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This study investigates our hypothesis that argininosuccinate synthase (AS), the rate-limiting enzyme for arginine (l-arg) regeneration from citrulline (l-cit), plays a pivotal role in supplying l-arg to endothelial (eNOS), but not inducible (iNOS) nitric oxide synthase, for nitric oxide (NO) production. Transgenic rat blood-brain barrier (TR-BBB) endothelial cells were used as a model to elucidate the accessibility of the l-arg compartments for NOS isozymes. NO production via eNOS or iNOS, with or without α-methyl-dl-aspartic acid (MDLA), an AS inhibitor, was measured by a fluorometric method. NO production via eNOS was activated by the calcium ionophore A23187, while via iNOS was induced by cytokines. AS activity was assayed by the amount of argininosuccinate regenerated from radioactive aspartic acid from cell extracts. Upon increased AS activity (5.9-fold) in cells grown in l-arg-free/l-cit-supplemented medium, A23187-activated NO production also significantly increased, however cytokine-induced NO production was not detected. A23187-activated NO production was observed not only in l-arg containing medium, but also l-arg-free and l-arg-free/l-cit-supplemented medium, and was abolished by MDLA regardless of medium type. Cytokine-induced NO production was only observed in l-arg containing medium, not in l-arg-free or l-arg-free/l-cit-supplemented medium, and it was not inhibited by MDLA in the l-arg containing medium. Our results indicate that extracellular l-arg was the only l-arg pool for cytokine-induced NO production and intracellular l-arg regenerated from l-cit via AS pathway was the major l-arg pool for A23187-activated NO production in TR-BBB endothelial cells. Therefore, modulation of AS activity could be a promising strategy to selectively alter NO production via eNOS, but not iNOS.