O'Brien E T, Kinch M, Harding T W, Epstein D L
Department of Ophthalmology, Duke University Medical Center, Durham, NC 27710, USA.
Exp Eye Res. 1997 Oct;65(4):471-83. doi: 10.1006/exer.1997.0357.
Ethacrynic acid (ECA) increases aqueous humor outflow facility in human and animal model systems, and causes cellular retraction in cultured trabecular meshwork (TM) cells. ECA-induced retraction, a possible correlate to the opening of spaces in the outflow pathway in vivo, takes place coincident with disruption of cell-cell attachments and actin stress fibers. Tyrosine phosphorylated proteins are located predominantly where actin filaments terminate at sites of cell-to-cell and cell-to-substrate adhesion, and are understood to regulate cellular adhesions and filamentous (F) actin organization in many cell types. In the present study we investigated whether ECA might affect cell adhesions and F-actin in TM cells by altering levels of phosphotyrosine. We analysed levels of phosphotyrosine in cultured human TM and calf pulmonary artery endothelial cells after exposure to ECA. Using immunoflourescence microscopy and antibodies to phosphotyrosinated proteins we found a rapid decrease in phosphotyrosine levels at the focal contacts of cells treated with ECA. Immunoblots of whole cell extracts showed a decrease in phosphotyrosine predominantly in a band running at about 120 kD, with a more subtle decrease in a band about 65 kD. Reprobing the blot with antibodies to pp120 focal adhesion kinase (FAK) or paxillin indicated that the 120 kD band was FAK and the 65 kD band was likely paxillin. Immunoprecipitation of FAK or paxillin and probing the resulting blot with antibodies to phosphotyrosine confirmed that these proteins were rapidly dephosphorylated after ECA addition. Loss of FAK and paxillin proteins in cells was then confirmed using immunofluorescence microscopy. Dephosphorylation of these proteins was detected before the onset of retraction, stress fiber disruption, or complete disruption of focal adhesions. A pure microtubule inhibitor (colchicine), did not cause stress fiber disruption or decrease focal adhesion phosphorylation. We postulate that dephosphorylation of FAK and paxillin by ECA disrupts signaling pathways that normally maintain the stability of the actin cytoskeleton and cellular adhesions, and that this action leads both to cell shape change in culture, and to facility changes in vivo.
依他尼酸（ECA）可增加人体和动物模型系统中的房水流出率，并导致培养的小梁网（TM）细胞发生细胞回缩。ECA诱导的回缩可能与体内流出途径中空间的开放有关，它与细胞间连接和肌动蛋白应力纤维的破坏同时发生。酪氨酸磷酸化蛋白主要位于肌动蛋白丝在细胞间和细胞与底物粘附部位终止的地方，并且被认为在许多细胞类型中调节细胞粘附和丝状（F）肌动蛋白的组织。在本研究中，我们调查了ECA是否可能通过改变磷酸酪氨酸水平来影响TM细胞中的细胞粘附和F-肌动蛋白。我们分析了培养的人TM细胞和小牛肺动脉内皮细胞在暴露于ECA后的磷酸酪氨酸水平。使用免疫荧光显微镜和针对磷酸化蛋白的抗体，我们发现用ECA处理的细胞的粘着斑处磷酸酪氨酸水平迅速下降。全细胞提取物的免疫印迹显示，磷酸酪氨酸主要在约120 kD处的条带中减少，在约65 kD处的条带中减少更为细微。用针对pp120粘着斑激酶（FAK）或桩蛋白的抗体对印迹进行再检测表明，120 kD条带是FAK，65 kD条带可能是桩蛋白。对FAK或桩蛋白进行免疫沉淀，并用针对磷酸酪氨酸的抗体检测所得印迹，证实这些蛋白在添加ECA后迅速去磷酸化。然后使用免疫荧光显微镜确认细胞中FAK和桩蛋白的丢失。在回缩、应力纤维破坏或粘着斑完全破坏之前就检测到了这些蛋白的去磷酸化。一种纯微管抑制剂（秋水仙碱）不会导致应力纤维破坏或降低粘着斑磷酸化。我们推测，ECA使FAK和桩蛋白去磷酸化会破坏通常维持肌动蛋白细胞骨架稳定性和细胞粘附的信号通路，并且这种作用导致培养中的细胞形状改变以及体内流出率改变。
