Ionic basis of noradrenaline depolarization and hyperpolarization in toad dorsal root ganglia neurones

Summary The membrane conductance and reversal potential were estimated for neurones in toad dorsal root ganglia with intracellular recording technique during depolarization or hyperpolarization induced by noradrenaline (NA). The effects of blocking agents for potassium or calcium ion current on NA-induced membrane potential responses were observed as well. The NA depolarization was accompanied by a 32.6% decrease of membrane conductance. In a few neurones, the membrane conductance showed an initial increase and a following decrease. The NA hyperpolarization was associated with an increase of membrane conductance by 16.2%. The mean reversal potential of NA depolarization was -88.5 ± 0.9mV. The NA hyperpolarization responses were nullified at -89 to -92 mV of membrane potential. TEA superfusion enhanced NA depolarization amplitude by 73.7 ± 11.9% and depressed NA hyperpolarization amplitude by 40.5%. CsCl (intracelluar injection) increased phenylephrine depolarization by 34.5 %. MnCl₂ superfusion decreased the amplitudes of NA depolarization by 50.5 ± 9.9%, and of NA hyperpolarizalion by 89.5±4.9% respectively. The results suggest that depolarization or hyperpolarization induced by NA might be mediated by the alteration of K or Ca channels.     

离体脊髓运动神经元谷氨酸反应的细胞电生理特征

目的观察离体脊髓运动神经元(MN)谷氨酸(Glu)反应的细胞电生理特征。方法应用新生大鼠(7~19d)脊髓切片MN细胞内记录和细胞旁压力喷射Glu(100 mmol/L,200 kPa,4~300 ms)技术,测量Glu反应的细胞电生理参数,并观察相关药物的影响。结果在23个MN的Glu反应幅度为(14.7±8.8)mV,时程为(24.1±17.3)s,呈现明显的浓度依赖性,反应期间膜电阻减小了(13.1±6.3)%(P<0.001),Glu反应在超极化时增大。用河豚毒素(TTX,0.3μmol/L)预处理后,Glu反应降低为对照的(39.2±28.3)%(P<0.05,n=5)。用TTX和印防己毒素(50μmol/L)预处理后,Glu反应幅度增大为对照的(175.7±67.1)%(P<0.05,n=5);而用TTX、毕扣扣灵(50μmol/L)和士的宁(1μmol/L)预处理后,Glu反应的变化没有显著性意义(P>0.05,n=3)。结论外源性Glu对脊髓MN具有直接兴奋作用,并受到抑制性氨基酸受体的调制。     

Noradrenaline depolarization and hyperpolarization mediated by alpha-adrenergic receptors in the soma of dorsal root ganglion neurons

Summary Intracellular recordings were made on perfused preparation isolated from toad dorsal root ganglion (DRG). Of the 51 neurones examined, 46 were of type A, and the remaining 5 of type C cell. The resting membrane potential of these two types of cells was ™60.06±1.30 mV ( ±SE). When the preparations were superfused with noradrenaline (NA; 10⁻⁴ ™ 10⁻³M), the changes of Rp were as follows: 1) hyperpolarization, with amplitude of 8.38 ± 1.42mV ( ±SE; 20/48); 2) depolarization, with amplitude of 9.39±1.24mv ( ±SE; 23/48); 3) no effect (5/48). The alteration in membrane potential mentioned above could be neither mimicked by application of isoproterenol, nor blocked by propranolol. Therefore, the possibility of the effect being mediated by β-adrenoceptor could be excluded. Application of phenylephrine and clonidine induced depolarization and hyperpolarization of membrane potential respectively, while application of prazosin eand yohimbine blocked depolarization and hyperpolarization response induced by NA, respectively. From the results mentioned above, it may be concluded that depolarization and hyperpolarization induced by NA are mediated by α₁- and α₂-adrenoceptors on the soma of DRG neurones respectively.     

大戟二萜醇酯对大鼠颈上神经节胆碱能传递的影响

应用细胞内生物电记录技术观察了两种大戟二萜醇酯对大鼠颈上神经节胆碱能传递的影响.灌流5或10μmol/L 的4β-大戟二萜醇-12,13-双丁酯(PDB)增大膜电阻而不影响膜电位,对刺激节前神经诱发的快兴奋性突触后电位(f-EPSP)及外源性乙酰胆碱(ACh)引起的 ACh电位均有浓度依赖性的增强作用,并诱导自发性 EPSP,表明 PDB 通过突触前、后机制易化交感神经节的胆碱能传递.4β-大戟二萜醇-12-肉豆蔻酯-13-乙酯(PMA,10μmol/L)灌流30min 对神经节细胞的电生理性质及其胆碱能传递无明显影响.     

阿扑吗啡对离体蟾蜍交感神经节细胞的作用

应用离体蟾蜍椎旁神经节（ＰＶＧ）细胞内记录技术，初步观察了阿扑吗啡（ＡＰＯ）对交感神经节细胞的作用。ＡＰＯ１，３和１０ｍｍｏｌ／Ｌ灌流３０ｓ分别使６４％（ｎ＝２５），８３％（ｎ＝２４）和１００％（ｎ＝１１）的ＰＶＧ细胞产生浓度依赖性的缓慢去极化反应，在部分细胞可伴有输入膜电阻减少和去极化幅度随膜超极化而增大，但在全部受试细胞均未记录到确切的超极化反应。ＡＰＯ０．１～３ｍｍｏｌ／Ｌ灌流１５ｍｉｎ不仅使全部受试细胞产生去极化反应（ｎ＝２０），还使电刺激交感节前纤维诱发的顺行动作电位起始段上升速率减慢，超极化后电位增大、去极化后电位减小，并在０．３ｍｍｏｌ／Ｌ以上的浓度使顺行动作电位转为兴奋性突触后电位或完全取消突触反应，但对直接动作电位无明显影响。另外，ＡＰＯ还可逆地降低外源性Ａｃｈ电位的幅度（ｎ＝１０）。结果表明ＡＰＯ对离体蟾蜍交感神经节细胞有去极化作用并抑制其胆碱能突触传递。     

离体新生大鼠运动神经元的电学特性

在新生大鼠脊髓切片,对经逆行激活鉴定的运动神经元进行常规细胞内记录,测得静息电位-67±3mV,膜电阻91±50MΩ,时间常数4.7±1.6ms,I-V曲线显示轻度正常整流性质。直接动作电位幅值74±4mV,阈电位-52±6mV,I-F曲线表明紧张型放电频率随去极化电流增大而升高。在4个细胞记录到自发的锋电位发放,其频率随膜超极化而降低,是否节律性活动有待进一步研究。     

大鼠脊髓背角胶状质神经元的去极化反跳及调控机制

目的 研究大鼠脊髓背角胶状质(SG)神经元的去极化反跳及调控机制,以期对去极化反跳相关疾病的临床治疗提供参考.方法 选取3～5周龄SD大鼠,制作离体脊髓纵切片,应用全细胞膜片钳技术记录SG神经元的电生理学特点及接受超极化刺激后的反应,并观察超极化激活环核苷酸门控阳离子(HCN)通道阻断剂和T型钙(Cav3)通道阻断剂对去极化反跳的作用.结果 共记录了63个SG神经元的电活动,其中23个无去极化反跳,19个为去极化反跳无放电,21个为去极化反跳伴放电.无去极化反跳组SG神经元的动作电位阈值(-28.7±1.6 mY)明显高于去极化反跳伴放电组(-36.0±2.0 mV)(P＜0.05).HCN通道阻断剂氯化铯和ZD7288可显著延长去极化反跳伴放电的潜伏期,分别从45.9±11.6 ms增加到121.6±51.3ms(P＜0.05)和从36.2±10.3 ms增加到73.6±13.6ms(P＜0.05);ZD7288也能显著延长去极化反跳不伴放电的潜伏期,从71.9±35.1 ms增加到267.0±68.8 ms (P＜0.05),而T型钙通道阻断剂氯化镍和米贝地尔可显著降低去极化反跳伴放电的振幅,分别从19.9±46.3 mV降到9.5±4.5 mV(P＜0.05)和从26.1±9.4 mV降到15.5±5.0mV(P＜0.05),米贝地尔同样能显著降低去极化反跳不伴放电的振幅,从14.3±3.0 mV降低至7.9±2.0 mY(P＜0.05).结论 近2/3的SG神经元有去极化反跳,其潜伏期和振幅分别受HCN通道和T型钙通道调控.     

大鼠海马脑片锥体神经元的细胞内记录

从成年大鼠分离海马结构并用震动切片机制备500μm 厚的海马脑片,应用玻璃微电极技术进行海马锥体层神经元的细胞内记录.对 CAl 锥体神经元的被动膜电性质、I-V 关系曲线、动作电位等进行了观察,发现灌流 Immol/L 的谷氨酸钠或乙酰胆碱均引起去极化,并伴有强烈放电.结果证明海马脑片神经元的细胞内记录是中枢神经电生理和药理学研究的一种稳定可靠的方法.     

下丘脑冠状切片视上核神经元的电生理特性

目的了解下丘脑冠状切片视上核神经元的电生理特性。方法在下丘脑冠状切片（厚５００μｍ）对视上核神经元进行细胞内生物电记录。结果测得静息电位（－５８±７） ｍＶ（ｘ± ｓ, ｎ＝４４）,膜斜率电阻（１９５±８３）ＭΩ,时间常数（１０．８± ５．０）ｍｓ,膜电容（６０±２０）ｐＦ,动作电位幅度（６７± ９）ｍＶ,阈电位（－４２ ±８）ｍＶ,半幅时程（２．１±１．１）ｍｓ,超射（１１±５）ｍＶ。在记录的１１３个细胞中,３１．９％的细胞有自发锋电位发放,其中５．５％为持续型（紧张型）放电,７０．６％为时相型放电,其余为不规则放电。在时相型放电的细胞可观察到自发的慢去极化电位。长时程去极化时锋电位发放呈明显的适应现象。顺行电刺激可诱发兴奋性突触后电位,并具有刺激强度依赖性。结论提示视上核神经元作为神经内分泌细胞具有与其他神经元不同的电生理特性。     

A study of calcium ion-selective PVC membrane electrode based on neutral carrier N,n,n’,n’-tetracyclo-3-oxapetanediamide

Summary A calcium ion-selective PVC membrane electrode based on neutral carrier n,n,n’,n’-tetracyclohexyl-3-oxapetanediamide, using di-(2-ethylhexyl) phthalate as the plasticizer and potassium tetrakis (4-chlorophenyl) borate as the additive is reported in this paper. The ion selective membrane consists of 1 wt % of the Ca²⁺ selective ligand, 65 wt % of the plasticizer, 1 wt % of the additive and 33 wt % of poly (vinyl chloride) powder. The electrode has the linear response range of 2.0 × 10^™7 —10^™1 mol/L with the Nernstian slope of 28 mV/decade at 25 °C and the detection limit of 2.0 × 10^™8 mol/L. The response time of the calcium ion-selective electrode is 4s as the concentration of calcium ion is rapidly shifted from 10^™5 to 10^™4 mol/L. The potential stability and reproducibility are good. The free calcium in blood serum was determined by the calcium ion-selective electrode with satisfactory results.     

Electrophysiological properties of neurons of guinea pig celiac ganglia

Summary The present study was carried out to investigate electrophysiological properties of neurons of guinea pig celiac ganglia in vitro. The mean value of resting membrane potential was-59.3 ±5.6 mV (n = 29). The mean values of membrane resistance, time constant and membrane capacity were 53.7±19.8mΩ, 18.4±8.5 ms, and 375.9±175.0 pF (n = 24) respectively. When depolarizing electrotonic potentials induced by application of depolarizing current pulses reached the threshold level, all of the neurons could be made to discharge action potentials. The mean values of amplitude, overshoot, duration of the spikes were 68.0±15.3 mV, 11.7 ±4.6mV, and 2.7±0.6ms (n = 29) respectively. 75% of the neurons tested (n = 29) produced tonic (slow adapting) firing in response to depolarizing current lasting 5 s. The remaining neurons (25%) produced phasic (fast adapting) firing. In addition, fast excitatory postsynaptic potentials or orihodromic action potentials were recorded from 80% of the neurons, antidromic action potentials were recorded from ihe remaining neurons during stimulation of the splanchnic nerves.     

山莨菪碱对离体坐骨神经-腓肠肌烟碱型胆碱受体的阻断作用

目的探讨山莨菪碱对运动终板膜上烟碱型乙酰胆碱受体的作用。方法以离体蟾蜍坐骨神经－腓肠肌为研究对象，用浓度为０．１、０．３和１．０ｍｍｏｌ·Ｌ－１的盐酸山莨菪碱灌流２６．０±８．５ｍｉｎ（ｎ＝２７），观察电刺激坐骨神经后，骨骼肌单收缩曲线的变化。结果浓度为０．３和１．０ｍｍｏｌ·Ｌ－１的山莨菪碱均可使骨骼肌收缩期和舒张期时程缩短，收缩幅度下降，且可逆，但曲线不能完全消失，潜伏期时程无变化。结论高浓度的山莨菪碱对离体坐骨神经－腓肠肌烟碱型乙酰胆碱受体有一定阻断作用。     

非对称性二甲基精氨酸对人阻力血管EDVD的抑制作用

目的探讨非对称性二甲基精氨酸(ADMA)对人离体阻力血管环内皮依赖性舒张(endothelium-dependent vasodilatation,EDVD)的抑制作用。方法在血液透析患者行动-静脉内瘘成形术时留取桡动脉,制备血管环,用机械法去内皮并设立内皮存在组和内皮缺失组。应用离体血管张力记录仪观察2组血管环在不同质量浓度ADMA(10-7mol/L、10-6mol/L、10-5mol/L、10-4mol/L和10-3mol/L)作用下张力的变化。内皮存在组用10-5mol/L苯肾上腺素(phenylephrine,PE)引发血管环收缩,再用质量浓度为10-5mol/L的乙酰胆碱(ACh)引发EDVD,然后用不同质量浓度ADMA处理,观察ADMA在内皮存在情况下对ACh所引起EDVD的抑制作用。内皮缺失组用质量浓度为10-5mol/L的PE引发血管环收缩后,再用质量浓度为10-7mol/L的硝普钠(SNP)引发非内皮依赖性血管舒张(EIVD),然后用不同质量浓度ADMA处理,观察ADMA在内皮缺失情况下对SNP引起EIVD的抑制作用。结果内皮存在组用10-5mol/L的ACh处理可使67.10%±18.63%因PE(10-5mol/L)引起收缩的血管舒张,ADMA对ACh引起的EDVD呈浓度依赖性抑制作用,相对收缩幅度依次为EDVD幅度的7.32%±8.60%(10-7mol/L)、20.03%±13.49%(10-6mol/L)、29.93%±11.78%(10-5mol/L)、43.30%±11.29%(10-4mol/L)和80.21%±18.16%(10-3mol/L)。在内皮缺失组,ADMA对SNP引起的EIVD呈微弱的抑制作用,且不表现为浓度依赖性。相对收缩幅度为EIVD的2.76%±1.98%(10-7mol/L)、2.27%±1.82%(10-6mol/L)、3.38%±2.99%(10-5mol/L)、3.59%±3.66%(10-4mol/L)和4.16%±3.67%(10-3mol/L)。结论ADMA可以有效地抑制ACh引发的EDVD反应,该抑制作用呈浓度依赖性和内皮依赖性。     

二十二碳六烯酸对大鼠心肌细胞通道的影响

目的:探讨二十二碳六烯酸(DHA)对大鼠心室肌细胞动作电位(AP)及钠通道电流(INa)的影响。阐述DHA抗心律失常的机制。方法:采用酶消化法获得大鼠心室肌细胞,用膜片钳技术分别记录0、20、40、60、80、100和120μmol/L DHA对大鼠心室肌细胞AP复极25%、50%和90%时程(APD25、APD50和APD90),对AP最大上升速率(Vmax)、幅值(APA)、超射值(OS)及INa的影响。结果:①不同浓度DHA对APD25、APD50和APD90呈浓度依赖性延长(P<0.05,n=20),对Vmax、APA和OS的影响无显著性差异(P>0.05,n=20)。②不同浓度DHA对INa呈浓度依赖性阻滞、I-V曲线上移、稳态失活曲线左移、失活后恢复时间延长,对稳态激活曲线无影响。在指令电压-30 mV时,上述浓度DHA对INa阻滞分别为(1.51±1.32)%、(21.13±4.62)%、(51.61±5.73)%、(67.62±6.52)%、(73.49±7.59)%和(79.95±7.62)%(P<0.05,n=20),DHA对INa的半效作用浓度(EC50)为(47.91±1.57)mol/L。结论:DHA对APD的延长及对钠通道的抑制作用可能是其抗心律失常机制之一。     

海马脑片锥体细胞的膜电性质和突触反应

应用成年大鼠海马脑片锥体神经元细胞内记录技术,测得CA_1区锥体细胞的静息电位为-78±6mV(n=16),膜电阻69±18MΩ,时间常数5.7±2.1ms,由I-V曲线求得斜率电阻57±15MΩ(n=8)。I-F曲线表明锋电位发放频率随去极化电流增大而升高(n=4).刺激Schaffer侧支通路可透发潜伏期为5±2ms(n=7)的兴奋性突触后电位,其幅值呈等级性,为低钙高镁溶液取消,其迟后成分在无镁溶液中增大,提示兴奋性氨基酸可能为介导递质。     

Effects of WIN 55,212-2 on IK Current in Cultured Trigeminal Ganglion Neurons of Rat

Summary To investigate the effects of WIN 55, 212-2 onI _K in cultured rat trigeminal ganglion (TG) neurons, whole-cell patch clamp techniques were used to record theI _K before and after WIN 55,212-2 perfusion at different concentrations. 30 μmol/L WIN 55,212-2 markedly (35.7%±7.3%P<0.01,n=8) inhibitedI _K currents, and the currents were partially recovered after washing. 30 μmol/L WIN 55, 212-2 also induced a significant depolarizing shift in conductance-voltage parameters (control: V_0.5=10.43±4.25 mV, k=16.27±3.86; WIN 55,212-2: V_0.5=24.71±3.91 mV, k=16.69±2.75;n=8,P<0.01 for V_0.5). 0.01 μmol/L WIN 55,212-2 slightly (27.0%±7.9%,P<0.05,n=7) increasedI _K currents, but had no significant change in conductance-voltage parameters (control: V_0.5=10.74±5.27 mV, k=17. 33±2.96; WIN 55, 212-2: V_0.5=11.06±2.05 mV, k=19.69±6.60;n=7,P>0.05 for V_0.5 and k). These results suggested that WIN 55, 212-2 has dual action, which might be through different receptors. MING Zhangyin, male, born in 1968, Doctorial Candidate This project was supported by a grant from National Natural Sciences Foundation of China (No. 30271500).     

氟灭酸对豚鼠耳蜗螺旋动脉平滑肌细胞膜电位和兴奋性接头电位的作用

目的:观察氯通道阻断剂氟灭酸(NFA)对耳蜗螺旋动脉平滑肌细胞膜电位和电刺激血管周围交感神经纤维引起兴奋性接头电位(EJP)的影响。方法:在耳蜗螺旋动脉平滑肌离体标本上,运用细胞内微电极记录技术研究非甾体类抗炎药物对平滑肌细胞的作用。结果:100μmol/L NFA引起低静息膜电位平滑肌细胞产生了(14.7±4.4)mV的超极化反应,引起高静息膜电位平滑肌细胞仅产生了(1.4±0.8)mV的超极化反应(P<0.01)。NFA引起低静息膜电位平滑肌细胞的反应是浓度依赖性的,这一超极化反应能被钙激活钾通道的阻断剂北非蝎毒素(charybdotoxin)和iberiotoxin阻断。另外,在高静息膜电位的平滑肌细胞,NFA对刺激引起的平滑肌细胞EJP有更明显的抑制作用(P<0.01),但对低静息膜电位的平滑肌细胞引起的EJP作用不明显(P>0.05)。结论:NFA既能通过阻断钙激活的氯通道抑制平滑肌细胞产生的EJP,又能激活钙激活钾通道使平滑肌细胞产生超极化反应,表现出对平滑肌细胞作用的多样性。     

Delayed rectifier potassium current in dissociated bullfrog primary afferent neurons

Cultured bullfrog dorsal root ganglion cells were voltage-clamped in the whole-cell configuration. The classical delayed rectifier potassium current (IK) was separated from other ionic currents. Tetraethylammonium (1-50 mM) depressed the amplitude of IK in a concentration-dependent manner, a complete block occurring with 30 mM. With the concentration of potassium ions in the superfusate at 20 mM, the reversal potential of IK amounted to about -30mV. IK was activated between -30 and +70 mV. The half activation of IK occurred at +15 mV. The amplitude of IK was increased e-fold with 13.6 mV depolarization. The time constant of IK de-activation was shortened with membrane hyperpolarization (tau congruent to 4 ms at -100 mV). Finally, reciprocal time constant (tau -1) of the de-activating IK was increased e-fold with congruent to 13 mV hyperpolarization. It appears that the properties of IK in amphibian afferent neurons are comparable to those which have been observed with respect to the IK of the squid giant axons (Hodgkin and Huxley, 1952).     

Effect of Nitric Oxide on Potassium Channels of Rat Airway Smooth Muscle Cells

Summary: The effect of nitric oxide donor sodium nitroprusside (SNP) on resting membrane potential (Em) and potassium currents of the bronchial smooth muscle cells from rats was investigated. All experiments were conducted in conventional whole-cell configuration. The changes of Em and potassium currents after addition of 0. 1 mmol/L SNP were measured under the current-clamp mode and the voltage-clamp mode respectively. Results showed that (1) SNP could decrease the Em from --33. 8±7.4 mV to -43. 7±6. 7mV (n=10, P＜0. 01); (2) SNP could increase the Ca2+-activated K+ channel peak currents under ramp protocol from 466.9±180. 1 pA to 597. 7±237. 6 pA (n= 7, P＜0. 01), and the currents under pulse protocol at +50 mV were increased from 544.2±145.4 pA to 678.1±206. 2 pA (n=6, P＜0.05); (3) SNP also could increase voltage-gated K+ channel peak currents under ramp protocol from 389. 6±84. 1 pA to 526. 7±98. 7 pA (n=7, P＜0. 01), the currents under pulse protocol at +50 mV were increased from 275.7±85.2 pA to 444.3±128.5 pA(n=6,P＜0. 01). It was concluded that SNP increases the activities of Ca2+-activated K+ channels and voltage-gated K+ channels and leads to K+ efflux and hyperpolarization of the cell membrane, resulting in a decrease of the cell excitement.     

Frequency- and state-dependent blockade of human ether-a-go-go-related gene K+ channel by arecoline hydrobromide

Background  The rapidly activating delayed rectifier potassium current (I_Kr), whose pore-forming alpha subunit is encoded by the human ether-a-go-go-related gene (hERG), is a key contributor to the third phase of action potential repolarization. The aim of this study was to investigate the effect and mechanism of arecoline hydrobromide induced inhibition of hERG K⁺ current (I_hERG).

Methods  Transient transfection of hERG channel cDNA plasmid pcDNA3.1 into the cultured HEK293 cells was performed using Lipofectamine. A standard whole-cell patch-clamp technique was used to record the I_hERG before and after the exposure to arecoline.

Results  Arecoline decreased the amplitude and the density of the I_hERG in a concentration-dependent manner (IC₅₀=9.55 mmol/L). At test potential of +60 mV, the magnitude of I_hERG tail at test pulse of -40 mV was reduced from (151.7±6.2) pA/pF to (84.4±7.6) pA/pF (P <0.01, n=20) and the magnitude of I_hERG tail at test pulse of -110 mV was reduced from (−187.5±9.8) pA/pF to (−97.6±12.6) pA/pF (P <0.01, n=20). The blockade of arecoline in the open and inactivated state was significant in a state-dependent manner. The maximal blockade was achieved in the inactivated state. Studies of gating mechanism showed that the steady-state activation curve of I_hERG was significantly negatively shifted by arecoline. Time constants of activation were shortened. Steady-state inactivation curve and time constants of fast inactivation were not significantly affected by arecoline. Furthermore, the inhibition of I_hERG by arecoline was characterized markedly by a frequency-dependent manner from 0.03 to 1.00 Hz pulse.

Conclusion  Arecoline could potently block I_hERG in both frequency and state-dependent manner.