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Sustained Nitric Oxide Production in Macrophages Requires the Arginine Transporter CAT2

2001; Elsevier BV; Volume: 276; Issue: 19 Linguagem: Inglês

10.1074/jbc.m010030200

1083-351X

Benjamin Nicholson, Cathyryne K. Manner, Jeanine Kleeman, Carol L. MacLeod,

Amino Acid Enzymes and Metabolism

The aberrant production of nitric oxide (NO) contributes to the pathogenesis of diseases as diverse as cancer and arthritis. Sustained NO production via the inducible enzyme, nitric-oxide synthase 2 (NOS2), requires extracellular arginine uptake. Three closely related cationic amino acid transporter genes (Cat1–3) encode the transporters that mediate most arginine uptake in mammalian cells. Because CAT2 is induced coordinately with NOS2 in numerous cell types, we investigated a possible role for CAT2-mediated arginine transport in regulating NO production. The complexity of arginine transport systems and their biochemically similar transport properties called for a genetic approach to determine the role of CAT2. CAT2-deficient mice were generated and found to be healthy and fertile in contrast toCat1 −/− animals. Analysis of cytokine-activated macrophages from Cat2 −/−mice revealed a 92% reduction in NO production and a 95% reduction inl-Arg uptake. The reduction in NO production was not due to differences in NOS2 protein expression, NOS2 activity, or intracellularl-arginine content. In conclusion, our results show that sustained abundant NO synthesis by macrophages requires arginine transport via the CAT2 transporter. The aberrant production of nitric oxide (NO) contributes to the pathogenesis of diseases as diverse as cancer and arthritis. Sustained NO production via the inducible enzyme, nitric-oxide synthase 2 (NOS2), requires extracellular arginine uptake. Three closely related cationic amino acid transporter genes (Cat1–3) encode the transporters that mediate most arginine uptake in mammalian cells. Because CAT2 is induced coordinately with NOS2 in numerous cell types, we investigated a possible role for CAT2-mediated arginine transport in regulating NO production. The complexity of arginine transport systems and their biochemically similar transport properties called for a genetic approach to determine the role of CAT2. CAT2-deficient mice were generated and found to be healthy and fertile in contrast toCat1 −/− animals. Analysis of cytokine-activated macrophages from Cat2 −/−mice revealed a 92% reduction in NO production and a 95% reduction inl-Arg uptake. The reduction in NO production was not due to differences in NOS2 protein expression, NOS2 activity, or intracellularl-arginine content. In conclusion, our results show that sustained abundant NO synthesis by macrophages requires arginine transport via the CAT2 transporter. nitric oxide nitric-oxide synthase nitric-oxide synthase 2 embryonic stem reverse transcription polymerase chain reaction interferon γ lipopolysaccharide cationic amino acid transporter The production and release of nitric oxide (NO)1 are involved in numerous cellular processes. Overproduction of NO by inflammatory cells is implicated in the pathogenesis of diseases as diverse as cancer, endotoxic shock, atherosclerosis, and arthritis (1Moncada S. Higgs E.A. FASEB J. 1995; 9: 1319-1330Crossref PubMed Scopus (720) Google Scholar, 2MacMicking J.D. Xie Q. Nathan C. Annu. Rev. Immunol. 1997; 15: 323-350Crossref PubMed Scopus (3446) Google Scholar, 3Thomsen L.L. Miles D.W. Cancer Metastasis Rev. 1998; 17: 107-118Crossref PubMed Scopus (271) Google Scholar, 4Stichtenoth D.O. Frèolich J.C. Br. J. Rheumatol. 1998; 37: 246-257Crossref PubMed Scopus (166) Google Scholar, 5Kilbourn R.G. Jubran A. Gross S.S. Griffith O.W. Levi R. Adams J. Lodato R.F. Biochem. Biophys. Res. Commun. 1990; 172: 1132-1138Crossref PubMed Scopus (556) Google Scholar, 6Detmers P.A. Hernandez M. Mudgett J. Hassing H. Burton C. Mundt S. Chun S. Fletcher D. Card D.J. Lisnock J. Weikel R. Bergstrom J.D. Shevell D.E. Hermanowski-Vosatka A. Sparrow C.P. Chao Y.S. Rader D.J. Wright S.D. Pure E. J. Immunol. 2000; 165: 3430-3435Crossref PubMed Scopus (195) Google Scholar, 7Knowles R.G. Moncada S. Biochem. J. 1994; 298: 249-258Crossref PubMed Scopus (2487) Google Scholar). NO is synthesized from arginine by three related enzymes. Two of these enzymes, the Ca2+-dependent neuronal nitric-oxide synthase and endothelial NOS, generate small amounts of NO over short periods of time (7Knowles R.G. Moncada S. Biochem. J. 1994; 298: 249-258Crossref PubMed Scopus (2487) Google Scholar). The Ca2+-independent inducible NOS (NOS2) produces large amounts of NO over sustained periods of time (1Moncada S. Higgs E.A. FASEB J. 1995; 9: 1319-1330Crossref PubMed Scopus (720) Google Scholar, 2MacMicking J.D. Xie Q. Nathan C. Annu. Rev. Immunol. 1997; 15: 323-350Crossref PubMed Scopus (3446) Google Scholar). Activated macrophages produce copious quantities of NO via NOS2 over extended periods of time that contribute to tissue injury (2MacMicking J.D. Xie Q. Nathan C. Annu. Rev. Immunol. 1997; 15: 323-350Crossref PubMed Scopus (3446) Google Scholar). Extracellular l-arginine is not required for endothelial NO synthase-mediated NO production in human endothelial cells (8Closs E.I. Scheld J.S. Sharafi M. Förstermann U. Mol. Pharmacol. 2000; 57: 68-74PubMed Google Scholar). In contrast, extracellular arginine is required for sustained NO production via NOS2 in macrophages (8Closs E.I. Scheld J.S. Sharafi M. Förstermann U. Mol. Pharmacol. 2000; 57: 68-74PubMed Google Scholar, 9Bogle R.G. Baydoun A.R. Pearson J.D. Moncada S. Mann G.E. Biochem. J. 1992; 284: 15-18Crossref PubMed Scopus (212) Google Scholar). In addition, increased arginine transport is known to accompany NO production via NOS2 (1Moncada S. Higgs E.A. FASEB J. 1995; 9: 1319-1330Crossref PubMed Scopus (720) Google Scholar, 2MacMicking J.D. Xie Q. Nathan C. Annu. Rev. Immunol. 1997; 15: 323-350Crossref PubMed Scopus (3446) Google Scholar, 9Bogle R.G. Baydoun A.R. Pearson J.D. Moncada S. Mann G.E. Biochem. J. 1992; 284: 15-18Crossref PubMed Scopus (212) Google Scholar, 10Hibbs J. Vavrin Z. Taintor R. J. Immunol. 1987; 138: 550-565PubMed Google Scholar).Among the several transport systems that mediate l-arginine uptake (y+, B0+, bo,+, and y+L) (2MacMicking J.D. Xie Q. Nathan C. Annu. Rev. Immunol. 1997; 15: 323-350Crossref PubMed Scopus (3446) Google Scholar, 11MacLeod C.L. Biochem. Soc. Trans. 1996; 24: 846-852Crossref PubMed Scopus (46) Google Scholar, 12Van Winkle L. Biochim. Biophys. Acta. 1993; 1154: 157-172Crossref PubMed Scopus (59) Google Scholar, 13Devés R. Boyd C.A.R. Physiol. Rev. 1998; 78: 487-545Crossref PubMed Scopus (444) Google Scholar), system y+ is widely expressed and considered to be the major arginine transporter in most tissues and cells (11MacLeod C.L. Biochem. Soc. Trans. 1996; 24: 846-852Crossref PubMed Scopus (46) Google Scholar). Encoded by cationic amino acid transporters Cat1(14Kim J.W. Closs E.I. Albritton L.M. Cunningham J.M. Nature. 1991; 352: 725-728Crossref PubMed Scopus (428) Google Scholar, 15Wang H. Kavanaugh M.P. North R.A. Kabat D. Nature. 1991; 352: 729-731Crossref PubMed Scopus (350) Google Scholar), Cat2 (16Kakuda D.K. Finley K.D. Dionne V.E. MacLeod C.L. Transgene. 1993; 1: 91-101Google Scholar, 17MacLeod C. Finley K. Kakuda D. Kozak C. Wilkinson M. Mol. Cell. Biol. 1990; 10: 3663-3674Crossref PubMed Scopus (80) Google Scholar), and Cat3 (18Ito K. Groudine M. J. Biol. Chem. 1997; 272: 26780-26786Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), system y+ is a Na+-independent high affinity cationic amino acid transport system (19White M.F. Gazzola G.C. Christensen H.N. J. Biol. Chem. 1982; 257: 4443-4449Abstract Full Text PDF PubMed Google Scholar). One or more Cat gene family members are expressed in most mammalian cells (11MacLeod C.L. Biochem. Soc. Trans. 1996; 24: 846-852Crossref PubMed Scopus (46) Google Scholar). With the exception of liver, Cat1 is expressed virtually ubiquitously (11MacLeod C.L. Biochem. Soc. Trans. 1996; 24: 846-852Crossref PubMed Scopus (46) Google Scholar) and is required for viability (20Perkins 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), whereas Cat2 andCat3 genes are expressed in a more restricted number of tissues (11MacLeod C.L. Biochem. Soc. Trans. 1996; 24: 846-852Crossref PubMed Scopus (46) Google Scholar, 16Kakuda D.K. Finley K.D. Dionne V.E. MacLeod C.L. Transgene. 1993; 1: 91-101Google Scholar, 18Ito K. Groudine M. J. Biol. Chem. 1997; 272: 26780-26786Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Due to differential splicing of the CAT2 mRNA, two isoforms of CAT2 exist: CAT2A, a low affinity transporter that is expressed primarily in the liver (21Closs E.I. Albritton L.M. Kim J.W. Cunningham J.M. J. Biol. Chem. 1993; 268: 7538-7544Abstract Full Text PDF PubMed Google Scholar), and the high affinity CAT2 (CAT2B) (16Kakuda D.K. Finley K.D. Dionne V.E. MacLeod C.L. Transgene. 1993; 1: 91-101Google Scholar, 17MacLeod C. Finley K. Kakuda D. Kozak C. Wilkinson M. Mol. Cell. Biol. 1990; 10: 3663-3674Crossref PubMed Scopus (80) Google Scholar).The first indication that CAT2 might provide NOS2 with its substrate came from the observation that CAT2 and NOS2 transcripts were co-induced in concert with increased system y+ activity (8Closs E.I. Scheld J.S. Sharafi M. Förstermann U. Mol. Pharmacol. 2000; 57: 68-74PubMed Google Scholar,11MacLeod C.L. Biochem. Soc. Trans. 1996; 24: 846-852Crossref PubMed Scopus (46) Google Scholar, 22Stevens 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, 23Gill D.J. Low B.C. Grigor M.R. J. Biol. Chem. 1996; 271: 11280-11283Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 24Kakuda D.K. Finley K.D. Maruyama M. MacLeod C.L. Biochim. Biophys. Acta. 1998; 1414: 75-84Crossref PubMed Scopus (44) Google Scholar) after appropriate cytokine stimulation in a variety of cell types. The requirement for arginine uptake stimulated our investigation into the identity of the relevant transporter and the possibility that it might regulate NO synthesis. Because of the genetic complexity of arginine transport systems (y+, y+L, b0,+, and B0,+), we chose a genetic approach to test our hypothesis that CAT2-mediated arginine transport plays a role in regulating NO production by inflammatory cells. In this paper we demonstrate that Cat2 −/− mice are viable and fertile and that CAT2 arginine transport function is required for sustained NO production in inflammatory peritoneal macrophages. The production and release of nitric oxide (NO)1 are involved in numerous cellular processes. Overproduction of NO by inflammatory cells is implicated in the pathogenesis of diseases as diverse as cancer, endotoxic shock, atherosclerosis, and arthritis (1Moncada S. Higgs E.A. FASEB J. 1995; 9: 1319-1330Crossref PubMed Scopus (720) Google Scholar, 2MacMicking J.D. Xie Q. Nathan C. Annu. Rev. Immunol. 1997; 15: 323-350Crossref PubMed Scopus (3446) Google Scholar, 3Thomsen L.L. Miles D.W. Cancer Metastasis Rev. 1998; 17: 107-118Crossref PubMed Scopus (271) Google Scholar, 4Stichtenoth D.O. Frèolich J.C. Br. J. Rheumatol. 1998; 37: 246-257Crossref PubMed Scopus (166) Google Scholar, 5Kilbourn R.G. Jubran A. Gross S.S. Griffith O.W. Levi R. Adams J. Lodato R.F. Biochem. Biophys. Res. Commun. 1990; 172: 1132-1138Crossref PubMed Scopus (556) Google Scholar, 6Detmers P.A. Hernandez M. Mudgett J. Hassing H. Burton C. Mundt S. Chun S. Fletcher D. Card D.J. Lisnock J. Weikel R. Bergstrom J.D. Shevell D.E. Hermanowski-Vosatka A. Sparrow C.P. Chao Y.S. Rader D.J. Wright S.D. Pure E. J. Immunol. 2000; 165: 3430-3435Crossref PubMed Scopus (195) Google Scholar, 7Knowles R.G. Moncada S. Biochem. J. 1994; 298: 249-258Crossref PubMed Scopus (2487) Google Scholar). NO is synthesized from arginine by three related enzymes. Two of these enzymes, the Ca2+-dependent neuronal nitric-oxide synthase and endothelial NOS, generate small amounts of NO over short periods of time (7Knowles R.G. Moncada S. Biochem. J. 1994; 298: 249-258Crossref PubMed Scopus (2487) Google Scholar). The Ca2+-independent inducible NOS (NOS2) produces large amounts of NO over sustained periods of time (1Moncada S. Higgs E.A. FASEB J. 1995; 9: 1319-1330Crossref PubMed Scopus (720) Google Scholar, 2MacMicking J.D. Xie Q. Nathan C. Annu. Rev. Immunol. 1997; 15: 323-350Crossref PubMed Scopus (3446) Google Scholar). Activated macrophages produce copious quantities of NO via NOS2 over extended periods of time that contribute to tissue injury (2MacMicking J.D. Xie Q. Nathan C. Annu. Rev. Immunol. 1997; 15: 323-350Crossref PubMed Scopus (3446) Google Scholar). Extracellular l-arginine is not required for endothelial NO synthase-mediated NO production in human endothelial cells (8Closs E.I. Scheld J.S. Sharafi M. Förstermann U. Mol. Pharmacol. 2000; 57: 68-74PubMed Google Scholar). In contrast, extracellular arginine is required for sustained NO production via NOS2 in macrophages (8Closs E.I. Scheld J.S. Sharafi M. Förstermann U. Mol. Pharmacol. 2000; 57: 68-74PubMed Google Scholar, 9Bogle R.G. Baydoun A.R. Pearson J.D. Moncada S. Mann G.E. Biochem. J. 1992; 284: 15-18Crossref PubMed Scopus (212) Google Scholar). In addition, increased arginine transport is known to accompany NO production via NOS2 (1Moncada S. Higgs E.A. FASEB J. 1995; 9: 1319-1330Crossref PubMed Scopus (720) Google Scholar, 2MacMicking J.D. Xie Q. Nathan C. Annu. Rev. Immunol. 1997; 15: 323-350Crossref PubMed Scopus (3446) Google Scholar, 9Bogle R.G. Baydoun A.R. Pearson J.D. Moncada S. Mann G.E. Biochem. J. 1992; 284: 15-18Crossref PubMed Scopus (212) Google Scholar, 10Hibbs J. Vavrin Z. Taintor R. J. Immunol. 1987; 138: 550-565PubMed Google Scholar). Among the several transport systems that mediate l-arginine uptake (y+, B0+, bo,+, and y+L) (2MacMicking J.D. Xie Q. Nathan C. Annu. Rev. Immunol. 1997; 15: 323-350Crossref PubMed Scopus (3446) Google Scholar, 11MacLeod C.L. Biochem. Soc. Trans. 1996; 24: 846-852Crossref PubMed Scopus (46) Google Scholar, 12Van Winkle L. Biochim. Biophys. Acta. 1993; 1154: 157-172Crossref PubMed Scopus (59) Google Scholar, 13Devés R. Boyd C.A.R. Physiol. Rev. 1998; 78: 487-545Crossref PubMed Scopus (444) Google Scholar), system y+ is widely expressed and considered to be the major arginine transporter in most tissues and cells (11MacLeod C.L. Biochem. Soc. Trans. 1996; 24: 846-852Crossref PubMed Scopus (46) Google Scholar). Encoded by cationic amino acid transporters Cat1(14Kim J.W. Closs E.I. Albritton L.M. Cunningham J.M. Nature. 1991; 352: 725-728Crossref PubMed Scopus (428) Google Scholar, 15Wang H. Kavanaugh M.P. North R.A. Kabat D. Nature. 1991; 352: 729-731Crossref PubMed Scopus (350) Google Scholar), Cat2 (16Kakuda D.K. Finley K.D. Dionne V.E. MacLeod C.L. Transgene. 1993; 1: 91-101Google Scholar, 17MacLeod C. Finley K. Kakuda D. Kozak C. Wilkinson M. Mol. Cell. Biol. 1990; 10: 3663-3674Crossref PubMed Scopus (80) Google Scholar), and Cat3 (18Ito K. Groudine M. J. Biol. Chem. 1997; 272: 26780-26786Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), system y+ is a Na+-independent high affinity cationic amino acid transport system (19White M.F. Gazzola G.C. Christensen H.N. J. Biol. Chem. 1982; 257: 4443-4449Abstract Full Text PDF PubMed Google Scholar). One or more Cat gene family members are expressed in most mammalian cells (11MacLeod C.L. Biochem. Soc. Trans. 1996; 24: 846-852Crossref PubMed Scopus (46) Google Scholar). With the exception of liver, Cat1 is expressed virtually ubiquitously (11MacLeod C.L. Biochem. Soc. Trans. 1996; 24: 846-852Crossref PubMed Scopus (46) Google Scholar) and is required for viability (20Perkins 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), whereas Cat2 andCat3 genes are expressed in a more restricted number of tissues (11MacLeod C.L. Biochem. Soc. Trans. 1996; 24: 846-852Crossref PubMed Scopus (46) Google Scholar, 16Kakuda D.K. Finley K.D. Dionne V.E. MacLeod C.L. Transgene. 1993; 1: 91-101Google Scholar, 18Ito K. Groudine M. J. Biol. Chem. 1997; 272: 26780-26786Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Due to differential splicing of the CAT2 mRNA, two isoforms of CAT2 exist: CAT2A, a low affinity transporter that is expressed primarily in the liver (21Closs E.I. Albritton L.M. Kim J.W. Cunningham J.M. J. Biol. Chem. 1993; 268: 7538-7544Abstract Full Text PDF PubMed Google Scholar), and the high affinity CAT2 (CAT2B) (16Kakuda D.K. Finley K.D. Dionne V.E. MacLeod C.L. Transgene. 1993; 1: 91-101Google Scholar, 17MacLeod C. Finley K. Kakuda D. Kozak C. Wilkinson M. Mol. Cell. Biol. 1990; 10: 3663-3674Crossref PubMed Scopus (80) Google Scholar). The first indication that CAT2 might provide NOS2 with its substrate came from the observation that CAT2 and NOS2 transcripts were co-induced in concert with increased system y+ activity (8Closs E.I. Scheld J.S. Sharafi M. Förstermann U. Mol. Pharmacol. 2000; 57: 68-74PubMed Google Scholar,11MacLeod C.L. Biochem. Soc. Trans. 1996; 24: 846-852Crossref PubMed Scopus (46) Google Scholar, 22Stevens 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, 23Gill D.J. Low B.C. Grigor M.R. J. Biol. Chem. 1996; 271: 11280-11283Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 24Kakuda D.K. Finley K.D. Maruyama M. MacLeod C.L. Biochim. Biophys. Acta. 1998; 1414: 75-84Crossref PubMed Scopus (44) Google Scholar) after appropriate cytokine stimulation in a variety of cell types. The requirement for arginine uptake stimulated our investigation into the identity of the relevant transporter and the possibility that it might regulate NO synthesis. Because of the genetic complexity of arginine transport systems (y+, y+L, b0,+, and B0,+), we chose a genetic approach to test our hypothesis that CAT2-mediated arginine transport plays a role in regulating NO production by inflammatory cells. In this paper we demonstrate that Cat2 −/− mice are viable and fertile and that CAT2 arginine transport function is required for sustained NO production in inflammatory peritoneal macrophages. We thank Dr. Karen Arden (Ludwig Institute, UCSD) for karyotyping several targeted ES cell clones, the UCSD Cancer Center transgenic mouse core for blastocyst injections, the UCSD Center for AIDS Research molecular biology core for DNA sequence determinations, and Dr. Guoyao Wu (Texas A&M) forl-arginine determinations. We also thank Drs. R. Venema, V. Ganapathy, L. Van Winkle, G. Mann, R. Cardiff, and J. Feramisco for helpful comments. The research was conducted in part by the Clayton Foundation for Research, California Division.