S-nitrosylation Of CaMK? In Spinal Cord Up-regulates Its Activity Associated With The Aggravation Of Mechanical Allodynia In Rat SNI Model.

Objective:Neuropathic pain?NP?refers to the pain arising as a direct consequence of a lesion or disease affecting the somatosensory system,which has multiple causes and complex pathogenesis.Neuropathic pain which is characterized by severe pain,long disease duration,is different from acute pain which is caused by tissue damage.The existing therapies and drugs can't bring satisfactory effects,therefore,the research on pathogenesis has been an important task for researchers.It is thought central sensitization of spinal cord is the essential cause of NP,in which Ca²⁺/calmodulin-dependent protein kinase ??CaMK??plays an increasingly important role,and how to inhibit the activity of CaMK? becomes the focus of research.Nitricoxide?NO?,as an significant messenger,participates in the pathogenesis of neuropathic pain in two pathways:traditional NO/sGC-cGMP-PKG and S-nitrosylation.The purpose of this paper is to investigate whether NO can regulate the activity of CaMK? through the S-nitrosylation pathway,and influence the pain behavior in the development of neuropathic pain.Method:All the rats?n=104?were randomly divided into 15 groups:Naive group?n=4?,sham group?n=24?,1 day after SNI operation group?D1??n=4?,4 days after SNI operation group?D4??n=4?,7 days after SNI operation group?D7??n=4?,10 days after SNI operation group?D10??n=4?,14 days after SNI operation group?D14??n=4?,DMSO+SNI operation group?DMSO??intrathecal injection:10ul/rat,n=20?,KN93+SNI operation group?KN93??30uM/10ul/rat,n=4?,tatCN21+SNI operation group?CN21??20uM/10ul/rat,n=4?,L-NAME+SNI operation group?L-NAME??50ug/10ul/rat,n=4?,1400W+SNI operation group?1400W??30ug/10ul/rat,n=4?,7-NI+SNI operation group?7-NI??30ug/10ul/rat,n=4?,GSNO+SNI operation group?GSNO??50ug/10ul/rat,n=12?,ODQ+GSNO+SNI operation group?ODQ+GSNO??20ug/5ul+50ug/5ul/rat,n=4?.The paw withdrawal threshold?PWT?of all the rats were collected before operation.All the animals must be implemented chronic lumbar catheterization of the subarachnoid space.After 7 days recovery,SNI surgery was performed.PWT was collected daily after the operation,and dayly intrathecal injection was started on the 7th day after SNI surgery,and lasted for 4 days?until to 10th day post-operation?.Autophosphorylation on Thr 286 of CaMK??CaMK?-T286?,phosphorylation on Ser831 of GluA1?GluA1-S831?,and S-nitrosylation of CaMK??S-CaMK??were detected by WB at each time point.The SNO-sites of CaMK? in SHAM,DMSO and GSNO groups were detected by LC-MS/MS and compared with each other after the drug administration was completed.Results:Part ?:KN93 and tatCN21 were used to verify whether CaMK? intrinsic activity was mediated by CaMK?-T286 and to confirm whether there were some other mechanisms involved in the regulation of activity.Based on the results,following conclusions were obtained:Changes in PWT showed that intrathecal injection of KN93and tatCN21 significantly allivated mechanical allodynia in SNI rats,with a greater effect of tatCN21?Figure 1A,p<0.05?;The down-regulation of GluA1-S831 caused by tatCN21 was consistent with the changes of PWT and was more significant than KN93?Figure 1B,C,p<0.05?;On the contrary,the down-regulation of CaM?-T286 caused by tatCN21 was weaker than KN93.This result indicated that the CaMK?-T286 did not accurately indicate the intrinsic activity and there were some other?patho-?physiological reactions that can affect the activity of CaMK? in SNI rats.Part ?:The changes of expression on CaMK?-T286,GluA1-S831 and S-CaMK? at different time points after SNI were detected to confirm which was consistent with the rats pain behavior caused by SNI.PWT of rats decreased rapidly,an extremely low PWT value was reached on day 10,and then the pain gradually alleviated,however,the mechanical hyperalgesia remained severe until day 14?FIG.2A,p<0.01?;The changes of GluA1-S831 showed same tendency as the PWT values,and began to decline slowly after reaching the peak on day 7?FIG.2B,C?;The changes of S-Ca MK? was also correlated with the animal behavioral,and began to decline slowly after reaching the peak on day 10?Figure 2B,E?;The CaMK?-T286 increased rapidly afterSNI,and kept at an extremely high level without a downward trend throughout the whole observation period?FIG.2B,D?.The results suggested GluA1-S831 and S-CaMK? could represent the intrinsic activity of CaMK? which was correlated with the animal mechanical allodynia.Part ?:In order to confirm which NOS mediated the S-CaMK?,effects of different NOS inhibitors on PWT values and S-CaMK? was observed.All the three inhibitors of NOS significantly ameliorated the PWT values and generated analgesic effects.As expected,nonselective inhibitor L-NAME reached an maximum analgesic effect,but,surprisingly,the selective inhibitor of iNOS,1400W,achieved a better analgesic effect compared with the nNOS inhibitor,7-NI?FIG.3A,p<0.01?;With the reduction of NO production?FIG.3B,p<0.01?,the down-regulation of S-CaMK? was observed?FIG.3C,F,p<0.01?,so was the down-regulation of GluA1-S831?FIG.3C,D,p<0.01?;CaMK?-T286 was not correlated with the changes of PWT values and GluA1-S831?FIG.3C,E?.This revealed all the three inhibitors of NOS were involved in the regulations of S-CaMK?,and the S-CaMK? could reflect the intrinsic activity of CaMK?,but not the CaMK?-T286.Part ?:GSNO and ODQ were used to verify whether exogenous NO was involved in the development of neuropathic pain through the regulation of S-CaMK?,in addition to the traditional NO/sGS-c GMP-PKG pathway.PWT values indicated GSNO significantly exacerbated mechanical allodynia and this effect was not abolished by ODQ?FIG.4A,p<0.05?.The up-regulation of S-CaMK? was also observed after GSNO application?FIG.4B,E?,and so was the GluA1-S831?FIG.4B,C?,but not the CaMK?-T286?FIG.4B,D?.Taken altogether,exogenous NO exacerbated mechanical allodynia via upregulation of the S-nitrosylation towards CaMK?.The SNO-sites of CaMK? in SHAM,DMSO and GSNO groups were identified by the combination use of cross-linked immunoprecipitation and mass spectrometry.The MS analysis revealed a total of 5SNO-Cys sites on CaMK?,respectively,C6,C30,C64,C280,C289?FIG.5?.No nitrosylation sites was identified in SHAM group.In DMSO group,C6,C30,C280 and C289 sites were nitrosylated.After GSNO administration,a new SNO-site,SNO-C64,was detected,and the ratio of SNO-C280 and SNO-C289 increased obviously,while the ratio of SNO-C6 and SNO-C30 showed no significant changes?Table1?.This results further demonstrated exogenous NO exacerbated mechanical allodynia via up-regulation of S-nitrosylation towards CaMK? associated with the up-regulation of its activity.Conclusion:1.S-nitrosylation could occur on CaMK? in rat SNI model.2.S-nitrosylation of CaMK? which mediated by NO participated in the regulation of CaMK? activity and could reflect the intrinsic activity of CaMK? better than T286autophosphorylation in rat SNI model.3.Besides the NO/cGMP-PKG pathway,non-selective NOS inhibitor,L-NAME could allivated mechanical allodynia via down-regulation of S-nitrosylation towards CaMK? in rat SNI model.4.SNO-sites of CaMK? were Cys6,30,64,280 and 289 in rat SNI model.5.S-nitrosylation of CaMK? which mediated by NO could up-regualte its activity which aggravated mechanical allodynia in rat SNI model.