内皮素-1引起搔痒的分子机制研究
|
摘要
内皮素-1(endothelin-1, ET-1)最初由猪主动脉内皮细胞培养液中分离得到的一种活性肽,是目前已知最强的缩血管活物质。除内皮细胞外,还发现角质形成细胞、血管平滑肌细胞、白细胞、心肌细胞及间质细胞及一些肿瘤细胞也发现能分泌ET-1。ET-1通过两种G蛋白耦联的受体(G-protein-coupled receptor, GPCR),ETA和ETB受体发挥作用。ETA受体主要在血管内皮、神经元及肥大细胞发现有表达,而ETB受体主要在角质形成细胞、血管平滑肌细胞及内皮细胞、神经胶质细胞等表达。
辣椒素可引起新鲜分离的背根神经节(dorsal root ganglion, DRG)神经元细胞内向电流及细胞内游离钙浓度上升,ET-1可通过蛋白激酶C (protein kinaseC,PKC)途径增强DRG神经元细胞内钙对辣椒素的反应,辣椒素引起的内向电流也明显增强。动物局部应用ET-1出现伤害性行为表现,而人局部应用ET-1出现疼痛,近年来发现人及动物局部应用ET-1也出现搔痒。
由同一种物质同时引起疼痛和搔痒,机体如何处理疼痛和搔痒的信息仍不得而知。ET-1引起搔痒的分子机制尚不清楚,脊髓中的胃泌素释放肽受体(Gastrin-releasing peptide receptor, GRPR)是搔痒感受分子,选择性去除小鼠脊髓Ⅰ层表达GRPR的神经元,动物对所有测试的致痒剂(包含ET-1在内)均无反应,提示初级神经元可能释放不同的神经递质,激活脊髓中痛或者痒特异性感受神经元,本文主要研究ET-1引起搔痒分子信号通路。
方法
实验动物
实验选用雄性C57BL/6J小鼠,体重20-25g,由中山大学实验动物中心提供,本研究及实验动物的使用与处理方法得到广东医学科学院动物试验伦理委员会的书面同意,并按照规定遵守动物福利及尽可能减少使用动物的数量。
颈背部注射搔痒模型
小鼠于实验前一天颈背部皮肤剃毛,小鼠于实验前30min放置于透明的塑料盒子里预适应。应用30G针头进行颈背部皮内注射,注射药物后立即放入塑料盒子里面观察,观察小鼠用后肢搔抓注射部位次数,小鼠完成从举起后肢搔抓注射部位到放下后肢全过程为一次搔抓,观察30min。
脸颊部注射行为学观察
实验前一天小鼠脸颊部剃毛,实验前放置于透明的塑料盒子预适应30min,小鼠从盒子里面取出后用微量注射器(将30G针头与微量注射器相连)进行脸颊部皮内注射,注射容量为10μl。注射药物后立即放入塑料笼子里面观察,记录30min内动物分别用前肢拂和后肢搔抓注射部位的次数,用注射侧前肢拂注射部位是疼痛相关的行为学表现,用后肢搔抓注射部位是搔痒相关的行为学表现。
热痛觉阈值
热痛觉阈值测量实验前一天,小鼠在实验台适应1h,测定热痛觉阈值时,用热辐射刺激仪照射小鼠足底紧贴玻璃板部位。记录照射开始至小鼠出现明显逃避的时间,调节光照强度,使注射药物前逃避时间(paw withdraw time,PWT)为(8-12)s。为避免热损伤,光照时间不超过20s。
去TRPV1神经元小鼠制作
雄性新生小鼠于出生后第二天七氟醚麻醉后皮下注射辣椒素(50mg/kg),对照组注射同样容量的溶剂(PBS含10%ethanol与10%Tween-80),动物在SPF动物房里面饲养6周后进行实验。新生期注射辣椒素能够去除表达TRPV1神经元,用角膜刺激实验验证去除TRPV1神经元的效果,用0.01%辣椒素20μl喷到小鼠眼睛上,小鼠会用前肢拂眼睛,记数1min内用前肢拂眼睛的次数,如1min内用前肢拂眼睛的次数少于5次就认为已经成功去除TRPV1神经元。
背根神经节神经元细胞培养
C57BL/6J小鼠用七氟醚麻醉后背部皮肤消毒,剪下脊柱后快速转移至放置于冰上培养皿内,将脊柱泡在冷的L15内,无菌状态下取出DRG,用0.25%胶原酶Ⅳ于37℃水浴振荡1h,机械吹打后用低温离心机在下离心(1000rpm)5min。培养液为含10%胎牛血清的Dulbecco's modified Eagle's medium (DMEM),加入青霉素G钠盐和硫酸链霉素各10μ g/ml,并且加入神经生长因子50ng/ml,细胞接种于多聚左旋赖氨酸包被的玻片或培养板上,置于37℃5%CO2培养箱内培养。
PKCε膜转位
细胞接种于多聚左旋赖氨酸包被的直径为10mm玻片上,用0.01M磷酸盐缓冲液(phosphate-buffered saline, PBS)洗脱培养液后,加入PBS或ET-1后放入37℃及5%CO2培养箱内60s后吸出上清,用4%PFA固定20min,漂洗后使用含10%的山羊血清室温下作用1h,加入第一抗体,分别为兔来源抗PKCε抗体(1：500)及豚鼠来源的抗TRPV1抗体(1：3000),并加入0.2%Triton4℃下孵育16h,漂洗后加入结合有Alexa Fluor594抗兔IgG和结合有Alexa Fluor488抗豚鼠IgG的第二抗体孵育2h。用激光共焦显微镜(Zeiss, Germany)观察PKCs膜转位情况。
细胞内cAMP检测
细胞接种于多聚左旋赖氨酸包被的24孔培养板上,倒去培养液,用PBS冲洗后,加入含磷酸二酯酶抑制3-异丁基-1-甲基黄嘌呤(3-isobutyl-1-methylxanthine, IBMX,2.5mM)的PBS(含0.1%葡萄糖)在37℃培养箱内孵育10min,吸出培养液,加入含IBMX (2.5mM)及ET-1(10nM)的PBS(对照组只加入IBMX)继续孵育60s,倒去培养液,每孔加入细胞裂解液250μ1(0.1M HC1,0.8%Triton X-100),室温下放置10min,吸出后离心(10000rpm)2min取上清,上清100μl应用酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)测量标本中cAMP水平,采用小鼠cAMP单克隆抗体ELISA检测试剂盒检测。用纯化的山羊抗小鼠抗体IgG包被96孔板,制成固相抗体,在实验前20min准备好所有试剂及酶联板放置于室温,往包被抗体的96孔板中依次加入cAMP标准品或样品、小鼠单克隆cAMP抗体、辣根过氧化物酶(horse-radish peroxidase, HRP)结合的cAMP,经过彻底洗涤后用底物四甲基联苯胺(3,3',5,5'-tetramethylbenzidine,TMB)显色。TMB在过氧化物酶的催化下转化成蓝色,并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的cGMP呈正相关。用在450nm波长下测定吸光度(OD值),根据标准品浓度吸光度曲线测量样品浓度。另取上清50μ l应用二喹啉甲酸蛋白质试剂盒(bicinchoninic acid protein assay kit, BCA protein assay kit)测量蛋白含量,测量562nm处吸光度,根据标准品浓度吸光度曲线计算样品蛋白浓度。我们将细胞内cAMP浓度根据蛋白含量进行校正比较,最终细胞内cAMP浓度单位表达为pmol/mg蛋白。
结果
1ET-1通过激活ETA受体引起搔痒
当颈背部皮内注射100μl浓度为10μM(总量为1000pmol)的ET-1时产生明显的搔抓,30min内小鼠用后肢抓注射部位的次数达到360次,依次稀释10倍发现颈背部皮内注射总量为1pmol(此时浓度仅为0.01μM)ET-1仍可引起明显搔抓,当注射总量为100pmol时30min内引起搔抓187次,从ET-1引起搔痒的剂量反应关系可以看到这一剂量(1μM,100μl)引起中等程度的搔抓次数,我们以后的研究中除非特别说明均采用这一剂量的ET-1(1μM,100μl)。
脸颊部注射ET-110μM(容量为10μl)可引起明显后肢搔抓与前肢拂注射部位,注射ET-11μM引起明显后肢搔抓,但前肢拂注射部位不明显,而注射ET-10.1μM不能引起明显的后肢搔抓与前肢拂注射部位。
特异性ETA受体拮抗剂BQ123及特异性ETB受体拮抗剂BQ788单独颈背部皮内注射时并不引起明显的搔抓反应,BQ123100nmol与100pmol ET-1共同注射时可明显抑制ET-1引起的搔抓,而BQ78830nmol与100pmol ET-1共同注射时可明显增加ET-1引起的搔抓,说明ET-1通过ETA受体引起搔痒,而ETB受体可能发挥止痒作用。
2PKC途径介导ET-1引起的搔痒
双吲哚亚酰胺I (Bisindolylmaleimide I, BIM)是蛋白激酶C (protein kinaseC,PKC)抑制剂,BIM2μg或10μg与ET-1共同注射均能明显抑制ET-1引起的搔痒,说明ET-1引起的搔痒和PKC途径有关。培养的DRG神经元用ET-1(10nM)处理后可见PKCε主要分布在细胞膜上,而未用ET-1处理的DRG神经元可见PKCε主要分布在细胞浆内。
巴豆醇-12-十四烷酸酯-13-乙酸酯(phorbol-12-myristate-13-acetate,PMA)是常用的PKC激活剂,颈背部皮内注射PMA1μM和10μM时(总量分别为100pmol与1000pmol)时引起搔痒,而注射10pmol时不引起明显搔痒。
足底注射PMA与ET-1均能引起热痛觉过敏,小鼠足底注射PMA100pmol/10μl一直出现抬足等行为,不能测量热痛觉阈值,10pmol/10μl引起明显的热痛觉过敏,1pmol/10μl引起的热痛觉过敏ET-1100pmol/10μl所引起的热痛觉过敏相当,足底注射1μg BIM15min后可以完全抑制同一部位注射ET-1和PMA引起的热痛觉过敏。
3cAMP-PKC途径介导ET-1引起的搔痒
由磷酯酶C (phospholipase C, PLC)激活PKC是PKC激活的经典通路,我们应用PLC阻滞剂U73122观察PLC通路的作用。颈背部注射U73122(10pmol,100μl)不引起明显搔抓及其他异常表现,但与预期结果相反,U73122(100pmol)与ET-1共同注射明显增加ET-1引起的搔抓次数。提示可能由非经典通路环单磷酸腺苷(cyclic adenosine monophosphate, cAMP)激活PKC从而介导ET-1引起的搔痒。
ET-1(10nM)刺激培养的DRG神经元60s后,应用cAMP单克隆抗体ELISA法测量细胞内cAMP浓度的变化,ET-1处理神经元后明显增加细胞内cAMP浓度。直接应用腺苷酸环化酶(Adenylyl cyclase, AC)抑制剂SQ22536对ET-1引起搔痒的作用。SQ22536与ET-1共同注射可剂量依赖性地抑制ET-1引起的搔痒。AC激活后引起细胞内cAMP浓度升高,cAMP浓度升高引起蛋白激酶A(protein kinase A, PKA)激活,PKA阻滞剂H89与ET-1共同皮内注射对ET-1引起的搔痒无明显作用。
为了排除U73122的非特异性作用,我们研究了更高浓度的U73122对ET-1及复合物48/80(compound48/80, C48/80)引起搔痒的影响。C48/80可刺激培养的肥大细胞脱颗粒释放组胺,U73122(10μM)可明显抑制肥大细胞的脱颗粒。C48/80皮内注射可引起明显的搔痒,与U73122(1000pmol)共同注射后引起的搔痒可明显减轻,而U73122(1000pmol)仍然增加ET-1引起的搔痒。皮内注射phosphatidylcholine specific phospholipase C(磷脂酰胆碱磷酯酶C, PC-PLC)抑制剂D609,15min再在同一部位注射ET-1或者C48/80,D609可抑制ET-1引起的搔痒,而D对C48/80引起的搔痒无明显影响。
4TRPV1、H1R、TRP、TRPA1在ET-1引起搔痒中的作用
腹腔注射TRPV1拮抗剂辣椒平(4mg/kg)或者H1R拮抗剂美吡拉敏(mepyramine,40mg/kg)对ET-1引起的搔痒无明显影响。新生期注射辣椒素后能去除TRPV1神经元,ET-1在新生期注射辣椒素成年动物不引起明显搔痒,而对照组成年动物对ET-1引起的搔痒反应无明显影响。非选择性TRP抑制剂钌红(ruthenium red, RR,5nmol)共同注射可增加ET-1引起的搔痒,但同样剂量的RR抑制组胺引起的搔痒。TRPA1拮抗剂AP18(100nmol)共同注射可增加ET-1引起的搔痒,同样剂量的AP18对组胺引起的搔痒无明显作用。ET-1引起的疼痛可能与TRPA1有关,RR与AP18增加ET-1引起的搔痒可能与其减少疼痛有关。在皮内注射ET-1前15min皮下注射吗啡(5mg/kg),吗啡对ET-1引起的搔痒无明显影响,提示ET-1引起的搔痒与疼痛无关。
5ETB受体的止痒作用
皮下注射纳洛酮0.5mg/kg可明显抑制15min后皮内注射ET-1所引起的搔痒,而小剂量的纳洛酮(2nmol)皮下注射对ET-1引起的搔痒无明显影响,当纳洛酮与ET-1共同一起皮内注射时,两种剂量的纳洛酮(2nmol与0.5mg/kg)均明显增加ET-1引起的搔痒,这种作用与纳洛酮的全身作用无关,因为全身应用纳洛酮的效果与此相反或者在小剂量的时候无明显效果。
外周存在μ、κ、δ三种阿片类受体亚型,我们应用选择性亚型拮抗剂来观察各亚型的作用,大剂量KOR拮抗剂Nor-BNI(超过15nmol/50μl时)颈背部皮内注射时可引起搔痒,而5nmol/50μl时不引起明显搔痒,我们选择了这一剂量进行研究,其他的剂量参考以前的研究,MOR拮抗剂CTOP或DOR拮抗剂naltrindole一起注射对ET-1引起的搔痒无明显影响,而Nor-BNI明显增加ET-1引起的搔痒,可见外周KOR介导了ETB受体的止痒作用。
KOR激动剂的抗伤害作用与一氧化氮有关,一氧化氮合酶(nitric oxide synthase)有内皮型(endothelia)、神经型(neuronal)和诱导型(inducible)三种亚型,nNOS抑抑剂可拮抗KOR激动剂的止痛作用。应用非选择性NOS抑制剂发现L-NAME(200nmol)明显增加ET-1引起的搔痒,而D-NAME对ET-1引起的搔痒无明显影响。NOS有三种亚型分别为内皮型、神经型、诱导型,应用特异的选择性亚型抑制剂与ET-1共同注射,eNOS抑制剂(L-NIO,200nmol)可明显增加ET-1引起的搔痒,而诱导型iNOS抑制剂(AMT,5nmol)、神经型nNOS亚型抑制剂(Nco-Propyl-L-arginine,20nmol)对ET-1引起的搔痒无明显作用。由于神经元中主要存在nNOS,角质形成细胞中主要存在eNOS, eNOS抑制剂也能增加ET-1引起的搔痒说明KOR激动剂可能通过角质形成细胞起作用。
结论
1ET-1通过ETA/AC/PKC途径引起搔痒。
2由cAMP激活PKC是非经典途径,这个通路通过Epac(exchange proteins activated directly by cyclic AMP, cAMP直接激活的交换蛋白)激活下游PLC与PLD,在体内与体外实验均发现从cAMP激活PKC离不开PLC与PLD的作用,本研究中PLC抑制剂U73122与预期相反的作用,说明ET-1引起搔痒的cAMP激活PKC通路与以前发现的通路不同,我们发现了一个全新的由cAMP激活PKC通路,该通路可能与PC-PLC通路有关。
3ETB受体发挥止痒作用,外周阿片类受体κ亚型通过eNOS介导这一止痒作用,角质形成细胞释放的NO也可能发挥止痒作用。
Endothelin-1(ET-1) is secreted by a variety of cell types, including keratinocytes, vascular smooth muscle cells, leukocytes, cardiomyocytes and mesangial cells, and some tumor cell lines since its first isolation from the cultures of porcine aortic endothelial cell cultures. It is the most potent vasoconstrictor known. The signaling of ET-1is mediated via two main membrane G-protein-coupled receptor (GPCR) subtypes, ETA and ETB receptors. The expression of ETA receptor is largely confined to the vascular endothelium, as well as neurons and mast cells, while ETB receptor is mainly expressed by keratinocytes, smooth muscle cells and vascular endothelium.
ET-1enhances capsaicin-evoked increases of intracellular calcium levels in freshly dissociated DRG neurons in a protein kinase C (PKC) dependent manner, and potentiation of capsaicin-induced currents. Local administration of ET-1induces nociceptive behaviors in animals and causes pain in humans. ET-1has been demonstrated to elicit pruritus in mice and humans also. How to cope with the messages of itch and pain inuuced by the same agent, ET-1. Little is known about the molecular mechanisms of the pruritogenic action of ET-1. Gastrin-releasing peptide receptor (GRPR) is an itch-specific molecule in the spinal cord [28]. Mice selectively ablated lamina I neurons expressing GRPR in the spinal cord showed profound scratching deficits in response to all of the itching (pruritogenic) stimuli (including ET-1) tested [29]. It is suggested that different neurotransmitters are released by the primary neurons to excite the pain-or itch-specicif second neurons in the spinal. The current study was designed to elucidate the molecular signals of itch induced by ET-1.
Methods
Animals
Male C57BL/6J mice, weighing20-22g, were purchased from the Center for Laboratory Animals, Sun Yat-Shen University. The experimental procedures and the animal use and care protocols were approved by the Committee on Ethical Use of Animals of Guangdong Academy of Medical Sciences. All efforts were made to minimize animal suffering and to reduce the number of animals used.
Pruritus model of neck injection
One day after shaving the rostral part of the back of the neck, mice were placed into a small plastic chamber30min before the experiment. For drug administration, mice were briefly removed from the chamber, and each test drug was intradermally injected with a30-gauge needle. Then the mice were returned to the chamber, and the hind limb scratching directed towards the shaved area at the back of the neck was observed and recorded for30min. One scratch was defined as a lift of the hind limb towards the injection site and then a reposition of the limb back to the floor, regardless of the scratching strokes that took place between the two movements.
Cheek injection model
One day after shaving the cheek, mice were placed into a small plastic chamber30min before the experiment. For drug administration, mice were briefly removed from the chamber, and each test drug was intradermally injected with a30-gauge needle.
A volume of10μl was injected intradermally into the cheek. Then the mice were returned to the chamber, number of hindpaw scratches directed to the injected area as an indicator of itch and number of ipsilateral forelimb wipes directed to the injected area as an indicator of pain were recorded for a period of30min after the injection. Paw withdrawal latency
Mice were placed into a small plastic chamber on glass table with the positioned in an heat stimulation apparatus for radiant heat stimulation on the plantar surface. Mice were placed into the small plastic chamber for1h one day before the experiment. Intensity of heat stimulus was adjusted for paw withdrawal latency of8-12s in normal animals. A cut-off time was set at20s to avoid injury to the hindpaw.
Depletion of TRPV1neurons in neonatal mice
Male neonatal mice (aged2days) were anesthetized with sevoflurane and injected subcutaneously with capsaicin (50mg/kg) or the vehicle (10%ethanol,10%Tween-80and80%PBS) as described previously. Animals were included in the study6weeks after the injection of capsaicin or the vehicle. The effects of capsaicin were expected to cause depletion of TRPV1neurons. It was verified by the eye-wiping test. For this test,0.01%capsaicin at the volume of20μl was sprayed into the eye and the number of wiping movements that occurred within1min was counted. The animal was considered to be desensitized to TRPV1by neonatal capsaicin treatment when the animal wiped its eyes no more than five times.
Cell culture of dorsal root ganglion
Adult male C57BL/6J mice were anesthetized with sevoflurane and the skin of the back were disinfected. The spinal were removed and transfered into cold L15medium and placed on ice. DRGs were removed bilaterally from lumbar and thoracic spinal levels under sterile conditions and incubated with0.25%collagenase type IV at37℃for60min with gentle agitation. Then, DRGs were centrifuged at1000rpm for5min in4℃after mechanically dissociated with pipette. Cells were resuspended with Dulbecco's modified Eagle's medium containing nerve growth factor (50ng/ml, Invitrogen),10%fetal bovine serum (Invitrogen), penicillin (100units/ml, In-vitrogen) and streptomycin (100g/ml, Invitrogen) and plated onto glass coverslips or plates precoated with poly-L-lysine and cultured in incubator containing5%CO2at37℃. Membrane translocation of PKCs
Cells were plated on10-mm diameter glass coverslips and were washed with0.01M PBS and then exposed to0.01M PBS or10nM ET-1for60s in incubator containing5%CO2at37℃, and immediately fixed with4%paraformaldehyde for20min, rinsed using PBS, and then incubated with10%normal goat for1h. Fixed slips were incubated with primary antibodies in0.2%Triton for16h at4℃. Then slips were incubated with second antibodies conjugated to Alexa Fluor488or Alexa Fluor594.
Measurement of cAMP
Cells were plated in24wells plate and washed with PBS before test, and preincubated at37℃for10min in the presence of the phosphodiesterase inhibitor3-isobutyl-l-methylxanthine (2.5mM) in PBS containing0.1%glucose. Cells were incubated for an additional60s at37℃with or without ET-1(10nM in2.5mM IBMX in PBS). Then PBS was removed, and250μl of HCl(0.1M) with0.8%Triton X-100(Sigma-Aldrich) was added to the plates. After10-min incubation at room temperature, the lysate was removed from the plates and centrifuged for2min. The supernatant was collected and100u1of it was measureded for cAMP concentration using a monoclonal cAMP enzyme immunoassay kit.
All reagents and plate are prepared at room temperature20min before test. Sample of cell extracts and standard cAMP are added to96-well plate coated with goat-anti-mouse serum. cAMP will competitively bind to the monoclonal anti-cAMP antibody in the presence of fixed amounts of cAMP-conjugated horse-radish peroxidase. Standard cAMP are used to generate the calculation curve. After a short incubation, the excess reagents are washed away and substrate (3,3',5,5'-tetramethylbenzidine, TMB) is added. The multiwell plates are then read on a microplate reader at450nm. The intensity of the yellow color is inversely proportional to the concentration of cAMP in samples. The measured optical density is used to calculate the concentration of cAMP in samples based on the curve from the cAMP standards.
The production of cAMP was normalized to the protein concentration in each well (pmol/mg protein). Protein content was determined using a bicinchoninic acid protein assay kit according to the manufacturer's instructions and determined at562nm.
Results
1ET-1induces scratches through activation of ETA receptor
Intradermal injection of ET-1to the back evoked the number of scratches in a dose-dependent manner, with a significant increase starting from1pmol (in100u1PBS) of ET-1. It induced an average of360bouts of scratches at the dose of1000pmol. At100pmol, ET-1induced a moderate number of scratches, within the linear range of ET-1effects. Therefore, the dose of100pmol (in100μl PBS) was selected for subsequent experiments.
Intradermal injection of ET-1to the cheek an average of18bouts of wipes at the dose of100pmol (in10μ1PBS). No wipes was evoked when the dose of ET-1lower than10pmol. Cheek injection of ET-1dose-dependently evoked scratching response when the dose of ET-1higher than10pmol (in10μl PBS).
Co-injection of BQ-123(selective antagonists for ETA) with ET-1at100nmol significantly reduced the number of scratches, while BQ-788(selective antagonists for ETB receptor) at30nmol increased the number of scratches induced by ET-1. These results show that ETA receptor mediates the pruritogenic effects, and the ETB receptor exerts anti-pruritic effects.
2PKC mediates pruritogenic effects of ET-1
Co-injection of Bisindolylmaleimide I (PKC inhibitor) at the dose of2u g or10μg reduced the scratching response induced by ET-1. Membrane translocation of PKCs were found in cultured dorsal root ganglion neurons after ET-1(10nM for60s) stimulation.
Scratching response was also induced by the PKC activator phorbol-12-myristate-13-acetate (PMA) at the dose above100pmol. Intraplantar injection of PMA at the dose of1pmol (in10μl PBS) reduced the paw withdraw latency from heat stimulation about that induced by ET-1at the dose of100pmol (in10μl PBS). Thermal hyperalgesia induced by PMA and ET-1were inhibited by premedication with Bisindolylmaleimide I.
3cAMP-PKC mediates pruritogenic effects of ET-1
PKC is downstream of phospholipaseC (PLC). Co-injection of U73122(PLC inhibitor) with ET-1increased the scratching number. PKC activated by cAMP, a non-classical pathway was evaluated. The concentration of cyclic adenosine monophosphate (cAMP) in dorsal root ganglion ganglion neurons were increased after ET-1(lOnM for60s)stimulation. Co-injection of SQ22536(adenylyl cyclase inhibitor) reduced the number of scratches in a dose-dependent manner. Activation of AC causes increases of intracellular cAMP concentration, protein kinase A (PKA)is activated accordingly. Co-injection of H89(PKA inhibitor) didn't affect the response to ET-1.
The effects of high concentration of U73122on scratching response induced by ET-1and compound48/80(C48/80) was evaluated to exclude the non-specific effects of U73122on PLC which might affect the results. Histamine is released by mast cell in culture upon the stimulation of C48/80. U73122at the concentration of10μM is sufficient to block the release of histamine evoked by C48/80. Scratching response was induced by intradermal injection of C48/80, which is inhibited by co-injection with U73122(10μM,1000pmol). Co-injection of U73122(10μM,1000pmol) with ET-1increased the scratching number induced by ET-1. Premedication with D609(phosphatidylcholine specific phospholipase C inhibitor,500nmol, intradermal injection) at the same site before administration of ET-1reduced the number of scratches, while it didn't affect the scratching response induced by C48/80..
4Role of TRPV1,H1R,TRP and TRPA1in pruritus inuced by ET-1
Intraperitoneal mepyramine (H1R antagonist,40mg/kg), capsazepine (TRPV1antagonist,4mg/kg) didn't affect the response to ET-1. Scratching response was inhibited in capsaicin treated-mice in neonatal period. Co-injection of ruthenium red (RR,5nmol) and AP18(100nmol) with ET-1increased the scratching number. TRPA1is involved in the pain-like behavior induced by ET-1. Co-injection of RR or AP18with ET-1inhibited the pain-like behavior induced by ET-1. To examined the effect of RR and AP18on pruritus caused by ET-1could be attributed to pain relief, morphine (5mg/kg) was injected subcutaneously15min before the administration of ET-1. Morphine had no effect on the number of scratches caused by ET-1. This provided evidence that ET-1induced itch is not related to the pain.
5Antipruritic effect of ETB
Pretreatment with systemically administered naloxone0.5mg/kg significantly reduced the number of scratches induced by ET-1. Co-injection of naloxone2nmol or0.5mg/kg augmented the effect of ET-1. Thus, local, but not systemic naloxone, prevented the antipruritic effect induced by activation of the ETB receptor, suggesting the involvement of peripheral opioid receptors in pruritis. We next examined the effects of CTOP, nor-BNI and naltrindole (μ-, κ-and8-opioid receptor antagonists, respectively) on the scratching response induced by ET-1. Intradermal injections of nor-BNI evoked dose-dependent scratching bouts. Five nmol of nor-BNI was selected because it did not cause a noticeable scratching response. Co-injection of nor-BNI significantly increased the number of scratches induced by ET-1, CTOP and naltrindole did not alter the scratching response to ET-1.
Nitric oxide (NO) is involved in the antinociception effects of KOR agonist. There are enothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS). Inhibitor of nNOS abolished the analgesia effects of KOR agonist. Co-injection of L-NAME (non-selective NOS inhibitor,200nmol) or together with selective inhibitors for eNOS (L-NIO,200nmol), with ET-1increased the scratching number. Co-injection of AMT (iNOS inhibitor,5nmol) and nNOS (Nco-Propyl-L-arginine,20nmol) did not alter the scratching response to ET-1. This resluts suggest that keratinocyte mediates the antipruitic effects of KOR agonist as neuron expresses mainly nNOS, while keratinocyte expresses mainly eNOS,
Conclusions
1ET-1induces pruritus through ETA/AC/PKC pathway.
2PKC activated by cAMP is a non-classical pathway. The cAMP-to-PKC signaling is mediated through the cAMP-activated guanine exchange factor Epac, which signals downstream to PI-PLC and PLD, both are necessary for activation of PKC in vitro and in vivo. However, the PKC signaling did not seem to descend from the PI-PLC pathway in our study, since blocking PI-PLC by U73122produced an opposite effect from blocking PKC. Our results reveal a new pathway for the cAMP-to-PKC signaling that is PI-PLC-independent, possibly via PC-PLC pathway.
3ETB exerts antipruritic effects, and peripheral KOR mediate the antipruritic effects through eNOS. Nitric oxide released from keratinocytes are involved in the antipruritic effects possibly.
辣椒素可引起新鲜分离的背根神经节(dorsal root ganglion, DRG)神经元细胞内向电流及细胞内游离钙浓度上升,ET-1可通过蛋白激酶C (protein kinaseC,PKC)途径增强DRG神经元细胞内钙对辣椒素的反应,辣椒素引起的内向电流也明显增强。动物局部应用ET-1出现伤害性行为表现,而人局部应用ET-1出现疼痛,近年来发现人及动物局部应用ET-1也出现搔痒。
由同一种物质同时引起疼痛和搔痒,机体如何处理疼痛和搔痒的信息仍不得而知。ET-1引起搔痒的分子机制尚不清楚,脊髓中的胃泌素释放肽受体(Gastrin-releasing peptide receptor, GRPR)是搔痒感受分子,选择性去除小鼠脊髓Ⅰ层表达GRPR的神经元,动物对所有测试的致痒剂(包含ET-1在内)均无反应,提示初级神经元可能释放不同的神经递质,激活脊髓中痛或者痒特异性感受神经元,本文主要研究ET-1引起搔痒分子信号通路。
方法
实验动物
实验选用雄性C57BL/6J小鼠,体重20-25g,由中山大学实验动物中心提供,本研究及实验动物的使用与处理方法得到广东医学科学院动物试验伦理委员会的书面同意,并按照规定遵守动物福利及尽可能减少使用动物的数量。
颈背部注射搔痒模型
小鼠于实验前一天颈背部皮肤剃毛,小鼠于实验前30min放置于透明的塑料盒子里预适应。应用30G针头进行颈背部皮内注射,注射药物后立即放入塑料盒子里面观察,观察小鼠用后肢搔抓注射部位次数,小鼠完成从举起后肢搔抓注射部位到放下后肢全过程为一次搔抓,观察30min。
脸颊部注射行为学观察
实验前一天小鼠脸颊部剃毛,实验前放置于透明的塑料盒子预适应30min,小鼠从盒子里面取出后用微量注射器(将30G针头与微量注射器相连)进行脸颊部皮内注射,注射容量为10μl。注射药物后立即放入塑料笼子里面观察,记录30min内动物分别用前肢拂和后肢搔抓注射部位的次数,用注射侧前肢拂注射部位是疼痛相关的行为学表现,用后肢搔抓注射部位是搔痒相关的行为学表现。
热痛觉阈值
热痛觉阈值测量实验前一天,小鼠在实验台适应1h,测定热痛觉阈值时,用热辐射刺激仪照射小鼠足底紧贴玻璃板部位。记录照射开始至小鼠出现明显逃避的时间,调节光照强度,使注射药物前逃避时间(paw withdraw time,PWT)为(8-12)s。为避免热损伤,光照时间不超过20s。
去TRPV1神经元小鼠制作
雄性新生小鼠于出生后第二天七氟醚麻醉后皮下注射辣椒素(50mg/kg),对照组注射同样容量的溶剂(PBS含10%ethanol与10%Tween-80),动物在SPF动物房里面饲养6周后进行实验。新生期注射辣椒素能够去除表达TRPV1神经元,用角膜刺激实验验证去除TRPV1神经元的效果,用0.01%辣椒素20μl喷到小鼠眼睛上,小鼠会用前肢拂眼睛,记数1min内用前肢拂眼睛的次数,如1min内用前肢拂眼睛的次数少于5次就认为已经成功去除TRPV1神经元。
背根神经节神经元细胞培养
C57BL/6J小鼠用七氟醚麻醉后背部皮肤消毒,剪下脊柱后快速转移至放置于冰上培养皿内,将脊柱泡在冷的L15内,无菌状态下取出DRG,用0.25%胶原酶Ⅳ于37℃水浴振荡1h,机械吹打后用低温离心机在下离心(1000rpm)5min。培养液为含10%胎牛血清的Dulbecco's modified Eagle's medium (DMEM),加入青霉素G钠盐和硫酸链霉素各10μ g/ml,并且加入神经生长因子50ng/ml,细胞接种于多聚左旋赖氨酸包被的玻片或培养板上,置于37℃5%CO2培养箱内培养。
PKCε膜转位
细胞接种于多聚左旋赖氨酸包被的直径为10mm玻片上,用0.01M磷酸盐缓冲液(phosphate-buffered saline, PBS)洗脱培养液后,加入PBS或ET-1后放入37℃及5%CO2培养箱内60s后吸出上清,用4%PFA固定20min,漂洗后使用含10%的山羊血清室温下作用1h,加入第一抗体,分别为兔来源抗PKCε抗体(1：500)及豚鼠来源的抗TRPV1抗体(1：3000),并加入0.2%Triton4℃下孵育16h,漂洗后加入结合有Alexa Fluor594抗兔IgG和结合有Alexa Fluor488抗豚鼠IgG的第二抗体孵育2h。用激光共焦显微镜(Zeiss, Germany)观察PKCs膜转位情况。
细胞内cAMP检测
细胞接种于多聚左旋赖氨酸包被的24孔培养板上,倒去培养液,用PBS冲洗后,加入含磷酸二酯酶抑制3-异丁基-1-甲基黄嘌呤(3-isobutyl-1-methylxanthine, IBMX,2.5mM)的PBS(含0.1%葡萄糖)在37℃培养箱内孵育10min,吸出培养液,加入含IBMX (2.5mM)及ET-1(10nM)的PBS(对照组只加入IBMX)继续孵育60s,倒去培养液,每孔加入细胞裂解液250μ1(0.1M HC1,0.8%Triton X-100),室温下放置10min,吸出后离心(10000rpm)2min取上清,上清100μl应用酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)测量标本中cAMP水平,采用小鼠cAMP单克隆抗体ELISA检测试剂盒检测。用纯化的山羊抗小鼠抗体IgG包被96孔板,制成固相抗体,在实验前20min准备好所有试剂及酶联板放置于室温,往包被抗体的96孔板中依次加入cAMP标准品或样品、小鼠单克隆cAMP抗体、辣根过氧化物酶(horse-radish peroxidase, HRP)结合的cAMP,经过彻底洗涤后用底物四甲基联苯胺(3,3',5,5'-tetramethylbenzidine,TMB)显色。TMB在过氧化物酶的催化下转化成蓝色,并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的cGMP呈正相关。用在450nm波长下测定吸光度(OD值),根据标准品浓度吸光度曲线测量样品浓度。另取上清50μ l应用二喹啉甲酸蛋白质试剂盒(bicinchoninic acid protein assay kit, BCA protein assay kit)测量蛋白含量,测量562nm处吸光度,根据标准品浓度吸光度曲线计算样品蛋白浓度。我们将细胞内cAMP浓度根据蛋白含量进行校正比较,最终细胞内cAMP浓度单位表达为pmol/mg蛋白。
结果
1ET-1通过激活ETA受体引起搔痒
当颈背部皮内注射100μl浓度为10μM(总量为1000pmol)的ET-1时产生明显的搔抓,30min内小鼠用后肢抓注射部位的次数达到360次,依次稀释10倍发现颈背部皮内注射总量为1pmol(此时浓度仅为0.01μM)ET-1仍可引起明显搔抓,当注射总量为100pmol时30min内引起搔抓187次,从ET-1引起搔痒的剂量反应关系可以看到这一剂量(1μM,100μl)引起中等程度的搔抓次数,我们以后的研究中除非特别说明均采用这一剂量的ET-1(1μM,100μl)。
脸颊部注射ET-110μM(容量为10μl)可引起明显后肢搔抓与前肢拂注射部位,注射ET-11μM引起明显后肢搔抓,但前肢拂注射部位不明显,而注射ET-10.1μM不能引起明显的后肢搔抓与前肢拂注射部位。
特异性ETA受体拮抗剂BQ123及特异性ETB受体拮抗剂BQ788单独颈背部皮内注射时并不引起明显的搔抓反应,BQ123100nmol与100pmol ET-1共同注射时可明显抑制ET-1引起的搔抓,而BQ78830nmol与100pmol ET-1共同注射时可明显增加ET-1引起的搔抓,说明ET-1通过ETA受体引起搔痒,而ETB受体可能发挥止痒作用。
2PKC途径介导ET-1引起的搔痒
双吲哚亚酰胺I (Bisindolylmaleimide I, BIM)是蛋白激酶C (protein kinaseC,PKC)抑制剂,BIM2μg或10μg与ET-1共同注射均能明显抑制ET-1引起的搔痒,说明ET-1引起的搔痒和PKC途径有关。培养的DRG神经元用ET-1(10nM)处理后可见PKCε主要分布在细胞膜上,而未用ET-1处理的DRG神经元可见PKCε主要分布在细胞浆内。
巴豆醇-12-十四烷酸酯-13-乙酸酯(phorbol-12-myristate-13-acetate,PMA)是常用的PKC激活剂,颈背部皮内注射PMA1μM和10μM时(总量分别为100pmol与1000pmol)时引起搔痒,而注射10pmol时不引起明显搔痒。
足底注射PMA与ET-1均能引起热痛觉过敏,小鼠足底注射PMA100pmol/10μl一直出现抬足等行为,不能测量热痛觉阈值,10pmol/10μl引起明显的热痛觉过敏,1pmol/10μl引起的热痛觉过敏ET-1100pmol/10μl所引起的热痛觉过敏相当,足底注射1μg BIM15min后可以完全抑制同一部位注射ET-1和PMA引起的热痛觉过敏。
3cAMP-PKC途径介导ET-1引起的搔痒
由磷酯酶C (phospholipase C, PLC)激活PKC是PKC激活的经典通路,我们应用PLC阻滞剂U73122观察PLC通路的作用。颈背部注射U73122(10pmol,100μl)不引起明显搔抓及其他异常表现,但与预期结果相反,U73122(100pmol)与ET-1共同注射明显增加ET-1引起的搔抓次数。提示可能由非经典通路环单磷酸腺苷(cyclic adenosine monophosphate, cAMP)激活PKC从而介导ET-1引起的搔痒。
ET-1(10nM)刺激培养的DRG神经元60s后,应用cAMP单克隆抗体ELISA法测量细胞内cAMP浓度的变化,ET-1处理神经元后明显增加细胞内cAMP浓度。直接应用腺苷酸环化酶(Adenylyl cyclase, AC)抑制剂SQ22536对ET-1引起搔痒的作用。SQ22536与ET-1共同注射可剂量依赖性地抑制ET-1引起的搔痒。AC激活后引起细胞内cAMP浓度升高,cAMP浓度升高引起蛋白激酶A(protein kinase A, PKA)激活,PKA阻滞剂H89与ET-1共同皮内注射对ET-1引起的搔痒无明显作用。
为了排除U73122的非特异性作用,我们研究了更高浓度的U73122对ET-1及复合物48/80(compound48/80, C48/80)引起搔痒的影响。C48/80可刺激培养的肥大细胞脱颗粒释放组胺,U73122(10μM)可明显抑制肥大细胞的脱颗粒。C48/80皮内注射可引起明显的搔痒,与U73122(1000pmol)共同注射后引起的搔痒可明显减轻,而U73122(1000pmol)仍然增加ET-1引起的搔痒。皮内注射phosphatidylcholine specific phospholipase C(磷脂酰胆碱磷酯酶C, PC-PLC)抑制剂D609,15min再在同一部位注射ET-1或者C48/80,D609可抑制ET-1引起的搔痒,而D对C48/80引起的搔痒无明显影响。
4TRPV1、H1R、TRP、TRPA1在ET-1引起搔痒中的作用
腹腔注射TRPV1拮抗剂辣椒平(4mg/kg)或者H1R拮抗剂美吡拉敏(mepyramine,40mg/kg)对ET-1引起的搔痒无明显影响。新生期注射辣椒素后能去除TRPV1神经元,ET-1在新生期注射辣椒素成年动物不引起明显搔痒,而对照组成年动物对ET-1引起的搔痒反应无明显影响。非选择性TRP抑制剂钌红(ruthenium red, RR,5nmol)共同注射可增加ET-1引起的搔痒,但同样剂量的RR抑制组胺引起的搔痒。TRPA1拮抗剂AP18(100nmol)共同注射可增加ET-1引起的搔痒,同样剂量的AP18对组胺引起的搔痒无明显作用。ET-1引起的疼痛可能与TRPA1有关,RR与AP18增加ET-1引起的搔痒可能与其减少疼痛有关。在皮内注射ET-1前15min皮下注射吗啡(5mg/kg),吗啡对ET-1引起的搔痒无明显影响,提示ET-1引起的搔痒与疼痛无关。
5ETB受体的止痒作用
皮下注射纳洛酮0.5mg/kg可明显抑制15min后皮内注射ET-1所引起的搔痒,而小剂量的纳洛酮(2nmol)皮下注射对ET-1引起的搔痒无明显影响,当纳洛酮与ET-1共同一起皮内注射时,两种剂量的纳洛酮(2nmol与0.5mg/kg)均明显增加ET-1引起的搔痒,这种作用与纳洛酮的全身作用无关,因为全身应用纳洛酮的效果与此相反或者在小剂量的时候无明显效果。
外周存在μ、κ、δ三种阿片类受体亚型,我们应用选择性亚型拮抗剂来观察各亚型的作用,大剂量KOR拮抗剂Nor-BNI(超过15nmol/50μl时)颈背部皮内注射时可引起搔痒,而5nmol/50μl时不引起明显搔痒,我们选择了这一剂量进行研究,其他的剂量参考以前的研究,MOR拮抗剂CTOP或DOR拮抗剂naltrindole一起注射对ET-1引起的搔痒无明显影响,而Nor-BNI明显增加ET-1引起的搔痒,可见外周KOR介导了ETB受体的止痒作用。
KOR激动剂的抗伤害作用与一氧化氮有关,一氧化氮合酶(nitric oxide synthase)有内皮型(endothelia)、神经型(neuronal)和诱导型(inducible)三种亚型,nNOS抑抑剂可拮抗KOR激动剂的止痛作用。应用非选择性NOS抑制剂发现L-NAME(200nmol)明显增加ET-1引起的搔痒,而D-NAME对ET-1引起的搔痒无明显影响。NOS有三种亚型分别为内皮型、神经型、诱导型,应用特异的选择性亚型抑制剂与ET-1共同注射,eNOS抑制剂(L-NIO,200nmol)可明显增加ET-1引起的搔痒,而诱导型iNOS抑制剂(AMT,5nmol)、神经型nNOS亚型抑制剂(Nco-Propyl-L-arginine,20nmol)对ET-1引起的搔痒无明显作用。由于神经元中主要存在nNOS,角质形成细胞中主要存在eNOS, eNOS抑制剂也能增加ET-1引起的搔痒说明KOR激动剂可能通过角质形成细胞起作用。
结论
1ET-1通过ETA/AC/PKC途径引起搔痒。
2由cAMP激活PKC是非经典途径,这个通路通过Epac(exchange proteins activated directly by cyclic AMP, cAMP直接激活的交换蛋白)激活下游PLC与PLD,在体内与体外实验均发现从cAMP激活PKC离不开PLC与PLD的作用,本研究中PLC抑制剂U73122与预期相反的作用,说明ET-1引起搔痒的cAMP激活PKC通路与以前发现的通路不同,我们发现了一个全新的由cAMP激活PKC通路,该通路可能与PC-PLC通路有关。
3ETB受体发挥止痒作用,外周阿片类受体κ亚型通过eNOS介导这一止痒作用,角质形成细胞释放的NO也可能发挥止痒作用。
Endothelin-1(ET-1) is secreted by a variety of cell types, including keratinocytes, vascular smooth muscle cells, leukocytes, cardiomyocytes and mesangial cells, and some tumor cell lines since its first isolation from the cultures of porcine aortic endothelial cell cultures. It is the most potent vasoconstrictor known. The signaling of ET-1is mediated via two main membrane G-protein-coupled receptor (GPCR) subtypes, ETA and ETB receptors. The expression of ETA receptor is largely confined to the vascular endothelium, as well as neurons and mast cells, while ETB receptor is mainly expressed by keratinocytes, smooth muscle cells and vascular endothelium.
ET-1enhances capsaicin-evoked increases of intracellular calcium levels in freshly dissociated DRG neurons in a protein kinase C (PKC) dependent manner, and potentiation of capsaicin-induced currents. Local administration of ET-1induces nociceptive behaviors in animals and causes pain in humans. ET-1has been demonstrated to elicit pruritus in mice and humans also. How to cope with the messages of itch and pain inuuced by the same agent, ET-1. Little is known about the molecular mechanisms of the pruritogenic action of ET-1. Gastrin-releasing peptide receptor (GRPR) is an itch-specific molecule in the spinal cord [28]. Mice selectively ablated lamina I neurons expressing GRPR in the spinal cord showed profound scratching deficits in response to all of the itching (pruritogenic) stimuli (including ET-1) tested [29]. It is suggested that different neurotransmitters are released by the primary neurons to excite the pain-or itch-specicif second neurons in the spinal. The current study was designed to elucidate the molecular signals of itch induced by ET-1.
Methods
Animals
Male C57BL/6J mice, weighing20-22g, were purchased from the Center for Laboratory Animals, Sun Yat-Shen University. The experimental procedures and the animal use and care protocols were approved by the Committee on Ethical Use of Animals of Guangdong Academy of Medical Sciences. All efforts were made to minimize animal suffering and to reduce the number of animals used.
Pruritus model of neck injection
One day after shaving the rostral part of the back of the neck, mice were placed into a small plastic chamber30min before the experiment. For drug administration, mice were briefly removed from the chamber, and each test drug was intradermally injected with a30-gauge needle. Then the mice were returned to the chamber, and the hind limb scratching directed towards the shaved area at the back of the neck was observed and recorded for30min. One scratch was defined as a lift of the hind limb towards the injection site and then a reposition of the limb back to the floor, regardless of the scratching strokes that took place between the two movements.
Cheek injection model
One day after shaving the cheek, mice were placed into a small plastic chamber30min before the experiment. For drug administration, mice were briefly removed from the chamber, and each test drug was intradermally injected with a30-gauge needle.
A volume of10μl was injected intradermally into the cheek. Then the mice were returned to the chamber, number of hindpaw scratches directed to the injected area as an indicator of itch and number of ipsilateral forelimb wipes directed to the injected area as an indicator of pain were recorded for a period of30min after the injection. Paw withdrawal latency
Mice were placed into a small plastic chamber on glass table with the positioned in an heat stimulation apparatus for radiant heat stimulation on the plantar surface. Mice were placed into the small plastic chamber for1h one day before the experiment. Intensity of heat stimulus was adjusted for paw withdrawal latency of8-12s in normal animals. A cut-off time was set at20s to avoid injury to the hindpaw.
Depletion of TRPV1neurons in neonatal mice
Male neonatal mice (aged2days) were anesthetized with sevoflurane and injected subcutaneously with capsaicin (50mg/kg) or the vehicle (10%ethanol,10%Tween-80and80%PBS) as described previously. Animals were included in the study6weeks after the injection of capsaicin or the vehicle. The effects of capsaicin were expected to cause depletion of TRPV1neurons. It was verified by the eye-wiping test. For this test,0.01%capsaicin at the volume of20μl was sprayed into the eye and the number of wiping movements that occurred within1min was counted. The animal was considered to be desensitized to TRPV1by neonatal capsaicin treatment when the animal wiped its eyes no more than five times.
Cell culture of dorsal root ganglion
Adult male C57BL/6J mice were anesthetized with sevoflurane and the skin of the back were disinfected. The spinal were removed and transfered into cold L15medium and placed on ice. DRGs were removed bilaterally from lumbar and thoracic spinal levels under sterile conditions and incubated with0.25%collagenase type IV at37℃for60min with gentle agitation. Then, DRGs were centrifuged at1000rpm for5min in4℃after mechanically dissociated with pipette. Cells were resuspended with Dulbecco's modified Eagle's medium containing nerve growth factor (50ng/ml, Invitrogen),10%fetal bovine serum (Invitrogen), penicillin (100units/ml, In-vitrogen) and streptomycin (100g/ml, Invitrogen) and plated onto glass coverslips or plates precoated with poly-L-lysine and cultured in incubator containing5%CO2at37℃. Membrane translocation of PKCs
Cells were plated on10-mm diameter glass coverslips and were washed with0.01M PBS and then exposed to0.01M PBS or10nM ET-1for60s in incubator containing5%CO2at37℃, and immediately fixed with4%paraformaldehyde for20min, rinsed using PBS, and then incubated with10%normal goat for1h. Fixed slips were incubated with primary antibodies in0.2%Triton for16h at4℃. Then slips were incubated with second antibodies conjugated to Alexa Fluor488or Alexa Fluor594.
Measurement of cAMP
Cells were plated in24wells plate and washed with PBS before test, and preincubated at37℃for10min in the presence of the phosphodiesterase inhibitor3-isobutyl-l-methylxanthine (2.5mM) in PBS containing0.1%glucose. Cells were incubated for an additional60s at37℃with or without ET-1(10nM in2.5mM IBMX in PBS). Then PBS was removed, and250μl of HCl(0.1M) with0.8%Triton X-100(Sigma-Aldrich) was added to the plates. After10-min incubation at room temperature, the lysate was removed from the plates and centrifuged for2min. The supernatant was collected and100u1of it was measureded for cAMP concentration using a monoclonal cAMP enzyme immunoassay kit.
All reagents and plate are prepared at room temperature20min before test. Sample of cell extracts and standard cAMP are added to96-well plate coated with goat-anti-mouse serum. cAMP will competitively bind to the monoclonal anti-cAMP antibody in the presence of fixed amounts of cAMP-conjugated horse-radish peroxidase. Standard cAMP are used to generate the calculation curve. After a short incubation, the excess reagents are washed away and substrate (3,3',5,5'-tetramethylbenzidine, TMB) is added. The multiwell plates are then read on a microplate reader at450nm. The intensity of the yellow color is inversely proportional to the concentration of cAMP in samples. The measured optical density is used to calculate the concentration of cAMP in samples based on the curve from the cAMP standards.
The production of cAMP was normalized to the protein concentration in each well (pmol/mg protein). Protein content was determined using a bicinchoninic acid protein assay kit according to the manufacturer's instructions and determined at562nm.
Results
1ET-1induces scratches through activation of ETA receptor
Intradermal injection of ET-1to the back evoked the number of scratches in a dose-dependent manner, with a significant increase starting from1pmol (in100u1PBS) of ET-1. It induced an average of360bouts of scratches at the dose of1000pmol. At100pmol, ET-1induced a moderate number of scratches, within the linear range of ET-1effects. Therefore, the dose of100pmol (in100μl PBS) was selected for subsequent experiments.
Intradermal injection of ET-1to the cheek an average of18bouts of wipes at the dose of100pmol (in10μ1PBS). No wipes was evoked when the dose of ET-1lower than10pmol. Cheek injection of ET-1dose-dependently evoked scratching response when the dose of ET-1higher than10pmol (in10μl PBS).
Co-injection of BQ-123(selective antagonists for ETA) with ET-1at100nmol significantly reduced the number of scratches, while BQ-788(selective antagonists for ETB receptor) at30nmol increased the number of scratches induced by ET-1. These results show that ETA receptor mediates the pruritogenic effects, and the ETB receptor exerts anti-pruritic effects.
2PKC mediates pruritogenic effects of ET-1
Co-injection of Bisindolylmaleimide I (PKC inhibitor) at the dose of2u g or10μg reduced the scratching response induced by ET-1. Membrane translocation of PKCs were found in cultured dorsal root ganglion neurons after ET-1(10nM for60s) stimulation.
Scratching response was also induced by the PKC activator phorbol-12-myristate-13-acetate (PMA) at the dose above100pmol. Intraplantar injection of PMA at the dose of1pmol (in10μl PBS) reduced the paw withdraw latency from heat stimulation about that induced by ET-1at the dose of100pmol (in10μl PBS). Thermal hyperalgesia induced by PMA and ET-1were inhibited by premedication with Bisindolylmaleimide I.
3cAMP-PKC mediates pruritogenic effects of ET-1
PKC is downstream of phospholipaseC (PLC). Co-injection of U73122(PLC inhibitor) with ET-1increased the scratching number. PKC activated by cAMP, a non-classical pathway was evaluated. The concentration of cyclic adenosine monophosphate (cAMP) in dorsal root ganglion ganglion neurons were increased after ET-1(lOnM for60s)stimulation. Co-injection of SQ22536(adenylyl cyclase inhibitor) reduced the number of scratches in a dose-dependent manner. Activation of AC causes increases of intracellular cAMP concentration, protein kinase A (PKA)is activated accordingly. Co-injection of H89(PKA inhibitor) didn't affect the response to ET-1.
The effects of high concentration of U73122on scratching response induced by ET-1and compound48/80(C48/80) was evaluated to exclude the non-specific effects of U73122on PLC which might affect the results. Histamine is released by mast cell in culture upon the stimulation of C48/80. U73122at the concentration of10μM is sufficient to block the release of histamine evoked by C48/80. Scratching response was induced by intradermal injection of C48/80, which is inhibited by co-injection with U73122(10μM,1000pmol). Co-injection of U73122(10μM,1000pmol) with ET-1increased the scratching number induced by ET-1. Premedication with D609(phosphatidylcholine specific phospholipase C inhibitor,500nmol, intradermal injection) at the same site before administration of ET-1reduced the number of scratches, while it didn't affect the scratching response induced by C48/80..
4Role of TRPV1,H1R,TRP and TRPA1in pruritus inuced by ET-1
Intraperitoneal mepyramine (H1R antagonist,40mg/kg), capsazepine (TRPV1antagonist,4mg/kg) didn't affect the response to ET-1. Scratching response was inhibited in capsaicin treated-mice in neonatal period. Co-injection of ruthenium red (RR,5nmol) and AP18(100nmol) with ET-1increased the scratching number. TRPA1is involved in the pain-like behavior induced by ET-1. Co-injection of RR or AP18with ET-1inhibited the pain-like behavior induced by ET-1. To examined the effect of RR and AP18on pruritus caused by ET-1could be attributed to pain relief, morphine (5mg/kg) was injected subcutaneously15min before the administration of ET-1. Morphine had no effect on the number of scratches caused by ET-1. This provided evidence that ET-1induced itch is not related to the pain.
5Antipruritic effect of ETB
Pretreatment with systemically administered naloxone0.5mg/kg significantly reduced the number of scratches induced by ET-1. Co-injection of naloxone2nmol or0.5mg/kg augmented the effect of ET-1. Thus, local, but not systemic naloxone, prevented the antipruritic effect induced by activation of the ETB receptor, suggesting the involvement of peripheral opioid receptors in pruritis. We next examined the effects of CTOP, nor-BNI and naltrindole (μ-, κ-and8-opioid receptor antagonists, respectively) on the scratching response induced by ET-1. Intradermal injections of nor-BNI evoked dose-dependent scratching bouts. Five nmol of nor-BNI was selected because it did not cause a noticeable scratching response. Co-injection of nor-BNI significantly increased the number of scratches induced by ET-1, CTOP and naltrindole did not alter the scratching response to ET-1.
Nitric oxide (NO) is involved in the antinociception effects of KOR agonist. There are enothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS). Inhibitor of nNOS abolished the analgesia effects of KOR agonist. Co-injection of L-NAME (non-selective NOS inhibitor,200nmol) or together with selective inhibitors for eNOS (L-NIO,200nmol), with ET-1increased the scratching number. Co-injection of AMT (iNOS inhibitor,5nmol) and nNOS (Nco-Propyl-L-arginine,20nmol) did not alter the scratching response to ET-1. This resluts suggest that keratinocyte mediates the antipruitic effects of KOR agonist as neuron expresses mainly nNOS, while keratinocyte expresses mainly eNOS,
Conclusions
1ET-1induces pruritus through ETA/AC/PKC pathway.
2PKC activated by cAMP is a non-classical pathway. The cAMP-to-PKC signaling is mediated through the cAMP-activated guanine exchange factor Epac, which signals downstream to PI-PLC and PLD, both are necessary for activation of PKC in vitro and in vivo. However, the PKC signaling did not seem to descend from the PI-PLC pathway in our study, since blocking PI-PLC by U73122produced an opposite effect from blocking PKC. Our results reveal a new pathway for the cAMP-to-PKC signaling that is PI-PLC-independent, possibly via PC-PLC pathway.
3ETB exerts antipruritic effects, and peripheral KOR mediate the antipruritic effects through eNOS. Nitric oxide released from keratinocytes are involved in the antipruritic effects possibly.
引文
[1]Yanagisawa M, Kurihara H, Kimura S, et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells[J]. Nature,1988,332(6163): 411-415.
[2]Inoue A, Yanagisawa M, Kimura S, et al. The human endothelin family:three structurally and pharmacologically distinct isopeptides predicted by three separate genes[J]. Proceedings of the National Academy of Sciences,1989, 86(8):2863-2867.
[3]Saida K, Mitsui Y, Ishida N. A novel peptide, vasoactive intestinal contractor, of a new (endothelin) peptide family. Molecular cloning, expression, and biological activity[J]. Journal of Biological Chemistry,1989,264(25): 14613-14616.
[4]Bloch K D, Hong C C, Eddy R L, et al. cDNA cloning and chromosomal assignment of the endothelin 2 gene:vasoactive intestinal contractor peptide is rat endothelin 2[J]. Genomics,1991,10(1):236-242.
[5]Dhaun N, Pollock D M, Goddard J, et al. Selective and mixed endothelin receptor antagonism in cardiovascular disease[J]. Trends in pharmacological sciences,2007,28(11):573-579.
[6]Uchide T, Masuda H, Mitsui Y, Saida K. Gene expression of vasoactive intestinal contractor/endothelin-2 in ovary, uterus and embryo:comprehensive gene expression profiles of the endothelin ligand-receptor system revealed by semi-quantitative reverse transcription-polymerase chain reaction analysis in adult mouse tissues and during late embryonic development [J]. J Mol Endocrinol.1999;22(2):161-71.
[7]Matsumoto H, Suzuki N, Onda H, et al. Abundance of endothelin-3 in rat intestine, pituitary gland and brain [J]. Biochemical and biophysical research communications,1989,164(1):74-80.
[8]Agapitov AV, Haynes WG. Role of endothelin in cardiovascular disease [J]. J Renin Angiotensin Aldosterone Syst.2002;3(1):1-15.
[9]Sessa W C, Kaw S, Hecker M, et al. The biosynthesis of endothelin-1 by human polymorphonuclear leukocytes[J]. Biochemical and biophysical research communications,1991,174(2):613-618.
[10]Ehrenreich H, Anderson RW, Fox CH, et al. Endothelins, peptides with potent vasoactive properties, are produced by human macrophages [J]. J Exp Med. 1990;172:1741-1748.
[11]Hahn AW, Resink TJ, Scott-Burden T, et al. Stimulation of endothelin mRNA and secretion in rat vascular smooth muscle cells:a novel autocrine function [J]. Cell Regul.1990; 1:649-659.
[12]Ito H, Hirata Y, Adachi S, et al. Endothelin-1 is an autocrine/paracrine factor in the mechanism of angiotensin II-induced hypertrophy in cultured rat cardiomyocytes [J]. J Clin Invest.1993;92:398-403.
[13]Fujisaki H, Ito H, Hirata Y, et al. Natriuretic peptides inhibit angiotensin Ⅱ-induced proliferation of rat cardiac fibroblasts by blocking endothelin-1 gene expression [J]. J Clin Invest.1995;96:1059-1065.
[14]Fukunaga M, Fujiwara Y, Ochi S, et al. Stimulatory effect of thrombin on endothelin-1 production in isolated glomeruli and cultured mesangial cells of rats [J]. J Cardiovasc Pharmacol.1991;17:S411-S413.
[15]Nakamura T, Ebihara I, Fukui M, et al. Renal expression of mRNAs for endothelin-1, endothelin-3 and endothelin receptors in NZB/WF1 mice [J]. Ren Physiol Biochem.1993; 16:233-243.
[16]Giaid A, Gibson SJ, Ibrahim BN, Legon S, Bloom SR, Yanagisawa M, Masaki T, Varndell IM, Polak JM. Endothelin 1, an endothelium-derived peptide, is expressed in neurons of the human spinal cord and dorsal root ganglia [J]. Proc Natl Acad Sci U S A.1989 Oct;86(19):7634-8.
[17]MacCumber MW, Ross CA and Snyder SH. Endothelin in brain:receptors, mitogenesis, and biosynthesis in glial cells. Proc Natl Acad Sci U S A 87:2359-2363,1990.
[18]Gandhi CR, Berkowitz DE, Watkins WD. Endothelins. Biochemistry and pathophysiologic actions. Anesthesiology 1994;80:892-905.
[19]Hans G, Deseure K, Adriaensen H. Endothelin-1-induced pain and hyperalgesia: a review of pathophysiology, clinical manifestations and future therapeutic options. Neuropeptides 2008;42:119-132.
[20]Alberts GF, Peifley KA, Johns A, et al. Constitutive endothelin-1 overexpression promotes smooth muscle cell proliferation via an external autocrine loop. J Biol Chem.1994;269:10112-10118.
[21]Barton M, Shaw S, d'Uscio LV, et al. Angiotensin Ⅱ increases vascular and renal endothelin-1 and functional endothelin converting enzyme activity in vivo: role of ETA-receptors for endothelin regulation. Biochem Biophys Res Commun.1997;238:861-865.
[22]Lee ME, Bloch KE, Clifford JA, et al. Functional analysis of the endothelin-1 gene promotor:evidence for an endothelial cell specific cis-acting enzyme. J Biol Chem.1993;265:10446-10450.
[23]Hirata Y, Kanno K, Watanabe TX, et al. Receptor binding and vasoconstrictor activity of big endothelin. Eur J Pharmacol.1990; 176:225-228.
[24]Xu D, Emoto N, Giaid A, et al. ECE-1:a membrane-bound metalloprotease that catalyzes the proteolytic activation of big endothelin-1. Cell.1994;78:473-485.
[25]Emoto N, Yanagisawa M. Endothelin-converting enzyme-2 is a membrane-bound, phosphoramidon-sensitive metalloprotease with acidic pH optimum. J Biol Chem.1995;270:15262-15268.
[26]Wypij DM, Nichols JS, Novak PJ, et al. Role of mast cell chymase in the extracellular processing of big-endothelin-1 to endothelin-1 in the perfused rat lung. Biochem Pharmacol.1992;43:845-853.
[27]Nakano A, Kishi F, Ninami K, et al. Selective conversion of big endothelins to tracheal smooth muscle-constricting 31-amino acid-length endothelins by chymase from human mast cells. J Immunol.1997; 159:1987-1992.
[28]Hasegawa H, Hiki K, Sawamura T, et al. Purification of a novel endothelin-converting enzyme specific for big endothelin-3. FEBS Lett. 1998;428:394-398.
[29]Maguire JJ, Johnson CM, Mockridge JW, et al. Endothelin converting enzyme (ECE) activity in human vascular smooth muscle. Br J Pharmacol. 1997; 122:1647-1654.
[30]Kobayashi T, Miyauochi T, Sakai S, et al. Endothelin converting enzyme (ECE) and angiotensin-converting enzyme in failing hearts of rats with myocardial infarction. J Cardiovasc Pharmacol.1998;31:S417-S420.
[31]Minamino T, Kurihara H, Takahashi M, et al. Endothelin-converting enzyme expression in the rat vascular injury model and human coronary atherosclerosis. Circulation.1997;95:221-230.
[32]Ohwaki T, Sakai H, Hirata Y. Endothelin-converting enzyme activity in human serum lipoprotein fraction. FEBS Lett.1993;320:165-168.
[33]Hans G, Deseure K, Robert D and De Hert S. Neurosensory changes in a human model of endothelin-1 induced pain:a behavioral study. Neurosci Lett 418:117-121,2007.
[34]Dahlof B, Gustafsson D, Hedner T, et al. Regional haemodynamic effects of endothelin-1 in rat and man:unexpected adverse reaction. J Hypertens. 1990;8(9):811-7.
[35]Zonnenberg BA, Groenewegen G, Janus TJ, et al. Phase I dose-escalation study of the safety and pharmacokinetics of atrasentan:an endothelin receptor antagonist for refractory prostate cancer. Clin Cancer Res.2003;9(8):2965-72.
[36]Carducci MA, Padley RJ, Breul J, et al.Effect of endothelin-A receptor blockade with atrasentan on tumor progression in men with hormone-refractory prostate cancer a randomized, phase II, placebo-controlled trial, J Clin Oncol 2003,21:679-689.
[37]Raffa RB,Jacoby HI. Endothelin-1,-2 and-3 directly and big-endothelin-1 indirectly elicit an abdominal constriction response in mice.Life Sci. 1991;48:PL85-90.
[38]Gokin AP, Fareed MU, Pan HL, et al. Local injection of endothelin-1 produces pain-like behavior and excitation of nociceptors in rats.J Neurosci. 2001;21:5358-5366.
[39]Ferreira SH,Romitelli M,NucciG de.Endothelin-1 participation in overt and inflammatory pain.J Cardiovasc Pharmacol 13:S220-S222.
[40]Baamonde A, Lastra A, Villazon M, et al. Involvement of endogenous endothelins in thermal and mechanical inflammatory hyperalgesia in mice.Naunyn Schmiedebergs Arch Pharmacol.2004;369:245-251.
[41]Piovezan AP, D'Orleans-Juste P, Tonussi CR, et al. Effects of endothelin-1 on capsaicin-induced nociception in mice. Eur J Pharmacol.1998;351(1):15-22.
[42]Liang J, Bi H, Ji W.Involvement of TRPA1 in ET-1-induced pain-like behavior in mice. Neuroreport 2010,21(3):201-205.
[43]Ahern GP, Brooks IM, Miyares RL, et al. Extracellular cations sensitize and gate capsaicin receptor TRPV1 modulating pain signaling.J Neurosci. 2005;25:5109-5116.
[44]洪迅,梁杰贤,季文进,等.腹腔内注射内皮素-1对硫酸镁引起腹痛的影响.热带医学杂志.2006,6(4)：455-456.
[45]Arai H, Hori S, Aramori I, et al. Cloning and expression of a cDNA encoding an endothelin receptor. Nature.1990;348:730-732.104.
[46]Lin HY, Kaji EH, Winkel G, et al. Cloning and expression of a vascular smooth muscle endothelin-1 receptor. Proc Natl Acad Sci U S A.1991;88:3185-3189.
[47]Sakurai T, Yanagisawa M, Takuwa Y, et al. Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor. Nature. 1990;348:732-735.
[48]Sakamoto A, Yanagisawa M, Sakurai T, et al. Cloning and functional expression of human cDNA for the ETB endothelin receptor. Biochem Biophys Res Commun.1991;178:656-663.
[49]Karne S, Jayawickreme CK, Lerner MR. Cloning and characterization of an endothelin-3 specific receptor (ETC receptor) from Xenopus laevis dermal melanophores. J Biol Chem.1993;268:19126-19133.
[50]Sakurai T, Yanagisawa M, Masaki T. Molecular characterization of endothelin receptors. Trends Pharmacol Sci 1992;13:103-108.
[51]Seo B, Oemar BS, Siebenmann R, et al. Both ETA and ETB receptors mediate contraction to endothelin-1 in human blood vessels. Circulation. 1994;89:1203-1208.
[52]Elshourbagy NA, Lee JA, Korman DR, et al.. Molecular cloning and characterization of the major endothelin receptor subtype in porcine cerebellum. Mol Pharmacol.1992;41:465-473.
[53]Boivin S, Tessier S, Aubin J,et al. Identification of a binding domain of the endothelin-B receptor using a selective IRL-1620-derived photoprobe. Biochemistry.2004;43:11516-11525.
[54]Krystek SR, Jr, Patel PS, Rose PM, et al. Mutation of peptide binding site in transmembrane region of a G protein-coupled receptor accounts for endothelin receptor subtype selectivity. J Biol Chem.1994;269:12383-12386.
[55]De-Melo JD, Tonussi CR, D'Orleans-Juste P, et al. Effects of endothelin-1 on inflammatory incapacitation of the rat knee joint. J Cardiovasc Pharmacol 1998;31(Suppl1):S518-520.
[56]Maxwell MJ, Goldie RG, Henry PJ. Ca2+signalling by endothelin receptors in rat and human cultured airway smooth muscle cells. Br J Pharmacol. 1998;125:1768-1778.
[57]Warner TD, Mitchell JA, de Nucci G, et al. Endothelin-1 and endothelin-3 release EDRF from isolated perfused arterial vessels of the rat and rabbit. J Cardiovasc Pharmacol.1989;13:S85-S88.
[58]Hirata Y, Emori T, Eguchi S, et al. Endothelin receptor subtype B mediates synthesis of nitric oxide by cultured bovine endothelial cells. J Clin Invest. 1993;91:1367-1373.
[59]Pomonis JD, Rogers SD, Peters CM, et al. Expression and localization of endothelin receptors:implications for the involvement of peripheral glia in nociception. J Neurosci.2001;21(3):999-1006.
[60]Peters CM, Lindsay TH, Pomonis JD,et al. Endothelin and the tumorigenic component of bone cancer pain. Neuroscience.2004;126(4):1043-52.
[61]Gold MS,LevineJD,Correa AM. Modulation of TTX-R INa by PKC and PKA and their role in PGE2-induced sensitization of rat sensory neurons in vitro, J Neurosci 1998;18:10345-10355.
[62]Zhang YH,Kenyon JL,Nicol GD. Phorbol ester-induced inhibition of potassium currents in rat sensory neurons requires voltage-dependent entry of calcium. J Neurophysiol.2001;85:362-373.
[63]Cheng TH, Chang CY, Wei J, et al. Effects of endothelin-1 on calcium and sodium currents in isolated human cardiac myocytes. Can J Physiol Pharmacol,1995; 73:1774-1783.
[64]Fjell J,Hjelmstrom P,Hormuzdiar W,et al.Localization of the tetrodotoxin-resistant sodium channel NaN in nociceptors. NeuroReport, 2000; 11:199-202.
[65]Zhou Z, Davar G, Strichartz G. Endothelin-1 (ET-1) selectively enhances the activation gating of slowly inactivating tetrodotoxin-resistant sodium currents in rat sensory neurons:a mechanism for the pain-inducing actions of ET-1.J Neurosci.2002;22:6325-6330.
[66]Cardenas LM, Cardenas CG, Scroggs RS.5HT Increases excitability of nociceptor-like rat dorsal root ganglion neurons via cAMP-coupled TTX-resistant Na+channels. J Neurophysiol,2001; 86:241-248.
[67]Renganathan M, Cummins TR, Waxman SG. Contribution of Na(v)1.8 sodium channels to action potential electrogenesis in DRG neurons. J Neurophysiol,2001;86:629-640.
[68]Trentin PG, Fernandes MB, D'Orleans-Juste P, et al. Endothelin-1 causes pruritus in mice. Exp Biol Med (Maywood) 2006; 231:1146-1151.
[69]McQueen DS, Noble MA, Bond SM. Endothelin-1 activates ETA receptors to cause reflex scratching in BALB/c mice. Br J Pharmacol 2007; 151:278-284.
[70]Sun YG, Zhao ZQ, Meng XL, et al. Cellular Basis of Itch Sensation.Science 2009; 325:1531-1534.
[71]Imamachi N, Park GH, Lee H, et al. TRPV1-expressing primary afferents generate behavioral responses to pruritogens via multiple mechanisms. Proc Natl Acad Sci U S A 2009;106:11330-11335.
[72]Davidson S, Giesler GJ.The multiple pathways for itch and their interactions with pain. Trends Neurosci 2010; 33:550-558.
[73]Schmelz M. Itch and pain. Neurosci Biobehav Rev 2010;34:171-176.
[74]Schmelz M. How pain becomes itch. Pain 2009; 144:14-15.
[75]v.Frey M. Zur Physiologie der Juckempfindung. Arch Neerl Physiol 1922;7:142-5.
[76]Chaney MA. Side effects of intrathecal and epidural opioids. Can J Anaesth. 1995;42(10):891-903.
[77]王春晓,季文进,林派冲.硬膜外腔及蛛网膜下腔注射吗啡诱发搔痒的比较.中华麻醉学杂志.1998,18(7)：441-442.
[78]Ikoma A, Steinhoff M, Stander S, et al. The neurobiology of itch. Nat Rev Neurosci.2006;7:535-47.
[79]Schmelz M, Schmidt R, Bickel A, et al. Specific C-receptors for itch in human skin. J Neurosci.1997;17(20):8003-8.
[80]Jeffry J, Kim S, Chen ZF. Itch signaling in the nervous system. Physiology (Bethesda).2011;26(4):286-92.
[81]Bandell M, Patapoutian A.Itching for Insight. Cell 2009;325:1224-1225.
[82]Liu Q, Tang Z, Surdenikova L,et al. Sensory Neuron-Specific GPCR Mrgprs Are Itch Receptors Mediating Chloroquine-Induced Pruritus. Cell 2009; 139: 1353-1365.
[83]Sun YG, Chen ZF. A gastrin-releasing peptide receptor mediates the itch sensation in the spinal cord. Nature.2007;448:700-3.
[84]Roesler, R., Henriques, J. A.& Schwartsmann, G Gastrin-releasing peptide receptor as a molecular target for psychiatric and neurological disorders. CNS Neurol. Disord. Drug Targets 5,197-204 (2006).
[85]Battey, J.& Wada, E. Two distinct receptor subtypes for mammalian bombesinlike peptides. Trends Neurosci.14,524-528 (1991).
[86]Gmerek, D. E.& Cowan, A. Bombesin-a central mediator of pruritus? Br. J. Dermatol.109,239(1983).
[87]O'Donohue TL, Massari VJ, Pazoles CJ, et al. A role for bombesin in sensory processing in the spinal cord. J Neurosci.1984;4(12):2956-62.
[88]Moody TW, Merali Z. Bombesin-like peptides and associated receptors within the brain:distribution and behavioral implications. Peptides. 2004;25(3):511-20.
[89]Sun YG, Zhao ZQ, Meng XL, et al. Cellular Basis of Itch Sensation.Science 2009; 325:1531-1534.
[90]Caterina MJ, Schumacher MA, Tominaga M, et al. The capsaicin receptor:a heat-activated ion channel in the pain pathway. Nature.1997;389:816-24.
[91]Helliwell RJ, McLatchie LM, Clarke M, et al. Capsaicin sensitivity is associated with the expression of the vanilloid (capsaicin) receptor (VR1) mRNA in adult rat sensory ganglia. Neurosci Lett.1998;250(3):177-80.
[92]Michael GJ, Priestley JV. Differential expression of the mRNA for the vanilloid receptor subtype 1 in cells of the adult rat dorsal root and nodose ganglia and its downregulation by axotomy. J Neurosci.1999; 19(5):1844-54.
[93]Davis JB, Gray J, Gunthorpe MJ, et al. Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia. Nature.2000;405(6783):183-7.
[94]Caterina MJ, Leffler A, Malmberg AB, et al. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science. 2000;288(5464):306-13.
[95]Drossman DA,Camilleri M,Mayer EA,et al. AGA technical review on irritable bowel syndrome.Gastroenterology.2002,123:2108-2131.
[96]Chan CL, FacerP, Davis JB, etal.Sensory fibres expressing capsaicin receptor TRPV1 in patients with rectal hypersensitivity and faecal urgency.Lancet. 2003,361:385-91.
[97]Mizumura K, Koda H. Potentiation and suppression of the histamine response by raising and lowering the temperature in canine visceral polymodal receptors in vitro. Neurosci Lett.1999;266:9-12.
[98]Nicolson TA, Bevan S, Richards CD. Characterisation of the calcium responses to histamine in capsaicin-sensitive and capsaicin-insensitive sensory neurones. Neuroscience.2002;110:329-38.
[99]Kim BM, Lee SH, Shim WS, et al. Histamine-induced Ca2+influx via the PLA(2)/lipoxygenase/TRPV1 pathway in rat sensory neurons. Neurosci Lett 361:159-162,2004.
[100]Shim WS, Tak MH, Lee MH, et al. TRPV1 mediates histamine-induced itching via the activation of phospholipase A2 and 12-lipoxygenase.J Neurosci. 2007;27:2331-7.
[101]Premkumar LS, Ahern GP. Induction of vanilloid receptor channel activity by protein kinase C. Nature.2000;408:985-90.
[102]Planells-Cases R, Garcia-Sanz N, Morenilla-Palao C, et al. Functional aspects and mechanisms of TRPV1 involvement in neurogenic inflammation that leads to thermal hyperalgesia. Pflugers Arch.2005;451:151-9.
[103]Moriyama T, Higashi T, Togashi K, et al. Sensitization of TRPV1 by EP1 and IP reveals peripheral nociceptive mechanism of prostaglandins. Mol Pain. 2005;1:3.
[104]Tang HB, Inoue A, Oshita K, et al. Sensitization of vanilloid receptor 1 induced by bradykinin via the activation of second messenger signaling cascades in rat primary afferent neurons. Eur J Pharmacol.2004;498:37-43.
[105]Amadesi S, Nie J, Vergnolle N, et al. Protease-activated receptor 2 sensitizes the capsaicin receptor transient receptor potential vanilloid receptor 1 to induce hyperalgesia. J Neurosci.2004;24:4300-12.
[106]梁杰贤,季文进,洪迅,等.内皮素-1引起热痛觉过敏的研究.热带医学杂志2006,6(6)：708-709.
[107]Kawamata T, Ji W, Yamamoto J, et al. Contribution of transient receptor potential vanilloid subfamily 1 to endothelin-1-induced thermal hyperalgesia. Neuroscience 2008; 154:1067-1076.
[108]梁杰贤,郭中敏,季文进,等.蛋白激酶C在ET-1引起热痛觉过敏中的作用.热带医学杂志2006,6(12)：1293-1294.
[109]季文进,梁杰贤,赵国栋.辣椒素受体在内皮素-1引起热痛觉过敏中的作用.南方医科大学学报.2007,27(1)：101-103.
[110]Yamamoto H, Kawamata T, Ninomiya T, et al. Endothelin-1 enhances capsaicin-evoked intracellular Ca2+response via activation of endothelin a receptor in a protein kinase Cepsilon-dependent manner in dorsal root ganglion neurons. Neuroscience 2006;137:949-960.
[111]Cortright DN, Szallasi A. TRP channels and pain. Curr Pharm Des. 2009; 15(15):1736-49.
[112]Steinhoff M, Bienenstock J, Schmelz M, et al. Neurophysiological, neuroimmunological, and neuroendocrine basis of pruritus. J Invest Dermatol. 2006;126(8):1705-18.
[113]Kawamata T, Ji W, Yamamoto J, et al. Involvement of transient receptor potential vanilloid subfamily 1 in endothelin-1-induced pain-like behavior. Neuroreport 2009; 20:233-237.
[114]梁杰贤,季文进,刘培庆,等.内皮素-1引起的热痛觉过敏和自发痛与辣椒素受体的关系.中国疼痛医学杂志.2008,14(1)：21-23.
[115]Liang J, Bi H, Ji W. Involvement of TRPA1 in ET-1-induced pain-like behavior in mice. Neuroreport 2010; 21:201-205.
[116]Wacnik PW, Eikmeier LJ, Ruggles TR, et al. Functional interactions between tumor and peripheral nerve:morphology, algogen identification, and behavioral characterization of a new murine model of cancer pain. J Neurosci 2001; 21:9355-9366.
[117]Story GM, Peier AM, Reeve AJ, et al. ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 2003; 112:819-829.
[118]Bautista DM, Movahed P, Hinman A,et al. Pungent products from garlic activate the sensory ionchannel TRPA1. Proc Natl Acad Sci U S A 2005; 102:12248-12252.
[119]Karashima Y, Prenen J, Meseguer V, et al. Modulation of the transient receptor potential channel TRPA1 by phosphatidylinositol 4,5-biphosphate manipulators. Pflugers Arch.2008;457(1):77-89.
[120]Smith I, White PF, Nathanson M and Gouldson R. Propofol. An update on its clinical use. Anesthesiology 81:1005-1043,1994.
[121]Matta JA, Cornett PM, Miyares RL, Abe K, Sahibzada N, Ahern GP. General anesthetics activate a nociceptive ion channel to enhance pain and inflammation. Proc Natl Acad Sci U S A 105:8784-8789,2008.
[122]Obata K, Katsura H, Mizushima T, et al. TRPA1 induced in sensory neurons contributes to cold hyperalgesia after inflammation and nerve injury. J Clin Invest.2005;115(9):2393-401.
[123]Wilson SR, Gerhold KA, Bifolck-Fisher A, et al. TRPA1 is required for histamine-independent, Mas-related G protein-coupled receptor-mediated itch. Nat Neurosci.2011;14(5):595-602.
[124]Bickers DR, Athar M. Oxidative stress in the pathogenesis of skin disease. J Invest Dermatol.2006;126:2565-2575.
[125]Kitada T, Seki S, Iwai S, et al. In situ detection of oxidative DNA damage, 8-hydroxydeoxyguanosine, in chronic human liver disease. J Hepatol. 2001;35:613-618.
[126]Tarng DC, Huang TP, Wei YH, et al.8-hydroxy-2'-deoxyguanosine of leukocyte DNA as a marker of oxidative stress in chronic hemodialysis patients. Am J Kidney Dis.2000;36:934-944.
[127]Liu T, Ji RR. Oxidative stress induces itch via activation of transient receptor potential subtype ankyrin 1 in mice. Neurosci Bull.2012;28(2):145-54.
[128]Seals DR, Jablonski KL, Donato AJ. Aging and vascular endothelial function in humans. Clin Sci (Lond).2011;120(9):357-75.
[129]Duerrschmidt N, Wippich N, Goettsch W, et al. Endothelin-1 induces NAD(P)H oxidase in human endothelial cells. Biochem Biophys Res Commun. 2000;269(3):713-7.
[130]Hunter AR, Turner AJ. Expression and localization of endothelin converting enzyme-1 isoforms in human endothelial cells. Exp Biol Med (Maywood) 2006;231:718-722.
[131]Shimada SG, Shimada KA, Collins JG. Scratching behavior in mice induced by the proteinase-activated receptor-2 agonist, SLIGRL-NH2. Eur J Pharmacol 2006; 530(3):281-3.
[132]Shimada SG, LaMotte RH. Behavioral differentiation between itch and pain in mouse. Pain 2008; 139:681-687.
[133]Rashid MH, Inoue M, Kondo S, Kawashima T, Bakoshi S and Ueda H. Novel expression of vanilloid receptor 1 on capsaicin-insensitive fibers accounts for the analgesic effect of capsaicin cream in neuropathic pain. J Pharmacol Exp Ther 304:940-948,2003.
[134]Scholze T, Moskvina E, Mayer M, et al. Sympathoexcitation by bradykinin involves Ca2+-independent protein kinase C. J Neurosci.2002;22(14):5823-32.
[135]Ji RR, Samad TA, Jin SX, et al. p38 MAPK activation by NGF in primary sensory neurons after inflammation increases TRPV1 levels and maintains heat hyperalgesia. Neuron.2002;36(1):57-68.
[136]梁杰贤,洪迅,季文进,等.辣椒素受体在腹腔注射内皮素-1引起内脏痛中的作用.中国疼痛医学杂志.2006,12(3)：167-169.
[137]Khodorova A, Navarro B, Jouaville LS, et al. Endothelin-B receptor activation triggers an endogenous analgesic cascade at sites of peripheral injury. Nat Med 9:1055-1061,2003.
[138]Kuraishi Y, Nagasawa T, Hayashi K, et al. Scratching behavior induced by pruritogenic but not algesiogenic agents in mice. Eur J Pharmacol. 1995;275:229-33.
[139]Yeo JF, Ling SF, Tang N, et al. Antinociceptive effect of CNS peroxynitrite scavenger in a mouse model of orofacial pain. Exp Brain Res.2008; 184:435-8.
[140]Yeo JF, Ong WY, Ling SF, et al. Intracerebroventricular injection of phospholipases A2 inhibitors modulates allodynia after facial carrageenan injection in mice. Pain.2004;112:148-55.
[141]Gomes LO, Hara DB, Rae GA. Endothelin-1 induces itch and pain in the mouse cheek model. Life Sci.2012;91(13-14):628-33.
[142]Dekker LV, Parker PJ. Protein kinase C--a question of specificity. Trends Biochem Sci.1994;19(2):73-7.
[143]Khasar SG, Lin YH, Martin A, et al. A novel nociceptor signaling pathway revealed in protein kinase C epsilon mutant mice. Neuron.1999;24(1):253-60.
[144]Cesare P, Dekker LV, Sardini A, et al. Specific involvement of PKC-epsilon in sensitization of the neuronal response to painful heat. Neuron. 1999;23(3):617-24.
[145]Ferreira J, da Silva GL, Calixto JB. Contribution of vanilloid receptors to the overt nociception induced by B2 kinin receptor activation in mice. Br J Pharmacol.2004;141(5):787-94.
[146]Hagermark O. Peripheral and central mediators of itch. Skin Pharmacol. 1992;5:1-8.
[147]Bolcskei K, Helyes Z, Szabo A, et al. Investigation of the role of TRPV1 receptors in acute and chronic nociceptive processes using gene-deficient mice. Pain 2005,117:368-76.
[148]Ferreira J, Triches KM, Medeiros R, et al:The role of kinin B1 receptors in the nociception produced by peripheral protein kinase C activation in mice. Neuropharmacology 2008; 54:597-604
[149]Liang J, He Y, Ji W. Bradykinin-evoked scratching responses in complete Freund's adjuvant-inflamed skin through activation of B1 receptor. Exp Biol Med (Maywood).2012;237:318-326.
[150]Motta E M, Calixto J B, Rae G A. Mechanical hyperalgesia induced by endothelin-1 in rats is mediated via phospholipase C, protein kinase C, and MAP kinases[J]. Experimental Biology and Medicine,2006,231(6): 1141-1145.
[151]Hucho TB, Dina OA, Levine JD. Epac mediates a cAMP-to-PKC signaling in inflammatory pain:an isolectin B4(+) neuron-specific mechanism. J Neurosci 2005; 25(26):6119-26.
[152]Southall MD, Vasko MR. Prostaglandin receptor subtypes, EP3C and EP4, mediate the prostaglandin E2-induced cAMP production and sensitization of sensory neurons. J Biol Chem.2001;276(19):16083-91.
[153]Kawanabe Y, Okamoto Y, Miwa S, et al. Molecular mechanisms for the activation of voltage-independent Ca2+channels by endothelin-1 in Chinese hamster ovary cells stably expressing human endothelin(A) receptors. Mol Pharmacol.2002;62(1):75-80.
[154]Hu HJ, Bhave G, Gereau RW 4th. Prostaglandin and protein kinase A-dependent modulation of vanilloid receptor function by metabotropic glutamate receptor 5:potential mechanism for thermal hyperalgesia. J Neurosci. 2002;22(17):7444-52.
[155]Stempelj M, Ferjan I. Signaling pathway in nerve growth factor induced histamine release from rat mast cells. Inflamm Res.2005;54(8):344-9.
[156]Schutze S, Potthoff K, Machleidt T, et al. TNF activates NF-kappa B by phosphatidylcholine-specific phospholipase C-induced "acidic" sphingomyelin breakdown. Cell.1992;71(5):765-76.
[157]梁杰贤,季文进,洪迅,吉锦泉,梅莉.辣椒素受体在肠易激惹综合征内脏痛觉过敏中的作用.热带医学杂志2007,7(4)：342-344.
[158]Ikeda Y, Ueno A, Naraba H,et al. Involvement of vanilloid receptor VR1 and prostanoids in the acid-induced writhing responses of mice. Life Sci 69, 2911-2919,2001.
[159]Ji W, Cui C, Zhang Z, et al. Paradoxic effects of propofol on visceral pain induced by different TRPV1 agonists. Exp Ther Med 2013,5(4):1259-1263.
[160]Bergasa NV, Alling DW, Talbot TL, et al. Effects of naloxone infusions in patients with the pruritus of cholestasis. A double-blind, randomized, controlled trial. Ann Intern Med 1995; 123:161-167.
[161]Luger NM, Sabino MA, Schwei MJ, et al. Efficacy of systemic morphine suggests a fundamental difference in the mechanisms that generate bone cancer vs inflammatory pain. Pain.2002; 99:397-406.
[162]Reichert JA, Daughters RS, Rivard R, et al. Peripheral and preemptive opioid antinociception in a mouse visceral pain model. Pain 2001; 89:221-227.
[163]Laird JM, Martinez-Caro L, Garcia-Nicas E, et al. A new model of visceral pain and referred hyperalgesia in the mouse. Pain 2001; 92:335-342.
[164]Leung DY, Boguniewicz M, Howell MD, et al. New insights into atopic dermatitis. J Clin Invest.2004; 113(5):651-7.
[165]Hosogi M, Schmelz M, Miyachi Y, Ikoma A. Bradykinin is a potent pruritogen in atopic dermatitis:a switch from pain to itch. Pain 2006; 126:16-23.
[166]Liang J, Xiao G, Ji W. Capsaicin induces reflex scratching in inflamed skin. Pharmacology 2011;88:82-87.
[167]Khodorova A, Zou S, Ren K, et al. Dual Roles for Endothelin-B Receptors in Modulating Adjuvant-Induced Inflammatory Hyperalgesia in Rats. Open Pain J. 2009; 2:30-40.
[168]Bouzigon E, Forabosco P, Koppelman GH, et al. Meta-analysis of 20 genome-wide linkage studies evidenced new regions linked to asthma and atopy. Eur J Hum Genet 2010;18:700-706.
[169]Quang PN and Schmidt BL. Peripheral endothelin B receptor agonist-induced antinociception involves endogenous opioids in mice. Pain 149:254-262,2010.
[170]Rittner HL, Hackel D, Voigt P, et al. Mycobacteria attenuate nociceptive responses by formyl peptide receptor triggered opioid peptide release from neutrophils. PLoS Pathog 5:e1000362,2009.
[171]Taniuchi S, Kojima T, Hara M, et al. Increased serum nitrate levels in infants with atopic dermatitis. J Invest Dermatol.2001; 56:693-695.
[172]Morita H, Semma M., Hori M, et al. Clinical application of nitric oxide synthase inhibitor for atopic dermatitis. Int J Dermatol.1995; 34:294-295.
[173]Andoh T, Kuraishi Y. Nitric oxide enhances substance P-induced itch-associated responses in mice. Br J Pharmacol.2003;138(1):202-8.
[174]Amarante LH, Duarte ID. The kappa-opioid agonist (+/-)-bremazocine elicits peripheral antinociception by activation of the L-arginine/nitric oxide/cyclic GMP pathway. Eur J Pharmacol.2002;454(1):19-23.
[175]Cury Y, Picolo G, Gutierrez V P, et al. Pain and analgesia:The dual effect of nitric oxide in the nociceptive system[J]. Nitric Oxide,2011,25(3):243-254.
[176]Cury Y, Picolo G, Gutierrez V P, et al. Pain and analgesia:The dual effect of nitric oxide in the nociceptive system[J]. Nitric Oxide,2011,25(3):243-254.
[177]Shimizu Y, Sakai M, Umemura Y, et al. Immunohistochemical localization of nitric oxide synthase in normal human skin:expression of endothelial-type and inducible-type nitric oxide synthase in keratinocytes. J Dermatol. 1997;24(2):80-7.
[178]Deliconstantinos G, Villiotou V, Stavrides JC. Alterations of nitric oxide synthase and xanthine oxidase activities of human keratinocytes by ultraviolet B radiation. Potential role for peroxynitrite in skin inflammation. Biochem Pharmacol.1996;51(12):1727-38.
[179]Romero T R L, Resende L C, Duarte I D G. The neuronal NO synthase participation in the peripheral antinociception mechanism induced by several analgesic drugs[J]. Nitric Oxide,2011,25(4):431-435.
[180]Kalani M. The importance of endothelin-1 for microvascular dysfunction in diabetes. Vasc Health Risk Manag 2008; 4(5):1061-8.
[181]Schiffrin EL. Role of endothelin-1 in hypertension and vascular disease. Am J Hypertens 2001; 14(6 Pt 2):83S-9S.
[182]Davie NJ, Schermuly RT, Weissmann N, Grimminger F, Ghofrani HA. The science of endothelin-1 and endothelin receptor antagonists in the management of pulmonary arterial hypertension:current understanding and future studies. Eur J Clin Invest 2009; 39 S(2):38-49.
[183]Matsushima H, Yamada N, Matsue H, et al. The effects of endothelin-1 on degranulation, cytokine, and growth factor production by skin-derived mast cells[J]. European journal of immunology,2004,34(7):1910-1919.
[184]Khodorova A, Montmayeur J P, Strichartz G. Endothelin receptors and pain[J]. The Journal of Pain,2009,10(1):4-28.
[185]Mobarakeh J I, Sakurada S, Katsuyama S, et al. Role of histamine H1 receptor in pain perception:a study of the receptor gene knockout mice[J]. European journal of pharmacology,2000,391(1):81-89.
[186]Hill S J, Ganellin C R, Timmerman H, et al. International Union of Pharmacology. XIII. Classification of histamine receptors [J]. Pharmacological reviews,1997,49(3):253-278.
[187]Han S K, Mancino V, Simon M I. Phospholipase Cβ3 mediates the scratching response activated by the histamine H1 receptor on C-fiber nociceptive neurons [J]. Neuron,2006,52(4):691-703.
[188]Xie W, Samoriski G M, McLaughlin J P, et al. Genetic alteration of phospholipase Cβ3 expression modulates behavioral and cellular responses to μ opioids[J]. Proceedings of the National Academy of Sciences,1999,96(18): 10385-10390.
[189]Metz M, Lammel V, Gibbs B F, et al. Inflammatory murine skin responses to UV-B light are partially dependent on endothelin-1 and mast cells[J]. The American journal of pathology,2006,169(3):815-822.
[190]Matsushima H, Yamada N, Matsue H, et al. The effects of endothelin-1 on degranulation, cytokine, and growth factor production by skin-derived mast cells[J]. European journal of immunology,2004,34(7):1910-1919.
[191]Yamamura H, Nabe T, Kohno S, et al. Endothelin-1 induces release of histamine and leukotriene C< sub> 4 from mouse bone marrow-derived mast cells[J]. European journal of pharmacology,1994,257(3):235-242.
[192]Yamamura H, Nabe T, Kohno S, et al. Endothelin-1, one of the most potent histamine releasers in mouse peritoneal mast cells[J]. European journal of pharmacology,1994,265(1):9-15.
[193]Brain S D, Thomas G, Crossman D C, et al. Endothelin-1 induces a histamine-dependent flare in vivo, but does not activate human skin mast cells in vitro[J]. British Journal of Clinical Pharmacology,1992,33(1):117.
[194]Katugampola R, Church M K, Clough G F. The neurogenic vasodilator response to endothelin-1:a study in human skin in vivo[J]. Experimental physiology,2000,85(6):839-846.
[195]Steinhoff M, Neisius U, Ikoma A, et al. Proteinase-activated receptor-2 mediates itch:a novel pathway for pruritus in human skin[J]. The Journal of neuroscience,2003,23(15):6176-6180.
[196]Costa R, Manjavachi M N, Motta E M, et al. The role of kinin B1 and B2 receptors in the scratching behaviour induced by proteinase-activated receptor-2 agonists in mice[J]. British journal of pharmacology,2010,159(4): 888-897.
[197]Tsujii K, Andoh T, Lee J B, et al. Activation of proteinase-activated receptors induces itch-associated response through histamine-dependent and-independent pathways in mice[J]. Journal of pharmacological sciences,2008,108(3): 385-388.
[198]Rowley D A, Benditt E P.5-Hydroxytryptamine and histamine as mediators of the vascular injury produced by agents which damage mast cells in rats[J]. The Journal of experimental medicine,1956,103(4):399-412.
[199]Dymshitz J, Vasko M R. Endothelin-1 enhances capsaicin-induced peptide release and cGMP accumulation in cultures of rat sensory neurons [J]. Neuroscience letters,1994,167(1):128-132.
[200]Vaalasti A, Suomalainen H, Rechardt L. Calcitonin gene-related peptide immunoreactivity in prurigo nodularis:a comparative study with neurodermatitis circumscripta[J]. British Journal of Dermatology,2006,120(5): 619-623.
[201]Weidner C, Klede M, Rukwied R, et al. Acute effects of substance P and calcitonin gene-related peptide in human skin-a microdialysis study[J]. Journal of investigative dermatology,2000,115(6):1015-1020.
[202]Yamamoto A, Kuyama S, Kamei C, et al. Characterization of scratching behavior induced by intradermal administration of morphine and fentanyl in mice[J]. European journal of pharmacology,2010,627(1):162-166.
[203]Yamamoto A, Sugimoto Y. Involvement of peripheral mu opioid receptors in scratching behavior in mice[J]. European journal of pharmacology,2010, 649(1):336-341.
[204]朱江婷,程飚,刘宏伟,等.p-内啡肽在增生性瘢痕组织中的表达及其与瘢痕瘙痒关系的研究[J].中国修复重建外科杂志,2012,26(6)：732.
[205]Bigliardi-Qi M, Sumanovski L T, Buchner S, et al. Mu-opiate receptor and Beta-endorphin expression in nerve endings and keratinocytes in human skin[J]. Dermatology,2004,209(3):183-189.
[206]Tominaga M, Ogawa H, Takamori K. Histological characterization of cutaneous nerve fibers containing gastrin-releasing peptide in NC/Nga mice:an atopic dermatitis model [J]. Journal of Investigative Dermatology,2009, 129(12):2901-2905.
[207]朱江婷,程飚,刘宏伟.阿片肽系统在增生性瘢痕感觉异常中充当的角色[J].中国美容医学,2012,21(4)：676-679.
[208]Liu X Y, Liu Z C, Sun Y G, et al. Unidirectional cross-activation of GRPR by MOR1D uncouples itch and analgesia induced by opioids[J]. Cell,2011,147(2): 447-458.
[209]da Cunha J M, Rae G A, Ferreira S H, et al. Endothelins induce ET< sub> B receptor-mediated mechanical hypernociception in rat hindpaw:roles of cAMP and protein kinase C[J]. European journal of pharmacology,2004, 501(1):87-94.
[210]Wang S, Dai Y, Fukuoka T, et al. Phospholipase C and protein kinase A mediate bradykinin sensitization of TRPA1:a molecular mechanism of inflammatory pain[J]. Brain,2008,131(5):1241-1251.
[211]Sultana R, Newman S, Mohmmad-Abdul H, et al. Protective effect of the xanthate, D609, on Alzheimer's amyloid β-peptide (1-42)-induced oxidative stress in primary neuronal cells[J]. Free radical research,2004,38(5):449-458.
[212]Sultana R, Newman S F, Abdul H M, et al. Protective effect of D609 against amyloid-betal-42-induced oxidative modification of neuronal proteins: Redox proteomics study [J]. Journal of neuroscience research,2006,84(2): 409-417.
[213]Touyz R M, Yao G, Viel E, et al. Angiotensin Ⅱ and endothelin-1 regulate MAP kinases through different redox-dependent mechanisms in human vascular smooth muscle cells[J]. Journal of hypertension,2004,22(6): 1141-1149.
[214]Fei J, Viedt C, Soto U, et al. Endothelin-1 and smooth muscle cells induction of Jun amino-terminal kinase through an oxygen radical-sensitive mechanism [J]. Arteriosclerosis, thrombosis, and vascular biology,2000,20(5):1244-1249.
[215]Luberto C, Hannun Y A. Sphingomyelin synthase, a potential regulator of intracellular levels of ceramide and diacylglycerol during SV40 transformation[J]. Journal of Biological Chemistry,1998,273(23): 14550-14559.
[216]Sprague A H, Khalil R A. Inflammatory cytokines in vascular dysfunction and vascular disease[J]. Biochemical pharmacology,2009,78(6):539.
[217]Nguyen B T, Dessauer C W. Relaxin stimulates protein kinase C ζ translocation: requirement for cyclic adenosine 3',5'-monophosphate production[J]. Molecular Endocrinology,2005,19(4):1012-1023.
[218]Kircik L H, Del Rosso J Q, Aversa D. Evaluating clinical use of a ceramide-dominant, physiologic lipid-based topical emulsion for atopic dermatitis [J]. The Journal of clinical and aesthetic dermatology,2011,4(3):34.
[219]Tominaga M, Takamori K. Recent advances in pathophysiological mechanisms of itch[J]. Expert Review of Dermatology,2010,5(2):197-212.
[220]Kamei J, Nagase H. Norbinaltorphimine, a selective κ-opioid receptor antagonist, induces an itch-associated response in mice[J]. European journal of pharmacology,2001,418(1):141-145.
[221]Inan S, Cowan A. Kappa opioid agonists suppress chloroquine-induced scratching in mice[J]. European journal of pharmacology,2004,502(3): 233-237.
[222]Satoh M, Minami M. Molecular pharmacology of the opioid receptors[J]. Pharmacology & therapeutics,1995,68(3):343-364.
[223]Cheng B, Liu H W, Fu X B, et al. Coexistence and upregulation of three types of opioid receptors, mu, delta and kappa, in human hypertrophic scars[J]. British Journal of Dermatology,2008,158(4):713-720.
[224]Ward L, Wright E, McMahon S B. A comparison of the effects of noxious and innocuous counterstimuli on experimentally induced itch and pain[J]. Pain, 1996,64(1):129-138.
[225]Shim W S, Oh U. Histamine-induced itch and its relationship with pain[J]. Mol Pain,2008,4:29.
[226]季文进,魏涧琦,梁杰贤,等.外周内源性大麻受体激动剂及其受体2亚型参与搔痒调节的研究[J].热带医学杂志,2009,9(011)：1231-1232.
[227]Mishra S K, Tisel S M, Orestes P, et al. TRPV1-lineage neurons are required for thermal sensation[J]. The EMBO Journal,2010,30(3):582-593.
[228]Lagerstrom M C, Rogoz K, Abrahamsen B, et al. VGLUT2-dependent sensory neurons in the TRPV1 population regulate pain and itch[J]. Neuron,2010,68(3): 529-542.
[229]Liu Y, Abdel Samad O, Zhang L, et al. VGLUT2-dependent glutamate release from nociceptors is required to sense pain and suppress itch[J]. Neuron,2010, 68(3):543-556.
[2]Inoue A, Yanagisawa M, Kimura S, et al. The human endothelin family:three structurally and pharmacologically distinct isopeptides predicted by three separate genes[J]. Proceedings of the National Academy of Sciences,1989, 86(8):2863-2867.
[3]Saida K, Mitsui Y, Ishida N. A novel peptide, vasoactive intestinal contractor, of a new (endothelin) peptide family. Molecular cloning, expression, and biological activity[J]. Journal of Biological Chemistry,1989,264(25): 14613-14616.
[4]Bloch K D, Hong C C, Eddy R L, et al. cDNA cloning and chromosomal assignment of the endothelin 2 gene:vasoactive intestinal contractor peptide is rat endothelin 2[J]. Genomics,1991,10(1):236-242.
[5]Dhaun N, Pollock D M, Goddard J, et al. Selective and mixed endothelin receptor antagonism in cardiovascular disease[J]. Trends in pharmacological sciences,2007,28(11):573-579.
[6]Uchide T, Masuda H, Mitsui Y, Saida K. Gene expression of vasoactive intestinal contractor/endothelin-2 in ovary, uterus and embryo:comprehensive gene expression profiles of the endothelin ligand-receptor system revealed by semi-quantitative reverse transcription-polymerase chain reaction analysis in adult mouse tissues and during late embryonic development [J]. J Mol Endocrinol.1999;22(2):161-71.
[7]Matsumoto H, Suzuki N, Onda H, et al. Abundance of endothelin-3 in rat intestine, pituitary gland and brain [J]. Biochemical and biophysical research communications,1989,164(1):74-80.
[8]Agapitov AV, Haynes WG. Role of endothelin in cardiovascular disease [J]. J Renin Angiotensin Aldosterone Syst.2002;3(1):1-15.
[9]Sessa W C, Kaw S, Hecker M, et al. The biosynthesis of endothelin-1 by human polymorphonuclear leukocytes[J]. Biochemical and biophysical research communications,1991,174(2):613-618.
[10]Ehrenreich H, Anderson RW, Fox CH, et al. Endothelins, peptides with potent vasoactive properties, are produced by human macrophages [J]. J Exp Med. 1990;172:1741-1748.
[11]Hahn AW, Resink TJ, Scott-Burden T, et al. Stimulation of endothelin mRNA and secretion in rat vascular smooth muscle cells:a novel autocrine function [J]. Cell Regul.1990; 1:649-659.
[12]Ito H, Hirata Y, Adachi S, et al. Endothelin-1 is an autocrine/paracrine factor in the mechanism of angiotensin II-induced hypertrophy in cultured rat cardiomyocytes [J]. J Clin Invest.1993;92:398-403.
[13]Fujisaki H, Ito H, Hirata Y, et al. Natriuretic peptides inhibit angiotensin Ⅱ-induced proliferation of rat cardiac fibroblasts by blocking endothelin-1 gene expression [J]. J Clin Invest.1995;96:1059-1065.
[14]Fukunaga M, Fujiwara Y, Ochi S, et al. Stimulatory effect of thrombin on endothelin-1 production in isolated glomeruli and cultured mesangial cells of rats [J]. J Cardiovasc Pharmacol.1991;17:S411-S413.
[15]Nakamura T, Ebihara I, Fukui M, et al. Renal expression of mRNAs for endothelin-1, endothelin-3 and endothelin receptors in NZB/WF1 mice [J]. Ren Physiol Biochem.1993; 16:233-243.
[16]Giaid A, Gibson SJ, Ibrahim BN, Legon S, Bloom SR, Yanagisawa M, Masaki T, Varndell IM, Polak JM. Endothelin 1, an endothelium-derived peptide, is expressed in neurons of the human spinal cord and dorsal root ganglia [J]. Proc Natl Acad Sci U S A.1989 Oct;86(19):7634-8.
[17]MacCumber MW, Ross CA and Snyder SH. Endothelin in brain:receptors, mitogenesis, and biosynthesis in glial cells. Proc Natl Acad Sci U S A 87:2359-2363,1990.
[18]Gandhi CR, Berkowitz DE, Watkins WD. Endothelins. Biochemistry and pathophysiologic actions. Anesthesiology 1994;80:892-905.
[19]Hans G, Deseure K, Adriaensen H. Endothelin-1-induced pain and hyperalgesia: a review of pathophysiology, clinical manifestations and future therapeutic options. Neuropeptides 2008;42:119-132.
[20]Alberts GF, Peifley KA, Johns A, et al. Constitutive endothelin-1 overexpression promotes smooth muscle cell proliferation via an external autocrine loop. J Biol Chem.1994;269:10112-10118.
[21]Barton M, Shaw S, d'Uscio LV, et al. Angiotensin Ⅱ increases vascular and renal endothelin-1 and functional endothelin converting enzyme activity in vivo: role of ETA-receptors for endothelin regulation. Biochem Biophys Res Commun.1997;238:861-865.
[22]Lee ME, Bloch KE, Clifford JA, et al. Functional analysis of the endothelin-1 gene promotor:evidence for an endothelial cell specific cis-acting enzyme. J Biol Chem.1993;265:10446-10450.
[23]Hirata Y, Kanno K, Watanabe TX, et al. Receptor binding and vasoconstrictor activity of big endothelin. Eur J Pharmacol.1990; 176:225-228.
[24]Xu D, Emoto N, Giaid A, et al. ECE-1:a membrane-bound metalloprotease that catalyzes the proteolytic activation of big endothelin-1. Cell.1994;78:473-485.
[25]Emoto N, Yanagisawa M. Endothelin-converting enzyme-2 is a membrane-bound, phosphoramidon-sensitive metalloprotease with acidic pH optimum. J Biol Chem.1995;270:15262-15268.
[26]Wypij DM, Nichols JS, Novak PJ, et al. Role of mast cell chymase in the extracellular processing of big-endothelin-1 to endothelin-1 in the perfused rat lung. Biochem Pharmacol.1992;43:845-853.
[27]Nakano A, Kishi F, Ninami K, et al. Selective conversion of big endothelins to tracheal smooth muscle-constricting 31-amino acid-length endothelins by chymase from human mast cells. J Immunol.1997; 159:1987-1992.
[28]Hasegawa H, Hiki K, Sawamura T, et al. Purification of a novel endothelin-converting enzyme specific for big endothelin-3. FEBS Lett. 1998;428:394-398.
[29]Maguire JJ, Johnson CM, Mockridge JW, et al. Endothelin converting enzyme (ECE) activity in human vascular smooth muscle. Br J Pharmacol. 1997; 122:1647-1654.
[30]Kobayashi T, Miyauochi T, Sakai S, et al. Endothelin converting enzyme (ECE) and angiotensin-converting enzyme in failing hearts of rats with myocardial infarction. J Cardiovasc Pharmacol.1998;31:S417-S420.
[31]Minamino T, Kurihara H, Takahashi M, et al. Endothelin-converting enzyme expression in the rat vascular injury model and human coronary atherosclerosis. Circulation.1997;95:221-230.
[32]Ohwaki T, Sakai H, Hirata Y. Endothelin-converting enzyme activity in human serum lipoprotein fraction. FEBS Lett.1993;320:165-168.
[33]Hans G, Deseure K, Robert D and De Hert S. Neurosensory changes in a human model of endothelin-1 induced pain:a behavioral study. Neurosci Lett 418:117-121,2007.
[34]Dahlof B, Gustafsson D, Hedner T, et al. Regional haemodynamic effects of endothelin-1 in rat and man:unexpected adverse reaction. J Hypertens. 1990;8(9):811-7.
[35]Zonnenberg BA, Groenewegen G, Janus TJ, et al. Phase I dose-escalation study of the safety and pharmacokinetics of atrasentan:an endothelin receptor antagonist for refractory prostate cancer. Clin Cancer Res.2003;9(8):2965-72.
[36]Carducci MA, Padley RJ, Breul J, et al.Effect of endothelin-A receptor blockade with atrasentan on tumor progression in men with hormone-refractory prostate cancer a randomized, phase II, placebo-controlled trial, J Clin Oncol 2003,21:679-689.
[37]Raffa RB,Jacoby HI. Endothelin-1,-2 and-3 directly and big-endothelin-1 indirectly elicit an abdominal constriction response in mice.Life Sci. 1991;48:PL85-90.
[38]Gokin AP, Fareed MU, Pan HL, et al. Local injection of endothelin-1 produces pain-like behavior and excitation of nociceptors in rats.J Neurosci. 2001;21:5358-5366.
[39]Ferreira SH,Romitelli M,NucciG de.Endothelin-1 participation in overt and inflammatory pain.J Cardiovasc Pharmacol 13:S220-S222.
[40]Baamonde A, Lastra A, Villazon M, et al. Involvement of endogenous endothelins in thermal and mechanical inflammatory hyperalgesia in mice.Naunyn Schmiedebergs Arch Pharmacol.2004;369:245-251.
[41]Piovezan AP, D'Orleans-Juste P, Tonussi CR, et al. Effects of endothelin-1 on capsaicin-induced nociception in mice. Eur J Pharmacol.1998;351(1):15-22.
[42]Liang J, Bi H, Ji W.Involvement of TRPA1 in ET-1-induced pain-like behavior in mice. Neuroreport 2010,21(3):201-205.
[43]Ahern GP, Brooks IM, Miyares RL, et al. Extracellular cations sensitize and gate capsaicin receptor TRPV1 modulating pain signaling.J Neurosci. 2005;25:5109-5116.
[44]洪迅,梁杰贤,季文进,等.腹腔内注射内皮素-1对硫酸镁引起腹痛的影响.热带医学杂志.2006,6(4)：455-456.
[45]Arai H, Hori S, Aramori I, et al. Cloning and expression of a cDNA encoding an endothelin receptor. Nature.1990;348:730-732.104.
[46]Lin HY, Kaji EH, Winkel G, et al. Cloning and expression of a vascular smooth muscle endothelin-1 receptor. Proc Natl Acad Sci U S A.1991;88:3185-3189.
[47]Sakurai T, Yanagisawa M, Takuwa Y, et al. Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor. Nature. 1990;348:732-735.
[48]Sakamoto A, Yanagisawa M, Sakurai T, et al. Cloning and functional expression of human cDNA for the ETB endothelin receptor. Biochem Biophys Res Commun.1991;178:656-663.
[49]Karne S, Jayawickreme CK, Lerner MR. Cloning and characterization of an endothelin-3 specific receptor (ETC receptor) from Xenopus laevis dermal melanophores. J Biol Chem.1993;268:19126-19133.
[50]Sakurai T, Yanagisawa M, Masaki T. Molecular characterization of endothelin receptors. Trends Pharmacol Sci 1992;13:103-108.
[51]Seo B, Oemar BS, Siebenmann R, et al. Both ETA and ETB receptors mediate contraction to endothelin-1 in human blood vessels. Circulation. 1994;89:1203-1208.
[52]Elshourbagy NA, Lee JA, Korman DR, et al.. Molecular cloning and characterization of the major endothelin receptor subtype in porcine cerebellum. Mol Pharmacol.1992;41:465-473.
[53]Boivin S, Tessier S, Aubin J,et al. Identification of a binding domain of the endothelin-B receptor using a selective IRL-1620-derived photoprobe. Biochemistry.2004;43:11516-11525.
[54]Krystek SR, Jr, Patel PS, Rose PM, et al. Mutation of peptide binding site in transmembrane region of a G protein-coupled receptor accounts for endothelin receptor subtype selectivity. J Biol Chem.1994;269:12383-12386.
[55]De-Melo JD, Tonussi CR, D'Orleans-Juste P, et al. Effects of endothelin-1 on inflammatory incapacitation of the rat knee joint. J Cardiovasc Pharmacol 1998;31(Suppl1):S518-520.
[56]Maxwell MJ, Goldie RG, Henry PJ. Ca2+signalling by endothelin receptors in rat and human cultured airway smooth muscle cells. Br J Pharmacol. 1998;125:1768-1778.
[57]Warner TD, Mitchell JA, de Nucci G, et al. Endothelin-1 and endothelin-3 release EDRF from isolated perfused arterial vessels of the rat and rabbit. J Cardiovasc Pharmacol.1989;13:S85-S88.
[58]Hirata Y, Emori T, Eguchi S, et al. Endothelin receptor subtype B mediates synthesis of nitric oxide by cultured bovine endothelial cells. J Clin Invest. 1993;91:1367-1373.
[59]Pomonis JD, Rogers SD, Peters CM, et al. Expression and localization of endothelin receptors:implications for the involvement of peripheral glia in nociception. J Neurosci.2001;21(3):999-1006.
[60]Peters CM, Lindsay TH, Pomonis JD,et al. Endothelin and the tumorigenic component of bone cancer pain. Neuroscience.2004;126(4):1043-52.
[61]Gold MS,LevineJD,Correa AM. Modulation of TTX-R INa by PKC and PKA and their role in PGE2-induced sensitization of rat sensory neurons in vitro, J Neurosci 1998;18:10345-10355.
[62]Zhang YH,Kenyon JL,Nicol GD. Phorbol ester-induced inhibition of potassium currents in rat sensory neurons requires voltage-dependent entry of calcium. J Neurophysiol.2001;85:362-373.
[63]Cheng TH, Chang CY, Wei J, et al. Effects of endothelin-1 on calcium and sodium currents in isolated human cardiac myocytes. Can J Physiol Pharmacol,1995; 73:1774-1783.
[64]Fjell J,Hjelmstrom P,Hormuzdiar W,et al.Localization of the tetrodotoxin-resistant sodium channel NaN in nociceptors. NeuroReport, 2000; 11:199-202.
[65]Zhou Z, Davar G, Strichartz G. Endothelin-1 (ET-1) selectively enhances the activation gating of slowly inactivating tetrodotoxin-resistant sodium currents in rat sensory neurons:a mechanism for the pain-inducing actions of ET-1.J Neurosci.2002;22:6325-6330.
[66]Cardenas LM, Cardenas CG, Scroggs RS.5HT Increases excitability of nociceptor-like rat dorsal root ganglion neurons via cAMP-coupled TTX-resistant Na+channels. J Neurophysiol,2001; 86:241-248.
[67]Renganathan M, Cummins TR, Waxman SG. Contribution of Na(v)1.8 sodium channels to action potential electrogenesis in DRG neurons. J Neurophysiol,2001;86:629-640.
[68]Trentin PG, Fernandes MB, D'Orleans-Juste P, et al. Endothelin-1 causes pruritus in mice. Exp Biol Med (Maywood) 2006; 231:1146-1151.
[69]McQueen DS, Noble MA, Bond SM. Endothelin-1 activates ETA receptors to cause reflex scratching in BALB/c mice. Br J Pharmacol 2007; 151:278-284.
[70]Sun YG, Zhao ZQ, Meng XL, et al. Cellular Basis of Itch Sensation.Science 2009; 325:1531-1534.
[71]Imamachi N, Park GH, Lee H, et al. TRPV1-expressing primary afferents generate behavioral responses to pruritogens via multiple mechanisms. Proc Natl Acad Sci U S A 2009;106:11330-11335.
[72]Davidson S, Giesler GJ.The multiple pathways for itch and their interactions with pain. Trends Neurosci 2010; 33:550-558.
[73]Schmelz M. Itch and pain. Neurosci Biobehav Rev 2010;34:171-176.
[74]Schmelz M. How pain becomes itch. Pain 2009; 144:14-15.
[75]v.Frey M. Zur Physiologie der Juckempfindung. Arch Neerl Physiol 1922;7:142-5.
[76]Chaney MA. Side effects of intrathecal and epidural opioids. Can J Anaesth. 1995;42(10):891-903.
[77]王春晓,季文进,林派冲.硬膜外腔及蛛网膜下腔注射吗啡诱发搔痒的比较.中华麻醉学杂志.1998,18(7)：441-442.
[78]Ikoma A, Steinhoff M, Stander S, et al. The neurobiology of itch. Nat Rev Neurosci.2006;7:535-47.
[79]Schmelz M, Schmidt R, Bickel A, et al. Specific C-receptors for itch in human skin. J Neurosci.1997;17(20):8003-8.
[80]Jeffry J, Kim S, Chen ZF. Itch signaling in the nervous system. Physiology (Bethesda).2011;26(4):286-92.
[81]Bandell M, Patapoutian A.Itching for Insight. Cell 2009;325:1224-1225.
[82]Liu Q, Tang Z, Surdenikova L,et al. Sensory Neuron-Specific GPCR Mrgprs Are Itch Receptors Mediating Chloroquine-Induced Pruritus. Cell 2009; 139: 1353-1365.
[83]Sun YG, Chen ZF. A gastrin-releasing peptide receptor mediates the itch sensation in the spinal cord. Nature.2007;448:700-3.
[84]Roesler, R., Henriques, J. A.& Schwartsmann, G Gastrin-releasing peptide receptor as a molecular target for psychiatric and neurological disorders. CNS Neurol. Disord. Drug Targets 5,197-204 (2006).
[85]Battey, J.& Wada, E. Two distinct receptor subtypes for mammalian bombesinlike peptides. Trends Neurosci.14,524-528 (1991).
[86]Gmerek, D. E.& Cowan, A. Bombesin-a central mediator of pruritus? Br. J. Dermatol.109,239(1983).
[87]O'Donohue TL, Massari VJ, Pazoles CJ, et al. A role for bombesin in sensory processing in the spinal cord. J Neurosci.1984;4(12):2956-62.
[88]Moody TW, Merali Z. Bombesin-like peptides and associated receptors within the brain:distribution and behavioral implications. Peptides. 2004;25(3):511-20.
[89]Sun YG, Zhao ZQ, Meng XL, et al. Cellular Basis of Itch Sensation.Science 2009; 325:1531-1534.
[90]Caterina MJ, Schumacher MA, Tominaga M, et al. The capsaicin receptor:a heat-activated ion channel in the pain pathway. Nature.1997;389:816-24.
[91]Helliwell RJ, McLatchie LM, Clarke M, et al. Capsaicin sensitivity is associated with the expression of the vanilloid (capsaicin) receptor (VR1) mRNA in adult rat sensory ganglia. Neurosci Lett.1998;250(3):177-80.
[92]Michael GJ, Priestley JV. Differential expression of the mRNA for the vanilloid receptor subtype 1 in cells of the adult rat dorsal root and nodose ganglia and its downregulation by axotomy. J Neurosci.1999; 19(5):1844-54.
[93]Davis JB, Gray J, Gunthorpe MJ, et al. Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia. Nature.2000;405(6783):183-7.
[94]Caterina MJ, Leffler A, Malmberg AB, et al. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science. 2000;288(5464):306-13.
[95]Drossman DA,Camilleri M,Mayer EA,et al. AGA technical review on irritable bowel syndrome.Gastroenterology.2002,123:2108-2131.
[96]Chan CL, FacerP, Davis JB, etal.Sensory fibres expressing capsaicin receptor TRPV1 in patients with rectal hypersensitivity and faecal urgency.Lancet. 2003,361:385-91.
[97]Mizumura K, Koda H. Potentiation and suppression of the histamine response by raising and lowering the temperature in canine visceral polymodal receptors in vitro. Neurosci Lett.1999;266:9-12.
[98]Nicolson TA, Bevan S, Richards CD. Characterisation of the calcium responses to histamine in capsaicin-sensitive and capsaicin-insensitive sensory neurones. Neuroscience.2002;110:329-38.
[99]Kim BM, Lee SH, Shim WS, et al. Histamine-induced Ca2+influx via the PLA(2)/lipoxygenase/TRPV1 pathway in rat sensory neurons. Neurosci Lett 361:159-162,2004.
[100]Shim WS, Tak MH, Lee MH, et al. TRPV1 mediates histamine-induced itching via the activation of phospholipase A2 and 12-lipoxygenase.J Neurosci. 2007;27:2331-7.
[101]Premkumar LS, Ahern GP. Induction of vanilloid receptor channel activity by protein kinase C. Nature.2000;408:985-90.
[102]Planells-Cases R, Garcia-Sanz N, Morenilla-Palao C, et al. Functional aspects and mechanisms of TRPV1 involvement in neurogenic inflammation that leads to thermal hyperalgesia. Pflugers Arch.2005;451:151-9.
[103]Moriyama T, Higashi T, Togashi K, et al. Sensitization of TRPV1 by EP1 and IP reveals peripheral nociceptive mechanism of prostaglandins. Mol Pain. 2005;1:3.
[104]Tang HB, Inoue A, Oshita K, et al. Sensitization of vanilloid receptor 1 induced by bradykinin via the activation of second messenger signaling cascades in rat primary afferent neurons. Eur J Pharmacol.2004;498:37-43.
[105]Amadesi S, Nie J, Vergnolle N, et al. Protease-activated receptor 2 sensitizes the capsaicin receptor transient receptor potential vanilloid receptor 1 to induce hyperalgesia. J Neurosci.2004;24:4300-12.
[106]梁杰贤,季文进,洪迅,等.内皮素-1引起热痛觉过敏的研究.热带医学杂志2006,6(6)：708-709.
[107]Kawamata T, Ji W, Yamamoto J, et al. Contribution of transient receptor potential vanilloid subfamily 1 to endothelin-1-induced thermal hyperalgesia. Neuroscience 2008; 154:1067-1076.
[108]梁杰贤,郭中敏,季文进,等.蛋白激酶C在ET-1引起热痛觉过敏中的作用.热带医学杂志2006,6(12)：1293-1294.
[109]季文进,梁杰贤,赵国栋.辣椒素受体在内皮素-1引起热痛觉过敏中的作用.南方医科大学学报.2007,27(1)：101-103.
[110]Yamamoto H, Kawamata T, Ninomiya T, et al. Endothelin-1 enhances capsaicin-evoked intracellular Ca2+response via activation of endothelin a receptor in a protein kinase Cepsilon-dependent manner in dorsal root ganglion neurons. Neuroscience 2006;137:949-960.
[111]Cortright DN, Szallasi A. TRP channels and pain. Curr Pharm Des. 2009; 15(15):1736-49.
[112]Steinhoff M, Bienenstock J, Schmelz M, et al. Neurophysiological, neuroimmunological, and neuroendocrine basis of pruritus. J Invest Dermatol. 2006;126(8):1705-18.
[113]Kawamata T, Ji W, Yamamoto J, et al. Involvement of transient receptor potential vanilloid subfamily 1 in endothelin-1-induced pain-like behavior. Neuroreport 2009; 20:233-237.
[114]梁杰贤,季文进,刘培庆,等.内皮素-1引起的热痛觉过敏和自发痛与辣椒素受体的关系.中国疼痛医学杂志.2008,14(1)：21-23.
[115]Liang J, Bi H, Ji W. Involvement of TRPA1 in ET-1-induced pain-like behavior in mice. Neuroreport 2010; 21:201-205.
[116]Wacnik PW, Eikmeier LJ, Ruggles TR, et al. Functional interactions between tumor and peripheral nerve:morphology, algogen identification, and behavioral characterization of a new murine model of cancer pain. J Neurosci 2001; 21:9355-9366.
[117]Story GM, Peier AM, Reeve AJ, et al. ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 2003; 112:819-829.
[118]Bautista DM, Movahed P, Hinman A,et al. Pungent products from garlic activate the sensory ionchannel TRPA1. Proc Natl Acad Sci U S A 2005; 102:12248-12252.
[119]Karashima Y, Prenen J, Meseguer V, et al. Modulation of the transient receptor potential channel TRPA1 by phosphatidylinositol 4,5-biphosphate manipulators. Pflugers Arch.2008;457(1):77-89.
[120]Smith I, White PF, Nathanson M and Gouldson R. Propofol. An update on its clinical use. Anesthesiology 81:1005-1043,1994.
[121]Matta JA, Cornett PM, Miyares RL, Abe K, Sahibzada N, Ahern GP. General anesthetics activate a nociceptive ion channel to enhance pain and inflammation. Proc Natl Acad Sci U S A 105:8784-8789,2008.
[122]Obata K, Katsura H, Mizushima T, et al. TRPA1 induced in sensory neurons contributes to cold hyperalgesia after inflammation and nerve injury. J Clin Invest.2005;115(9):2393-401.
[123]Wilson SR, Gerhold KA, Bifolck-Fisher A, et al. TRPA1 is required for histamine-independent, Mas-related G protein-coupled receptor-mediated itch. Nat Neurosci.2011;14(5):595-602.
[124]Bickers DR, Athar M. Oxidative stress in the pathogenesis of skin disease. J Invest Dermatol.2006;126:2565-2575.
[125]Kitada T, Seki S, Iwai S, et al. In situ detection of oxidative DNA damage, 8-hydroxydeoxyguanosine, in chronic human liver disease. J Hepatol. 2001;35:613-618.
[126]Tarng DC, Huang TP, Wei YH, et al.8-hydroxy-2'-deoxyguanosine of leukocyte DNA as a marker of oxidative stress in chronic hemodialysis patients. Am J Kidney Dis.2000;36:934-944.
[127]Liu T, Ji RR. Oxidative stress induces itch via activation of transient receptor potential subtype ankyrin 1 in mice. Neurosci Bull.2012;28(2):145-54.
[128]Seals DR, Jablonski KL, Donato AJ. Aging and vascular endothelial function in humans. Clin Sci (Lond).2011;120(9):357-75.
[129]Duerrschmidt N, Wippich N, Goettsch W, et al. Endothelin-1 induces NAD(P)H oxidase in human endothelial cells. Biochem Biophys Res Commun. 2000;269(3):713-7.
[130]Hunter AR, Turner AJ. Expression and localization of endothelin converting enzyme-1 isoforms in human endothelial cells. Exp Biol Med (Maywood) 2006;231:718-722.
[131]Shimada SG, Shimada KA, Collins JG. Scratching behavior in mice induced by the proteinase-activated receptor-2 agonist, SLIGRL-NH2. Eur J Pharmacol 2006; 530(3):281-3.
[132]Shimada SG, LaMotte RH. Behavioral differentiation between itch and pain in mouse. Pain 2008; 139:681-687.
[133]Rashid MH, Inoue M, Kondo S, Kawashima T, Bakoshi S and Ueda H. Novel expression of vanilloid receptor 1 on capsaicin-insensitive fibers accounts for the analgesic effect of capsaicin cream in neuropathic pain. J Pharmacol Exp Ther 304:940-948,2003.
[134]Scholze T, Moskvina E, Mayer M, et al. Sympathoexcitation by bradykinin involves Ca2+-independent protein kinase C. J Neurosci.2002;22(14):5823-32.
[135]Ji RR, Samad TA, Jin SX, et al. p38 MAPK activation by NGF in primary sensory neurons after inflammation increases TRPV1 levels and maintains heat hyperalgesia. Neuron.2002;36(1):57-68.
[136]梁杰贤,洪迅,季文进,等.辣椒素受体在腹腔注射内皮素-1引起内脏痛中的作用.中国疼痛医学杂志.2006,12(3)：167-169.
[137]Khodorova A, Navarro B, Jouaville LS, et al. Endothelin-B receptor activation triggers an endogenous analgesic cascade at sites of peripheral injury. Nat Med 9:1055-1061,2003.
[138]Kuraishi Y, Nagasawa T, Hayashi K, et al. Scratching behavior induced by pruritogenic but not algesiogenic agents in mice. Eur J Pharmacol. 1995;275:229-33.
[139]Yeo JF, Ling SF, Tang N, et al. Antinociceptive effect of CNS peroxynitrite scavenger in a mouse model of orofacial pain. Exp Brain Res.2008; 184:435-8.
[140]Yeo JF, Ong WY, Ling SF, et al. Intracerebroventricular injection of phospholipases A2 inhibitors modulates allodynia after facial carrageenan injection in mice. Pain.2004;112:148-55.
[141]Gomes LO, Hara DB, Rae GA. Endothelin-1 induces itch and pain in the mouse cheek model. Life Sci.2012;91(13-14):628-33.
[142]Dekker LV, Parker PJ. Protein kinase C--a question of specificity. Trends Biochem Sci.1994;19(2):73-7.
[143]Khasar SG, Lin YH, Martin A, et al. A novel nociceptor signaling pathway revealed in protein kinase C epsilon mutant mice. Neuron.1999;24(1):253-60.
[144]Cesare P, Dekker LV, Sardini A, et al. Specific involvement of PKC-epsilon in sensitization of the neuronal response to painful heat. Neuron. 1999;23(3):617-24.
[145]Ferreira J, da Silva GL, Calixto JB. Contribution of vanilloid receptors to the overt nociception induced by B2 kinin receptor activation in mice. Br J Pharmacol.2004;141(5):787-94.
[146]Hagermark O. Peripheral and central mediators of itch. Skin Pharmacol. 1992;5:1-8.
[147]Bolcskei K, Helyes Z, Szabo A, et al. Investigation of the role of TRPV1 receptors in acute and chronic nociceptive processes using gene-deficient mice. Pain 2005,117:368-76.
[148]Ferreira J, Triches KM, Medeiros R, et al:The role of kinin B1 receptors in the nociception produced by peripheral protein kinase C activation in mice. Neuropharmacology 2008; 54:597-604
[149]Liang J, He Y, Ji W. Bradykinin-evoked scratching responses in complete Freund's adjuvant-inflamed skin through activation of B1 receptor. Exp Biol Med (Maywood).2012;237:318-326.
[150]Motta E M, Calixto J B, Rae G A. Mechanical hyperalgesia induced by endothelin-1 in rats is mediated via phospholipase C, protein kinase C, and MAP kinases[J]. Experimental Biology and Medicine,2006,231(6): 1141-1145.
[151]Hucho TB, Dina OA, Levine JD. Epac mediates a cAMP-to-PKC signaling in inflammatory pain:an isolectin B4(+) neuron-specific mechanism. J Neurosci 2005; 25(26):6119-26.
[152]Southall MD, Vasko MR. Prostaglandin receptor subtypes, EP3C and EP4, mediate the prostaglandin E2-induced cAMP production and sensitization of sensory neurons. J Biol Chem.2001;276(19):16083-91.
[153]Kawanabe Y, Okamoto Y, Miwa S, et al. Molecular mechanisms for the activation of voltage-independent Ca2+channels by endothelin-1 in Chinese hamster ovary cells stably expressing human endothelin(A) receptors. Mol Pharmacol.2002;62(1):75-80.
[154]Hu HJ, Bhave G, Gereau RW 4th. Prostaglandin and protein kinase A-dependent modulation of vanilloid receptor function by metabotropic glutamate receptor 5:potential mechanism for thermal hyperalgesia. J Neurosci. 2002;22(17):7444-52.
[155]Stempelj M, Ferjan I. Signaling pathway in nerve growth factor induced histamine release from rat mast cells. Inflamm Res.2005;54(8):344-9.
[156]Schutze S, Potthoff K, Machleidt T, et al. TNF activates NF-kappa B by phosphatidylcholine-specific phospholipase C-induced "acidic" sphingomyelin breakdown. Cell.1992;71(5):765-76.
[157]梁杰贤,季文进,洪迅,吉锦泉,梅莉.辣椒素受体在肠易激惹综合征内脏痛觉过敏中的作用.热带医学杂志2007,7(4)：342-344.
[158]Ikeda Y, Ueno A, Naraba H,et al. Involvement of vanilloid receptor VR1 and prostanoids in the acid-induced writhing responses of mice. Life Sci 69, 2911-2919,2001.
[159]Ji W, Cui C, Zhang Z, et al. Paradoxic effects of propofol on visceral pain induced by different TRPV1 agonists. Exp Ther Med 2013,5(4):1259-1263.
[160]Bergasa NV, Alling DW, Talbot TL, et al. Effects of naloxone infusions in patients with the pruritus of cholestasis. A double-blind, randomized, controlled trial. Ann Intern Med 1995; 123:161-167.
[161]Luger NM, Sabino MA, Schwei MJ, et al. Efficacy of systemic morphine suggests a fundamental difference in the mechanisms that generate bone cancer vs inflammatory pain. Pain.2002; 99:397-406.
[162]Reichert JA, Daughters RS, Rivard R, et al. Peripheral and preemptive opioid antinociception in a mouse visceral pain model. Pain 2001; 89:221-227.
[163]Laird JM, Martinez-Caro L, Garcia-Nicas E, et al. A new model of visceral pain and referred hyperalgesia in the mouse. Pain 2001; 92:335-342.
[164]Leung DY, Boguniewicz M, Howell MD, et al. New insights into atopic dermatitis. J Clin Invest.2004; 113(5):651-7.
[165]Hosogi M, Schmelz M, Miyachi Y, Ikoma A. Bradykinin is a potent pruritogen in atopic dermatitis:a switch from pain to itch. Pain 2006; 126:16-23.
[166]Liang J, Xiao G, Ji W. Capsaicin induces reflex scratching in inflamed skin. Pharmacology 2011;88:82-87.
[167]Khodorova A, Zou S, Ren K, et al. Dual Roles for Endothelin-B Receptors in Modulating Adjuvant-Induced Inflammatory Hyperalgesia in Rats. Open Pain J. 2009; 2:30-40.
[168]Bouzigon E, Forabosco P, Koppelman GH, et al. Meta-analysis of 20 genome-wide linkage studies evidenced new regions linked to asthma and atopy. Eur J Hum Genet 2010;18:700-706.
[169]Quang PN and Schmidt BL. Peripheral endothelin B receptor agonist-induced antinociception involves endogenous opioids in mice. Pain 149:254-262,2010.
[170]Rittner HL, Hackel D, Voigt P, et al. Mycobacteria attenuate nociceptive responses by formyl peptide receptor triggered opioid peptide release from neutrophils. PLoS Pathog 5:e1000362,2009.
[171]Taniuchi S, Kojima T, Hara M, et al. Increased serum nitrate levels in infants with atopic dermatitis. J Invest Dermatol.2001; 56:693-695.
[172]Morita H, Semma M., Hori M, et al. Clinical application of nitric oxide synthase inhibitor for atopic dermatitis. Int J Dermatol.1995; 34:294-295.
[173]Andoh T, Kuraishi Y. Nitric oxide enhances substance P-induced itch-associated responses in mice. Br J Pharmacol.2003;138(1):202-8.
[174]Amarante LH, Duarte ID. The kappa-opioid agonist (+/-)-bremazocine elicits peripheral antinociception by activation of the L-arginine/nitric oxide/cyclic GMP pathway. Eur J Pharmacol.2002;454(1):19-23.
[175]Cury Y, Picolo G, Gutierrez V P, et al. Pain and analgesia:The dual effect of nitric oxide in the nociceptive system[J]. Nitric Oxide,2011,25(3):243-254.
[176]Cury Y, Picolo G, Gutierrez V P, et al. Pain and analgesia:The dual effect of nitric oxide in the nociceptive system[J]. Nitric Oxide,2011,25(3):243-254.
[177]Shimizu Y, Sakai M, Umemura Y, et al. Immunohistochemical localization of nitric oxide synthase in normal human skin:expression of endothelial-type and inducible-type nitric oxide synthase in keratinocytes. J Dermatol. 1997;24(2):80-7.
[178]Deliconstantinos G, Villiotou V, Stavrides JC. Alterations of nitric oxide synthase and xanthine oxidase activities of human keratinocytes by ultraviolet B radiation. Potential role for peroxynitrite in skin inflammation. Biochem Pharmacol.1996;51(12):1727-38.
[179]Romero T R L, Resende L C, Duarte I D G. The neuronal NO synthase participation in the peripheral antinociception mechanism induced by several analgesic drugs[J]. Nitric Oxide,2011,25(4):431-435.
[180]Kalani M. The importance of endothelin-1 for microvascular dysfunction in diabetes. Vasc Health Risk Manag 2008; 4(5):1061-8.
[181]Schiffrin EL. Role of endothelin-1 in hypertension and vascular disease. Am J Hypertens 2001; 14(6 Pt 2):83S-9S.
[182]Davie NJ, Schermuly RT, Weissmann N, Grimminger F, Ghofrani HA. The science of endothelin-1 and endothelin receptor antagonists in the management of pulmonary arterial hypertension:current understanding and future studies. Eur J Clin Invest 2009; 39 S(2):38-49.
[183]Matsushima H, Yamada N, Matsue H, et al. The effects of endothelin-1 on degranulation, cytokine, and growth factor production by skin-derived mast cells[J]. European journal of immunology,2004,34(7):1910-1919.
[184]Khodorova A, Montmayeur J P, Strichartz G. Endothelin receptors and pain[J]. The Journal of Pain,2009,10(1):4-28.
[185]Mobarakeh J I, Sakurada S, Katsuyama S, et al. Role of histamine H1 receptor in pain perception:a study of the receptor gene knockout mice[J]. European journal of pharmacology,2000,391(1):81-89.
[186]Hill S J, Ganellin C R, Timmerman H, et al. International Union of Pharmacology. XIII. Classification of histamine receptors [J]. Pharmacological reviews,1997,49(3):253-278.
[187]Han S K, Mancino V, Simon M I. Phospholipase Cβ3 mediates the scratching response activated by the histamine H1 receptor on C-fiber nociceptive neurons [J]. Neuron,2006,52(4):691-703.
[188]Xie W, Samoriski G M, McLaughlin J P, et al. Genetic alteration of phospholipase Cβ3 expression modulates behavioral and cellular responses to μ opioids[J]. Proceedings of the National Academy of Sciences,1999,96(18): 10385-10390.
[189]Metz M, Lammel V, Gibbs B F, et al. Inflammatory murine skin responses to UV-B light are partially dependent on endothelin-1 and mast cells[J]. The American journal of pathology,2006,169(3):815-822.
[190]Matsushima H, Yamada N, Matsue H, et al. The effects of endothelin-1 on degranulation, cytokine, and growth factor production by skin-derived mast cells[J]. European journal of immunology,2004,34(7):1910-1919.
[191]Yamamura H, Nabe T, Kohno S, et al. Endothelin-1 induces release of histamine and leukotriene C< sub> 4 from mouse bone marrow-derived mast cells[J]. European journal of pharmacology,1994,257(3):235-242.
[192]Yamamura H, Nabe T, Kohno S, et al. Endothelin-1, one of the most potent histamine releasers in mouse peritoneal mast cells[J]. European journal of pharmacology,1994,265(1):9-15.
[193]Brain S D, Thomas G, Crossman D C, et al. Endothelin-1 induces a histamine-dependent flare in vivo, but does not activate human skin mast cells in vitro[J]. British Journal of Clinical Pharmacology,1992,33(1):117.
[194]Katugampola R, Church M K, Clough G F. The neurogenic vasodilator response to endothelin-1:a study in human skin in vivo[J]. Experimental physiology,2000,85(6):839-846.
[195]Steinhoff M, Neisius U, Ikoma A, et al. Proteinase-activated receptor-2 mediates itch:a novel pathway for pruritus in human skin[J]. The Journal of neuroscience,2003,23(15):6176-6180.
[196]Costa R, Manjavachi M N, Motta E M, et al. The role of kinin B1 and B2 receptors in the scratching behaviour induced by proteinase-activated receptor-2 agonists in mice[J]. British journal of pharmacology,2010,159(4): 888-897.
[197]Tsujii K, Andoh T, Lee J B, et al. Activation of proteinase-activated receptors induces itch-associated response through histamine-dependent and-independent pathways in mice[J]. Journal of pharmacological sciences,2008,108(3): 385-388.
[198]Rowley D A, Benditt E P.5-Hydroxytryptamine and histamine as mediators of the vascular injury produced by agents which damage mast cells in rats[J]. The Journal of experimental medicine,1956,103(4):399-412.
[199]Dymshitz J, Vasko M R. Endothelin-1 enhances capsaicin-induced peptide release and cGMP accumulation in cultures of rat sensory neurons [J]. Neuroscience letters,1994,167(1):128-132.
[200]Vaalasti A, Suomalainen H, Rechardt L. Calcitonin gene-related peptide immunoreactivity in prurigo nodularis:a comparative study with neurodermatitis circumscripta[J]. British Journal of Dermatology,2006,120(5): 619-623.
[201]Weidner C, Klede M, Rukwied R, et al. Acute effects of substance P and calcitonin gene-related peptide in human skin-a microdialysis study[J]. Journal of investigative dermatology,2000,115(6):1015-1020.
[202]Yamamoto A, Kuyama S, Kamei C, et al. Characterization of scratching behavior induced by intradermal administration of morphine and fentanyl in mice[J]. European journal of pharmacology,2010,627(1):162-166.
[203]Yamamoto A, Sugimoto Y. Involvement of peripheral mu opioid receptors in scratching behavior in mice[J]. European journal of pharmacology,2010, 649(1):336-341.
[204]朱江婷,程飚,刘宏伟,等.p-内啡肽在增生性瘢痕组织中的表达及其与瘢痕瘙痒关系的研究[J].中国修复重建外科杂志,2012,26(6)：732.
[205]Bigliardi-Qi M, Sumanovski L T, Buchner S, et al. Mu-opiate receptor and Beta-endorphin expression in nerve endings and keratinocytes in human skin[J]. Dermatology,2004,209(3):183-189.
[206]Tominaga M, Ogawa H, Takamori K. Histological characterization of cutaneous nerve fibers containing gastrin-releasing peptide in NC/Nga mice:an atopic dermatitis model [J]. Journal of Investigative Dermatology,2009, 129(12):2901-2905.
[207]朱江婷,程飚,刘宏伟.阿片肽系统在增生性瘢痕感觉异常中充当的角色[J].中国美容医学,2012,21(4)：676-679.
[208]Liu X Y, Liu Z C, Sun Y G, et al. Unidirectional cross-activation of GRPR by MOR1D uncouples itch and analgesia induced by opioids[J]. Cell,2011,147(2): 447-458.
[209]da Cunha J M, Rae G A, Ferreira S H, et al. Endothelins induce ET< sub> B receptor-mediated mechanical hypernociception in rat hindpaw:roles of cAMP and protein kinase C[J]. European journal of pharmacology,2004, 501(1):87-94.
[210]Wang S, Dai Y, Fukuoka T, et al. Phospholipase C and protein kinase A mediate bradykinin sensitization of TRPA1:a molecular mechanism of inflammatory pain[J]. Brain,2008,131(5):1241-1251.
[211]Sultana R, Newman S, Mohmmad-Abdul H, et al. Protective effect of the xanthate, D609, on Alzheimer's amyloid β-peptide (1-42)-induced oxidative stress in primary neuronal cells[J]. Free radical research,2004,38(5):449-458.
[212]Sultana R, Newman S F, Abdul H M, et al. Protective effect of D609 against amyloid-betal-42-induced oxidative modification of neuronal proteins: Redox proteomics study [J]. Journal of neuroscience research,2006,84(2): 409-417.
[213]Touyz R M, Yao G, Viel E, et al. Angiotensin Ⅱ and endothelin-1 regulate MAP kinases through different redox-dependent mechanisms in human vascular smooth muscle cells[J]. Journal of hypertension,2004,22(6): 1141-1149.
[214]Fei J, Viedt C, Soto U, et al. Endothelin-1 and smooth muscle cells induction of Jun amino-terminal kinase through an oxygen radical-sensitive mechanism [J]. Arteriosclerosis, thrombosis, and vascular biology,2000,20(5):1244-1249.
[215]Luberto C, Hannun Y A. Sphingomyelin synthase, a potential regulator of intracellular levels of ceramide and diacylglycerol during SV40 transformation[J]. Journal of Biological Chemistry,1998,273(23): 14550-14559.
[216]Sprague A H, Khalil R A. Inflammatory cytokines in vascular dysfunction and vascular disease[J]. Biochemical pharmacology,2009,78(6):539.
[217]Nguyen B T, Dessauer C W. Relaxin stimulates protein kinase C ζ translocation: requirement for cyclic adenosine 3',5'-monophosphate production[J]. Molecular Endocrinology,2005,19(4):1012-1023.
[218]Kircik L H, Del Rosso J Q, Aversa D. Evaluating clinical use of a ceramide-dominant, physiologic lipid-based topical emulsion for atopic dermatitis [J]. The Journal of clinical and aesthetic dermatology,2011,4(3):34.
[219]Tominaga M, Takamori K. Recent advances in pathophysiological mechanisms of itch[J]. Expert Review of Dermatology,2010,5(2):197-212.
[220]Kamei J, Nagase H. Norbinaltorphimine, a selective κ-opioid receptor antagonist, induces an itch-associated response in mice[J]. European journal of pharmacology,2001,418(1):141-145.
[221]Inan S, Cowan A. Kappa opioid agonists suppress chloroquine-induced scratching in mice[J]. European journal of pharmacology,2004,502(3): 233-237.
[222]Satoh M, Minami M. Molecular pharmacology of the opioid receptors[J]. Pharmacology & therapeutics,1995,68(3):343-364.
[223]Cheng B, Liu H W, Fu X B, et al. Coexistence and upregulation of three types of opioid receptors, mu, delta and kappa, in human hypertrophic scars[J]. British Journal of Dermatology,2008,158(4):713-720.
[224]Ward L, Wright E, McMahon S B. A comparison of the effects of noxious and innocuous counterstimuli on experimentally induced itch and pain[J]. Pain, 1996,64(1):129-138.
[225]Shim W S, Oh U. Histamine-induced itch and its relationship with pain[J]. Mol Pain,2008,4:29.
[226]季文进,魏涧琦,梁杰贤,等.外周内源性大麻受体激动剂及其受体2亚型参与搔痒调节的研究[J].热带医学杂志,2009,9(011)：1231-1232.
[227]Mishra S K, Tisel S M, Orestes P, et al. TRPV1-lineage neurons are required for thermal sensation[J]. The EMBO Journal,2010,30(3):582-593.
[228]Lagerstrom M C, Rogoz K, Abrahamsen B, et al. VGLUT2-dependent sensory neurons in the TRPV1 population regulate pain and itch[J]. Neuron,2010,68(3): 529-542.
[229]Liu Y, Abdel Samad O, Zhang L, et al. VGLUT2-dependent glutamate release from nociceptors is required to sense pain and suppress itch[J]. Neuron,2010, 68(3):543-556.