| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Journal of Physiology (2001), 534.3, pp. 837-848
© Copyright 2001 The Physiological Society
 |
ABSTRACT |
|---|
 TopAbstract IntroductionMethodsResultsDiscussionReferences |
|---|
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.
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Cl--HCO3- exchangers (AEs) are widely expressed and are involved in the regulation of intracellular pH, cell volume, cell migration, and transepithelial acid/base transport (reviewed in Kopito, 1990; Alper, 1994). Full length sequence information has been obtained from several species for three isoforms of the AE gene family: AE1, AE2 and AE3. For all three ae genes two or more 5' variant mRNAs are known (Kopito & Lodish, 1985; Kudrycki & Shull, 1993; Wang et al. 1996), which are transcribed from different promoters. For rat AE2, Wang et al. (1996) described three alternatively initiated subtypes named AE2a, AE2b and AE2c and showed that all three mRNAs are expressed in rat stomach. The rat AE2b protein contains three N-terminal amino acids, which replace the 17 N-terminal amino acids of rat AE2a. Besides this difference the two proteins show 100 % sequence identity. Rat AE2c is identical to AE2a, except for the absence of the N-terminal 199 amino acids.
AE2 mRNA has been detected in nearly all tissues and species examined (Kudrycki et al. 1990; Chow et al. 1992; Wang et al. 1996; Alper et al. 1999) and is thought to participate in the regulation of intracellular pH and cell volume. In the stomach, however, AE2 mRNA is highly expressed compared to other organs and is thought to encode the parietal cell basolateral Cl--HCO3- exchanger (Stuart-Tilley et al. 1994), responsible for uptake of Cl- ions destined for HCl secretion and extrusion of HCO3- ions, which are generated intracellularly at a very high rate during acid secretion (Muallem et al. 1988; Paradiso et al. 1989; Thomas & Machen, 1991; Seidler et al. 1992).
Recently, several groups found that the expression pattern of the AE2 subtypes is tissue specific. AE2a predominates in most organs studied, whereas in the rat stomach, AE2a, AE2b and AE2c are all expressed at very high levels (Wang et al. 1996; Stuart-Tilley et al. 1998; Alper et al. 1999). This observation led to suggestions that the alternative promoters could be crucial for organ-specific expression of the three subtypes, or that the different N-termini of the AE2 variants may be involved in membrane sorting of the AE2 protein or regulation of its anion exchange activity (Wang et al. 1996; Stuart-Tilley et al. 1998; Chow, 1998, 1999).
Northern hybridization experiments suggested to us that AE2 mRNA is expressed not only in isolated rabbit parietal cells but also in isolated rabbit mucous cells, and that these two cell types display distinct patterns of AE2 transcript sizes. We hypothesized that the different AE2 transcript sizes in rabbit parietal and mucous cells correspond to different patterns of AE2 promoter usage, and that the anion exchange in parietal and mucous cells might be differentially membrane sorted and/or regulated.
|
METHODS |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Materials
All reagents were purchased from Sigma-Aldrich (Deisenhofen, Germany) or Merck (Darmstadt, Germany), unless stated otherwise.
Animals
All experiments were carried out according to the guidelines laid down by the local Animal Care Committees (Regierungspräsidium Tübingen, Referat 37).
New Zealand White rabbits (2-3 kg) were anaesthetized by an I.M. injection of Ketanest 50 (Parke-Davis, Morris Plains, NJ, USA; 20 mg kg-1), Rompun (Bayer AG, Leverkusen, Germany; 5 mg kg-1) and atropine sulfate (Pharma-Hameln, Hameln, Germany; 0.2 mg kg-1). After opening the abdomen, the rabbits were killed by an I.V. injection of an overdose of the same mixture.
Adult male CD1 mice were anaesthetized with diethylether and underwent cardiac perfusion with Hanks' balanced salt solution followed by PLP (2 % paraformaldehyde-75 mM lysine-10 mM sodium periodate) fixation (see below).
Rabbit gastric cell purification for the molecular biology studies
Parietal, chief and mucous cells were purified from rabbit gastric mucosa (Rossmann et al. 1999), and the homogeneity of the three cell fractions was assessed by light microscopy after staining cytospin preparations as described previously (Seidler et al. 1989). The mucous cell fraction consisted of 90-95 % periodic acid-Schiff stain (PAS) granule-positive cells, whereas the parietal cell fraction showed a purity of 95-98 % and the chief cell population contained less than 2 % contaminating cells. These findings were confirmed by the expression level of the H+-K+-ATPase in the different cell fractions (Rossmann et al. 1999).
RNA isolation, cloning of an AE2 cDNA fragment, and Northern analysis
Oligodeoxynucleotide primers designed from the rabbit AE2 cDNA sequence (GenBank accession number (GB Acc.) S45791) were used to PCR-amplify a rabbit AE2 cDNA fragment present in all AE2 transcripts (Table 1). Exons 1b and 1c and intron 5 were PCR amplified from rabbit genomic DNA using primers deduced from the rat sequence (GB Acc. U45887). PCR cloning, DNA sequencing, isolation of total and poly(A+) RNA (Chomczynski & Sacchi, 1987) and Northern analysis were carried out as described (Rossmann et al. 1999). Northern blots were exposed to autoradiography films (Hyperfilm MP, Amersham Pharmacia Biotech (APB), Freiburg, Germany) and digitized by a Sharp JX-325 scanner. Bands were analysed by ImageMaster software (APB).
Semiquantitative RT-PCR
Primers were deduced from the rabbit sequence obtained and are presented in Table 1. Semiquantiative PCR was performed as described in Rossmann et al. (1999), and the results were expressed as means ± S.E.M. of at least three experiments. The identities of the AE2a, AE2b and AE2c fragments were confirmed by hybridization with a 32P-labelled exon 6 oligonucleotide (nucleotides 673-722 of S45791), present in all rabbit AE2 transcripts.
Purification and culture of rabbit parietal and mucous cells
The cell culture technique was modified from Chew et al. (1989), as previously described (Bachmann et al. 1998). Acid formation of parietal cells was periodically assessed by cellular uptake of [14C]aminopyrine (AP; APB) as previously described (Bachmann et al. 1998). Mucous cells were evaluated optically.
Fluorescence microscopy for determination of pHi
Intracellular pH (pHi) measurements are described elsewhere in detail (Bachmann et al. 1998). Cultured cells were loaded with 5 µM BCECF AM (Molecular Probes, Leiden, The Netherlands) for 30 min in buffer A (120 mM NaHCO3, 14 mM Hepes, 7 mM Tris, 3 mM KH2PO4, 1.2 mM CaCl2, 1.2 mM MgSO4, 20 mM glucose, pH 7.4, gassed with 5 % CO2-95 % O2) and then alternately excited at 440 ± 10 nm and 490 ± 10 nm at a rate of 100 s-1. The emission wavelength was 530 nm. At the end of each experiment the 490 nm/440 nm ratio was calibrated to pHi after clamping pHi to pHo using the high potassium/nigericin method (Bachmann et al. 1998). In Cl--free buffers, NaCl was replaced by sodium gluconate.
Determination of the intrinsic buffer capacity
Intrinsic buffer capacity of parietal and mucous cells was determined according to Boyarsky et al. (1988), and used to calculate proton/base flux rates. The values for the intrinsic buffer capacity for cultured parietal and mucous cells (
i) are shown elsewhere (Rossmann et al. 1999).
Preparation of rabbit gastric apical and basolateral membranes
Rabbit tubulo-vesicular and basolateral membrane vesicles were prepared by a combination of differential- and density-gradient centrifugation as described (Lamprecht et al. 1993). The apical tubulo-vesicular membrane fraction was 14.1 ± 1.6-fold enriched in H+-K+-ATPase activity (Na+-K+-ATPase: 1.9 ± 1.1; n = 5). The basolateral fraction was 18.1 ± 3.1-fold enriched in Na+-K+-ATPase activity (H+-K+-ATPase: 2.0 ± 0.7; n = 5).
SDS-PAGE and Western analysis
Crude cell lysates (lysis buffer: 1 mM EDTA, 0.1 % SDS, 1 % Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride (PMSF) in phosphate-buffered saline (PBS), pH 7.3) or purified apical or basolateral membranes were resolved on SDS-PAGE (Laemmli et al. 1970). Proteins were electroblotted to nitrocellulose or polyvinylidene difluoride (PVDF) membranes (APB). Membranes were stained by Ponceau S, blocked in 5 % non-fat dry milk in Tris-buffered saline (TBS, pH 7.5) plus 0.02 % Triton X-100 for 1 h and incubated with an anti-AE2 antibody (anti-SA6, 1:2000; Alper et al. 1994) or a monoclonal anti-H+-K+ ATPase antibody (Ab 12.18, 1:1000, Fain et al. 1988) for 4 h at room temperature. After washing in TBS Triton, membranes were incubated with a horseradish peroxidase-conjugated goat anti-rabbit IgG (1:6000, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) or sheep anti-mouse IgG secondary antibody (1:5000) for 1 h. Bands were detected by enhanced chemiluminescence (APB).
Immunohistochemistry
CD1 mice were perfusion-fixed with Hanks' balanced salt solution followed by 2 % paraformaldehyde-75 mM lysine-10 mM sodium periodate (PLP) as described (Stuart-Tilley et al. 1994, 1998). The stomach pieces were post-fixed overnight in PLP at 4 °C, washed in PBS and stored in PBS containing 0.02 % azide until use. Fixed tissue blocks were infiltrated with 30 % sucrose in PBS, frozen in liquid nitrogen, and sectioned at 5-7 µm thickness on a Reichert-Jung Frigocut model 2300N cryostat (Leica Microsystems, Exton, PA, USA). After epitope unmasking by SDS pretreatment, immunostaining with affinity-purified rabbit polyclonal anti-mouse AE2 amino acids 1224-1237 (dilution 1:400, incubation 1-2 h) as primary antibody and Cy3-coupled donkey anti-rabbit IgG (Jackson Immunochemicals, West Grove, PA, USA; concentration 10-15 µg ml-1, incubation 1 h) as secondary antibody was performed as previously described (Stuart-Tilley et al. 1998). Irrelevant peptide and peptide antigen were included in the antibody incubation mix at 12 µg ml-1. Sections were examined and photographed with an Olympus BH2 epifluorescence photomicroscope (Olympus, Melville, NY, USA), using Kodak TMAX 400 film push-processed to 1600 ASA (Kodak, Rochester, NY, USA). Images of scanned negatives were compiled and annotated in Adobe Photoshop.
Statistics
Results are given as means ± S.E.M. Proton fluxes were calculated by performing linear regression analysis on individual pHi traces during the first 1-2 min of recovery (linear phase). Statistical analyses of data were performed using analysis of variance.
|
RESULTS |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Determination of AE2 subtype expression and AE2 transcript sizes in rabbit gastric parietal and mucous cells by Northern analysis and RT-PCR
The complete coding sequence of rabbit AE2a (Chow et al. 1992) along with sequence information from rabbit exons 1b and 1c, and intron 5 suggested that the rabbit AE2 gene resembles that of rat and mouse, and predicted a length of rabbit AE2 transcripts of about 4.2 kb (AE2a), 4.0 kb (AE2b), and 3.5 kb (AE2c). Accordingly, three AE2 mRNAs were detected in poly(A+) RNA preparations from rabbit gastric mucosa by hybridization with a homologous AE2 cDNA fragment (bp 2484-2742 of S45791, Fig. 1A and B). Only the largest of the three bands was detected in rabbit kidney (Fig. 1A, lower panel) and only AE2a was amplified by RT-PCR. Therefore, the 4.2 kb fragment represents AE2a. AE2a and AE2b, but not AE2c cDNAs were amplified from rabbit colon (data not shown), and only the two larger transcripts were detected by Northern analysis in rabbit colon (Fig. 1A, lower panel). Thus, the 4.0 kb band represents AE2b. Three bands were detected in rabbit stomach, and PCR amplification detected AE2a, AE2b and AE2c (Fig. 2). Thus, the 3.5 kb transcript represents AE2c.
 |
View larger version [in this window] [in a new window] |
|
|
Figure 1. Poly(A+) RNA Northern blots (A and B) and analysis (C) of the band pattern in parietal cells, mucous cells and gastric mucosa by densitometry Hybridization of (A) ~5 µg poly(A+) RNA from rabbit kidney cortex (lane 1), colonic mucosa (lane 2), gastric mucosa (lane 3), parietal cell (lane 4), chief cell (lane 5) and mucous cell (lane 6) fractions and of (B) ~4 µg poly(A+) RNA of rabbit antrum mucosa (lane 1), gastric mucosa (lane 2) and parietal cells (lane 3) with a 32P-labelled AE2 cDNA fragment (bp 2484-2742 of S45791, common to rabbit AE2a, AE2b and AE2c) as described in Methods. Exposure times (expo.) are shown in parentheses beside each blot (in hours (h) and days (d)). C, the bands were analysed by densitometry, which showed no optical saturation. Mucous cell sample: blot A, long exposure; gastric mucosa and parietal cell sample: short exposure of blot B. | ||
 |
View larger version [in this window] [in a new window] |
|
|
Figure 2. RT-PCR analysis of AE2 mRNA variants, expressed in rabbit gastric mucosa, using specific forward primers for AE2a (exon 2), AE2b (exon 1b) and AE2c (exon 1c) Upper panel, ethidium bromide (EtBr)-stained AE2 amplification products (28 cycles) of RNA samples from rabbit gastric mucosa (lanes 1, 4, 7), rabbit mucous cells (lanes 2, 5, 8), and parietal cells (lanes 3, 6, 9). Histone 3.3a amplimers (lane 10: gastric mucosa; lane 11: mucous cells; lane 12: parietal cells) underwent 22 cycles of amplification. Lane 13 contains the H2O control, lane 14 the molecular weight standard. All lanes were loaded with a 13 µl aliquot of a 50 µl PCR reaction. Lower panel, the cDNAs shown in the upper panel were transferred to a nylon membrane and hybridized to a 32P-labelled internal AE2 oligonucleotide probe (exon 6, nucleotides (nt) 673-722 of S45791) encoding a sequence present in all AE2 variants. | ||
RT-PCR proved the expression of AE2a, b and c in rabbit gastric mucosa as well as parietal and mucous cells (Fig. 2). Two splice variants are described for rat and mouse AE2c: AE2c2 contains an intronic sequence, which is spliced out from AE2c1 (Wang et al. 1996; Stuart-Tilley et al. 1998; Alper et al. 1999). Our primer pair was designed to detect both potential AE2c splice variants (Table 1). In contrast to the situation in rat and mouse, only AE2c1 was detected in rabbit stomach (Fig. 2).
As AE2a, AE2b and AE2c mRNAs are different only in their N-terminal sequence, one AE2 cDNA fragment hybridizes with all three AE2 transcripts. Therefore, Northern analysis was used to compare the expression level of AE2a, AE2b and AE2c within one cell fraction. Figure 1A and B demonstrates the different AE2 band patterns in gastric epithelial cell types; Fig. 1C shows the quantitative analysis of the blots: AE2b was the predominant transcript in rabbit parietal cells, whereas it was expressed at a markedly lower level than AE2a in mucous cells. AE2c showed substantial expression in parietal cells but was almost undetectable in mucous cells. Since parietal and mucous cells originate from the same gastric stem cells (Karam et al. 1997), the findings demonstrate that AE2 subtype expression in the stomach is cell type and not organ specific.
Comparison of AE2a, AE2b and AE2c expression levels in rabbit gastric epithelial cell types by semiquantitative RT-PCR
We used a semiquantitative RT-PCR technique to study the expression level of each transcript (AE2a or AE2b or AE2c) in relation to histone 3.3a within the different cell fractions (Fig. 3), because it is the more sensitive method of clarifying this question compared to our poly(A+) Northern blot protocol. Histone 3.3a mRNA is expressed similarly in all gastric epithelial cell types and is therefore a suitable control for RT-PCR (Rossmann et al. 1999). As expected, all AE2 variant transcripts were most highly expressed in parietal cells, but AE2a was also substantially expressed in mucous cells. Since the mucous cell fraction can contain up to 5 % contaminating parietal cells, we calculated the percentage of AE2 subtype expression in the mucous cell fraction that could be attributable to the estimated 5 % of contaminating parietal cells, and subtracted this value. Therefore, the shaded bars are likely to represent an accurate estimate of AE2 variant transcript levels in mucous cells compared to the other gastric cell fractions. The results of the PCR studies prove the expression of the AE2 subtypes in the gastric cell types and confirm the different subtype expression pattern in parietal vs. mucous cells. The quantitative distribution of the different variants within one cell type as a percentage of total AE2 expression is reflected more accurately by Northern analysis (Fig. 1) than by our semiquantitative RT-PCR method, because oligo(dT) primed reverse transcription of different mRNAs is influenced by the different distance of the target from the poly(A) tail and the potential presence of (different) secondary structures and PCR amplification is affected by the length and composition of the PCR product. We therefore prefer semiquantitative RT-PCR for comparison of the expression of one transcript in different samples, but Northern blots for quantitative analysis of different transcripts in one sample.
 |
View larger version [in this window] [in a new window] |
|
|
Figure 3. Semiquantitative RT-PCR analysis of rabbit AE2 variants A, the ratios AE2a/histone 3.3a (representing the relative expression level of AE2a), AE2b/histone 3.3a and AE2c/histone 3.3a were plotted for gastric mucosa, the different cell fractions and kidney cortex. The shaded columns represent the expression of AE2a, AE2b and AE2c in rabbit mucous cells after subtraction of that part of the AE2 signal which is caused by a 5 % contamination of the mucous cell fraction with parietal cells (as explained in Results). n = 3 from three different cell preparations. B, representative curves illustrating the amplification of each AE2 subtype (AE2a, AE2b, AE2c) and control (histone 3.3a) cDNA fragments from rabbit parietal cell RNA. The ratio of the gene of interest/histone 3.3a was determined during the exponential phase of both reactions. | ||
Membrane and cellular localization of AE2 polypeptide by immunoblot and immunohistochemistry
The membrane localization of rabbit AE2 was examined by immunoblot of total rabbit gastric mucosal homogenate, gastric apical/tubulo-vesicular membranes and basolateral membranes (Fig. 4, upper panel), and compared with the content of H+-K+-ATPase (middle panel). Ponceau S staining of the filter confirmed equal loading in all lanes (lower panel). A very strong ~164 kDa AE2 signal was detected in the basolateral membrane fraction, suggesting a basolateral location for the AE2 protein. The very faint AE2 band in the apical membrane fraction is likely to represent basolateral membrane contamination, in view of the low but measurable enrichment of Na+-K+-ATPase activity in this apical membrane fraction. As our anti-AE2 antibody was raised in rabbit, cryosections of mouse stomach were used for immunohistochemistry. Figure 5 shows bright saturating staining of the parietal cell basolateral membrane. In mucous cells immunostaining is weaker, but substantial in the basal and especially the lateral membrane compartment. Thus, we conclude that both AE2a and AE2b are basolateral proteins in parietal and mucous cells.
 |
View larger version [in this window] [in a new window] |
|
|
Figure 4. Membrane localization of the AE2 protein by Western blot analysis of purified rabbit gastric tubulo-vesicular and basolateral membrane fractions Protein (100 µg) from total mucosal homogenate (lane 3), tubulo-vesicular membranes (lane 2) and basolateral membranes (lane 1) isolated from rabbit gastric mucosa were separated by 10 % SDS-PAGE, electroblotted, and stained by Ponceau S (to ensure equal loading in all lanes and to check protein quality, lower panel). The membrane shown in the upper panel was incubated with a rabbit polyclonal anti-AE2 antibody. The middle panel demonstrates the same filter, stripped, and incubated with an anti-H+-K+-ATPase | ||
 |
View larger version [in this window] [in a new window] |
|
|
Figure 5. AE2 immunolocalization in mouse gastric mucosa, detected by antibody to mouse C-terminal amino acids 1224-1237 in the presence of irrelevant peptide (a and c) or of peptide antigen (b and d) AE2 is present at modest abundance in basolateral membranes of surface mucous cells (a) and at very high abundance (saturating pixel intensity) in basolateral membranes of parietal cells (panel c and panel a, lower right). The identical exposures in all panels were selected to optimize antibody signal in the surface mucous cells (a). In exposures chosen to optimize parietal cell staining (Stuart-Tilley et al. 1994), surface mucous cell staining was undetectable. Scale bar, 15 µm. | ||
Fluorescence optical detection of anion exchange activity in cultured rabbit parietal and mucous cells
To investigate if the different AE2 expression levels in parietal and mucous cells give rise to different anion exchange activities in their membranes, we compared maximal anion gradient-driven Cl--HCO3- exchange activities of cultured rabbit parietal and mucous cells. Maximal Cl--HCO3- exchange rates were assessed by measuring pHi changes upon Cl- removal and restoration in the absence and presence of the anion exchange inhibitor 4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) (Fig. 6). The IC50 for DIDS inhibition of the parietal cell anion exchanger was determined by measuring the initial pHi change in the first minute after re-addition of 120 mM Cl- in the presence of DIDS at concentrations between 10 and 1000 µM and multiplying 
pHi with the intracellular buffer capacity at the given pHi value. The IC50 for DIDS was 94 µM in the presence of 120 mM [Cl-]o (n = 27, data not shown), in good agreement with the IC50 value for DIDS inhibition of AE2 when expressed in HEK 293 cells (Lee et al. 1991) and consistent with that measured in Xenopus oocytes (Humphreys et al. 1994). DIDS-inhibitable Cl--HCO3- exchange rates were ~12-fold lower in mucous than parietal cells (Fig. 6B and D), consistent with much lower AE2 expression levels in mucous cells.
 |
View larger version [in this window] [in a new window] |
|
|
Figure 6. Proton/base flux rates in cultured parietal (A and B) and mucous cells (C and D) in the presence of CO2-HCO3- and in the presence and absence of Cl- and/or 1 mM DIDS, an anion exchange inhibitor Parietal cells (A) and mucous cells (C) alkalized in Cl- free medium. Addition of Cl--containing medium was associated with a quick pHi recovery, which was inhibited by 1 mM DIDS (A and C). pHi recovery rates (indicated by dashed lines) and the intracellular buffering capacity at the appropriate pHi were used to calculate the proton/base flux rates shown in B and D. Proton/base flux rates were ~12-fold higher in parietal cells than in mucous cells, but in both cell types the addition of 1 mM DIDS blocked more than 91 % of the proton/base flux (n = 4-6 in each group, * P < 0.05, ** P < 0.01). | ||
Differential regulation of anion exchange in parietal and mucous cells
AE2a and AE2b differ in their N-terminal amino acid sequence (Table 2), and PROSITE predicted potential phosphorylation sites within rabbit AE2a (one highly conserved protein kinase C (PKC) consensus site, defined as one possibly phosphorylated serine or threonine, followed by one arbitrary amino acid and then at least one basic residue (Fig. 7) and one atypical cAMP dependent protein kinase (PKA) consensus site, described by Wang et al. (1996)), which are both absent from AE2b. We therefore hypothesized that the two AE2 subtypes may be differentially regulated by PKC. As the AE2b transcript predominates in parietal cells and the AE2a transcript in mucous cells, we speculated that a differential regulation of the two AE2 subtypes may be reflected by a differential effect of PKC or PKA activation on maximal anion gradient-driven Cl--HCO3- exchange. Indeed, activation of PKC by 12-O-tetradecanoyl-phorbol 13-acetate (TPA) stimulated maximal anion exchange activity 2-fold in mucous cells (Fig. 8F), but had no effect on parietal cell anion exchange rates (which were very high already) (Fig. 8C). PKA activation by forskolin had no effect on maximal Cl--HCO3- exchange rates in either cell type (Fig. 8C and F). Obviously, other intracellular events than the PKC consensus sequence in the AE2a subtype may explain the PKC activation of anion exchange in mucous but not parietal cells. Nevertheless, the predominance of AE2a in mucous but not parietal cells and the PKC consensus site in AE2a but not AE2b and AE2c is one feasible explanation for this phenomenon.
 |
View larger version [in this window] [in a new window] |
|
|
Figure 7. Multiple sequence alignment (MALIGN, Husar 5.0 Sequence Analysis, EMBL, Heidelberg, Germany) of the 40 N-terminal amino acids of AE2 From top to bottom: mouse (Mus musculus), rat (Rattus norvegicus), guinea-pig (Cavia porcellus), man (Homo sapiens) and rabbit (Oryctolagus cuniculus) AE2a sequence. Serine 14 is conserved in all known species. It is predicted to be phosphorylated by PKC (consensus pattern: serine or threonine-one arbitrary amino acid-one basic amino acid (lysine, arginine, or histidine); the presence of additional basic residues at the N- or C-terminus of the target amino acid enhances the Vmax and Km of the phosphorylation reaction (appendix to PROSITE, Husar 5.0 Sequence Analysis) in mouse, rat, guinea-pig and rabbit. Even in man, where the consensus pattern is atypical, a possible phosphorylation of serine 14 is not excluded. Furthermore Wang et al. (1965) described an atypical PKA consensus site at serine 10 of the rat sequence, which is conserved in mouse, rabbit and guinea-pig. | ||
 |
View larger version [in this window] [in a new window] |
|
|
Figure 8. Proton/base flux rates (C and F) and exemplary pHi traces (A, B, D and E) in cultured parietal (A, B and C) and mucous cells (D, E and F) in the presence of CO2-HCO3- and in the absence and presence of forskolin (A and D) and TPA (B and E), respectively, and [14C]aminopyrine uptake rates in parietal cells (G) A, B, D and E show pHi traces. pHi recovery rates (indicated by dashed lines) and the intracellular buffering capacity (data not shown) at the appropriate pHi were used to calculate the proton/base flux rates summarized in C and F. While forskolin (an agonist of cAMP-dependent protein kinase) had no influence on proton/base flux in both studied epithelial cell types, TPA (an agonist of PKC) stimulated proton/base flux 2-fold compared to control in mucous cells, but not in parietal cells (n = 4-8 in parietal cells, n = 5-10 in mucous cells, ** P < 0.01). G shows [14C]aminopyrine uptake rates in parietal cells expressed as the n-fold of the basal (non-treated) control. Forskolin and histamine caused a marked increase in [14C]aminopyrine uptake, whereas the values of TPA alone or in combination with histamine were not statistically different from the control (n = 4-5, * P < 0.05). | ||
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
We examined AE2 subtype mRNA expression levels in rabbit gastric epithelial cell types by Northern blot and RT-PCR and determined Cl--HCO3- exchange rates in cultured rabbit parietal and mucous cells by BCECF fluorescence ratio measurements. We found that the major cell types of rabbit gastric mucosa display distinct profiles of AE2 transcript variants and distinct patterns of regulation of Cl--HCO3- exchange activity.
Expression of AE2 subtype mRNAs and measurement of anion exchange activity in rabbit parietal and mucous cells
Northern hybridization using an AE2 probe revealed three transcripts in rabbit gastric mucosa and RT-PCR results confirmed the identity of these mRNAs as AE2a (4.2 kb), AE2b (4.0 kb), and AE2c (3.5 kb). Total AE2 mRNA expression was extremely high in parietal cells, ~11-fold lower in mucous cells and low in chief cells. DIDS-sensitive Cl--HCO3- exchange rates in cultured rabbit parietal cells were approximately 12-fold higher than in mucous cells, which corresponds well with the much higher AE2 expression levels in this cell type. These data suggest a good correlation between anion exchange activity and AE2 mRNA levels in both gastric parietal and mucous cells. While AE2 expression levels in mucous cells are comparable to those of other pHi-regulating ion transporters such as Na+-H+ exchangers and Na+-HCO3- cotransporters, in this and other gastrointestinal cell types AE2 expression levels in parietal cells are extraordinarily high, which is likely to be due to the important role of AE2 during acid secretion. As determined by Northern analysis, AE2b was more abundant than AE2c and AE2a in parietal cells, whereas AE2a was the predominant AE2 subtype in mucous cells.
Membrane localization of variant AE2 polypeptides
The observed cell type-specific differences in AE2 variant transcript profile could reflect isoform-specific targeting of AE2 variant polypeptides to different membrane compartments as suggested by Wang et al. (1996) for rat AE2 and as shown for chicken AE1 by Adair-Kirk et al. (1999). Immunoblot analysis (Fig. 4) of apical and basolateral membrane vesicles isolated from rabbit gastric mucosa using an AE2 antibody showed a strong AE2 signal in the basolateral fraction. An extremely weak signal in the apical fraction was consistent with measured low-level contamination with basolateral membrane. If the N-terminal amino acid sequences of AE2a and AE2b indeed encode distinct sorting signals directing delivery to opposite cellular surface membranes, then the high expression of both isoforms in gastric mucosa would predict the presence of apical AE2 polypeptide at levels much higher than observed. We therefore conclude that all AE2 subtypes exhibit steady-state localization to the basolateral membrane of the three major gastric cell types. This is consistent with immunohistochemical localization of AE2 to the basolateral membrane in mouse gastric parietal and mucous cells. As in rabbit three AE2 subtypes are expressed in mouse stomach, but have not been analysed at a cellular level.
Regulation of gastric anion exchange activity
The rat AE2a but not the AE2b N-terminus contains typical PKC consensus sites and one atypical PKA consensus site (described by Wang et al. 1996). When all available sequence information from different species is compared (Fig. 7), the PKC phosphorylation site at serine 14 is highly conserved, whereas a conserved PKA consensus site in the AE2a N-terminus cannot be found. One possible explanation for the distinct candidate phosphorylation sites in the N-terminal sequences of AE2a, AE2b and AE2c (Table 2) may be a differential regulation by protein kinases. We therefore studied the effect of PKA and PKC activation on maximal anion exchange rates in cultured rabbit parietal and surface cells. Neither parietal nor mucous cell stimulation by forskolin showed any influence on maximal anion exchange rates in these cell types. Although consistent with the lack of PKA consensus sites in the AE2 sequence, this result is nevertheless somewhat surprising because agents which increase intracellular cAMP are the strongest acid secretagogues. Basolateral Cl--HCO3- exchange is essential for the maintenance of acid secretion, and a concomitant activation of acid secretion and basolateral Cl--HCO3- exchange activity by cAMP-dependent agonists has been described (Muallem et al. 1988; Thomas & Machen, 1991; Seidler et al. 1992). Since no increase in the maximal transport rate was observed during forskolin stimulation, stimulation of anion exchange rates during acid secretion is possibly secondary to changes in the driving force such as agonist-induced Cl- loss (Thomas & Machen, 1991), or secondary to cellular shrinkage (Sonnentag et al. 2000).
A twofold stimulation of maximal anion exchange rates by TPA was observed in mucous cells, whereas no stimulation was seen in parietal cells. We knew from previous experiments that PKC activation does not influence basal acid secretion and even inhibits PKA-activated acid secretion (Muallem et al. 1986; Brown & Chew, 1987; Nandi et al. 1994; this paper), whereas it stimulates the synthesis and secretion of gastric mucus in vivo and in vitro (Terano et al. 1986; Seidler & Sewing, 1989; Seno et al. 1995). Although a role for AE2 in mucus secretion is unknown, a role in the control of mucous cell volume and/or pHi is reasonable. One possible explanation for the predominance of AE2a (which is the only AE2 subtype with a conserved PKC consensus site present in its N-terminus) expression in mucous cells may be a necessity for anion exchange activation during PKC-mediated secretory activity in this cell type.
AE2 variant expression pattern changes during the differentiation of the gastric epithelial cell types
All cell types of the gastric epithelium originate from one presumptive multipotent stem cell within a proliferative zone of the gland isthmus (Karam & Leblond, 1993; Li et al. 1996; Karam et al. 1997). Though arising from a single stem cell, rabbit parietal and mucous cells display distinct AE2 transcript patterns. We conclude that AE2 variant expression pattern is not organ but cell type specific. It is of note that both the predominant AE2 subtype and the approximate AE2 expression level in mucous cells are shared by many other cells, whereas the AE2 subtype pattern and the very high AE2 expression level in parietal cells are unique. Possibly the usage of more than one promoter allows the parietal cells to generate the very high AE2 levels needed for acid secretion. In addition, the lack of consensus sites and the possible absence of other as yet unidentified binding regions for regulatory proteins in AE2b and AE2c may make the activity of these subtypes independent of regulatory factors which interact with AE2a. One such example may be the observed lack of stimulation of maximal anion exchange activity by TPA in parietal but not mucous cells.
In summary, we have shown that the different cell types of rabbit gastric mucosa express different patterns of AE2 variant transcripts. This demonstrates that AE2 isoform expression pattern is not organ specific but cell type specific. Our results suggest that all AE2 polypeptide variants exhibit basolateral localization. The parietal cell is the only cell type so far described expressing higher mRNA levels of AE2b than AE2a. Significant AE2c mRNA is also found only in this cell type. The AE2b promoter is likely to contain regulatory sequences that foster particularly high levels of expression in the parietal cell, and the AE2c promoter is likely to contain regulatory elements that confer parietal cell-specific expression. The fact that PKC activation doubled maximal anion exchange rates in surface mucous cells, but did not influence parietal cell exchange rates, suggests that AE2 variant polypeptides are differentially regulated. Perhaps parietal cells require their particular profile of AE2 subtype expression in order to sustain, when needed, their extremely high rates of transcellular Cl- transport independent of regulatory factors, which might regulate AE2 in other cells.
|
REFERENCES |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
| ADAIR-KIRK T. L., COX, K. H. & COX, J. V. (1999). Intracellular trafficking of variant chicken kidney AE1 anion exchangers: role of alternative NH2 termini in polarized sorting and Golgi recycling. Journal of Cell Biology 147, 1237-1248 | [Abstract/Full Text] |
| ALPER S. L. (1994). The band 3-related AE anion exchanger gene family. Cellular Physiology and Biochemistry 4, 265-281 | |
| ALPER S. L., ROSSMANN, H., WILHELM, S., STUART-TILLEY, A. K., SHMUKLER, B. E. & SEIDLER, U. (1999). Expression of AE2 anion exchanger in mouse intestine. American Journal of Physiology 40, G321-332 | |
| ALPER S. L., STUART-TILLEY, A., SIMMONS, C. F., BROWN, D. & DRENCKHAHN, D. (1994). The fodrin-ankyrin cytoskeleton of choroid plexus preferentially colocalizes with apical Na+/K+ ATPase rather than with basolateral anion exchanger AE2. Journal of Clinical Investigation 93, 1430-1438 | [Medline] |
| BACHMANN O., SONNENTAG, TH., SIEGEL, W.-K., LAMPRECHT, G., WEICHERT, A., GREGOR, M. & SEIDLER, U. (1998). Different secretagogues activate different Na+/H+ exchanger isoforms in rabbit parietal cells. American Journal of Physiology 275, G185-193 | |
| BOYARSKY G., GANZ, M. B., STERZEL, R. B. & BORON, W. F. (1988). pH regulation in single glomerular mesangial cells. I. Acid extrusion in absence and presence of HCO3-. American Journal of Physiology 255, C844-856 | [Medline] |
| BROWN M. R. & CHEW, C. S. (1987). Multiple effects of phorbol ester on secretory activity in rabbit gastric glands and parietal cells. Canadian Journal of Physiology and Pharmacology 65, 1840-1847 | [Medline] |
| CHEW C. S., LJUNGSTROM, M., SMOLKA, A. & BROWN, M. R. (1989). Primary culture of secretagogue-responsive parietal cells from rabbit gastric mucosa. American Journal of Physiology 256, G254-263 | [Medline] |
| CHOMCZYNSKI P. & SACCHI, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry 162, 156-159 | [Medline] |
| CHOW A. (1998). AE2a and AE2b expression have distinct tissue-related and developmental regulations. Gastroenterology A359, G1466 | |
| CHOW A. (1999). Identification of cell type specific AE2b promoter and an upstream possitive regulatory region. Gastroenterology A869, G3779 | |
| CHOW A., DOBBINS, J. W., ARONSON, P. S. & IGARASHI, P. (1992). cDNA cloning and localization of a band 3-related protein from ileum. American Journal of Physiology 263, G345-352 | [Medline] |
| FAIN G. L., SMOLKA, A., CILLUFFO, M. C., FAIN, M. J., LEE, D. A., BRECHA, N. C. & SACHS, G. (1988). Monoclonal antibodies to the H+/K+ ATPase of gastric mucosa selectively stain the non-pigmented cells of the rabbit ciliary body epithelium. Investigative Ophthalmology and Visual Science 29, 785-794 | [Abstract] |
| HUMPHREYS B. D., JIANG, L., CHERNOVA, M. N. & ALPER, S. L. (1994). Functional characterization and regulation by pH of murine AE2 anion exchanger expressed in Xenopus oocytes. American Journal of Physiology 267, C1295-1307 | [Medline] |
| KARAM S. M. & LEBLOND, C. P. (1993). Dynamics of epithelial cells in the corpus of the mouse stomach. I. Identification of proliferative cell types and pinpointing of the stem cell. Anatomical Record 236, 259-279 | [Medline] |
| KARAM S. M., LI, Q. & GORDON, J. I. (1997). Gastric epithelial morphogenesis in normal and transgenic mice. American Journal of Physiology 272, G1209-1220 | [Medline] |
| KOPITO R. R. (1990). Molecular biology of the anion exchanger gene family. International Review of Cytology 123, 177-199 | [Medline] |
| KOPITO R. R. & LODISH, H. F. (1985). Primary structure and transmembrane orientation of the murine anion exchange protein. Nature 316, 234-238 | [Medline] |
| KUDRYCKI K. E., NEWMAN, P. R. & SHULL, G. E. (1990). cDNA cloning and tissue distribution of mRNAs for two proteins that are related to the band 3 Cl-/HCO3- exchanger. Journal of Biological Chemistry 265, 462-471 | [Abstract] |
| KUDRYCKI K. E. & SHULL, G. E. (1993). Rat kidney band 3 Cl-/HCO3- exchanger mRNA is transcribed from an alternative promoter. American Journal of Physiology 264, F540-547 | [Medline] |
| LAEMMLI U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277, 680-685 | |
| LAMPRECHT G., SEIDLER, U. & CLASSEN, M. (1993). Intracellular pH-regulating ion transport mechanisms in parietal cell basolateral membrane vesicles. American Journal of Physiology 265, G903-910 | [Medline] |
| LEE B. S., GUNN, R. B. & KOPITO, R. R. (1991). Functional differences among nonerythroid anion exchangers expressed in a transfected human cell line. Journal of Biological Chemistry 266, 11448-11454 | [Abstract] |
| LI Q., KARAM, S. M. & GORDON, J. I. (1996). Diphtheria toxin-mediated ablation of parietal cells in the stomach of transgenic mice. Journal of Biological Chemistry 271, 3671-3676 | [Abstract/Full Text] |
| MUALLEM S., BLISSARD, D., CRAGOE, E. J. JR & SACHS, G. (1988). Activation of the Na+/H+ and Cl-/HCO3- exchange by stimulation of acid secretion in the parietal cell. Journal of Biological Chemistry 263, 14703-14711 | [Abstract] |
| MUALLEM S., FIMMEL, C. J., PANDOL, S. J. & SACHS, G. (1986). Regulation of free cytosolic Ca2+ in the peptic and parietal cells of the rabbit gastric gland. Journal of Biological Chemistry 261, 2660-2667 | [Abstract] |
| NANDI J., CROCKETT, J. & LEVINE, R. A. (1994). A possible role of protein kinase C in augmenting H+ secretion by nonsteroidal anti-inflammatory drugs. Journal of Pharmacology and Experimental Therapeutics 269, 932-940 | [Abstract] |
| PARADISO A. M., TOWNSLEY, M. C., WENZL, E. & MACHEN, T. E. (1989). Regulation of intracellular pH in resting and in stimulated parietal cells. American Journal of Physiology 257, C554-561 | [Medline] |
| ROSSMANN H., BACHMANN, O., VIEILLARD-BARON, D., GREGOR, M. & SEIDLER, U. (1999). Na+/HCO3- cotransport and expression of NBC1 and NBC2 in rabbit gastric parietal and mucous cells. Gastroenterology 116, 1389-1398 | [Medline] |
| SEIDLER U., BEINBORN, M. & SEWING, K. F. (1989). Inhibition of acid formation in rabbit parietal cells by prostaglandins is mediated by the prostaglandin E2 receptor. Gastroenterology 96, 314-320 | [Abstract] |
| SEIDLER U., ROITHMAIER, S., CLASSEN, M. & SILEN, W. (1992). Influence of acid secretory state on Cl--base and Na+/H+ exchange and pHi in isolated rabbit parietal cells. American Journal of Physiology 262, G81-91 | [Medline] |
| SEIDLER U. & SEWING, K. F. (1989). Ca2+-dependent and -independent secretagogue action on gastric mucus secretion in rabbit mucosal explants. American Journal of Physiology 256, G739-746 | [Medline] |
| SENO K., JOH, T., YOKOYAMA, Y. & ITOH, M. (1995). Role of mucus in gastric mucosal injury induced by local ischemia/reperfusion. Journal of Laboratory and Clinical Medicine 126, 287-293 | [Medline] |
| SONNENTAG T., SIEGEL, W. K., BACHMANN, O., ROSSMANN, H., MACK, A., WAGNER, H. J., GREGOR, M. & SEIDLER, U. (2000). Agonist-induced cytoplasmic volume changes in cultured rabbit parietal cells. American Journal of Physiology 279, G40-48 | |
| STUART-TILLEY A., SARDET, C., POUYSSEGUR, J., SCHWARTZ, M. A., BROWN, D. & ALPER, S.L. (1994). Immunolocalization of anion exchanger AE2 and cation exchanger NHE-1 in distinct adjacent cells of gastric mucosa. American Journal of Physiology 266, C559-568 | [Medline] |
| STUART-TILLEY A., SHMUKLER, B. E., BROWN, D. & ALPER, S. L. (1998). Immunolocalization and tissue-specific splicing of AE2 anion exchanger in mouse kidney. Journal of the American Society of Nephrology 9, 946-959 | [Medline] |
| TERANO A., SHIGA, J., HIRAISHI, H., OTA, S. & SUGIMOTO, T. (1986). Protective action of tetraprenylacetone against ethanol-induced damage in rat gastric mucosa. Digestion 35, 182-188 | [Medline] |
| THOMAS H. A. & MACHEN, T. E. (1991). Regulation of Cl-/HCO3- exchange in gastric parietal cells. Cellular Regulation 2, 727-737 | |
| WANG Z., SCHULTHEIS, P. J. & SHULL, G. E. (1996). Three N-terminal variants of the AE2 Cl-/HCO3- exchanger are encoded by mRNAs transcribed from alternative promoters. Journal of Biological Chemistry 271, 7835-7843 | [Abstract/Full Text] |
Acknowledgements
We gratefully acknowledge the technical help of Dorothee Vieillard-Baron. We also thank Barbara Seidler for providing gastric cell type poly(A+) RNA for the Northern blot, Petra Jacob for providing gastric basolateral membranes for immunoblots, Wolf-Kristian Siegel and Thorsten Sonnentag for their help with the fluorometric experiments, and Alexander Zolotarev for helpful discussion. This work was supported by grants Se 460/2-5, Se 460/9-1, 9-2 and 9-3 from the Deutsche Forschungsgemeinschaft and IZKF grant IIIC1 from the German Ministry for Education and Research (BMBF).
Heidi Rossmann and Oliver Bachmann contributed equally to this work.
Corresponding author
U. Seidler: Abteilung Innere Medizin I, Universitätsklinikum Schnarrenberg, Eberhard-Karls Universität Tübingen, Otfried-Müller Strasse 10, 72076 Tübingen, Germany.
Email: ursula.seidler{at}uni-tuebingen.de
This article has been cited by other articles:
|
|
 |
|
  T. Fujii, Y. Takahashi, Y. Itomi, K. Fujita, M. Morii, Y. Tabuchi, S. Asano, K. Tsukada, N. Takeguchi, and H. Sakai K+-Cl- Cotransporter-3a Up-regulates Na+,K+-ATPase in Lipid Rafts of Gastric Luminal Parietal Cells J. Biol. Chem., March 14, 2008; 283(11): 6869 - 6877. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Heitzmann and R. Warth No Potassium, No Acid: K+ Channels and Gastric Acid Secretion Physiology, October 1, 2007; 22(5): 335 - 341. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Pushkin and I. Kurtz SLC4 base (HCO3-, CO32-) transporters: classification, function, structure, genetic diseases, and knockout models Am J Physiol Renal Physiol, March 1, 2006; 290(3): F580 - F599. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. McDaniel, A. J. Pace, S. Spiegel, R. Engelhardt, B. H. Koller, U. Seidler, and C. Lytle Role of Na-K-2Cl cotransporter-1 in gastric secretion of nonacidic fluid and pepsinogen Am J Physiol Gastrointest Liver Physiol, September 1, 2005; 289(3): G550 - G560. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Xu, J. Henriksnas, S. Barone, D. Witte, G. E. Shull, J. G. Forte, L. Holm, and M. Soleimani SLC26A9 is expressed in gastric surface epithelial cells, mediates Cl-/HCO3- exchange, and is inhibited by NH4+ (Report) Am J Physiol Cell Physiol, August 1, 2005; 289(2): C493 - C505. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. R. Gawenis, J. M. Greeb, V. Prasad, C. Grisham, L. P. Sanford, T. Doetschman, A. Andringa, M. L. Miller, and G. E. Shull Impaired Gastric Acid Secretion in Mice with a Targeted Disruption of the NHE4 Na+/H+ Exchanger J. Biol. Chem., April 1, 2005; 280(13): 12781 - 12789. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Komlosi, S. Frische, A. L. Fuson, A. Fintha, A. Zsembery, J. Peti-Peterdi, and P. D. Bell Characterization of basolateral chloride/bicarbonate exchange in macula densa cells Am J Physiol Renal Physiol, February 1, 2005; 288(2): F380 - F386. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. R. Gawenis, C. Ledoussal, L. M. Judd, V. Prasad, S. L. Alper, A. Stuart-Tilley, A. L. Woo, C. Grisham, L. P. Sanford, T. Doetschman, et al. Mice with a Targeted Disruption of the AE2 Cl-/HCO3- Exchanger Are Achlorhydric J. Biol. Chem., July 16, 2004; 279(29): 30531 - 30539. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Frische, A. S. Zolotarev, Y.-H. Kim, J. Praetorius, S. Alper, S. Nielsen, and S. M. Wall AE2 isoforms in rat kidney: immunohistochemical localization and regulation in response to chronic NH4Cl loading Am J Physiol Renal Physiol, June 1, 2004; 286(6): F1163 - F1170. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Xu, S. Barone, S. Petrovic, Z. Wang, U. Seidler, B. Riederer, K. Ramaswamy, P. K. Dudeja, G. E. Shull, and M. Soleimani Identification of an apical Cl-/HCO3- exchanger in gastric surface mucous and duodenal villus cells Am J Physiol Gastrointest Liver Physiol, December 1, 2003; 285(6): G1225 - G1234. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Zhou, C. Watson, C. Fu, X. Yao, and J. G. Forte Myosin II is present in gastric parietal cells and required for lamellipodial dynamics associated with cell activation Am J Physiol Cell Physiol, September 1, 2003; 285(3): C662 - C673. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Petrovic, X. Ju, S. Barone, U. Seidler, S. L. Alper, H. Lohi, J. Kere, and M. Soleimani Identification of a basolateral Cl-/HCO3- exchanger specific to gastric parietal cells Am J Physiol Gastrointest Liver Physiol, June 1, 2003; 284(6): G1093 - G1103. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. N. Chernova, A. K. Stewart, L. Jiang, D. J. Friedman, Y. Z. Kunes, and S. L. Alper Structure-function relationships of AE2 regulation by Ca2+i-sensitive stimulators NH+4 and hypertonicity Am J Physiol Cell Physiol, May 1, 2003; 284(5): C1235 - C1246. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Coskun, H. K. Baumgartner, S. Chu, and M. H. Montrose Coordinated regulation of gastric chloride secretion with both acid and alkali secretion Am J Physiol Gastrointest Liver Physiol, November 1, 2002; 283(5): G1147 - G1155. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Petrovic, Z. Wang, L. Ma, U. Seidler, J. G. Forte, G. E. Shull, and M. Soleimani Colocalization of the apical Cl-/HCO3- exchanger PAT1 and gastric H-K-ATPase in stomach parietal cells Am J Physiol Gastrointest Liver Physiol, November 1, 2002; 283(5): G1207 - G1216. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME |
-subunit antibody.