Overexpression of CuZn superoxide dismutase protects RAW 264.7 macrophagesagainst nitric oxide cytotoxicity

Initiation of nitric oxide (NO.)-mediated apoptotic cell death in RAW 264.7 macrophages is associated with up-regulation of mitochondrial manganese su peroxide dismutase (MnSOD; SOD2) and down-regulation of cytosolic copper zi nc superoxide dismutase (CuZnSOD; SOD1) at their individual mRNA and protei n levels. To evaluate the decreased CuZnSOD expression and the initiation o f apoptosis we stably transfected macrophages to overexpress human CuZnSOD. Individual clones revealed a 2-fold increase in CuZnSOD activity. Expressi on of a functional and thus protective CuZnSOD was verified by attenuated s uperoxide (O-2(.-))-mediated apoptotic as well as necrotic cell death. In t his study we showed that SOD-overexpressing macrophages (R-SOD 1-12) were a lso protected against NO.-initiated programmed cell death. Protection was s ubstantial towards NO. derived from exogenously added NO donors or when NO. was generated by inducible NO synthase activation, and was evident at the level of p53 accumulation, caspase activation and DNA fragmentation. Stimul ation of parent and SOD-overexpressing cells with a combination of lipopoly saccharide and murine interferon gamma produced equivalent amounts of nitri te/nitrate, which ruled out attenuated inducible NO. synthase activity duri ng protection. Because protection by a O2(.-)-scavenging system during NO.- intoxication implies a role of NO. and O-2(.-) in the progression of cell d amage, we used uric acid to delineate the role of peroxynitrite during NO.- elicited apoptosis. The peroxynitrite scavenger uric acid left S-nitrosoglu tathione or spermine-NO-elicited apoptosis unaltered, blocking only 3-morph olinosydnonimine-mediated cell death. As a result we exclude peroxynitrite from contributing, to any major extent, to NO.-mediated apoptosis. Therefor e protection observed with CuZnSOD overexpression is unlikely to stem from interference with peroxynitrite formation and/or action. Unequivocally, the down-regulation of CuZnSOD is associated with NO. cytotoxicity, whereas Cu ZnSOD overexpression protects macrophages from apoptosis.