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• W1998271882 abstract "Gastric parietal cells migrate from the luminal to the basal region of the gland, and they gradually lose acid secretory activity. So far, distribution and function of K+-Cl- cotransporters (KCCs) in gastric parietal cells have not been reported. We found that KCC3a but not KCC3b mRNA was highly expressed, and KCC3a protein was predominantly expressed in the basolateral membrane of rat gastric parietal cells located in the luminal region of the glands. KCC3a and the Na+,K+-ATPase α1-subunit (α1NaK) were coimmunoprecipitated, and both of them were highly localized in a lipid raft fraction. The ouabain-sensitive K+-dependent ATP-hydrolyzing activity (Na+,K+-ATPase activity) was significantly inhibited by a KCC inhibitor (R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid (DIOA)). The stable exogenous expression of KCC3a in LLC-PK1 cells resulted in association of KCC3a with endogenous α1NaK, and it recruited α1NaK in lipid rafts, accompanying increases of Na+,K+-ATPase activity and ouabain-sensitive Na+ transport activity that were suppressed by DIOA, whereas the total expression level of α1NaK in the cells was not significantly altered. On the other hand, the expression of KCC4 induced no association with α1NaK. In conclusion, KCC3a forms a functional complex with α1NaK in the basolateral membrane of luminal parietal cells, and it up-regulates α1NaK in lipid rafts, whereas KCC3a is absent in basal parietal cells. Gastric parietal cells migrate from the luminal to the basal region of the gland, and they gradually lose acid secretory activity. So far, distribution and function of K+-Cl- cotransporters (KCCs) in gastric parietal cells have not been reported. We found that KCC3a but not KCC3b mRNA was highly expressed, and KCC3a protein was predominantly expressed in the basolateral membrane of rat gastric parietal cells located in the luminal region of the glands. KCC3a and the Na+,K+-ATPase α1-subunit (α1NaK) were coimmunoprecipitated, and both of them were highly localized in a lipid raft fraction. The ouabain-sensitive K+-dependent ATP-hydrolyzing activity (Na+,K+-ATPase activity) was significantly inhibited by a KCC inhibitor (R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid (DIOA)). The stable exogenous expression of KCC3a in LLC-PK1 cells resulted in association of KCC3a with endogenous α1NaK, and it recruited α1NaK in lipid rafts, accompanying increases of Na+,K+-ATPase activity and ouabain-sensitive Na+ transport activity that were suppressed by DIOA, whereas the total expression level of α1NaK in the cells was not significantly altered. On the other hand, the expression of KCC4 induced no association with α1NaK. In conclusion, KCC3a forms a functional complex with α1NaK in the basolateral membrane of luminal parietal cells, and it up-regulates α1NaK in lipid rafts, whereas KCC3a is absent in basal parietal cells. The cation-chloride cotransporter gene family (SLC12) includes Na+-Cl- cotransporters, Na+-K+-2Cl- cotransporters (NKCCs) 2The abbreviations used are:NKCCNa+-K+-2Cl- cotransporterKCCK+-Cl- cotransporterα1NaKNa+,K+-ATPase α1-subunitAQPaquaporinMβCDmethyl-β-cyclodextrinDIOAR-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acidMES2-morpholinoethanesulfonic acidCHAPS3-[(3-cholamidopropyl)dimethyl-ammonio]propanesulfonic acidDRMdetergent resistance membraneEPphosphoenzyme.2The abbreviations used are:NKCCNa+-K+-2Cl- cotransporterKCCK+-Cl- cotransporterα1NaKNa+,K+-ATPase α1-subunitAQPaquaporinMβCDmethyl-β-cyclodextrinDIOAR-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acidMES2-morpholinoethanesulfonic acidCHAPS3-[(3-cholamidopropyl)dimethyl-ammonio]propanesulfonic acidDRMdetergent resistance membraneEPphosphoenzyme. and K+-Cl- cotransporters (KCCs). KCCs mediate an electroneutrally coupled transport of K+ and Cl- and contribute to transepithelial transport and cell volume regulation (1Gamba G. Physiol. Rev. 2005; 85: 423-493Crossref PubMed Scopus (615) Google Scholar, 2Adragna N.C. Fulvio Di M. Lauf P.K. J. Membr. Biol. 2004; 201: 109-137Crossref PubMed Scopus (179) Google Scholar). KCCs are encoded by at least four homologues genes (KCC1–KCC4), and KCC3 has three isoforms (KCC3a–KCC3c) generated by the difference of first coding exons (3Mount D.B. Mercado A. Song L. Xu J. George Jr., A.L. Delpire E. Gamba G. J. Biol. Chem. 1999; 274: 16355-16362Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 4Race J.E. Makhlouf F.N. Logue P.J. Wilson F.H. Dunham P.B. Holtzman E.J. Am. J. Physiol. 1999; 277: C1210-C1219Crossref PubMed Google Scholar, 5Pearson M.M. Lu J. Mount D.B. Delpire E. Neuroscience. 2001; 103: 481-491Crossref PubMed Scopus (118) Google Scholar). KCC1 is widely expressed, whereas KCC2 and KCC3b are restricted in neuron and kidney, respectively. KCC3a and KCC4 are mainly expressed in epithelial cells (3Mount D.B. Mercado A. Song L. Xu J. George Jr., A.L. Delpire E. Gamba G. J. Biol. Chem. 1999; 274: 16355-16362Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 4Race J.E. Makhlouf F.N. Logue P.J. Wilson F.H. Dunham P.B. Holtzman E.J. Am. J. Physiol. 1999; 277: C1210-C1219Crossref PubMed Google Scholar, 5Pearson M.M. Lu J. Mount D.B. Delpire E. Neuroscience. 2001; 103: 481-491Crossref PubMed Scopus (118) Google Scholar).Among the SLC12 family, Na+-K+-2Cl- cotransporter-1 (NKCC1) is present in the basolateral membrane of gastric parietal cells (6McDaniel N. Lytle C. Am. J. Physiol. 1999; 276: G1273-G1278PubMed Google Scholar). NKCC1 was reported to be not involved in the gastric acid (HCl) secretion but involved in nonacidic electrogenic ion secretions in mouse parietal cells (7Flagella M. Clarke L.L. Miller M.L. Erway L.C. Giannella R.A. Andringa A. Gawenis L.R. Kramer J. Duffy J.J. Doetschman T. Lorenz J.N. Yamoah E.N. Cardell E.L. Shull G.E. J. Biol. Chem. 1999; 274: 26946-26955Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 8McDaniel N. Pace A.J. Spiegel S. Engelhardt R. Koller B.H. Seidler U. Lytle C. Am. J. Physiol. 2005; 289: G550-G560Crossref PubMed Scopus (46) Google Scholar). So far, the distribution and function of KCCs have not been reported in the gastric parietal cells.Lipid rafts are glycosphingolipid- and cholesterol-enriched microdomains and are thought to be the functional domains involved in membrane trafficking and signal transduction (9Simons K. Vaz W.L.C. Annu. Rev. Biophys. Biomol. Struct. 2004; 33: 269-295Crossref PubMed Scopus (1343) Google Scholar, 10Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8019) Google Scholar, 11Polishchuk R. Pentima Di A. Lippincott-Schwartz J. Nat. Cell Biol. 2004; 6: 297-307Crossref PubMed Scopus (182) Google Scholar). Recently, it has been reported that some transporters and channels, such as Na+,K+-ATPase (12Lingwood D. Harauz G. Ballantyne J.S. J. Biol. Chem. 2005; 280: 36545-36550Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 13Welker P. Geist B. Frühauf J.-H. Salanova M. Groneberg D.A. Krause E. Bachmann S. Am. J. Physiol. 2007; 292: R1328-R1337Crossref PubMed Scopus (37) Google Scholar), Na+/H+ exchanger 3 (14Li X. Galli T. Leu S. Wade J.B. Weinman E.J. Leung G. Cheong A. Louvard D. Donowitz M. J. Physiol. 2001; 537: 537-552Crossref PubMed Scopus (118) Google Scholar, 15Murtazina R. Kovbasnjuk O. Donowitz M. Li X. J. Biol. Chem. 2006; 281: 17845-17855Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), and epithelial Na+ channel (16Balut C. Steels P. Radu M. Ameloot M. Driessche Van W. Jans D. Am. J. Physiol. 2006; 290: C87-C94Crossref PubMed Scopus (37) Google Scholar), are localized in lipid rafts of several tissues. However, little is known about lipid rafts of gastric parietal cells.In the present study, we studied distribution of KCC3a and association of KCC3a with Na+,K+-ATPase α1-subunit (α1NaK) in the gastric mucosa. In addition, we studied recruitment of α1NaK by KCC3a in lipid rafts using the cells stably expressing exogenous KCC3a.EXPERIMENTAL PROCEDURESMaterials—Anti-rat KCC3 rabbit polyclonal antibody was generated with keyhole limpet hemocyanin-coupled peptides against 19 amino acids corresponding to the N-terminal sequence of KCC3 (KKARNAYLNNSNYEEGDEY). Both KCC3a and b contain this N-terminal sequence. This antigen peptide has no homology with the sequences of KCC1, 2, and 4. Anti-Na+,K+-ATPase α1-subunit (α1NaK) mouse monoclonal antibody was obtained from Upstate Biotechnology, Inc.(Lake Placid, NY). Anti-H+,K+-ATPase α-subunit mouse monoclonal antibody (1H9) was from Medical & Biological Laboratories Co. (Nagoya, Japan). Anti-flotillin-2 mouse monoclonal antibody (B-6) and anti-human aquaporin-4 (AQP4) goat polyclonal antibody (H-19) were from Santa Cruz Biotechnology (Santa Cruz, CA). Lipofectamine 2000, anti-Xpress mouse monoclonal antibody, Alexa Fluor 488/546-conjugated anti-mouse IgG antibody, Alexa Fluor 488/546-conjugated anti-rabbit IgG antibody, and Alexa Fluor 546-conjugated anti-goat IgG antibody were from Invitrogen. Ouabain, SCH 28080, methyl-β-cyclodextrin (MβCD), and R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid (DIOA) were from Sigma-Aldrich. Protein A-agarose beads was from Pierce. Hygromycin B and blasticidin S were obtained from Wako Pure Chemical Industries (Osaka, Japan) and Kaken Pharmaceutical Co. (Tokyo, Japan), respectively. 22NaCl was obtained from PerkinElmer Life Sciences.Isolation of Tissues and Cells—The cell suspension rich in parietal cells and the gastric gland suspension were prepared from rabbit stomach as previously described (17Sakai H. Ohira Y. Tanaka A. Suzuki T. Ikari A. Morii M. Takeguchi N. J. Physiol. 2003; 551: 207-217Crossref PubMed Scopus (10) Google Scholar). Human gastric mucosa was obtained from surgical resection of Japanese patients at Toyama University Hospital in accordance with the recommendations of the Declaration of Helsinki and with the ethics committee approval. All of the patients gave informed consent.Northern Blotting—Poly(A)+ RNA of the cells was prepared by using the PolyATtract mRNA isolation system II (Promega, Madison, WI). The amplified products were sequenced and used for the preparation of the 32P-labeled cDNA probes. The rabbit KCC3 probe was 712 bp long and corresponded to nucleotides 806–1507 of the KCC3a cDNA. The poly(A)+ RNA (2.5 μg) was separated on a 1% agarose/formaldehyde gel and transferred onto a nylon membrane (Zeta-probe GT; Bio-Rad). The membrane was hybridized with the 32P-labeled cDNA fragment overnight at 65 °C in 250 mm NaH2PO4-Na2HPO4, pH 7.2, 7% SDS; washed in 40 mm NaH2PO4-Na2HPO4, pH 7.2, 1% SDS at 65 °C; and exposed to the Imaging Plate (Fuji Film) for 20 h.Plasmid Construction—Full-length cDNAs encoding rat KCC3a and rat KCC4 were inserted into pcDNA4/His vector (Invitrogen) by using KpnI/NotI and EcoRI/XbaI restriction sites, respectively (KCC3a-pcDNA4/His vector and KCC4-pcDNA4/His vector), and also inserted into pcDNA5/TO vector (Invitrogen) by using AflII/NotI and AflII/XbaI restriction sites, respectively (KCC3a-pcDNA5/TO vector and KCC4-pcDNA5/TO vector).Expression of KCC3a and KCC4 in Cultured Cells—To establish the tetracycline-regulated expression system of KCC3a or KCC4 in LLC-PK1 cells, the cells were cotransfected with pcDNA6/TR vector (Invitrogen), KCC3a-pcDNA5/TO vector, or KCC4-pcDNA5/TO vector by using Lipofectamine 2000 and cultured for 24 h in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The transfected cells were selected in the presence of 800 units/ml hygromycin B and 5 μg/ml blasticidin S. For transient expression of KCC3a in HEK293 cells stably expressing gastric H+,K+-ATPase (18Kimura T. Tabuchi Y. Takeguchi N. Asano S. J. Biol. Chem. 2002; 277: 20671-20677Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), the cells were transfected with the KCC3a-pcDNA4/His vector by using Lipofectamine 2000 and cultured for 24 h in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum.Western Blotting—Preparation of membrane fractions and Western blotting were carried out as described previously (19Fujii T. Ohira Y. Itomi Y. Takahashi Y. Asano S. Morii M. Takeguchi N. Sakai H. Eur. J. Pharmacol. 2007; 560: 123-126Crossref PubMed Scopus (18) Google Scholar). The signals were visualized with the ECL Plus system (GE Healthcare, Buckinghamshire, UK). To quantify the chemiluminescence signals on the membranes, a FujiFilm LAS-1000 system and the MultiGauge software were used. Anti-KCC3, anti-Xpress, and anti-H+,K+-ATPase (1H9) antibodies were used at 1:1,000 dilution, and anti-α1NaK antibody was used at 1:10,000 dilution. Anti-flotillin-2 antibody was used at 1:2,000 dilution. For negative control, one volume of each primary antibody was preincubated with five volumes of the corresponding blocking peptide. Horseradish peroxidase-conjugated anti-mouse, anti-rabbit, or anti-goat IgG was used as a secondary antibody (1:2,500 dilution).Immunohistochemistry—The gastric mucosa isolated from rat stomach was embedded in the O.C.T. compound (Sakura Finetechnical Co., Tokyo, Japan) and was cut at 8 μm. The sections were fixed in ice-cold methanol for 7 min at room temperature and were pretreated with the 3% bovine serum albumin for 1 h at room temperature to block nonspecific binding of antibody. Then they were incubated with anti-KCC3, anti-H+,K+-ATPase (1H9), anti-α1NaK, or anti-AQP4 antibody (1:100 dilution) overnight at 4 °C. Alexa Fluor 488-conjugated and Alexa Fluor 546-conjugated anti-IgG antibodies (1:100 dilution) were used as secondary antibodies. Immunofluorescence images were visualized by using a Zeiss LSM 510 laser scanning confocal microscope.Immunocytochemistry—LLC-PK1 cells were fixed with ice-cold methanol for 7 min and permeabilized with phosphate-buffered saline containing 0.3% Triton X-100 and 0.1% bovine serum albumin for 15 min at room temperature. Nonspecific binding was blocked by 3% bovine serum albumin. The permeabilized cells were incubated with the anti-Xpress or anti-α1NaK antibody (1:100 dilution) overnight at 4 °C and then with the Alexa Fluor 488-conjugated or Alexa Fluor 546-conjugated anti-IgG antibody (1:100 dilution) for 1 h at room temperature. Immunofluorescence images were visualized by using a Zeiss LSM 510 laser scanning confocal microscope.Isolation of Lipid Rafts—The membrane proteins were lysed with the ice-cold MBS buffer (150 mm NaCl, 25 mm MES-NaOH, pH 6.5) containing 1% CHAPS, 10 μg/ml aprotinin, 10 μg/ml phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 1 μg/ml pepstatin A for 15 min. The solution was mixed with equal volume of 66% sucrose in MBS buffer, the mixture was placed at the bottom of an ultracentrifuge tube, and a discontinuous gradient was formed by overlaying with the 30% sucrose and the 5% sucrose solutions. The sample was centrifuged at 261,000 × g in SW41Ti rotor (Beckman) for 18 h at 4 °C. Ten fractions of 1 ml each were collected from the top of the gradient, and proteins were precipitated by acetone before SDS-polyacrylamide gel electrophoresis and Western blotting. For depletion of cholesterol, the cells were treated with 10 mm MβCD for 30 min at 37 °C before harvesting the cells.Immunoprecipitation—Membrane fractions of rat gastric mucosa (500 μg of protein) and KCC3a-expressing LLC-PK1 cells (2.5 mg of protein) were solubilized in the 500 μl of lysis buffer (phosphate-buffered saline containing 0.5% Triton X-100, 0.1% bovine serum albumin and 1 mm EDTA) for 30 min on ice and centrifuged at 90,000 × g for 30 min at 4 °C. The lysate was precleared with protein A-agarose beads, and the supernatant was incubated with anti-KCC3 antibody or control rabbit IgG for 24 h at 4 °C with end-over-end rotation. The antibody-antigen complexes were incubated with protein A-agarose beads for 4 h at 4 °C with end-over-end rotation. Then the beads were washed three times with the lysis buffer and suspended in SDS sample buffer. The samples were used for Western blotting.Measurement of Na+,K+-ATPase Activity—Na+,K+-ATPase activity of LLC-PK1 (30 μg of protein) and membrane fractions of the rabbit gastric glands (30 μg of protein) and rabbit kidney (3 μg of protein) was measured in a 1 ml of solution containing 120 mm NaCl, 15 mm KCl, 3 mm MgSO4, 1 or 3 mm ATP, 50 μm SCH 28080, and 40 mm Tris-HCl, pH 7.4, in the presence or absence of 100 μm ouabain. After incubation for 10 or 30 min at 37 °C, the reaction was terminated by addition of the ice-cold stop solution containing 12% perchloric acid and 3.6% ammonium molybdate, and inorganic phosphate released was measured (20Yoda A. Hokin L.E. Biochem. Biophys. Res. Commun. 1970; 40: 880-886Crossref PubMed Scopus (189) Google Scholar). The Na+,K+-ATPase activity was calculated as the difference between the activities in the presence and absence of ouabain.Measurement of Phosphorylation Level of Na+,K+-ATPase—Ten μg of membrane proteins were phosphorylated in a solution comprising 2 μm [γ-32P]ATP (4 × 106 cpm), 20 mm NaCl, 0.43 mm MgCl2, and 25 mm imidazole-HCl (pH 6.0) for 20 s at 0 °C. The reaction was quenched by the solution containing 10% trichloroacetic acid, 10 mm inorganic phosphate, and 1 mm ATP. Then the samples were centrifuged at 13,000 × g for 3 min at 4 °C, and the pellet was washed with ice-cold 10% trichloroacetic acid, solubilized in a sample buffer comprising 2% SDS, 2.5% dithiothreitol, 10% glycerol, and 50 mm Tris-HCl, pH 6.8, and subjected to the 5% SDS-polyacrylamide gel electrophoresis under acidic conditions at pH 6.0 (21Teramachi S. Imagawa T. Kaya S. Taniguchi K. J. Biol. Chem. 2002; 277: 37394-37400Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 22Imagawa T. Yamamoto T. Kaya S. Sakaguchi K. Taniguchi K. J. Biol. Chem. 2005; 280: 18736-18744Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The radioactivity associated with the Na+,K+-ATPaseα-subunit separated on the gel was visualized and quantified by a digital autoradiography of the dried gel using Bio-Imaging Analyzer BAS2000 (Fuji Film).Measurement of H+,K+-ATPase Activity—H+,K+-ATPase activity of HEK293 cells (30 μg of protein) was measured in a 1-ml solution containing 15 mm KCl, 3 mm MgSO4, 1 mm ATP, 5 mm NaN3, 2 mm ouabain, and 40 mm Tris-HCl, pH 6.8, in the presence or absence of 50 μm SCH 28080, a specific inhibitor of H+,K+-ATPase. After incubation for 30 min at 37 °C, the reaction was terminated by addition of the ice-cold stop solution containing 12% perchloric acid and 3.6% ammonium molybdate, and the inorganic phosphate released was measured (20Yoda A. Hokin L.E. Biochem. Biophys. Res. Commun. 1970; 40: 880-886Crossref PubMed Scopus (189) Google Scholar).Measurement of 22Na+ Transport Activity—Na+ transport activity of LLC-PK1 cells was estimated by measuring intracellular 22Na+ content (23Kometiani P. Liu L. Askari A. Mol. Pharmacol. 2005; 67: 929-936Crossref PubMed Scopus (191) Google Scholar, 24Liang M. Tian J. Liu L. Pierre S. Liu J. Shapiro J. Xie Z.-J. J. Biol. Chem. 2007; 282: 10585-10593Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). In each well of 12-well culture plates, 1 × 105 cells equipped with the tetracycline-regulated expression system of KCC3a were seeded and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum for 2 days, followed by treatment with or without 2 μg/ml tetracycline for 24 h. The treated cells were incubated at 37 °C in the culture medium containing trace amounts of 22Na+ (0.5 μCi/ml) for 120 min to fully equilibrate exchangeable intracellular Na+ with 22Na+. The 22Na+-loaded cells were incubated at 37 °C for additional 30 min in the presence and absence of 10 μm ouabain. The cells were then washed three times with ice-cold solution of 100 mm MgCl2 and dissolved in 1 ml of the solution containing 1% SDS. 22Na+ contents in the samples were measured in a γ-counter (Aloka Accu-FLEXγ7001, Aloka Co., Tokyo, Japan). The Na+ transport activity of Na+,K+-ATPase was estimated as the difference between the intracellular 22Na+ contents in the presence and absence of ouabain.Statistics—The results are shown as the means ± S.E. Differences between groups were analyzed by one-way analysis of variance, and correction for multiple comparisons was made by using Dunnett's multiple comparison test. Comparison between the two groups was made by using Student's t test. Statistically significant differences were assumed at p < 0.05.RESULTSExpression of KCC3a in Gastric Parietal Cells—Northern blotting with the KCC3 cDNA probe gave a strong band of ∼8 kb and faint band of ∼7 kb in the preparation of rabbit gastric parietal cells (Fig. 1A). The transcript sizes of KCC3a and KCC3b were reported to be around 8 and 7 kb, respectively (25Mercado A. Vázquez N. Song L. Cortés R. Enck A.H. Welch R. Delpire E. Gamba G. Mount D.B. Am. J. Physiol. 2005; 289: F1246-F1261Crossref PubMed Scopus (72) Google Scholar). Western blotting with the anti-KCC3 antibody showed a single band of ∼180 kDa in the samples from rats, rabbits, and humans (Fig. 1B, upper panel). The specificity of the antibody for the 180-kDa band was confirmed by using the corresponding blocking peptide (Fig. 1B, lower panel). KCC3b protein was reported to be expressed abundantly in kidney (5Pearson M.M. Lu J. Mount D.B. Delpire E. Neuroscience. 2001; 103: 481-491Crossref PubMed Scopus (118) Google Scholar); thus we compared the band size of KCC3 expressed in the gastric mucosa with that expressed in the kidney of rats (Fig. 1C). The band size of the gastric mucosa (180 kDa) was apparently greater than that in the kidney (150 kDa). These results suggest that KCC3a mRNA and protein are predominantly expressed in gastric mucosa.In the immunohistochemistry of isolated rat gastric mucosa, the distribution pattern of KCC3a (Fig. 2, A, C, D, and F) was apparently different from that of H+,K+-ATPase (Fig. 2, B, C, E, and F). KCC3a was colocalized with Na+,K+-ATPase α1-subunit (α1NaK) (Fig. 2, J–L), suggesting that it is present in the basolateral membrane of gastric parietal cells. The specificity of anti-KCC3 antibody for positive staining was confirmed by using the blocking peptide (Fig. 2, G–I). AQP4 was abundantly expressed in the basal parietal cells (Fig. 2, M and N) as previously reported (26Carmosino M. Procino G. Nicchia G.P. Mannucci R. Verbavatz J.-M. Gobin R. Svelto M. Valenti G. J. Cell Biol. 2001; 154: 1235-1243Crossref PubMed Scopus (39) Google Scholar).FIGURE 2Immunostaining for KCC3a in isolated rat gastric mucosa. A–C show the same tissue under a microscope (similarly, D–F, G–I, and J–L). Double immunostaining was performed with isolated rat gastric mucosa by using anti-KCC3 plus anti-H+,K+-ATPase antibodies (A–I), anti-KCC3 plus anti-Na+,K+-ATPase antibodies (J–L), anti-KCC3 plus anti-AQP4 antibodies (M), and anti-H+,K+-ATPase plus anti-AQP4 antibodies (N). A–F, localizations of KCC3a (A and D), H+,K+-ATPase (B and E), and KCC3a plus H+,K+-ATPase (merged image; C and F) are shown. G–I, anti-KCC3 antibody was pretreated with the blocking peptide. Localizations of KCC3a (G), H+,K+-ATPase (H), and KCC3a plus H+,K+-ATPase (merged image; I) are shown. Positive KCC3a staining disappeared (G). J–L, localizations of KCC3a (J), Na+,K+-ATPase (K), and KCC3a plus Na+,K+-ATPase (merged image; L). M, localizations of KCC3a (green) plus AQP4 (red). N, localizations of H+,K+-ATPase (green) plus AQP4 (red). Original magnification: ×20 (A–C, M, and N) and ×40 (D–L). Scale bars, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Previously, younger parietal cells in the luminal region of the glands have been reported to secrete acid more actively than do older parietal cells in the basal region (27Bamberg K. Nylander S. Helander K.G. Lundberg L.G. Sachs G. Helander H.F. Biochim. Biophys. Acta. 1994; 1190: 355-359Crossref PubMed Scopus (22) Google Scholar, 28Karam S.M. Yao X. Forte J.G. Am. J. Physiol. 1997; 272: G161-G171PubMed Google Scholar, 29Sachs G. Eur. J. Gastroenterol. Hepatol. 2001; 13: S35-S41PubMed Google Scholar). Interestingly, the present double immunostaining of KCC3a and AQP4 in rat gastric mucosa has shown clearly that KCC3a is predominantly expressed in the luminal parietal cells (Fig. 2M).Association of KCC3a with α1NaK in the Gastric Mucosa—To study whether KCC3a is associated with α1NaK in rat gastric mucosa, immunoprecipitation was performed by using an anti-KCC3 antibody. The subsequent Western blotting of the immune pellets with an anti-α1NaK antibody gave a clear band for α1NaK (100 kDa), whereas that with an anti-H+,K+-ATPase antibody gave no band for H+,K+-ATPase (95 kDa) (Fig. 3A). These results suggest that KCC3a is associated with α1NaK in gastric parietal cells.FIGURE 3Association of KCC3a and α1NaK in rat gastric mucosa. A, immunoprecipitation (IP) was performed with the detergent extracts of rat gastric mucosa by using anti-KCC3 antibody and protein A-agarose. The detergent extracts (input; 1/25 (for KCC3) or 1/200 (for α1NaK and αHK) of total protein) and immunoprecipitation samples obtained with and without the antibody (1/20 of immunoprecipitation samples) were detected by Western blotting (WB) using antibodies for KCC3 (top panel), Na+,K+-ATPase α1-subunit (α1NaK; middle panel) and H+,K+-ATPase α-subunit (αHK; bottom panel). B, lipid raft fractions (DRM) and nonraft fractions (non-DRM) were isolated from membrane fractions of rat gastric mucosa by sucrose gradient (5–40%) as described under “Experimental Procedures.” Western blotting was performed by using antibodies for KCC3, α1NaK, and flotillin-2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Distribution of KCC3a and α1NaK in Lipid Rafts in Rat Gastric Mucosa—Lipid rafts are known to be insoluble for treatment with nonionic detergents such as Triton X-100 and CHAPS at low temperature and form detergent resistance membrane (DRM) fractions with low density (9Simons K. Vaz W.L.C. Annu. Rev. Biophys. Biomol. Struct. 2004; 33: 269-295Crossref PubMed Scopus (1343) Google Scholar). In the present study, lipid rafts were isolated from rat gastric mucosa by using CHAPS and sucrose gradient. Flotillin-2 was used as a marker for lipid rafts (30Bickel P.E. Scherer P.E. Schnitzer J.E. Oh P. Lisanti M.P. Lodish H.F. J. Biol. Chem. 1997; 272: 13793-13802Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar, 31Volonté D. Galbiati F. Li S. Nishiyama K. Okamoto T. Lisanti M.P. J. Biol. Chem. 1999; 274: 12702-12709Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). As shown in Fig. 3B, most of KCC3a and α1NaK were distributed in the DRM fractions, in which flotillin-2 was also expressed (Fig. 3B).Tetracycline-regulated Stable Expression of KCC3a in LLC-PK1 Cells—Next, we constructed the tetracycline-regulated stable expression system of KCC3a in LLC-PK1 cells, a hog kidney proximal tubular cell line (Fig. 4). No significant expression of endogenous KCC3a was observed in control LLC-PK1 cells (data not shown). In this heterologous expression system, exogenous expression of KCC3a protein was assessed by using an anti-Xpress antibody. Expression of KCC3a (180 kDa) was observed in the cells treated with tetracycline (Tet-on cells), whereas no significant expression of KCC3a was observed in the cells treated without tetracycline (Tet-off cells) (Fig. 4A). Both KCC3a and α1NaK were found to be present in the basolateral membrane of the Tet-on cells (Fig. 4, F and G). The expression pattern of α1NaK in the Tet-on cells was similar to that in the Tet-off cells (Fig. 4, D and E).FIGURE 4Tetracycline-regulated expression system of KCC3a in LLC-PK1 cells. The tetracycline-regulated expression system of KCC3a was introduced to LLC-PK1 cells. The cells were treated with (Tet-on) or without (Tet-off) 2 μg/ml tetracycline. The expression of KCC3a was confirmed by Western blotting (A) and immunocytochemistry (B and C) using anti-Xpress antibody. In the cells, α1NaK was localized in the basolateral membrane (D, E, and G). In the Tet-on cells, KCC3a was localized in the basolateral membrane (F). Scale bars, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Distribution of KCC3a and α1NaK in Lipid Rafts in the Heterologous Expression System—Lipid rafts were isolated from the Tet-on and Tet-off cells (Fig. 5). In the Tet-off cells, 33.0 ± 4.1% of total α1NaK was in DRM fractions in which flotillin-2 was localized, and 67.0 ± 4.1% of total α1NaK was in non-DRM fractions (Fig. 5, A and C). In the Tet-on cells, KCC3a was predominantly distributed in the DRM fractions, and an increased amount of α1NaK (76.9 ± 2.1%) was localized in the DRM fractions (Fig. 5, B and C). These findings suggest that the presence of KCC3a induces a significant recruitment of α1NaK from non-DRM fractions into the DRM fractions. Furthermore, depletion of cholesterol with MβCD (10 mm) shifted the distributions of both KCC3a and α1NaK from the DRM fractions to the non-DRM fractions (Fig. 5, B and D).FIGURE 5Lipid raft analysis of the tetracycline-regulated expression system of KCC3a. A and B, lipid raft fractions (DRM) and nonraft fractions (non-DRM) were isolated from membrane fractions of the LLC-PK" @default.
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• W1998271882 title "K+-Cl- Cotransporter-3a Up-regulates Na+,K+-ATPase in Lipid Rafts of Gastric Luminal Parietal Cells" @default.
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