神经元M通道、Na通道、TRPV1通道功能调节及神经元兴奋性调节的研究
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摘要
神经元兴奋性(neuroal excitability)是指神经元受到适宜刺激后爆发动作电位的能力,这是神经元的一个基本特性。若神经元受到阈上刺激时,神经元爆发动作电位(action potential),即神经元产生兴奋。简而言之,整个兴奋过程可以分为以下三个过程,即:除极化期(很短时间内细胞膜电位由负变正,形成动作电位的上升支)、复极化期(短时间内细胞膜电位由正变负,形成动作电位的下降支)和静息期(一定时间内细胞膜电位保持稳定,此时的膜电位称为静息膜电位)。
离子通道的活动是神经元产生兴奋的基础。一般来说,钠离子通道开放,钠离子内流导致神经元除极化,形成动作电位的上升支,爆发动作电位。随即,钠离子通道失活,钾离子通道激活而开放,钾离子外流导致神经元复极化,形成动作电位的下降支。此后,经过钠钾泵的作用使细胞膜两侧各离子恢复不均衡分布的状态,为下一次爆发动作电位做准备。除钠离子通道外,其他离子通道的开放也可导致细胞膜去极化进而影响神经元兴奋性。例如,在感觉神经元如DRG神经元,不同的刺激因素可诱发由不同离子通道承载的内向电流,膜电位的变化可以引起电压门控钠离子通道的开放,化学物质的刺激可以引起化学门控通道的开放(如,辣椒素Capsaicin可引起TRPV1通道的开放),温度的刺激或pH值的变化,均能引起相应的离子通道(这两种刺激也是TRPV1通道的适宜刺激)的开放,等等。这些内向离子通道的开放,均能引起神经元膜电位的除极化,达到阈电位即可爆发动作电位。
M通道是一种电压门控性,时间依赖性,慢激活、非失活的外向钾离子通道,因其能被毒蕈碱受体(muscranic receptor, M1)强烈抑制而得名。M通道介导的电流即M电流。M通道广泛分布于哺乳动物的神经系统中,包括中枢神经元和外周神经元。M通道的分子基础是KCNQ2/3通道构成的异四聚体,KCNQ2/3的突变可引起良性家族性新生儿惊厥症(benign familial neonatal convulsions, BFNC)。M通道一般在-60 mV左右激活,受多种因素的调节,在稳定细胞膜电位和调节细胞兴奋性等方面具有重要的生理意义。
电压门控钠离子通道是由α和β两个亚基构成的复合体,其中α亚基含有电压感受器和离子选择性孔区,β亚基为辅助亚基。迄今为止,已确定有9种α亚基,并广泛分布于哺乳动物的各种组织,它们分别是Nav1.1-Nav1.9。电压门控钠离子通道受很多因素的调节,其中丝/苏氨酸磷酸化是G蛋白偶联受体(G protein-coupled receptors, GPCRs)调节钠离子通道功能的主要机制。在颈上交感神经节神经元(SCG神经元),主要分布的是Nav1.7通道,其编码基因为SCN9A,后者的一个错位突变可导致原发性红斑性肢痛病(primary erythermalgia)。
TRPV1通道即香草酸1型瞬时电位感受器通道,为一种非选择性阳离子通道,可允许Ca2+、Mg2+和Na+等的内流。TRPV1通道主要分布于伤害性神经元如背根神经节(DRG神经元)的伤害性神经元,可感受伤害性刺激并引起痛觉。TRPV1通道本身也是辣椒素的受体,与辣椒素结合可立即激活该通道,介导阳离子内流,增强神经元兴奋性。TRPV1通道同时也是温热刺激(>43℃)、酸性刺激、机械压觉的受体,在致痛和镇痛方面占至关重要重要的地位。
本实验分别以原代培养的SD大鼠的SCG神经元和DRG神经元为研究平台,对神经元兴奋性及影响神经元兴奋性的M通道、Na通道(SCG神经元)和TRPV1通道(DRG神经元)的功能调节及相关机制进行了研究。
一、神经生长因子抑制M/KCNQ电流并增强SCG神经元的兴奋性
目的:在大鼠SCG神经元分别记录M/KCNQ电流和神经元动作电位,观察神经生长因子(nerve growth factor, NGF)对M/KCNQ电流和神经元动作电位的调节作用,并进行二者相关性研究及M/KCNQ电流调节机制的研究。
方法:(1)细胞培养。取3-5周龄的SD大鼠SCG神经节进行原代神经元培养。颈椎脱臼处死动物,快速打开手术野取出两侧SCG神经节,手术剪剪碎后用胶原酶(1 mg/ml)和胰蛋白酶(2.5 mg/ml)分别依次消化30分钟。终止消化后,将细胞团吹打成细胞悬液,然后种植在铺有多聚赖氨酸的圆玻片上。放入生化培养箱中,保持37°C并通5% CO2 + 95%空气的混合气。12小时后以Neurobasal A+B27培养基置换,继续培养。48小时内进行记录。(2)电生理实验。利用穿孔膜片钳技术和传统全细胞式膜片钳技术,电压钳制模式记录SCG神经元的M/KCNQ电流。硼硅玻璃电极拉制抛光后,充灌电极内液测定其电阻为3-5 M?可用。用Axon200B放大器结合pClamp9.0软件进行实验记录。采样率设定为5 KHz、信号过滤设定为2 KHz。将SCG神经元的膜电位钳制在-20 mV,每隔4秒给予超极化脉冲至-60 mV,维持0.8 s,以-60 mV时M/KCNQ尾电流的起始处10-20 ms与结束处10-20 ms电流平均值之差为统计对象,利用30μM Linopirdine (M/KCNQ电流的特异性抑制剂,此浓度能将M/KCNQ电流完全抑制)确定M/KCNQ电流的基线。利用穿孔膜片钳技术,电流钳钳制记录SCG神经元的动作电位。持续钳制电流为0,适时注入适宜电流2 s以引发神经元动作电位。记录M/KCNQ电流的细胞外液为(mM):NaCl 120,KCl 3,HEPES 5,NaHCO3 23,Glucose 11,MgCl2 1.2,CaCl2 2.5,TTX 0.00005,用NaOH调pH至7.4;穿孔膜片钳时,记录M/KCNQ电流的电极内液为(mM):KAc 90,KCl 40,HEPES 20,MgCl2 3,用KOH调pH至7.3-7.4。用传统全细胞膜片钳时,电极内液中加入3 mM Na2ATP和5 mM EGTA。记录神经元动作电位的的细胞外液与记录M/KCNQ电流的细胞外液相同,只是不含有TTX。记录神经元动作电位的电极内液与穿孔膜片钳记录M/KCNQ电流的电极内液相同。利用细胞贴附模式记录SCG神经元M/KCNQ单通道电流,此记录模式首先需要清楚跨膜片的电位,在使用高K+(63 mM)细胞外液的情况下,神经元静息电位约为-20 mV (根据Nerst公式),根据Vm=Vrest -Vpipette,钳制膜片的跨膜电位即为静息电位与通过电极给予的钳制电位之差,即当钳制电位为0 mV时,则钳制膜片跨膜电位为-20 mV,此时M/KCNQ通道应处于开放状态。单通道电流记录信号先经0.5-2 kHz虑过,而后的采样率设定为5-10 kHz。单通道记录所用电极拉制抛光后涂sylgard以降低电极介质噪声,电极电阻为8-10 MΩ。记录M/KCNQ单通道电流的细胞外液为(mM):NaCl 65,KCl 63,CaCl2 0.5,MgCl2 1.2,HEPES 10,Glucose 11,并加入CgTx-GVIA 250 nM,nifedipine 10μM,tetrodotoxin 250 nM,以KOH调pH至7.3;记录M/KCNQ单通道电流的电极内液为(mM):NaCl 125,KCl 3,MgCl2 1.2,HEPES 10,Glucose 11,并加入apamin 200 nM,charybdotoxin 100 nM,α和βdendrotoxins 300 nM以及TTX 250 nM,以NaOH调pH至7.3。(3)以Clamfit和Origin软件来分析、统计数据。
结果:(1)SD大鼠SCG神经元中存在三种不同电生理类型的神经元。根据神经元爆发动作电位的特征,可将SCG神经元分为三个类型,即Phasic-1、Phasic-2和Tonic神经元。Phasic-1神经元,只爆发一个动作电位(2 s内,下同),动作电位数目不随注入电流的增大而增加;此类神经元占SCG神经元总数的36%。Phasic-2神经元,爆发2~6个动作电位,动作电位数目随注入电流的增大而增加,甚至可转变成Tonic神经元;此类神经元占SCG神经元总数的54%。Tonic神经元,持续爆发动作电位,且动作电位数目随注入电流增大而增加;此类神经元占SCG神经元总数的10%。三类神经元中,Phasic-1及Phasic-2神经元的静息电位高于Tonic神经元,其统计学差异有显著性,而Phasic-1与Phasic-2神经元静息电位之间无统计学差异。Phasic-1及Phasic-2神经元的峰电位数目远低于Tonic神经元,其统计学差异有显著性,而Phasic-1与Phasic-2神经元峰电位数目之间无统计学差异。(2)SCG神经元的类型与神经元的M/KCNQ电流密切相关。先在电流钳钳制模式下确定SCG神经元的类型,然后改为电压钳钳制模式记录相应类型SCG神经元的M/KCNQ电流。Phasic-1与Phasic-2神经元的M/KCNQ电流形态、电流幅度相似。Phasic-1与Phasic-2神经元的M/KCNQ尾电流幅度较大,而Tonic神经元的M/KCNQ尾电流幅度较小,三者在-60 mV时的尾电流密度分别为2.8±0.2,2.3±0.2和0.9±0.1 pA/pF,Phasic-1与Phasic-2神经元之间没有统计学差异,而两种Phasic神经元与Tonic神经元之间有显著的统计学差异。三种神经元的电流-电压关系曲线(I-V曲线)的特征也有不同:三种神经元的半数激活电压(V1/2)分别是-30±1, -29±1和-15±3 mV,其中两种Phasic神经元之间的V1/2相似,没有统计学差异,而Tonic神经元的电流-电压关系曲线明显右移,其V1/2与两种Phasic神经元神经元相比明显降低,统计学差异有显著性。以-60 mV时的M/KCNQ尾电流密度为横坐标,以三种神经元爆发动作电位的次数为纵坐标做动作电位-M/KCNQ电流关系散点图,可以看出,三种神经元动作电位的分布呈明显的区域性,随着M/KCNQ电流的增大,动作电位爆发的次数明显减少,此为两种Phasic神经元的特征;相反随着M/KCNQ电流的减小,动作电位爆发的次数明显增多,此为Tonic神经元的特征;而且从散点图中可以看到一条明显的分界线位于约1pA/pF的M/KCNQ电流处,两种Phasic神经元大多位于分界线的右侧,而Tonic神经元大多位于分界线的左侧。三种神经元M/KCNQ电流的动力学表现也不同,表现在-20 mV时电流的激活速度和-60 mV时电流的去活速度均不同,电流的激活速度和去活速度可用相应的电流激活时间常数和去活时间常数代表。Phasic-1、Phasic-2和Tonic三种神经元-20 mV时的激活时间常数分别是60±5、64±4和99±12 ms,-60 mV时去活时间常数分别是51±2、60±6和96±5 ms。不论是激活时间常数还是去活时间常数,Phasic-1神经元与Phasic-2神经元之间无统计学差异,而两种Phasic神经元均与Tonic神经元有显著的统计学差异。(3)NGF抑制大鼠SCG神经元的M/KCNQ电流。穿孔膜片钳技术记录SCG神经元的M/KCNQ电流,然后给予生理浓度的NGF,观察NGF对M/KCNQ电流的作用。不管是Phasic神经元还是Tonic神经元,生理浓度的NGF(20 ng/ml)均能显著抑制其M/KCNQ电流,抑制率分别是25±2%和26±3%,两者之间没有显著性差异。为了证明被NGF抑制的电流就是M/KCNQ电流,我们在持续应用5μM的Linopirdine(M/KCNQ特异阻断剂)的情况下,再观察NGF的作用。这种情况下,NGF对M/KCNQ电流的抑制作用消失,说明NGF敏感电流也对Linopirdine敏感,NGF敏感电流应为M/KCNQ电流。在传统全细胞式膜片钳记录情况下,生理浓度NGF同样抑制M/KCNQ电流,抑制率达34±4%。做NGF抑制M/KCNQ电流的量-效关系曲线,利用Hill方程拟合曲线得出NGF抑制M/KCNQ电流的半数有效浓度(EC50)为0.7±0.1 ng/ml,Hill方程参数为0.9±0.1。电极内液中加入NGF受体Trk A的特异性阻断剂AG879,可明显减弱NGF的抑制作用,将其抑制率从34±4%降至17±3%。灌流给予酪氨酸蛋白酶抑制剂Genistein,也显著降低NGF对M/KCNQ电流的抑制作用,将其抑制率从34±4%降至7±4%。灌流给予磷脂酶C(PLC)抑制剂U73122也显著降低NGF对M/KCNQ电流的抑制作用,将其抑制率从34±4%降至12±5%。这些数据提示NGF通过激活Trk A受体及其下有信号通路而抑制M/KCNQ电流,可能通过酪氨酸磷酸化和膜磷脂PI(4,5)P2水解两条信号通路来抑制M/KCNQ电流。(4)NGF显著抑制M/KCNQ单通道电流。细胞贴附式记录SCG神经元的M/KCNQ单通道电流,灌流给予20 ng/ml的NGF可显著抑制M/KCNQ单通道电流的开放概率(Open properbility,Po),抑制率达29±3%,与全细胞模式下的NGF的抑制率一致。做为阳性对照,3μM M1受体激动剂Oxo-M强烈抑制M/KCNQ单通道电流的Po,抑制率达89±2%。NGF不改变M/KCNQ单通道的电导,给予NGF前后的电导分别是6.1±0.2 pS和6.5±0.2 pS,两者之间没有统计学差异。阳性对照药Oxo-M同样不改变M/KCNQ单通道的电导----在Oxo-M存在情况下,M/KCNQ单通道电导为6.4±0.3 pS,与给药前相比没有统计学差异。从-80 mV每隔20 mV增加至+40 mV,做M/KCNQ单通道的开放概率-电压关系曲线,并用Boltzmann方程拟合,发现NGF和Oxo-M均能在各个电压下降低M/KCNQ单通道的开放概率,但只有NGF将M/KCNQ单通道的半数激活电压(V1/2)从-32±3 mV降至-25±2 mV,两者之间有显著性差异。(5)NGF显著增强Tonic神经元的兴奋性,而对两种Phasic神经元兴奋性无影响。20 ng/ml NGF显著增强Tonic神经元的兴奋性,使其动作电位次数从12±2增至20±2,但NGF对该类型神经元的静息电位无显著性影响(给予NGF前后,该类型神经元的静息电位分别是-47±3和-47±4 mV,二者之间无显著性差异)。阳性对照药Oxo-M(10μM)和Linopirdine(30μM)显著增强Tonic神经元的兴奋性,分别使其动作电位次数从12±2和12±5增至28±4和30±4。给予Oxo-M和Linopirdine后,静息膜电位发生除极化,但给药前后相比并无显著性差异。对于Phasic-1和Phasic-2神经元,20 ng/ml NGF既没有不能改变他们的兴奋性,也不能明显改变他们的静息电位水平。阳性对照药Oxo-M(10μM)和Linopirdine(30μM)既显著增加了Phasic-1神经元的兴奋性(两者分别使该类型神经元的动作电位次数从1增至4.7±1.2和3.2±1,统计学差异均具有显著性)也显著增加了Phasic-2神经元的兴奋性(两者分别使该类型神经元的动作电位次数从3.2±0.7和3.1±0.5增至31±6和16±2,统计学差异均具有显著性)。Oxo-M(10μM)和Linopirdine(30μM)均能使Phasic-2神经元的静息电位发生明显的除极化(两者分别使该类型神经元的静息电位从-58±2 mV和-50±3 mV除极化至-49±2 mV和-43±2 mV,用药前后相比,统计学差异均有显著性)。Oxo-M(10μM)和Linopirdine(30μM)随能使Phasic-1神经元发生除极化,但统计学差异无显著性。(6)SCG神经元兴奋性与其M/KCNQ功能成负相关。这部分实验是在NGF(20 ng/ml)和Oxo-M(10μM)交替顺序共同应用的给药方式下,观察两者对SCG神经元的兴奋性的增强作用和M/KCNQ电流的抑制作用是否一致。首先灌流应用NGF,M/KCNQ电流被抑制约20±2%。在NGF存在的情况下,给予Oxo-M,M/KCNQ电流被抑制约62±7%。同样的给药方式,NGF没有改变Phasic神经元的兴奋性,而随后给予Oxo-M,却显著增强此神经元的兴奋性(动作电位次数由4.4±0.7增至22±5,用药前后相比,统计学差异有显著性)。交换顺序,首先灌流应用Oxo-M,神经元M/KCNQ电流被抑制约72±8%,在Oxo-M存在的情况下,随后给予NGF没有进一步抑制该神经元M/KCNQ电流(NGF给药前后相比,没有统计学差异)。同样的给药方式,Oxo-M显著增强神经元兴奋性(动作电位次数由3.4±1.2增至12±5,有显著的统计学差异),随后给予NGF并没有进一步影响神经元的兴奋性。(7)对M/KCNQ电流有相同抑制效应的低剂量Linopirdine能重现NGF分别影响SCG不同类型神经元兴奋性的效应。首先做Linopirdine抑制M/KCNQ电流的量-效关系曲线,以Hill方程拟合得出Linopirdine抑制M/KCNQ电流的半数有效剂量(EC50)为2.1±0.2μM,Hill方程参数为1.2±0.1。根据该量-效关系曲线可得出抑制M/KCNQ电流约25%时的Linopirdine的剂量应该是0.7μM,而Linopirdine的这一抑制效应与NGF(20 ng/ml)对M/KCNQ电流的抑制效应相类似。0.7μM Linopirdine能明显增强Tonic神经元的兴奋性,使其动作电位次数由12±1增至18±2,其统计学差异有显著性,而该浓度的Linopirdine不改变Phasic-1和Phasic-2两种神经元的兴奋性。
结论:(1)从电生理特性看,根据大鼠SCG神经元神经元所表达的M/KCNQ电流密度的大小可将神经元分为Phasic-1、Phasic-2和Tonic三种神经元。Phasic-1和Phasic-2神经元表达M/KCNQ电流密度大,稳定膜电位作用较强,故其兴奋性较低,不易爆发动作电位;Tonic神经元表达M/KCNQ电流密度小,稳定膜电位作用较弱,故其兴奋性较高,较易爆发动作电位。(2)生理浓度的NGF能显著抑制SCG神经元M/KCNQ电流,其抑制M/KCNQ电流的机制可能是通过激活Trk A受体及其下游酪氨酸蛋白激酶磷酸化和PLC水解PI(4,5)P2两条信号通路。在三种神经元上,NGF抑制M/KCNQ电流的效应相似,但NGF只增强Tonic神经元的兴奋性,而对两种Phasic神经元兴奋性无影响。这可能与Phasic神经元M/KCNQ电流被NGF抑制后仍余留较大的电流能稳定其膜电位有关。(3)与NGF抑制M/KCNQ电流相同效应的低剂量Linopirdine能完全重现NGF对三种神经元兴奋性的效应。进一步提示NGF抑制Phasic神经元M/KCNQ电流后余留较大电流能稳定其膜电位。(4)M/KCNQ通道在癫痫、惊厥以及老年性痴呆等疾病发病中起重要作用,本研究证明NGF是M/KCNQ通道的一种新的调节因素,丰富了NGF的生物学功能,同时也为M/KCNQ通道的功能调节提供了新的思路。
二、Genistein通过酪氨酸蛋白激酶依赖性和非依赖性两个信号通路抑制SCG神经元的电压门控性钠电流。
目的:在大鼠SCG神经元记录电压门控性Na通道电流(VGSC电流)和神经元动作电位,观察Genistein对VGSC电流的调节作用,研究其调节机制。Genistein是广泛应用的非特异性酪氨酸蛋白激酶抑制剂,可广泛抑制细胞内酪氨酸蛋白激酶(或带有酪氨酸蛋白激酶活性的蛋白质)的活性。
方法:(1)细胞培养。大鼠SCG神经元的的培养同上部分。(2)电生理实验。利用穿孔膜片钳技术,电压钳钳制模式记录SCG神经元的VGSC电流,电流钳钳制模式记录SCG神经元的动作电位。硼硅玻璃电极拉制抛光后,充灌电极内液测定其电阻为3-5 M?可用。串联电阻补偿达80-90%后,电极最大接入电阻为2 M?左右。信号采样率分别设定为10 KHz(VGSC电流)和2.5 KHz(动作电位)。神经元钳制在-70 mV每隔3秒给予除极化脉冲至0 mV,持续20毫秒。电流钳钳制神经元在0 pA,然后注入约2倍于阈值的除极化电流0.1 pA以引发神经元动作电位。记录VGSC电流的神经元细胞外液为(mM):NaCl 120,KCl 3,HEPES 5,NaHCO3 23,glucose 11,MgCl2 1.2,CaCl2 2.5,BaCl2 0.2,CdCl2 0.2,用NaOH调pH至7.4;记录VGSC电流的电极内液为(mM):CsCl 90,KCl 40,HEPES 20,MgCl2 3,用CsOH调pH至7.3-7.4。记录神经元动作电位的细胞外液为(mM):NaCl 120,KCl 3,HEPES 5,NaHCO3 23,glucose 11,MgCl2 1.2,CaCl2 2.5,用NaOH调pH至7.4;记录神经元动作电位的电极内液为(mM):KAC 90,KCl 40,HEPES 20,MgCl2 3用KOH调pH至7.3-7.4。(3)细胞免疫荧光化学实验。细胞用4%多聚甲醛固定、0.1%的Triton X-100打孔、3%的BSA封闭30分钟,孵育家兔来源的一抗anti-Nav1.7抗体或anti-Nav1.1抗体,4°C,过夜。用PBS洗涤后孵育二抗羊抗兔IgG-TRITC抗体30分钟。共聚焦显微镜观察,TRITC激发光波长为564 nm,吸收光波长为570 nm。(4)用Clamfit和Origin软件来分析、统计数据。
结果:(1)大鼠SCG神经元表达的电压门控性Na通道(VGSC)主要是Nav1.7通道。细胞免疫荧光化学实验表明,大鼠SCG神经元主要表达TTX-敏感的,电压门控性Nav1.7通道;微弱表达TTX-敏感的,电压门控性Nav1.1通道。(2)Genistein显著抑制SCG神经元的TTX-敏感的电压门控性Na通道电流。100μM Genistein抑制VGSC电流达73.3±5.4%,而且Genistein对VGSC电流的抑制呈明显的双相型----快相紧跟一个慢相。双指数方程拟合此抑制过程得出快相和慢相的时间常数分别是10.6±1.2 s和55.9±2.4 s。做Genistein抑制VGSC电流的量-效关系曲线,并以Hill方程拟合得到Genistein抑制VGSC电流的半数有效量(EC50)为9.1±0.9μM,其方程系数为1.1±0.2。SCG神经元的VGSC电流为TTX-敏感的Na电流,0.05μM TTX能完全抑制之。做VGSC的电流-电压关系曲线(I-V曲线),50μM Genistein明显抑制各个电压下的VGSC的电流,且最大激活电压范围内(-20 mV~ 0 mV)的抑制最明显。50μM Genistein使VGSC的电压依赖性激活曲线明显右移,使其半数激活电压(V1/2)由-32.4±0.2 mV降至-20.6±0.1 mV,二者统计学差异有显著性。50μM Genistein对VGSC的电压依赖性失活曲线没有影响,给药前后的半数失活电压分别为-41.8±0.3 mV和-41.3±0.4 mV,两者之间无统计学差异。(3)酪氨酸蛋白激酶依赖性和酪氨酸蛋白激酶非依赖性两种机制参与了Genistein对VGSC电流的抑制。灌流给予Genistein的无活性结构类似物Daidzein(10μM~ 300μM),做Daidzein抑制VGSC电流的量-效关系曲线。以Hill方程拟合此曲线,可以看出100μM Daidzein即可达到最大抑制(抑制率为28.5±3.1%),其抑制VGSC电流的半数有效量(EC50)为20.7±0.1μM,方程系数为1.2±0.2。单独给予100μM Daidzein可抑制VGSC电流达27.8±3.6%,在100μM Daidzein存在的情况下,紧接着给予100μM Genistein,可进一步抑制VGSC电流,二者合用对VGSC电流的抑制率达57.3±10.1%。这提示酪氨酸蛋白激酶非依赖性机制参与了Genistein对VGSC电流的抑制,可能是一种直接作用。100μM Daidzein虽抑制VGSC电流(此剂量已达最大抑制),但没有完全取代100μM Genistein的抑制作用,这提示可能还有其他机制参与Genistein对VGSC电流的调节作用。Vanadate是一种广泛应用的酪氨酸蛋白磷酸酶的抑制剂,它能间接加强底物发生的磷酸化,而Genistein则抑制底物产生磷酸化,所以二者的细胞内作用恰恰相反。单独给予100μM Genistein可抑制VGSC电流达57±5.4%,在Genistein存在的情况下紧接着给予Vanadate(1 mM),Vanadate可部分反转Genistein的抑制作用,使其抑制率降至39.5±4.7%。预先应用Vanadate(1 mM),然后在Vanadate存在的情况下紧接着给予100μM Genistein,1 mM Vanadate可降低Genistein对VGSC电流的抑制作用达18.7±3.3%。这两情况下,Vanadate干预前后,Genistein对VGSC电流的抑制率均有显著性差异。这部分实验提示,酪氨酸蛋白激酶依赖性机制也参与了Genistein对VGSC电流的调节作用。以饱和剂量分别给予Genistein的三种功能类似物(均是常用的酪氨酸蛋白激酶抑制剂)Tyrphostin 23(100μM)、Erbstatin analog(100μM)和PP2(1μM),发现三者均能模拟Genistein对VGSC电流的抑制作用,其抑制率分别是14.6±0.5%、19.3±1.0%和17.8±1.9%。虽然三者对VGSC电流的抑制率较低,但均具有显著性统计学差异。这部分实验仍提示酪氨酸蛋白激酶依赖性机制参与了Genistein对VGSC电流的调节作用。(4)Genistein降低SCG神经元的兴奋性,降低其除极速率,但不改变其静息电位水平。灌流给予100μM Genistein可抑制SCG神经元的兴奋性,使其动作电位次数由8.7±2.1降至1.3±0.3;然而,给药前后神经元的静息电位水平分别是56.1±3.1 mV和54.7±4.1 mV,没有显著性统计学差异。100μM Genistein还显著减慢SCG神经元动作电位的除极速率,使其由7.0±1.3 V/s降至4.4±0.9 V/s,给药前后除极速率的变化有显著性统计学差异。
结论:(1)Genistein通过酪氨酸蛋白激酶依赖性和酪氨酸蛋白激酶非依赖性两种机制抑制SCG神经元的VGSC电流。(2)Genistein抑制SCG神经元的VGSC电流导致其降低神经元兴奋性和动作电位除极速率。
三、Gq蛋白偶联受体激活诱导的DRG神经元细胞内钙变化
目的:研究同为G(q)蛋白偶联受体(G(q)PCRs)激活后对DRG神经元细胞内钙信号影响的异同,建立进一步研究细胞内钙信号在膜受体调节DRG神经元离子通道功能中的作用的基础。
方法:(1)细胞培养。DRG神经节取自14天鼠龄的SD大鼠。原代培养大鼠DRG神经元的方法与上述培养大鼠SCG的方法相似。(2)钙信号测定。用Fura-2 AM(5μM,辅以20%的pluronic F127)标记细胞内游离的Ca2+。利用Hamamatsu光电子Ca2+显像系统,使用F340/F380双波长比例测钙法测定DRG神经元细胞内钙信号的变化。用simplePCT 6.0软件进行数据分析。(3)DRG细胞外液。正常DRG细胞外液为(mM):NaCl 145,KCl 5,MgCl2 2,CaCl2 2,HEPES 10,Glucose 10,用NaOH调pH至7.4。无钙细胞外液中,去除Ca2+并加入3 mM的EGTA。(4)药物。以缓激肽(Bradykinin,BK)为阳性对照药物,以辣椒素(Capsaicin,CAP)和丙烯醛(Acrolein,ACR)确定DRG神经元的类型,以高K+液(50 mM KCl)确定细胞内钙升高。其他几种GPCRs分别以各自相应的激动剂来激活。(5)实验数据用Origin软件做统计处理。
结果:(1)DRG神经元鉴定。有钙外液情况下,共测定DRG神经元349例,按对受体激动有细胞内钙升高(阳性)的比例计算,其中BK阳性神经元为109例,占总数的31.2%;CAP阳性神经元为74例,占总数的21.2%;ACR阳性神经元为62例,占总数的17.8%。在109例BK阳性神经元中,CAP阳性神经元为31例,占28.4%;ACR阳性神经元为29例,占26.6%。无钙外液情况下,共测定DRG神经元57例,其中BK阳性神经元为4例,占总数的7%;CAP阳性神经元为1例,占总数的1.8%;ACR阳性神经元为1例,占总数的1.8%。在4例BK阳性神经元中,CAP阳性神经元为1例,占25%;ACR阳性神经元为1例,占25%。(2)缩胆囊素受体(Cholecystokinin receptors,CCK)。缩胆囊素受体用CCK-8(1μM)来激活。有钙外液情况下,CCK能使131例DRG神经元中的46例神经元内钙升高,占35.1%。无钙外液情况下,共测定DRG神经元57例,但没有神经元的细胞内钙能被CCK升高。(3)内皮素受体(Endothelin receptors,ET receptors)。内皮素受体用ET-1(100 nM)来激活。有钙外液情况下,内皮素能使101例DRG神经元中的49例神经元内钙升高,占总数的48.5%。无钙外液情况下,共测定DRG神经元50例,其中有5例神经元能被内皮素升高细胞内钙。(4)Mrg D受体。Mrg D受体是一种孤儿蛋白,在DRG神经元丰富表达。Mrg D受体可被丙氨酸(β-Alanine,ALA,500μM)激活。有钙外液情况下,ALA能使134例DRG神经元中的12例神经元内钙升高。无钙外液情况下没有神经元的细胞内钙能被ALA升高。(5)组胺受体(Histamine receptors)。组胺受体可用组胺(100μM)来激活。有钙外液情况下,组胺仅能使95例DRG神经元中的10例神经元内钙升高。(6)P物质受体(Substance P receptors,SP receptors)。P物质受体也称为NK1受体,可用P物质(SP, 1μM)来激活。有钙外液情况下,共测定DRG神经元55例,只有1例神经元能被SP升高内钙,绝大部分不能被SP升高细胞内钙。(7)5羟色胺受体(5-HT receptors)。5羟色胺受体用10μM的5-HT来激活。有钙外液情况下,共测定DRG神经元55例,5-HT也不能升高细胞内钙。(8)血管紧张素II受体(Angiotensin II receptors,AT receptors)。血管紧张素II受体可用100 nM的血管紧张素II来激活。有钙外液情况下,共测定44例DRG神经元,仅有2例神经元能被血管紧张素II升高细胞内钙。(9)嘌呤能受体(purinergic Y receptors,P2Y receptors)。P2Y受体可用100 nM的ATP来激活。有钙外液情况下,共测定43例DRG神经元,没有神经元能被ATP升高细胞内钙。
结论:几种G蛋白偶联受体中,CCK受体、ET-1受体、Μrg D受体和组胺受体激活后能升高DRG神经元细胞内钙。而SP受体、AT II受体、5-HT受体和P2Y受体不能升高DRG神经元细胞内钙。
四、辣椒素和CCK对DRG神经元TRPV1电流的调节。
目的:文献报道和本实验室前期工作表明BK可引发TRPV1电流和Ca2+激活的氯电流(Ca2+-activated Cloride currents , CaCC ,TMEM16A-dependent)。二者均为内向电流,可使膜电位除极化而爆发动作电位。本部分实验拟观察研究内容三中所涉及GPCRs激活可否引发DRG神经元内向电流以及电流成分和激活机制,同时研究GPCRs激活对TRPV1通道的功能调节。
方法:(1)细胞培养。按上述方法培养大鼠DRG神经元。(2)电生理实验。利用穿孔膜片钳技术,将DRG神经元钳制在-60 mV,分别给予BK和其他几种G蛋白偶联受体激动剂(能升高细胞内钙者),观察内向电流有否被激活,然后分别给予TRPV1或CaCC特异性阻断剂确定其通道性质。硼硅玻璃电极拉制抛光后,充灌电极内液测定其电阻为3-5 M?可用。Clampex采样率设定为2.5 KHz,信号过滤设定为0.5 KHz。记录内向电流的细胞外液为(mM):NaCl 145,KCl 5,MgCl2 2,CaCl2 2,HEPES 10,Glucose 10,用NaOH调pH至7.4;电极内液为(mM):KCl 145,MgCl22,HEPES 10,用KOH调pH至7.4。无钙外液时,细胞外液中的Ca2+被去除,并加入0.1 mM EGTA。
结果:(1)辣椒素(capsaicin,CAP,1μM)激活CAP阳性神经元TRPV1电流并很快脱敏。CAP激活的内向电流可被TRPV1通道特异性阻断剂capsazepine(CZP,100μM)迅速完全抑制,证明CAP引发的内向电流就是TRPV1电流。给予CAP 20-30秒后,TRPV1电流激活达最大值,持续时间短,约10秒钟,然后很快发生脱敏现象。给予CAP 5分钟末的TRPV1电流远小于给药20-30秒时的TRPV1最大电流,二者的比例为30.5%±3.6%。第一次给予1μM CAP 30秒,此时TRPV1电流达最大值,冲洗5分钟,然后第二次给与1μM CAP 30秒,第二次给药引发的TRPV1电流显著小于第一次给药引发的TRPV1电流,二者比例为50.7%±3.8%。(2)CCK-8在DRG神经元能引发微小的内向电流并能阻断CAP引起的TRPV1电流的脱敏现象。灌流给予1μM的CCK-8,在CAP阳性神经元或者CAP阴性神经元均能激活微弱的内向电流。用CCK预处理DRG神经元1 min,然后在CCK存在情况下给予1μM CAP 5分钟,CCK可明显减弱TRPV1电流的快速衰减,只发生微小衰减,5分钟末的TRPV1电流(在CCK-8存在的情况下)占起始处TRPV1最大电流的68.4%±3.4%,与对照情况下相比,有显著的统计学差异。在第二次给予CAP之前,预先给予1μM的CCK-8处理细胞1分钟,第二次给予CAP所引发的TRPV1电流幅度与第一次CAP所引发的TRPV1电流幅度相似,而二者比例为91.2%±7.4%,与对照情况下相比,有显著的统计学差异。(3)其他受体激活后能否引发内向电流,还在实验中。
总结
1.从电生理角度看,大鼠颈上交感神经节(SCG)神经元根据神经元所表达的M/KCNQ电流的大小可分为Phasic-1、Phasic-2和Tonic三种神经元。Phasic-1和Phasic-2神经元表达M/KCNQ电流密度大,稳定膜电位作用较强,故其兴奋性较低,不易爆发动作电位;Tonic神经元表达M/KCNQ电流密度小,稳定膜电位作用较弱,故其兴奋性较高,较易爆发动作电位。
2.生理浓度NGF显著抑制SCG神经元M/KCNQ电流,可能是通过激活TrkA受体及其下游酪氨酸蛋白激酶磷酸化和PLC-PI(4,5)P2水解两条信号通路。在三种神经元上,NGF抑制M/KCNQ电流的效应相似,但NGF只增强Tonic神经元的兴奋性,而对两种Phasic神经元兴奋性无影响。这可能与Phasic神经元M/KCNQ电流被NGF抑制后仍余留较大的电流能稳定其膜电位有关。与NGF抑制M/KCNQ电流相同效应的低剂量Linopirdine能完全重现NGF对三种神经元兴奋性的效应。进一步提示NGF抑制Phasic神经元M/KCNQ电流后余留较大电流能稳定其膜电位。
3. Genistein通过酪氨酸蛋白激酶依赖性和酪氨酸蛋白激酶非依赖性两种机制抑制SCG神经元的电压依赖性钠通道(VGSC)电流。Genistein抑制SCG神经元的VGSC电流导致神经元兴奋性降低,动作电位除极速率减慢。
4.几种G蛋白偶联受体中,BK受体、缩胆囊素受体(CCK)、内皮素受体、Μrg D受体和组胺受体能升高背根神经节(DRG)神经元细胞内钙,而SP受体、血管紧张素II受体、5羟色胺受体和ATP受体不能升高DRG神经元细胞内钙。
5.辣椒素(CAP)激活TRPV1电流,并很快发生脱敏现象。CCK能在DRG神经元引发轻微的内向电流,同时能抑制CAP引起的TRPV1通道的脱敏现象。
Neuronal excitability is one of the basic characteristics of neurons. Neurons will fire action potentials when they receive adequate stimulus. Generally, the whole period of neuronal excitation can be divided into three processes, depolarization period (membrane potential was reduced in very short time, a process forming the upslope of the action potential), repolarization period (membrane potential was increased negatively in short time, a process forming the downslope of the action potential) and recovery period or resting period (membrane potential maintains relatively stable, normally called resting potential).
Neuronal excitation is founded on the activity of ion channels. In general, sodium ions flux into the cells when sodium channels are open, which leads to depolarization of neurons and forms the upslope of action potential. Soon after, sodium channels become inactivated and closed, and at the same time potassium channels are activated and open, which allow potassium ions flux out of the cell, which results in repolarization of neurons and forms the downslope of action potential. The upslope and downslope of action potential make a spike potential. After each action potential, displaced ions are recovered to their original disproportional distribution across the membrane by the action of sodium-potassium pump. This disproportional distribution of sodium and potassim across the mebrane prepares the cell to fire next action potential when the cell receives again a suprathreshold stimulus. Apart from sodium currents, other inward currents can also induce membrane depolarization action potential. For example, in sensory neurons like dorsal root ganglion (DRG) neurons, capsaicin can lead to the activation and opening of TRPV1, which are also sensitive to thermal stimulus and pH.
M-type potassium channel or M/KCNQ channel is a voltage-gated channel with characteristics of slow-activation and deactivation and non-inactivation. Activation of muscranic receptor strongly inhibitits this potassium channel, hence, it is called M-type potassium channel. The molecular basis of M/KCNQ channel is the tetraheterologus of KCNQ2 and KCNQ3 channels. Mutations of KCNQ2/3 lead to benign familial neonatal convulsions. M/KCNQ channel distributes widely in the nerve system in mammalian, including the central nerve system and peripheral nerve system. Generally, M/KCNQ channel is activated around– 60 mV, a membrane potential near the resting membrane potential. Thus M/KCNQ channels are believed to play a key role in stabilizing membrane potential and regulating neuronal excitability. M/KCNQ channel can be regulated by many factors such as G protein-coupled receptors, receptor tyrosine kinase, and other factors.
Voltage-gated sodium channels (VGSC) are composed of a complex of aαsubunit that forms the voltage-sensitive and ion-selective pore, in association with one or more auxiliaryβsubunits. To date, nineαsubunits of the VGSC superfamily, Nav1.1-Nav1.9, have been identified, which are widely distributed in mammalian tissue. Among these VGSC, Nav1.7 channel is dominantly expressed in superior cervical ganglion (SCG) neurons. Nav1.7 channel is coded by gene, SCN9A, a missense mutation in which underlies the primary erythermalgia. Many factors modulate VGSC, including G-protein coupled receptors and tyrosine kinases.
Transient receptor potential vanillicacid 1 channel, simply called TRPV1 channel, is a kind of non-selective cation channels, which allows calcium, magnesium and sodium ions flux into cells. TRPV1 channel mainly distributes in nociceptive neurons such as the nociceptive neurons in DRG. TRPV1 channel can be activated responding to nociceptive stimulus. TRPV1 channel also is the receptor of capsaicin, a kind of extractive agent from capsicum chilli. Capsaicin binds to TRPV1 channel and then activates this channel and induces influx of cations, which enhances neuronal excitability. TRPV1 channel is also sensitive to thermal stimulus, pH and machinery pressure. Many factors can modulate the activatiy of TRPV1 channel, such as membrane PI(4,5)P2, Ca2+ and Calmodulin, etc.
The present study will investigate the functional regulation of M/KCNQ channel, voltage-gated sodium channel and TRPV1 channel, and related neuronal excitability using primary cultured neurons from rat SCG and DRG. We also study the intrcellular calcium signals induced by activation several G protein-coupled receptors expressed in DRG neurons.
1. The regulation of M/KCNQ currents and neuronal excitability by NGF. Aim: (1) To record M/KCNQ currents and neuronal action potential in rat SCG neurons, and to study the mechanism of regulation of M/KCNQ currents and neuronal exctibality produced by nerve growth factor (NGF).
Methods: (1) Cell culture. Primary cultures of neurons were prepared from SCG from 3- to 5-week-old Sprague-Dawley rats. Briefly, ganglia were digested with collagenase (1 mg/ml) and next trypsin (2.5 mg/ml), and then ganglia were dissociated into a suspension of individual cells and planted on poly-lysine coated glass coverslips. Cellas were incubated at 37°C with a 5% CO2 + 95% air. The DMEM medium plus 10% serum were changed to neurobasal A medium plus 2% B27 supplement after 12 hr and cells were used within 48 hr. (2) Electrophysiology. Perforated patch and conventional whole-cell patch were used to record the neuronal M/KCNQ currents under voltage-clamping mode. Pipettes were pulled from borosilicate glass capillaries and had resistances of 3-5 M? when filled with pipette solution. Currents and action potentials were recorded using an Axon 200B amplifier and pClamp 9.0 software, and were filtered at 2 KHz. The protocol for recording of M/KCNQ currents is as follows: SCG neurons were held at -20 mV followed a 0.8 s hyperpolarization step to -60 mV every 4 s. The amplitude of M/KCNQ currents was defined as the outward currents sensitive to 30μM linopirdine, a specific M/KCNQ channel blocker, and was measured from deactivation currents records at -60 mV as the difference between the average of an initial 10-ms segment, taken 10-20 ms into the hyperpolarizing step, and the average during the last 10 ms of that step. Perforated patch was used to record neuronal action potentials under current-clamping mode. The protocol to record action potential is as follows: SCG neurons were held at 0 current level and the action potentials were elicited by injection of a depolarizing current for 2 s. The external solution used to record M/KCNQ currents contained (in mM): NaCl 120, KCl 3, HEPES 5, NaHCO3 23, Glucose 11, MgCl2 1.2, CaCl2 2.5 and TTX 0.00005, (adjusted to pH 7.4 with NaOH). The pipette solution for perforated patch recording consisted of (in mM): KAc 90, KCl 40, HEPES 20 and MgCl2 3 (adjusted to pH 7.3-7.4 with KOH). Na2ATP (3 mM) and EGTA (5 mM) were added to the above internal solution for conventional whole-cell recording. The external solution used to record neuronal action potentials was the same as that used for M/KCNQ currents recording, but did not contain TTX. The internal solution for action potential recording was also the same solution as that used for M/KCNQ currents using perforated patch recording. Single M/KCNQ current was recorded under cell-attached patch. The protocol for recording single M/KCNQ current is as follows: Cells bath perfused with high K+ external solution (63 mM) had a resting membrane potential near -20 mV. Membrane potentials (Vm) were therefore calculated as Vm=Vrest -Vpipette, where Vrest was taken to be -20 mV and Vpipette was the voltage applied. When applied 0 mV, the patch membrane will be clamped at -20 mV and at this level, single M/KCNQ channel will be open. The data were sampled at 5-10 kHz after filtered at 0.5-2 kHz. Transitions between open and closed states were detected by setting the threshold to 50% of the open channel level. Sylgard-coated pipettes had resistances of 8-10 MΩwhen filled with pipette solution consisting of (mM): NaCl 125, KCl 3, MgCl2 1.2, HEPES 10, Glucose 11 and apamin 200 nM, charybdotoxin 100 nM,αandβdendrotoxins 300 nM, tetrodotoxin 250 nM (pH 7.3). The extracellular solution consisted of (mM): NaCl 65, KCl 63, CaCl2 0.5, MgCl2 1.2, HEPES 10, Glucose 11 and CgTx-GVIA 250 nM, nifedipine 10μM, tetrodotoxin 250 nM (pH 7.3 with KOH). (3) Data were analyzed using clamfit and origin software.
Results: (1) Rat SCG neurons can be divided into three electrophysiological phenotypes based on their action potential firing patterns: Phasic-1, Phasic-2 and Tonic neurons. Phasic-1 neurons, making up 36% of total SCG neurons studied, fired only one spike during the period of stimulation even with increased current injection. Phasic-2 neurons, seen in 54% of neurons, fired two to six spikes, but fired more frequently in response to the increased current injection, and the phasic firing pattern could be converted to a tonic firing pattern. Tonic neurons were seen in 10% of SCG neurons. Tonic neurons fired action potentials in a sustained manner even with a minimal stimulus, and the number of spikes increased with increased current injection. The resting potential between phasic-1 and phasic-2 neurons had no significant difference, but they were higher than that of tonic neurons and there had a significant difference between phasic and tonic neurons. The spike number of tonic neurons was much more than that of phasic-1 and phasic-2 neurons, and there had significant difference between tonic and phasic neurons, whereas, there was no significant diferrence between the two phasic neurons. (2) Three kinds of neuronal excitatory pattern in SCG neurons were tightly related to their M/KCNQ currents. We first identified the excitatory pattern of SCG neurons under current-clamping mode, and then use voltage-clamping mode to record the neuronal M/KCNQ currents in the same cell. The shape and amplitude of M/KCNQ currents were similar in phasic-1 and phasic-2 neurons, and the amplitude of tail M/KCNQ current at -60 mV was relatively big. On other hand, the amplitude of M/KCNQ current from tonic neurons was relatively small. The density of tail M/KCNQ currents at -60 mV from phasic-1, phasic-2 and tonic neurons were 2.8±0.2,2.3±0.2 and 0.9±0.1 pA/pF, respectively. The two phasic neurons were significantly different from tonic neurons but no significant difference existed between two phasic neurons. The current-voltage (I-V) relationship curves of the three type neurons were also different. The half-activation voltages for phasic-1, phasic-2 and tonic neurons were -30±1, -29±1 and -15±3 mV, respectively. Compared with phasic neurons, the I-V curve of tonic neurons was positively shifted. A detailed analysis of the relationship between spike number and the density of M/KCNQ tail currents was made. It appeared that the density of M/KCNQ tail currents was diagnostic in separating SCG neurons into either phasic or tonic neurons. Specifically, a line of demarcation was located at an M/KCNQ current density level of -1 pA/pF. We used single exponential equation to fit the kenitics of M/KCNQ currents activation (at -20 mV) and deactivation (at -60 mV) and obtained the the corresponding time constants, respectively. The time constants of activation for phasic-1, phasic-2 and tonic neurons were 60±5, 64±4 and 99±12 ms, respectively. The time constants of deactivation for phasic-1, phasic-2 and tonic neurons were 51±2, 60±6 and 96±5 ms, respenctively. Both activation and deactivation of tonic neurons were significantly slower than phasic neurons, but no significant differences were found between phasic-1 and phasix-2 neurons. (3) NGF inhibited M/KCNQ currents in rat SCG neurons. NGF at concentration of 20 ng/ml (within the range of reported physiological concentration of NGF in mammalians) inhibited the M/KCNQ currents of SCG neurons both in phasic and tonic neurons. NGF inhibited M/KCNQ currents by 25±2% and 26±3% in phasic and tonic neurons, respectively, and there had no significant differences between them. Application of NGF in the presence of 5μM linopirdine after the M/KCNQ currents was inhibited by linopirdine did not further inhibit the currents. The inhibition was 86.3±3.2% and 86.6±3.3% before and after application of NGF, and these were not significantly different. Oxo-M, a muscranic receptor agonist, also strongly inhibited M/KCNQ currents. NGF also inhibited M/KCNQ currents by 34±4% under the conventional whole-cell patch mode. Under this condition, we established the concentration-response curve for NGF-induced inhibition of M/KCNQ currents and fitted this curve by Hill function. The half-maximal inhibition (IC50) was 0.7±0.1 ng/ml and the coefficient was 0.9±0.1. AG879 (50μM), a specific inhibitor of Trk A receptor (one of the two NGF receptors, Trk A receptor and p75 receptor), when applied in pipette solution, siginificantly reduced NGF-induced inhibition of M/KCNQ currents from 34±4% to 17± 3%, whereas it had no effect on oxo-M-dediated inhibition. These results suggested that NGF inhibited M/KCNQ currents through activation of TrkA receptor. Bath applied genistein (100μM), a broad spectrum inhibitor for cellular protein tyrosine kinase, reduced significantly NGF-induced inhibition of M/KCNQ currents from 34±4% to 7±4%. Bath application of U73122, a commonly used inhibitor of phospholipase C (PLC), also significantly reduced the inhibitory effect on M/KCNQ currents mediated by NGF from 34±4% to 12±5%. These data suggested that NGF inhibited M/KCNQ currents through Trk A receptor and its downstream signal pathways, possibly involving both tyrosine phosphorylation and PI(4,5)P2 hydrolysis. (4) NGF inhibited single M/KCNQ current in cell-attached patches. NGF significantly reduced M/KCNQ channel Po by 29±3%. Oxo-M (3μM) strongly reduced the M/KCNQ channels Po by 89±2%. These data were consisitent with the whole-cell experiment. NGF did not change the conductance of the single M/KCNQ channel. The conductances were 6.1±0.2 and 6.5±0.3 pS, before and after applied NGF and had no significant difference. Oxo-M did not change the channel conductance either. The conductance was 6.4±0.3 pS in the presence of oxo-M. Both NGF and oxo-M reduced the M/KCNQ channel Po at each voltage level but only NGF significantly decreased the half-activation voltage (V1/2) from -32±3 mV to -25±2 mV. (5) NGF increased the excitability of tonic neurons but not phasic neurons. NGF significantly increased the number of spikes fired in tonic neurons from 12±2 to 20±2. NGF did not significantly change the resting potential of tonic neurons (the resting potential are -47±3 mV and -47±4 mV, before and after NGF, respectively). In the same batch of tonic neurons, oxo-M (10μM) and linopirdine (30μM) rapidly and significantly increased the spike number from 12±2 and 12±5 to 28±4 and 30±4, respectively. Oxo-M and linopirdine also induced small depolarization, but the changes did not reach the statistical significance. In the case of phasic-1 and phasic-2 neurons, NGF neither significantly changed their excitability nor significantly changed their resting potential level. On other hand, oxo-M and linopirdine significantly increased the excitability of both phasic-1 neurons (oxo-M and linopirdine enhanced the spike number from 1 to 4.7±1.2 and 3.2±1, respectively) and phasic-2 neurons (oxo-M and linopirdine enhanced the spike number from 3.2±0.7 and 3.1±0.5 to 31±6 and 16±2, respectively). Both Oxo-M and linopirdine significantly depolarized the resting potential of phasic-2 neurons (the two agent depolarized membrane from -58±2 mV and -50±3 mV to -49±2 mV and -43±2 mV, respectively); they did not affect the resting membrane potentials of phasic-1 neurons. (6) Modulation of neuronal excitability was tightly related to function of neuronal M/KCNQ channels. In phasic neurons, NGF (20 ng/ml) alone inhibited M/KCNQ currentd by 20±2%, whereas following adminstration of oxo-M (10μM) inhibited M/KCNQ currents by 62±7% in the presence of NGF. Under this condition, NGF alone did not change the excitability of phasic neurons but a significant enhancement of the neuronal excitability was produced by oxo-M in the presence of NGF; Oxo-M increased the spike number from 4.4±0.7 to 22±5 in the presence of NGF. In a reversed sequential application of these two drugs, oxo-M alone inhibited M/KCNQ currents by 72±8% and following adminstration of NGF did not further inhibit M/KCNQ currents in the presence of oxo-M. Oxo-M alone increased the spike number of phasic-2 neurons from 3.4±1.2 to 12±5, whereas, additional application of NGF did not show a significant effect. (7) Small inhibition of M/KCNQ currents by low concentration of linopirdine mimiced the effect of NGF on neuronal excitability. That NGF failed to modulate the excitability of phasic neurons may be due to its insufficient inhibition of the relative large M/KCNQ current densities. We choose linopirdine, a specific M/KCNQ channel blocker, to verify this hypothesis. We first established the concentration-response relationship curve of linopirdine-induced inhibition of M/KCNQ currents to find a proper concentration of linopirdine with a similar inhibitory effect to that seen with NGF. Linopirdine began to inhibit M/KCNQ currents at 0.3μM and reached its maximal inhibition at 30μM. The half-maximum inhibitory concentration was 2.1±0.2μM. According to this curve, 0.7μM linopirdine would inhibit M/KCNQ current by 25%, similar to the inhibition mediated by NGF (20 ng/ml). Linopirdine at concentration of 0.7μM significantly enhanced the excitability of tonic neurons by increasing its spike number from 12±1 to 18±2. However, this concentration of linopirdine did not alter the excitability of phasic-1 and phasic-2 neurons. Thus, the selective modulation of excitability of tonic neurons by NGF was likely due to ite moderate capability in inhibiting M/KCNQ current.
Conclusion: (1) Rat SCG neurons can be divided into three electrophysiological phenotypes based on their action potential firing patterns: phasic-1, phasic-2 and tonic neurons. Phasic-1 and phasic-2 neurons expressed relatively large M/KCNQ current density, which will render the cell low excitability and difficulty to fire action potentials. Tonic neurons expressed relatively small M/KCNQ current density, thus will have high excitability and easy to fire action potentials. (2) NGF at physiological concentration can significantly inhibit M/KCNQ currents of SCG neurons. NGF may inhibit M/KCNQ current through activation of Trk A receptor and its downstream signal pathways, possibly involving both tyrosine phosphorylation and PI(4,5)P2 hydrolysis. NGF inhibited M/KCNQ currents in a similar degree among phasic-1, phasic-2 and tonic neurons. (3) NGF only increased neuronal excitability of tonic neurons. Phasic neurons may still have relative large M/KCNQ currents left after inhibition induced by NGF, and the residual M/KCNQ currents were sufficient to stabilize cellular membrane potential. (4) Low concentration of linopirdine, who had the similar inhibitory effect to NGF, mimiced the effects of NGF on neuronal excitability.
2. The regulation of voltage-gated sodium currents and neuronal excitability by genistein.
Aim: To study the mechanisms involve in the regulation of VGSC currents induced by genistein, a broad spectrum inhibitor of cellular protein tyrosine kinase.
Method: Voltage-gated sodium currents (VGSC) and neuronal action potentials were recorded from rat SCG neurons. (1) Cell culture. SCG neurons were primary cultured with the same procedur describered in part one. (2) Electrophysiology. Using perforated patch clamp technique to record VGSC currents under voltage-clamping mode and record neuronal action potentials under current-clamping mode. Pipettes were pulled from borosilicate glass capillaries and had a resistance of 3-5 M? when filled with internal solution. Series resistance compensation has always been used and up to 80-90% compensation can be reached in our condition. Under this condition, the maximum access resistance was about 2 M?. The sampling rate was 10 KHz for membrane currents and was 2.5 KHz for membrane potential recordings. The protocol used to record the VGSC currents was as follows: the cells were held at -70 mV and a 20 ms depolarizing step to 0 mV was applied every 3 s. The action potentials were elicited by an approximate two-fold threshold depolarizing current of 0.1 nA. The external solution used to record the VGSC currents contained (in mM): NaCl 120, KCl 3, HEPES 5, NaHCO3 23, glucose 11, MgCl2 1.2, CaCl2 2.5, BaCl2 0.2, CdCl2 0.2, (adjusted to pH 7.4 with NaOH). The internal solution for VGSC currents recording consisted of (in mM): CsCl 90, KCl 40, HEPES 20, MgCl2 3 (adjusted to pH 7.3-7.4 with CsOH). The external solution used to record neuronal action potentials contained (in mM): NaCl 120, KCl 3, HEPES 5, NaHCO3 23, glucose 11, MgCl2 1.2, CaCl2 2.5, (adjusted to pH 7.4 with NaOH). The internal solution for action potential recording contained (in mM): KAC 90, KCl 40, HEPES 20, MgCl2 3 (adjusted to pH 7.3-7.4 with KOH). (3) Cell immunofluorescence and confocal imaging. Briefly, neurons were incubated with primary rabbit anti-Nav1.7 Ab or anti-Nav1.1 Ab overnight at 4°C, followed by three washes with PBS and incubation with goat anti-rabbit IgG-TRITC sencondary Ab for 30 min at 37°C. Cells were examined on an inverted laser-scanning microscope. TRITC was excited at 564 nm and the emitted fluorescence signal was at 570 nm. (4) Data were analyzed using clamfit and origin software.
Results: (1) Predominantly expressed voltage-gated sodium channel in rat SCG neurons was Nav1.7 channel. Cell immunofluorescence and confocal imaging experiment indicated that, Nav1.7 channel was dominantly expressed in rat SCG neurons. Nav1.1 channel, another voltage-gated sodium channel, was also weakly expressed in SCG neurons. (2) Genistein significantly inhibited the voltage-gated sodium currents in SCG neurons. 100μM genistein strongly inhibited VGSC currents by 73.3±5.4%. A double phase inhibition was evident for genistein-induced inhibition: an initial fast inhibition followed by a relatively slow inhibition. These two processes were fitted with a double exponential function and time constants were obtained. The fast time constant was 10.6±1.2 s, and the slow time constant was 55.9±2.4 s, respectively. Genistein inhibited VGSC currents in a concentration dependent manner. The concentration of half-maximal (IC50) inhibition was 9.1±0.9μM and the coefficient was 1.1±0.2. VGSC currents in SCG neurons were TTX-sensitive currents since 0.05μM TTX reversally and compeletly inhibited them. Voltage-dependence of genistein-indcued inhibtion was tested. 50μM genistein obviously inhibited VGSC currents at all voltages tested, more significantly at the range from -20 mV to 0 mV, the range of maximal activation. Genistein significantly shifted the voltage-dependent activation of VGSC current to the right, but did not affect voltage-dependent inactivation. The half-maximal activation voltage (V1/2) was significantly more positive for genistein-treated neurons (-20.6±0.1 mV) than in control cells (-32.4±0.2 mV). The half-maximal inactivation voltage were -41.3±0.4 mV for genistein and -41.8±0.3 mV for controls, respectively. (3) Genistein-indeuced inhibition of VGSC currents involved two mechanisms: PTK-independent and PTK-dependnent mechanisms. Daidzein is an inactive structural analog of genistein. Daidzein also inhibited VGSC currents in a concentration dependent manner and reached its maximal inhibitory effect at 100μM (the inhibition is 28.5±3.1 %). Fitting the curve with Hill function produced an IC50 of 20.7±0.1μM and a coefficient of 1.2±0.2. When daidzein and genistein were co-applied, daidzein (100μM) alone inhibited VGSC currents by 27.8±3.6%, additional applied genistein (100μM) inhibited VGSC currents by 57.3±10.1%. These data suggested that genistein inhibited VGSC currents through PTK-independent and other menchanisms. We tested the effect of Vanadate, a broadly used inhibitor of cellular protein tyrosine phosphotase. The effect of vanadate will antagonize the effect of genistein. Genistein alone inhibited VGSC currents by 57±5.4%, additional application of vanadate (1 mM, a saturate concentration) partly rescued the genistein-induced inhibition from 57±5.4% to 39.5±4.7%. Pretreatment with vanadate significantly reduced the genistein-induced inhibition of VGSC current by 18.7±3.3%. These data suggested that anti-PTK activity were partly responsible for the VGSC current inhibition produced by genistein. We also tested other three functional analog of genistein, tyrphotin 23, erbstatin analog and PP2 in their saturate concentration of 100μM, 100μM and 1μM, respectively. The three agents all weakly but significantly inhibited VGSC currents, and their inhibitory effect were 14.6±0.5%, 19.3±1.0 and 17.8±1.9%, respectively. At parallel experiments, gensitein inhibited VGSC currents by 54-58%. These data further suggested that genistein inhibited VGSC currents partly involved the PTK-dependent mechanism. (4) Genistein depressed the neuronal excitability and reduced the depolarized rate of action potential but did not change the resting potential of SCG neurons. Bath application of genistein (100μM) immediately suppressed the neuronal excitability by reducing the action potential number from 8.7±2.1 to 1.3±0.3. However, genistein did not significantly change the neuronal resting potential; the resting potentials were 56.1±3.1 and 54.7±4.1 before and after genistein application, respectively. Genistein significantly reduced the depolarization rate of action potential from 7.0±1.3 to 4.4±0.9 V/s.
Conclusion: (1) Genistein inhibited VGSC currents through PTK-dependent and PTK-independent mechanisms. (2) Genistein suppressed neuronal excitability and reduced the depolarization rate of SCG neurons.
3. Detection of intracellular Ca2+ signals in DRG neurons induced by G(q) protein-coupled receptors.
Aim: Do study the intracellular Ca2+ signals induced by activation of G(q) protein-coupled receptors expressed in rat DRG neurons.
Methods: (1) Cell culture. The procedur of primary culture for rat DRG neurons is similar to SCG neurons described above. DRG neurons were prepared from 14 day-old Sprague-Dawley rats. (2) Ca2+ imaging. Neurons were loaded with fura-2 AM (5μM) in the presence of pluronic F-127 (20%) and imaged using a Nikon TE-2000 microscope equipped with a Hamamatsu photonics Ca2+ imaging system. Fura-2 was excited at 340 and 380 nm and images were analyzed with simplePCT 6.0. (3) Solutions. Bath solution of DRG neurons contained (mM): NaCl 145, KCl 5, MgCl2 2, CaCl2 2, HEPES 10, Glucose 10 and adjusted to pH 7.4 with NaOH. In the Ca2+-free bath solution, Ca2+ was omitted and 3 mM EGTA was added. (4) Drugs. In this part of eaperiment, bradykinin (BK, 100 nM) was used as a positive control, capsaicin (CAP, 1μM) and acrolein (ACR, 0.1 mM) was used to identify the noceptive DRG neurons. KCl (50 mM) was used to identify the DRG neurons.
Results: (1) Identification of the DRG neurons. When bathed with normal extracellular solution (with Ca2+), 349 DRG neurons (identified by 50 mM KCl) were studied. In the following description of the results, the number of neurons which show an intracellular Ca2+ rising in responding to a particular agonist will be called as the agonist-sensitive neurons. Among 349 DRG neurons, 109 neurons were BK-sensitive neurons, making up 31.2% of the total neurons. 74 neurons were CAP-sensitive neurons, or 21.2% of the total DRG neurons. 62 neurons were ACR-sensitive neurons, 17.8% of the total DRG neurons. Among BK-sensitive DRG neurons, CAP- and ACR-sensitive neurons were 31 and 29, making up 28.4% and 26.6% of the BK-sensitive neurons, respectively. When bathed with Ca2+-free external solution, 57 DRG neurons were studied. Among the 57 neurons, 4 neurons, making up 7% of the total, were sensitive to BK; 1 neuron, making up of 1.8%, was sensitive to CAP; 1 neuron, making up of 1.8%, was sensitive to ACR, respectively. Among the 4 BK-sensitive DRG neurons, CAP- and ACR-sensitive neurons were 1 and 1, making up 25% and 25%, respectively. (2) Cholecystokinin receptors (CCK receptors). CCK receptors were activated by CCK-8 (1μM). In 131 DRG neurons bathed with normal external solution, CCK-8 elicited Ca2+ signals in 46 neurons, or 35.1% of the total neurons. Under Ca2+-free condition, No neurons out of 57 DRG neurons studied were sensitive to CCK. (3) Endothelin (ET) receptors. ET receptors were activated by ET-1 (100 nM). In Ca2+ extracellular solution, ET-1 elicited intracellular Ca2+ signals in 49 neurons out of 101 DRG neurons studied, making up a ratio of 48.5%. Among 50 DRG neurons bathed in Ca2+-free condition, 5 neurons were sensitive to ET-1. (4) Mrg D receptors. Mrg D receptors, a kind of orphan proteins, were abundantly expressed in DRG neurons and were activatied byβ-Alanine (500μM). In Ca2+ extracellular solution, 12 neurons out of 134 DRG neurons studied were sensitive toβ-Alanine. In Ca2+-free condition, No neurons were sensitive toβ-Alanine. (5) Histamine (H1) receptors. H1 receptors were activatied by Histamine (100μM). In normal external solution, Histamine only elicited Ca2+ signals in 10 neurons out of 95 DRG neurons. (6) Substance P receptors. Substance P receptors, also called NK1 receptors, were activated by substance P (SP, 1μM). When bathed with normal extracellular solution, 55 DRG neurons (identified by 50 mM KCl) were studied. Only one neuron was sensitive to SP. (7) 5-HT receptors. 5-HT receptors were activated by 5-HT (10μM).When bathed with normal solution, 55 DRG neurons were studied. No neurons were sensitive to 5-HT. (8) Angiotensin II receptors (AT1 receptors). AT1 receptors were activated by angiotensin II (100 nM). When bathed with normal external solution, 44 DRG neurons were studied. Among the detected 44 neurons, only 2 neurons were sensitive to angiotensin II. (9) Purinergic Y receptors (P2Y receptors). P2Y receptors were activated by adenosine triphsphate (ATP, 100 nM). In normal extracellular solution, 43 DRG neurons were studied, but no neurons were sensitive to ATP.
Conlusion: Among agonists of Gq-copupled receptors we stuided, agonits of BK receptors, CCK receptors, ET receptors, MrgD receptors and histamine receptors elicited intracellular Ca2+ signals, whereas agonists of substance P receptors, angiotensin II receptors, 5-HT receptors and purinergic receptors could not elicit the intracellular Ca2+ signals.
4. Modulation of TRPV1 currents in DRG neurons by capsaicin and CCK.
Aim: To study modulation of TRPV1 currents in DRG neurons by GPCR.
Methods: (1) Cell culture. Primary culture of rat DRG neurons was described above. (2) Electrophysiology. Perforated whole-cell patch was used to record currents. The protocol used to record TRPV1 and other inward currents was as follows: neurons were held at -60 mV constantly, BK and other GPCRs agonists were applied to elicit inward currents, and specific blockers of TRPV1 and Ca2+-activated chloride currents (CaCC) were used to verify the identity of the inward currents. Pipettes were pulled from borosilicate glass capillaries and had a resistance of 3-5 M? when filled with internal solution. The sampling rate was 2.5 KHz after filtered at 0.5 KHz. The extracellular solution contained (mM): NaCl 145, KCl 5, MgCl2 2, CaCl2 2, HEPES 10, Glucose 10 and adjusted to pH 7.4 with NaOH. In the Ca2+-free bath solution, Ca2+ was replaced by 0.1 mM EGTA.
Results: (1) Capsaicin elicited fast desensitizing TRPV1 currents in DRG neurons. Capsaicin (CAP, 1μM) elicited TRPV1 currents in CAP-sensitive nociceptive DRG neurons. Application of capsazepine (CZP, 100μM), a specific TRPV1 channel blocker, eliminated the capsaicin-induced inward currents at -60 mV. TRPV1 current reached its maximal value within 20 ~ 30 s, and then desensitized rapidly. The current amplitude at 5min was significantly smaller than the initial TRPV1 currents, about 30.5%±3.6% of the initial amplitude of the TRPV1 currents. The peak currents of TRPV1 from two sequential 30 s applications of CAP interrupted by a 5-6 min period of no CAP gave very different values The amplitude of the second CAP-induced TRPV1 currents was only about 50.7%±3.8% of that of the first CAP-induced TRPV1 currents. (2) CCK-8 elicited a small inward current in DRG
离子通道的活动是神经元产生兴奋的基础。一般来说,钠离子通道开放,钠离子内流导致神经元除极化,形成动作电位的上升支,爆发动作电位。随即,钠离子通道失活,钾离子通道激活而开放,钾离子外流导致神经元复极化,形成动作电位的下降支。此后,经过钠钾泵的作用使细胞膜两侧各离子恢复不均衡分布的状态,为下一次爆发动作电位做准备。除钠离子通道外,其他离子通道的开放也可导致细胞膜去极化进而影响神经元兴奋性。例如,在感觉神经元如DRG神经元,不同的刺激因素可诱发由不同离子通道承载的内向电流,膜电位的变化可以引起电压门控钠离子通道的开放,化学物质的刺激可以引起化学门控通道的开放(如,辣椒素Capsaicin可引起TRPV1通道的开放),温度的刺激或pH值的变化,均能引起相应的离子通道(这两种刺激也是TRPV1通道的适宜刺激)的开放,等等。这些内向离子通道的开放,均能引起神经元膜电位的除极化,达到阈电位即可爆发动作电位。
M通道是一种电压门控性,时间依赖性,慢激活、非失活的外向钾离子通道,因其能被毒蕈碱受体(muscranic receptor, M1)强烈抑制而得名。M通道介导的电流即M电流。M通道广泛分布于哺乳动物的神经系统中,包括中枢神经元和外周神经元。M通道的分子基础是KCNQ2/3通道构成的异四聚体,KCNQ2/3的突变可引起良性家族性新生儿惊厥症(benign familial neonatal convulsions, BFNC)。M通道一般在-60 mV左右激活,受多种因素的调节,在稳定细胞膜电位和调节细胞兴奋性等方面具有重要的生理意义。
电压门控钠离子通道是由α和β两个亚基构成的复合体,其中α亚基含有电压感受器和离子选择性孔区,β亚基为辅助亚基。迄今为止,已确定有9种α亚基,并广泛分布于哺乳动物的各种组织,它们分别是Nav1.1-Nav1.9。电压门控钠离子通道受很多因素的调节,其中丝/苏氨酸磷酸化是G蛋白偶联受体(G protein-coupled receptors, GPCRs)调节钠离子通道功能的主要机制。在颈上交感神经节神经元(SCG神经元),主要分布的是Nav1.7通道,其编码基因为SCN9A,后者的一个错位突变可导致原发性红斑性肢痛病(primary erythermalgia)。
TRPV1通道即香草酸1型瞬时电位感受器通道,为一种非选择性阳离子通道,可允许Ca2+、Mg2+和Na+等的内流。TRPV1通道主要分布于伤害性神经元如背根神经节(DRG神经元)的伤害性神经元,可感受伤害性刺激并引起痛觉。TRPV1通道本身也是辣椒素的受体,与辣椒素结合可立即激活该通道,介导阳离子内流,增强神经元兴奋性。TRPV1通道同时也是温热刺激(>43℃)、酸性刺激、机械压觉的受体,在致痛和镇痛方面占至关重要重要的地位。
本实验分别以原代培养的SD大鼠的SCG神经元和DRG神经元为研究平台,对神经元兴奋性及影响神经元兴奋性的M通道、Na通道(SCG神经元)和TRPV1通道(DRG神经元)的功能调节及相关机制进行了研究。
一、神经生长因子抑制M/KCNQ电流并增强SCG神经元的兴奋性
目的:在大鼠SCG神经元分别记录M/KCNQ电流和神经元动作电位,观察神经生长因子(nerve growth factor, NGF)对M/KCNQ电流和神经元动作电位的调节作用,并进行二者相关性研究及M/KCNQ电流调节机制的研究。
方法:(1)细胞培养。取3-5周龄的SD大鼠SCG神经节进行原代神经元培养。颈椎脱臼处死动物,快速打开手术野取出两侧SCG神经节,手术剪剪碎后用胶原酶(1 mg/ml)和胰蛋白酶(2.5 mg/ml)分别依次消化30分钟。终止消化后,将细胞团吹打成细胞悬液,然后种植在铺有多聚赖氨酸的圆玻片上。放入生化培养箱中,保持37°C并通5% CO2 + 95%空气的混合气。12小时后以Neurobasal A+B27培养基置换,继续培养。48小时内进行记录。(2)电生理实验。利用穿孔膜片钳技术和传统全细胞式膜片钳技术,电压钳制模式记录SCG神经元的M/KCNQ电流。硼硅玻璃电极拉制抛光后,充灌电极内液测定其电阻为3-5 M?可用。用Axon200B放大器结合pClamp9.0软件进行实验记录。采样率设定为5 KHz、信号过滤设定为2 KHz。将SCG神经元的膜电位钳制在-20 mV,每隔4秒给予超极化脉冲至-60 mV,维持0.8 s,以-60 mV时M/KCNQ尾电流的起始处10-20 ms与结束处10-20 ms电流平均值之差为统计对象,利用30μM Linopirdine (M/KCNQ电流的特异性抑制剂,此浓度能将M/KCNQ电流完全抑制)确定M/KCNQ电流的基线。利用穿孔膜片钳技术,电流钳钳制记录SCG神经元的动作电位。持续钳制电流为0,适时注入适宜电流2 s以引发神经元动作电位。记录M/KCNQ电流的细胞外液为(mM):NaCl 120,KCl 3,HEPES 5,NaHCO3 23,Glucose 11,MgCl2 1.2,CaCl2 2.5,TTX 0.00005,用NaOH调pH至7.4;穿孔膜片钳时,记录M/KCNQ电流的电极内液为(mM):KAc 90,KCl 40,HEPES 20,MgCl2 3,用KOH调pH至7.3-7.4。用传统全细胞膜片钳时,电极内液中加入3 mM Na2ATP和5 mM EGTA。记录神经元动作电位的的细胞外液与记录M/KCNQ电流的细胞外液相同,只是不含有TTX。记录神经元动作电位的电极内液与穿孔膜片钳记录M/KCNQ电流的电极内液相同。利用细胞贴附模式记录SCG神经元M/KCNQ单通道电流,此记录模式首先需要清楚跨膜片的电位,在使用高K+(63 mM)细胞外液的情况下,神经元静息电位约为-20 mV (根据Nerst公式),根据Vm=Vrest -Vpipette,钳制膜片的跨膜电位即为静息电位与通过电极给予的钳制电位之差,即当钳制电位为0 mV时,则钳制膜片跨膜电位为-20 mV,此时M/KCNQ通道应处于开放状态。单通道电流记录信号先经0.5-2 kHz虑过,而后的采样率设定为5-10 kHz。单通道记录所用电极拉制抛光后涂sylgard以降低电极介质噪声,电极电阻为8-10 MΩ。记录M/KCNQ单通道电流的细胞外液为(mM):NaCl 65,KCl 63,CaCl2 0.5,MgCl2 1.2,HEPES 10,Glucose 11,并加入CgTx-GVIA 250 nM,nifedipine 10μM,tetrodotoxin 250 nM,以KOH调pH至7.3;记录M/KCNQ单通道电流的电极内液为(mM):NaCl 125,KCl 3,MgCl2 1.2,HEPES 10,Glucose 11,并加入apamin 200 nM,charybdotoxin 100 nM,α和βdendrotoxins 300 nM以及TTX 250 nM,以NaOH调pH至7.3。(3)以Clamfit和Origin软件来分析、统计数据。
结果:(1)SD大鼠SCG神经元中存在三种不同电生理类型的神经元。根据神经元爆发动作电位的特征,可将SCG神经元分为三个类型,即Phasic-1、Phasic-2和Tonic神经元。Phasic-1神经元,只爆发一个动作电位(2 s内,下同),动作电位数目不随注入电流的增大而增加;此类神经元占SCG神经元总数的36%。Phasic-2神经元,爆发2~6个动作电位,动作电位数目随注入电流的增大而增加,甚至可转变成Tonic神经元;此类神经元占SCG神经元总数的54%。Tonic神经元,持续爆发动作电位,且动作电位数目随注入电流增大而增加;此类神经元占SCG神经元总数的10%。三类神经元中,Phasic-1及Phasic-2神经元的静息电位高于Tonic神经元,其统计学差异有显著性,而Phasic-1与Phasic-2神经元静息电位之间无统计学差异。Phasic-1及Phasic-2神经元的峰电位数目远低于Tonic神经元,其统计学差异有显著性,而Phasic-1与Phasic-2神经元峰电位数目之间无统计学差异。(2)SCG神经元的类型与神经元的M/KCNQ电流密切相关。先在电流钳钳制模式下确定SCG神经元的类型,然后改为电压钳钳制模式记录相应类型SCG神经元的M/KCNQ电流。Phasic-1与Phasic-2神经元的M/KCNQ电流形态、电流幅度相似。Phasic-1与Phasic-2神经元的M/KCNQ尾电流幅度较大,而Tonic神经元的M/KCNQ尾电流幅度较小,三者在-60 mV时的尾电流密度分别为2.8±0.2,2.3±0.2和0.9±0.1 pA/pF,Phasic-1与Phasic-2神经元之间没有统计学差异,而两种Phasic神经元与Tonic神经元之间有显著的统计学差异。三种神经元的电流-电压关系曲线(I-V曲线)的特征也有不同:三种神经元的半数激活电压(V1/2)分别是-30±1, -29±1和-15±3 mV,其中两种Phasic神经元之间的V1/2相似,没有统计学差异,而Tonic神经元的电流-电压关系曲线明显右移,其V1/2与两种Phasic神经元神经元相比明显降低,统计学差异有显著性。以-60 mV时的M/KCNQ尾电流密度为横坐标,以三种神经元爆发动作电位的次数为纵坐标做动作电位-M/KCNQ电流关系散点图,可以看出,三种神经元动作电位的分布呈明显的区域性,随着M/KCNQ电流的增大,动作电位爆发的次数明显减少,此为两种Phasic神经元的特征;相反随着M/KCNQ电流的减小,动作电位爆发的次数明显增多,此为Tonic神经元的特征;而且从散点图中可以看到一条明显的分界线位于约1pA/pF的M/KCNQ电流处,两种Phasic神经元大多位于分界线的右侧,而Tonic神经元大多位于分界线的左侧。三种神经元M/KCNQ电流的动力学表现也不同,表现在-20 mV时电流的激活速度和-60 mV时电流的去活速度均不同,电流的激活速度和去活速度可用相应的电流激活时间常数和去活时间常数代表。Phasic-1、Phasic-2和Tonic三种神经元-20 mV时的激活时间常数分别是60±5、64±4和99±12 ms,-60 mV时去活时间常数分别是51±2、60±6和96±5 ms。不论是激活时间常数还是去活时间常数,Phasic-1神经元与Phasic-2神经元之间无统计学差异,而两种Phasic神经元均与Tonic神经元有显著的统计学差异。(3)NGF抑制大鼠SCG神经元的M/KCNQ电流。穿孔膜片钳技术记录SCG神经元的M/KCNQ电流,然后给予生理浓度的NGF,观察NGF对M/KCNQ电流的作用。不管是Phasic神经元还是Tonic神经元,生理浓度的NGF(20 ng/ml)均能显著抑制其M/KCNQ电流,抑制率分别是25±2%和26±3%,两者之间没有显著性差异。为了证明被NGF抑制的电流就是M/KCNQ电流,我们在持续应用5μM的Linopirdine(M/KCNQ特异阻断剂)的情况下,再观察NGF的作用。这种情况下,NGF对M/KCNQ电流的抑制作用消失,说明NGF敏感电流也对Linopirdine敏感,NGF敏感电流应为M/KCNQ电流。在传统全细胞式膜片钳记录情况下,生理浓度NGF同样抑制M/KCNQ电流,抑制率达34±4%。做NGF抑制M/KCNQ电流的量-效关系曲线,利用Hill方程拟合曲线得出NGF抑制M/KCNQ电流的半数有效浓度(EC50)为0.7±0.1 ng/ml,Hill方程参数为0.9±0.1。电极内液中加入NGF受体Trk A的特异性阻断剂AG879,可明显减弱NGF的抑制作用,将其抑制率从34±4%降至17±3%。灌流给予酪氨酸蛋白酶抑制剂Genistein,也显著降低NGF对M/KCNQ电流的抑制作用,将其抑制率从34±4%降至7±4%。灌流给予磷脂酶C(PLC)抑制剂U73122也显著降低NGF对M/KCNQ电流的抑制作用,将其抑制率从34±4%降至12±5%。这些数据提示NGF通过激活Trk A受体及其下有信号通路而抑制M/KCNQ电流,可能通过酪氨酸磷酸化和膜磷脂PI(4,5)P2水解两条信号通路来抑制M/KCNQ电流。(4)NGF显著抑制M/KCNQ单通道电流。细胞贴附式记录SCG神经元的M/KCNQ单通道电流,灌流给予20 ng/ml的NGF可显著抑制M/KCNQ单通道电流的开放概率(Open properbility,Po),抑制率达29±3%,与全细胞模式下的NGF的抑制率一致。做为阳性对照,3μM M1受体激动剂Oxo-M强烈抑制M/KCNQ单通道电流的Po,抑制率达89±2%。NGF不改变M/KCNQ单通道的电导,给予NGF前后的电导分别是6.1±0.2 pS和6.5±0.2 pS,两者之间没有统计学差异。阳性对照药Oxo-M同样不改变M/KCNQ单通道的电导----在Oxo-M存在情况下,M/KCNQ单通道电导为6.4±0.3 pS,与给药前相比没有统计学差异。从-80 mV每隔20 mV增加至+40 mV,做M/KCNQ单通道的开放概率-电压关系曲线,并用Boltzmann方程拟合,发现NGF和Oxo-M均能在各个电压下降低M/KCNQ单通道的开放概率,但只有NGF将M/KCNQ单通道的半数激活电压(V1/2)从-32±3 mV降至-25±2 mV,两者之间有显著性差异。(5)NGF显著增强Tonic神经元的兴奋性,而对两种Phasic神经元兴奋性无影响。20 ng/ml NGF显著增强Tonic神经元的兴奋性,使其动作电位次数从12±2增至20±2,但NGF对该类型神经元的静息电位无显著性影响(给予NGF前后,该类型神经元的静息电位分别是-47±3和-47±4 mV,二者之间无显著性差异)。阳性对照药Oxo-M(10μM)和Linopirdine(30μM)显著增强Tonic神经元的兴奋性,分别使其动作电位次数从12±2和12±5增至28±4和30±4。给予Oxo-M和Linopirdine后,静息膜电位发生除极化,但给药前后相比并无显著性差异。对于Phasic-1和Phasic-2神经元,20 ng/ml NGF既没有不能改变他们的兴奋性,也不能明显改变他们的静息电位水平。阳性对照药Oxo-M(10μM)和Linopirdine(30μM)既显著增加了Phasic-1神经元的兴奋性(两者分别使该类型神经元的动作电位次数从1增至4.7±1.2和3.2±1,统计学差异均具有显著性)也显著增加了Phasic-2神经元的兴奋性(两者分别使该类型神经元的动作电位次数从3.2±0.7和3.1±0.5增至31±6和16±2,统计学差异均具有显著性)。Oxo-M(10μM)和Linopirdine(30μM)均能使Phasic-2神经元的静息电位发生明显的除极化(两者分别使该类型神经元的静息电位从-58±2 mV和-50±3 mV除极化至-49±2 mV和-43±2 mV,用药前后相比,统计学差异均有显著性)。Oxo-M(10μM)和Linopirdine(30μM)随能使Phasic-1神经元发生除极化,但统计学差异无显著性。(6)SCG神经元兴奋性与其M/KCNQ功能成负相关。这部分实验是在NGF(20 ng/ml)和Oxo-M(10μM)交替顺序共同应用的给药方式下,观察两者对SCG神经元的兴奋性的增强作用和M/KCNQ电流的抑制作用是否一致。首先灌流应用NGF,M/KCNQ电流被抑制约20±2%。在NGF存在的情况下,给予Oxo-M,M/KCNQ电流被抑制约62±7%。同样的给药方式,NGF没有改变Phasic神经元的兴奋性,而随后给予Oxo-M,却显著增强此神经元的兴奋性(动作电位次数由4.4±0.7增至22±5,用药前后相比,统计学差异有显著性)。交换顺序,首先灌流应用Oxo-M,神经元M/KCNQ电流被抑制约72±8%,在Oxo-M存在的情况下,随后给予NGF没有进一步抑制该神经元M/KCNQ电流(NGF给药前后相比,没有统计学差异)。同样的给药方式,Oxo-M显著增强神经元兴奋性(动作电位次数由3.4±1.2增至12±5,有显著的统计学差异),随后给予NGF并没有进一步影响神经元的兴奋性。(7)对M/KCNQ电流有相同抑制效应的低剂量Linopirdine能重现NGF分别影响SCG不同类型神经元兴奋性的效应。首先做Linopirdine抑制M/KCNQ电流的量-效关系曲线,以Hill方程拟合得出Linopirdine抑制M/KCNQ电流的半数有效剂量(EC50)为2.1±0.2μM,Hill方程参数为1.2±0.1。根据该量-效关系曲线可得出抑制M/KCNQ电流约25%时的Linopirdine的剂量应该是0.7μM,而Linopirdine的这一抑制效应与NGF(20 ng/ml)对M/KCNQ电流的抑制效应相类似。0.7μM Linopirdine能明显增强Tonic神经元的兴奋性,使其动作电位次数由12±1增至18±2,其统计学差异有显著性,而该浓度的Linopirdine不改变Phasic-1和Phasic-2两种神经元的兴奋性。
结论:(1)从电生理特性看,根据大鼠SCG神经元神经元所表达的M/KCNQ电流密度的大小可将神经元分为Phasic-1、Phasic-2和Tonic三种神经元。Phasic-1和Phasic-2神经元表达M/KCNQ电流密度大,稳定膜电位作用较强,故其兴奋性较低,不易爆发动作电位;Tonic神经元表达M/KCNQ电流密度小,稳定膜电位作用较弱,故其兴奋性较高,较易爆发动作电位。(2)生理浓度的NGF能显著抑制SCG神经元M/KCNQ电流,其抑制M/KCNQ电流的机制可能是通过激活Trk A受体及其下游酪氨酸蛋白激酶磷酸化和PLC水解PI(4,5)P2两条信号通路。在三种神经元上,NGF抑制M/KCNQ电流的效应相似,但NGF只增强Tonic神经元的兴奋性,而对两种Phasic神经元兴奋性无影响。这可能与Phasic神经元M/KCNQ电流被NGF抑制后仍余留较大的电流能稳定其膜电位有关。(3)与NGF抑制M/KCNQ电流相同效应的低剂量Linopirdine能完全重现NGF对三种神经元兴奋性的效应。进一步提示NGF抑制Phasic神经元M/KCNQ电流后余留较大电流能稳定其膜电位。(4)M/KCNQ通道在癫痫、惊厥以及老年性痴呆等疾病发病中起重要作用,本研究证明NGF是M/KCNQ通道的一种新的调节因素,丰富了NGF的生物学功能,同时也为M/KCNQ通道的功能调节提供了新的思路。
二、Genistein通过酪氨酸蛋白激酶依赖性和非依赖性两个信号通路抑制SCG神经元的电压门控性钠电流。
目的:在大鼠SCG神经元记录电压门控性Na通道电流(VGSC电流)和神经元动作电位,观察Genistein对VGSC电流的调节作用,研究其调节机制。Genistein是广泛应用的非特异性酪氨酸蛋白激酶抑制剂,可广泛抑制细胞内酪氨酸蛋白激酶(或带有酪氨酸蛋白激酶活性的蛋白质)的活性。
方法:(1)细胞培养。大鼠SCG神经元的的培养同上部分。(2)电生理实验。利用穿孔膜片钳技术,电压钳钳制模式记录SCG神经元的VGSC电流,电流钳钳制模式记录SCG神经元的动作电位。硼硅玻璃电极拉制抛光后,充灌电极内液测定其电阻为3-5 M?可用。串联电阻补偿达80-90%后,电极最大接入电阻为2 M?左右。信号采样率分别设定为10 KHz(VGSC电流)和2.5 KHz(动作电位)。神经元钳制在-70 mV每隔3秒给予除极化脉冲至0 mV,持续20毫秒。电流钳钳制神经元在0 pA,然后注入约2倍于阈值的除极化电流0.1 pA以引发神经元动作电位。记录VGSC电流的神经元细胞外液为(mM):NaCl 120,KCl 3,HEPES 5,NaHCO3 23,glucose 11,MgCl2 1.2,CaCl2 2.5,BaCl2 0.2,CdCl2 0.2,用NaOH调pH至7.4;记录VGSC电流的电极内液为(mM):CsCl 90,KCl 40,HEPES 20,MgCl2 3,用CsOH调pH至7.3-7.4。记录神经元动作电位的细胞外液为(mM):NaCl 120,KCl 3,HEPES 5,NaHCO3 23,glucose 11,MgCl2 1.2,CaCl2 2.5,用NaOH调pH至7.4;记录神经元动作电位的电极内液为(mM):KAC 90,KCl 40,HEPES 20,MgCl2 3用KOH调pH至7.3-7.4。(3)细胞免疫荧光化学实验。细胞用4%多聚甲醛固定、0.1%的Triton X-100打孔、3%的BSA封闭30分钟,孵育家兔来源的一抗anti-Nav1.7抗体或anti-Nav1.1抗体,4°C,过夜。用PBS洗涤后孵育二抗羊抗兔IgG-TRITC抗体30分钟。共聚焦显微镜观察,TRITC激发光波长为564 nm,吸收光波长为570 nm。(4)用Clamfit和Origin软件来分析、统计数据。
结果:(1)大鼠SCG神经元表达的电压门控性Na通道(VGSC)主要是Nav1.7通道。细胞免疫荧光化学实验表明,大鼠SCG神经元主要表达TTX-敏感的,电压门控性Nav1.7通道;微弱表达TTX-敏感的,电压门控性Nav1.1通道。(2)Genistein显著抑制SCG神经元的TTX-敏感的电压门控性Na通道电流。100μM Genistein抑制VGSC电流达73.3±5.4%,而且Genistein对VGSC电流的抑制呈明显的双相型----快相紧跟一个慢相。双指数方程拟合此抑制过程得出快相和慢相的时间常数分别是10.6±1.2 s和55.9±2.4 s。做Genistein抑制VGSC电流的量-效关系曲线,并以Hill方程拟合得到Genistein抑制VGSC电流的半数有效量(EC50)为9.1±0.9μM,其方程系数为1.1±0.2。SCG神经元的VGSC电流为TTX-敏感的Na电流,0.05μM TTX能完全抑制之。做VGSC的电流-电压关系曲线(I-V曲线),50μM Genistein明显抑制各个电压下的VGSC的电流,且最大激活电压范围内(-20 mV~ 0 mV)的抑制最明显。50μM Genistein使VGSC的电压依赖性激活曲线明显右移,使其半数激活电压(V1/2)由-32.4±0.2 mV降至-20.6±0.1 mV,二者统计学差异有显著性。50μM Genistein对VGSC的电压依赖性失活曲线没有影响,给药前后的半数失活电压分别为-41.8±0.3 mV和-41.3±0.4 mV,两者之间无统计学差异。(3)酪氨酸蛋白激酶依赖性和酪氨酸蛋白激酶非依赖性两种机制参与了Genistein对VGSC电流的抑制。灌流给予Genistein的无活性结构类似物Daidzein(10μM~ 300μM),做Daidzein抑制VGSC电流的量-效关系曲线。以Hill方程拟合此曲线,可以看出100μM Daidzein即可达到最大抑制(抑制率为28.5±3.1%),其抑制VGSC电流的半数有效量(EC50)为20.7±0.1μM,方程系数为1.2±0.2。单独给予100μM Daidzein可抑制VGSC电流达27.8±3.6%,在100μM Daidzein存在的情况下,紧接着给予100μM Genistein,可进一步抑制VGSC电流,二者合用对VGSC电流的抑制率达57.3±10.1%。这提示酪氨酸蛋白激酶非依赖性机制参与了Genistein对VGSC电流的抑制,可能是一种直接作用。100μM Daidzein虽抑制VGSC电流(此剂量已达最大抑制),但没有完全取代100μM Genistein的抑制作用,这提示可能还有其他机制参与Genistein对VGSC电流的调节作用。Vanadate是一种广泛应用的酪氨酸蛋白磷酸酶的抑制剂,它能间接加强底物发生的磷酸化,而Genistein则抑制底物产生磷酸化,所以二者的细胞内作用恰恰相反。单独给予100μM Genistein可抑制VGSC电流达57±5.4%,在Genistein存在的情况下紧接着给予Vanadate(1 mM),Vanadate可部分反转Genistein的抑制作用,使其抑制率降至39.5±4.7%。预先应用Vanadate(1 mM),然后在Vanadate存在的情况下紧接着给予100μM Genistein,1 mM Vanadate可降低Genistein对VGSC电流的抑制作用达18.7±3.3%。这两情况下,Vanadate干预前后,Genistein对VGSC电流的抑制率均有显著性差异。这部分实验提示,酪氨酸蛋白激酶依赖性机制也参与了Genistein对VGSC电流的调节作用。以饱和剂量分别给予Genistein的三种功能类似物(均是常用的酪氨酸蛋白激酶抑制剂)Tyrphostin 23(100μM)、Erbstatin analog(100μM)和PP2(1μM),发现三者均能模拟Genistein对VGSC电流的抑制作用,其抑制率分别是14.6±0.5%、19.3±1.0%和17.8±1.9%。虽然三者对VGSC电流的抑制率较低,但均具有显著性统计学差异。这部分实验仍提示酪氨酸蛋白激酶依赖性机制参与了Genistein对VGSC电流的调节作用。(4)Genistein降低SCG神经元的兴奋性,降低其除极速率,但不改变其静息电位水平。灌流给予100μM Genistein可抑制SCG神经元的兴奋性,使其动作电位次数由8.7±2.1降至1.3±0.3;然而,给药前后神经元的静息电位水平分别是56.1±3.1 mV和54.7±4.1 mV,没有显著性统计学差异。100μM Genistein还显著减慢SCG神经元动作电位的除极速率,使其由7.0±1.3 V/s降至4.4±0.9 V/s,给药前后除极速率的变化有显著性统计学差异。
结论:(1)Genistein通过酪氨酸蛋白激酶依赖性和酪氨酸蛋白激酶非依赖性两种机制抑制SCG神经元的VGSC电流。(2)Genistein抑制SCG神经元的VGSC电流导致其降低神经元兴奋性和动作电位除极速率。
三、Gq蛋白偶联受体激活诱导的DRG神经元细胞内钙变化
目的:研究同为G(q)蛋白偶联受体(G(q)PCRs)激活后对DRG神经元细胞内钙信号影响的异同,建立进一步研究细胞内钙信号在膜受体调节DRG神经元离子通道功能中的作用的基础。
方法:(1)细胞培养。DRG神经节取自14天鼠龄的SD大鼠。原代培养大鼠DRG神经元的方法与上述培养大鼠SCG的方法相似。(2)钙信号测定。用Fura-2 AM(5μM,辅以20%的pluronic F127)标记细胞内游离的Ca2+。利用Hamamatsu光电子Ca2+显像系统,使用F340/F380双波长比例测钙法测定DRG神经元细胞内钙信号的变化。用simplePCT 6.0软件进行数据分析。(3)DRG细胞外液。正常DRG细胞外液为(mM):NaCl 145,KCl 5,MgCl2 2,CaCl2 2,HEPES 10,Glucose 10,用NaOH调pH至7.4。无钙细胞外液中,去除Ca2+并加入3 mM的EGTA。(4)药物。以缓激肽(Bradykinin,BK)为阳性对照药物,以辣椒素(Capsaicin,CAP)和丙烯醛(Acrolein,ACR)确定DRG神经元的类型,以高K+液(50 mM KCl)确定细胞内钙升高。其他几种GPCRs分别以各自相应的激动剂来激活。(5)实验数据用Origin软件做统计处理。
结果:(1)DRG神经元鉴定。有钙外液情况下,共测定DRG神经元349例,按对受体激动有细胞内钙升高(阳性)的比例计算,其中BK阳性神经元为109例,占总数的31.2%;CAP阳性神经元为74例,占总数的21.2%;ACR阳性神经元为62例,占总数的17.8%。在109例BK阳性神经元中,CAP阳性神经元为31例,占28.4%;ACR阳性神经元为29例,占26.6%。无钙外液情况下,共测定DRG神经元57例,其中BK阳性神经元为4例,占总数的7%;CAP阳性神经元为1例,占总数的1.8%;ACR阳性神经元为1例,占总数的1.8%。在4例BK阳性神经元中,CAP阳性神经元为1例,占25%;ACR阳性神经元为1例,占25%。(2)缩胆囊素受体(Cholecystokinin receptors,CCK)。缩胆囊素受体用CCK-8(1μM)来激活。有钙外液情况下,CCK能使131例DRG神经元中的46例神经元内钙升高,占35.1%。无钙外液情况下,共测定DRG神经元57例,但没有神经元的细胞内钙能被CCK升高。(3)内皮素受体(Endothelin receptors,ET receptors)。内皮素受体用ET-1(100 nM)来激活。有钙外液情况下,内皮素能使101例DRG神经元中的49例神经元内钙升高,占总数的48.5%。无钙外液情况下,共测定DRG神经元50例,其中有5例神经元能被内皮素升高细胞内钙。(4)Mrg D受体。Mrg D受体是一种孤儿蛋白,在DRG神经元丰富表达。Mrg D受体可被丙氨酸(β-Alanine,ALA,500μM)激活。有钙外液情况下,ALA能使134例DRG神经元中的12例神经元内钙升高。无钙外液情况下没有神经元的细胞内钙能被ALA升高。(5)组胺受体(Histamine receptors)。组胺受体可用组胺(100μM)来激活。有钙外液情况下,组胺仅能使95例DRG神经元中的10例神经元内钙升高。(6)P物质受体(Substance P receptors,SP receptors)。P物质受体也称为NK1受体,可用P物质(SP, 1μM)来激活。有钙外液情况下,共测定DRG神经元55例,只有1例神经元能被SP升高内钙,绝大部分不能被SP升高细胞内钙。(7)5羟色胺受体(5-HT receptors)。5羟色胺受体用10μM的5-HT来激活。有钙外液情况下,共测定DRG神经元55例,5-HT也不能升高细胞内钙。(8)血管紧张素II受体(Angiotensin II receptors,AT receptors)。血管紧张素II受体可用100 nM的血管紧张素II来激活。有钙外液情况下,共测定44例DRG神经元,仅有2例神经元能被血管紧张素II升高细胞内钙。(9)嘌呤能受体(purinergic Y receptors,P2Y receptors)。P2Y受体可用100 nM的ATP来激活。有钙外液情况下,共测定43例DRG神经元,没有神经元能被ATP升高细胞内钙。
结论:几种G蛋白偶联受体中,CCK受体、ET-1受体、Μrg D受体和组胺受体激活后能升高DRG神经元细胞内钙。而SP受体、AT II受体、5-HT受体和P2Y受体不能升高DRG神经元细胞内钙。
四、辣椒素和CCK对DRG神经元TRPV1电流的调节。
目的:文献报道和本实验室前期工作表明BK可引发TRPV1电流和Ca2+激活的氯电流(Ca2+-activated Cloride currents , CaCC ,TMEM16A-dependent)。二者均为内向电流,可使膜电位除极化而爆发动作电位。本部分实验拟观察研究内容三中所涉及GPCRs激活可否引发DRG神经元内向电流以及电流成分和激活机制,同时研究GPCRs激活对TRPV1通道的功能调节。
方法:(1)细胞培养。按上述方法培养大鼠DRG神经元。(2)电生理实验。利用穿孔膜片钳技术,将DRG神经元钳制在-60 mV,分别给予BK和其他几种G蛋白偶联受体激动剂(能升高细胞内钙者),观察内向电流有否被激活,然后分别给予TRPV1或CaCC特异性阻断剂确定其通道性质。硼硅玻璃电极拉制抛光后,充灌电极内液测定其电阻为3-5 M?可用。Clampex采样率设定为2.5 KHz,信号过滤设定为0.5 KHz。记录内向电流的细胞外液为(mM):NaCl 145,KCl 5,MgCl2 2,CaCl2 2,HEPES 10,Glucose 10,用NaOH调pH至7.4;电极内液为(mM):KCl 145,MgCl22,HEPES 10,用KOH调pH至7.4。无钙外液时,细胞外液中的Ca2+被去除,并加入0.1 mM EGTA。
结果:(1)辣椒素(capsaicin,CAP,1μM)激活CAP阳性神经元TRPV1电流并很快脱敏。CAP激活的内向电流可被TRPV1通道特异性阻断剂capsazepine(CZP,100μM)迅速完全抑制,证明CAP引发的内向电流就是TRPV1电流。给予CAP 20-30秒后,TRPV1电流激活达最大值,持续时间短,约10秒钟,然后很快发生脱敏现象。给予CAP 5分钟末的TRPV1电流远小于给药20-30秒时的TRPV1最大电流,二者的比例为30.5%±3.6%。第一次给予1μM CAP 30秒,此时TRPV1电流达最大值,冲洗5分钟,然后第二次给与1μM CAP 30秒,第二次给药引发的TRPV1电流显著小于第一次给药引发的TRPV1电流,二者比例为50.7%±3.8%。(2)CCK-8在DRG神经元能引发微小的内向电流并能阻断CAP引起的TRPV1电流的脱敏现象。灌流给予1μM的CCK-8,在CAP阳性神经元或者CAP阴性神经元均能激活微弱的内向电流。用CCK预处理DRG神经元1 min,然后在CCK存在情况下给予1μM CAP 5分钟,CCK可明显减弱TRPV1电流的快速衰减,只发生微小衰减,5分钟末的TRPV1电流(在CCK-8存在的情况下)占起始处TRPV1最大电流的68.4%±3.4%,与对照情况下相比,有显著的统计学差异。在第二次给予CAP之前,预先给予1μM的CCK-8处理细胞1分钟,第二次给予CAP所引发的TRPV1电流幅度与第一次CAP所引发的TRPV1电流幅度相似,而二者比例为91.2%±7.4%,与对照情况下相比,有显著的统计学差异。(3)其他受体激活后能否引发内向电流,还在实验中。
总结
1.从电生理角度看,大鼠颈上交感神经节(SCG)神经元根据神经元所表达的M/KCNQ电流的大小可分为Phasic-1、Phasic-2和Tonic三种神经元。Phasic-1和Phasic-2神经元表达M/KCNQ电流密度大,稳定膜电位作用较强,故其兴奋性较低,不易爆发动作电位;Tonic神经元表达M/KCNQ电流密度小,稳定膜电位作用较弱,故其兴奋性较高,较易爆发动作电位。
2.生理浓度NGF显著抑制SCG神经元M/KCNQ电流,可能是通过激活TrkA受体及其下游酪氨酸蛋白激酶磷酸化和PLC-PI(4,5)P2水解两条信号通路。在三种神经元上,NGF抑制M/KCNQ电流的效应相似,但NGF只增强Tonic神经元的兴奋性,而对两种Phasic神经元兴奋性无影响。这可能与Phasic神经元M/KCNQ电流被NGF抑制后仍余留较大的电流能稳定其膜电位有关。与NGF抑制M/KCNQ电流相同效应的低剂量Linopirdine能完全重现NGF对三种神经元兴奋性的效应。进一步提示NGF抑制Phasic神经元M/KCNQ电流后余留较大电流能稳定其膜电位。
3. Genistein通过酪氨酸蛋白激酶依赖性和酪氨酸蛋白激酶非依赖性两种机制抑制SCG神经元的电压依赖性钠通道(VGSC)电流。Genistein抑制SCG神经元的VGSC电流导致神经元兴奋性降低,动作电位除极速率减慢。
4.几种G蛋白偶联受体中,BK受体、缩胆囊素受体(CCK)、内皮素受体、Μrg D受体和组胺受体能升高背根神经节(DRG)神经元细胞内钙,而SP受体、血管紧张素II受体、5羟色胺受体和ATP受体不能升高DRG神经元细胞内钙。
5.辣椒素(CAP)激活TRPV1电流,并很快发生脱敏现象。CCK能在DRG神经元引发轻微的内向电流,同时能抑制CAP引起的TRPV1通道的脱敏现象。
Neuronal excitability is one of the basic characteristics of neurons. Neurons will fire action potentials when they receive adequate stimulus. Generally, the whole period of neuronal excitation can be divided into three processes, depolarization period (membrane potential was reduced in very short time, a process forming the upslope of the action potential), repolarization period (membrane potential was increased negatively in short time, a process forming the downslope of the action potential) and recovery period or resting period (membrane potential maintains relatively stable, normally called resting potential).
Neuronal excitation is founded on the activity of ion channels. In general, sodium ions flux into the cells when sodium channels are open, which leads to depolarization of neurons and forms the upslope of action potential. Soon after, sodium channels become inactivated and closed, and at the same time potassium channels are activated and open, which allow potassium ions flux out of the cell, which results in repolarization of neurons and forms the downslope of action potential. The upslope and downslope of action potential make a spike potential. After each action potential, displaced ions are recovered to their original disproportional distribution across the membrane by the action of sodium-potassium pump. This disproportional distribution of sodium and potassim across the mebrane prepares the cell to fire next action potential when the cell receives again a suprathreshold stimulus. Apart from sodium currents, other inward currents can also induce membrane depolarization action potential. For example, in sensory neurons like dorsal root ganglion (DRG) neurons, capsaicin can lead to the activation and opening of TRPV1, which are also sensitive to thermal stimulus and pH.
M-type potassium channel or M/KCNQ channel is a voltage-gated channel with characteristics of slow-activation and deactivation and non-inactivation. Activation of muscranic receptor strongly inhibitits this potassium channel, hence, it is called M-type potassium channel. The molecular basis of M/KCNQ channel is the tetraheterologus of KCNQ2 and KCNQ3 channels. Mutations of KCNQ2/3 lead to benign familial neonatal convulsions. M/KCNQ channel distributes widely in the nerve system in mammalian, including the central nerve system and peripheral nerve system. Generally, M/KCNQ channel is activated around– 60 mV, a membrane potential near the resting membrane potential. Thus M/KCNQ channels are believed to play a key role in stabilizing membrane potential and regulating neuronal excitability. M/KCNQ channel can be regulated by many factors such as G protein-coupled receptors, receptor tyrosine kinase, and other factors.
Voltage-gated sodium channels (VGSC) are composed of a complex of aαsubunit that forms the voltage-sensitive and ion-selective pore, in association with one or more auxiliaryβsubunits. To date, nineαsubunits of the VGSC superfamily, Nav1.1-Nav1.9, have been identified, which are widely distributed in mammalian tissue. Among these VGSC, Nav1.7 channel is dominantly expressed in superior cervical ganglion (SCG) neurons. Nav1.7 channel is coded by gene, SCN9A, a missense mutation in which underlies the primary erythermalgia. Many factors modulate VGSC, including G-protein coupled receptors and tyrosine kinases.
Transient receptor potential vanillicacid 1 channel, simply called TRPV1 channel, is a kind of non-selective cation channels, which allows calcium, magnesium and sodium ions flux into cells. TRPV1 channel mainly distributes in nociceptive neurons such as the nociceptive neurons in DRG. TRPV1 channel can be activated responding to nociceptive stimulus. TRPV1 channel also is the receptor of capsaicin, a kind of extractive agent from capsicum chilli. Capsaicin binds to TRPV1 channel and then activates this channel and induces influx of cations, which enhances neuronal excitability. TRPV1 channel is also sensitive to thermal stimulus, pH and machinery pressure. Many factors can modulate the activatiy of TRPV1 channel, such as membrane PI(4,5)P2, Ca2+ and Calmodulin, etc.
The present study will investigate the functional regulation of M/KCNQ channel, voltage-gated sodium channel and TRPV1 channel, and related neuronal excitability using primary cultured neurons from rat SCG and DRG. We also study the intrcellular calcium signals induced by activation several G protein-coupled receptors expressed in DRG neurons.
1. The regulation of M/KCNQ currents and neuronal excitability by NGF. Aim: (1) To record M/KCNQ currents and neuronal action potential in rat SCG neurons, and to study the mechanism of regulation of M/KCNQ currents and neuronal exctibality produced by nerve growth factor (NGF).
Methods: (1) Cell culture. Primary cultures of neurons were prepared from SCG from 3- to 5-week-old Sprague-Dawley rats. Briefly, ganglia were digested with collagenase (1 mg/ml) and next trypsin (2.5 mg/ml), and then ganglia were dissociated into a suspension of individual cells and planted on poly-lysine coated glass coverslips. Cellas were incubated at 37°C with a 5% CO2 + 95% air. The DMEM medium plus 10% serum were changed to neurobasal A medium plus 2% B27 supplement after 12 hr and cells were used within 48 hr. (2) Electrophysiology. Perforated patch and conventional whole-cell patch were used to record the neuronal M/KCNQ currents under voltage-clamping mode. Pipettes were pulled from borosilicate glass capillaries and had resistances of 3-5 M? when filled with pipette solution. Currents and action potentials were recorded using an Axon 200B amplifier and pClamp 9.0 software, and were filtered at 2 KHz. The protocol for recording of M/KCNQ currents is as follows: SCG neurons were held at -20 mV followed a 0.8 s hyperpolarization step to -60 mV every 4 s. The amplitude of M/KCNQ currents was defined as the outward currents sensitive to 30μM linopirdine, a specific M/KCNQ channel blocker, and was measured from deactivation currents records at -60 mV as the difference between the average of an initial 10-ms segment, taken 10-20 ms into the hyperpolarizing step, and the average during the last 10 ms of that step. Perforated patch was used to record neuronal action potentials under current-clamping mode. The protocol to record action potential is as follows: SCG neurons were held at 0 current level and the action potentials were elicited by injection of a depolarizing current for 2 s. The external solution used to record M/KCNQ currents contained (in mM): NaCl 120, KCl 3, HEPES 5, NaHCO3 23, Glucose 11, MgCl2 1.2, CaCl2 2.5 and TTX 0.00005, (adjusted to pH 7.4 with NaOH). The pipette solution for perforated patch recording consisted of (in mM): KAc 90, KCl 40, HEPES 20 and MgCl2 3 (adjusted to pH 7.3-7.4 with KOH). Na2ATP (3 mM) and EGTA (5 mM) were added to the above internal solution for conventional whole-cell recording. The external solution used to record neuronal action potentials was the same as that used for M/KCNQ currents recording, but did not contain TTX. The internal solution for action potential recording was also the same solution as that used for M/KCNQ currents using perforated patch recording. Single M/KCNQ current was recorded under cell-attached patch. The protocol for recording single M/KCNQ current is as follows: Cells bath perfused with high K+ external solution (63 mM) had a resting membrane potential near -20 mV. Membrane potentials (Vm) were therefore calculated as Vm=Vrest -Vpipette, where Vrest was taken to be -20 mV and Vpipette was the voltage applied. When applied 0 mV, the patch membrane will be clamped at -20 mV and at this level, single M/KCNQ channel will be open. The data were sampled at 5-10 kHz after filtered at 0.5-2 kHz. Transitions between open and closed states were detected by setting the threshold to 50% of the open channel level. Sylgard-coated pipettes had resistances of 8-10 MΩwhen filled with pipette solution consisting of (mM): NaCl 125, KCl 3, MgCl2 1.2, HEPES 10, Glucose 11 and apamin 200 nM, charybdotoxin 100 nM,αandβdendrotoxins 300 nM, tetrodotoxin 250 nM (pH 7.3). The extracellular solution consisted of (mM): NaCl 65, KCl 63, CaCl2 0.5, MgCl2 1.2, HEPES 10, Glucose 11 and CgTx-GVIA 250 nM, nifedipine 10μM, tetrodotoxin 250 nM (pH 7.3 with KOH). (3) Data were analyzed using clamfit and origin software.
Results: (1) Rat SCG neurons can be divided into three electrophysiological phenotypes based on their action potential firing patterns: Phasic-1, Phasic-2 and Tonic neurons. Phasic-1 neurons, making up 36% of total SCG neurons studied, fired only one spike during the period of stimulation even with increased current injection. Phasic-2 neurons, seen in 54% of neurons, fired two to six spikes, but fired more frequently in response to the increased current injection, and the phasic firing pattern could be converted to a tonic firing pattern. Tonic neurons were seen in 10% of SCG neurons. Tonic neurons fired action potentials in a sustained manner even with a minimal stimulus, and the number of spikes increased with increased current injection. The resting potential between phasic-1 and phasic-2 neurons had no significant difference, but they were higher than that of tonic neurons and there had a significant difference between phasic and tonic neurons. The spike number of tonic neurons was much more than that of phasic-1 and phasic-2 neurons, and there had significant difference between tonic and phasic neurons, whereas, there was no significant diferrence between the two phasic neurons. (2) Three kinds of neuronal excitatory pattern in SCG neurons were tightly related to their M/KCNQ currents. We first identified the excitatory pattern of SCG neurons under current-clamping mode, and then use voltage-clamping mode to record the neuronal M/KCNQ currents in the same cell. The shape and amplitude of M/KCNQ currents were similar in phasic-1 and phasic-2 neurons, and the amplitude of tail M/KCNQ current at -60 mV was relatively big. On other hand, the amplitude of M/KCNQ current from tonic neurons was relatively small. The density of tail M/KCNQ currents at -60 mV from phasic-1, phasic-2 and tonic neurons were 2.8±0.2,2.3±0.2 and 0.9±0.1 pA/pF, respectively. The two phasic neurons were significantly different from tonic neurons but no significant difference existed between two phasic neurons. The current-voltage (I-V) relationship curves of the three type neurons were also different. The half-activation voltages for phasic-1, phasic-2 and tonic neurons were -30±1, -29±1 and -15±3 mV, respectively. Compared with phasic neurons, the I-V curve of tonic neurons was positively shifted. A detailed analysis of the relationship between spike number and the density of M/KCNQ tail currents was made. It appeared that the density of M/KCNQ tail currents was diagnostic in separating SCG neurons into either phasic or tonic neurons. Specifically, a line of demarcation was located at an M/KCNQ current density level of -1 pA/pF. We used single exponential equation to fit the kenitics of M/KCNQ currents activation (at -20 mV) and deactivation (at -60 mV) and obtained the the corresponding time constants, respectively. The time constants of activation for phasic-1, phasic-2 and tonic neurons were 60±5, 64±4 and 99±12 ms, respectively. The time constants of deactivation for phasic-1, phasic-2 and tonic neurons were 51±2, 60±6 and 96±5 ms, respenctively. Both activation and deactivation of tonic neurons were significantly slower than phasic neurons, but no significant differences were found between phasic-1 and phasix-2 neurons. (3) NGF inhibited M/KCNQ currents in rat SCG neurons. NGF at concentration of 20 ng/ml (within the range of reported physiological concentration of NGF in mammalians) inhibited the M/KCNQ currents of SCG neurons both in phasic and tonic neurons. NGF inhibited M/KCNQ currents by 25±2% and 26±3% in phasic and tonic neurons, respectively, and there had no significant differences between them. Application of NGF in the presence of 5μM linopirdine after the M/KCNQ currents was inhibited by linopirdine did not further inhibit the currents. The inhibition was 86.3±3.2% and 86.6±3.3% before and after application of NGF, and these were not significantly different. Oxo-M, a muscranic receptor agonist, also strongly inhibited M/KCNQ currents. NGF also inhibited M/KCNQ currents by 34±4% under the conventional whole-cell patch mode. Under this condition, we established the concentration-response curve for NGF-induced inhibition of M/KCNQ currents and fitted this curve by Hill function. The half-maximal inhibition (IC50) was 0.7±0.1 ng/ml and the coefficient was 0.9±0.1. AG879 (50μM), a specific inhibitor of Trk A receptor (one of the two NGF receptors, Trk A receptor and p75 receptor), when applied in pipette solution, siginificantly reduced NGF-induced inhibition of M/KCNQ currents from 34±4% to 17± 3%, whereas it had no effect on oxo-M-dediated inhibition. These results suggested that NGF inhibited M/KCNQ currents through activation of TrkA receptor. Bath applied genistein (100μM), a broad spectrum inhibitor for cellular protein tyrosine kinase, reduced significantly NGF-induced inhibition of M/KCNQ currents from 34±4% to 7±4%. Bath application of U73122, a commonly used inhibitor of phospholipase C (PLC), also significantly reduced the inhibitory effect on M/KCNQ currents mediated by NGF from 34±4% to 12±5%. These data suggested that NGF inhibited M/KCNQ currents through Trk A receptor and its downstream signal pathways, possibly involving both tyrosine phosphorylation and PI(4,5)P2 hydrolysis. (4) NGF inhibited single M/KCNQ current in cell-attached patches. NGF significantly reduced M/KCNQ channel Po by 29±3%. Oxo-M (3μM) strongly reduced the M/KCNQ channels Po by 89±2%. These data were consisitent with the whole-cell experiment. NGF did not change the conductance of the single M/KCNQ channel. The conductances were 6.1±0.2 and 6.5±0.3 pS, before and after applied NGF and had no significant difference. Oxo-M did not change the channel conductance either. The conductance was 6.4±0.3 pS in the presence of oxo-M. Both NGF and oxo-M reduced the M/KCNQ channel Po at each voltage level but only NGF significantly decreased the half-activation voltage (V1/2) from -32±3 mV to -25±2 mV. (5) NGF increased the excitability of tonic neurons but not phasic neurons. NGF significantly increased the number of spikes fired in tonic neurons from 12±2 to 20±2. NGF did not significantly change the resting potential of tonic neurons (the resting potential are -47±3 mV and -47±4 mV, before and after NGF, respectively). In the same batch of tonic neurons, oxo-M (10μM) and linopirdine (30μM) rapidly and significantly increased the spike number from 12±2 and 12±5 to 28±4 and 30±4, respectively. Oxo-M and linopirdine also induced small depolarization, but the changes did not reach the statistical significance. In the case of phasic-1 and phasic-2 neurons, NGF neither significantly changed their excitability nor significantly changed their resting potential level. On other hand, oxo-M and linopirdine significantly increased the excitability of both phasic-1 neurons (oxo-M and linopirdine enhanced the spike number from 1 to 4.7±1.2 and 3.2±1, respectively) and phasic-2 neurons (oxo-M and linopirdine enhanced the spike number from 3.2±0.7 and 3.1±0.5 to 31±6 and 16±2, respectively). Both Oxo-M and linopirdine significantly depolarized the resting potential of phasic-2 neurons (the two agent depolarized membrane from -58±2 mV and -50±3 mV to -49±2 mV and -43±2 mV, respectively); they did not affect the resting membrane potentials of phasic-1 neurons. (6) Modulation of neuronal excitability was tightly related to function of neuronal M/KCNQ channels. In phasic neurons, NGF (20 ng/ml) alone inhibited M/KCNQ currentd by 20±2%, whereas following adminstration of oxo-M (10μM) inhibited M/KCNQ currents by 62±7% in the presence of NGF. Under this condition, NGF alone did not change the excitability of phasic neurons but a significant enhancement of the neuronal excitability was produced by oxo-M in the presence of NGF; Oxo-M increased the spike number from 4.4±0.7 to 22±5 in the presence of NGF. In a reversed sequential application of these two drugs, oxo-M alone inhibited M/KCNQ currents by 72±8% and following adminstration of NGF did not further inhibit M/KCNQ currents in the presence of oxo-M. Oxo-M alone increased the spike number of phasic-2 neurons from 3.4±1.2 to 12±5, whereas, additional application of NGF did not show a significant effect. (7) Small inhibition of M/KCNQ currents by low concentration of linopirdine mimiced the effect of NGF on neuronal excitability. That NGF failed to modulate the excitability of phasic neurons may be due to its insufficient inhibition of the relative large M/KCNQ current densities. We choose linopirdine, a specific M/KCNQ channel blocker, to verify this hypothesis. We first established the concentration-response relationship curve of linopirdine-induced inhibition of M/KCNQ currents to find a proper concentration of linopirdine with a similar inhibitory effect to that seen with NGF. Linopirdine began to inhibit M/KCNQ currents at 0.3μM and reached its maximal inhibition at 30μM. The half-maximum inhibitory concentration was 2.1±0.2μM. According to this curve, 0.7μM linopirdine would inhibit M/KCNQ current by 25%, similar to the inhibition mediated by NGF (20 ng/ml). Linopirdine at concentration of 0.7μM significantly enhanced the excitability of tonic neurons by increasing its spike number from 12±1 to 18±2. However, this concentration of linopirdine did not alter the excitability of phasic-1 and phasic-2 neurons. Thus, the selective modulation of excitability of tonic neurons by NGF was likely due to ite moderate capability in inhibiting M/KCNQ current.
Conclusion: (1) Rat SCG neurons can be divided into three electrophysiological phenotypes based on their action potential firing patterns: phasic-1, phasic-2 and tonic neurons. Phasic-1 and phasic-2 neurons expressed relatively large M/KCNQ current density, which will render the cell low excitability and difficulty to fire action potentials. Tonic neurons expressed relatively small M/KCNQ current density, thus will have high excitability and easy to fire action potentials. (2) NGF at physiological concentration can significantly inhibit M/KCNQ currents of SCG neurons. NGF may inhibit M/KCNQ current through activation of Trk A receptor and its downstream signal pathways, possibly involving both tyrosine phosphorylation and PI(4,5)P2 hydrolysis. NGF inhibited M/KCNQ currents in a similar degree among phasic-1, phasic-2 and tonic neurons. (3) NGF only increased neuronal excitability of tonic neurons. Phasic neurons may still have relative large M/KCNQ currents left after inhibition induced by NGF, and the residual M/KCNQ currents were sufficient to stabilize cellular membrane potential. (4) Low concentration of linopirdine, who had the similar inhibitory effect to NGF, mimiced the effects of NGF on neuronal excitability.
2. The regulation of voltage-gated sodium currents and neuronal excitability by genistein.
Aim: To study the mechanisms involve in the regulation of VGSC currents induced by genistein, a broad spectrum inhibitor of cellular protein tyrosine kinase.
Method: Voltage-gated sodium currents (VGSC) and neuronal action potentials were recorded from rat SCG neurons. (1) Cell culture. SCG neurons were primary cultured with the same procedur describered in part one. (2) Electrophysiology. Using perforated patch clamp technique to record VGSC currents under voltage-clamping mode and record neuronal action potentials under current-clamping mode. Pipettes were pulled from borosilicate glass capillaries and had a resistance of 3-5 M? when filled with internal solution. Series resistance compensation has always been used and up to 80-90% compensation can be reached in our condition. Under this condition, the maximum access resistance was about 2 M?. The sampling rate was 10 KHz for membrane currents and was 2.5 KHz for membrane potential recordings. The protocol used to record the VGSC currents was as follows: the cells were held at -70 mV and a 20 ms depolarizing step to 0 mV was applied every 3 s. The action potentials were elicited by an approximate two-fold threshold depolarizing current of 0.1 nA. The external solution used to record the VGSC currents contained (in mM): NaCl 120, KCl 3, HEPES 5, NaHCO3 23, glucose 11, MgCl2 1.2, CaCl2 2.5, BaCl2 0.2, CdCl2 0.2, (adjusted to pH 7.4 with NaOH). The internal solution for VGSC currents recording consisted of (in mM): CsCl 90, KCl 40, HEPES 20, MgCl2 3 (adjusted to pH 7.3-7.4 with CsOH). The external solution used to record neuronal action potentials contained (in mM): NaCl 120, KCl 3, HEPES 5, NaHCO3 23, glucose 11, MgCl2 1.2, CaCl2 2.5, (adjusted to pH 7.4 with NaOH). The internal solution for action potential recording contained (in mM): KAC 90, KCl 40, HEPES 20, MgCl2 3 (adjusted to pH 7.3-7.4 with KOH). (3) Cell immunofluorescence and confocal imaging. Briefly, neurons were incubated with primary rabbit anti-Nav1.7 Ab or anti-Nav1.1 Ab overnight at 4°C, followed by three washes with PBS and incubation with goat anti-rabbit IgG-TRITC sencondary Ab for 30 min at 37°C. Cells were examined on an inverted laser-scanning microscope. TRITC was excited at 564 nm and the emitted fluorescence signal was at 570 nm. (4) Data were analyzed using clamfit and origin software.
Results: (1) Predominantly expressed voltage-gated sodium channel in rat SCG neurons was Nav1.7 channel. Cell immunofluorescence and confocal imaging experiment indicated that, Nav1.7 channel was dominantly expressed in rat SCG neurons. Nav1.1 channel, another voltage-gated sodium channel, was also weakly expressed in SCG neurons. (2) Genistein significantly inhibited the voltage-gated sodium currents in SCG neurons. 100μM genistein strongly inhibited VGSC currents by 73.3±5.4%. A double phase inhibition was evident for genistein-induced inhibition: an initial fast inhibition followed by a relatively slow inhibition. These two processes were fitted with a double exponential function and time constants were obtained. The fast time constant was 10.6±1.2 s, and the slow time constant was 55.9±2.4 s, respectively. Genistein inhibited VGSC currents in a concentration dependent manner. The concentration of half-maximal (IC50) inhibition was 9.1±0.9μM and the coefficient was 1.1±0.2. VGSC currents in SCG neurons were TTX-sensitive currents since 0.05μM TTX reversally and compeletly inhibited them. Voltage-dependence of genistein-indcued inhibtion was tested. 50μM genistein obviously inhibited VGSC currents at all voltages tested, more significantly at the range from -20 mV to 0 mV, the range of maximal activation. Genistein significantly shifted the voltage-dependent activation of VGSC current to the right, but did not affect voltage-dependent inactivation. The half-maximal activation voltage (V1/2) was significantly more positive for genistein-treated neurons (-20.6±0.1 mV) than in control cells (-32.4±0.2 mV). The half-maximal inactivation voltage were -41.3±0.4 mV for genistein and -41.8±0.3 mV for controls, respectively. (3) Genistein-indeuced inhibition of VGSC currents involved two mechanisms: PTK-independent and PTK-dependnent mechanisms. Daidzein is an inactive structural analog of genistein. Daidzein also inhibited VGSC currents in a concentration dependent manner and reached its maximal inhibitory effect at 100μM (the inhibition is 28.5±3.1 %). Fitting the curve with Hill function produced an IC50 of 20.7±0.1μM and a coefficient of 1.2±0.2. When daidzein and genistein were co-applied, daidzein (100μM) alone inhibited VGSC currents by 27.8±3.6%, additional applied genistein (100μM) inhibited VGSC currents by 57.3±10.1%. These data suggested that genistein inhibited VGSC currents through PTK-independent and other menchanisms. We tested the effect of Vanadate, a broadly used inhibitor of cellular protein tyrosine phosphotase. The effect of vanadate will antagonize the effect of genistein. Genistein alone inhibited VGSC currents by 57±5.4%, additional application of vanadate (1 mM, a saturate concentration) partly rescued the genistein-induced inhibition from 57±5.4% to 39.5±4.7%. Pretreatment with vanadate significantly reduced the genistein-induced inhibition of VGSC current by 18.7±3.3%. These data suggested that anti-PTK activity were partly responsible for the VGSC current inhibition produced by genistein. We also tested other three functional analog of genistein, tyrphotin 23, erbstatin analog and PP2 in their saturate concentration of 100μM, 100μM and 1μM, respectively. The three agents all weakly but significantly inhibited VGSC currents, and their inhibitory effect were 14.6±0.5%, 19.3±1.0 and 17.8±1.9%, respectively. At parallel experiments, gensitein inhibited VGSC currents by 54-58%. These data further suggested that genistein inhibited VGSC currents partly involved the PTK-dependent mechanism. (4) Genistein depressed the neuronal excitability and reduced the depolarized rate of action potential but did not change the resting potential of SCG neurons. Bath application of genistein (100μM) immediately suppressed the neuronal excitability by reducing the action potential number from 8.7±2.1 to 1.3±0.3. However, genistein did not significantly change the neuronal resting potential; the resting potentials were 56.1±3.1 and 54.7±4.1 before and after genistein application, respectively. Genistein significantly reduced the depolarization rate of action potential from 7.0±1.3 to 4.4±0.9 V/s.
Conclusion: (1) Genistein inhibited VGSC currents through PTK-dependent and PTK-independent mechanisms. (2) Genistein suppressed neuronal excitability and reduced the depolarization rate of SCG neurons.
3. Detection of intracellular Ca2+ signals in DRG neurons induced by G(q) protein-coupled receptors.
Aim: Do study the intracellular Ca2+ signals induced by activation of G(q) protein-coupled receptors expressed in rat DRG neurons.
Methods: (1) Cell culture. The procedur of primary culture for rat DRG neurons is similar to SCG neurons described above. DRG neurons were prepared from 14 day-old Sprague-Dawley rats. (2) Ca2+ imaging. Neurons were loaded with fura-2 AM (5μM) in the presence of pluronic F-127 (20%) and imaged using a Nikon TE-2000 microscope equipped with a Hamamatsu photonics Ca2+ imaging system. Fura-2 was excited at 340 and 380 nm and images were analyzed with simplePCT 6.0. (3) Solutions. Bath solution of DRG neurons contained (mM): NaCl 145, KCl 5, MgCl2 2, CaCl2 2, HEPES 10, Glucose 10 and adjusted to pH 7.4 with NaOH. In the Ca2+-free bath solution, Ca2+ was omitted and 3 mM EGTA was added. (4) Drugs. In this part of eaperiment, bradykinin (BK, 100 nM) was used as a positive control, capsaicin (CAP, 1μM) and acrolein (ACR, 0.1 mM) was used to identify the noceptive DRG neurons. KCl (50 mM) was used to identify the DRG neurons.
Results: (1) Identification of the DRG neurons. When bathed with normal extracellular solution (with Ca2+), 349 DRG neurons (identified by 50 mM KCl) were studied. In the following description of the results, the number of neurons which show an intracellular Ca2+ rising in responding to a particular agonist will be called as the agonist-sensitive neurons. Among 349 DRG neurons, 109 neurons were BK-sensitive neurons, making up 31.2% of the total neurons. 74 neurons were CAP-sensitive neurons, or 21.2% of the total DRG neurons. 62 neurons were ACR-sensitive neurons, 17.8% of the total DRG neurons. Among BK-sensitive DRG neurons, CAP- and ACR-sensitive neurons were 31 and 29, making up 28.4% and 26.6% of the BK-sensitive neurons, respectively. When bathed with Ca2+-free external solution, 57 DRG neurons were studied. Among the 57 neurons, 4 neurons, making up 7% of the total, were sensitive to BK; 1 neuron, making up of 1.8%, was sensitive to CAP; 1 neuron, making up of 1.8%, was sensitive to ACR, respectively. Among the 4 BK-sensitive DRG neurons, CAP- and ACR-sensitive neurons were 1 and 1, making up 25% and 25%, respectively. (2) Cholecystokinin receptors (CCK receptors). CCK receptors were activated by CCK-8 (1μM). In 131 DRG neurons bathed with normal external solution, CCK-8 elicited Ca2+ signals in 46 neurons, or 35.1% of the total neurons. Under Ca2+-free condition, No neurons out of 57 DRG neurons studied were sensitive to CCK. (3) Endothelin (ET) receptors. ET receptors were activated by ET-1 (100 nM). In Ca2+ extracellular solution, ET-1 elicited intracellular Ca2+ signals in 49 neurons out of 101 DRG neurons studied, making up a ratio of 48.5%. Among 50 DRG neurons bathed in Ca2+-free condition, 5 neurons were sensitive to ET-1. (4) Mrg D receptors. Mrg D receptors, a kind of orphan proteins, were abundantly expressed in DRG neurons and were activatied byβ-Alanine (500μM). In Ca2+ extracellular solution, 12 neurons out of 134 DRG neurons studied were sensitive toβ-Alanine. In Ca2+-free condition, No neurons were sensitive toβ-Alanine. (5) Histamine (H1) receptors. H1 receptors were activatied by Histamine (100μM). In normal external solution, Histamine only elicited Ca2+ signals in 10 neurons out of 95 DRG neurons. (6) Substance P receptors. Substance P receptors, also called NK1 receptors, were activated by substance P (SP, 1μM). When bathed with normal extracellular solution, 55 DRG neurons (identified by 50 mM KCl) were studied. Only one neuron was sensitive to SP. (7) 5-HT receptors. 5-HT receptors were activated by 5-HT (10μM).When bathed with normal solution, 55 DRG neurons were studied. No neurons were sensitive to 5-HT. (8) Angiotensin II receptors (AT1 receptors). AT1 receptors were activated by angiotensin II (100 nM). When bathed with normal external solution, 44 DRG neurons were studied. Among the detected 44 neurons, only 2 neurons were sensitive to angiotensin II. (9) Purinergic Y receptors (P2Y receptors). P2Y receptors were activated by adenosine triphsphate (ATP, 100 nM). In normal extracellular solution, 43 DRG neurons were studied, but no neurons were sensitive to ATP.
Conlusion: Among agonists of Gq-copupled receptors we stuided, agonits of BK receptors, CCK receptors, ET receptors, MrgD receptors and histamine receptors elicited intracellular Ca2+ signals, whereas agonists of substance P receptors, angiotensin II receptors, 5-HT receptors and purinergic receptors could not elicit the intracellular Ca2+ signals.
4. Modulation of TRPV1 currents in DRG neurons by capsaicin and CCK.
Aim: To study modulation of TRPV1 currents in DRG neurons by GPCR.
Methods: (1) Cell culture. Primary culture of rat DRG neurons was described above. (2) Electrophysiology. Perforated whole-cell patch was used to record currents. The protocol used to record TRPV1 and other inward currents was as follows: neurons were held at -60 mV constantly, BK and other GPCRs agonists were applied to elicit inward currents, and specific blockers of TRPV1 and Ca2+-activated chloride currents (CaCC) were used to verify the identity of the inward currents. Pipettes were pulled from borosilicate glass capillaries and had a resistance of 3-5 M? when filled with internal solution. The sampling rate was 2.5 KHz after filtered at 0.5 KHz. The extracellular solution contained (mM): NaCl 145, KCl 5, MgCl2 2, CaCl2 2, HEPES 10, Glucose 10 and adjusted to pH 7.4 with NaOH. In the Ca2+-free bath solution, Ca2+ was replaced by 0.1 mM EGTA.
Results: (1) Capsaicin elicited fast desensitizing TRPV1 currents in DRG neurons. Capsaicin (CAP, 1μM) elicited TRPV1 currents in CAP-sensitive nociceptive DRG neurons. Application of capsazepine (CZP, 100μM), a specific TRPV1 channel blocker, eliminated the capsaicin-induced inward currents at -60 mV. TRPV1 current reached its maximal value within 20 ~ 30 s, and then desensitized rapidly. The current amplitude at 5min was significantly smaller than the initial TRPV1 currents, about 30.5%±3.6% of the initial amplitude of the TRPV1 currents. The peak currents of TRPV1 from two sequential 30 s applications of CAP interrupted by a 5-6 min period of no CAP gave very different values The amplitude of the second CAP-induced TRPV1 currents was only about 50.7%±3.8% of that of the first CAP-induced TRPV1 currents. (2) CCK-8 elicited a small inward current in DRG
引文
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