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Rapid S-nitrosylation of actin by NO-generating donors and in inflammatory pain model mice.

Lu J, Katano T, Uta D, Furue H, Ito S - Mol Pain (2011)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Chemistry, Kansai Medical University, Moriguchi, Japan.

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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Nitrosocysteine immunoreactivity in the spinal cord of inflammatory pain models. A. Immunoreactivity of nitrosocysteine in the spinal cord. Lumbar transverse sections (20 μm) of spinal cords prepared from before and 5 min, 6 h, and 24 h after injection of formalin, carrageenan, and CFA, respectively, were fixed and stained with anti-nitrosocysteine antibody as described in "Method". Bar = 100 μm. B. Quantification of S-nitrosocysteine immunostaining. Fluorescence intensity of the spinal cord was quantified by ImageJ. The intensity of the spinal cord in inflammatory pain models was normalized to that of naive mice, and the intensity (mean ± SEM, n = 23-24) of naive mice was taken as "1." **P < 0.01.
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Figure 6: Nitrosocysteine immunoreactivity in the spinal cord of inflammatory pain models. A. Immunoreactivity of nitrosocysteine in the spinal cord. Lumbar transverse sections (20 μm) of spinal cords prepared from before and 5 min, 6 h, and 24 h after injection of formalin, carrageenan, and CFA, respectively, were fixed and stained with anti-nitrosocysteine antibody as described in "Method". Bar = 100 μm. B. Quantification of S-nitrosocysteine immunostaining. Fluorescence intensity of the spinal cord was quantified by ImageJ. The intensity of the spinal cord in inflammatory pain models was normalized to that of naive mice, and the intensity (mean ± SEM, n = 23-24) of naive mice was taken as "1." **P < 0.01.

Mentions: Then we immunostained the spinal cord of inflammatory pain models with the anti-nitrosocysteine antibody. Transverse sections were prepared from the spinal cords before and 5 min, 6 h, and 24 h after injection of formalin, carrageenan and CFA, respectively. The immunoreactivity of nitrosocysteine was detected in the superficial layer of the spinal cord (Figure 6A). The intensity of immunoreactivity was significantly increased 1.29-fold by formalin, but not by carrageenan and CFA (Figure 6B). Consistent with the results of immunoblotting (Figure 5A), the immunohistochemical studies support rapid S-nitrosylation of proteins in the spinal cord in vivo in inflammatory pain model.


Rapid S-nitrosylation of actin by NO-generating donors and in inflammatory pain model mice.

Lu J, Katano T, Uta D, Furue H, Ito S - Mol Pain (2011)

Nitrosocysteine immunoreactivity in the spinal cord of inflammatory pain models. A. Immunoreactivity of nitrosocysteine in the spinal cord. Lumbar transverse sections (20 μm) of spinal cords prepared from before and 5 min, 6 h, and 24 h after injection of formalin, carrageenan, and CFA, respectively, were fixed and stained with anti-nitrosocysteine antibody as described in "Method". Bar = 100 μm. B. Quantification of S-nitrosocysteine immunostaining. Fluorescence intensity of the spinal cord was quantified by ImageJ. The intensity of the spinal cord in inflammatory pain models was normalized to that of naive mice, and the intensity (mean ± SEM, n = 23-24) of naive mice was taken as "1." **P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3295738&req=5

Figure 6: Nitrosocysteine immunoreactivity in the spinal cord of inflammatory pain models. A. Immunoreactivity of nitrosocysteine in the spinal cord. Lumbar transverse sections (20 μm) of spinal cords prepared from before and 5 min, 6 h, and 24 h after injection of formalin, carrageenan, and CFA, respectively, were fixed and stained with anti-nitrosocysteine antibody as described in "Method". Bar = 100 μm. B. Quantification of S-nitrosocysteine immunostaining. Fluorescence intensity of the spinal cord was quantified by ImageJ. The intensity of the spinal cord in inflammatory pain models was normalized to that of naive mice, and the intensity (mean ± SEM, n = 23-24) of naive mice was taken as "1." **P < 0.01.
Mentions: Then we immunostained the spinal cord of inflammatory pain models with the anti-nitrosocysteine antibody. Transverse sections were prepared from the spinal cords before and 5 min, 6 h, and 24 h after injection of formalin, carrageenan and CFA, respectively. The immunoreactivity of nitrosocysteine was detected in the superficial layer of the spinal cord (Figure 6A). The intensity of immunoreactivity was significantly increased 1.29-fold by formalin, but not by carrageenan and CFA (Figure 6B). Consistent with the results of immunoblotting (Figure 5A), the immunohistochemical studies support rapid S-nitrosylation of proteins in the spinal cord in vivo in inflammatory pain model.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Chemistry, Kansai Medical University, Moriguchi, Japan.

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.

Show MeSH
Related in: MedlinePlus