Cat2 L-arginine transporter-deficient fibroblasts can sustain nitric oxide production.

High-output nitric oxide (NO) production by nitric oxide synthase 2 (NOS2) contributes to normal cellular processes and pathophysiological conditions. The transport of L-arginine, the substrate for NOS2, is required for sustained NO production by NOS2. L-Arginine can be transported by several kinetically defined transport systems, although the majority of arginine uptake is mediated by transport system y(+), encoded by the Cat1-3 gene family. Using macrophages from Cat2-deficient mice, we previously determined that arginine uptake via CAT2 is absolutely required for sustained NO production. Because NO production by fibroblasts is important in wound healing, we sought to determine whether CAT2 is required for NO production in cytokine-stimulated Cat2-deficient and wild-type embryonic fibroblasts. Although macrophages and fibroblasts both required extracellular L-arginine for NO production, NO synthesis by activated Cat2(-/-) fibroblasts was reduced only 19%, whereas Cat2(-/-) macrophages were virtually unable to produce NO. As expected, activated Cat2(-/-) fibroblasts had reduced system y(+)-mediated arginine uptake. However, their reduced NO output was not the result of a significant difference in intracellular L-arginine levels following cytokine stimulation. Uptake experiments revealed that the L-arginine transport system y(+)L was the major cationic amino acid carrier in fibroblasts of both genotypes. We conclude that NO production in embryonic fibroblasts is only partially dependent on CAT2 and that other compensating transporters provide arginine for NOS2-mediated NO synthesis. The data demonstrate that fibroblasts and macrophages have differential dependence on CAT2-mediated L-arginine transport for NO synthesis. The important physiological implication of this finding is discussed.     

CAT2 arginine transporter deficiency significantly reduces iNOS-mediated NO production in astrocytes

We have previously demonstrated that genetic ablation of cationic amino acid transporter 2 (Cat2) significantly inhibits nitric oxide (NO) production by inducible nitric oxide synthase (iNOS) in activated macrophages. Here we report that iNOS activity is impaired by 84% in activated Cat2-deficient astrocytes. Cat2 ablation appears to reduce astrocyte NO synthesis by decreasing the uptake of the sole precursor, arginine, as well as by reducing the expression of iNOS following activation. Excessive or dysregulated NO production by activated astrocytes and other CNS cell types has been implicated in the pathogenesis of neurological disorders. Our results support the idea that manipulation of CAT2 transporter function might be useful for the therapeutic modulation of iNOS activity.     

Regulation of nitric oxide production by arginine metabolic enzymes

Nitric oxide (NO) is synthesized from arginine by NO synthase (NOS), and the availability of arginine is one of the rate-limiting factors in cellular NO production. Citrulline, which is formed as a by-product of the NOS reaction, can be recycled to arginine by successive actions of argininosuccinate synthetase (AS) and argininosuccinate lyase (AL), forming the citrulline-NO cycle. AS and sometimes AL have been shown to be coinduced with inducible NOS (iNOS) in various cell types including activated macrophages, vascular smooth muscle cells, glial cells, neuronal PC12 cells, and pancreatic beta-cells. Cationic amino acid transporter (CAT)-2 is induced in activated macrophages but not in PC12 cells. On the other hand, arginase can downregulate NO production by decreasing intracellular arginine concentrations. iNOS and arginase activities are regulated reciprocally in macrophages by cytokines, and this may guarantee the efficient production of NO. In contrast, iNOS and arginase isoforms (type I and II) are coinduced in lipopolysaccharide (LPS)-activated macrophages. These results indicate that NO production is modulated by the uptake, recycling, and degradation of arginine.     

Characterization of L-arginine transporters in rat renal inner medullary collecting duct

Previous work from our laboratory has demonstrated that the inner medullary collecting duct (IMCD) expresses a large amount of nitric oxide synthase (NOS) activity. The present study was designed to characterize the transport of NOS substrate, L-arginine, in a suspension of bulk-isolated IMCD cells from the Sprague-Dawley rat kidney. Biochemical transport studies demonstrated an L-arginine transport system in IMCD cells that was saturable and Na(+) independent (n = 6). L-Arginine uptake by IMCD cells was inhibited by the cationic amino acids L-lysine, L-homoarginine, and L-ornithine (10 mmol/l each) and unaffected by the neutral amino acids L-leucine, L-serine, and L-glutamine. Both L-ornithine (n = 6) and L-lysine (n = 6) inhibited NOS enzymatic activity in a dose-dependent manner in IMCD cells, supporting the important role of L-arginine transport for NO production by this tubular segment. Furthermore, RT-PCR of microdissected IMCD confirmed the presence of cationic amino acid transporter CAT1 mRNA, whereas CAT2A, CAT2B, and CAT3 were not detected. These results indicate that L-arginine uptake by IMCD cells occurs via system y(+), is encoded by CAT1, and may participate in the regulation of NO production in this renal segment.     

Cationic amino acid transporter 2 enhances innate immunity during Helicobacter pylori infection

Once acquired, Helicobacter pylori infection is lifelong due to an inadequate innate and adaptive immune response. Our previous studies indicate that interactions among the various pathways of arginine metabolism in the host are critical determinants of outcomes following infection. Cationic amino acid transporter 2 (CAT2) is essential for transport of L-arginine (L-Arg) into monocytic immune cells during H. pylori infection. Once within the cell, this amino acid is utilized by opposing pathways that lead to elaboration of either bactericidal nitric oxide (NO) produced from inducible NO synthase (iNOS), or hydrogen peroxide, which causes macrophage apoptosis, via arginase and the polyamine pathway. Because of its central role in controlling L-Arg availability in macrophages, we investigated the importance of CAT2 in vivo during H. pylori infection. CAT2(-/-) mice infected for 4 months exhibited decreased gastritis and increased levels of colonization compared to wild type mice. We observed suppression of gastric macrophage levels, macrophage expression of iNOS, dendritic cell activation, and expression of granulocyte-colony stimulating factor in CAT2(-/-) mice suggesting that CAT2 is involved in enhancing the innate immune response. In addition, cytokine expression in CAT2(-/-) mice was altered from an antimicrobial Th1 response to a Th2 response, indicating that the transporter has downstream effects on adaptive immunity as well. These findings demonstrate that CAT2 is an important regulator of the immune response during H. pylori infection.     

Effects of dietary salt intake on plasma arginine

Because L-arginine is degraded by hepatic arginase to ornithine and urea and is transported by the regulated 2A cationic amino acid y(+) transporter (CAT2A), hepatic transport may regulate plasma arginine concentration. Groups of rats (n = 6) were fed a diet of either low salt (LS) or high salt (HS) for 7 days to test the hypothesis that dietary salt intake regulates plasma arginine concentration and renal nitric oxide (NO) generation by measuring plasma arginine and ornithine concentrations, renal NO excretion, and expression of hepatic CAT2A, and arginase. LS rats had lower excretion of NO metabolites and cGMP, lower plasma arginine concentration (LS: 83 +/- 7 vs. HS: 165 +/- 10 micromol/l, P < 0.001), but higher plasma ornithine concentration (LS: 82 +/- 6 vs. HS: 66 +/- 4 micromol/l, P < 0.05) and urea excretion. However, neither the in vitro hepatic arginase activity nor the mRNA for hepatic arginase I was different between groups. In contrast, LS rats had twice the abundance of mRNA for hepatic CAT2A (LS: 3.4 +/- 0.4 vs. HS: 1.6 +/- 0.5, P < 0.05). The reduced plasma arginine concentration with increased plasma ornithine concentration and urea excretion during LS indicates increased arginine metabolism by arginase. This cannot be ascribed to changes in hepatic arginase expression but may be a consequence of increased hepatic arginine uptake via CAT2A.     

Characterization of l-Arginine and Aminoguanidine Uptake into Isolated Rat Choroid Plexus: Differences in Uptake Mechanisms and Inhibition by Nitric Oxide Synthase Inhibitors

Abstract: Recent reports suggest that nitric oxide (NO) may contribute to several neurodegenerative diseases, e.g., focal cerebral ischemia, N -methyl- d -aspartate-mediated neurotoxicity, and experimental autoimmune encephalomyelitis. Accordingly, an understanding of the CNS transport processes of NO synthase (NOS) inhibitors has important therapeutic implications. The objective of the present study was to characterize the in vitro transport processes governing the uptake of l -[¹⁴C]arginine and the NOS inhibitor [¹⁴C]aminoguanidine in rat choroid plexus tissue. Consistent with previous reports, the uptake of l -[¹⁴C]arginine was mediated by both saturable and nonsaturable processes and was inhibited by the NOS inhibitors N ^G-methyl- l -arginine, N ^G-amino- l -arginine, and N ⁵-imidoethyl- l -ornithine. l -[¹⁴C]Arginine uptake was not inhibited by aminoguanidine or N ^G-nitro- l -arginine. Because aminoguanidine is an organic cation that bears some structural similarity to l -arginine, aminoguanidine might be transported by either an organic cation transporter or by the basic amino acid transporter governing arginine uptake. However, there was no evidence of a saturable uptake process for [¹⁴C]aminoguanidine in isolated rat choroid plexus, in contrast to that observed for l -[¹⁴C]arginine.     

An inhibitor of macrophage arginine transport and nitric oxide production (CNI-1493) prevents acute inflammation and endotoxin lethality.

BACKGROUND: Nitric oxide (NO), a small effector molecule produced enzymatically from L-arginine by nitric oxide synthase (NOS), is a mediator not only of important homeostatic mechanisms (e.g., blood vessel tone and tissue perfusion), but also of key aspects of local and systemic inflammatory responses. Previous efforts to develop inhibitors of NOS to protect against NO-mediated tissue damage in endotoxin shock have been unsuccessful, largely because such competitive NOS antagonists interfere with critical vasoregulatory NO production in blood vessels and decrease survival in endotoxemic animals. Accordingly, we sought to develop a pharmaceutical approach to selectively inhibit NO production in macrophages while sparing NO responses in blood vessels. MATERIALS AND METHODS: The process of cytokine-inducible L-arginine transport and NO production were studied in the murine macrophage-like cell line (RAW 264.7). A series of multivalent guanylhydrazones were synthesized to inhibit cytokine-inducible L-arginine transport. One such compound (CNI-1493) was studied further in animal models of endothelial-derived relaxing factor (EDRF) activity, carrageenan inflammation, and lethal lipopolysaccharide (LPS) challenge. RESULTS: Upon activation with cytokines, macrophages increase transport of L-arginine to support the production of NO by NOS. Since endothelial cells do not require this additional arginine transport to produce NO, we reasoned that a competitive inhibitor of cytokine-inducible L-arginine transport would not inhibit EDRF activity in blood vessels, and thus might be effectively employed against endotoxic shock. CNI-1493, a tetravalent guanylhydrazone, proved to be a selective inhibitor of cytokine-inducible arginine transport and NO production, but did not inhibit EDRF activity. In mice, CNI-1493 prevented the development of carrageenan-induced footpad inflammation, and conferred protection against lethal LPS challenge. CONCLUSIONS: A selective inhibitor of cytokine-inducible L-arginine transport that does not inhibit vascular EDRF responses is effective against endotoxin lethality and significantly reduces inflammatory responses.     

Arginine transport via cationic amino acid transporter 2 plays a critical regulatory role in classical or alternative activation of macrophages

Arginine is processed by macrophages in response to the cytokines to which these cells are exposed. Th1-type cytokines induce NO synthase 2, which metabolizes arginine into nitrites, while the Th2-type cytokines produce arginase, which converts arginine into polyamines and proline. Activation of bone marrow-derived macrophages by these two types of cytokines increases L-arginine transport only through the y(+) system. Analysis of the expression of the genes involved in this system showed that Slc7A1, encoding cationic amino acid transporters (CAT)1, is constitutively expressed and is not modified by activating agents, while Slc7A2, encoding CAT2, is induced during both classical and alternative activation. Macrophages from Slc7A2 knockout mice showed a decrease in L-arginine transport in response to the two kinds of cytokines. However, while NO synthase 2 and arginase expression were unmodified in these cells, the catabolism of arginine was impaired by both pathways, producing smaller amounts of nitrites and also of polyamines and proline. In addition, the induction of Slc7A2 expression was independent of the arginine available and of the enzymes that metabolize it. In conclusion, the increased arginine transport mediated by activators is strongly regulated by CAT2 expression, which could limit the function of macrophages.     

Role of nitric oxide synthase type 2 in acute infection with murine cytomegalovirus

Whether or not NO plays a critical role in murine CMV (MCMV) infection has yet to be elucidated. In this study, we examined the role of NO in acute infection with MCMV using NO synthase type 2 (NOS2)-deficient mice. NOS2(-/-) mice were more susceptible to lethal infection with MCMV than NOS2(+/+) mice and generated a much higher peak virus titer in the salivary gland after acute infection. A moderate increase in the MCMV titer was also observed in other organs of NOS2(-/-) mice such as the spleen, lung, and liver. The immune responses to MCMV infection including NK cell cytotoxicity and CTL response in NOS2(-/-) mice were comparable with those of NOS2(+/+) mice. Moreover, the ability to produce IFN-gamma is not impaired in NOS2(-/-) mice after MCMV infection. The peritoneal macrophages from NOS2(-/-) mice, however, exhibited a lower antiviral activity than those from NOS2(+/+) mice, resulting in an enhanced viral replication in macrophages themselves. Treatment of these cells from NOS2(+/+) mice with a selective NOS2 inhibitor decreased the antiviral activity to a level below that obtained with NOS2(-/-) mice. In addition, the absence of NOS2 and NOS2-mediated antiviral activity of macrophages resulted in not only an enhanced MCMV replication and a high mortality but also a consequent risk of the latency. It was thus concluded that the NOS2-mediated antiviral activity of macrophages via NO plays a protective role against MCMV infection at an early and late stage of the infection.     

Lipopolysaccharide-induced apoptosis of macrophages determines the up-regulation of concentrative nucleoside transporters Cnt1 and Cnt2 through tumor necrosis factor-alpha-dependent and -independent mechanisms

In murine bone marrow macrophages, lipopolysaccharide (LPS) induces apoptosis through the autocrine production of tumor necrosis factor-alpha (TNF-alpha), as demonstrated by the fact that macrophages from TNF-alpha receptor I knock-out mice did not undergo early apoptosis. In these conditions LPS up-regulated the two concentrative high affinity nucleoside transporters here shown to be expressed in murine bone marrow macrophages, concentrative nucleoside transporter (CNT) 1 and 2, in a rapid manner that is nevertheless consistent with the de novo synthesis of carrier proteins. This effect was not dependent on the presence of macrophage colony-stimulating factor, although LPS blocked the macrophage colony-stimulating factor-mediated up-regulation of the equilibrative nucleoside transport system es. TNF-alpha mimicked the regulatory response of nucleoside transporters triggered by LPS, but macrophages isolated from TNF-alpha receptor I knock-out mice similarly up-regulated nucleoside transport after LPS treatment. Although NO is produced by macrophages after LPS treatment, NO is not involved in these regulatory responses because LPS up-regulated CNT1 and CNT2 transport activity and expression in macrophages from inducible nitric oxide synthase and cationic amino acid transporter (CAT) 2 knock-out mice, both of which lack inducible nitric oxide synthesis. These data indicate that the early proapoptotic responses of macrophages, involving the up-regulation of CNT transporters, follow redundant regulatory pathways in which TNF-alpha-dependent- and -independent mechanisms are involved. These observations also support a role for CNT transporters in determining extracellular nucleoside availability and modulating macrophage apoptosis.     

Cationic amino acid transporter-2 regulates immunity by modulating arginase activity

Cationic amino acid transporters (CAT) are important regulators of NOS2 and ARG1 activity because they regulate L-arginine availability. However, their role in the development of Th1/Th2 effector functions following infection has not been investigated. Here we dissect the function of CAT2 by studying two infectious disease models characterized by the development of polarized Th1 or Th2-type responses. We show that CAT2(-/-) mice are significantly more susceptible to the Th1-inducing pathogen Toxoplasma gondii. Although T. gondii infected CAT2(-/-) mice developed stronger IFN-gamma responses, nitric oxide (NO) production was significantly impaired, which contributed to their enhanced susceptibility. In contrast, CAT2(-/-) mice infected with the Th2-inducing pathogen Schistosoma mansoni displayed no change in susceptibility to infection, although they succumbed to schistosomiasis at an accelerated rate. Granuloma formation and fibrosis, pathological features regulated by Th2 cytokines, were also exacerbated even though their Th2 response was reduced. Finally, while IL-13 blockade was highly efficacious in wild-type mice, the development of fibrosis in CAT2(-/-) mice was largely IL-13-independent. Instead, the exacerbated pathology was associated with increased arginase activity in fibroblasts and alternatively activated macrophages, both in vitro and in vivo. Thus, by controlling NOS2 and arginase activity, CAT2 functions as a potent regulator of immunity.     

Apolipoprotein E acts to increase nitric oxide production in macrophages by stimulating arginine transport

Previous studies have shown that apolipoprotein E (apoE) plays a role in immune function by modulating tissue redox balance. Using a mouse macrophage cell line (RAW 264.7), we have examined the mechanism by which apoE regulates nitric oxide (NO) production in macrophages. ApoE potentiates NO production in immune activated RAW cells in combination with lipopolysaccharide or polyinosinic:polycytidylic acid (PIC), agents known to induce expression of inducible nitric oxide synthase mRNA and protein. The effect is not observed with apolipoprotein B or heat-inactivated apoE. The combination of PIC plus apoE produced more NO than the level expected from an additive effect of PIC and apoE alone. Furthermore, this increase was observed at submaximal extracellular arginine concentrations, suggesting that apoE altered arginine (substrate) availability. Examination of [(3)H]arginine uptake across the cell membrane demonstrated that arginine uptake was increased by PIC but further increased by PIC plus apoE. Treatment of RAW cells with apoE was associated with an increased apparent V(max) and decreased affinity for arginine as well as a switch in the induction of mRNA for subtypes of cationic amino acid transporters (CAT). Treatment of RAW cells with PIC plus apoE resulted in the loss of detectable CAT1 mRNA and expression of CAT2 mRNA. Regulation of arginine availability is a novel action of apoE on the regulation of macrophage function and the immune response.     

Pertussis toxin activates L-arginine uptake in pulmonary endothelial cells through downregulation of PKC-alpha activity

Pertussis toxin (PTX) induces activation of l-arginine transport in pulmonary artery endothelial cells (PAEC). The effects of PTX on l-arginine transport appeared after 6 h of treatment and reached maximal values after treatment for 12 h. PTX-induced changes in l-arginine transport were not accompanied by changes in expression of cationic amino acid transporter (CAT)-1 protein, the main l-arginine transporter in PAEC. Unlike holotoxin, the beta-oligomer-binding subunit of PTX did not affect l-arginine transport in PAEC, suggesting that Galpha(i) ribosylation is an important step in the activation of l-arginine transport by PTX. An activator of adenylate cyclase, forskolin, and an activator of protein kinase A (PKA), Sp-cAMPS, did not affect l-arginine transport in PAEC. In addition, inhibitors of PKA or adenylate cyclase did not change the activating effect of PTX on l-arginine uptake. Long-term treatment with PTX (18 h) induced a 40% decrease in protein kinase C (PKC)-alpha but did not affect the activities of PKC-epsilon and PKC-zeta in PAEC. An activator of PKC-alpha, phorbol 12-myristate 13-acetate, abrogated the activation of l-arginine transport in PAEC treated with PTX. Incubation of PTX-treated PAEC with phorbol 12-myristate 13-acetate in combination with an inhibitor of PKC-alpha (Go 6976) restored the activating effects of PTX on l-arginine uptake, suggesting PTX-induced activation of l-arginine transport is mediated through downregulation of PKC-alpha. Measurements of nitric oxide (NO) production by PAEC revealed that long-term treatment with PTX induced twofold increases in the amount of NO in PAEC. PTX also increased l-[(3)H]citrulline production from extracellular l-[(3)H]arginine without affecting endothelial NO synthase activity. These results demonstrate that PTX increased NO production through activation of l-arginine transport in PAEC.     

Reduced citrulline availability by OTC deficiency in mice is related to reduced nitric oxide production

The amino acid arginine is the sole precursor for nitric oxide (NO) synthesis. We recently demonstrated that an acute reduction of circulating arginine does not compromise basal or LPS-inducible NO production in mice. In the present study, we investigated the importance of citrulline availability in ornithine transcarbamoylase-deficient spf(ash) (OTCD) mice on NO production, using stable isotope techniques and C57BL6/J (wild-type) mice controls. Plasma amino acids and tracer-to-tracee ratios were measured by LC-MS. NO production was measured as the in vivo conversion of l-[guanidino-(15)N(2)]arginine to l-[guanidine-(15)N]citrulline; de novo arginine production was measured as conversion of l-[ureido-(13)C-5,5-(2)H(2)]citrulline to l-[guanidino-(13)C-5,5-(2)H(2)]arginine. Protein metabolism was measured using l-[ring-(2)H(5)]phenylalanine and l-[ring-(2)H(2)]tyrosine. OTC deficiency caused a reduction of systemic citrulline concentration and production to 30-50% (P < 0.001), reduced de novo arginine production (P < 0.05), reduced whole-body NO production to 50% (P < 0.005), and increased net protein breakdown by a factor of 2-4 (P < 0.001). NO production was twofold higher in female than in male OTCD mice in agreement with the X-linked location of the OTC gene. In response to LPS treatment (10 mg/kg ip), circulating arginine increased in all groups (P < 0.001), and NO production was no longer affected by the OTC deficiency due to increased net protein breakdown as a source for arginine. Our study shows that reduced citrulline availability is related to reduced basal NO production via reduced de novo arginine production. Under basal conditions this is probably cNOS-mediated NO production. When sufficient arginine is available after LPS stimulated net protein breakdown, NO production is unaffected by OTC deficiency.     

Increased L-arginine uptake and inducible nitric oxide synthase activity in aortas of rats with heart failure

L-Arginine crosses the cell membrane primarily through the system y(+) transporter. The aim of this study was to investigate the role of L-arginine transport in nitric oxide (NO) production in aortas of rats with heart failure induced by myocardial infarction. Tumor necrosis factor-alpha levels in aortas of rats with heart failure were six times higher than in sham rats (P < 0.01). L-Arginine uptake was increased in aortas of rats with heart failure compared with sham rats (P < 0.01). Cationic amino acid transporter-2B and inducible (i) nitric oxide synthase (NOS) expression were increased in aortas of rats with heart failure compared with sham rats (P < 0.05). Aortic strips from rats with heart failure treated with L-arginine but not D-arginine increased NO production (P < 0.05). The effect of L-arginine on NO production was blocked by L-lysine, a basic amino acid that shares the same system y(+) transporter with L-arginine, and by the NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). Treatment with L-lysine and L-NAME in vivo decreased plasma nitrate and nitrite levels in rats with heart failure (P < 0.05). Our data demonstrate that NO production is dependent on iNOS activity and L-arginine uptake and suggest that L-arginine transport plays an important role in enhanced NO production in heart failure.     

Coinduction of endothelial nitric oxide synthase and arginine recycling enzymes in aorta of diabetic rats.

Decreased availability of arginine and impaired production of NO (nitric oxide) have been implicated in the development of endothelial dysfunction. Citrulline formed by the NOS reaction is recycled to arginine by the citrulline-NO cycle, which is composed of NOS, argininosuccinate synthetase (AS), and argininosuccinate lyase. Therefore, we investigated the alterations of these enzymes in the aorta of streptozotocin (STZ)-induced diabetic rats. eNOS and AS mRNAs were increased by three- to fourfold 1-2 weeks after STZ treatment and decreased at 4 weeks. AL mRNA was weakly induced. Induction of eNOS and AS proteins was also observed. Cationic amino acid transporter (CAT)-1 mRNA remained little changed, and CAT-2 mRNA was not detected. The plasma nitrogen oxide levels were increased 1-2 weeks after STZ treatment and decreased at 4 weeks. Transforming growth factor-beta1 (TGF-beta1) mRNA in the aorta was also induced. TGF-beta1 induced eNOS and AS mRNAs in human umbilical vein endothelial cells but inhibited the proliferation of HUVEC. These results indicate that eNOS and AS are coinduced in the aorta in early stages of STZ-induced diabetic rats and that the induction is mediated by TGF-beta1. The results also suggest that TGF-beta1 works antiatherogenically at early stages of diabetes by increasing NO production, whereas prolonged elevation of TGF-beta1 functions atherogenically by inhibiting endothelial cell growth.