1, and Pdzk1-gene-deleted mice were studied. PDZK1 mRNA and protein expression was strongly decreased in inflamed human and murine intestinal tissue as compared to inactive disease or control tissue, whereas that of NHE3 or NHERF1 was not. Inflamed human and murine intestinal tissues displayed correct brush border localization of NHE3 but reduced acid-activated NHE3 transport activity. A similar NHE3 transport defect was observed when PDZK1 protein content was decreased by shRNA knockdown in Caco-2BBe cells or when enterocyte PDZK1 protein content was decreased to similar levels as found in inflamed mucosa by heterozygote breeding of Pdzk1-gene-deleted and WT mice. We conclude that a decrease in PDZK1 expression, whether induced by inflammation, shRNA-mediated knockdown, or heterozygous breeding, is associated with a decreased NHE3 transport rate in human and murine enterocytes. We therefore hypothesize that inflammation-induced loss of PDZK1 expression may contribute to the NHE3 dysfunction observed in the inflamed intestine.
Assuntos
Proteínas de Transporte/metabolismo , Colite/metabolismo , Colo/metabolismo , Enterócitos/metabolismo , Ileíte/metabolismo , Íleo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Biópsia , Células CACO-2 , Proteínas de Transporte/genética , Colite/induzido quimicamente , Colite/genética , Colite/patologia , Colo/patologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Sulfato de Dextrana , Modelos Animais de Doenças , Regulação para Baixo , Enterócitos/patologia , Humanos , Ileíte/induzido quimicamente , Ileíte/genética , Ileíte/patologia , Íleo/patologia , Mediadores da Inflamação/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana , Camundongos da Linhagem 129 , Camundongos Knockout , Microvilosidades/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , Estudos Retrospectivos , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/genética , Transfecção , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Na(+)/H(+) exchanger regulatory factor 3 (NHERF3) is a PSD-95/discs large/ZO-1 (PDZ)-based adaptor protein that regulates several membrane-transporting proteins in epithelia. However, the in vivo physiologic role of NHERF3 in transepithelial transport remains poorly understood. Multidrug resistance protein 4 (MRP4) is an ATP binding cassette transporter that mediates the efflux of organic molecules, such as nucleoside analogs, in the gastrointestinal and renal epithelia. Here, we report that Nherf3 knockout (Nherf3(-/-)) mice exhibit profound reductions in Mrp4 expression and Mrp4-mediated drug transport in the kidney. A search for the binding partners of the COOH-terminal PDZ binding motif of MRP4 among several epithelial PDZ proteins indicated that MRP4 associated most strongly with NHERF3. When expressed in HEK293 cells, NHERF3 increased membrane expression of MRP4 by reducing internalization of cell surface MRP4 and consequently, augmented MRP4-mediated efflux of adefovir, a nucleoside-based antiviral agent and well known substrate of MRP4. Examination of wild-type and Nherf3(-/-) mice revealed that Nherf3 is most abundantly expressed in the kidney and has a prominent role in modulating Mrp4 levels. Deletion of Nherf3 in mice caused a profound reduction in Mrp4 expression at the apical membrane of renal proximal tubules and evoked a significant increase in the plasma and kidney concentrations of adefovir, with a corresponding decrease in the systemic clearance of this drug. These results suggest that NHERF3 is a key regulator of organic transport in the kidney, particularly MRP4-mediated clearance of drug molecules.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas de Transporte/fisiologia , Rim/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Adenina/análogos & derivados , Adenina/farmacocinética , Animais , Células HEK293 , Humanos , Proteínas de Membrana , Camundongos , Organofosfonatos/farmacocinética , Regulação para CimaRESUMO
The mixing of gastric and pancreatic juice subjects the jejunum to unique ionic conditions with high luminal CO2 tension and HCO3 − concentration. We investigated the role of the small intestinal apical anion exchangers PAT-1 (Slc26a6) and DRA (Slc26a3) in basal and CO2/HCO3 −-stimulated jejunal fluid absorption. Single pass perfusion of jejunal segments was performed in anaesthetised wild type (WT) as well as in mice deficient in DRA, PAT-1, Na+/H+ exchanger 3 (NHE3) or NHE2, and in carbonic anhydrase II (CAII). Unbuffered saline (pH 7.4) perfusion of WT jejunum resulted in fluid absorption and acidification of the effluent. DRA-deficient jejunum absorbed less fluid than WT, and acidified the effluent more strongly, consistent with its action as a Cl−/HCO3 − exchanger. PAT-1-deficient jejunum also absorbed less fluid but resulted in less effluent acidification. Switching the luminal solution to a 5 % CO2/HCO3 − buffered solution (pH 7.4), resulted in a decrease in jejunal enterocyte pHi in all genotypes, an increase in luminal surface pH and a strong increase in fluid absorption in a PAT-1- and NHE3- but not DRA-, CAII, or NHE2-dependent fashion. Even in the absence of luminal Cl−, luminal CO2/HCO3 − augmented fluid absorption in WT, CAII, NHE2- or DRA-deficient, but not in PAT-1- or NHE3-deficient mice, indicating the likelihood that PAT-1 serves to import HCO3 − and NHE3 serves to import Na+ under these circumstances. The results suggest that PAT-1 plays an important role in jejunal Na+HCO3 reabsorption, while DRA absorbs Cl− and exports HCO3 − in a partly CAII-dependent fashion. Both PAT-1 and DRA significantly contribute to intestinal fluid absorption and enterocyte acid/base balance but are activated by different ion gradients.
Assuntos
Antiporters/metabolismo , Absorção Intestinal/fisiologia , Equilíbrio Hidroeletrolítico/fisiologia , Animais , Concentração de Íons de Hidrogênio , Imuno-Histoquímica , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Reação em Cadeia da Polimerase , Transportadores de SulfatoRESUMO
Pseudomonas aeruginosa infections of the airway cells decrease apical expression of both wild-type (wt) and F508del CFTR through the inhibition of apical endocytic recycling. CFTR endocytic recycling is known to be regulated by its interaction with PDZ domain containing proteins. Recent work has shown that the PDZ domain scaffolding protein NHERF1 finely regulates both wt and F508delCFTR membrane recycling. Here, we investigated the effect of P. aeruginosa infection on NHERF1 post-translational modifications and how this affects CFTR expression in bronchial epithelial cells and in murine lung. Both in vitro in bronchial cells, and in vivo in mice, infection reduced CFTR expression and increased NHERF1 molecular weight through its hyper-phosphorylation and ubquitination as a consequence of both bacterial pilin- and flagellin-mediated host-cell interaction. The ability of P. aeruginosa to down-regulate mature CFTR expression was reduced both in vivo in NHERF1 knockout mice and in vitro after silencing NHERF1 expression or mutations blocking its phosphorylation at serines 279 and 301. These studies provide the first evidence that NHERF1 phosphorylation may negatively regulate its action and, therefore, the assembly and function of multiprotein NHERF1 complexes in response to infection. The identification of molecular mechanisms responsible for these effects could identify novel targets to block potential P. aeruginosa interference with the efficacy of potentiator and/or corrector compounds.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fosfoproteínas/metabolismo , Processamento de Proteína Pós-Traducional , Infecções por Pseudomonas/metabolismo , Mucosa Respiratória/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Brônquios/citologia , Brônquios/metabolismo , Brônquios/microbiologia , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Pulmão/citologia , Pulmão/metabolismo , Pulmão/microbiologia , Camundongos , Mutação , Fosfoproteínas/genética , Fosforilação , Pseudomonas aeruginosa , Mucosa Respiratória/microbiologia , Trocadores de Sódio-Hidrogênio/genética , UbiquitinaçãoRESUMO
OBJECTIVES: A common potentially fatal disease of the pancreas is acute pancreatitis, for which there is no treatment. Most studies of this disorder focus on the damage to acinar cells since they are assumed to be the primary target of multiple stressors affecting the pancreas. However, increasing evidence suggests that the ducts may also have a crucial role in induction of the disease. To test this hypothesis, we sought to determine the specific role of the duct in the induction of acute pancreatitis using well-established disease models and mice with deletion of the Na/H exchanger regulatory factor-1 that have selectively impaired ductal function. DESIGN: Randomized animal study. SETTING: Animal research laboratory. SUBJECTS: Wild-type and Na/H exchanger regulatory factor-1 knockout mice. INTERVENTIONS: Acute necrotizing pancreatitis was induced by i.p. administration of cerulein or by intraductal administration of sodium taurocholate. The pancreatic expression of Na/H exchanger regulatory factor-1 and cystic fibrosis transmembrane conductance regulator (a key player in the control of ductal secretion) was analyzed by immunohistochemistry. In vivo pancreatic ductal secretion was studied in anesthetized mice. Functions of pancreatic acinar and ductal cells as well as inflammatory cells were analyzed in vitro. MEASUREMENTS AND MAIN RESULTS: Deletion of Na/H exchanger regulatory factor-1 resulted in gross mislocalization of cystic fibrosis transmembrane conductance regulator, causing marked reduction in pancreatic ductal fluid and bicarbonate secretion. Importantly, deletion of Na/H exchanger regulatory factor-1 had no deleterious effect on functions of acinar and inflammatory cells. Deletion of Na/H exchanger regulatory factor-1, which specifically impaired ductal function, increased the severity of acute pancreatitis in the two mouse models tested. CONCLUSIONS: Our findings provide the first direct evidence for the crucial role of ductal secretion in protecting the pancreas from acute pancreatitis and strongly suggest that improved ductal function should be an important modality in prevention and treatment of the disease.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Ductos Pancreáticos/metabolismo , Pancreatite Necrosante Aguda/metabolismo , Pancreatite Necrosante Aguda/patologia , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Animais , Biomarcadores/metabolismo , Distribuição de Qui-Quadrado , Modelos Animais de Doenças , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Pâncreas/metabolismo , Pâncreas/fisiologia , RNA Mensageiro/metabolismo , Distribuição Aleatória , Valores de Referência , Regeneração/fisiologia , Sensibilidade e Especificidade , Simportadores/metabolismoRESUMO
AIM: Trafficking, membrane retention, and signal-specific regulation of the Na+/H+ exchanger 3 (NHE3) are modulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adapter proteins. This study explored the assembly of NHE3 and NHERF2 with the cGMP-dependent kinase II (cGKII) within detergent-resistant membrane microdomains (DRMs, "lipid rafts") during in vivo guanylate cycle C receptor (Gucy2c) activation in murine small intestine. METHODS: Small intestinal brush border membranes (siBBMs) were isolated from wild type, NHE3-deficient, cGMP-kinase II-deficient, and NHERF2-deficient mice, after oral application of the heat-stable Escherichia coli toxin (STa) analog linaclotide. Lipid raft and non-raft fractions were separated by Optiprep density gradient centrifugation of Triton X-solubilized siBBMs. Confocal microscopy was performed to study NHE3 redistribution after linaclotide application in vivo. RESULTS: In the WT siBBM, NHE3, NHERF2, and cGKII were strongly raft associated. The raft association of NHE3, but not of cGKII, was NHERF2 dependent. After linaclotide application to WT mice, lipid raft association of NHE3 decreased, that of cGKII increased, while that of NHERF2 did not change. NHE3 expression in the BBM shifted from a microvillar to a terminal web region. The linaclotide-induced decrease in NHE3 raft association and in microvillar abundance was abolished in cGKII-deficient mice, and strongly reduced in NHERF2-deficient mice. CONCLUSION: NHE3, cGKII, and NHERF2 form a lipid raft-associated signal complex in the siBBM, which mediates the inhibition of salt and water absorption by Gucy2c activation. NHERF2 enhances the raft association of NHE3, which is essential for its close interaction with the exclusively raft-associated activated cGKII.
Assuntos
Microdomínios da Membrana , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio , Animais , Camundongos , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Intestino Delgado/metabolismo , Microdomínios da Membrana/metabolismo , Microvilosidades/metabolismo , Trocador 3 de Sódio-Hidrogênio/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Proteína Quinase Dependente de GMP Cíclico Tipo II/metabolismoRESUMO
The duodenal villus brush border membrane expresses several ion transporters and/or channels, including the solute carrier 26 anion transporters Slc26a3 (DRA) and Slc26a6 (PAT-1), the Na(+)/H(+) exchanger isoform 3 (NHE3), as well as the anion channels cystic fibrosis transmembrane conductance regulator (CFTR) and Slc26a9. Using genetically engineered mouse models lacking Scl26a3, Slc26a6, Slc26a9 or Slc9a3 (NHE3), the study was carried out to assess the role of these transporters in mediating the protective duodenal bicarbonate secretory response (DBS-R) to luminal acid; and to compare it to their role in DBS-R elicited by the adenylyl cyclase agonist forskolin. While basal DBS was reduced in the absence of any of the three Slc26 isoforms, the DBS-R to forskolin was not altered. In contrast, the DBS-R to a 5 min exposure to luminal acid (pH 2.5) was strongly reduced in the absence of Slc26a3 or Slc26a9, but not Slc26a6. CFTR inhibitor [CFTR(Inh)-172] reduced the first phase of the acid-induced DBS-R, while NHE3 inhibition (or knockout) abolished the sustained phase of the DBS-R. Luminal acid exposure resulted in the activation of multiple intracellular signalling pathways, including SPAK, AKT and p38 phosphorylation. It induced a biphasic trafficking of NHE3, first rapidly into the brush border membrane, followed by endocytosis in the later stage. We conclude that the long-lasting DBS-R to luminal acid exposure activates multiple duodenocyte signalling pathways and involves changes in trafficking and/or activity of CFTR, Slc26 isoforms Slc26a3 and Slc26a9, and NHE3.
Assuntos
Antiporters/metabolismo , Bicarbonatos/metabolismo , Duodeno/metabolismo , Transporte de Íons , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Antiporters/genética , Benzoatos/farmacologia , Colforsina/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Endocitose , Concentração de Íons de Hidrogênio , Mucosa Intestinal/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Fosforilação , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Trocadores de Sódio-Hidrogênio/genética , Transportadores de Sulfato , Tiazolidinas/farmacologiaRESUMO
Duodenal epithelial cells need efficient defence strategies during gastric acidification of the lumen, while colonic mucosa counteracts damage by pathogens by building up a bacteria-free adherent mucus layer. Transport of HCO3(-) is considered crucial for duodenal defence against acid as well as for mucus release and expansion, but the transport pathways involved are incompletely understood. This study investigated the significance of the electroneutral Na(+)-HCO3(-) cotransporter NBCn1 for duodenal defence against acid and colonic mucus release. NBCn1 was localized to the basolateral membrane of duodenal villous enterocytes and of colonic crypt cells, with predominant expression in goblet cells. Duodenal villous enterocyte intracellular pH was studied before and during a luminal acid load by two-photon microscopy in exteriorized, vascularly perfused, indicator (SNARF-1 AM)-loaded duodenum of isoflurane-anaesthetized, systemic acid-base-controlled mice. Acid-induced HCO3(-) secretion was measured in vivo by single-pass perfusion and pH-stat titration. After a luminal acid load, NBCn1-deficient duodenocytes were unable to recover rapidly from intracellular acidification and could not respond adequately with protective HCO3(-) secretion. In the colon, build-up of the mucus layer was delayed, and a decreased thickness of the adherent mucus layer was observed, suggesting that basolateral HCO3(-) uptake is essential for optimal release of mucus. The electroneutral Na(+)-HCO3(-) cotransporter NBCn1 displays a differential cellular distribution in the murine intestine and is essential for HCO3(-)-dependent mucosal protective functions, such as recovery of intracellular pH and HCO3(-) secretion in the duodenum and secretion of mucus in the colon.
Assuntos
Colo/metabolismo , Duodeno/metabolismo , Muco/metabolismo , Simportadores de Sódio-Bicarbonato/fisiologia , Equilíbrio Ácido-Base , Animais , Bicarbonatos/metabolismo , Feminino , Concentração de Íons de Hidrogênio , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos KnockoutRESUMO
BACKGROUND/AIMS: Trafficking, brush border membrane (BBM) retention, and signal-specific regulation of the Na+/H+ exchanger NHE3 is regulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adaptor proteins, which enable the formation of multiprotein complexes. It is unclear, however, what determines signal specificity of these NHERFs. Thus, we studied the association of NHE3, NHERF1 (EBP50), NHERF2 (E3KARP), and NHERF3 (PDZK1) with lipid rafts in murine small intestinal BBM. METHODS: Detergent resistant membranes ("lipid rafts") were isolated by floatation of Triton X-incubated small intestinal BBM from a variety of knockout mouse strains in an Optiprep step gradient. Acid-activated NHE3 activity was measured fluorometrically in BCECF-loaded microdissected villi, or by assessment of CO2/HCO3(-) mediated increase in fluid absorption in perfused jejunal loops of anethetized mice. RESULTS: NHE3 was found to partially associate with lipid rafts in the native BBM, and NHE3 raft association had an impact on NHE3 transport activity and regulation in vivo. NHERF1, 2 and 3 were differentially distributed to rafts and non-rafts, with NHERF2 being most raft-associated and NHERF3 entirely non-raft associated. NHERF2 expression enhanced the localization of NHE3 to membrane rafts. The use of acid sphingomyelinase-deficient mice, which have altered membrane lipid as well as lipid raft composition, allowed us to test the validity of the lipid raft concept in vivo. CONCLUSIONS: The differential association of the NHERFs with the raft-associated and the non-raft fraction of NHE3 in the brush border membrane is one component of the differential and signal-specific NHE3 regulation by the different NHERFs.
Assuntos
Intestino Delgado/citologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Microvilosidades/metabolismo , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Intestino Delgado/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Jejuno/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfoproteínas/genética , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/genética , Esfingomielina Fosfodiesterase/genéticaRESUMO
Duodenocyte pHi control and HCO3 − secretion protects the proximal duodenum against damage by gastric acid. The molecular details of duodenocyte pH control are not well understood. A selective duodenal expression (within the upper GI tract) has been reported for the electroneutral Na+:HCO3 − cotransporter NBCn1 (Slc4a7). We aimed to determine the role of NBCn1 and NBCe2 in duodenocyte intracellular pH regulation as well as basal and agonist-stimulated duodenal bicarbonate secretion (JHCO3 −), exploiting mouse models of genetic slc4a7 and slc4a5 disruption. Basal and forskolin (FSK)-stimulated JHCO3 − was measured by single-pass perfusion in the duodenum of slc4a7−/− and slc4a7+/+ as well as slc4a5−/− and slc4a5+/+ mice in vivo, and by pH-stat titration in isolated duodenal mucosa in vitro. Duodenocyte HCO3 − uptake rates were fluorometrically assessed after acidification of intact villi and of isolated duodenocytes. Slc4a7−/− mice displayed significantly lower basal and FSK-stimulated duodenal HCO3 − secretion than slc4a7+/+ littermates in vivo. FSK-stimulated HCO3 − secretion was significantly reduced in slc4a7−/− isolated duodenal mucosa. Na+- and HCO3 −-dependent base uptake rates were significantly decreased in slc4a7−/− compared with slc4a7+/+ villus duodenocytes when measured in intact villi. Carbonic anhydrase (CA)-mediated CO2 hydration played no apparent role as a HCO3 − supply mechanism for basal or FSK-stimulated secretion in the slc4a7+/+ duodenum, but was an important alternative HCO3 − supply mechanism in the slc4a7−/− duodenum. NBCe2 (Slc4a5) displayed markedly lower duodenal mRNA expression levels, and its disruption did not interfere with duodenal HCO3 − secretion. The electroneutral Na+:HCO3 − cotransporter NBCn1 (slc4a7) is a major duodenal HCO3 − importer that supplies HCO3 − during basal and FSK-stimulated HCO3 − secretion.
Assuntos
Bicarbonatos/metabolismo , Duodeno/metabolismo , Mucosa Intestinal/metabolismo , Simportadores de Sódio-Bicarbonato/metabolismo , Animais , Dióxido de Carbono/metabolismo , Anidrases Carbônicas/metabolismo , Colforsina/farmacologia , Concentração de Íons de Hidrogênio , Mucosa Intestinal/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/biossíntese , Sódio/metabolismo , Simportadores de Sódio-Bicarbonato/genéticaRESUMO
Rapid exchange of metabolites between different cell types is crucial for energy homeostasis of the brain. Besides glucose, lactate is a major metabolite in the brain and is primarily produced in astrocytes. In the present study, we report that carbonic anhydrase 2 (CAII) enhances both influx and efflux of lactate in mouse cerebellar astrocytes. The augmentation of lactate transport is independent of the enzyme's catalytic activity, but requires direct binding of CAII to the C-terminal of the monocarboxylate transporter MCT1, one of the major lactate/proton cotransporters in astrocytes and most tissues. By employing its intramolecular proton shuttle, CAII, bound to MCT1, can act as a 'proton collecting antenna' for the transporter, suppressing the formation of proton microdomains at the transporter-pore and thereby enhancing lactate flux. By this mechanism CAII could enhance transfer of lactate between astrocytes and neurons and thus provide the neurons with an increased supply of energy substrate.
Assuntos
Astrócitos/metabolismo , Anidrase Carbônica II/metabolismo , Cerebelo/metabolismo , Ácido Láctico/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Simportadores/metabolismo , Animais , Anidrase Carbônica II/deficiência , Anidrase Carbônica II/genética , Células Cultivadas , Feminino , Camundongos , Camundongos Knockout , Transportadores de Ácidos Monocarboxílicos/genética , Oócitos , RNA Interferente Pequeno/genética , Simportadores/genética , Xenopus laevisRESUMO
This study investigated whether expression of the common cystic fibrosis transmembrane conductance regulator (CFTR) mutant F508del in the apical membrane of enterocytes confers increased bicarbonate secretory capacity on the intestinal epithelium of F508del mutant mice compared to that of CFTR knockout (KO) mice. CFTR KO mice, F508del mutant mice (F508del) and wild-type (WT) littermates were bred on the FVB/N background. F508del isolated brush border membrane (BBM) contained approximately 5-10% fully glycosylated band C protein compared to WT BBM. Similarly, the forskolin (FSK)-induced, CFTR-dependent short-circuit current (I(sc)) of F508del mucosa was approximately 5-10% of WT, whereas the HCO(3)(-) secretory response ( ) was almost half that of WT in both duodenum and mid-colon studied in vitro and in vivo. While WT intestine retained full FSK-induced in the absence of luminal Cl(-), the markedly higher than I(sc) in F508del intestine was dependent on the presence of luminal Cl(-), and was blocked by CFTR inhibitors. The Ste20-related proline-alanine-rich kinases (SPAK/OSR1), which are downstream of the with-no-lysine (K) protein kinases (WNK), were rapidly phosphorylated by FSK in WT and F508del, but significantly more slowly in CFTR KO intestine. In conclusion, the data demonstrate that low levels of F508del membrane expression in the intestine of F508del mice significantly increased FSK-induced HCO(3)(-) secretion mediated by Cl(-)/HCO(3)(-) exchange. However, in WT mucosa FSK elicited strong SPAK/OSR1 phosphorylation and Cl(-)-independent HCO(3)(-) efflux. This suggests that therapeutic strategies which deliver F508del to the apical membrane have the potential to significantly enhance epithelial HCO(3)(-) secretion.
Assuntos
Bicarbonatos/metabolismo , Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Mucosa Intestinal/metabolismo , Animais , Membrana Celular/metabolismo , Colforsina/farmacologia , Colo/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Duodeno/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos CFTR , Microvilosidades/metabolismo , Mutação , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Kir4.1 channels were found to colocalize with the H(+)/K(+)-ATPase throughout the parietal cell (PC) acid secretory cycle. This study was undertaken to explore their functional role. Acid secretory rates, electrophysiological parameters, PC ultrastructure, and gene and protein expression were determined in gastric mucosae of 7-8-day-old Kir4.1-deficient mice and WT littermates. Kir4.1(-/-) mucosa secreted significantly more acid and initiated secretion significantly faster than WT mucosa. No change in PC number but a relative up-regulation of H(+)/K(+)-ATPase gene and protein expression (but not of other PC ion transporters) was observed. Electron microscopy revealed fully fused canalicular membranes and a lack of tubulovesicles in resting state Kir4.1(-/-) PCs, suggesting that Kir4.1 ablation may also interfere with tubulovesicle endocytosis. The role of this inward rectifier in the PC apical membrane may therefore be to balance between K(+) loss via KCNQ1/KCNE2 and K(+) reabsorption by the slow turnover of the H(+)/K(+)-ATPase, with consequences for K(+) reabsorption, inhibition of acid secretion, and membrane recycling. Our results demonstrate that Kir4.1 channels are involved in the control of acid secretion and suggest that they may also affect secretory membrane recycling.
Assuntos
Ácido Gástrico/metabolismo , Regulação da Expressão Gênica , Células Parietais Gástricas/citologia , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Animais , Transporte Biológico , Eletrofisiologia/métodos , Endocitose , Mucosa Gástrica/metabolismo , ATPase Trocadora de Hidrogênio-Potássio/química , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão/métodos , Microscopia de Fluorescência/métodos , Potássio/química , Canais de Potássio Corretores do Fluxo de Internalização/biossínteseRESUMO
The incidence of duodenal ulcer, especially Helicobacter pylori-negative duodenal ulcer, strongly increases with age. In humans, telomere length shortening is considered to be one critical factor in cellular senescence and organ survival. In this study, we compared basal and stimulated gastric acid and duodenal HCO(3)(-) secretory rates in aged late-generation (G(3)) telomerase-deficient (mTERC(-/-)) mice, which are characterized by severe telomere dysfunction due to the inability to elongate telomeres during cell division. We found that basal and forskolin-stimulated HCO(3)(-) secretion and short-circuit current (I(sc)) in isolated duodenal mucosa of G(3) mTERC(-/-) mice were markedly reduced compared with age-matched wild-type mice. In contrast, basal and forskolin-stimulated acid secretory rates in isolated G(3) mTERC(-/-) gastric mucosa were not significantly altered. Correspondingly, duodenal mucosa of G(3) mTERC(-/-) mice showed slimming and shortening of villi, whereas gastric mucosal histology was not significantly altered. However, the ratios of cystic fibrosis transmembrane conductance regulator (CFTR) and solute-linked carrier 26 gene family (Slc26a6) mRNA expression in relation to cytokeratin-18 were not altered in duodenal mucosa. The further knockout of p21, which is a downstream effector of telomere shortening-induced senescence, rescued villus atrophy of duodenal mucosa, and basal and forskolin-stimulated duodenal HCO(3)(-) secretion and I(sc) in mTERC(-/-) p21(-/-) double-knockout mice were not different from wild-type controls. In conclusion, genetic ablation of telomerase resulted in p21-dependent duodenal mucosal atrophy and reduced duodenal HCO(3)(-) secretory capacity, whereas gastric morphology and acid secretory function were preserved. This suggests that telomere shortening during aging may result in an imbalance between aggressive and protective secretions against duodenal mucosa and thus predispose to ulcer formation.