INTESTINAL SECRETION & INFLAMMATION - IMPACT OF AMMONIA

肠道分泌

• 批准号: 6196979
• 负责人: JEFFREY B. MATTHEWS
• 金额: $ 34.8万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 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 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6196979
• 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，都以上皮 C1 分泌失调为特征。该项目最初发现铵离子（NH4+，通常在结肠腔中以高浓度存在）可能是一种新型的内源性 C1- 分泌调节剂，通过对 K+ 通道的影响，并开始定义 NH4+ 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，当前的申请考虑了 K+ 通道调节的改变如何影响各种肠道疾病状态。初步数据表明，氨源氧化剂一氯胺 (NH2Cl) 可能通过增强 Ca2+ 依赖性 K+ 通道而导致结肠炎腹泻。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 疗法进行临床评估。初步研究结果表明，肌动蛋白细胞骨架在功能上改变 Ca2+ 依赖性 K+ 通道，相反，这些 K+ 通道可以调节细胞功能，例如涉及肌动蛋白重塑的上皮恢复。提出了三组研究。首先，将在培养的上皮细胞和人结肠粘膜制剂中进一步表征氨对结肠上皮转运的影响，重点关注 NH4+ 与基底外侧 Na+-k+-2Cl- 协同转运蛋白、Na+_K+ ATP 酶和 K+ 通道的相互作用。其次，将使用培养的上皮细胞作为模型系统来探索 cAMP 和 NH2Cl 对基底外侧 Ca+2 依赖性 K+ 通道的增强作用，目的是通过这些看似不同的刺激来定义 K+ 通道增强的共同机制。将探索基底外侧 K + 通道治疗调节的潜力，特别检查二十二碳六烯酸 (DHA) 是否可以增强 T84 细胞和人结肠中 Ca2+ 依赖性 Cl- 分泌，如果可以，则确定其作用机制。最后，这些研究将定义 F-肌动蛋白的化学操作对 Ca2+ 依赖性 K+ 通道调节的影响，并扩展初步发现，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，并增强了它们作为新药物设计目标的潜力。