Tetrahydrobiopterin compounds prolong allograft survival independently of their effect on nitric oxide synthase activity

Background. In previous work, the four-amino analogue of tetrahydrobiopterin, a novel, selective inhibitor of inducible nitric oxide synthase, has been shown to prolong survival of murine cardiac allografts. Methods. To further elucidate the underlying Molecular immunosuppressive mechanism, we compared the effect of four-amino tetrahydrobiopterin with that of the unsubstituted parent compound tetrahydrobiopterin and of N-6- (iminoethyl)-L-lysine (L-NIL), a nonpterin inhibitor of inducible nitric oxide synthase using a murine cardiac transplant model. We analyzed allograft survival, intragraft gene expression in grafts by microarray and real-time polymerase chain reaction, graft nitrotyrosine staining by immunohistochemistry and plasma nitrite plus nitrate levels by high-performance liquid chromatography. Results. Allograft Survival was significantly prolonged by tetrahydrobiopterin and cyclosporin A, but not by L-NIL although decreased plasma nitrite plus nitrate levels confirmed nitric oxide synthase inhibition in vivo. As compared to allogeneic untreated controls, intragraft peroxynitrite formation and hence nitrotyrosine staining was lowered in all groups except in cyclosporine A-treated animals. Gene expression profiles obtained by microarray analysis demonstrated that cyclosporine A was able to Counteract the expression changes of more than half of the genes differently expressed in syngeneic grafts versus allografts, whereas tetrahydrobiopterin compounds and L-NIL showed only smaller influences on gene expression profiles. Conclusions. These results demonstrate that the four-amino Substitution, which is essential for inhibition of nitric oxide synthase, is not required for the immunosuppressive effect of tetrahydrobiopterin compounds. We describe a novel immunosuppressive role of pharmacologically applied tetrahydrobiopterin.