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

众所周知，在各种细胞中，机械刺激引起细胞内钙浓度的短暂增加和核苷酸的分泌，如ATP、UTP和UDP。释放的核苷酸诱导邻近细胞内钙波扩散。钙的最大来源是光滑内质网，也有容性钙内流和线粒体。在本项目中，我们明确了乳腺秘书上皮细胞和乳腺肌上皮细胞之间通过钙波进行细胞间通讯的存在。在本项目前期，我们报道了孕早期增殖中电容钙电流的增加。我们分析了核苷酸和钙诱导的钾通道的激活所激发的外向电流。最近，我们得出结论，存在另一种向内电流，由相同的激动剂诱导。我们认为这是由于Cl通道的激活。此外，UTP诱导ATP释放被Cl通道阻滞剂硝夫酸抑制。细胞内ATP可能通过某些Cl通道释放到细胞外液。我们解决了单通道活动的记录使用内外膜片钳，ATP在内部和外部。目前，我们还不能得出Cl/ATP通道存在的结论。我们在乳腺肿瘤细胞系A23587中克隆了ClC3通道基因，并转染到神经母细胞瘤/胶质瘤细胞系NG108-15中。ClC3过表达对动作电位无显著影响。

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.