Inhibition of actin polymerization by peroxynitrite modulates neutrophil functional responses

Peroxynitrite, a potent oxidant generated in inflammatory tissues, can nitrate tyrosine residues on a variety of proteins. Based on previous studies suggesting that actin might be a potential target for peroxynitrite‐mediated nitration in neutrophils, we investigated the effects of peroxynitrite on actin function. We show here that peroxynitrite and the peroxynitrite generator (SIN‐1) modified actin in a concentration‐dependent manner, resulting in an inhibition of globular‐actin polymerization and filamentous‐actin depolymerization in vitro. The effects of peroxynitrite were inhibited by the pyrrolopyrimidine antioxidant PNU‐101033E, which has been shown previously to specifically block peroxynitrite‐mediated tyrosine nitration. Furthermore, spectrophotometric and immunoblot analysis of peroxynitrite‐treated actin demonstrated a concentration‐dependent increase in nitrotyrosine, which was also blocked by PNU‐101033E. Activation of neutrophils in the presence of a nitric oxide donor (S‐nitroso‐N‐acetylpenicillamine) resulted in nitration of exogenously added actin. Nitrated actin was also found in peroxynitrite‐treated neutrophils, suggesting that actin may be an important intracellular target during inflammation. To investigate this issue, we analyzed the effect of peroxynitrite treatment on a number of actin‐dependent neutrophil processes. Indeed, neutrophil actin polymerization, migration, phagocytosis, and respiratory burst activity were all inhibited by SIN‐1 treatment in a concentration‐dependent manner. Therefore, the ability of peroxynitrite to inhibit actin dynamics has a significant effect on actin‐dependent, cellular processes in phagocytic cells and may modulate their host defense function.