RESUMEN

Increased intestinal permeability is a manifestation of cystic fibrosis (CF) in people with CF (pwCF) and in CF mouse models. CF transmembrane conductance regulator knockout (Cftr KO) mouse intestine exhibits increased proliferation and Wnt/ß-catenin signaling relative to wild-type mice (WT). Since the Rho GTPase Cdc42 plays a central role in intestinal epithelial proliferation and tight junction remodeling, we hypothesized that Cdc42 may be altered in the Cftr KO crypts. Immunofluorescence showed distinct tight junction localization of Cdc42 in Cftr KO fresh crypts and enteroids, the latter indicating an epithelial-autonomous feature. Quantitative PCR and immunoblots revealed similar expression of Cdc42 in the Cftr KO crypts/enteroids relative to WT, whereas pulldown assays showed increased GTP-bound (active) Cdc42 in proportion to total Cdc42 in Cftr KO enteroids. Cdc42 activity in the Cftr KO and WT enteroids could be reduced by inhibition of the Wnt transducer Disheveled. With the use of a dye permeability assay, Cftr KO enteroids exhibited increased paracellular permeability to 3 kDa dextran relative to WT. Leak permeability and Cdc42 tight junction localization were reduced to a greater extent by inhibition of Wnt/ß-catenin signaling with endo-IWR1 in Cftr KO relative to WT enteroids. Increased proliferation or inhibition of Cdc42 activity with ML141 in WT enteroids had no effect on permeability. In contrast, inhibition of Cdc42 with ML141 increased permeability to both 3 kDa dextran and tight junction impermeant 500 kDa dextran in Cftr KO enteroids. These data suggest that increased constitutive Cdc42 activity may alter the stability of paracellular permeability in Cftr KO crypt epithelium.NEW & NOTEWORTHY Increased tight junction localization and GTP-bound activity of the Rho GTPase Cdc42 was identified in small intestinal crypts and enteroids of cystic fibrosis (CF) transmembrane conductance regulator knockout (Cftr KO) mice. The increase in epithelial Cdc42 activity was associated with increased Wnt signaling. Paracellular flux of an uncharged solute (3 kDa dextran) in Cftr KO enteroids indicated a moderate leak permeability under basal conditions that was strongly exacerbated by Cdc42 inhibition. These findings suggest increased activity of Cdc42 in the Cftr KO intestine underlies alterations in intestinal permeability.

Asunto(s)

Regulador de Conductancia de Transmembrana de Fibrosis Quística , Mucosa Intestinal , Uniones Estrechas , Proteína de Unión al GTP cdc42 , Animales , Ratones , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP cdc42/genética , Fibrosis Quística/metabolismo , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células Epiteliales/metabolismo , Mucosa Intestinal/metabolismo , Ratones Noqueados , Permeabilidad , Uniones Estrechas/metabolismo , Vía de Señalización Wnt

RESUMEN

Nitrogen fertilization is vital for productive agriculture and efficient land use. However, globally, approximately 50% of the nitrogen applied is lost to the environment, causing inefficiencies, pollution, and greenhouse gas emissions. Rainfall and its effect on soil moisture are the major components controlling nitrogen losses in agriculture. Thus, changing rainfall patterns could accelerate nitrogen inefficiencies. We used a mechanistic modeling platform to determine how precipitation-optimal nitrogen fertilization timings and resulting crop nitrogen uptake have changed historically (1950-2020) and how they are predicted to change under the RCP8.5 climate scenario (2021-2069) in the South East of England. We found that historically, neither precipitation-optimal fertilization timings nor resulting plant uptake changed significantly. However, there were large year-to-year variations in both. In the 2030s, where it is projected to get wetter, precipitation-optimal fertilization timings are predicted to be later in the season and the resulting plant uptake noticeably lower. After 2040, the precipitation-optimal uptakes are projected to increase with earlier precipitation-optimal timings closer to historical values, corresponding to the projected mean daily rainfall rates decreasing to the historical values in these growing seasons. It seems that the interannual variation in precipitation-optimal uptake is projected to increase. Ultimately, projected changes in precipitation patterns will affect nitrogen uptake and precipitation-optimal fertilization timings. We argue that the use of bespoke fertilization timings in each year can help recuperate the reduced N uptake due to changing precipitation.

RESUMEN

Goblet cell hyperplasia is an important manifestation of cystic fibrosis (CF) disease in epithelial-lined organs. Explants of CF airway epithelium show normalization of goblet cell numbers; therefore, we hypothesized that small intestinal enteroids from Cftr knockout (KO) mice would not exhibit goblet cell hyperplasia. Toll-like receptors 2 and 4 (Tlr2 and Tlr4) were investigated as markers of inflammation and influence on goblet cell differentiation. Ex vivo studies found goblet cell hyperplasia in Cftr KO jejunum compared with wild-type (WT) mice. IL-13, SAM pointed domain-containing ETS transcription factor (Spdef), Tlr2, and Tlr4 protein expression were increased in Cftr KO intestine relative to WT. In contrast, WT and Cftr KO enteroids did not exhibit differences in basal or IL-13-stimulated goblet cell numbers, or differences in expression of Tlr2, Tlr4, and Spdef. Ileal goblet cell numbers in Cftr KO/Tlr4 KO and Cftr KO/Tlr2 KO mice were not different from Cftr KO mice, but enumeration was confounded by altered mucosal morphology. Treatment with Tlr4 agonist LPS did not affect goblet cell numbers in WT or Cftr KO enteroids, whereas the Tlr2 agonist Pam3Csk4 stimulated goblet cell hyperplasia in both genotypes. Pam3Csk4 stimulation of goblet cell numbers was associated with suppression of Notch1 and Neurog3 expression and upregulated determinants of goblet cell differentiation. We conclude that goblet cell hyperplasia and inflammation of the Cftr KO small intestine are not exhibited by enteroids, indicating that this manifestation of CF intestinal disease is not epithelial-automatous but secondary to the altered CF intestinal environment.NEW & NOTEWORTHY Studies of small intestinal organoids from cystic fibrosis (CF) mice show that goblet cell hyperplasia and increased Toll-like receptor 2/4 expression are not primary manifestations of the CF intestine. Intestinal goblet cell hyperplasia in the CF mice was not strongly altered by genetic ablation of Tlr2 and Tlr 4, but could be induced in both wild-type and CF intestinal organoids by a Tlr2-dependent suppression of Notch signaling.

Asunto(s)

Células Caliciformes/metabolismo , Hiperplasia/metabolismo , Inflamación/metabolismo , Intestinos/metabolismo , Animales , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Ratones Noqueados , Organoides/metabolismo , Transducción de Señal/fisiología

RESUMEN

Small-molecule modulators of cystic fibrosis transmembrane conductance regulator (CFTR) biology show promise in the treatment of cystic fibrosis (CF). A Cftr knockout (Cftr KO) mouse expressing mutants of human CFTR would advance in vivo testing of new modulators. A bacterial artificial chromosome (BAC) carrying the complete hCFTR gene including regulatory elements within 40.1 kb of DNA 5' and 25 kb of DNA 3' to the gene was used to generate founder mice expressing hCFTR. Whole genome sequencing indicated a single integration site on mouse chromosome 8 (8qB2) with ~6 gene copies. hCFTR+ offspring were bred to murine Cftr KO mice, producing hCFTR+/mCftr- (H+/m-) mice, which had normal survival, growth and goblet cell function as compared to wild-type (WT) mice. Expression studies showed hCFTR protein and transcripts in tissues typically expressing mCftr. Functionally, nasal potential difference and large intestinal short-circuit (Isc) responses to cAMP stimulation were similar in magnitude to WT mice, whereas small intestinal cAMP ΔIsc responses were reduced. A BAC transgenic mouse with functional hCFTR under control of its regulatory elements has been developed to enable the generation of mouse models of hCFTR mutations by gene editing for in vivo testing of new CF therapies.

Asunto(s)

Cromosomas Artificiales Bacterianos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Secuencias Reguladoras de Ácidos Nucleicos , Transgenes , Animales , Exocitosis , Edición Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos

RESUMEN

BACKGROUND & AIMS: Cystic fibrosis (CF) patients and CF mouse models have increased risk for gastrointestinal tumors. CF mice show augmented intestinal proliferation of unknown etiology and an altered intestinal environment. We examined the role of the cystic fibrosis transmembrane conductance regulator (Cftr) in Wnt/ß-catenin signaling, stem cell proliferation, and its functional expression in the active intestinal stem cell (ISC) population. Dysregulation of intracellular pH (pHi) in CF ISCs was investigated for facilitation of Wnt/ß-catenin signaling. METHODS: Crypt epithelia from wild-type (WT) and CF mice were compared ex vivo and in intestinal organoids (enteroids) for proliferation and Wnt/ß-catenin signaling by standard assays. Cftr in ISCs was assessed by immunoblot of sorted Sox9 enhanced green fluorescent protein(EGFP) intestinal epithelia and pHi regulation by confocal microfluorimetry of leucine-rich G-protein-coupled receptor 5 ISCs. Plasma membrane association of the Wnt transducer Dishevelled 2 (Dvl2) was assessed by fluorescence imaging of live enteroids from WT and CF mice crossed with Dvl2-EGFP/ACTB-tdTomato,-EGFP)Luo/J (RosamT/mG) mice. RESULTS: Relative to WT, CF intestinal crypts showed an ∼30% increase in epithelial and Lgr5+ ISC proliferation and increased Wnt/ß-catenin signaling. Cftr was expressed in Sox9EGFPLo ISCs and loss of Cftr induced an alkaline pHi in ISCs. CF crypt-base columnar cells showed a generalized increase in plasma membrane Dvl2-EGFP association as compared with WT. Dvl2-EGFP membrane association was charge- and pH-dependent and increased in WT crypt-base columnar cells by Cftr inhibition. CONCLUSIONS: CF intestine shows increased ISC proliferation and Wnt/ß-catenin signaling. Loss of Cftr increases pHi in ISCs, which stabilizes the plasma membrane association of the Wnt transducer Dvl, likely facilitating Wnt/ß-catenin signaling. Absence of Cftr-dependent suppression of ISC proliferation in the CF intestine may contribute to increased risk for intestinal tumors.

Asunto(s)

Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Epitelio/fisiología , Animales , Proliferación Celular , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células Epiteliales/fisiología , Eliminación de Gen , Regulación de la Expresión Génica , Concentración de Iones de Hidrógeno , Ratones , Ratones Noqueados , Ratones Transgénicos

RESUMEN

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), an anion channel providing a major pathway for Cl(-) and HCO3 (-) efflux across the apical membrane of the epithelium. In the intestine, CF manifests as obstructive syndromes, dysbiosis, inflammation, and an increased risk for gastrointestinal cancer. Cftr knockout (KO) mice recapitulate CF intestinal disease, including intestinal hyperproliferation. Previous studies using Cftr KO intestinal organoids (enteroids) indicate that crypt epithelium maintains an alkaline intracellular pH (pHi). We hypothesized that Cftr has a cell-autonomous role in downregulating pHi that is incompletely compensated by acid-base regulation in its absence. Here, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein microfluorimetry of enteroids showed that Cftr KO crypt epithelium sustains an alkaline pHi and resistance to cell acidification relative to wild-type. Quantitative real-time PCR revealed that Cftr KO enteroids exhibit downregulated transcription of base (HCO3 (-))-loading proteins and upregulation of the basolateral membrane HCO3 (-)-unloader anion exchanger 2 (Ae2). Although Cftr KO crypt epithelium had increased Ae2 expression and Ae2-mediated Cl(-)/HCO3 (-) exchange with maximized gradients, it also had increased intracellular Cl(-) concentration relative to wild-type. Pharmacological reduction of intracellular Cl(-) concentration in Cftr KO crypt epithelium normalized pHi, which was largely Ae2-dependent. We conclude that Cftr KO crypt epithelium maintains an alkaline pHi as a consequence of losing both Cl(-) and HCO3 (-) efflux, which impairs pHi regulation by Ae2. Retention of Cl(-) and an alkaline pHi in crypt epithelium may alter several cellular processes in the proliferative compartment of Cftr KO intestine.

Asunto(s)

Bicarbonatos/metabolismo , Antiportadores de Cloruro-Bicarbonato/metabolismo , Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Mucosa Intestinal/metabolismo , Animales , Antiportadores de Cloruro-Bicarbonato/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Concentración de Iones de Hidrógeno , Ratones , Ratones Noqueados

RESUMEN

Cystic fibrosis (CF) intestinal disease is associated with the pathological manifestation mucoviscidosis, which is the secretion of tenacious, viscid mucus that plugs ducts and glands of epithelial-lined organs. Goblet cells are the principal cell type involved in exocytosis of mucin granules; however, little is known about the exocytotic process of goblet cells in the CF intestine. Using intestinal organoids from a CF mouse model, we determined that CF goblet cells have altered exocytotic dynamics, which involved intrathecal granule swelling that was abruptly followed by incomplete release of partially decondensated mucus. Some CF goblet cells exhibited an ectopic granule location and distorted cellular morphology, a phenotype that is consistent with retrograde intracellular granule movement during exocytosis. Increasing the luminal concentration of bicarbonate, which mimics CF transmembrane conductance regulator-mediated anion secretion, increased spontaneous degranulation in WT goblet cells and improved exocytotic dynamics in CF goblet cells; however, there was still an apparent incoordination between granule decondensation and exocytosis in the CF goblet cells. Compared with those within WT goblet cells, mucin granules within CF goblet cells had an alkaline pH, which may adversely affect the polyionic composition of the mucins. Together, these findings indicate that goblet cell dysfunction is an epithelial-autonomous defect in the CF intestine that likely contributes to the pathology of mucoviscidosis and the intestinal manifestations of obstruction and inflammation.

Asunto(s)

Fibrosis Quística/patología , Exocitosis , Células Caliciformes/metabolismo , Animales , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Concentración de Iones de Hidrógeno , Mucosa Intestinal/patología , Ratones Noqueados , Mucinas/metabolismo , Vesículas Secretoras/metabolismo

RESUMEN

Physiological studies of intact crypt epithelium have been limited by problems of accessibility in vivo and dedifferentiation in standard primary culture. Investigations of murine intestinal stem cells have recently yielded a primary intestinal culture in three-dimensional gel suspension that recapitulates crypt structure and epithelial differentiation (Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, Van Es JH, Abo A, Kujala P, Peters PJ, Clevers H. Nature 459: 262-265, 2009). We investigated the utility of murine intestinal crypt cultures (termed "enteroids") for physiological studies of crypt epithelium by focusing on the transport activity of the cystic fibrosis transmembrane conductance regulator Cftr. Enteroids had multiple crypts with well-differentiated goblet and Paneth cells that degranulated on exposure to the muscarinic agonist carbachol. Modified growth medium provided a crypt proliferation rate, as measured by 5-ethynyl-2'-deoxyuridine labeling, which was similar to proliferation in vivo. Immunoblots demonstrated equivalent Cftr expression in comparisons of freshly isolated crypts with primary and passage 1 enteroids. Apparent enteroid differences in mRNA expression of other transporters were primarily associated with villous epithelial contamination of freshly isolated crypts. Microelectrode analysis revealed cAMP-stimulated membrane depolarization in enteroid epithelium from wild-type (WT) but not Cftr knockout (KO) mice. Morphological and microfluorimetric studies, respectively, demonstrated Cftr-dependent cell shrinkage and lower intracellular pH in WT enteroid epithelium in contrast to Cftr KO epithelium or WT epithelium treated with Cftr inhibitor 172. We conclude that crypt epithelium of murine enteroids exhibit Cftr expression and activity that recapitulates crypt epithelium in vivo. Enteroids provide a primary culture model that is suitable for physiological studies of regenerating crypt epithelium.

Asunto(s)

Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Mucosa Intestinal/citología , Mucosa Intestinal/fisiología , Intestino Delgado/citología , Intestino Delgado/fisiología , Animales , Ratones , Ratones Endogámicos CFTR , Ratones Noqueados , Técnicas de Cultivo de Órganos/métodos

RESUMEN

The recent proposal that Dra/Slc26a3 mediates electrogenic 2Cl(-)/1HCO(3)(-) exchange suggests a required revision of classical concepts of electroneutral Cl(-) transport across epithelia such as the intestine. We investigated 1) the effect of endogenous Dra Cl(-)/HCO(3)(-) activity on apical membrane potential (V(a)) of the cecal surface epithelium using wild-type (WT) and knockout (KO) mice; and 2) the electrical properties of Cl(-)/(OH(-))HCO(3)(-) exchange by mouse and human orthologs of Dra expressed in Xenopus oocytes. Ex vivo (36)Cl(-) fluxes and microfluorometry revealed that cecal Cl(-)/HCO(3)(-) exchange was abolished in the Dra KO without concordant changes in short-circuit current. In microelectrode studies, baseline V(a) of Dra KO surface epithelium was slightly hyperpolarized relative to WT but depolarized to the same extent as WT during luminal Cl(-) substitution. Subsequent studies indicated that Cl(-)-dependent V(a) depolarization requires the anion channel Cftr. Oocyte studies demonstrated that Dra-mediated exchange of intracellular Cl(-) for extracellular HCO(3)(-) is accompanied by slow hyperpolarization and a modest outward current, but that the steady-state current-voltage relationship is unaffected by Cl(-) removal or pharmacological blockade. Further, Dra-dependent (36)Cl(-) efflux was voltage-insensitive in oocytes coexpressing the cation channels ENaC or ROMK. We conclude that 1) endogenous Dra and recombinant human/mouse Dra orthologs do not exhibit electrogenic 2Cl(-)/1HCO(3)(-) exchange; and 2) acute induction of Dra Cl(-)/HCO(3)(-) exchange is associated with secondary membrane potential changes representing homeostatic responses. Thus, participation of Dra in coupled NaCl absorption and in uncoupled HCO(3)(-) secretion remains compatible with electroneutrality of these processes, and with the utility of electroneutral transport models for predicting epithelial responses in health and disease.

Asunto(s)

Antiportadores/metabolismo , Antiportadores de Cloruro-Bicarbonato/metabolismo , Animales , Antiportadores/genética , Bicarbonatos/metabolismo , Ciego/metabolismo , Antiportadores de Cloruro-Bicarbonato/genética , Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Femenino , Humanos , Masculino , Potenciales de la Membrana , Ratones , Ratones Noqueados , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transportadores de Sulfato

RESUMEN

The majority of dietary amino acids are absorbed via the H(+)-di-/tripeptide transporter Pept1 of the small intestine. Proton influx via Pept1 requires maintenance of intracellular pH (pH(i)) to sustain the driving force for peptide absorption. The apical membrane Na(+)/H(+) exchanger Nhe3 plays a major role in minimizing epithelial acidification during H(+)-di-/tripeptide absorption. However, the contributions of HCO(3)(-)-dependent transporters to this process have not been elucidated. In this study, we investigate the role of putative anion transporter-1 (Pat-1), an apical membrane anion exchanger, in epithelial pH(i) regulation during H(+)-peptide absorption. Using wild-type (WT) and Pat-1(-) mice, Ussing chambers were employed to measure the short-circuit current (I(sc)) associated with Pept1-mediated glycyl-sarcosine (Gly-Sar) absorption. Microfluorometry was used to measure pH(i) and Cl(-)/HCO(3)(-) exchange in the upper villous epithelium. In CO(2)/HCO(3)(-)-buffered Ringers, WT small intestine showed significant Gly-Sar-induced I(sc) and efficient pH(i) regulation during pharmacological inhibition of Nhe3 activity. In contrast, epithelial acidification and reduced I(sc) response to Gly-Sar exposure occurred during pharmacological inhibition of Cl(-)/HCO(3)(-) exchange and in the Pat-1(-) intestine. Pat-1 interacts with carbonic anhydrase II (CAII), and studies using CAII(-) intestine or the pharmacological inhibitor methazolamide on WT intestine resulted in increased epithelial acidification during Gly-Sar exposure. Increased epithelial acidification during Gly-Sar exposure also occurred in WT intestine during inhibition of luminal extracellular CA activity. Measurement of Cl(-)/HCO(3)(-) exchange in the presence of Gly-Sar revealed an increased rate of Cl(-)(OUT)/HCO(3)(-)(IN) exchange that was both Pat-1 dependent and CA dependent. In conclusion, Pat-1 Cl(-)/HCO(3)(-) exchange contributes to pH(i) regulation in the villous epithelium during H(+)-dipeptide absorption, possibly by providing a HCO(3)(-) import pathway.

Asunto(s)

Antiportadores/fisiología , Dipéptidos/metabolismo , Animales , Bicarbonatos/metabolismo , Anhidrasa Carbónica II/metabolismo , Duodeno/metabolismo , Concentración de Iones de Hidrógeno , Ratones , Transportadores de Sulfato

RESUMEN

BACKGROUND & AIMS: The current model of duodenal HCO(3)(-) secretion proposes that basal secretion results from Cl(-)/HCO(3)(-) exchange, whereas cyclic adenosine monophosphate (cAMP)-stimulated secretion depends on a cystic fibrosis transmembrane conductance regulator channel (Cftr)-mediated HCO(3)(-) conductance. However, discrepancies in applying the model suggest that Cl(-)/HCO(3)(-) exchange also contributes to cAMP-stimulated secretion. Of 2 candidate Cl(-)/HCO(3)(-) exchangers, studies of putative anion transporter-1 knockout (KO) mice find little contribution of putative anion transporter-1 to basal or cAMP-stimulated secretion. Therefore, the role of down-regulated in adenoma (Dra) in duodenal HCO(3)(-) secretion was investigated using DraKO mice. METHODS: Duodenal HCO(3)(-) secretion was measured by pH stat in Ussing chambers. Apical membrane Cl(-)/HCO(3)(-) exchange was measured by microfluorometry of intracellular pH in intact villous epithelium. Dra expression was assessed by immunofluorescence. RESULTS: Basal HCO(3)(-) secretion was reduced approximately 55%-60% in the DraKO duodenum. cAMP-stimulated HCO(3)(-) secretion was reduced approximately 50%, but short-circuit current was unchanged, indicating normal Cftr activity. Microfluorimetry of villi demonstrated that Dra is the dominant Cl(-)/HCO(3)(-) exchanger in the lower villous epithelium. Dra expression increased from villous tip to crypt. DraKO and wild-type villi also demonstrated regulation of apical Na(+)/H(+) exchange by Cftr-dependent cell shrinkage during luminal Cl(-) substitution. CONCLUSIONS: In murine duodenum, Dra Cl(-)/HCO(3)(-) exchange is concentrated in the lower crypt-villus axis where it is subject to Cftr regulation. Dra activity contributes most basal HCO(3)(-) secretion and approximately 50% of cAMP-stimulated HCO(3)(-) secretion. Dra Cl(-)/HCO(3)(-) exchange should be considered in efforts to normalize HCO(3)(-) secretion in duodenal disorders such as ulcer disease and cystic fibrosis.

Asunto(s)

Antiportadores/genética , Antiportadores/metabolismo , Bicarbonatos/metabolismo , Duodeno/metabolismo , Ácidos/metabolismo , Animales , Antiportadores de Cloruro-Bicarbonato/farmacocinética , AMP Cíclico/metabolismo , Regulación hacia Abajo/fisiología , Concentración de Iones de Hidrógeno , Mucosa Intestinal/metabolismo , Ratones , Ratones Noqueados , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Intercambiadores de Sodio-Hidrógeno/metabolismo , Transportadores de Sulfato

RESUMEN

BACKGROUND & AIMS: Electroneutral NaCl absorption across small intestine contributes importantly to systemic fluid balance. Disturbances in this process occur in both obstructive and diarrheal diseases, eg, cystic fibrosis, secretory diarrhea. NaCl absorption involves coupling of Cl(-)/HCO(3)(-) exchanger(s) primarily with Na(+)/H(+) exchanger 3 (Nhe3) at the apical membrane of intestinal epithelia. Identity of the coupling Cl(-)/HCO(3)(-) exchanger(s) was investigated using mice with gene-targeted knockout (KO) of Cl(-)/HCO(3)(-) exchangers: Slc26a3, down-regulated in adenoma (Dra) or Slc26a6, putative anion transporter-1 (Pat-1). METHODS: Intracellular pH (pH(i)) of intact jejunal villous epithelium was measured by ratiometric microfluoroscopy. Ussing chambers were used to measure transepithelial (22)Na(36)Cl flux across murine jejunum, a site of electroneutral NaCl absorption. Expression was estimated using immunofluorescence and quantitative polymerase chain reaction. RESULTS: Basal pH(i) of DraKO epithelium, but not Pat-1KO epithelium, was alkaline, whereas pH(i) in the Nhe3KO was acidic relative to wild-type. Altered pH(i) was associated with robust Na(+)/H(+) and Cl(-)/HCO(3)(-) exchange activity in the DraKO and Nhe3KO villous epithelium, respectively. Contrary to genetic ablation, pharmacologic inhibition of Nhe3 in wild-type did not alter pH(i) but coordinately inhibited Dra. Flux studies revealed that Cl(-) absorption was essentially abolished (>80%) in the DraKO and little changed (<20%) in the Pat-1KO jejunum. Net Na(+) absorption was unaffected. Immunofluorescence demonstrated modest Dra expression in the jejunum relative to large intestine. Functional and expression studies did not indicate compensatory changes in relevant transporters. CONCLUSIONS: These studies provide functional evidence that Dra is the major Cl(-)/HCO(3)(-) exchanger coupled with Nhe3 for electroneutral NaCl absorption across mammalian small intestine.

Asunto(s)

Adenoma/genética , Antiportadores de Cloruro-Bicarbonato/genética , Regulación hacia Abajo , Absorción Intestinal/fisiología , Yeyuno/metabolismo , ARN Neoplásico , Cloruro de Sodio/metabolismo , Adenoma/metabolismo , Adenoma/patología , Animales , Antiportadores de Cloruro-Bicarbonato/metabolismo , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente , Concentración de Iones de Hidrógeno , Líquido Intracelular/metabolismo , Transporte Iónico/fisiología , Neoplasias del Yeyuno/genética , Neoplasias del Yeyuno/metabolismo , Neoplasias del Yeyuno/patología , Yeyuno/patología , Ratones , Ratones Mutantes , Reacción en Cadena de la Polimerasa

RESUMEN

Basal HCO(3)(-) secretion across the duodenum has been shown in several species to principally involve the activity of apical membrane Cl(-)/HCO(3)(-) exchanger(s). To investigate the identity of relevant anion exchanger(s), experiments were performed using wild-type (WT) mice and mice with gene-targeted deletion of the following Cl(-)/HCO(3)(-) exchangers localized to the apical membrane of murine duodenal villi: Slc26a3 [down-regulated in adenoma (DRA)], Slc26a6 [putative anion transporter 1 (PAT-1)], and Slc4a9 [anion exchanger 4 (AE4)]. RT-PCR of the isolated villous epithelium demonstrated PAT-1, DRA, and AE4 mRNA expression. Using the pH-sensitive dye BCECF, anion exchange rates were measured across the apical membrane of epithelial cells in the upper villus of the intact duodenal mucosa. Under basal conditions, Cl(-)/HCO(3)(-) exchange activity was reduced by 65-80% in the PAT-1(-) duodenum, 30-40% in the DRA(-) duodenum, and <5% in the AE4(-) duodenum compared with the WT duodenum. SO(4)(2-)/HCO(3)(-) exchange was eliminated in the PAT-1(-) duodenum but was not affected in the DRA(-) and AE4(-) duodenum relative to the WT duodenum. Intracellular pH (pH(i)) was reduced in the PAT-1(-) villous epithelium but increased to WT levels in the absence of CO(2)/HCO(3)(-) or during methazolamide treatment. Further experiments under physiological conditions indicated active pH(i) compensation in the PAT-1(-) villous epithelium by combined activities of Na(+)/H(+) exchanger 1 and Cl(-)-dependent transport processes at the basolateral membrane. We conclude that 1) PAT-1 is the major contributor to basal Cl(-)/HCO(3)(-) and SO(4)(2-)/HCO(3)(-) exchange across the apical membrane and 2) PAT-1 plays a role in pH(i) regulation in the upper villous epithelium of the murine duodenum.

Asunto(s)

Antiportadores/metabolismo , Bicarbonatos/metabolismo , Antiportadores de Cloruro-Bicarbonato/metabolismo , Cloruros/metabolismo , Duodeno/metabolismo , Mucosa Intestinal/metabolismo , Animales , Antiportadores/deficiencia , Antiportadores/genética , Proteínas de Transporte de Catión/metabolismo , Antiportadores de Cloruro-Bicarbonato/deficiencia , Antiportadores de Cloruro-Bicarbonato/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Cámaras de Difusión de Cultivos , Duodeno/citología , Expresión Génica , Concentración de Iones de Hidrógeno , Mucosa Intestinal/citología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Intercambiador 1 de Sodio-Hidrógeno , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/metabolismo , Transportadores de Sulfato , Sulfatos/metabolismo , Factores de Tiempo , Técnicas de Cultivo de Tejidos

RESUMEN

Intestinal disease in cystic fibrosis (CF) mice closely mirrors aspects of obstructive syndromes in CF patients. The pathogenesis involves accumulation of mucoid debris in the crypts that fuse with intestinal content to form obstructing mucofeculant impactions. Treatment involves modalities that increase the fluidity of the luminal content, such as osmotic laxatives and liquid diets. We investigated the effects of talniflumate (Lomucin, Genaera Corporation, Plymouth Meeting, PA), a compound that may be beneficial to treatment of CF intestinal disease based on three mechanisms of action: mucus synthesis inhibition by blockade of the murine calcium-activated chloride channel 3 (mCLCA3), nonsteroidal anti-inflammatory effects, and inhibition of Cl(-)/HCO (-)(3) exchanger(s) involved in intestinal NaCl absorption. Cohorts of CF mice were fed control diet or diets containing either talniflumate (0.4 mg/g chow) or ibuprofen (0.4 mg/g chow) for 21 days to assess survival. Talniflumate significantly increased CF mouse survival from 26 to 77%, whereas ibuprofen had no effect (22% survival). Oral talniflumate did not alter crypt goblet cell numbers or change intestinal expression of mCLCA3 but tended to decrease crypt mucoid impaction. Ussing chamber studies indicated that talniflumate slightly increased the basal short-circuit current of CF intestine, but the change was not sensitive to secretagogue stimulation or bumetanide inhibition. In contrast, intracellular pH measurements of intact intestinal villous epithelium indicated that talniflumate significantly inhibited apical membrane Cl(-)/HCO (-)(3) exchange by >50%. We conclude that oral talniflumate increases the survival of CF mice, possibly by the beneficial effects of decreasing small intestinal NaCl absorption through the inhibition of apical membrane Cl(-)/HCO (-)(3) exchanger(s).

Asunto(s)

Antiinflamatorios no Esteroideos/uso terapéutico , Benzofuranos/uso terapéutico , Fibrosis Quística/complicaciones , Obstrucción Intestinal/tratamiento farmacológico , Piridinas/uso terapéutico , Animales , Recuento de Células , Canales de Cloruro/biosíntesis , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Modelos Animales de Enfermedad , Concentración de Iones de Hidrógeno , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Obstrucción Intestinal/etiología , Obstrucción Intestinal/metabolismo , Intestino Delgado/citología , Intestino Delgado/efectos de los fármacos , Intestino Delgado/metabolismo , Transporte Iónico/efectos de los fármacos , Ratones , Mucinas/biosíntesis , Mucoproteínas/biosíntesis , Mutación

RESUMEN

OBJECTIVE: To describe workplace limitations and the validity and reliability of the Work Limitations Questionnaire (WLQ) in persons with rheumatoid arthritis (RA). METHODS: A total of 836 employed persons with RA reported clinical and work related measures and completed the WLQ, a 25 item questionnaire that assesses the impact of chronic health conditions on job performance and productivity. Limitations are categorized into 4 domains: physical demands (PDS), mental demands (MDS), time management demands (TMS), and output demands (ODS), which are then used to calculate the WLQ index. RESULTS: Of the 836 completed WLQ, about 10% (85) could not be scored, as more than half the items in each domain were not applicable to the patient's job. Demographic and clinical variables were associated with missing WLQ scores including older age (OR 1.7, 95% CI 1.3-2.1), male sex (OR 1.9, 95% CI 1.2-3.0), and Health Assessment Questionnaire (HAQ) scores (OR 1.4, 95% CI 1.0-2.0). Work limitations were present in all work domains: PDS (27.5%), MDS (15.7%), ODS (19.4%), and TMS (28.6%), resulting in a mean WLQ index of 5.9 (SD 5.6), which corresponds to a 4.9% decrease in productivity and a 5.1% increase in work hours to compensate for productivity loss. The WLQ index was inversely associated with Medical Outcomes Study Short Form 36 (SF-36) Mental Component Score (MCS; r = -0.60) and Physical Component Score (PCS; r = -0.49). Fatigue (0.5), pain (0.46), and HAQ (0.56) were also significantly associated with the WLQ index. Weaker associations were seen with days unable to perform (0.29), days activities cut down (0.38), and annual income (-0.10). CONCLUSION: The WLQ is a reliable tool for assessing work productivity. However, persons with RA tend to select jobs that they can do with their RA limitations, with the result that the WLQ does not detect functional limitations as well as the HAQ and SF-36. The WLQ provides special information that is not available using conventional measures of assessment, and can provide helpful knowledge about individual patient problems in the workplace. Whether this information will have greater predictive ability and clinical relevance compared with surrogate measures such as the HAQ and SF-36 has not been determined, but should be the subject of future studies.

Asunto(s)

Absentismo , Artritis Reumatoide/fisiopatología , Evaluación de la Discapacidad , Eficiencia , Encuestas y Cuestionarios , Evaluación de Capacidad de Trabajo , Artritis Reumatoide/economía , Artritis Reumatoide/rehabilitación , Empleo , Femenino , Indicadores de Salud , Humanos , Masculino , Persona de Mediana Edad , Psicometría , Reproducibilidad de los Resultados , Índice de Severidad de la Enfermedad , Resultado del Tratamiento

RESUMEN

To address gaps in the quality of care for osteo-arthritis, the authors developed a Web-based computer program to provide patients with personalized feedback designed to improve the quality of their osteoarthritis care. The current study was designed to examine satisfaction as well as the potential effects of the feedback on patients' perceptions of their osteoarthritis care by randomizing patients to use the site before or after they answered questions about the quality of their osteoarthritis care. On average, participants received 8.7 recommendations to change their osteoarthritis care. Satisfaction with osteo-arthritis care was similar between subjects in both groups. Most subjects believed that the Web site would help them get better care from their doctor (77.7%), and most would recommend it to others (94.3%). Overall, the Web site is well accepted and has no negative effect on patients' satisfaction with their osteo-arthritis care.

Asunto(s)

Internet , Osteoartritis/terapia , Satisfacción del Paciente/estadística & datos numéricos , Gestión de la Calidad Total , Femenino , Humanos , Masculino , Persona de Mediana Edad , Participación del Paciente , Programas Informáticos , Encuestas y Cuestionarios , Estados Unidos

RESUMEN

Villi of the proximal duodenum are situated for direct exposure to gastric acid chyme. However, little is known about active bicarbonate secretion across villi that maintains the protective alkaline mucus barrier, a process that may be compromised in cystic fibrosis (CF), i.e., in the absence of a functional CF transmembrane conductance regulator (CFTR) anion channel. We investigated Cl(-)/HCO(3)(-) exchange activity across the apical membrane of epithelial cells located at the midregion of villi in intact duodenal mucosa from wild-type (WT) and CF mice using the pH-sensitive dye BCECF. Under basal conditions, the Cl(-)/HCO(3)(-) exchange rate was reduced by approximately 35% in CF compared with WT villous epithelium. Cl(-)/HCO(3)(-) exchange in WT and CF villi responded similarly to inhibitors of anion exchange, and membrane depolarization enhanced rates of Cl(-)(out)/HCO(3)(-)(in) exchange in both epithelia. In anion substitution studies, anion(in)/HCO(3)(-)(out) exchange rates were greater in WT epithelium using Cl(-) or NO(3)(-), but decreased to the level of the CF epithelium using the CFTR-impermeant anion, SO(4)(2-). Similarly, treatment of WT epithelium with the CFTR-selective blocker glybenclamide decreased the Cl(-)/HCO(3)(-) exchange rate to the level of CF epithelium. The mRNA expression of Slc26a3 (downregulated in adenoma) and Slc26a6 (putative anion exchanger-1) was similar between WT and CF duodena. From these studies of murine duodenum, we conclude 1) characteristics of Cl(-)/HCO(3)(-) exchange in the villous epithelium are most consistent with Slc26a6 activity, and 2) Cl(-) channel activity of CFTR facilitates apical membrane Cl(-)(in)/HCO(3)(-)(out) exchange by providing a Cl(-) "leak" under basal conditions.

Asunto(s)

Bicarbonatos/farmacocinética , Cloro/farmacocinética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/farmacología , Fibrosis Quística/fisiopatología , Duodeno/fisiología , Intercambio Iónico , Animales , Antiportadores/genética , Antiportadores/farmacología , Regulación hacia Abajo , Duodeno/patología , Electrofisiología , Mucosa Intestinal/citología , Mucosa Intestinal/fisiología , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CFTR , ARN Mensajero/biosíntesis , Transportadores de Sulfato

RESUMEN

To examine the role of the p53 homolog p73 in brain development, we studied p73-/-, p73+/-, E2F1-/-, and reeler mutant mice. p73 in developing brain is expressed in Cajal-Retzius (CR) cells, the cortical hem, and the choroid plexus. p73-expressing CR cells are lost in p73-/- embryos, although Reelin is faintly expressed in the marginal zone. Ectopic neurons in the p73-/- preplate and cortical hem at embryonic day 12 implicate p73 in the early developmental program of the cortex; however, preplate partition and early cortical plate formation are not disturbed. Postnatal p73-/- mice show a mild hypoplasia of the rostral cortex and a severely disrupted architecture of the posterior telencephalon. In the developing p73-/- hippocampus, the most striking abnormality is the absence of the hippocampal fissure, suggesting a role of p73 in cortical folding. p73+/- mice have a less severe cortical phenotype; they display a dorsal shift of the entorhinal cortex and a reduced size of occipital and posterior temporal areas, which acquire entorhinal-like features such as Reelin-positive cells in layer II. CR cells appear unaffected by heterozygosity. We relate the malformations of the posterior pole in p73 mutant mice to alterations of p73 expression in the cortical hem and suggest that p73 forms part of an early signaling network that controls neocortical and archicortical regionalization. In mice deficient for the transcription factor E2F1, a main activator of the TAp73 (transactivating p73) isoform, we find a defect of the caudal cortical architecture resembling the p73+/- phenotype along with reduced TAp73 protein levels and propose that an E2F1-TAp73 dependent pathway is involved in cortical patterning.

Asunto(s)

Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Corteza Cerebral/citología , Proteínas de Unión al ADN/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuronas/fisiología , Proteínas Nucleares/fisiología , Animales , Encéfalo/anomalías , Moléculas de Adhesión Celular Neuronal/biosíntesis , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiología , Corteza Cerebral/anomalías , Corteza Cerebral/embriología , Corteza Cerebral/crecimiento & desarrollo , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Proteínas de la Matriz Extracelular/biosíntesis , Genes Supresores de Tumor , Sistema Límbico/anomalías , Sistema Límbico/embriología , Sistema Límbico/crecimiento & desarrollo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes Neurológicos , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Fenotipo , Isoformas de Proteínas/fisiología , Proteína Reelina , Serina Endopeptidasas/biosíntesis , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Proteína Tumoral p73 , Proteínas Supresoras de Tumor

RESUMEN

The major disease-causing mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of phenylalanine 508 (DeltaF508), which adversely affects processing and plasma membrane targeting of CFTR. Under conditions predicted to stabilize protein folding, DeltaF508 CFTR is capable of trafficking to the plasma membrane and retains cAMP-regulated anion channel activity. Overexpression is one factor that increases CFTR trafficking; therefore, we hypothesized that expression of a domain mimic of the first nucleotide-binding fold (NBF1) of CFTR, i.e., the site of F508, may be sufficient to overwhelm the quality control process or otherwise stabilize DeltaF508 CFTR and thereby restore cAMP-stimulated anion secretion. In epithelial cells expressing recombinant DeltaF508 human (h)CFTR, expression of wild-type NBF1 increased the amount of both core-glycosylated and mature protein to a greater extent than expression of DeltaF508 NBF1. Expression of wild-type NBF1 in the DeltaF508 hCFTR cells increased whole cell Cl(-) current density to approximately 50% of that in cells expressing wild-type hCFTR. Expression of NBF1 in polarized epithelial monolayers from a DeltaF508/DeltaF508 cystic fibrosis mouse (MGEF) restored cAMP-stimulated transepithelial anion secretion but not in monolayers from a CFTR-null mouse (MGEN). Restoration of anion secretion was sustained in NBF1-expressing MGEF for >30 passages, whereas MGEN corrected with hCFTR progressively lost anion secretion capability. We conclude that expression of a NBF1 domain mimic may be useful for correction of the DeltaF508 CFTR protein trafficking defect in cystic fibrosis epithelia.

Asunto(s)

Aniones/metabolismo , AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/metabolismo , Animales , División Celular , Células Cultivadas , Cloruros/metabolismo , Fibrosis Quística/patología , Fibrosis Quística/fisiopatología , Conductividad Eléctrica , Femenino , Humanos , Ratones , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo