Rapid S-Nitrosylation of Actin by NO-Generating Donors and in Inflammatory Pain Model Mice

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 ( t 1/2 = 1.8 min) and much less by NOR3 ( t 1/2 = 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.