CFTR AND PH IN TGN PROCESSING AND PSEUDOMONAS BINDING

TGN 处理和假单胞菌结合中的 CFTR 和 PH

• 批准号: 6153603
• 负责人: TERRY E MACHEN
• 金额: $ 3.82万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-30 至 2000-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6153603
• 关键词: Golgi apparatus; Pseudomonas aeruginosa; acid base balance; chloride channels; cystic fibrosis; host organism interaction; human tissue; respiratory epithelium; tissue /cell culture

DESCRIPTION (Taken directly from the application) It is unknown how in Cystic Fibrosis (CF) a "simple" reduction of Cl permeability could account for preferential binding and colonization by Pseudomonas aeruginosa in CF lungs. Two hypotheses, not mutually exclusive, that could account for the pathologies are based on the fact that the CFTR Cl channel could exert its effect through alterations of pH of organelles or at the plasma membrane. The organelle pH hypothesis is that pH of trans-Golgi network (pHTGN ) and other organelles is altered due to altered Cl and HCO3 permeability of organelle membranes. Changes in pHTGN in turn lead to altered sialyltransferase activity, reduced sialylation of GM1 and increased binding of Pseudomonas aeruginosa to asialo-GM1. Alternatively, the cytoplasmic/airway pH hypothesis states that cytoplasmic pH (pHC) is elevated while epithelial apical pH (pH apical) is reduced due to reduced Cl and HCO3 exit from the cell. Elevated cytosolic pH (pHC) increases rate of vesicular transport out of Golgi/TGN, which reduces the residence time of GM1 in the secretory pathway and hence time for sialylation reactions. In addition, airway fluid will be more acidic, which may influence Pseudomonas colonization. Using molecular targeting, digital imaging microscopy and biochemical analyses, we will test the role of CFTR in control of pH apical, pHC, pHTGN, rates of TGN vesicle traffic, GM1 sialylation and Pseudomonas binding of airway epithelial cells. We will: 1. Determine role of CFTR in controlling apical fluid pH. The cytoplasmic/airway pH hypothesis predicts pH apical should be reduced in CF cells and in normal cells when CFTR is inhibited. 2. Determine role of CFTR in control of pHC (digital imaging of cytosolic, pH-sensitive dye) and pHTGN (digital imaging of pH-sensitive dyes targeted solely to the TGN) in control and in untreated and CFTR-rescued CF airway cells. The organelle pH hypothesis predicts that pHTGN of CF cells is greater than the pHTGN of CFTR-rescued cells. 3. Determine role of CFTR, pHC and pHTGN in TGN vesicular traffic, GM1 sialylation and Pseudomonas binding in untreated and CFTR-rescued CF airway cells. According to the cytoplasmic pH hypothesis, export from the TGN should be accelerated in CF compared to control or CFTR-rescued cells, and the CF phenotype should be reversed by slowing transport out of the TGN using inhibitors or by lowering pHC. The cytoplasmic/airway hypothesis also predicts that alterations of pH airway or pHC should predictably control GM1 sialylation and Pseudomonas binding. The organelle pH hypothesis predicts that sialylation of GM1 is impaired in CF and that Pseudomonas binding should be altered when pHTGN is altered.

描述(直接从应用程序中获取) 目前尚不清楚囊性纤维化(CF)中氯的“简单”还原是如何发生的 渗透性可以解释优先结合和殖民的原因 CF肺感染铜绿假单胞菌两个不相互排斥的假设， 这可以解释这些病理是基于这样一个事实，即CFTR CL通道的作用可能是通过改变细胞器的pH或 在质膜上。细胞器的pH假说是 反式高尔基体网络(PHTGN)和其他细胞器因改变而改变 细胞器膜的CL和HCO3通透性。PHTGN依次发生变化 导致唾液酸转移酶活性改变，减少GM1唾液酸化和 铜绿假单胞菌与asialo-GM1的结合增加。或者， 细胞质/呼吸道pH假说指出，细胞质pH(PHC)是 上皮尖pH升高，而上皮尖pH(pH尖端)由于氯降低而降低 并且HCO3从细胞中退出。升高的胞浆pH(PHC)增加了 囊泡转运出高尔基体/TGN，减少了滞留时间 GM1在分泌途径中，因此唾液酸化反应的时间。在……里面 此外，呼吸道液体会更酸性，这可能会影响假单胞菌 殖民主义。使用分子靶向、数字成像显微镜和 生化分析，我们将测试CFTR在根尖pH控制中的作用， PHC、pHTGN、TGN囊泡流量、GM1唾液酸化和假单胞菌 呼吸道上皮细胞的结合。我们会： 1.确定CFTR在控制根尖周液pH中的作用。这个 细胞质/气道pH假说预测CF应降低尖端pH 当CFTR被抑制时，在正常细胞中也是如此。2.确定角色 控制PHC(胞浆、pH敏感染料的数字成像)和 PHTGN(仅针对TGN的pH敏感染料的数字成像) 对照组和未经处理和CFTR挽救的CF呼吸道细胞中。细胞器的pH值 假设预测CF细胞的pHTGN大于pHTGN Cftr-被拯救的细胞。3.确定CFTR、PHC和pHTGN在TGN中的作用 未处理和未治疗患者的囊泡交通、GM1唾液酸化和假单胞菌结合 CFTR-挽救的CF呼吸道细胞。根据细胞质pH假说， 在CF中，从TGN导出的速度应比控制或 CFTR-挽救的细胞，应通过放慢速度逆转CF型 使用抑制剂或通过降低PHC将其转运出TGN。这个 细胞质/气道假说还预测，pH、气道或 PHC应可预测地控制GM1唾液酸化和假单胞菌结合。这个 细胞器pH假说预测CFGM1唾液酸化受损 当pHTGN改变时，假单胞菌的结合也应该改变。