Analysis of origin and spreading of Ca^<2+> transients by digital imaging system in isolated cardiac cells.

为了研究电刺激心肌细胞后钙瞬变的起源和传播，构建了一种能够以足够的时间和空间分辨率记录细胞内钙分布的系统。该系统由计算机控制的倒置荧光显微镜和数字图像分析仪组成。使用该系统，可以每4毫秒采集一张荧光图像。本研究以豚鼠和大鼠心脏单个心肌细胞为研究对象。使用Fura-2作为Ca^lt；2+&gt；指示剂，监测钙瞬变。这是一项从1992年到1993年进行的为期两年的研究。实验结果总结如下：电刺激触发的钙瞬变起源于心肌细胞的一个或几个部位，或几乎整个心肌细胞。当钙瞬变在一个部位开始时，大约需要30毫秒才能传播到心肌细胞的另一端。在电刺激后约80毫秒，观察到钙瞬变的峰值。激活钙通道的异丙肾上腺素可增加起始点的数目，而抑制钙通道的异搏定可减少起始点的数目。哇巴因能增加细胞内Ca~(2+)浓度，但对Ca~(2+)~(2+)通道有直接作用，但不能改变起始点的数目。阿霉素抑制肌浆网钙释放机制，但不改变起始点的数目，但显著延迟了钙瞬变的峰值时间。这些结果表明，每个细胞都有钙瞬变的优势部位(S)，钙瞬变起始于膜去极化反应。Cz^lt；2+>可能由Ca^lt；2+>通过钙通道内流而启动，并随着钙离子从肌浆网释放的浪潮在细胞内传播。

To examine the origin and spreading of the Ca^<2+> transients following electrical stimulation of isolated myocyte, a system capable of recording intracellular Ca^<2+> distribution with sufficient temporal and spatial resolution was constructed. The system consists of an inverted epifuorescent microscope with a computer-controlled CCD camera and a digital image analyzer. With ths system, it is possible to collect a fluorescent picture every 4 msec. In this study, single myocytes obtained from guinea pig or rat hearts were used. Ca^<2+> transient was monitored using fura-2 as the Ca^<2+> indicator. This was a two year study conducted from 1992 throught 1993. Experimental results are summarized as follows.Ca^<2+> transients triggered by electrical stimulation originated from one or a few sites, or almost entire part of a myocyte. When the Ca^<2+> transient initiated at one site, it took approximately 30 msec to propagate to the other end of the myocyte. A peak of the Ca^<2+> transient was observed about 80 msec after electrical stimulation. The mumber of the initiating sites was increased by isoproterenol which stimulates Ca^<2+> channels, and decreased by verapamil which inhibits Ca^<2+> channels. Ouabain, which increases intracellular Ca^<2+> concentration but has n direct effect on Ca^<2+> channels, failed to alter the number of initiating sites. Doxorubicin, shown to inhibit the Ca^<2+> release mechanism of sarcoplasmic reticulum, did not alter the number of initiating sites athough this agent delayd time to peak Ca^<2+> transients remarkably.These results ndicate that each cell has preferential site(s) at which Ca^<2+> transients originate responding to membrane depolarization. Cz^<2+> transients may be initiated by Ca^<2+> influx via Ca^<2+> channels, and propagate within a cell as the wave of Ca^<2+> induced Ca^<2+> release from sarcoplasmic reticulum.