Physiol. Genomics Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Physiol. Genomics 24: 124-132, 2006. First published November 8, 2005; doi:10.1152/physiolgenomics.00133.2005
1094-8341/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
24/2/124    most recent
00133.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (10)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jain, R. N.
Right arrow Articles by Samuelson, L. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jain, R. N.
Right arrow Articles by Samuelson, L. C.
Received 8 June 2005; accepted in final form 4 November 2005.
Physiological Genomics 24:124-132 (2006)
1094-8341/05 $8.00 © 2006 American Physiological Society

Gene expression profiling of gastrin target genes in parietal cells

Renu N. Jain1, Cynthia S. Brunkan1, Catherine S. Chew2 and Linda C. Samuelson1

1 Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan
2 Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Previous studies demonstrated that mice with a null mutation in the gene encoding the hormone gastrin have impaired gastric acid secretion. Hence, the aim of this study was to evaluate changes in the acid-secreting parietal cell in gastrin-deficient (GAS-KO) mice. Analysis of several transcripts encoding parietal cell proteins involved in gastric acid secretion showed reduced abundance in the GAS-KO stomach, including H+,K+-ATPase {alpha}- and ß-subunits, KCNQ1 potassium channel, aquaporin-4 water channel, and creatine kinase B, which were reversed by gastrin infusion for 1 wk. Although mRNA and protein levels of LIM and SH3 domain-containing protein-1 (LASP-1) were not greatly changed in the mutant, there was a marked reduction in phosphorylation, consistent with its proposed role as a cAMP signal adaptor protein associated with acid secretion. A more comprehensive analysis of parietal cell gene expression in GAS-KO mice was performed using the Affymetrix U74AV2 chip with RNA from parietal cells purified by flow cytometry to >90%. Comparison of gene expression in GAS-KO and wild-type mice identified 47 transcripts that differed by greater than or equal to twofold, suggesting that gastrin affects parietal cell gene expression in a specific manner. The differentially expressed genes included several genes in signaling pathways, with a substantial number (20%) known to be target genes for Wnt and Myc.

stomach; gastric acid secretion; gene expression microarray; gastrin-deficient mice; fundic mucosa


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
THE PRINCIPAL HORMONE regulating gastric acid secretion is gastrin. Eating a meal stimulates gastrin release from endocrine G cells in the distal portion of the stomach, and circulating gastrin stimulates acid release from parietal cells in the fundus of the stomach. Gastrin stimulation of acid secretion involves both direct and indirect stimulation of parietal cells. In the stomach, gastrin (CCK2) receptors are found on both parietal and enterochromaffin-like (ECL) cells (19, 31, 41). ECL cells regulate parietal cells and acid secretion by releasing the potent acid secretagogue histamine, which binds to H2 receptors on parietal cells. Gastrin stimulates both the synthesis and secretion of histamine in ECL cells (32). Thus gastrin induces acid secretion directly by binding to CCK2 receptors on parietal cells and indirectly by stimulating histamine release from ECL cells (33). Both direct and indirect stimulation by gastrin contribute unique components to the regulation of acid secretion and other aspects of the parietal cell.

In addition to inducing acid secretion, gastrin plays a role as a growth factor for the gastric mucosa. Elevated gastrin increases the number of both parietal and ECL cells (10, 27), and sustained hypergastrinemia has been associated with the development of ECL cell carcinoid tumors (29). Gastrin has been shown to increase the expression and secretion of certain growth factors in parietal cells, including heparin-binding epidermal growth factor (HB-EGF) (2, 37, 40) and amphiregulin (37, 40). Gastrin also induces regenerating gene protein (Reg-1) expression in ECL cells (1, 15), which is proposed to be a growth factor related to stress or healing of the gastric mucosa (23).

Generation of gastrin-deficient (GAS-KO) mice by gene targeting in embryonic stem cells confirmed the importance of gastrin in acid secretion (13, 25). GAS-KO mice are viable and fertile, but have significantly reduced basal and secretagogue-induced gastric acid secretion (13). Parietal cells and ECL cells are formed in the mutant, demonstrating that gastrin is not required for development of these cell lineages. However, gastrin contributes to the functional maturation of both parietal and ECL cells. Parietal cells appear immature in GAS-KO mice, since they are smaller in size and have decreased gastric acid secretion (13, 19). Moreover, GAS-KO parietal cells have impaired migration along the gastric glands (24). Gastrin replacement using osmotic minipumps can at least partially restore basal acid secretion in GAS-KO mice, demonstrating that the mutant parietal cells have the capacity to respond to gastrin and that the impairment in the acid secretory system can be repaired (5, 13).

Previous studies have shown that gastrin regulates both parietal and ECL cell gene expression. In ECL cells, gastrin enhances the expression of a number of genes involved in histamine production, including histidine decarboxylase, vesicular monoamine transporter-2, and chromogranin A (12, 16, 20). More limited information exists on how gastrin regulates parietal cell gene expression. Gastrin has been shown to increase the abundance of H+,K+-ATPase-{alpha} and carbonic anhydrase II mRNAs in isolated canine parietal cells and in animal models with elevated gastrin (4, 22, 39). However, a comprehensive analysis of gastrin regulation of parietal cell gene expression in vivo has not been described. In this study, GAS-KO mice were used to assess changes in the parietal cell resulting from the loss of gastrin. Expression levels of several parietal cell-specific genes involved in acid secretion were examined in GAS-KO mice and compared with expression levels in wild-type controls and with GAS-KO mice treated with gastrin. In addition, gene expression profiling using Affymetrix microarrays was performed on parietal cells purified by flow cytometry to analyze changes in gene expression resulting from the loss of gastrin.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mice.
The generation of GAS-KO mice by gene targeting in embryonic stem cells was described previously (13). Mutant mice and wild-type (WT) controls were maintained by homozygous breeding on strain background 129X1/SvJ in a specific pathogen-free facility. Experiments were performed on male mice 8–10 wk of age. Before death, mice were fasted overnight with free access to water. Animal use was approved by the University of Michigan Committee on Use and Care of Animals.

Parietal cell purification.
Flow cytometry was used to purify parietal cells based on their large size and structural complexity, as described in Hinkle et al. (19). Briefly, stomachs were excised from 10–14 mice, the fundus was dissected and dissociated into single cells with pronase, and parietal cells were sorted based on light scatter properties using a Becton Dickinson Elite flow cytometer, yielding ~106 parietal cells. To assess purity of the sorted population, cells were cytospun onto microscope slides and immunostained with a monoclonal antibody for the {alpha}-subunit of H+,K+-ATPase (1:500 dilution, Medical and Biological Laboratories), followed by incubation with Cy2-conjugated donkey anti-mouse IgG (1:200 dilution, Jackson Immunoresearch Laboratories) and the nuclear stain 4,6-diamidino-2-phenylindole (DAPI; 1 µg/ml, Sigma). Sorted fractions >90% pure were stored at –80°C for subsequent RNA isolation and microarray analysis.

Microarray analysis.
RNA was extracted from sorted parietal cell preparations using Trizol (Invitrogen) and purified by RNeasy (Qiagen). Three independent RNA samples from pooled WT and GAS-KO parietal cells were prepared and used for microarray analysis with the U74AV2 Affymetrix chip, which contains 12,422 probe sets representing 8,700 genes. Microarray Suite 4.0 software (Affymetrix) was used for obtaining probe pair hybridization signals. Data analysis was performed as described previously (21, 35). Detailed data analysis methods are available at http://dot.ped.med.umich.edu:2000/ourimage/pub/shared/Affymethods.html. Data was quantile normalized to adjust for differences in the probe intensity distribution across the six chips. Fold change (FC) was calculated between WT and GAS-KO by replacing mean expression values <100 units with 100 to avoid negative values or spuriously large fold changes. The complete data set is deposited in the Gene Expression Omnibus (GEO) database (accession no. GSE2772) at the following site: http://www.ncbi.nlm.nih.gov/geo/.

Gastrin replacement analysis.
Gastrin (5 µg·kg–1·h–1 rat gastrin-17, Bachem) or vehicle (1% BSA in 0.9% NaCl) was infused for 7 days, using 0.2-ml capacity miniosmotic pumps (1 µl/h; 2001 model, Alzet), to GAS-KO mice (n = 4/group) as described previously (13). Filled pumps were primed overnight in normal saline before inserting them subcutaneously in mice. After 7 days, the animals were killed with the pumps intact, the fundic mucosa was scraped, and RNA was extracted and analyzed by quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR).

QRT-PCR analysis.
RNA from fundic mucosal scrapings was isolated using Trizol (Invitrogen), purified, and DNase treated using RNeasy (Qiagen), and 200 ng were reverse transcribed in a 10-µl volume using either the Taqman Gold RT-PCR kit (Invitrogen) or Iscript (BioRad), following manufacturers' protocols. QRT-PCR was performed using an Icycler with SYBR Green dye (Molecular Probes). Each 20-µl reaction contained 2 µl of reverse-transcribed product, BioRad PCR buffer, 5.5 mM MgCl2, 100 nM each primer, SYBR Green, 10 nM fluorescein, 200 µM dNTPs, and 0.025 U Platinum Taq polymerase (Invitrogen). The following amplification conditions were used: 3 min at 95°C, 40 cycles of 9 s at 95°C and 1 min at 60°C, followed by 1 min at 55°C. Melt curve analysis was used to assess product purity. Beacon software (BioRad) was used to design primer sequences (Table 1). Primer pairs were validated by sequencing the amplification products and by confirming quantitative amplification with a dilution of control tissue RNAs. Data for each gene were normalized to the expression of Gapdh, which remained the same in WT and GAS-KO samples.


View this table:
[in this window]
[in a new window]
  		Table 1. QRT-PCR primer sequences

 
QRT-PCR analysis of parietal cell gene expression was performed on cells purified ≥90% from WT (4–9 mice/sort) or GAS-KO (4–5 mice/sort) mice by flow sorting, as described above. RNA was purified and DNase treated using the RNeasy mini kit (Qiagen). RNA was isolated from three independent cell sorts for each genotype. Purity of the sort was determined by staining for the parietal specific marker H+,K+-ATPase, as described above. RNA quality and quantity were determined using the Agilent 2100 Bioanalyzer picochip, followed by reverse transcription (20 ng/10 µl) using Iscript (BioRad). QRT-PCR was performed using 2 µl of the reverse-transcribed product, as described above.

Protein expression analysis.
Fundic mucosal scrapings from two to four mice were pooled and homogenized with a syringe and increasingly smaller needles (18–25 gauge) in 0.25 ml of buffer [50 mM ß-glycerophosphate, pH 7.3, 1.5 mM EGTA, 5 mM EDTA, 1 mM DTT, 0.2 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, and a protease inhibitor cocktail containing leupeptin (2 µg/ml), antipain (2 µg/ml), benzamidine (20 µg/ml), aprotinin (0.01–0.02 trypsin inhibitor U/ml), chymostatin (2 µg/ml), and pepstatin-A (2 µg/ml)]. An aliquot of the homogenate was saved for protein determination (BioRad Lowry assay), and an equal volume of 2x SDS-stop solution (4% SDS, 0.125 M Tris·HCl, pH 6.8, 2 mM EDTA, 20% glycerol, 200 mM DTT, and 0.004% bromophenol blue) was added to the remainder and boiled for 10 min. The samples were cooled on ice and stored at –20°C until further use. Western analysis of protein homogenates (20 µg/lane) resolved on 10% SDS polyacrylamide gels (BioRad) included electrotransfer onto polyvinylidene difluoride (PVDF)-plus membrane (Osmonics) and chemiluminescence detection (Pierce Supersignal West Pico Substrate). Antibodies for H+,K+-ATPase {alpha}- and ß-subunits (Medical and Biological Laboratories) and GAPDH (Chemicon) were used at 1:1,000 dilution. Anti-mouse horseradish peroxidase (HRP; Amersham) and anti-rabbit HRP (Jackson Immunoresearch labs) were used as secondary antibodies at 1:5,000 dilution. Western blots were quantitated on the Fluoromax Image Analyzer using Quantity One software (BioRad).

LIM and SH3 domain-containing protein-1 phosphorylation analysis.
Protein extracts were prepared from GAS-KO and WT mice under basal (treated with the H2 receptor antagonist ranitidine) or stimulated (histamine treated) conditions. Three mice per genotype for each condition were pooled for analysis. Basal group mice were injected intraperitoneally with ranitidine (10 mg/kg) 24, 14, and 2.5 h before death, and the histamine group was injected with PBS at the same time intervals, followed by histamine injection (20 mg/kg) 30 min before death. Two-dimensional (2D) Western blot analyses were performed on paired samples as previously described (7). In brief, homogenates of the fundic mucosa were prepared as described above, and an equal volume of SDS-ßME (3% SDS, 10% ß-mercaptoethanol) was added. Samples were immediately frozen in liquid N2 and stored at –80°C. For 2D gel analysis, protein was precipitated with acetone at room temperature, and pellets (250 µg of protein) were dissolved in rehydration buffer [8 M urea, 2% CHAPS, 18 mM DTT, 0.001% bromophenol blue, 0.5% immobilized pH gradient (IPG) buffer, pH 3–10; Amersham-Pharmacia] and subjected to isoelectric focusing with an IPGPhor [IPG strips, pH 3–10 nonlinear (NL); Amersham-Pharmacia], followed by SDS-polyacrylamide gel electrophoresis. Resolved proteins were transferred to nitrocellulose for Western blot using a LIM and SH3 domain-containing protein-1 (LASP-1) monoclonal antibody (clone 8C6) and enhanced chemiluminescence detection (Amersham-Pharmacia). Changes in LASP-1 protein phosphorylation were defined based on the acidic shift of entrained spots (9), which were quantitated with a CCD-based Syngene GeneGnome system (7).

Immunostaining.
Dispersed fundic mucosal cells were prepared from GAS-KO stomach using pronase as described in Hinkle et al. (19). The cell suspension was fixed and permeabilized using the Cytofix-Cytoperm kit (Pharmingen) and stained with polyclonal antibodies (1:10 dilution) against annexin A1 (Santa Cruz), Nmyc (Santa Cruz C19), or Myc (Santa Cruz N262) using a Cy3-conjugated secondary antibody (1:500 dilution, Jackson Immunoresearch Laboratories). Cells were co-stained with a parietal cell-specific H+,K+-ATPase {alpha}-subunit monoclonal antibody (1:100 dilution, Medical and Biological Laboratories) using a Cy2-conjugated donkey anti-mouse secondary antibody (1:500 dilution, Jackson Immunoresearch Laboratories). Stained cells were cytospun onto slides, covered with Fluoromount-G (Southern Biotechnology Assoc.) containing the nuclear stain DAPI (1 µg/ml, Sigma), and visualized using a Nikon Eclipse 800 microscope equipped with a Spot digital camera (Diagnostic Instruments).

Statistical analysis.
Data are presented as means ± SE (n = 3–4) and were analyzed using Student's t-test, with P < 0.05 considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Reduced expression of parietal cell components of acid secretion.
To determine how loss of gastrin affects cells in the stomach, we examined critical components of the acid secretory system in GAS-KO mice. Our analysis first focused on parietal cell-specific genes. RNA and protein samples were isolated from fundic mucosal scraping, and expression in GAS-KO mice was compared with wild-type controls and with GAS-KO mice in which gastrin was replaced for 1 wk with osmotic minipumps. QRT-PCR analysis of H+,K+-ATPase {alpha}- and ß-subunit mRNAs showed that the transcripts for the proton pump subunits were both modestly reduced in GAS-KO mice to 80% of wild-type levels (Fig. 1A). Similarly, Western blot analysis of protein expression demonstrated lower H+,K+-ATPase in the mutant, with {alpha}- and ß-subunits reduced to 60 and 40% of normal, respectively (Fig. 1, B and C). The reduction in proton pump expression is actually larger when normalized to cell number, since the proportion of parietal cells in the fundic mucosa is increased approximately twofold in GAS-KO mice of this age (19). Gastrin infusion for 1 wk in GAS-KO mice increased both {alpha}- and ß-H+,K+-ATPase mRNA by 2- and 1.5-fold, respectively, confirming gastrin responsiveness of these genes (Fig. 1A).



View larger version (31K):
[in this window]
[in a new window]
  		Fig. 1. Reduced H+,K+-ATPase expression in gastrin-deficient (GAS-KO) mice. A: abundance of mRNAs for the {alpha}- and ß-H+,K+-ATPase genes was measured by quantitative RT-PCR (QRT-PCR). Three independent fundic mucosal RNA samples from wild-type (WT) mice (solid bars), GAS-KO mice (open bars), and gastrin-replaced mice (GR; gray bars) were evaluated. Data were normalized to Gapdh expression in the same samples and reported as fold change (mean ± SE) from levels in WT mice (*P < 0.05). B: Western blot analysis of H+,K+-ATPase {alpha}- and ß-subunits in WT and GAS-KO mice. Representative blots from fundic mucosal protein homogenates are shown with the GAPDH loading control. C: quantitation of Western blots for H+,K+-ATPase {alpha}- and ß-subunits. Three independent fundic mucosal protein homogenates were analyzed. Protein levels were quantitated using the Fluoromax Image Analyzer, normalized to GAPDH signals in each sample, and represented as fold change (mean ± SE) from WT levels (*P < 0.05).

 
We next evaluated LASP-1, which is a parietal cell cytoskeletal-associated adaptor protein regulated by PKA phosphorylation (9). Similar to the proton pump subunits, the abundance of LASP-1 RNA and protein was modestly reduced to 80% of wild-type levels in GAS-KO mice (data not shown). However, measurement of LASP-1 protein phosphorylation showed a marked change under both basal (ranitidine treated) and stimulated (histamine treated) conditions (Fig. 2). Three different LASP-1 isoforms were identified in fundic mucosal extracts by two-dimensional electrophoresis that have differing charge characteristics consistent with differential phosphorylation. The most acidic, hyperphosphorylated form c was predominate after histamine treatment in wild-type mice. This form increased from undetectable levels in basal conditions to 51% of the total LASP-1 protein after stimulation. In contrast, form c was not observed in histamine-stimulated GAS-KO mice, suggesting that histamine treatment did not increase phosphorylation. In addition, LASP-1 was hypophosphorylated in unstimulated GAS-KO mice, with an increase in the more basic form a compared with wild-type mice from 27 to 39% of the total LASP-1 protein (Fig. 2). Thus GAS-KO mice lack the normal phosphorylation response to histamine stimulation and have reduced basal LASP-1 phosphorylation.



View larger version (31K):
[in this window]
[in a new window]
  		Fig. 2. LIM and SH3 domain-containing protein-1 (LASP-1) is hypophosphorylated in GAS-KO mice. Two-dimensional (2D) LASP-1 Western blot of fundic mucosal scrapings from WT and GAS-KO mice treated with the H2 receptor blocker ranitidine (Ran) or stimulator histamine (His). Three LASP-1 forms were identified (forms a, b, and c), with form a being the most basic and least phosphorylated form and form c the most acidic and highly phosphorylated form. The proportion of the total signal represented by each isoform (circled spot) is indicated below. Histamine stimulation shifts LASP-1 to the more highly phosphorylated form c in WT mice but not in GAS-KO mice.

 
The KCNQ1 potassium channel, which co-localizes with the proton pump at the apical membrane, has been proposed to be responsible for the K+ conductance associated with acid secretion (11, 17, 28). Quantitation of Kcnq1 transcripts in fundic mucosa demonstrated markedly lower expression in GAS-KO mice to 30% of wild-type levels, with partial recovery after gastrin replacement (Fig. 3). Similar results were seen with the energy-coupling protein creatine kinase B, which has been localized on H+,K+-ATPase-enriched membranes in the parietal cell (38). GAS-KO gastric mucosa contained ~25% of normal levels of creatine kinase B mRNA, which was partially restored after gastrin replacement (Fig. 3). The basolaterally located water channel aquaporin-4 (14) was similarly decreased to 30% of normal levels in the mutant, with partial restoration after gastrin replacement. Thus changes in parietal cell gene expression in GAS-KO mice include channels/transporters that are proposed to play a role in acid secretion at the apical, acid-secreting membrane as well as the basolateral membrane.



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 3. Expression of parietal cell genes involved in acid secretion. QRT-PCR was used to measure the abundance of mRNAs for the parietal cell-specific genes Kcnq1, Ckb, and Aqp4 in total RNA isolated from fundic mucosal scrapings from WT (solid bars), GAS-KO (open bars), and GAS-KO mice treated with gastrin (gray bars). Three independent samples were analyzed, and values were normalized to Gapdh expression and shown as fold change (mean ± SE) compared with WT levels (*P < 0.05).

 
Microarray analysis of parietal cells.
To gain a more comprehensive view of changes in parietal cell gene expression in GAS-KO mice, microarray analysis was performed on purified cells. Flow cytometry was used to isolate viable parietal cells to >90% purity from GAS-KO and wild-type mice (Fig. 4). RNAs from cells isolated from three independent pools of mice for each genotype were analyzed on Affymetrix murine U74Av2 chips. Comparison of the microarray data from GAS-KO and wild-type parietal cells revealed a number of genes with altered expression in the gastrin-deficient mutant. Of the 12,422 probe sets on the U74Av2 chip, 47 transcripts for known genes were changed greater than or equal to twofold (P < 0.05) in the GAS-KO mice. These genes are listed in Table 2 together with a few transcripts of functional interest with more modest, yet statistically significant, changes. Of these 50 genes, 31 are upregulated (62%) and 19 are downregulated (38%) in the mutant.



View larger version (49K):
[in this window]
[in a new window]
  		Fig. 4. Purification of parietal cells by flow cytometry. Live parietal cells were purified from dispersed fundic mucosal cell preparations based on their large size and structural complexity. Shown are representative cells sorted from WT (A) and GAS-KO mice (B). Cells were stained with an antibody to the parietal cell marker H+,K+-ATPase {alpha}-subunit (green), and 4,6-diamidino-2-phenylindole (DAPI; blue) was used to stain nuclei. Greater than 90% of the cells stained for the H+,K+-ATPase marker, demonstrating the high purity of the preparation. Note that the images are shown at equal magnification and that GAS-KO mice have smaller parietal cells. Bar = 20 µm.

 

View this table:
[in this window]
[in a new window]
  		Table 2. Gastrin-regulated parietal cell transcripts*

 
A significant fraction (10/50, 20%) of the differentially expressed genes identified by microarray analysis are known targets for Wnt and/or Myc signaling. We measured mRNA abundance for four of these genes by QRT-PCR to validate the microarray results (Fig. 5A). Parietal cells were purified by flow sorting from wild-type and GAS-KO mice and tested for expression of Arl4, Myc, Nmyc (Mycn), and Emp1. In concordance with the microarray data, expression of Arl4, Myc, and Nmyc was significantly increased in GAS-KO mice; Emp1 expression, although increased in GAS-KO parietal cells, did not reach significance. Immunohistochemical staining of dispersed gastric mucosal cells confirmed that Myc and Nmyc proteins were expressed in parietal cells, with Nmyc expression limited to cells that co-expressed H+,K+-ATPase {alpha}-subunit (Fig. 6). Thus Nmyc appears to be parietal cell specific in the mouse fundic mucosa.



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 5. QRT-PCR validation of gastrin-responsive parietal cell genes. A: quantitation of gene expression in purified parietal cells. Parietal cells were purified by flow sorting to ≥90%, RNA was isolated, and expression of Arl4, Myc, Nmyc and Emp1 was measured by QRT-PCR. Results are shown as fold change (mean ± SE) compared with WT levels (*P < 0.05), with n = 3 parietal cell RNA preparations per genotype. B: expression of the parietal cell-specific genes Anxa1, Pigr, Pthlh, and Itpr was determined in RNA prepared from fundic mucosal scrapings of WT, GAS-KO, and GR mice. Results are shown as fold change (mean ± SE) compared with WT levels (*P < 0.05), with n = 3 mucosal RNA preparations per group.

 


View larger version (49K):
[in this window]
[in a new window]
  		Fig. 6. Immunostaining of dispersed fundic cells. Nmyc (top), Myc (middle), and annexin A1 (bottom) proteins were detected in dispersed fundic mucosal cells by immunostaining with polyclonal antibodies and Cy3 (red)-conjugated secondary antibodies (left). Co-staining the parietal cell-specific H+,K+-ATPase {alpha}-subunit with a monoclonal antibody and Cy2 (green)-conjugated secondary antibody allowed identification of parietal cells in the mixed fundic cell preparation (right). The nuclear stain DAPI (right, blue) was used to mark all cells. Both N-myc and annexin A1 only stained parietal cells, while Myc is expressed in parietal cells and a subset of nonparietal cells (arrow).

 
Further validation of the microarray results was performed on RNA isolated from fundic mucosal scrapings from wild-type, GAS-KO, and GAS-KO mice treated with gastrin. Selected genes that have been characterized to be parietal cell specific were analyzed. The microarray data showed a 2.4-fold increase in expression of the anti-inflammatory gene annexin A1 in GAS-KO mice. We demonstrated that annexin A1 is parietal cell specific by co-staining dispersed fundic mucosal cells with antibodies against annexin A1 and H+,K+-ATPase {alpha}-subunit (Fig. 6). QRT-PCR analysis of fundic mucosal RNA showed increased Anxa1 expression in GAS-KO mice in correspondence with the microarray results. Moreover, administration of gastrin decreased gene expression to levels similar to wild-type mice, confirming that this gene is gastrin responsive (Fig. 5B).

We next examined the expression of three genes shown by Mills et al. (30) to be highly enriched in parietal cells, including the polymeric immunoglobulin receptor (Pigr), which was increased twofold in the microarray data, and parathyroid hormone-like hormone (Pthlh) and inositol 1,4,5-triphosphate receptor-2 (Itpr2), which were both decreased in GAS-KO. The results of QRT-PCR analysis of fundic mucosal RNA were in agreement with the microarray results (Fig. 5B). Pigr mRNA abundance was increased threefold in GAS-KO fundic mucosa, with normalization to wild-type levels after gastrin replacement. Similar to the microarray results, Pthlh expression was markedly reduced in GAS-KO mice to ~10% of wild-type levels. Gastrin infusion increased expression fivefold, confirming gastrin responsiveness. Similarly, Itpr2 was decreased to 40% of wild-type expression levels, with partial restoration after gastrin infusion.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we report a comparative analysis of critical components of the acid secretory system in wild-type and GAS-KO mice. Previous studies showed that gastrin-deficient mice have a severe reduction in gastric acid, including both basal and induced secretion (13, 25). The reduced acid secretion in GAS-KO mice is associated with morphological and functional changes in parietal cells, including smaller size (19), and altered migration in the gastric glands (24). We describe here an analysis of the molecular changes in the GAS-KO parietal cell, examining target molecules, as well as characterization of transcriptional differences by gene expression microarray analysis. Several molecules known to be important for acid secretion were reduced in GAS-KO parietal cells, including both {alpha}- and ß-subunits of the proton pump, with expression recovered after gastrin administration. This result agrees with earlier studies showing that animal models with elevated gastrin, as well as isolated parietal cells treated with gastrin peptides, increase expression of the {alpha}- and ß-subunits of H+,K+-ATPase (4, 22, 39).

In addition to the subunits of the proton pump, we discovered that several other key molecules thought to participate in gastric acid secretion are gastrin regulated, with reduced expression in GAS-KO mice and increased expression after gastrin administration. KCNQ1 has been suggested to be a critical potassium channel in the apical membrane, with activity directly coupled to proton extrusion (26). We observed decreased expression of Kcnq1 in GAS-KO mice. We also observed downregulation of expression of the basolateral water channel gene Aqp4, suggesting that expression of membrane transport molecules at both the apical and basolateral surfaces is regulated by gastrin. Acid secretion consumes a large amount of energy, and parietal cells are known to contain numerous mitochondria. Our data show that components of the energy supply system in the parietal cell are also reduced in GAS-KO mice. There is a marked reduction in expression of creatine kinase B (Crb), which has been proposed to supply ATP to the proton pump at the apical membrane (38), as well as reduced expression of Cox7a1 and Cox6b2, critical components of the mitochondrial electron transport chain. Together these data suggest that parietal cells in gastrin-deficient mice are functionally immature, with reduced expression of several different components of the acid secretory system that are characteristic of mature parietal cells.

The cytoskeletal adaptor protein LASP-1 is markedly hypophosphorylated in GAS-KO mice, with reduced basal phosphorylation and loss of histamine-stimulated phosphorylation. Histamine stimulation of the parietal cell is known to increase intracellular cAMP, and previous studies in rabbit isolated gastric glands demonstrated a strong association of PKA-induced LASP-1 phosphorylation with acid secretion and translocation to the acid secretory canalicular membrane (7–9). This is the first in vivo report of association of LASP-1 phosphorylation with acid secretion. GAS-KO mice lacked the normal phosphorylation response to histamine and also had reduced basal LASP-1 phosphorylation. The lack of a phosphorylation response to histamine stimulation suggests that one or more components of cAMP signaling and/or PKA phosphorylation are abnormal in GAS-KO mice. Previous studies showed that GAS-KO mutant mice do not increase acid secretion in response to acute histamine administration, although they do express normal levels of the H2 receptor mRNA in the stomach (13). Thus, in addition to the previously described defects in histamine synthesis in the ECL cells of GAS-KO mice (6, 13), our LASP-1 data show that the mutant parietal cells have a postreceptor defect in response to histamine stimulation.

We confirmed increased expression of the protooncogenes Myc and Nmyc, which are known to be direct transcriptional targets of Wnt signaling (18, 36). The microarray data contained 10 different gene targets for Wnt and/or Myc that were significantly upregulated in the GAS-KO parietal cells (Table 2). We confirmed increased expression of several of these targets by QRT-PCR analysis. We also showed that Nmyc is specific for parietal cells in the fundic mucosa. Nmyc is also expressed in differentiated epithelial cells in the intestine (3). The importance of Wnt or Myc signaling for parietal cell development and/or function has not been determined. Our data suggest that Wnt signaling might play an important role in parietal cell maturation, since the GAS-KO parietal cells are functionally immature and also exhibited increased expression of Wnt target genes.

Increased numbers of parietal cells have been reported in young adult GAS-KO mice (19). However, increased parietal cell number is not a consistent observation for GAS-KO mice (13, 25). As a consequence of the hypochlorhydria, bacterial overgrowth and inflammation occur in these mice as they age, which can lead to eventual parietal cell atrophy (42). Thus the parietal cell number can vary depending on the inflammatory status of the mice. The studies reported in this paper used young mice (8–10 wk of age) to minimize confounding inflammatory changes. Although there is no histological evidence of inflammation in these young GAS-KO mice (data not shown), we observed an upregulation of some immune/defense-related genes such as annexin A1 (Anxa1) and the polymeric immunoglobulin receptor (Pigr), which could indicate early inflammatory changes in the absence of evident histological changes.

This study demonstrates that gastrin deficiency results in limited, but significant, changes in parietal cell gene expression in vivo. These changes could reflect a direct consequence of loss of gastrin stimulation to the parietal cell as well as an indirect effect due to reduced histamine signaling resulting from impaired ECL cell function, or, perhaps, due to early inflammatory changes in the GAS-KO stomach. The observed repair of the acid secretory defect after administration of gastrin (13) suggests that the alterations in gene expression and the apparent immaturity of the GAS-KO parietal cells are not permanent. Thus gastrin can act in a dynamic way to sculpt the function of parietal cells. Analysis of changes in gene expression in the fundus of GAS-KO mice, including ECL cells and other gastric cells, would be helpful to obtain a more complete picture of the importance of gastrin for overall stomach function.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-56882 (to L. C. Samuelson) and a Pilot Project Award from the University of Michigan Gastrointestinal Peptide Center (P30-DK-34933; to R. N. Jain).


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the assistance of Thomas Saunders for implantation of the osmotic minipumps; David Misek, Rork Kuick, and Pascal Lescure for expert gene expression microarray analysis; and University of Michigan Cancer Center Flow Cytometry Core for assistance with purification of mouse parietal cells. We thank Andrea Todisco, Lymari Lopez-Diaz, and Kelli VanDussen for careful reading of the manuscript.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: L. C. Samuelson, Dept. of Molecular and Integrative Physiology, Univ. of Michigan, Ann Arbor, MI 48109-0622 (e-mail: lcsam{at}umich.edu)

10.1152/physiolgenomics.00133.2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Ashcroft FJ, Varro A, Dimaline R, and Dockray GJ. Control of expression of the lectin-like protein Reg-1 by gastrin: role of the Rho family GTPase RhoA and a C-rich promoter element. Biochem J 381: 397–403, 2004.[CrossRef][ISI][Medline]
  2. Beales IL. Gastrin and interleukin-1beta stimulate growth factor secretion from cultured rabbit gastric parietal cells. Life Sci 75: 2983–2995, 2004.[CrossRef][ISI][Medline]
  3. Bettess MD, Dubois N, Murphy MJ, Dubey C, Roger C, Robine S, and Trumpp A. c-Myc is required for the formation of intestinal crypts but dispensable for homeostasis of the adult intestinal epithelium. Mol Cell Biol 25: 7868–7878, 2005.[Abstract/Free Full Text]
  4. Campbell VW and Yamada T. Acid secretagogue-induced stimulation of gastric parietal cell gene expression. J Biol Chem 264: 11381–11386, 1989.[Abstract/Free Full Text]
  5. Chen D, Zhao CM, Dockray GJ, Varro A, Van Hoek A, Sinclair NF, Wang TC, and Koh TJ. Glycine-extended gastrin synergizes with gastrin 17 to stimulate acid secretion in gastrin-deficient mice. Gastroenterology 119: 756–765, 2000.[CrossRef][ISI][Medline]
  6. Chen D, Zhao CM, Hakanson R, Samuelson LC, Rehfeld JF, and Friis-Hansen L. Altered control of gastric acid secretion in gastrin-cholecystokinin double mutant mice. Gastroenterology 126: 476–487, 2004.[CrossRef][ISI]
  7. Chew CS, Chen X, Parente JA Jr, Tarrer S, Okamoto C, and Qin HY. Lasp-1 binds to non-muscle F-actin in vitro and is localized within multiple sites of dynamic actin assembly in vivo. J Cell Sci 115: 4787–4799, 2002.[Medline]
  8. Chew CS, Parente JA Jr, Chen X, Chaponnier C, and Cameron RS. The LIM and SH3 domain-containing protein, lasp-1, may link the cAMP signaling pathway with dynamic membrane restructuring activities in ion transporting epithelia. J Cell Sci 113: 2035–2045, 2000.[Abstract]
  9. Chew CS, Parente JA Jr, Zhou C, Baranco E, and Chen X. Lasp-1 is a regulated phosphoprotein within the cAMP signaling pathway in the gastric parietal cell. Am J Physiol Cell Physiol 275: C56–C67, 1998.[Abstract/Free Full Text]
  10. Crean GP, Marshall MW, and Rumsey RD. Parietal cell hyperplasia induced by the administration of pentagastrin (ICI 50,123) to rats. Gastroenterology 57: 147–155, 1969.[ISI][Medline]
  11. Dedek K and Waldegger S. Colocalization of KCNQ1/KCNE channel subunits in the mouse gastrointestinal tract. Pflügers Arch 442: 896–902, 2001.[CrossRef][ISI][Medline]
  12. Dimaline R and Sandvik AK. Histidine decarboxylase gene expression in rat fundus is regulated by gastrin. FEBS Lett 281: 20–22, 1991.[CrossRef][ISI][Medline]
  13. Friis-Hansen L, Sundler F, Li Y, Gillespie PJ, Saunders TL, Greenson JK, Owyang C, Rehfeld JF, and Samuelson LC. Impaired gastric acid secretion in gastrin-deficient mice. Am J Physiol Gastrointest Liver Physiol 274: G561–G568, 1998.[Abstract/Free Full Text]
  14. Fujita A, Horio Y, Nielsen S, Nagelhus EA, Hata F, Ottersen OP, and Kurachi Y. High-resolution immunogold cytochemistry indicates that AQP4 is concentrated along the basal membrane of parietal cell in rat stomach. FEBS Lett 459: 305–309, 1999.[CrossRef][ISI][Medline]
  15. Fukui H,Kinoshita Y, Maekawa T, Okada A, Waki S, Hassan S, Okamoto H, and Chiba T. Regenerating gene protein may mediate gastric mucosal proliferation induced by hypergastrinemia in rats. Gastroenterology 115: 1483–1493, 1998.[CrossRef][ISI][Medline]
  16. Gerhard M, Neumayer N, Presecan-Siedel E, Zanner R, Lengyel E, Cramer T, Hocker M, and Prinz C. Gastrin induces expression and promoter activity of the vesicular monoamine transporter subtype 2. Endocrinology 142: 3663–3672, 2001.[Abstract/Free Full Text]
  17. Grahammer F, Herling AW, Lang HJ, Schmitt-Graff A, Wittekindt OH, Nitschke R, Bleich M, Barhanin J, and Warth R. The cardiac K+ channel KCNQ1 is essential for gastric acid secretion. Gastroenterology 120: 1363–1371, 2001.[CrossRef][ISI][Medline]
  18. He TC, Sparks AB, Rago C, Hermeking H, Zawel L, da Costa LT, Morin PJ, Vogelstein B, and Kinzler KW. Identification of c-MYC as a target of the APC pathway. Science 281: 1509–1512, 1998.[Abstract/Free Full Text]
  19. Hinkle KL, Bane GC, Jazayeri A, and Samuelson LC. Enhanced calcium signaling and acid secretion in parietal cells isolated from gastrin-deficient mice. Am J Physiol Gastrointest Liver Physiol 284: G145–G153, 2003.[Abstract/Free Full Text]
  20. Hocker M, Raychowdhury R, Plath T, Wu H, O'Connor DT, Wiedenmann B, Rosewicz S, and Wang TC. Sp1 and CREB mediate gastrin-dependent regulation of chromogranin A promoter activity in gastric carcinoma cells. J Biol Chem 273: 34000–34007, 1998.[Abstract/Free Full Text]
  21. Ji B, Chen XQ, Misek DE,Kuick R, Hanash S, Ernst S, Najarian R, and Logsdon CD. Pancreatic gene expression during the initiation of acute pancreatitis: identification of EGR-1 as a key regulator. Physiol Genomics 14: 59–72, 2003.[Abstract/Free Full Text]
  22. Kaise M, Muraoka A, Seva C, Takeda H, Dickinson CJ, and Yamada T. Glycine-extended progastrin processing intermediates induce H+,K(+)-ATPase alpha-subunit gene expression through a novel receptor. J Biol Chem 270: 11155–11160, 1995.[Abstract/Free Full Text]
  23. Kinoshita Y, Ishihara S,Kadowaki Y, Fukui H, and Chiba T. Reg protein is a unique growth factor of gastric mucosal cells. J Gastroenterol 39: 507–513, 2004.[ISI][Medline]
  24. Kirton CM, Wang T, and Dockray GJ. Regulation of parietal cell migration by gastrin in the mouse. Am J Physiol Gastrointest Liver Physiol 283: G787–G793, 2002.[Abstract/Free Full Text]
  25. Koh TJ, Goldenring JR, Ito S, Mashimo H,Kopin AS, Varro A, Dockray GJ, and Wang TC. Gastrin deficiency results in altered gastric differentiation and decreased colonic proliferation in mice. Gastroenterology 113: 1015–1025, 1997.[CrossRef][ISI][Medline]
  26. Lambrecht NW, Yakubov I, Scott D, and Sachs G. Identification of the K efflux channel coupled to the gastric H-K-ATPase during acid secretion. Physiol Genomics 21: 81–91, 2005.[Abstract/Free Full Text]
  27. Larsson H, Carlsson E, Mattsson H, Lundell L, Sundler F, Sundell G, Wallmark B, Watanabe T, and Hakanson R. Plasma gastrin and gastric enterochromaffinlike cell activation and proliferation. Studies with omeprazole and ranitidine in intact and antrectomized rats. Gastroenterology 90: 391–399, 1986.[ISI][Medline]
  28. Lee MP, Ravenel JD, Hu RJ, Lustig LR, Tomaselli G, Berger RD, Brandenburg SA, Litzi TJ, Bunton TE, Limb C, Francis H, Gorelikow M, Gu H, Washington K, Argani P, Goldenring JR, Coffey RJ, and Feinberg AP. Targeted disruption of the Kvlqt1 gene causes deafness and gastric hyperplasia in mice. J Clin Invest 106: 1447–1455, 2000.[ISI][Medline]
  29. Mattsson H, Havu N, Brautigam J, Carlsson K, Lundell L, and Carlsson E. Partial gastric corpectomy results in hypergastrinemia and development of gastric enterochromaffinlike-cell carcinoids in the rat. Gastroenterology 100: 311–319, 1991.[ISI][Medline]
  30. Mills JC, Syder AJ, Hong CV, Guruge JL, Raaii F, and Gordon JI. A molecular profile of the mouse gastric parietal cell with and without exposure to Helicobacter pylori. Proc Natl Acad Sci USA 98: 13687–13692, 2001.[Abstract/Free Full Text]
  31. Ochi Y, Horie S, Maruyama T, Watanabe K, and Yano S. Necessity of intracellular cyclic AMP in inducing gastric acid secretion via muscarinic M(3) and cholecystokinin(2) receptors on parietal cells in isolated mouse stomach. Life Sci 77: 2040–2050, 2005.[CrossRef][Medline]
  32. Prinz C, Zanner R, and Gratzl M. Physiology of gastric enterochromaffin-like cells. Annu Rev Physiol 65: 371–382, 2003.[CrossRef][ISI][Medline]
  33. Samuelson LC and Hinkle KL. Insights into the regulation of gastric acid secretion through analysis of genetically engineered mice. Annu Rev Physiol 65: 383–400, 2003.[CrossRef][ISI][Medline]
  34. Schoenfeld JR, Vasser M, Jhurani P, Ng P, Hunter JJ, Ross J Jr, Chien KR, and Lowe DG. Distinct molecular phenotypes in murine cardiac muscle development, growth, and hypertrophy. J Mol Cell Cardiol 30: 2269–2280, 1998.[CrossRef][ISI][Medline]
  35. Shedden K, Chen W,Kuick R, Ghosh D, Macdonald J, Cho KR, Giordano TJ, Gruber SB, Fearon ER, Taylor JM, and Hanash S. Comparison of seven methods for producing Affymetrix expression scores based on False Discovery Rates in disease profiling data. BMC Bioinformatics 6: 26, 2005.[CrossRef][Medline]
  36. Shu W, Guttentag S, Wang Z, Andl T, Ballard P, Lu MM, Piccolo S, Birchmeier W, Whitsett JA, Millar SE, and Morrisey EE. Wnt/beta-catenin signaling acts upstream of N-myc, BMP4, and FGF signaling to regulate proximal-distal patterning in the lung. Dev Biol 283: 226–239, 2005.[CrossRef][ISI][Medline]
  37. Sinclair NF, Ai W, Raychowdhury R, Bi M, Wang TC,Koh TJ, and McLaughlin JT. Gastrin regulates the heparin-binding epidermal-like growth factor promoter via a PKC/EGFR-dependent mechanism. Am J Physiol Gastrointest Liver Physiol 286: G992–G999, 2004.[Abstract/Free Full Text]
  38. Sistermans EA,Klaassen CH, Peters W, Swarts HG, Jap PH, De Pont JJ, and Wieringa B. Co-localization and functional coupling of creatine kinase B and gastric H+/K(+)-ATPase on the apical membrane and the tubulovesicular system of parietal cells. Biochem J 311: 445–451, 1995.[Medline]
  39. Tari A, Wu V, Sumii M, Sachs G, and Walsh JH. Regulation of rat gastric H+/K(+)-ATPase alpha-subunit mRNA by omeprazole. Biochim Biophys Acta 1129: 49–56, 1991.[Medline]
  40. Tsutsui S, Shinomura Y, Higashiyama S, Higashimoto Y, Miyazaki Y,Kanayama S, Hiraoka S, Minami T,Kitamura S, Murayama Y, Miyagawa J, Taniguchi N, and Matsuzawa Y. Induction of heparin binding epidermal growth factor-like growth factor and amphiregulin mRNAs by gastrin in the rat stomach. Biochem Biophys Res Commun 235: 520–523, 1997.[CrossRef][ISI][Medline]
  41. Wank SA. G protein-coupled receptors in gastrointestinal physiology. I. CCK receptors: an exemplary family. Am J Physiol Gastrointest Liver Physiol 274: G607–G613, 1998.[Abstract/Free Full Text]
  42. Zavros Y, Rieder G, Ferguson A, Samuelson LC, and Merchant JL. Genetic or chemical hypochlorhydria is associated with inflammation that modulates parietal and G-cell populations in mice. Gastroenterology 122: 119–133, 2002.[CrossRef][ISI][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
C. S. Chew, X. Chen, H. Zhang, E. A. Berg, and H. Zhang
Calcium/calmodulin-dependent phosphorylation of tumor protein D52 on serine residue 136 may be mediated by CAMK2{delta}6
Am J Physiol Gastrointest Liver Physiol, December 1, 2008; 295(6): G1159 - G1172.
[Abstract] [Full Text] [PDF]

Home page
 Exp PhysiolHome page
A. Pagliocca, P. Hegyi, V. Venglovecz, S. A. Rackstraw, Z. Khan, G. Burdyga, T. C. Wang, R. Dimaline, A. Varro, and G. J. Dockray
Identification of ezrin as a target of gastrin in immature mouse gastric parietal cells
Exp Physiol, November 1, 2008; 93(11): 1174 - 1189.
[Abstract] [Full Text] [PDF]

Home page
 PhysiologyHome page
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]

Home page
 Physiol. GenomicsHome page
R. N. Jain and L. C. Samuelson
Transcriptional profiling of gastrin-regulated genes in mouse stomach
Physiol Genomics, March 14, 2007; 29(1): 1 - 12.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
R. N. Jain and L. C. Samuelson
Differentiation of the Gastric Mucosa II. Role of gastrin in gastric epithelial cell proliferation and maturation.
Am J Physiol Gastrointest Liver Physiol, November 1, 2006; 291(5): G762 - G765.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
T. K. Roepke, A. Anantharam, P. Kirchhoff, S. M. Busque, J. B. Young, J. P. Geibel, D. J. Lerner, and G. W. Abbott
The KCNE2 Potassium Channel Ancillary Subunit Is Essential for Gastric Acid Secretion
J. Biol. Chem., August 18, 2006; 281(33): 23740 - 23747.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
24/2/124    most recent
00133.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (10)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jain, R. N.
Right arrow Articles by Samuelson, L. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jain, R. N.
Right arrow Articles by Samuelson, L. C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.