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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. [ABSTRACT FROM AUTHOR]

• CFTR inhibition by toxic factors is common in everyday life and may exacerbate pancreatitis. • CF-associated decrease in intestinal luminal fluidity may be ameliorated by apical Na+/ H + exchange inhibition. • Loss of CFTR function may reduce renal HCO 3 −secretion, causing metabolic alkalosis and reducing ventilatory drive. With increased longevity of patients suffering from cystic fibrosis, and widespread lung transplantation facilities, the sequelae of defective CFTR in other organs than the airways come to the fore. This minireview highlights recent scientific progress in the understanding of CFTR function in the pancreas, the intestine and the kidney, and explores potential therapeutic strategies to combat defective CFTR function in these organs. [Display omitted] [ABSTRACT FROM AUTHOR]

Background: Several members of the SLC26A family of transporters, including SLC26A3 (DRA), SLC26A5 (prestin), SLC26A6 (PAT-1; CFEX) and SLC26A9, form multi-protein complexes with a number of molecules (e.g., cytoskeletal proteins, anchoring or adaptor proteins, cystic fibrosis transmembrane conductance regulator, and protein kinases). These interactions provide regulatory signals for these molecules. However, the identity of proteins that interact with the Cl−/HCO3− exchanger, SLC26A4 (pendrin), have yet to be determined. The purpose of this study is to identify the protein(s) that interact with pendrin.Methods: A yeast two hybrid (Y2H) system was employed to screen a mouse kidney cDNA library using the C-terminal fragment of SLC26A4 as bait. Immunofluorescence microscopic examination of kidney sections, as well as co-immunoprecipitation assays, were performed using affinity purified antibodies and kidney protein extracts to confirm the co-localization and interaction of pendrin and the identified binding partners. Co-expression studies were carried out in cultured cells to examine the effect of binding partners on pendrin trafficking and activity.Results: The Y2H studies identified IQ motif-containing GTPase-activating protein 1 (IQGAP1) as a protein that binds to SLC26A4’s C-terminus. Co-immunoprecipitation experiments using affinity purified anti-IQGAP1 antibodies followed by western blot analysis of kidney protein eluates using pendrin-specific antibodies confirmed the interaction of pendrin and IQGAP1. Immunofluorescence microscopy studies demonstrated that IQGAP1 co-localizes with pendrin on the apical membrane of B-intercalated cells, whereas it shows basolateral expression in A-intercalated cells in the cortical collecting duct (CCD). Functional and confocal studies in HEK-293 cells, as well as confocal studies in MDCK cells, demonstrated that the co-transfection of pendrin and IQGAP1 shows strong co-localization of the two molecules on the plasma membrane along with enhanced Cl−/HCO3− exchanger activity.Conclusion: IQGAP1 was identified as a protein that binds to the C-terminus of pendrin in B-intercalated cells. IQGAP1 co-localized with pendrin on the apical membrane of B-intercalated cells. Co-expression of IQGAP1 with pendrin resulted in strong co-localization of the two molecules and increased the activity of pendrin in the plasma membrane in cultured cells. We propose that pendrin’s interaction with IQGAP1 may play a critical role in the regulation of CCD function and physiology, and that disruption of this interaction could contribute to altered pendrin trafficking and/or activity in pathophysiologic states.

The nonselective anion exchanger Slc26a6, also known as putative anion transporter 1 and chloride/formate exchanger, is thought to play a major role in HCO3- transport in exocrine glands. In this study, Slc26a6 null mice were used to explore the function of Slc26a6 in the exocrine pancreas. Slc26a6 primarily localized to the apical membrane of pancreatic exocrine acinar cells. The volume of stimulated juice secretion by the ex vivo pancreas was significantly reduced ~35% in Slc26a6-/- mice, but no changes occurred in the gross structure or gland weights of Slc26a6 null mice. The secretion of pancreatic juice by Slc26a6+/+ mice was dependent on HCO3- while, in contrast, fluid secretion by Slc26a6-/- mice was independent of HCO3-, suggesting that Slc26a6 mediates the HCO3--dependent component of fluid secretion. Consistent with these observations, disruption of Slc26a6 also significantly reduced HCO3- secretion by the pancreas ~35%. Taken together, these results demonstrate that the apical Slc26a6 anion exchanger in acinar cells is involved in HCO3--dependent fluid secretion but that another major HCO3--independent pathway is the primary driver of the fluid secretion process in the mouse pancreas. [ABSTRACT FROM AUTHOR]

Background: Although ClC-2 channels are important in colonic Cl- secretion, it is unclear about their roles in small intestinal anion secretion. Therefore, we sought to examine whether ClC-2 channels play important roles in anion secretion, particularly duodenal bicarbonate secretion (DBS).Methods: Duodenal mucosae from mice were stripped of seromuscular layers and mounted in Ussing chambers. Both duodenal short-circuit current (Isc) and HCO3- secretion in vitro were simultaneously recorded. DBS in vivo was measured by a CO2-sensitive electrode.Results: Lubiprostone, a selective ClC-2 activator, concentration-dependently increased both duodenal Isc and DBS only when applied basolaterally, but not when applied apically. Removal of extracellular Cl- abolished lubiprostone-induced duodenal Isc, but did not alter HCO3- secretion even in the presence of DIDS, a Cl-/HCO3- exchanger inhibitor. However, further addition of glibenclamide, a CFTR channel blocker, abolished lubiprostone-evoked HCO3- secretion. Moreover, lubiprostone-induced HCO3- secretion was impaired in CFTR-/- mice compared to wild-type littermates. Luminal perfusion of duodenal lumen with lubiprostone did not alter basal DBS in vivo, but lubiprostone (i.p.) was able to induce DBS, which was also significantly inhibited by Cd2+, a ClC-2 channel blocker. [Ca2+]cyt level, Ca2+-activated K+ channel- and cAMP-mediated duodenal Isc, and HCO3- secretion were unchanged by lubiprostone.Conclusions: We have provided the first evidence for the novel functional role of basolateral ClC-2 channels in the regulation of duodenal anion secretion. [ABSTRACT FROM AUTHOR]

Pancreatic ductular epithelial cells comprise the majority of duct cells in pancreas, control cystic fibrosis transmembrane conductance regulator (CFTR)-dependent bicarbonate ([Formula: see text]) secretion, but are difficult to grow as a polarized monolayer. Using NIH-3T3-J2 fibroblast feeder cells and a Rho-associated kinase inhibitor, we produced well-differentiated and polarized porcine pancreatic ductular epithelial cells. Cells grown on semipermeable filters at the air-liquid interface developed typical epithelial cell morphology and stable transepithelial resistance and expressed epithelial cell markers (zona occludens-1 and β-catenin), duct cell markers (SOX-9 and CFTR), but no acinar (amylase) or islet cell (chromogranin) markers. Polarized cells were studied in Ussing chambers bathed in Krebs-Ringer [Formula: see text] solution at 37°C gassed with 5% CO2 to measure short-circuit currents ( Isc). Ratiometric measurement of extracellular pH was performed with fluorescent SNARF-conjugated dextran at 5% CO2. Cells demonstrated a baseline Isc (12.2 ± 3.2 μA/cm2) that increased significantly in response to apical forskolin-IBMX (∆ Isc: 35.4 ± 3.8 μA/cm2, P < 0.001) or basolateral secretin (∆ Isc: 31.4 ± 2.5 μA/cm2, P < 0.001), both of which increase cellular levels of cAMP. Subsequent addition of apical GlyH-101, a CFTR inhibitor, decreased the current (∆ Isc: 20.4 ± 3.8 μA/cm2, P < 0.01). Extracellular pH and [Formula: see text] concentration increased significantly after forskolin-IBMX (pH: 7.18 ± 0.23 vs. 7.53 ± 0.19; [Formula: see text] concentration, 14.5 ± 5.9 vs. 31.8 ± 13.4 mM; P < 0.05 for both). We demonstrate the development of a polarized pancreatic ductular epithelial cell epithelium with CFTR-dependent [Formula: see text] secretion in response to secretin and cAMP. This model is highly relevant, as porcine pancreas physiology is very similar to humans and pancreatic damage in the cystic fibrosis pig model recapitulates that of humans. NEW & NOTEWORTHY Pancreas ductular epithelial cells control cystic fibrosis transmembrane conductance regulator (CFTR)-dependent bicarbonate secretion. Their function is critical because when CFTR is deficient in cystic fibrosis bicarbonate secretion is lost and the pancreas is damaged. Mechanisms that control pancreatic bicarbonate secretion are incompletely understood. We generated well-differentiated and polarized porcine pancreatic ductular epithelial cells and demonstrated feasibility of bicarbonate secretion. This novel method will advance our understanding of pancreas physiology and mechanisms of bicarbonate secretion. [ABSTRACT FROM AUTHOR]

The diffuse chemosensory system (DCS) is well developed in the apparatuses of endodermal origin like gastrointestinal (GI) tract. The primary function of the GI tract is the extraction of nutrients from the diet. Therefore, the GI tract must possess an efficient surveillance system that continuously monitors the luminal contents for beneficial or harmful compounds. Recent studies have shown that specialized cells in the intestinal lining can sense changes in the luminal content. The chemosensory cells in the GI tract belong to the DCS which consists of enteroendocrine and related cells. These cells initiate various important local and remote reflexes. Although neural and hormonal involvements in ion transport in the GI tract are well documented, involvement of the DCS in the regulation of intestinal ion transport is much less understood. Since activation of luminal chemosensory receptors is a primary signal that elicits changes in intestinal ion transport and motility and failure of the system causes dysfunctions in host homeostasis, as well as functional GI disorders, study of the regulation of GI function by the DCS has become increasingly important. This review discusses the role of the DCS in epithelial ion transport, with particular emphasis on the involvement of free fatty acid receptor 2 (FFA2) and free fatty acid receptor 3 (FFA3). [ABSTRACT FROM AUTHOR]

Serotonin (5-HT), predominantly synthesized and released by enterochromaffin cells, is implicated in gastrointestinal symptoms such as emesis, abdominal pain, and diarrhea. Because luminal short-chain fatty acids (SCFAs) release 5-HT from enterochromaffin cells, which express the SCFA receptor free fatty acid receptor 2 (FFA2) in rat duodenum, we examined the effects of the selective FFA2 agonist phenylacetamide-1 (PA1) on duodenal 5-HT release with consequent bicarbonate secretion [duodenal bicarbonate secretion (DBS)] and on indomethacin (IND)-induced enteropathy. Intestinal injury was induced by IND (10 mg/kg sc) with or without PA1. We measured DBS in vivo in a duodenal loop perfused with PA1 while measuring 5-HT released in the portal vein. Duodenal blood flow was measured by laser-Doppler flowmetry. IND induced small intestinal ulcers with duodenal sparing. PA1 given with IND (IND + PA1) dose dependently induced duodenal erosions. IND + PA1-induced duodenal lesions were inhibited by the FFA2 antagonist GLPG-0974, ondansetron, or omeprazole but not by RS-23597 or atropine. Luminal perfusion of PA1 augmented DBS accompanied by increased portal blood 5-HT concentrations with approximately eight times more release at 0.1 mM than at 1 μM, with the effects inhibited by coperfusion of GLPG-0974. Luminal PA1 at 1 μM increased, but at 0.1 mM diminished, duodenal blood flow. Cosuperfusion of PA1 (0.1 mM) decreased acid-induced hyperemia, further reduced by IND pretreatment but restored by ondansetron. These results suggest that, although FFA2 activation enhances duodenal mucosal defenses, FFA2 overactivation during ulcerogenic cyclooxygenase inhibition may increase the vulnerability of the duodenal mucosa to gastric acid via excessive 5-HT release and 5-HT3 receptor activation, implicated in foregut-related symptoms such as emesis and epigastralgia. [ABSTRACT FROM AUTHOR]

Osmoregulation in fish is a complex process that requires the orchestrated cooperation of many tissues. In fish facing hyperosmotic environments, the intestinal absorption of some monovalent ions and the secretion of bicarbonate are key processes to favor water absorption. In the present study, we showed that bicarbonate levels in the intestinal fluid are several fold higher in seawater than in freshwater acclimated tilapia ( Oreochromis mossambicus ). In addition, we analyzed gene expression of the main molecular mechanisms involved in HCO 3 – movements i.e. slc26a6 , slc26a3 , slc4a4 and v-type H-ATPase sub C in the intestine of tilapia acclimated to both seawater and freshwater. Our results show an anterior/posterior functional regionalization of the intestine in tilapia in terms of expression patterns, which is affected by environmental salinity mostly in the anterior and mid intestine. Analysis of bicarbonate secretion using pH-Stat in tissues mounted in Ussing chambers reveals high rates of bicarbonate secretion in tilapia acclimated to seawater from anterior intestine to rectum ranging between ~ 900 and ~ 1700 nmol HCO 3 – cm − 2 h − 1 . However, a relationship between the expression of slc26a6 , slc26a3 , slc4a4 and the rate of bicarbonate secretion seems to be compromised in the rectum. In this region, the low expression of the bicarbonate transporters could not explain the high bicarbonate secretion rates here described. However, we postulate that the elevated v-type H-ATPase mRNA expression in the rectum could be involved in this process. [ABSTRACT FROM AUTHOR]

Key points The present study suggests that the electrogenic sodium-bicarbonate cotransporter, NBCe1, supported by carbonic anhydrase II, CAII, provides an efficient mechanism of bicarbonate sensing in cortical astrocytes. This mechanism is proposed to play a major role in setting the pHi responses to extracellular acid/base challenges in astrocytes., A decrease in extracellular [HCO3−] during isocapnic acidosis and isohydric hypocapnia, or an increase in intracellular [HCO3−] during hypercapnic acidosis, was effectively sensed by NBCe1, which carried bicarbonate out of the cells under these conditions, and caused an acidification and sodium fall in WT astrocytes, but not in NBCe1-knockout astrocytes., Isocapnic acidosis, hypercapnic acidosis and isohydric hypocapnia evoked inward currents in NBCe1- and CAII-expressing Xenopus laevis oocytes, but not in native oocytes, suggesting that NBCe1 operates in the outwardly directed mode under these conditions consistent with our findings in astrocytes., We propose that bicarbonate sensing of astrocytes may have functional significance during extracellular acid/base disturbances in the brain, as it not only alters intracellular pH/[HCO3−]-dependent functions of astrocytes, but also modulates the extracellular pH/[HCO3−] in brain tissue., Abstract Extracellular acid/base status of the mammalian brain undergoes dynamic changes during many physiological and pathological events. Although intracellular pH (pHi) of astrocytes responds to extracellular acid/base changes, the mechanisms mediating these changes have remained unresolved. We have previously shown that the electrogenic sodium-bicarbonate cotransporter, NBCe1, is a high-affinity bicarbonate carrier in cortical astrocytes. In the present study, we investigated whether NBCe1 plays a role in bicarbonate sensing in astrocytes, and in determining the pHi responses to extracellular acid/base challenges. We measured changes in intracellular H+ and Na+ in astrocytes from wild-type (WT) and from NBCe1-knockout (KO) mice, using ion-selective dyes, during isocapnic acidosis, hypercapnic acidosis and hypocapnia. We also analysed NBCe1-mediated membrane currents in Xenopus laevis oocytes under similar conditions. Comparing WT and NBCe1-KO astrocytes, we could dissect the contribution of NBCe1, of diffusion of CO2 across the cell membrane and, after blocking carbonic anhydrase (CA) activity with ethoxyzolamide, of the role of CA, for the amplitude and rate of acid/base fluxes. Our results suggest that NBCe1 transport activity in astrocytes, supported by CA activity, renders astrocytes bicarbonate sensors in the mouse cortex. NBCe1 carried bicarbonate into and out of the cell by sensing the variations of transmembrane [HCO3−], irrespective of the changes in intra- and extracellular pH, and played a major role in setting pHi responses to the extracellular acid/base challenges. We propose that bicarbonate sensing of astrocytes may have potential functional significance during extracellular acid/base alterations in the brain. [ABSTRACT FROM AUTHOR]

Key points The ductal system of the pancreas secretes large volumes of alkaline fluid containing HCO3− concentrations as high as 140 m m during hormonal stimulation., A computational model has been constructed to explore the underlying ion transport mechanisms. Parameters were estimated by fitting the model to experimental data from guinea-pig pancreatic ducts., The model was readily able to secrete 140 m m HCO3−. Its capacity to do so was not dependent upon special properties of the cystic fibrosis transmembrane conductance regulator (CFTR) anion channels and solute carrier family 26 member A6 (SLC26A6) anion exchangers., We conclude that the main requirement for secreting high HCO3− concentrations is to minimize the secretion of Cl− ions., These findings help to clarify the mechanism responsible for pancreatic HCO3− secretion, a vital process that prevents the formation of protein plugs and viscous mucus in the ducts, which could otherwise lead to pancreatic disease., Abstract A computational model of guinea-pig pancreatic duct epithelium was developed to determine the transport mechanism by which HCO3− ions are secreted at concentrations in excess of 140 m m. Parameters defining the contributions of the individual ion channels and transporters were estimated by least-squares fitting of the model predictions to experimental data obtained from isolated ducts and intact pancreas under a range of experimental conditions. The effects of cAMP-stimulated secretion were well replicated by increasing the activities of the basolateral Na+-HCO3− cotransporter (NBC1) and apical Cl−/HCO3− exchanger (solute carrier family 26 member A6; SLC26A6), increasing the basolateral K+ permeability and apical Cl− and HCO3− permeabilities (CFTR), and reducing the activity of the basolateral Cl−/HCO3− exchanger (anion exchanger 2; AE2). Under these conditions, the model secreted ∼140 m m HCO3− at a rate of ∼3 nl min−1 mm−2, which is consistent with experimental observations. Alternative 1:2 and 1:1 stoichiometries for Cl−/HCO3− exchange via SLC26A6 at the apical membrane were able to support a HCO3−-rich secretion. Raising the HCO3−/Cl− permeability ratio of CFTR from 0.4 to 1.0 had little impact upon either the secreted HCO3− concentration or the volume flow. However, modelling showed that a reduction in basolateral AE2 activity by ∼80% was essential in minimizing the intracellular Cl− concentration following cAMP stimulation and thereby maximizing the secreted HCO3− concentration. The addition of a basolateral Na+-K+-2Cl− cotransporter (NKCC1), assumed to be present in rat and mouse ducts, raised intracellular Cl− and resulted in a lower secreted HCO3− concentration, as is characteristic of those species. We conclude therefore that minimizing the driving force for Cl− secretion is the main requirement for secreting 140 m m HCO3−. [ABSTRACT FROM AUTHOR]

Background The mechanisms for Helicobacter pylori ( H. pylori)-induced duodenal ulcerogenesis are not fully understood. In this study, we investigated the effects of H. pylori infection on the expressions and functional activities of human duodenal mucosal bicarbonate transport proteins and hope to further clarify the pathogenesis of H. pylori-associated duodenal ulcer. Materials and Methods The experiments were performed in the patients with H. pylori-associated duodenal ulcers, H. pylori-associated chronic gastritis, and H. pylori-negative healthy subjects. Duodenal mucosal bicarbonate secretion was measured by Ussing Chamber technology. Results The expressions of duodenal mucosal bicarbonate transport proteins, CFTR (cystic fibrosis transmembrane conductance regulator) and SLC26A6 (solute-linked carrier 26 gene A6), in the patients with H. pylori-associated duodenal ulcers were markedly lower than those in healthy controls. Basal and both forskolin- and prostaglandin E2-stimulated duodenal mucosal bicarbonate secretions in the patients with H. pylori-associated duodenal ulcers were also lower than those in healthy controls. After anti- H. pylori treatment for H. pylori-associated duodenal ulcers, duodenal mucosal bicarbonate secretion and CFTR and SLC26A6 expressions in H. pylori-eradicated patients recovered to levels comparable to healthy controls, but those were found to be not significantly altered in non- H. pylori-eradicated patients. The further results showed that decreases in the H. pylori-induced CFTR and SLC26A6 expression were related to the severity and virulent factors of H. pylori infection. Conclusion H. pylori infection impairs the expressions and functional activities of duodenal mucosal bicarbonate transport proteins, CFTR and SLC26A6, which contributes to the development of duodenal ulcer. [ABSTRACT FROM AUTHOR]

Intracellular pH (pH) regulation fundamentally participates in maintaining HCO release from HCO -secreting epithelia. We used parotid intralobular ducts loaded with BCECF to investigate the contributions of a carbonic anhydrase (CA), anion channels and a Na-H exchanger (NHE) to pH regulation for HCO secretion by cAMP and Ca signals. Resting pH was dispersed between 7.4 and 7.9. Forskolin consistently decreased pH showing the dominance of pH-lowering activities, but carbachol gathered pH around 7.6. CA inhibition suppressed the forskolin-induced decrease in pH, while it allowed carbachol to consistently increase pH by revealing that carbachol prominently activated NHE via Ca-calmodulin. Under NHE inhibition, forskolin and carbachol induced the remarkable decreases in pH, which were slowed predominantly by CA inhibition and by CA or anion channel inhibition, respectively. Our results suggest that forskolin and carbachol primarily activate the pH-lowering CA and pH-raising NHE, respectively, to regulate pH for HCO secretion. [ABSTRACT FROM AUTHOR]

Application of mild irritants (1 M NaCl; pH 2.0) on the gastric mucosa potentiates the protective secretion of bicarbonates by epithelial cells. This response is mainly mediated by capsaicin-sensitive afferent nerve endings located in the submucosa. It was shown that activation of vanilloid type 1 receptors (TRPV1) induced by exogenous acidification of GM is not sufficient to potentiate the production of HCO3, including production depending on neuronal NO synthase. However, the effect of exogenous acid on TRPV1 leads to activation of endothelial NO synthase that restrict the gastric secretion of HCO 3 − . [ABSTRACT FROM AUTHOR]

The diffuse chemosensory system (DCS) is well developed in the apparatuses of endodermal origin like gastrointestinal (GI) tract. The primary function of the GI tract is the extraction of nutrients from the diet. Therefore, the GI tract must possess an efficient surveillance system that continuously monitors the luminal contents for beneficial or harmful compounds. Recent studies have shown that specialized cells in the intestinal lining can sense changes in the luminal content. The chemosensory cells in the GI tract belong to the DCS which consists of enteroendocrine and related cells. These cells initiate various important local and remote reflexes. Although neural and hormonal involvements in ion transport in the GI tract are well documented, involvement of the DCS in the regulation of intestinal ion transport is much less understood. Since activation of luminal chemosensory receptors is a primary signal that elicits changes in intestinal ion transport and motility and failure of the system causes dysfunctions in host homeostasis, as well as functional GI disorders, study of the regulation of GI function by the DCS has become increasingly important. This review discusses the role of the DCS in epithelial ion transport, with particular emphasis on the involvement of free fatty acid receptor 2 (FFA2) and free fatty acid receptor 3 (FFA3).

The pH of airway epithelial secretions influences bacterial killing and mucus properties and is reduced by acidic pollutants, gastric reflux, and respiratory diseases such as cystic fibrosis (CF). The effect of acute acid loads depends on buffer capacity, however the buffering of airway secretions has not been well characterized. In this work we develop a method for titrating micro-scale (30 µl) volumes and use it to study fluid secreted by the human airway epithelial cell line Calu-3, a widely used model for submucosal gland serous cells. Microtitration curves revealed that HCO3- is the major buffer. Peak buffer capacity (β) increased from 17 to 28 mM/pH during forskolin stimulation, and was reduced by >50% in fluid secreted by cystic fibrosis transmembrane conductance regulator (CFTR)-deficient Calu-3 monolayers, confirming an important role of CFTR in HCO3- secretion. Back-titration with NaOH revealed non-volatile buffer capacity due to proteins synthesized and released by the epithelial cells. Lysozyme and mucin concentrations were too low to buffer Calu-3 fluid significantly, however model titrations of porcine gastric mucins at concentrations near the sol-gel transition suggest that mucins may contribute to the buffer capacity of ASL in vivo. We conclude that CFTR-dependent HCO3- secretion and epithelially-derived proteins are the predominant buffers in Calu-3 secretions.

Background Pancreatic ductal HCO 3 - secretion is critically dependent on the cystic fibrosis transmembrane conductance regulator chloride channel (CFTR) and the solute-linked carrier 26 member 6 anion transporter (SLC26A6). Deterioration of HCO 3 - secretion is observed in chronic pancreatitis (CP), and CFTR mutations increase CP risk. Therefore, SLC26A6 is a reasonable candidate for a CP susceptibility gene, which has not been investigated in CP patients so far. Methods As a first screening cohort, 106 subjects with CP and 99 control subjects with no pancreatic disease were recruited from the Hungarian National Pancreas Registry. In 60 non-alcoholic CP cases the entire SLC26A6 coding region was sequenced. In the Hungarian cohort variants c.616G > A (p.V206M) and c.1191C > A (p.P397=) were further genotyped by restriction fragment length polymorphism analysis. In a German replication cohort all exons were sequenced in 40 non-alcoholic CP cases and variant c.616G > A (p.V206M) was further analyzed by sequencing in 321 CP cases and 171 controls. Results Sequencing of the entire coding region revealed four common variants: intronic variants c.23 + 78_110del, c.183-4C > A, c.1134 + 32C > A, and missense variant c.616G > A (p.V206M) which were found in linkage disequilibrium indicating a conserved haplotype. The distribution of the haplotype did not show a significant difference between patients and controls in the two cohorts. A synonymous variant c.1191C > A (p.P397=) and two intronic variants c.1248 + 9_20del and c.-10C > T were detected in single cases. Conclusion Our data show that SLC26A6 variants do not alter the risk for the development of CP. [ABSTRACT FROM AUTHOR]

Slc26a9 is an anion transporter that is strongly expressed in the stomach and lung. Slc26a9 variants were recently found associated with a higher incidence of meconium ileus in cystic fibrosis (CF) infants, raising the question whether Slc26a9 is expressed in the intestine and what its functional role is. Slc26a9 messenger RNA (mRNA) was found highly expressed in the mucosae of the murine and human upper gastrointestinal tract, with an abrupt decrease in expression levels beyond the duodenum. Absence of SLC26a9 expression strongly increased the intestinally related mortality in cystic fibrosis transmembrane conductance regulator (CFTR)-deficient mice. Proximal duodenal J and fluid secretion were reduced in the absence of Slc26a9 expression. In the proximal duodenum of young Slc26a9 KO mice, the glands and villi/crypts were elongated and proliferation was enhanced. This difference was lost with ageing, as were the alterations in fluid movement, whereas the reduction in J remained. Laser dissection followed by qPCR suggested Slc26a9 expression to be crypt-predominant in the duodenum. In summary, deletion of Slc26a9 caused bicarbonate secretory and fluid absorptive changes in the proximal duodenal mucosa and increased the postweaning death rates in CFTR-deficient mice. Functional alterations in the duodenum were most prominent at young ages. We assume that the association of meconium ileus and Slc26a9 variants may be related to maldigestion and impaired downstream signaling caused by loss of upper GI tract digestive functions, aggravating the situation of lack of secretion and sticky mucus at the site of obstruction in CF intestine. [ABSTRACT FROM AUTHOR]

Colonic bicarbonate (HCO3-) secretion is a well-established physiological process that is closely linked to overall fluid and electrolyte movement in the mammalian colon. These present studies show that extracellular calcium-sensing receptor (CaSR), a fundamental mechanism for sensing and regulating ionic and nutrient compositions of extracellular milieu in the small and large intestine, regulates HCO3- secretion. Basal and induced HCO3- secretory responses to CaSR agonists were determined by pH stat techniques used in conjunction with shortcircuit current measurements in mucosa from rat distal colon mounted in Ussing chambers. R568, a specific CaSR activator, stimulated lumen Cl- and short-chain fatty acid (SCFA)-dependent HCO3- secretion but inhibited cyclic nucleotide-activated HCO3- secretion. Consequently, at physiological conditions (either at basal or during lumen acid challenge) when electroneutral Cl/HCO3- and SCFA/ HCO3- exchangers dominate, CaSR stimulates HCO3- secretion; in contrast, in experimental conditions that stimulate fluid and HCO3- secretion, e.g., when forskolin activates electrogenic cystic fibrosis transmembrane conductance regulator-mediated HCO3- conductance, CaSR activation inhibits HCO3- secretion. Corresponding changes in JHCO3 (μeq·h-1·cm-2, absence vs. presence of R568) were 0.18 ± 0.03 vs. 0.31 ± 0.08 under basal nonstimulated conditions and 1.85 ± 0.23 vs. 0.45 ± 0.06 under forskolin-stimulated conditions. Similarly, activation of CaSR by R568 stimulated Cl-- and SCFA-dependent HCO3- secretion and inhibited cAMP-dependent HCO3- secretion in colon mucosa of wild-type mice; such effects were abolished in CaSR-null mice. These results suggest a new paradigm for regulation of intestinal ion transport in which HCO3- secretion may be fine-tuned by CaSR in accordance with nutrient availability and state of digestion and absorption. The ability of CaSR agonists to inhibit secretagogueinduced intestinal HCO3- secretion suggests that modulation of CaSR activity may provide a new therapeutic approach to correct HCO3- deficit and metabolic acidosis, a primary cause of morbidity and mortality in acute infectious diarrheal illnesses. [ABSTRACT FROM AUTHOR]

Water ingestion by drinking is fundamental for ion homeostasis in marine fish. However, the fluid ingested requires processing to allow net water absorption in the intestine. The formation of luminal carbonate aggregates impacts on calcium homeostasis and requires epithelial HCO 3 − secretion to enable water absorption. In light of its endocrine importance in calcium handling and the indication of involvement in HCO 3 − secretion the present study was designed to expose the role of the parathyroid hormone-related protein (PTHrP) in HCO 3 − secretion, water absorption and the regulation of aqp1 gene expression in the anterior intestine of the sea bream. HCO 3 − secretion rapidly decreased when PTHrP(1–34) was added to anterior intestine of the sea bream mounted in Ussing chambers. The effect achieved a maximum inhibition of 60% of basal secretion rates, showing a threshold effective dose of 0.1 ng ml −1 compatible with reported plasma values of PTHrP. When applied in combination with the adenylate cyclase inhibitor (SQ 22.536, 100 μmol l −1 ) or the phospholipase C inhibitor (U73122, 10 μmol l −1 ) the effect of PTHrP(1–34) on HCO 3 − secretion was reduced by about 50% in both cases. In parallel, bulk water absorption measured in intestinal sacs was sensitive to inhibition by PTHrP. The inhibitory action conforms to a typical dose–response curve in the range of 0.1–1000 ng ml −1 , achieves a maximal effect of 60–65% inhibition from basal rates and shows threshold significant effects at hormone levels of 0.1 ng ml −1 . The action of PTHrP in water absorption was completely abolished in the presence of the adenylate cyclase inhibitor (SQ 22.536, 100 μmol l −1 ) and was insensitive to the phospholipase C inhibitor (U73122, 10 μmol l −1 ). In vivo injections of PTHrP(1–34) or the PTH/PTHrP receptor antagonist PTHrP(7–34) evoked respectively, a significant decrease or increase of aqp1ab , but not aqp1a . Overall the present results suggest that PTHrP acts as a key regulator of carbonate aggregate formation in the intestine of marine fish via its actions on water absorption, calcium regulation and HCO 3 − secretion. [ABSTRACT FROM AUTHOR]

The gastric epithelium is protected from the highly acidic luminal content by alkaline mucus which is secreted from specialized epithelial cells. In the stomach of mice strong secretion of alkaline fluid was observed at the gastric groove, the border between corpus and fundus mucosa. Since this region is characterized by numerous brush cells it was proposed that these cells might secrete alkaline solution as suggested for brush cells in the bile duct. In fact, it was found that in this region multiple cells express elements which are relevant for the secretion of bicarbonate, including carbonic anhydrase (CAII), the cystic fibrosis transmembrane conductance regulator (CFTR) and the Na+/H+ exchanger (NHE1). However, this cell population was distinct from brush cells which express the TRP-channel TRPM5 and are considered as putative sensory cells. The location of both cell populations in close proximity implies the possibility for a paracrine interaction. This view was substantiated by the finding that brush cells express prostaglandin synthase-1 (COX-1) and the neighbouring cells a specific receptor type for prostaglandins. The notion that brush cells may be able to sense a local acidification was supported by the observation that they express the channel PKD1L3 which contributes to the acid responsiveness of gustatory sensory cells. The results support the concept that brush cells may sense the luminal content and influence via prostaglandins the secretion of alkaline solution.

Chronic pancreatitis (CP) is a progressive inflammatory disease in which the pancreatic secretory parenchyma is destroyed and replaced by fibrosis. The presence of intraductal pancreatic stone(s) is important for the diagnosis of CP; however, the precise molecular mechanisms of pancreatic stone formation in CP were left largely unknown. CFTR is a chloride channel expressed in the apical plasma membrane of pancreatic duct cells and plays a central role in HCO3- secretion. In previous studies, we have found that CFTR is largely mislocalized to the cytoplasm of pancreatic duct cells in all forms of CP and corticosteroids normalizes the localization of CFTR to the proper apical membrane at least in autoimmune pancreatitis. From these observations, we could conclude that the mislocalization of CFTR is a cause of protein plug formation in CP, subsequently resulting in pancreatic stone formation.Considering our observation that the mislocalization of CFTR also occurs in alcoholic or idiopathic CP, it is very likely that these pathological conditions can also be treated by corticosteroids, thereby preventing pancreatic stone formation in these patients. Further studies are definitely required to clarify these fundamental issues.

Marine teleost fish secrete bicarbonate (HCO) into the intestine to aid osmoregulation and limit Ca uptake by carbonate precipitation. Intestinal HCO secretion is associated with an equimolar transport of protons (H) into the blood, both being proportional to environmental salinity. We hypothesized that the H-sensitive haemoglobin (Hb) system of seawater teleosts could be exploited via the Bohr and/or Root effects (reduced Hb-O affinity and/or capacity with decreasing pH) to improve O delivery to intestinal cells during high metabolic demand associated with osmoregulation. To test this, we characterized H equilibria and gas exchange properties of European flounder ( Platichthys flesus) haemoglobin and constructed a model incorporating these values, intestinal blood flow rates and arterial-venous acidification at three different environmental salinities (33, 60 and 90). The model suggested red blood cell pH (pH) during passage through intestinal capillaries could be reduced by 0.14-0.33 units (depending on external salinity) which is sufficient to activate the Bohr effect (Bohr coefficient of −0.63), and perhaps even the Root effect, and enhance tissue O delivery by up to 42 % without changing blood flow. In vivo measurements of intestinal venous blood pH were not possible in flounder but were in seawater-acclimated rainbow trout which confirmed a blood acidification of no less than 0.2 units (equivalent to −0.12 for pH). When using trout-specific values for the model variables, predicted values were consistent with measured in vivo values, further supporting the model. Thus this system is an elegant example of autoregulation: as the need for costly osmoregulatory processes (including HCO secretion) increases at higher environmental salinity, so does the enhancement of O delivery to the intestine via a localized acidosis and the Bohr (and possibly Root) effect. [ABSTRACT FROM AUTHOR]

The pH of airway epithelial secretions influences bacterial killing and mucus properties and is reduced by acidic pollutants, gastric reflux, and respiratory diseases such as cystic fibrosis (CF). The effect of acute acid loads depends on buffer capacity, however the buffering of airway secretions has not been well characterized. In this work we develop a method for titrating micro-scale (30 μl) volumes and use it to study fluid secreted by the human airway epithelial cell line Calu-3, a widely used model for submucosal gland serous cells. Microtitration curves revealed that HCO-3 is the major buffer. Peak buffer capacity (β) increased from 17 to 28 mM/pH during forskolin stimulation, and was reduced by >50% in fluid secreted by cystic fibrosis transmembrane conductance regulator (CFTR)-deficient Calu-3 monolayers, confirming an important role of CFTR in HCO-3 secretion. Back-titration with NaOH revealed non-volatile buffer capacity due to proteins synthesized and released by the epithelial cells. Lysozyme and mucin concentrations were too low to buffer Calu-3 fluid significantly, however model titrations of porcine gastric mucins at concentrations near the sol-gel transition suggest that mucins may contribute to the buffer capacity of ASL in vivo. We conclude that CFTR-dependent HCO-3 secretion and epithelially-derived proteins are the predominant buffers in Calu-3 secretions. [ABSTRACT FROM AUTHOR]

In marine fish, high epithelial bicarbonate secretion by the intestine generates luminal carbonate precipitates of divalent cations that play a key role in water and ion homeostasis. In vitro studies highlight the involvement of the calciotropic hormones PTHrP (parathyroid hormone-related protein) and stanniocalcin (STC) in the regulation of epithelial bicarbonate transport. The present study tested the hypothesis that calciotropic hormones have a regulatory role in carbonate precipitate formation in vivo. Sea bream (Sparus aurata) juveniles received single intraperitoneal injections of piscine PTHrP(1-34), the PTH/PTHrP receptor antagonist PTHrP(7-34) or purified sea bream STC, or were passively immunized with polyclonal rabbit antisera raised against sea bream STC (STC-Ab). Endocrine effects on the expression of the basolateral sodium bicarbonate cotransporter (Slc4a4.A), the apical anion exchangers Slc26a6.A and Slc26a3.B, and the V-type proton pump ß-subunit (Atp6v1b) in the anterior intestine were evaluated. In keeping with their calciotropic nature, the hypocalcaemic factors PTHrP(7-34) and STC upregulated gene expression of all transporters. In contrast, the hypercalcaemic factor PTHrP(1-34) and STC antibodies downregulated transporters involved in the bicarbonate secretion cascade. Changes in intestine luminal precipitate contents provoked by calcaemic endocrine factors validated these results: 24 h postinjection either PTHrP(1-34) or immunization with STC-Ab reduced the carbonate precipitate content in the sea bream intestine. In contrast, the PTH/PTHrP receptor antagonist PTHrP(7-34) increased not only the precipitated fraction but also the concentration of HCO3 - equivalents in the intestinal fluid. These results confirm the hypothesis that calciotropic hormones have a regulatory role in carbonate precipitate formation in vivo in the intestine of marine fish. Furthermore, they illustrate for the first time in fish the counteracting effect of PTHrP and STC, and reveal an unexpected contribution of calcaemic factors to acid-base balance. [ABSTRACT FROM AUTHOR]

The gastric epithelium is protected from the highly acidic luminal content by alkaline mucus which is secreted from specialized epithelial cells. In the stomach of mice strong secretion of alkaline fluid was observed at the "gastric groove," the border between corpus and fundus mucosa. Since this region is characterized by numerous brush cells it was proposed that these cells might secrete alkaline solution as suggested for brush cells in the bile duct. In fact, it was found that in this region multiple cells express elements which are relevant for the secretion of bicarbonate, including carbonic anhydrase (CAII), the cystic fibrosis transmembrane conductance regulator (CFTR) and the Na+/H+ exchanger (NHE1). However, this cell population was distinct from brush cells which express the TRP-channel TRPM5 and are considered as putative sensory cells. The location of both cell populations in close proximity implies the possibility for a paracrine interaction. This view was substantiated by the finding that brush cells express prostaglandin synthase-1 (COX-1) and the neighboring cells a specific receptor type for prostaglandins. The notion that brush cells may be able to sense a local acidification was supported by the observation that they express the channel PKD1L3 which contributes to the acid responsiveness of gustatory sensory cells. The results support the concept that brush cells may sense the luminal content and influence via prostaglandins the secretion of alkaline solution. [ABSTRACT FROM AUTHOR]

Background and Purpose Our recent studies on human airway serous-like Calu-3 cells showed that cAMP agonists stimulated a HCO3− rich secretion containing up to 80 mM HCO3−. This alkaline secretion relied on a coordinated switch in the activity of distinct Cl−- HCO3− anion exchangers ( AE) located at different regions of the cell. At the apical membrane, cAMP agonists activated the electroneutral AE pendrin ( SLC26A4), together with cystic fibrosis transmembrane conductance regulator ( CFTR), while at the basolateral membrane the agonists inhibited AE2 ( SLC4A2). However, the underlying mechanism(s) that orchestrates this cAMP-dependent switch in AE activity has not been elucidated. Experimental Approach Apical and basolateral Cl−- HCO3− exchange was assessed by measuring Cl−-dependent changes in intracellular pH ( pHi). Key Results We show that protein phosphatase 1 ( PP1), together with CFTR, play central roles in this reciprocal regulation of AE activity. Activation of pendrin by cAMP agonists, but not inhibition of the basolateral exchanger, was protein kinase A-dependent. Knocking down CFTR expression, or blocking its activity with GlyH-101, led to incomplete inhibition of the basolateral AE by cAMP, supporting a role for CFTR in this process. Addition of the PP1/ 2A inhibitor, okadaic acid, but not the PP2A specific inhibitor fostreicin, mimicked the effect of cAMP stimulation. Furthermore, okadaic acid-treated Calu-3 monolayers produced a more alkaline fluid than untreated cells, which was comparable with that produced by cAMP stimulation. Conclusions and Implications These results identify PP1 as a novel regulator of AE activity which, in concert with CFTR, coordinates events at both apical and basolateral membranes, crucial for efficient HCO3− secretion from Calu-3 cells. [ABSTRACT FROM AUTHOR]

Background and Purpose To test a hypothesis that: (i) duodenal pH and osmolarity are individually controlled at constant set points by negative feedback control centred in the enteric nervous system ( ENS); (ii) the purinergic P2Y1 receptor subtype is expressed by non-cholinergic secretomotor/vasodilator neurons, which represent the final common excitatory pathway from the ENS to the bicarbonate secretory glands. Experimental Approach Ussing chamber and pH-stat methods investigated involvement of the P2Y1 receptor in neurogenic stimulation of mucosal bicarbonate ( HCO3−) secretion in guinea pig duodenum. Key Results ATP increased HCO3− secretion with an EC50 of 160 nM. MRS2179, a selective P2Y1 purinergic receptor antagonist, suppressed ATP-evoked HCO3− secretion by 47% and Cl− secretion by 63%. Enteric neuronal blockade by tetrodotoxin or exposure to a selective vasoactive intestinal peptide ( VIP, VPAC1) receptor antagonist suppressed ATP-evoked HCO3− secretion by 61 and 41%, respectively, and Cl- by 97 and 70% respectively. Pretreatment with the muscarinic antagonist, scopolamine did not alter ATP-evoked HCO3− or Cl− secretion. Conclusion and Implications Whereas acid directly stimulates the mucosa to release ATP and stimulate HCO3− secretion in a cytoprotective manner, neurogenically evoked HCO3− secretion accounts for feedback control of optimal luminal pH for digestion. ATP stimulates duodenal HCO3− secretion through an excitatory action at purinergic P2Y1 receptors on neurons in the submucosal division of the ENS. Stimulation of the VIPergic non-cholinergic secretomotor/vasodilator neurons, which are one of three classes of secretomotor neurons, accounts for most, if not all, of the neurogenic secretory response evoked by ATP. [ABSTRACT FROM AUTHOR]

Background: Several members of the SLC26A family of transporters, including SLC26A3 (DRA), SLC26A5 (prestin), SLC26A6 (PAT-1; CFEX) and SLC26A9, form multi-protein complexes with a number of molecules (e.g., cytoskeletal proteins, anchoring or adaptor proteins, cystic fibrosis transmembrane conductance regulator, and protein kinases). These interactions provide regulatory signals for these molecules. However, the identity of proteins that interact with the Cl - /HCO 3 - exchanger, SLC26A4 (pendrin), have yet to be determined. The purpose of this study is to identify the protein(s) that interact with pendrin. Methods: A yeast two hybrid (Y2H) system was employed to screen a mouse kidney cDNA library using the C-terminal fragment of SLC26A4 as bait. Immunofluorescence microscopic examination of kidney sections, as well as co-immunoprecipitation assays, were performed using affinity purified antibodies and kidney protein extracts to confirm the co-localization and interaction of pendrin and the identified binding partners. Co-expression studies were carried out in cultured cells to examine the effect of binding partners on pendrin trafficking and activity. Results: The Y2H studies identified IQ motif-containing GTPase-activating protein 1 (IQGAP1) as a protein that binds to SLC26A4's C-terminus. Co-immunoprecipitation experiments using affinity purified anti-IQGAP1 antibodies followed by western blot analysis of kidney protein eluates using pendrin-specific antibodies confirmed the interaction of pendrin and IQGAP1. Immunofluorescence microscopy studies demonstrated that IQGAP1 co-localizes with pendrin on the apical membrane of B-intercalated cells, whereas it shows basolateral expression in A-intercalated cells in the cortical collecting duct (CCD). Functional and confocal studies in HEK-293 cells, as well as confocal studies in MDCK cells, demonstrated that the co-transfection of pendrin and IQGAP1 shows strong co-localization of the two molecules on the plasma membrane along with enhanced Cl - /HCO 3 - exchanger activity. Conclusion: IQGAP1 was identified as a protein that binds to the C-terminus of pendrin in B-intercalated cells. IQGAP1 co-localized with pendrin on the apical membrane of B-intercalated cells. Co-expression of IQGAP1 with pendrin resulted in strong co-localization of the two molecules and increased the activity of pendrin in the plasma membrane in cultured cells. We propose that pendrin's interaction with IQGAP1 may play a critical role in the regulation of CCD function and physiology, and that disruption of this interaction could contribute to altered pendrin trafficking and/or activity in pathophysiologic states., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Xu, Barone, Varasteh Kia, Holliday, Zahedi and Soleimani.)

In the marine fish intestine luminal, HCO can remove divalent ions (calcium and magnesium) by precipitation in the form of carbonate aggregates. The process of epithelial HCO secretion is under endocrine control, therefore, in this study we aimed to characterize the involvement of transmembrane (tmACs) and soluble (sACs) adenylyl cyclases on the regulation of bicarbonate secretion (BCS) and water absorption in the intestine of the sea bream ( Sparus aurata). We observed that all sections of sea bream intestine are able to secrete bicarbonate as measured by pH-Stat in Ussing chambers. In addition, gut sac preparations reveal net water absorption in all segments of the intestine, with significantly higher absorption rates in the anterior intestine that in the rectum. BCS and water absorption are positively correlated in all regions of the sea bream intestinal tract. Furthermore, stimulation of tmACs (10 μM FK + 500 μM IBMX) causes a significant decrease in BCS, bulk water absorption and short circuit current (Isc) in a region dependent manner. In turn, stimulation of sACs with elevated HCO results in a significant increase in BCS, and bulk water absorption in the anterior intestine, an action completely reversed by the sAC inhibitor KH7 (200 μM). Overall, the results reveal a functional relationship between BCS and water absorption in marine fish intestine and modulation by tmACs and sAC. In light of the present observations, it is hypothesized that the endocrine effects on intestinal BCS and water absorption mediated by tmACs are locally and reciprocally modulated by the action of sACs in the fish enterocyte, thus fine-tuning the process of carbonate aggregate production in the intestinal lumen. [ABSTRACT FROM AUTHOR]

The pituitary hormone prolactin is a pleiotropic endocrine factor that plays a major role in the regulation of ion balance in fish, with demonstrated actions mainly in the gills and kidney. The role of prolactin in intestinal ion transport remains little studied. In marine fish, which have high drinking rates, epithelial bicarbonate secretion in the intestine produces luminal carbonate aggregates believed to play a key role in water and ion homeostasis. The present study was designed to establish the putative role of prolactin in the regulation of intestinal bicarbonate secretion in a marine fish. Basolateral addition of prolactin to the anterior intestine of sea bream mounted in Ussing chambers caused a rapid (<20min) decrease of bicarbonate secretion measured by pH-stat. A clear inhibitory dose-response curve was obtained, with a maximal inhibition of 60-65% of basal bicarbonate secretion. The threshold concentration of prolactin for a significant effect on bicarbonate secretion was 10ngml-1, which is comparable with putative plasma levels in seawater fish. The effect of prolactin on apical bicarbonate secretion was independent of the generation route for bicarbonate, as shown in a preparation devoid of basolateral HC03-/C02 buffer. Specific inhibitors of JAK2 (AG-490, 50μmoll-1), PI3K (LY-294002, 75μmoll-1) or MEK (U-012610, 10μmoll-1) caused a 50-70% reduction in the effect of prolactin on bicarbonate secretion, and demonstrated the involvement of prolactin receptors. In addition to rapid effects, prolactin has actions at the genomic level. Incubation of intestinal explants of anterior intestine of the sea bream in vitro for 3h demonstrated a specific effect of prolactin on the expression of the Slc4a4A Na+-HC03- co-transporter, but not on the Slc26a6A or Slc26a3B Cl-HC03- exchanger. We propose a new role for prolactin in the regulation of bicarbonate secretion, an essential function for ion/water homeostasis in the intestine of marine fish. [ABSTRACT FROM AUTHOR]

Chronic pancreatitis (CP) is a progressive inflammatory disease in which the pancreatic secretory parenchyma is destroyed and replaced by fibrosis. The presence of intraductal pancreatic stone(s) is important for the diagnosis of CP; however, the precise molecular mechanisms of pancreatic stone formation in CP were left largely unknown. CFTR is a chloride channel expressed in the apical plasma membrane of pancreatic duct cells and plays a central role in HCO3 secretion. In previous studies, we have found that CFTR is largely mislocalized to the cytoplasm of pancreatic duct cells in all forms of CP and corticosteroids normalizes the localization of CFTR to the proper apical membrane at least in autoimmune pancreatitis. From these observations, we could conclude that the mislocalization of CFTR is a cause of protein plug formation in CP, subsequently resulting in pancreatic stone formation. Considering our observation that the mislocalization of CFTR also occurs in alcoholic or idiopathic CP, it is very likely that these pathological conditions can also be treated by corticosteroids, thereby preventing pancreatic stone formation in these patients. Further studies are definitely required to clarify these fundamental issues. [ABSTRACT FROM AUTHOR]

Marine fish drink seawater and eliminate excess salt by active salt transport across gill and gut epithelia. Euryhaline pufferfish (Takifugu obscurus, mefugu) forms a CaCO3 precipitate on the luminal gut surface after transitioning to seawater. NBCe1 (Slc4a4) at the basolateral membrane of intestinal epithelial cell plays a major role in transepithelial intestinal HCO3 - secretion and is critical for mefugu acclimation to seawater. We assayed fugu-NBCe1 (fNBCe1) activity in the Xenopus oocyte expression system. Similar to NBCe1 found in other species, fNBCe1 is an electrogenic Na+/HCO3- cotransporter and sensitive to the stilbene inhibitor DIDS. However, our experiments revealed several unique and distinguishable fNBCe1 transport characteristics not found in mammalian or other teleost NBCe1-orthologs: electrogenic Li+/nHCO3- cotransport; HCO3- independent, DIDS-insensitive transport; and increased basal intracellular Na+ accumulation. fNBCe1 is a voltage-dependent Na+/nHCO3- cotransporter that rectifies, independently from the extracellular Na+ or HCO3- concentration, around -60 mV. Na+ removal (0Na+ prepulse) is necessary to produce the true HCO3--elicited current. HCO3- addition results in huge outward currents with quick current decay. Kinetic analysis of HCO3- currents reveals that fNBCe1 has a much higher transport capacity (higher maximum current) and lower affinity (higher Km) than human kidney NBCe1 (hkNBCe1) does in the physiological range (membrane potential = -80 mV; [HCO3-] = 10 mM). In this state, fNBCe1 is in favor of operating as transepithelial HCO3- secretion, opposite of hkNBCe1, from blood to the luminal side. Thus, fugu-NBCe1 represents the first ortholog-based tool to study amino acid substitutions in NBCe1 and how those change ion and voltage dependence. [ABSTRACT FROM AUTHOR]