N0 has been identified as a secretory product mediating diverse functions in mammalian systems including as a regulator of blood flow, as a mediator for the neurotransmitter glutamate, and as a cytotoxic mediator of macrophages. N0 is synthesized by the enzyme nitric oxide synthase (N0S) which exists in three isoforms, commonly referred to as cN0S, eN0S, and iN0S. A key to the successful prevention of toxic N0 formation is the development of inhibitors of N0S that are isoform selective, cell permeable, and non-toxic in vivo. This laboratory has pioneered the development and characterization of a novel class of N0S inhibitors, the imidazoleindazole class, which include the agents 7- nitroindazole and 1-phenylimidazole. Further, the investigator has recently identified that aminoguanidine is an isoform selective, non- toxic mechanism based inactivator of iN0S. The current grant proposes to examine the relationship of structure with the mechanism of inhibition and the isoform selectivity of aminoarginine analogs. Further, using C14-aminoguanidine, it is planned to determine the chemical mechanism of N0S inhibition both in vitro and in vivo in cells known to contain the cN0S and iN0S isoforms. Studies will be extended to compare the inhibition of isolated affinity purified N0S isoforms with that in intact cellular systems. These studies will clarify the factors operating in living cells that govern the sensitivity of the iN0S to inhibition by these agents.
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