Rapid S-nitrosylation of actin by NO-generating donors and in inflammatory pain model miceReportar como inadecuado




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Molecular Pain

, 7:101

First Online: 22 December 2011Received: 23 June 2011Accepted: 22 December 2011

Abstract

BackgroundS-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.

ResultsDopamine 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.

ConclusionsThe 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.

Keywordsdopamine release F-actin inflammatory pain nitric oxide PC12 cell S-nitrosylation spinal cord in vivo patch-clamp recordings Abbreviationsbiotin-HPDPN-6-biotinamidohexyl-3-2-pyridyldithiopropionamide

8-Br-cGMP8-bromo-cGMP

CFAcomplete Freund-s adjuvant

cGMPguanosine 3-, 5-cyclic monophosphate

CRP4cysteine-rich protein 4

F-actinfilamentous actin

GSNOS-nitroso-glutathione

IPSCinhibitory postsynaptic current

MMTSS-methyl methanethiosulfonate

NADPH-dNADPH-diaphorase

NMDAN-methyl-D-aspartate

nNOSneuronal NOS, NO: nitric oxide

NOR1±-E-4-methyl-2-E-hydroxyimino-5-nitro-6-methoxy-3-hexenamide

NOR3±-E-4-ethyl-2-E-hydroxyimino-5-nitro-3-hexenamide

NOSNO synthase

ODQ1H-1, 2, 4oxadiazolo-4,3-aquinoxalin-1-one

PACAPpituitary adenylate cyclase-activating polypeptide

PAGEpolyacrylamide gel electrophoresis

PKGcGMP-dependent protein kinase

SDSsodium dodecyl sulfate

SGsubstantia gelatinosa

sGCsoluble guanylyl cyclase

SNAPS-nitroso-N-acetyl-DL- penicillamine.

Electronic supplementary materialThe online version of this article doi:10.1186-1744-8069-7-101 contains supplementary material, which is available to authorized users.

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Autor: Jingshan Lu - Tayo Katano - Daisuke Uta - Hidemasa Furue - Seiji Ito

Fuente: https://link.springer.com/







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