体外培养新生大鼠大脑皮层星形神经元钾通道的特性及膜外钾离子浓度对其影响

用膜片钳技术中的细胞贴附方式和内面向外方式,首次在新生大鼠大脑皮层星形神经元胞体膜上记录到一类电压依赖性钾通道。此通道可被２０ｍｍｏｌ／Ｌ ＴＥＡ,５ｍｍｏｌ／Ｌ Ｂａ＾２＋,１４０ｍｍｏｌ／Ｌ Ｃｓ＾＋阻断,不受２０ｍｍｏｌ／Ｌ４－ＡＰ影响,其激活不依赖Ｃａ＾２＋。膜外钾离子浓度对通道的特性有显著的影响,逆转电位随Ｋ＾＋的增大而增大,并表现出一定的饱和现象,两者的对数呈线性关系;同一驱动电位下,     

细胞外低钠灌流对豚鼠心室肌细胞内钾离子活度的影响

本实验应用钾离子选择微电极观察了不同浓度的低钠对豚鼠心室肌细胞内钾离子活度（ａｉＫ）的影响。发现细胞外低钠（低［Ｎａ］ｏ）灌流引起明显的ａｉＫ下降,下降幅度随灌流液中Ｎａ＋浓度的降低而加大。Ｃｓ＋（５ｍｍｏｌ／Ｌ）及低钾（２．５ｍｍｏｌ／Ｌ）灌流均可明显减小这种ａｉＫ的下降。Ｃａ２＋通道阻断剂Ｃｄ２＋、Ｎｉ２＋、Ｍｎ２＋及Ｖｅｒａｐａｍｉｌ对此无影响;而长时间的Ｃａｆｅｉｎｅ（１０ｍｍｏｌ／Ｌ,３０ｍｉｎ）灌流可减小低钠引起的ａｉＫ下降。鉴于上述结果,我们认为：低［Ｎａ］ｏ引起的ａｉＫ下降与细胞膜钠泵活动的改变关系不大,而很可能与细胞膜对Ｋ＋的通透性及细胞膜上钙调节Ｋ＋通道的活动改变有关。     

钙对生长素诱导的玉米胚芽鞘切段延长生长的影响

１０μｇ／ｇ的ＩＡＡ溶液强烈地促进玉米胚芽鞘切段的延长生长和质子分泌,但这两种效应的启动时间有别．Ｃａ２＋在高浓度下（２—５ｍｍｏｌ／Ｌ）强烈抑制ＩＡＡ诱导的延长生长,但在低浓度下（０．５ｍｍｏｌ／Ｌ）则有轻微的促进作用．ＩＡＡ增大质膜相对透性,而Ｃａ２＋则有稳定膜的作用,且适宜的浓度较低（０．５ｍｍｏｌ／Ｌ）．     

培养乳鼠心肌细胞自发性〔Ca~（2＋）〕_i瞬间变化的实验模型

使用Ｃａ￣（２＋）敏感的荧光指示剂Ｆｕｒａ－２／ＡＭ,对培养的心肌细胞测定细胞内Ｃａ￣（２＋）的瞬间变化。结果为,在一个心搏周期中,经过大约１８０ｍｓ的时间,［Ｃａ￣（２＋）］＿ｉ瞬间变化达到最大值,然后恢复到基线水平（最小值）,经历了２６０ｍｓ。测得平均［Ｃａ￣（２＋）］＿ｉ瞬间变化的最大值及最小值分别为３４６±３８ｎｍｏｌ／Ｌ和１０２±１８ｎｍｏｌ／Ｌ。血管紧张素Ⅱ１００ｎｍｏｌ／Ｌ引起［Ｃａ￣（２＋）］＿ｉ瞬间变化最大值明显增加,但对［Ｃａ￣（２＋）］＿ｉ瞬间变化的基线水平没有明显的影响。ｒｙａｎｏｄｉｎｅ３μｍｏｌ／Ｌ使［Ｃａ￣（２＋）］＿ｉ瞬间变化的幅度明显降低。ＫＣｌ５０ｍｍｏｌ／Ｌ使［Ｃａ￣（２＋）］＿ｉ瞬间变化完全停止,并明显增加［ｃａ￣（２＋）］＿ｉ的基线水平。结果提示,本实验模型可用来观察［Ｃａ￣（２＋）］＿ｉ的瞬间变化。     

用Fura－2双波长荧光法测定神经细胞内游离钙

采用新型Ｃａ＾２＋荧光指示剂Ｆｕｒａ－２建立双波长荧光法测定分离的大鼠神经细胞内游离钙浓度（［Ｃａ＾２＋］ｉ）。结果显示,在静息状态下,其［Ｃａ＾２＋］ｉ为１０９±１２ｎｍｏｌ／Ｌ．３０ｍｍｏｌ／ＬＫＣｌ可显增加［Ｃａ＾２＋］ｉ,并且ＫＣｌ的这种效应呈一定的剂量依赖关系,提示该法灵敏、可靠。     

POTENTIATION OF CAFFEINEINDUCED CONTRACTURE BY RAISING EXTRACELLULAR POTASSIUM IN FROG SKELETAL MUSCLE

用蛙胫前肌小束为材料,研究了提高胞外钾［Ｋ＋］Ｏ对咖啡因挛缩的作用。［Ｋ＋］Ｏ从２ｍｍｏｌ／Ｌ提高到１０或２５ｍｍｏｌ／Ｌ,由３ｍｍｏｌ／Ｌ咖啡因引起的挛缩明显增强。以ＰＫＣ／ＰＣ（ＰＫＣ和ＰＣ分别为在高钾和正常钾条件下的咖啡因挛缩）表示的咖啡因挛缩增强,依赖［Ｋ＋］Ｏ和高钾作用时间。随着１０ｍｍｏｌ／Ｌ［Ｋ＋］Ｏ作用时间延长,直至１０ｍｉｎ,增强逐渐增加。但是,２５ｍｍｏｌ／Ｌ［Ｋ＋］Ｏ作用１ｍｉｎ时增强达到最大,然后下降到对照。ＰＫＣ／ＰＣ变化时程不能用高钾引起的去极化解释,而与由相似［Ｋ＋］Ｏ引起的胞浆自由钙变化时程相符。提示,至少在蛙骨骼肌,高钾引起的咖啡因挛缩增强主要是由胞浆自由钙升高引起的。     

SNP抑制5-HT诱导的胞内游离钙浓度升高和内钙释放

用Ｆｕｒａ － ２／ＡＭ 荧光测量技术研究了５ － 羟色胺（５－ ＨＴ） 诱导的大鼠尾动脉平滑肌细胞胞内钙升高和一氧化氮（ＮＯ） 的抑制效应。实验表明, 胞外０ｍ ｍｏｌ／ Ｌ Ｃａ２ ＋ 时胞内静息［Ｃａ２ ＋ ］ ｉ 为２０ ．２±８ ．６ｎｍｏｌ／Ｌ（ｎ ＝ ８） 。１０μｍｏｌ／Ｌ ５－ ＨＴ 可诱导出胞内钙库释放引起的瞬态［Ｃａ２ ＋］ｉ 升高,其峰值达２４５ ．７ ±７１．６ｎｍｏｌ／ Ｌ（ｎ ＝ ６） 。１０ － ７ ｍｏｌ／Ｌ 硝普钠（ＳＮＰ） 可抑制５－ ＨＴ 诱导的［Ｃａ２ ＋］ｉ 升高,其峰值浓度降为７５．１±３５ ．９ｎｍｏｌ／Ｌ（ｎ ＝ ５） 。当细胞浴液含２．５ｍ ｍｏｌ／Ｌ Ｃａ２ ＋ 时,静息［Ｃａ２ ＋］ｉ为１１２ ．８ ±１０ ．３ｎｍｏｌ／ Ｌ（ｎ ＝ ５） , 这时１０μｍｏｌ／ Ｌ ５ － ＨＴ 可诱导［Ｃａ２ ＋ ］ ｉ 的峰值为２５２ ．３ ±８０ ．６ｎｍｏｌ／Ｌ（ｎ ＝ ４） ,以及其后平台浓度为１４３ ．０ ±３７ ．６ｎｍｏｌ／Ｌ（ｎ ＝ ４） ,略大于［Ｃａ２ ＋］ｉ 为１１２．８ ±１０ ．３ｎｍｏｌ／Ｌ 的静息浓度,为外钙内流引起。１０ － ７ ｍｏｌ／Ｌ ＳＮＰ 也可抑制５－ ＨＴ 诱导［Ｃａ２ ＋ ］ｉ 平台相浓度。平台浓度由１４３ ±４７     

大鼠纹状体神经元中乙酰胆碱激活的离子通道

用膜片钳技术对培养的纹状体神经元膜上的乙酰胆碱激活的离子通道进行了研究。结果表明,单通道电流为内向电流,随着超极化程度增大而增加,随着去极化程度增大而减少;翻转电位为１５．０±６．１ｍＶ,电导为８２．７１±１６．１７ｐＳ;通道的开放时间分布直方图和关闭时间分布直方图均需双指数拟合,开放时间分布直方图的时间常数分别为１．９３ｍｓ和５．７３ｍｓ,关闭时间分布直方图的时间常数则为０．５２８ｍｓ和２．３２ｍｓ;通道的平均开放时间和开放概率均与超极化程度无明显相关关系。由于电极液中含ＡＣｈ和Ｋ￣＋通道阻断剂Ｃｓ￣＋而不含Ｎａ￣＋或Ｃａ￣（２＋）,且浴槽液中含Ｎａ￣＋通道阻断剂河豚毒素,因此可排除内向Ｎａ￣＋流、Ｃａ￣（２＋）流和Ｋ￣＋流,提示内向电流为ＡＣｈ激活的通道电流。     

腺苷对缺氧时海马神经元内游离Ca^2＋的影响

实验用Ｆｌｕｏ－３负载细胞,在激光扫描共聚焦显微镜下直接监测缺氧后分散培养的大鼠海马ＣＡ１区神经元内游离Ｃａ＾２＋浓度（［Ｃａ＾２＋］ｉ）的变化,观察腺苷对这种变化的影响并初步探讨其作用机制。结果发现,急性缺氧使海马神经元［Ｃａ＾２＋］ｉ显著升高;腺苷（１００μｍｏｌ／Ｌ）明显抑制缺氧引起的［Ｃａ＾２＋］ｉ增高,腺苷Ａ１受体拮抗剂ＣＰＴ以及Ｋ＾＋通道阻断剂４－ＡＰ和ＡＴＰ敏感性Ｋ＾＋通道阻断剂ｇｌ     

脉冲电场对鸡胚脑细胞Ca~(2 )和cAMP含量的影响

利用ｆｕｒａ－２／ＡＭ荧光方法和放射免疫方法分别研究了脉冲电场对１１天和１４天胚龄的悬浮鸡胚脑细胞内Ｃａ２＋和环腺苷酸（ｃＡＭＰ）含量的影响,并探讨了在脉冲电场作用下这两种信号物质变化的相关性。实验结果表明：当电激液中［Ｃａ２＋］为１ｍｍｏｌ／Ｌ时,在宽度为１００微秒．强度为０．５,１．０,２．０ｋＶ／ｃｍ的脉冲电场作用下,１１天胚龄的鸡胚脑细胞内钙离子浓度（以［Ｃａ２＋］ｉ表示）与对照相比显著上升,其升高值随着脉冲强度的增加而增加,而细胞内ｃＡＭＰ含量与对照相比显著降低;用ＥＧＴＡ络合电激液中游离Ｃａ２＋以后,虽然细胞内［Ｃａ２＋］ｉ显著上升,细胞内ｃＡＭＰ含量没有显著变化。１４天胚龄的鸡胚脑细胞内［Ｃａ２＋］ｉ在脉冲电场作用下显著上升,而细胞内ｃＡＭＰ含量没有显著变化。脉冲电场对１１天和１４天胚龄的鸡胚脑细胞膜和细胞内Ｃａ２＋库Ｃａ２＋转移系统的开放均有显著作用,此作用具有对脉冲强度的依赖性。为了解物理信号跨细胞膜传导的方式提供了初步的实验根据     

中华大蟾蜍卵母细胞质膜的外向整流型钾离子通道

我们用电压箝方法研究了中华大蟾蜍卵母细胞的膜生理特性。发现卵母细胞膜去极化至-30mV及更偏正时,有一持续的外向电流出现,该电流与去极化程度约呈正比增加,当膜电位箝在20mV时其峰值达3.7±1.4μA。该电流被钾离子通道拮抗剂TEA和4-AP抑制,TEA半抑制浓度为2.6mmol/L。氯通道拮抗剂9-AC(2.5mmol/L)无抑制作用。无钙的或钙离子浓度增加三倍的胞外灌流液均对该电流无影响、该外向电流的逆转电位随胞外钾离子浓度的改变而变化。胞外钾离子浓度增加十倍,逆转电位约增加47.3mV,而胞外钠、钙或氯离子浓度的改变对逆转电位基本上无影响,因此该电流可被认为主要是电压依赖性钾离子流。取自冬眠蟾蜍的卵母细胞经孕酮诱发成熟后,电压依赖性钾离子流减小,仅为原来的1/20-1/30,而取自全年在高温饲养的蟾蜍的卵母细胞经孕酮处理后未见成熟,其电压依赖性钾离子流仅减小至原来的三分之一。     

电压激活的钾通道阻断剂抑制山莨菪碱松弛去甲肾上腺素预收缩的兔主动脉平滑肌

研究显示,山莨菪碱预处理不改变高钾引起的兔主动脉环收缩,但可明显减弱去甲肾上腺素(noradrenaline,NA)、组织胺或5-羟色胺引起的收缩,且其减弱作用不受去除血管内皮影响。本实验观察了几种钾通道阻断剂对山良菪碱松弛：NA预收缩的兔主动脉环的影响。结果表明,1、3、10μmol／L山莨菪碱作用8min,可使0．01μmol／L NA预收缩的兔主动脉环松弛(P＜O．01)。10mmol／L,CsCl、1mmol／L 4-氨基吡啶、10μmol／L BaCl2、10μmol/L格列本脲、3μmol／L charybdotoxin和3μmol／L蜂毒明从分别与0．0lμmol／L NA同时加入,可增强后者收缩兔主动脉环的作用(P＜0．01)。10、30mmol／L CsCl或10、30mmol／L 4-氨基吡啶存在时,10μmol／L山茛菪碱对NA预收缩的兔主动脉环的松弛作用减弱,松弛率与对照组比较分别有极显著差异(P＜0．01);10、30μmol／L BaCl2,10、30μmol/L格列本脲,3μmol/L charybdotoxin或3μmol／L蜂毒明肽存在时,山莨菪碱对NA预收缩的兔主动脉环的松弛作用不受影响(P＞O．05)。本研究表明,电压激活的钾通道阻断剂抑制山莨菪碱松弛NA预收缩的兔主动脉平滑肌,初步提示血管平滑肌细胞膜上电压激活的钾通道参与山莨菪碱扩血管作用。     

兔血管平滑肌延迟整流钾通道研究及其与克隆Kv1.5通道的电生理特性比较

本文用膜片箝全细胞技术比较了研究了单个兔肺动脉血管平滑肌细胞上延迟整流钾通道与克隆Ｋｖ１．５通道的电生理及药理学特性。将平滑肌细胞箝制在－４０ｍＶ,以１０ｍＶ的步跨阶跃去极化（０￣６０ｍＶ）可产生一系列快速上升的外向电流,几无衰减,其激活曲线的Ｖ１／２为２７．２ｍＶ。灌流液中加入１００ｍｍｏｌ／Ｌ和ＴＥＡ １ｍｍｏｌ／Ｌ ４ＡＰ,电流幅度均明显减小,细胞外Ｃａ＾２＋水平由１．５ｍｍｏｌ／Ｌ降至０．     

The Ca2+-activated K+ channel and its functional roles in smooth muscle cells of guinea pig taenia coli

Currents through single potassium channels were studied in cell-attached or inside-out patches from collagenase-dispersed smooth muscle cells of the guinea pig taenia coli. Under conditions mimicking the physiological state with [K+]i = 135 mM: [K+]o = 5.4 mM, three distinct types of K+ channel were identified with conductances around 0 mV of 147, 94, and 63 pS. The activities of the 94- and 63-pS channel were observed infrequently. The 147-pS channel was most abundant. It has a reversal potential of approximately -75 mV. It is sensitive to [Ca2+]i and to membrane potential. At -30 mV, the probability of a channel being open is at a minimum. At more positive voltages, the probability follows Boltzman distribution. A 10-fold change in [Ca2+]i causes a 25-mV negative shift of the voltage where half of the channels are open; an 11.3-mV change in membrane potential produces an e-fold increase in the probability of the channel being open when P is low. At voltages between -30 and -50 mV, the open probability increases in an anomalous manner because of a large decrease of the channel closed time without much change in the channel open time. This anomalous activity may play a regulatory role in maintaining the resting potential. The histograms of channel open and closed time fit well, respectively, with single and double exponential distributions. Upon step depolarizations by 100-ms pulses, the 147-pS channel opens with a brief delay. The delay shortens and both the number of open channels and the open time increase with increasing positivity of the potential. The averaged currents during the step depolarizations closely resemble the delayed rectifying outward K+ currents in whole-cell recordings.     

四氯化碳中毒对大鼠离体再生肝细胞钾离子外漏的影响

本工作用三种剂量四氯化碳(CCl_4,10,15和20mmol/L)损伤正常大鼠离体肝细胞,分别在5,10,15和20min测定细胞内K~+和GPT漏出量。实验观察到细胞内K~+和GPT漏出量与CCl_4染毒的剂量和时间有明显关系,而且K~+漏出量较GPT更能灵敏地反映细胞的损伤程度;用中等剂量CCl_4(15mmol/L)损伤离体再生肝细胞20min后,细胞内K~+漏出的变化百分数明显低于正常肝细胞。这些结果表明,大鼠离体再生肝细胞具有较强的抗CCl_4损伤作用,其机制可能与再生肝细胞膜稳定性较强有关。     

不同钾水平对钾饥饿墨兰生长发育和生理的影响

墨兰钾饥饿两个月后,培养在低于１０ｍｍｏｌ／Ｌ的不同钾水平的营养液中．以比较各处理植株的生长发育、抗病和生理反应．结果表明：在正常生长状态下,墨兰体内钾含量高于氮和磷．Ｎ、Ｐ、Ｋ三者比例为６：１：９,因此栽培墨兰要重视钾肥．墨兰在缺钾（０ｍｍｏｌ／Ｌ）或低钾（０．１ｍｍｏｌ／Ｌ）条件下生长不良、花数少,叶片褐斑病较多;当供给较高钾含量（１、５或１０ｍｍｏｌ／Ｌ）时,植株生长良好,光合速率和呼吸速率亦高,根系活力较强,叶片生长快,花数多．褐斑病发病率低．作者认为,５ｍｍｏｌ／Ｌ钾水平的营养液较适合于墨兰生长发育．     

ATP interaction with the open state of the K(ATP) channel

The mechanism of ATP-sensitive potassium (K(ATP)) channel closure by ATP is unclear, and various kinetic models in which ATP binds to open or to closed states have previously been presented. Effects of phosphatidylinositol bisphosphate (PIP2) and multiple Kir6.2 mutations on ATP inhibition and open probability in the absence of ATP are explainable in kinetic models where ATP stabilizes a closed state and interaction with an open state is not required. Evidence that ATP can in fact interact with the open state of the channel is presented here. The mutant Kir6.2[L164C] is very sensitive to Cd2+ block, but very insensitive to ATP, with no significant inhibition in 1 mM ATP. However, 1 mM ATP fully protects the channel from Cd2+ block. Allosteric kinetic models in which the channel can be in either open or closed states with or without ATP bound are considered. Such models predict a pedestal in the ATP inhibition, i.e., a maximal amount of inhibition at saturating ATP concentrations. This pedestal is predicted to occur at >50 mM ATP in the L164C mutant, but at >1 mM in the double mutant L164C/R176A. As predicted, ATP inhibits Kir6.2[L164C/R176A] to a maximum of approximately 40%, with a clear plateau beyond 2 mM. These results indicate that ATP acts as an allosteric ligand, interacting with both open and closed states of the channel.     

Phosphoinositides decrease ATP sensitivity of the cardiac ATP-sensitive K(+) channel. A molecular probe for the mechanism of ATP-sensitive inhibition.

Anionic phospholipids modulate the activity of inwardly rectifying potassium channels (Fan, Z., and J.C. Makielski. 1997. J. Biol. Chem. 272:5388-5395). The effect of phosphoinositides on adenosine triphosphate (ATP) inhibition of ATP-sensitive potassium channel (K(ATP)) currents was investigated using the inside-out patch clamp technique in cardiac myocytes and in COS-1 cells in which the cardiac isoform of the sulfonylurea receptor, SUR2, was coexpressed with the inwardly rectifying channel Kir6.2. Phosphoinositides (1 mg/ml) increased the open probability of K(ATP) in low [ATP] (1 microM) within 30 s. Phosphoinositides desensitized ATP inhibition with a longer onset period (>3 min), activating channels inhibited by ATP (1 mM). Phosphoinositides treatment for 10 min shifted the half-inhibitory [ATP] (K(i)) from 35 microM to 16 mM. At the single-channel level, increased [ATP] caused a shorter mean open time and a longer mean closed time. Phosphoinositides prolonged the mean open time, shortened the mean closed time, and weakened the [ATP] dependence of these parameters resulting in a higher open probability at any given [ATP]. The apparent rate constants for ATP binding were estimated to be 0.8 and 0.02 mM(-1) ms(-1) before and after 5-min treatment with phosphoinositides, which corresponds to a K(i) of 35 microM and 5.8 mM, respectively. Phosphoinositides failed to desensitize adenosine inhibition of K(ATP). In the presence of SUR2, phosphoinositides attenuated MgATP antagonism of ATP inhibition. Kir6.2DeltaC35, a truncated Kir6.2 that functions without SUR2, also exhibited phosphoinositide desensitization of ATP inhibition. These data suggest that (a) phosphoinositides strongly compete with ATP at a binding site residing on Kir6.2; (b) electrostatic interaction is a characteristic property of this competition; and (c) in conjunction with SUR2, phosphoinositides render additional, complex effects on ATP inhibition. We propose a model of the ATP binding site involving positively charged residues on the COOH-terminus of Kir6.2, with which phosphoinositides interact to desensitize ATP inhibition.     

Ion channels in human endothelial cells.

Ion channels were studied in human endothelial cells from umbilical cord by the patch clamp technique in the cell attached mode. Four different types of ion channels were recorded: i) potassium channel current that rectifies at positive potentials in symmetrical potassium solutions (inward rectifier); ii) low-conductance non-selective cation channel with a permeability ratio K:Na:Ca = 1:0.9:0.2; iii) high-conductance cation-selective channel that is about 100 times more permeable for calcium than for sodium or potassium; iv) high-conductance potassium channel with a permeability ratio K:Na = 1:0.05. The extrapolated reversal potential of the inwardly rectifying current was near to the potassium equilibrium potential. The slope conductance decreased from 27 pS in isotonic KCl solution to 7 pS with 5.4 mmol/l KCl and 140 mmol/l NaCl in the pipette but 140 mmol/l KCl in the bath. The low-conductance non-selective cation channel showed a single-channel conductance of 26 pS with 140 mmol/l Na outside, 28 pS with 140 mmol/l K outside, and rectified in inward direction in the presence of Ca (60 mmol/l Ca, 70 mmol/l Na, 2.7 mmol/l K in the pipette) at negative potentials. The current could be observed with either chloride or aspartate as anion. The high-conductance non-selective channel did not discriminate between Na and K. The single-channel conductance was about 50 pS. The extrapolated reversal potential was more positive than +40 mV (140 K or 140 Na with 5 Ca outside). Both the 26 and 50 pS channel showed a run-down, and they rapidly disappeared in excised patches. The high-conductance potassium channel with a single-channel conductance of 170 pS was observed only rarely. It reversed near the expected potassium equilibrium potential. The 26 pS channel could be stimulated with histamine and thrombin from outside in the cell-attached mode. Both the 26 pS as well as the 50 pS channel can mediate calcium flux into the endothelial cell.