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Biochimica et Biophysica Acta 1745 (2005) 65–73 http://www.elsevier.com/locate/bba

Y+ and y+L transporters in neuronal cells expressing hydroxylase

S.Y. Bae, Q. Xu, D. Hutchinson, C.A. Colton*

Division of Neurology, Box 2900, Bryan Research Bldg, Duke University Medical Center, Durham, NC 27710, United States

Received 23 August 2004; received in revised form 28 December 2004; accepted 28 December 2004 Available online 25 February 2005

Abstract

Arginine is a semi-essential amino acid that serves as sole substrate for involved in diverse cell processes including redox balance via (NOS) and cell proliferation via . Neurons that express nNOS require intracellular arginine to generate nitric oxide (NO). Using a TH+ neuronal cell line (CAD cells), we show that neuronal NO production is largely dependent on extracellular arginine. Although a small intracellular pool exists in CAD cells, the lack of mRNA for argininosuccinate synthase (AS), a rate limiting for arginine recycling, suggests that intracellular pools are not re-supplied by this mechanism in this sub-class of neurons. Rather, arginine is taken up from the extracellular media by two primary transport systems, the y+ and the y+L systems. The expression of CAT1, CAT3, y+LAT1 and y+LAT2 mRNAs supports the presence of each system. CAD cell arginine transport is depressed by increased extracellular K+ levels and demonstrates that variations in membrane potential control neuronal arginine uptake. Short term exposure to the oxidizing agents, rotenone and Angeli’s salt, but not FeSO4, increases arginine transport. The regulation of arginine uptake by physiological factors suggests that arginine supply adapts in a moment-to-moment fashion to the changing needs of the neuron. D 2005 Elsevier B.V. All rights reserved.

Keywords: Arginine transport; Neuronal ; Cationic amino acid transporter; Citrulline–NO cycle; Rotenone;

1. Introduction in the CNS and are thought to be co-localized with arginine uptake mechanisms that provide arginine to intracellular Arginine is a semi-essential amino acid and is a key stores [3–6]. Arginine uptake has been reported in synapto- component of multiple biochemical pathways, including somes and in primary cultures of neurons, microglia and protein synthesis, the urea cycle, polyamine synthesis and astrocytes [7–14]. In addition, mRNA expression has been nitric oxide production. As a result, arginine is involved in observed in the brain for members of the cationic amino acid diverse cellular processes ranging from redox balance to cell transporter family (CAT1, CAT2B and CAT3) that generate cycle [1]. The impact of arginine on the y+ transport activity [3,15–17]. The y+ transport system is the physiological functions of a cell depends on the pattern of most common, specific uptake system for arginine in cells enzymes that utilize arginine at any one time and the and is one of at least 4 arginine transport systems, including intracellular availability of arginine mediated by arginine y+,L, B0,+ and the b0,+. The characterization and identifica- transport proteins. Not all cells are created equal in this tion of these systems are based on kinetics, dependence on regard and cell and tissue specific patterns of enzyme and Na+ and specificity for cationic amino acids [18,19]. The y+, transport protein expression and activity provide unique the y+,L and the b0,+ systems are Na+ independent while the functional profiles for arginine utilization [1,2]. B0,+ system is Na+ dependent. Nitric oxide synthase (NOS), arginase 1 (AGI) and The paucity of information on neuronal arginine uptake arginase II (AGII) are the primary enzymes that use arginine mechanisms prompted us to examine arginine uptake in a neuronal cell model that expresses both arginase I and II and * Corresponding author. Tel.: +1 919 668 2758; fax: +1 919 684 6514. nNOS. We have used the CAD cell line, a variant of a CNS E-mail address: [email protected] (C.A. Colton). monoaminergic neuronal cell line [20]. CAD cells undergo

0167-4889/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2004.12.006 66 S.Y. Bae et al. / Biochimica et Biophysica Acta 1745 (2005) 65–73 morphological differentiation to resemble a CNS neuron buffer was then replaced with uptake buffer containing upon serum deprivation including the generation of long, various concentrations of l-arginine (from 3 AM to 1 mM) neuronal-like processes with varicosities typical of catecho- plus [3H] l-arginine (0.05 ACi/ml [3H] l-arginine per 10 AM laminergic neurons [21]. In addition, we have previously arginine specific activity). Uptake was followed for 4 min at shown that NO production can be accurately measured from 37 8C and stopped by washing with ice cold uptake buffer intact CAD cells, thus allowing us to examine the direct containing 10 mM TRIS, 10 mM HEPES and 137 mM NaCl relationship between extracellular arginine levels and NO at pH 7.4. Uptake time was pre-determined in separate production by nNOS [22]. Our data demonstrate that CAD control studies by measuring specific uptake as a function of cell NO production is dependent on extracellular arginine. time. Uptake for 4 min was chosen as the experimental time We also show that y+,L and y+ transport systems are the point and was within the linear region of the curve. Cells were predominant form of arginine uptake mechanisms. Further- then lysed in 1.0% Triton X-100 plus 0.05% NaN3 and more, the transport of arginine in this neuronal cell line is radioactivitymeasuredbyliquidscintillationcounting. altered by membrane potential and redox factors. Specific arginine uptake was determined from the difference between uptake at 4 8C (non-specific) and uptake at 37 8C (total uptake). At least 6 wells from 3 different culture groups 2. Methods were assayed and uptake values were normalized to Ag pro- tein/min. Data are presented as the average uptake in nmol/ 2.1. Materials mg protein/min (FS.E.) and significance was determined using an unpaired Students’ t test or ANOVAwith GraphPad Vinyl-l-NIO (N5-(1-imino-3-butenyl)-l-) (v-l- Prism 3.02 (GraphPad Software Inc., San Diego, CA). NIO) was purchased from Alexis Biochemicals (San Diego, To determine the Na+ dependence of the transport CA). N-Nitro-l-arginine methyl ester (l-NAME), N-l- mechanisms and to differentiate between transport systems, monomethyl arginine (NMMA) and ferrous sulfate were the uptake buffer was modified. For certain experiments, purchased from Sigma Chemical Co. (St. Louis, MO). chloride (137 mM) replaced NaCl (Na+-free buffer) Ionomycin was purchased from Calbiochem-Novabiochem or 5 mM leucine or 5 mM alanine was added to either Na+- + Corp. (San Diego, CA). Angeli’s salt (Na2N2O3; sodium containing or Na -free uptake buffer. trioxodinitrate) was a generous gift of Dr. Jon Fukuto, UCLA, Los Angeles, CA. Stock solutions were dissolved in 9 M 2.4. RT-PCR NaOH and diluted into media at the appropriate concentration immediately prior to the pretreatment period. Rotenone Total RNA from differentiated CAD cells was extracted (Calbiochem Corp., San Diego CA) was dissolved into using the RNeasy Mini kit (Qiagen, Valencia, CA). For this 100% DMSO for storage at 4 8C and was diluted into normal process, 1 Ag of total RNA was treated with RQ1 DNase media immediately prior to the pretreatment of cells. (Promega Biosciences Inc., San Luis Obispo, CA) to remove DNA contamination and then reverse-transcribed using 2.2. CAD cell culture AMV transcriptase (Promega Biosciences Inc., San Luis Obispo, CA) and random primers. The reverse transcription CAD cells were generously provided by Dr. D. Chikar- reactions were incubated at room temperature for 10 min, and aishi, Department of Neurobiology, Duke University Med- then at 42 8C for 40 min. The transcriptase was subsequently ical Center, Durham NC. CAD cells were cultured in a inactivated by heating at 99 8C for 5 min and cooling to 4 8C humidified 5% CO2/95% air atmosphere at 37 8C in DMEM/ for 5 min. Samples lacking reverse-transcriptase were also F12 medium supplemented with 8% fetal bovine serum used to ensure that genomic DNA was not amplified in the (FBS) (Hyclone, Logan, UT) and 100 U/ml penicillin and PCR reaction. The resulting cDNA templates were then 100 Ag/ml streptomycin as described previously [21]. For the mixed with primer/probe sets (Table 1) for the murine experiments, cells were plated onto poly-d- (mol. wt. cationic amino acid transporter 1gene (CAT1, also known as N300,000)-coated 24-well tissue culture plates at a density of SLC7A1), for a common sequence of both isoforms of the 5Â104 cells/well and cultured in the presence of serum until cationic amino acid transporter 2 (CAT2A and B, also confluent (usually 48 h). Once confluent, the cells were known as SLC7A2), the cationic amino acid transporter 3 placed into serum-free media for 15–24 h to differentiate. gene (CAT3, also known as SLC7A3), the arginase I gene (AGI), the arginase II gene (AGII), the nitric oxide synthase 1 2.3. Arginine uptake gene (NOS1), the argininosuccinate synthase (AS)and argininosuccinate (AL), for the y+L-type amino Arginine uptake was measured essentially as described by acid transporter gene ( y+LAT1, also known as SLC7A7), for Nicholson et al. [23]. Immediately prior to the experiment, the y+ L-type amino acid transporter gene ( y+LAT2, also cells were pre-equilibrated for 30 min in an arginine-free known as SLC7A6) and for the brelated to b0,+ amino acid uptake buffer (137 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, transportQ gene (rBAT, also known as SLC7A9) [22,23]. The 1.2 mM MgSO4, 10 mM HEPES pH 7.4). The pre-incubation PCR amplification was programmed to heat at 94 8C for 4 S.Y. Bae et al. / Biochimica et Biophysica Acta 1745 (2005) 65–73 67

Table 1 Primers used for the detection of arginine transport proteins and arginine utilization Target gene Forward primers Reverse primers Product length (bp) CAT1 5V-CACCTGTATCAATGTCCTGGTCTTG-3V 5V-ATGCAGTCAAAGCCCACGAAGG-3V 216 CAT2 5V-CCAAATACGTTGTCGCAGCAG-3V 5V-CCATGAGGGTGCCAATAGACATC-3V 250 CAT3 5V-GAAGCAGAGAAGCTGACTGTCCAG-3V 5V-TCACCAGTGGGAGTAGAGGAACAG-3V 298 AGI 5V-ACAAGACAGGGCTCCTTTCAGG-3V 5V-GGAGAAGGCGTTTGCTTAGTTCTG-3V 252 AGII 5V-CATAATACAGGGTTGCTGTCAGCTC-3V 5V-GGGAGTTTTACAGTTGTGAGAGTG-3V 354 AS 5V-AGAGCCCCTGGAGTATGGAT-3V 5V-ATGAGCGTGGTAAAGGATGG-3V 356 AL 5V-GTGTATGACCCACCTCAGCAG-3V 5V-CAGCCTCCTTGTCTTCCTGTA-3V 251 NOS1 5V-AATGATCTGTGGGGGAAGGGCAAC-3V 5V-TGTGATGGGATGGCAGCATGATG-3V 253 rBAT 5V-GAGAAGCTGGACTATATCACTGC-3V 5V-CAGATCCTTCGCTTCCAGAAG-3V 536 Y+LAT1 5V-AGCTGTGGCGCTCCCTATGCC-3V 5V-CAATGTCACCCATTGCAAAGG-3V 237 Y+LAT2 5V-ACCTGTGATCCTCCATATGTG-3U 5V-AGAGGTCTCCCACATTCCACG-3V 237

min, followed by 30 cycles of denaturing at 94 8C for 30 s, the intracellular levels of arginine. In turn, intracellular annealing at 60 8C for 30 s and extension at 72 8C for 40 s. arginine pools are supplied by arginine entering the cell The reactions were further incubated at 72 8C for 10 min and across the plasma membrane and from intracellular stores then chilled at 4 8C. The PCR products were analyzed by of arginine that may include arginine released from electrophoresis, stained with ethidium bromide and imaged protein breakdown and from recycling of citrulline to with a Kodak imager (Eastman Kodak Co., Rochester, NY). arginine [24,25]. To determine the relative contribution of these sources to NO production by CAD cells, we 2.5. Nitric oxide measurement measured NO production as a function of extracellular arginine levels. CAD cells were pre-equilibrated for 2 h The production of nitric oxide by CAD cells was in media containing 0 mM arginine and then exposed to measured as previously described [22]. Essentially, cells media containing varying concentrations of arginine (0– were treated at 37 8C in a humidified 5% CO2/95% air 300 AM). Ionomycin (3 AM) alone or in combination atmosphere with 3 AM ionomycin in arginine-free, serum- with 50 AM ruthenium red (RuR) was used to stimulate free MEM (ICN Biomedicals Inc., Costa Mesa, CA) to NO production by increasing intracellular Ca2+ levels which varying concentrations of l-arginine (5–300 AM) [22]. RuR blocks mitochondrial Ca2+ uptake and were added. Cells were treated for 5 h to ensure increases the duration of time that Ca2+ is available for accumulation. After 5 h, supernatants from each well were the enzymatic activation of nNOS [26]. The enhanced collected and assayed for nitrite content. production of NO promoted the detection of any changes NO production was determined from the supernatant in NO levels with changes in arginine. As shown in Fig. level of nitrite, the stable oxidation product of NO in 1, NO production significantly increased with increasing biological solutions, using a Seivers 280 Nitric Oxide extracellular arginine concentrations. In the absence of Analyzer that converts nitrite in the medium to NO by a extracellular arginine (Arginine=0 AM), ionomycin-stimu- reaction between ozone and NaI. The NO was detected by lated NO production was significantly higher (11.5%) chemiluminescence and the concentration determined from than the background levels and was 35% higher when a standard curve using sodium nitrite. Protein content was RuR was added to enhance intracellular Ca2+ levels. determined using the BCA assay (Pierce Biotechnology, Rockford, IL) and the data normalized to nM nitrite/Ag 3.2. Detection of mRNA for arginine transporters and protein. Average values were obtained from 4 to 6 wells arginine recycling enzymes in CAD cells from each plate for a minimum of 3 different culture groups. Significant differences were determined using an unpaired The expression of mRNAs for the known arginine Students’ t test or ANOVA using GraphPad Prism 3.02 uptake mechanisms was determined using RT-PCR and (GraphPad Software Inc., San Diego, CA). isoform specific primers for CAT1, CAT2, CAT3, rBAT, y+LAT1 and y+LAT2. Message was detected for CAT1, CAT3, and the y+LAT component of the y+L transport 3. Results systems but was not observed for CAT2A or B, using a primer sequence that detects both isoforms, or rBAT (Fig. 3.1. CAD cell NO production is dependent on extracellular 2). In addition, CAD cells expressed mRNA for NOS1, arginine AGI, AGII and AL but not AS. mRNA expression for CAT2, rBAT and AS was observed in control tissue The enzymatic production of NO by nNOS utilizes samples from the kidney (rBAT) and liver (CAT2, AS) arginine as the sole substrate and thus is dependent on (data not shown). 68 S.Y. Bae et al. / Biochimica et Biophysica Acta 1745 (2005) 65–73

cell membrane into the neuron. To further identify the transport mechanisms and their functional characteristics, we measured arginine uptake in CAD cells using radio- labeled l-arginine. Specific [H3]-l-arginine uptake was determined in Na+-containing medium over varying extracellular arginine concentrations (5– 1000 AM). The relationship between the initial rates of uptake (measured at 4 min) and extracellular arginine concentration is shown in Fig. 3A. Further analysis using an Eadie-Hofstee transformation of the hyperbolic data (Fig. 3B) demon- strates that CAD cell arginine uptake has multiple transport components. A non-linear relationship is observed for + Fig. 1. Extracellular arginine is required for NO production by nNOS. CAD arginine uptake in an Na containing medium and suggests A cells were treated with 3 M inonomycin to stimulate NO production in at least 2 distinct slopes with apparent Km values of 95 and media containing varying concentrations of l-arginine (0–300 AM). A 2+ 36 M and corresponding relative Vmax values of 1.5 and Ruthenium red (RuR) was added to block mitochondrial Ca uptake and 1.0 (nmol/mg/min). prolong the ionomycin-mediated activation of nNOS. NO production was significantly increased with increasing arginine concentrations for ionomy- The relative contribution of the known arginine transport cin alone (#Pb0.0001) or ionomycin+RuR (#Pb0.0001). Significance was systems to the uptake of arginine by CAD cells was determined using a one-way ANOVA with the Bonferroni correction. NO determined by measuring [H3]-l-arginine uptake (a) in levels were significantly higher in the absence of arginine in ionomycin Na+-containing media; (b) in Na+-free (choline) media; (c) b treated cells compared to untreated (*P 0.03) and in ionomycin+RuR in Na+-free media containing 5 mM alanine; and (d) in Na+- treated cells compared to untreated (**=0.001). Significance was deter- mined using an unpaired Student’s t test. containing media containing 5 mM leucine. The initial rate of arginine uptake is shown for CAD cells equilibrated with 3.3. Arginine uptake in CAD cells each of the above solutions in media containing 300 AM arginine (Table 2, column 3). To determine the specific Our data demonstrate that ionomycin-mediated NO uptake system corresponding to each experimental condition, production by nNOS in CAD cells depends on extrac- ellular arginine and the movement of arginine across the

Fig. 2. Arginine utilization pathway components are expressed in CAD cells. RT-PCR was used to detect the expression of mRNA for arginine transporters (CAT1, CAT2 (common sequence for A and B isoforms), CAT3, rBAT, y+LAT1 and y+LAT2), for NOS1, for arginase enzymes (AGI Fig. 3. Kinetics of arginine uptake into CAD cells. (A) Linear plot of and AGII) and for citrulline–arginine recycling enzymes (AS and AL). specific [H3]-l-arginine uptake as a function of varying concentrations of Representative bands indicate the presence of mRNA for the corresponding extracellular arginine. (B) Eadie-Hofstee transformation of the hyperbolic gene. The expression of mRNAs for rBAT, CAT2 and AS was not observed. data indicates two or more transport components. S.Y. Bae et al. / Biochimica et Biophysica Acta 1745 (2005) 65–73 69

Table 2 Characteristics of CAD cell arginine uptake systems Assay condition l-Arginine uptake l-Arginine uptakea Determination of transport Arginine uptake for system observed (nmol/mg protein/min) system utilized each transport system (nmol/mg protein/min) (a) Na+ containing y+;y+,L;b0,+;B0,+ 3.4F0.05 (8) (a)À(b)=B0,+ 0.36F0.14 10.6% (b) Na+ free y+;y+,L;b0,+ 3.0F0.14 (8) (b)À(c)=b0,+ 0.13F0.18 3.8% (c) Na+ free plus alanine y+;y+,L; 2.88F0.1 (8) (c)À(d)=y+,L 1.96F0.1 57.6% (d) Na+ containing plus leucine y+ 0.92F0.07 (8) (d) only=y+ 0.92F0.07 27% a These data are measured experimentally and are used to derive the relative contribution (%) of the arginine transport systems as shown in columns 4 and 5. we used previously described characteristic relationships for and N-Nitro-l-arginine methyl ester (l-NAME; 500 AM), the known arginine uptake systems [18,23] (Table 2, column known NOS inhibitors. No significant change in arginine 4). For example, the contribution of the B0,+ cationic amino uptake was observed (data not shown). acid transport system to arginine uptake in CAD neuronal Arginine transport systems are electrogenic in nature and cells is identified by subtracting uptake in Na+ containing arginine flux is altered when the resting membrane potential media from uptake in Na+-free media (conditions a–b; Table is hyperpolarized or depolarized [27–29]. To determine if 2). Using this analysis technique, our data demonstrate that neuronal arginine transport is also regulated by changes in the y+L cationic amino acid transport is the predominant membrane potential, arginine uptake was determined for transporter (N50% of the uptake in Na+ containing media) CAD cells in the presence of uptake media containing 1 or for arginine in our cells. The y+ transporter also contributes 50 mM KCl in place of the normal value of 5 mM KCl. significantly to total arginine uptake (approximately 27%) Osmolar balance was maintained by substituting KCl for while residual uptake appears to be mediated by the B0,+ NaCl in the high K+ media. Although there was a tendency system. Arginine uptake via the b0,+ transporter was not for increased arginine transport, reducing KCl from 5 to 1 significantly different from background. mM KCl had no statistically significant effect on [H3]-l- arginine uptake (Fig. 4A). However, arginine uptake in the 3.3.1. Regulation of neuronal arginine uptake presence of 50 mM KCl was significantly reduced. To confirm that a known transport inhibitor blocked Exposure of astrocytes to peroxynitrite, a biologically CAD cell arginine uptake, we measured arginine uptake in generated agent known to modify cellular redox status, the presence of 500 AM N-monomethyl arginine (NMMA). increased arginine transport [30]. To determine if neuronal Arginine uptake decreased by 88% (from 2.9F0.16 to arginine transporter systems are also altered by redox active 0.35F0.02 nmol/mg protein/min). We also tested vinyl-l- agents, we exposed CAD cells to FeSO4 (10,100 AM), to NIO (N5-(1-imino-3-butenyl)-l-ornithine) (v-l-NIO; 5 AM) HNO (nitroxyl) generated by the decomposition of Angeli’s

Fig. 4. Regulation of arginine uptake in CAD cells. (A) Changes in extracellular KCl alter CAD cell arginine uptake. CAD cell specific [H3]-l-arginine uptake was measured in media containing 100 or 300 AM arginine plus 1 mM KCl (hyperpolarizing conditions) or 50 mM KCl (depolarizing conditions). *Pb0.03 using unpaired Student’s t test. (B) CAD cells were pre-treated (10 min) with FeSO4 (10 or 100 AM), Angeli’s salt (0.3 or 3 mM), an HNO donor or rotenone (1 or 10 nM). Arginine uptake was then measured in media containing 300 AM arginine plus the oxidizing agents. A significant increase in uptake was observed for 3 mM Angeli’s salt and 10 nM rotenone. *Pb0.02 using unpaired Student’s t test. 70 S.Y. Bae et al. / Biochimica et Biophysica Acta 1745 (2005) 65–73 salt (0.3, 3 mM) and to rotenone, a mitochondrial nucleus accumbens robustly express AL but AS mRNA uncoupling agent (1.0, 10 nM), for 10 min prior to expression is sparse. measuring [H3]-l-arginine uptake. As shown in Fig. 4B, The addition of ruthenium red to enhance intracellular 2+ short-term pretreatment with FeSO4 had no significant effect Ca levels decreased the dependence of NO production on on [H3]-l-arginine uptake. However, Angeli’s salt (3 mM) extracellular arginine. Although the mechanism for this and rotenone (10 nM) significantly increased arginine change in not known, it is possible that the activation of uptake compared to untreated values. Ca2+-dependent proteases promoted protein breakdown within the CAD cell and the subsequent release of protein-bound arginine, thus increasing intracellular argi- 4. Discussion nine levels. Simon et al. [24] have demonstrated that intracellular protein catabolism is a key factor in the The movement of arginine from the extracellular to creation of non-exchangeable intracellular arginine pools intracellular domain across the cell membrane is a rate- in endothelial cells. limiting factor in the downstream outcomes of arginine Based on the combination of transport properties and utilization processes such as arginase enzymatic activity analysis of [31,34], the primary transport or NOS enzymatic activity. For immune activated system for arginine in CAD cells is the y+L transport that express iNOS, the dependence of system. Y+L transporters are high affinity, low capacity NOS activity and NO production on extracellular transporters formed as disulfide-linked heterodimers of arginine is unequivocal. Using CAT2 knock out mice, 4F2hc, a glycoprotein from the SLC3 family, and y+LAT1 Nicholson et al. [31] demonstrated that sustained NO or y+LAT2, members of the glycoprotein associated amino production is absent in activated immune cells lacking acid transport family (SLC7A5-8) [18,19,35,36].The CAT2, indicating that intracellular arginine levels expression of mRNA for the 4F2hc subunit has also been required for iNOS activity in macrophages are derived observed in hypothalamic neurons [7]. However, transport from extracellular sources of arginine. Cells that express kinetic and ion substitution studies failed to demonstrate a constitutive forms of NOS (eNOS and presumably functional role for the y+L system in these neurons. Y+L nNOS), however, have a restricted, intracellular store of transport activity has also been observed in cortical arginine available to arginine-requiring enzymes that is synaptosomes [10]. not freely exchangeable with extracellular arginine [25]. Unlike the CAT transporters, the SLC7A6/7 (4F2hc/ Closs et al. [24,25] have shown that eNOS activation in y+LAT1/2) transporters are obligatory exchange transporters endothelial cells continues in the absence of extracellular [19,34]. In the absence of Na+, the y+L transport system arginine. exchanges cationic amino acids, and in the presence of Na+, Our data from CAD cells, a cell model for mono- moves cationic amino acids in exchange for neutral amino aminergic ( positive; TH+) neurons, acids [19,34,36]. Although not specific for arginine, these directly and clearly demonstrate that NO production by transporters regulate the internal pools of arginine within nNOS utilizes extracellular arginine. Decreasing arginine many types of cells. from the value found in the normal media (approximately The y+ system is an arginine-specific uptake mechanism 300 AM) to the value observed in cerebral spinal fluid that has been observed in multiple cell types including (approximately 30 AM) [32] decreased NO levels by 36% neurons [7,9–11,19,34]. CAD cells also utilize this arginine (from 38 nmol/mg protein for 300 AM arginine to 24 nmol/ uptake mechanism. CAT1 and CAT3, two isoforms of the mg protein for 30 AM arginine). However, a very low, but cationic amino acid transporter family (CAT; SLC7A1-4 detectable level of NO production is observed in the family) are the most likely candidates for transport proteins arginine-free media. CAD cells, thus, also have a restricted subserving the y+ transport activity observed in CAD intracellular arginine pool that supplies arginine for NO neurons. Both of these transporters are expressed in CAD production in the absence of extracellular arginine. cells. However, CAT2 transporters are not detected in CAD Importantly, this pool is not dependent on citrulline to cells although in situ hybridization has demonstrated mRNA arginine recycling since CAD cells express only arginino- for CAT2B in rat neurons in whole brain sections [17]. Our succinate lyase (AL) and do not express argininosuccinate data also suggest that the B0,+ transport system remains as synthase (AS). The sequential action of these enzymes is an elusive candidate mechanism for neuronal arginine required for the conversion of citrulline to arginine via the uptake [18]. However, we have not observed b0,+ activity citrulline–arginine cycle (also known as the citrulline–NO or rBAT mRNA expression, indicating that this transport cycle) [33]. The lack of mRNA expression for AS in CAD system does not play a role in the uptake of arginine in CAD cells may reflect the arginine-utilization machinery within cells. this specialized sub-class of monoaminergic, TH+ neurons. The dependence of NOS and arginase activities on CAD cells are derived from SV40 transformed CNS arginine uptake has important physiological and patho- monoaminergic neurons [20,21]. Braissant et al. [3] have physiological consequences [2,33,37]. Thus, it is not shown that neurons in monoamine rich areas such as the surprising that arginine uptake is regulated by cellular S.Y. Bae et al. / Biochimica et Biophysica Acta 1745 (2005) 65–73 71 events. Transcriptional and post-transcriptional changes transport by oxidative events or by prolonged fluctuations have been observed in arginine transport systems. For in resting membrane potential are likely to impact NO example, the expression of CAT1 or CAT2 mRNA in production and, in turn, impact NO-mediated regulation macrophages, vascular , hepatic cells and of the basal activity and responsiveness of neurons to endothelial cells is either increased or decreased by a synaptic inputs. number of cellular factors. These include immune activa- tors such as LPS and gIFN, glucocorticoids, stress, insulin, glucose, hypoxia and cytokines such as IL-10 Acknowledgements (see Mann et al. [38] for a recent review) [13,39–44]. 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