Nitric oxide has been shown to inhibit apoptosis of human umbilical venous endothelial cells (HUVEC). Therefore we investigated the effect of different NO donors, PAPA NONOate (NOC-15; NO.) and nitrosodium tetrafluoroborate (NOBF4, NO+), and the reaction product of NO and O2-, peroxynitrite (ONOO- ), on TNF-alpha- or serum depletion-induced apoptosis of HUVEC. TNF-alpha-induced DNA fragmentation, determined by ELISA, was inhibited by NOC-15, NOBF4, and ONOO- in a concentration-dependent manner (maximal effects with 10 microM NO. and ONOO- and 100 microM NO+). The inhibition of apoptosis correlated with a protective effect on cell viability. The caspases, a cysteine protease family, play an important role in apoptotic processes. To determine whether the different NO donors and ONOO- regulate this enzyme, caspase-3-like activity was measured in homogenates of TNF-alpha-treated HUVEC. The TNF-alpha-induced enzyme activity was abrogated by NO., NO+, and ONOO-. Furthermore, caspase-3 activity was determined in vitro by reconstitution of the separately cloned, bacterially expressed, and purified active p17 and p12 subunits. The reconstituted caspase-3 exhibited enzyme activity, which was suppressed by the different NO donors and ONOO- with an IC50 of 50 microM for NOC-15, 1 mM for NOBF4, and 50 microM for ONOO-. The inhibition of caspase-3 activity correlated with a S-nitrosylation of the reactive cysteine residue and was reversed by further addition of dithiothreitol. This study suggests that the cellular regulatory processes of NO to protect cells from apoptosis may be independent of the redox state and that low concentrations of NO and ONOO- inhibit the cellular suicide program in HUVEC via S-nitrosylation of members of the caspase family.