Identification of a basolateral Cl-/HCO3- exchanger specific to gastric parietal cells

The basolateral Cl(-)/HCO(3)(-) exchanger in parietal cells plays an essential role in gastric acid secretion mediated via the apical gastric H(+)-K(+)-ATPase. Here, we report the identification of a new Cl(-)/HCO(3)(-) exchanger, which shows exclusive expression in mouse stomach and kidney, with expression in the stomach limited to the basolateral membrane of gastric parietal cells. Tissue distribution studies by RT-PCR and Northern hybridizations demonstrated the exclusive expression of this transporter, also known as SLC26A7, to stomach and kidney, with the stomach expression significantly more abundant. No expression was detected in the intestine. Cellular distribution studies by RT-PCR and Northern hybridizations demonstrated predominant localization of SLC26A7 in gastric parietal cells. Immunofluorescence labeling localized this exchanger exclusively to the basolateral membrane of gastric parietal cells, and functional studies in oocytes indicated that SLC26A7 is a DIDS-sensitive Cl(-)/HCO(3)(-) exchanger that is active in both acidic and alkaline pH(i). On the basis of its unique expression pattern and function, we propose that SLC26A7 is a basolateral Cl(-)/HCO(3)(-) exchanger in gastric parietal cells and plays a major role in gastric acid secretion.     

Cl/HCO3 exchange in the basolateral membrane domain of rat jejunal enterocyte

Summary Basolateral membrane vesicles isolated from rat jejunal enterocyte and well purified from brush border contamination were tested to examine Cl and HCO₃ movements. Uptake experiments provided no evidence for a coupling between Na and HCO₃ fluxes; K–HCO₃ and K–Cl cotransports also could be excluded. Transport studies revealed the presence of a Cl/HCO₃ exchanger accepting other anions and inhibitable by the disulfonic stilbenes SITS and DIDS. We can exclude that the evidenced HCO₃-dependent Cl uptake is due to brush border contamination, since in jejunal brush border membranes this mechanism, if present, has a very low transport rate. Besides the Cl/HCO₃ antiporter, a Cl-conductive pathway seems to exist in jejunal basolateral membranes.     

Basolateral Cl/HCO3 exchange in rat jejunum: The effect of sodium

Basolateral membrane vesicles isolated from rat jejunum were used to characterize a Cl/HCO₃ exchange mechanism previously evidenced. Cl uptake experiments provided no evidence for Cl/OH countertransport, confirming anyhow the presence of Cl/HCO₃ antiport, which was inhibited by 2 mm furosemide and unaffected by 2 mm amiloride. An outwardly directed Na gradient stimulated Cl uptake and this effect was increased if Na was present at both vesicle surfaces. To investigate the mechanism of coupling between Na and the transport protein, we performed Na uptake experiments. Na uptake was unaffected by cis-bicarbonate and trans-Cl gradients; the reversal of anion gradients was still ineffective. Similar results were obtained when a pH difference across the membrane vesicles was imposed. This study seems to suggest that Na is not transported by the Cl/HCO₃ exchanger and that another mode of Na dependence must be taken into account.     

Colocalization of the apical Cl-/HCO3- exchanger PAT1 and gastric H-K-ATPase in stomach parietal cells

The apical Cl-/HCO exchanger called the putative anion transporter (PAT1; SLC26A6) is expressed on apical membranes of villus cells in the duodenum, but its location in the stomach remains unknown. Here we examined the cell distribution and membrane location of PAT1 in mouse stomach. Immunofluorescence labeling studies with anti-PAT1 antibodies and Dolichos biflorus agglutinin indicated the exclusive expression of PAT1 in gastric parietal cells. Double immunocytochemical staining revealed colocalization of PAT1 with the gastric H-K-ATPase, consistent with expression in tubulovesicles and/or the secretory canaliculus. Radiolabeled 36Cl flux studies demonstrated the functional presence of Cl-/HCO exchange in purified tubulovesicles of parietal cells. The expression of PAT1 was significantly decreased in parietal cells of gastric H-K-ATPase-null mice, which exhibit a sharp reduction in tubulovesicle membranes. These data indicate that the Cl-/HCO exchanger PAT1 is localized on tubulovesicular membranes, and they are consistent with the hypothesis that it functions in the maintenance of intravesicular ion concentrations in the resting state and dehydration of vesicles derived from the secretory membranes following the transition from the stimulated to the resting state.     

Basolateral membrane Cl/HCO3 exchange in the rat proximal convoluted tubule. Na-dependent and -independent modes

To examine whether Cl-coupled HCO3 transport mechanisms were present on the basolateral membrane of the mammalian proximal tubule, cell pH was measured in the microperfused rat proximal convoluted tubule using the pH-sensitive, intracellularly trapped fluorescent dye (2',7')- bis(carboxyethyl)-(5,6)-carboxyfluorescein. Increasing the peritubular Cl concentration from 0 to 128.6 meq/liter caused cell pH to decrease from 7.34 +/- 0.04 to 7.21 +/- 0.04 (p less than 0.001). With more acid extracellular fluid (pH 6.62), a similar increase in the peritubular Cl concentration caused cell pH to decrease by a similar amount from 6.97 +/- 0.04 to 6.84 +/- 0.05 (p less than 0.001). This effect was blocked by 1 mM SITS. To examine the Na dependence of Cl/HCO3 exchange, the above studies were repeated in the absence of luminal and peritubular Na. In alkaline Na-free solutions, peritubular Cl addition caused cell pH to decrease from 7.57 +/- 0.06 to 7.53 +/- 0.06 (p less than 0.025); in acid Na-free solutions, peritubular Cl addition caused cell pH to decrease from 7.21 +/- 0.04 to 7.19 +/- 0.04 (p less than 0.05). The effect of Cl on cell pH was smaller in the absence of luminal and peritubular Na than in its presence. To examine whether the previously described Na/(HCO3)n greater than 1 cotransporter was coupled to or dependent on Cl, the effect of lowering the peritubular Na concentration from 147 to 25 meq/liter was examined in the absence of ambient Cl. Cell pH decreased from 7.28 +/- 0.03 to 7.08 +/- 0.03, a response similar to that observed previously in the presence of Cl. The results demonstrate that Cl/HCO3 (or Cl/OH) exchange is present on the basolateral membrane. Most of Cl/HCO3 exchange is dependent on the presence of Na and may be coupled to it. The previously described Na/(HCO3)n greater than 1 cotransporter is the major basolateral membrane pathway for the coupling of Na and HCO3 and is not coupled to Cl.     

cAMP activates Cl−/HCO3− exchange for regulation of intracellular pH in renal epithelial cells

The role of cAMP in regulation of intracellular pH in the confluent LLC-PK₁ cells was investigated. DibutyrylcAMP and forskolin induce intracellular acidification. This acidification is inhibited by DIDS and ethacrynic acid, inhibitors of Na⁺-independent Cl^?/HCO₃^? exchange, and by removal of extracellular Cl^?. In addition, Bt₂ cAMP causes Cl^? entry into LLC-PK₁ cells. These results suggest that cAMP activates Cl^? transport, namely Na⁺-independent Cl^?/HCO₃^? exchange, which participates in pH_i regulation.     

Functional and molecular adaptation of Cl/HCO3- exchanger to chronic alkaline media in renal cells.

The Cl(-)/HCO3- exchanger (AE) is one of the mechanisms that cells have developed to adjust pH Despite its importance, the role of AE isoforms in controlling steady-state pH during alkalosis has not been widely investigated. In the present study, we have evaluated whether conditions simulating acute and chronic metabolic alkalosis affected the transport activity and protein levels of Cl-/HCO3- exchangers in a rat cortical collecting duct cell line (RCCD1). pH(i) was monitored using the fluorescent dye BCECF in monolayers grown on permeable supports. Anion exchanger function was assessed by the response of pH(i) to acute chloride removal. RT-PCR and immunoblot assays were also performed. Our results showed that RCCD1 cells express two members of the anion exchanger gene family: AE2 and AE4. Functional studies demonstrated that while in acute alkalosis pH(i) became alkaline and was not regulated, after 48 h adaptation; steady-state pH(i) reached a value similar to the physiological one. Chronic treated cells also resulted in a 3-fold rise in Cl(-)/HCO3- exchange activity together with a 2.2-fold increase in AE2, but not AE4, protein abundance. We conclude that RCCD1 cells can adapt to chronic extracellular alkalosis reestablishing its steady-state pH(i) and that AE2 would play a key role in cell homeostasis.     

Independence of apical Cl-/HCO3- exchange and anion conductance in duodenal HCO3- secretion

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.     

Intracellular pH regulation in cultured embryonic chick heart cells. Na(+)-dependent Cl-/HCO3- exchange

The contribution of Cl-/HCO3- exchange to intracellular pH (pHi) regulation in cultured chick heart cells was evaluated using ion-selective microelectrodes to monitor pHi, Na+ (aiNa), and Cl- (aiCl) activity. In (HCO3- + CO2)-buffered solution steady-state pHi was 7.12. Removing (HCO3- + CO2) buffer caused a SITS (0.1 mM)-sensitive alkalinization and countergradient increase in aiCl along with a transient DIDS-sensitive countergradient decrease in aiNa. SITS had no effect on the rate of pHi recovery from alkalinization. When (HCO3- + CO2) was reintroduced the cells rapidly acidified, aiNa increased, aiCl decreased, and pHi recovered. The decrease in aiCl and the pHi recovery were SITS sensitive. Cells exposed to 10 mM NH4Cl became transiently alkaline concomitant with an increase in aiCl and a decrease in aiNa. The intracellular acidification induced by NH4Cl removal was accompanied by a decrease in aiCl and an increase in aiNa that led to the recovery of pHi. In the presence of (HCO3- + CO2), addition of either amiloride (1 mM) or DIDS (1 mM) partially reduced pHi recovery, whereas application of amiloride plus DIDS completely inhibited the pHi recovery and the decrease in aiCl. Therefore, after an acid load pHi recovery is HCO3o- and Nao- dependent and DIDS sensitive (but not Ca2+o dependent). Furthermore, SITS inhibition of Na(+)-dependent Cl-/HCO3- exchange caused an increase in aiCl and a decrease in the 36Cl efflux rate constant and pHi. In (HCO3- + CO2)-free solution, amiloride completely blocked the pHi recovery from acidification that was induced by removal of NH4Cl. Thus, both Na+/H+ and Na(+)-dependent Cl-/HCO3- exchange are involved in pHi regulation from acidification. When the cells became alkaline upon removal of (HCO3- + CO2), a SITS-sensitive increase in pHi and aiCl was accompanied by a decrease of aiNa, suggesting that the HCO3- efflux, which can attenuate initial alkalinization, is via a Na(+)-dependent Cl-/HCO3- exchange. However, the mechanism involved in pHi regulation from alkalinization is yet to be established. In conclusion, in cultured chick heart cells the Na(+)-dependent Cl-/HCO3- exchange regulates pHi response to acidification and is involved in the steady-state maintenance of pHi.     

Cl-/HCO3- exchange modulates intracellular pH in rat type II alveolar epithelial cells

The role of an anion exchange pathway in modulating intracellular pH (pHi) under steady-state and alkaline load conditions was investigated in confluent monolayers of rat type II alveolar epithelial cells using the pH-sensitive fluorescent probe 2'-7'-biscarboxy-ethyl-5,6-carboxylfluorescein. Under steady-state conditions in the presence of 25 mM HCO3-, 5% CO2 at pHo 7.4, pHi was 7.32 in a Na+-replete medium and 7.33 in the absence of Na+. Steady-state pHi was 7.19 in a nominally HCO3(-)-free medium at pHo 7.4, and 7.52 in a Cl(-)-free medium, with both values significantly different from that obtained in the presence of both HCO3- and Cl-. Monolayers in which pHi was rapidly elevated by removal of HCO3-/CO2 from the bathing medium demonstrated an absolute requirement for Cl- to recover toward base-line pHi. The Km of Cl- for the external site of the exchange pathway was 11 +/- 1 mM. Recovery of pHi from the alkaline load in the presence of Cl- was inhibited 60% by the stilbene derivative 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Removal of Cl- from the medium of cells bathed in HCO3-/CO2 resulted in a rapid increment in pHi which returned to base line when Cl- was reintroduced into the bathing medium. In contrast, pHi was not perturbed by removal or addition of Cl- to monolayers bathed in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered medium, indicating that HCO3- was the preferred species for transport. Recovery of pHi from an alkaline load was not affected by the presence or absence of Na+. These findings define the transport pathway as Na+-independent Cl-/HCO3- exchange. This pathway contributes importantly to determining resting pHi of pneumocytes and enables the cell to recover from an alkaline load.     

Regulation and function of COX-2 gene expression in isolated gastric parietal cells

We examined expression, function, and regulation of the cyclooxygenase (COX)-2 gene in gastric parietal cells. COX-2-specific mRNA was isolated from purified (>95%) canine gastric parietal cells in primary culture and measured by Northern blots using a human COX-2 cDNA probe. Carbachol was the most potent inducer of COX-2 gene expression. Gastrin and histamine exhibited minor stimulatory effects. Carbachol-stimulated expression was inhibited by intracellular Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (90%), protein kinase C (PKC) inhibitor GF-109203X (48%), and p38 kinase inhibitor SB-203580 (48%). Nuclear factor (NF)-kappaB inhibitor 1-pyrrolidinecarbodithioic acid inhibited carbachol-stimulated expression by 80%. Similar results were observed in the presence of adenoviral vector Ad.dom.neg.IkappaB, which expresses a repressor of NF-kappaB. Addition of SB-203580 with Ad.dom.neg.IkappaB almost completely blocked carbachol stimulation of COX-2 gene expression. We examined the effect of carbachol on PGE(2) release by enzyme-linked immunoassay. Carbachol induced PGE(2) release. Ad.dom.neg.IkappaB, alone or with SB-203580, produced, respectively, partial (70%) and almost complete (>80%) inhibition of carbachol-stimulated PGE(2) production. Selective COX-2 inhibitor NS-398 blocked carbachol-stimulated PGE(2) release without affecting basal PGE(2) production. In contrast, indomethacin inhibited both basal and carbachol-stimulated PGE(2) release. Carbachol induces COX-2 gene expression in the parietal cells through signaling pathways that involve intracellular Ca(2+), PKC, p38 kinase, and activation of NF-kappaB. The functional significance of these effects seems to be stimulation of PGE(2) release.     

Regulation of Na+/H+ and Cl-/HCO3- antiports in Vero cells

The effect of serum, phorbol-12-myristate-13-acetate (TPA), and forskolin on the activity Na+/H+ antiport and the Na(+)-coupled and Na(+)-independent Cl-/HCO3- antiport was studied in Vero cells by measuring 22Na+ and 36Cl- fluxes and changes in cytosolic pH (pHi). The Na(+)-independent Cl-/HCO3- antiport, which acts as an acidifying mechanism, is strongly pH-sensitive. In serum-starved cells it is activated at alkaline cytosolic pH, with a half-maximal activity at pHi approximately 7.20. Incubation with serum increased the activity of the Na(+)-independent Cl-/HCO3- antiport at pHi values from 6.8 to 7.2. Thus serum appeared to alter the pHi sensitivity of this antiporter such that the threshold value for activation of the antiport was shifted to a more acidic value. Na+/H+ antiport was somewhat stimulated initially by addition of serum, but further incubation with serum (greater than 45 min) decreased its activity. The activity of the Na(+)-coupled Cl-/HCO3- antiport, which is the major alkalinizing antiport in Vero cells, was not altered by short-term incubation with serum (less than 10 min) but decreased after prolonged incubation (greater than 45 min). Our findings with TPA and forskolin indicate that the effect of serum is partly mediated by the protein kinase C pathway, whereas the cyclic adenosine monophosphate pathway does not appear to play an important role. The net effect of serum on the pHi-regulating antiports was a slight decrease in intracellular pH.     

Activation of the Na+/H+ and Cl-/HCO3- exchange by stimulation of acid secretion in the parietal cell

Upon stimulation, the gastric parietal cell secretes a large quantity of isotonic HCl across its apical membrane which must be accompanied by the generation of base in the cytosol. The ability of this cell type to regulate cytosolic pH (pHi) was examined as a function of stimulation of acid secretion by histamine or forskolin. The pHi was estimated from the change of fluorescence of the trapped dye, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein-bis-carboxyethylcarbo xy fluorescein in a purified cell suspension of rabbit parietal cells. Stimulation of the cell suspension raised pHi by an average of 0.13 +/- 0.038 pH units. The H+,K+-ATPase inhibitor, SCH28080 (2-methyl-8-[phenyl-methoxy]-imidazo-(1,2)-pyridine-3-acetonitrile) had only a small effect on the increase of pHi, therefore, was largely independent of H+,K+-ATPase activity. In Na+-free medium, where Na+/H+ exchange would be absent, the rise of pHi was only 0.03 pH units. This increase was blocked by SCH28080, showing that this small increment was the result of acid secretion. In Na+-containing medium, 90% of the increase was inhibited by an inhibitor of Na+/H+ exchange, dimethyl amiloride (DMA). This compound also blocked changes in pHi due to changes in extracellular Na+. Accordingly, most of the change in pHi upon stimulation of acid secretion by histamine and forskolin is due to activation of Na+/H+ exchange in the parietal cell basal-lateral membrane. The addition of DMA to stimulated, but not resting cells, gave a rapid acidification that was blocked by inhibition of anion exchange by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), showing that anion exchange was also activated by stimulation. In single cell recording, canalicular and cytosolic pH were monitored simultaneously using 9-amino acridine and dimethyl carboxyfluorescein, respectively. Cytosolic alkalinization correlated with acid accumulation in the secretory canaliculus until a set point was reached. Thereafter, acidification continued without further change in pHi. To determine the role of Na+/H+ and Cl-/HCO3- exchange in acid secretion, Cl(-)-depleted cells were suspended in medium containing 40 mM Cl-. DMA and DIDS each blocked acid secretion by about 40%, but in combination, acid secretion was blocked by more than 90%. Thus, basal-lateral Na+/H+ and Cl-/HCO3- exchange activities are necessary for acid secretion across the apical membrane of the parietal cell.     

Regulation of parietal cell calcium signaling in gastric glands

The ligands interacting with enterochromaffin-like (ECL) and parietal cells and the signaling interactions between these cells were investigated in rabbit gastric glands using confocal microscopy. Intracellular calcium concentration ([Ca(2+)](i)) changes were used to monitor cellular responses. Histamine and carbachol increased [Ca(2+)](i) in parietal cells. Gastrin (1 nM) increased [Ca(2+)](i) in ECL cells and adjacent parietal cells. Only the increase of [Ca(2+)](i) in parietal cells was inhibited by H(2) receptor antagonists (H(2)RA). Gastrin (10 nM) evoked an H(2)RA-insensitive [Ca(2+)](i) increase in parietal cells. Carbachol produced large H(2)RA- and somatostatin-insensitive signals in parietal cells. Pituitary adenylate cyclase-activating peptide (PACAP, 100 nM) elevated [Ca(2+)](i) in ECL cells and adjacent parietal cells. H(2)RAs abolished the PACAP-stimulated [Ca(2+)](i) increase in adjacent parietal cells. Somatostatin did not inhibit the increase of [Ca(2+)](i) in parietal cells stimulated with histamine, high gastrin concentrations, or carbachol but abolished ECL cell calcium responses to gastrin or PACAP. Hence, rabbit parietal cells express histaminergic, muscarinic, and CCK-B receptors coupled to calcium signaling but insensitive to somatostatin, whereas rabbit and rat ECL cells express PACAP and CCK-B calcium coupled receptors sensitive to somatostatin.     

Regulation and function of p38 protein kinase in isolated canine gastric parietal cells

We examined the regulation and functional role of p38 kinase in gastric acid secretion. p38 kinase was immunoprecipitated from cell lysates of highly purified gastric parietal cells in primary culture, and its activity was quantitated by in vitro kinase assay. Carbachol effects were dose- and time-dependent, with a maximal 10-fold stimulatory effect detected after 30 min of incubation. SB-203580, a highly selective inhibitor of p38 kinase, blocked carbachol induction of p38 kinase activity, with maximal inhibition at 10 microM. Stimulation by carbachol was unaffected by preincubation of parietal cells with the intracellular Ca(2+) chelator BAPTA-AM, but incubation of cells in Ca(2+)-free medium led to a 50% inhibition of carbachol induction of p38 kinase activity. Because some of the effects of carbachol are mediated by the small GTP-binding protein Rho, we examined the role of Rho in carbachol induction of p38 kinase activity. We tested the effect of exoenzyme C3 from Clostridium botulinum (C3), a toxin known to ADP-ribosylate and specifically inactivate Rho. C3 led to complete ADP-ribosylation of Rho, and it inhibited carbachol induction of p38 kinase by 50%. We then tested the effect of SB-203580 and C3 on carbachol-stimulated uptake of [(14)C]aminopyrine (AP). Inhibition of p38 kinase by SB-203580 led to a dose-dependent increase in AP uptake induced by carbachol, with maximal (threefold) effect at 10 microM SB-203580. Similarly, preincubation of parietal cells with C3 led to a twofold increase in AP uptake induced by carbachol. Thus carbachol induces a cascade of events in parietal cells that results in activation of p38 kinase through signaling pathways that are at least in part dependent on Rho activation and on the presence of extracellular Ca(2+). p38 kinase appears to inhibit gastric acid secretion.     

Polarized distribution of actin isoforms in gastric parietal cells.

The actin genes encode several structurally similar, but perhaps functionally different, protein isoforms that mediate contractile function in muscle cells and determine the morphology and motility in nonmuscle cells. To reveal the isoform profile in the gastric monomeric actin pool, we purified actin from the cytosol of gastric epithelial cells by DNase I affinity chromatography followed by two-dimensional gel electrophoresis. Actin isoforms were identified by Western blotting with a monoclonal antibody against all actin isoforms and two isoform-specific antibodies against cytoplasmic beta-actin and gamma-actin. Densitometry revealed a ratio for beta-actin/gamma-actin that equaled 0.73 +/- 0.09 in the cytosol. To assess the distribution of actin isoforms in gastric glandular cells in relation to ezrin, a putative membrane-cytoskeleton linker, we carried out double immunofluorescence using actin-isoform-specific antibodies and ezrin antibody. Immunostaining confirmed that ezrin resides mainly in canaliculi and apical plasma membrane of parietal cells. Staining for the beta-actin isoform was intense along the entire gland lumen and within the canaliculi of parietal cells, thus predominantly near the apical membrane of all gastric epithelial cells, although lower levels of beta-actin were also identified near the basolateral membrane. The gamma-actin isoform was distributed heavily near the basolateral membrane of parietal cells, with much less intense staining of parietal cell canaliculi and no staining of apical membranes. Within parietal cells, the cellular localization of beta-actin, but not gamma-actin, isoform superimposed onto that of ezrin. In a search for a possible selective interaction between actin isoforms and ezrin, we carried out immunoprecipitation experiments on gastric membrane extracts in which substantial amounts of actin were co-eluted with ezrin from an anti-ezrin affinity column. The ratio of beta-actin/gamma-actin in the immunoprecipitate (beta/gamma = 2.14 +/- 0.32) was significantly greater than that found in the cytosolic fraction. In summary, we have shown that beta- and gamma-actin isoforms are differentially distributed in gastric parietal cells. Furthermore, our data suggest a preferential, but not exclusive, interaction between beta-actin and ezrin in gastric parietal cells. Finally, our results suggest that the beta- and gamma-actin-based cytoskeleton networks might function separately in response to the stimulation of acid secretion.     

State of actin in gastric parietal cells

Remodeling of theapical membrane-cytoskeleton has been suggested to occur when gastricparietal cells are stimulated to secrete HCl. The present experimentsassayed the relative amounts of F-actin and G-actin in gastric glandsand parietal cells, as well as the changes in the state of actin onstimulation. Glands and cells were treated with a Nonidet P-40extraction buffer for separation into detergent-soluble (supernatant)and detergent-insoluble (pellet) pools. Two actin assays were used toquantitate actin: the deoxyribonuclease I binding assay to measureG-actin and F-actin content in the two pools and a simple Western blotassay to quantitate the relative amounts of actin in the pools.Functional secretory responsiveness was assayed by aminopyrineaccumulation. About 5% of the total parietal cell protein is actin,with about 90% of the actin present as F-actin. Stimulation of acidsecretion resulted in no measurable change in the relative amounts ofG-actin and cytoskeletal F-actin. Treatment of gastric glands withcytochalasin D inhibited acid secretion and resulted in a decrease inF-actin and an increase in G-actin. No inhibition of parietal cellsecretion was observed when phalloidin was used to stabilize actinfilaments. These data are consistent with the hypothesis thatmicrofilamentous actin is essential for membrane recruitment underlyingparietal cell secretion. Although the experiments do not eliminate theimportance of rapid exchange between G- and F-actin for the secretoryprocess, the parietal cell maintains actin in a highly polymerizedstate, and no measurable changes in the steady-state ratio of G-actin to F-actin are associated with stimulation to secrete acid.

     

CFTR induces the expression of DRA along with Cl(-)/HCO(3)(-) exchange activity in tracheal epithelial cells

Thickening of airway mucus and lungdysfunction in cystic fibrosis (CF) results, at least in part, fromabnormal secretion of Cl and HCO₃across the tracheal epithelium. The mechanism of the defect in HCO₃ secretion is ill defined; however, a lack ofapical Cl/HCO₃ exchange may exist inCF. To test this hypothesis, we examined the expression ofCl/HCO₃ exchangers in trachealepithelial cells exhibiting physiological features prototypical ofcystic fibrosis [CFT-1 cells, lacking a functional cystic fibrosistransmembrane conductance regulator (CFTR)] or normal trachea (CFT-1cells transfected with functional wild-type CFTR, termed CFT-WT). Cellswere grown on coverslips and were loaded with the pH-sensitive dye2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, andintracellular pH was monitored. Cl/HCO₃exchange activity increased by ~300% in cells transfected with functional CFTR, with activities increasing from 0.034 pH/min in CFT-1cells to 0.11 in CFT-WT cells (P < 0.001, n = 8). This activity was significantly inhibited byDIDS. The mRNA expression of the ubiquitous basolateral AE-2Cl/HCO₃ exchanger remained unchanged.However, mRNA encoding DRA, recently shown to be aCl/HCO₃ exchanger (Melvin JE, Park K,Richardson L, Schultheis PJ, and Shull GE. J Biol Chem 274:22855-22861, 1999.) was abundantly expressed in cells expressingfunctional CFTR but not in cells that lacked CFTR or that expressedmutant CFTR. In conclusion, CFTR induces the mRNA expression of"downregulated in adenoma" (DRA) and, as a result, upregulates theapical Cl/HCO₃ exchanger activity intracheal cells. We propose that the tracheal HCO₃secretion defect in patients with CF is partly due to thedownregulation of the apical Cl/HCO₃exchange activity mediated by DRA.

     

Muscarinic responses of gastric parietal cells

Summary Isolated rabbit gastric glands were used to study the nature of the muscarinic cholinergic responses of parietal cells. Carbachol (CCh, 100 m) stimulation of acid secretion, as measured by the accumulation of aminopyrine, was inhibited by the M1 antagonist, pirenzepine, with an IC₅₀ of 13 m; by the M2 antagonist, 11,2-(diethylamino)methyl-1 piperidinyl acetyl-5,11-dihydro-6H-pyrido 2,3-b 1,4 benzodiazepin-6-one (AF-DX 116), with an IC₅₀ of 110 m; and by the M1/M3 antagonist, diphenylacetoxy-4-methylpiperidinemethiodide (4-DAMP), with an IC₅₀ of 35nm. The three antagonists displayed equivalent IC₅₀ values for the inhibition of carbachol-stimulated production of¹⁴CO₂ from radiolabeled glucose, which is a measure of the turnover of the H,K-ATPase, the final step of acid secretion. Intracellular calcium levels were measured in gastric glands loaded with FURA 2. Carbachol was shown to both release calcium from an intracellular pool and to promote calcium entry across the plasma membrane. The calcium entry was inhibitable by 20 m La³⁺. The relative potency of the three muscarinic antagonists for inhibition of calcium entry was essentially the same as for inhibition of acid secretion or pump related glucose oxidation. Image analysis of the glands showed the effects of carbachol, and of the antagonists, on intracellular calcium were occurring largely in the parietal cell. The rise in cell calcium due to release of calcium from intracellular stores was inhibited by 4-DAMP with an IC₅₀ of 1,7nm, suggesting that the release pathway was regulated by a low affinity M3 muscarinic receptor or state; Ca entry and acid secretion are regulated by a high affinity M3 muscarinic receptor or state, inhibited by higher 4-DAMP concentrations (>30nm), suggesting that it is the steady-state elevation of Ca that is related to parietal cell function rather than the [Ca] _i transient. Displacement of³H N-methyl scopolamine (NMS) binding to purified parietal cells by CCh showed the presence of two affinities for CCh, but only a single affinity for 4-DAMP and lower affinity for pirenzepine and AFDX 116, providing further evidence for the parietal cell location of the [Ca] _i response. Elevation of steady-state [Ca] _i levels with either ionomycin or arachidonic acid did not replicate M3 stimulation of acid secretion or glucose oxidation, hence elevation of [Ca] _i is necessary but not sufficient for acid secretion.