The nitric oxide donor SIN-1 protects endothelial cells from tumor necrosis factor-α-mediated cytotoxicity: Possible role for cyclic GMP and heme oxygenase

In cultured endothelial cells, incubation with TNF-α (50 ng/ml) for 72 h markedly reduced viability of endothelial cells. A 6-h pre-incubation with the nitric oxide (NO) donor linsidomine (SIN-1, 10-150 μM) protected endothelial cells in a concentration-dependent manner and increased viability by up to 59% of control. The unmetabolized parent compound molsidomine and the NO-free metabolite of SIN-1 3-morpholinoiminoacetonitrile (SIN-1C) were without cytoprotective effect. Cytoprotection by SIN-1 was completely abolished by the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO, 30 μM). A cytoprotective effect comparable to SIN-1 was observed when preincubating the cells with dibutyryl cyclic GMP (10-100 μM). Moreover, no protection by SIN-1 occurred in the presence of cycloheximide (1 μM) or 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ, 0.1 μM), a selective inhibitor of soluble guanylyl cyclase. Tin protoporphyrin-IX (SnPP, 25 μM), an inhibitor of heme oxygenase, was found to attenuate SIN-1-induced cytoprotection. Our results demonstrate that SIN-1 produces a long-term endothelial protection against cellular injury by TNF-α, presumably via a cyclic GMP-dependent pathway leading to up-regulation of protective proteins such as heme oxygenase.