Effects Of PICK1 Knockout On The Inflammatory Pain And Related Mechanisms

Partâ… Effects of PICK1 gene knockout on the inflammatory painAim:PICK1 (protein interacting with Cαkinase 1),a membrane-bound proteinresided in cytoplasm shows high protein sequence conservation in many species fromcaenorhabditis elegans to human.Since been found in 1995,it has interaction with morethan twenty proteins.PICK1 expresses widely in many tissues and participates in somediseases,such as cancer,schizophrenia,pain and Parkinson's disease.Inflammation is a common pathological condition,in which the impaired region canproduce chronically rest pain and hyperalgia.It has been reported that the PICK1 knockoutmice showed attenuated reaction to mechanical irritation,but its influence on inflammatorystimulus remained unclear.The aim of our research is to observe the behavior of PICK1knockout mice whether change in inflammatory pain.Methods:Hypodermic injection 4% formalin in wild type and PICK1 knockout miceto cause the inflammatory pain model,and then observe the different behavior between thetwo groups.Results:The behaviors of inflammatory pain changed obviously in PICK1 knockoutmice.They showed more tolerance to inflammatory pain.1.Effects of PICK1 gene knockout on the behaviors of orofacial formalin test.After 4% formalin was injected subcutaneously into the center of the right vibrissa padas quickly as possible,the number of senconds that the animals spent grooming the injectedareawith the ipsilateral fore-or hindpaw in each 3 min block was obviously decreased inPICK1 gene knockout mice.2.Effects of PICK1 gene knockout on the behaviors of hot plate test and traditionalyformalin test.A.The reaction time of the first evoked behavior to the hot plate test in PICK1 gene knockout mice were prolonged obviously compare to wild type mice.B.After 4% formalin was injected subcutaneously into the dorsum of right foot,thereaction time of the first evoked behavior to the hot plate test in PICK1 gene knockout micewere prolonged obviously compare to wild type mice.Conclusion:1.The behaviors of inflammatory pain changed obviously in PICK1 knockout mice.The absence of PICK1 can enhance the tolerance to inflammatory pain induced by orofacialformalin test.PICK1 disruption not only can modulate the direct irritant effect of theinflamed stimulant mediated through peripheral nociception,but also can enhanced thetolerance to the combination of ongoing sensory input and central sensitization mediatedthrough central nociception.2.Before and after 4% formalin was injected subcutaneously into the dorsum of rightfoot,the reaction time of the first evoked behavior to the hot plate test was both prolongedobviously in PICK1 gene knockout mice compare to wild type mice.It illustrated that thedisruption of PICK1 can cause more tolerance to physiological and inflammatory hot pain.Partâ…¡Effects of PICK1 gene knockout on the functions andexpressions of ASICs in TG,DRG and spinal dorsal hornAim:Acid-sensing ion channels (ASICs),one of the members of thedegenerin/epithelial Na⁺ channel superfamily (DEG/ENaC),can be activated by a drop ofthe extracellular pH or an increase of proton concentration.They express widely inperipheral and central nerve system and participate in many important physiological andpathological processes,such as learning and memroy,synaptic plasticity,cerebral ischemiaand pain.Activation of ASICs by protons can depolarize the neurons and generate action potentials.The acid-induced cutaneous pain elicited by moderate pH decrease appears to belargely mediated by ASICs and the pain associated with more acidic pH also involves thecapsaicin receptor TRPV1.Four genes (ASIC1-ASIC4) encoding six subunits have beenidentified;they are ASIC1a,ASIC1b,ASIC2a,ASIC2b,ASIC3 and ASIC4.PICK1 (proteinthat interacts with C kinase 1)is recently shown to interact with ASIC1 and ASIC2 throughits PDZ domain,and play critical role in ASICs' function.But the specific action betweenPICK1 and ASICs in peripheral and central nerve system,the mechanism of PICK1knockout caused strengthened tolerance to inflammatory pain,these problem remainunsolved.Methods:The effects of PICK1 gene on the functions and expressions of ASICs inTG,DRG and dorsal horn were observed by using PICK1 knockout mice,together with thewestern blotting,whole-cell patch clamp,calcium imaging techniques andimmunofluorescent histochemistry methods.Results:The functions and exPressions of ASIC1 decreased significantly after PICK1knocking out.While the functions and expressions of ASIC3 remained unchanged.1.ASIC1 currents of littermate mice TG neurons decreased after PICK1 knocked out.The amplitudes were 2.37±0.46nA in wild type,1.21±0.35 nA (n=7,p＜0.05 vs wildtype) in homozygote mice,respectively.The acid-induced increasing of [Ca²⁺]_i in littermatemice TG neurons were down-regulated by PICK1 knocked out.When changed theextracellular solution from pH 7.4 to pH 6.0,theΔ[Ca²⁺]/[Ca²⁺] were 0.218±0.034 (n=11from 2 animals) in wild type,0.131±0.020 (n=10 from 2 animals,p＜0.05 vs wild type) inhomozygote,respectively.When changed the extracellular solution from pH 7.4 to pH 5.0,theΔ[Ca²⁺]/[Ca²⁺] were 0.818±0.204 (n=18 from 3 animals) in wild type,0.379±0.091(n=18 from 4 animals,p＜0.01 vs wild type) in homozygote,respectively.2.The protein expression of ASIC1 decreased in TG,DRG and dorsal horn of PICK1knockout mice,so the cellular distribution of ASIC1 decreased uniformity,but thesubcellular localization remained unchanged(n=6). 3.The protein expressions and functions of ASIC3 and TRPV1 had no change in TGneuron of PICK1 knockout mice,they distribute uniformly in cytoplasm and membrane(n=6 for ASIC3 and n=3 for TRPV1).The amplitude of ASIC3 and TRPV1 currents had noobvious change yet(ASIC3:WT 2.59±0.41nA vs KO 2.43±0.39 nA,n=8,p＞0.05;TRPV1:WT 1314±268.3pA vs KO 1256±272.4pA,n=4,p＞0.05).Conclusion:1.The disruption of PICK1 caused the damage of ASICs' function.The amplitude ofASIC1 currents in TG neurons decreased after PICK1 knocked out.The acid-inducedincreasing of [Ca²⁺]_i through ASIC1 in TG neurons were down-regulated by PICK1knocked out.On the basis of different character of ASICs,it is supposed that the action isachieved through ASIC1a homopolymer,and has no relationship to TRPV1 and ASIC3.2.The damage of ASICs' function caused by PICK1 disruption may due to thedecrease of protein expression of ASIC1,so the cellular distribution of ASIC1 decreaseduniformity,but the subcellular localization remained unchanged.Partâ…¢Effects of PICK1 on the action of ASICs in inflammatorypain and the related mechanismAim:Inflammation is a common pathological condition,in which the impaired regioncan produce chronically rest pain and hyperalgia.The generation of inflammatory pain duesto the release of some pain producing substance (PPS),such as bradykinin,PGs,SP,CGRPon one hand;On the other hand,because the pH step down in inflammatory region,thegenerated H⁺ can activate the periphery nociceptors and generate long time coursedepolarization and then promote hyperalgia.In the inflammatory pain model,the expression of ASIC1a in dorsal horn increases significantly.Inhibition of this augment hasevident analgesic effect.In nervous system,PICK1 interacts with PKCαwith its PDZdomain.On one hand this action promotes the location and effect of PICK1;on the otherhand PICK1 plays an important role in PKC's function.Same as PKC,the modulation ofPKA to ASICs also is mediated by PICK1.We had confirmed that the behaviors of inflammatory pain changed obviously inPICK1 knockout mice.They showed more tolerance to inflammatory pain.But the specialmechanism remains unknown.The previous results hinted that the absence of PICK1 couldaffect the protein expression,cellular location and function of ASICs.Then,how about theeffect of PICK1 on ASICs in inflammation? Does ASICs participate in the effect of PICK1on inflammatory pain? Does some SSP and PKA,PKC also participate in the effect ofPICK1 on inflammatory pain? These problems need us to investigate further.Methods:The effects of PICK1 on the distribution and expressions of ASICs,PKA,PKC,SP,CGRP before and after 4% formalin injection by using PICK1 knockout mice,together with animal inflammatory model and immunofluorescent histochemistry methods.Results:1.After been injected with 4% formalin,the expressions of ASIC1 increasedsignificantly in wild type mice TG,DRG and dorsal horn.The absence of PICK1 couldinhibit this augment,but did not change the cellular distribution of ASIC1.2.After been injected with 4% formalin,the expressions of SP increased significantlyin wild type mice TG and DRG neurons.The absence of PICK1 could inhibit this augment,but did not change the cellular distribution of SP.Different from SP,we didn't find thechange of CGRP in PICK1 knockout mice TG and DRG neurons,but discovered thecontent of CGRP decreased in dorsal horn of PICK1 knockout mice.3.After been injected with 4% formalin,the fluorescence intensity of PKC and PKAenhanced obviously in TG,DRG and dorsal horn.The absence of PICK1 could inhibit theenhancement of PKC,but did not change the fluorescence intensity of PKA compare to wild type mice.Conclusion:1.After been injected with 4% formalin,the expressions of ASIC1 and ASIC3increased significantly.The absence of PICK1 could inhibit the enhancement of ASIC1 butnot ASIC3 regardless of control group or inflammation group.2.After been injected with 4% formalin,the amount of SP increased obviously in TGand DRG,and the absence of PICK1 could abolish this increase in inflammatory pain.3.After been injected with 4% formalin,the expression of PKC and PKA increasedobviously in TG,DRG and dorsal horn,and the absence of PICK1 could abolish theaugment of PKC but not PKA in inflammatory pain.