Sustained Nitric Oxide Production in Macrophages Requires the Arginine Transporter CAT2*

Benjamin NicholsonDagger, Cathyryne K. Manner§, Jeanine Kleeman, and Carol L. MacLeod

From the Cancer Center and Department of Medicine, University of California, San Diego, La Jolla, California 92093-0064

Received for publication, November 3, 2000, and in revised form, January 22, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

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 to Cat1-/- animals. Analysis of cytokine-activated macrophages from Cat2-/- mice revealed a 92% reduction in NO production and a 95% reduction in L-Arg uptake. The reduction in NO production was not due to differences in NOS2 protein expression, NOS2 activity, or intracellular L-arginine content. In conclusion, our results show that sustained abundant NO synthesis by macrophages requires arginine transport via the CAT2 transporter.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

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 (1-7). 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 (7). The Ca2+-independent inducible NOS (NOS2) produces large amounts of NO over sustained periods of time (1, 2). Activated macrophages produce copious quantities of NO via NOS2 over extended periods of time that contribute to tissue injury (2). Extracellular L-arginine is not required for endothelial NO synthase-mediated NO production in human endothelial cells (8). In contrast, extracellular arginine is required for sustained NO production via NOS2 in macrophages (8, 9). In addition, increased arginine transport is known to accompany NO production via NOS2 (1, 2, 9, 10).

Among the several transport systems that mediate L-arginine uptake (y+, B0+, bo,+, and y+L) (2, 11-13), system y+ is widely expressed and considered to be the major arginine transporter in most tissues and cells (11). Encoded by cationic amino acid transporters Cat1 (14, 15), Cat2 (16, 17), and Cat3 (18), system y+ is a Na+-independent high affinity cationic amino acid transport system (19). One or more Cat gene family members are expressed in most mammalian cells (11). With the exception of liver, Cat1 is expressed virtually ubiquitously (11) and is required for viability (20), whereas Cat2 and Cat3 genes are expressed in a more restricted number of tissues (11, 16, 18). 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 (21), and the high affinity CAT2 (CAT2B) (16, 17).

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 (8, 11, 22-24) 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.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Generation of Cat2-/- Mice-- The targeting vector for the Cat2/2A gene was constructed by replacing a 1.9-kilobase fragment including exon 2 (25) with a 3.1-kilobase Neomycin cassette. AB2.2 ES cells (Lexicon Genetics), transformed with the linearized targeting construct, were selected in 180 µg ml-1 G418 and 200 nM 1-(1-2-deoxy-2-fluoro-beta -D-arabinofuranosyl)-5-iodouracil (FIAU) for 9 days. Southern blotting of DNA from 384 drug-resistant clones identified 7 containing the correct insert. Three diploid clones were injected into C57Bl/6 blastocysts, and they all gave chimeric males; chimeras from only one ES cell clone transmitted the mutant allele to its progeny. The mice were handled according to National Institutes of Health and University of California, San Diego guidelines for humane treatment.

RT/PCR and DNA Sequence Analysis-- Cat2 transcripts from the liver of Cat2-/- mice were amplified by RT/PCR using oligonucleotides 1 and 3 or 2 and 3 (their positions on the targeted construct are shown in Fig. 2B). The oligonucleotide sequences were: 1) 5'-TGT CTG CGC GGA TCT GGA AAG G-3', 2) 5'-GCC CTG AAT GAA CTG CAG GAC G-3', and 3) 5'-CGC GAA TTC GTC TGG ATA CTC TGC CAG-3'. After purification using the QIAquick PCR purification kit (Qiagen), automated DNA sequence analysis was performed using the same oligonucleotide pairs as sequencing primers.

RNA Extraction and Northern Analysis-- RNA from mouse tissues and peritoneal macrophages was prepared with TRIzol (Life Technologies, Inc.). Prior to RNA preparation, macrophages were incubated in the presence or absence of 20 units ml-1 IFNgamma (Sigma) for 2 h followed by the addition of 100 ng ml-1 LPS (Escherichia coli 055:B5, Sigma) to the primed cells for an additional 6 h. 10 µg of tissue or 5 µg of macrophage RNA was analyzed with Cat1, Cat2, and glyceraldehyde-3-phosphate dehydrogenase probes (26). A 1.3-kilobase BstX1/XhoI fragment of Cat3 was isolated for random-prime labeling (26). The NOS2 cDNA probe was cloned by RT/PCR from mouse mammary tumor cDNA using the primers 5'-CAG TGC CCT GCT TTG TGC GAA GT-3' and 5'-AAC GTT TCT GGC TCT TGA GCT GGA A-3'; the product then was ligated into the vector pGEM-T Easy (Promega), and the sequence was verified using flanking T7 and SP6 primers. Densitometry was performed as described (26). Nucleotide size markers (Life Technologies, Inc.) determined the relative size of each message.

Analysis of Peritoneal Macrophages-- Inflammatory peritoneal macrophages were isolated as described (27). After culture for 2 h, cells were washed to remove unattached cells and, where indicated, primed with 20 units ml-1 IFNgamma . 2 h later 100 ng ml-1 LPS was added and incubated for 17 h. The culture media contained ~400 µM L-arginine, representing a 2-5-fold excess of the reported Km values for system y+ (28). After activation, triplicate L-Arg transport measurements were performed as described previously (26) except the final L-Arg concentration was 400 µM. Ca2+-independent NOS2 activities were measured using the NOSdetect assay kit (Stratagene). Protein lysates were prepared from control and activated macrophages after the overnight incubation as described (24). Lysates were concentrated using Centricon-30 concentrators (Amicon). Protein amounts were measured using the Bio-Rad DC assay, and 20 µg of protein lysate was separated on 10% SDS-polyacrylamide gel electrophoresis under reducing conditions. Western blots were incubated 2 h with 0.25 µg ml-1 polyclonal NOS2 antisera (Transduction Laboratories), washed repeatedly, and then incubated with 1:10,000 goat anti-rabbit horseradish peroxidase conjugate (Bio-Rad). The protein was detected by chemiluminescence (PerkinElmer Life Sciences). After a 19-h incubation in the presence or absence of cytokines, cell lysates were prepared and shipped on dry ice to Dr. Guoyao Wu for L-arginine determinations (29). Prior to measuring NO<UP><SUB>2</SUB><SUP>−</SUP></UP> and NO<UP><SUB>3</SUB><SUP>−</SUP></UP> (the stable end products of NO metabolism) the macrophages were washed and incubated in fresh cytokine-free media for an additional 24 h. NO<UP><SUB>2</SUB><SUP>−</SUP></UP> and NO<UP><SUB>3</SUB><SUP>−</SUP></UP> then were determined as described (30). Macrophages were derived from mice of mixed C57Bl/6-129 background except C57Bl/6 Cat2+/+ macrophages were used to generate data in Fig. 3, B, C, and E.

    RESULTS AND DISCUSSION
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Production and Characterization of the Cat2 Knockout Mice-- To test directly whether CAT2-mediated arginine transport is required for NO production by NOS2, the gene encoding CAT2/2a was disrupted by homologous recombination (Fig. 1A). Three independent AB2.2 embryonic stem (ES) cell clones were used to generate chimeric males (Fig. 1, B and C) that were crossed with C57BL/6 females to produce Cat2+/- C57BL/6-129 hybrid progeny (Fig. 1D). The heterozygous mice were crossed to produce Cat2+/+, Cat2+/-, and Cat2-/- progeny that were obtained in the predicted Mendelian ratios. Unlike Cat1-/- mice (20), Cat2-/- animals are completely viable and fertile and have no gross abnormalities, providing evidence that Cat2 is a dispensable transporter gene.


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Fig. 1.   Targeted disruption of Cat2. A, construction of the Cat2 targeting vector. Upper, the genomic structure surrounding the first coding exon of Cat2. Center, the 5' and 3' homology regions of the targeting construct, the Neomycin (Neo) cassette, and flanking herpes simplex virus thymidine kinase genes (HTK). Lower, the flanking probes and expected fragment sizes after restriction enzyme digests. The restriction sites indicated are: S, SstI; H, HindIII; K, KpnI; and E, EcoRI. B and C, Southern blot analysis of ES cell genomic DNA with the 5'- (B) and 3'- (C) flanking probes. Rearr., chromosomal rearrangement. D, Southern blot analysis of mouse genomic DNA using the 5'-flanking probe.

We examined the message levels of other CAT mRNAs to learn whether the expression of another CAT family member was increased to compensate for the functional loss of CAT2 transport. No detectable up-regulation of other CAT family members was observed in Cat2-/- muscle, brain, liver, lung, or kidney in the absence of catalytically active CAT2/2A (Fig. 2A). Inspection of the Northern blots, however, revealed detectable truncated CAT2 transcripts in the liver of Cat2-/- mice (Fig. 2A) that were 400 base pairs shorter than wild-type 4.5- and 8.5-kilobase mRNAs (shown more clearly in Fig. 3B). Such aberrantly sized transcripts have been reported previously in other knockout models (for example see Ref. 31). However, to determine categorically that Cat2-/- mice were indeed lacking a functional CAT2, the truncated transcripts from Cat2-/- liver were assessed in RT/PCRs to verify the absence of the 400-base pair exon 2 (the first coding exon (25)), as indicated in Fig. 2B. DNA sequence analysis of the PCR products confirmed the absence of exon 2 and revealed the truncated transcripts initiated at either the promoter adjacent to the noncoding exon 1A or within the Neo construct. These transcripts both spliced correctly into exon 3 (Fig. 2B). Because exon 1A is noncoding (25), any initiating methionine in the Cat2-/- transcripts must lie within or downstream of exon 3. Inspection of the exon 3 DNA sequence disclosed that the first long open reading frame is located +466 into the Cat2 coding sequence, constituting a loss of 24% of the CAT2 protein if the transcript were translated. The missing portion encodes the most conserved Cat sequences (11) and includes a conserved glutamate residue previously shown to be essential for CAT1 function (32). This extensive loss makes it extremely unlikely that a functional CAT2/2A transporter can be synthesized.


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Fig. 2.   Cat gene expression. A, Northern blot of RNA from the indicated Cat2+/+ and Cat2-/- mouse tissues was probed sequentially with Cat1, Cat2, Cat3, and gapdh-specific cDNAs. B, DNA sequence analysis of RT/PCR products after amplification of truncated Cat2 mRNAs from Cat2-/- liver. Primers were specific for exon 1A (1) and exon 3 (3) or Neo (2) and exon 3 (3) as indicated. The predicted protein sequence is displayed also. The lowercase letters refer to noncoding sequences from the Neo-targeting vector.


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Fig. 3.   Functional analysis of Cat2-/- peritoneal macrophages. A, L-Arg uptake measured in Cat2+/+ and Cat2-/- macrophages activated with IFNgamma and LPS with or without 20 mM L-Lys as indicated (mean ± S.E. of three determinations for each condition). **, p < 0.01 versus activated Cat2-/- macrophages; ***, p < 0.001 versus activated Cat2+/+ macrophages. B, Northern blot analysis of RNA from control (-) and activated (+) Cat2-/- and Cat2+/+ macrophages. C, nitrite and nitrate production of control and activated Cat2-/-, Cat2+/-, Cat2+/+, and Nos2-/- macrophages (mean ± S.E. of triplicate determinations). ***, p < 0.001 versus activated Cat2+/+ macrophages. D, Ca2+-independent NOS activity of control and activated Cat2+/+ and Cat2-/- macrophages (mean ± S.E. of three determinations). *, p < 0.05 versus activated Cat2+/+ macrophages. E, Western blot analysis of NOS2 expression in control (-) and activated (+) Cat2-/- and Cat2+/+ macrophages. Protein size markers (New England BioLabs) are indicated. Results presented are representative examples of duplicate or greater determinations.

Cat2-deficient Macrophages Have Significantly Reduced NO Production and System y+ Transport-- To determine whether inflammatory peritoneal macrophages from Cat2-/- mice have diminished system y+ transport activity, L-arginine uptake was measured in control and activated Cat2+/+ and Cat2-/- macrophages (Fig. 3A). The cells were activated by priming with interferon gamma  (IFNgamma ) for 2 h followed by the addition of lipopolysaccharide (LPS) for an additional 17 h according to the protocol described previously (33). The initial rate of system y+-mediated L-Arg transport (26) into wild-type macrophages increased 3.6-fold after activation with IFNgamma and LPS (Fig. 3A). The uptake was inhibited competitively by L-Lys, a system y+ substrate (19). Importantly, system y+ transport activity was reduced significantly in both Cat2-/- control (50% inhibition) and activated macrophages (95% reduction) compared with their wild-type counterparts. The reduction in system y+-mediated arginine uptake provided strong evidence that CAT2 function was ablated effectively. The decrease in arginine uptake in activated Cat2-/- cells corresponds to the known decrease in Cat1 expression when macrophages (24, 33) and vascular smooth muscle cells (23) are activated. The observation that truncated CAT2 transcripts (Fig. 3B) were induced in activated Cat2-/- macrophages contemporaneous with a decrease in y+ transport activity further supports our conclusion that the Cat2 mutation is a null allele. These transport experiments were performed in the absence of sodium and the presence of 5 mM L-Leu to inhibit transport mediated by the competing systems y+L, b0,+, and B0,+. In addition, the maintenance of cellular viability was demonstrated by [35S]methionine metabolic labeling studies. Such methods revealed that macrophages of both genotypes synthesize equivalent amounts of protein in the presence or absence of cytokines (data not shown). Hence it appears that competing cationic amino acid transport systems adequately compensate for the lack of functional CAT2 and preserve the intracellular concentrations of L-Arg and L-Lys.

Production of NO by NOS2 was investigated in control and activated peritoneal macrophages isolated from Cat2+/+, Cat2+/-, Cat2-/-, and Nos2-/- (34) mice. Cat2+/+ and Cat2+/- macrophages produced comparable amounts of NO, whereas macrophages from Nos2-/- mice failed to synthesize detectable amounts of NO after activation (2, 31, 34) (Fig. 3C). IFNgamma /LPS-stimulated Cat2-/- macrophages made only 8% of the NO generated by wild-type cells (Fig. 3C). Similar rates of inhibition were observed in Cat2-/- macrophages in the presence of LPS alone (data not shown), indicating that the requirement for CAT2 is not unique to IFNgamma /LPS induction. NO synthesis in these cells was derived solely from NOS2 because neither endothelial NO synthase nor neuronal nitric-oxide synthase protein was detected in either wild-type or mutant cells (data not shown). In vitro NOS2 assays were performed using extracts from wild-type and mutant macrophages previously activated with cytokines to determine whether macrophages from Cat2-/- mice have functional NOS2 enzyme activity. The data reveal that Cat2-/- macrophages exhibited 81% of wild-type NOS2 activity (Fig. 3D) and expressed 90% of the wild-type amount of NOS2 protein (Fig. 3E). These small differences in the amount of NOS2 protein and/or activity are not sufficient to account for the 92% reduction in NO<UP><SUB>2</SUB><SUP>−</SUP></UP> and NO<UP><SUB>3</SUB><SUP>−</SUP></UP> production by the mutant macrophages.

Nos2 transcripts and NOS2 protein accumulated to a similar extent after macrophage activation in both genotypes, although levels were lower in Cat2-/- cells (Fig. 3, B and E). Cat1 was not perceptible by Northern analysis but was detected by RT/PCR in both Cat2+/+ and Cat2-/- macrophages (data not shown). Because CAT1 but not CAT3 mRNA was detectable by RT/PCR, it seems that CAT1 protein accounts for the basal rate of system y+ transport activity in Cat2-/- macrophages.

After an overnight incubation in the presence or absence of IFNgamma /LPS, the intracellular L-arginine levels were determined by high pressure liquid chromatography (29). In Cat2-/- cells the L-arginine content was unchanged between the control (0.81 ± 0.18 nmol/mg (mean ± S.E.)) and IFNgamma /LPS-stimulated macrophages (0.84 ± 0.24 nmol/mg). In wild-type macrophages the decrease in L-arginine from 0.71 ± 0.34 to 0.47 ± 0.1 nmol/mg after activation was not significant. Recently published data reported the intracellular L-Arg concentrations in a murine macrophage-like cell line (8). In this paper Closs et al. demonstrate that the intracellular L-Arg concentration is reduced upon activation of NOS2 or after depletion with excess extracellular L-lysine. However, after L-Arg depletion, the macrophages generated negligible NO even though the intracellular L-Arg concentration is still at least 35-fold greater than the Km of NOS2 for L-Arg. The authors propose the existence of two defined pools of L-arginine, only one of which is accessible to NOS2. Based on these observations, the requirement of NOS2 for extracellular L-Arg (8, 9), and our data, we would predict that the vast majority of the L-arginine in the wild-type and Cat2-/- macrophages was in the second nonaccessible pool. Hence, the steady-state levels of L-arginine are not necessarily a reliable indicator of the ability of the cell to generate NO via NOS2. On the contrary, the ability of the cell to transport arginine via CAT2 seems to be the key regulator of NOS2 activity in macrophages.

The substantial reduction in NO production in Cat2-/- macrophages documents that CAT2 transport is linked functionally to NOS2 for NO production. Similarly, if the two proteins compose a functional unit at the cell membrane, NOS2 might derive its substrate from the transporter directly. Such a physical association between CAT1 and endothelial NO synthase was implied by their colocalization in caveolar structures at the membrane (35). In addition, a putative association between CAT2 and NOS2 might serve to enhance the stability of the proteins. The observation that Cat2-/- macrophages have a reduced steady-state amount of NOS2 protein and have lower Ca2+-independent NOS activity may be consistent with the hypothesis that CAT2 and NOS2 coassociate. Although immunofluorescence and immunoprecipitation data from Cat2-/- mouse embryonic fibroblasts indicate that there is no physical interaction between CAT2 and NOS2 (data not shown), this possible mechanism is currently being investigated further.

The 95% reduction in system y+ transport activity, accompanied by a 92% reduction in NO production in Cat2-deficient activated macrophages, together provide strong genetic evidence that CAT2-mediated L-arginine transport limits NO production in these cells. However, preliminary experiments testing NOS2 protein expression in wild-type and Cat2 null mouse embryonic fibroblasts suggest that in mouse embryonic fibroblasts NO production depends more on the level of NOS2 protein and less on the presence of functional CAT2 (data not shown). Hence, not all cell types require CAT2 for maximal NO production. This difference may be a function of cell type-specific regulation or developmental differences. For example, the expression of the Cat genes is quite distinct in embryos compared with adult mice (18). Further, there is little information on the functional importance of NO production by embryonic fibroblasts, and their capacity to make NO in response to a variety of cytokine combinations is much more limited. In contrast, the role of NOS2 in macrophages is clearly important (1, 2, 7), and they have the capacity to produce copious amounts of NO. Experiments are being performed currently to directly compare the regulation of NOS2 function in the two cell types. Finally, the absence of CAT2 has little effect on the metabolic activity of macrophages (as demonstrated by [35S]methionine labeling) or intracellular L-arginine content. These findings support the idea that the arginine required for protein synthesis is completely adequate in Cat2-/- macrophages. This arginine supply is likely to be maintained by the activity of other cationic amino acid transport systems.

In conclusion, there is a clear functional association between CAT2 and NOS2 in peritoneal macrophages. Regardless of the mechanisms that underlie this functional association, the findings potentially are important because the overproduction of NO by macrophages mediates a number of pathophysiological conditions (1-7). This finding is of particular interest because Cat2 gene function is clearly dispensable for normal viability and fertility in mice. Hence the CAT2 transporter may offer a particularly suitable new drug target for the clinical management of aberrant NO synthesis.

    ACKNOWLEDGEMENTS

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) for L-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.

    FOOTNOTES

* This work was supported by the Susan G. Komen Breast Cancer Foundation, American Institute of Cancer Research Grant 9724, National Institutes of Health Grant CA81376, and the California Breast Cancer Research Program. Support for early phases of this study was provided by the UCSD Academic Senate and the California Research Coordinating Committee.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Susan G. Komen Breast Cancer Research Foundation Postdoctoral Fellow.

§ Howard Hughes Medical Institute Predoctoral Fellow.

Clayton Foundation Investigator. To whom correspondence should be addressed: UCSD Cancer Center, 9500 Gilman Dr., Mail Code 0064, La Jolla, CA 92093-0064. Tel.: 858-534-7251; Fax: 858-534-7340; E-mail: cmacleod@ucsd.edu.

Published, JBC Papers in Press, February 16, 2001, DOI 10.1074/jbc.M010030200

    ABBREVIATIONS

The abbreviations used are: NO, nitric oxide; NOS, nitric-oxide synthase; NOS2, nitric-oxide synthase 2; ES, embryonic stem; RT, reverse transcription; PCR, polymerase chain reaction; IFNgamma , interferon gamma ; LPS, lipopolysaccharide; CAT, cationic amino acid transporter.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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