CORONARY MUSCLE CELL FREE CALCIUM BUFFERING

肌浆网（SR）可以缓冲（减弱） 静止（静息）细胞中平均支原体游离Ca（Ca/m）， 从表面SR中缓慢释放钙以挤出（卸载） 进入细胞外空间 这一过程被称为“SR Ca卸载” 因为它导致减弱的激动剂诱导的Ca释放。 网Ca 卸载表明SR Ca的密切功能关联（定位） 释放位点和肌膜钙外流机制，如钙泵 和/或Na-Ca交换器，这将导致肌膜下Ca（Ca/s） 本地化 我们已经证明， 运动训练（EX），非久坐（SED），猪显示SR Ca卸载 依赖于ryanodine敏感的Ca释放通道。 的 该建议的一般假设是EX细胞中的SR释放 Ca从浅表SR流出，从而导致净Ca流出（Ca卸载） 从细胞中几乎没有增加Ca/m。 具体目标是测试 以下假设：1）EX诱导的SR Ca卸载需要 Ryanodine敏感的钙释放通道，发生变化极小 但肌膜下室游离钙增加 (Ca/s）。 细胞将从心外膜冠状动脉分散 （导管尺寸）的EX和SED尤卡坦小型猪。 全细胞电压 钳将允许使用钙激活的钾电流或膜结合 fura-2（FFP 18）用于监测Ca/s;支原体fura-2盐将监测 Ca/m。 2)EX诱导的SR Ca卸载导致从细胞膜的净Ca流出。 细胞，而不是易位（重新分配）到咖啡因不敏感的商店。 将通过以下方法监测细胞悬液的净Ca流出量： 细胞外，膜不渗透的fura-2 3）EX诱导的SR Ca卸载 需要在表面SR位点从细胞局部流出Ca。 来自完整细胞的Ca流出的相对定位将是 使用细胞外fluo-3进行数字成像监测， 切除的（由内而外）钙激活钾通道片，用作“钙激活钾通道”。 电极贴片”。 SR位置将显示为绿色 荧光SR标记DiOC 6。 4)EX诱导的SR Ca卸载需要 局部钠钙交换 将确定在EX细胞中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 数据将提供证据，证明缓慢的 局部浅表SR部位的Ca释放和Na- 换卡。 5)SR Ca释放与收缩成正比 动脉环的反应。 钙/米和钙/米的同时fura-2测量 动脉环中的收缩记录将确定 完整动脉中SR Ca释放对血管收缩功能的影响。 的 这项工作的主要意义在于功能数据的整合 从冠状动脉组织和亚细胞水平的研究中获得， 动脉平滑肌

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.