Localization of ClC-2 Clminus channels in rabbit gastric mucosa

Ann M. Sherry1, Danuta H. Malinowska1, Randal E. Morris2, Georgianne M. Ciraolo2, and John Cuppoletti1

Departments of 1 Molecular and Cellular Physiology and 2 Cell Biology, Neurobiology, and Anatomy, University of Cincinnati College of Medicine, Cincinnati, Ohio 43267-0576


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

HCl secretion across the parietal cell apical secretory membrane involves the H+-K+-ATPase, the ClC-2 Cl- channel, and a K+ channel. In the present study, the cellular and subcellular distribution of ClC-2 mRNA and protein was determined in the rabbit gastric mucosa and in isolated gastric glands. ClC-2 mRNA was localized to parietal cells by in situ hybridization and by direct in situ RT-PCR. By immunoperoxidase microscopy, ClC-2 protein was concentrated in parietal cells. Immunofluorescent confocal microscopy suggested that the ClC-2 was localized to the secretory canalicular membrane of stimulated parietal cells and to intracellular structures of resting parietal cells. Immunogold electron microscopy confirmed that ClC-2 is in the secretory canalicular membrane of stimulated cells and in tubulovesicles of resting parietal cells. These findings, together with previous functional characterization of the native and recombinant channel, strongly indicate that ClC-2 is the Cl- channel, which together with the H+-K+-ATPase and a K+ channel, results in HCl secretion across the parietal cell secretory membrane.

gastric chloride channel; hydrochloric acid secretion; secretory canalicular membrane; tubulovesicles


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

HYDROGEN CHLORIDE SECRETION across the secretory apical membrane of the gastric parietal cell involves the H+-K+-ATPase (25), a Cl- channel (7), and a K+ channel (6). The H+-K+-ATPase has been localized to tubulovesicular membranes in resting parietal cells and to apical canalicular membranes in stimulated parietal cells (29).

ClC-2, a member of the ClC family of Cl- channels, has been cloned from rabbit gastric mucosa (16), and its electrophysiological characteristics are similar to those of the native channel studied in H+-K+-ATPase-containing vesicles (7). Channel characteristics include activation by voltage, low extracytosolic pH, and phosphorylation by cAMP-dependent protein kinase (PKA) (7, 16, 31, 32). Activation of the channel by these mechanisms is consistent with it playing an essential role in HCl secretion. Although Cl- channel function has been studied in an H+-K+-ATPase-containing membrane vesicle population, the native ClC-2 protein has not yet been localized using structural probes. The present study uses in situ hybridization and direct in situ reverse transcript polymerase chain reaction (RT-PCR) to examine ClC-2 mRNA distribution and immunohistochemistry and immunogold electron microscopy to examine ClC-2 Cl- channel protein distribution in rabbit gastric glands.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Rabbit stomach and gastric gland preparation. Adult and fetal (day 22 and day 29) rabbit stomachs were removed, washed with cold phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde, and paraffin embedded. Sections (5 µm) were cut and used as described below. Rabbit gastric glands were isolated by collagenase digestion as previously described (3) and incubated at 37°C for 30 min with 10-4 M cimetidine or 10-4 M histamine plus 10-5 M IBMX to make them resting or stimulated, respectively. The glands were then either washed, fixed, paraffin embedded, and sectioned as described above for experiments that subsequently used light microscopy, or were washed, fixed in 4% paraformaldehyde at 22°C for 20 min, washed, and stored at 4°C for subsequent fluorescent immunohistochemistry and confocal microscopy (13).

Immunostaining with anti-H+-K+-ATPase alpha -subunit antibody. Tissue sections were loaded onto poly-D-lysine-coated slides. Paraffin was removed with xylene and ethanol washes. Sections were stained with hematoxylin and eosin (H and E) or 0.1% nuclear fast red in 5% aluminum sulfate, mounted, and photographed. Coverslips were then removed, endogenous peroxidase activity was quenched using 0.5% hydrogen peroxidase, and sections were blocked overnight at 4°C with PBS (81 mM Na2HPO4, 22 mM NaH2PO4, and 154 mM NaCl) containing 0.3% Triton X-100 and 2% goat serum (Vector Laboratories). Sections were incubated for 2 h at 22°C with a mouse monoclonal antibody to the H+-K+- ATPase alpha -subunit (1:200,000; Chemicon) diluted in the same solution. Biotinylated goat anti-mouse immunoglobulin G (IgG; Vector Laboratories) was the secondary antibody (1:2,000), and an avidin-biotinylated horseradish peroxidase complex (Vector Laboratories) was used for detection. The reaction was visualized with nickel diaminobenzidine in 0.1 M acetate buffer, followed by enhancement with tris(hydroxymethyl)aminomethane (Tris) cobalt, giving a black precipitate (34). Controls included absence of primary antibody and absence of secondary antibody.

In situ hybridization. Sections were loaded onto salinated slides, pretreated with proteinase K, and acetylated immediately before use (37). The full-length rabbit ClC-2 cDNA (adult tissue) or a 261-bp cDNA fragment of the ClC-2 COOH-terminal domain (fetal tissue) was used as a template for the preparation of antisense and sense control 35S-labeled cRNA fragments. Hybridization was performed essentially as described (37) with hybridization at 55°C followed by high-stringency washes. Autoradiography was performed for 12 wk, and results were analyzed with a Nikon microscope and a dark field filter. For adult gastric mucosa, slides were rehydrated, stained with nuclear fast red to view the silver grains, and photographed. Tissue sections were then stained with H and E and viewed under bright field. Silver grains were counted over parietal cells and non-parietal cells in the middle and lower regions of the glands in sections hybridized with antisense and sense control cRNAs. Specific ClC-2 antisense labeling was calculated by subtracting background labeling (sense controls) from the antisense labeling.

Direct in situ RT-PCR. Sections of paraffin-embedded gastric glands were loaded onto slides (Elmeco) pretreated with RNase-Off (CPG, Lincoln Park, NJ). Immediately before use, sections were pretreated with proteinase K and acetylated. Sections were then incubated with RNase-free DNase I (Roche) overnight at 37°C, heated for 2 min at 94°C to deactivate the enzyme, washed with diethyl pyrocarbonate (DEPC)-treated water, and placed in a ThermoSlide Block (Elmeco) for the RT reaction. First-strand cDNA synthesis was performed directly on the tissue sections (11, 21) using the antisense primer to the ClC-2 COOH-terminal domain (27) and SuperScript II RT (GIBCO BRL). Sections were rinsed with DEPC-treated water and placed again in the ThermoSlide Block for PCR amplification of ClC-2 COOH-terminal domain cDNA using ExTaq polymerase (PanVera) and digoxigenin (DIG)-11-dUTP (Roche). Hot-start PCR was performed using TaqStart antibody (Clontech). PCR conditions consisted of 25 cycles of denaturing for 45 s at 94°C, annealing for 45 s at 53°C, and elongating for 2 min at 72°C. After PCR, sections were rinsed with DEPC-treated water, blocked with 150 mM NaCl and 0.2% BSA for 10 min at 50°C, and incubated with alkaline phosphatase-conjugated anti-DIG antibody (1:150; Roche) in medium (0.1 M NaCl and 0.1 M Tris · HCl, pH 7.4) for 30 min at 37°C. After being rinsed, the color reaction was developed with 1:200 nitro blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolylphosphate-p-toluidine salt (BCIP) stock solution:medium (18.75 mg/ml NBT/9.4 mg/ml BCIP; Roche) for >2 h at 37°C. The following controls were prepared in parallel to the test sample: no RT, no ExTaq polymerase, no DIG-11-dUTP, and no DNase pretreatment. Samples were photographed under bright field using a Nikon microscope.

Immunohistochemistry. Sections of isolated fixed paraffin-embedded gastric glands were loaded onto slides as described above. Antigenic sites were unmasked using the antigen retrieval method (9, 28) in which the sections were microwaved in 0.01 M citrate buffer (1.9 mM citric acid and 8.2 mM sodium citrate, pH 6.0) at high power for 7 min to boil and at medium-high power for 15 min. Sections were then blocked overnight at 4°C in PBS with Triton X-100 containing 2% goat serum and goat anti-rabbit Fab fragments (1:130, Jackson ImmunoResearch). Sections were incubated with a rabbit polyclonal antibody (1:10) to the rat ClC-2 COOH terminus (amino acids 888-906; Alomone) and biotinylated goat anti-rabbit IgG (1:2,000; Vector Laboratories). Reaction product was visualized using peroxidase as described above. Sections were counterstained with nuclear fast red. Controls included preincubation of the primary antibody with its antigenic peptide, absence of primary antibody, and absence of secondary antibody.

For immunohistochemistry using fluorescently labeled antibodies, isolated, fixed rabbit gastric glands (resting and stimulated) were permeabilized with 0.5% Triton X-100 in PBS for 20 min and washed three times in PBS before settling onto poly-D-lysine-coated coverslips (13). Glands were then blocked (1% BSA and 0.05% Tween 20 in PBS) for 30 min and incubated with anti-ClC-2 antibody (1:10) or anti-H+-K+-ATPase alpha -subunit antibody (1:200,000) for 1 h. After each incubation, glands were washed three times for 15 s with PBS containing 0.05% Tween 20. Primary antibodies were detected using goat anti-rabbit (ClC-2) or goat anti-mouse (H+-K+-ATPase alpha -subunit) IgG labeled with Alexa Fluor 546 (1:1,000; Molecular Probes) and mounted with Gel/Mount (Biomedia). To visualize F-actin, glands were counterstained with Alexa Fluor 488-labeled phalloidin (20 U/ml; Molecular Probes).

Confocal microscopy. Stained gastric glands were examined by confocal microscopy. Images were obtained using a laser scanning confocal microscope (LSM 510; Zeiss) that was equipped with argon and HeNe lasers, an Axioplan upright microscope, and water-immersion objectives. Digitized images were processed using LSM 510 software and Adobe Photoshop. Alexa 546 (red fluorescence) was visualized by excitation with the 546-nm HeNe laser and examination of emissions from 590 to 620 nm. Alexa 488 (green fluorescence) was visualized by excitation with the 488-nm argon laser and examination of emissions from 500 to 540 nm.

Western blot analysis. Rabbit gastric vesicles (8), 250 µg/lane, were boiled for 30 s, separated by SDS-PAGE, and transblotted onto Hybond enhanced chemiluminescence (ECL) nitrocellulose paper (Amersham). Nonspecific sites on the nitrocellulose replicates were blocked for 1 h at 22°C with TBS (20 mM Tris · HCl and 500 mM NaCl, pH 7.5) containing 0.1% Tween 20, 5% blotting-grade nonfat dry milk (Bio-Rad), and goat anti-rabbit Fab fragments (1:130). Nitrocellulose replicates were incubated with or without anti-ClC-2 antibody (1:200), followed by horseradish peroxidase-conjugated goat anti-rabbit antibody (1:1,000; Amersham) in TBS containing 0.1% Tween 20 and 1% milk for 1 h each. Reaction was detected using ECL Western blotting analysis system (Amersham) and visualized on autoradiographic film.

Immunogold electron microscopy. Resting and stimulated gastric glands were washed, fixed, embedded, and labeled as described (18). Briefly, glands were fixed in Hanks' balanced salt solution (HBSS; Sigma) containing 4% paraformaldehyde (Electron Microscopy Sciences; EMS) and 0.5% glutaraldehyde (EMS) for 30 min at 4°C and then washed three times in cold HBSS and postfixed for 60 min at 4°C in HBSS containing 1% osmium tetroxide (EMS). After postfixation, samples were dehydrated, infiltrated, embedded in Eponate 12 (Ted Pella), and then polymerized for 48 h at 60°C. Ultrathin sections (<80 nm in thickness) were cut with a Reichert-Jung 4E ultramicrotome using a diamond knife (Diatome) and picked up on nickel grids (EMS). To expose antigenic sites, the samples were incubated with 3% sodium metaperiodate (Sigma). Endogenous biotin sites were blocked with streptavidin (50 µg/ml, Sigma) and D-biotin (50 µg/ml, Sigma). All of the immunochemical steps were done by floating the grids on 50-µl droplets of primary antibody (1:50), biotinylated goat anti-rabbit (1:100; Kirkegaard and Perry), and streptavidin-5 nm gold (1:10). After labeling, samples were washed with distilled water, stained for 5 min with 2% uranyl acetate, and viewed and photographed in a JEOL 100 CX II electron microscope. Controls included antibody block by its antigenic peptide, irrelevant antibody from the same species as the primary antibody (rabbit anti-horse spleen ferritin), no primary antibody (antibody replaced by carbonate buffer), and a streptavidin-gold control (no primary antibody and no secondary or bridging antibody). Gold particles were digitally enhanced on micrographs with Photoshop 3.0 (1).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ClC-2 mRNA in adult rabbit gastric mucosa by in situ hybridization. Sections of adult rabbit gastric mucosa were labeled with H and E, photographed, and then stained with anti-H+-K+-ATPase alpha -subunit antibody as shown in Fig. 1A. Parietal cells were large and had a characteristic "fried egg" appearance (2) with pink cytoplasm and a dark blue nucleus when stained with H and E (left). The same cells (1:1 correspondence) showed a black precipitate, indicating the H+-K+- ATPase alpha -subunit (right). Similar sections of adult rabbit gastric mucosa were hybridized with antisense and sense control 35S-labeled ClC-2 cRNA fragments and then stained with nuclear fast red and photographed (Fig. 1B). The same view was photographed after staining the same section with H and E (Fig. 1C). ClC-2 mRNA (silver grains) were present mainly over pink parietal cells (antisense, left), and some background hybridization was evident in the sense control (right). H and E staining allowed identification of parietal and non-parietal cells. Silver grains, easily seen with nuclear fast red staining, were then counted over parietal cells and non-parietal cells in both antisense and sense control experiments. After subtracting background sense control numbers of silver grains for parietal and non-parietal cells, there was a significant (P < 0.001), approximately fourfold higher number of silver grains over parietal cells (3.64 ± 0.40, n = 73) than over non-parietal cells (1.06 ± 0.21, n = 85), indicating that ClC-2 mRNA was predominantly present in parietal cells.


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Fig. 1.   In situ hybridization of ClC-2 mRNA in adult rabbit gastric mucosa. A: adult gastric mucosa was stained with hematoxylin and eosin (H and E; left) and then with anti-H+-K+-ATPase alpha -subunit antibody (right) to identify parietal cells. The large cells with pink cytoplasm and blue nuclei (H and E) were parietal cells because they contained the H+-K+-ATPase alpha -subunit (black precipitate). Scale bar: 10 µm. B: adult rabbit gastric mucosa was hybridized with antisense and sense control 35S-labeled ClC-2 cRNA fragments, and the sections were then counterstained with nuclear fast red. Scale bar: 10 µm. C: sections in B were rehydrated and stained with H and E to show parietal cells more clearly. Silver grains are evident mainly over parietal cells. Scale bar: 10 µm.

ClC-2 mRNA in fetal rabbit stomach by in situ hybridization. In other studies, ClC-2 mRNA has been shown to be higher in fetal tissue compared with adult (19, 20, 27). Therefore, localization of ClC-2 mRNA was also investigated in fetal stomach at two ages: day 22 (early) and day 29 (late, gestation is usually 32 days) and compared with H+-K+-ATPase alpha -subunit to mark parietal cells. Sections from day 29 (left) and day 22 (right) fetal stomachs were stained with nuclear fast red followed by anti-H+-K+-ATPase alpha -subunit antibody (Fig. 2A) or hybridized with ClC-2 antisense and sense control cRNA fragments and then stained with H and E (Fig. 2, B and C). In both day 29 and day 22 fetal stomachs, H+-K+-ATPase alpha -subunit (black precipitate) was detected in random cells in the lower regions of the developing gastric epithelial layer, while ClC-2 mRNA was detected throughout the epithelial layer. At fetal day 22, the parietal cells may be "preoxyntic" (17). Maturation of the secretory glands occurs late in gestation (day 31 to after birth) in the fetal rabbit (14). However, HCl secretion begins at day 23 (35) and increases until birth (36).


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Fig. 2.   In situ hybridization of ClC-2 mRNA in fetal rabbit stomach. A: rabbit stomachs from fetal day 29 (left) and fetal day 22 (right) were stained with anti-H+-K+-ATPase alpha -subunit antibody and counterstained with nuclear fast red. The black precipitate indicates H+-K+-ATPase alpha -subunit, identifying (pre)parietal cells. B: H and E staining of the fetal stomach sections used for in situ hybridization. C: hybridization with antisense and sense control 35S-labeled ClC-2 cRNA fragments visualized under dark field. L, stomach lumen. Scale bar: 100 µm.

Localization of ClC-2 Cl- channel mRNA in gastric glands by direct in situ RT-PCR. Direct in situ RT-PCR was also used to investigate the cellular site of ClC-2 mRNA expression in the gastric mucosa using isolated rabbit gastric glands, a simpler tissue preparation consisting mainly of parietal and peptic cells. Figure 3A shows H and E staining (left) and staining for the H+-K+-ATPase alpha -subunit (right) in the same gastric gland. The parietal cells had pink cytoplasm and a blue nucleus (H and E) or a black precipitate indicating H+-K+-ATPase alpha -subunit. Peptic cells were stained dark blue with H and E and did not stain for H+-K+-ATPase alpha -subunit. Figure 3B shows ClC-2 mRNA localization in gastric glands by direct in situ RT-PCR. ClC-2 PCR cDNA product (black precipitate) was concentrated in the parietal cells of the gland and not in the peptic cells (Fig. 3, Ba). Lighter staining of the parietal cells was detected in negative controls that did not contain all of the RT-PCR components: no reverse transcriptase (Fig. 3, Bb), no ExTaq polymerase (Fig. 3, Bc), and no DIG-11-dUTP label (Fig. 3, Bd). In the positive control (Fig. 3, Be), the section was not pretreated with DNase, and as expected, staining was slightly increased in all cells due to the additional amplification of ClC-2 from genomic DNA (11, 21). ClC-2 mRNA was thus localized to the parietal cell in the intact gastric mucosa and in isolated gastric glands.


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Fig. 3.   Detection of ClC-2 Cl- channel mRNA in gastric glands by direct in situ RT-PCR. A: gastric glands were stained with H and E (left) followed by staining with anti-H+-K+-ATPase alpha -subunit antibody (right). Parietal cells had pink cytoplasm and blue nuclei (H and E) and the black precipitate identifying the H+-K+-ATPase. Scale bar: 10 µm. B: direct in situ RT-PCR was carried out on isolated adult rabbit gastric glands. Ba contained all RT-PCR components. Parietal cells are stained dark purple. Bb-e are controls; no reverse transcriptase (b; RT), no ExTaq polymerase (c), no digoxigenin (DIG)-11-dUTP (d), and no DNase pretreatment (e). Scale bar: 10 µm.

Immunohistochemical localization of ClC-2 in gastric glands. Localization of the ClC-2 Cl- channel protein in isolated gastric glands was then investigated using an immunoperoxidase method with a polyclonal antibody made to the rat ClC-2 COOH terminus (Fig. 4A). Major staining was present in parietal cells and not in peptic cells of the gland (Fig. 4, Aa). Staining was blocked by preincubating the antibody with its antigenic peptide (Fig. 4, Ab), in the absence of primary antibody (Fig. 4, Ac), or in the absence of secondary antibody (Fig. 4, Ad). However, distribution of ClC-2 to specific membranes of the parietal cell could not be determined with peroxidase staining. Therefore, immunofluorescence together with confocal microscopy was used to investigate the subcellular location of ClC-2 within the parietal cell. ClC-2 was detected in resting and stimulated fixed, permeabilized gastric glands using anti-ClC-2 antibody and Alexa Fluor 546-labeled goat anti-rabbit antibody. Figure 4B shows the results with stimulated glands. In Fig. 4, Ba, the red fluorescence due to ClC-2 appeared evident only in parietal cells (left). The Nomarski image is shown (middle), and when the red fluorescence was superimposed on the Nomarski view (right), ClC-2 appeared associated with intracellular structures that resembled the secretory canalicular membrane of stimulated parietal cells. No staining was evident at the basal-lateral membrane. Staining was blocked by the antigenic peptide (Fig. 4, Bb) and was absent when anti-ClC-2 antibody was omitted (Fig. 4, Bc). Results with resting gastric glands are shown in Fig. 4C. For comparison, localization of the H+-K+-ATPase alpha -subunit and F-actin was also examined. Gastric glands were stained with Alexa Fluor 546-labeled anti-ClC-2 or anti-H+-K+-ATPase alpha -subunit antibodies (red fluorescence) and then counterstained with Alexa Fluor 488-labeled phalloidin to detect F-actin (green fluorescence). F-actin clearly delineates the parietal cell with a ring just inside of the basal-lateral membrane and the lumen of the gland. In addition, there is a concentrated amount of F-actin at the apical membrane adjoining the lumen. ClC-2 (left) and H+-K+-ATPase alpha -subunit (right) appear to have similar subcellular distribution, as seen by punctate staining within the parietal cell and not at the basal-lateral membrane.


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Fig. 4.   Immunohistochemical detection of ClC-2 Cl- channel in isolated gastric glands. A: ClC-2 Cl- channel was detected with an immunoperoxidase method in parietal cells of isolated gastric glands (a). Immunostaining was absent by preincubating anti-ClC-2 antibody with its antigenic peptide (b), without anti-ClC-2 antibody (c), and without secondary antibody (d). Glands were counterstained with nuclear fast red. Scale bar: 10 µm. B: fluorescence and Nomarski micrographs of stimulated gastric glands. Stimulated glands were labeled with anti-ClC-2 antibody followed by Alexa Fluor 546-labeled goat anti-rabbit IgG (a; red fluorescence). Shown are fluorescence alone (left), Nomarski image (middle), and fluorescence imposed on the Nomarski image (right). Red fluorescence present predominantly in parietal cells seemed associated with an intracellular membrane structure resembling the secretory canaliculus of stimulated parietal cells. Immunofluorescent staining was absent with anti-ClC-2 antibody preincubated with antigenic peptide (b) and no anti-ClC-2 antibody (c). Scale bar: 10 µm. C: fluorescence/Nomarski micrographs of resting gastric glands. Fluorescent image of a gland stained with anti-ClC-2 antibody (a; left) or anti-H+-K+-ATPase alpha -subunit antibody (right) labeled with Alexa Fluor 546 (red fluorescence). The glands were counterstained with Alexa Fluor 488-labeled phalloidin (b; green fluorescence) to visualize F-actin; a and b views superimposed (c), Nomarski image alone (d), and c and d superimposed (e). Scale bar: 10 µm. D: rabbit gastric vesicles were separated by 10% SDS-PAGE, transferred to nitrocellulose, immunoblotted with anti-ClC-2 antibody, and visualized by chemiluminescence. Lane 1: + anti-ClC-2 antibody; lane 2- anti-ClC-2 antibody. Arrow indicates ClC-2 at 93 kDa.

Figure 4D shows a Western blot of rabbit gastric vesicles with (lane 1) and without (lane 2) anti-ClC-2 antibody. A 93-kDa protein band was detected by the anti-ClC-2 antibody in rabbit gastric vesicles. A similar size for ClC-2 has been reported (20), although there appears to be some variability (80-100 kDa) (19, 24, 26).

Immunogold electron microscopy of ClC-2 in parietal cells. Precise intracellular localization of the ClC-2 Cl- channel in parietal cells could not be determined by immunohistochemistry. Therefore, immunogold electron microscopy was carried out using both resting and stimulated gastric glands. ClC-2 Cl- channel proteins were localized predominantly to the secretory, canalicular membrane of stimulated parietal cells (Fig. 5A). In contrast, the channel protein was predominantly localized to intracellular tubulovesicles in the resting, nonsecreting, parietal cells (Fig. 5B). There was significantly greater gold per unit area over membranes compared with cytoplasm. The values (membrane vs. cytoplasm) were 4.15 ± 1.2 vs. 0.6 ± 0.2 (n = 11) with P < 0.001 for stimulated parietal cells and 2.9 ± 1.5 vs. 0.7 ± 0.3 (n = 4) with P < 0.05 for resting parietal cells, with no significant difference between stimulated and resting cells. Only background levels of gold were detected over non-parietal (peptic) cells (data not shown). Controls were all negative (data not shown). They included antibody block by its antigenic peptide, irrelevant antibody from the same species as the primary antibody (rabbit anti-horse spleen ferritin), no primary antibody (antibody replaced by carbonate buffer), and a streptavidin-gold control (no primary antibody and no secondary or bridging antibody).


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Fig. 5.   Subcellular localization of ClC-2 in stimulated and resting parietal cells using immunogold electron microscopy. A: a secretory, canalicular membrane of a stimulated (secreting) parietal cell. Inset shows low-power magnification of the cell. Gold particles (large black dots) indicating ClC-2 are evident predominantly close to the secretory, canalicular membrane. Scale bar: 500 nm; inset scale bar: 180 nm. B: resting parietal cell containing many tubulovesicles and an apical membrane. Inset shows low-power magnification of the cell. Gold particles (large black dots) indicating ClC-2 were detected predominantly close to tubulovesicles compared with the apical membrane. Scale is the same as in A.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The object of the present studies was to determine whether ClC-2 Cl- channels were localized to the parietal cell of the gastric mucosa, and if so, whether they were localized to the secretory canalicular membranes of stimulated cells. Previous studies had demonstrated that the gastric mucosa contained mRNA for ClC-2 and a pH- and PKA-activated Cl- channel present in membrane preparations from rabbit gastric mucosa that were enriched in H+-K+-ATPase (7, 16). This Cl- channel's properties were consistent with a role for it in regulated HCl secretion. ClC-2 was cloned from rabbit gastric mucosa and human tissues (5, 16, 27), and it is active at low pH (16, 26, 27, 31, 32) and activated by PKA (16, 27, 32). A similar pH-activated Cl- channel is present in rat tissues (15, 33). ClC-2 has not been previously localized within the gastric mucosa. The distribution of ClC-2 mRNA was studied using in situ hybridization and direct in situ RT-PCR. ClC-2 protein distribution was studied using an antibody to ClC-2.

In situ hybridization was used with sections of the gastric mucosa, but this approach had the disadvantage of long periods of exposure (12 wk) due to low abundance of the mRNA. The H+-K+-ATPase alpha -subunit, specifically expressed in gastric parietal cells (29), was used as a marker to ensure correct identification of parietal cells in gastric mucosal tissue sections and in isolated gastric glands. In situ hybridization of adult gastric mucosa showed similar distribution of ClC-2 as the H+-K+-ATPase over the lower two-thirds of the epithelium (data not shown). Counts of silver grains over parietal cells and non-parietal cells indicated approximately fourfold more silver grains over parietal cells vs. non-parietal cells.

Direct in situ PCR has been used to localize ion channels and receptors in other tissues (4, 10). It is rapid and more sensitive than in situ hybridization because of amplification of the signal, but has the disadvantage that relatively harsh conditions, including repeated exposure to high temperature, are required, potentially compromising the quality of the specimen. Despite these difficulties, direct in situ RT-PCR demonstrated unequivocally that mRNA for ClC-2 was localized to the parietal cell.

Immunocytochemistry using peroxidase staining was useful in localization of the ClC-2 protein to the parietal cell, as expected from the in situ hybridization studies. However, this method is insufficient to demonstrate staining associated with any membrane within the parietal cell. Therefore, to further elucidate the distribution of ClC-2 within the parietal cell, immunofluorescent confocal microscopy of detergent-permeabilized parietal cells was used. This approach has been successfully used by others to show parietal cell cytoskeletal elements and the H+-K+-ATPase beta -subunit associated with the secretory canaliculi with a variety of fluorescent probes (12, 22, 23, 30). Using stimulated gastric glands, ClC-2 was found to be predominantly associated with an intracellular structure that closely resembled the secretory, canalicular membrane that contains the H+-K+-ATPase (12, 22, 23, 29) and across which HCl is secreted. ClC-2 was not associated with the basal-lateral membrane. As shown previously by others (12, 23, 30), F-actin (fluorescently labeled with phalloidin-Alexa Fluor 488) in resting gastric glands clearly delineated the glandular lumen, apical membrane adjoining the lumen, and the parietal cells and was also observed to be associated with small intracellular structures. F-actin was just inside the basal-lateral membrane of the parietal cell as was also reported by others (12), while ClC-2 appeared intracellular, similar to the distribution of H+-K+-ATPase and definitively not in the basal-lateral membrane. These findings suggest that ClC-2 has a similar distribution as the H+-K+-ATPase. F-actin's distribution relative to both ClC-2 and the H+-K+-ATPase is expected since F-actin is known to be involved in the translocation of tubulovesicles containing H+-K+-ATPase (and the K+ and Cl- ion channels) to the secretory canaliculus upon stimulation (12, 22, 23, 30).

To examine more precisely and definitively the subcellular location of the ClC-2 Cl- channel in stimulated and resting parietal cells, immunogold electron microscopy was used. ClC-2 protein was predominantly in the secretory canaliculus of stimulated parietal cells and in the tubulovesicles of resting parietal cells. This distribution of ClC-2 is thus similar to that reported for the H+-K+-ATPase (12, 22, 23, 29). These findings may be of importance to understanding the cellular mechanisms underlying activation of HCl secretion.

In the present studies, ClC-2 Cl- channel mRNA and protein have been localized to the rabbit parietal cell. Detection of the ClC-2 Cl- channel in the secretory canalicular membrane of the stimulated parietal cell and its unique function (activation) at low extracytosolic pH (7, 16, 27, 31, 32) provides further support that ClC-2 is the channel responsible for providing Cl- equivalents for HCl secretion across the secretory membrane of the parietal cell.


    ACKNOWLEDGEMENTS

We thank Dr. Susan Wert at The Children's Hospital Research Foundation for training A. M. Sherry in the in situ hybridization technique, Dr. J. A. Whitsett at The Children's Hospital Research Foundation for use of equipment, Drs. C. S. Chew and J. G. Forte for helpful advice and discussions, and Dr. Nancy Koster-Kleene for training A. M. Sherry and D. H. Malinowska in confocal microscopy.


    FOOTNOTES

This work was supported by National Institutes of Health Grants DK-43816 and HL-58399 (to J. Cuppoletti and D. H. Malinowska).

Address for reprint requests and other correspondence: J. Cuppoletti, Dept. of Molecular and Cellular Physiology, Univ. of Cincinnati College of Medicine, PO Box 670576, Cincinnati, OH 45267-0576 (E-mail: John.Cuppoletti{at}uc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8 May 2000; accepted in final form 22 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Broomall, K. Computer-assisted enhancement permits the use of five-nanometer gold probes in low magnification transmission electron microscopy. J Electron Microsc (Tokyo) 47: 355-357, 1998[Abstract].

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