PHARMACOLOGY OF QUANTAL INTRACELLULAR CALCIUM RELEASE

两个主要的细胞内钙释放通道参与 细胞内的钙动员是兰尼定和肌醇1，4，5- 三磷酸(InsP3)受体。它们表现出高度的有序性 同源。InsP3受体介导的钙释放是已知的 不同寻常的是，它可以被连续触发几次 以小幅度提高InsP3浓度。与此不同的是 通过常规的受体脱敏，这些受体可以重新 通过进一步增加配体浓度而激活，而不去除 中间的配基。这种释放最初被称为 “量子”，因为它表现出像钙离子释放这样的特征 来自不同种类的囊泡，每个囊泡都释放出它们的钙离子 一种要么全有要么全不的方式，但自那以后提出了其他假设 解释这一现象。虽然自1989年以来进行了广泛的调查，但没有 对于潜在的机制，已经有了明确的解释。在1993年和 显然，肌肉兰尼定受体也有类似的过程报道。 本提案将系统地检验针对以下方面的假设 回答以下问题：一)钙离子是否参与脑组织两侧的 膜？II.)是否还有另一种蛋白质参与？三、)这种行为是一种 细胞内钙释放通道本身的固有特性？IV.) 这种现象在心血管疾病的细胞水平上是否明显 系统，以及如何在原地加速？InsP3和兰诺定 将对受体进行测试，特别是最近对兰诺定的研究结果 受体对最初提出的某些假设表示怀疑 InsP3受体。将用于大多数药物的制剂 这些研究包括心脏微粒体和浓缩程度最高的 已知的每种受体的天然膜来源，骨骼肌SR 兰尼定受体的终端池和小脑微体 InsP3受体。实验将利用药理学 操作、分光光度、同位素通量和平面脂双分子层 单声道记录技术。将开发一种计算机模型来 对这种不寻常的行为是如何发生的做出连贯的解释， 它的原位意义将通过细胞内[Ca2+]来评估 心血管组织、心室肌细胞和肠系膜细胞的测量 阻力动脉。 这些研究应该能揭示其潜在的分子机制。 新发现的调节肌肉收缩、发出信号的过程 中枢神经系统内的转导及其控制的过程 Ca~(2+)在可兴奋和不可兴奋细胞中都有振荡。改建 在这个过程中可能会导致各种病理状态， 尤其是恶性高热和心律失常的发作 约由细胞内钙超载所致。

The two principal intracellular calcium release channels involved in calcium mobilization inside cells are the ryanodine and inositol 1,4,5- trisphosphate (InsP3) receptors. They exhibit a high degree of sequence homology. Ca2+ release mediated by the InsP3 receptor is known to be unusual in that it can be triggered several times in succession by elevating the InsP3 concentration in small increments. Unlike the case with conventional receptor desensitization, these receptors can be re- activated by further increases in ligand concentration without removal of the ligand in between. Such release was originally referred to as "quantal" because it manifested features like that of a release of Ca2+ from a heterogeneous population of vesicles each releasing their Ca2+ in an all-or-none fashion, but other hypotheses have since been proposed to explain the phenomenon. While investigated extensively since 1989, no clear explanation has emerged as to the underlying mechanism. In 1993 an apparently similar process was reported for the muscle ryanodine receptor. The present proposal will systematically test hypotheses directed at answering the following questions: I.) Is Ca2+ involved on either side of the membrane? II.) Is another protein involved? III.) Is the behavior an inherent property of the intracellular Ca2+ release channel itself? IV.) Is the phenomenon manifest at the cellular level in the cardiovascular system, and how might it be accelerated in situ? Both InsP3 and ryanodine receptors will be tested, especially since recent findings with ryanodine receptors cast doubt on certain hypotheses originally proposed for the InsP3 receptor. The preparations that will be used for the majority of these studies include cardiac microsomes and the most highly enriched source of native membranes known for each receptor, skeletal muscle SR terminal cisternae for the ryanodine receptor, and cerebellar microsomes for the InsP3 receptor. Experiments will utilize pharmacologic manipulation, spectrophotometry, isotope flux and planar lipid bilayer single channel recording techniques. A computer model will be developed to generate a coherent explanation of how this unusual behavior takes place, and its in situ significance will be assessed with intracellular [Ca2+] measurements on cardiovascular tissue, ventricular myocytes and mesenteric resistance artery. These studies should reveal the underlying molecular mechanism for this newly discovered process that regulates muscle contraction, signal transduction within the central nervous system and processes controlled by Ca2+ oscillations in both excitable and non-excitable cells. Alterations in this process are likely to contribute to various pathologic states, particularly episodes of malignant hyperthermia and arrhythmias brought about by intracellular Ca2+ overload.