[show abstract][hide abstract] ABSTRACT: Cell migration is crucial for immune defence, wound healing or formation of tumour metastases. It has been shown that the activity of the Na(+)-H(+) exchanger (NHE1) plays an important role in cell migration. However, so far it is unknown whether Na(+)- HCO(3)(-) cotransport (NBC), which has similar functions in the regulation of intracellular pH (pH(i)) as NHE1, is also involved in cell migration. We therefore isolated NHE-deficient Madin-Darby canine kidney (MDCK-F) cells and tested whether NBC compensates for NHE in pH(i) and cell volume regulation as well as in migration. Intracellular pH was measured with the fluorescent pH indicator 2'7'-bis(carboxyethyl)-5-carboxyfluorescein (BCECF). The expression of NBC isoforms was determined with semiquantitative PCR. Migration was monitored with time-lapse video microscopy and quantified as the displacement of the cell centre. We found that MDCK-F cells express the isoform NBC1 (SLCA4A gene product) at a much higher level than the isoform kNBC3 (SLCA4A8 gene product). This difference is even more pronounced in NHE-deficient cells so that NBC1 is likely to be the major acid extruder in these cells and the major mediator of propionate-induced cell volume increase. NHE-deficient MDCK-F cells migrate more slowly than normal MDCK-F cells. NBC activity promotes migration during an acute intracellular acid load and increases migratory speed and displacement on a short timescale (< 30 min) whereas it has no effect on the long-term behaviour of migrating MDCK-F cells. Taken together, our results show that NBC actvity, despite many functional similarities, does not have the same importance for cell migration as NHE1 activity.
The Journal of Physiology 10/2005; 568(Pt 2):445-58. · 4.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: The Na(+)/H(+) exchanger isoform NHE2 is highly expressed in the intestinal tract, but its physiological role has remained obscure. The aim of this study was to define its expression, location, and regulatory properties in murine colon and to look for the compensatory changes in NHE2 (-/-) colon that allow normal histology and absorptive function. To this end, we measured murine proximal colonic surface and crypt cell NHE1, NHE2, and NHE3 expression levels, transport rates in response to acid, hyperosmolarity and cAMP in murine proximal colonic crypts, as well as changes in transcript levels and acid-activated NHE activity in NHE2 (-/-) crypts. We found that NHE2 was expressed most abundantly in crypts, NHE1 equally in crypts and surface cells, and NHE3 much stronger in surface cells. NHE2, like NHE1, was activated by low intracellular pH (pH(i)), hyperosmolarity, and cAMP, whereas NHE3 was activated only by low pH(i). Crypts isolated from NHE2 (-/-) mice displayed increased acid-activated NHE1- and NHE3-attributable Na(+)/H(+) exchange activity, no change in NHE1 expression, and NHE3 expression levels twice as high as in normal littermates. No change in cellular ultrastructure was found in NHE2 (-/-) colon. Our results demonstrate high NHE2 expression in the crypts and suggest a role for NHE2 in cryptal pH(i) and volume homeostasis.
[show abstract][hide abstract] ABSTRACT: Reduced gastrointestinal HCO3- secretion contributes to malabsorption and obstructive syndromes in cystic fibrosis. The apical HCO3- transport pathways in these organs have not been defined. We therefore assessed the involvement of apical Cl-/HCO3- exchangers and anion conductances in basal and cAMP-stimulated duodenal HCO3- secretion. Muscle-stripped rat and rabbit proximal duodena were mounted in Ussing chambers, and electrical parameters, HCO3- secretion rates, and 36Cl-, 22Na+, and 3H+ mannitol fluxes were assessed. mRNA expression levels were measured by a quantitative PCR technique. Removal of Cl- from or addition of 1 mM DIDS to the luminal perfusate markedly decreased basal HCO3- secretion but did not influence the HCO3- secretory response to 8-bromo-cAMP, which was inhibited by luminal 5-nitro-2-(3-phenylpropylamino)-benzoate. Bidirectional 22Na+ and 36Cl- flux measurements demonstrated an inhibition rather than a stimulation of apical anion exchange during cAMP-stimulated HCO3- secretion. The ratio of Cl- to HCO3- in the anion secretory response was compatible with both Cl- and HCO3- being secreted via the CFTR anion channel. CFTR expression was very high in the duodenal mucosa of both species. We conclude that in rat and rabbit duodena, an apical Cl-/HCO3- exchanger mediates a significant part of basal HCO3- secretion but is not involved in the HCO3- secretory response to cAMP analogs. The inhibitor profile, the strong predominance of Cl- over HCO3- in the anion secretory response, and the high duodenal CFTR expression levels suggest that a major portion of cAMP-stimulated duodenal HCO3- secretion is directly mediated by CFTR.
[show abstract][hide abstract] ABSTRACT: Defective regulation and/or reduced expression of the Na+-K+-2Cl- cotransporter NKCC1 may contribute to the severe secretory defect that is observed in cystic fibrosis, but data concerning the expression and function of NKCC1 in cystic fibrosis transmembrane conductance regulator (CFTR)-deficient cells are equivocal. We therefore investigated NKCC1 mRNA expression, Na+-K+-2Cl- cotransport activity and regulation by cAMP in crypts isolated from the proximal colon of CFTR-containing (CFTR (+/+)) and CFTR-deficient (CFTR (-/-)) mice. mRNA expression levels were determined by semiquantitative PCR, transport rates were measured fluorometrically in 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetomethylester (BCECF)-loaded crypts, cytoplasmic volume changes were assessed by confocal microscopy, and [Cl-]i changes were examined by N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) quenching. NKCC1 mRNA expression levels were not significantly reduced in CFTR (-/-) crypts compared to controls. Azosemide-sensitive NH4+ influx (used as a measure of Na+-K+-2Cl- cotransport) was 2.23 +/- 0.72 vs. 1.56 +/- 0.16 mM min-1, and increased by 63.6 % in (+/+) and 87.3 % in (-/-) crypts upon stimulation for 5 min with forskolin. After 20 min of stimulation with forskolin, the Na+-K+-2Cl- cotransport rates in (-/-) and (+/+) crypts were identical. Crypt cross-sectional area and [Cl-]i decreased only in (+/+) crypts upon stimulation. In conclusion, normal NKCC1 expression levels, somewhat reduced Na+-K+-2Cl- cotransport rates, but preserved activation by cAMP were found in colonic crypts from CFTR (-/-) mice, ruling out a severe dysfunction of the Na+-K+-2Cl- cotransporter in the CF intestine. Furthermore, these studies establish the existence of a direct, cell-volume- and [Cl-]i-independent activation of colonic NKCC1 by an increase in intracellular cAMP.
The Journal of Physiology 07/2003; 549(Pt 2):525-36. · 4.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: Basolateral Na(+)-HCO(3)(-) cotransport is essential for intestinal anion secretion, and indirect evidence suggests that it may be stimulated by a rise of intracellular cAMP. We therefore investigated the expression, activity, and regulation by cAMP of the Na(+)-HCO(3)(-) cotransporter isoforms NBC1 and NBCn1 in isolated murine colonic crypts. Na(+)-HCO(3)(-) transport rates were measured fluorometrically in BCECF-loaded crypts, and mRNA expression levels and localization were determined by semiquantitative PCR and in situ hybridization. Acid-activated Na(+)-HCO(3)(-) cotransport rates were 5.07 +/- 0.7 mM/min and increased by 62% after forskolin stimulation. NBC1 mRNA was more abundant in colonic crypts than in surface cells, and crypts expressed far more NBC1 than NBCn1. To investigate whether the cAMP-induced Na(+)-HCO(3)(-) cotransport activation was secondary to secretion-associated changes in HCO(3)(-) or cell volume, we measured potential forskolin-induced changes in intracellular pH and assessed Na(+)-HCO(3)(-) transport activity in CFTR -/- crypts (in which no forskolin-induced cell shrinkage occurs). We found 30% reduced Na(+)-HCO(3)(-) transport rates in CFTR -/- compared with CFTR +/+ crypts but similar Na(+)-HCO(3)(-) cotransport activation by forskolin. These studies establish the existence of an intracellular HCO(3)(-) concentration- and cell volume-independent activation of colonic NBC by an increase in intracellular cAMP.
[show abstract][hide abstract] ABSTRACT: 1. The anion exchanger isoform 2 (AE2) gene encodes three subtypes (AE2a, b and c), which have different N-termini and tissue distributions. AE2 is expressed at high levels in the stomach, where it is thought to mediate basolateral base exit during acid production. The present study investigated if the three AE2 subtypes are differentially expressed and regulated in different cell types within the gastric mucosa. 2. The cloning strategy to obtain rabbit AE2a, b and c cDNAs combined genomic PCR and RT-PCR based on primers deduced from the rat sequences. Semiquantitative RT-PCR using homologous primers revealed much higher AE2 mRNA expression in rabbit parietal cells (PCs) than in mucous cells (MCs). The subtype expression pattern was AE2b > AE2c > or = AE2a in PCs and AE2a >AE2b > AE2c in MCs. Sequence analysis revealed the presence of a highly conserved protein kinase C (PKC) consensus sequence in the AE2a alternative N-terminus. 3. Maximal Cl(-)-HCO(3)(-) exchange rates, measured fluorometrically in BCECF-loaded cultured gastric cells, were much higher in PCs than MCs. PKC activation by phorbol ester stimulated maximal Cl(-)-HCO(3)(-) exchange rates in MCs but not in PCs, whereas forskolin had no effect in each cell type. 4. In summary, rabbit PCs and MCs, which originate from the same gastric stem cell population, display a completely different AE2 subtype expression pattern. Therefore, AE2 subtype expression is not organ specific but cell type specific. The different regulation of anion exchange in parietal and mucous cells suggests that AE2 subtypes may be differentially regulated.
The Journal of Physiology 09/2001; 534(Pt 3):837-48. · 4.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: Several Na(+)/H(+) exchanger (NHE) isoforms are expressed in the stomach, and NHE1 and NHE2 knockout mice display gastric mucosal atrophy. This study investigated the cellular distribution of the NHE isoforms NHE1, NHE2, NHE3, and NHE4 in rabbit gastric epithelial cells and their regulation by intracellular pH (pH(i)), hyperosmolarity, and an increase in cAMP. Semiquantitative RT-PCR and Northern blot experiments showed high NHE1 and NHE2 mRNA levels in mucous cells and high NHE4 mRNA levels in parietal and chief cells. Fluorescence optical measurements in cultured rabbit parietal and mucous cells using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein and NHE isoform-specific inhibitors demonstrated that in both cell types, intracellular acidification activates NHE1 and NHE2, whereas hyperosmolarity activates NHE1 and NHE4. The relative contribution of the different isoforms to pH(i)- and hyperosmolarity-activated Na(+)/H(+) exchange in the different cell types paralleled their relative expression levels. cAMP elevation also stimulated NHE4, whereas an increase in osmolarity above a certain threshold further increased NHE1 and not NHE4 activity. We conclude that in rabbit gastric epithelium, NHE1 and NHE4 regulate cell volume and NHE1 and NHE2 regulate pH(i). The high NHE1 and NHE2 expression levels in mucous cells may reflect their special need for pH(i) regulation during high gastric acidity. NHE4 is likely involved in volume regulation during acid secretion.
[show abstract][hide abstract] ABSTRACT: In a search for the HCO(3)(-) supply mechanisms to the enterocyte we cloned and sequenced an intestinal subtype of the Na(+)HCO(3)(-) cotransporter isoform I (dNBC1), which turned out to be identical to the pancreatic NBC1 subtype (pNBC1). Within the intestine, we found particularly high NBC1 expression levels in the duodenum and proximal colon. Experiments with stripped rabbit duodenum in Ussing-chambers revealed that Na(+)HCO(3)(-) cotransport (NBC) and CO(2) hydration/Na(+)/H(+) exchange were equally important duodenal HCO(3)(-) supply pathways and were both upregulated during cAMP-mediated secretion. In the proximal colon, however, HCO(3)(-) secretion was low but NBC1 expression even higher than in the duodenum. Ussing-chamber experiments with an NBC-specific inhibitor revealed that NBC, coupled to basolateral Cl(-)/HCO(3)(-) exchange, was an important alternative Cl(-) supply pathway to Na(+)K(+)2Cl(-) cotransport (NKCC) during cAMP-stimulated colonic Cl(-) secretion. To investigate the functional integrity of anion uptake pathways in the absence of cystic fibrosis transmembrane conductance regulator (CFTR), we fluorometrically assessed NBC and NKCC transport rates and cell volume before and during forskolin-stimulation in isolated colonic crypts from normal and CFTR (-/-) mice. Although forskolin stimulation decreased cell volume only in normal, not in CFTR (-/-) crypts, it activated NBC and NKCC to a similar degree in both normal and CFTR (-/-) crypts. We conclude that, depending on the intestinal segment, NBC1 plays an important role in basolateral HCO(3)(-) or Cl(-) uptake. Expression and activation by cAMP is preserved in CFTR (-/-) intestine.
JOP: Journal of the pancreas 08/2001; 2(4 Suppl):247-56.
[show abstract][hide abstract] ABSTRACT: Concomitant Na(+)/H(+) and Cl(-)/HCO(3)(-) exchange activation occurs during stimulation of acid secretion in cultured rabbit parietal cells, possibly related to a necessity for volume regulation during the secretory process. We investigated whether cytoplasmic volume changes occur during secretagogue stimulation of cultured rabbit parietal cells. Cells were loaded with the fluorescent dye calcein, and the calcein concentration within a defined cytoplasmic volume was recorded by confocal microscopy. Forskolin at 10(-5) M, carbachol at 10(-4) M, and hyperosmolarity (400 mosmol) resulted in a rapid increase in the cytoplasmic dye concentration by 21 +/- 6, 9 +/- 4, and 23 +/- 5%, respectively, indicative of cell shrinkage, followed by recovery to baseline within several minutes, indicative of regulatory volume increase (RVI). Depolarization by 5 mM barium resulted in a decrease of the cytoplasmic dye concentration by 10 +/- 2%, indicative of cell swelling, with recovery within 15 min, and completely prevented forskolin- or carbachol-induced cytoplasmic shrinkage. Na(+)/H(+) exchange inhibitors slightly reduced the initial cell shrinkage and significantly slowed the RVI, whereas 100 microM bumetanide had no significant effect on either parameter. We conclude that acid secretagoguges induce a rapid loss of parietal cell cytoplasmic volume, followed by RVI, which is predominantly mediated by Na(+)/H(+) and Cl(-)/HCO(3)(-) exchange.
[show abstract][hide abstract] ABSTRACT: HCO(3)(-) supply to the enterocyte is rate limiting for duodenal HCO(3)(-) secretion (J(HCO3-)). This study defines the molecular nature of the major HCO(3)(-) uptake pathways in rabbit duodenocytes and investigates their physiologic significance and regulation during basal and stimulated J(HCO3-).
pH gradient-driven (22)Na(+) uptake into duodenal basolateral membrane vesicles was partly HCO(3)(-) dependent, stilbene sensitive, and therefore mediated by Na(+)HCO(3)(-) cotransport, and partly HCO(3)(-) independent, Hoechst 642 sensitive, and therefore mediated by the Na(+)/H(+) exchanger isoform NHE1. Semiquantitative polymerase chain reaction (PCR) revealed high duodenal expression levels for the NBC1 isoform of the Na(+)HCO(3)(-) cotransporter gene family and NHE1. Cloning and comparison of full-length rabbit with human gastrointestinal and kidney NBC1 subtype revealed a conserved protein kinase A consensus sequence in the cytoplasmic N-terminus of the gastrointestinal NBC1. Inhibition of either Na(+)HCO(3)(-) cotransport or carbonic anhydrase reduced ouabain-sensitive J(HCO3-) in in vitro rabbit duodenal mucosae by approximately 50%, but did not affect 8-Br-cAMP-induced DeltaJ(HCO3-), suggesting cAMP-mediated up-regulation of the alternative pathway. However, inhibition of both Na(+)HCO(3)(-) cotransport and either carbonic anhydrase or NHE1 strongly reduced DeltaJ(HCO3-).
NBC1 and NHE1 are the major base importers in rabbit duodenocytes. Na(+)HCO(3)(-) cotransport and CO(2) hydration/Na(+)/H(+) exchange are equally important pathways for duodenal HCO(3)(-) supply and are up-regulated during cAMP-mediated stimulation.
[show abstract][hide abstract] ABSTRACT: 1. It was postulated that swelling dependent chloride channels are involved in the proton secretion of parietal cells. Since omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 are structurally related to phenol derivatives known to block swelling dependent chloride channels, we set out to test, whether these substances--which are known to block the H,K-ATPase--could also lead to an inhibition of swelling-dependent chloride channels. Swelling-dependent chloride channels--characterized in many different cell types--show highly conserved biophysical and pharmacological features, therefore we investigated the effect of omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 on swelling-dependent chloride channels elicited in fibroblasts, after the reduction of the extracellular osmolarity. 2. Omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 are able to block swelling-dependent chloride channels (IClswell). 3. Lansoprazole and its protonated metabolite AG2000 act on at least two different sites of the IClswell protein: on an extracellular site which seems to be in a functional proximity to the nucleotide binding site, and on an intracellular site which allows the formation of disulfide-bridges. 4. The inhibition of the proton pump and the simultaneous blocking of chloride channels by omeprazole, lansoprazole and its acid activated sulphenamide form AG2000, as described here could be an effective mode to restrict proton secretion in parietal cells.
British Journal of Pharmacology 03/2000; 129(3):598-604. · 5.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: The stomach, duodenum, colon, and pancreas secrete HCO3- ions into the lumen. Although the importance of HCO3- secretion for the maintenance of mucosal integrity, a normal digestion, and the reabsorption of Cl- has been well established, the molecular nature of the apical and basolateral HCO3- transporting proteins has remained largely unknown. Functional studies have suggested that a Na+HCO3- cotransport system, similar but not identical to the well-characterized Na+HCO3- cotransporter in the basolateral membrane of the kidney proximal tubule, is present in duodenal and colonic enterocytes, pancreatic ducts cells, and gastric cells and involved in HCO3- uptake from the interstitium. This report describes our work towards understanding the molecular nature, cellular origin, and functional relevance of the Na+HCO3- cotransporter(s) in the stomach and intestine and reviews work by others on the function and localization of Na+HCO3- cotransport processes in the gastrointestinal tract.
Annals of the New York Academy of Sciences 02/2000; 915:1-14. · 4.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: AE2 is one of three known isoforms of the anion exchanger (AE) gene family. The use of alternative promoters, resulting in a tissue-specific transcript pattern, was reported for all AE genes. Three N-terminal variant AE2 subtypes are described: AE2a, AE2b, and AE2c. Although the basolaterally located parietal cell anion exchanger is known to be an AE2, the molecular identity of the basolateral and apical anion exchangers throughout the gut are still unknown. This article summarizes functional, immunohistochemical, and Western blot data demonstrating the basolateral localization of the gastric and intestinal AE2 in rabbit, mouse, and rat, and showing the AE2 subtype mRNA expression pattern in the stomach and along the intestine of rabbit and mouse: AE2a is expressed in all studied tissues, but most strongly in the colon; AE2b is expressed mainly in the stomach; and AE2c is detected nearly exclusively in the stomach. Further investigation is necessary to characterize the apical anion transport protein involved in NaCl absorption and HCO3- secretion in the gut.
Annals of the New York Academy of Sciences 02/2000; 915:81-91. · 4.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have characterized expression of anion exchanger 2 (AE2) mRNA and protein in the mouse intestine. AE2 mRNA abundance was higher in colon than in more proximal segments. AE2a mRNA was more abundant than AE2b mRNA throughout the intestine, and AE2c mRNA was expressed at very low levels. This AE2 mRNA pattern contrasted with that in mouse stomach, in which AE2c > AE2b > AE2a. AE2 polypeptide abundance as detected by immunoblot qualitatively paralleled that of mRNA, whereas AE2 immunostaining exhibited a more continuous decrease in intensity from colon to duodenum. AE2 polypeptide was more abundant in colonic surface cells than in crypts, whereas ileal crypts and villi exhibited similar AE2 abundance. AE2 was also observed in mural and vascular smooth muscle. Localization of AE2 epitopes was restricted to the basolateral membranes of epithelial cells throughout the intestine with three exceptions. Under mild fixation conditions, anti-AE2 amino acids (aa) 109-122 detected nonpolarized immunostaining of ileal enterocytes and of Paneth cell granule membranes. An epitope detected by anti-AE2 aa 1224-1237 was also localized to subapical regions of Brunner's gland ducts of duodenum and upper jejunum. These localization studies will aid in the interpretation of anion exchanger function measured in epithelial sheets, isolated cells, and membrane vesicles.
The American journal of physiology 09/1999; 277(2 Pt 1):G321-32. · 3.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: The gastric epithelium protects itself against luminal acid by secreting HCO3--rich fluid into the mucous layer and by HCO3--dependent intracellular pH regulation, but the basolateral HCO3- uptake mechanisms are incompletely characterized. This study examined the expression and functional significance of the Na+/HCO3- cotransporters NBC1 and NBC2 in rabbit gastric epithelial cells.
Rabbit NBC1 and NBC2 complementary DNA fragments were cloned and sequenced, and cellular expression levels were assessed by semiquantitative polymerase chain reaction. Na+/HCO3- cotransport activity was measured fluorometrically in cultured rabbit parietal and mucous cells.
NBC1 expression was 4.5-fold lower in the stomach than kidney cortex and 5.5-fold higher in mucous than parietal cells. NBC2 expression in the stomach was much lower than in the eye, approximately 4-fold lower than NBC1 expression in the stomach, and 2.5-fold higher in mucous than parietal cells. The Na+- and HCO3--dependent, dimethylamiloride-insensitive (which at 500 micromol/L completely inhibits all Na+/H+ exchanger isoforms) base influx rates were 4.6 +/- 0.02 and 16.2 +/- 0.04 mmol/L/min in acidified parietal and mucous cells, respectively, and were not significantly different in the absence of Cl-.
This study shows that NBC1 and NBC2 are expressed in rabbit stomach, with high levels in mucous cells where Na+/HCO3- cotransport is the major base-importing mechanism in the presence of CO2/HCO3-.