INTRODUCTION

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THE PLASMA MEMBRANES of virtually all eukaryotic cell types contain Na⁺/H⁺ exchangers (NHEs) that participate in diverse cellular functions such as intracellular pH regulation, transepithelial ion transport, regulation of cell volume, and cellular responses to mitogens and growth factors (12, 13, 15). Four specific cDNAs (8, 14, 21, 26, 29-32, 34) that encode mammalian Na⁺/H⁺ exchangers (NHE1-NHE4) have been identified. Recently, partial sequence for a fifth mammalian isoform (NHE5) has been described (18). All of the isoforms have a high degree of similarity in the NH₂-terminal hydrophobic domain that contains multiple predicted membrane-spanning segments, but the similarity is appreciably less in the COOH-terminal hydrophilic region that contains several putative regulatory domains.

By Northern blot hybridization (14, 21, 31), NHE1 is ubiquitously expressed in many types of both epithelial and nonepithelial tissues, consistent with the physiological role of a housekeeping, growth factor-activable isoform. Furthermore, immunocytochemical studies have shown that in polarized epithelia such as the ileum (31) and renal tubular cells (2, 3, 23), NHE1 is localized to the basolateral membrane. In contrast, expression of NHE3 transcript in both rat and rabbit is epithelial tissue specific with highest levels found in intestine and kidney (21, 29). Immunocytochemical localization of NHE3 protein indicates that this isoform is present on the apical membrane (brush border) of renal proximal tubule (1, 2, 27), loop of Henle (1, 27), and small intestine (4, 16).

Isoforms NHE2 and NHE4 have not been studied as extensively as NHE1 and NHE3. Northern blot hybridization in both rat and rabbit indicates NHE2 message expression is highest in tissues such as intestine and stomach, but lower levels of expression are found in tissues such as kidney, testes, skeletal muscle, uterus, and brain (8, 30, 34). Recent immunocytochemical and Western blot analysis has demonstrated that in small intestine and colon, NHE2 is localized to brush-border membranes (16).

Finally, NHE4 message expression is highest in stomach but is detectable in intestine, uterus, kidney, brain, and skeletal muscle (21). In situ hybridization studies (5) suggest mRNA for NHE4 in rat kidney is highest in inner medullary collecting tubules. However, immunochemical characterization has not been reported.¹ In the present study we exploited the low sequence similarity among NHEs in the COOH-terminal hydrophilic domain to construct a fusion protein consisting of 111 amino acids of NHE4. Isoform-specific monoclonal antibodies were produced, characterized, and used to describe the localization of NHE4 in rat using immunoblot analysis.

	METHODS

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Preparation of the fusion protein construct. A portion of the COOH-terminal hydrophilic domain of NHE4 was amplified using techniques previously described (23). Briefly, cDNA was synthesized from 1-4 µg poly(A)⁺ RNA prepared from rat stomach. A pair of degenerate primers beginning forward at 1519 on the sense strand (5' GAY GTV TGY GGX CAR TGG AG 3') and reverse at 2102 on the antisense strand (5' CCC CAD ATY TTY TCX ACC AT 3') were used to amplify a cDNA using PCR. The resultant product was cloned into the EcoR V restriction site in pBluescript KS⁺ (Stratagene, La Jolla, CA). A fusion protein containing amino acids 565-675 of NHE4 (fpNHE4AA565-675) was constructed using the maltose-binding protein (pMALc) fusion vector (New England Biolabs, Beverly, MA) and a Sac I and Hind III fragment of the NHE4 clone. The Sac I site at the 5' end of the construct cloned in-frame in pMALc, whereas the 3' Hind III site from pBluescript KS⁺ added four additional nucleotides that were in-frame with a stop codon in the vector. Subsequent expression and isolation of fusion proteins were carried out according to manufacturer's protocol. Plasmids were propagated by transformation of competent Escherichia coli strain XL1-Blue as described by Sambrook et al. (24). The 5' cloning site of the fusion protein construct was sequenced using the dideoxynucleotide chain termination method (25) to check for frame-shift errors. Antigen concentration was determined by Lowry assay (20) with BSA as the standard.

Antibody production. Methods for the subsequent production of monoclonal anti-fpNHE4AA565-675 have been described in detail (17). Mice were initially immunized with 50 µg of fpNHE4AA565-675 in complete Freund's adjuvant intraperitoneally and once a month thereafter with 50 µg of fpNHE4AA565-675 in incomplete Freund's adjuvant. After the fifth boost, spleen cells were fused with AG8 myeloma cells. Indirect ELISA was performed as described previously (33) using detection with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Organon Teknika-Cappel). Hybridoma supernatants were compared in parallel ELISAs performed using purified maltose-binding protein and fpNHE4AA565-675. Sixteen supernatants reacted as positive against the fpNHE4AA565-675 but negative against the purified maltose-binding protein. Eight of the ELISA-positive supernatants also reacted positive for fpNHE4AA565-675 but not the maltose-binding protein by Western blot analysis. These eight supernatants were cloned by limiting dilution. Purified monoclonal antibodies were produced by growing hybridoma cells to confluent density and selecting IgGs on a flow-through protein G affinity cartridge (Sigma Chemical, St. Louis, MO) per manufacturer's recommendation. Immunoglobulins were dialyzed overnight against PBS-50% glycerol to prevent freezing at 20°C and were concentrated to a range of 5-10 µg/ml using Centricon-30 microconcentrators. Since monoclonal 11H11 was found to work the best for immunoblotting, it was used throughout the study. The other seven monoclonal reagents gave qualitatively similar results (not shown).

Cloning of NHEs for expression. The cloning of three overlapping cDNAs, which together contain the entire coding region of rabbit NHE1 (2,448 bp), plus 726 bp of the 5'-untranslated region and 178 bp of the 3'-untranslated region have been described previously (14). For expression, a single cDNA was constructed in pBluescript KS⁺ as follows: a construct containing the middle coding sequence of NHE1 in pBluescript KS⁺ was digested with Acc I and BamH I, and the resulting fragment was ligated into the altered vector deleting pBluescript sequence outward from the EcoR V site up to the Acc I and BamH I sites on either side. A second clone containing the 3' coding and 3'-untranslated regions of NHE1 in pBluescript KS⁺ was digested with Acc I, and the resulting fragment was cloned into the first cDNA construct regenerating the pBluescript sequence from the EcoR V site downstream to the Acc I. The 5'-untranslated region was deleted and replaced with restriction sites for BamH I, EcoR I, and Kpn I in sequence upstream of the start methionine using a third clone as template and standard PCR techniques (24). The product was blunt-end cloned into EcoR V cut pBluescript KS⁺. A BamH I fragment was removed and subcloned into the 3' construct, regenerating the pBluescript sequence from the EcoR V site upstream to the BamH I site. The final cDNA construct, in-frame behind the T7 promoter, was sequenced completely in both directions as described above.

The cloning of rabbit NHE3 into pBluescript KS⁺ for expression has been reported (2). Rat NHE2 and NHE4 were a gift from Dr. Gary Shull (University of Cincinnati, OH). NHE2 and NHE4 clones were transferred from pBR322 to pBluescript KS⁺ as follows: NHE2 (clone RSNHE10-3) was excised from pBR322 with Nco I, which cleaves at the start ATG and also in the 3'-untranslated region. The resulting 3-kb cDNA was first cloned into pBlueBac III (Invitrogen, San Diego, CA) and then removed with BamH I and Hind III and directionally cloned into pBluescript, in-frame behind the T7 promoter for expression using vaccinia. NHE4 was obtained as two overlapping clones (5'-RSNHE3-1 and 3'-RSNHE10-2) that were removed from pBR322 using Pst I and individually ligated into the Pst I site in pBluescript KS⁺. Each clone was subsequently digested with Xho I and at a unique internal restriction site (Nsi I) present in the overlapping region. The Xho I/Nsi I fragment containing the 5' end of NHE4 was directionally cloned into the pBluescript KS⁺ containing the 3' construct to yield a full-length cDNA in-frame behind the T7 promoter. The cloning junctions of the final constructs were sequenced as described above.

Vaccinia-T7-induced expression. NHE1-NHE4 were expressed in a mouse L-cell line deficient in Na⁺/H⁺ exchange activity (10) provided by Dr. Jacques Pouysségur (Center de Biochimie, Nice, France), using the bacteriophage T7/vaccinia virus expression system as described previously (2). LAP1 cells were maintained in -minimal essential media supplemented with 10% fetal calf serum, 50 U/ml penicillin, and 50 µg/ml streptomycin at 37°C in 5% CO₂-95% air. Cells, 80%-90% confluent, were infected with 40 plaque-forming units/cell of vaccinia virus (VTF-7) for 1 h and subsequently transfected with 5 µg of appropriate NHE plasmid using Lipofectin (GIBCO-BRL, Grand Island, NY). After a 24-h incubation period at 37°C, cells were harvested in sample buffer for SDS-PAGE and Western blot analysis as described below.

Membrane preparation. Membranes were prepared from female Sprague-Dawley rats, euthanized by intraperitoneal injection of pentobarbital sodium. Crude membranes were prepared from various organs as follows. Tissues were removed (1 g/10 ml) and minced in ice-cold buffer (250 mM sucrose, 10 mM HEPES, pH 7.5) containing protease inhibitors pepstatin A, leupeptin, phenylmethylsulfonyl fluoride and benzamidine at a final concentration of 2 mM. All subsequent steps were performed on ice or at 4°C in a refrigerated centrifuge. Minced tissues were homogenized using a Polytron (Brinkmann Instruments, Westbury, NY), at setting 3, for 15 s. Crude membranes were separated by differential centrifugation as follows. The homogenate was centrifuged at 750 g for 30 min at 4°C in a Sorvall RC-5B (SS34 rotor), and the supernatant was centrifuged in a Beckman LB-55M Ultra (70.1 Ti rotor) at 100,000 g for 1 h at 4°C. The final pellet was resuspended in sucrose buffer, and the protein concentration was determined as indicated above. Kidney membranes were prepared from renal cortex as described (11) with the following modifications. The final pellet containing both apical and basolateral membranes was resuspended in sucrose buffer with 12% Percoll and was centrifuged at 200,000 g for 1 h at 4°C. After separation, the gradient was divided into 14 (2 ml) fractions starting at the top and analyzed using SDS-PAGE and immunoblotting as described below. Fractions enriched in either -glutamyl transferase (apical) or -subunit of Na⁺-K⁺-ATPase (basolateral) as determined by Western blot analysis, were pooled (2 × 2 ml each), and protein concentration determined as indicated above.

SDS-PAGE and immunoblotting. Membrane fractions were solubilized and separated by SDS-PAGE using 7.5% polyacrylamide gels according to Laemmli (19). In some instances gels were stained with Coomassie brilliant blue. For immunoblotting, proteins were transferred to Immobilon-P (300 mA for 6-10 h at 4°C) and stained with Ponceau S in 0.5% trichloroacetic acid. Immunoblotting was performed as follows. Strips of Immobilon-P were incubated first in Blotto (5% nonfat dry milk in PBS, pH 7.4) for 1-3 h to block nonspecific binding of antibody followed by overnight incubation in primary antibody diluted 1:2,000. Blocking experiments were performed by preincubating 100 µg of monoclonal antibody with 100 µg of fpNHE4AA565-675 in 1 ml of Blotto for 1 h prior to immunoblotting. The strips were then washed three times in Blotto and incubated with appropriate HRP-conjugated secondary (see below) for 1 h. The strips were washed three times in Blotto, and bound secondary was detected with the Renaissance chemiluminescence reagent (DuPont NEN, Boston, MA) and Hyperfilm MP (Amersham, Arlington Heights, IL) according to the manufacturer's protocols.

Immunocytochemistry. Of eight clones positively identified by both ELISA and Western blotting criteria, none gave a signal in immunocytochemical experiments in the kidney. However, 11H11 was found to work in stomach using the following procedure. Rat stomach was fixed by vascular perfusion using paraformaldehyde-lysine-periodate fixative as described previously (2). Blocks of fixed stomach were embedded in paraffin and sectioned at 5 µm by the Histology Laboratory in the Pathology Department at Yale University on a fee-for-service basis. Sections were deparaffinized in xylene, washed in graded ethanols, and hydrated to PBS. Sections were then incubated in Antigen Unmasking Solution (Vector Laboratories, Burlingame, CA) and heated in a microwave oven according to manufacturer's protocol. Sections were washed in PBS and blocked for 30 min in goat serum dilution buffer (GSDB) containing 15% goat serum, 0.3% Triton X-100, 20 mM sodium phosphate (pH 7.4), and 0.9% NaCl. MAb 11H11 and control MAb were diluted to 10 µg/ml in GSDB and incubated with the sections for 2 h. Bound antibody was detected using a Vectastain ABC Kit (Vector), which utilizes a biotinylated secondary IgG in conjunction with HRP-conjugated avidin. This kit was used according to manufacturer's protocol. In addition, ImmunoPure peroxidase suppressor (Pierce Chemicals, Rockford, IL) was used to inhibit endogenous peroxidase activity. HRP reaction product was developed using ImmunoPure metal-enhanced diaminobenzidine substrate kit (Pierce Chemicals). After staining, sections were washed briefly in distilled H₂O, mounted in 95% glycerol, 5% PBS, and examined with a Zeiss Axiophot microscope.

Antibodies and antibody conjugates. Guinea pig antiserum (anti-fp347A) raised to the COOH-terminal domain (amino acids 778-818) of pig NHE1 reacts with a 95- to 110-kDa polypeptide in rat and was used following affinity purification at a dilution of 1:5,000 for immunoblotting (3). A second guinea pig antiserum (anti-fpNHE2C688-813) raised to the COOH-terminal 125 amino acids of NHE2 was used following affinity purification at a dilution 1:1,000 for immunoblotting (D. Biemesderfer, unpublished observations). A third guinea pig antiserum raised to the COOH-terminal 40 amino acids of NHE3 (anti-fpNHE3-C40) was used following affinity purification at a dilution of 1:5,000 for immunoblotting (2). A monoclonal antibody prepared against the -subunit of dog Na⁺-K⁺-ATPase (17) was a gift from Dr. Michael Caplan and was used at 1:10,000. Rabbit polyclonal antisera against -glutamyl transferase (anti-GGT) was a gift from Dr. David Castle (University of Virginia) and was used at 1:5,000 (6). HRP-conjugated rabbit anti-guinea pig IgG (heavy and light chain specific), goat anti-rabbit, and goat anti-mouse sera were purchased from Zymed Laboratories (San Francisco, CA) and used at 0.5 µg/ml.

Materials and reagents. All enzymes for molecular biology and amylose affinity resin were from New England Biolabs. BSA type V was purchased from Sigma Chemical. Pentobarbital sodium was purchased from the Butler (Columbus, OH). Immobilon-P was purchased from Millipore (Bedford, MA). Percoll was obtained from Pharmacia Biotec (Piscataway NJ). Centricon 30 microconcentrators were purchased from Amicon (Beverly, MA).

Animals. Female Sprague-Dawley rats and BALB/c mice were purchased from Charles River Laboratories (Wilmington, MA).

	RESULTS

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Specificity of monoclonal antibody (11H11) for fusion protein. In the present study, a fusion protein construct containing the maltose-binding protein and amino acids 565-675 of rat NHE4 was produced as antigen for the generation of monoclonal antibodies. Therefore, the first experiment was designed to confirm the specificity of subsequently generated antibodies for the NHE4 portion of the antigen. Purified maltose-binding protein and fpNHE4AA565-675 were prepared, separated by SDS-PAGE, and either Coomassie stained (Fig. 1, left) or immunoblotted (Fig. 1, middle and right) as shown (Fig. 1). Although three bands were noted on Coomassie-stained gel of purified fpNHE4AA565-675, with a lower form in the region of 55 kDa and upper bands at 65 kDa and 110 kDa (lane A), the actual predicted molecular mass for fpNHE4AA565-675 is 55 kDa. The apparent molecular mass of the maltose-binding protein on Coomassie-stained gel is 45 kDa (lane B). When an immunoblot was prepared from the gel and probed with the monoclonal anti-fpNHE4AA565-675 11H11 (Fig. 1, middle), the results show specific recognition of the NHE4-derived epitopes since there is no labeling of the maltose-binding protein (lane D). Specific reactivity toward fpNHE4AA565-675 at 55 kDa is demonstrated (lane C). Additionally, the minor bands in lane A are recognized by 11H11 and therefore probably represent aggregates of the fusion protein. Preabsorption of 100 µg of 11H11 with 100 µg of fpNHE4AA565-675 for 1 h completely blocked reactivity with fpNHE4AA565-675 (lane E).

View larger version (51K): [in this window] [in a new window]   Fig. 1.   SDS-PAGE and immunoblot characterization of anti-fpNHE4AA565-675 (11H11) specificity for fpNHE4AA565-675 versus maltose binding protein. Ten micrograms (lanes A and B) or 1 µg (lanes C-F) of either maltose-binding protein (lanes B, D, and F) or fpNHE4AA565-675 (lanes A, C, and E) were separated by 7.5% SDS-PAGE and either stained with Coomassie blue (lanes A and B) or transferred to Immobilon and immunoblotted with anti-fpNHE4AA565-675 clone 11H11 as indicated (lanes C-F). Preabsorption of 100 µg of 11H11 with 100 µg of fpNHE4AA565-675 (lanes E and F) blocks staining. Molecular weights, expressed as 103 × Mr, are presented on the right.

Identification of NHE4 polypeptide after transient expression in LAP1 cells. Next, 11H11 reactivity and specificity for NHE4 polypeptide was tested after transient expression of NHE isoforms in LAP1 cells, which are deficient in NHE activity (10). Figure 2 shows an immunoblot of cell lysates prepared from LAP1 cells transfected with NHE1-NHE4 as indicated (designated 1, 2, 3, and 4, respectively, in Fig. 2). The blot was sequentially probed with antibodies as indicated above each panel of Fig. 2. Incubation with the anti-NHE4 monoclonal antibody labeled a major band with an apparent molecular mass between 60 and 66 kDa in NHE4 transfected cell lysates but not in cell lysates from cells transfected with NHE1, NHE2, or NHE3. When anti-NHE4 monoclonal antibody was preabsorbed against fpNHE4AA565-675, reactivity was completely blocked (data not shown). Subsequently, the blot was stripped and reprobed with antibodies to NHE1, NHE2, and NHE3. In each case immunoblotting with an antibody raised against a specific isoform produced specific staining in the corresponding cell lysate with no cross-reactivity with any other isoforms. LAP1 cells transfected with rabbit NHE1 and probed with anti-NHE1 showed major bands at 80-85 kDa and 95-110 kDa, as described previously (3). Similarly, LAP1 cells transfected with rabbit NHE3 and immunoblotted using affinity-purified anti-fpNHE3C791-831 demonstrated a major band at 82 kDa, as previously described (1, 2). Finally, cells transfected with rat NHE2 and immunoblotted using affinity-purified anti-fpNHE2C689-813 showed a major band at 75-80 kDa. These findings indicate that NHE1-NHE3 were each expressed in these experiments and therefore that the anti-NHE4 monoclonal antibody does not cross-react with any of these isoforms.

View larger version (37K): [in this window] [in a new window]   Fig. 2.   Immunoblot characterization of NHE protein expression in LAP1 cells. LAP1 cells were grown to 80-90% confluence in 35-mm plastic dishes and infected with vaccinia virus, then transfected with cDNAs for NHE1-NHE4 as indicated by the numbers at bottom. Cells were solubilized in 400 µl of sample buffer, and 100 µl from each sample was separated by 7.5% SDS-PAGE and transferred to Immobilon. Sequential immunoblotting was performed with anti-NHE antibodies as indicated at top. After each immunoblotting reaction, the membrane was stripped, and the removal of antibody was verified by incubation in secondary conjugate alone. Molecular weights, expressed as 103 × Mr, are presented on the right.

Characterization of tissue expression by immunoblotting. Having confirmed the specificity of 11H11 for NHE4 polypeptide, we next investigated the tissue distribution of NHE4 in the rat by preparing crude membranes from rat organs identified as being sites for expression of NHE4 mRNA (21). Preliminary results of immunoblots indicated NHE4 protein was highly enriched in crude membranes prepared from stomach, consistent with previously reported Northern blot data (21). Thus, in Fig. 3, 10-fold less crude membrane protein (5 µg) was used for stomach compared with other tissues. As can be seen in Fig. 3 (top), the anti-NHE4 monoclonal antibody recognized a major band at 65-70 kDa that is highly expressed in the stomach. NHE4 protein expression was also noted with lower abundance in skeletal muscle, kidney, uterus, heart, and liver. Barely detectable amounts were seen in brain and spleen. Minor bands were noted in most tissues at 45-50 kDa, which probably represents a proteolytic fragment. Additionally, minor bands at 75 kDa were seen in stomach and kidney. Labeling of both the upper and lower minor bands was specific, since the labeling was blocked when 11H11 was preincubated with fpNHE4AA565-675 (data not shown). For comparison, the blot was stripped and reprobed with anti-NHE1 antibody to determine expression of NHE1 in the same rat tissues (Fig. 3, bottom). A major band at 95-110 kDa was observed in all tissues, consistent with the ubiquitous expression of NHE1 mRNA previously reported for the rat (21). This is also consistent with an apparent molecular mass of 110 kDa reported for NHE1 from rat intestine (4). High levels of NHE1 expression were observed in stomach (attenuated 10-fold) as well as in brain, spleen, heart, kidney, and uterus. Much lower levels of expression of NHE1 protein were observed in liver and skeletal muscle. A minor band at 66 kDa was noted in most tissues and probably represents a proteolytic product based on its variability and the absence of known splice variants of NHE1.

View larger version (57K): [in this window] [in a new window]   Fig. 3.   Immunoblot characterization of NHE4 and NHE1 tissue distribution in rat. Fifty micrograms of crude membrane protein from rat kidney, uterus, brain, liver, heart, skeletal muscle, and spleen, as well as 5 µg from rat stomach, was separated by SDS-PAGE and transferred to Immobilon for immunoblotting. Blot was first probed with 11H11 at 1:5,000 (top) and then was stripped and reprobed with anti-NHE1 at 1:5,000 (bottom). Molecular weights, expressed as 103 × Mr, are presented on the right.

Renal expression of NHE4 protein. Having identified the rat kidney as a site of expression of NHE4 protein, we next explored the intrarenal distribution in more detail. In the first series of experiments, Western blot analysis was performed to compare kidney whole homogenate with crude membranes prepared from medulla and cortex (Fig. 4). The blot was probed sequentially with anti-NHE4 monoclonal antibody, 11H11 (Fig. 4, left), the anti-NHE1 antiserum (Fig. 4, middle), and an antibody generated against rat -glutamyl transferase (Fig. 4, right), as indicated above each blot. The results suggest that NHE4 (Fig. 4, left), again identified as a major band of 65-70 kDa with the proteolytic fragment at 45-50 kDa, is enriched in cortical membranes compared with either the whole kidney homogenate or membranes prepared from the medulla. Similarly, shown in Fig. 4, right, -glutamyl transferase is enriched in cortical membranes compared with medullary membranes. Studies by Castle et al. (6) have demonstrated that on SDS-PAGE -glutamyl transferase from rat kidney runs as a major band between 50 and 55 kDa and is localized to, and highly enriched in, brush-border membranes of the proximal tubule. In contrast, NHE1 (Fig. 4, middle) is enriched in membrane fractions from both cortex and medulla relative to whole kidney homogenate. In the present study, NHE1 from rat kidney has an apparent molecular mass between 95 and 110 kDa, consistent with reported values for NHE1 from rabbit renal cortical membranes (3). The fact that a strong signal for NHE1 protein was detected in renal medullary membranes suggests that the relatively reduced abundance of NHE4 in these membranes is not due to greater proteolysis of medullary compared with cortical membranes.

View larger version (45K): [in this window] [in a new window]   Fig. 4.   Immunoblot characterization of NHE4, NHE1, and -glutamyl transferase (GGT) protein expression in rat kidney. Fifty micrograms of whole kidney homogenate or crude membrane protein prepared from either rat kidney cortex or medulla was separated on 7.5% SDS-PAGE and transferred to Immobilon. Sequential immunoblotting was performed with antibodies as indicated at top. Molecular weights, expressed as 103 × Mr, are presented on the right.

In the second series of experiments cortical membranes were prepared as described in the METHODS and further separated on a Percoll gradient. The gradient was divided into 14 fractions, separated by SDS-PAGE, and analyzed by immunoblotting with antibody markers for the apical (-glutamyl transferase) and basolateral (Na⁺-K⁺-ATPase) membranes. The fractions most enriched for apical and basolateral membrane markers (-glutamyl transferase and Na⁺-K⁺-ATPase, respectively) were selected, and an immunoblot was prepared (Fig. 5). The immunoblot was sequentially probed with antibodies as indicated on the top of each blot in Fig. 5. The rat -subunit of Na⁺-K⁺-ATPase was labeled as a protein of 97-110 kDa, which served as a marker for the basolateral membrane fraction. NHE1 was identified as a 95- to 110-kDa protein highly enriched in basolateral compared with apical membranes, confirming previous findings in rabbit kidney membranes (2). NHE4 was identified as a 65- to 70-kDa protein also enriched in basolateral membranes. In contrast, -glutamyl transferase was relatively enriched in the apical membranes and was almost completely absent from the basolateral membrane fraction.

View larger version (37K): [in this window] [in a new window]   Fig. 5.   Immunoblot localization of NHE4, NHE1, Na+-K+-ATPase, and GGT protein expression in rat kidney apical and basolateral membranes. Rat kidney crude membranes were further separated on a Percoll gradient, and 50 µg of specific fractions determined to be enriched in either apical or basolateral membranes were separated on 7.5% SDS-PAGE and transferred to Immobilon. Sequential immunoblotting was performed with antibodies indicated at top. Molecular weights, expressed as 103 × Mr, are presented on the right.

Despite an exhaustive search of fixatives and other preparative techniques, suitable conditions to perform immunocytochemistry in rat kidney could not be determined. However, since the stomach expresses 50-fold higher levels of NHE4 protein, experiments were repeated in this tissue. By use of an antigen unmasking protocol, specific staining of gastric mucosa was observed (Fig. 6). The anti-NHE4 monoclonal antibody 11H11 labeled the basolateral membrane of a population of cells at the base of the gastric mucosa. Specific staining was observed along the lateral plasma membranes of many cells of the gastric glands. Unfortunately, the conditions employed with the antigen unmasking technique were not suitable for immunocytochemistry with either monoclonal or polyclonal antibodies directed against NHE1, so double labeling studies to colocalize the isoforms were not possible.

View larger version (152K): [in this window] [in a new window]   Fig. 6.   Immunolocalization of NHE4 in rat stomach. Sections of rat stomach were stained with MAb 11H11 as described in METHODS. Shown here is a region containing several gastric glands at the base of the mucosa. Specific (black) reaction product can be seen along the lateral plasma membranes of many cells of the gastric glands. Arrows point to examples of this staining pattern. L, lumen of a gastric gland; sm, submucosa. Magnification, ×390.

	DISCUSSION

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The molecular identification (8, 18, 21, 26, 29-32, 34) of at least five distinct isoforms of NHEs (NHE1-NHE5) has necessitated the development of immunochemical reagents for specific cellular and subcellular localization of protein expression. The purpose of the present study was to begin to establish the physiological role for rat NHE4 by describing the distribution of the protein. Our strategy included the generation of monoclonal antibodies to a fusion protein construct containing amino acids 565-675, because in general the COOH-terminal hydrophilic domains of the four isoforms of NHEs share the lowest amino acid similarity.

The first experiment was designed to verify the specificity of the anti-NHE4 monoclonal antibodies. Since the entire fusion protein construct (i.e., containing the maltose-binding protein) was used as immunogen, it was important to demonstrate that anti-NHE4 antibodies exclusively recognized fpNHE4AA565-675 but not the maltose-binding protein (Fig. 1). NHE4 shares 46% identity with NHE2 in the COOH-terminal hydrophilic domain. Thus a second important criterion for specificity is that anti-NHE4 antibodies should not cross-react with either NHE2 polypeptide or other NHE isoforms. Accordingly, the specificity of the anti-NHE4 monoclonal for NHE4 polypeptide was tested by transiently expressing NHE4 in a mouse fibroblast cell (LAP1) devoid of endogenous NHE activity (10) using the vaccinia-T7 system. The anti-NHE4 antibody was found to react only with NHE4 polypeptide (Fig. 2) and not with NHE1, NHE2, or NHE3.

Characterization of native NHE4 by SDS-PAGE and immunoblotting revealed the apparent molecular mass of 65-70 kDa (Figs. 3-5), which is 15% below the predicted value of 81 kDa based on the amino acid sequence. Similarly, NHE1 protein devoid of O-linked oligosaccharide but containing the N-linked high-mannose oligosaccharide runs smaller than the predicted molecular mass at 85 kDa (9). NHE3, which is not glycosylated (9), was identified as an 82- to 85-kDa protein when expressed using the vaccinia-T7 system, or in native rat (9) or rabbit (2, 16) brush-border membranes, 10% below the predicted molecular mass of 93 kDa (32). Thus NHE4 protein may migrate with faster mobility by SDS-PAGE than predicted for actual molecular mass, as is the case for NHE1 and NHE3.

In addition to the major band at 65-70 kDa, a minor band was noted in most tissues at 45-50 kDa, and a larger form at 75 kDa was noted in both stomach and kidney. Although the basis for these minor forms is not known, NHE4 does have two potential N-linked glycosylation sites. The Asn³⁴² site is conserved in all four isoforms, but is not used in NHE1 or NHE3 (9). Still it is possible that the 75-kDa band might represent a small component of the NHE4 protein that is glycosylated, perhaps in a tissue-specific manner. A second possibility is that the 75-kDa band represents an alternatively spliced isoform. The identification of NHE1 on the same blot as a major band between 95 and 110 kDa (Fig. 3, bottom) argues against general protein degradation to yield the 65- to 70-kDa species as a proteolytic fragment of the 75-kDa species of NHE4. Conversely, the smaller 45- to 50-kDa band probably represents a degradation product, since it is more uniformly expressed in proportion to the major band at 65-70 kDa. Recently, Bookstein et al. (5), have reported identification of a 100-kDa protein in PS120 cells stably expressing rat NHE4. The reason for the discrepancy between the apparent molecular mass of NHE4 in the study of Bookstein et al. (5) and in the present study is unknown.

Examination of the tissue distribution of NHE4 protein in the rat revealed highest levels of expression in stomach with much lower levels in kidney, uterus, and skeletal muscle, consistent with expression of NHE4 transcript (21). However, NHE4 protein was not detected in brain, where detectable levels of mRNA were noted. Additionally, detectable levels of NHE4 protein were observed in heart, liver, and spleen, where NHE4 mRNA was not detectable. These data suggest that there may be differences in mRNA stability between tissues or that low-level protein expression results from levels of mRNA expression below the level of detection in previous studies. The high level of expression in gastric glands as well as significant expression in other tissues that are not facing hyperosmotic stress (i.e., uterus, heart, and skeletal muscle) argue against the previous suggestion (5) that NHE4 is uniquely expressed in regions of high osmolarity where it plays a specialized role in cell volume regulation.

Appreciable amounts of NHE4 protein were detectable in rat kidney. Recently, the renal distribution of NHE4 mRNA was examined by in situ hybridization (5). The cRNA probe hybridized to tubules in renal cortex and outer medulla with the highest levels of mRNA expression detected in the inner medulla, an area rich in collecting duct tubules. In contrast, we found that NHE4 protein (Fig. 4) was preferentially expressed in renal cortex. Although the reason for the discrepancy between mRNA and protein expression is not obvious, it is possible that differences in mRNA stability and/or protein turnover may underlie these observations. In contrast, NHE1 protein was abundant in crude membranes from the renal medulla as well as the renal cortex, which is consistent with immunocytochemical data indicating NHE1 expression along the basolateral membranes of medullary thick ascending limb and medullary collecting duct (3), as well as Northern analysis indicating NHE1 transcript abundance in both renal medulla and cortex (14).

The distribution of NHE4 in apical and basolateral membranes was examined. Immunoblotting and immunocytochemical studies have shown that both NHE1 (3) and Na⁺-K⁺-ATPase (17) are localized to basolateral membranes in renal epithelia. Our results (Fig. 5) confirm these data and suggest that NHE4 is also enriched in basolateral membrane fractions. Given the ubiquitous expression of NHE1 along the nephron, it is possible that NHE1 and NHE4 are coexpressed in certain cell types. Alternatively, NHE4 might account for the basolateral Na⁺/H⁺ exchange activity noted in intercalated cells of the connecting tubule and cortical collecting duct (35), where NHE1 expression could not be detected (3).

In further support of the basolateral distribution of NHE4, immunocytochemical experiments with 11H11 revealed labeling of the basolateral membrane of a population of epithelial cells at the base of the gastric gland in an area where NHE1 was not detected (28). Indeed, Stuart-Tilley et al. (28) reported that NHE1 is present along the basolateral membranes of the mucous neck cells, interdigitated between the parietal cells of the gastric glands, and in the basolateral membranes of the surface mucous cells. They found that NHE1 staining was weak or undetectable near the base of the gland. Thus NHE4 may be the isoform accounting for basolateral Na⁺/H⁺ exchange activity in parietal cells, the predominant cell type in gastric glands. Precise localization using double labeling will require more compatible immunoreagents. In any event, the basolateral localization of NHE4 determined by immunocytochemical staining in gastric cells is consistent with the basolateral localization of NHE4 determined by immunoblotting of kidney membrane fractions.

In summary, we have used isoform-specific anti-NHE4 monoclonal antibodies to identify NHE4 as a protein of approximate molecular mass 65-70 kDa that is most abundantly expressed in stomach and that is also detected in skeletal muscle, heart, kidney, uterus, and liver. In the kidney, NHE4 polypeptide abundance as determined by immunoblot is greater in the cortex than in the medulla, and membranes containing NHE4 protein cofractionate in sucrose density gradients with basolateral membrane markers NHE1 and Na⁺-K⁺-ATPase. In the stomach, NHE4 is present on the basolateral membranes of a population of cells near the base of the gastric gland, some of which may include parietal cells.