The relation between Ca^<2+>-transient and Ca^<2+>-handling energy in excitation-contraction coupling

为建立分离心肌细胞的方法，采用Langendorff灌流法，用胶原酶溶液灌流豚鼠心脏。我们对原方法进行了多点改进，得到了相当高比例的完整心肌细胞。然而，为了测量细胞悬液的钙荧光，我们试图获得越来越多的完整心肌细胞的产量。然而，我们得出结论，不能达到高于70-80%的产率。因此，既不能测量细胞悬浮液的钙荧光也不能测量细胞悬浮液的耗氧量。然后，我们将通过使用心肌切片建立细胞内钙和氧消耗的测量。同时测定细胞内钙和氧的消耗是非常困难的。同时，我们决定分别测量它们。首先建立了耗氧量的测量系统。近年来，氧耗量的合理值被认为是基础代谢和兴奋-收缩偶联中钙处理能的总和。然后，利用本公司购买的CAF 110，我们在340 nm和380 nm处检测了pCa-荧光强度关系。在pCa = 8.5和7.5之间，340 nm与380 nm的比率逐渐增加。然后，我们做了以下实验。我们将犬左心室解剖成大块，并将其放入冰冷的KB溶液中。取出心外膜部分后，将这些块切成小块（几mm）。用KB溶液中的FuraII-AM负载来自正常心脏和Ca过载心脏的小块。3-4小时后，洗出小片并测量其细胞内钙浓度。正常心脏和钙超载衰竭心脏细胞内钙浓度无差异。

To establish the method to obtain isolated cardiac myocytes, we perfused guinea pig heart with collagenase solution by Langendorff perfusion method. We improved many points in principle method, and got intact myocytes at considerably high percentage. However, to measure calcium fluorescence of cell suspension, we tried to get more and more yields of intact myocytes. However, we concluded that higher than 70-80% of yield could not be attained. Therefore, neither calcium fluorescence nor oxygen consumption of cell suspension could be measure. Then, we are going to establish the measurement of intracellular calcium and oxygen consumption by using cardiac muscle slices. Simultaneous measurement of intracellular calcium and oxygen consumption was very difficult. Also, we determined to measure them separately. At first, the measuring system for oxygen consumption could be established. Recently, we got reasonable values of oxygen consumption, which is supposed to be the sum of basal metabolism and calcium handling energy in excitation-contraction coupling. Next, by using CAF110 which was bought by this grand, we examined pCa-intensity of fluorescence relation at 340nm and 380nm. The ratio of 340nm to 380nm gradually increased between pCa = 8.5 and 7.5. Then, we did the following experiments. We dissected canine left ventricles into big blocks and put them into ice-cold KB solution. After removal of epicardial parts, these blocks were cut into small pieces (several mm). Small pieces from normal hearts and Ca overload hearts were loaded with FuraII-AM in KB solution. After 3-4 hrs, small pieces were washed out and measured their intracellular calcium concentration. No difference was observed between intracellular calcium concentration of normal hearts and Ca overload failing hearts.