Peroxynitrite Stimulates l-Arginine Transport Systemy+ in Glial Cells
2002; Elsevier BV; Volume: 277; Issue: 33 Linguagem: Inglês
10.1074/jbc.m203728200
ISSN1083-351X
AutoresVictoria Vega‐Agapito, Ángeles Almeida, Maria Hatzoglou, Juan P. Bolaños,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoWe have reported previously that peroxynitrite stimulates l-arginine release from astrocytes, but the mechanism responsible for such an effect remains elusive. To explore this issue, we studied the regulation ofl-[3H]arginine transport by either exogenous or endogenous peroxynitrite in glial cells. A 2-fold peroxynitrite-mediated stimulation of l-arginine release in C6 cells was found to be Na+-independent, was prevented by 5 mm l-arginine and, although only in the presence of Na+, was blocked by 5 mm l-alanine or l-leucine. Peroxynitrite-mediated stimulation of l-arginine uptake was trans-stimulated by 10 mm l-arginine and was inhibited in a dose-dependent fashion (ki of ∼40 μm) by the system y+ inhibitorN-ethylmaleimide in C6 cells. Endogenous production of peroxynitrite in lipopolysaccharide-treated astrocytes triggered an increased l-arginine transport activity without affectingCat1l-arginine transporter mRNA levels. However, Western blot analyses of peroxynitrite-treated astrocytes and C6 glial cells revealed a 3-nitrotyrosinated anti-Cat1-immunopositive band, strongly suggesting peroxynitrite-mediated Cat1 nitration. Furthermore, peroxynitrite stimulation of l-arginine release was abolished in fibroblast cells homozygous for a targeted inactivation of the Cat1 gene. Finally, peroxynitrite-triggered l-arginine released from astrocytes was efficiently taken up by neurons in an insert-based co-culture system. These results strongly suggest that peroxynitrite-mediated activation of the Cat1 transporter in glial cells may serve as a mechanism focused to replenish l-arginine in the neighboring neurons. We have reported previously that peroxynitrite stimulates l-arginine release from astrocytes, but the mechanism responsible for such an effect remains elusive. To explore this issue, we studied the regulation ofl-[3H]arginine transport by either exogenous or endogenous peroxynitrite in glial cells. A 2-fold peroxynitrite-mediated stimulation of l-arginine release in C6 cells was found to be Na+-independent, was prevented by 5 mm l-arginine and, although only in the presence of Na+, was blocked by 5 mm l-alanine or l-leucine. Peroxynitrite-mediated stimulation of l-arginine uptake was trans-stimulated by 10 mm l-arginine and was inhibited in a dose-dependent fashion (ki of ∼40 μm) by the system y+ inhibitorN-ethylmaleimide in C6 cells. Endogenous production of peroxynitrite in lipopolysaccharide-treated astrocytes triggered an increased l-arginine transport activity without affectingCat1l-arginine transporter mRNA levels. However, Western blot analyses of peroxynitrite-treated astrocytes and C6 glial cells revealed a 3-nitrotyrosinated anti-Cat1-immunopositive band, strongly suggesting peroxynitrite-mediated Cat1 nitration. Furthermore, peroxynitrite stimulation of l-arginine release was abolished in fibroblast cells homozygous for a targeted inactivation of the Cat1 gene. Finally, peroxynitrite-triggered l-arginine released from astrocytes was efficiently taken up by neurons in an insert-based co-culture system. These results strongly suggest that peroxynitrite-mediated activation of the Cat1 transporter in glial cells may serve as a mechanism focused to replenish l-arginine in the neighboring neurons. nitric oxide nitric oxide synthase 1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine cationic amino acid transporter broad spectrum amino acid transporter Dulbecco's modified Eagle's medium fetal calf serum lipopolysaccharide phosphate-buffered saline The biosynthesis of the central nervous system physiological messenger nitric oxide (⋅NO)1 (1Palmer R.M.J. Ferrige A.G. Moncada S. Nature. 1987; 327: 524-526Crossref PubMed Scopus (9288) Google Scholar, 2Garthwaite J. Charles S.L. Chess-Williams R. Nature. 1988; 336: 385-387Crossref PubMed Scopus (2270) Google Scholar) requiresl-arginine for the ⋅NO synthase (NOS)-catalyzed reaction (3Knowles R.G. Palacios M. Palmer R.M.J. Moncada S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5159-5162Crossref PubMed Scopus (1363) Google Scholar). In neurons and astrocytes, stimulation of glutamate receptors leads to a Ca2+-dependent activation of neuronal NOS (nNOS) (2Garthwaite J. Charles S.L. Chess-Williams R. Nature. 1988; 336: 385-387Crossref PubMed Scopus (2270) Google Scholar, 4Agulló L. Garcı́a A. Biochem. Biophys. Res. Commun. 1992; 182: 1362-1368Crossref PubMed Scopus (85) Google Scholar), although ⋅NO biosynthesis in neurons depends on a correct l-arginine supply (5Westergaard N. Beart P.M. Schousboe A. J. Neurochem. 1993; 61: 364-367Crossref PubMed Scopus (86) Google Scholar, 6Schmidlin A. Wiesinger H. Glia. 1994; 11: 262-268Crossref PubMed Scopus (59) Google Scholar, 7Stevens B.R. Kakuda D.K., Yu, K. Waters M., Vo, C.B. Raizada M.K. J. Biol. Chem. 1996; 271: 24017-24022Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), possibly provided from the glia (8Grima G. Cuénod M. Pfeiffer S. Mayer B. Do K.Q. J. Neurochem. 1998; 71: 2139-2144Crossref PubMed Scopus (37) Google Scholar). Glial cells can be activated to endogenously produce ⋅NO through the induction of the inducible NOS isoform (iNOS) (9Simmons M.L. Murphy S. J. Neurochem. 1992; 59: 897-905Crossref PubMed Scopus (481) Google Scholar, 10Galea E. Feinstein D.L. Reis D.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10945-10949Crossref PubMed Scopus (466) Google Scholar, 11Galea E. Reis D.J. Feinstein D.L. J. Neurosci. Res. 1994; 37: 406-414Crossref PubMed Scopus (187) Google Scholar, 12Corradin S.B. Mauël J. Donini S.D. Quattrocchi E. Ricciardi- Castagnoli P. Glia. 1993; 7: 255-262Crossref PubMed Scopus (206) Google Scholar, 13Merrill J.E. Murphy S.P. Mitrovic B. Mackenzie-Graham A. Dopp J.C. Ding M. Griscavage J. Ignarro L.J. Lowenstein C.J. J. Neurosci. Res. 1997; 48: 372-384Crossref PubMed Scopus (82) Google Scholar). Within the brain, free l-arginine is predominantly located in astrocytes (14Aoki E. Semba R. Mikoshiba K. Kashiwamata S. Brain Res. 1991; 547: 190-192Crossref PubMed Scopus (106) Google Scholar), which produce large amounts of ⋅NO upon iNOS (15Brown G.C. Bolaños J.P. Heales S.J.R. Clark J.B. Neurosci. Lett. 1995; 193: 201-204Crossref PubMed Scopus (181) Google Scholar) and l-arginine transporter (7Stevens B.R. Kakuda D.K., Yu, K. Waters M., Vo, C.B. Raizada M.K. J. Biol. Chem. 1996; 271: 24017-24022Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) induction. However, unlike in astrocytes, l-arginine content in neurons is very low and is limiting for nNOS activity (16Garthwaite J. Garthwaite G. Palmer R.M.J. Moncada S. Eur. J. Pharmacol. 1989; 172: 413-416Crossref PubMed Scopus (782) Google Scholar, 17Culcasi M. Lafon-Cazal M. Pietri S. Bockaert J. J. Biol. Chem. 1994; 269: 12589-12593Abstract Full Text PDF PubMed Google Scholar). Consistent with this, glial-derived l-arginine has been shown to be increased upon activation of ionotropic glutamate (non-N-methyl-d- aspartate) receptors (18Grima G. Benz B. Do K.Q. Eur. J. Neurosci. 1997; 9: 2248-2258Crossref PubMed Scopus (44) Google Scholar) and peroxynitrite (19Vega-Agapito V. Almeida A. Heales S.J.R. Medina J.M. Bolaños J.P. J. Neurochem. 1999; 73: 1446-1452Crossref PubMed Scopus (20) Google Scholar), suggesting a neuronal-astrocytic signaling transduction pathway focused to provide NOS substrate for the neurons. However, direct demonstration of such a pathway and the elucidation of the precise transport system involved remain elusive. Plasma membrane l-arginine transport is brought about by two families of cationic amino acid transport proteins: Cat (cationic amino acid transporter) and Bat (broad scope amino acid transporter). The Cat family of transporters comprises three different genes encoding four isoform proteins (Cat1, Cat2, Cat2a, and Cat3) (20MacLeod C.L. Finley K.D. Kakuda D.K. J. Exp. Biol. 1994; 196: 109-121Crossref PubMed Google Scholar, 21Aulak K.S. Liu J., Wu, J. Hyatt S.L. Puppi M. Henning S.J. Hatzoglou M. J. Biol. Chem. 1996; 271: 29799-29806Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), commonly referred to as the system y+ (22Devés R. Boyd C.A.R. Physiol. Rev. 1998; 78: 487-545Crossref PubMed Scopus (445) Google Scholar), which is mostly selective for cationic amino acids, although it does show a weak interaction with neutral amino acids in the presence of Na+ (23White M.F. Gazzola G.C. Christensen H.N. J. Biol. Chem. 1982; 257: 4443-4449Abstract Full Text PDF PubMed Google Scholar). Glial cells mainly, but not exclusively, express the high affinity (kt for l-arginine = 40–250 μm) (24Malandro M.S. Kilberg M.S. Annu. Rev. Biochem. 1996; 65: 305-336Crossref PubMed Scopus (180) Google Scholar) 67-kDa Cat1 (constitutive) (25Stoll J. Wadhwani K.C. Smith Q.R. J. Neurochem. 1993; 60: 1956-1959Crossref PubMed Scopus (112) Google Scholar) and the 71.8-kDa Cat2 (inducible) (26MacLeod C.L. Finley K.D. Kakuda D.K. Kozak C. Wilkinson M. Mol. Cell. Biol. 1990; 10: 3663-3674Crossref PubMed Scopus (80) Google Scholar) system y+ proteins (7Stevens B.R. Kakuda D.K., Yu, K. Waters M., Vo, C.B. Raizada M.K. J. Biol. Chem. 1996; 271: 24017-24022Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar,27Kilberg M.S. Stevens B.R. Novak D.A. Annu. Rev. Nutr. 1993; 13: 137-165Crossref PubMed Scopus (179) Google Scholar). The Bat family of constitutive transporter proteins is found in systems bo,+, Bo,+, and y+L (y+Lat1 and y+Lat2), which are mainly expressed in kidney and intestine, except for y+Lat2, which is expressed in astrocytes (28Broër A. Carsten A. Wagner Lang F. Broer S. Biochem. J. 2000; 349: 787-795Crossref PubMed Scopus (160) Google Scholar). We have reported previously that the neurotoxic ⋅NO derivative, peroxynitrite anion (ONOO−), specifically stimulatesl-arginine release from astrocytes (19Vega-Agapito V. Almeida A. Heales S.J.R. Medina J.M. Bolaños J.P. J. Neurochem. 1999; 73: 1446-1452Crossref PubMed Scopus (20) Google Scholar). Although the ONOO−-mediated stimulatory effect was inhibited byl-lysine, hence suggesting the involvement of systemy+ (19Vega-Agapito V. Almeida A. Heales S.J.R. Medina J.M. Bolaños J.P. J. Neurochem. 1999; 73: 1446-1452Crossref PubMed Scopus (20) Google Scholar), an in-depth study focused on elucidating the precise mechanism responsible forl-arginine transport activation has not yet been carried out. In view of the potential critical role of glial cells as neuronall-arginine suppliers for ⋅NO biosynthesis (8Grima G. Cuénod M. Pfeiffer S. Mayer B. Do K.Q. J. Neurochem. 1998; 71: 2139-2144Crossref PubMed Scopus (37) Google Scholar, 18Grima G. Benz B. Do K.Q. Eur. J. Neurosci. 1997; 9: 2248-2258Crossref PubMed Scopus (44) Google Scholar,29Wiesinger H. Progr. Neurobiol. (N. Y.). 2001; 64: 365-391Crossref PubMed Scopus (295) Google Scholar), we were prompted to investigate the mechanism through which ONOO− modulates l-arginine transport across the glial cell plasma membrane as well as the potential relevance of such modulation for neuronal l-arginine uptake. Peroxynitrite was synthesized and quantified spectrophotometrically (ε302 = 1,670m−1 × cm−1) as described previously (30Hughes M.N. Nicklin H.G. J. Chem. Soc. Abstr. 1968; : 450-452Crossref Google Scholar). Alkaline stock solutions, with an approximate ONOO− concentration of 0.3–0.4 m, were stable at −70 °C for at least 3–4 months. Dulbecco's modified Eagle's medium (DMEM), lipopolysaccharide (LPS), amino acids, and N-ethylmaleimide were obtained from Sigma. Fetal calf serum (FCS) was purchased from Roche Diagnostics.l-[2,3,4,5-3H]Arginine and [α-32P]dCTP were obtained from AmershamBiosciences. The iNOS inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) and the ⋅NO donor 1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) were purchased from Alexis Corp. (San Diego, CA). Plastic tissue culture dishes were purchased from Nunc (Roskilde, Denmark). Cell culture inserts (Millicell-PCF; 4.2 cm2 of effective membrane area; 0.4-mm membrane pore size) were obtained from Millipore (Bedford, MA). Hybond® nitrocellulose membrane was purchased from Amersham Biosciences. Anti-Cat1 antiserum was a generous gift from Dr. M. Kilberg (Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, FL). Anti-3-Nitrotyrosine antibody was kindly provided by Dr. J. Beckman (Department of Anesthesiology, University of Alabama at Birmingham, AL). Anti-rabbit or anti-mouse secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Other substrates, enzymes, and coenzymes were purchased from Sigma, Roche Diagnostics or Merck. Astroglia-rich primary cultures derived from neonatal 1-day-old Wistar rats were prepared as described previously (31Bolaños J.P. Peuchen S. Land J.M. Clark J.B. Heales S.J.R. Brain Res. Protoc. 1997; 1: 258-262Crossref PubMed Scopus (12) Google Scholar). Cell suspensions were plated in DMEM supplemented with 10% (v/v) fetal calf serum at a density of 1.25 × 105cells/cm2 in 150-cm2 flasks. Cells were maintained in a humidified incubator under an atmosphere of 5% CO2/95% air at 37 °C with a change of medium twice a week. After 12–14 days, astrocytes reached confluence, and cells were collected by trypsinization, reseeded in DMEM/10% FCS at a density of 2.5 × 105 cells/cm2 either in 6-well plates or in cell culture inserts as described previously (32Bolaños J.P. Heales S.J.R. Peuchen S. Barker J.E. Land J.M. Clark J.B. Free Radic. Biol. Med. 1996; 21: 995-1001Crossref PubMed Scopus (256) Google Scholar), and used after 24 h. Neuronal-rich primary cultures were prepared from fetal (embryonic day 17) rats as described previously (33Delgado-Esteban M. Almeida A. Bolaños J.P. J. Neurochem. 2000; 75: 1618-1624Crossref PubMed Scopus (65) Google Scholar) and grown at a density of 2.5 × 105 cells/cm2 in poly-d-lysine-coated dishes in DMEM/10% FCS. Forty-eight h after plating, the medium was replaced with DMEM supplemented with 5% horse serum and 20 mm d-glucose. On day 4 of culture, cytosine arabinoside (10 μm) was added to prevent non-neuronal proliferation, and neurons were used on day 8. For astrocytic-neuronal co-cultures, astrocyte-containing inserts were placed on the top of 8-day-old neuronal cultures and bathed with Hanks' buffer. C6 glioma cells (passage number 48) were kindly provided by Prof. B. Hamprecht (Tübingen, Germany) and were maintained in DMEM/10% FCS. Cells were passaged twice per week and were seeded at a density of 1.5 × 105 cells/cm2 24 h before experiments. Mouse fibroblast cell line K047, which is homozygous for inactivation of the Cat1 gene (Cat1−/−) (34Perkins C.P. Mar V. Shutter J.R. del Castillo J. Danilenko D.M. Medlock E.S. Ponting I.L. Graham M. Stark K.L. Zuo Y. Cunningham J.M. Bosselman R.A. Genes Dev. 1997; 11: 914-925Crossref PubMed Scopus (65) Google Scholar), as well as the wild type were maintained in DMEM/10% FCS/1% non-essential amino acids. Cells were passaged twice per week and were seeded at a density of 1.5 × 105 cells/cm2 24 h before experiments. Release experiments were carried out essentially as described previously (19Vega-Agapito V. Almeida A. Heales S.J.R. Medina J.M. Bolaños J.P. J. Neurochem. 1999; 73: 1446-1452Crossref PubMed Scopus (20) Google Scholar). Briefly, 24 h after reseeding, the culture medium was removed, and cells were washed once with prewarmed (∼37 °C) Hanks' buffer (5.26 mm KCl, 0.43 mmKH2PO4, 132.4 mm NaCl, 4.09 mm NaHCO3, 0.33 mmNa2HPO4, 5.44 mm glucose, 2 mm CaCl2, and 20 mm HEPES, pH 7.4) or Na+-free Hanks' buffer (NaCl was replaced by 132.4 mm choline chloride). Cells were then incubated in fresh Hanks' buffer (with or without Na+) containing 1 μCi/mll-[2,3,4,5-3H]arginine (50 μm) at 37 °C for 30 min. Extensive washing was performed until a constant basal l-[3H]arginine release was found. Then, ONOO− (100 μm) was added, and the radioactivity released from the cells was measured after 5 min. When appropriate, all incubations were carried out in the presence of 5 mm l-arginine, l-alanine, orl-leucine. The final buffer pH varied between 7.3 and 7.4. Control cells were treated in the same way except that the ONOO− solution was degraded previously in Hanks' buffer at 37 °C for 15 min before being added to the cells. In some experiments, astrocytes were preincubated for 24 h with LPS (1 μg/ml) alone, LPS in combination with AMT (50 μm), or DETA-NO alone (0.1 mm, which continuously releases 0.28 μm⋅NO for ∼24 h as measured with an ⋅NO electrode; ISO-NO, World Precision Instruments). In these experiments,l-arginine release experiments were carried out as above except that exogenous peroxynitrite was not included. Results were expressed as percentages of l-arginine release obtained in each treatment as compared with controls (degraded ONOO−-treated cells), which were arbitrarily given a value of 100%. Uptake experiments were carried out as described previously (5Westergaard N. Beart P.M. Schousboe A. J. Neurochem. 1993; 61: 364-367Crossref PubMed Scopus (86) Google Scholar, 6Schmidlin A. Wiesinger H. Glia. 1994; 11: 262-268Crossref PubMed Scopus (59) Google Scholar). Briefly, 24 h after reseeding, the culture medium was removed, and cells were washed once with prewarmed Na+-free Hanks' buffer. Cells were preincubated in Na+-free Hanks' buffer containing 50 μm l-arginine at 37 °C for 5 min. For trans-stimulation studies, cells were preincubated in Na+-free Hanks' buffer containing 10 mm l-arginine at 37 °C for 30 min. Uptake experiments were performed in fresh Na+-free Hanks' buffer containing 0.25 μCi/ml l-[2,3,4,5-3H]-arginine (50 μm). In experiments focused on inhibiting they+l-arginine transporter, all incubations were carried out in the presence ofN-ethylmaleimide at the indicated concentrations. Peroxynitrite additions (100 μm) and the appropriate controls were done as in the release experiments, and cells were incubated at 37 °C. After 5 min, the buffer was aspirated, and cells were rapidly washed three times with ice-cold Hanks' buffer. Cells were then lysed with 0.5 ml of 10 mm NaOH/0.1% Triton X-100, and the radioactivity present in the cell lysates was measured. In some experiments, astrocytes were preincubated for 24 h with LPS (1 μg/ml) alone, LPS in combination with AMT (50 μm), or DETA-NO alone (0.1 mm). In these experiments, l-arginine uptake experiments were carried out as above except that exogenous peroxynitrite was not included. Blanks were obtained from cells briefly (∼2 s) exposed tol-[3H]arginine medium on ice (∼0–4 °C) whose radioactivity was subtracted from sample values. Results were expressed as percentages of l-arginine uptake obtained in each treatment as compared with the controls (degraded ONOO−-treated cells), which were arbitrarily given a value of 100%. To study the neuronal uptake of l-arginine released from ONOO−-stimulated astrocytes, a co-culture system was used (32Bolaños J.P. Heales S.J.R. Peuchen S. Barker J.E. Land J.M. Clark J.B. Free Radic. Biol. Med. 1996; 21: 995-1001Crossref PubMed Scopus (256) Google Scholar). Astrocytes seeded in cell culture inserts were loaded with 5 μCi/ml l-[2,3,4,5-3H]arginine (50 μm) in Hanks' buffer at 37 °C for 30 min. After extensive washing, astrocyte-containing inserts were placed on the top of 8-day-old neuronal cultures and bathed with Hanks' buffer. Peroxynitrite (100 μm) was added, and the co-culture system was further incubated at 37 °C for 5 min. After this incubation, inserts were removed, and neurons washed and lysed with 0.5 ml of 10 mm NaOH/0.1% Triton X-100 to determine the radioactivity present in the neuronal lysates. Blanks were performed as described above, and results were expressed as pmol ofl-arginine taken up/min/mg of protein. Controls were carried out using degraded ONOO−. In some experiments, neurons were depleted of l-arginine by incubating these cells in DMEM containing arginase (2 units/ml; Sigma) for 24 h as described previously (35Schmidlin A. Wiesinger H. J. Neurochem. 1995; 65: 590-594Crossref PubMed Scopus (36) Google Scholar) before the co-incubation with astrocytes. Cells were scraped off the plastic dishes with lysis buffer (12.5 mmNa2HPO4, 116 mm NaCl, 0.5m EDTA, 1% (by volume) Triton X-100, 0.1% (w/v) sodium dodecylsulfate, 100 μmN-α-p-tosyl-l-lysine chloromethylketone, 100 μm phenylmethylsulfonyl fluoride, 1 mmphenanthroline, 10 μg/ml pepstatin A, 100 μmN-tosyl-l-phenylalanine chloromethylketone, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 10 μg/ml soybean trypsin inhibitor, pH 7) and 100 μg of protein from each sample, extemporarily determined following Bradford (36Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214351) Google Scholar), using ovalbumin as standard, and the BenchMarkTM prestained protein ladder (Invitrogen) were electrophoresed on 8% acrylamide gel (MiniProtan®, Bio-Rad) and transferred to a Hybond® nitrocellulose membrane (Amersham Biosciences). Membranes were blocked with 5% (w/v) low fat milk Tween Tris buffer solution (20 mm Tris, 500 mm NaCl, 0.1% (w/v) Tween 20 (pH 7.5)) for 1 h and then incubated in the presence of the appropriate antibody (either anti-nitrotyrosine at 1:100 dilution or anti-Cat1 at 1:2000 dilution) at 4 °C overnight. After washing, membranes were further incubated in 2% (w/v) low fat milk Tween Tris buffer solution for 45 min at room temperature in the presence of the appropriate (anti-rabbit or anti-mouse) IgG-horseradish peroxidase secondary antibodies (at 1:40,000 or 1:1,000 dilution, respectively) and immediately incubated with the luminol chemiluminescence reagent (Santa Cruz Biotechnology) for 1 min before exposure to HyperfilmTM chemiluminescence film for 5 min. Northern blotting analysis was carried out in total RNA samples isolated from the cells by the guanidinium isothiocyanate method as described previously (37Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63084) Google Scholar). The samples were electrophoresed (22 μg of RNA/line) on a 1% (w/v) agarose-formaldehyde gel. After transfer to a GeneScreen Plus membrane (PerkinElmer Life Sciences) and cross-linking with ultraviolet irradiation (UV Stratalinker, Model 2400, Genetic Research Instruments, Essex, UK), membranes were hybridized for 18 h at 65 °C in the presence of the appropriate random-primed [α-32P]dCTP-radiolabelled cDNA probes and exposed to Kodak XAR-5 film. As cDNA probes, we used either a 0.9-kbiNOS cDNA fragment (a generous gift of Dr. Elena Galea, University of Illinois, Chicago, IL) or a 0.9-kb Cat1cDNA fragment (generously provided by Dr. Manuel Palacı́n, University of Barcelona, Barcelona, Spain). Immunocytochemistry was carried out on astrocytes grown on glass coverslips, which were fixed for 30 min in PBS containing 4% paraformaldehyde, rinsed with PBS, and permeabilized for 10 min with methanol at −20 °C. Cells were then incubated at room temperature in PBS containing blocking serum (10% normal goat serum) plus anti-3-nitrotyrosine antibody for 3 h. After washing with PBS, cells were incubated in PBS containing the secondary antibody (anti-mouse-IgG-fluorescein isothiocyanate) for 2 h. Finally, coverslips were washed and developed using the SlowFade®light antifade kit (Molecular Probes, Eugene, OR) for fluorescence microphotographs. Data are expressed as mean ± S.E. values for the number of culture preparations indicated in the figure legends. Statistical significance was evaluated by one-way analysis of variance followed by the least significant difference multiple range test. p < 0.05 was considered significant. To elucidate the mechanism involved in ONOO−-mediated l-arginine release (19Vega-Agapito V. Almeida A. Heales S.J.R. Medina J.M. Bolaños J.P. J. Neurochem. 1999; 73: 1446-1452Crossref PubMed Scopus (20) Google Scholar), we first studied the Na+ dependence on this effect in C6 glial cells. Cells were loaded withl-[3H]arginine and incubated either in the absence (Na+-free, choline chloride-containing Hanks' buffer) or in the presence (Hanks' buffer) of Na+at 37 °C for 25 min and then exposed to 100 μmONOO− or to degraded ONOO−. After 5 min,l-arginine release was determined as reported previously (19Vega-Agapito V. Almeida A. Heales S.J.R. Medina J.M. Bolaños J.P. J. Neurochem. 1999; 73: 1446-1452Crossref PubMed Scopus (20) Google Scholar). As shown in Fig. 1, ONOO− increased l-arginine release by about 2-fold both in the presence (Fig. 1A) and in the absence (Fig. 1B) of Na+. Supplementation of excess (5 mm) l-arginine, l-alanine, orl-leucine partially or fully prevented ONOO−-stimulated l-arginine release in the presence of Na+ (Fig. 1A). However, onlyl-arginine, but not l-alanine orl-leucine supplementation, was able to prevent ONOO−-mediated l-arginine release in the absence of Na+ (Fig. 1B). To further investigate the transport system involved in the ONOO−-mediated activation of l-arginine release in glial cells, we tested whether the release was trans-stimulated by l-arginine. To simplify the experimental design, l-arginine uptake instead of efflux was measured in the absence or presence of a large excess (10 mm) of extracellular l-arginine. Fig.1C shows that ONOO− significantly stimulated (by ∼1.6-fold) l-arginine uptake in the absence of 10 mm l-arginine. Moreover, in the presence of 10 mm l-arginine, ONOO−-mediated stimulation of l-arginine uptake was further increased by a factor of ∼1.4. Peroxynitrite-mediated stimulation ofl-arginine uptake was dose dependently abolished by the system y+-specific inhibitorN-ethylmaleimide (38Devés R. Angelo S. Chavez P. J. Physiol. 1993; 468: 753-766Crossref PubMed Scopus (86) Google Scholar) (Fig. 1D). Polynomial (n = 2) transformation of the data (not shown) yielded a ki of ∼40 μm forN-ethylmaleimide. In view of the possibility that system y+ might be involved in ONOO−-mediated stimulation of l-arginine transport, we were prompted to investigate the effect of ONOO− in cells lacking this type of transporter system. For this purpose, we used Cat1−/− fibroblast cells, which functionally lack this type of transporter (34Perkins C.P. Mar V. Shutter J.R. del Castillo J. Danilenko D.M. Medlock E.S. Ponting I.L. Graham M. Stark K.L. Zuo Y. Cunningham J.M. Bosselman R.A. Genes Dev. 1997; 11: 914-925Crossref PubMed Scopus (65) Google Scholar), and the effect was compared with those observed in wild-type fibroblast cells, C6, and astrocytic cells, which constitutively express the Cat1 transporter (7Stevens B.R. Kakuda D.K., Yu, K. Waters M., Vo, C.B. Raizada M.K. J. Biol. Chem. 1996; 271: 24017-24022Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 27Kilberg M.S. Stevens B.R. Novak D.A. Annu. Rev. Nutr. 1993; 13: 137-165Crossref PubMed Scopus (179) Google Scholar). We found that the stimulation caused by ONOO− on l-arginine release from wild-type fibroblast cells was very similar to that found in C6 cells and astrocytes (Fig. 2). However, this effect was fully abolished in homozygous (Cat1−/−)Cat1 knock-out fibroblast cells (Fig. 2). The functional protein modification by ONOO−-mediated nitration of 3-nitrotyrosine residues (3-nitrotyrosination) is now widely documented (39Radi R. Beckman J.S. Bush K.M. Freeman B.A. J. Biol. Chem. 1991; 266: 4244-4250Abstract Full Text PDF PubMed Google Scholar, 40Estévez A.G. Spear N. Manuel S.M. Barbeito L. Radi R. Beckman J.S. Prog. Brain Res. 1998; 118: 269-280Crossref PubMed Google Scholar). In view of the rapid effect brought about by ONOO− onl-arginine transport activity, we wondered whether Cat1 3-nitrotyrosination might be taking place under our own conditions. A 5-min incubation of intact C6 or astrocytic cells in the presence of 100 μm ONOO− revealed intense protein 3-nitrotyrosination, as judged by Western blotting using monoclonal anti-3-nitrotyrosine antibody in both cell types (Fig.3). Interestingly, among other 3-nitrotyrosinated bands, in both C6 cells and astrocytes, a protein was also found to be anti-Cat1-immunopositive, as judged by parallel Western blotting using an antiserum raised against Cat1 protein (Fig.3). Either immunoprecipitation with anti-Cat1 and Western blotting with anti-3-nitrotyrosine or immunoprecipitation with anti-3-nitrotyrosine and Western blotting with anti-Cat1 were not successful, probably due to the very low level of nitrated Cat1 protein. In view of the evidence showing l-arginine transport activity stimulation by exogenous ONOO− and to elucidate the possible physiological relevance of this phenomenon, we were prompted to investigate the possible role of endogenous ONOO− formation on l-arginine transport activity. Accordingly, we used astrocytes in primary culture incubated with LPS (1 μg/ml), an endotoxin that is well known to induce iNOS in these cells (9Simmons M.L. Murphy S. J. Neurochem. 1992; 59: 897-905Crossref PubMed Scopus (481) Google Scholar, 10Galea E. Feinstein D.L. Reis D.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10945-10949Crossref PubMed Scopus (466) Google Scholar) and to stimulate iNOS-dependent ONOO− formation (41Xia Y. Roman I.J. Masters B.S. Zweier J.L. J. Biol. Chem. 1998; 273: 22635-22639Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar). We have corroborated iNOS induction (Fig. 4D) as well as functional iNOS activity by measuring LPS-mediated, AMT- (50 μm) inhibitable nitrite released to the culture medium (results not shown) (42Cidad P. Garcı́a-Nogales P. Almeida A. Bolaños J.P. J. Neurochem. 2001; 79: 17-24Crossref PubMed Scopus (37) Google Scholar). Furthermore, endogenous ONOO−formation was also confirmed by immunocytochemical evidence for LPS-mediated 3-nitrotyrosine formation in these cells (Fig.5B). Since ONOO−synthesis requires ⋅NO reaction with O2⨪ (43Beckman J.S. Beckman T.W. Chen J. Marshall P.A. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1620-1624Crossref PubMed Scopus (66
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