CONTROL OF CARDIAC CA2+ BY ENDOGENOUS SPHINGOLIPIDS

内源性鞘脂对心脏 CA2 的控制

• 批准号: 6107589
• 负责人: Roger A Sabbadini
• 金额: --
• 依托单位: SAN DIEGO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-02-01 至 1999-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6107589
• 关键词: calcium channel; calcium flux; high performance liquid chromatography; immunocytochemistry; laboratory rabbit; muscle contraction; myocardium; protein kinase; receptor binding; receptor coupling; sarcoplasmic reticulum; second messengers; sphingolipids; striated muscles

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.

几种肌肉萎缩症，包括杜兴氏贝克氏症 和强直性肌营养不良的特征是膜异常， 功能 例如，在杜氏肌营养不良症中， 钙水平上升，导致兴奋收缩中断 (EC)偶联，钙超载的发展和激活的Ca- 产生肌肉坏死的蛋白酶。 一个完整的 了解肌肉功能障碍需要了解如何 骨骼肌控制钙。 肌肉产生力量的能力（或能力）取决于 很大程度上依赖于肌浆中钙离子的释放 肌细胞的网状（SR）膜。 在EC偶联期间，钙 通过钙释放通道从SR中释放出来， 兰尼碱受体 我们的实验室一直在研究 内源性鞘脂引起的肌肉收缩。 的主要目标 建议的研究是阐明骨骼肌EC- 耦合，特别是自然发生的第二个作用， 信使，鞘氨醇（SPH）和其他鞘脂在调节 SR膜钙释放通道的钙通量。 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， Ryanodine受体和SPH作用可能是生理上的 调节释放的相关机制。 我们想测试一下 通过研究SPH对受体的影响来验证这一假说。 我们有 表明SPH可以显著改变收缩行为， 骨骼肌细胞和分离的心肌细胞。 我们解释了 SPH对细胞内钙释放通道的作用; 因此，我们打算测量钙释放的动力学， 分离的SR末端池（TC）囊泡响应SPH和其他 鞘脂 我们打算将SPH作用与快速 钙释放动力学及其对[3 H]-兰尼碱结合的影响 分离骨骼TC囊泡，因为ryanodine结合直接 与受体开放钙通道的水平有关。 我们将 采用我们实验室最近开发的HPLC方法来评估 肌肉细胞质和各种其他肌肉中的鞘脂水平 以确定SPH和其他第二信使是否在 在这个范围内，我们观察它们对钙释放的生理作用。 因为我们的初步研究表明肌肉的T型小管 拥有SPH生产的酶机制，我们计划实验 旨在测量各种肌肉亚组分中关键的 参与鞘脂代谢的酶。 这些数据将 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和其他鞘脂是否 是内源性的第二信使， 调节横纹肌的收缩力。 MBRS学生将 参与研究的各个方面。