Nitric oxide (NO) has been shown to be endothelium-derived relaxation factor, a ubiquitous substance that has numerous physiological functions as a result of a novel signal transduction mechanisms that links extracellular stimuli to the biosynthesis of the second messenger cGMP in nearby cells. Some of the activities of NO that have been established to date include mediation of brain development, learning, and memory, neuronal degeneracy and stroke, smooth muscle relaxation leading to blood pressure lowing and antithrombotic effects, mediation of the analgesic effect of acetylcholine, cytotoxicity against tumor cells and microorganisms, and chemical mediation of penile erection. Inhibition of NO synthase appears to be an effective approach for the blockage of neurotoxicity resulting from stroke, Huntington, and Alzheimer diseases, in the treatment of septic shock, long-term depression, and long-term potentiation, in protection against immune complex-induced vascular injury, and in the treatment of priapism. The objectives of the proposed research are to elucidate the chemical mechanisms of inactivators of brain NO synthase which should aid in the design of new potential inactivators of NO synthase. Mechanisms for the inactivation of NO synthase by Nomega-methyl-L-Arg, Nomega-nitro-L-Arg, Nomega-amino-L-Arg, N-iminoethyl-L-ornithine, and Nomega-allyl-L-ARg will be investigated by radioactively-labeling the various inactivators and determining if the inactivation is the result of covalent attachment. For those that become covalently attached, the part(s) of the molecules that become covalently bound will be determined and the structures of the adducts elucidated. The second part of the proposal involves the design of new potential inactivators of NO synthase. Nomega-Propargyl-L-Arg and Nomega-trimethylsilylmethyl-L-Arg will be synthesized and it will be determined if they inactivate NO synthase. If so, then they will be synthesized with radioactive labels in order to determine if a covalent inactivation is involved, the stoichiometry of binding, what part of the molecule becomes attached to the enzyme, and the structure of the adduct. Nomega-Nitro-L-Arg will be substituted for L-Arg in dipeptides that are known to be substrates for NO synthase from brain and macrophages to attain selectivity in inactivation of the brain enzyme over the macrophage enzyme.
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