The supply of arginine may become rate limiting for enzymatic reactions that use this semiessential amino acid as a substrate (e.g., nitric oxide, agmatine, creatine, and urea synthesis), particularly under conditions of high demand such as growth, sepsis, or wound healing. In addition, arginine acts as a signaling molecule that regulates essential cellular functions such as protein synthesis, apoptosis, and growth. In the past decade, a number of carrier proteins for amino acids have been identified on the molecular level. They belong to different gene families, exhibit overlapping but distinctive substrate specificities, and can further be distinguished by their requirement for the cotransport or countertransport of inorganic ions. A number of these transporters function as exchangers rather than uniporters. Uptake of amino acids by these transporters therefore depends largely on the intracellular substrate composition. Hence, there is a complex crosstalk between transporters for cationic and neutral amino acids as well as for peptides. This article briefly reviews current knowledge regarding mammalian plasma membrane transporters that accept arginine as a substrate.

The expression of NOS isoforms was studied in guinea pig skeletal muscle at the mRNA and protein level, and the effect of NO on contractile response was examined. Ribonuclease protection analyses demonstrated NOS I and NOS II mRNAs in diaphragm and gastrocnemius muscle. In Western blots, NOS I and NOS II immunoreactivities were found in the particulate but not the soluble fraction of skeletal muscle. NOS activity was found almost exclusively in the particulate fraction. About 50% of this activity was Ca2+ independent. In immunohistochemistry, the anti-NOS I antibody stained distinct membrane regions of muscle fibers. The most intense staining was seen in neuromuscular endplates identified by labeling with alpha-bungarotoxin. The anti-NOS II antibody labeled muscle fibers that contained alkali-labile myosin ATPase (type I fibers). NOS II was located to intracellular structures and was also seen in "specific pathogen-free" animals. Pretreatment of guinea pigs with bacterial lipopolysaccharide (LPS) markedly intensified NOS II staining. Significant NOS III immunoreactivity was detected only in vascular endothelium. In functional experiments, tetanic muscle contractions were induced in diaphragm and gastrocnemius muscle by electrical stimulation of the innervating nerves. Pretreatment of guinea pigs with LPS or addition of S-nitroso-N-acetyl-D,L-penicillamine to the organ bath markedly decreased tetanic contractions. N(G)-nitro-L-arginine, on the other hand, increased contractile force and reversed the effect of LPS. Our data indicate that NOS II and NOS I are expressed in different structures of skeletal muscle and are involved in the regulation of contractile response.

Interfering with tumor metabolism by specifically restricting the availability of extracellular nutrients is a rapidly emerging field of cancer research. A variety of tumor entities depend on the uptake of the amino acid arginine since they have lost the ability to synthesize it endogenously, that is they do not express the rate limiting enzyme for arginine synthesis, argininosuccinate synthase (ASS). Arginine transport through the plasma membrane of mammalian cells is mediated by eight different transporters that belong to two solute carrier (SLC) families. In the present study we found that the proliferation of primary as well as immortalized chronic lymphocytic leukemia (CLL) cells depends on the availability of extracellular arginine and that primary CLL cells do not express ASS and are therefore arginine-auxotrophic. The cationic amino acid transporter-1 (CAT-1) was the only arginine importer expressed in CLL cells. Lentiviral-mediated downregulation of the CAT-1 transporter in HG3 CLL cells significantly reduced arginine uptake, abolished cell proliferation and impaired cell viability. In a murine CLL xenograft model, tumor growth was significantly suppressed upon induced downregulation of CAT-1 in the CLL cells. Our results suggest that inhibition of CAT-1 is a promising new therapeutic approach for CLL.

Myeloid cell arginase-mediated arginine depletion with consecutive inhibition of T cell functions is a key component of tumor immune escape. Both, granulocytic myeloid-derived suppressor cells (G-MDSC) and conventional mature human polymorphonuclear neutrophil granulocytes (PMN) express high levels of arginase 1 and can act as suppressor cells of adaptive anti-cancer immunity. Here we demonstrate that pharmacological inhibition of PMN-derived arginase 1 not only prevents the suppression of T cell functions but rather leads to a strong hyperactivation of T cells. Human PMN were incubated in cell culture medium in the absence or presence of an arginase inhibitor. T cells from healthy donors were then activated either polyclonally or in an antigen-specific manner in the supernatants of the PMN cultures at different PMN-T cell ratios. T cell proliferation was completely suppressed in these supernatants in the absence of an arginase inhibitor. Arginase inhibition led to a strong hyperinduction of T cell proliferation, which exceeded control activation conditions up to 25-fold. The hyperinduction was correlated with higher PMN-T cell ratios and was only apparent when PMN arginase activity was blocked sufficiently. The T cell stimulatory factor was liberated very early by PMN and was present in the < 3 kDa fraction of the PMN supernatants. Increased T cell production of specific proinflammatory cytokines by PMN supernatant in the presence of arginase inhibitor was apparent. Upon arginase inhibition, downregulation of important T cell membrane activation and costimulation proteins was completely prevented or de novo induction accelerated. Antigen-specific T cell cytotoxicity against tumor cells was enhanced by PMN supernatant itself and could be further increased by PMN arginase blockade. Finally, we analyzed anergic T cells from multiple myeloma patients and noticed a complete reversal of anergy and the induction of strong proliferation upon T cell activation in PMN supernatants by arginase inhibition. In summary, we discovered a potent PMN-mediated hyperactivation of human T cells, which is apparent only when PMN arginase-mediated arginine depletion is concurrently inhibited. Our findings are clearly relevant for the analysis and prevention of human tumor immune escape in conjunction with the application of arginase inhibitors already being developed clinically.

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