Electron Spin Resonance Analysis of Heme-Nitrosyl and Reduced Iron-Sulfur Centered Complexes in Allogeneic, Heterotopic Cardiac Transplants: Effects of Treatment with Pyrrolidine Dithiocarbamate

Abstract

Inhibition of inducible nitric oxide synthase (iNOS) prolongs allograft survival suggesting a role for nitric oxide ( •NO) in allograft rejection. Induction of iNOS is regulated by the oxidant-sensitive, nuclear factor kappa B (NF-κB) in many cell types. In the present study using electron spin resonance (ESR) spectroscopy, we evaluated whether pyrrolidine dithiocarbamate (PDTC), a metal chelator and antioxidant, might limit •NO production during the development of rejection in cardiac allografts. We performed either isogeneic (Lewis to Lewis) or allogeneic (Wistar–Furth to Lewis) heterotopic abdominal cardiac transplantation. Allograft recipients received daily injections of PDTC or aminoguanidine (a known inhibitor of iNOS). At postoperative days 4 or 6, grafted and native hearts of transplant recipients were flushed with cardioplegic solution to remove blood contamination. ESR data of allografts revealed a triplet nitrogen signal ( a N = 17.5 G) and centered at g = 2.012 and an additional broad signal at g = 2.08. This signal was not seen in either isografts or native hearts of either isograft or allograft recipients. Based upon these parameters, these signals are attributed to nitrosomyoglobin. This signal was inhibited by treatment with aminoguanidine or PDTC. Under these conditions, PDTC also prolonged graft survival from 6.6 ± 0.2 to 11.7 ± 0.3 days. Thus, it is conceivable that nitrosylmyoglobin formation precedes rejection in cardiac allografts and inhibition of nitrosomyoglobin with agents such as PDTC contribute to improved graft survival.