豚鼠耳蜗离体外毛细胞的膜电位和离子电流

利用膜片钳技术对分离的豚鼠耳蜗外毛细胞进行了研究,结果表明：（１）新分离的正常ＯＨＣ呈术状,胞膜光滑,胞核位于底部,静纤毛由顶端表皮板伸出,４小时内形态无明显变化。（２）全细胞电压钳记录结合通道阻断剂实验表明,ＯＨＣ膜电流主要由电压依赖性钾离子流组成。（３）利用全细胞记录方式得到的ＯＨＣ静息电位值为－２６±９ｍＶ．     

微丝在低渗牵张诱导毒蕈碱电流增加中的作用

在急性分离的豚鼠胃窦平滑肌细胞上 ,利用膜片钳技术的传统全细胞模式记录离子电流的方法 ,探讨微丝在低渗牵张诱导毒蕈碱电流增加中的作用。当豚鼠胃窦平滑肌细胞的膜电位钳制在 - 2 0mV时 ,灌流液中 5 0μmol/L 卡巴胆碱 (carbachol,CCh)或电极内液中 0 5mmol/LGTPγS均可引导毒蕈碱电流 (muscariniccurrentICCh) ,低渗牵张 ( 2 0 2mOsmol/L)分别使其增加 145± 2 7%和 183± 3 0 % ;当电极内液中加入 2 0 μmol/L的细胞松弛B (一种微丝骨架的解聚剂 )时 ,低渗牵张使ICCh只增加 70± 6% ;而电极内液中加入 2 0 μmol/L的鬼笔环肽 (一种微丝骨架的稳定剂 )则使ICCh增加了 5 45± 81%。结果表明 ,低渗牵张可增加由卡巴胆碱或GTPγS诱导的毒蕈碱电流 ,微丝参与调节低渗牵张诱导豚鼠胃窦平滑肌细胞ICCh增加的作用     

蝎毒耐热蛋白对大鼠急性分离海马神经元兴奋性的影响

应用全细胞膜片钳记录技术在电流钳模式下观察经持续高温等特殊处理后分离纯化的30～50 kDa蝎毒耐热蛋白(scorpion venom heat resistant protein,SVHRP)(国家发明专利,专利号ZL01 106166.92)对急性分离大鼠海马神经元兴奋性的影响.结果发现SVHRP可致海马神经元兴奋性降低.神经元经1×10-2 μg/mL SVHRP处理后动作电位发放模式改变,发放频率减少.在52个受检细胞中,有45个细胞产生位相放电(占86.54%);7个细胞产生重复放电(占13.46%).在产生位相放电的45个细胞中,有8个细胞在SVHRP处理后仍可以诱发出位相放电(占17.78%);37个细胞在SVHRP处理后无法诱导出位相放电(占82.22%),SVHRP处理后动作电位的产生与处理前相比,有显著差异(P＜0.01,n=45);在产生重复放电的7个细胞中,在1×10-2μg/mL SVHRP作用后均不能再次诱发出重复放电,而是产生一个动作电位或不再产生动作电位,药物处理前产生的动作电位个数为14.57±1.00,SVHRP处理后产生动作电位的个数为0.57±0.20,二者之间有显著性差异(P＜0.01,n=7).1×10-4 μg/mLSVHRP处理后,诱发动作电位产生的基强度由(75.10±8.99)pA增加到(119.85±12.73)pA(P＜0.01,n=8);阈电位由(-41.17±2.15)mV升至(-32.40±1.48)mV(P＜0.01,n=8);动作电位峰值由(68.49±2.33)mV下降至(54.71±0.81)mV(P＜0.01,n=8).由于神经元超兴奋性被认为是癫痫发作的基本机制之一,因此上述结果表明SVHRP有可能通过降低海马神经元兴奋性发挥其抗癫痫作用,这为蝎毒药物的进一步开发提供理论依据.     

不饱和脂肪酸在低渗牵张加强豚鼠胃窦平滑肌细胞毒蕈碱电流中的作用

利用全细胞膜片钳技术在急性分离的胃窦平滑肌细胞上记录离子电流的方法 ,探讨外源性不饱和脂肪酸是否参与低渗牵张加强毒蕈碱电流的过程。在豚鼠胃窦平滑肌细胞上膜电位被钳制在 - 2 0 0mV等渗状态时 ,5 0 μmol/L卡巴胆碱 (carbachol,CCh)引起的毒蕈碱电流 (ICCh)作为对照 ,发现低渗牵张可以使ICCh明显增加到对照的 2 2 6 0±2 1 0 %。当用含 5 μmol花生四烯酸 (arachidacid ,AA)、亚麻酸 (linoleicacid ,LA)或亚油酸 (oleicacid,OA)细胞外液灌流时 ,ICCh分别被抑制在对照的 3 8± 0 6%、3 5 2± 0 8%和 66 6± 0 6%。在这种情况下 ,低渗牵张刺激可以使ICCh分别增加到 10 6 0± 2 5 %、173 2± 6 8%和 2 2 2 1± 11 0 %。 5 μmol/LAA抑制低渗牵张增加的毒蕈碱电流 5 1 2± 3 8% ,而在等渗状态下抑制ICCh为 96 2± 1 6%。上述结果提示 ,不饱和脂肪酸中双键数目越多 ,抑制效应越强 ;但不饱和脂肪酸不参与低渗刺激加强毒蕈碱电流的过程。     

肾性高血压大鼠肺动脉平滑肌细胞钾通道的研究

目的:观察肾性高血压大鼠(RHR)肺动脉平滑肌细胞膜电容(Em)、膜电流(I)、电流密度(pA/pF)、膜电位和I-V曲线的变化及盐酸埃他卡林对正常血压及肾性高血压大鼠肺动脉平滑肌钾通道的影响。方法:用内径为0.2~0.3 mm的银夹夹住大鼠左肾肾动脉起始部,制成两肾一夹RHR模型,血压以无创性套尾法测量。急性分离大鼠肺内动脉平滑肌细胞,用全细胞记录技术记录细胞钾电流、膜电容并计算电流密度。结果:RHR肺动脉平滑肌细胞膜电容均值为(3.43±1.16)pF,比正常血压大鼠(NTR,4.98±0.62pF)降低31.1%;钾电流值为(0.54±0.26)nA,比正常大鼠(1.70±0.67nA)降低68.2%;电流密度值为(180±90)pA/pF,比正常大鼠(350±80 pA/pF)降低48.6%;膜电位为(-26.96±7.23)mV,比正常大鼠(-27.66±7.1 mV)降低2.5%。盐酸埃他卡林在0.1-100μmol.L-1浓度下,可显著增强正常血压大鼠动脉平滑肌钾电流;在1.0-100μmol.L-1浓度下,可显著增强肾性高血压大鼠动脉平滑肌钾电流。结论:RHR的膜电容、膜电流、电流密度比正常血压大鼠低,I-V曲线下移。盐酸埃他卡林对正常血压大鼠及肾性高血压大鼠动脉平滑肌钾电流都有增强作用。     

细胞外pH值降低可增强豚鼠心室肌细胞持续性钠电流

应用全细胞和单通道(贴附式)膜片钳技术观察胞外pH值降低对心室肌细胞持续性钠电流(persistent sodium current,ⅠNa.P)的影响,探讨其作用机制。结果显示:全细胞记录模式下,细胞外pH值降低可明显增大ⅠNa.P,且呈H+浓度依赖性增强。当细胞外pH值从对照值的7．4降低为6．5时,ⅠNa.P的电流密度从(0．347±0．067)pAJpF增加到(0．817±0．137)pA/pF(P< 0．01,n=6),而加入还原剂1,4-二硫甙苏糖醇(dithiothreitiol,DTT,1 mmol／L)后可使,ⅠNa.P的电流密度回落到(0．233±0．078)pA／pF (P<0．01 vs pH 6．5,n=6)。单通道记录模式中,当细胞外pH值从对照值的7．4降低为6．5时,持续性钠通道的开放概率和开放时间分别从0．021±0．007和(0．899±0．074)ms增加到0．205±0．023和(1．593±0．158)ms(P<0．叭,n=6),再加入还原剂DTT(1 mmol／L)使开放概率和开放时间分别回落到0．019±0．005和(0．868±0．190)ms(P<0．01 vs pH 6．5,n=6);加入蛋白激酶C(protein kinase C,PKC)抑制剂bisindolylmaleimide(BIM,5μmol/L)可使pH 6．5时增大的,ⅠNa.P明显减小,开放概率和开放时间分别从0．214±0．024和(1．634±0．137)ms回落到0．025±0．006和(0．914±0．070)ms(P<0．01 vs pH 6．5,n=6)。结果表明,细胞外pH值降低可诱发心室肌细胞ⅠNa.P增大,其机制可能与PKC的激活有关。     

大鼠结肠平滑肌细胞的钙库操纵性通道

目的:探讨大鼠结肠平滑肌细胞是否存在钙库操纵性通道(SOC)。方法:荧光探针Fura-2/AM标记细胞内游离Ca2+后,用荧光分光光度计检测毒胡萝卜素(thapsigargin)和咖啡因(caffeine)耗竭胞内钙库后激活的SOC通道对酶解分离的大鼠结肠平滑肌细胞[Ca2+]i的影响。结果:在无Ca2+缓冲液中,thapsigargin(1μmol/L)以及caf-feine(10 mmol/L)分别使[Ca2+]i由静息时(68.32±3.43)nmol/L升高至(240.85±12.65)nmol/L(、481.25±34.77)nmol/L,继之,向细胞外液中引入两种浓度的Ca2+(1.5 mmol/L和3.0 mmol/L),导致[Ca2+]i进一步升高,分别为(457.55±19.80)nmol/L、(1005.93±54.62)nmol/L;(643.88±34.65)nmol/L、(920.16±43.25)nmol/L。且上述升高效应对维拉帕米(verapamil,5μmol/L)以及KCl引起的细胞膜去极化不敏感,但可被La3+(1 mmol/L)抑制。结论:在酶解分离的大鼠结肠平滑肌细胞上,存在胞内钙库耗竭激活的SOC通道,为支持在电兴奋性细胞上存在库容性Ca2+内流提供了实验和理论依据。     

多异瓢虫的发育与温度的关系

测定多异瓢虫Hippodamia variegata(Goeze)在13,17,21,25,29和33℃下各虫态的发育历期。结果表明,在不同温度梯度下,多异瓢虫全世代历期依次为:(62.23±3.42)d,(48.31±2.96)d,(25.01±2.53)d,(20.78±1.83)d,(13.68±0.67)d和(10.92±0.58)d。利用最小二乘法确定出多异瓢虫各虫态的发育起点温度和有效积温,其全世代的分别为10.85℃和249.79日.度;由微分法求出Logistic曲线方程中的最大发育速率K,进而由最小二乘法求出a、r,从而确定出多异瓢虫各虫态的发育速率与温度的Logistic曲线关系,其全世代的为:V=0.101156/[1+exp(3.905041-0.183509T)];由Lagrangc中值定理求出各虫态的发育最适温度、适宜温区,其全世代的分别为:Tmid(最适温度)=21.28℃、Tmin(适宜温区下限)=11.55℃、Tmax(适宜温区上限)=31.01℃。     

甲硫—脑啡肽对大鼠DRG分离神经元ATP—激活内向电流的抑制

本研究探讨了甲硫-脑啡肽(met-Enk)对ATP-激活电流(IATP)的调制作用.实验在大鼠新鲜分离背根神经节(DRG)神经元上进行.应用全细胞膜片钳技术所记录的IATP为内向电流.在被检测的DRG神经元中,90.0%(45/50)的细胞对ATP有反应.在45个对ATP敏感的细胞中对大部分细胞(29/45)施加met-Enk(10-9～10-5mol/L)也引起一内向电流;少部分细胞(9/45)为外向电流;其余的细胞(7/45)未引起可检测的膜反应.预加met-Enk后IATP明显地被抑制,此种抑制作用为剂量依赖性的.在预加10-9、10-8、10-7、10-6、10-5mol/Lmet-Enk后,IATP的抑制分别为13.2±5.4%(n=5)、39.2±8.6%(n=8)、54.1±8.6%(n=8)、43.3±7.9%(n=7);43.1±7.9%(n=7)(mean±SKM).阿片肽拮抗剂纳洛酮能翻转此种抑制效应.IATP的量-效关系表明,预加met-Enk后曲线明显压低,在浓度为10-3mol/L时IATP下降约25%,而Kd值几乎不变.应用二次钳压技术胞内透析H-9(PKA抑制剂)能取消此种抑制作用.上述结果提示met-Enk对IATP的抑制效应为非竞争性抑制作用,可能是由于阿片受体激活后,经相应的胞内信号转导途径使ATP受体磷酸化所致.     

S

在45只切断双侧缓冲神经的Sprague-Dawley 大鼠,应用细胞外记录方法, 观察了颈动脉内注射腺苷对76 个最后区 (AP) 神经元自发放电活动的影响。所得结果如下(1) 在记录到的42个自发放电单位中, 颈动脉内注射腺苷(25 μg/kg) 引起其中29个单位的放电频率由6.26±0.75 下降至4.74±0.76 spikes/s (P<0.01), 6 个单位放电频率由4.13±0.77增加至4.72±0.83 spikes/s (P<0.05),另外7个单位放电频率无明显变化, 而血压和心率在实验中无变化; (2)在应用非选择性腺苷受体拮抗剂8-苯茶碱(8-phenyltheophylline, 15 μg/kg) 的10个单位, 腺苷对放电的抑制效应可被完全阻断; (3) 应用选择性腺苷A     

Electromotility of outer hair cells from the cochlea of the echolocating bat,Carollia perspicillata

Isolated outer hair cells (OHCs) and explants ot the organ of Corti were obtained from the cochlea of the echolocating bat, Carollia perspicillata, whose hearing range extends up to about 100 kHz. The OHCs were about 10–30 m long and produced resting potentials between-30 to -69 mV. During stimulation with a sinusoidal extracellular voltage field (voltage gradient of 2 mV/m) cyclic length changes were observed in isolated OHCs. The displacements were most prominent at the level of the cell nucleus and the cuticular plate. In the organ of Corti explants, the extracellular electric field induced a radial movement of the cuticular plate which was observed using video subtraction and photodiode techniques. Maximum displacements of about 0.3–0.8 m were elicited by stimulus frequencies below 100 Hz. The displacement amplitude decreased towards the noise level of about 10–30 nm for stimulus frequencies between 100–500 Hz, both in apical and basal explants. This compares well with data from the guinea pig, where OHC motility induced by extracellular electrical stimulation exhibits a low pass characteristic with a corner frequency below 1 kHz. The data indicate that fast OHC movements presumably are quite small at ultrasonic frequencies and it remains to be solved how they participate in amplifying and sharpening cochlear responses in vivo.Abbreviations BM basilar membrane - FFT fast Fourier Transfer - IHC inner hair cell - OHC outer hair cell     

Effect of diamide on force generation and axial stiffness of the cochlear outer hair cell.

We found that diamide, which affects spectrin, reduces the axial stiffness of the cochlear outer hair cell, the cylindrically shaped mechanoreceptor cell with a unique voltage-sensitive motility. This effect thus provides a means of examining the relationship between the stiffness and the motility of the cell. For measuring axial stiffness and force production, we used an experimental configuration in which an elastic probe was attached to the cell near the cuticular plate and the other end of the cell was held with a patch pipette in the whole-cell recording mode. Diamide at concentrations of up to 5 mM reduced the axial stiffness in a dose-dependent manner to 165 nN per unit strain from 502 nN for untreated cells. The isometric force elicited by voltage pulses under whole-cell voltage clamp was also reduced to 35 pN/mV from 105 pN/mV for untreated cells. Thus the isometric force was approximately proportional to the axial stiffness. Our observations suggest a series connection between the motor and cytoskeletal elements and can be explained by the area motor model previously proposed for the outer hair cell.     

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously^1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.     

On the effect of prestin on the electrical breakdown of cell membranes

The voltage-dependent activity of prestin, the outer hair cell (OHC) motor protein essential for its electromotility, enhances the mammalian inner ear's auditory sensitivity. We investigated the effect of prestin's activity on the plasma membrane's (PM) susceptibility to electroporation (EP) via cell-attached patch-clamping. Guinea pig OHCs, TSA201 cells, and prestin-transfected TSA cells were subjected to incremental 50 mus and/or 50 ms voltage pulse trains, or ramps, at rates from 10 V/s to 1 kV/s, to a maximum transmembrane potential of +/-1000 mV. EP was determined by an increase in capacitance to whole-cell levels. OHCs were probed at the prestin-rich lateral PM or prestin-devoid basal portion; TSA cells were patched at random points. OHCs were consistently electroporated with 50 ms pulses, with significant resistance to depolarizing pulses. Although EP rarely occurred with 50 mus pulses, prior stimulation with this protocol had a significant effect on the sensitivity to EP with 50 ms pulses, regardless of polarity or PM domain. Consistent with these results, resistance to EP with depolarizing 10-V/s ramps was also found. Our findings with TSA cells were comparable, showing resistance to EP with both depolarizing 50-ms pulses and 10 V/s ramps. We conclude prestin significantly affects susceptibility to EP, possibly via known biophysical influences on specific membrane capacitance and/or membrane stiffness.     

新鲜分离小鼠胃ICC样细胞的鉴定及其电生理学特性

将免疫荧光及传统全细胞膜片钳技术应用于新鲜分离的小鼠胃Cajal 间质细胞样细胞上,探讨了小鼠胃Cajal 间质细胞样细胞形态和电生理学特性。经胶原酶消化得到的Cajal间质细胞样细胞胞体呈短梭形,且自胞体发出多个较短的毛刺状突起。免疫细胞化学结果表明,Cajal 间质细胞样细胞胞体和突起酪氨酸激酶受体c-kit表达呈阳性。在传统全细胞记录模式、膜电位钳制在-60 mV 的条件下,可以记录到自发、节律性内向电流,即起搏电流。钙调蛋白抑制剂W-7 (50µmol/L）明显增强了起搏电流幅度并引发明显的内向钳制电流。当电极内液中EGTA 的浓度由0.1 mmol/L增加到10 mmol/L时,也明显增强了起搏电流幅度并引发明显的内向钳制电流。实验结果提示,新鲜分离的小鼠胃Cajal 间质细胞样细胞可以产生自发、自律性内向电流,而这种电流对胞内低钙或钙调蛋白抑制剂敏感。这种具有自发性电活动的Cajal 间质细胞样细胞可能就是胃Cajal 间质细胞。     

Voltage-dependent inactivation of the potassium current of embryonic chick hepatocytes

The whole-cell patch electrode voltage clamp technique was used to study the inactivation properties of the delayed rectifying potassium current of single cultured embryonic chick hepatocytes at 20 degrees C. The potassium current activates maximally within 250-500 ms of membrane depolarization, after which it decays with a monoexponential time course. Both steady-state activation and inactivation are voltage dependent. Steady-state inactivation declines from 100% at -5 mV to 0 near -70 mV. with half inactivation at -41 mV. At the resting potential (EM) of these cells (-21.5 +/- 6.0 mV, n = 36) 6-18% of the IK channels are not inactivated and less than 5% are open. Development and removal of inactivation follow single exponential time courses. The inactivation time constant attains a maximum of around 30 s at -35 mV and is sharply voltage dependent at the EM of these cells. Measurement of EM under current clamp shows random oscillations of 5-10 mV amplitude. We suggest that the voltage- and time-dependent properties of IK, in tandem with a time- and voltage-independent, non-selective current also seen here, would provide the mechanism for a fluctuating EM.     

Membrane tension directly shifts voltage dependence of outer hair cell motility and associated gating charge.

The unique electromotility of the outer hair cell (OHC) is believed to promote sharpening of the passive mechanical vibration of the mammalian basilar membrane. The cell also presents a voltage-dependent capacitance, or equivalently, a nonlinear gating current, which correlates well with its mechanical activity, suggesting that membrane-bound voltage sensor-motor elements control OHC length. We report that the voltage dependence of the gating charge and motility are directly related to membrane stress induced by intracellular pressure. A tracking procedure was devised to continuously monitor the voltage at peak capacitance (VpkCm) after obtaining whole cell voltage clamp configuration. In addition, nonlinear capacitance was more fully evaluated with a stair step voltage protocol. Upon whole cell configuration, VpkCm was typically near -20 mV. Negative patch pipette pressure caused a negative shift in VpkCm, which obtained a limiting value near the normal resting potential of the OHC (approximately -70 mV) at the point of cell collapse. Positive pressure in the pipette caused a positive shift that could reach values greater than 0 mV. Measures of the mechanical activity of the OHC mirrored those of charge movement. Similar membrane-tension dependent peak shifts were observed after the cortical cytoskeletal network was disrupted by intracellular dialysis of trypsin from the patch pipette. We conclude that unlike stretch receptors, which may sense tension through elastic cytoskeletal elements, the OHC motor senses tension directly. Furthermore, since the voltage dependence of the OHC nonlinear capacitance and motility is directly regulated by intracellular turgor pressure, we speculate that modification of intracellular pressure in vivo provides a mechanism for controlling the gain of the mammalian "cochlear amplifier".     

Non-uniform Distribution of Outer Hair Cell Transmembrane Potential Induced by Extracellular Electric Field

Intracochlear electric fields arising out of sound-induced receptor currents, silent currents, or electrical current injected into the cochlea induce transmembrane potential along the outer hair cell (OHC) but its distribution along the cells is unknown. In this study, we investigated the distribution of OHC transmembrane potential induced along the cell perimeter and its sensitivity to the direction of the extracellular electric field (EEF) on isolated OHCs at a low frequency using the fast voltage-sensitive dye ANNINE-6plus. We calibrated the potentiometric sensitivity of the dye by applying known voltage steps to cells by simultaneous whole-cell voltage clamp. The OHC transmembrane potential induced by the EEF is shown to be highly nonuniform along the cell perimeter and strongly dependent on the direction of the electrical field. Unlike in many other cells, the EEF induces a field-direction-dependent intracellular potential in the cylindrical OHC. We predict that without this induced intracellular potential, EEF would not generate somatic electromotility in OHCs. In conjunction with the known heterogeneity of OHC membrane microdomains, voltage-gated ion channels, charge, and capacitance, the EEF-induced nonuniform transmembrane potential measured in this study suggests that the EEF would impact the cochlear amplification and electropermeability of molecules across the cell.     

Non-uniform Distribution of Outer Hair Cell Transmembrane Potential Induced by Extracellular Electric Field

Intracochlear electric fields arising out of sound-induced receptor currents, silent currents, or electrical current injected into the cochlea induce transmembrane potential along the outer hair cell (OHC) but its distribution along the cells is unknown. In this study, we investigated the distribution of OHC transmembrane potential induced along the cell perimeter and its sensitivity to the direction of the extracellular electric field (EEF) on isolated OHCs at a low frequency using the fast voltage-sensitive dye ANNINE-6plus. We calibrated the potentiometric sensitivity of the dye by applying known voltage steps to cells by simultaneous whole-cell voltage clamp. The OHC transmembrane potential induced by the EEF is shown to be highly nonuniform along the cell perimeter and strongly dependent on the direction of the electrical field. Unlike in many other cells, the EEF induces a field-direction-dependent intracellular potential in the cylindrical OHC. We predict that without this induced intracellular potential, EEF would not generate somatic electromotility in OHCs. In conjunction with the known heterogeneity of OHC membrane microdomains, voltage-gated ion channels, charge, and capacitance, the EEF-induced nonuniform transmembrane potential measured in this study suggests that the EEF would impact the cochlear amplification and electropermeability of molecules across the cell.