RECEPTOR ACTIVATED CHLORIDE SECRETION IN HT 29 CELLS

活跃的氯化物分泌是正常体液平衡的基础， 肠，病理后果由以下任一原因引起： 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，并了解 囊性纤维化跨膜传导调节因子（CFTR） 是制定治疗方案的关键一步 治疗各种分泌失调 我们发现了一个外表上 在结肠癌衍生细胞系HT 29 -18中纠正Cl-传导， Cl，其由cAMP和Ca 2+介导的信号转导激活 途径。 我们将利用电生理（经上皮，整个 细胞和单通道膜片钳技术），细胞生物学 （细胞内Ca 2+和Cl-的荧光成像），生物化学，和 分子生物学（反义寡核苷酸）方法， 实现两个目标。 (1)我们将决定是否在表面上纠正 HT 29 -18-C1细胞的Cl-传导由CFTR介导。 我们将 表征单通道处的向外整流Cl-电导 水平;我们将用CFTR反义寡脱氧核苷酸处理细胞， 确定CFTR蛋白水平是否平行降低， 向外整流氯电导;我们将使用佛波醇酯， 下调CFTR并确定这是否也消除了 整流Cl-电导。 如果我们观察到两个CFTR平行下降， 和向外整流Cl-电导，我们将假设CFTR是 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蛋白和外向整流氯离子传导的水平 是不相关的，我们将进一步表征的性质， 向外整流Cl-传导，因为它代表肠Cl- 在CF中不受影响的通道，因此其药理学 调节可以为CF患者提供治疗益处。 (2)我们将 确定Ca 2+和cAMP介导的信号转导途径 激活向外整流的Cl-传导。 在单层， 整个细胞和单通道水平，我们将定义第二信使， 蛋白激酶和磷酸酶参与激活的外向 整流Cl-电导。

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.