熊果酸激活低分化鼻咽癌细胞氯通道和减小细胞容积

本文旨在研究熊果酸对低分化鼻咽癌细胞氯通道的激活作用,以及熊果酸对其细胞容积的影响。采用膜片钳技术记录熊果酸激活的鼻咽癌细胞(CNE-2Z)全细胞氯电流,应用离子置换、改变细胞外渗透压、氯通道阻断剂等观察熊果酸诱导的氯电流的特性,活细胞动态图像分析技术测量细胞容积变化。结果显示,等渗条件下可记录到CNE-2Z细胞微弱且稳定的背景氯电流,细胞外灌流熊果酸可浓度依赖性(1～100nmol/L)诱发氯电流的产生,在±80mV电压钳制下,100nmol/L熊果酸激活的氯电流的平均电流密度为(78.92±6.39)pA/pF和(59.86±4.86)pA/pF,该电流具有较明显的外向优势,不表现明显的时间依赖性和电压依赖性失活。该电流翻转电位为(4.83±0.30)mV,较接近Cl平衡电位(0.9mV)。熊果酸激活的氯通道对不同阴离子的通透性为:Cl-=I->Br->葡萄酸根离子。该电流具有容积敏感性,可被细胞外高渗透压显著抑制;氯通道阻断剂他莫昔芬(tamoxifen)、5-硝基-2-(3-苯丙胺)苯甲酸[(5-nitro-2-(3-phenylpro-pylamino)benzoic acid,NPPB]可抑制该电流。细胞外灌流熊果酸1h后,细胞容积减小,氯通道阻断剂NPPB可抑制该容积变化。以上结果提示,熊果酸可以激活低分化鼻咽癌细胞的氯通道,使Cl外流,进而引起细胞容积减小。     

睫状体色素上皮细胞容积激活性氯电流

为研究睫状体色素上皮 (pigmentedciliaryepithelial,PCE)细胞容积激活性Cl-电流的特性 ,用膜片箝全细胞记录技术记录了猪的低渗液诱发的容积激活性Cl-电流。此电流外向占优势 ,几乎没有时间依赖性失活 ,电流 电压曲线显示此电流反转电位 (- 6 3± 0 5mV)很接近氯离子平衡电位的计算值 (ECl=0mV)。电流的激活依赖于细胞内ATP ,细胞外ATP抑制外向电流和内向电流 ,但外向电流抑制率大于内向电流抑制率 (92 %比 74% ,P <0 0 1)。氯离子通道阻断剂tamoxifen抑制外向电流和内向电流 ,两个抑制率几乎相等 (85 %比 87% ,P >0 0 5 )。此电流特性与其他类型细胞的P糖蛋白相关电流很相似。结果提示PCE细胞容积激活性Cl-电流的形成可能与P糖蛋白有关     

人胎儿鼻咽上皮细胞的背景氯电流

采用膜片钳和图像分析技术,研究人胎儿鼻咽上皮细胞背景电流的特性及其与容积激活性氯电流的关系。在等张溶液中,可记录到一背景电流,该电流呈微弱的外向整流性,无明显时间依赖性失活,其翻转电位为(?0.73±1.7)mV(n=21),接近氯离子平衡电位(?0.9mV)。细胞外高张刺激(440mOsmol/L)明显抑制此电流(59.6±7.1)%,而低张刺激(160mOsmol/L)则诱发细胞产生容积激活性氯电流。氯通道阻断剂tamoxifen和5-硝基-2-(3-苯丙胺基)苯甲酸[5-nitro-2-(3-phenylpropylamino)benzoicacid,NPPB]显著地抑制背景电流并使细胞基础容积增大。上述结果表明,人胎儿鼻咽上皮细胞的背景Cl?电流是背景电流的重要成分,此Cl?电流与容积激活性氯电流及细胞基础容积调节有关。     

迁移的鼻咽癌细胞容积激活性氯电流

用膜片钳技术研究了Transwell小室趋化迁移后的鼻咽癌CNE-2Z细胞容积激活性CT电流。47％低渗刺激迁移后的CNE-2Z细胞诱发容积激活性氯电流,与未迁移细胞相比,其特性以及其对氯通道阻断剂的敏感性发生明显的变化,此电流的密度明显高于未迁移细胞,而且该电流几乎完全被氯通道阻断剂adenosine-5'-triphosphate(ATP,10 mmol/L)、5-nitro-2-3-phenylpropylamino benzoic acid(NPPB,100μmol/L)和他莫昔芬(30μmol/L)抑制,其中NPPB和他莫昔芬对迁移细胞的抑制作用明显强于未迁移细胞。迁移后的CNE-2Z细胞容积激活性氯通道对阴离子的通透性为:Br>Cl>I>葡萄糖酸,与未迁移细胞(I>Br>Cl>葡萄糖酸)不同。结果提示,容积激活性氯通道可能参与CNE-2Z细胞的迁移过程。     

海马脑片锥体神经元钙离子通道的盲法膜片钳研究

目的和方法 :采用大鼠海马脑片盲法膜片钳全细胞记录技术研究CA1区锥体神经元电压门控性Ca2 通道的动力学特征。结果 :大鼠海马脑片CA1区锥体神经元电压门控性Ca2 通道电流具有如下特点 :①激活的阈电位偏低 ,为 (- 4 9.3± 8.6 )mV ,范围为 - 6 5～ - 30mV(n =2 3)。②衰减时间常数τ值较大 ,且变化范围大 (10 0～ 70 0ms) (n =12 ) ,并且衰减具有Ca2 电流幅值的依赖性 ,③稳态失活呈现电压依赖性 ,半失活电压为 (- 5 5 .4± 9.7)mV ,斜率因子为 (5 .3± 0 .9)mV(n =10 )。④当细胞外Ca2 浓度为 2 .5mmol/L时 ,Ca2 通道的反转电位为 (5 5±13)mV(n =10 )。⑤尾电流成分较为单一 ,不表现电压依赖性。另外 ,Ca2 电流对戊脉胺及双氢吡啶类化合物硝苯地平均不敏感。结论 :根据上述Ca2 电流特征 ,海马脑片CA1区锥体神经元上的Ca2 通道主要以N型为主     

鼻咽癌细胞CIC-3在细胞周期中的表达

用免疫荧光、激光共聚焦显微镜图像分析及膜片钳等技术研究了鼻咽癌上皮CNE-2Z细胞容积激活性氯通道候选基因CIC-3的表达及其在细胞周期中与容积激活性氯电流及细胞容积调节性回缩(regulatory volume decrease,RVD)的关系。结果显示,CNE-2Z细胞表达CIC-3。CIC-3蛋白主要位于细胞内而不是在细胞膜上,其表达水平及其在细胞中的分布呈细胞周期依赖性。G1期细胞的CIC-3表达水平较低而S期则较高,M期细胞的表达水平中等。在细胞周期中,CIC-3表达水平与细胞RVD能力及容积激活性氯电流水平呈反比。上述观察结果提示,CIC-3可能参与细胞周期的调节,但CNE-2Z细胞中的CIC-3可能不是与RVD有关的氯通道。     

鼻咽癌细胞ClC-3在细胞周期中的表达

用免疫荧光、激光共聚焦显微镜图像分析及膜片钳等技术研究了鼻咽癌上皮CNE-2Z细胞容积激活性氯通道候选基因C1C-3的表达及其在细胞周期中与容积激活性氯电流及细胞容积调节性回缩(regulatoryvolumedecrease,RVD)的关系.结果显示,CNE-2Z细胞表达ClC-3.ClC-3蛋白主要位于细胞内而不是在细胞膜上,其表达水平及其在细胞中的分布呈细胞周期依赖性.G1期细胞的ClC-3表达水平较低而S期则较高,M期细胞的表达水平中等.在细胞周期中,ClC-3表达水平与细胞RVD能力及容积激活性氯电流水平呈反比.上述观察结果提示,ClC-3可能参与细胞周期的调节,但CNE-2Z细胞中的ClC-3可能不是与RVD有关的氯通道.     

豚鼠Ⅰ型前庭毛细胞胆碱能受体通道特性

本文旨在探讨豚鼠Ⅰ型前庭毛细胞上有无胆碱能受体存在,并对其相应的离子通道特性进行研究.应用全细胞膜片钳技术检测急性分离的豚鼠Ⅰ型前庭毛细胞对乙酰胆碱(acetylcholine, ACh)的反应.结果显示,7.5%(21/279)的Ⅰ型前庭毛细胞对10～1000μmol/L ACh敏感,引发明显的外向电流.该电流对ACh的反应呈浓度依赖性,半数激活浓度(EC50)为(63.78±2.31)μmol/L,但该电流为非电压依赖性.在-50mV钳制电压和正常细胞外液中,100μmol/L ACh激活一持久缓慢的外向电流,电流幅值为(170±15)pA,该电流幅值依赖于胞外钙离子浓度,可被胞外给予的钙依赖性钾通道拮抗剂TEA阻断.Ⅰ型前庭毛细胞的再次激活时间不小于1min.长时间暴露在ACh的情况下,受体离子通道不会发生自发性关闭.以上结果提示,部分豚鼠Ⅰ型前庭毛细胞上存在胆碱能受体,胞外给予ACh可激活一持久缓慢的外向电流,其胆碱能受体通道对于ACh的作用呈浓度依赖性和外钙依赖性、非电压依赖性或失敏性.本研究结果对于阐明前庭传出神经的功能及其作用机制,证实并揭示Ⅰ型前庭毛细胞上存在传出神经递质受体以及日后临床指导眩晕疾病的康复治疗具有重要的意义.     

大鼠肺动脉平滑肌细胞钙激活氯通道电流的电生理检测

目的：研究大鼠肺动脉平滑肌细胞钙激活氯通道电流的电生理特性。方法：膜片钳全细胞和膜内向外记录模式检测大鼠肺动脉平滑肌细胞上钙激活氯通道全细胞电流和单通道电流。结果：大鼠肺动脉平滑肌细胞记录到稳定的钙激活氯通道电流（ICl（Ca））;ICl（Ca）表现出典型的外向整流特性和电压时间依赖性激活。结论：大鼠肺动脉平滑肌细胞膜上存在电压、时间依赖性氯通道电流,钙激活氯通道通过促进肺动脉平滑肌细胞去极化而成为调节肺动脉特性的关键调节因子。     

三磷酸腺苷对蟾蜍交感神经节细胞的作用及机制分析

目的　观察ATP所引起的蟾蜍椎旁交感神经节细胞电位变化和有关因素的影响 ,以探讨ATP在神经节内的作用及其机制。方法　采用细胞外微电极技术记录离体灌流神经节电位。结果　外源性ATP(2 0 0 μmol L)可引起去极化 (n =1 1 0 ) ,超极化 (n =33) ,以及去极后伴随一超极过程的双相反应 (n =1 2 )。P2 嘌呤受体拮抗剂ci bacronblue 3GA(30 0 μmol/L)和奎尼丁 (30 0 μmol/L) ,均可抑制ATP所致去极反应 ,其幅值分别减小 (2 4 .2± 1 7.5 ) %(n =1 8,P <0 .0 1 )和 (32 .5± 1 1 .5 ) % (n =2 1 ,P <0 .0 1 )。P1 嘌呤受体拮抗剂氨茶碱 (2 0 0 μmol L) ,可抑制ATP所致超极反应 ,幅值减小 (6 5 .0± 2 2 .9) % (n =9,P <0 .0 1 )。在无钠溶液中 ,ATP的去极幅值变化为 (1 3.6± 1 7.5 ) % (n =1 0 ,P >0 .0 5 ) ,无统计学意义。在无钙溶液中或同时加用EDTA(1mol L)以去除溶液的Ca2 ,可使ATP所致去极幅值减小 (2 3.6± 1 8.3) % (n =1 5 ,P <0 .0 1 )。电压依赖性钾通道阻断剂 4—氨基吡啶 (3mmol L)和ATP敏感性钾通道阻断剂格列苯脲 (1 0 0mol L) ,均可可抑制ATP所致超极反应 ,其幅值分别减小 (6 4 .5± 2 1 .9) % (n =1 1 ,P <0 .0 1 )和(6 6 .4± 2 2 .4 ) % (n =6 ,P <0 .0 1 )。前列腺素合成酶抑制剂吲     

Extracellular ATP-induced inward current in isolated epithelial cells of the endolymphatic sac.

Using whole-cell patch-clamp technique and Fura-2 fluorescence measurement, the presence of ATP-activated ion channels and its dependence on intracellular Ca2+ concentration ([Ca2+]i) in the epithelial cells of the endolymphatic sac were investigated. In zero current-clamp configuration, the average resting membrane potential was -66.8+/-1.3 mV (n=18). Application of 30 microM ATP to the bath induced a rapid membrane depolarization by 43.1+/-2.4 mV (n=18). In voltage-clamp configuration, ATP-induced inward current at holding potential (VH) of -60 mV was 169.7+/-6.3 pA (n=18). The amplitude of ATP-induced currents increased in sigmoidal fashion over the concentration range between 0.3 and 300 microM with a Hill coefficient (n) of 1.2 and a dissociation constant (Kd) of 11.7 microM. The potency order of purinergic analogues in ATP-induced current, which was 2MeSATP>ATPgammas>/=ATP>alpha, beta-ATP>ADP=AMP>/=adenosine=UTP, was consistent with the properties of the P2Y receptor. The independence of the reversal potential of the ATP-induced current from Cl- concentration suggests that the current is carried by a cation channel. The relative ionic permeability ratio of the channel modulated by ATP for cations was Ca2+>Na+>Li+>Ba2+>Cs+=K+. ATP (10 microM) increased [Ca2+]i in an external Ca2+-free solution to a lesser degree than that in the external solution containing 1.13 mM CaCl2. ATP-induced increase in [Ca2+]i can be mimicked by application of ionomycin in a Ca2+-free solution. These results indicate that ATP increases [Ca2+]i through the P2Y receptor with a subsequent activation of the non-selective cation channel, and that these effects of ATP are dependent on [Ca2+]i and extracellular Ca2+.     

Volume-activated chloride currents in interstitial cells of Cajal

Interstitial cells of Cajal (ICC) undergo marked morphological changes on contraction of the musculature, making it essential to understand properties of mechanosensitive ion channels. The whole cell patch-clamp technique was used to identify and to characterize volume-activated Cl- currents in ICC cultured through the explant technique. Hypotonic solutions (approximately 210 mosM) activated an outwardly rectifying current, which reversed near the equilibrium potential for Cl-. Time-dependent inactivation occurred only at pulse potentials of +80 mV, with a time constant of 478 +/- 182 ms. The degree of outward rectification was calculated using a rectification index, the ratio between the slope conductances of +65 and -55 mV, which was 13.9 +/- 1.5 at 76 mM initial extracellular Cl- concentration. The sequence of relative anion permeability of the outwardly rectifying Cl- channel was I- > Cl- > aspartate-. The chloride channel blockers, DIDS and 5-nitro-2-(3-phenlypropl-amino)benzoic acid, caused a voltage-dependent block of the outwardly rectifying Cl- current, inhibition occurring primarily at depolarized potentials. On exposure to hypotonic solution, the slope conductance significantly increased at the resting membrane potential (-70 mV) from 1.2 +/- 0.2 to 2.0 +/- 0.4 nS and at the slow-wave plateau potential (-35 mV) from 2.1 +/- 0.3 to 5.0 +/- 1.0 nS. The current was constitutively active in ICC and contributed to the resting membrane potential and excitability at the slow-wave plateau. In conclusion, swelling or volume change will depolarize ICC through activation of outwardly rectifying chloride channels, thereby increasing cell excitability.     

Pharmacological and biophysical properties of swelling-activated chloride channel in mouse cardiac myocytes

The present study was designed to observe the properties of swelling-activated chloride channel (ICl.swell) in mouse cardiac myocytes using patch clamp techniques. In whole-cell recordings, hypotonic solution activated a chloride current that exhibited outward rectification, weak voltage-dependent inactivation, and anion selectivity with permeability sequence of I- > Br- > Cl-. The current was sensitive to Cl- channel blockers tamoxifen, NPPB and DIDS. In single-channel recordings, cell swelling activated a single channel current which showed outward rectification with open probability of 0.76 +/- 0.08 and conductance of 38.1 +/- 2.5 pS at +100 mV under [Cl-] symmetrical condition. I-V relation revealed the reversal potential as expected for a Cl(-)-selective channel. These results suggested that in mouse cardiac myocytes, swelling-activated, outward rectifying chloride channel with a single channel conductance of 38.1 +/- 2.5 pS (at +100 mV under [Cl-] symmetrical condition) underlies the volume regulatory Cl- channel.     

ClC-3 is a main component of background chloride channels activated under isotonic conditions by autocrine ATP in nasopharyngeal carcinoma cells

In this study, the activation mechanisms of the background chloride current and the role of the current in maintaining of basal cell volume were investigated in human nasopharyngeal carcinoma CNE-2Z cells. Under isotonic conditions, a background chloride current was recorded by the patch clamp technique. The current presented the properties similar to those of the volume-activated chloride current in the same cell line and was inhibited by chloride channel blockers or by cell shrinkage induced by hypertonic challenges. Extracellular applications of reactive blue 2, a purinergic receptor antagonist, suppressed the background chloride current in a concentration-dependent manner under isotonic conditions. Depletion of extracellular ATP with apyrase or inhibition of ATP release from cells by gadolinium chloride decreased the background current. Extracellular applications of micromolar concentrations of ATP activated a chloride current which was inhibited by chloride channel blockers and hypertonic solutions. Extracellular ATP could also reverse the action of gadolinium chloride. Transfection of CNE-2Z cells with ClC-3 siRNA knocked down expression of ClC-3 proteins, attenuated the background chloride current and prevented activation of the ATP-induced current. Furthermore, knockdown of ClC-3 expression or exposures of cells to ATP (10 mM), the chloride channel blockers 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and tamoxifen, or reactive blue 2 increased cell volume under isotonic conditions. The results suggest that ClC-3 protein may be a main component of background chloride channels which can be activated under isotonic conditions by autocrine/paracrine ATP through purinergic receptor pathways; the background current is involved in maintenance of basal cell volume.     

Basolateral Cl- channels in the larval bullfrog skin epithelium

The addition of 150 U/ml nystatin to the mucosal surface of isolated skin from larval bullfrogs increases apical membrane permeability and allows a voltage clamp to be applied to the basolateral membrane. With identical Ringer's solutions bathing either side of the tissue the short-circuit current (I(SC)) averaged 7.60+/-0.78 micro A/cm2, and this current could be increased or decreased by imposing a Cl- concentration gradient. Fluctuation analysis of the I(SC) gave power spectra that could be fit with low- and high-frequency Lorentzian functions having corner frequencies of 1.48+/-0.06 Hz and 48.5+/-11.4 Hz, respectively. The Lorentzian plateau was minimal at the lowest I(SC) and increased as the I(SC) became greater in the positive or negative direction. Current-voltage plots with identical Ringer's on either side of the tissue showed a pattern of outward rectification. Cell attached patches of cells isolated from the skin with collagenase-trypsin treatment showed spontaneous channel activity with a conductance of 20.9 pS at a pipette potential, -Vp=20 mV. Current-voltage plots of single channels showed a similar pattern of rectification to that of the intact skin, and partial replacement of Cl- by gluconate in the pipette solution shifted the reversal potential from zero to about 40 mV, which is close to the expected shift of the reversal potential of the chloride current through a Cl- selective ion channel. These results suggest that the basolateral Cl- conductance of the larval skin is mediated by a channel with properties that resemble a volume-sensing outward-rectifier anion channel that has been described in a variety of cell types     

ATP activates a Ca2+-dependent Cl− current in the rat thyroid cell line,FRTL-5

The effect of external ATP on both the membrane potential and the transmembrane current of the thyroid cell line FRTL-5 has been investigated in the patch-clamp whole-cell recording configuration. In the resting situation the membrane potential is around -70 mV and the membrane acts like a K(+)-sensitive electrode. Application of ATP at concentrations higher than 1 microM elicited an increase in Cl- conductance, responsible for a membrane depolarization which could be blocked by preincubation with the P2-antagonist quinidine. Chelation of intracellular Ca2+ also blocked the ATP induced changes in membrane potential and Cl- current. Intracellular perfusion with inositol trisphosphate (IP3) (50 microM) also stimulated a Cl- current which mimicked the response induced by ATP. ATP is able to initiate a response in the absence of extracellular Ca2+, but also opens a Ca(2+)-influx pathway, as demonstrated by a secondary response upon Ca2+ readmission in the external medium, in the continued presence of ATP. ADP and ATP gamma S were able to mimic the ATP response, whereas AMP and adenosine were unable to elicit a Cl- current. The P2X receptor agonist alpha,beta-methyleneATP was without effect as was the P2Y receptor agonist 2-methylthio ATP. We conclude that ATP is able to elicit a large IP3-mediated Ca(2+)-dependent Cl- current and membrane depolarization via a novel P2-type purinergic receptor.     

Chloride channels activated by osmotic stress in T lymphocytes

We have used whole-cell and perforated-patch recording techniques to characterize volume-sensitive Cl- channels in T and B lymphocytes. Positive transmembrane osmotic pressure (intracellular osmolality > extracellular osmolality) triggers the slow induction of a Cl- conductance. Membrane stretch caused by cellular swelling may underlie the activation mechanism, as moderate suction applied to the pipette interior can reversibly oppose the induction of Cl- current by an osmotic stimulus. Intracellular ATP is required for sustaining the Cl- current. With ATP-free internal solutions, the inducibility of Cl- current declines within minutes of whole-cell recording, while in whole- cell recordings with ATP or in perforated-patch experiments, the current can be activated for at least 30 min. The channels are anion selective with a permeability sequence of I- > SCN- > NO3-, Br- > Cl- > MeSO3- > acetate, propionate > ascorbate > aspartate and gluconate. GCl does not show voltage- and time-dependent gating behavior at potentials between -100 and +100 mV, but exhibits moderate outward rectification in symmetrical Cl- solutions. Fluctuation analysis indicates a unitary chord conductance of approximately 2 pS at -80 mV in the presence of symmetrical 160 mM Cl-. The relationship of mean current to current variance during the osmotic activation of Cl- current implies that each cell contains on the order of 10(4) activatable Cl- channels, making it the most abundant ion channel in lymphocytes yet described. The current is blocked in a voltage-dependent manner by DIDS and SITS (Ki = 17 and 89 microM, respectively, at +40 mV), the degree of blockade increasing with membrane depolarization. The biophysical and pharmacological properties of this Cl- channel are consistent with a role in triggering volume regulation in lymphocytes exposed to hyposmotic conditions.     

Permeation of both cations and anions through a single class of ATP-activated ion channels in developing chick skeletal muscle.

Micromolar concentrations of extracellular adenosine 5'-triphosphate (ATP) elicit a rapid excitatory response in developing chick skeletal muscle. Excitation is the result of a simultaneous increase in membrane permeability to sodium, potassium, and chloride ions. In the present study we quantify the selectivity of the ATP response, and provide evidence that a single class of ATP-activated ion channels conducts both cations and anions. Experiments were performed on myoballs using the whole-cell patch-clamp technique. We estimated permeability ratios by measuring the shift in reversal potential when one ion was substituted for another. We found that monovalent cations, divalent cations, and monovalent anions all permeate the membrane during the ATP response, and that there was only moderate selectivity between many of these ions. Calcium was the most permeant ion tested. To determine if ATP activates a single class of channels that conducts both cations and anions, or if ATP activates separate classes of cation and anion channels, we analyzed the fluctuations about the mean current induced by ATP. Ionic conditions were arranged so that the reversal potential for cations was +50 mV and the reversal potential for anions was -50 mV. Under these conditions, if ATP activates a single class of channels, ATP should not evoke an increase in noise at the reversal potential of the ATP current. However, if ATP activates separate classes of cation and anion channels, ATP should evoke a significant increase in noise at the reversal potential of the ATP current. At both +40 and -50 mV ATP elicited a clear increase in noise, but at the reversal potential of the ATP current (-5 mV), no increase in noise above background was seen. These results indicate that there is only a single class of excitatory ATP-activated channels, which do not select by charge. Based on analysis of the noise spectrum, the conductance of individual channels is estimated to be 0.2-0.4 pS.     

P2X2 receptors are essential for [Ca2+]i increases in response to ATP in cultured rat myenteric neurons

We characterized ATP-induced changes in intracellular Ca2+ concentration ([Ca2+]i) and membrane current in cultured rat myenteric neurons using ratiometric Ca2+ imaging with fura-2 and the whole cell patch-clamp technique, respectively. Neuronal cells were functionally identified by [Ca2+]i responses to high K+ and nicotine, which occurred only in cells positive for neuron-specific protein gene product 9.5 immunoreactivity. ATP evoked a dose-dependent increase of [Ca2+]i that was greatly decreased by the removal of extracellular Ca2+ concentration ([Ca2+]o). The amplitude of the [Ca2+]i response to ATP was reduced by half in the presence of voltage-dependent Ca2+ channel blockers. In [Ca2+]o-free solution, ATP produced a small transient rise in [Ca2+]i similar to that induced by P2Y agonists. At -60 mV, ATP evoked a slowly inactivating inward current that was suppressed by the removal of extracellular Na+ concentration. The current-voltage relation for ATP showed an inward rectification with the reversal potential of about 0 mV. The apparent rank order of potency for the purinoceptor agonist-induced increases of [Ca2+]i was ATP > or = adenosine 5'-O-3-triphosphate > or = CTP > or = 2-methylthio-ATP > benzoylbenzoyl-ATP. A similar potency order was obtained with current responses to these agonists. P2 antagonists inhibited inward currents induced by ATP. Ca2+ and Mg2+ suppressed the ATP-induced current, and Zn2+, Cu2+, and protons potentiated it. RT-PCR and immunocytochemical studies showed the expression of P2X2 receptors in cultured rat myenteric neurons. These results suggest that ATP mainly activates ionotropic P2X2 receptors, resulting in a [Ca2+]i increase dependent on [Ca2+]o in rat myenteric neurons. A small part of the ATP-induced [Ca2+]i increase may be also mediated via a P2Y receptor-related mechanism.     

A quantitative assessment of the use of 36Cl- distribution to measure plasma membrane potential in isolated hepatocytes

The plasma membrane potential of isolated rat hepatocytes was clamped at different values between 0 and -68 mV by addition of valinomycin in the presence of different extracellular concentrations of K+, and measured by the distribution of 86Rb+ between cells and medium. 36Cl- distribution came to steady state in 10-15 min. This steady-state distribution was compared to the plasma membrane potential over a range of values. 36Cl- distribution provided an accurate measurement of plasma membrane potential between -4 and -40 mV. At higher potentials intracellular chloride concentration is less than 20% of the extracellular concentration and errors due to uncertainties in the measurement of intracellular volume and of the contamination of cell pellets by extracellular medium precluded accurate determination of membrane potential: thus in our experiments 36Cl- underestimated the plasma membrane potential at -68 mV by 8 mV.