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ryanodine和肌醇1，4，5- 三磷酸（InsP 3）受体。它们表现出高度的序列性 同源性已知由InsP 3受体介导的Ca 2+释放是 不寻常的是，它可以连续触发几次， 以小增量提高InsP 3浓度。的情况不同 通过常规的受体脱敏，这些受体可以被重新激活， 通过进一步增加配体浓度而不除去 中间的配体这种释放最初被称为 “量子”，因为它表现出的特点，如释放的Ca 2 + 从一个异质群体的囊泡，每个释放其Ca 2+， 一种全有或全无的方式，但此后又提出了其他假设， 解释这个现象。 虽然自1989年以来进行了广泛的调查， 关于基本机制已经有了明确的解释。1993年， 对于肌肉兰尼碱受体报道了明显类似的过程。 本提案将系统地检验针对以下问题的假设： 回答以下问题：（一）Ca 2+是否参与了 膜？二.）是否涉及其他蛋白质？三.）这种行为是 细胞内Ca 2+释放通道本身的固有特性？四.） 这种现象是否在心血管疾病的细胞水平上表现出来？ 系统，以及如何在原地加速？InsP 3和兰尼碱 受体将被测试，特别是因为最近的研究结果与ryanodine 接受者对最初提出的某些假设表示怀疑。 InsP 3受体。将用于大多数 这些研究包括心脏微粒体， 已知每种受体的天然膜来源，骨骼肌SR Ryanodine受体的终末池和小脑微粒体 InsP 3受体。实验将利用药理学 操作，分光光度法，同位素通量和平面脂双层 单通道记录技术。将开发一个计算机模型， 对这种不寻常的行为是如何发生的给出一个连贯的解释， 其原位意义将通过细胞内[Ca 2 +] 心血管组织、心室肌细胞和肠系膜 阻力动脉 这些研究应该揭示这一现象的潜在分子机制 新发现的调节肌肉收缩的过程，信号 中枢神经系统内的转导和由 Ca 2+在可兴奋和不可兴奋细胞中的振荡。改变 在这一过程中可能会导致各种病理状态， 特别是恶性高热和心律失常的发作 细胞内钙超载。

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.