在inside-out膜片上caffeine对猪冠状动脉平滑肌细胞钙激活钾通道的调节作用及ryanodine的影响

目的:利用膜片钳技术,研究咖啡因对钙激活钾通道(calcium-activated potassium channel,KCa)的作用机制,及咖啡因存在时兰尼定(ryanodine)对KCa的影响以阐明冠状动脉平滑肌的调控机制。方法:采用急性酶分离方法,应用膜片钳单通道电流记录技术记录猪冠状动脉平滑肌钿胞上KCa电流活动。电流信号经放大、滤波及A/D、D/A转换后输入微机进行采样和储存。应用PCLAMP9.0软件系统进行数据采集及分析。结果:在内面向外式(inside-out)膜片下,咖啡因(0.1、0.5、1.0、5.0mmol/L)可浓度依赖性地增加通道开放概率,而对电流幅值无明显影响,开放概率增加是通过明显缩短平均关闭时间实现的(n=8,P0.01);洗去药物后通道活性可以恢复到对照水平;5.0mmol/L咖啡因对KCa激活作用最大(P0.01)。在细胞贴附式(cell-attached)膜片上,咖啡因激活KCa后,ryanodine(10-40μmol/L)浓度依赖性地抑制通道开放概率,开放时间缩短,关闭时间延长,对电流幅度无明显影响。结论:在inside-out膜片下,咖啡因能够直接激活猪冠状动脉平滑肌细胞KCa通道。在cell-atta-ched构型上,ryanodine可通过胞内一定的信号通路浓度依赖性间接抑制咖啡因对KCa激活。     

糖尿病胃轻瘫大鼠胃平滑肌细胞BK_(Ca)通道的变化

目的:探讨糖尿病胃轻瘫(DGP)的发病机制与胃平滑肌细胞大电导钙激活钾通道(BKCa)变化之间的关系。方法:将大鼠分为对照组和糖尿病胃轻瘫模型组。应用木瓜蛋白酶液急性酶分离大鼠胃平滑肌细胞,采用单通道膜片钳技术记录大鼠胃平滑肌细胞BKCa电流,免疫组织化学技术测定BKCa蛋白KCNMA和KCNMB1的表达。结果:大鼠胃平滑肌细胞BKCa电流的开放具有电压依赖性和胞内钙离子浓度依赖性,可被钾离子通道阻滞剂Ib TX阻断;模型组大鼠胃平滑肌细胞BKCa开放概率及开放幅度增加(P0.05),平均开放时间及平均关闭时间减少(P0.01);模型组大鼠胃平滑肌细胞KCNMB1蛋白表达增加(P0.01),KCNMA蛋白表达无明显变化。结论:DGP发病与胃平滑肌细胞BKCaβ1亚基蛋白表达上调及通道功能活动增强有关,β1亚基蛋白可能通过调节BKCa功能活动参与DGP的发生。     

Cell-attached膜片上,咖啡因对猪冠状动脉平滑肌细胞KCa的调节作用

目的:研究咖啡因对猪冠状动脉平滑肌细胞KCa通道的调控机理,以期揭示胞内钙库RyR激活后,局部钙离子浓度升高和KCa的关系。方法:采用急性酶分离方法,应用膜片钳单通道电流记录技术记录大脑皮层神经元猪冠状动脉平滑肌细胞上KCa通道电流活动。电流信号经放大、滤波及A/D、D/A转换后输入微机进行采样和储存。实验数据应用CLAMP9.0软件系统进行数据采集及分析。结果:在cell-attached膜片上咖啡因对KCa通道有明显的作用,咖啡(0.1-5.0 mM)可以增加通道的开放概率(NPo),呈现出浓度依赖性,开放时间延长,关闭时间也随之缩短,而对电流幅值无明显影响,开放概率的增加是通过明显缩短平均关闭时间实现的(n=8,P<0.05);洗去药物后通道活性可以一定程度的恢复到对照水平,再加入一定浓度咖啡因(如1.0mM)可再次激活KCa,激活程度与洗脱前较接近。结论:在细胞贴附式构型上咖啡因浓度依赖性地激活KCa,有饱和性。可能是通过影响胞内信号转导过程而调控KCa活性。     

Genistein对大鼠冠状动脉平滑肌细胞BKCa调控机制研究

目的 探讨Genistein对大鼠冠状动脉平滑肌细胞BKCa的调控机制.方法 通过急性酶分离法获取SD大鼠冠状动脉平滑肌细胞,并经由全细胞膜片钳技术分析Genistein对BKCa通道电流的调控机制.结果 电流幅度、通道开放概率、平均开放时间与膜电位相关(P<0.05);有钙外液中行10-5 mol/L Genistein干预后外向电流(+60 mV)为(303.58±70.36)pA,显著高于对照的(173.02±42.68)pA(P<0.05),而无钙外液孵育0.5 h后行Genistein干预后外向电流与对照比较无显著差异(P>0.05);四乙胺+Genistein干预后外向电流(65.32±6.34)pA,显著低于Genistein干预的(303.58±70.36)pA(P<0.05);BAPTA-AM加入后给予Genistein干预,两组外向电流比较无显著差异(P>0.05).结论 大鼠冠状动脉平滑肌细胞BKCa单通道开放概率、电流幅度与膜电位密切相关;Genistein能快速激活BKCa通道,可能与促使内钙释放有关.     

丹参素对猪冠脉平滑肌细胞钙激活钾通道的作用

目的观察丹参素(DS-182)对原代培养猪冠脉平滑肌细胞钙激活钾通道(KCa)的作用,从单个钾通道水平揭示其扩冠机制。方法采用膜片钳单通道电流记录技术。结果①猪冠脉平滑肌细胞KCa的单通道电导值高,为(291.74±4.89)pS;对膜电位变化及细胞内钙离子浓度变化敏感,能被5~20 mmol/L膜内侧四乙基铵(TEA)所阻断。②在细胞贴附式膜片方式下,细胞外应用10~20μmol/L的DS-182对通道具明显的激活效应;而在内面向外式膜片方式下,细胞内应用5~30μmol/L的DS-182对通道均无明显作用。结论DS-182对通道的激活作用是非直接的,需要一系列的胞内过程。     

内源性一氧化氮对哮喘大鼠支气管平滑肌细胞钾通道的影响

目的：观察体内应用一氧化氮（NO）前体左旋精氨酸（L-Arg）对哮喘大鼠支气管平滑肌细胞（BSMC）钾通道电生理特性的影响，为哮喘治疗提供理论基础。 方法： 雄性SD大鼠，随机分为对照组、哮喘组和哮喘L-Arg(300 mg/kg)治疗组（L-Arg组）。急性酶消化法分离获得大鼠单个BSMC。用常规全细胞膜片钳技术记录3组BSMC的静息膜电位（Em）、钙激活钾通道(BKCa)电流和电压依赖性钾通道（Kv）电流的差异。 结果： （1）哮喘组的静息膜电位为(-29.4±5.6) mV，显著低于对照组(-34.8±6.2)mV, (P<0.05)；L-Arg治疗组的静息膜电位为(-36.1±6.8) mV, 与哮喘组差异显著(P<0.05)。（2）钙激活钾通道电流：+50 mV电压刺激时，方波刺激模式下哮喘组大鼠BSMC的BKCa峰值电流密度［(43.8±16.5) pA/pF, n=8］低于对照组［(72.5±19.9)pA/pF, n=8］,(P<0.01)；L-Arg组的BKCa电流密度［(58.7±12.4) pA/pF, n=8］则高于哮喘组(P<0.05)。（3）电压依赖性钾通道电流：+50 mV电压刺激时，方波刺激模式下哮喘组大鼠BSMC的Kv峰值电流密度为［(32.4±8.7)pA/pF, n=8］，显著低于对照组［(57.7±9.8)pA/pF, n=8］ (P<0.01)；L-Arg组的Kv峰值电流密度［(43.6±7.9)pA/pF, n=8］，显著高于哮喘组［(32.4±8.7)pA/pF, n=8］ (P<0.05)。结论： 体内应用L-Arg可增加钙激活钾通道和电压依赖性钾通道电流，明显改善哮喘大鼠BSMC的静息膜电位，从而降低气道平滑肌细胞的兴奋性，可能具有抑制气道高反应性的作用。     

大电导钙激活钾通道与动脉粥样硬化

动脉粥样硬化(AS)形成是一个复杂炎症反应的过程.多种致AS的危险因素,尤其是氧化型低密度脂蛋白存在,导致内皮细胞、单核巨噬细胞、血管平滑肌细胞及血小板等的大电导钙激活钾通道(BKCa)激活,后者促使细胞功能障碍,进而促进AS的形成.本文简要综述BKCa参与AS形成的研究进展.     

原代培养猪冠脉平滑肌细胞的电压依赖和钙激活的钾通道

作者应用改良的细胞膜片钳制技术对原代培养的猪冠状动脉平滑肌细胞的电压依赖性和钙激活的钾通道的特性进行了研究。整个实验均在计算机的控制下进行,各种膜片中引导出的电流,通过膜片钳制放大器后,均输入至计算机进行采样、存贮和分析。结果显示:①这种通道活动呈明显的电压依赖性,随膜的去极化而激活,电流-电压关系     

钙激活钾通道在重症失血性休克血管反应性下降中的作用

目的探讨大电导钙激活钾通道(largeconductancecalci um_activatedpotassiumchannel,BKCa)在重症失血性休克中的变化及其特异性阻断剂charybodotoxin(CTX)对休克血管反应性的恢复作用。方法1.复制大鼠重症失血性休克模型 ,分离一侧股静脉用以测量血压和放血 ;2.酶法分离出单个肠系膜A2、A3血管平滑肌细胞 ;3.应用膜片钳的细胞贴附式记录法记录休克组及假手术组大电导钙激活钾通道电流变化 ,并用 pClamp7.0软件对所记录通道的相关数据进行分析和处理 ;4.整体动物静脉给药比较观察生理盐水和CTX对休克动物平均动脉压(MAP)的影响及对去甲肾上腺素(NE)升压作用的影响 ,并计算对比休克动物24h存活率和平均存活时间的变化。结果大鼠重症失血性休克后BKCa 异常激活 ,在高钾细胞外液中 ,单通道电导由对照组的(131±4)pS(n=52)增加为休克时的(172±6)pS(n=38,P<0.01) ;通道的开放概率(Po)在膜电位为20、40和60mV时 ,比正常组增加 ,但没有显著意义(P>0.05) ,而随着膜电位的增加 ,休克组开放概率的增加速率明显大于对照组(使Po增加e倍的电压增幅由14mV降为11mV) ,当膜电位去极化为80和100mV时 ,Po分别由对照组的(15.5±2.8) %和(19.6±3.8) %增加为休克组的(21.2±2.6) %(P<0.05)和(30.3±3.4) %(P<0.01)。休克120m     

cAMP依赖的蛋白激酶激活猪冠脉平滑肌钙激活的钾通道

许多蛋白质包括离子通道能被磷酸化作用所调节。本文应用细胞膜片钳制技术观察了环磷酸腺苷(cAMP)对原代培养猪冠状动脉平滑肌细胞的150pS电导的钙激活的钾通道的影响。实验结果:①在“细胞吸附”膜片中,向浴槽溶液中加入Forskolin 10μM,可通过激活腺苷酸环化酶和增加细胞内cAMP浓度,来激活钙激活的钾通道。②在“内     

羟基红花黄色素A通过激活Kv通道舒张大鼠冠状动脉

目的:研究羟基红花黄色素A (hydroxysafflor yellow A,HSYA)对离体大鼠冠状动脉(rat coronary artery,RCA)的舒张作用,并探讨该作用与电压门控性钾通道(Kv通道)的关系。方法:采用DMT离体血管环技术明确HSYA对RCA的肌源性作用及血管内皮对该作用的影响;采用全细胞膜片钳技术观察HSYA对RCA平滑肌细胞Kv电流的影响;采用Western blot技术探讨HSYA对RCA平滑肌细胞Kv1.2和Kv1.5通道蛋白表达的影响。结果:10μmol/L和30μmol/L HSYA能够舒张60 mmol/L KCl或0.1 mmol/L U46619预收缩的RCA(P<0.05),但其对预收缩的内皮完整组和去内皮组RCA舒张作用的差异无统计学显著性(P>0.05);HSYA(3μmol/L、10μmol/L和30μmol/L)可使RCA平滑肌细胞Kv电流显著增加(P<0.05);10μmol/L和30μmol/L HSYA能够增加RCA平滑肌细胞Kv1.2和Kv1.5通道蛋白的表达。结论:HSYA可舒张预收缩的RCA,该作用与血管内皮无关,其可能的机制与增强Kv通道功能和蛋白表达有关。     

依托咪酯对新生大鼠皮质神经元钙激活性钾通道的作用

采用膜片钳技术的吸附式和内面向外式两种构型研究了依托咪酯对增减的ＳＤ新生鼠皮持 钙激活性钾（ＫＣａ）通道的作用。结果：（１）新生ＳＤ大鼠皮质神经元上存在有单元电导为１７６．６５±３７．９７ｐＳ的ＫＣａ通道;（２）依托咪酯０．８、４、１０、２０四个不同剂量在吸附式构型时浓度依赖性激活ＫＣａ通道,其开放概率较对照组均明显增大,（３）依托咪酯０．８μｍｏｌ／Ｌ在内面向外式构型时对ＫＣａ通道也有明显激活作     

Effect of acidosis on Ca2+-activated K+ channels in cultured porcine coronary artery smooth muscle cells

 Although acidosis induces vasodilation, the vascular responses mediated by large-conductance Ca²⁺-activated K⁺ (K_Ca) channels have not been investigated in coronary artery smooth muscle cells. We therefore investigated the response of porcine coronary arteries and smooth muscle cells to acidosis, as well as the role of K_Ca channels in the regulation of muscular tone. Acidosis (pH 7.3–6.8), produced by adding HCl to the extravascular solution, elicited concentration-dependent relaxation of precontracted, endothelium-denuded arterial rings. Glibenclamide (20 μM) significantly inhibited the vasodilatory response to acidosis (pH 7.3-6.8). Charybdotoxin (100 nM) was effective only at pH 6.9–6.8. When we exposed porcine coronary artery smooth muscle cells to a low-pH solution, K_Ca channel activity in cell-attached patches increased. However, pretreatment of these cells with 10 or 30 μM O, O′-bis(2-aminophenyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl)ester (BAPTA-AM), a Ca²⁺ chelator for which the cell membrane is permeable, abolished the H⁺-mediated activation of K_Ca channels in cell-attached patches. Under these circumstances H⁺ actually inhibited K_Ca channel activity. When inside-out patches were exposed to a [Ca²⁺] of 10^–6 M [adjusted with ethyleneglycolbis(β-aminoethylester)-N,N,N′,N′-tetraacetic acid (EGTA) at pH 7.3], K_Ca channels were activated by H⁺ concentration dependently. However, when these patches were exposed to a [Ca²⁺] of 10^–6 M adjusted with BAPTA at pH 7.3, H⁺ inhibited K_Ca channel activity. Extracellular acidosis had no significant direct effect on K_Ca channels, suggesting that extracellular H⁺ exerts its effects after transport into the cell, and that K_Ca channels are regulated by intracellular H⁺ and by cytosolic free Ca²⁺ modulated by acute acidosis. These results indicate that the modulation of K_Ca channel kinetics by acidosis plays an important role in the determination of membrane potential and, hence, coronary arterial tone. Received: 20 January 1998 / Received after revision: 9 April 1998 / Accepted: 22 April 1998     

Calcium channels in smooth muscle cells from cerebral precapillary arterioles activate at more negative potentials than those from basilar artery

We compared Ca²⁺ channels in cell-attached patches of smooth muscle cells from cerebral precapillary arterioles and basilar artery of guinea pig. Patches were studied without Ca²⁺ channel activators in the pipette solution. In both preparations, a 23 pS channel (40 mM Ba²⁺) sensitive to block by nifedipine was identified. In arteriolar but not in basilar artery patches, channel activity was recorded without apparent inactivation at potentials of –40 to –20 mV. Values for the number of channels in a patch x probability of channel opening (n·P_o) at various potentials were fit to a Boltzmann function. For the arteriolar patches (n=5) and for patches from basilar artery (n=5), the midpoint potentials for the voltage dependence of n·P_o were –9.3 mV and +8.9 mV, and maximum values of n·P_o at positive potentials were 1.23 and 0.33. At potentials 0 mV, the average for the maximum number of superimposed openings in basilar artery patches was 1.7 (n=17) and in arteriolar patches was 6.5 (n=6). For both preparations, histograms of channel open times at –10 mV required two time constants, 0.48 and 3.95 ms, and the shorter open state accounted for 88% of openings. Our data indicate that Ca²⁺ channel activity is likely to be more prominent near resting membrane potentials in arteriolar cells than in basilar artery cells.     

Hyperpolarization-activated cationic channels in smooth muscle cells are stretch sensitive

The properties of hyperpolarization-activated channels were studied in single smooth muscle cells from the stomach of the toad, Bufo marinus, using the patchclamp technique. In cell-attached patches, inward channel currents were activated by hyperpolarizing pulses from a holding potential of –20 mV to potentials more negative than –60 mV. The activity of the channels increased and their latency of activation decreased as the hyperpolarization was increased. The slope conductance of the channels with standard high sodium concentration pipette solution was 64.2±9.1 pS (SD, n=17). Stretching the patch, by suction applied to the back of the patch pipette, also increased the activity and shortened the latency of activation. We designate these channels as HA-SACs (hyperpolarization- and stretch-activated channels). HA-SACs were observed in 83% (175/210) of the patches studied. HA-SAC currents were carried by sodium and potassium ions, but their amplitude was increased by replacing extracellular sodium with potassium. Extracellular magnesium and calcium ions significantly reduced the single-channel conductance of HA-SACs. These permeation characteristics and the single-channel conductance of HA-SACs were indistinguishable from those of stretch-activated channels (SACs) previously described in these cells. The following observations are consistent with HA-SACs being a subset of SACs. First, SACs were at times found in cell-attached patches which lacked HA-SACs. Second, the number of channels in a cell-attached patch simultaneously activated by stretch (usually 5–10 and often more) exceeded by far the number simultaneously activated by hyperpolarization (usually one or two). Third, in excised insideout patches, the ability of hyperpolarization to activat HA-SACs was lost within 3–5 min, whereas stretch continued to activate a large number of channels. The last observation also suggests that activation by hyperpolarization requires an intracellular agent, whereas stretch activation does not.     

Number of K(Ca) channels underlying spontaneous miniature outward currents (SMOCs) in mudpuppy cardiac neurons

Spontaneous miniature outward currents (SMOCs) in parasympathetic neurons from mudpuppy cardiac ganglia are caused by activation of TEA- and iberiotoxin-sensitive, Ca(2+)-dependent K(+) (BK) channels. Previously we reported that SMOCs are activated by Ca(2+)-induced Ca(2+) release (CICR) from caffeine- and ryanodine-sensitive intracellular Ca(2+) stores. In the present study, we analyzed the single channel currents that contribute to SMOC generation in mudpuppy cardiac neurons. The slope conductance of BK channels, determined from the I-V relationship of single-channel currents recorded with cell-attached patches in physiological K(+) concentrations, was 84 pS. The evidence supporting the identity of this channel as the channel involved in SMOC generation was its sensitivity to internal Ca(2+), external TEA, and caffeine. In cell-attached patch recordings, 166 microM TEA applied in the pipette reduced single-channel current amplitude by 32%, and bath-applied caffeine increased BK channel activity. The ratio between the averaged SMOC amplitude and the single-channel current amplitude was used to estimate the average number of channels involved in SMOC generation. The estimated number of channels involved in generation of an averaged SMOC ranged from 18 to 23 channels. We also determined that the Po of the BK channels at the peak of a SMOC remains constant at voltages more positive than -20 mV, suggesting that the transient rise in intracellular Ca(2+) from ryanodine-sensitive intracellular stores in the vicinity of the BK channel reached concentrations most likely exceeding 40 microM.