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• W2070513189 abstract "The heteromeric amino acid transporters b0,+AT-rBAT (apical), y+LAT1-4F2hc, and possibly LAT2-4F2hc (basolateral) participate to the (re)absorption of cationic and neutral amino acids in the small intestine and kidney proximal tubule. We show now by immunofluorescence that their expression levels follow the same axial gradient along the kidney proximal tubule (S1>S2≫S3). We reconstituted their co-expression in MDCK cell epithelia and verified their polarized localization by immunofluorescence. Expression of b0,+AT-rBAT alone led to a net reabsorption of l-Arg (given together withl-Leu). Coexpression of basolateral y+LAT1-4F2hc increased l-Arg reabsorption and reversed l-Leu transport from (re)absorption to secretion. Similarly, l-cystine was (re)absorbed when b0,+AT-rBAT was expressed alone. This net transport was further increased by the coexpression of 4F2hc, due to the mobilization of LAT2 (exogenous and/or endogenous) to the basolateral membrane. In summary, apical b0,+AT-rBAT cooperates with y+LAT1-4F2hc or LAT2-4F2hc for the transepithelial reabsorption of cationic amino acids and cystine, respectively. The fact that the reabsorption of l-Arg led to the secretion of l-Leu demonstrates that the implicated heteromeric amino acid transporters function in epithelia as exchangers coupled in series and supports the notion that the parallel activity of unidirectional neutral amino acid transporters is required to drive net amino acid reabsorption. The heteromeric amino acid transporters b0,+AT-rBAT (apical), y+LAT1-4F2hc, and possibly LAT2-4F2hc (basolateral) participate to the (re)absorption of cationic and neutral amino acids in the small intestine and kidney proximal tubule. We show now by immunofluorescence that their expression levels follow the same axial gradient along the kidney proximal tubule (S1>S2≫S3). We reconstituted their co-expression in MDCK cell epithelia and verified their polarized localization by immunofluorescence. Expression of b0,+AT-rBAT alone led to a net reabsorption of l-Arg (given together withl-Leu). Coexpression of basolateral y+LAT1-4F2hc increased l-Arg reabsorption and reversed l-Leu transport from (re)absorption to secretion. Similarly, l-cystine was (re)absorbed when b0,+AT-rBAT was expressed alone. This net transport was further increased by the coexpression of 4F2hc, due to the mobilization of LAT2 (exogenous and/or endogenous) to the basolateral membrane. In summary, apical b0,+AT-rBAT cooperates with y+LAT1-4F2hc or LAT2-4F2hc for the transepithelial reabsorption of cationic amino acids and cystine, respectively. The fact that the reabsorption of l-Arg led to the secretion of l-Leu demonstrates that the implicated heteromeric amino acid transporters function in epithelia as exchangers coupled in series and supports the notion that the parallel activity of unidirectional neutral amino acid transporters is required to drive net amino acid reabsorption. +AT, broad specificity, Na+-independent neutral and cationic amino acid transporter Madin-Darby canine kidney cells related to b0,+ amino acid transport Free amino acids need to be transported from the lumen of the intestine as well as from the urinary filtrate of kidney tubules into the extracellular space. This transcellular (re)absorption involves the passage across both the luminal and the basolateral membrane of epithelial cells. Most neutral amino acids are taken up through the luminal membrane of these cells by a Na+-dependent electrogenic transport system that was named B0 or B for kidney and intestine, respectively, and that is not molecularly defined as yet (1Palacin M. Estevez R. Bertran J. Zorzano A. Physiol. Rev. 1998; 78: 969-1054Crossref PubMed Scopus (715) Google Scholar, 2Verrey F. Meier C. Rossier G. Kuhn L. Pflugers Arch. 2000; 440: 503-512Crossref PubMed Google Scholar). The import of cationic amino acids and of l-cystine depends on the expression of a heteromeric transporter that is composed of a multitransmembrane span catalytic subunit (glycoprotein-associated amino acid transporter, light chain) named b0,+AT1 (broad specificity, Na+-independent neutral and cationic amino acid transporter) and the covalently associated type II glycoprotein (heavy chain) rBAT (related to b0,+ amino acid transport) (3Chairoungdua A. Segawa H. Kim J.Y. Miyamoto K. Haga H. Fukui Y. Mizoguchi K. Ito H. Takeda E. Endou H. Kanai Y. J. Biol. Chem. 1999; 274: 28845-28848Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 4Feliubadalo L. Font M. Purroy J. Rousaud F. Estivill X. Nunes V. Golomb E. Centola M. Aksentijevich I. Kreiss Y. Goldman B. Pras M. Kastner D.L. Pras E. Gasparini P. Bisceglia L. Beccia E. Gallucci M. de Sanctis L. Ponzone A. Rizzoni G.F. Zelante L. Bassi M.T. George A.L. Manzoni M. Palacin M. et al.Nat. Genet. 1999; 23: 52-57Crossref PubMed Scopus (297) Google Scholar, 5Mizoguchi K. Cha S.H. Chairoungdua A. Kim D. Shigeta Y. Matsuo H. Fukushima J. Awa Y. Akakura K. Goya T. Ito H. Endou H. Kanai Y. Kidney Int. 2001; 59: 1821-1833Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 6Pfeiffer R. Loffing J. Rossier G. Bauch C. Meier C. Eggermann T. Loffing-Cueni D. Kuhn L. Verrey F. Mol. Biol. Cell. 1999; 10: 4135-4147Crossref PubMed Scopus (112) Google Scholar). Genetic studies have shown that defects in the genes encoding either subunit lead to cystinuria (4Feliubadalo L. Font M. Purroy J. Rousaud F. Estivill X. Nunes V. Golomb E. Centola M. Aksentijevich I. Kreiss Y. Goldman B. Pras M. Kastner D.L. Pras E. Gasparini P. Bisceglia L. Beccia E. Gallucci M. de Sanctis L. Ponzone A. Rizzoni G.F. Zelante L. Bassi M.T. George A.L. Manzoni M. Palacin M. et al.Nat. Genet. 1999; 23: 52-57Crossref PubMed Scopus (297) Google Scholar, 7Calonge M.J. Volpini V. Bisceglia L. Rousaud F. Desanctis L. Beccia E. Zelante L. Testar X. Zorzano A. Estivill X. Gasparini P. Nunes V. Palacin M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9667-9671Crossref PubMed Scopus (99) Google Scholar, 8Font M. Feliubadalo L. Estivill X. Nunes V. Golomb E. Kreiss Y. Pras E. Bisceglia L. d'Adamo A.P. Zelante L. Gasparini P. Bassi M.T. George Jr A.L. Manzoni M. Riboni M. Ballabio A. Borsani G. Reig N. Fernandez E. Zorzano A. Bertran J. Palacin M. Hum. Mol. Genet. 2001; 10: 305-316Crossref PubMed Scopus (115) Google Scholar). The function of this transporter has been characterized in Xenopus oocytes first by investigating the function of exogenous rBAT associated with an endogenous b0,+AT and then, upon identification of the mammalian b0,+AT subunit, by measuring the function of the mammalian heterodimer expressed as fusion protein in Xenopusoocytes or in COS-7 cells (3Chairoungdua A. Segawa H. Kim J.Y. Miyamoto K. Haga H. Fukui Y. Mizoguchi K. Ito H. Takeda E. Endou H. Kanai Y. J. Biol. Chem. 1999; 274: 28845-28848Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 5Mizoguchi K. Cha S.H. Chairoungdua A. Kim D. Shigeta Y. Matsuo H. Fukushima J. Awa Y. Akakura K. Goya T. Ito H. Endou H. Kanai Y. Kidney Int. 2001; 59: 1821-1833Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 6Pfeiffer R. Loffing J. Rossier G. Bauch C. Meier C. Eggermann T. Loffing-Cueni D. Kuhn L. Verrey F. Mol. Biol. Cell. 1999; 10: 4135-4147Crossref PubMed Scopus (112) Google Scholar, 9Busch A.E. Herzer T. Waldegger S. Schmidt F. Palacin M. Biber J. Markovich D. Murer H. Lang F. J. Biol. Chem. 1994; 269: 25581-25586Abstract Full Text PDF PubMed Google Scholar, 10Coady M.J. Jalal F. Chen X.Z. Lemay G. Berteloot A. Lapointe J.Y. FEBS Lett. 1994; 356: 174-178Crossref PubMed Scopus (47) Google Scholar, 11Chillaron J. Estevez R. Mora C. Wagner C.A. Suessbrich H. Lang F. Gelpi J.L. Testar X. Busch A. Zorzano A. Palacin M. J. Biol. Chem. 1996; 271: 17761-17770Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). These studies indicated that b0,+AT-rBAT functions as an obligatory exchanger and suggested that its major mode of transport is, at a normal membrane potential, the uptake of extracellular cationic amino acids orl-cystine that are exchanged against intracellular neutral amino acids. These neutral amino acids could in turn be recycled into the cell by system B/B0. Recently, we have characterized the biosynthesis, localization and uptake function of b0,+AT-rBAT expressed in MDCK cells (12Bauch C. Verrey F. Am. J. Physiol. Renal Physiol. 2002; 283: F181-F189Crossref PubMed Scopus (51) Google Scholar). This study has shown in an epithelial context that the surface expression of b0,+AT and rBAT depends on their association, which is necessary for the maturation and stabilization of rBAT. This study has also confirmed that b0,+AT-rBAT displays extracellularly a high apparent affinity for l-cystine > cationic amino acids (l-Arg) > large neutral amino acids (l-Leu). The basolateral efflux of amino acids is as yet less well understood than the apical influx. The two heteromeric amino acid transporters, composed of the glycoprotein subunit (heavy chain) 4F2hc(CD98) and an associated catalytic subunit, y+LAT1 or LAT2, that are highly expressed in the proximal kidney tubule and the small intestine, were shown in Xenopus oocytes to function as obligatory exchangers (13Pfeiffer R. Rossier G. Spindler B. Meier C. Kuhn L. Verrey F. EMBO J. 1999; 18: 49-57Crossref PubMed Scopus (239) Google Scholar, 14Kanai Y. Fukasawa Y. Cha S.H. Segawa H. Chairoungdua A. Kim D.K. Matsuo H. Kim J.Y. Miyamoto K. Takeda E. Endou H. J. Biol. Chem. 2000; 275: 20787-20793Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 15Meier C. Ristic Z. Klauser S. Verrey F. EMBO J. 2002; 21: 580-589Crossref PubMed Scopus (257) Google Scholar, 16Pineda M. Fernandez E. Torrents D. Estevez R. Lopez C. Camps M. Lloberas J. Zorzano A. Palacin M. J. Biol. Chem. 1999; 274: 19738-19744Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, 17Segawa H. Fukasawa Y. Miyamoto K. Takeda E. Endou H. Kanai Y. J. Biol. Chem. 1999; 274: 19745-19751Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar). The physiological function of y+LAT1-4F2hc appears to be the electroneutral efflux of intracellular cationic amino acids that are exchanged for extracellular large neutral amino acids together with Na+. This mode of transport is based on the results of expression experiments made in Xenopus oocytes and is compatible with the phenotype of patients carrying the genetic disease lysinuric protein intolerance that was shown to be due to a defect in the corresponding gene (13Pfeiffer R. Rossier G. Spindler B. Meier C. Kuhn L. Verrey F. EMBO J. 1999; 18: 49-57Crossref PubMed Scopus (239) Google Scholar, 14Kanai Y. Fukasawa Y. Cha S.H. Segawa H. Chairoungdua A. Kim D.K. Matsuo H. Kim J.Y. Miyamoto K. Takeda E. Endou H. J. Biol. Chem. 2000; 275: 20787-20793Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 18Borsani G. Bassi M.T. Sperandeo M.P., De Grandi A. Buoninconti A. Riboni M. Manzoni M. Incerti B. Pepe A. Andria G. Ballabio A. Sebastio G. Nat. Genet. 1999; 21: 297-301Crossref PubMed Scopus (193) Google Scholar, 19Torrents D. Mykkanen J. Pineda M. Feliubadalo L. Estevez R. de Cid R. Sanjurjo P. Zorzano A. Nunes V. Huoponen K. Reinikainen A. Simell O. Savontaus M.L. Aula P. Palacin M. Nat. Genet. 1999; 21: 293-296Crossref PubMed Scopus (240) Google Scholar). As this system needs to function in the context of the (re)absorption of all amino acids, we postulate that an additional export system that recycles the imported neutral amino acids needs to be expressed in the basolateral membrane. The function of the second heteromeric exchanger, LAT2-4F2hc, has been extensively studied inXenopus oocytes (15Meier C. Ristic Z. Klauser S. Verrey F. EMBO J. 2002; 21: 580-589Crossref PubMed Scopus (257) Google Scholar, 16Pineda M. Fernandez E. Torrents D. Estevez R. Lopez C. Camps M. Lloberas J. Zorzano A. Palacin M. J. Biol. Chem. 1999; 274: 19738-19744Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, 17Segawa H. Fukasawa Y. Miyamoto K. Takeda E. Endou H. Kanai Y. J. Biol. Chem. 1999; 274: 19745-19751Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar, 20Rossier G. Meier C. Bauch C. Summa V. Sordat B. Verrey F. Kuhn L.C. J. Biol. Chem. 1999; 274: 34948-34954Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). This transporter preferentially exchanges middle-sized and large neutral amino acids across the membrane. Importantly, its apparent affinities for various amino acids is much lower inside compared with outside of the cells (with an exception for Gly), suggesting that its activity depends on the intracellular amino acid availability (15Meier C. Ristic Z. Klauser S. Verrey F. EMBO J. 2002; 21: 580-589Crossref PubMed Scopus (257) Google Scholar). It was suggested that its physiological role is to equilibrate the relative concentration of the various intracellular neutral amino acids, in particular by transporting some amino acids out of the cell that would not be substrates of the putative unidirectional efflux pathway (20Rossier G. Meier C. Bauch C. Summa V. Sordat B. Verrey F. Kuhn L.C. J. Biol. Chem. 1999; 274: 34948-34954Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). Because LAT2-4F2hc efficiently exchanges intracellularl-Cys against other extracellular neutral amino acids, this transporter was proposed to play a major role in the basolateral efflux of this amino acid, a part of which is produced intracellularly by the reduction of l-cystine (16Pineda M. Fernandez E. Torrents D. Estevez R. Lopez C. Camps M. Lloberas J. Zorzano A. Palacin M. J. Biol. Chem. 1999; 274: 19738-19744Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, 17Segawa H. Fukasawa Y. Miyamoto K. Takeda E. Endou H. Kanai Y. J. Biol. Chem. 1999; 274: 19745-19751Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar, 20Rossier G. Meier C. Bauch C. Summa V. Sordat B. Verrey F. Kuhn L.C. J. Biol. Chem. 1999; 274: 34948-34954Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). To investigate the function of the basolateral exchangers y+LAT1-4F2hc and LAT2-4F2hc, alone, together and in conjunction with the apical exchanger b0,+AT-rBAT in the context of transepithelial transport, we expressed them in MDCK cells, a recipient cell line that forms a tight epithelium and does not express a high amount of endogenous epithelial amino acid transporters itself. MDCK cells (strain II) were cultured at 37 °C and 5% CO2 in Dulbecco's modified Eagle's medium (cat. 41965, Invitrogen, Basel, Switzerland) with 5 × 104 units/liter penicillin, 50 mg/liter streptomycin, 2 mm l-glutamine, 1% non-essential amino acids (cat. 11140-035), Invitrogen) and 10% fetal calf serum. Phoenix amphotropic retrovirus producer cells, kindly provided by Dr. G. Nolan (Baxter Laboratory for Genetic Pharmacology, Dept. of Microbiology and Immunology, Dept. of Molecular Pharmacology, Stanford University), were cultured at 37 °C and 5% CO2 in Dulbecco's modified Eagle's medium (cat. 41966, Invitrogen, Basel, Switzerland) with 1 × 103units/liter penicillin, 100 mg/liter streptomycin, 2 mm l-glutamine, 1% non-essential amino acids (cat. 11140-035, Invitrogen) and 10% fetal calf serum. The MDCK cells transfected with hrBAT and mb0,+AT were previously described (12Bauch C. Verrey F. Am. J. Physiol. Renal Physiol. 2002; 283: F181-F189Crossref PubMed Scopus (51) Google Scholar). The h4F2hc, mLAT2 and my+LAT1 cDNA (13Pfeiffer R. Rossier G. Spindler B. Meier C. Kuhn L. Verrey F. EMBO J. 1999; 18: 49-57Crossref PubMed Scopus (239) Google Scholar, 20Rossier G. Meier C. Bauch C. Summa V. Sordat B. Verrey F. Kuhn L.C. J. Biol. Chem. 1999; 274: 34948-34954Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar) coding sequences were subcloned in the vector LZRSpBMN-Z (kindly provided by Dr. G. Nolan) in place of the lacZ sequence. Production of supernatants containing the pseudoviruses and subsequent transduction of MDCK target cells was performed according to protocols provided by Dr. G. Nolan (www.stanford.edu/group/nolan/protocols/pro_helper_free.html), which were adapted from Ref. 21Pear W. Scott M. Nolan G.P. Robbins P. Methods in Molecular Medicine: Gene Therapy Protocols. Humana Press, Totowa, NJ1997: 41-57Google Scholar. Briefly, Phoenix amphotropic retrovirus producer cells were transfected with the abovementioned constructs using the FuGENE 6 transfection reagent (Roche Molecular Biochemicals, Basel, Switzerland) according to the manufacturer's protocol. 16 h after transfection, the medium was exchanged, and after another 48 h at 32 °C, the supernatant was harvested and filtered. MDCK target cells were plated at 5–10% confluency, and viral supernatants containing 4 μg/ml polybrene (Sigma) were added 24 h later. The freshly transduced cells were centrifuged for 30 min at 1000 rpm and 32 °C immediately thereafter. After 24 h at 32 °C the medium was exchanged, and cells were further cultivated in normal medium at 37 °C. This procedure was repeated 8–10 times to achieve a maximal expression of the transport proteins to be investigated. Polyclonal rabbit antibodies were raised against synthetic peptides corresponding to the NH2 terminus of human rBAT, MAEDKSKRDSIEMSMKGC, the COOH terminus of mouse b0,+AT, CHLQMLEVVPEKDPE, the NH2 terminus of mouse y+LAT1, QHEADDGSALGDGASPC, the COOH terminus of mouse y+LAT1, CDLEDGELSKQDPKSK, and the COOH terminus of mouse LAT2, CPIFKPTPVKDPDSEEQP, coupled to keyhole limpet hemocyanin (Eurogentech, Seraing, Belgium). The monoclonal anti-human 4F2hc antibody used was previously described in Ref. 22Haynes B.F. Hemler M.E. Mann D.L. Eisenbarth G.S. Shelhamer J. Mostowski H.S. Thomas C.A. Strominger J.L. Fauci A.S. J. Immunol. 1981; 126: 1409-1414PubMed Google Scholar. Cells were seeded on filters (24-mm Corning Costar Transwell filters, cat. Nr. 3412) at 100% confluence and cultivated for 7 days preceding experiments. rBAT expression was induced 3 days prior to experiment with 1 μm dexamethasone. Filters were washed three times using phosphate-buffered saline. Cells were fixed using 3% paraformaldehyde and 0.2% Triton X-100 for 15 min at room temperature. Filters were washed three times and cut into squares. 4F2hc-rBAT, b0,+AT-4F2hc, y+LAT1-4F2hc, and LAT2-4F2hc double immunofluorescence was performed with a mix of the respective antibodies 4F2hc (1:1000), rBAT (SZ564; 1:50), b0,+AT (SZ557; 1:500), y+LAT1 (SZ398; 1:200), LAT2 (SZ560; 1:200) in phosphate-buffered saline containing 0.5% bovine serum albumin overnight at 4 °C. After washing, filter pieces were incubated for 6 h at room temperature with fluorescein isothiocyanate-labeled anti-rabbit-IgG antibody (Sigma) and CY3-labeled anti-mouse IgG antibody (Sigma). After another round of washing, the filters were mounted in DAKO-glycergel (DAKO, Glostrup, Denmark) containing 2.5% 1,4-diazabicyclo (2Verrey F. Meier C. Rossier G. Kuhn L. Pflugers Arch. 2000; 440: 503-512Crossref PubMed Google Scholar, 2Verrey F. Meier C. Rossier G. Kuhn L. Pflugers Arch. 2000; 440: 503-512Crossref PubMed Google Scholar, 2Verrey F. Meier C. Rossier G. Kuhn L. Pflugers Arch. 2000; 440: 503-512Crossref PubMed Google Scholar) octane (DABCO) as fading retardant. Confocal images were taken using a Leica laser scan microscope (TCSSP, Wetzlar, Germany) equipped with a ×63 oil immersion objective. The appropriate controls were performed without the first and/or second primary antibodies. Kidneys of anesthetized male mice (NMRI; RCC, Füllinsdorf, Switzerland) were fixed for 5 min by intravascular perfusion through the abdominal aorta as previously described (23Loffing J. Loffing-Cueni D. Valderrabano V. Klausli L. Hebert S.C. Rossier B.C. Hoenderop J.G. Bindels R.J. Kaissling B. Am. J. Physiol. Renal Physiol. 2001; 281: F1021-F1027Crossref PubMed Scopus (296) Google Scholar). Coronal slices (1–2-mm thick) of the kidney were frozen in liquid propane and stored at −80 °C until use. Serial cryosection (4–5 μm) were cut and placed on Chromium(III) potassium sulfate-coated glass slides. Sections were preincubated for 10 min with 10% normal goat serum in phosphate-buffered saline, 2% bovine serum albumin. Afterward, sections were sequentially incubated with primary antibodies against LAT2 (SZ559) 1:1000, y+LAT1 (SZ553) 1:200–1:500, and bo,+AT (SZ400) 1:500 for 16 h at 4 °C. Binding sites of primary antibodies were revealed with Cy3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). Sections were studied by epifluorescence with a Polyvar microscope (Reichert Jung, Vienna, Austria). For controls, consecutive cryosections were incubated in preimmune serum. Images were acquired with a charge-coupled device camera (Visicam 1280, Visitron System, Puching, Germany) and processed by Image-Pro Plus version 3.0 (Media Cybernetics, Silver Spring, MD) and Corel Photoshop software. MDCK cells were passaged to 24-mm Corning Costar Transwell filters at 100% confluence and cultivated for 7 days. rBAT expression was induced 24 h prior to experiment with 1 μm dexamethasone. Integrity of the monolayer was checked by resistance measurement using the Millicell device (Millipore, Bedford, MA). Filters were washed three times with uptake buffer (150 mm NaCl, 10 mm HEPES pH 7.4, 1 mm CaCl2, 5 mm KCl, 1 mm MgCl2, 10 mm glucose) at 37 °C and incubated in uptake buffer for 30 min. The buffer was replaced unilaterally with buffer supplemented with amino acid at the indicated concentrations and the corresponding 3H-labeledl-amino acid as tracer (except forl-[14C]cystine); the contralateral compartment received the same solution without the labeledl-amino acid tracer. Uptake experiments were performed for the times indicated. DTNB (5,5′-dithiobis(2-nitrobenzoic acid)) (100 μm) (Sigma) was added to the solution for experiments with l-cystine. The uptake was stopped by replacing the amino acid uptake solution with ice-cold uptake buffer and washed four times. The filters were excised and placed into scintillation vials containing scintillation fluid (Packard, Meriden, CT). After shaking overnight at room temperature, radioactivity was determined by scintillation counting. RT-PCR was performed to identify the dog LAT2 transporter. First-strand cDNA was synthesized from 100 ng of total RNA from wild-type MDCK cells, LAT2-transfected cells serving as the internal positive control, with or without MMRV reverse transcriptase (Promega, Madison, WI) and 50 pmol of random hexamer primers (Invitrogen). 1:10 of the first-strand cDNA was used as a template for PCR amplification using 50 pmol of degenerate LAT2 primers (forward primer 5′-GGTCAGYGCCTGTGGTATCA-3′, reverse primer 5′-GCAGCACRTAGTTGGAGAAG-3′ (Microsynth, Balgach, Switzerland)) and 2 units of recombinant Taq polymerase (Promega, Madison, WI). The cycling parameters were the following: 3 min 94 °C, 35 cycles of 1 min 94 °C, 30 s 57 °C, and 1 min 72 °C, followed by 10 min at 72 °C. PCR products were separated on agarose gel, the band cut out and extracted with QIAquick Gel Extraction kit (Qiagen, Hilden, Germany). DNA sequencing was performed at Microsynth (Balgach, Switzerland) using the above PCR primers. Data are expressed as means ± S.E. The difference between control and test values was evaluated using analysis of variance (one-way) with Bonferroni's multiple comparison test. The fact that b0,+AT, LAT2 and y+LAT1 are colocalized in the proximal tubule of the kidney in vivo is shown in Fig. 1. As yet, the in vivolocalization of y+LAT1 had not been published. Interestingly, the present images of serial kidney sections indicate that the localizations of b0,+AT, LAT2, and y+LAT1 along the proximal tubule are superimposable, all showing the same axial gradient along the proximal tubule segments (S1>S2≫S3). Thus, in view of the fact that all b0,+AT expressed in the kidney was shown to be associated with rBAT (24Fernandez E. Carrascal M. Rousaud F. Abian J. Zorzano A. Palacin M. Chillaron J. Am. J. Physiol. Renal Physiol. 2002; 283: F540-F548Crossref PubMed Scopus (85) Google Scholar), the three heteromeric transporter b0,+AT-rBAT, LAT2-4F2hc, and y+LAT1-4F2hccolocalize along the kidney proximal tubule with a very similar axial gradient. Only for y+LAT1, the staining along the S1 and S2 segments appears to terminate earlier. Interestingly, some LAT2 staining is visible along the distal tubules as well. To perform a functional analysis of coexpressed heteromeric amino acid transporters of the proximal tubule and intestine, the MDCK cell line that is of distal nephron origin was chosen as recipient, because it does not express a high baseline transepithelial amino acid transport activity. Nevertheless, for the interpretation of the data obtained on transfected/transduced MDCK cells, one has to keep in mind that they do express some endogenous transporters. For instance, experiments performed in the eighties suggested the presence of endogenous system A and L (mostly basolateral), ASC (basolateral and apical), and of an apical Na+/amino acid symporter with a broad substrate selectivity (25Boerner P. Evans-Laying M., U, H.S. Saier M.H. J. Biol. Chem. 1986; 261: 13957-13962Abstract Full Text PDF PubMed Google Scholar). The MDCK cell line already expressing the apical heteromeric transporter b0,+AT-rBAT as well as untransfected MDCK cells were sequentially transduced with amphotropic pseudoretrovirus encoding the subunits of the heteromeric transporters y+LAT1-4F2hc and/or LAT2-4F2hc. The steady-state localization of these gene products was then analyzed by immunofluorescence confocal microscopy. Fig. 2, A and B shows that 4F2hc, when expressed with LAT2 in b0,+AT-rBAT-expressing cells, localizes mainly to the lateral membrane. Only little intracellular and no apical 4F2hc staining can be seen. At the cell surface, there is also no overlap with rBAT (panel A, apical staining) or b0,+AT (panel B, intracellular and apical staining). LAT2 and 4F2hc co-localize in the basolateral membrane (Fig. 2 C), as expected from their localization in mouse kidney and small intestine (20Rossier G. Meier C. Bauch C. Summa V. Sordat B. Verrey F. Kuhn L.C. J. Biol. Chem. 1999; 274: 34948-34954Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). In this figure, made at a stage at which only ∼50% of the cells expressed both 4F2hc and LAT2, some cells exhibited LAT2 staining only, that, interestingly, was localized both intracellularly and at the lateral membrane. Because LAT2 was previously shown to necessitate association with 4F2hc for functional surface expression in Xenopus oocytes, the partial membrane localization of LAT2 in the absence of visible 4F2hc indirectly suggests the presence of some endogenous canine 4F2hc that is not recognized by the species-specific monoclonal antibody. The fact that coexpression of exogenous 4F2hc leads to an essentially basolateral immunolocalization of LAT2 confirms the hypothesis that 4F2hc is the limiting factor for LAT2 surface expression in MDCK cells. The subcellular localization of y+LAT1 has been visualized as yet only in Xenopus oocytes and in non-polarized transfected HEK293 cells (26Mykkanen J. Torrents D. Pineda M. Camps M. Yoldi M.E. Horelli-Kuitunen N. Huoponen K. Heinonen M. Oksanen J. Simell O. Savontaus M.L. Zorzano A. Palacin M. Aula P. Hum. Mol. Genet. 2000; 9: 431-438Crossref PubMed Scopus (55) Google Scholar, 27Toivonen M. Mykkanen J. Aula P. Simell O. Savontaus M.L. Huoponen K. Biochem. Biophys. Res. Commun. 2002; 291: 1173-1179Crossref PubMed Scopus (14) Google Scholar). Here we demonstrate that exogenous y+LAT1 behaves as LAT2, namely that it colocalizes with exogenous 4F2hc in the basolateral membrane of MDCK cells (Fig. 2 D) and that in the absence of exogenous 4F2hc, only a small fraction of it appears at the basolateral surface (putatively associated with endogenous 4F2hc). To test the role and the cooperation of b0,+AT-rBAT and/or y+LAT1-4F2hc on transepithelial cationic amino acid transport, we added l-Arg and l-Leu together to both sides of the epithelia, to allow amino acid exchange to proceed in the absence of a transepithelial concentration gradient.l-Arg was chosen because it is, on the one hand, a good influx substrate for the apical transporter b0,+AT-rBAT that exchanges preferentially extracellular cationic amino acids orl-cystine against intracellular neutral amino acids and, on the other hand, a good efflux substrate for the basolateral transporter y+LAT1 known to preferentially exchange intracellular cationic amino acids against extracellular neutral amino acids plus Na+ (13Pfeiffer R. Rossier G. Spindler B. Meier C. Kuhn L. Verrey F. EMBO J. 1999; 18: 49-57Crossref PubMed Scopus (239) Google Scholar, 14Kanai Y. Fukasawa Y. Cha S.H. Segawa H. Chairoungdua A. Kim D.K. Matsuo H. Kim J.Y. Miyamoto K. Takeda E. Endou H. J. Biol. Chem. 2000; 275: 20787-20793Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). l-Leu was taken as second substrate because it is a good influx substrate for y+LAT1 and a suitable efflux substrate for b0,+AT-rBAT (5Mizoguchi K. Cha S.H. Chairoungdua A. Kim D. Shigeta Y. Matsuo H. Fukushima J. Awa Y. Akakura K. Goya T. Ito H. Endou H. Kanai Y. Kidney Int. 2001; 59: 1821-1833Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 13Pfeiffer R. Rossier G. Spindler B. Meier C. Kuhn L. Verrey F. EMBO J. 1999; 18: 49-57Crossref PubMed Scopus (239) Google Scholar,14Kanai Y. Fukasawa Y. Cha S.H. Segawa H. Chairoungdua A. Kim D.K. Matsuo H. Kim J.Y. Miyamoto K. Takeda E. Endou H. J. Biol. Chem. 2000; 275: 20787-20793Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Radioactive tracer of either amino acid was added on separate filter cultures to each side of the epithelia and the amount of labeled amino acid in the cell and at the contralateral side was me" @default.
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