RECEPTOR ACTIVATED CHLORIDE SECRETION IN HT 29 CELLS

HT 29 细胞中受体激活的氯离子分泌

• 批准号: 3754614
• 负责人: GERDA BREITWIESER
• 金额: --
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3754614
• 关键词: active transport; antisense nucleic acid; biological signal transduction; chloride channels; chlorine; cystic fibrosis; ion transport; phorbols; protein biosynthesis; protein kinase; tissue /cell culture; voltage /patch clamp

Active chloride secretion is fundamental to normal fluid balance in the intestine, with pathological consequences resulting from either hypersecretion, as in diarrhea, or hyposecretion, as in cystic fibrosis. Defining the pathways involved in active Cl- secretion, and understanding the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in the process is a crucial step in the development of treatment regimes for a variety of secretory disorders. We have identified an outwardly rectifying Cl- conductance in a colon carcinoma-derived cell line, HT29-18- Cl, which is activated by both cAMP and Ca2+-mediated signal transduction pathways. We will utilize electrophysiological (transepithelial, whole cell and single channel patch clamp techniques), cell biological (fluorescence imaging of intracellular Ca2+ and Cl-), biochemical, and molecular biological (antisense oligodeoxynucleotides) approaches to address two aims. (1) We will determine whether the outwardly rectifying Cl- conductance of HT29-18-Cl cells is mediated by CFTR. We will characterize the outwardly rectifying Cl- conductance at the single channel level; we will treat cells with CFTR antisense oligodeoxynucleotides and determine whether there is a parallel decrease in CFTR protein levels and outwardly rectifying Cl- conductance; and we will use phorbol esters to down-regulate CFTR and determine whether this also eliminates the outwardly rectifying Cl- conductance. If we observe parallel decreases in both CFTR and the outwardly rectifying Cl- conductance, we will assume that CFTR is mediating the current, and we will then undertake a characterization of the signal transduction pathways controlling CFTR activation. If we determine that the levels of CFTR protein and outwardly rectifying Cl- conductance are not correlated, we will further characterize the properties of the outwardly rectifying Cl- conductance, since it represents an intestinal Cl- channel which is not affected in CF, and hence its pharmacological modulation may provide therapeutic benefits to CF patients. (2) We will define the signal transduction pathways for Ca2+ and cAMP-mediated activation of the outwardly rectifying Cl- conductance. At the monolayer, whole cell and single channel level, we will define second messengers, protein kinases and phosphatases involved in activation of the outwardly rectifying Cl- conductance.

活性氯的分泌是体内正常液体平衡的基础 肠道，由以下任一原因引起的病理后果 分泌过多，如腹泻，或分泌不足，如囊性纤维化。 定义参与活性 Cl- 分泌的途径，并理解 囊性纤维化跨膜电导调节器 (CFTR) 的作用 在此过程中是制定治疗方案的关键一步 用于多种分泌障碍。 我们已经确定了一个外在的 纠正结肠癌衍生细胞系 HT29-18- 中的 Cl- 电导 Cl，由 cAMP 和 Ca2+ 介导的信号转导激活 途径。 我们将利用电生理学（跨上皮、整体 细胞和单通道膜片钳技术），细胞生物学 （细胞内 Ca2+ 和 Cl- 的荧光成像）、生化和 分子生物学（反义寡脱氧核苷酸）方法 解决两个目标。 (1) 判断是否对外整改 HT29-18-Cl细胞的Cl-电导由CFTR介导。 我们将 表征单通道的外向整流 Cl- 电导 等级;我们将用 CFTR 反义寡脱氧核苷酸处理细胞 确定 CFTR 蛋白水平是否平行下降，以及 外向整流Cl-电导；我们将使用佛波酯 下调 CFTR 并确定这是否也消除了外部 校正 Cl- 电导。 如果我们观察到 CFTR 平行下降 和外向整流 Cl- 电导，我们假设 CFTR 为 调解电流，然后我们将进行表征 控制 CFTR 激活的信号转导途径。 如果我们确定 CFTR蛋白水平和外向整流Cl-电导 不相关，我们将进一步表征 向外整流 Cl- 电导，因为它代表肠道 Cl- CF 中不受影响的通道，因此其药理作用 调节可以为 CF 患者提供治疗益处。 (2) 我们将 定义 Ca2+ 和 cAMP 介导的信号转导途径 激活向外整流的 Cl-电导。 在单层， 全细胞和单通道水平，我们将定义第二信使， 参与外在激活的蛋白激酶和磷酸酶 校正 Cl- 电导。