SLC4A2 anion exchanger promotes tumour cell malignancy via enhancing net acid efflux across golgi membranes.

Proper functioning of each secretory and endocytic compartment relies on its unique pH micro-environment that is known to be dictated by the rates of V-ATPase-mediated H+ pumping and its leakage back to the cytoplasm via an elusive "H+ leak" pathway. Here, we show that this proton leak across Golgi membranes is mediated by the AE2a (SLC4A2a)-mediated bicarbonate-chloride exchange, as it is strictly dependent on bicarbonate import (in exchange for chloride export) and the expression level of the Golgi-localized AE2a anion exchanger. In the acidic Golgi lumen, imported bicarbonate anions and protons then facilitate a common buffering reaction that yields carbon dioxide and water before their egress back to the cytoplasm via diffusion or water channels. The flattened morphology of the Golgi cisternae helps this process, as their high surface-volume ratio is optimal for water and gas exchange. Interestingly, this net acid efflux pathway is often upregulated in cancers and established cancer cell lines, and responsible for their markedly elevated Golgi resting pH and attenuated glycosylation potential. Accordingly, AE2 knockdown in SW-48 colorectal cancer cells was able to restore these two phenomena, and at the same time, reverse their invasive and anchorage-independent growth phenotype. These findings suggest a possibility to return malignant cells to a benign state by restoring Golgi resting pH.

Unique Regulation of Intestinal Villus Epithelial Cl-/HCO3- Exchange by Cyclooxygenase Pathway Metabolites of Arachidonic Acid in a Mouse Model of Spontaneous Ileitis.

Electrolytes (NaCl) and fluid malabsorption cause diarrhea in inflammatory bowel disease (IBD). Coupled NaCl absorption, mediated by Na+/H+ and Cl-/HCO3- exchanges on the intestinal villus cells brush border membrane (BBM), is inhibited in IBD. Arachidonic acid metabolites (AAMs) formed via cyclooxygenase (COX) or lipoxygenase (LOX) pathways are elevated in IBD. However, their effects on NaCl absorption are not known. We treated SAMP1/YitFc (SAMP1) mice, a model of spontaneous ileitis resembling human IBD, with Arachidonyl Trifluoro Methylketone (ATMK, AAM inhibitor), or with piroxicam or MK-886, to inhibit COX or LOX pathways, respectively. Cl-/HCO3- exchange, measured as DIDS-sensitive 36Cl uptake, was significantly inhibited in villus cells and BBM vesicles of SAMP1 mice compared to AKR/J controls, an effect reversed by ATMK. Piroxicam, but not MK-886, also reversed the inhibition. Kinetic studies showed that inhibition was secondary to altered Km with no effects on Vmax. Whole cell or BBM protein levels of Down-Regulated in Adenoma (SLC26A3) and putative anion transporter-1 (SLC26A6), the two key BBM Cl-/HCO3- exchangers, were unaltered. Thus, inhibition of villus cell Cl-/HCO3- exchange by COX pathway AAMs, such as prostaglandins, via reducing the affinity of the exchanger for Cl-, and thereby causing NaCl malabsorption, could significantly contribute to IBD-associated diarrhea.

Intracellular Ca2+-Mediated AE2 Is Involved in the Vectorial Movement of HaCaT Keratinocyte.

Keratinocyte migration is initiated toward the wound skin barrier as a crucial process in wound healing. However, the migratory machinery used by keratinocytes is relatively unknown. Histamine signaling, including an increase in the Ca2+ signal, mediated the enhanced protein expression and chloride/bicarbonate exchange activity of anion exchanger AE2 in keratinocytes. In this study, we applied an agarose spot assay to induce a vectorial motion. The vectorial stimulation of the histamine-containing agarose spot enhanced the HaCaT keratinocyte migration, compared to non-directional stimulation. AE2 is associated with the vectorial movement of HaCaT keratinocytes. Enhanced expression of AE2 was mainly associated with an increase in Ca2+ and was abolished by the treatment with the Ca2+ chelating agent BAPTA-AM. These findings revealed that the directionality of Ca2+-exerted stimulation can play a prominent role in facilitating migration through the involvement of AE2 as a migratory machinery in HaCaT keratinocytes.

Decreased SLC26A3 expression and function in intestinal epithelial cells in response to Cryptosporidium parvum infection.

The protozoan parasite Cryptosporidium parvum (CP) causes cryptosporidiosis, a diarrheal disease worldwide. Infection in immunocompetent hosts typically results in acute, self-limiting, or recurrent diarrhea. However, in immunocompromised individuals infection can cause fulminant diarrhea, extraintestinal manifestations, and death. To date, the mechanisms underlying CP-induced diarrheal pathogenesis are poorly understood. Diarrheal diseases most commonly involve increased secretion and/or decreased absorption of fluid and electrolytes. We and others have previously shown impaired chloride absorption in infectious diarrhea due to dysregulation of SLC26A3 [downregulated in adenoma (DRA)], the human intestinal apical membrane Cl-/HCO3- exchanger protein. However, there are no studies on the effects of CP infection on DRA activity. Therefore, we examined the expression and function of DRA in intestinal epithelial cells in response to CP infection in vitro and in vivo. CP infection (0.5 × 106 oocysts/well in 24-well plates, 24 h) of Caco-2 cell monolayers significantly decreased Cl-/HCO3- exchange activity (measured as DIDS-sensitive 125I uptake) as well as DRA mRNA and protein levels. Substantial downregulation of DRA mRNA and protein was also observed following CP infection ex vivo in mouse enteroid-derived monolayers and in vivo in the ileal and jejunal mucosa of C57BL/6 mice for 24 h. However, at 48 h after infection in vivo, the effects on DRA mRNA and protein were attenuated and at 5 days after infection DRA returned to normal levels. Our results suggest that impaired chloride absorption due to downregulation of DRA could be one of the contributing factors to CP-induced acute, self-limiting diarrhea in immunocompetent hosts.

Targeting the anion exchanger 2 with specific peptides as a new therapeutic approach in B lymphoid neoplasms.

Regulatory T (Treg) cells can weaken antitumor immune responses, and inhibition of their function appears to be a promising therapeutic approach in cancer patients. Mice with targeted deletion of the gene encoding the Cl-/HCO3- anion exchanger AE2 (also termed SLC4A2), a membrane-bound carrier involved in intracellular pH regulation, showed a progressive decrease in the number of Treg cells. We therefore challenged AE2 as a potential target for tumor therapy, and generated linear peptides designed to bind the third extracellular loop of AE2, which is crucial for its exchange activity. Peptide p17AE2 exhibited optimal interaction ability and indeed promoted apoptosis in mouse and human Treg cells, while activating effector T-cell function. Interestingly, this linear peptide also induced apoptosis in different types of human leukemia, lymphoma and multiple myeloma cell lines and primary malignant samples, while it showed only moderate effects on normal B lymphocytes. Finally, a macrocyclic AE2 targeting peptide exhibiting increased stability in vivo was effective in mice xenografted with B-cell lymphoma. These data suggest that targeting the anion exchanger AE2 with specific peptides may represent an effective therapeutic approach in B-cell malignancies.

EPEC effector EspF promotes Crumbs3 endocytosis and disrupts epithelial cell polarity.

Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system to inject effector proteins into host intestinal epithelial cells causing diarrhoea. EPEC infection redistributes basolateral proteins ß1-integrin and Na+ /K+ ATPase to the apical membrane of host cells. The Crumbs (Crb) polarity complex (Crb3/Pals1/Patj) is essential for epithelial cell polarisation and tight junction (TJ) assembly. Here, we demonstrate that EPEC displaces Crb3 and Pals1 from the apical membrane to the cytoplasm of cultured intestinal epithelial cells and colonocytes of infected mice. In vitro studies show that EspF, but not Map, alters Crb3, whereas both effectors modulate Pals1. EspF perturbs polarity formation in cyst morphogenesis assays and induces endocytosis and apical redistribution of Na+ /K+ ATPase. EspF binds to sorting nexin 9 (SNX9) causing membrane remodelling in host cells. Infection with ΔespF/pespFD3, a mutant strain that ablates EspF binding to SNX9, or inhibition of dynamin, attenuates Crb3 endocytosis caused by EPEC. In addition, infection with ΔespF/pespFD3 has no impact on Na+ /K+ ATPase endocytosis. These data support the hypothesis that EPEC perturbs apical-basal polarity in an EspF-dependent manner, which would contribute to EPEC-associated diarrhoea by disruption of TJ and altering the crucial positioning of membrane transporters involved in the absorption of ions and solutes.

Inhibitors of pendrin anion exchange identified in a small molecule screen increase airway surface liquid volume in cystic fibrosis.

Pendrin (SLC26A4) is a Cl(-)/anion exchanger expressed in the epithelium of inflamed airways where it is thought to facilitate Cl(-) absorption and HCO3 (-) secretion. Studies using pendrin knockout mice and airway epithelial cells from hearing-impaired subjects with pendrin loss of function suggest involvement of pendrin in inflammatory lung diseases, including cystic fibrosis (CF), perhaps by regulation of airway surface liquid (ASL) volume. Here we identified small-molecule pendrin inhibitors and demonstrated their efficacy in increasing ASL volume. A cell-based, functional high-throughput screen of ∼36,000 synthetic small molecules produced 3 chemical classes of inhibitors of human pendrin. After structure-activity studies, tetrahydropyrazolopyridine and pyrazolothiophenesulfonamide compounds reversibly inhibited pendrin-facilitated Cl(-) exchange with SCN(-), I(-), NO3 (-), and HCO3 (-) with drug concentration causing 50% inhibition down to ∼2.5 µM. In well-differentiated primary cultures of human airway epithelial cells from non-CF and CF subjects, treatment with IL-13, which causes inflammation with strong pendrin up-regulation, strongly increased Cl(-)/HCO3 (-) exchange and the increase was blocked by pendrin inhibition. Pendrin inhibition significantly increased ASL depth (by ∼8 µm) in IL-13-treated non-CF and CF cells but not in untreated cells. These studies implicate the involvement of pendrin-facilitated Cl(-)/HCO3 (-) in the regulation of ASL volume and suggest the utility of pendrin inhibitors in inflammatory lung diseases, including CF.-Haggie, P. M., Phuan, P.-W., Tan, J.-A., Zlock, L., Finkbeiner, W. E., Verkman, A. S. Inhibitors of pendrin anion exchange identified in a small molecule screen increase airway surface liquid volume in cystic fibrosis.

Role of anion exchangers in Cl- and HCO3- secretion by the human airway epithelial cell line Calu-3.

Despite the importance of airway surface liquid pH in the lung's defenses against infection, the mechanism of airway HCO3- secretion remains unclear. Our aim was to assess the contribution of apical and basolateral Cl-/HCO3- exchangers to Cl- and HCO3- transport in the Calu-3 cell line, derived from human airway submucosal glands. Changes in intracellular pH (pHi) were measured following substitution of Cl- with gluconate. Apical Cl- substitution led to an alkalinization in forskolin-stimulated cells, indicative of Cl-/HCO3- exchange. This was unaffected by the anion exchange inhibitor DIDS but inhibited by the CFTR blocker CFTRinh-172, suggesting that the HCO3- influx might occur via CFTR, rather than a solute carrier family 26 (SLC26) exchanger, as recently proposed. The anion selectivity of the recovery process more closely resembled that of CFTR than an SLC26 exchanger, and quantitative RT-PCR showed only low levels of SLC26 exchanger transcripts relative to CFTR and anion exchanger 2 (AE2). For pHi to rise to observed values (∼7.8) through HCO3- entry via CFTR, the apical membrane potential must reverse to at least +20 mV following Cl- substitution; this was confirmed by perforated-patch recordings. Substitution of basolateral Cl- evoked a DIDS-sensitive alkalinization, attributed to Cl-/HCO3- exchange via AE2. This appeared to be abolished in forskolin-stimulated cells but was unmasked by blocking apical efflux of HCO3- via CFTR. We conclude that Calu-3 cells secrete HCO3- predominantly via CFTR, and, contrary to previous reports, the basolateral anion exchanger AE2 remains active during stimulation, providing an important pathway for basolateral Cl- uptake.

Translational repression of SLC26A3 by miR-494 in intestinal epithelial cells.

SLC26A3 [downregulated in adenoma (DRA)] is a Cl(-)/HCO3(-) exchanger involved in electroneutral NaCl absorption in the mammalian intestine. Altered DRA expression levels are associated with infectious and inflammatory diarrheal diseases. Therefore, it is critical to understand the regulation of DRA expression. MicroRNAs (miRNAs) are endogenous, small RNAs that regulate protein expression via blocking the translation and/or promoting mRNA degradation. To investigate potential modulation of DRA expression by miRNA, five different in silico algorithms were used to predict the miRNAs that target DRA. Of these miRNAs, miR-494 was shown to have a highly conserved putative binding site in the DRA 3'-untranslated region (3'-UTR) compared with other DRA-targeting miRNAs in vertebrates. Transfection with pmirGLO dual luciferase vector containing DRA 3'-UTR (pmirGLO-3'-UTR DRA) resulted in a significant decrease in relative luciferase activity compared with empty vector. Cotransfection of the DRA 3'-UTR luciferase vector with a miR-494 mimic further decreased luciferase activity compared with cells transfected with negative control. The transfection of a miR-494 mimic into Caco-2 and T-84 cells significantly increased the expression of miR-494 and concomitantly decreased the DRA protein expression. Mutation of the seed sequences for miR-494 in 3'-UTR of DRA abrogated the effect of miR-494 on 3'-UTR. These data demonstrate a novel regulatory mechanism of DRA expression via miR-494 and indicate that targeting this microRNA may serve to be a potential therapeutic strategy for diarrheal diseases.

Efflux of glutathione and glutathione complexes from human erythrocytes in response to vanadate.

The main objective of the present study was to investigate if vanadate is extruded from the cells in a glutathione dependent manner resulting in the appearance of extracellular glutathione and complexes of glutathione with vanadium. Vanadate significantly depleted intracellular non-protein sulfhydryl (NPSH) levels in a time- and concentration-dependent manner. The intracellular NPSH level was decreased to 0.0 ± 0.0 µmol/ml erythrocyte when exposed to 10 mM of vanadate for 4h. Extracellular NPSH level was increased concomitantly with the intracellular decrease and reached to 0.1410 ± 0.005 µmol/ml erythrocyte in 4h. Intracellular decrease and extracellular increase in NPSH levels were significantly inhibited in the presence of DIDS, a chloride-bicarbonate exchanger which also mediates phosphate and arsenate transport in erythrocytes. In parallel with the increase in extracellular NPSH levels, significant increases in extracellular glutathione levels were detected following exposure to vanadate. Extracellular glutathione levels reached to 0.0150 ± 0.0.001, 0.0330 ± 0.001, and 0.0576 ± 0.002 µmol/ml erythrocyte with 1, 5, and 10 mM of vanadate respectively. Dimercaptosuccinic acid treatment of supernatants significantly increased the glutathione levels measured in the extracellular media. Utilization of MK571 an MRP inhibitor decreased the rate of glutathione efflux from erythrocytes suggesting a role for this membrane transporter in the process. A known methylation inhibitor periodate oxidized adenosine decreased the rate of glutathione efflux from erythrocytes. This observed decrease in extracellular GSH levels suggests that GSH release partly requires a proper cellular methylation process and that part of GSH detected in the extracellular media may arise from GSH-vandium complexes. The results of the present study indicate that human erythrocyte efflux glutathione in reduced free form and in conjugated form/s that can be recovered with dimercaptosuccinic acid when exposed to vanadate.

DPOFA, a Cl⁻/HCO3⁻ exchanger antagonist, stimulates fluid absorption across basolateral surface of the retinal pigment epithelium.

BACKGROUND: Retinal detachment is a disorder of the eye in which sensory retina separates from the retinal pigment epithelium (RPE) due to accumulation of fluid in subretinal space. Pharmacological stimulation of fluid reabsorption from subretinal space to choroid across the RPE has been suggested as a treatment strategy for retinal detachment. DPOFA, (R)-(+)-(5,6-dichloro 2,3,9,9a-tetrahydro 3-oxo-9a-propyl-1H-fluoren-7-yl)oxy]acetic acid, is an abandoned drug capable of inhibiting Cl⁻/HCO3⁻ exchanger activity. We hypothesized that DPOFA may increase fluid absorption across basolateral surface of the RPE. METHODS: Reverse transcription polymerase chain reaction (RT-PCR) analysis of mRNA for six different transporters that may act as Cl⁻/HCO3⁻ exchangers was conducted in bovine and human RPE to confirm that RPE from two species expresses the same repertoire of Cl⁻/HCO3⁻ exchanger isoforms. The degree of amino acid homology between orthologous human and bovine RPE-specific isoforms was calculated after performing protein alignments. Transport of fluid across bovine RPE-choroid explants mounted in the Ussing chamber was used to assess the ability of DPOFA to modulate fluid absorption across the RPE. RESULTS: Using RT-PCR we showed that three isoforms (SLC4A2, SLC4A3, and SLC26A6) are strongly expressed in human and bovine RPE preparations. Amino acid comparisons conducted for RPE-specific isoforms support the use of bovine RPE-choroid explants as an adequate experimental system for assessing fluid absorption activity for DPOFA. Our data is consistent with the fact that DPOFA stimulates fluid absorption across the RPE in bovine RPE-choroid explants. CONCLUSIONS: DPOFA seems to stimulate transport of water across the RPE in bovine RPE-choroid explants. Additional experiments are required to establish dose-dependent effect of DPOFA on fluid absorption in the bovine RPE-choroid experimental system.

SLC26 anion exchangers of guinea pig pancreatic duct: molecular cloning and functional characterization.

The secretin-stimulated human pancreatic duct secretes HCO(3)(-)-rich fluid essential for normal digestion. Optimal stimulation of pancreatic HCO(3)(-) secretion likely requires coupled activities of the cystic fibrosis transmembrane regulator (CFTR) anion channel and apical SLC26 Cl(-)/HCO(3)(-) exchangers. However, whereas stimulated human and guinea pig pancreatic ducts secrete ∼140 mM HCO(3)(-) or more, mouse and rat ducts secrete ∼40-70 mM HCO(3)(-). Moreover, the axial distribution and physiological roles of SLC26 anion exchangers in pancreatic duct secretory processes remain controversial and may vary among mammalian species. Thus the property of high HCO(3)(-) secretion shared by human and guinea pig pancreatic ducts prompted us to clone from guinea pig pancreatic duct cDNAs encoding Slc26a3, Slc26a6, and Slc26a11 polypeptides. We then functionally characterized these anion transporters in Xenopus oocytes and human embryonic kidney (HEK) 293 cells. In Xenopus oocytes, gpSlc26a3 mediated only Cl(-)/Cl(-) exchange and electroneutral Cl(-)/HCO(3)(-) exchange. gpSlc26a6 in Xenopus oocytes mediated Cl(-)/Cl(-) exchange and bidirectional exchange of Cl(-) for oxalate and sulfate, but Cl(-)/HCO(3)(-) exchange was detected only in HEK 293 cells. gpSlc26a11 in Xenopus oocytes exhibited pH-dependent Cl(-), oxalate, and sulfate transport but no detectable Cl(-)/HCO(3)(-) exchange. The three gpSlc26 anion transporters exhibited distinct pharmacological profiles of (36)Cl(-) influx, including partial sensitivity to CFTR inhibitors Inh-172 and GlyH101, but only Slc26a11 was inhibited by PPQ-102. This first molecular and functional assessment of recombinant SLC26 anion transporters from guinea pig pancreatic duct enhances our understanding of pancreatic HCO(3)(-) secretion in species that share a high HCO(3)(-) secretory output.

Functional coupling of apical Cl-/HCO3- exchange with CFTR in stimulated HCO3- secretion by guinea pig interlobular pancreatic duct.

Pancreatic ductal epithelium produces a HCO(3)(-)-rich fluid. HCO(3)(-) transport across ductal apical membranes has been proposed to be mediated by both SLC26-mediated Cl(-)/HCO(3)(-) exchange and CFTR-mediated HCO(3)(-) conductance, with proportional contributions determined in part by axial changes in gene expression and luminal anion composition. In this study we investigated the characteristics of apical Cl(-)/HCO(3)(-) exchange and its functional interaction with Cftr activity in isolated interlobular ducts of guinea pig pancreas. BCECF-loaded epithelial cells of luminally microperfused ducts were alkalinized by acetate prepulse or by luminal Cl(-) removal in the presence of HCO(3)(-)-CO(2). Intracellular pH recovery upon luminal Cl(-) restoration (nominal Cl(-)/HCO(3)(-) exchange) in cAMP-stimulated ducts was largely inhibited by luminal dihydro-DIDS (H(2)DIDS), accelerated by luminal CFTR inhibitor inh-172 (CFTRinh-172), and was insensitive to elevated bath K(+) concentration. Luminal introduction of CFTRinh-172 into sealed duct lumens containing BCECF-dextran in HCO(3)(-)-free, Cl(-)-rich solution enhanced cAMP-stimulated HCO(3)(-) secretion, as calculated from changes in luminal pH and volume. Luminal Cl(-) removal produced, after a transient small depolarization, sustained cell hyperpolarization of approximately 15 mV consistent with electrogenic Cl(-)/HCO(3)(-) exchange. The hyperpolarization was inhibited by H(2)DIDS and potentiated by CFTRinh-172. Interlobular ducts expressed mRNAs encoding CFTR, Slc26a6, and Slc26a3, as detected by RT-PCR. Thus Cl(-)-dependent apical HCO(3)(-) secretion in pancreatic duct is mediated predominantly by an Slc26a6-like Cl(-)/HCO(3)(-) exchanger and is accelerated by inhibition of CFTR. This study demonstrates functional coupling between Cftr and Slc26a6-like Cl(-)/HCO(3)(-) exchange activity in apical membrane of guinea pig pancreatic interlobular duct.

Nhe1 is a luminal Na+/H+ exchanger in mouse choroid plexus and is targeted to the basolateral membrane in Ncbe/Nbcn2-null mice.

The choroid plexus epithelium (CPE) secretes the major fraction of the cerebrospinal fluid (CSF). The Na(+)-HCO(3)(-) transporter Ncbe/Nbcn2 in the basolateral membrane of CPE cells is important for Na(+)-dependent pH(i) increases and probably for CSF secretion. In the current study, the anion transport inhibitor DIDS had no effect on the residual pH(i) recovery in acidified CPE from Ncbe/Nbcn2 knockout mouse by 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-fluorescence microscopy in the presence of CO(2)/HCO(3)(-) (Ncbe/Nbcn2-ko+DIDS 109% of control, P = 0.76, n = 5). Thus Ncbe/Nbcn2 mediates the DIDS-sensitive Na(+)-dependent pH(i) recovery in the CPE. The Na(+)/H(+) exchanger-1 Nhe1 is proposed to mediate similar functions as Ncbe/Nbcn2 in CPE. Here, we immunolocalize the Nhe1 protein to the luminal membrane domain in mouse and human CPE. The Na(+)-dependent pH(i) recovery of Nhe1 wild-type (Nhe1-wt) mice in the absence of CO(2)/HCO(3)(-) was abolished in the Nhe1 knockout CPE (Nhe1-ko 0.37% of Nhe1-wt, P = 0.0007, n = 5). In Ncbe/Nbcn2-ko mice, Nhe1 was targeted to the basolateral membrane. Nevertheless, the luminal Na(+)-dependent pH(i) recovery was increased in Ncbe/Nbcn2-ko compared with wild-type littermates (Nhe1-ko 146% of Nhe1-wt, P = 0.007, n = 5). Whereas the luminal Nhe activity was inhibited by the Nhe blocker EIPA (10 microM) in the Ncbe/Nbcn2-wt, it was insensitive to the inhibitor in Ncbe/Nbcn2-ko (Ncbe/Nbcn2-ko+EIPA 100% of control, P = 0.98, n = 5). This indicates that a luminal EIPA-insensitive Nhe was induced in Ncbe/Nbcn2-ko CPE and that EIPA-sensitive Nhe activity was basolateral. The Nhe1 translocation in Ncbe/Nbcn2-ko CPE may reflect a compensatory response, which provides the cells with better means of regulating pH(i) or transporting Na(+) after Ncbe/Nbcn2 disruption.

Vectorial bicarbonate transport by Par-C10 salivary cells.

Salivary glands produce a HCO(3)(-)-rich fluid that is important for the neutral milieu in the upper gastrointestinal tract. The molecular mechanism of this secretion is poorly understood. Par-C10, an immortalized rat parotid acinar line, has been shown to secrete Cl(-)- in response to Ca(2+-)-mobilizing stimuli. Our aim was to assess the capacity of polarized monolayers of Par-C10 cells to transport and secrete HCO(3)(-)-. Transepithelial electrolyte movement was evaluated by short-circuit current measurements. Intracellular pH (pH(i)) was measured by microfluorometry in cells loaded with BCECF. Monolayers of Par-C10 cells, grown on Transwell membranes, developed high transepithelial resistance and exhibited vectorial anion secretion which was activated by both ATP and forskolin. The currents were partially inhibited by bumetanide and by withdrawal of HCO(3)(-) indicating the dependence of ion movements on NKCC and on HCO(3)(-) ions, respectively. In HCO(3)(-)-free solutions the recovery of pH(i) from acid loading was abolished by EIPA. In the presence of HCO(3)(-) there was a strong EIPA-insensitive recovery from acid loading which was inhibited by H(2)DIDS. ATP and forskolin stimulated HCO(3)(-) efflux from the cells. Furthermore, HCl(-) withdrawal experiments showed the presence of DNDS-sensitive basolateral anion exchange. In conclusion Par-C10 cells achieve transepithelial transport that is sensitive to both intracellular Ca(2+)- and cAMP-dependent stimulation. We identified Na(+)/H(+) exchange, Na(+)-HCO(3)(-) cotransport and anion exchange at the basolateral side of the cells as being involved in intracellular pH regulation and vectorial HCO(3)(-) secretion. This cell line offers a good model for further studies to understand the molecular mechanisms of salivary HCO(3)(-) secretion.

Changes of HCO3-/Cl- exchanger activity during meiotic maturation in Balb/c strain mouse oocytes and zygotes.

Intracellular pH-regulatory mechanisms are acquired by growing mouse oocytes with meiotic competence, and these mechanisms become fully active when the oocytes develop to the germinal vesicle (GV) stage as shown in CF1 and Balb/c strains mice. On the other hand, there is some evidence showing that intracellular pH-regulatory mechanisms are inhibited at the stages of Metaphase I (MI) and II (MII) oocytes in the CF1 strain mouse and hamster. Since it has been shown that the intracellular pH regulatory mechanism can be functionally different among mouse strains (e.g., CF1, Balb/c), the aim of this study was to investigate the activity of HCO3-/Cl- exchanger (anion exchanger, AE), which protects cells against alkalosis during the meiotic maturation process, in the GV oocyte up to the pronuclear (PN) zygote derived from the Balb/c strain mouse. Intracellular pH (pHi) was recorded using a microspectrofluorometric technique during meiotic maturation stages. KSOM-based solutions were used as culture and recording solutions. AE activity was determined using a Cl- removal assay and was reported as the change in pHi per minute. AE activity was high in GV stage oocytes but was significantly inhibited at the MI and MII stages. AE activity was higher in the PN zygote stage. This activity was significantly inhibited in all oocyte and zygote stages by 4,4'-Diisocyanatostilbene-2,2'-disulfonic acid disodium salt. After alkalosis induction, the pHi of MI and MII stage oocytes did not completely recover; however, almost complete recovery occurred in the GV stage oocytes and PN zygotes. These results suggest that AE is inhibited during the meiotic maturation process in the Balb/c strain mouse.

Acute inflammation alters bicarbonate transport in mouse ileum.

T-cell mediated acute inflammation of the ileum may occur during Crohn's disease exacerbations. During ileal inflammation, absorption of nutrients and electrolytes by villus cells is decreased with a concomitant increase in crypt and/or villus fluid secretion. These alterations lead to fluid accumulation and the subsequent diarrhoea. Net intestinal fluid secretion consists of HCO3--rich plasma-like fluid. However, the regulation and mechanisms of HCO3- secretion in normal and acutely inflamed ileum are not clearly understood. To study this phenomenon, anti-CD3 monoclonal antibody (mAb)- induced in vivo ileal inflammatory mouse models was used for in vitro functional studies with Ussing chamber and pH stat techniques. Three hours after anti-CD3 mAb injection, ileal mucosa stripped of muscular and serosal layers showed a significant increase in short circuit current (Isc) (0.58+/-0.07 microEq h(-1) cm2 versus 1.63+/-0.14 microEq h(-1) cm2). The cAMP-stimulated Isc component was sensitive to glibenclamide but not to DIDS, suggesting that a cystic fibrosis transmembrane conductance regulator (Cftr)-mediated anion conductance was responsible. Basal Cl--dependent HCO3- secretion, measured using a pH stat technique, was decreased significantly in anti-CD3-injected mice, with a simultaneous increase in Cl--independent HCO3- secretion that was also inhibited by glibenclamide. Experiments using Cftr-/- mice showed neither an increase in Isc nor an increase in HCO3- secretion, confirming the role for Cftr protein in stimulating anion secretion following anti-CD3 treatment. Western blot analysis indicated that Cftr protein levels were unaltered by anti-CD3 treatment, at least acutely. Finally, an immunoassay for cAMP showed significant increases in intracellular cAMP in villus cells, but not in crypt cells. These studies therefore suggest a shift from a predominantly electroneutral Cl-HCO3- exchange in normal mice, to a predominantly electrogenic anion secretion including HCO3- that occurs via functional Cftr during anti-CD3-mediated acute inflammation.

Selective inhibition of ion transport mechanisms regulating intracellular pH reduces proliferation and induces apoptosis in cholangiocarcinoma cells.

BACKGROUND: Cells within the acidic extracellular environment of solid tumours maintain their intracellular pH through the activity of the Na(+)/H(+) exchanger and the Na(+) dependent Cl(-)/HCO(3)(-) exchanger. The inhibition of these mechanisms could therefore inhibit cancer cell growth. AIM: We evaluated the effect of two selective inhibitors of these transporters (cariporide and S3705) on proliferation and apoptosis of human cholangiocarcinoma cells (HUH-28 and Mz-ChA-1 cells) as a function of external pH (7.4 and 6.8). METHODS/RESULTS: HUH-28 cells incubated for 24h at external pH 7.4 or 6.8 without inhibitors maintained intracellular pH at physiological level, whereas incubation with cariporide and/or S3705 caused the intracellular pH of cells to drop. Incubation of HUH-28 cells with cariporide and/or S3705 was able to reduce proliferation, evaluated by a colorimetric ELISA method, and to induce apoptosis, evaluated by measuring caspase-3 activity and Annexin-V staining, and these effects were more evident at external pH 6.8. S3705 but not cariporide was able to inhibit serum-induced phosphorylation of ERK1/2, AKT and BAD, intracellular molecules involved in cancer cell proliferation and survival. Similar results were obtained in Mz-ChA-1 cells. CONCLUSIONS: (1) Inhibition of intracellular pH regulatory mechanisms by cariporide and S3705 reduces proliferation and induces apoptosis in cholangiocarcinoma cells; and (2) these drugs might have potential therapeutic value against cholangiocarcinoma.

Delay of acute intracellular pH recovery after acidosis decreases endothelial cell activation.

Reperfusion after ischemic conditions induces massive endothelial cell (EC) activation, an initial step of reperfusion injury. Reperfusion is characterized by reoxygenation, realkalinization and a localized increase of inflammatory stimuli. In this study, we focused on the influence of extracellular realkalinization on human umbilical vein endothelial cell (HUVEC) activation. We examined intracellular pH (pH(in)) and intracellular free calcium concentration ([Ca(2+)](in)), a second messenger known to mediate von Willebrand factor (VWF) exocytosis in endothelium, upon realkalinization. Furthermore, we measured the agonist-stimulated exocytosis of VWF, Interleukin-8 and soluble P-selectin (sP-Selectin) as markers of EC activation. To verify a morphological correlate of EC activation, we finally observed platelet-endothelial adherence during realkalinization using shear flow. Realkalinization of HUVEC was simulated by switching from bicarbonate buffered Ringer solution of an acidotic pH(ex) of 6.4 to a physiologic pH(ex) of 7.4. Extracellular realkalinization was accompanied by pH(in) recovery from 6.5 to 7.2 within 10 min. Application of cariporide, an inhibitor of the Na(+)/H(+) exchanger subtype 1 (NHE), during extracellular realkalinization significantly delayed the early kinetics of intracellular realkalinization. Histamine stimulated [Ca(2+)](in) was significantly increased upon realkalinization compared to control cells. Also agonist-stimulated release of VWF, Interleukin-8 and sP-Selectin was massively enhanced during pH(in) recovery in comparison to control. Furthermore, we observed an increased platelet binding to endothelium. Interestingly, each of these realkalinization-induced effects were significantly reduced by early application of cariporide. Therefore, delay of acute NHE-dependent pH(in) recovery may represent a promising mechanism for inhibition of EC activation upon reperfusion.

Dopamine D1-like receptor-mediated inhibition of Cl/HCO3- exchanger activity in rat intestinal epithelial IEC-6 cells is regulated by G protein-coupled receptor kinase 6 (GRK 6).

The present study investigated the effect of dopamine D1-like receptor stimulation on the Cl-/HCO3- exchange activity in rat intestinal epithelial IEC-6 cells. The Cl-/HCO3- exchange activity was found to be a chloride-dependent, DIDS-sensitive and niflumate-insensitive process. The presence of the SLC26A6 anion exchanger was detected by both RT-PCR and immunoblotting analysis in IEC-6 cells, in which three different small interfering RNAs (siRNAs) targeting SLC26A6 markedly inhibited Cl-/HCO3- exchange. Activation of dopamine D1-like receptors with SKF 38393 inhibited Cl-/HCO3- exchanger activity, this being antagonized by the D1 selective antagonist SKF 83566. However, effects of SKF 38393 were maximal at 5 min of exposure to the agonist and rapidly diminished with no effect at 15 min, suggestive of agonist-induced desensitization of D1-like receptors. Pretreatment of cells with heparin, a non-selective inhibitor of G protein-coupled receptor kinases (GRKs), prevented the observed attenuation of SKF 38393-induced inhibition of Cl-/HCO3- exchange. Overnight pretreatment with anti-GRK6A and anti-GRK6B, but not with anti-GRK4 antibodies, prevented the loss of SKF 38393-mediated effects. Both PKA and PKC signaling pathways participate in SKF 38393-mediated inhibition of Cl-/HCO3- exchange. These findings suggest that SLC26A6 is at least one of the anion exchanger's family members responsible for Cl-/HCO3- exchange in IEC-6 cells. Dopamine D1 receptors in IEC-6 rapidly desensitize to D1-like agonist stimulation and GRK 6, but not GRK 4, appear to be involved in agonist-mediated responsiveness and desensitization.