L-NAME hydrochloride (L-NAME HCl), a cell-permeable NO synthase inhibitor, displays Ki values of 4.4 μM, 15 nM, and 39 nM, for iNOS (mouse) , nNOS (bovine) , and eNOS (human), respectively.

eNOS:39 nM(Ki), nNOS:15 nM(Ki)

L-NAME (0.03-300 mg kg-1, i.v.) induces a dose-dependent increase in mean systemic arterial blood pressure accompanied by bradycardia. L-NAME (100 mg kg-1, i.v.) inhibits significantly the hypotensive responses to ACh and bradykinin. The increase in blood pressure and bradycardia produced by L-NAME is reversed by L-arginine (30-100 mg kg-1, i.v.) in a dose-dependent manner. [2]

Enzyme Assay: The oxidation of L-arginine is monitored by the conversion of [3H]- or [14C]-arginine to L-citrulline which separates L-citrulline from L-arginine by Dowex 50x8-200 (Na) chromatography. Typical reaction mixtures (100 pL) contains 50 mM HEPES, pH 7.0, 8 pM tetrahydrobiopterin, 1 mM CaC12, 0.01 mg/mL calmodulin, 0.5 mM EDTA, 0.450 pM [14C]-arginine (30000 cpm), and 100-200 pM NADPH. The cNOS-catalyzed oxidation of NADPH to NADP+ is monitored by the reduction of absorbance at 340 nm with a Kontron 860 spectrophotometer in a volume of 300 pL. All reactions are at 30 ℃ unless otherwise indicated.

rMC-1 cells are incubated in 5 or 25 mM glucose, with or without l-NAME (1 mM). Media is changed every other day for up to 5 days. BREC cells are incubated in 5 or 25 mM glucose as well as inhibitor as described above for 5 days. Cell death is determined by light microscopy using a hemocytometer and a 0.4% trypan blue dye exclusion method. The number of cells that do not exclude the dye is expressed per 1,000 total cells. A minimum of 800 cells is counted per assay (8 dishes, >100 cells counted per dish), and the assay is replicated three times on different days. (Only for Reference)