Immunohistochemistry in the identification of nitric oxide synthase isoenzymes in myocardial infarction

Abstract

Objective: Inducible nitric oxide synthase (iNOS) activity, as measured by conversion of L-¹⁴C-arginine to L-¹⁴C-citrulline is significantly increased in infarcted rabbit myocardium as compared to healthy myocardium 48 h after coronary occlusion. The aim of this study was to localise the nitric oxide synthase (NOS) isoforms in normal myocardium and compare these findings with NOS activity in cells of the infarcted region. Methods: Activities of NOS isoforms were enzymatically assayed in normal and infarcted myocardium. Localisation of NOS was performed on identical sections using specific monoclonal IgG antibodies against endothelial constitutive (cNOS) and macrophage inducible (iNOS) nitric oxide synthase. In addition, macrophages were identified using fluorescein conjugated ChromPure rabbit IgG, Fc fragment. Results: iNOS activity increased significantly in infarcted as compared to normal myocardium [0.42(SEM 0.03) v 0.85(0.08) fmol·μg⁻¹·min⁻¹, P = 0.02, respectively]. However, cNOS did not increase significantly in infarcted regions [0.18(0.04) v 0.24(0.06) fmol·μg⁻¹·min⁻¹; P = 0.16, respectively]. cNOS was immunohistochemically localised in endothelial and endocardial cells in normal and infarcted tissues. The presence of iNOS activity in macrophages in infarcted myocardium was identified immunohistochemically. Cardiomyocytes and neutrophils did not label with the antibodies to cNOS and iNOS. Conclusions: (1) Infiltrating macrophages are the main site of increased iNOS activity in infarcted rabbit myocardium. (2) cNOS activity is not significantly increased in infarcted tissues as compared to normal myocardium. (3) Neutrophils and cardiomyocytes do not express NOS imunoreactivity in infarcted and normal rabbit myocardium.