ARGININE TRANSPORT AND NITRIC OXIDE PRODUCTION

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Activated macrophages demonstrate arginine-dependent cytotoxicity for tumor cells and intracellular pathogens mediated by the free radical, nitric oxide (NO). These cells increase arginine uptake, the substrate for synthesis of NO, when activated by cytokines. Preliminary studies have identified coordinate expression of CAT-2B, a new member of a family of cationic amino acid transporters (CAT), including arginine, and of nitric oxide synthase (NOS) in the gamma interferon (IFN-gamma) and lipopolysaccharide (LPS)-activated macrophage cell line, RAW 264.7. This proposal summarizes experiments designed to investigate the importance of CAT-2B in providing arginine for synthesis of NO in macrophages and other tissues that produce NO in response to cytokines. Specific Aim #1: Examine the structure/function of CAT-2B A. Obtain cDNA encoding CAT-2B from a library of RAW 264.7 cells activated by IFN-gamma/LPS. B. Perform a survey of amino acid uptake in frog oocytes injected with cRNA encoding CAT-2B. C. Compare the properties of CAT-2B to previously identified transporters, CAT-2A and CAT-1. Specific Aim #2: Investigate the dependence of nitric oxide production on CAT-2B expression. A. Correlate CAT-2B and NOS expression induced by cytokines in macrophages and in other tissues. B. Determine if NO production is dependent upon CAT-2B expression in reconstruction experiments performed in oocytes and mammalian cell lines. C. Determine if CAT-2B expression is coordinate with NOS expression or secondary to intracellular arginine depletion. D. Examine the feasibility of deleting the CAT-2B-specific exon from the mouse genome with the long term goal of determining the susceptibility to infection and tumor formation of a CAT-2B deficient mouse.