Goblet cell hyperplasia is not epithelial-autonomous in the Cftr knockout intestine.

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.

Cellular chloride and bicarbonate retention alters intracellular pH regulation in Cftr KO crypt epithelium.

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.

Functional Cftr in crypt epithelium of organotypic enteroid cultures from murine small intestine.

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.

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.

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.

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.

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.

A BAC Transgene Expressing Human CFTR under Control of Its Regulatory Elements Rescues Cftr Knockout Mice.

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.

Cftr Modulates Wnt/ß-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/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.

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.

PAT-1 (Slc26a6) is the predominant apical membrane Cl-/HCO3- exchanger in the upper villous epithelium of the murine duodenum.

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.

Talniflumate increases survival in a cystic fibrosis mouse model of distal intestinal obstructive syndrome.

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).

Chloride conductance of CFTR facilitates basal Cl-/HCO3- exchange in the villous epithelium of intact murine duodenum.

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.

An alternate pathway of cAMP-stimulated Cl secretion across the NKCC1-null murine duodenum.

BACKGROUND & AIMS: Adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated anion secretion across the duodenal epithelium requires the cystic fibrosis transmembrane conductance regulator (CFTR) in the apical membrane and anion uptake proteins in the basolateral membrane. NKCC1, the epithelial Na(+)/K(+)/2Cl(-) cotransporter, is the major protein responsible for Cl(-) uptake. In this study, we evaluate the role of NKCC1 in determining the relative rates of transepithelial Cl(-) and HCO(3)(-) secretion during cAMP stimulation of the duodenum. METHODS: Bicarbonate and chloride secretion across duodenal mucosa was measured in Ussing chambers by pH stat and (36)Cl flux methods using mice with either gene-targeted deletion of NKCC1 (NKCC1-/-) or bumetanide blockade of NKCC1. RESULTS: Total anion secretion stimulated by forskolin treatment of NKCC1-null duodenum resulted from approximately equivalent rates of electrogenic chloride, electrogenic bicarbonate, and electroneutral bicarbonate secretion. Evaluation of the alternate chloride secretory pathway indicated chloride uptake by a basolateral membrane anion exchange process with characteristics consistent with the anion exchanger isoform AE2. CONCLUSIONS: Chloride uptake by basolateral anion exchanger activity (AE2) supports intracellular cAMP-stimulated chloride secretion in the NKCC1-null duodenum. A model for the alternate chloride secretion pathway is proposed whereby chloride uptake via AE2 is coupled to basolateral NaHCO(3) cotransport to support CFTR-mediated chloride and bicarbonate secretion.

A domain mimic increases DeltaF508 CFTR trafficking and restores cAMP-stimulated anion secretion in cystic fibrosis epithelia.

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.

Lateral intercellular space volume as a determinant of CFTR-mediated anion secretion across small intestinal mucosa.

Studies of full-thickness, small intestinal preparations have shown that maximal anion secretion [indexed by short-circuit current (I(sc))] during intracellular cAMP (cAMP(i)) stimulation is transient and followed by a decline toward baseline. Declining I(sc) is preceded by decreases in transepithelial conductance (G(t)), which in the small intestine reflects the lateral intercellular space (LIS) volume of the paracellular pathway. We hypothesized that decreases in LIS volume limit the magnitude and duration of cAMP(i)-stimulated anion secretion. Experimental manipulations to increase the patency of the LIS (assessed by G(t) and electron microscopy) were investigated for an effect on the magnitude of cAMP(i)-stimulated anion secretion (assessed by the I(sc) and isotopic fluxes) across murine small intestine. In control studies, changes of G(t) after cAMP(i) stimulation were associated with a morphological "collapse" of the LIS, which did not occur in intestine of CFTR-null mice. Removal of the outer intestinal musculature, exposure to a serosal hypertonic solution, or increased serosal hydrostatic pressure minimized reductions in G(t) and increased the cAMP(i)-stimulated I(sc) response. Increased I(sc) primarily resulted from increased Cl(-) secretion that was largely bumetanide sensitive. However, bumetanide-insensitive I(sc) was also increased, and similar increases occurred in the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1)-null intestine, indicating that activities of non-NKCC1 anion uptake proteins are also affected by LIS volume. Thus LIS patency is an important determinant of the magnitude and duration of CFTR-mediated anion secretion in murine small intestine. Decreases in LIS volume may limit the pool of available anions to basolateral transporters involved in transepithelial secretion.

Intestinal NaCl transport in NHE2 and NHE3 knockout mice.

Sodium/proton exchangers [Na(+)/H(+) (NHEs)] play an important role in salt and water absorption from the intestinal tract. To investigate the contribution of the apical membrane NHEs, NHE2 and NHE3, to electroneutral NaCl absorption, we measured radioisotopic Na(+) and Cl(-) flux across isolated jejuna from wild-type [NHE(+)], NHE2 knockout [NHE2(-)], and NHE3 knockout [NHE3(-)] mice. Under basal conditions, NHE(+) and NHE2(-) jejuna had similar rates of net Na(+) (approximately 6 microeq/cm(2) x h) and Cl(-) (approximately 3 microeq/cm(2) x h) absorption. In contrast, NHE3(-) jejuna had reduced net Na(+) absorption (approximately 2 microeq/cm(2) x h) but absorbed Cl(-) at rates similar to NHE(+) and NHE2(-) jejuna. Treatment with 100 microM 5-(N-ethyl-N-isopropyl) amiloride (EIPA) completely inhibited net Na(+) and Cl(-) absorption in all genotypes. Studies of the Na(+) absorptive flux (J) indicated that J in NHE(+) jejunum was not sensitive to 1 microM EIPA, whereas J in NHE3(-) jejunum was equally sensitive to 1 and 100 microM EIPA. Treatment with forskolin/IBMX to increase intracellular cAMP (cAMP(i)) abolished net NaCl absorption and stimulated electrogenic Cl(-) secretion in all three genotypes. Quantitative RT-PCR of epithelia from NHE2(-) and NHE3(-) jejuna did not reveal differences in mRNA expression of NHE3 and NHE2, respectively, when compared with jejunal epithelia from NHE(+) siblings. We conclude that 1) NHE3 is the dominant NHE involved in small intestinal Na(+) absorption; 2) an amiloride-sensitive Na(+) transporter partially compensates for Na(+) absorption in NHE3(-) jejunum; 3) cAMP(i) stimulation abolishes net Na(+) absorption in NHE(+), NHE2(-), and NHE3(-) jejunum; and 4) electroneutral Cl(-) absorption is not directly dependent on either NHE2 or NHE3.

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.

Abnormal Paneth cell granule dissolution and compromised resistance to bacterial colonization in the intestine of CF mice.

Paneth cells of intestinal crypts contribute to host defense by producing antimicrobial peptides that are packaged as granules for secretion into the crypt lumen. Here, we provide evidence using light and electron microscopy that postsecretory Paneth cell granules undergo limited dissolution and accumulate within the intestinal crypts of cystic fibrosis (CF) mice. On the basis of this finding, we evaluated bacterial colonization and expression of two major constituents of Paneth cells, i.e., alpha-defensins (cryptdins) and lysozyme, in CF murine intestine. Paneth cell granules accumulated in intestinal crypt lumens in both untreated CF mice with impending intestinal obstruction and in CF mice treated with an osmotic laxative that prevented overt clinical symptoms and mucus accretion. Ultrastructure studies indicated little change in granule morphology within mucus casts, whereas granules in laxative-treated mice appear to undergo limited dissolution. Protein extracts from CF intestine had increased levels of processed cryptdins compared with those from wild-type (WT) littermates. Nonetheless, colonization with aerobic bacteria species was not diminished in the CF intestine and oral challenge with a cryptdin-sensitive enteric pathogen, Salmonella typhimurium, resulted in greater colonization of CF compared with WT intestine. Modest downregulation of cryptdin and lysozyme mRNA in CF intestine was shown by microarray analysis, real-time quantitative PCR, and Northern blot analysis. Based on these findings, we conclude that antimicrobial peptide activity in CF mouse intestine is compromised by inadequate dissolution of Paneth cell granules within the crypt lumens.