Contreras R G, Flores-Maldonado C, Lázaro A, Shoshani L, Flores-Benitez D, Larré I, Cereijido M
Department of Physiology, Biophysics and Neurosciences, Av., Center for Research & Advanced Studies, Instituto Politécnico Nacional 2508, 07300, México, D.F., Mexico.
J Membr Biol. 2004 Apr 1;198(3):147-58. doi: 10.1007/s00232-004-0670-2.
Abstract. In previous work we described a "P-->A mechanism" that transduces occupancy of the pump ( P) by ouabain into changes in phosphorylation, stimulation of mitogen-activated protein kinase (MAPK), and endocytosis of cell-cell- and cell-substrate-attaching molecules ( A), thereby causing a release of the cell from the monolayer. In the present work we try to understand the mechanism of this effect; whether, in order to trigger the P-->A mechanism, ouabain should block the pumping activity of Na(+),K(+)-ATPase as pump, or whether it would suffice that the drug occupies this enzyme as a receptor. We assay a series of drugs known to act on the pump, such as ouabain, digoxin, digitoxin, palytoxin, oligomycin, strophanthidin, neothyoside-A, proscillaridin-A, etc. We gauge their ability to block the pump by measuring the K(+) content in the cells, and their ability to detach the cells from the monolayer by determining the amount of protein remaining in the culturing well. None of the drugs tested was able to cause detachment without stopping the pump. Ouabain also enhances phosphorylation, yet pump inhibition and signal transduction do not seem to be intimately associated in a causal chain, but to occur simultaneously. To investigate the response of the site of cell attachment, we analyze the position of beta-catenin by fluorescence confocal microscopy, and find that this adherent junction-associated molecule is sent to the nucleus, where it is known to act as a transcriptional cofactor.
摘要。在之前的工作中，我们描述了一种“P→A机制”，该机制将哇巴因对泵（P）的占据转化为磷酸化变化、丝裂原活化蛋白激酶（MAPK）的激活以及细胞间和细胞与底物附着分子（A）的内吞作用，从而导致细胞从单层中释放出来。在本工作中，我们试图了解这种效应的机制；为了触发P→A机制，哇巴因是应该作为泵阻断Na⁺,K⁺-ATP酶的泵浦活性，还是该药物作为受体占据这种酶就足够了。我们检测了一系列已知作用于该泵的药物，如哇巴因、地高辛、洋地黄毒苷、岩沙海葵毒素、寡霉素、毒毛花苷元、新甲状腺素-A、海葱苷-A等。我们通过测量细胞中的K⁺含量来评估它们阻断泵的能力，并通过测定培养孔中剩余的蛋白量来评估它们使细胞从单层中脱离的能力。所测试的药物中没有一种能够在不停止泵的情况下导致细胞脱离。哇巴因还能增强磷酸化作用，然而泵抑制和信号转导似乎并非在因果链中紧密相关，而是同时发生。为了研究细胞附着位点的反应，我们通过荧光共聚焦显微镜分析β-连环蛋白的位置，发现这种与黏附连接相关的分子被转运到细胞核，在那里它作为转录辅因子发挥作用。
