Bay K8644及nifedipine对大鼠下丘脑神经元L-型Ca~(2 )通道特性的影响

采用神经元急性分离和膜片箝技术以及细胞贴附式方式记录通道活动 ,探讨DHP类Ca2 通道激动剂BayK8644及拮抗剂nifedipine对下丘脑神经元L 型Ca2 通道的影响。结果显示 ,在BayK8644作用下 ,通道开放形式发生变化 ,明显可见多级开放 ;通道平均开放时间、平均开放概率显著增加 ,但单通道电导无明显变化。nifedipine的作用与BayK8644相反。结果提示 ,BayK8644对下丘脑神经元L 型Ca2 通道有明显激动作用 ,nifedip ine有显著抑制作用     

新生大鼠下丘脑神经元L—Ca^2＋通道电流活动特征

目的研究新生大鼠下丘脑神经元L-Ca2+通道单通道特性;Ca2+通道激动剂BayK8644对Ca2+通道单通道特性的影响.方法采用神经元急性分离技术;用膜片钳细胞贴附式记录方式进行研究.结果大鼠下丘脑神经元L-Ca2+通道是一种电导相对较大的Ca2+通道,其电导为(29.5±3.1)pS,平均开放时间(τ0)为0.28ms,平均关闭时间的短关闭时间常数(τc1)为2.91ms,长关闭时间常数(τc2)为53.22ms.此通道几乎不存在时间依赖性失活.BayK8644显著增加通道的开放概率,通道平均开放时间增加为1.61ms.结论下丘脑神经元存在L-Ca2+通道,该通道具有明显电压依赖性,而无显著的时间依赖性.通道特征与文献报道的其它神经元上L-Ca2+通道相似,也有明显不同,显示下丘脑神经元L-Ca2+钙通道的独特性.     

乙醇对大脑皮层神经元延迟整流型钾通道的影响

本文用膜片箝技术（细胞贴附式和内面向外式）研究了乙醇对大脑皮层锥体神经元膜上延迟整流型钾通道（Ｉｋ）开放概率、开放频率和开放幅度的影响。１５０～５５０ｍｍｏｌ／Ｌ的乙醇抑制Ｉｋ通道开放概率、开放频率,随乙醇浓度增大,抑制作用增强,但不影响通道的电流幅度。４５０ｍｍｏｌ／Ｌ乙醇对Ｉｋ通道开放概率和开放频率的抑制程度在两种记录模式之间差异无显著性。５０ｍｍｏｌ／Ｌ的乙醇不影响Ｉｋ通道的动力学特性。本文结果为阐明乙醇对神经系统的作用机制提供有关单离子通道方面的资料     

Ca^2＋预处理对热胁迫下辣椒叶肉细胞中Ca^2＋—ATP酶活性的影响

在常温下生长的辣椒（Capsicum annuum L.）叶肉细胞中Ca^2 －ATP酶主要分布于质膜、液泡膜上,叶绿体的基质和基粒片层上也有少量分布;在40℃下热胁迫不同的时间,酶活性逐渐下降,直到叶绿体超微结构解体。同样条件下,经过Ca^2 预处理后,分布在上述细胞器膜或片层上的酶活性大大提高,表明Ca^2 预处理对该活性具有激活作用;Ca^2 预处理对热胁迫下的超微结构的完整性具有一定的保护作用,并且能使Ca^2 －ATP酶在热胁迫下维持较高活性。结果表明,Ca^2 预处理增强辣椒幼苗的抗热性,可能与其稳定细胞膜、从而使Ca^2 －ATP酶在热迫下保护较高活性有一定关系。     

力竭游泳对红细胞膜MDA含量、Na^＋—K^＋—ATPase和Ca^2＋—ATPase活性的影响

运动性贫血在运动训练中经常发生 ,不仅常发于耐力性运动员中 ,而且在技巧、速度性等项目中也较为常见 ,它严重影响运动员的机能水平和运动成绩。本实验通过对力竭运动大鼠红细胞膜ＭＤＡ含量、Ｎａ Ｋ ＡＴＰａｓｅ和Ｃａ2 ＡＴＰａｓｅ活性的研究 ,旨在探讨运动性贫血的发生机理 ,为预防和治疗运动性贫血提供一定的理论依据。1　材料与方法(1)实验动物与运动方式　实验选用ＳＤ大鼠 2 4只 ,体重为 2 2 0～ 2 5 0ｇ ,由上海实验动物中心提供。大鼠随机分为 4组 ,每组 6只。即 :对照组 (Ｃ) ;运动后即刻组 (ＥＸ1) ;运动后 1ｈ组 (Ｅ…     

β—淀粉样蛋白31—35片段对海马神经元分离膜片Ca^2＋激活大电导钾通道的抑制

为了确定β－淀粉样蛋白（AβP）在影响神经元电生理特性并导致神经毒作用时的最短活性序列,实验采用片钳技术,在急性分离的大鼠海马CA1区锥体细胞的“内面向外”式膜片上,观察了AβP的31－35和25－35片段对C^2 a激活大电导钾（BK）通道活动的影响,结果显示,浴液中给预5umol/L的AβP31－35后,BK通道的平均开放概率（P0）和开放频率在1－3min内分别减少了85．8％（P＜0．01）和72．1％（P＜0．01）,平均开放时间减少了41．1％（P＜0．01）,平均电流幅度则无明显改变（P＞0．05）,给予同样摩尔浓度的AβP25－35后,BK通道平均P0减少了85．5％（P＜0．01）,平均开放时间减少51．4％,（P＜0．05）,结果提示：两种AβP片段对海马神经元BK通道具有抑制作用,。这可能与AβP的神经性作用有关,AβP－31－35片段可能是AβP分子中影响细胞电生理特性的最小活性序列。     

光周期对光敏胞质不育小麦花药发育过程中Ca^2＋—ATPase分布的影响

应用铅沉淀法研究了不同光照条件下泖负质不育小麦Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ Ｌ．）可育花药和不育药药发育过程中Ｃａ＾２＋－ＡＴＰａｓｅ的分布。短日可育条件下,单核早期至成熟花粉,Ｃａ＾２＋－ＡＴＰａｓｅ在花粉表面,外壁内先增加后 和,在花粉内壁及质膜上逐渐增加,在南内分布较少,成熟花粉的营养细胞核仁内有大量Ｃａ＾２＋－ＡＴＰａｓｅ会面２。精细胞核仁内亦有Ｃａ＾２＋－ＡＴＰａｓｅ分布。乌氏体上     

L-type Ca2+ channels access multiple open states to produce two components of Bay K 8644-dependent current in GH3 cells

To determine the number of L-channel populations responsible for producing the two components of whole-cell L-type Ca2+ channel current revealed by Bay K 8644 (Fass, D.M., and E.S. Levitan. 1996. J. Gen. Physiol. 108:1-11), L-type Ca2+ channel activity was recorded in cell- attached patches. Ensemble tail currents from most (six out of nine) single-channel patches had double-exponential time courses, with time constants that were similar to whole-cell tail current decay values. Also, in single-channel patches subjected to two different levels of depolarization, ensemble tail currents exactly reproduced the voltage dependence of activation of the two whole-cell components: The slow component is activated at more negative potentials than the fast component. In addition, deactivation of Bay K 8644-modified whole-cell L-current was slower after long (100-ms) depolarizations than after short (20-ms) depolarizations, and this phenomenon was also evident in ensemble tail currents from single L-channels. Thus, a single population of L-channels can produce the two components of macroscopic L-current deactivation. To determine how individual L-channels produce multiple macroscopic tail current components, we constructed ensemble tail currents from traces that contained a single opening upon repolarization and no reopenings. These ensemble tails were biexponential. This type of analysis also revealed that reopenings do not contribute to the slowing of tail current deactivation after long depolarizations. Thus, individual L-channels must have access to several open states to produce multiple macroscopic current components. We also obtained evidence that access to these open states can vary over time. Use of several open states may give L-channels the flexibility to participate in many cell functions.     

L-type Ca2+ channels in Ca2+ channelopathies

Voltage-gated L-type Ca2+ channels (LTCCs) mediate depolarization-induced Ca2+ entry in electrically excitable cells, including muscle cells, neurons, and endocrine and sensory cells. In this review we summarize the role of LTCCs for human diseases caused by genetic Ca2+ channel defects (channelopathies). LTCC dysfunction can result from structural aberrations within pore-forming alpha1 subunits causing incomplete congenital stationary night blindness, malignant hyperthermia sensitivity or hypokalemic periodic paralysis. However, studies in mice revealed that LTCC dysfunction also contributes to neurological symptoms in Ca2+ channelopathies affecting non-LTCCs, such as Ca(v)2.1 alpha1 in tottering mice. Ca2+ channelopathies provide exciting molecular tools to elucidate the contribution of different LTCC isoforms to human diseases.     

Coassembly of big conductance Ca2+-activated K+ channels and L-type voltage-gated Ca2+ channels in rat brain

Based on electrophysiological studies, Ca(2+)-activated K(+) channels and voltage-gated Ca(2+) channels appear to be located in close proximity in neurons. Such colocalization would ensure selective and rapid activation of K(+) channels by local increases in the cytosolic calcium concentration. The nature of the apparent coupling is not known. In the present study we report a direct coassembly of big conductance Ca(2+)-activated K(+) channels (BK) and L-type voltage-gated Ca(2+) channels in rat brain. Saturation immunoprecipitation studies were performed on membranes labeled for BK channels and precipitated with antibodies against alpha(1C) and alpha(1D) L-type Ca(2+) channels. To confirm the specificity of the interaction, precipitation experiments were carried out also in reverse order. Also, additive precipitation was performed because alpha(1C) and alpha(1D) L-type Ca(2+) channels always refer to separate ion channel complexes. Finally, immunochemical studies showed a distinct but overlapping expression pattern of the two types of ion channels investigated. BK and L-type Ca(2+) channels were colocalized in various compartments throughout the rat brain. Taken together, these results demonstrate a direct coassembly of BK channels and L-type Ca(2+) channels in certain areas of the brain.     

Effects of BAY K 8644, nifedipine, and low Ca2+ on halothane and caffeine potentiation.

The purpose of this investigation was to examine the effects of the Ca2+ agonist BAY K 8644 and the Ca2+ antagonist nifedipine on halothane- and caffeine-induced twitch potentiation of mammalian skeletal muscle. Muscle fiber bundles were taken from normal Landrace pigs and exposed to BAY K 8644 (10 microM), nifedipine (1 microM), and low Ca2+ media administered alone and in combination with halothane (3%) or with increasing concentrations of caffeine (0.5-8.0 mM). Both BAY K 8644 and halothane potentiated twitches by approximately 80%; when they were administered in combination, twitch potentiation was nearly double that caused by either drug alone. In the presence of nifedipine, halothane increased twitches by less than 30%. Low Ca2+ significantly depressed twitches by approximately 25% but also inhibited halothane's inotropic effect. BAY K 8644 augmented caffeine potentiation but only at low caffeine concentrations (0.5-2.0 mM). Nifedipine and low Ca2+ failed to inhibit caffeine's inotropic effects. These results suggest that halothane potentiates twitches via a mechanism that involves or is influenced by extracellular Ca2+.     

45Ca2+ uptake in rat brain neurons: absence of sensitivity to the Ca2+ channel ligands nitrendipine and Bay K 8644

K+-stimulated 45Ca2+ uptake into intact rat brain cells was biphasic, consisting of a fast first phase and a slow second phase; the latter was Na+ dependent. Cobalt and cadmium at 10(-4) and 10(-3) M produced 19-97% block of first phase 45Ca2+ uptake, but nitrendipine (to 10(-6) M) and Bay K 8644 (to 10(-6) M) were without effect on uptake and were similarly without effect in cells prepared in the presence of ATP, cAMP, Mg2+, and protease inhibitors. The second phase of K+-stimulated 45Ca2+ uptake was inhibited by 3,4-dichlorobenzamil (IC50, 29.6 microM). Depolarization-induced 45Ca2+ uptake into intact rat brain cells occurs by at least two different mechanisms. The first phase probably represents uptake through 1,4-dihydropyridine-insensitive Ca2+ channels, while the second phase is probably due to Na+-Ca2+ exchange.     

Potentiated L-type Ca2+ channels rectify

Strong depolarization and dihydropyridine agonists potentiate inward currents through native L-type Ca2+ channels, but the effect on outward currents is less clear due to the small size of these currents. Here, we examined potentiation of wild-type alpha1C and two constructs bearing mutations in conserved glutamates in the pore regions of repeats II and IV (E2A/E4A-alpha1C) or repeat III (E3K-alpha1C). With 10 mM Ca2+ in the bath and 110 mM Cs+ in the pipette, these mutated channels, expressed in dysgenic myotubes, produced both inward and outward currents of substantial amplitude. For both the wild-type and mutated channels, we observed strong inward rectification of potentiation: strong depolarization had little effect on outward tail currents but caused the inward tail currents to be larger and to decay more slowly. Similarly, exposure to DHP agonist increased the amplitude of inward currents and decreased the amplitude of outward currents through both E2A/E4A-alpha1C and E3K-alpha1C. As in the absence of drug, strong depolarization in the presence of dihydropyridine agonist had little effect on outward tail currents but increased the amplitude and slowed the decay of inward tail currents. We tested whether cytoplasmic Mg2+ functions as the blocking particle responsible for the rectification of potentiated L-type Ca2+ channels. However, even after complete removal of cytoplasmic Mg2+, (-)BayK 8644 still potentiated inward current and partially blocked outward current via E2A/E4A-alpha1C. Although zero Mg2+ did not reveal potentiation of outward current by DHP agonist, it did have two striking effects, (a) a strong suppression of decay of both inward and outward currents via E2A/E4A-alpha1C and (b) a nearly complete elimination of depolarization-induced potentiation of inward tail currents. These results can be explained by postulating that potentiation exposes a binding site in the pore to which an intracellular blocking particle can bind and produce inward rectification of the potentiated channels.     

Role of Cav1.2 L-type Ca2+ channels in vascular tone: effects of nifedipine and Mg2+

Ca(2+) entry via L-type voltage-gated Ca(2+) channels (LVGCs) is a key factor in generating myogenic tone (MT), as dihydropyridines (DHPs) and other LVGC blockers, including Mg(2+), markedly reduce MT. Recent reports suggest, however, that elevated external Mg(2+) concentration and DHPs may also inhibit other Ca(2+)-entry pathways. Here, we explore the contribution of LVGCs to MT in intact, pressurized mesenteric small arteries using mutant mice (DHP(R/R)) expressing functional but DHP-insensitive Ca(v)1.2 channels. In wild-type (WT), but not DHP(R/R), mouse arteries, nifedipine (0.3-1.0 microM) markedly reduced MT and vasoconstriction induced by high external K(+) concentrations ([K(+)](o)), a measure of LVGC-mediated Ca(2+) entry. Blocking MT and high [K(+)](o)-induced vasoconstriction by <1 microM nifedipine in WT but not in DHP(R/R) arteries implies that Ca(2+) entry via Ca(v)1.2 LVGCs is obligatory for MT and that nifedipine inhibits MT exclusively by blocking LVGCs. We also examined the effects of Mg(2+) on MT and LVGCs. High external Mg(2+) concentration (10 mM) blocked MT, slowed the high [K(+)](o)-induced vasoconstrictions, and decreased their amplitude in WT and DHP(R/R) arteries. To verify that these effects of Mg(2+) are due to block of LVGCs, we characterized the effects of extracellular and intracellular Mg(2+) on LVGC currents in isolated mesenteric artery myocytes. DHP-sensitive LVGC currents are inhibited by both external and internal Mg(2+). The results indicate that Mg(2+) relaxes MT by inhibiting Ca(2+) influx through LVGCs. These data provide new information about the central role of Ca(v)1.2 LVGCs in generating and maintaining MT in mouse mesenteric small arteries.