CONTROL OF CARDIAC CA2+ BY ENDOGENOUS SPHINGOLIPIDS

几种肌营养不良症，包括杜兴的贝克尔 强直性肌营养不良以膜异常为特征。 功能。例如，在杜兴氏肌营养不良症中，细胞内 钙水平升高，导致兴奋收缩中断。 (EC)偶联、钙超载的发生和钙激活 产生肌肉坏死的依赖的蛋白水解酶。因此，一个完整的 对肌肉功能障碍的理解需要了解如何 骨骼肌控制着钙。 肌肉产生力量的能力(或无能)取决于 很大程度上取决于肌浆中钙离子的释放 肌细胞的网状(SR)膜。在EC偶联过程中，钙 是通过钙释放通道从SR释放的，称为 兰尼定受体。我们的实验室一直在调查控制措施 内源性鞘脂引起的肌肉收缩。这次会议的主要目标是 拟开展的研究旨在阐明骨骼肌内皮细胞损伤的机制。 耦合，特别是自然发生的秒的作用 信使、鞘氨醇(SPH)和其他鞘磷脂在调节中的作用 钙离子通过SR膜钙释放通道的通量。基于 初步工作，我们假设内源性产生的SPH块 通过直接作用于SR钙释放通道释放钙， 兰尼定受体，而SPH的作用可能是一种生理上的 调节释放的相关机制。我们希望测试一下这一点 通过检测SPH对受体的影响来提出假说。我们有 表明SPH可以显著改变血管的收缩行为。 骨骼肌细胞和分离的心肌细胞。我们已经解读了 SPH对细胞内钙释放通道的影响； 因此，我们打算测量钙释放的动力学。 分离的SR终池(TC)囊泡对SPH和其他 狮身人面像的脂类。我们打算将SPH的快速行动与 钙释放动力学及其对[~3H]-兰尼定结合的影响 分离骨骼TC囊泡，因为ryanodine结合是直接的 与受体开放钙通道的水平有关。我们会 使用我们实验室最新开发的高效液相色谱方法来评估 肌浆和其他各种肌肉中的鞘磷脂水平 用于确定SPH和其他第二信使是否在 我们观察它们对钙释放的生理作用的范围。 因为我们的初步工作表明，肌肉的T小管 拥有生产大豆分离蛋白的酶促设备，我们计划进行实验 设计用于在不同的肌肉亚部分中测量关键字的存在 与鞘脂代谢有关的酶。这些数据将是 与SMase的原位免疫细胞化学定位相关。 我们计划的实验将确定SPH和其他狮身人面像脂质 是与生理相关的内源性第二信使 调节横纹肌的收缩能力。MBRS的学生将成为 参与研究的方方面面。

Several of the muscular dystrophies, including the Duchenne's Becker's and Myotonic Dystrophies are characterized by abnormalities in membrane function. In Duchenne's Muscular Dystrophy, for example, intracellular calcium levels rise, resulting in a disruption of excitation-contraction (EC) coupling, the development of calcium overload and activation of Ca- dependent proteases which produce muscle necrosis. Thus, a complete understanding of muscle dysfunction necessitates an understanding of how skeletal muscle controls calcium. The ability (or inability) of the muscle to produce force depends in large part upon the release of calcium ions from the sarcoplasmic reticulum (SR) membranes of the muscle cell. During EC-coupling, calcium is released from SR through a calcium release channel called the ryanodine receptor. Our laboratory has been investigating the control of muscle contraction by endogenous sphingolipids. The major goal of the proposed research is to elucidate the mechanism skeletal muscle EC- coupling, and in particular, the role of the naturally occurring second messenger, sphingosine (SPH), and other sphingolipids in modulating calcium flux through SR membrane calcium release channels. Based on preliminary work, we hypothesize that endogenously produced SPH blocks calcium release via a direct effect on the SR calcium release channel, the ryanodine receptor, and that SPH's action may be a physiologically relevant mechanism for modulating release. We wish to test this hypothesis by examining the effects of SPH on the receptor. We have shown that SPH can substantially alter the contractile behavior of skeletal muscle cells and isolated cardiac myocyte. We have interpreted the effect of SPH as an action on intracellular calcium release channels; consequently, we intend to measure the kinetics of calcium release from isolated SR terminal cisternae (TC) vesicles in response to SPH and other sphingoid lipids. We intend to correlate SPH action on the rapid kinetics of calcium release with its effects on [3H]-ryanodine binding to isolate skeletal TC vesicles, since ryanodine binding is directly related to the level of calcium channel opening by the receptor. We will employ HPLC methods recently developed in our laboratory to assess sphingolipid levels in the muscle cytosol and various other muscle subfractions to determine if SPH and other second messengers are in the range where we observe their physiological actions on calcium release. Because our preliminary work indicates that the T-tubules of the muscle possesses the enzymatic machinery for SPH production, we plan experiments designed to measure in various muscle subfractions the presence of key enzymes involved in sphingolipid metabolism. These data will be correlated with the in situ immunocytochemical localization of SMase. Our planned experiments will determine if SPH and other sphingoid lipids are endogenous second messengers with physiological relevance in modulating contractility in striated muscle. MBRS students will be involved in all aspects of the research.