* Department of Veterinary Molecular Biology, Montana State University, Bozeman; and
Department of Biology, Boise State University, Idaho
Correspondence: Mark T. Quinn, Ph.D., Department of Veterinary Molecular Biology, Montana State University, P.O. Box 173610, Bozeman, MT 59717. E-mail: mquinn{at}montana.edu
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.
Key Words: polymorphonuclear leukocytes • NADPH oxidase • nitrotyrosine • cytoskeleton
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