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: 2770602
• 负责人: TERRY E MACHEN
• 金额: $ 16.19万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-30 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2770602
• 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（直接取自应用程序） 目前尚不清楚在囊性纤维化（CF）中， 渗透性可以解释优先结合和殖民化 CF肺中的铜绿假单胞菌。 两个假设，并不相互排斥， 可以解释病理的事实是基于CFTR Cl-通道通过改变细胞器pH值发挥作用， 在质膜上。 细胞器pH假说认为， trans-Golgi网络（pHTGN）和其他细胞器由于改变而改变。 细胞器膜的Cl和HCO 3渗透性。 pHTGN的变化依次为 导致唾液酸转移酶活性改变、GM 1的唾液酸化减少， 铜绿假单胞菌与去唾液酸GM 1的结合增加。 可选择地， 细胞质/气道pH假说指出，细胞质pH（pHC） 升高，而上皮顶端pH值（顶端pH值）由于Cl减少而降低 和HCO 3离开细胞。 升高的胞质pH值（pHC）增加 囊泡运输出高尔基体/TGN，这减少了 分泌途径中的GM 1，因此唾液酸化反应的时间。 在 此外，气道液体将更酸性，这可能会影响假单胞菌 殖民化 使用分子靶向，数字成像显微镜和 通过生物化学分析，我们将测试CFTR在控制顶端pH中的作用， pHC、pHTGN、TGN囊泡运输速率、GM 1唾液酸化和假单胞菌 气道上皮细胞的结合。 我们将： 1. 确定CFTR在控制根尖液pH中的作用。 细胞质/气道pH假设预测CF中顶端pH应降低 当CFTR被抑制时，在细胞和正常细胞中。 2. 确定角色 CFTR控制pHC（细胞溶质pH敏感染料的数字成像）， pHTGN（仅针对TGN的pH敏感染料的数字成像）， 对照组和未处理的和CFTR-拯救的CF气道细胞。 细胞器pH 假设预测CF细胞的pHTGN大于 CFTR拯救的细胞 3. 确定CFTR、pHC和pHTGN在TGN中的作用 囊泡交通，GM 1唾液酸化和假单胞菌结合， CFTR拯救的CF气道细胞。 根据细胞质pH假说， 与对照组相比，在CF中应加速从TGN的出口，或 CFTR拯救的细胞，CF表型应该通过减缓 使用抑制剂或通过降低pHC将其转运出TGN。 的 细胞质/气道假说还预测气道pH的改变， pHC应可预测地控制GM 1唾液酸化和假单胞菌结合。 的 细胞器pH假说预测CF中GM 1的唾液酸化受损 并且当pHTGN改变时，假单胞菌的结合也应该改变。