title: Rapid S-nitrosylation of actin by NO-generating donors and in inflammatory pain model mice.
abstract: BACKGROUND
  S-Nitrosylation, the reversible post-translational modification of reactive cysteine residues in proteins, has emerged as an important mechanism by which NO acts as a signaling molecule. We recently demonstrated that actin is a major S-nitrosylated protein in the spinal cord and suggested that NO directly attenuates dopamine release from PC12 cells by causing the breakdown of F-actin. However, the occurrence of S-nitrosylation of actin remained unclarified in animal pain model. Kinetic analysis of S-nitrosylation of actin in the present study was made by using NO-generating donors. The biotin-switch assay and purification on streptavidin-agarose were employed for identification of S-nitrosylated actin.
  
  
  RESULTS
  Dopamine release from PC12 cells was markedly attenuated by NOR1 (t1/2 = 1.8 min) and much less by NOR3 (t1/2 = 30 min), but not by S-nitroso-glutathione, an endogenous NO donor. A membrane-permeable cGMP analogue could not substitute for NOR1 as a suppressor nor could inhibitors of soluble guanylate cyclase and cGMP-dependent protein kinase attenuate the suppression. S-Nitrosylated actin was detected by the biotin-switch assay at 5 min after the addition of NOR1. Consistent with the kinetic analysis, actin in the spinal cord was rapidly and maximally S-nitrosylated in an inflammatory pain model at 5 min after the injection of 2% formalin into the hind paws. In vivo patch-clamp recordings of the spinal dorsal horn, NOR3 showed an inhibitory action on inhibitory synaptic transmission in interneurons of the substantia gelatinosa.
  
  
  CONCLUSIONS
  The present study demonstrates that rapid S-nitrosylation of actin occurred in vitro in the presence of exogenous NO-generating donors and in vivo in inflammatory pain model mice. Our data suggest that, in addition to the well-known cGMP-dependent protein kinase pathway, S-nitrosylation is involved in pain transmission via disinhibition of inhibitory neurons.
authors.affiliation: Department of Medical Chemistry, Kansai Medical University, Moriguchi, Japan.
authors.forename: Jingshan
authors.initials: J
authors.lastname: Lu
authors.forename: Tayo
authors.initials: T
authors.lastname: Katano
authors.forename: Daisuke
authors.initials: D
authors.lastname: Uta
authors.forename: Hidemasa
authors.initials: H
authors.lastname: Furue
authors.forename: Seiji
authors.initials: S
authors.lastname: Ito
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D006152:Cyclic GMP / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D004195:Disease Models, Animal; D007249:Inflammation / Q000378:metabolism; D051379:Mice; D009474:Neurons / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000378:metabolism*; D016716:PC12 Cells; D010146:Pain / Q000378:metabolism*; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide / Q000378:metabolism; D051381:Rats
chemical_list: D000199:Actins; D020030:Nitric Oxide Donors; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP