INTESTINAL SECRETARY MECHANISMS

肠道秘书机制

• 批准号: 6107604
• 负责人: Kim Elaine Barrett
• 金额: $ 5.51万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6107604
• 关键词: calcium flux; calmodulin dependent protein kinase; cell cell interaction; chloride channels; chlorine; clone cells; cyclic AMP; diarrhea; gastrointestinal epithelium; image processing; inositol phosphates; intracellular transport; ion transport; phosphorylation; second messengers; tissue /cell culture; voltage /patch clamp

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.

该提案中描述的调查的长期目标是 定义负责控制的精确细胞内机制 通过肠道的氯离子的主动跨传输 上皮。这种运输机制是 水进入肠腔，因此其过度 表达是分泌性腹泻的主要基础。研究 在当前资金期间完成了两者 调节的正面和负面的细胞内信号或使者 对激素的氯化物分泌的总体水平 增加细胞内细胞质钙的水平。现在建议 检查这些信号的确切性质，如何生成 在细胞内，以及它们发挥积极的机制或 负面影响。将测试两个具体的假设。首先，那 肌醇（3,4,5,6）四磷酸（INSP4）是一个负信号 通过钙动员。 磷脂酶C-Gamma的酪氨酸磷酸化，该分子 可能与基底外侧钾通道和/或氯化根顶相互作用 限制分泌的频道。其次，循环放大器（营地）可以增强 卡尔巴乔尔通过修改推定的生产的分泌作用 负信号，例如细胞外钙的涌入 环境和/或INSP4的合成。一般策略将 被用来解决这些假设将是与生化相关的 和生理参数，在良好的细胞培养模型中 肠上皮，T84细胞系。要使用的技术 包括使用室，同位素通量和离子的斑块夹具研究 运输，二介醇和酪氨酸的生化分析 细胞内钙的磷酸化和荧光成像 浓度。研究的意义在于它们的潜力 提高我们对细胞异常的理解 分泌性腹泻。最终，改善了这种异常的知识 可能期望提出更好的治疗方法 健康）状况。