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.
Clements
Mark K
MK
Department of Veterinary Molecular Biology, Montana State University, Bozeman, 59717, USA.
Siemsen
Daniel W
DW
Swain
Steve D
SD
Hanson
Angela J
AJ
Nelson-Overton
Laura K
LK
Rohn
Troy T
TT
Quinn
Mark T
MT
D000199:Actins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002633:Chemotaxis / Q000187:drug effects; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008981:Molsidomine / Q000031:analogs & derivatives* / Q000494:pharmacology; D018375:Neutrophil Activation; D009504:Neutrophils / Q000187:drug effects* / Q000502:physiology / Q000648:ultrastructure; D020030:Nitric Oxide Donors; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D010587:Phagocytosis / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D016897:Respiratory Burst / Q000187:drug effects; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
D000199:Actins; D020030:Nitric Oxide Donors; D017382:Reactive Oxygen Species; D030421:Peroxynitrous Acid; C002744:3-nitrotyrosine; D014443:Tyrosine; C002385:linsidomine; D008981:Molsidomine