Nancy M. Walker

Down-regulated in Adenoma Cl/HCO3 Exchanger Couples With Na/H Exchanger 3 for NaCl Absorption in Murine Small Intestine

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.

Role of Down-Regulated in Adenoma Anion Exchanger in HCO3 - Secretion Across Murine Duodenum

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.

cAMP inhibition of murine intestinal Na/H exchange requires CFTR-mediated cell shrinkage of villus epithelium.

BACKGROUND AND AIMS Unlike the intestine of normal subjects, small-intestinal epithelia of cystic fibrosis patients and cystic fibrosis transmembrane conductance regulator protein-null (CFTR(-)) mice do not respond to stimulation of intracellular cyclic adenosine monophosphate with inhibition of electroneutral NaCl absorption. Because CFTR-mediated anion secretion has been associated with changes in crypt cell volume, we hypothesized that CFTR-mediated cell volume reduction in villus epithelium is required for intracellular cyclic adenosine monophosphate inhibition of Na(+)/H(+) exchanger (primarily Na(+)/H(+) exchanger 3) activity in the proximal small intestine. METHODS Transepithelial (22)Na flux across the jejuna of CFTR(+), CFTR(-), the basolateral membrane Na(+)/K(+)/2Cl(-) co-transporter protein NKCC1(+), and NKCC1(-) mice were correlated with changes in epithelial cell volume of the midvillus region. RESULTS Stimulation of intracellular cyclic adenosine monophosphate resulted in cessation of Na(+)/H(+) exchanger-mediated Na(+) absorption (J(ms)(NHE)) in CFTR(+) jejunum but had no effect on J(ms)(NHE) across CFTR(-) jejunum. Cell volume indices indicated an approximately 30% volume reduction of villus epithelial cells in CFTR(+) jejunum but no changes in CFTR(-) epithelium after intracellular cyclic adenosine monophosphate stimulation. In contrast, cell shrinkage induced by hypertonic medium inhibited J(ms)(NHE) in both CFTR(+) and CFTR(-) mice. Bumetanide treatment to inhibit Cl(-) secretion by blockade of the Na(+)/K(+)/2Cl(-) co-transporter, NKCC1, of stimulated CFTR(+) jejunum prevented maximal volume reduction of villus epithelium and recovered approximately 40% of J(ms)(NHE). Likewise, J(ms)(NHE) and cell volume were unaffected by intracellular cyclic adenosine monophosphate stimulation in NKCC1(-) jejuna. CONCLUSIONS These findings show a previously unrecognized role of functional CFTR expressed in villus epithelium: regulation of Na(+)/H(+) exchanger 3-mediated Na(+) absorption by alteration of epithelial cell volume.

Desensitization of P2Y2receptor-activated transepithelial anion secretion

Desensitization of P2Y2 receptor-activated anion secretion may limit the usefulness of extracellular nucleotides in secretagogue therapy of epithelial diseases, e.g., cystic fibrosis (CF). To investigate the desensitization process for endogenous P2Y2 receptors, freshly excised or cultured murine gallbladder epithelia (MGEP) were mounted in Ussing chambers to measure short-circuit current (Isc), an index of electrogenic anion secretion. Luminal treatment with nucleotide receptor agonists increased the Isc with a potency profile of ATP = UTP > 2-methylthioATP >> alpha,beta-methylene-ATP. RT-PCR revealed the expression of P2Y2 receptor mRNA in the MGEP cells. The desensitization of anion secretion required a 10-min preincubation with the P2Y2 receptor agonist UTP and increased in a concentration-dependent manner (IC50 approximately 10(-6) M). Approximately 40% of the anion secretory response was unaffected by maximal desensitizing concentrations of UTP. Recovery from UTP-induced desensitization was rapid (<10 min) at preincubation concentrations less than the EC50 (1.9 x 10(-6) M) but required progressively longer time periods at greater concentrations. UTP-induced total inositol phosphate production and intracellular Ca2+ mobilization desensitized with a concentration dependence similar to that of anion secretion. In contrast, maximal anion secretion induced by Ca2+ ionophore ionomycin was unaffected by preincubation with a desensitizing concentration of UTP. It was concluded that 1) desensitization of transepithelial anion secretion stimulated by the P2Y2 receptor agonist UTP is time and concentration dependent; 2) recovery from desensitization is prolonged (>90 min) at UTP concentrations >10(-5) M; and 3) UTP-induced desensitization occurs before the operation of the anion secretory mechanism.Desensitization of P2Y2 receptor-activated anion secretion may limit the usefulness of extracellular nucleotides in secretagogue therapy of epithelial diseases, e.g., cystic fibrosis (CF). To investigate the desensitization process for endogenous P2Y2 receptors, freshly excised or cultured murine gallbladder epithelia (MGEP) were mounted in Ussing chambers to measure short-circuit current ( I sc), an index of electrogenic anion secretion. Luminal treatment with nucleotide receptor agonists increased the I sc with a potency profile of ATP = UTP > 2-methylthioATP >> α,β-methylene-ATP. RT-PCR revealed the expression of P2Y2 receptor mRNA in the MGEP cells. The desensitization of anion secretion required a 10-min preincubation with the P2Y2receptor agonist UTP and increased in a concentration-dependent manner (IC50 ≈ 10-6 M). Approximately 40% of the anion secretory response was unaffected by maximal desensitizing concentrations of UTP. Recovery from UTP-induced desensitization was rapid (<10 min) at preincubation concentrations less than the EC50 (1.9 × 10-6 M) but required progressively longer time periods at greater concentrations. UTP-induced total inositol phosphate production and intracellular Ca2+ mobilization desensitized with a concentration dependence similar to that of anion secretion. In contrast, maximal anion secretion induced by Ca2+ ionophore ionomycin was unaffected by preincubation with a desensitizing concentration of UTP. It was concluded that 1) desensitization of transepithelial anion secretion stimulated by the P2Y2 receptor agonist UTP is time and concentration dependent; 2) recovery from desensitization is prolonged (>90 min) at UTP concentrations >10-5 M; and 3) UTP-induced desensitization occurs before the operation of the anion secretory mechanism.

Native and recombinant Slc26a3 (downregulated in adenoma, Dra) do not exhibit properties of 2Cl-/1HCO3- exchange.

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.

Defective goblet cell exocytosis contributes to murine cystic fibrosis–associated intestinal disease

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.

Putative anion transporter-1 (Pat-1, Slc26a6) contributes to intracellular pH regulation during H+-dipeptide transport in duodenal villous epithelium

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.

Functional Activity of Pat-1 (Slc26a6) Cl−/HCO3− Exchange in the Lower Villus Epithelium of Murine Duodenum

Aims:  The apical membrane anion exchanger putative anion transporter‐1 (Pat‐1) is expressed at significant levels in the lower villus epithelium of murine duodenum. However, previous studies of Cl−/HCO3− exchange in the lower villus have failed to demonstrate Pat‐1 function. Those studies routinely included luminal glucose which induces Na+‐coupled glucose transport and acidifies the villus epithelium. Since Pat‐1 has been proposed to be an electrogenic 1Cl−/2HCO3− exchanger, membrane depolarization or cell acidification during glucose transport may obscure Pat‐1 activity. Therefore, we investigated the effects of luminal glucose on Cl−IN/HCO3−OUT exchange activity in the lower villus epithelium.

Su1994 Loss of CFTR Results in Intestinal Stem Cell Hyperproliferation

Standard methods such as western blot and flow-cytometry (protein expression); quantitative real time polymerase chain reaction (qPCR) (mRNA levels) and TCF/LEF reporter assay (βcatenin transcription) were used. Chromatin immunoprecipitation assay was performed with anti-CtBP2 antibody on spheroids. Results: Spheroid showed significant up regulation of CSCs factors (CD133, CXCR4, LGR5 and C-MYC), TCF/LEF activity as well as increased NADH/NAD ratio and enhanced binding of CtBP on p21 (a known CtBP target) promoter compared to monolayer controls. Depletion of CtBP2 inhibited, while its overexpression enhanced CSC growth (1° spheroid) and self-renewal (2°/3° spheroids). Similarly, MTOB caused a robust inhibition of CSC growth and self-renewal in a dose dependent manner (IC50: 250-700μM) in 4/5 cells tested. The corresponding IC50 for monolayer growth was 6-fold higher, suggesting selective targeting of CSCs. Moreover, MTOB treatment (1mM) caused robust apoptosis induction only in spheroids. Additionally, MTOB inhibited expression of CSCmarkers (CD133, CD44 and LGR5) and self-renewal factor (C-MYC) in spheroids. Moreover, MTOB inhibited basal as well as induced (GSK-3β inhibitor) TCF/LEF activity while inhibiting mRNA and protein levels of several β-catenin target genes (CD44, Snail, C-MYC and LGR5) and significantly reduced CtBP binding to LGR5 (a TCF/LEF target) and p21 promoters. Lastly, CtBP physically interacted with β-catenin. Conclusion: We have discovered a novel role of CtBP in promotion of CSC growth and self-renewal through direct regulation of TCF/LEF transcription. Moreover, small molecular inhibition of its function can selectively target CSCs, presenting a paradigm-shifting treatment approach for colorectal cancer.

Cftr Modulates Wnt/β-Catenin Signaling and Stem Cell Proliferation in Murine Intestine

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/β-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/β-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/β-catenin signaling by standard assays. Cftr in ISCs was assessed by immunoblot of sorted Sox9enhanced 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/β-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/β-catenin signaling. Loss of Cftr increases pHi in ISCs, which stabilizes the plasma membrane association of the Wnt transducer Dvl, likely facilitating Wnt/β-catenin signaling. Absence of Cftr-dependent suppression of ISC proliferation in the CF intestine may contribute to increased risk for intestinal tumors.