基于钙响应钟型曲线构建的钙振荡模型

细胞质钙离子(Ca~(2+))常以浓度振荡变化的方式控制许多生理活动,内质网上的三磷酸肌醇受体(inositol 1,4,5-trisphosphate receptor,IP_3R)通道是产生钙振荡的关键因素,因此研究其振荡机制具有重要意义.虽然钙振荡已被广泛建模,但大部分模型细胞质与内质网中钙振荡的范围与实验观测数据差异较大.通过整合最近的实验结果,基于IP_3R通道活性对细胞质Ca~(2+)浓度依赖的钟型曲线构建了一个新的钙振荡模型.模型结果除了可以很好地模拟实验中关于钙振荡范围的数据,还可重复多种实验现象.对模型进行参数敏感性分析后,进一步采用单参数分岔分析分别研究了两个高敏感参数,即细胞质内Ca~(2+)缓冲蛋白的总浓度和IP_3R通道激活Ca~(2+)的解离常数(K_1)对钙振荡的影响,发现它们对钙信号有相反的作用.对三磷酸肌醇(IP_3)浓度和K_1进行双参数分岔分析的结果表明K_1对IP_3能产生钙振荡的区域及其振幅有重要影响,揭示可以通过改变K_1影响细胞对外界刺激的响应及其钙振荡模式.该研究有助于理解钙振荡机制,并可为后续理论研究提供框架.     

细胞膜钾通道激活作用的模拟和肝素抑制效应的研究

本工作用离子单通道膜片钳全细胞电流技术。对单细胞进行内外灌流，证明（1，4，5）三磷酸肌醇和（1，3，4，5）四磷酸肌醇细胞内联用能模拟乙酰胆脸细胞外灌流时所诱发的钾通道电流的增加，并通过细胞内加用肝素，观察到肝素阻断了这种模拟性的钾电流激活，提示肝素可能作为一种多磷酸肌醇的拮抗剂，抑制腺泡细胞的刺激分泌偶联。     

植物脱落酸信号转导研究进展

本文从脱落酸(ABA)结合位点与受体、Ca~(2 )信号系统、蛋白质的可逆磷酸化、磷酸肌醇系统及 G 蛋白等几个方面介绍了脱落酸信号转导的研究进展。     

糖皮质激素对原代培养的海马细胞中p44/42的快速非基因组激活作用

用Western blot方法研究了皮质酮(B)对原代培养海马细胞15 min刺激下p44/42的快速激活作用.主要结果有(1)在5～15 min的时间内,皮质酮可以激活p44/42.当激活时间为15 min时,p44/42升得最高,30 min后恢复到正常水平,呈钟形的瞬时激活特点.(2)B激活p44/42的作用,不能被GR阻断剂RU38486所阻断.根据快速、GR受体阻断剂不能阻断两点,基本上可以认为,所观察到的p44/42的激活属于GC的快速非基因组作用.(3)酪氨酸激酶阻断剂Genistein不能阻断,而GDPβs可以阻断此作用,提示GC的这一效应不是通过TRK受体,而是通过G-蛋白耦联受体(GPCR).(4)PKA的阻断剂H89不能阻断,而PKC阻断剂G(o)6976及MEK(即MAPKK)阻断剂PD98059能够阻断这一效应,所以B快速激活p44/42的信号转导通路可能是GC→GPCR→PKC→MEK→p44/42.     

三氟氯氰菊酯对棉铃虫中枢神经细胞高电压激活Ca~(2+)通道激活的影响

用全细胞膜片钳技术分析了棉铃虫Helicoverpa armigera(Hübner)幼虫中枢神经细胞电压门控Ca~(2+)通道的电生理学特性,并检测了三氟氯氰菊酯(cyhalothrin,Cyh)对Ca~(2+)通道功能特性的影响.结果表明,Ca~(2+)通道电流(I_(Ca))激活阈值-40 mV,峰值电压介于-10 mV-10 mV.约82%细胞的I_(Ca)在-20 mV激活,93%细胞的I_(Ca)在0 mV左右达峰值.棉铃虫表达高电压激活、对Cd~(2+)敏感的Ca~(2+)通道和高电压激活、对Cd~(2+)不敏感的Ca~(2+)通道.Cyh作用后,I_(Ca)在幅值减小的同时,I-V曲线和激活曲线均向超极化方向移动10-20 mV,表明Cyh不仅对I_(Ca)有抑制作用,对Ca~(2+)通道的激活也有影响,棉铃虫幼虫腹神经索中枢神经细胞高电压门控Ca~(2+)通道也是Cyh的作用靶标之一.     

植物细胞第二信使研究进展

介绍了植物细胞信号转导中的主要第二信使钙离子、三磷酸肌醇、二酯酰甘油、环腺苷酸的研究进展，特别对它们的产生及其在信号转导中的地位和作用进行了重点介绍。     

荧光光谱法研究钙调神经磷酸酶与其激活剂的相互作用

应用荧光光谱法研究了钙调神经磷酸酶 (CN)激活剂JJYD对CN的激活作用 .实验表明 ,JJYD确实对CN有一定的激活效应 ,其最佳激活质量浓度为 0 .2g·L- 1,激活率为 (14 9.9±0 772 ) % ;JJYD对CN的内源荧光有较强的猝灭作用 ,形成的复合物所产生的静态猝灭是引起CN荧光猝灭的主要原因 ,进一步依据荧光猝灭结果确定了JJYD CN复合物的结合常数K及结合位点数n ,证明二者有较强的结合力 .     

SKF38393对DRG神经元GABA-激活电流的抑制

在大鼠新鲜分离DRG神经元标本上应用全细胞膜片作记录,观察了多巴胺D_1受体的选择性激动剂(±)SKF38393HCI对GABA-激活电流的作用。大部分受检细胞(60/70)对GABA敏感。10~(-6)～10~(-3)mol/L GABA可引起呈剂量依赖性的明显去敏感作用的内向电流。在60个对GABA敏感的细胞中,预加SKF38393引起的膜反应如下:(1)外向电流(7/60);(2)内向电流(5/60);(3)无反应(48/60)。与GABA激活的内向电流相比,SKF38393激活电流幅值较小,无明显去敏感现象。预加SKF38393 30秒对GABA-激活电流幅值的影响如下:产生抑制作用的为88.33％(53/60),增强的为1.66％(1/60),无明显作用的为10.0％(6/0)。SKF38393对10~(-4)mol/L的GABA-激活电流的抑制作用依赖于SKF38393的浓度,在其浓度为10~(-4)、10~(-5)、10~(-6)、10~(-7)mol/L时对GABA-激活电流的抑制(±)分别为46.5％±2.3(n=8)、35.5％±1.2(n=8)、26.8％±1.5(n=7)、24.8％±2.6(n=7)。通过应用二次膜片作(repatch)技术在同一细胞(n=2)以及不同细胞组间(n=14)进行对比,证明:细胞内加蛋白激酶抑制剂H-7后,SKF38393对GABA的抑制作用完全清除。结果提示:SKF38393对GABA-激活电流的抑制作用可能是由于SKF38393激活D_1受体后通过胞内转导机制,使GABA受体通道复合体胞内磷酸化所致。     

牵牛子提取物对CN的激活及对东莨菪碱致记忆获得性障碍小鼠的影响

研究了牵牛子 (Pharbitisnil)提取物 (EPN)对钙调神经磷酸酶 (CN)的激活作用以及对东莨菪碱所致记忆获得性障碍小鼠的影响 .结果表明 ,在EPN终质量浓度 ρ≥ 0 .0 0 8g·L- 1时 ,对CN有激活作用 ,在 ρ =0 .0 4 g·L- 1时达到最高激活率为 118.3% .利用东莨菪碱造成小鼠学习记忆障碍 ,采用小鼠跳台法检测学习记忆功能 .结果表明 ,EPN低剂量组与东莨菪碱造模组比较 ,小鼠触电潜伏期明显延长 (P <0 .0 5 ) ,错误次数有降低趋势 ,说明EPN低剂量组对东莨菪碱所致小鼠记忆获得性障碍有比较明显的改善作用 .     

中华管鞭虾多酚氧化酶生化特性研究

对中华管鞭虾中多酚氧化酶(PPO)的活性及其影响因素进行研究,讨论了最适反应温度、pH、特异性底物及其酶动力学性质,激活剂对酚氧化酶的激活作用,抑制剂对酚氧化酶的抑制作用。结果表明:中华管鞭虾酚氧化酶的最适反应温度为45℃,最适反应pH为8.0;十二烷基硫酸钠(SDS)对PPO的激活作用最大,其次为胰凝乳酶和β-葡聚糖,激活率分别为71.9%、62.4%和33.4%;4-己基间苯二酚对PPO抑制作用最强。     

Occurrence and extracellular actions of inositol pentakis- and hexakisphosphate in mammalian brain

Although inositol 1,3,4,5,6-pentakisphosphate (InsP5) and hexakisphosphate (InsP6) have been recognized for some time as naturally-occurring metabolites of inositol, their occurrence in mammalian cell types, including one of neural origin, has only recently been documented. This is of interest because of the recognized second messenger role of inositol 1,4,5-trisphosphate (InsP3) in intracellular signalling; coupling surface stimuli to cytoplasmic calcium discharge. The metabolism, existence in normal mature tissues, and possible functional roles of these inositol polyphosphates are unknown. Here we report evidence that InsP5 and InsP6 are synthesized in intact brain after labelling with [3H]inositol in vivo. We also show that local infusion of InsP5 and InsP6 into a discrete brain stem nucleus implicated in cardiovascular regulation, results in dose-dependent changes in heart rate and blood pressure.     

Neuropeptide modulation of single calcium and potassium channels detected with a new patch clamp configuration

Calcium channel activity is crucial for secretion and synaptic transmission, but it has been difficult to study Ca2+ channel modulation because survival and regulation of some of these channels require cytoplasmic constituents that are lost with the formation of cell-free patches. Here we report a new patch clamp configuration in which activity and regulation of channels are maintained after removal from cells. A pipette containing the pore-forming agent nystatin is sealed onto a cell and withdrawn to form an enclosed vesicle. The resulting perforated vesicle, formed from pituitary tumour cells, contains Ca2+ and K+ channels. Ca2(+)-activated K+ channels in the vesicle are activated by cyclic AMP analogues, and by a neuropeptide (thyrotropin-releasing hormone) that stimulates phosphatidylinositol turnover and inositol trisphosphate-gated Ca2+ release from intracellular organelles. Thus, the perforated vesicle retains signal transduction systems necessary for ion channel modulation. Functional dihydropyridine-sensitive Ca2+ channels (L-type) are maintained in the vesicle, and their gating is inhibited by thyrotropin-releasing hormone. Hence, this new patch clamp configuration has allowed a direct detection of the single-channel basis of transmitter-induced inhibition of L-type Ca2+ channels. The modulation of Ca2(+)-channel gating may be an important mechanism for regulating hormone secretion from pituitary cells.     

硅胶负载碱金属磷酸二氢盐催化乳酸甲酯转化

以硅胶为载体,碱金属磷酸二氢盐MH₂PO₄ (M=Li,Na,K)为原料,利用浸渍法制备了3种硅胶负载的含有不同碱金属阳离子的聚合磷酸盐催化剂MH₂PO₄/SiO₂。在固定床连续流动反应器上考察了不同碱金属阳离子对聚合磷酸盐催化乳酸甲酯转化生成丙烯酸和丙烯酸甲酯的影响。3种催化剂中,NaH₂PO₄/SiO₂催化生成丙烯酸和丙烯酸甲酯的总选择性最高,380℃时,乳酸甲酯转化率为99.5%,目标产物选择性可达52.0%。催化剂表征及量子化学计算的结果表明,碱金属阳离子的性质影响聚合磷酸盐链末端P—OH的酸性和催化剂的表面酸量,导致3种催化剂上乳酸甲酯转化成丙烯酸和丙烯酸甲酯的选择性的差异。     

基于蚁群算法的信道分配优化模型与仿真

借由对蜂窝系统的分析,建立了一个固定信道分配的数学模型。在基本蚁群算法的基础上,改进了转移策略和信息素更新策略,提高了收敛速度并有效防止局部最优。在单一信道问题中,得到各种(K1,K2)约束的最优循环分配方案;且约束为(K,K)的图例,总能通过1 iK这7个不同信道,分配出可无限延伸的最佳方案。而以21小区费城问题为实例的多信道分配,也得到较好的结果。通过实际图例给出的性能仿真结果, 节约了过多的频率资源占用和计算时间。     

Regulation of fast inactivation of cloned mammalian IK(A) channels by cysteine oxidation

Modulation of neuronal excitability by regulation of K+ channels potentially plays a part in short-term memory but has not yet been studied at the molecular level. Regulation of K+ channels by protein phosphorylation and oxygen has been described for various tissues and cell types; regulation of fast-inactivating K+ channels mediating IK(A) currents has not yet been described. Functional expression of cloned mammalian K+ channels has provided a tool for studying their regulation at the molecular level. We report here that fast-inactivating K+ currents mediated by cloned K+ channel subunits derived from mammalian brain expressed in Xenopus oocytes are regulated by the reducing agent glutathione. This type of regulation may have a role in vivo to link metabolism to excitability and to regulate excitability in specific membrane areas of mammalian neurons.     

Alpha 1-adrenoceptor subtypes linked to different mechanisms for increasing intracellular Ca2+ in smooth muscle

Receptor-mediated increases in intracellular Ca2+ levels can be caused by release from intracellular organelles and/or influx from the extracellular fluid. Noradrenaline (NA) released from sympathetic nerves acts on alpha 1-adrenoceptors to increase cytosolic Ca2+ and promote smooth muscle contraction. In many cells activation of alpha 1-adrenoceptors causes formation of inositol 1,4,5-trisphosphate which promotes Ca2+ release from intracellular stores. The mechanism by which receptor activation opens cell surface Ca2+ channels is not known, although in some cases it may be secondary to formation of inositol phosphates or release of stored intracellular Ca2+ (ref. 3). However, alpha 1-adrenoceptors have recently been shown to have different pharmacological properties in different tissues, and it has been proposed that different alpha 1-adrenoceptor subtypes may control mobilization of intracellular Ca2+ and gating of extracellular Ca2+ influx. We here report evidence for two subtypes of alpha 1-adrenoceptors which cause contractile responses through different molecular mechanisms. One subtype stimulates inositol phosphate (InsP) formation and causes contractions which are independent of extracellular Ca2+, and the other does not stimulate inositol phosphate formation and causes contractions which require the influx of extracellular Ca2+ through dihydropyridine-sensitive channels. These results suggest that neurotransmitters and hormones may control Ca2+ release from intracellular stores and influx through voltage-gated membrane channels through distinct receptor subtypes.     

Activation of Ca2+ entry into acinar cells by a non-phosphorylatable inositol trisphosphate.

In many cell types, receptor activation of phosphoinositidase C results in an initial release of intracellular Ca2+ stores followed by sustained Ca2+ entry across the plasma membrane. Inositol 1,4,5-trisphosphate is the mediator of the initial Ca2+ release, although its role in the mechanism underlying Ca2+ entry remains controversial. We have now used two techniques to introduce inositol phosphates into mouse lacrimal acinar cells and measure their effects on Ca2+ entry: microinjection into cells loaded with Fura-2, a fluorescent dye which allows the measurement of intracellular free calcium concentration by microspectrofluorimetry, and perfusion of patch clamp pipettes in the whole-cell configuration while monitoring the activity of Ca(2+)-activated K+ channels as an indicator of intracellular Ca2+. We report here that inositol 1,4,5-trisphosphate serves as a signal that is both necessary and sufficient for receptor activation of Ca2+ entry across the plasma membrane in these cells.     

myo-Inositol polyphosphate may be a messenger for visual excitation in Limulus photoreceptors

Photoreceptor excitation begins with the absorption of a photon by rhodopsin and proceeds through an unknown sequence of steps that leads to changes in specific ionic conductances. These conductance changes produce the receptor potential. It has been proposed that hydrolysis of phosphoinositides is involved in the control of a variety of physiological processes. Recent studies have implicated inositol 1,4,5-trisphosphate as an intracellular messenger in the cascade mediating hormone-stimulated secretion. We propose that one of the steps in the excitatory cascade in Limulus ventral photoreceptors may be an increase in intracellular concentration of myo-inositol polyphosphates, derived from hydrolysis of the membrane component phosphatidylinositol bisphosphate by a phospholipase. Here we present biochemical and electrophysiological evidence that an inositol polyphosphate may be an intracellular messenger in the cascade mediating excitation, based on the following criteria: the cells possess the synthetic and degradative metabolism for the messenger; the natural stimulus leads to a change in the concentration of the messenger within the cells; and intracellular injection of exogenous messenger mimics naturally occurring electrophysiological events.     

Inositol trisphosphate-dependent periodic activation of a Ca(2+)-activated K+ conductance in glucose-stimulated pancreatic beta-cells.

Glucose-stimulated insulin secretion is associated with the appearance of electrical activity in the pancreatic beta-cell. At intermediate glucose concentrations, beta-cell electrical activity follows a characteristic pattern of slow oscillations in membrane potential on which bursts of action potentials are superimposed. The electrophysiological background of the bursting pattern remains unestablished. Activation of Ca(2+)-activated large-conductance K+ channels (KCa channel) has been implicated in this process but seems unlikely in view of recent evidence demonstrating that the beta-cell electrical activity is unaffected by the specific KCa channel blocker charybdotoxin. Another hypothesis postulates that the bursting arises as a consequence of two components of Ca(2+)-current inactivation. Here we show that activation of a novel Ca(2+)-dependent K+ current in glucose-stimulated beta-cells produces a transient membrane repolarization. This interrupts action potential firing so that action potentials appear in bursts. Spontaneous activity of this current was seen only rarely but could be induced by addition of compounds functionally related to hormones and neurotransmitters present in the intact pancreatic islet. K+ currents of the same type could be evoked by intracellular application of GTP, the effect of which was mediated by mobilization of Ca2+ from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores. These observations suggest that oscillatory glucose-stimulated electrical activity, which is correlated with pulsatile release of insulin, results from the interaction between the beta-cell and intraislet hormones and neurotransmitters. Our data also provide evidence for a close interplay between ion channels in the plasma membrane and InsP3-induced mobilization of intracellular Ca2+ in an excitable cell.     

Detection of K+ and Cl-channels from calf cardiac sarcolemma in planar lipid bilayer membranes

The ionic currents underlying the cardiac action potential are believed to be much more complex than those in nerve. During the cardiac action potential, various membrane channels control the flow of K+, Na+, Ca2+ and Cl- across the sarcolemma of cardiac muscle cells. Thus, it has become increasingly clear that a detailed knowledge of the mechanisms that activate (or inactivate) heart channels is required to understand cardiac excitability. We report here the use of planar lipid bilayer techniques to detect and characterize K+ and Cl- channels in purified heart sarcolemma membrane vesicles. We have identified four different types of channel on the basis of their selectivity, conductance and gating kinetics. We present in some detail the properties of a K+ channel and a Cl- channel. We have tentatively identified the K+ channel with the ix type of current found in Purkinje, myocardial ventricular and atrial fibres. The chloride channel might be related to the transient chloride current found in Purkinje fibres.