The Role And Regulatory Mechanism Of Spinal GlunN1Subunit In Acute Remifentanil-induced Hyperalgesia

INTRODUTIONOpioids were classical drugs to treat of a variety of noxious stimuli(surgery, trauma, acute pain, chronic pain), with a lot of side effects,forexample drug addiction,tolerance and opioid induced hyperalgesia(OIH).The ultra-short μ-opioids receptor antagonist,Remifentanil, waswidely used to alleviate pain in general anesthesia.it was reported thatremifentanil has a higher incidence of hyperalgesia than other μ-opioidsreceptor antagonists, The function and quantity of NMDA receptor changeshave played an important role in the formation of remifentanilhyperalgesia.But spinal dorsal horn NMDA receptor subunit GluN1functional change and key protein kinase in remifentanil hyperalgesiamechanisms relations were seldom reported.This study aims to study theimpact of remifentanil on NMDA receptor electrophysiological changesfrom different levels of the whole, sub-cellular, molecular, etc.;remifentanil, ketamine, naloxone activating phosphorylation of GluN1 impact; protein kinase C (PKC) and calcium/calmodulin-dependentprotein kinase (CaMKII) phosphorylation effects on GluN1three aspects ofremifentanil-induced GluN1subunit activity, and explore GluN1phosphorylation mechanism and role of OIH thus infer glutamate receptorregulation and its place in the role of OIH and maintenance phase. In orderto further study the opioid receptors, NMDA receptors in neuronaldegeneration OIH and even the role and mechanism of the development ofnew pain treatments foundation.NMDA receptor is one of ionotropic receptor of glutamate receptorfamily,it is an excitatory amino acid and is the majorexcitatory neurotransmitter found throughout the whole of thenervous system. Since it is released by primary afferent fibres,by dorsal horn neurones, and by neurones in the brain, glutamate isessential for nociceptive signalling at every anatomical level. The thirdfamily in this class are the NMDA receptors, which againform tetramers from combinations of seven different subunits, NR1,NR2A-D and NR3A-B. In the dorsal horn, a combination ofthe GluN1subunit and one GluN2subunit to form thefunctional receptors. opening of the channel pore isvoltage-dependent since at normal resting membrane potentials, thechannel pore is blocked by extracellular Mg2+， After block effect of Mg2+disappeared by stimulus signal, significant Ca2+influx occurs through NMDA receptor channels and subsequently activatesnumerous downstream intracellular signalling mechanisms.Acute OIH isrelevant to remifentanil exposure.Phosphorylation is the most commonly studied posttranslationalmodification and plays a key role in modulating trafficking and especiallyNMDAR surface expression and function,showed ketamine, naloxoneimpact on GluN1subunit phosphorylation.Effect of protein kinase C (PKC)and calcium/calmodulin-dependent protein kinase II(CaMKII) activity afteracute remifentanil exposure and the modulation of phosphorylation of thespinal NMDA receptor GluN1subunit by PKC and CaMKII.Revealschanges in NMDA receptor function and number of mechanisms in theformation of remifentanil hyperalgesia in the regulation.Enhanced NMDAreceptor currents induced by remifentanil is the electrophysiological basisof acute OIH; over-phosphorylation of GluN1is the molecular basis ofsuch electrophysiological changes; PKC and CaMKII are key proteinkinase mediated over-phosphorylation of GluN1,which can becometargets for prevention and treatment of OIHPart I The Changes of Electrophysiologic characteristics ofSpinal N-Methyl-D-aspartate Receptor after Acute Remifentanilexposure Objective:To explore the concentration and time-dependence effect ofelectrophysiologic characteristics of N-methyl-D-aspartate (GluN) receptorof rat dorsal horn neurons after acute remifentanil exposure, anddetermine the concentration of remifentanil corresponding to the maximumcurrent.Methods: The primary spinal dorsal horn neurons were divided into4groups.Control group (0.9%NS),R1group(4nM Remifentanil),R2group(6nM Remifentanil)and R3group(8nM Remifentanil)respectively.Whole cell patch clamp recordings of NMDA current weremade from cultured rat dorsal horn neurons treated with remifentanil at4,6,8nM concentrations for60min in each group. Identified theconcentration of remifentanil which induced the maximum current ofNMDA.Results: Remifentanil at4,6, and8nM(R1,R2,R3group), but nothigher or lower concentrations, caused significant mean increases inNMDA peak current amplitude respectively,showedtime-dependence,compared with control group. Concentration ofremifentanil response relation for the enhancement of NMDA responses,R1group(4nM remifentanil) induced maximum NMDA current responsescompared with R2and R3groups and revealed short-acting and long-lasttrend. Conclusions: The increases in NMDA peak current amplitudeinduced by4nM remifentanil was more significant compared with6、8nMremifentanil.Clinically relevant concentrations of remifentanil induce rapid,persistent increases in NMDA responses that mirror the electrophysiologicbasis of remifentanil-induced hyperalgesia.The following researchconducted based4nM remifentanil.Part II Phosphorylation of Spinal NMDA Receptor GluN1subunit by Remifentanil ActionObjective:To research the phosphorylation of GluN1subunits ofrat spinal dorsal horn neurons after acute remifentanil-exposure andthe time-dependent characteristics, over-phosphorylation of GluN1isthe molecular basis of such electrophysiological changes.The effectof ketamine and naloxone on phosphorylation of GluN1.Methods:All primary spinal dorsal horn neurons were divided into6groups.Group A: remifentanil+glycine,Group B:glycine;GroupC:remifentanil+glycine+ketamine,Group D:glycine+ketamine;GroupE:remifentanil+glycine+naloxone,Group F:glycine+naloxone.After60minof4nM remifentanil exposure, Western-blotting experiments were made toreveal the presence of GluN1and phosphorylated GluN1receptor.Reverse transcriptase polymerase chain reaction(RT-PCR) were made to reveal theeffect of remifentanil,naloxone and ketamine on GluN1mRNA expression.Results: GluN1of group A expression were significantly lower thangroup B (P <0.01) in Western Blot test at all four time points(1h,2h,4h,24h),in group A the p-GluN1expression at4h and24h significantly increasedcompared to1h and2h (P <0.05); GluN1expression of Group A and Bwere lower significantly compared with group C and Group D respectively;the expression of GluN1at2,4,24h showed significant differences betweenEF groups. p-GluN1of group A increased slowly from1h24h reach peakconcentration; within the E group compared with p-GluN1at2h,4h,24h1h difference increased (P <0.05) GluN1mRNA expression of each groupalso showed significant time-dependent in rt-PCR test,expression ofGluN1mRNA were significantly reduced at1,2,4,24h in six groups afterremifentanil exposure (P <0.05), with4h and24h inhibited the mostobvious; C, D groups is even more evident with time prolonged inhibitionof expression of GluN1mRNA continued until24h, and the A, C groupscompared with ketamine added to GluN1mRNA more pronouncedinhibition of expression (P <0.05); E, F groups in addition to GluN1mRNA2h no significant effect on the expression of foreign The remaining time isstill displayed on GluN1expression were inhibited.Conclusion: The expression of GluN1decreased on rat spinal dorsalhorn neuronal membrane after remifentanil exposure, increased phosphorylation of GluN1and inhibition of expression of GluN1mRNAare the molecular basis of increase inward current of NMDA and showtime-dependent manner. μ receptor played an important role in GluN1activation, the increased phosphorylation of GluN1is one of theunderlying molecular mechanisms of OIH.Part III Effect of protein kinase C andcalcium/calmodulin-dependent protein kinase II onremifentanil-induced spinal NMDA receptor GluN1subunitphosphorylationObjective: Effect of protein kinase C (PKC) andcalcium/calmodulin-dependent protein kinase II(CaMKII) activity afteracute remifentanil exposure and the modulation of phosphorylation of thespinal NMDA receptor GluN1subunit by PKC and CaMKII.Methods: The concentration of PKC and CaMKII were measuredafter the primary cultured rat dorsal horn neurons treated with remifentanilat4nM concentrations for60min and120min. Then,50and100umo/L ofchelerythrine the inhibitor of PKC,KN93the inhibitor of CaMKII wereadded after remifentanil-treated for60min. Western-blotting experimentswere made to identified the expression of GluN1and phosphorylated GluN1after60and120min after remifentanil exposure.Results: The concentration of PKC of rat spinal dorsal horn neuronswas significantly increased after1h and last to2h (P <0.05), theconcentration of CaMKII1h concentrations did not change significantly,began to increase2h after exposure to4nm remifentanil.PKCconcentrations began to decrease CaMKII concentration increases at2hafter exposure to4nm remifentanil. GluN1expression changes over timegradually decreased, but there was no significant difference; p-GluN1theexpression gradually increased over time. The concentration of p-GluN1ofgroups A1and A2, A1group was significantly lower (0.42vs0.21; P <0.05),A2group (0.49vs0.33; P <0.05), after exposure to KN93, antagonist ofCaMKII, GluN1phosphorylation was inhibited significantly. Theconcentration of p-GluN1of groups A1and A2, A1group wassignificantly lower (0.42vs0.26; P <0.05), B2group (0.49vs0.36; P <0.05),after exposure to chelerythrine, antagonist of PKC, GluN1phosphorylationwas inhibited significantly.Conclusion: PKC and CaMKII are two key kinases participated thephosphorylation of GluN1,Phosphorylation of GluN1by PKC mediate theoccurrence of acute OIH, by CaMKII mediate the maintaince of acuteOIH; selective inhibition of PKC or CaMKII is the target of treating OIH.