CORONARY MUSCLE CELL FREE CALCIUM BUFFERING

冠状肌细胞无钙缓冲

• 批准号: 6296872
• 负责人: Michael Sturek
• 金额: $ 27.57万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6296872
• 关键词: buffers; calcium channel; calcium flux; confocal scanning microscopy; coronary artery; coronary disorder; digital imaging; disease /disorder model; electrophysiology; exercise; fluorimetry; immunofluorescence technique; miniature swine; muscle contraction; potassium chloride; ryanodine; sarcolemma; sarcoplasmic reticulum; vascular smooth muscle; voltage /patch clamp

The sarcoplasmic reticulum (SR) may buffer (attenuate) increases in averaged mycoplasmic free Ca (Ca/m) in quiescent (resting) cells by slowly releasing Ca from the superficial SR to be extruded (unloaded) into the extracellular space. This process is termed "SR Ca unloading" because it results in attenuated agonist-induced ca release. SR Ca unloading suggests close functional association (localization) of SR Ca release sites and sarcolemmal Ca efflux mechanisms, such as the Ca pump and/or Na-Ca exchanger, which would result in subsarcolemmal Ca (Ca/s) localization. We have shown that coronary smooth muscle cells of exercise trained (EX), not sedentary (SED), pigs show SR Ca unloading that is dependent on ryanodine-sensitive Ca release channels. The general hypothesis of this proposal is that the SR in EX cells releases Ca from superficial SR, thus resulting in net Ca efflux (Ca unloading) from the cell with almost no increase in Ca/m. Specific aims are to test the following hypotheses: 1) EX-induced SR Ca unloading requires ryanodine-sensitive Ca release channels and occurs with minimal change in Ca/m, but an increase in free Ca in a subsarcolemmal compartment (Ca/s). Cells will be dispersed from epicardial coronary arteries (conduit size) of EX and SED Yucatan miniature pigs. Whole-cell voltage clamp will allow the use of Ca-activated K current or membrane-bound fura-2 (FFP18) for monitoring Ca/s; mycoplasmic fura-2 salt will monitor Ca/m. 2) EX-induced SR Ca unloading results in net Ca efflux from the cell, not translocation (redistribution) to a caffeine-insensitive store. Net Ca efflux from suspensions of cells will be monitored by extracellular, membrane-impermeant fura-2 3) EX-induced SR Ca unloading requires localized efflux of Ca from the cell at superficial SR sites. The relative localization of Ca efflux from an intact cell will be monitored with digital imaging using extracellular fluo-3 and with an excised (inside-out) patch of Ca-activated K channels used as a "Ca electrode patch". SR locations will be visualized with the green fluorescent SR marker DiOC6. 4) EX-induced SR Ca unloading requires localized Na-Ca exchange. It will be determined whether in EX cells Na- Ca exchange immunofluorescence co-localizes with Ca efflux sites determined with the Ca electrode patch and extracellular fluo-3. These data will provide evidence for a tight functional coupling between slow Ca release from localized, superficial SR sites and Ca extrusion via Na- Ca exchange. 5) SR Ca release is proportional to the contractile response of arterial rings. Simultaneous fura-2 measures of Ca/m and contraction recordings in arterial rings will determine the relevance of SR Ca release to vascular contractile function in intact arteries. The main significance of this work is the integration of functional data obtained from studies at the tissue and subcellular levels of coronary artery smooth muscle.

肌浆网 (SR) 可以缓冲（减弱） 静息细胞中的平均支原体游离 Ca (Ca/m) 从表面 SR 缓慢释放 Ca 并被挤出（卸载） 进入细胞外空间。 这个过程被称为“SR Ca 卸载” 因为它会导致激动剂诱导的 ca 释放减弱。 SR钙 卸载表明 SR Ca 的紧密功能关联（定位） 释放位点和肌膜 Ca 流出机制，例如 Ca 泵 和/或 Na-Ca 交换器，这将导致肌膜下 Ca (Ca/s) 本土化。 我们已经证明冠状动脉平滑肌细胞 运动训练（EX），不久坐（SED），猪显示 SR Ca 卸载 这取决于兰尼碱敏感的 Ca 释放通道。 这 该提案的一般假设是 EX 细胞中的 SR 释放 来自表面 SR 的 Ca，从而导致 Ca 净流出（Ca 卸载） 来自细胞的 Ca/m 几乎没有增加。 具体目标是测试 以下假设：1) EX 诱导的 SR Ca 卸载需要 兰尼碱敏感的 Ca 释放通道，变化极小 Ca/m，但肌膜下室中游离 Ca 增加 （钙/秒）。 细胞将从心外膜冠状动脉分散 EX 和 SED 尤卡坦小型猪（导管尺寸）。 全细胞电压 钳将允许使用 Ca 激活 K 电流或膜结合 fura-2 (FFP18) 用于监测 Ca/s；支原体fura-2盐会监测 钙/米。 2) EX诱导的SR Ca卸载导致Ca从 细胞，而不是易位（重新分配）到对咖啡因不敏感的商店。 细胞悬浮液中的净 Ca 流出将通过以下方式监测 细胞外、膜非渗透性的 fura-2 3) EX 诱导的 SR Ca 卸载 需要Ca从表面SR位点的细胞局部流出。 来自完整细胞的 Ca 流出的相对定位为 使用细胞外 Fluo-3 进行数字成像监测 切除（由内而外）的 Ca 激活 K 通道斑块，用作“Ca 电极贴片”。SR 位置将以绿色显示 荧光 SR 标记 DiOC6。 4) EX引起的SR Ca卸载需要 局部Na-Ca交换。 将确定 EX 细胞中是否存在 Na- Ca 交换免疫荧光与 Ca 流出位点共定位 用 Ca 电极贴片和细胞外 Fluo-3 测定。 这些 数据将为慢速之间的紧密功能耦合提供证据 Ca 从局部表面 SR 位点释放，并通过 Na- 挤出 Ca 钙交换。 5) SR Ca 释放与收缩力成正比 动脉环的反应。 同时进行 Fura-2 测量 Ca/m 和 动脉环中的收缩记录将确定以下相关性 SR Ca 释放对完整动脉中血管收缩功能的影响。 这 这项工作的主要意义是功能数据的整合 从冠状动脉组织和亚细胞水平的研究中获得 动脉平滑肌。