【作者】 季文进；

【导师】 张智伟；

【作者基本信息】 南方医科大学 ， 内科学（专业学位）， 2013， 博士

【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。更多还原

【Abstract】 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.MethodsAnimalsMale 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 injectionOne 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 modelOne 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 latencyMice 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 miceMale 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 ganglionAdult 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 PKCsCells 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 cAMPCells 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.Results1ET-1induces scratches through activation of ETA receptorIntradermal 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-1Co-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-1PKC 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-1Intraperitoneal 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 ETBPretreatment 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,Conclusions1ET-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.更多还原