PICK1基因敲除对炎性疼痛的作用及相关机制
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摘要
第一部分PICK1基因敲除对炎性痛觉行为学的影响
目的：PICK1(蛋白激酶Cα相互作用蛋白1，protein interacting with Cαkinase 1)是从线虫到人的所有生物中非常保守的一类存在于细胞质中的膜结合蛋白，自1995年被发现以来，已报道能与20多种蛋白发生相互作用。PICK1在人和动物的组织和细胞内均可表达，广泛分布于机体的各种组织。PICK1参与一些疾病的发生，如：癌症、精神分裂症、疼痛、帕金森综合症等。虽然PICK1在这些疾病发生过程中的具体作用机制现在还不清楚，但其广泛的结合特性使其可能通过与疾病相关蛋白的相互作用进而在疾病发生过程中扮演一定角色。
炎症是一种常见的病理情况，在组织炎症时，病变部位可产生慢性持续性疼痛和痛觉过敏，不仅使患者感到不适或痛苦，并且可造成诸多生理功能障碍和精神疾病，如睡眠失调、抑郁症、注意力下降等。已有研究显示PICK1基因敲除小鼠对机械性刺激反应减弱，但其对炎性疼痛的影响还不清楚。本实验的目的就是观察PICK1基因敲除后小鼠炎性痛觉行为是否改变。
方法：应用PICK1基因敲除小鼠，皮下注射4％福尔马林溶液造成炎性疼痛模型，与同窝野生型小鼠对比，观察炎性痛觉行为的变化情况。
结果：PICK1基因敲除后，其炎性痛觉行为学发生明显变化，表现为对炎性疼痛更加耐受。
1．PICK1基因敲除对头面部炎性痛觉行为学的影响。
单侧面部触须垫皮下注射4％福尔马林溶液后，与同窝野生型小鼠比较，PICK1基因敲除小鼠每3 min时间内用同侧前肢或后肢摩擦注射部位的行为明显减少。
2．PICK1基因敲除对足部炎性痛觉行为学的影响。
(1)采用热板法测小鼠基础痛阈，发现与同窝野生型小鼠比较，PICK1基因敲除小鼠基础痛阈值明显增高。
(2)单侧足背皮下注射4％福尔马林溶液后，与同窝野生型小鼠比较，PICK1基因敲除小鼠痛阈值明显增高。
结论：
1．PICK1基因敲除小鼠对福尔马林导致的面部炎性疼痛的两期耐受性均增加，即不仅对第一相的急痛反应耐受性增加，对炎症反应所导致的第二相中枢敏感化也有抑制作用。
2．PICK1基因敲除小鼠单侧足背皮下注射4％福尔马林溶液前后对热刺激引发的缩足反应时间均明显延长，提示PICK1基因敲除对生理性和炎性热敏性疼痛耐受性均增强。
第二部分PICK1基因敲除对TG、DRG和脊髓背角ASICs通道功能和表达的影响
目的：酸敏感离子通道(acid-sensing ion channels，ASICs)是ENaC／DEG(epithelial Na~+channel／degenerin)通道超家族的成员之一，能被细胞外pH下降或H~+浓度上升直接激活。它广泛分布于中枢及外周神经系统的神经元，参与了很多重要的生理过程和病理反应，与学习记忆、突触可塑性、脑缺血、疼痛等关系密切。H~+激活ASICs可使神经元去极化，产生动作电位，参与了组织酸化如缺血、炎症、肿瘤、癫痫等时痛觉的感受。由中度pH降低所诱发的皮肤表面酸诱导疼痛主要是由ASICs介导的，而更强的酸化引起的疼痛则是ASICs和TRPV1共同参与。目前已克隆出的ASICs主要有ASIC1a、ASIC1b、ASIC2a、ASIC2b、ASIC3和ASIC4。PICK1通过其PDZ结构域与ASIC1和ASIC2相互作用，对ASICs功能的发挥起到重要作用，但PICK1与外周和中枢感觉神经元ASICs之间具体作用如何以及有何意义?在上文中我们已经证实PICK1基因敲除小鼠对炎性疼痛的耐受性增强，它的具体机制是什么?是否与ASICs有关?这些都值得我们去进一步探讨。
方法：采用western blotting、全细胞膜片钳、钙影像技术和免疫荧光组织化学技术，观察PICK1基因敲除前后，外周初级感觉神经元和脊髓背角上ASICs的蛋白表达和功能的变化情况，探讨可能的机制。
结果：PICK1基因敲除后AISC1的功能和蛋白表达均降低而AISC3的功能和蛋白表达均无变化。
1．PICK1基因敲除小鼠TG神经元上AISC1电流幅度减小，全细胞膜片钳结果显示野生型AISC1电流幅度为2．37±0．46nA，PICK1基因敲除后AISC1电流幅度减少为1．21±0．35 nA(n=7，p＜0．05 vs wild type)。PICK1基因敲除小鼠TG神经元上通过ASIC1通道诱导的胞内钙升高减少。将细胞外液由pH 7．4迅速转换成pH 6．0时，野生型△[Ca~(2+)]／[Ca~(2+)]比值为0．218±0．034(n=11 from 2 animals)，纯合子为0．131±0．020(n=10 from 2 animals，p＜0．05 vs wild type)。将细胞外液由pH 7．4迅速转换成pH5．0时，野生型△[Ca~(2+)]／[Ca~(2+)]比值为0．818±0．204 (n=18 from 3 animals)，纯合子为0．379±0．091 (n=18 from 4 animals，p＜0．01 vs wild type)。
2．PICK1缺失造成的ASIC1功能损伤可能是由于ASIC1蛋白表达总量的减少所致，使其在细胞内的分布均匀减少，但其亚细胞定位并无改变。Western blotting和免疫荧光的结果均显示，小鼠TG、DRG和脊髓背角上ASIC1蛋白表达总量和荧光强度均减少，但分布并未改变，仍在细胞膜上和胞浆内均匀分布(n=6)。
3．PICK1基因敲除小鼠TG神经元上ASIC3和TRPV1的表达和功能无变化。膜片钳实验结果显示：PICK1基因敲除小鼠TG神经元上ASIC3和TRPV1电流幅值均无明显变化(ASIC3：WT 2．59±0．41nA vs KO 2．43±0．39 nA，n=8，P＞0．05；TRPV1：WT 1314±268．3pAvs KO 1256±272．4pA，n=4，p＞0．05)；Western blotting和免疫荧光的结果均显示，小鼠TG、DRG上无论是ASIC3还是TRPV1的蛋白表达量都未发生改变；ASIC3和TRPV1在细胞膜上和胞浆内均匀分布(n=6 for ASIC3 and n=3 forTRPV1)。
结论：
1．PICK1基因敲除造成ASICs的功能损伤。PICK1基因敲除小鼠TG神经元上AISC1电流幅度减小，通过ASIC1通道诱导的胞内钙升高减少。根据不同ASIC亚单位的分布和特性，推测这一作用主要是通过ASIC1a同聚体来实现，而与TRPV1和ASIC3无关。
2．PICK1缺失造成的ASICs功能损伤可能是由于ASIC1蛋白表达总量的减少所致，使其在细胞内的分布均匀减少，但其亚细胞定位并无改变。
第三部分PICK1对炎性痛时ASICs的作用及相关机制
目的：炎性疼痛的产生一是由于一些致痛物质的释放，如缓激肽、前列腺素、P物质、降钙素基因相关肽等；另一方面，由于炎症局部pH降低，可激活外周伤害感受器，引起伤害性感受器神经元产生失活速率较慢的长时程去极化，促使痛觉过敏的产生。在外周炎症模型中，脊髓背角神经元中ASIC1a蛋白表达明显增加，抑制其表达可产生明显镇痛效果。在神经系统，PICK1通过其特有的PDZ区域与PKCα结合，一方面可以促进PICK1的定位和作用发挥，另一方面，PICK1在PKC功能的发挥中也有重要作用。与PKC相同的是，PKA对ASICs功能的调节作用也需要PICK1来介导。
我们通过行为学实验已经证实，PICK1基因敲除小鼠对福尔马林导致的面部炎性疼痛和足部炎性热敏性疼痛耐受性均增加，但是其具体机制还不是很清楚。前面实验结果提示PICK1基因的缺失会影响生理情况下ASICs蛋白表达、细胞定位、电流幅度以及钙的释放，那么，在炎症时PICK1对ASICs的作用是怎样的?是否参与了PICK1对炎性痛觉行为学的作用?除了ASICs外一些炎性致痛物质和PKA、PKC是否也参与了PICK1对炎性痛觉行为学的作用?这些也值得我们去进一步探讨。
方法：采用整体动物实验和免疫荧光组织化学技术，观察PICK1基因敲除前后，ASICs、PKA、PKC、SP、CGRP的表达和分布情况，探讨PICK1影响炎性痛觉行为学可能的机制。
结果：
1．福尔马林造模后，野生型小鼠TG、DRG和脊髓背角的ASIC1和ASIC3表达均明显增加。PICK1基因敲除后，与野生型相比，这些部位ASIC1表达增加被明显抑制，但ASIC1在细胞内的分布并未改变，仍在细胞膜上和胞浆内均匀分布。PICK1基因敲除对ASIC3的表达增加无抑制作用。
2．4％福尔马林皮下注射后，野生型小鼠TG、DRG SP表达明显增强，同时PICK1敲除小鼠TG、DRG上SP表达较野生型小鼠明显减少。与之不同的是，在单纯比较对照组时我们在PICK1敲除小鼠TG、DRG上并未发现CGRP含量的变化，却发现其脊髓背角CGRP含量减少。
3．给予4％福尔马林皮下注射后，小鼠TG、DRG和脊髓背角PKC和PKA的荧光强度均明显增强，PICK1敲除后PKC的增强作用被取消而PKA的荧光强度无变化。
结论：
1．给予4％福尔马林皮下注射后，小鼠TG、DRG和脊髓背角ASIC1和ASIC3的表达均增加。不论是生理情况还是炎症时，PICK1基因敲除可减少TG、DRG和脊髓背角ASIC1的表达和分布，但是PICK1缺失对ASIC3的表达和分布无影响。
2．4％福尔马林皮下注射后，小鼠TG、DRG SP的含量明显增加，而PICK1敲除可取消炎症时的SP含量增加。
3．给予4％福尔马林皮下注射后，小鼠TG、DRG和脊髓背角PKC和PKA的表达均明显增加，PICK1敲除后PKC的增强作用被取消而PKA无变化。
PartⅠEffects of PICK1 gene knockout on the inflammatory pain
Aim: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.
PartⅡEffects of PICK1 gene knockout on the functions andexpressions of ASICs in TG、DRG and spinal dorsal horn
Aim: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 [Ca~(2+)]_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[Ca~(2+)]/[Ca~(2+)] 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[Ca~(2+)]/[Ca~(2+)] 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 [Ca~(2+)]_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.
PartⅢEffects of PICK1 on the action of ASICs in inflammatorypain and the related mechanism
Aim: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.
目的：PICK1(蛋白激酶Cα相互作用蛋白1，protein interacting with Cαkinase 1)是从线虫到人的所有生物中非常保守的一类存在于细胞质中的膜结合蛋白，自1995年被发现以来，已报道能与20多种蛋白发生相互作用。PICK1在人和动物的组织和细胞内均可表达，广泛分布于机体的各种组织。PICK1参与一些疾病的发生，如：癌症、精神分裂症、疼痛、帕金森综合症等。虽然PICK1在这些疾病发生过程中的具体作用机制现在还不清楚，但其广泛的结合特性使其可能通过与疾病相关蛋白的相互作用进而在疾病发生过程中扮演一定角色。
炎症是一种常见的病理情况，在组织炎症时，病变部位可产生慢性持续性疼痛和痛觉过敏，不仅使患者感到不适或痛苦，并且可造成诸多生理功能障碍和精神疾病，如睡眠失调、抑郁症、注意力下降等。已有研究显示PICK1基因敲除小鼠对机械性刺激反应减弱，但其对炎性疼痛的影响还不清楚。本实验的目的就是观察PICK1基因敲除后小鼠炎性痛觉行为是否改变。
方法：应用PICK1基因敲除小鼠，皮下注射4％福尔马林溶液造成炎性疼痛模型，与同窝野生型小鼠对比，观察炎性痛觉行为的变化情况。
结果：PICK1基因敲除后，其炎性痛觉行为学发生明显变化，表现为对炎性疼痛更加耐受。
1．PICK1基因敲除对头面部炎性痛觉行为学的影响。
单侧面部触须垫皮下注射4％福尔马林溶液后，与同窝野生型小鼠比较，PICK1基因敲除小鼠每3 min时间内用同侧前肢或后肢摩擦注射部位的行为明显减少。
2．PICK1基因敲除对足部炎性痛觉行为学的影响。
(1)采用热板法测小鼠基础痛阈，发现与同窝野生型小鼠比较，PICK1基因敲除小鼠基础痛阈值明显增高。
(2)单侧足背皮下注射4％福尔马林溶液后，与同窝野生型小鼠比较，PICK1基因敲除小鼠痛阈值明显增高。
结论：
1．PICK1基因敲除小鼠对福尔马林导致的面部炎性疼痛的两期耐受性均增加，即不仅对第一相的急痛反应耐受性增加，对炎症反应所导致的第二相中枢敏感化也有抑制作用。
2．PICK1基因敲除小鼠单侧足背皮下注射4％福尔马林溶液前后对热刺激引发的缩足反应时间均明显延长，提示PICK1基因敲除对生理性和炎性热敏性疼痛耐受性均增强。
第二部分PICK1基因敲除对TG、DRG和脊髓背角ASICs通道功能和表达的影响
目的：酸敏感离子通道(acid-sensing ion channels，ASICs)是ENaC／DEG(epithelial Na~+channel／degenerin)通道超家族的成员之一，能被细胞外pH下降或H~+浓度上升直接激活。它广泛分布于中枢及外周神经系统的神经元，参与了很多重要的生理过程和病理反应，与学习记忆、突触可塑性、脑缺血、疼痛等关系密切。H~+激活ASICs可使神经元去极化，产生动作电位，参与了组织酸化如缺血、炎症、肿瘤、癫痫等时痛觉的感受。由中度pH降低所诱发的皮肤表面酸诱导疼痛主要是由ASICs介导的，而更强的酸化引起的疼痛则是ASICs和TRPV1共同参与。目前已克隆出的ASICs主要有ASIC1a、ASIC1b、ASIC2a、ASIC2b、ASIC3和ASIC4。PICK1通过其PDZ结构域与ASIC1和ASIC2相互作用，对ASICs功能的发挥起到重要作用，但PICK1与外周和中枢感觉神经元ASICs之间具体作用如何以及有何意义?在上文中我们已经证实PICK1基因敲除小鼠对炎性疼痛的耐受性增强，它的具体机制是什么?是否与ASICs有关?这些都值得我们去进一步探讨。
方法：采用western blotting、全细胞膜片钳、钙影像技术和免疫荧光组织化学技术，观察PICK1基因敲除前后，外周初级感觉神经元和脊髓背角上ASICs的蛋白表达和功能的变化情况，探讨可能的机制。
结果：PICK1基因敲除后AISC1的功能和蛋白表达均降低而AISC3的功能和蛋白表达均无变化。
1．PICK1基因敲除小鼠TG神经元上AISC1电流幅度减小，全细胞膜片钳结果显示野生型AISC1电流幅度为2．37±0．46nA，PICK1基因敲除后AISC1电流幅度减少为1．21±0．35 nA(n=7，p＜0．05 vs wild type)。PICK1基因敲除小鼠TG神经元上通过ASIC1通道诱导的胞内钙升高减少。将细胞外液由pH 7．4迅速转换成pH 6．0时，野生型△[Ca~(2+)]／[Ca~(2+)]比值为0．218±0．034(n=11 from 2 animals)，纯合子为0．131±0．020(n=10 from 2 animals，p＜0．05 vs wild type)。将细胞外液由pH 7．4迅速转换成pH5．0时，野生型△[Ca~(2+)]／[Ca~(2+)]比值为0．818±0．204 (n=18 from 3 animals)，纯合子为0．379±0．091 (n=18 from 4 animals，p＜0．01 vs wild type)。
2．PICK1缺失造成的ASIC1功能损伤可能是由于ASIC1蛋白表达总量的减少所致，使其在细胞内的分布均匀减少，但其亚细胞定位并无改变。Western blotting和免疫荧光的结果均显示，小鼠TG、DRG和脊髓背角上ASIC1蛋白表达总量和荧光强度均减少，但分布并未改变，仍在细胞膜上和胞浆内均匀分布(n=6)。
3．PICK1基因敲除小鼠TG神经元上ASIC3和TRPV1的表达和功能无变化。膜片钳实验结果显示：PICK1基因敲除小鼠TG神经元上ASIC3和TRPV1电流幅值均无明显变化(ASIC3：WT 2．59±0．41nA vs KO 2．43±0．39 nA，n=8，P＞0．05；TRPV1：WT 1314±268．3pAvs KO 1256±272．4pA，n=4，p＞0．05)；Western blotting和免疫荧光的结果均显示，小鼠TG、DRG上无论是ASIC3还是TRPV1的蛋白表达量都未发生改变；ASIC3和TRPV1在细胞膜上和胞浆内均匀分布(n=6 for ASIC3 and n=3 forTRPV1)。
结论：
1．PICK1基因敲除造成ASICs的功能损伤。PICK1基因敲除小鼠TG神经元上AISC1电流幅度减小，通过ASIC1通道诱导的胞内钙升高减少。根据不同ASIC亚单位的分布和特性，推测这一作用主要是通过ASIC1a同聚体来实现，而与TRPV1和ASIC3无关。
2．PICK1缺失造成的ASICs功能损伤可能是由于ASIC1蛋白表达总量的减少所致，使其在细胞内的分布均匀减少，但其亚细胞定位并无改变。
第三部分PICK1对炎性痛时ASICs的作用及相关机制
目的：炎性疼痛的产生一是由于一些致痛物质的释放，如缓激肽、前列腺素、P物质、降钙素基因相关肽等；另一方面，由于炎症局部pH降低，可激活外周伤害感受器，引起伤害性感受器神经元产生失活速率较慢的长时程去极化，促使痛觉过敏的产生。在外周炎症模型中，脊髓背角神经元中ASIC1a蛋白表达明显增加，抑制其表达可产生明显镇痛效果。在神经系统，PICK1通过其特有的PDZ区域与PKCα结合，一方面可以促进PICK1的定位和作用发挥，另一方面，PICK1在PKC功能的发挥中也有重要作用。与PKC相同的是，PKA对ASICs功能的调节作用也需要PICK1来介导。
我们通过行为学实验已经证实，PICK1基因敲除小鼠对福尔马林导致的面部炎性疼痛和足部炎性热敏性疼痛耐受性均增加，但是其具体机制还不是很清楚。前面实验结果提示PICK1基因的缺失会影响生理情况下ASICs蛋白表达、细胞定位、电流幅度以及钙的释放，那么，在炎症时PICK1对ASICs的作用是怎样的?是否参与了PICK1对炎性痛觉行为学的作用?除了ASICs外一些炎性致痛物质和PKA、PKC是否也参与了PICK1对炎性痛觉行为学的作用?这些也值得我们去进一步探讨。
方法：采用整体动物实验和免疫荧光组织化学技术，观察PICK1基因敲除前后，ASICs、PKA、PKC、SP、CGRP的表达和分布情况，探讨PICK1影响炎性痛觉行为学可能的机制。
结果：
1．福尔马林造模后，野生型小鼠TG、DRG和脊髓背角的ASIC1和ASIC3表达均明显增加。PICK1基因敲除后，与野生型相比，这些部位ASIC1表达增加被明显抑制，但ASIC1在细胞内的分布并未改变，仍在细胞膜上和胞浆内均匀分布。PICK1基因敲除对ASIC3的表达增加无抑制作用。
2．4％福尔马林皮下注射后，野生型小鼠TG、DRG SP表达明显增强，同时PICK1敲除小鼠TG、DRG上SP表达较野生型小鼠明显减少。与之不同的是，在单纯比较对照组时我们在PICK1敲除小鼠TG、DRG上并未发现CGRP含量的变化，却发现其脊髓背角CGRP含量减少。
3．给予4％福尔马林皮下注射后，小鼠TG、DRG和脊髓背角PKC和PKA的荧光强度均明显增强，PICK1敲除后PKC的增强作用被取消而PKA的荧光强度无变化。
结论：
1．给予4％福尔马林皮下注射后，小鼠TG、DRG和脊髓背角ASIC1和ASIC3的表达均增加。不论是生理情况还是炎症时，PICK1基因敲除可减少TG、DRG和脊髓背角ASIC1的表达和分布，但是PICK1缺失对ASIC3的表达和分布无影响。
2．4％福尔马林皮下注射后，小鼠TG、DRG SP的含量明显增加，而PICK1敲除可取消炎症时的SP含量增加。
3．给予4％福尔马林皮下注射后，小鼠TG、DRG和脊髓背角PKC和PKA的表达均明显增加，PICK1敲除后PKC的增强作用被取消而PKA无变化。
PartⅠEffects of PICK1 gene knockout on the inflammatory pain
Aim: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.
PartⅡEffects of PICK1 gene knockout on the functions andexpressions of ASICs in TG、DRG and spinal dorsal horn
Aim: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 [Ca~(2+)]_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[Ca~(2+)]/[Ca~(2+)] 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[Ca~(2+)]/[Ca~(2+)] 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 [Ca~(2+)]_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.
PartⅢEffects of PICK1 on the action of ASICs in inflammatorypain and the related mechanism
Aim: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.
引文
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6. Chu XP, Wemmie JA, Wang WZ, Zhu XM, Saugstad JA, Price MP, Simon RP, Xiong ZG. (2004) Subunit-dependent high-affinity zinc inhibition of acid-sensing ion channels. J Neurosci 24: 8678-8689.
7. Wang W, Yu Y, Xu TL. (2007) Modulation of acid-sensing ion channels by Cu(2+) in cultured hypothalamic neurons of the rat. Neuroscience. 145(2): 631-41.
8. Voilley N, Welle J, Mamet J, et al. (2001)Nonsteroid anti-inflammatory drugs inhibit both the activity and the inflammation-induced expression of ASICs in niciceptors. J.Neurosci, 21(20):8026-8033.
9. Duggan A, Garcia-Anoveros J, Corey DP. (2002) The PDZ domain protein PICK1 and the sodium channel BNaCl interact and localize at mechanosensory terminals of dorsal root ganglion neurons and dendrites of central neurons. J. Biol. Chem. 277(7):5203-5208.
10. Hruska-Hageman AM, Wemmie JA, Price MP, Welsh MJ. (2002) Interaction of the synaptic protein PICK1 (protein interacting with C kinase 1) with the non-voltage gated sodium channels BNC1 (brain Na+ channel 1) and ASIC (acid-sensing ion channel)．Biochem J．361(Pt 3)： 443-450．
11．Deval， E．； Salinas， M．； Baron， A．； Lingueglia， E．； Lazdunski， M．ASIC2b-dependent Regulation of ASIC3， an Essential Acid-sensing Ion Channel Subunit in Sensory Neurons via the Partner Protein PICK1．J．Biol．Chem．2004， 279， 19531-19539．
12．Xiong ZG， Zhu XM， Chu XP， et al．(2004) Neuroprotection in ischemia： blocking calcium permeable acid-sensing ion channels．Cell．118 (6)： 687 -698．
13．伍龙军徐天乐。(2006)大鼠脊髓背角神经元中酸敏感离子通道的特性和功能研究。生理科学进展，37 (2)： 135-137．
1．H(?)ctor I．Rocha-Gonz(?)leza， Emma B．Herrejon-Abreub， Francisco J．L(?)pez-Santill(?)nc， et al．(2009) Acid increases inflammatory pain in rats： Effect of local peripheral ASICs inhibitors．European Journal of Pharmacology， 603(1-3)： 56-61．
2．Reeh PW， Kress M．(2001) Molecular physiology of proton transduction in nociceptors．Curr Opin Pharmacol， 1(1)：45-51．
3．Bo Duan， Long-JunWu， Tian-Le Xu， et al．(2007) Upregulation of acid-sensing ion channel ASIC1a in spinal dorsal horn neurons contributes to inflammatory pain hypersensitivity．J．Neurosci， 27(41)： 11139 -11148．
4．Deval E， Salinas M， Baron A， et al．(2004)ASIC2b-dependent Regulation of ASIC3， an Essential Acid-sensing Ion Channel Subunit in Sensory Neurons via the Partner Protein PICK-1．J．Biol．Chem．279(19)： 19531-19539．
5．Hruska-Hageman AM， Wemmie JA， Price MP， Welsh MJ．(2002)Interaction of the synaptic protein PICK1 (protein interacting with C kinase 1) with the non-voltage gated sodium channels BNC1 (brain Na+ channel 1) and ASIC (acid-sensing ion channel)．Biochem J．361(Pt 3)： 443-450．
6．Duggan A， Garcia-Anoveros J， Corey DP．(2002)The PDZ domain protein PICK1 and the sodium channel BNaC1 interact and localize at mechanosensory terminals of dorsal root ganglion neurons and dendrites of central neurons．J．Biol．Chem．277(7)：5203-5208．
7．MaggiCA．(1997)The effects of tachykininson inflammatory and immune cells．Regul Pept， 70： 75-90．
8．Jang JH．(2004)Involvement of peripherally released substance P and calcitonin gene-related peptide in mechanical hyperalesia in a traumatic neuropathy model of the rat．Neurosci Lett， 360(3)： 129-132．
9．Zhang L， Hoff AO， Wimalawansa SJ， et al．(2001)Arthritic calcitonin/alpha calcitonin gene-related peptide knockout mice have reduced nociceptive hypersensitivity．Pain，89(223)： 2652-2731．
10．Staudinger J， Zhou J， Burgess R， Elledge SJ， Olson EN．(1995) PICK1： a perinuclear binding protein and substrate for protein kinase C isolated by the yeast two-hybrid system．J．Cell．Biol．128： 263-271．2．
11．Masukawa K， Sakai N， Ohiho S， Shirai Y and Saito N．(2006)．Spatiotemporal analysis of the molecular interaction between PICK1 and PKC．Acta Histochem．Cytochem．39(6)： 173-181．
12．Baron A， Deval E， Salinas M， Lingueglia E， Voilley N， Lazdunski M．(2002) Protein kinaseCstimulatestheacid-sensingionchannelASIC2aviathePDZ domain-containing protein PICK1．J Biol Chem．277(52)： 50463-50468．
13．Gerard PA， Louis SP．(2002)Voltate-dependent priming of rat vanilloid receptor： effects fo agonist and protein kinase C activation．J．Physiol．545：441-451．
1. Tadeya R, Tadeshige K, Sumimoto H. Interaction of the PDZ domain of human PICKl with class Ⅰ ADP-ribosylation factors. Biochem Biophys Res Commun, 2000, 267(1):149-155.
2. Staudinger J, Zhou J, Burgess R, et al. PICKl: a perinuclear binding protein and substrate for protein kinase C isolated by the yeast two-hybrid system. J Cell Biol,1995,128(3): 263-271.
3. Dev KK. Making protein interactions druggable: targeting PDZ domains. Nat Rev Drug Discov, 2004, 3(12): 1047-1056.
4. Hanley JG, Khatri L, Hanson PI, et al. NSF ATPase and alpha-/beta-SNAPs disassemble the AMPA receptor-PICKl complex. Neuron, 2002, 28; 34(1): 53-67.
5. Kenneth LM, Thijs Beuming, Masha YN, et al. Molecular determinants for the complex binding specificity of the PDZ domain in PICKl. J.Biol.Chem. 2005, 280:20539-20548.
6. Perez JL, Khatri L, Chang C, et al. PICKl targets activated protein kinase Calpha to AMPA receptor clusters in spines of hippocampal neurons and reduces surface levels of the AMPA-type glutamate receptor subunit 2. J Neurosci. 2001, 21(15): 5417-28.
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31. Torres GE, Yao WD, Mohn AR, et al. Functional interaction between monoamine plasma membrane transporters and the synaptic PDZ domain-containing protein PICKl.Neuron, 2001, 30: 121-134.
32. Deval E, Salinas M, Baron A, et al. ASIC2b-dependent regulation of ASIC3, an essential acid-sensing ion channel subunit in sensory neurons via the partner protein PICK-1. J Biol Chem, 2004, 279: 19531-19539.
33. Baron E Deval, M Salinas, E Lingueglia, et al. Protein kinase C stimulates the acid-sensing ion channel ASIC2a via the PDZ domain-containing protein PICK1. J Biol Chem, 2002, 277(52): 50463-50468.
34. S Leonard, O Yermolaieva, MJ Welsh, et al. cAMP-dependent protein kinase phosphorylation of the acid-sensing ion channel-1 regulates its binding to the protein interacting with C-kinase-1 PNAS, 2003, 100(4): 2029-2034.
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39. Jourdi H, Iwakura Y, Narisawa-Saito M, et al. Brain-derived neurophic factor signal enhances and maintains the expression of AMPA receptor-associated PDZ proteins in developing cortical neurons. Dev Biol, 2003, 263(2): 216-230.
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41. Excoffon K J, Hruska-Hageman A, Klotz M, et al. A role for the PDZ-binding domain of coxsackie B virus and adenovirus receptor(CAR) in cell adhesion and growth. J Cell Sci, 2004,117(Pt 19): 4401-4409.
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44. Xiong ZG, Zhu XM, Chu XP, et al. Neuroprotection in ischemia: blocking calcium permeable acid-sensing ion channels. Cell, 2004,118 (6): 687-698.
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51. Piserchil A, Salinas G D, Li T, et al. Targeting specific PDZ domains of PSD-95: structural basis for enhanced affinity and enzymatic stability of a cyclic peptide. Chem Biol, 2004, 11(4): 469-473.
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53. Fujii N, Haresco JJ, Novak KA, et al. A selective irreversible inhibitor targeting a PDZ protein interaction domain. J Am Chem Soc, 2003,125: 12074-12075.
2. Reeh PW, Kress M. Molecular physiology of proton transduction in nociceptors. Curr Opin Pharmacol, 2001; 1(1):45-51.
3. A Leffler, B Monter, and M Koltzenburg. The role of the capsaicin receptor TRPV1 and acid-sensing ion channels (ASICS) in proton sensitivity of subpopulations of primary nociceptive neurons in rats and mice. Neuroscience, 2006; 139(2): 699-709.
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7. Bo Duan, Long-Jun Wu, Yao-Qing Yu, et al. Upregulation of acid-sensing ion channel ASICla in spinal dorsal horn neurons contributes to inflammatory pain hypersensitivity.J. NeuroscL, 2007; 27(41):11139-11148.
8. Krishtal, O. The ASICs: signaling molecules? Modulators? Trends Neurosci. 2003; 26:477-483.
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12. Anne Baron, Nicolas Voilley, Michel Lazdunski, et al. Acid sensing ion channels in dorsal spinal cord neurons. J. Neurosci., 2008; 28(6):1498-1508.
13. Nicolas Voilley, Jan de Weille, Julien Mamet, and Michel Lazdunski. Nonsteroid anti-inflammatory drugs inhibit both the activity and the inflammation-induced expression of acid-sensing ion channels in nociceptors. J. Ncurosci, 2001; 21(20):8026-803.
14. Monica Joch, Ariel R. Ase, Carol X.-Q. Chen, et al. Parkin-mediated monoubiquitmation of the PDZ protein PICKl regulates the activity of acid-sensing ion channels. Mol. Biol. Cell, 2007; 18: 3105-3118.
15. Duggan A, Garcia-Anoveros J, Corey DP. (2002) The PDZ domain protein PICKl and the sodium channel BNaCl interact and localize at mechanosensory terminals of dorsal root ganglion neurons and dendrites of central neurons. J. Biol. Chem. 277(7):5203-5208.
16. Hruska-Hageman AM, Wemmie JA, Price MP, Welsh MJ. (2002) Interaction of the synaptic protein PICKl (protein interacting with C kinase 1) with the non-voltage gated sodium channels BNC1 (brain Na+ channel 1) and ASIC (acid-sensing ionchannel). Biochem J. 361(Pt 3): 443-450
17. Anne Baron, Emmanuel Deval, Miguel Salinas, et al. Protein kinase C stimulates the acid-sensing ion channel ASIC2a via the PDZ domain-containing protein PICKl. J. Biol. Chem., 2002; 277: 50463-50468.
1．HaoS，TakahataO，IwasakiH．(2000)Intrathecalendomorphin-1produces antimociceptive activities modulated by alpha 2-adrenoceptors in the rat tail flick， tail pressure and formalin tests．Life Sci， 66(15)：PL195．
2．Neubert JK， King C， Malphurs W， Wong F， et al．(2008)Characterization of mouse orofacial pain and the effects of lesioning TRPV1-expressing neurons on operant behavior．Mol Pain．1(4)：43．
3．Reeh PW， Kress M．(2001) Molecular physiology of proton transduction in nociceptors Curr Opin Pharmacol， 1(1)：45-51．
4．Lee KH， Choi EM．(2008)Analgesic and anti-inflammatory effects of Ligularia fischeri leaves in experimental animals．J Ethnopharmacol．30；120(1)：103-7．
5．Philippe Luccarini， Anne Childeric， Anne-Marie Gaydier， et al．(2006)The Orofacial Formalin Test in the Mouse： A Behavioral Modelfor Studying Physiology and Modulation of Trigeminal Nociception．J Pain， 7： 908-914．
6．PatrickRaboissonb，RadhouaneDallel．(2004)Theorofacialformalintest．Neuroscience and Biobehavioral Reviews， 28： 219-226．
7．Kumlesh K．Dev．(2007) PDZ domain protein-protein interactions： A case study with PICK1．Current Topics in Medicinal Chemistry， 7： 3-20．
8．Monica Joch， Ariel R．Ase， Carol X．-Q．Chen， et al．(2007) Parkin-mediated monoubiquitination of the PDZ protein PICK1 regulates the activity of acid-sensing ion channels．Mol．Biol．Cell， 18： 3105-3118．
9．Anne Baron， Emmanuel Deval， Miguel Salinas， et al．(2002) Protein kinase C stimulates the acid-sensing ion channel ASIC2a via the PDZ domain-containing protein PICK1．J．Biol．Chem．， 277： 50463-50468．
10．Hanley， J．G．； Khatri， L．； Hanson， P．I．； Ziff， E．B．(2002)NSF ATPase and alpha-/ beta-SNAPs disassemble the AMPA receptor-PICK1 complex．Neuron 34， 53-67．
11．Lu， W．； Ziff， E．B．(2005) PICK1 interacts with ABP/GRIP to regulate AMPA receptor trafficking．Neuron， 47， 407-421．
1．G ．R．Dube， Sonya G．Lehto， Nicole M．Breese， et al．Electrophysiological and in vivo characterization of A-317567， a novel blocker of acid sensing ion channels．Pain 2005， 117: 88-96.
2. Bo Duan, Long-JunWu, Tian-Le Xu, et al. Upregulation of acid-sensing ion channel ASICla in spinal dorsal horn neurons contributes to inflammatory pain hypersensitivity.The Journal of Neuroscience, 2007, 27(41): 11139-11148.
3. Reeh PW, Kress M. Molecular physiology of proton transduction in nociceptors. Curr. Opin. Pharmcol, 2001,1: 45-51.
4. Dube GR, Lehto SG, Breese NM, et al. Electrophysiological and in vivo characterization of A-317567, a novel blocker of acid sensing ion channels. Pain. 2005,117(l-2):88-96.
5. Gao J, Wu LJ, Xu L, Xu TL. (2004) Properties of the proton-evoked currents and their modulation by Ca2+ and Zn2+ in the acutely dissociated hippocampus CA1 neurons.Brain Res 1017: 197-207.
6. Chu XP, Wemmie JA, Wang WZ, Zhu XM, Saugstad JA, Price MP, Simon RP, Xiong ZG. (2004) Subunit-dependent high-affinity zinc inhibition of acid-sensing ion channels. J Neurosci 24: 8678-8689.
7. Wang W, Yu Y, Xu TL. (2007) Modulation of acid-sensing ion channels by Cu(2+) in cultured hypothalamic neurons of the rat. Neuroscience. 145(2): 631-41.
8. Voilley N, Welle J, Mamet J, et al. (2001)Nonsteroid anti-inflammatory drugs inhibit both the activity and the inflammation-induced expression of ASICs in niciceptors. J.Neurosci, 21(20):8026-8033.
9. Duggan A, Garcia-Anoveros J, Corey DP. (2002) The PDZ domain protein PICK1 and the sodium channel BNaCl interact and localize at mechanosensory terminals of dorsal root ganglion neurons and dendrites of central neurons. J. Biol. Chem. 277(7):5203-5208.
10. Hruska-Hageman AM, Wemmie JA, Price MP, Welsh MJ. (2002) Interaction of the synaptic protein PICK1 (protein interacting with C kinase 1) with the non-voltage gated sodium channels BNC1 (brain Na+ channel 1) and ASIC (acid-sensing ion channel)．Biochem J．361(Pt 3)： 443-450．
11．Deval， E．； Salinas， M．； Baron， A．； Lingueglia， E．； Lazdunski， M．ASIC2b-dependent Regulation of ASIC3， an Essential Acid-sensing Ion Channel Subunit in Sensory Neurons via the Partner Protein PICK1．J．Biol．Chem．2004， 279， 19531-19539．
12．Xiong ZG， Zhu XM， Chu XP， et al．(2004) Neuroprotection in ischemia： blocking calcium permeable acid-sensing ion channels．Cell．118 (6)： 687 -698．
13．伍龙军徐天乐。(2006)大鼠脊髓背角神经元中酸敏感离子通道的特性和功能研究。生理科学进展，37 (2)： 135-137．
1．H(?)ctor I．Rocha-Gonz(?)leza， Emma B．Herrejon-Abreub， Francisco J．L(?)pez-Santill(?)nc， et al．(2009) Acid increases inflammatory pain in rats： Effect of local peripheral ASICs inhibitors．European Journal of Pharmacology， 603(1-3)： 56-61．
2．Reeh PW， Kress M．(2001) Molecular physiology of proton transduction in nociceptors．Curr Opin Pharmacol， 1(1)：45-51．
3．Bo Duan， Long-JunWu， Tian-Le Xu， et al．(2007) Upregulation of acid-sensing ion channel ASIC1a in spinal dorsal horn neurons contributes to inflammatory pain hypersensitivity．J．Neurosci， 27(41)： 11139 -11148．
4．Deval E， Salinas M， Baron A， et al．(2004)ASIC2b-dependent Regulation of ASIC3， an Essential Acid-sensing Ion Channel Subunit in Sensory Neurons via the Partner Protein PICK-1．J．Biol．Chem．279(19)： 19531-19539．
5．Hruska-Hageman AM， Wemmie JA， Price MP， Welsh MJ．(2002)Interaction of the synaptic protein PICK1 (protein interacting with C kinase 1) with the non-voltage gated sodium channels BNC1 (brain Na+ channel 1) and ASIC (acid-sensing ion channel)．Biochem J．361(Pt 3)： 443-450．
6．Duggan A， Garcia-Anoveros J， Corey DP．(2002)The PDZ domain protein PICK1 and the sodium channel BNaC1 interact and localize at mechanosensory terminals of dorsal root ganglion neurons and dendrites of central neurons．J．Biol．Chem．277(7)：5203-5208．
7．MaggiCA．(1997)The effects of tachykininson inflammatory and immune cells．Regul Pept， 70： 75-90．
8．Jang JH．(2004)Involvement of peripherally released substance P and calcitonin gene-related peptide in mechanical hyperalesia in a traumatic neuropathy model of the rat．Neurosci Lett， 360(3)： 129-132．
9．Zhang L， Hoff AO， Wimalawansa SJ， et al．(2001)Arthritic calcitonin/alpha calcitonin gene-related peptide knockout mice have reduced nociceptive hypersensitivity．Pain，89(223)： 2652-2731．
10．Staudinger J， Zhou J， Burgess R， Elledge SJ， Olson EN．(1995) PICK1： a perinuclear binding protein and substrate for protein kinase C isolated by the yeast two-hybrid system．J．Cell．Biol．128： 263-271．2．
11．Masukawa K， Sakai N， Ohiho S， Shirai Y and Saito N．(2006)．Spatiotemporal analysis of the molecular interaction between PICK1 and PKC．Acta Histochem．Cytochem．39(6)： 173-181．
12．Baron A， Deval E， Salinas M， Lingueglia E， Voilley N， Lazdunski M．(2002) Protein kinaseCstimulatestheacid-sensingionchannelASIC2aviathePDZ domain-containing protein PICK1．J Biol Chem．277(52)： 50463-50468．
13．Gerard PA， Louis SP．(2002)Voltate-dependent priming of rat vanilloid receptor： effects fo agonist and protein kinase C activation．J．Physiol．545：441-451．
1. Tadeya R, Tadeshige K, Sumimoto H. Interaction of the PDZ domain of human PICKl with class Ⅰ ADP-ribosylation factors. Biochem Biophys Res Commun, 2000, 267(1):149-155.
2. Staudinger J, Zhou J, Burgess R, et al. PICKl: a perinuclear binding protein and substrate for protein kinase C isolated by the yeast two-hybrid system. J Cell Biol,1995,128(3): 263-271.
3. Dev KK. Making protein interactions druggable: targeting PDZ domains. Nat Rev Drug Discov, 2004, 3(12): 1047-1056.
4. Hanley JG, Khatri L, Hanson PI, et al. NSF ATPase and alpha-/beta-SNAPs disassemble the AMPA receptor-PICKl complex. Neuron, 2002, 28; 34(1): 53-67.
5. Kenneth LM, Thijs Beuming, Masha YN, et al. Molecular determinants for the complex binding specificity of the PDZ domain in PICKl. J.Biol.Chem. 2005, 280:20539-20548.
6. Perez JL, Khatri L, Chang C, et al. PICKl targets activated protein kinase Calpha to AMPA receptor clusters in spines of hippocampal neurons and reduces surface levels of the AMPA-type glutamate receptor subunit 2. J Neurosci. 2001, 21(15): 5417-28.
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