抑制氯通道阻抑鼻咽癌细胞周期和细胞增殖

目的:研究Cl^-通道在鼻咽癌细胞调节性容积回缩(RVD)、增殖及细胞周期分布中的作用。方法:活细胞图像分析低分化鼻咽癌细胞(CNE－2Z)RVD,用台盼蓝拒染法检测细胞存活率,MTT法检测细胞增殖能力。用流式细胞仪测定细胞周期不同时相细胞百分率。结果:Cl^-通道抑制剂硝基苯丙胺基苯甲酸(NPPB)剂量依赖性抑制RVD和细胞增殖,100μmol/LNPPB明显阻抑细胞周期进程,使细胞停滞于G₁期,G₁期细胞百分率从54%提高到71%,但对细胞存活率没有显著性影响。结论:阻抑Cl^-通道可阻滞细胞于G₁期而抑制细胞增殖。提示Cl^-通道和RVD的激活是促进细胞从G₁期进入S期和维持增殖所必需的因素。     

反义多重耐药性基因抑制眼睫状体非色素上皮细胞容积激活性氯电流

目的:研究多重耐药性(MDR1)基因及其产物P糖蛋白(P-gp)与容积激活性Cl^-电流的关系。方法:用膜片钳全细胞记录技术记录牛眼睫状体非色素上皮(NPCE)细胞容积激活性Cl^-电流,反义寡核苷酸阻抑细胞MDR1基因表达,在激光共聚焦显微镜下检测细胞P-gp免疫荧光。结果:P-gp免疫荧光与人源反义MDR1呈剂量依赖性减弱关系,容积激活性Cl^-电流被人源反义MDR1特异性地部分阻抑,电流潜伏期延长,峰电流值减少,电流抑制率与人源反义MDR1呈现剂量依赖性增强关系,(r=0.99, P＜0.01)。 P-gp表达抑制率和容积激活性Cl^-电流抑制率高度正相关(r=0.99, P＜0.01)。结论: MDR1基因及其产物P-gp参与NPCE细胞容积激活性Cl^-电流的形成。     

低渗诱导高分化鼻咽癌细胞CNE-1容积激活性氯电流

目的： 研究细胞外低渗诱导的高分化鼻咽癌细胞CNE-1的容积激活性氯电流。方法：全细胞膜片钳记录氯电流,通过应用氯通道阻断剂、离子置换和改变细胞容积方法研究该电流的特性。结果：当细胞在等渗环境中背景电流微弱且稳定,细胞外给予47%低渗刺激后电流迅速增大,呈外向优势,对阴离子通透性的大小为：I->Br->Cl->葡萄糖酸。氯通道阻断剂ATP和NPPB可逆性地抑制此电流,ATP的抑制作用在外向电流显著强于内向电流。此电流对细胞容积改变敏感,细胞肿胀时被激活,细胞发生皱缩时则被抑制。结论：细胞外低渗诱导CNE-1 细胞产生氯电流,此电流对细胞容积的改变敏感,在CNE-1细胞容积调节中起重要作用。     

顺铂激活的低分化鼻咽癌细胞氯电流

目的： 研究抗癌药顺铂对低分化鼻咽癌细胞（CNE-2Z）氯通道的激活作用以及该电流的特性。方法：采用膜片钳技术记录顺铂激活的CNE-2Z细胞全细胞电流;离子置换法等方法分析通道的特性。结果：细胞外灌流5 μmol/L的顺铂诱发CNE-2Z细胞产生一个有明显外向优势的电流,电流的翻转电位为(-6.69 ± 0.51) mV,接近氯离子平衡电位(-0.9 mV)。该电流的激活依赖于细胞内ATP。顺铂激活的细胞膜氯通道对阴离子的通透性顺序为：I-≥Br->Cl->gluconate。氯通道阻断剂tamoxifen完全抑制该电流。结论：抗癌药顺铂可以激活一个电流特征与容积激活性氯通道相似的氯通道。     

细胞外低渗诱导大鼠胚胎心肌细胞H9c2容积激活性氯电流和调节性细胞容积回缩

目的:研究细胞外低渗诱导的大鼠胚胎心肌细胞(H9c2)容积激活性氯电流和调节性容积回缩(regulatory volumede crease,RVD)。方法:采用全细胞膜片钳技术记录低渗激活的H9c2细胞氯电流并分析电流特性;用实时活细胞影像系统拍摄细胞图像,测量细胞容积,探讨氯通道在H9c2细胞调节性容积回缩(RVD)过程中的作用。结果:等渗灌流下,可在H9c2细胞记录到一个较小的背景电流。47%低渗液灌流可迅速诱发一个具有外向优势的电流,该电流无明显时间依赖性失活和电压依赖性失活;在+80mV和-80mV电压钳制下,细胞的平均电流密度分别为(47.77±3.80)pA/pF和(-33.36±2.80)pA/pF;翻转电位为(-9.02±0.61)mV,接近氯离子的平衡电位(-0.9mV)。高渗灌流液可以完全抑制该电流。此外,该电流可被氯通道阻断剂他莫昔芬、5-硝基-2-(3-苯丙胺)苯甲酸(NPPB)和ATP不同程度抑制。同时,细胞外灌流47%低渗液可诱发H9c2细胞产生RVD,100μmol/L的NPPB几乎完全抑制低渗诱发的RVD。结论:细胞外低渗刺激可以诱导H9c2细胞容积激活性氯电流和RVD。容积激活性氯通道在H9c2细胞RVD中起重要作用。     

氯通道在三氧化二砷诱导鼻咽癌细胞凋亡中的作用

目的:探讨氯通道在三氧化二砷(arsenic trioxide,As_2O_3)诱导人鼻咽癌CNE-2Z细胞凋亡中的作用。方法:采用流式细胞术检测CNE-2Z细胞经As_2O_3作用24及48 h的凋亡率;应用膜片钳技术记录As_2O_3激活的细胞膜电流,并分析其电流特性;采用流式细胞术检测氯通道阻断剂DIDS对As_2O_3诱导的CNE-2Z细胞凋亡的抑制作用。结果:(1)5μmol/L As_2O_3能够时间依赖性地诱导的CNE-2Z细胞凋亡;(2)CNE-2Z细胞外灌流含5μmol/L As_2O_3的等渗溶液能够激活一个具有外向整流特征的电流,激活的电流无明显的时间及电压依赖性失活,翻转电位接近氯平衡电位;(3)As_2O_3激活的电流能够被氯通道阻断剂DIDS和NPPB完全抑制,细胞外灌流47%的高渗溶液能够完全抑制As_2O_3激活的电流;(4)氯通道阻断剂DIDS能够抑制As_2O_3诱导的CNE-2Z细胞凋亡。结论:As_2O_3可以激活CNE-2Z细胞的容积敏感性氯通道。氯通道在As_2O_3诱导的CNE-2Z凋亡中发挥重要作用。     

顺铂激活的低分化鼻咽癌细胞氯电流为非钙激活氯电流*

 目的：探讨顺铂激活的低分化鼻咽癌细胞（CNE-2Z）氯通道电流是否为钙激活的氯电流。方法：采用膜片钳全细胞记录技术记录细胞内/外无Ca2+及钙通道阻断剂对顺铂激活氯电流的影响,并用高渗灌流液观察顺铂激活氯电流的容积敏感性。结果：去除细胞外液的Ca2+后,5 μmol/L顺铂能诱发氯电流,且电流大小与细胞外有Ca2+ 时无明显差异,但潜伏期与达峰时间延长。细胞内外均无Ca2+ 对顺铂激活氯电流未产生影响。钙通道阻断剂nifedipine未能抑制顺铂诱发的氯电流。但细胞外灌流高渗液几乎可完全抑制顺铂激活的氯电流。结论: 顺铂激活的氯通道开放不依赖于细胞内/外的Ca2+,该通道不是钙激活氯通道而很可能是容积敏感性氯通道。     

阻塞性黄疸对肠上皮细胞氯离子分泌影响的研究

目的: 探讨阻塞性黄疸(OJ)对肠上皮细胞氯离子分泌的影响。方法: 建立OJ大鼠模型,分别于胆管结扎7 d(OJ1组)和14 d(OJ2组)后,检测实验动物外周血Cl^-浓度,取末端回肠上皮细胞体外培养,荧光分光光度法测量细胞内Cl^-浓度,全细胞膜片钳技术检测Cl^-电流的改变,并用Western blotting法分析电压门控氯离子通道蛋白2(ClC-2)表达的变化。结果: OJ1组和OJ2组的血清Cl^-浓度分别为(88.67±4.68)mmol/L 和(82.01±6.25)mmol/L,均低于对照组(均P＜0.05)。细胞内Cl^-相对浓度,OJ1组(3.14±0.38)和OJ2组(3.55±0.47)均高于对照组(均P＜0.05)。对照组肠上皮细胞Cl^-电流,从-80 mV的(-15.45±7.56)pA/pF升到80 mV的(5.85±0.81)pA/pF,呈外向整流趋势。OJ组Cl^-平均电流密度绝对值逐渐减小,与对照组比较差异显著(均P＜0.05)。Western blotting分析显示ClC-2蛋白表达条带在90 kD附近,对条带的定量分析表明,OJ1组为0.20±0.04,OJ2组为0.19±0.06,均低于对照组(0.27±0.06)(均P＜0.05),但OJ1、2组间比较没有显著差异(P＞0.05)。结论: OJ抑制肠腔氯离子分泌,缩小上皮细胞内外氯离子浓度差,减少Cl^-外向电流,此作用与细胞膜上氯离子通道蛋白 2的表达降低有关。     

人急性淋巴细胞白血病细胞容积激活性氯电流的研究

目的: 研究人急性淋巴细胞白血病细胞系(Molt4细胞)容积激活性氯电流。方法: 采用膜片钳全细胞方式记录细胞外低渗液(160 mOsmol/L)激活的Molt4细胞容积激活性氯电流,分析该电流的特性。结果: Molt4细胞在等渗溶液中背景电流较小且稳定,低渗溶液诱导激活容积激活性氯电流,该电流呈现较明显的外向优势,在钳制电压0 mV~±120 mV及脉冲波宽200 ms条件下,没有观察到明显的电压依赖性失活和时间依赖性失活。该电流具有容积敏感性,可被细胞外高渗刺激所抑制。氯通道阻断剂他莫昔芬显著抑制容积激活性氯电流, 对内向电流和外向电流的抑制作用无显著差异。结论: Molt4细胞膜存在容积敏感性氯通道;低渗刺激可激活该通道产生容积激活性氯电流;该通道对氯通道阻断剂他莫昔芬敏感。     

蟾蜍灵激活低分化鼻咽癌细胞氯通道

目的: 研究蟾蜍灵(bufalin)对低分化鼻咽癌(CNE-2Z)细胞氯通道的激活作用以及通道的特性。方法: 采用全细胞膜片钳技术记录蟾蜍灵激活CNE-2Z细胞膜电流并分析其电流特征。结果: 细胞外灌流1 μmol/L 的蟾蜍灵可诱发CNE-2Z细胞产生一个氯电流,该电流潜伏期较长,为(12.1 ± 6.4)min, 其翻转电位接近氯离子平衡电位。该电流具有较明显的外向优势,没有明显的时间依赖性失活和电压依赖性失活。氯通道阻断剂他莫昔芬(tamoxifen)可完全抑制该电流, 细胞外灌流高渗液也可完全抑制该电流。结论: 蟾蜍灵可以激活CNE-2Z细胞氯通道产生氯电流,与容积激活性氯电流相比,该电流的潜伏期较长,并且有更明显的外向优势。     

The role of bicarbonate in regulatory volume decrease (RVD) in the epithelial-derived human breast cancer cell line ZR-75-1

This study investigates the mechanisms involved in the regulatory volume decrease (RVD) in ZR-75-1 epithelial-derived human breast cancer cells. Cell volume changes were measured during osmotic shock using video imaging. In HEPES-buffered hypotonic solutions no RVD was observed; however, RVD was observed in HCO(3)(-)-buffered hypotonic solutions. Inhibition of RVD by 10 microM tamoxifen and 100 microM DIDS (inhibitors of volume-regulated anion channels; VRAC) and 2 mM TEA(+) (inhibitor of K(+) channels) indicates a role for these channels. In HCO(3)(-)-buffered Cl(-)-free solutions RVD was partially abolished indicating that HCO(3)(-) efflux can support RVD but also may have another role. Further experiments investigated whether HCO(3)(-) assists in the accumulation of Cl(-) via Cl(-)-HCO(3)(-) exchange. Regulatory volume increase (RVI) was also HCO(3)(-)-dependent and was inhibited by 500 microM DIDS and 10 microM 5-( N, N-dimethyl)-amiloride (DMA) indicating a role for coupled Cl(-)-HCO(3)(-) and Na(+)-H(+) exchange. Finally, in the presence of 10 microM DMA, RVD was partially inhibited providing further evidence for a role of Cl(-)-HCO(3)(-) exchange. Thus RVD in ZR-75-1 cells involves the activation of VRAC and K(+) channels. RVD is HCO(3)(-)-dependent and HCO(3)(-) efflux through VRAC appears to contribute directly to RVD. HCO(3)(-), however, also has another role in facilitating Cl(-) accumulation via Cl(-)-HCO(3)(-) exchange.     

Role of volume-stimulated osmolyte and anion channels in volume regulation by mammalian sperm

The ability to maintain cellular volume is an important general physiological function. Swelling induced by hypotonic stress results in the opening of channels, through which ions exit with accompanying water loss (regulatory volume decrease, RVD). RVD has been shown to occur in mammalian sperm, primarily through the opening of quinine-sensitive potassium channels. However, as yet, direct evidence for the participation of anion channels in sperm RVD has been lacking. The chloride channel type ClC-3 is believed to be involved in RVD in other cell types. Using electronic cell sizing for cell volume measurement, the following results were obtained. (i) The anion channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), tamoxifen and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) increased hypotonic swelling in concentration-dependent fashion, whereas verapamil (P-glycoprotein inhibitor) had little effect. The most potent, NPPB and DIDS, blocked RVD without affecting cell membrane integrity at effective concentrations. (ii) When gramicidin was included to dissipate Na+/K+ gradients, major secondary swelling was observed under hypotonic conditions. This secondary swelling could be reduced by NPPB, and suppressed completely by replacing chloride in the medium with sulphate, an ion which does not pass through chloride channels. It was deduced that the initial hypotonic swelling activated an anion channel through which chloride ions could then enter freely down a concentration gradient, owing to the lack of a counter-gradient of potassium. (iii) Taurine, an osmolyte often involved in RVD, does not appear to play a role in sperm RVD because lengthy preincubation with taurine did not alter sperm RVD response. Our observations provide direct evidence that a chloride channel (possibly ClC-3) is involved in the process of volume regulation in mammalian sperm.     

氯通道在不同生长周期的鼻咽癌细胞迁移中的作用

目的：研究不同生长周期的鼻咽癌低分化上皮细胞（CNE-2Z）的迁移能力及氯通道在迁移过程中所起的作用。 方法： 运用血清饥饿法、化学药物双阻断法、有丝分裂药物阻断及摇落法分别将CNE-2Z细胞同步化至细胞周期的G1、S、M期，流式细胞仪检测其同步化效果。结合应用迁移小室和图像分析法，测定迁移率。台盼蓝活细胞染色法测定药物的细胞毒性。 结果： 不同生长周期的细胞迁移能力不同，G1期细胞迁移能力最强，随后是M期， S期迁移能力最弱。氯通道阻断剂（ATP、NPPB、tamoxifen）抑制CNE-2Z细胞迁移，但不同的氯通道阻断剂对不同生长周期CNE-2Z细胞迁移的阻滞效应不相同。 结论： CNE-2Z细胞的迁移能力与其所在的生长周期密切相关，氯通道在各期CNE-2Z细胞的迁移过程中起着重要作用。     

Changes in element composition of A6 cells following hypotonic stress

Cellular element concentrations and dry weight contents were determined in A6 epithelia using electron microprobe analysis. This was done to assess the quantitative contributions of Na, K and Cl to the regulatory volume decrease (RVD) and isovolumetric regulation (IVR) after decreasing the basolateral osmolality from 260 to 140 mosmol/kg in a stepwise or gradual way. Two minutes after inducing acute hypotonic stress the cells behaved almost like ideal osmometers, as indicated by a pronounced increase in cell height and decreases in the cellular dry weight and concentrations of all measured elements by about the same degree. Sixty minutes after inducing acute hypotonic stress the dry weight and concentrations of the impermeant elements P, Mg and Ca had returned approximately to control values, indicating normalized cell volume. Na, K and Cl concentrations, however, remained greatly reduced. The cellular amounts of Na, K and Cl diminished during RVD by approximately 31%, 24% and 46%, respectively. The dry weights and element concentrations measured 60 min after inducing acute hypotonic stress were similar to those obtained after a continuous reduction of basolateral osmolality. The cellular loss of Na and K following hypotonic stress exceeded that of Cl by about 40 mmol/kg wet wt., suggesting the exit of an other anion and/or the titration of fixed negative charges. The contribution of Na, K and Cl to total cellular osmolality increased from about 75% under control conditions to about 85% during RVD and IVR. Since only approximately 70% of the loss of cellular osmolytes necessary for the observed RVD and IVR is accounted for by the cellular exit of Na, K and Cl, other osmolytes, possibly amino acids, must leave the cells following hypotonic stress.     

敲低IK1钾通道抑制ClC-3氯通道的表达和功能

 目的: 探讨ClC-3氯通道是否为IK1钾通道的调节靶点,重点研究鼻咽癌细胞IK1钾通道对ClC-3氯通道功能及蛋白表达的影响。方法: 采用siRNA转染技术抑制低分化鼻咽癌上皮细胞(CNE-2Z) IK1 基因的表达;real-time PCR技术检测ClC-3 mRNA的表达;Western blot检测ClC-3的蛋白表达;细胞免疫荧光结合激光共聚焦显微镜技术检测ClC-3和IK1蛋白在细胞内分布;全细胞膜片钳记录细胞氯电流。结果: IK1 siRNA可以成功转染CNE-2Z细胞,有效抑制鼻咽癌细胞IK1钾离子通道的表达;用IK1 siRNA抑制鼻咽癌细胞IK1钾离子通道的表达后, ClC-3的mRNA表达上调而ClC-3蛋白却表达减少:在低分化鼻咽癌上皮细胞,低渗刺激可激活氯通道,产生一个较大的氯电流,在成功转染IK1 siRNA的细胞,此氯电流明显减弱。结论: 敲低IK1钾离子通道可抑制ClC-3氯离子通道的表达和功能。     

Gadolinium as an opener of the outwardly rectifying Cl(-) channel (ORCC). Is there relevance for cystic fibrosis therapy?

There is indirect evidence that the plasmalemma-integrated eukaryotic porin (the voltage-dependent anion-selective channel, VDAC) functions as the outwardly rectifying chloride channel (ORCC). The channel, which is believed to play a role in cell volume regulation, appears to be relevant for cystic fibrosis (CF) therapy, in that it may function as an alternative Cl(-) channel. In the present study we showed first that Gd(3+) altered the voltage dependence of human type-1 porin incorporated into artificial planar lipid bilayers. Next, using a light-scattering approach on transformed normal or CF human B-lymphocytes in hypotonic Ringer solution, we found slightly differing regulatory volume decrease (RVD) curves for the cell lines under study. Addition of 15-60 microM GdCl3 in hypotonic Ringer increased light scattering, pointing to cell swelling beyond normal values. RVD was not observed in those experiments. A corresponding effect was seen in isotonic Ringer containing GdCl3. In either osmotic situation Gd(3+)-induced cell swelling was abolished by monoclonal mouse anti-human type-1 porin antibodies. Agonist and antibody effects were dose dependent. Finally, videocamera-monitored control experiments with adherent HeLa cells verified the direct effect of the agonist on cell swelling in hypo- or isotonic situations and its prevention by the antibodies. We conclude that GdCl3 opens plasmalemma-integrated porin channels, allowing ions to following their gradients, resulting in cell swelling. Since respiratory epithelium expresses porin channels in the apical membrane, the use of gadolinium to activate ORCC may represent a new therapeutic approach in CF.