Measurement of rapidly exchangeable cellular calcium in the perfused beating rat heart.
Measurement of rapidly exchangeable cellular calcium in the perfused beating rat heart.
复制标题
测量灌注跳动的大鼠心脏中快速交换的细胞钙。
DOI：
10.1073/pnas.78.9.5665
复制
发表时间：
1981
影响因子：
11.1
通讯作者：
H. Berkoff
中科院分区：
文献类型：
--
作者：
D. R. Hunter;R. Haworth;H. Berkoff
虽然钙离子早已被认为在心脏的兴奋-收缩耦合中起着至关重要的作用，但由于测量细胞不同隔室中钙离子运动的实验困难，对这种作用的细节的研究一直受到阻碍。一个主要的问题是区分相对少量的快速可交换的细胞Ca 2+与大量的血管和间质Ca 2+。我们在这里报告的方法，克服了这个问题。通过在37 ℃下用含有45 Ca 2+的培养基灌注来标记大鼠心脏。然后将它们冷却，并通过在6 ℃下用无Ca 2+培养基灌注来除去细胞外45 Ca 2+。通过冷却而被捕获在心脏中的细胞45 Ca 2+然后通过在37 ℃下用含有未标记Ca 2+的培养基再灌注而释放。通过使用[3 H]蔗糖证实了这种45 Ca 2+的细胞来源：当心脏也用[3 H]蔗糖标记时，非常少的[3 H]-蔗糖与45 Ca 2+峰一起释放。标记5分钟的心脏中可交换的细胞Ca 2+的量为125 +/- 13 nmol/g湿重。其释放的半衰期小于1分钟。此细胞内的钙离子至少包含两个池：一个快速交换池，需要细胞外的钙离子释放（池A，38%的总），和一个更慢的交换池，没有（池B，62%的总）。在5分钟标记期间用异丙肾上腺素刺激的心脏显示可交换的细胞Ca 2+总量增加46%;这种增加完全位于池A中。
Although Ca2+ has long been known to play a vital role in excitation--contraction coupling in the heart, investigation of the details of this role has been hampered by the experimental difficulty of measuring Ca2+ movements through the different compartments of the cell. A major problem has been to distinguish the relatively small amount of rapidly exchangeable cellular Ca2+ from the large amount of vascular and interstitial Ca2+. We report here a method that overcomes this problem. Rat hearts were labeled by perfusion at 37 degrees C with medium containing 45Ca2+. They were then cooled, and extracellular 45Ca2+ was removed by perfusion at 6 degrees C with Ca2+-free medium. Cellular 45Ca2+ that had been trapped in the hearts by cooling was then released by reperfusion at 37 degrees C with medium containing unlabeled Ca2+. The cellular origin of this 45Ca2+ was confirmed by using [3H]sucrose: When hearts were also labeled with [3H]sucrose, very little [3H]-sucrose was released with the 45Ca2+ peak. The amount of exchangeable cellular Ca2+ in hearts labeled for 5 min was 125 +/- 13 nmol/g of wet weight. The half-time for its release was less than 1 min. This cellular Ca2+ contained at least two pools: a rapidly exchanging pool that required extracellular Ca2+ for release (pool A, 38% of total), and a more slowly exchanging pool that did not (pool B, 62% of total). Hearts stimulated with isoproterenol during the 5-min labeling period showed an increase of 46% for the total amount of exchangeable cellular Ca2+; this increase was entirely located in pool A.