Peptide Release Regulated by Ca2+ from Neurosecretory Granules

描述(申请人提供)：本提案的总体目标是研究神经末梢内钙(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钙浓度的升高密切相关。虽然钙离子通过电压门控钙通道的内流无疑是钙离子影响释放的一个非常重要的来源，但从内库释放钙离子可能为这一过程提供了另一个重要的钙来源。直到最近，这些细胞内存储还相对较少引起注意，它们在神经末梢中的存在也是有争议的。最近的研究表明，在神经元准备中可以看到高度局部性的钙释放事件，比如肌肉中的钙火花。在神经垂体终末，这些钙释放事件似乎来自兰诺定敏感的细胞内钙池，去极化刺激导致它们的频率增加。尽管有这些信息，但释放的钙离子的来源以及这种现象的生理作用尚不清楚。初步证据表明，这些Ca~(2+)释放事件可能代表囊泡库中的Ca~(2+)被动员。如果是这样的话，胞吐作用精确位置的局部钙离子释放应该会调节释放。我们的目标是确定这些兰尼定敏感的钙释放事件的来源，并阐明这种钙离子的动员对神经肽分泌的生理作用。为了实现这一点，我们第一次能够开发出一种能够检测单个神经末梢的量子释放事件的技术。这项安培技术使我们能够同时进行钙离子成像，并从单个孤立神经末梢的指定区域监测递质释放。该项目旨在增加目前关于神经垂体终末生理学的知识体系，更广泛地说，将提供对细胞内钙所起的作用以及在中枢神经系统中发生大而密的核心颗粒融合的机制的更全面的了解。因此，这些知识可能被证明对理解和治疗突触病理很重要。公共卫生相关性：大脑内部和与其靶点的通信都是通过神经末梢的递质释放进行的，众所周知，这种释放依赖于细胞外钙的进入和随后的细胞内钙的升高。我们最近发现神经末梢有细胞内的钙库，在这项建议中，我们利用新的技术来确定这些终末内的库是什么，以及它们是否可以调节神经末梢递质的释放。这些知识可能被证明对理解和治疗突触疾病，如伊顿-兰伯特综合征和肌萎缩侧索硬化症，以及阿尔茨海默病和神经元老化很重要。

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.