P-281: Localization of protein nitration and immunoreactive endothelial nitric oxide (eNOS) in the wall layers of arteries from coronary artery disease patients

Objective: Nitric oxide (NO), generated by endothelial nitric oxide synthase (eNOS), can react with reactive oxygen species in tissues to form peroxynitrate. The latter by reacting with tyrosin-residues of proteins, leads to protein nitration. It remains, however, unclear whether and where this phenomenon occurs in the human artery wall. Thus, the purpose of this study was to locate eNOS and protein nitration in the human arterial wall ex vivo from patients with coronary artery disease. Methods: We collected (in isopentane precooled on dry ice), rings of human internal thoracic artery from 32 consecutive patients (16 female and 16 male, age 66±9 years), undergoing coronary by-pass grafting. Serial 10um slides were cut in a cryostat and fixed in formalin, washed in PBS and incubated with primary policlonal antibodies to eNOS. and to nitrotyrosine. Secondary antibody were peroxidase-conjugated goat anti-rabbit immunoglobulins. Detection was performed with diaminobenzidine. Negative controls were identically processed in parallel, but with omission the primary antibody. Results: We found immunoreactive eNOS to be expressed throughout the tunica media in 90% of all arteries. At variance, immunoreactive nitrosilated proteins were found at the endothelial level in 77% of the patients, and only sporadically in the tunica media. Conclusions: These results indicate that NO can be synthesized in the tunica media of human arteries from patients with coronary artery disease. Nonetheless, protein nitration seems to be confined to the endothelial layer, thus suggesting that peroxynitrate formation mainly occurs in vivo in these cells.