Kimura H, Oyamada Y, Ohshika H, Mori M, Oyamada M
Department of Pharmacology, Sapporo Medical University School of Medicine, Japan.
Exp Cell Res. 1995 Oct;220(2):348-56. doi: 10.1006/excr.1995.1325.
We analyzed by Fotonic Sensor, a fiber-optic displacement measurement instrument, the effects of heptanol on synchronized contraction of primary neonatal rat cardiac myocytes cultured at confluent density. We also examined the effect of heptanol on the changes in gap junctional intercellular communication by using the microinjection dye transfer method, and on intercellular Ca2+ fluctuation by confocal laser scanning microscopy of myocytes loaded with the fluorescent Ca2+ indicator fluo 3. In addition, we studied expression, phosphorylation, and localization of the major cardiac gap junction protein connexin 43 (Cx43) using immunofluorescence and Western blotting. At Day 6 of culture, numerous myocytes exhibited spontaneous, synchronous contractions, excellent dye coupling, and synchronized intracellular Ca2+ fluctuations. We treated the cells with 1.5, 2.0, 2.5, and 3.0 mmol/liter heptanol. With 1.5 mmol/liter heptanol, we could not observe significant effects on spontaneous contraction of myocytes. At 3.0 mmol/liter, the highest concentration used in the current experiment, heptanol inhibited synchronous contractions and even after washing out of heptanol, synchronous contraction was not rapidly recovered. On the other hand, at the intermediate concentrations of 2.0 and 2.5 mmol/liter, heptanol reversely inhibited synchronized contraction, gap junctional intercellular communication, and synchronization of intracellular Ca2+ fluctuations in the myocytes without preventing contraction and changes of intracellular Ca2+ in individual cells. Brief exposure (5-20 min) to heptanol (2.0 mmol/liter) did not cause detectable changes in the expression, phosphorylation, or localization of Cx43, despite strong inhibition of gap junctional intercellular communication. These results suggest that gap junctional intercellular communication plays an important role in synchronous intracellular Ca2+ fluctuations, which facilitate synchronized contraction of cardiac myocytes.
我们使用光纤位移测量仪器Fotonic Sensor分析了庚醇对以汇合密度培养的原代新生大鼠心肌细胞同步收缩的影响。我们还通过显微注射染料转移法研究了庚醇对缝隙连接细胞间通讯变化的影响，并通过对加载荧光钙指示剂fluo 3的心肌细胞进行共聚焦激光扫描显微镜观察了其对细胞间Ca2+波动的影响。此外，我们使用免疫荧光和蛋白质印迹法研究了主要心脏缝隙连接蛋白连接蛋白43（Cx43）的表达、磷酸化和定位。在培养的第6天，许多心肌细胞表现出自发性同步收缩、良好的染料偶联以及同步的细胞内Ca2+波动。我们用1.5、2.0、2.5和3.0 mmol/升的庚醇处理细胞。使用1.5 mmol/升庚醇时，我们未观察到对心肌细胞自发收缩有显著影响。在3.0 mmol/升（本实验使用的最高浓度）时，庚醇抑制了同步收缩，并且即使在洗脱庚醇后，同步收缩也未迅速恢复。另一方面，在2.0和2.5 mmol/升的中间浓度下，庚醇反向抑制了心肌细胞中的同步收缩、缝隙连接细胞间通讯以及细胞内Ca2+波动的同步化，而不影响单个细胞的收缩和细胞内Ca2+的变化。短暂暴露（5 - 20分钟）于2.0 mmol/升的庚醇，尽管强烈抑制了缝隙连接细胞间通讯，但并未导致Cx43的表达、磷酸化或定位出现可检测到的变化。这些结果表明，缝隙连接细胞间通讯在促进心肌细胞同步收缩的细胞内Ca2+同步波动中起重要作用。
