Physiological role of calcium wave and capacitative calcium current in mammary epithelial cells

众所周知，在不同的细胞中，机械刺激引起细胞内钙离子浓度的一过性升高和核苷酸的分泌，如ATP、UTP和UDP。释放的核苷酸引起邻近细胞内钙波的扩散。钙的最大来源是光滑的内质网，也有容量性的钙内流，还有线粒体。在这个项目中，我们通过钙波清除了乳腺分泌上皮细胞和乳腺肌上皮细胞之间存在的细胞间通讯。在这个项目的早期，我们报道了妊娠早期细胞增殖过程中电容钙电流的增加。我们分析了核苷酸刺激的外向电流，以及钙诱导的钾通道的激活。最近，我们得出结论，存在另一种内向电流，由相同的激动剂诱导。我们认为这是由于氯通道的激活所致。此外，氯通道阻断剂Nifulic acid可抑制UTP诱导的ATP释放。细胞内的三磷酸腺苷可能通过某种氯通道释放到细胞外液中。我们使用内向外膜片钳记录单通道活动，内、外均有ATP。目前，尚不能得出氯/三磷酸腺苷通道存在的结论。我们从乳腺肿瘤细胞系A23587中克隆了ClC3通道基因，并将其导入神经母细胞瘤/胶质瘤细胞系NG108-15。ClC3过表达对动作电位无明显影响。

It is well known that in various cells, mechanical stimulation caused transient increase of the intracellular calcium concentration and secretion of nucleotides, such as ATP, UTP and UDP.Released nucleotides induce spreading calcium wave in adjacent cells. The largest source of calcium is the smooth endoplasmic reticulum, and also there are capacitative calcium influx, and mitochondria. In this project, we cleared the presence of the intercellular communication between mammary secretary epithelial cells and mammary myoepithelial cells, via calcium wave. In the early of this project, we reported the increase of capacitative calcium current in the proliferation in early pregnant stage.We analysed the outward current stimulated by nucleotide, and by the activation of calcium-induced potassium channel. Recently, we concluded that there is another inward current, induced by same agonist. We concluded that this is due to the activation of Cl channel. Moreover, UTP induced ATP release inhibited by blocker of Cl channel, nifulmic acid. It is possible that intracellular ATP released to the extracellular fluid via some Cl channel. We tackled to the recording of single channel activity using inside out patch clamp, with ATP in both inside and outside. In the present, we could not reach any conclusion of presence of Cl/ATP channel. We cloned ClC3 channel gene in mammary tumor cell line, A23587, and transfected it in the neuroblastoma/glioma cell line, NG108-15. There was no difference in the action potential with overexpression of ClC3.