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.
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