CORONARY MUSCLE CELL FREE CALCIUM BUFFERING

冠状肌细胞无钙缓冲

• 批准号: 6242382
• 负责人: Michael Sturek
• 金额: $ 26.17万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 1998-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6242382
• 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)增加 静息细胞内平均肌浆游离钙(Ca/m) 从要挤压(卸载)的表层SR中缓慢释放钙 进入细胞外空间。这个过程被称为“SR钙卸载”。 因为它导致了减弱的激动剂诱导的钙释放。高级案例 卸载提示SR Case的密切功能关联(定位) 钙释放部位和肌膜钙外流机制，如钙泵 和/或钠钙交换器，这将导致肌膜下钙(钙/S) 本地化。我们已经证明，冠脉平滑肌细胞 运动训练(EX)，不久坐(SED)，猪表现出SR钙卸载 这依赖于兰诺定敏感的钙释放通道。这个 这一提议的一般假设是EX细胞中的SR释放 钙从表面SR中分离出来，从而导致净钙外流(钙卸载) 在几乎没有增加钙/钙的情况下来自细胞。具体目标是测试 假设：1)EX诱导的肌浆网钙卸载需要 Ryanodine敏感的钙释放通道，变化很小 在Ca/m中，但在肌膜下间隔中游离钙的增加 (CA/S)。细胞将从心外膜冠状动脉中分散出来 (导管大小)前和后尤卡坦小型猪。全电池电压 钳制将允许使用钙激活的钾电流或膜结合 Fura-2用于监测钙/S；支原体Fura-2盐将监测 Ca/m.2)ex诱导的肌浆网钙卸载导致钙净流出 细胞，而不是转移(重新分配)到咖啡因不敏感的存储。 细胞悬浮液的净钙外流将通过以下方式进行监测 胞外无膜Fura-2ex诱导的肌浆网钙卸载 需要从细胞表面SR部位局部外流钙。 一个完整细胞的钙外流的相对定位将是 使用细胞外Fluo-3进行数字成像监测，并使用 切除(由内向外)钙激活钾通道的斑块作为“钙” 电极贴片“。SR位置将用绿色显示 荧光SR标记DiOC6。4)EX诱导的SR钙卸载需要 局部钠钙交换。将确定在EX细胞中是否存在Na- 钙交换免疫荧光与钙外流部位共定位 用钙电极贴片和胞外Fluo-3测定。这些 数据将为Slow和Slow之间的紧密功能耦合提供证据 局部、浅层SR部位的钙释放和通过Na~-的钙排出 CA交易所。5)肌浆网钙释放与收缩成正比 动脉环的反应。Fura-2同时测定Ca/m和 动脉环上的收缩记录将决定 在完整的动脉中，SR钙释放到血管收缩功能中。这个 这项工作的主要意义在于功能数据的集成 从冠脉组织和亚细胞水平的研究获得 动脉平滑肌。