Nitric oxide has been identified as a secretory product mediating diverse functions in mammalian systems including regulation of blood pressure and flow, as a mediator of many of the actions of the neurotransmitter glutamate in the central nervous system, and as a cytotoxic mediator of macrophages involved in killing pathogenic organisms. Nitric oxide is synthesized by the enzyme nitric oxide synthase (NOS) that exists in three isoforms, commonly referred to as cNOS, eNOS, and iNOS. Under certain conditions, NO production by cNOS has been implicated in the pathogenesis of post stroke damage and hypoxia/reoxygenation injuries while NO production by iNOS has been implicated in the tissue damage of diverse autoimmune disorder including arthritis, diabetes, multiple sclerosis and ileitis. A key to the successful prevention of toxic NO formation is the development of inhibitors of NOS that are isoform selective, cell permeable and non-toxic in vivo. Our laboratory has pioneered the development and characterization of a novel class of NOS inhibitors the imidazole-indazole class which include the agents 7-nitroindazole and 1-phenylimidazole. Further, we have recently identified that amainoguanidine is an isoform selective, non toxic, mechanism based inactivator of iNOS. The current grant proposes to examine the relationship of structure to the mechanism of inhibition and the isoform selectivity of aminoguanidine homologs with an eye to identifying structural features related to cNOS isoform selectivity of inhibition. Further, using 14C-aminogluanidine, we plan to determine the chemical mechanism of NOS inhibition both in vitro and in situ in cells known to contain the cNOS and iNOS isoforms. Further, we plan to extend our studies conducted on isolated, affinity purified NOS isoforms to intact cellular systems including GH3 cells that contain the cNOS isoform and to RAW 264.7 macrophages that contain the cytokine-inducible iNOS isoform. These studies will clarify the factors operative in living cells that govern the sensitivity of the NOS isoform to inhibition by these agents.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Toxicology Subcommittee 2 (TOX)
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University of Medicine & Dentistry of NJ
Schools of Medicine
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Wolff, D J; Mialkowski, K; Richardson, C F et al. (2001) C60-Fullerene monomalonate adducts selectively inactivate neuronal nitric oxide synthase by uncoupling the formation of reactive oxygen intermediates from nitric oxide production. Biochemistry 40:37-45
Wolff, D J; Papoiu, A D; Mialkowski, K et al. (2000) Inhibition of nitric oxide synthase isoforms by tris-malonyl-C(60)-fullerene adducts. Arch Biochem Biophys 378:216-23
Cooper, G R; Mialkowski, K; Wolff, D J (2000) Cellular and enzymatic studies of N(omega)-propyl-l-arginine and S-ethyl-N-[4-(trifluoromethyl)phenyl]isothiourea as reversible, slowly dissociating inhibitors selective for the neuronal nitric oxide synthase isoform. Arch Biochem Biophys 375:183-94
Bryk, R; Lubeskie, A; Wolff, D J (1999) Studies of neuronal nitric oxide synthase inactivation by diverse suicide inhibitors. Arch Biochem Biophys 369:243-51
Bryk, R; Wolff, D J (1999) Pharmacological modulation of nitric oxide synthesis by mechanism-based inactivators and related inhibitors. Pharmacol Ther 84:157-78
Bryk, R; Wolff, D J (1998) Mechanism of inducible nitric oxide synthase inactivation by aminoguanidine and L-N6-(1-iminoethyl)lysine. Biochemistry 37:4844-52
Cooper, G R; Barr, A; Wolff, D J (1998) Neuronal nitric oxide synthase is refractory to mechanism-based inactivation in GH3 pituitary cells. Arch Biochem Biophys 357:195-206
Wolff, D J; Lubeskie, A; Gauld, D S et al. (1998) Inactivation of nitric oxide synthases and cellular nitric oxide formation by N6-iminoethyl-L-lysine and N5-iminoethyl-L-ornithine. Eur J Pharmacol 350:325-34
Wolff, D J; Gauld, D S; Neulander, M J et al. (1997) Inactivation of nitric oxide synthase by substituted aminoguanidines and aminoisothioureas. J Pharmacol Exp Ther 283:265-73
Wolff, D J; Lubeskie, A; Li, C (1997) Inactivation and recovery of nitric oxide synthetic capability in cytokine-induced RAW 264.7 cells treated with ""irreversible"" NO synthase inhibitors. Arch Biochem Biophys 338:73-82

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