Increased Superoxide Production in Coronary Arteries in Hyperhomocysteinemia: Role of TNFα, NAD(P)H Oxidase and iNOS

Methionine diet-induced hyperhomocysteinemia and isolation of coronary arteries Male Wistar rats (300-330 g, Charles River Co., www.criver.com) were used. In one group, moderate HHcy was induced by administration of L-methionine (10 g/L in the drinking water) for a period of four weeks. The septal artery was isolated from the hearts of control (C, n=40) and HHcy rats (n=40) as previously described. Serum homocysteine concentrations, measured with a commercially available kits (Abbott Diagnostics Co, Sigma Co) were 3.9±0.8 μmol/L and 29.9±5.5 μmol/L in C and HHcy rats, respectively. Measurement of vascular superoxide level Vascular O2 production was assessed in coronary arterial samples by the lucigenin chemiluminescence (CL) method, as previously described. In brief, coronary vessels from C and HHcy rats were placed in scintillation vials containing HEPES-buffered (10 mmol/L; pH 7.4) Krebs solution and lucigenin (10 μmol/L) CL was measured in a liquid scintillation counter (Beckman LS-6000IC) in the absence or presence of superoxide dismutase (SOD, 200 U/mL) or 4,5-dihydroxy-1,3-benzene-disulphonic acid (Tiron, a O2 scavenger known to eliminate superoxide in coronary vessels, 10 mmol/L). In separate experiments O2 production of HHcy vessels was determined in the absence or presence of rotenone (10 mol/L, a proximal inhibitor of the mitochondrial respiratory chain), or allopurinol (10 mol/L, an inhibitor of xanthine oxidase) or diphenyliodonium [DPI, 10 mol/L, an inhibitor of flavin-containing enzymes, including NAD(P)H oxidases] or apocynin (4-hydroxy-3-methoxy-acetophenone, 3x10 mol/L, an inhibitor of superoxide production by NAD(P)H oxidases), or apocynin plus aminoguanidine (10 mol/L, an inhibitor of iNOS), or SOD or PEG-SOD (200 U/mL). Scintillation counts were obtained 15 to 20 minutes after addition of vessels (averaged) and background-corrected values were normalized to tissue weight. In separate experiments O2 production of aortic segments from HHcy rats was also determined in the absence and presence (30 min incubation) of DPI (10 mol/L) or apocynin or SOD (200 U/mL) or after endothelium removal (by rubbing).