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

钙波和电容性钙电流在乳腺上皮细胞中的生理作用

• 批准号: 11670042
• 负责人: ENOMOTO Koh-ichi
• 金额: $ 2.3万
• 依托单位: Shimane Medical University (2000)Kyoto Institute of Technology (1999)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670042/
• 关键词: mechanical stimulus; calcium wave; intercellular communication; nucleotide; ATP; Cl channel; ATP channel; purinergic receptor

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.

众所周知，在各种细胞中，机械刺激引起细胞内钙离子浓度的瞬时增加和核苷酸的分泌，如ATP、UTP和UDP，释放的核苷酸在邻近细胞中引起扩散的钙波。钙的最大来源是滑面内质网，也有容量性钙内流和线粒体。本课题研究了乳腺分泌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可能性。本课题前期报道了在妊娠早期细胞增殖过程中，细胞的电容性钙电流增加，并分析了核苷酸刺激的外向电流和钙诱导的钾通道激活的外向电流。最近，我们得出结论，有另一个内向电流，由相同的激动剂诱导。我们认为这是由于Cl通道的激活。ATP释放可被Cl通道阻断剂硝氟酸所抑制。细胞内ATP可能通过某些Cl通道释放到细胞外液中。我们采用由内而外的膜片钳技术记录单通道的活动，在内部和外部都有ATP。目前，我们不能得出Cl/ATP通道存在的任何结论。我们在乳腺肿瘤细胞系A23587中克隆了ClC 3通道基因，并将其转染到神经母细胞瘤/胶质瘤细胞系NG 108 -15中。ClC 3过表达的动作电位无差异。

项目成果

Nakai S,Furuya K,Miyata S and Kiyohara T: "Intracellular Ca^<2+> responses to nucleotides, peptides, amines, amino acids and prostaglandins in cultured pituicytes from adult rat neurohypophysis"Neuroscience Letters. 266. 185-188 (1999)

Nakai S、Furuya K、Miyata S 和 Kiyohara T：“成年大鼠神经垂体培养的垂体细胞中细胞内 Ca^2 对核苷酸、肽、胺、氨基酸和前列腺素的反应”神经科学快报。

Kataoka H.: "Cloning and embryonic expression patterns of the zebrafish Runt domain genes, runxa and runxb."Mechanisms of Development. 98. 139-143 (2000)

Kataoka H.：“斑马鱼 Runt 结构域基因 runxa 和 runxb 的克隆和胚胎表达模式。”发育机制。

Miyata S.: "Morphological plasticity and rearrangement of cytoskeletons in pituicytes cultured from adult rat neurohypophysis."Neuroscience Research. 33. 299-306 (1999)

Miyata S.：“从成年大鼠神经垂体培养的垂体细胞中细胞骨架的形态可塑性和重排。”神经科学研究。

Enomoto K.: "Semi-quantitative analysis of expression of GABA-A receptor subunits involving alternative splicing in the embryonic chick brainstem."Japanese Journal of Physiology. (In press).

Enomoto K.：“涉及胚胎鸡脑干选择性剪接的 GABA-A 受体亚基表达的半定量分析。”日本生理学杂志。