Cytokines and Insulin Induce Cationic Amino Acid ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 271, No. 20, Issue of May 17, pp. 11694 –11702, 1996 Printed in U.S.A.

Cytokines and Insulin Induce Cationic Amino Acid Transporter (CAT) Expression in Cardiac Myocytes REGULATION OF L-ARGININE TRANSPORT AND NO PRODUCTION BY CAT-1, CAT-2A, AND CAT-2B* (Received for publication, September 21, 1995, and in revised form, January 2, 1996)

William W. Simmons‡, Ellen I. Closs§, James M. Cunningham§, Thomas W. Smith, and Ralph A. Kelly¶ From the Cardiovascular Division and the §Howard Hughes Medical Institute and Division of Hematology/Oncology, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115

The inducible isoform of nitric oxide synthase (iNOS or

* This work was supported in part by National Heart, Lung, and Blood Institute Grant R37-HL36141 (to T. W. S.), by Specialized Center of Research (SCOR) Award IP50-HL52320 in Heart Failure, and by National Institutes of Health Grant CA61246 (to J. M. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ Supported by a fellowship award from the Medical Research Council of Canada. ¶ To whom correspondence should be addressed: Cardiovascular Division, Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02115. Tel.: 617-732-7503; Fax: 617-732-5132; E-mail: rakelly@bics. bwh.harvard.edu.

NOS2)1 is expressed in a wide variety of cell types including neonatal and adult cardiac myocytes after exposure to inflammatory mediators (1– 6). Nitric oxide (NO) production by cardiac myocytes in vitro decreases spontaneous beating rate (a negative chronotropic effect) (5, 7) and the velocity and extent of shortening (a negative inotropic effect) (6 –12), and accelerates the velocity of re-lengthening (a positive lusitropic effect) (13, 14). We recently demonstrated that cytokine-induced NO production by adult rat ventricular myocytes (ARVM), and its effect to diminish the inotropic responsiveness of these cells in vitro to the b-adrenergic agonist isoproterenol, was insulin-dependent (15). The observation that this effect of insulin was not a consequence of changes in NOS activity suggested that insulin was affecting myocyte NO generation by additional mechanisms that were independent of the extent of increased expression and activity of NOS2 protein. Potential additional sites for regulating cellular NO production include the availability of intracellular arginine and NOS co-factors. Changes in arginine supply could result from changes in uptake and/or de novo synthesis. Cellular NO production by NOS2 is dependent on extracellular arginine both in vitro and in vivo (16 –19), and vascular hyporesponsiveness to vasoconstrictor agonists after in vivo lipopolysaccharide exposure also appears to be dependent on extracellular arginine (20). Arginine is obtained from exogenous sources via a plasma membrane cationic amino acid transport system termed “system y1” (21, 22). The transport activity of system y1 is characterized by a high-affinity for cationic amino acids, sodium independence, and stimulation of transport by substrate on the opposite (trans) side of the membrane (21, 22). Two transporters exhibiting y1 system properties were cloned from the mouse and were termed mouse cationic amino acid transporter-1 and -2B (CAT-1 and CAT-2B) (23–28). CAT-1 is widely expressed in murine tissues, whereas CAT-2B has been identified only in activated murine macrophages and lymphocytes (25, 28). A third member of the cationic amino acid transporter family, cloned from murine hepatocytes (CAT-2A), was distinguished by its lower affinity for cationic amino acids and insensitivity to trans-stimulation (i.e. absence of the “y1” phenotype) (29, 30). Consistent with previous reports that failed to detect y1 activity in hepatocytes (22), CAT-2A has been detected only in the liver of adult rodents, whereas the high-affinity CAT-1 was not (24, 29). Changes in the activity of these transporters could potentially alter NO production. 1 The abbreviations used are: iNOS, inducible NO synthase; NO, nitric oxide; NOS, nitric oxide synthase; MCAT, mouse cationic amino acid transporter; CAT, cationic amino acid transporter; BH4, 5,6,7,8tetrahydrobiopterin; bp, base pair(s); ARVM, adult rat ventricular myocytes; NRVM, neonatal rat ventricular myocytes; IL, interleukin; IFN, interferon; DMEM, Dulbecco’s modified Eagle’s medium.

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Cytokine-dependent production of nitric oxide (NO) by rat cardiac myocytes is a consequence of increased expression of the inducible isoform of nitric oxide synthase (iNOS or NOS2) and, in the presence of insulin, depresses the contractile function of these cells in vivo and in vitro. Experiments reported here show that Llysine, a competitive antagonist of L-arginine uptake, suppressed NO production (detected as nitrite accumulation) by interleukin (IL)-1b and interferon (IFN) g-pretreated cardiac myocytes by 70%, demonstrating that NO production is dependent on L-arginine uptake. Cardiac myocytes constitutively exhibit a high-affinity Larginine transport system (Km 5 125 mM; Vmax 5 44 pmol/2 3 105 cells/min). Following a 24-h exposure to IL-1b and IFNg, arginine uptake increases (Vmax 5 167 pmol/2 3 105 cells/min) and a second low-affinity L-arginine transporter activity appears (Km 5 1.2 mM). To examine the molecular basis for these cytokine-induced changes in arginine transport, we examined expression of three related arginine transporters previously identified in other cell types. mRNA for the high-affinity cationic amino acid transporter-1 (CAT-1) is expressed in resting myocytes and steady-state levels increase by 10-fold following exposure to IL-1b and IFNg. Only cytokine-pretreated myocytes expressed a second high-affinity L-arginine transporter, CAT-2B, as well as a lowaffinity L-arginine transporter, CAT-2A. In addition, insulin, which potentiated cytokine-dependent NO production independent of any change in NOS activity, increased myocyte L-arginine uptake by 2-fold and steadystate levels of CAT-1, but not CAT-2A or CAT-2B mRNA. Thus, NO production by cardiac myocytes exposed to IL-1b plus IFNg appears to be dependent on the coinduction of CAT-1, CAT-2A, and CAT-2B, while insulin independently augments L-arginine transport through CAT-1.

L-Arginine

Transport, CAT Expression, and NOS2 in Cardiac Myocytes

EXPERIMENTAL PROCEDURES

Cell Isolation and Culture—Calcium-tolerant ventricular myocytes (ARVM) were isolated from adult male Sprague-Dawley rats (225–275 g) as described previously (40). This included two density gradient sedimentations through 6% bovine serum albumin (Sigma) and differential attachment to laminin-coated tissue culture dishes to limit nonmyocyte contamination to less than 5% of the enriched myocyte population (40). ARVM were cultured in a defined medium (41) consisting of Dulbecco’s modified Eagle’s medium with 25 mM HEPES and 44 mM NaHCO3 with 4.2 mM L-glutamine (DMEM; Life Technologies, Inc.), supplemented with 100 IU/ml penicillin, 100 mg/ml streptomycin, 2 mg/ml bovine serum albumin, 2 mM L-carnitine, 5 mM creatine, 5 mM taurine, and 0.1 nM triiodothyronine. Where indicated, the defined medium was also supplemented with 100 nM insulin from bovine pancreas (Sigma). Experiments with ARVM were initiated 24 h after cell isolation. Neonatal rat ventricular myocytes (NRVM) were isolated from 1-dayold Sprague-Dawley pups as described previously (42, 43). Briefly, neonatal cardiac ventricular tissue was excised aseptically and then digested with 0.1% trypsin in Hanks’ balanced salt solution (Life Technologies, Inc.) overnight at 4 °C. Ventricular cells were then recovered by repeated digestions of the tissue in 10 ml of 0.1% collagenase in Hanks’ solution at 37 °C in a shaking water bath. The supernatants collected from each digestion were centrifuged at 1000 rpm for 4 min (4 °C). The pellets were then resuspended in ice-cold Hanks’ solution, pooled, and centrifuged at 1000 rpm for 4 min (4 °C). Cells were resuspended in DMEM supplemented with 7% fetal calf serum (Life Technologies, Inc.) and subsequently underwent two preplatings of 75 min each to minimize nonmyocyte contamination to less than 5% of the enriched myocyte population (43). Nonadherent cells were counted with a hemacytometer and plated at a density of 1000 cells/mm2. After 24 h, the culture medium was changed to serum-free DMEM supplemented where indicated with 100 nM insulin. All experiments were performed 24 h later at which time spontaneous contractile activity was evident. L-Arginine Transport Assay—Neonatal ventricular myocytes were

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plated at a density of 2 3 10 cells per well (1000 cells/mm ) in 24-well cluster trays (Costar Corp.). Transport assays were performed using cluster trays and other equipment as described by Gazzola et al. (44). Cells were washed with 1 ml of Dulbecco’s phosphate-buffered saline (pH 7.4) (Sigma), containing 0.9 mM CaCl2, 0.5 mM MgCl2, 2.7 mM KCl, 1.5 mM KH2PO4, 137 mM NaCl, 8.1 mM Na2HPO4 and supplemented with 0.1% (w/v) glucose. Cells were then incubated in 200 ml of phosphate-buffered saline with 0.1% glucose containing L-[3H]arginine (69 Ci/mmol; Amersham) at 37 °C for the times and external L-arginine concentrations indicated. In selected experiments, 10 mM lysine was added to the transport buffer. Incubations were terminated by rapidly decanting and shaking the uptake medium into a receiving vessel and immediately washing the cells three times with cold (4 °C) phosphatebuffered saline with 0.1% glucose containing 10 mM unlabeled L-arginine with the cluster tray placed on ice. Cells were detergent solubilized in 0.5% Nonidet P-40 and 0.1% SDS and the radioactivity in the extracts was quantified by liquid scintillation counting. Calculations of Km and Vmax of arginine uptake were determined using GraphPad Inplot, version 4.03 software (GraphPad Software, Inc.), to perform regression analyses and rectangular hyperbola transformations. Ribonuclease Protection Analysis—A 237-bp BamHI/KpnI of MCAT-1 cDNA (bp 1244 –1481) (23) with a BamHI recognition site previously introduced at bp 1244 –1249 and subcloned into pBluescript II SK(1) (25) was linearized with BamHI for use as a template in RNase protection analysis. An antisense riboprobe was transcribed with T7 RNA polymerase (Boehringer Mannheim) in the presence of [a-32P]UTP (DuPont, NEN, specific activity 800 Ci/mmol) and protected a 237-bp RNA fragment. The MCAT-2A RNase protection probe was generated from a 127-bp fragment of MCAT-2A cDNA (bp 1095–1222) (25, 29) subcloned into pSP64, linearized with EcoRI, and transcribed using SP6 RNA polymerase in the presence of [a-32P]UTP. A 151-bp template of MCAT-2B cDNA (bp 1074 –1225) (25) subcloned into pSP64 was linearized with BamHI and an antisense riboprobe transcribed with SP6 RNA polymerase in the presence of [a-32P]UTP. The 18 S ribosomal RNase protection probe was generated from the 80-bp antisense template of a highly conserved region of the human 18 S ribosomal RNA gene, obtained commercially (Ambion, pT7 18 S), and linearized with HindIII. A lower specific activity probe was generated by using T7 RNA polymerase in the presence of a 1:1000 dilution of [a-32P]UTP with the balance being unlabeled UTP (3.3 mM). Total RNA was isolated from adult and neonatal ventricular myocytes using the method of Chomczynski and Sacchi (45). RNase protection analysis was performed as described (46). Following gel purification of the probes, hybridization reactions were performed with 20 mg of total RNA in 50% formamide for 12 h at 50 °C using 2 3 105 cpm per reaction of the radiolabeled antisense RNA transcripts, except for the 18 S riboprobe where 104 cpm per reaction was co-hybridized with all samples. Samples were then digested with ribonuclease A and T1 (Boehringer Mannheim) and analyzed on 8% denaturing polyacrylamide gels, with adjacent RNA size markers (Ambion), followed by autoradiography. Total RNA from murine macrophage and liver was also analyzed by RNase protection on initial gels that verified the presence of protected fragments of the predicted size for the rat. Nitrite Assay—Nitrite accumulation in the medium was used as an indicator of cellular NO synthesis and was determined as described (47). Cells were treated with phenol red-free DMEM containing the desired test reagents for 24 h. The medium was then collected and centrifuged at 3,000 rpm for 10 min at 4 °C to remove cellular debris. The nitrite content in the supernatant was measured by combining 150 ml of medium with 900 ml of the Griess reagent (0.75% sulfanilamide in 0.5 N HCl, 0.075% naphthylethylenediamine dihydrochloride) and the concentration of the resulting chromophore was determined spectrophotometrically at 543 nm. Nitrite concentration was calculated from a standard curve constructed over the linear range of the assay (0.1 to 50 mM). Measurement of NO Synthase (NOS) Activity—NOS activity in myocyte homogenates was quantified by measuring the conversion of 3 3 L-[ H]arginine to L-[ H]citrulline in the presence of saturating concentrations of the enzyme’s substrate and co-factors (see below) as described previously (6). Total cellular homogenates were prepared from approximately 6 3 106 neonatal rat ventricular myocytes and 1 3 106 ARVM in 100-mm plates that were placed on ice, the medium aspirated, and the cells rinsed twice with ice-cold phosphate-buffered saline. Cells were then scraped free of the plate in 1 ml of phosphate-buffered saline with a rubber policeman. The cell suspension was centrifuged at 100 3 g for 5 min at 4 °C, and the pellet resuspended in 200 ml of lysis buffer containing 20 mM Tris-HCl (pH 7.4 at 4 °C), 0.5 mM EDTA, 0.5 mM EGTA, 1 mM dithiothreitol, 1 mM leupeptin, 1 mM pepstatin A, 2 mM 2


Arginine can also be generated within cells from intracellular protein degradation and by the endogenous synthesis of arginine (31). Many NO-producing cell types, including murine macrophages and bovine aortic endothelial cells, are capable of synthesizing arginine from citrulline by the sequential action of argininosuccinate synthetase and argininosuccinate lyase (32, 33). Expression of argininosuccinate synthetase is increased along with NOS2 in macrophages and vascular smooth muscle cells following exposure to soluble inflammatory mediators (34, 35) and is associated with increased synthesis of arginine from citrulline (32). Another potential site for regulation of cellular NO production by cytokines and insulin is the availability of tetrahydrobiopterin (BH4), an essential NOS co-factor (3). GTP cyclohydrolase I, the rate-limiting enzyme for the de novo synthesis of BH4, is co-induced with NOS2 by cytokines in a variety of cell types, including cardiac myocytes (6, 36 –38). Inhibitors of GTP cyclohydrolase I limited the production of NO by rat aortic smooth muscle cells in vitro following NOS2 induction, indicating that BH4 availability also may limit NO production in cytokine-pretreated cells (39). In the experiments described here, we addressed the mechanisms by which IL-1b and IFNg, and insulin regulate L-arginine availability. To this end, we measured the dependence of cytokine-induced NO production on L-arginine transport in rat cardiac myocytes and correlated this with mRNA levels for the three cloned cationic amino acid transporters. We also determined whether cytokine- and insulin-mediated augmentation of NO production in these cells is associated with changes in levels of mRNA coding for argininosuccinate synthetase and argininosuccinate lyase, enzymes for the de novo synthesis of arginine, or for GTP cyclohydrolase I, the rate-limiting enzyme for BH4 synthesis. These data support the conclusion that L-arginine transport into cardiac myocytes is rate-limiting for NO production by cardiac myocytes in the presence of insulin and these cytokines.

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b-mercaptoethanol, and 0.2 mM phenylmethylsulfonyl fluoride. Cells were lysed by three cycles of freeze-thawing and the homogenates were centrifuged at 1,500 3 g for 15 min at 4 °C. The protein content was determined using the Bradford technique (Bio-Rad) with albumin as a standard. Twenty-five ml of supernatant (approximately 50 mg of protein) were added to 125 ml of buffer containing 50 mM HEPES (pH 7.4, 37 °C), 1.25 mM CaCl2, 1 mM EDTA, 0.5 mM NADPH, 10 mM FAD, 5 mM FMN, 10 mM tetrahydrobiopterin, 10 mg/ml calmodulin, 1 mM dithiothreitol, and 50 mM L-arginine combined with L-[3H]arginine (69 Ci/ mmol; Amersham) for 1 h at 37 °C. The reaction was terminated by adding 2 ml of ice-cold 20 mM HEPES (pH 5.5) with 5 mM EDTA and the total volume was applied to a Dowex 50W-X8 column that had been pre-equilibrated with 20 mM HEPES (pH 5.5). L-[3H]Citrulline was eluted with 2 ml of deionized water and radioactivity was quantified by liquid scintillation counting. Northern Hybridization—Total RNA was isolated as described previously (45) and 15 mg of RNA separated by electrophoresis on a 1% formaldehyde-agarose gel. Following transfer to nylon membranes (GeneScreen Plus, DuPont) and fixation by UV cross-linking, hybridizations were performed overnight at 42 °C in 50% formamide with the 32 P-radiolabeled cDNAs for rat argininosuccinate synthetase (48), rat argininosuccinate lyase (49), murine macrophage iNOS (1), a fulllength cDNA of the rat GTP cyclohydrolase I (50), and rat liver arginase (51). Hybridized blots were sequentially washed for 30 min each with 2 3 SSC, O.1% SDS at 37 °C, followed by 1 3 SSC, 0.1% SDS at 37 °C, and 0.2 3 SSC, 0.1% SDS at 65 °C, and autoradiography was performed at 270 °C with an intensifier screen for 24 h. Relative mRNA abundance was quantified by measuring the density of the exposed film with a laser densitometer (Ultrascan 2202, LKB). mRNA levels were normalized to 18 S ribosomal RNA following re-hybridization of blots with a 32P-labeled oligonucleotide complementary to rat 18 S ribosomal RNA with repeat washing and autoradiography. Cell Respiration Assay—Cardiac myocytes were incubated in 24-well cluster trays at 37 °C in DMEM supplemented with 100 IU/ml penicillin, 100 mg/ml streptomycin, and 0.2 mg/ml 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide. After 90 min, culture medium was removed and cells were solubilized in 500 ml of dimethyl sulfoxide. The extent of reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan within cells, a measure of cellular respiration, was quantified by measurement of absorbance at 570 nm (52). Cell viability was not significantly diminished following 24 h treatment with the highest concentration of agents tested (i.e. IL-1b (4 ng/ml) plus IFNg (500 units/ml), L-lysine (10 mM), D-arginine (10 mM)). RESULTS

Nitrite Production, Arginine Transport, and NOS Activity in Cardiac Myocytes—Prior studies from this laboratory have demonstrated that incubation of ARVM with the combination of rhIL-1b and IFNg induced NOS2 expression, activity, and nitrite production (6, 15). This combination of cytokines for 24 h produced an increase in nitrite accumulation in neonatal myocyte-conditioned medium at 24 h that was clearly potentiated by the addition of insulin (approximately 2-fold) in four separate experiments (Fig. 1, upper panel) and is consistent with our previous observations in ARVM (15). Nitrite accumulation was undetectable in medium conditioned by cardiac myocytes in the absence of cytokine stimulation. The dependence of NO production in intact myocytes on L-arginine transport was studied by incubating neonatal cardiac myocytes for 24 h with IL-1b and IFNg in the presence and absence of L-lysine, a cationic amino acid that competes with L-arginine for transmembrane transport (21). Insulin and the combination of IL-1b and IFNg were both observed to increase 3 L-[ H]arginine (100 mM) uptake 2-fold compared to control (p , 0.001), and the effects on L-arginine uptake of insulin and the cytokines were additive (Fig. 1, lower panel). The 100 mM concentration of L-arginine was chosen as it is the approximate mammalian plasma concentration for this amino acid (53). A concentration of L-lysine (10 mM) was selected that was determined to inhibit myocyte L-arginine (100 mM) uptake by .95%. The addition of L-lysine to the medium reduced cytokine-induced nitrite production by two-thirds (Fig. 1, upper panel), whereas D-arginine (10 mM) had no effect on nitrite production

FIG. 1. Dependence of cytokine-induced nitrite production and its regulation by insulin on L-arginine uptake by cardiac myocytes. 24-h nitrite accumulation in medium (upper panel) conditioned by neonatal rat ventricular myocytes treated with IL-1b and IFNg (open bars) or the combination of IL-1b, IFNg, and insulin (black bars), in the absence and presence of 10 mM L-lysine. Nitrite accumulation was undetectable in medium conditioned by control myocytes or myocytes exposed to insulin alone. Following 24-h incubation with the indicated treatments, uptake of L-[3H]arginine (100 mM) was measured for 5 min in the absence and presence of 10 mM L-lysine (lower panel). Each point is the mean 6 S.E. from six repetitions and is from a single experiment that was representative of four separate experiments (*, p , 0.001 versus other groups; **, p , 0.001 versus control; #, p , 0.001 versus absence of L-lysine).

(data not shown). Addition of L-lysine did not completely abolish the effect of insulin to augment cytokine-induced nitrite production, but it did lead to a 15% greater reduction in lysineinhibitable (uptake-dependent) nitrite in the cytokine plus insulin group when compared to cytokines alone. Consistent with previous observations, as shown in Fig. 2, the combination of IL-1b and IFNg (but not insulin) produced

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FIG. 2. NOS2 activity in cytokine-treated cardiac myocytes. Whole-cell extracts from neonatal (A) and adult (B) rat ventricular myocytes were assayed for the enzymatic conversion of L-[3H]arginine to L-[3H]citrulline as described under “Experimental Procedures.” Cells were incubated for 24 h in control medium or in medium containing IL-1b and IFNg alone, or in combination with insulin (100 nM) in the absence or presence of L-lysine (10 mM). Extracts of the cells treated with the combination of cytokines and insulin were also incubated in either a reaction buffer alone or in a reaction buffer supplemented with 1 mM L-N-monomethylarginine (L-NMMA). Data are means 6 S.E. from two experiments (each in triplicate). *, p , 0.01 versus control or L-N-monomethylarginine.

comparable increases in maximal NOS activity at 24 h in cellular homogenates of neonatal and adult cardiac myocytes above low levels of baseline activity, as measured by the rate of conversion of L-[3H]arginine to L-[3H]citrulline in the presence of excess substrate and co-factors. In contrast to its effects on nitrite production by intact cardiac myocytes (Fig. 2), the 24-h incubation of cells with L-lysine (10 mM) added to cytokines did not diminish maximal NOS activity in myocyte homogenates. The addition of the NOS inhibitor L-N-monomethylarginine (1

mM) to the enzyme assay buffer decreased maximal NOS activity close to background levels. Effects of IL-1b/IFNg and Insulin on L-Arginine Transport— The dependence of cytokine-induced NO production on arginine uptake in cardiac myocytes and its regulation by cytokines and insulin led us to characterize further the time course and kinetics of arginine uptake. Since both the neonatal and adult rat ventricular myocyte phenotypes exhibit qualitatively similar responses in NOS activity and nitrite production (15) following treatment with IL-1b, IFNg, and insulin, these studies were performed in neonatal myocytes for technical reasons. Transport of L-arginine was approximately linear over 5 min in control and cytokine-pretreated cells. There was no detectable metabolism of L-arginine to L-citrulline in these cells during this 5-min linear portion of the time course for L-arginine uptake. Furthermore, we could not detect expression of arginase, another potential source of arginine metabolism, in these cells by Northern hybridization under any of the treatment conditions (data not shown). IL-1b and IFNg treatment for 24 h increased the rate of uptake of 100 mM L-[3H]arginine 2-fold compared to controls during the linear range of uptake (51.7 6 5 versus 26.4 6 2.0 pmol/2 3 105 cells/min for cytokine-treated and control myocytes, respectively at t 5 1 min; mean 6 S.E.; n 5 6 in both groups; p , 0.001) (Fig. 3). Insulin, which had also been shown to increase nitrite production by cardiac myocytes in response to cytokines (15), independently increased L-arginine uptake into ventricular myocytes to an extent comparable to that achieved with cytokines (Fig. 3). The effects of cytokines and insulin were additive, resulting in a 4-fold increase in L-arginine uptake compared to the control value. Qualitatively similar effects of the cytokines and insulin on arginine uptake were observed in the adult myocyte phenotype. Also, uptake of arginine in both cell types, under control and cytokine-pretreated conditions, was sodium-independent as substitution of choline chloride for sodium chloride in the uptake buffer had no effect on arginine uptake (data not shown). To characterize the kinetics of L-arginine transport in ventricular myocytes, saturable uptake of L-arginine (0 –5 mM) was measured. The plot of uptake of L-arginine as a function of the extracellular L-arginine concentration is shown in Fig. 4. A high-affinity transporter having a Km of 125 6 19 mM and a


FIG. 3. Time course of L-arginine uptake by cytokine- and insulin-treated cardiac myocytes. Neonatal rat ventricular myocytes were exposed to either control medium alone for 24 h (f), to control medium containing 4 ng/ml rhIL-1b and 500 units/ml rmIFNg (●), to medium supplemented with 100 nM insulin (E), or the combination of cytokines and insulin (D). The uptake of 100 mM L-[3H]arginine was then initiated as described under “Experimental Procedures.” The data shown are from a representative experiment (repeated twice with similar results). Each point represents mean 6 S.E. from six replicates. The S.E. is less than 8.0 pmol/2 3 105 cells on those data points where the S.E. bars are not visible.

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maximum transport velocity (Vmax) of 44 6 2.4 pmol/2 3 105 cells/min was present in control cells (Fig. 4, A and B). Following a 24-h exposure to IL-1b and IFNg, arginine uptake increased (Vmax, 167 6 22 pmol/2 3 105 cells/min; Fig. 4C) and a second low-affinity transporter activity appeared (Km 5 1.2 mM), in addition to the high-affinity transport activity (Km 5 54 mM), as is evident from the biphasic Eadie and Hofstee plot in Fig. 4D. This suggested that there was cytokine regulation of the cationic amino acid transporters, given the previous report of a low-affinity (CAT-2A) member of this family (29, 30). Effects of IL-1b/IFNg and Insulin on Cationic Amino Acid Transporter mRNA Levels—To determine whether cytokines and/or insulin increase levels of mRNA for CAT, total RNA from adult and neonatal rat ventricular myocytes was analyzed by RNase protection assay. The riboprobes for murine CAT-1, -2A, and -2B and for 18 S ribosomal RNA, generated from the template cDNAs described under “Experimental Procedures,” protected fragments of the predicted size. The mRNA for the high-affinity arginine transporter, CAT-1, was expressed in control cells as shown in Fig. 5. Treatment of neonatal cardiac myocytes with IL-1b and IFNg for 24 h increased steady-state CAT-1 mRNA levels, normalized to 18 S

mRNA, by 10-fold as determined by densitometry. Insulin independently increased the expression of CAT-1 transcript and amplified the induction of CAT-1 by IL-1b and IFNg by 5-fold in both neonatal and adult cardiac myocyte phenotype. An independent effect of IL-1b and IFNg to induce CAT-1 by at least 2-fold was also observed in separate experiments in adult cardiac myocytes (data not shown). The mRNA coding for CAT-2B, a high-affinity cationic amino acid transporter originally described in activated macrophages and lymphocytes (25, 28), was only consistently detected in myocytes after a 24-h exposure to IL-1b and IFNg (Fig. 6). Note that in contrast to CAT-1, insulin did not alter the levels of mRNA for CAT-2B following exposure to the cytokines. The expression of the low-affinity transporter CAT-2A had previously been identified only in hepatocytes (29). However, cardiac myocytes were also observed to express CAT-2A mRNA (Fig. 6), but only consistently following a 24-h treatment with IL-1b and IFNg. This paralleled the induction by cytokines of a low-affinity L-arginine transport system as assessed by kinetic studies (Fig. 4, C and D). As was noted for CAT-2B, insulin alone or in combination with cytokines did not appear to have an independent effect on the levels of mRNA for CAT-2A.


FIG. 4. Concentration dependence of L-arginine uptake by cardiac myocytes. A, uptake of L-[3H]arginine by control neonatal rat ventricular myocytes was measured for 5 min over a range of concentrations (0.03–750 mM). The data shown are from a single experiment that is representative of three separate experiments. Each point is the mean 6 S.E. from 12 replicates. B, Eadie-Hofstee transformation of the data from A gave a Km of 125 mM and a Vmax of 44 pmol/2 3 105 cells/min. C, the effect of 24-h pretreatment with IL-1b and IFNg on the uptake of 3 L-[ H]-arginine by neonatal rat ventricular myocytes. Uptake of L-arginine was measured for 5 min over a range of L-arginine concentrations (30 nM to 5 mM). A 24-h exposure to IL-1b and IFNg increased both the Km to 655 6 233 mM and Vmax to 167 6 22 pmol/2 3 105 cells/min. Each point shown is the mean 6 S.E. from 12 replicates from a single experiment that was representative of three separate experiments. D, Eadie-Hofstee transformation of data from C demonstrated both a high- and a low-affinity transport component for L-arginine uptake; the high-affinity Km is 54 mM and Vmax of 56 pmol/2 3 105 cells/min, and the low-affinity Km is 1.2 mM with a Vmax of 213 pmol/2 3 105 cells/min.

L-Arginine


FIG. 7. Effect of cytokines and insulin on mRNA levels for NOS2, argininosuccinate synthetase, argininosuccinate lyase, and GTP cyclohydrolase I. Northern blots of total RNA from neonatal rat ventricular myocytes were analyzed as described under “Experimental Procedures.” Myocytes were treated for 24 h with control medium alone or medium supplemented with insulin (100 nM) in the presence and absence of IL-1b and IFNg. Equal loading of samples was confirmed by hybridizing the same membrane to a labeled oligonucleotide probe for 18 S ribosomal RNA. These blots are representative of three independent experiments. Autoradiograph exposure time was 24 h for all samples, except 18 S, which was exposed for 5 min.

zyme in tetrahydrobiopterin synthesis, or enzymes necessary for the endogenous synthesis of arginine (i.e. argininosuccinate synthetase and argininosuccinate lyase), Northern hybridization was performed on total RNA from cytokine- and insulinpretreated cells. As shown in Fig. 7, insulin had no effect on cytokine-induced NOS2 mRNA levels. The mRNAs for GTP cyclohydrolase I and argininosuccinate synthetase were coinduced with NOS2 by cytokines as described previously for other cell types (34 –37). There was no independent effect of insulin on the extent of induction of either of these enzymes by cytokines. Argininosuccinate lyase mRNA was detected in cardiac myocytes and its mRNA levels were not affected by exposure to cytokines and/or insulin. DISCUSSION

FIG. 6. Regulation of CAT-2B and CAT-2A mRNA by cytokines in cardiac myocytes. Primary cultures of neonatal rat ventricular myocytes were incubated for 24 h in control medium, or treated with IL-1b and IFNg, insulin, or the combination of IL-1b, IFNg, and insulin. Total RNA was analyzed by RNase protection with DNA sequencing reactions or a 100-bp RNA ladder (not shown) used to determine fragment sizes. Correction for differences in loading of samples was performed by co-hybridizing with a riboprobe for 18 S ribosomal RNA. This experiment was repeated four times with similar results. Autoradiograph exposure time was 3 weeks for CAT-2B and CAT-2A and 6 h for 18 S.

Northern Blots of mRNA for NOS2, GTP Cyclohydrolase I, Argininosuccinate Synthetase, and Argininosuccinate Lyase in Cardiac Myocytes—To determine whether insulin could alter mRNA levels for GTP cyclohydrolase I, the rate-limiting en-

These results indicate that the increase in sarcolemmal Larginine transport that accompanies the induction of NOS2 by the cytokines IL-1b and IFNg in rat cardiac myocytes is associated with increased expression of the high-affinity cationic amino acid transporter CAT-1, and co-induction of at least two additional transporters, CAT-2A and CAT-2B. Approximately two-thirds of cytokine-induced NO production (measured as nitrite released) was dependent on the transport of extracellular L-arginine, as determined by the ability of L-lysine to competitively inhibit L-arginine uptake and nitrite production by cardiac myocytes. Insulin, which independently increased CAT-1 mRNA levels and L-arginine transport in control myocytes, also enhanced L-arginine transport and nitrite production in cytokine-pretreated cells. Whenever possible technically, experimental protocols were performed in both neonatal and adult ventricular myocyte primary cultures. No qualitative differences were observed between these two myocyte phenotypes with regard to the cytokine- and/or insulin-induced effects described here. Specifically, there were no qualitative differences in NOS activity, nitrite accumulation, arginine uptake, and CAT-1 mRNA expression. The enzymatic activity of NOS2 in homogenates of most cell types in which it has been examined appears to be regulated


FIG. 5. Regulation of CAT-1 mRNA by cytokines and insulin in cardiac myocytes. Total RNA from primary cultures of neonatal and adult rat ventricular myocytes was isolated following a 24-h incubation in control medium alone, or following treatment with IL-1b and IFNg, insulin, or the combination of IL-1b, IFNg, and insulin. CAT-1 mRNA was detected by RNase protection analysis. DNA sequencing reactions or a 100-bp RNA ladder (not shown) were used to determine fragment sizes. Correction for differences in loading of samples was performed by co-hybridizing with a riboprobe for 18 S ribosomal RNA. This experiment was repeated five times with similar results. Autoradiograph exposure time was 24 h for CAT-1 and 3 h for 18 S.

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M. Y. Park, E. I. Closs, and J. M. Cunningham, unpublished data.

types (60), the stimulation of cationic amino acid transport by insulin has not previously been documented. Interestingly, Larginine has long been known to regulate insulin secretion by pancreatic islet b cells, an effect that may be mediated by NO (61, 62). In cardiac myocytes, we observed that insulin-dependent L-arginine uptake was associated with enhanced expression of CAT-1 mRNA. Wu et al. (63) also reported that insulin increases the expression of the ecotropic retrovirus receptor, now known to be identical to CAT-1 (24, 26), in FTO2B rat hepatoma cells although these authors did not examine the transport of cationic amino acids. In cardiac myocytes, the effect of insulin on L-arginine uptake and CAT-1 mRNA levels was additive to that of IL-1b and IFNg. This effect of insulin to increase arginine transport contributed, in part, to increased cytokine-induced nitrite production in cardiac myocytes, although it appears likely that insulin may potentiate cellular NO production by additional mechanisms that are independent of NOS activity. This provides one possible mechanism for our previous observation that insulin is required for the impaired inotropic response of ARVM to the b-adrenergic agonist isoproterenol following the induction of NOS2 in vitro (15), and for the previously reported negative inotropic effect of insulin in an in vivo canine model of sepsis (64). Furthermore, enhanced L-arginine uptake into vascular smooth muscle cells, which are known to express NOS2, may contribute to the insulin-induced hypotension in vivo in the canine sepsis model (65). The additive effects of insulin and IL-1b plus IFNg to increase CAT-1 expression distinguish this transporter from the glucose transport system, in which cytokines such as IL-1 and tumor necrosis factor a, as well as lipopolysaccharide, impair insulin-mediated glucose uptake (66, 67). In vitro exposure to tumor necrosis factor a or IFNg have been shown to suppress insulin-induced tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 (68, 69), glucose uptake (64), and expression of the glucose transporters GLUT4 and GLUT1 (70, 71). This suggests post-receptor divergence of insulin signaling for the CAT-1 and glucose transport systems. Downstream divergence may occur at the ras-raf mitogen-activated protein kinase and phosphoinositide 3-kinase pathways, since both pathways are activated by insulin, but the latter has been shown to be required for glucose uptake via the insulin-sensitive GLUT4 glucose transporter and is independent of activation of mitogen-activated protein kinase (72, 73). The additive effects of insulin and IL-1b plus IFNg on CAT-1 expression is consistent with this explanation since the IL-1b plus IFNg also activates p44/p42 mitogen-activated protein kinase (ERK1/ERK2) in cardiac myocytes (74). The regulation of CAT-1-mediated L-arginine transport by insulin may have broader implications for the cardiovascular system as this transporter is constitutively expressed in a wide variety of tissues (24, 75), and y1 transport activity has been demonstrated both in endothelial cells (76, 77) and vascular smooth muscle cells (55, 78). Insulin has been demonstrated to cause vasodilation that is NO-dependent (79, 80), although the mechanism(s) by which it stimulates NO release have been unclear. It is possible that insulin-mediated L-arginine transport also could potentially contribute to the regulation of the generation of NO by the endothelial constitutive NO synthase (ecNOS, NOS3) and that abnormalities in insulin-induced Larginine transport could lead to altered vascular function and hypertension as in non-insulin-dependent diabetes mellitus. A limitation of NO release caused by decreased L-arginine transport provides one possible explanation for the improvement in endothelium-dependent vasodilation with the systemic administration of L-arginine producing plasma concentrations of L-


mainly at the transcriptional level, although post-transcriptional and post-translational regulatory mechanisms have been described (54). However, the observation that L-lysine diminishes cytokine-induced NO production by intact cells indicates that L-arginine transport is limiting in NO synthesis in cardiac myocytes. This dependence of myocyte NO generation on extracellular L-arginine has also been observed in activated murine macrophages (16, 17, 55) and in cytokine-pretreated rat vascular smooth muscle cells (18, 56). However, the specific cellular mechanisms responsible for increased arginine transport were unknown. In studies of the kinetics of nitrite production by NOS2 in activated macrophages over a range of extracellular L-arginine concentrations, the Km for L-arginine in intact cells was 73 to 150 mM (16, 17). This value in intact cells is significantly greater than the Km for L-arginine that has been reported for the isolated enzyme (57) and is close in magnitude to both the plasma arginine concentration (100 mM) (53) and the Km reported for the high-affinity L-arginine transporters CAT-1 and CAT-2B (24 –26, 30). These observations, combined with the data in this report, indicate that L-arginine transport may be an important regulatory mechanism for determining the rate of NO production by NOS2 in cardiac myocytes and other cell types as well. The transport properties of CAT-1, which is constitutively expressed in neonatal and adult cardiac myocytes, and the cytokine-inducible CAT-2B transporter, have previously been identified to be characteristic of the y1 amino acid transporter phenotype (24 –27, 30). CAT-1 mRNA is constitutively expressed in a variety of tissues, with the exception of the liver (24), but it has not previously been shown to be regulated by inflammatory cytokines. This enhanced expression of CAT-1 and NOS2 by cytokines in cardiac myocytes may have added significance due to the widespread tissue distribution of CAT-1, and the growing number of cell types in which NOS2 induction has been observed. Expression of CAT-2B was initially detected in activated murine macrophages and lymphocytes (25, 28), and the demonstration of its expression in cytokine-pretreated cardiac myocytes indicates that it also has a wider tissue distribution than original reports had indicated. In addition to these high-affinity cationic amino acid transporters, the kinetic studies reported here show that cytokinepretreated cardiac myocytes also exhibit induction of a lowaffinity uptake system for L-arginine that coincided with the expression of CAT-2A mRNA. CAT-2A has previously been shown to have the same specificity for cationic amino acids as CAT-1, but with at least a 10-fold greater Km and less sensitivity to trans-stimulation (29, 30). CAT-2A had been thought to be constitutively expressed only in hepatocytes, where it was presumed to mediate the uptake of cationic amino acids from the portal circulation after a protein meal (29). The regulation of CAT-2A by cytokines has also been observed in murine macrophages.2 However, the expression of the low-affinity cationic amino acid transporter in response to cytokines in neonatal cardiac myocytes (or any cell type other than hepatocytes) may be of little physiologic significance given that the highaffinity CAT-1 and CAT-2B transporters would mediate most of the uptake of L-arginine at usual mammalian plasma concentrations of this amino acid. In cardiac muscle, as in many other cell types, insulin regulates a variety of key metabolic functions including transmembrane amino acid transport and protein turnover (58, 59). While the transport of small aliphatic amino acids into cells by the sodium-dependent amino acid transporter System A has been shown to be up-regulated by insulin in a wide range of cell

L-Arginine


3

J. M. Cunningham, unpublished data.

In summary, cellular NO production by cytokine-pretreated cardiac myocytes is determined not only by the extent of transcriptional induction of NOS2, but also by intracellular arginine availability and is dependent on transmembrane arginine transport. Thus, there are multiple potential sites whereby NO production may be regulated in intact cells, and the actual site of regulation may vary for different cell types. This study indicates that increased L-arginine transport following cytokine exposure correlates with the coordinate induction of CAT-1, CAT-2B, and CAT-2A with NOS2 and that arginine transport is necessary for as much as two-thirds of cellular NO production. Furthermore, the fact that changes in NO production do not always reflect changes in NOS enzyme levels is exemplified by the effect of insulin to induce CAT-1 and stimulate L-arginine transport, which appears to contribute to increased cytokine-induced NO production in cardiac myocytes. Acknowledgment—We thank Mary Y. Park for technical assistance. REFERENCES 1. Lyons, C. R., Orloff, G. J., and Cunningham, J. M. (1992) J. Biol. Chem. 267, 6370 – 6374 2. Xie., Q., Cho, H. J., Calaycay, J., Mumford, R. A., Swiderek, K. M., Lee, T. D., Ding, A., Troso, T., and Nathan, C. (1992) Science 256, 225–228 3. Nathan, C., and Xie, Q. (1994) Cell 78, 915–918 4. Schulz, R., Nava, E., and Moncada, S. (1992) Br. J. Pharmacol. 105, 575–580 5. Roberts, A. B., Vodovotz, Y., Roche, N. S., Sporn, M. B., and Nathan, C. F. (1992) Mol. Endocrinol. 6, 1921–1930 6. Balligand, J.-L., Ungureanu-Longrois, D., Simmons, W. W., Pimental, D., Malinski, T. A., Kapturczak, M., Taha, Z., Lowenstein, C. J., Davidoff, A. J., Kelly, R. A., Smith, T. W., and Michel, T. (1994) J. Biol. Chem. 269, 27580 –27588 7. Balligand, J.-L., Kelly, R. A., Marsden, P. A., Smith, T. W., and Michel, T. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 347–351 8. Finkel, M. S., Oddis, C. V., Jacob, T. D., Watkins, S. C., Hattler, B. G., and Simmons. R. L. (1992) Science 257, 387–389 9. Brady, A. J. B., Poole-Wilson, P. A., Harding, S. E., and Warren, J. B. (1992) Am. J. Physiol. 263, H1963-H1966 10. Balligand, J.-L., Ungureanu, D., Kelly, R. A., Kobzik, L., Pimental, D., Michel, T., and Smith, T. W. (1993) J. Clin. Invest. 91, 2314 –2319 11. Brady, A. J. B., Warren, J. B., Poole-Wilson, P. A., Williams, T. J., and Harding, S. E. (1993) Am. J. Physiol. 265, H176-H182 12. Balligand, J.-L., Kobzik, L., Han, X., Kaye, D. M., Belhassen, L., O’Hara, D. S., Kelly, R. A., Smith, T. W., and Michel, T. (1995) J. Biol. Chem. 270, 14582–14586 13. Grocott-Mason, R., Fort, S., Lewis, M. J., and Shah, A. M. (1994) Am. J. Physiol. 266, H1699-H1705 14. Grocott-Mason, R., Anning, P., Evans, H., Lewis, M. J., and Shah, A. M. (1994) Am. J. Physiol. 267, H1804-H1813 15. Ungureanu-Longrois, D., Balligand, J.-L., Simmons, W. W., Okada, I., Kobzik, L., Lowenstein, C. J., Kunkel, S., Michel, T., Kelly, R. A., and Smith, T. W. (1995) Circ. Res. 77, 486 – 493 16. Iyengar, R., Stuehr, D. J., and Marletta, M. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 6369 – 6373 17. Granger, D. L., Hibbs, J. B., Jr., Perfect, J. R., and Durack, D. T. (1990) J. Clin. Invest. 85, 264 –273 18. Beasley, D., Schwartz, J. H., and Brenner, B. M. (1991) J. Clin. Invest. 87, 602– 608 19. Bune, A. J., Shergill, J. K., Cammack, R., and Cook, H. T. (1995) FEBS Lett. 366, 127–130 20. Schott, C. A., Gray, G. A., and Stoclet, J.-C. (1993) Br. J. Pharmacol. 108, 38 – 43 21. White, M. F., Gazzola, G. C., and Christensen, H. N. (1982) J. Biol. Chem. 257, 4443– 4449 22. White, M. F., and Christensen, H. N. (1982) J. Biol. Chem. 257, 4450 – 4457 23. Albritton, L. M., Tseng, L., Scadden, D., and Cunningham, J. M. (1989) Cell 57, 659 – 666 24. Kim, J. W., Closs, E. I., Albritton, L. M., and Cunningham, J. M. (1991) Nature 352, 725–728 25. Closs, E. I., Lyons, C. R., Kelly, C., and Cunningham, J. M. (1993) J. Biol. Chem. 268, 20796 –20800 26. Wang, H., Kavanaugh, M. P., North, R. A., and Kabat, D. (1991) Nature 352, 729 –731 27. Kavanaugh, M. P. (1993) Biochemistry 32, 5781–5785 28. MacLeod, C. L., Finley, K., Kakuda, D., Kozak, C. A., and Wilkinson, M. F. (1990) Mol. Cell. Biol. 10, 3663–3674 29. Closs, E. I., Albritton, L. M., Kim, J. W., and Cunningham, J. M. (1993) J. Biol. Chem. 268, 7538 –7544 30. Kavanaugh, M. P., Wang, H., Zhang, Z., Zhang, W., Wu, Y.-N., Dechant, E., North, R. A., and Kabat, D. (1994) J. Biol. Chem. 269, 15445–15450 31. Morris, S. M., Jr. (1992) Annu. Rev. Nutr. 12, 81–101 32. Wu, G., and Brosnan, J. T. (1992) Biochem. J. 281, 45– 48 33. Hecker, M., Sessa, W. C., Harris, H. J., Anggard, E. E., and Vane, J. R. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 8612– 8616 34. Nussler, A. K., Billiar, T. R., Liu, Z.-Z., and Morris, S. M., Jr. (1994) J. Biol. Chem. 269, 1257–1261


arginine far in excess of the Km for the purified NO synthase (81, 82). In addition to enhancing L-arginine transport, inflammatory cytokines and/or insulin could also affect the de novo synthesis of L-arginine within cardiac myocytes. The expression of mRNA for argininosuccinate synthetase, the rate-limiting enzyme in arginine synthesis, was induced in cardiac myocytes by IL-1b and IFNg, as has been previously reported in other cell types (34, 35). In contrast, argininosuccinate lyase mRNA was constitutively expressed in these cells and was unaffected by these cytokines. Insulin, either alone or in combination with cytokines, had no effect on mRNA levels for these two enzymes that together convert citrulline to arginine. Previous studies of cultured hepatocytes also have shown that insulin alone has no effect on the mRNA levels or on the activities of either argininosuccinate synthetase or argininosuccinate lyase (83), and the activities of these enzymes are not known to be modulated by post-translational modifications (31). Arginase mRNA was undetectable under any conditions in cardiac myocytes, consistent with the absence of a complete urea cycle in this cell type (31). Therefore, recycling of citrulline to arginine may be one source of arginine for the approximately one-third of cytokine-induced NO production that is transport-independent, although it is unlikely that insulin is mediating its effects through this mechanism. The regulation of GTP cyclohydrolase I, the rate-limiting enzyme for BH4 synthesis, a necessary co-factor for NOS2 activity, by cytokines and by insulin were also assessed in cardiac myocytes. Insulin-induced hypoglycemia in rats had previously been reported to increase the activity of GTP cyclohydrolase I, the rate-limiting enzyme in the de novo BH4 synthesis pathway, as well as BH4 levels in adrenal cells (84). However, a direct effect of insulin on BH4 or GTP cyclohydrolase I mRNA levels has not been addressed. Following exposure to IL-1b and IFNg, GTP cyclohydrolase I mRNA was co-induced with NOS2 in rat ventricular myocytes, as we have previously reported (6), but insulin had no effect on mRNA levels of either enzyme, with or without IL-1b and IFNg. Thus, increased expression of GTP cyclohydrolase I mRNA does not appear to be contributing to insulin’s action of augmenting cytokine-induced NO production. A limitation of the current study is that arginine uptake rates and arginine uptake-dependent NO production are only correlated with the expression of mRNA for CAT-1, CAT-2B, and CAT-2A. Support for this correlation representing a causal relationship between CAT-mediated L-arginine uptake and NO production in these cells includes the following. 1) The detection of CAT-2A transcripts, the only low affinity cationic amino acid transporter identified to date, in cytokine-pretreated myocytes coincided with the detection of a low affinity transporter based on L-arginine kinetic studies. 2) The sodium independence of arginine uptake is consistent with the CAT family of transporters. 3) Reconstitution experiments in Xenopus oocytes have demonstrated that the dependence of nitrite production by transfected NOS2 is dependent upon co-transfection and expression of the CAT transporters.3 4) While other transport systems have been defined in physiologic terms (60, 85), the only other cationic amino acid transporters cloned and sequenced to date, D2 (86) and 4F2 (87), are unlikely to have contributed to L-arginine uptake in our cells. D2 expression is limited to the kidney and intestine (86), and the 4F2 transporter has one-tenth the activity of the CAT family of transporters and could not account for the kinetic data in this report (87).

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Cell Biology and Metabolism: Cytokines and Insulin Induce Cationic Amino Acid Transporter (CAT) Expression in Cardiac Myocytes: REGULATION OF L-ARGININE TRANSPORT AND NO PRODUCTION BY CAT-1, CAT-2A, AND CAT-2B William W. Simmons, Ellen I. Closs, James M. Cunningham, Thomas W. Smith and Ralph A. Kelly

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J. Biol. Chem. 1996, 271:11694-11702. doi: 10.1074/jbc.271.20.11694