The electroneutral Na+-HCO3- cotransporter NBCn1 (SLC4A7) modulates colonic enterocyte pHi, proliferation and migration.

NBCn1 (SLC4A7) is one of the two major Na+-HCO3- cotransporters in the human colonic epithelium, expressed predominantly in the highly proliferating colonocytes at the cryptal base. Increased NBCn1 expression levels are reported in tumours, including colorectal cancer. The study explores its importance for maintenance of the intracellular pH (pHi),as well as the proliferative, adhesive, and migratory behavior of the self-differentiating Caco2bbe colonic tumour cell line. In the self-differentiating Caco2BBe cells, NBCn1 mRNA was highly expressed from the proliferative stage until full differentiation. The downregulation of NBCn1 expression by RNA interference affected proliferation and differentiation, and decreased intracellular pH (pHi)of the cells in correlation with the degree of knockdown. In addition, a disturbed cell adhesion and reduced migratory speed were associated with NBCn1 knockdown. Murine colonic Nbcn1-/- enteroids also displayed reduced proliferative activity. In the migrating Caco2BBe cells, NBCn1 was found at the leading edge and in colocalization with the focal adhesion markers vinculin and paxillin, which suggests that NBCn1 is involved in the establishment of cell-matrix adhesion. Our data highlight the physiological significance of NBCn1 in modulating epithelial pH-homeostasis and cell-matrix interactions in the proliferative region of the colonic epithelium, and unravel the molecular mechanism behind pathological overexpression of this transporter in human colorectal cancers.

Bicarbonate secretion and acid/base sensing by the intestine.

The transport of bicarbonate across the enterocyte cell membrane regulates the intracellular as well as the luminal pH and is an essential part of directional fluid movement in the gut. Since the first description of "active" transport of HCO3- ions against a concentration gradient in the 1970s, the fundamental role of HCO3- transport for multiple intestinal functions has been recognized. The ion transport proteins have been identified and molecularly characterized, and knockout mouse models have given insight into their individual role in a variety of functions. This review describes the progress made in the last decade regarding novel techniques and new findings in the molecular regulation of intestinal HCO3- transport in the different segments of the gut. We discuss human diseases with defects in intestinal HCO3- secretion and potential treatment strategies to increase luminal alkalinity. In the last part of the review, the cellular and organismal mechanisms for acid/base sensing in the intestinal tract are highlighted.

Reduced surface pH and upregulated AE2 anion exchange in SLC26A3-deleted polarized intestinal epithelial cells.

Loss of function mutations in the SLC26A3 gene cause chloride-losing diarrhea in mice and humans. Although systemic adaptive changes have been documented in these patients and in the corresponding knockout mice, how colonic enterocytes adapt to loss of this highly expressed and highly regulated luminal membrane anion exchanger remains unclear. To address this question, SLC26A3 was deleted in the self-differentiating Caco2BBe colonic cell line by the CRISPR/Cas9 technique. We selected a clone with loss of SLC26A3 protein expression and morphological features indistinguishable from those of the native cell line. Neither growth curves nor development of transepithelial electrical resistance (TEER) differed between wild-type (WT) and SLC26A3 knockout (KO) cells. Real-time qPCR and Western analysis in SLC26A3-KO cells revealed an increase in AE2 expression without significant change in NHE3 expression or localization. Steady-state pHi and apical and basolateral Cl-/HCO3- exchange activities were assessed fluorometrically in a dual perfusion chamber with independent perfusion of luminal and serosal baths. Apical Cl-/HCO3- exchange rates were strongly reduced in SLC26A3-KO cells, accompanied by a surface pH more acidic than that of WT cells. Steady-state pHi was not significantly different from that of WT cells, but basolateral Cl-/HCO3- exchange rates were higher in SLC26A3-KO than in WT cells. The data show that CRISPR/Cas9-mediated SLC26A3 deletion strongly reduced apical Cl-/HCO3- exchange rate and apical surface pH, but sustained a normal steady-state pHi due to increased expression and function of basolateral AE2. The low apical surface pH resulted in functional inhibition of NHE-mediated fluid absorption despite normal expression of NHE3 polypeptide.NEW & NOTEWORTHY SLC26A3 gene mutations cause chloride-losing diarrhea. To understand how colonic enterocytes adapt, SLC26A3 was deleted in Caco2BBe cells using CRISPR/Cas9. In comparison to the wild-type cells, SLC26A3 knockout cells showed similar growth and transepithelial resistance but substantially reduced apical Cl-/HCO3- exchange rates, and an acidic surface pH. Steady-state intracellular pH was comparable between the WT and KO cells due to increased basolateral AE2 expression and function.

The Role of Plasma Membrane Sodium/Hydrogen Exchangers in Gastrointestinal Functions: Proliferation and Differentiation, Fluid/Electrolyte Transport and Barrier Integrity.

The five plasma membrane Na+/H+ exchanger (NHE) isoforms in the gastrointestinal tract are characterized by distinct cellular localization, tissue distribution, inhibitor sensitivities, and physiological regulation. NHE1 (Slc9a1) is ubiquitously expressed along the gastrointestinal tract in the basolateral membrane of enterocytes, but so far, an exclusive role for NHE1 in enterocyte physiology has remained elusive. NHE2 (Slc9a2) and NHE8 (Slc9a8) are apically expressed isoforms with ubiquitous distribution along the colonic crypt axis. They are involved in pHi regulation of intestinal epithelial cells. Combined use of a knockout mouse model, intestinal organoid technology, and specific inhibitors revealed previously unrecognized actions of NHE2 and NHE8 in enterocyte proliferation and differentiation. NHE3 (Slc9a3), expressed in the apical membrane of differentiated intestinal epithelial cells, functions as the predominant nutrient-independent Na+ absorptive mechanism in the gut. The new selective NHE3 inhibitor (Tenapanor) allowed discovery of novel pathophysiological and drug-targetable NHE3 functions in cystic-fibrosis associated intestinal obstructions. NHE4, expressed in the basolateral membrane of parietal cells, is essential for parietal cell integrity and acid secretory function, through its role in cell volume regulation. This review focuses on the expression, regulation and activity of the five plasma membrane Na+/H+ exchangers in the gastrointestinal tract, emphasizing their role in maintaining intestinal homeostasis, or their impact on disease pathogenesis. We point to major open questions in identifying NHE interacting partners in central cellular pathways and processes and the necessity of determining their physiological role in a system where their endogenous expression/activity is maintained, such as organoids derived from different parts of the gastrointestinal tract.

Alterations of mucosa-attached microbiome and epithelial cell numbers in the cystic fibrosis small intestine with implications for intestinal disease.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Defective CFTR leads to accumulation of dehydrated viscous mucus within the small intestine, luminal acidification and altered intestinal motility, resulting in blockage. These changes promote gut microbial dysbiosis, adversely influencing the normal proliferation and differentiation of intestinal epithelial cells. Using Illumina 16S rRNA gene sequencing and immunohistochemistry, we assessed changes in mucosa-attached microbiome and epithelial cell profile in the small intestine of CF mice and a CF patient compared to wild-type mice and non-CF humans. We found increased abundance of pro-inflammatory Escherichia and depletion of beneficial secondary bile-acid producing bacteria in the ileal mucosa-attached microbiome of CFTR-null mice. The ileal mucosa in a CF patient was dominated by a non-aeruginosa Pseudomonas species and lacked numerous beneficial anti-inflammatory and short-chain fatty acid-producing bacteria. In the ileum of both CF mice and a CF patient, the number of absorptive enterocytes, Paneth and glucagon-like peptide 1 and 2 secreting L-type enteroendocrine cells were decreased, whereas stem and goblet cell numbers were increased. These changes in mucosa-attached microbiome and epithelial cell profile suggest that microbiota-host interactions may contribute to intestinal CF disease development with implications for therapy.

The Role of pHi in Intestinal Epithelial Proliferation-Transport Mechanisms, Regulatory Pathways, and Consequences.

During the maturation of intestinal epithelial cells along the crypt/surface axis, a multitude of acid/base transporters are differentially expressed in their apical and basolateral membranes, enabling processes of electrolyte, macromolecule, nutrient, acid/base and fluid secretion, and absorption. An intracellular pH (pHi)-gradient is generated along the epithelial crypt/surface axis, either as a consequence of the sum of the ion transport activities or as a distinctly regulated entity. While the role of pHi on proliferation, migration, and tumorigenesis has been explored in cancer cells for some time, emerging evidence suggests an important role of the pHi in the intestinal stem cells (ISCs) proliferative rate under physiological conditions. The present review highlights the current state of knowledge about the potential regulatory role of pHi on intestinal proliferation and differentiation.

Inhibition of Na+ /H+ exchanger isoform 3 improves gut fluidity and alkalinity in cystic fibrosis transmembrane conductance regulator-deficient and F508del mutant mice.

BACKGROUND AND PURPOSE: Constipation and intestinal obstructive episodes are major health problems in cystic fibrosis (CF) patients. Three FDA-approved drugs against constipation-prone irritable bowel syndrome were tested for their ability to increase luminal fluidity and alkalinity in cystic fibrosis transmembrane conductance regulator (CFTR) null (cftr-/- ) and F508del mutant (F508delmut/mut ) murine intestine. EXPERIMENTAL APPROACH: Guanylate cyclase C agonist linaclotide, PGE1 analogue lubiprostone and intestine-specific NHE3 inhibitor tenapanor were perfused through a ~3 cm jejunal, proximal or mid-distal colonic segment in anaesthetized cftr-/- , F508delmut/mut and WT mice. Net fluid balance was determined gravimetrically and alkaline output by pH-stat back titration. KEY RESULTS: Basal jejunal fluid absorptive rates were significantly higher and basal HCO3- output was significantly lower in cftr-/- and F508delmut/mut compared to WT mice. In cftr-/- and F508delmut/mut mice, all three drugs significantly inhibited the fluid absorptive rate and increased alkaline output in the jejunum and tenapanor and lubiprostone, but not linaclotide, in the colon. After tenapanor pre-incubation, linaclotide elicited a robust fluid secretory response in WT jejunum, while no further change in absorptive rates was observed in cftr-/- and F508delmut/mut jejunum, suggesting that the increase in gut fluidity and alkalinity by linaclotide in CF gut is mediated via NHE3 inhibition. Lubiprostone also inhibited fluid absorption in cftr-/- and F508delmut/mut jejunum via NHE3 inhibition but had a residual NHE3-independent effect. CONCLUSION AND IMPLICATIONS: Linaclotide, lubiprostone and tenapanor reduced fluid absorption and increased alkaline output in the CF gut. Their application may ameliorate constipation and reduce obstructive episodes in CF patients.

IL-1ß-Induced Downregulation of the Multifunctional PDZ Adaptor PDZK1 Is Attenuated by ERK Inhibition, RXRα, or PPARα Stimulation in Enterocytes.

Background: The PDZ adaptor protein PDZK1 modulates the membrane expression and function of a variety of intestinal receptors and ion/nutrient transporters. Its expression is strongly decreased in inflamed intestinal mucosa of mice and IBD patients. Aim and Methods: We investigated whether the inflammation-associated PDZK1 downregulation is a direct consequence of proinflammatory cytokine release by treating intestinal Caco-2BBE cells with TNF-α, IFN-γ, and IL-1ß, and analysing PDZK1 promotor activity, mRNA and protein expression. Results: IL-1ß was found to significantly decrease PDZK1 promoter activity, mRNA and protein expression in Caco-2BBE cells. A distal region of the hPDZK1 promoter was identified to be important for basal expression and IL-1ß-responsiveness. This region harbors the retinoid acid response element RARE as well as binding sites for transcription factors involved in IL-ß downstream signaling. ERK1/2 inhibition by the specific MEK1/2 inhibitors PD98059/U0126 significantly attenuated the IL-1ß mediated downregulation of PDZK1, while NF-κB, p38 MAPK, and JNK inhibition did not. Expression of the nuclear receptors RXRα and PPARα was decreased in inflamed colonic-mucosa of ulcerative colitis patients and in IL-1ß-treated Caco2-BBE cells. Moreover, the RAR/RXR ligand 9-cis retinoic acid and the PPARα-agonist GW7647 stimulated PDZK1 mRNA and protein expression and attenuated IL-1ß-mediated inhibition. Conclusions: The strong decrease in PDZK1 expression during intestinal inflammation may be in part a consequence of IL-1ß-mediated RXRα and PPARα repression and can be attenuated by agonists for either nuclear receptor, or by ERK1/2 inhibition. The negative consequences of inflammation-induced PDZK1 downregulation on epithelial transport-function may thus be amenable to pharmacological therapy.

Gastrointestinal HCO3- transport and epithelial protection in the gut: new techniques, transport pathways and regulatory pathways.

The concept of a protective alkaline gastric and duodenal mucus layer is a century old, yet it is amazing how much new information on HCO3(-) transport pathways has emerged recently, made possible by the extensive utilization of gene-deleted and transgenic mice and novel techniques to study HCO3(-) transport. This review highlights recent findings regarding the importance of HCO3(-) for mucosal protection of duodenum and other gastrointestinal epithelia against luminal acid and other damaging factors. Recently, methods have been developed to visualize HCO3(-) transport in vivo by assessing the surface pH in the mucus layer, as well as the epithelial pH. New information about HCO3(-) transport pathways, and emerging concepts about the intricate regulatory network that governs duodenal HCO3(-) secretion are described, and new perspectives for drug therapy discussed.

Secretagogue stimulation enhances NBCe1 (electrogenic Na(+)/HCO(3)(-) cotransporter) surface expression in murine colonic crypts.

A Na(+)/HCO(3)(-) cotransporter (NBC) is located in the basolateral membrane of the gastrointestinal epithelium, where it imports HCO(3)(-) during stimulated anion secretion. Having previously demonstrated secretagogue activation of NBC in murine colonic crypts, we now asked whether vesicle traffic and exocytosis are involved in this process. Electrogenic NBCe1-B was expressed at significantly higher levels than electroneutral NBCn1 in colonic crypts as determined by QRT-PCR. In cell surface biotinylation experiments, a time-dependent increase in biotinylated NBCe1 was observed, which occurred with a peak of +54.8% after 20 min with forskolin (P < 0.05) and more rapidly with a peak of +59.8% after 10 min with carbachol (P < 0.05) and which corresponded well with the time course of secretagogue-stimulated colonic bicarbonate secretion in Ussing chamber experiments. Accordingly, in isolated colonic crypts pretreated with forskolin and carbachol for 10 min, respectively, and subjected to immunohistochemistry, the NBCe1 signal showed a markedly stronger colocalization with the E-cadherin signal, which was used as a membrane marker, compared with the untreated control. Cytochalasin D did not change the observed increase in membrane abundance, whereas colchicine alone enhanced NBCe1 membrane expression without an additional increase after carbachol or forskolin, and LY294002 had a marked inhibitory effect. Taken together, our results demonstrate a secretagogue-induced increase of NBCe1 membrane expression. Vesicle traffic and exocytosis might thus represent a novel mechanism of intestinal NBC activation by secretagogues.

The C-terminus of the transmembrane mucin MUC17 binds to the scaffold protein PDZK1 that stably localizes it to the enterocyte apical membrane in the small intestine.

The membrane-bound mucins have a heavily O-glycosylated extracellular domain, a single-pass membrane domain and a short cytoplasmic tail. Three of the membrane-bound mucins,MUC3, MUC12 and MUC17, are clustered on chromosome 7 and found in the gastrointestinal tract. These mucins have C-terminal sequences typical of PDZ-domain-binding proteins. To identify PDZ proteins that are able to interact with the mucins,we screened PDZ domain arrays using YFP (yellow fluorescent protein)-tagged proteins. MUC17 exhibited a strong binding to PDZK1 (PDZ domain containing 1), whereas the binding toNHERF1 (Na+/H+-exchanger regulatory factor 1) was weak.Furthermore, we showed weak binding of MUC12 to PDZK1, NHERF1 and NHERF2. GST (glutathione transferase) pull-down experiments confirmed that the C-terminal tail of MUC17 coprecipitates with the scaffold protein PDZK1 as identified byMS. This was mediated through the C-terminal PDZ-interaction site in MUC17, which was capable of binding to three of the four PDZ domains in PDZK1. Immunostaining of wild-type or Pdzk1-/- mouse jejunum with an antiserum against Muc3(17),the mouse orthologue of human MUC17, revealed strong brushborder membrane staining in the wild-type mice compared with an intracellular Muc3(17) staining in the Pdzk1-/- mice. This suggests that Pdzk1 plays a specific role in stabilizing Muc3(17)in the apical membrane of small intestinal enterocytes.