Harpin_(Pss)诱导的烟草早期防卫反应及钙的参与

Harpin_(Pss)可引发烟草过敏反应。超氧化物歧化酶能抑制,而过氧化氢酶不能抑制harpin_(Pss)诱导产生的这个反应,表明是超氧阴离子(O_2~-)是harpin_(Pss)诱导的烟草过敏反应的必要因子。Harpin_(Pss)还可引起烟草悬浮细胞活性氧的释放和胞外碱性化这两个早期防卫反应。Diphenylene iodonium能消除这种活性氧的诱导,这提示harpin_(Pss)可能是通过诱导NADPH氧化酶而产生O_2~_的。EGTA在无Ca~(2 )培养基中,能消除harpin_(Pss)诱导的烟草悬浮培养物的这两种防卫反应,再向培养基加Ca~(2 ),可恢复harpin_(Pss)的效应。LaCl_3、verapamil、新霉素、U-73122和LiCl也能抑制harpin_(Pss)诱导的这些反应,这表明由Ca~(2 )通道介导的Ca~(2 )内流和胞内Ca~(2 )库中Ca~(2 )的释放也参与了harpin_(Pss)诱导的早期防卫反应和过敏反应。在加harpin_(Pss)后很长时期内加放线菌素D、环己亚胺,都能抑制由它引发的过敏性细胞坏死过程,但它们对harpin_(Pss)诱导的活性氧的产生没有影响。表明O_2~-只是harpin_(Pss)诱发过敏性细胞凋亡的一个促发信号,过敏反应是更复杂的过程,需有基因持续地表达。     

愈伤组织形成过程中钙离子与激素诱导效应的关系

实验研究了在胡萝卜、烟草愈伤组织形成过程中,激素诱导作用与钙离子的关系。结果表明:在含有正常浓度的激素和Ca~(2+)的培养基中,0.1—1mmol/L Ca~(2+)螯合剂EGTA抑制愈伤组织鲜重增长78.0%—88.4%; 10—50μmol/L尼群地平及10—60μmol/L异博定等细胞膜钙通道阻断剂分别抑制愈伤组织鲜重增长19.1%—81.9%及17.6%—70.3%。除去上述Ca~(2+)螯合剂及Ca~(2+)通道阻断剂后,受抑制的外植体基本上可恢复生长。在无激素培养基中,10—30μmol/L Ca~(2+)载体A_(23187)可使外植体膨大,使外植体脱分化细胞增多,并出现分生细胞团,初步说明A_(23187)诱导的Ca~(2+)内流可以部分地模拟激素的作用。以膜显示剂氯四环素探测胞内Ca~(2+)分布时发现分裂细胞、脱分化细胞、分生细胞团及愈伤组织区域的细胞荧光较强。以上事实说明在愈伤组织形成中激素诱导效应与细胞内Ca~(2+)有密切关系。     

cAMP参与烟草悬浮细胞的ABA信号转导

ABA诱导型启动子 (rd2 9A)重组到报告基因 (GUS)的上游构建表达载体。通过农杆菌介导转化烟草 ,获得转基因植株。将转基因植株诱导愈伤组织 ,建立稳定、均一的转rd2 9A_GUS融合基因的悬浮培养细胞系。用ABA处理悬浮细胞 2 4h后GUS活性显著升高 ,说明外源ABA能够诱导rd2 9A启动子的表达 ,获得了用于ABA信号转导研究的实用细胞系。在ABA激活表达的细胞介质中加入尼克酰胺 (cADPR合成酶的抑制剂 )或U73 12 2 (PLC抑制剂 )只能部分抑制ABA的效应 ,但如果加入蛋白激酶抑制剂K2 5 2a ,抑制效果达 95 %以上。用可跨膜的cAMP的类似物8_Br_cAMP处理细胞发现 ,它能代替ABA的作用 ;当介质中加入 1mmol/LIBMX(磷酸二酯酶的抑制剂 )增加cAMP的稳定性 ,发现低浓度的 8_Br_cAMP与ABA相同的效应。以上结果表明cAMP参与了烟草悬浮细胞中ABA信号的传递。     

Ca2+在水杨酸诱发的丹参培养细胞培养基碱化过程中的作用

利用质膜钙离子通道抑制剂LaCl3、异搏定(Verapamil,VP),钙离子载体A23187,内膜系统钙离子通道抑制剂2-APB和LiCl处理,研究水杨酸(SA)诱发的丹参培养细胞内Ca2+迸发在培养基碱化过程中的作用。结果显示:SA处理诱发丹参培养细胞培养基碱化,质膜钙离子通道抑制剂LaCl3和VP、内膜系统钙离子通道抑制剂2-APB和LiCl单独处理均可显著抑制SA处理诱发的培养基碱化过程,但质膜钙离子通道抑制剂对SA处理诱发的培养基碱化的抑制作用要显著强于内膜系统钙离子通道抑制剂;当两类钙离子通道抑制剂同时使用,培养基碱化过程被完全抑制,甚至培养基出现酸化趋势;钙离子载体A23187可以显著促进培养基碱化过程。以上结果说明,由水杨酸诱发的胞外Ca2+内流与胞内钙库Ca2+释放均参与了丹参培养基碱化的诱导过程,但胞外Ca2+内流的作用更重要。本研究揭示了SA诱发的Ca2+与丹参细胞培养基碱化之间的关系,为更深层次地阐明植物次生代谢调控机制提供理论基础。     

Ca2+参与茉莉酸诱导蚕豆气孔关闭的信号转导

以Fluo-3AM为Ca~(2 )荧光探针,结合激光共聚焦扫描显微技术,观察到在处理后数十秒内,气孔关闭之前,茉莉酸(JA)可引起[Ca~(2 )]cyt的迅速上升;叶照和JA的前体物亚麻酸(LA)几乎不能引起[Ca~(2 )]cyt的明显变化;钙的螯合剂EGTA预处理可完全阻断JA诱导气孔关闭的效应,并且JA不再引起保卫细胞[Ca~(2 )]cyt增加;质膜Ca~(2 )通道的抑制剂硝苯吡啶(nifedipine,NIF)可减弱JA诱导气孔关闭的效应,也使JA诱导保卫细胞[Ca~(2 )]cyt增加的幅度有所下降;胞内Ca~(2 )释放的抑制剂钌红不能明显改变JA诱导气孔关闭的趋势,但使JA引起的保卫细胞[Ca~(2 )]cyt增加有所降低。实验结果表明:Ca~(2 )参与JA诱导气孔关闭的信号转导;推测JA引起的[Ca~(2 )]cyt升高可能主要来源于胞外,但不能完全排除胞内Ca~(2 )的释放。     

致凋亡的声动力疗法诱导巨噬细胞线粒体钙升高的机制研究

目的:研究致凋亡的声动力疗法诱导巨噬细胞线粒体钙升高的机制。方法:应用佛波酯(PMA)诱导THP-1单核细胞分化为巨噬细胞进行实验研究。选用5-氨基酮戊酸(ALA)作为声敏剂,进行声动力治疗(SDT)。应用流式细胞术证实SDT显著促进了细胞凋亡;应用Rhod 2/AM实时监测线粒体Ca~(2+)水平;通过蛋白质免疫印迹对全细胞蛋白中的Bax、Cleaved-caspase3、电压依赖性阴离子通道1(VDAC1)和三磷酸肌醇Ⅲ型受体(IP3R-Ⅲ)进行检测;应用VDAC1抗体进行免疫共沉淀,检测VDAC1和IP3R-Ⅲ之间的相互作用;实时监测线粒体Ca~(2+)水平,检测VDAC抑制剂DIDS和IP3Rs抑制剂2-ABP对SDT效果的影响。结果:与对照组相比,仅SDT组出现了显著的细胞凋亡(P0.001)。与对照组相比,ALA对线粒体Ca~(2+)水平无明显影响,超声诱导了线粒体Ca~(2+)水平的明显升高,SDT诱导了线粒体Ca~(2+)水平快速且大幅度的升高,且去除超声后仍维持在较高水平。与对照组相比,超声对Bax、Cleaved-caspase3、VDAC1和IP3R-Ⅲ的表达无明显影响,ALA诱导了VDAC1(P0.01)和IP3R-Ⅲ表达量的增加(P0.05),SDT诱导了Bax(P0.001)、Cleaved-caspase3(P0.001)、VDAC1(P0.01)和IP3R-Ⅲ(P0.05)表达量的增加,VDAC1和IP3R-Ⅲ的增加幅度与ALA组接近;ALA和SDT均诱导了VDAC1和IP3R-Ⅲ之间相互作用的显著增强(P0.05)。DIDS和2-ABP均明显抑制了SDT诱导的线粒体Ca~(2+)增加。结论:在致凋亡的SDT作用于THP-1巨噬细胞的过程中,ALA诱导了线粒体外膜Ca~(2+)转运通道VDAC1和内质网重要Ca~(2+)转运通道IP3R-Ⅲ的表达量增加与二者间相互作用的增强,在内质网和线粒体之间建立了大量的Ca~(2+)转运通道,超声的作用则在于触发这些Ca~(2+)转运通道的开放,进而引发线粒体钙的迅速增加。这是后续线粒体凋亡通路启动的重要机制之一。     

钙参与铜诱导的小麦根中NADPH氧化酶活性的升高

研究钙离子(Ca~(2 ))在重金属铜诱导的小麦根质膜NADPH氧化酶活性变化中作用的结果表明,Ca~(2 )以剂量依赖的方式提高NADPH氧化酶活性,且这种增加效应可完全为Ca~(2 )螯合剂乙二醇-双-(2-氨基乙基)四乙酸(EGTA)所抑制.用Ca~(2 )通道阻断剂氯化镧和异搏定以及EGTA预处理小麦根可抑制铜诱导的NADPH氧化酶活性升高,这类抑制效应也是剂量依赖的。这些结果说明Ca~(2 )参与铜诱导小麦根NADPH氧化酶活性和活性氧产生的调节过程.     

Con A刺激致T淋巴细胞胞浆游离Ca~(2 )浓度升高

本文分别应用荧光Ca~(2 )指示剂Quin2和Indo-1研究了Con A刺激的T淋巴细胞[Ca~(2 )]i升高过程及其发生机制.结果表明Con A与T淋巴细胞作用可导致细胞[Ca~(2 )]i的迅速升高.这种增加的胞内游离Ca~(2 )不仅来自胞外Ca~(2 )的内流,也来源于胞内钙库的释放.其中Ca~(2 )内流与T细胞钙通道的开放有关.可被钙通道抑制剂戊脉胺抑制,细胞的去极化及钾通道阻断剂四乙胺均不能阻断Ca~(2 )的内流,提示Ca~(2 )内流不是通过电位操纵的钙通道实现的,也与拥通道的开闭无关.Ca~(2 )内流可能是通过Con A受体活化的受体操纵的钙通道而实现的.     

线粒体钙单向转运体和线粒体应激关系的研究进展

钙离子(calcium ion,Ca~(2+))在线粒体功能障碍及细胞损伤凋亡过程中发挥重要的细胞信号作用。近些年来关于Ca~(2+)通道以及其调控蛋白的研究越来越多,其中,线粒体单向转运体(uniporter)复合物的结构组成及其相关蛋白的分布特点成为主要研究热点。作为uniporter复合物中关键的通道蛋白,线粒体钙单向转运蛋白(mitochondrial calcium uniporter,MCU)可顺电化学梯度摄入Ca~(2+),将Ca~(2+)从胞质转运到线粒体基质并控制转运速率,其在胞内Ca~(2+)信号转导、Ca~(2+)稳态、线粒体能量代谢以及细胞凋亡方面具有重要意义。识别调控线粒体内Ca~(2+)信号的MCU及其相关蛋白可深入阐明线粒体应激在相关疾病中的发生发展,并为进一步的疾病治疗提供理论依据。     

cAMP 参与烟草悬浮细胞的ABA信号转导

ABA诱导型启动子(rd29A)重组到报告基因(GUS)的上游构建表达载体.通过农杆菌介导转化烟草,获得转基因植株.将转基因植株诱导愈伤组织,建立稳定、均一的转rd29A-GUS融合基因的悬浮培养细胞系.用ABA处理悬浮细胞24 h后GUS活性显著升高,说明外源ABA能够诱导rd29A启动子的表达,获得了用于ABA信号转导研究的实用细胞系.在ABA激活表达的细胞介质中加入尼克酰胺(cADPR合成酶的抑制剂)或U73122(PLC抑制剂)只能部分抑制ABA的效应,但如果加入蛋白激酶抑制剂K252a,抑制效果达95%以上.用可跨膜的cAMP的类似物8-Br-cAMP处理细胞发现,它能代替ABA的作用;当介质中加入1 mmol/L IBMX(磷酸二酯酶的抑制剂)增加cAMP的稳定性,发现低浓度的8-Br-cAMP与ABA相同的效应.以上结果表明cAMP参与了烟草悬浮细胞中ABA信号的传递.     

Involvement of anion channel in signal transduction of early defense responses elicited by harpinPss in tobacco]

No matter when anion channel inhibitors, DIDS (4, 4'-diisothiocyanatostilbene-2, 2'-disulfonic acid) and A9C (anthracene-9-carboxylic acid) added (before, at the same time of or after harpinPss treatment), they can inhibit harpinPss-induced hypersensitive response in tobacco seedlings and release of active oxygen and extracellular alkalinization in tobacco suspension cells. DIDS and A9C also inhibit harpinPss-induced Ca2+ influx. In all these cases, DIDS is more efficient than A9C. It is postulated that anion channel positively regulates calcium channel in plasma membrane, and harpinPss may function through signal transduction mediated by anion channel and calcium channel to regulate cellular Ca2+ concentration and defense responses.     

HarpinPss elicited early defense responses of tobacco cells and involvement of calcium]

HarpinPss can induce hypersensitive reaction (HR) in tobacco leaves. As superoxide dismutase can inhibit but catalase can not inhibit the development of HR induced by harpinPss, superoxide anion is required for this response. HarpinPss can also induce the release of active oxygen and extracellular alkalinization, two early defence responses in tobacco suspension culture. Diphenylene iodoium, can completely inhibit the induction of HR in tobacco leaves, and the release of active oxygen in the suspension culture system, superoxide anion in these system may be produced by the activation of NADPH oxidase. Ethyleneglycol-bis (beta-aminoethyl) N, N, N'N'-tetraacetic acid (EGTA) can inhibit the development of harpinPss-induced HR and these two early defence responses in suspension culture system. Adding Ca2+ into the medium again, these responses can return to normal level in a short time. Lanthanum chloride, verapamil, neomycin, U-73122, and LiCl can also inhibit these harpinPss-induced responses. Therefore, the influx of Ca2+ mediated by calcium channel and the release of Ca2+ from internal Ca2+ pool may be involved in the two early defense responses induced by harpinPss. Cycloheximide and actinomycin D have no effect on the release of active oxygen but can inhibit harpinPss-induced HR even added them in the intermediate process for inducing HR. It indicates superoxide is just a trigger for HR, and HR is a more complex process that needs the sustained expression of some genes.     

The effects of several ion channel blockers and calmodulin antagonists on fertilization-induced acid release and 45Ca2+ uptake in sea urchin eggs

The effects of calcium antagonists, diltiazem and verapamil, and calmodulin antagonists, chlorpromazine, N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride (W-5) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), were tested on two responses of the sea urchin egg to insemination: (1) H+ release; (2) Ca2+ uptake. It was found that calcium antagonists inhibited both processes, while calmodulin antagonists only inhibited H+ release but not Ca2+ uptake. Verapamil and diltiazem were effective to inhibit H+ release when added to the egg suspension up to 120 sec and W-7 was effective around 150 sec after insemination. Calcium antagonists became ineffective earlier than W-7 in inhibiting H+ release. A calmodulin-dependent step may thus occur linking the Ca2+ uptake and H+ release. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an anion channel blocker, also inhibited both Ca2+ uptake and H+ release. This result suggests that an uptake of anion(s) occurs along with Ca2+ uptake.     

Characteristics of anion channels in the tonoplast of the liverwort Conocephalum conicum

Isolated vacuoles of the liverwort Conocephalum conicum thallus cells were investigated using the patch-clamp technique. At high cytosolic Ca(2+) activities, slowly activating currents were evoked by positive potentials. The currents were conducted by the SV (slow-vacuolar) channel. When isolation of vacuoles was carried out at high Mg(2+) and low Ca(2+) concentration and the same proportion of the cations was kept in the bath, currents were recorded at negative potentials. Once activated, these currents persisted even after replacing Mg(2+) with K(+) in the bath. Sr(2+) and Ba(2+) were also effective activators of the currents. With a Cl(-) gradient, 10 mM in the bath and 100 mM in the lumen, currents were significantly reduced and the current-voltage characteristics shifted towards the reversal potential of Cl(-), indicating Cl(-) selectivity. Currents almost vanished after substituting Cl(-) with gluconate. They were strongly reduced by anion channel inhibitors 4,4'-diisothicyanatostilbene-2,2'-disulfonic acid (DIDS; 1 mM), anthracene-9-carboxylic acid (A9C; 2 mM) and ethacrinic acid (0.5 mM). Single-channel recordings revealed a 32 pS channel activating at negative voltages. It is concluded that the currents at negative potentials are carried by anion channels suitable for conducting anions from the cytosol to the vacuole. The anion channels were weakly calcium dependent, remaining active at physiological calcium concentration. The channels were almost equally permeable to Cl(-), NO3(-) and SO4(2-), and much less permeable to malate(2-). Anion channels did not respond to ATP addition. cAMP (10 microM) had a weak effect on anion channels. Protein kinase A (0.4 U) added to the medium caused no significant effect on anion channels.     

The role of anion channels in the mechanism of acrosome reaction in bull spermatozoa.

The involvement of anion channels in the mechanism of the acrosome reaction (AR) was investigated. The AR was induced by Ca2+ or by addition of the Ca2+ ionophore A23187. The occurrence of AR was determined by following the release of acrosin from the cells. In order to investigate the role of anion channels in the AR, several anion-channel inhibitors were tested, mainly DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid). Other blockers, like SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid), furosemide, probenecid and pyridoxal 5-phosphate, were also tested. We found that DIDS binds covalently to sperm plasma membrane in a time- and concentration-dependent manner. Maximal binding occurs after 2 h with 0.3 mM DIDS. DIDS and SITS inhibit AR in a concentration-dependent manner. The IC50 of DIDS and SITS in the presence of A23187 is 0.15 and 0.22 mM, respectively. Tributyltin chloride (TBTC), an Cl-/OH- exchanger, partially overcomes DIDS inhibition of the AR. HCO3- is required for a maximal acrosin release and Ca(2+)-uptake, in the presence or absence of A23187. It is known that HCO3- activates adenylate cyclase and therefore, increases the intracellular level of cAMP. The inhibition of the AR by DIDS decreases from 95 to 50% when (dibutyryl cyclic AMP (dbcAMP) was added, i.e., HCO3- is no longer required while elevating the level of cAMP in an alternative way. Moreover, we show that the stimulatory effect of HCO3- on Ca(2+)-uptake is completely inhibited by DIDS. We conclude that DIDS inhibits AR by blocking anion channels, including those that transport HCO3- into the cell.     

Disulfonic stilbene derivatives open the Ca2+ release channel of sarcoplasmic reticulum

Ca2+ channels of isolated sarcoplasmic reticulum were incorporated into a planar lipid bilayer and their pharmacological properties were studied. The results show that the channel is a Ca2+-induced Ca2+ release channel like that observed in skinned muscle fibers and isolated vesicles. (i) The open channel probability was increased by the addition of micromolar amounts of Ca2+ to the cis (myoplasmic) side and further increased by millimolar ATP. (ii) The channel was closed by millimolar Mg2+ and micromolar ruthenium red. We found that two disulfonic stilbene derivatives, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 4-acetoamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), when added to the cis side open the channel and lock it irreversibly at open without changing the single channel conductance. Ca2+ efflux from SR vesicles was also enhanced by SITS and DIDS, as monitored by a tracer assay. Further, Ag+ activated the channel transiently. These results suggest that certain amino and SH residues play important roles in gating the Ca2+ channel.     

An increase in free radical production by means of an anion channel blocker DIDS in mouse peritoneal neutrophils

DIDS (4, 4'-diisothiocyanostilbene-2, 2'-disulfonic acid) has been recognized as an anion channel blocker. In this study, we demonstrated that DIDS significantly enhanced the production of free radicals in mouse peritoneal neutrophils. By means of a luminol-chemiluminescence (LCL) monitoring system, DIDS markedly increased LCL which could be suppressed by SOD, sodium azide (NaN3), EGTA and BAPTA-AM and only slightly inhibited by staurosporine (STP). Depletion of the endoplasmic reticulum (ER)-Ca2+ store by means of thapsigargin (TG) had no effects on DIDS-enhanced LCL, but DIDS significantly increased the amount of intracellular free calcium as monitored by means of fura-2 staining. These results indicate that DIDS may enhance free radical production mediated by Ca2+ release from the mitochondria. Both phorbol-12-myristate-13-acetate (PMA) and DIDS can induce increased translocation of p47-phox of the neutrophil to the membrane fraction, which is inhibited by STP pretreatment. Since free radical generation could reduce the cytoplasmic pH (pHi), we further examined whether DIDS was capable of inducing intracellular acidification. The result indicated that DIDS certainly lowered the pHi which was also suppressed by pretreatment with either NaN3 or NaCN, but not by diphenyleneiodonium (DPI). These findings lead us to propose a working hypothesis that DIDS mainly induces superoxide production accompanied by decreasing pHi mediated through a Ca2+ -dependent effect on the mitochondria rather than on NADPH oxidase. Using the lipophilic fluorescent dye DiOC6(3), we showed that DIDS decreased the transitional mitochondrial membrane potential. NaN3, but not STP or pyrrolidine dithiocarbamate (PDTC), antagonized DIDS in the course of decreasing the mitochondrial membrane potential. Taken together, all of these findings imply a possible role of anion channels of the mitochondria in modulating free radical production and intracellular acidification of neutrophils through alteration of the mitochondrial transition membrane potential and Ca2+ -release.     

Mechanism for electrosilent Ca2+ transport to cause calcification of spicules in sea urchin embryos

Embryos of the sea urchin, Hemicentrotus pulcherrimus, kept in sea water containing the calcium antagonists, diltiazem and verapamil, or an anion transport inhibitor, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS), during a developmental period between the mesenchyme blastula and the pluteus corresponding stage, became abnormal plutei with poorly developed arms and quite small spicules. Treatment with ethacrynic acid and furosemide, inhibitors of chloride transport, during the same period of development yielded quasi-normal plutei with poor spicules and somewhat developed arms. In late gastrulae, the inhibitory effects of these calcium antagonists and DIDS on the uptake of 45Ca2+ in whole embryos were as strong as those on 45Ca deposition in spicules, whereas the effects of chloride transport inhibitors on calcium deposition in the spicules were markedly stronger than on its uptake in whole embryos. Electrosilent uptake of Ca2+ seems to be established mainly by coupled influx of chloride in the cells which mediate spicule calcification, and by concomitant influx of anions in the other cells. In swimming blastulae, 45Ca2+ uptake was inhibited by calcium antagonists and DIDS, but not by chloride transport inhibitors. Ca2+ uptake probably becomes coupled with chloride influx only in embryos in which spicule calcification occurs.     

Functional geometry of the permeation pathway of Ca2+-activated Cl-channels inferred from analysis of voltage-dependent block

We examined the voltage-dependent block of Ca(2+)-activated Cl(-) channels by anthacene-9-carboxylic acid (A9C), diphenylamine-2-carboxylic acid (DPC), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and niflumic acid (NFA) in excised inside-out and outside-out patches from Xenopus oocytes. The fraction of the voltage field (delta) experienced by the blocking drug was determined from the voltage dependence of block. All the drugs blocked by entering the channel from the outside. delta was 0.6 for A9C, 0.3 for DPC and DIDS, and <0.1 for NFA. Because the voltage dependence of the drugs differed, the order of potency was also voltage-dependent. At +100 mV the order of potency was NFA > A9C > DIDS > DPC (K(i) (microm) = 10.1, 18.3, 48, and 111, respectively). Because the drugs are hydrophobic, they can cross the bilayer when applied from the inside and block the channel from the outside. The equilibrium geometries of the blockers were determined by molecular modeling and compared with their blocking positions (delta). This analysis suggests that the channel is an elliptical cone with the largest opening facing the extracellular space. The selectivity filter has an apparent size of 0.33 x 0.75 nm, because C(CN)(3)-, which has these dimensions, permeates. The external opening is at least 0.60 x 0.94 nm, because DPC has these dimensions and penetrates the channel approximately 30%.     

Effect of disulfonic stilbene anion-channel blockers on the guinea-pig myocardium

The role of anions in the maintenance of tension in electrically driven left atria isolated from guinea pigs has been examined. The disulfonic stilbene anion-channel blockers SITS (4-acetamido-4'-isothiocyanostilbene 2'-disulfonate) and DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonate) decreased the contractile force developed in a time- and concentration-dependent manner. As in the red cell anion channel, DIDS was more potent than SITS, but the maximal inhibition of tension produced by N-(4-azido-2-nitrophenyl)-2-aminoethyl sulfonate (NAP-taurine) was considerably lower than the near maximal inhibition produced by SITS and DIDS. The inhibition by SITS and DIDS was irreversible, suggesting a covalent interaction, and could not be overcome by increasing the calcium concentration or the frequency of stimulation. Consistent with a requirement for chloride anion, substitution of chloride and bicarbonate by the impermeant anion gluconate did not support contraction, while only partial tension was maintained with the lipophilic anions acetate and thiocyanate. Incubation of atria with 400 microM SITS blocked both 36Cl and 45Ca uptake to a similar extent, whereas the efflux of both these ions was not affected by incubation of the atria with SITS. The blockade by disulfonic stilbene anion-channel blockers of the contraction of the guinea pig myocardium may result from impairment of excitation-contraction coupling.