INTESTINAL SECRETION & INFLAMMATION - IMPACT OF AMMONIA

肠道分泌

• 批准号: 6381296
• 负责人: JEFFREY B. MATTHEWS
• 金额: $ 30.6万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6381296
• 关键词: actins; adenosinetriphosphatase; ammonia; ammonium chloride; calcium; chloride channels; chlorine; colitis; colon; cyclic AMP; cystic fibrosis; electrophysiology; gastrointestinal epithelium; human tissue; intestines; ion transport; membrane lipids; membrane transport proteins; omega 3 fatty acid; potassium; potassium channel; secretion; sodium; tissue /cell culture; voltage /patch clamp

Numerous diseases affecting the GI tract, ranging from secretory diarrhea to cystic fibrosis, are characterized by dysregulation of epithelial C1- secretion. This project originally identified that ammonium ion (NH4+, normally present at high concentrations in the colonic lumen) may be a novel endogenous regulator of C1- secretion via effects via effects on K+ channels and begins to define the interaction of NH4+ with the basolateral membrane K+ transporters also required for Cl-secretion. Based on work already accomplished, the current application considers how altered K+ channel regulation may influence various intestinal disease states. Preliminary data indicate that the ammonia-derived oxidant monochloramine (NH2Cl) may contribute to the diarrhea of colitis by potentiating Ca2+- dependent K+ channels. Experiments also suggest that docosahexaenoic acid (DHA, a component of fish oil) can augment Ca+2- dependent K+ channels, finding of particular interest as DHA begins clinical evaluation as therapy in CF. Preliminary findings suggest that the actin cytoskeleton an functionally alter Ca2+-dependent K+ channels, and conversely, that these K+ channels can modulate cell functions such as epithelial restitution that involve actin remodeling. Three sets of studies are proposed. First, the impact of ammonia on colonic epithelial transport will be further characterized in cultured epithelial ells and in human colonic mucosal preparations, with attention to the interaction of NH4+ with the basolateral Na+-k+-2Cl- co- transporter, Na+_K+ ATPase, and K+ channels. Second, potentiation of basolateral Ca+2-dependent K+ channels by cAMP and NH2Cl will be explored using cultured epithelial cells as model systems with the goal of defining a common mechanism for K+ channel potentiation by these seemingly diverse stimuli. The potential for therapeutic modulation of basolateral K + channels will be explored, specifically examining wheth4r docosahexaenoic acid (DHA) can augment Ca2+-dependent Cl- secretion in T84 cells and human colon, and, if so, to determine its mechanism of action. Finally, the studies will define the effect of chemical manipulation of F-actin on Ca2+-dependent K+ channel regulation and extend preliminary findings suggesting that K+ channel regulation affects the actin-regulated process of epithelial restitution. These studies highlight the importance of basolateral K+ channels in the regulation of secretion and other epithelial functions and reinforce their potential as targets for new drug design.

许多影响胃肠道的疾病，从分泌性腹泻到囊性纤维化，其特征在于上皮细胞C1分泌失调。该项目最初确定铵离子（NH 4+，通常以高浓度存在于结肠腔中）可能是通过对K+通道的作用而影响Cl-分泌的新型内源性调节剂，并开始定义NH 4+与Cl-分泌所需的基底外侧膜K+转运蛋白的相互作用。基于已经完成的工作，本申请考虑了改变的K+通道调节如何影响各种肠道疾病状态。初步数据表明，氨衍生的氧化剂一氯胺（NH 2Cl）可能有助于结肠炎的腹泻，通过增强Ca 2+依赖性K+通道。实验还表明，二十二碳六烯酸（DHA，鱼油的一种成分）可以增强Ca+2依赖性K+通道，这一发现特别令人感兴趣，因为DHA开始作为CF治疗的临床评估。初步研究结果表明，肌动蛋白细胞骨架的功能改变Ca 2+依赖的K+通道，相反，这些K+通道可以调节细胞功能，如上皮细胞的恢复，涉及肌动蛋白重塑。提出了三组研究。首先，氨对结肠上皮转运的影响将在培养的上皮细胞和人结肠粘膜制备物中进一步表征，关注NH 4+与基底外侧Na+-K+-2Cl-共转运体、Na+_K+ ATP酶和K+通道的相互作用。第二，增强的基底外侧Ca+2依赖性K+通道cAMP和NH 2Cl将探讨培养的上皮细胞作为模型系统的目标，定义一个共同的机制K+通道增强这些看似不同的刺激。将探索基底外侧K +通道的治疗调节潜力，特别是检查二十二碳六烯酸（DHA）是否可以增加T84细胞和人结肠中的Ca 2+依赖性Cl-分泌，如果可以，确定其作用机制。最后，这些研究将确定化学操作F-肌动蛋白对Ca 2+依赖的K+通道调节的影响，并扩展初步研究结果，表明K+通道调节影响肌动蛋白调节的上皮恢复过程。这些研究强调了基底外侧K+通道在调节分泌和其他上皮功能中的重要性，并加强了其作为新药设计靶点的潜力。