Arginase-1 overexpression induces cationic amino acid transporter-1 in psoriasis

Free Radical Biology & Medicine 38 (2005) 1073 – 1079 www.elsevier.com/locate/freeradbiomed

Original Contribution

Arginase-1 overexpression induces cationic amino acid transporter-1 in psoriasis Oliver Schnorra,T, Maximilian Schuiera, Guido Kagemanna, Ronald Wolf c, Markus Walzc, Thomas Ruzickac, Ertan Mayatepekd, Maurice Laryead, Christoph V. Suschekb, Victoria Kolb-Bachofenb, Helmut Siesa a

Institute for Biochemistry and Molecular Biology I, Building 22.03, Heinrich-Heine-University Duesseldorf, Universitaetsstr.1, D-40225 Duesseldorf, Germany b Institute for Molecular Medicine, Heinrich-Heine-University, Duesseldorf, Germany c Department of Dermatology, Heinrich-Heine-University, Duesseldorf, Germany d Department of General Pediatrics, Heinrich-Heine-University, Duesseldorf, Germany Received 11 November 2004; revised 3 January 2005; accepted 5 January 2005 Available online 29 January 2005

Abstract Regulated uptake of extracellular l-arginine by cationic amino acid transporters (CATs) is required for inducible nitric oxide synthase and arginase activity. Both enzymes were recently recognized as important in the pathophysiology of psoriasis because of their contribution to epidermal hyperproliferation. We here characterize the expression pattern of CATs in psoriatic skin compared to healthy skin. CAT-1 mRNA expression was strongly upregulated in lesional and nonlesional areas of psoriatic skin compared to healthy skin, whereas expression of CAT2A and the inducible isoform CAT-2B was unaltered in psoriatic skin. Furthermore, we tested the hypothesis that arginase-1 overexpression regulates CAT expression via intracellular l-arginine concentration. In in vitro experiments with arginase-1 overexpressing HaCaT cells, CAT-1 mRNA expression was increased. Likewise, this occurs in l-arginine-starved HaCaT cells. Both CAT-2 isoforms were not affected. Arginase-1 overexpression limits the synthesis of NO at physiological, but not supraphysiological, l-arginine levels. Plasma l-arginine concentration was diminished in psoriasis patients and the arginase product l-ornithine was significantly increased compared to healthy controls. In summary, arginase-1 overexpression leads to upregulated CAT-1 expression in psoriatic skin, which is due to lowered intracellular l-arginine levels and limits NO synthesis at physiological l-arginine concentrations. D 2005 Elsevier Inc. All rights reserved. Keywords: Psoriasis; Cationic amino acid transporter; l-Arginine; Nitric oxide; Arginase; Free radicals

Introduction Psoriasis is a chronic inflammatory skin disease characterized by localized areas of epidermal hyperproliferation [1]. Although the molecular mechanisms leading to keratinocyte hyperproliferation remain unknown, it is thought that Abbreviations: CAT, cationic amino acid transporter; cNOS, constitutive nitric oxide synthase; iNOS, inducible nitric oxide synthase; RT-PCR, reverse-transcriptase–polymerase chain reaction. T Corresponding author. Fax: +49 211 8113029. E-mail address: [email protected] (O. Schnorr). 0891-5849/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2005.01.005

high-output nitric oxide synthesis via iNOS as a potent regulator of proliferation and differentiation contributes to the hyperproliferative disease state of psoriasis [2]. Concerning the role of iNOS, we have previously shown a consistent and significant arginase-1 overexpression in psoriatic skin lesions, suggesting a limitation of iNOSmediated NO synthesis in vivo, because of restricted larginine availability [3]. Recently, DNA microarray analysis by Zimmermann et al. [4] demonstrated increased arginase expression in asthma patients, challenging the conventional view that l-arginine is primarily metabolized by iNOS in asthmatic responses.

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The cationic amino acid l-arginine is the common substrate for iNOS as well as arginases, the latter catalyzing the conversion of l-arginine to urea and l-ornithine, a substrate for polyamine synthesis. l-Arginine requires regulated uptake in most cells, utilizing cationic amino acid transporters (CAT). There are at least four different genes (CAT1–CAT4) [5], exhibiting differential expression patterns and tissue localization [6]. Mammalian cells ubiquitously and constitutively express CAT-1, which mediates the basal larginine import and feeds an l-arginine pool serving constitutive NOS isoforms with substrate (see Verrey et al. [7]). In contrast, de novo expression of CAT-2B, the inducible isoform, is observed in many different cell types only under inflammatory conditions, providing increased l-arginine availability, in particular for iNOS [8]. Notable exceptions known so far are hepatocytes and smooth muscle cells with constitutive expression of the splice variant CAT-2A, the amino acid sequence of which differs in a stretch of 42 amino acids from that of CAT-2B, resulting in a 10-fold lower substrate affinity [9]. CAT-mediated l-arginine transport is an important mediator in regulating keratinocyte proliferation via two independent mechanisms. First, iNOS-mediated high-output NO synthesis inhibits keratinocyte proliferation [10] and second, increased l-arginine turnover to l-ornithine by arginase-1 abolishes the limiting role of polyamines in cell proliferation [11]. Psoriatic skin lesions are characterized by high larginine turnover as a consequence of coexpressed iNOS and arginase-1 in the epidermis, and l-arginine transport is crucial for both enzymes. Therefore, we here analyzed the expression pattern of CAT isoforms in psoriatic skin and investigated the effects of arginase-1 overexpression or larginine deprivation on CAT expression and NO synthesis in human keratinocytes in vitro. Additionally, we determined l-arginine and l-ornithine concentrations in the plasma of psoriasis patients. We show that arginase-1 overexpression has functional relevance for regulation of CAT-1 expression and iNOS activity in human skin-derived cells. The data substantiate the key role for CAT-mediated l-arginine transport for iNOS and arginase activity in psoriatic skin, both of which contribute to epidermal hyperproliferation of psoriatic keratinocytes.

Materials and methods Materials All chemicals were purchased from Sigma (Deisenhofen, Germany) except when stated otherwise. Patients and skin biopsies Psoriasis patients (n = 10) and healthy volunteers (n = 10) were recruited for this study. Patients were included if

afflicted by clinically manifest psoriasis, measured by the Psoriasis Area and Severity Index (PASI) [12]. The laboratory data of clinical parameters of the study population are summarized in Table 1. Age- and sex-matched healthy controls were recruited from the general population and were included in this study if without clinical or diagnostic evidence for psoriasis. Skin biopsies from five subjects of each group were taken, immediately snap-frozen in liquid nitrogen, and later used for reverse-transcriptase– polymerase chain reaction (RT-PCR). The study was approved by the Ethics Board of the Medical Faculty of the Heinrich-Heine-University Duesseldorf. RNA Isolation and reverse-transcriptase–PCR Total RNA was isolated using the RNeasy-KIT (Qiagen, Hilden, Germany) following the manufacturer’s recommendations including DNAse treatment. The RNA from each sample was dissolved in 30 Al of RNAse-free water and stored at –70oC. First-strand cDNA synthesis was performed in 20 Al volume using 0.05 Ag/Al total RNA, reverse transcriptase, oligo d(T)15 primer, and deoxyribonucleoside triphosphate. Quantitative gene expression analysis Real-time RT-PCR on the LightCycler (Roche Diagnostics, Mannheim, Germany) was performed in a total volume of 20 Al containing 2 Al cDNA, 2 Al Fast Start Reaction Mix SYBR Green I, 1.6 Al of 25 mM MgCl2, 2 Al of each primer 5 pmol/Al, and 10.4 Al H20. Primer and PCR conditions are listed in Table 2. Human keratinocytes were used as positive controls. For negative controls, the same RNA preparations were used with the omission of the reverse-transcriptase step. After completion of the cycling process, samples were subjected to a temperature ramp with continuous fluorescence monitoring for melting-curve analysis. For each PCR product, apart from primer–dimers, a single narrow peak was obtained by melting-curve analysis at the specific melting temperature and only a single band of the predicted size was observed by agarose gel electrophoresis, indicating specific amplification without significant by-products. Samples were quantified accordingly (LightCycler analysis software, version 3.5) using the housekeeping gene GAPDH as standard. Table 1 Laboratory data of healthy controls (n = 10) and psoriasis patients (n = 10) Age [years] Sex [m/f] Psoriasis Area and Severity Index (PASI) Cholesterol [mg/dl] C-reactive protein [mg/dl] Body Mass Index

Control

Psoriasis

34 F 8 7/3 0 142 F 34 b0.5 21.4 F 2.0

47 7/3 17.4 192 8.4 26.8

F 12 F F F F

9 30 4.6 2.6

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Table 2 Human primer sequences and cycling conditions Product/GenBank Accession No. Human CAT-1 NM _003045

Human CAT-2B U76369

Human CAT-2A U76368

Human GAPDH M17851

Sense Antisense Cycle protocol Sense Antisense Cycle protocol Sense Antisense Cycle protocol Sense Antisense Cycle protocol

Sequence/cycle protocol

Fragment size

5V-ATCTGCTTCATCGCCTACTT-3V(bases 1070–1089) 5V-TAGCAGTCCATCCTCAGCGATG-3V(bases1276–1297) 50  (1 sW 958C,10 sW 558C, 20 sW 728C) + 5 min 728C 5V-CCCAATGCCTCGTGTAATCT-3V(bases 68–87) 5V-TGCCACTGCACCCGATGATAAAGT-3V(bases 165–188) 50  (1 sW 958C,10 sW 558C,10 sW 728C) + 5 min 728C 5V-CAAGACGGGGTCTGCATATT -3V(bases 476–495) 5V-TGCCACATTTCCTTTCACAA -3V(bases 823–843) 50  (1 sW 958C,20 sW 588C,30 sW 728C) + 5 min 728C 5V-CAACTACATGGTTTACATGTTCC-3V(bases 153–175) 5V-GGACTGTGGTCATGAGTCCT-3V(bases 549–568) 50  (1 sW 958C,10 sW 588C,10 sW 728C) + 5 min 728C

228 bp

Sequencing of PCR fragments PCR products were collected after the light cycler run and purified with the QIAquick PCR purification Kit (Qiagen) according to the manufacturer’s recommendation. One hundred nanograms of purified DNA was used for sequence reactions with the same primer as used for the PCR. Sequencing was performed at the Biological-Medical Research Center (BMFZ, Heinrich-Heine-University, Duesseldorf, Germany). The sequence obtained was compared with the published sequences (see accession numbers, Table 2). Nitric oxide synthase activity iNOS activity was assayed indirectly by measuring nitrite accumulation in culture supernatants through the reaction of diaminonaphthalene to naphthotriazole (Alexis, Gruenberg, Germany) using NaNO2 as standard following the manufacturer’s protocol. For each experimental sample a control sample was coincubated, containing medium and respective additives without cells. Fluorescence was measured in a Fluorostar fluorometer (BMG, Offenburg, Germany) with kEx = 365 nm and kEm = 450 nm. Arginase activity Urea production was measured using the Urea Nitrogen Assay (Sigma). Briefly, urea was hydrolyzed by urease, and a further reaction of ammonia with alkaline hypochlorite and phenol in the presence of sodium nitroprusside leading to indophenol formation, which is measured spectrophotometrically in a microplate reader (Titertek Multiplan Plus, Flow Laboratories, Heidelberg, Germany) at 540 nm. The assay was modified to allow for measurement in 20 Al of culture supernatants.

120 bp

368 bp

416 bp

amplified by cloning into pCRII-Topo vector (Invitrogen, Karlsruhe, Germany). After cloning into the expression vector pCDNA3 (Invitrogen) the DNA was transiently transfected in HaCaT cells by Superfect Transfection Reagent (Qiagen). Transfected were 2 Ag pCDNA3 vector with or without arginase-1 construct (vector control) following the manufacturer’s recommendations. Cell activation and measurement of enzyme activities were performed as described above. Sequencing of Arginase-1 DNA fragments DNA bands were eluted from the agarose gel and the DNA was extracted with QIAEX II (Qiagen) according to manufacturer’s recommendations. Sequencing was performed at the Biological-Medical Research Center (BMFZ, Dqsseldorf). The sequence obtained was compared with the GenBank sequences. Amino acid analyses Blood samples were centrifuged immediately and plasma was deproteinized with 10% sulfosalicylic acid. Plasma amino acids were measured by automated ion-exchange chromatography using ninhydrin (LC-3000; EppendorfBiotronik, Hamburg, Germany). Statistical analyses Values are reported as means F SD. Statistical analysis was made by impaired Student’s t test and P b 0.05 was considered significant.

Results Arginase-1 overexpression limits iNOS activity

Arginase-1 transfection cDNA sequence of human arginase-1 (Accession No. M14502) was amplified by RT-PCR from HepG2 cells and

The impact of overexpressed arginase-1 on iNOS activity under physiological and supraphysiological l-arginine concentrations in vitro was examined in the human

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keratinocyte cell line HaCaT. Arginase-1 was cloned from the human hepatocyte cell line HepG2 and the protein overexpressed in HaCaT cells by transient transfection, leading to increased urea production (Fig. 1A). As shown in Fig. 1B, cytokine challenge of HaCaT cells overexpressing arginase-1 leads to coexpression of iNOS. Extracellular l-arginine concentrations in the physiological range between 50 and 200 AM led to lowered iNOS activity in arginase-1-transfected cells (62 F 4% decreased iNOS activity at 50 AM l-Arg and 35 F 9% at 200 AM l-Arg). In contrast, iNOS activity was unchanged with supraphysiological l-arginine (1 mM) in the culture medium (Fig. 1B).

(1 mM) or transiently transfected with arginase-1 for 48 h. Subsequently, mRNA expression of CAT isoforms was quantified by PCR. CAT-1 mRNA was strongly upregulated in both arginase-1 overexpressing cells (8.5 F 1.2-fold induction, n = 3, P b 0.005) and in l-arginine-deprived cells (6.9 F 0.8-fold induction, n = 3, P b 0.005). This increased CAT-1 mRNA levels occurred irrespective of the means by which l-arginine depletion was achieved. In contrast, both CAT-2 isoforms were not regulated by l-arginine availability (data not shown).

Intracellular L-arginine depletion leads to upregulated CAT-1 expression

Biopsies from lesional and nonlesional psoriatic skin were analyzed for CAT mRNA expression with real-time PCR, and the expression levels were compared to healthy controls. As shown in Fig. 2A, an increase in CAT-1 expression in lesional (10.7 F 3.3-fold induction, n = 3, P b 0.05) and interestingly, in nonlesional (11.3 F 2.0-fold induction, n = 3, P b 0.05) skin occurs in psoriasis patients, whereas both splice variants of the inducible isoform CAT-2 were unchanged (Figs. 2B and C). PCR fragments from the gene-specific amplifications were sequenced, and the alignment with the respective sequence in the database gives 100% identity (Table 2).

To investigate the potential effect of lowered intracellular l-arginine concentration on CAT expression, HaCaT cells were either cultured in the absence or presence of l-arginine

CAT-1 is upregulated in psoriatic skin

Plasma L-arginine is diminished in psoriasis patients The plasma amino acid concentrations in 10 patients suffering from psoriasis and 10 healthy controls were measured. Plasma l-arginine (Fig. 3A) was significantly lower in psoriasis patients in relation to healthy controls (55.2 F 8.6 AM vs 80.7 F 14.1 AM; P b 0.05), whereas conversely l-ornithine (Fig. 3B) was significantly increased (87.0 F 17.5 AM vs 58.5 F 12.9 AM; P b 0.05) in this group. The other 18 standard amino acids analyzed did not show significant concentration differences between psoriasis patients and healthy controls (Table 3).

Discussion Arginase-1 overexpression limits NO synthesis and induces CAT-1 expression Fig. 1. Arginase-1 overexpression limits iNOS activity at physiological larginine concentrations. HaCat cells, overexpressing arginase-1 by transient transfection, were activated with a cytokine mix (IL-1h, TNFa, and IFN-g, 500 U each) for iNOS induction and cultured in customerformulated RPMI without nitrite and nitrate for 48 h in the presence of different l-arginine concentrations. (A) Arginase-1 overexpression leads to increased urea concentration. Control cells transfected with the expression vector pCDNA3 did not show arginase activity. (B) iNOS activity was measured as nitrite production. Limitation of iNOS activity of 62 F 4 and 35 F 9% compared to controls was seen at l-arginine levels of 50 and 200 AM, whereas at supraphysiological l-arginine (1000 AM) iNOS activity was not limited. Bars represent mean values F SD (n = 4). TP b 0.05 and TTP b 0.005.

The role of overexpressed arginase-1 in vitro analyzing the effect on iNOS activity under various l-arginine concentrations was investigated. In the presence of supraphysiological l-arginine (1 mM), as present in most cell culture media, no effect on iNOS activity was observed, indicative of substrate saturation for both iNOS and arginase. In contrast, at the physiological concentrations of 50 and 200 AM of l-arginine (see Table 3) there is substrate limitation of iNOS activity. Thus, limited substrate availability for iNOS activity due to high arginase-1-mediated larginine turnover restricts NO synthesis.

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CAT-2A and CAT-2B remain unchanged. We conclude that upregulated CAT-1 mRNA expression in lesional skin is due to lowered l-arginine concentrations, because of a high larginine turnover by arginase-1 in these skin areas. This is viewed as a positive adaptive regulation of gene expression to l-arginine restriction. The unaltered CAT-2B expression (Fig. 2C) also came as a surprise, since this isoform is known to be induced under inflammatory conditions in vivo and in vitro in a broad range of tissues. This observation may point to a failure in the cytokine-induced NF-nB-regulated coinduction of iNOS and CAT-2B in psoriasis, which is

Fig. 2. Quantitative expression of cationic amino acid transporters in psoriatic skin. RNA was extracted from healthy controls (n = 5), lesional (n = 5), and nonlesional (n = 5) psoriatic skin biopsies, reverselytranscribed, and amplified by real-time PCR with primers specific for CAT-1 (228 bp), CAT-2A (368 bp) and CAT-2B (120 bp). Amplification levels were related to the housekeeping gene GAPDH. Bars represent mean values F SD (n = 3). TTP b 0.005.

Next, we tested for a potential regulatory effect of overexpressed arginase-1 on CAT expression in vitro. A strong upregulation of CAT-1 mRNA after arginase-1 overexpression, resulting in lowered intracellular l-arginine, was observed in HaCaT cells. Control experiments with larginine starvation confirm this observation and are in line with the report by Aulak et al. [13] of an amino acid concentration-dependent effect on CAT-1 expression in kidney cells, due to an increased mRNA stability in the 5V-UTR. Upregulated CAT-1 expression in psoriasis Analyses of psoriatic skin biopsies show highly upregulated CAT-1 gene transcripts, not only in lesional but surprisingly also in nonlesional psoriatic skin, whereas

Fig. 3. l-Arginine plasma concentration is diminished in psoriasis patients. Amino acid concentrations in plasma samples of healthy controls (n = 10) and psoriasis patients (n = 10) were determined by ion-exchange chromatography. Plasma l-arginine concentration (A) was significantly decreased in psoriasis patients as compared to healthy controls and, in contrast, l-ornithine (B) was significantly elevated in plasma from psoriasis patients. All of the other 18 standard amino acids analyzed did not show significant differences (Table 3). TP b 0.05.

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Table 3 No differences in plasma amino acid concentrations between psoriasis patients and healthy controls except l-arginine and l-ornithine Amino acid [Amol/L]

Controls (n = 10)

Psoriasis patients (n = 10)

Significance

l-Arginine l-Ornithine l-Threonine l-Serine l-Asparagine l-Glutamine l-Proline l-Alanine l-Citrulline l-Valine l-Cysteine l-Methionine l-Isoleucine l-Leucine l-Tyrosine l-Phenylalanine l-Histidine l-Tryptophan l-Lysine Taurine Glycine

80.7 58.5 136 101 53.1 641 194 367 31.1 244 53.5 29.5 76.3 137 64.2 57.7 86.6 44.6 190 43.3 253

55.2 F 87.0 F 134 F 114 F 42.3 F 641 F 190 F 376 F 32.2 F 234 F 51.6 F 25.8 F 66.3 F 129 F 62.2 F 57.3 F 83.8 F 58.2 F 185 F 42.1 F 265 F

P b 0.05 P b 0.05 n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.

F 14.1 F 12.9 F 23 F 16 F 7.5 F 61 F 98 F 87 F 8.7 F 57 F 4.8 F 5.8 F 16.9 F 23 F 10 F 8.4 F 8.5 F 8.3 F 35 F 21.1 F 58

8.6 17.5 27 28 12.4 122 71 82 6.4 40 9.7 5.8 12.9 24 14.9 12.8 12.7 19.6 27 8.6 109

known for many cell types [8,14]. Due to its specific role for the substrate supply of iNOS, this unchanged CAT-2B expression in psoriatic skin contributes to limited iNOS activity, despite increased CAT-1 expression. Unexpectedly, low expression levels of the low-affinity transporter CAT-2A were also detected in psoriatic as well as in normal skin, since CAT-2A was so far known as the bliver-typeQ transporter, exclusively expressed in hepatocytes and smooth muscle cells [15]. Due to the finding of an increased CAT-1 expression in nonlesional psoriatic skin, we searched for a systemic effect and analyzed circulating plasma amino acid concentrations in psoriasis patients and healthy controls. An inverse relationship of l-arginine and l-ornithine concentrations was seen in psoriasis patients (Fig. 3). l-ornithine was significantly increased and, in contrast, l-arginine diminished, whereas none of the other 18 standard amino acids showed significant concentration differences (Table 3). In recent work by Morris et al. [16], decreased l-arginine plasma concentrations in asthma patients were attributed to increased arginase activity. These authors discuss the possibility of lowered l-arginine bioavailability, contributing to limited NO synthesis and promoting cell proliferation by downstream products of arginase activity in asthma patients. This might also be true for psoriasis, where an identical pattern of l-argininemetabolizing enzymes is expressed [3]. We conclude that upregulated CAT-1 expression in psoriatic skin is due to low plasma l-arginine concentrations as well as arginase-1 overexpression in psoriatic skin in vivo. Local l-arginine deficiency and diminished systemic l-arginine concentrations in the circulation of psoriasis patients limit NO synthesis in vivo and, as a consequence,

these inappropriately low NO concentrations may promote proliferation of psoriatic keratinocytes, whereas iNOSmediated high-output NO synthesis is capable of restricting epidermal hyperproliferation. l-Arginine treatment is a new promising concept in the therapy of pulmonary hypertension in sickle cell disease patients [17], which are characterized by lowered NO bioavailability due to high arginase activity. It might also be a promising therapeutic strategy in psoriasis, because topically applied l-arginine might enhance iNOS-mediated NO production in psoriatic keratinocytes leading to an antiproliferative effect in psoriatic plaques. Taken together, l-arginine-metabolizing enzymes and transporters and l-arginine itself are promising therapeutic targets for controlling NO production in psoriasis.

Acknowledgments This study was supported by a grant to O.S. from the Forschungskommission of the Heinrich-Heine-University Duesseldorf. H.S. is a Fellow of the National Foundation for Cancer Research (NFCR), Bethesda, MD. This article contains data from the thesis work of M.S. and G.K. References [1] Kolb-Bachofen, V.; Bruch-Gerharz, D. Langerhans cells, nitric oxide, keratinocytes and psoriasis. Immunol. Today 20:289; 1999. [2] Bruch-Gerharz, D.; Ruzicka, T.; Kolb-Bachofen, V. Nitric oxide in human skin: current status and future prospects. J. Invest. Dermatol. 110:1 – 7; 1998. [3] Bruch-Gerharz, D.; Schnorr, O.; Suschek, C.; Beck, K. F.; Pfeilschifter, J.; Ruzicka, T.; Kolb-Bachofen, V. Arginase 1 overexpression in psoriasis— limitation of inducible nitric oxide synthase activity as a molecular mechanism for keratinocyte hyperproliferation. Am. J. Pathol. 162:203 – 211; 2003. [4] Zimmermann, N.; King, N. E.; Laporte, J.; Yang, M.; Mishra, A.; Pope, S. M.; Muntel, E. E.; Witte, D. P.; Pegg, A. A.; Foster, P. S.; Hamid, Q.; Rothenberg, M. E. Dissection of experimental asthma with DNA microarray analysis identifies arginase in asthma pathogenesis. J. Clin. Invest. 111:1863 – 1874; 2003. [5] Deves, R.; Boyd, C. A. Transporters for cationic amino acids in animal cells: discovery, structure, and function. Physiol. Rev. 78:487 – 545; 1998. [6] Vekony, N.; Wolf, S.; Boissel, J. P.; Gnauert, K.; Closs, E. I. Human cationic amino acid transporter hCAT-3 is preferentially expressed in peripheral tissues. Biochemistry 40:12387 – 12394; 2001. [7] Verrey, F.; Closs, E. I.; Wagner, C. A.; Palacin, M.; Endou, H.; Kanai, Y. CATs and HATs: the SLC7 family of amino acid transporters. Pflugers Arch. 447:532 – 542; 2004. [8] Kawahara, K.; Gotoh, T.; Oyadomari, S.; Kajizono, M.; Kuniyasu, A.; Ohsawa, K.; Imai, Y.; Kohsaka, S.; Nakayama, H.; Mori, M. Coinduction of argininosuccinate synthetase, cationic amino acid transporter-2, and nitric oxide synthase in activated murine microglial cells. Brain Res. Mol. Brain Res. 90:165 – 173; 2001. [9] Habermeier, A.; Wolf, S.; Martine, U.; Graf, P.; Closs, E. I. Two amino acid residues determine the low substrate affinity of human cationic amino acid transporter-2A. J. Biol. Chem. 278:19492 – 19499; 2003. [10] Krischel, V.; Bruch-Gerharz, D.; Suschek, C.; Kroncke, K. D.; Ruzicka, T.; Kolb-Bachofen, V. Biphasic effect of exogenous nitric

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