Molecular Pain (Dec 2011)
Rapid <it>S</it>-nitrosylation of actin by NO-generating donors and in inflammatory pain model mice
Abstract
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.
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