INTESTINAL SECRETARY MECHANISMS

本提案所述调查的长期目标是 定义负责控制的精确细胞内机制， 肠内氯离子的主动跨细胞转运 上皮这种运输机制是主要的驱动力， 水进入肠腔的运动，因此，它的过度- 表达是分泌性腹泻的主要基础。研究 在本供资期内完成的工作确定了 细胞内的正负信号，或信使， 氯化物分泌的总体水平响应激素， 增加细胞内钙水平。现提议 研究这些信号的确切性质，它们是如何产生的 在细胞内，以及它们发挥其积极或 负面影响。将检验两个具体假设。第一个 肌醇（3，4，5，6）四磷酸（InsP4）是一个负信号， 动员激动剂，如卡巴胆碱，通过 磷脂酶C-γ的酪氨酸磷酸化， 可能与基底外侧钾通道和/或顶端氯离子相互作用 限制分泌的通道。第二，环磷酸腺苷（cAMP）可以增强 卡巴胆碱的分泌作用，通过修改生产的推定 负信号，如钙离子从细胞外 环境和/或InsP4的合成。总体战略将 解决这些假设的方法将是将生物化学 和生理参数，在一个良好表征的细胞培养模型， 肠上皮细胞T84细胞系拟采用的技术 包括Ussing室、同位素通量和离子膜片钳研究 肌醇磷酸盐和酪氨酸的转运、生化分析 磷酸化和细胞内钙的荧光成像 浓度的这些研究的意义在于， 增加我们对细胞异常的理解， 分泌性腹泻最终，提高对这些异常的认识 可能会提出更好的治疗方法， 条件

The long-term goal of the investigations described in this proposal is to define the precise intracellular mechanisms responsible for the control of active transcellular transport of chloride ions by the intestinal epithelium. This transport mechanism is the major driving force for the movement of water into the intestinal lumen and, accordingly, its over- expression is the primary basis for secretory diarrhea. Studies accomplished during the current funding period have identified both positive and negative intracellular signals, or messengers, that regulate the overall level of chloride secretion in response to hormones that increase intracellular levels of cytoplasmic calcium. It is now proposed to examine the exact nature of these signals, how they are generated within the cell, and the mechanisms whereby they exert their positive or negative effects. Two specific hypotheses will be tested. First, that inositol (3,4,5,6) tetrakisphosphate (InsP4) is a negative signal that is mobilized by calcium.mobilizing agonists such as carbachol, via the tyrosine phosphorylation of phospholipase C-gamma, and that this molecule may interact with basolateral potassium channels and/or apical chloride channels to limit secretion. Second, that cyclic AMP (cAMP) can potentiate the secretory effects of carbachol by modifying the production of putative negative signals, such as the influx of calcium from the extracellular environment and/or the synthesis of InsP4. The general strategy that will be employed to address these hypotheses will be to correlate biochemical and physiological parameters in a well-characterized cell culture model of the intestinal epithelium, the T84 cell line. Techniques to be employed include Ussing chamber, isotope flux and patch clamp studies of ion transport, biochemical analyses of inositolphosphates and tyrosine phosphorylation, and fluorescence imaging of intracellular calcium concentrations. The significance of the studies lies in their potential to increase our understanding of the cellular abnormalities underlying secretory diarrhea. Ultimately, improved knowledge of such abnormalities might be expected to suggest better therapeutic approaches to this condition.