Peptide Release Regulated by Ca2+ from Neurosecretory Granules

描述（由申请人提供）：本提案的总体目标是检查神经末梢中内部钙（Ca 2+）储存的作用。众所周知，神经递质和激素的释放与细胞内游离Ca 2+浓度的升高紧密相关。虽然通过电压门控Ca 2+通道的Ca 2+内流无疑是影响释放的Ca 2+的一个非常重要的来源，但来自内部储存的Ca 2+释放可能为该过程提供另一个重要的Ca 2+来源。直到最近，这些细胞内储存才引起了相对较少的关注，它们在神经末梢中的存在也是有争议的。最近的工作表明，高度本地化的Ca 2+释放事件，如Ca 2+火花的肌肉，可以看到在神经元制剂。在神经垂体末梢，这些Ca 2+释放事件似乎是从ryanodine敏感的细胞内Ca 2+池发出的，去极化刺激诱导其频率增加。尽管所有这些信息，释放的Ca 2+的来源，以及这种现象的生理作用，是未知的。初步证据表明，这些Ca 2+释放事件可能代表动员的Ca 2+从囊泡商店。如果是这样的话，在胞吐的精确位置的局部Ca 2+释放应该调节释放。这是我们的目标，以确定这些ryanodine敏感的Ca 2+释放事件在神经垂体终端的来源，并阐明这种动员的Ca 2+对神经肽分泌的生理作用。为了实现这一点，我们已经能够开发，第一次，一种技术能够检测量子释放事件从个别神经末梢。这种安培技术使我们能够同时进行Ca 2+成像和监测发射器释放从一个单一的孤立的神经末梢的一个定义的区域。该项目旨在增加神经垂体末梢生理学的现有知识体系，并且更普遍地，将提供对细胞内钙所起的作用以及中枢神经系统中发生大致密核心颗粒融合的机制的更完整的理解。因此，这一知识可能被证明是重要的理解和治疗突触病理。公共卫生相关性：脑内和与其靶点的通信都是通过神经末梢处的递质释放进行的，已知这种释放依赖于细胞外钙的进入和随后的末梢内钙的升高。我们最近表明，神经末梢有细胞内钙储存，在这个建议中，我们利用新的技术来确定这些intraterminal商店是什么，以及它们是否可以调节神经末梢的递质释放。这些知识对于理解和治疗突触疾病，如Eaton-Lambert综合征和肌萎缩性侧索硬化症，以及阿尔茨海默病和神经元老化非常重要。

DESCRIPTION (provided by applicant): The overall aim of this proposal is to examine the role of internal calcium (Ca2+) stores in nerve terminals. It is well known that the release of neurotransmitters and hormones is tightly coupled to rises in the free intracellular Ca2+ concentration. While the influx of Ca2+ through voltage-gated Ca2+ channels is undoubtedly a very important source of Ca2+ affecting release, Ca2+ release from internal stores may provide another important source of Ca2+ for this process. Until recently these intracellular stores have attracted relatively little attention, and their very existence in nerve terminals was controversial. Recent work has shown that highly localized Ca2+ release events, like Ca2+ sparks of the muscle, can be seen in neuronal preparations. In neurohypophysial terminals, these Ca2+ release events appear to emanate from a ryanodine-sensitive intracellular Ca2+ pool, and depolarizing stimuli induce an increase in their frequency. In spite of all this information, the source of the released Ca2+, and a physiological role for this phenomenon, is unknown. Preliminary evidence suggests that these Ca2+ release events could represent mobilization of Ca2+ from vesicular stores. If so, localized Ca2+ release in the precise location of exocytosis should modulate release. It is our goal to determine the source of these ryanodine sensitive Ca2+ release events in neurohypophysial terminals, and to elucidate the physiological role of this mobilization of Ca2+ on neuropeptide secretion. To accomplish this we have been able to develop, for the first time, a technique capable of detecting quantal release events from individual nerve terminals. This amperometric technique allows us to simultaneously perform Ca2+ imaging and monitor transmitter release from a defined area of a single isolated nerve terminal. This project aims to add to the current body of knowledge of the physiology of the neurohypophysial terminals, and, more generally, will provide a more complete understanding of the role played by intracellular calcium and of the mechanism by which large dense core granular fusion occurs in the Central Nervous System. This knowledge could thus prove to be important for the understanding and treatment of synaptic pathologies. PUBLIC HEALTH RELEVANCE: Communication both within the brain and with its targets is via release of transmitters at nerve terminals, and such release is known to be dependent on the entry of extracellular calcium and the subsequent elevation of intraterminal calcium. We have recently shown that nerve terminals have intracellular calcium stores and in this proposal we utilize novel techniques to determine what are these intraterminal stores and whether they can regulate the release of transmitters from nerve terminals. This knowledge could prove to be important for the understanding and treatment of synaptic diseases such Eaton-Lambert Syndrome and Amyotrophic Lateral Sclerosis, as well as Alzheimer's disease and neuronal aging.