嗜铬细胞分泌的模型研究

在大鼠肾上腺嗜铬细胞上应用全细胞膜片钳和膜电容检测技术，对细胞分泌的模型进行了研究，对Ｈｅｉｎｅｍａｎｎ等提出的准备释放库Ｂ，进行了更为精确深入的分析，提出了新的论据和修改内容，证实在距细胞膜Ｃａ＾２＋通道３０ｎｍ处存在释放库，在距Ｃａ＾２＋通道３００ｎｍ处存在可释放库，通过内分泌细胞分泌模型分析了Ｃａ＾２＋的控制作用以及囊泡释放过程时序特征。     

￣（45）Ca跨膜流动测定技术用于中药钙拮抗作用的研究

应用￣（４５）Ｃａ跨膜流动测定技术研究钙拮抗剂ｖｅｒａｐａｍｉｌ、中药西红花（ＣｒｏｃｕｓＳａｔｉｖｕｓＬ．）、“地奥心血康”（ＤＡＸＸＫ）、川红花（ＣａｒｔｈａｍｕｓｔｉｎｃｔｏｒｉｕｓＬ．）及银杏叶（ＧｉｎｋｇｏｂｉｌｏｂａＬ．）等对大鼠主动脉￣（４５）Ｃａ跨膜内流的作用，以确定其对细胞膜Ｃａ￣（２＋）通道影响。实验结果表明：西红花、川红花、ＤＡＸＸＫ对平滑肌细胞膜上电压依赖Ｃａ￣（２＋）通道（ＰＤＣ）和受体操纵Ｃａ￣（２＋）通道（ＲＯＣ）都有阻滞作用，并以西红花的阻滞作用最强；而银杏叶对细胞膜Ｃａ￣（２＋）通道无阻滞作用。     

^4^5Ca跨膜流动测定技术用于中药钙拮抗作用的研究

应用４５Ｃａ跨膜流动测定研究的钙拮抗剂ｖｅｒａｐａｍｉｌ，中药西红花（Ｃｒｏｃｕｓ ＳａｔｉｖｕｓＬ．），“地奥心血康”（ＤＡＸＸＫ），川红花及银杏叶等对大鼠主动脉４５Ｃａ跨膜内流的作用，以确定其对细胞膜Ｃａ＾２＾＋通道影响。实验结果表明：西红花，川红花，ＤＡＸＸＫ对平滑肌细胞膜上电压依赖Ｃａ＾２＾＋通道（ＰＤＣ）和受体操纵Ｃａ＾２＾＋通道（ＲＯＣ）都有阻滞作用，并以西红花的阻滞作用最强；而银杏叶     

Ca_8Mg（SiO_4）_4Cl_2中Ce~（3＋）的光谱与晶体学格位

合成了新型蓝色光致发光材料Ｃａ８Ｍｇ（ＳｉＯ４）４Ｃｌ２：Ｃｅ（３＋），并对其进行了光谱测试．分析了Ｃｅ（３＋）在Ｃａ８Ｍｇ（ＳｉＯ４）４Ｃｌ２晶体中应占据的晶体学格位．测试结果表明，在紫外光激发下，Ｃａ８Ｍｇ（ＳｉＯ４）４Ｃｌ２：Ｃｅ（３＋）的发射光谱为一峰位在４１０ｎｍ～４２５ｎｍ之间的宽谱带，经分析确认作为发光中心的Ｃｅ（３＋）占据Ｃａ８Ｍｇ（ＳｉＯ４）４Ｃｌ２晶体中多面体八配位的Ｃａ（Ⅱ）格位．     

肌浆网摄取和释放钙的机制与运动的关系

肌浆网通过摄取与吸收Ｃａ２＋调节细胞内游离Ｃａ２＋浓度，在肌肉兴奋－收缩偶联中起重要作用．肌浆网Ｃａ２＋摄取主要由Ｃａ２＋－Ｍｇ２＋－ＡＴＰａｓｅ来完成．肌浆网Ｃａ２＋释放主要由１，４，５－三磷酸肌醇受体系统和ｒｙａｎｏｄｉｎｅ受体系统调控．     

Ca~（2＋）和亚精胺在莴苣种子萌发过程中的相互关系

莴苣（挂丝红品种）种子萌发过程中，内源亚精胺（Ｓｐｄ）含量逐渐增加．Ｃａ２＋通道抑制剂Ｃｏ２＋和Ｌ３＋、钙调素（ＣａＭ）拮抗剂氯两嗪（ＣＰＺ）处理抑制种子内源亚精胺含量的增加，Ｃａ２＋可部分逆转Ｃｏ２＋和Ｌａ３＋的抑制效应．外源亚精胺处理能部分逆转Ｃｏ２＋和Ｌａ３＋对萌发的抑制效应．表明Ｃａ２＋和ＣａＭ对种子萌发的调节作用与其对亚精胺含量的影响有关．     

人工虫草钙拮抗作用的研究

用４５Ｃａ跨膜测量方法,研究了人工虫草对大鼠主动脉平滑肌细胞膜三种Ｃａ２＋通道的阻滞作用,与西药钙拮抗剂维拉帕米比较的结果表明,当人工虫草的浓度为２５×１０－６ｇ／ｍＬ,２５０×１０－６ｇ／ｍＬ时,不影响静流,能降低ＲＯＣ和ＰＤＣ的Ｃａ２＋内流,其规律与维拉帕米的相似．     

大鼠嗜铬细胞分泌的膜片钳和 fura-2 联合检测

在原代培养的大鼠肾上腺嗜铬细胞上,综合运用细胞内钙测定法和全细胞膜片钳法,以检测膜电容变化为手段测定单一肾上腺嗜铬细胞的胞吐过程．通过施加＋２０ｍＶ去极化时引起的钙电流,对细胞膜电容的变化以及细胞内钙浓度［Ｃａ２＋］ｉ变化的同时检测,多方位地确定影响细胞分泌的因素．     

番茄碱对人细胞膜Ca^2＋—Mg^2＋ATPase活性影响的初步研究

本研究了番茄碱对人红细胞膜Ｃａ＾２＋－Ｍｇ＾２＋ＡＴＰａｓｅ活性的影响。实验结果表明，反应体系中ｔｏｍａｔｉｎｅ的浓度低于ｌｍｍｏｌ／Ｌ时，对不依赖钙调蛋白激活的Ｃａ＾２＋－Ｍｇ＾２＋ＡＴＰａｓｅ影响不大，浓度达ｌｍｍｏｌ／Ｌ时，该酶活性仍保持在９６％左右；而在此浓度范围内，ｔｏｍａｔｉｎｅ对依赖于ＣａＭ激活的Ｃａ＾２＋Ｍｇ＾２＋ＡＴＰａｓｅｅ有明显的抑制作用；其ＩＣ５０值为０．１４ｍｍｏｌ／Ｌ     

番茄碱对人红细胞膜Ca~（2＋）－Mg~（2＋）ATPase活性影响的初步研究

本文研究了番茄碱（ｔｏｍａｔｉｎｅ）对人红细胞膜Ｃａ~２＋－Ｍｇ~２＋ＡＴＰａｓｅ活性的影响。实验结果表明，反应体系中ｔｏｍａｔｉｎｅ的浓度低于１ｍｍｏｌ／Ｌ时，对不依赖钙调蛋白（ＣａＭ）激活的Ｃａ~２＋－Ｍｇ２＋ＡＴＰａｓｅ（称之基本活性）影响不大，浓度达１ｍｍｏｌ／Ｌ时，该酶活性仍保持在９６％左右；而在此浓度范围内，ｔｏｍａｔｉｎｅ对依赖于ＣａＭ激活的Ｃａ~２＋Ｍｇ~２＋ＡＴＰａｓｅｅ有明显的抑制作用；其ＩＣ５０值为０．１４ｍｍｏｌ／Ｌ     

Functional and spatial segregation of secretory vesicle pools according to vesicle age

Synaptic terminals and neuroendocrine cells are packed with secretory vesicles, only a few of which are docked at the plasma membrane and readily releasable. The remainder are thought to constitute a large cytoplasmic reserve pool awaiting recruitment into the readily releasable pool (RRP) for exocytosis. How vesicles are prioritized in recruitment is still unknown: the choice could be random, or else the oldest or the newest ones might be favoured. Here we show, using a fluorescent cargo protein that changes colour with time, that vesicles in bovine adrenal chromaffin cells segregate into distinct populations, based on age. Newly assembled vesicles are immobile (morphologically docked) at the plasma membrane shortly after biogenesis, whereas older vesicles are mobile and located deeper in the cell. Different secretagogues selectively release vesicles from the RRP or, surprisingly, selectively from the deeper cytoplasmic pool. Thus, far from being equal, vesicles are segregated functionally and spatially according to age.     

Hair cell synaptic ribbons are essential for synchronous auditory signalling

Hearing relies on faithful synaptic transmission at the ribbon synapse of cochlear inner hair cells (IHCs). At present, the function of presynaptic ribbons at these synapses is still largely unknown. Here we show that anchoring of IHC ribbons is impaired in mouse mutants for the presynaptic scaffolding protein Bassoon. The lack of active-zone-anchored synaptic ribbons reduced the presynaptic readily releasable vesicle pool, and impaired synchronous auditory signalling as revealed by recordings of exocytic IHC capacitance changes and sound-evoked activation of spiral ganglion neurons. Both exocytosis of the hair cell releasable vesicle pool and the number of synchronously activated spiral ganglion neurons co-varied with the number of anchored ribbons during development. Interestingly, ribbon-deficient IHCs were still capable of sustained exocytosis with normal Ca2+-dependence. Endocytic membrane retrieval was intact, but an accumulation of tubular and cisternal membrane profiles was observed in ribbon-deficient IHCs. We conclude that ribbon-dependent synchronous release of multiple vesicles at the hair cell afferent synapse is essential for normal hearing.     

Synaptotagmin I functions as a calcium regulator of release probability

In all synapses, Ca2+ triggers neurotransmitter release to initiate signal transmission. Ca2+ presumably acts by activating synaptic Ca2+ sensors, but the nature of these sensors--which are the gatekeepers to neurotransmission--remains unclear. One of the candidate Ca2+ sensors in release is the synaptic Ca2+-binding protein synaptotagmin I. Here we have studied a point mutation in synaptotagmin I that causes a twofold decrease in overall Ca2+ affinity without inducing structural or conformational changes. When introduced by homologous recombination into the endogenous synaptotagmin I gene in mice, this point mutation decreases the Ca2+ sensitivity of neurotransmitter release twofold, but does not alter spontaneous release or the size of the readily releasable pool of neurotransmitters. Therefore, Ca2+ binding to synaptotagmin I participates in triggering neurotransmitter release at the synapse.     

Fast vesicle replenishment allows indefatigable signalling at the first auditory synapse

Ribbon-type synapses in inner hair cells of the mammalian cochlea encode the complexity of auditory signals by fast and tonic release through fusion of neurotransmitter-containing vesicles. At any instant, only about 100 vesicles are tethered to the synaptic ribbon, and about 14 of these are docked to the plasma membrane, constituting the readily releasable pool. Although this pool contains about the same number of vesicles as that of conventional synapses, ribbon release sites operate at rates of about two orders of magnitude higher and with submillisecond precision. How these sites replenish their vesicles so efficiently remains unclear. We show here, using two-photon imaging of single release sites in the intact cochlea, that preformed vesicles derived from cytoplasmic vesicle-generating compartments participate in fast release and replenishment. Vesicles were released at a maximal initial rate of 3 per millisecond during a depolarizing pulse, and were replenished at a rate of 1.9 per millisecond. We propose that such rapid resupply of vesicles enables temporally precise and sustained release rates. This may explain how the first auditory synapse can encode with indefatigable precision without having to rely on the slow, local endocytic vesicle cycle.     

Impaired PtdIns(4,5)P2 synthesis in nerve terminals produces defects in synaptic vesicle trafficking

Phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) has an important function in cell regulation both as a precursor of second messenger molecules and by means of its direct interactions with cytosolic and membrane proteins. Biochemical studies have suggested a role for PtdIns(4,5)P2 in clathrin coat dynamics, and defects in its dephosphorylation at the synapse produce an accumulation of coated endocytic intermediates. However, the involvement of PtdIns(4,5)P2 in synaptic vesicle exocytosis remains unclear. Here, we show that decreased levels of PtdIns(4,5)P2 in the brain and an impairment of its depolarization-dependent synthesis in nerve terminals lead to early postnatal lethality and synaptic defects in mice. These include decreased frequency of miniature currents, enhanced synaptic depression, a smaller readily releasable pool of vesicles, delayed endocytosis and slower recycling kinetics. Our results demonstrate a critical role for PtdIns(4,5)P2 synthesis in the regulation of multiple steps of the synaptic vesicle cycle.     

Fast vesicle reloading and a large pool sustain high bandwidth transmission at a central synapse

What limits the rate at which sensory information can be transmitted across synaptic connections in the brain? High-frequency signalling is restricted to brief bursts at many central excitatory synapses, whereas graded ribbon-type synapses can sustain release and transmit information at high rates. Here we investigate transmission at the cerebellar mossy fibre terminal, which can fire at over 200 Hz for sustained periods in vivo, yet makes few synaptic contacts onto individual granule cells. We show that connections between mossy fibres and granule cells can sustain high-frequency signalling at physiological temperature. We use fluctuation analysis and pharmacological block of desensitization to identify the quantal determinants of short-term plasticity and combine these with a short-term plasticity model and cumulative excitatory postsynaptic current analysis to quantify the determinants of sustained high-frequency transmission. We show that release is maintained at each release site by rapid reloading of release-ready vesicles from an unusually large releasable pool of vesicles (approximately 300 per site). Our results establish that sustained vesicular release at high rates is not restricted to graded ribbon-type synapses and that mossy fibres are well suited for transmitting broad-bandwidth rate-coded information to the input layer of the cerebellar cortex.     

Single and multiple vesicle fusion induce different rates of endocytosis at a central synapse

During synaptic transmission, neurotransmitter-laden vesicles fuse with the presynaptic membrane and discharge their contents into the synaptic cleft. After fusion, the vesicular membrane is retrieved by endocytosis for reuse. This recycling mechanism ensures a constant supply of releasable vesicles at the nerve terminal. The kinetics of endocytosis have been measured mostly after intense or non-physiological stimulation. Here we use capacitance measurements to resolve the fusion and retrieval of single and multiple vesicles following mild physiological stimulation at a mammalian central synapse. The time constant of endocytosis after single vesicle fusion was 56 ms; after a single action potential or trains at < or = 2 Hz it was about 115 ms, but increased gradually to tens of seconds as the frequency and the number of action potentials increased. These results indicate that an increase in the rate of exocytosis at the active zone induces a decrease in the rate of endocytosis. Existing models, including inhibition of endocytosis by Ca(2+), could not account for these results our results suggest that an accumulation of unretrieved vesicles at the plasma membrane slows endocytosis. These findings may resolve the debate about the dependence of endocytosis kinetics on the stimulation frequency, and suggest a potential role of regulation of endocytosis in short-term synaptic depression.     

Intracellular calcium dependence of transmitter release rates at a fast central synapse

Calcium-triggered fusion of synaptic vesicles and neurotransmitter release are fundamental signalling steps in the central nervous system. It is generally assumed that fast transmitter release is triggered by elevations in intracellular calcium concentration ([Ca2+]i) to at least 100 microM near the sites of vesicle fusion. For synapses in the central nervous system, however, there are no experimental estimates of this local [Ca2+]i signal. Here we show, by using calcium ion uncaging in the large synaptic terminals of the calyx of Held, that step-like elevations to only 10 microM [Ca2+]i induce fast transmitter release, which depletes around 80% of a pool of available vesicles in less than 3 ms. Kinetic analysis of transmitter release rates after [Ca2+]i steps revealed the rate constants for calcium binding and vesicle fusion. These show that transient (around 0.5 ms) local elevations of [Ca2+]i to peak values as low as 25 microM can account for transmitter release during single presynaptic action potentials. The calcium sensors for vesicle fusion are far from saturation at normal release probability. This non-saturation, and the high intracellular calcium cooperativity in triggering vesicle fusion, make fast synaptic transmission very sensitive to modulation by changes in local [Ca2+]i.     

Synaptosomes possess an exocytotic pool of glutamate

There is increasing evidence that L-glutamate is a major excitatory neurotransmitter in the central nervous system. Immunocytochemical studies indicate that glutamate within nerve terminals may be concentrated in vesicles and glutamate-accumulating vesicles have recently been isolated. Exocytotic release of glutamate from synaptosomes (isolated nerve terminals) has not been convincingly demonstrated, however, and remains highly controversial. In order to study the kinetics of release of endogenous L-glutamate from guinea pig cerebral cortical synaptosomes we have devised a continuous enzymatic assay. This has enabled us to identify a pool, equivalent to 15-20% of the total synaptosomal glutamate, which is capable of rapid Ca2+-dependent exocytotic release.