INTESTINAL SECRETION & INFLAMMATION - IMPACT OF AMMONIA

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+，通常高浓度存在于结肠腔中）可能是一种新的内源性C1-分泌调节剂，通过对K+通道的影响，并开始定义NH4+与基底外侧膜K+转运体的相互作用，这也是cl分泌所必需的。基于已经完成的工作，目前的应用考虑了改变的K+通道调节如何影响各种肠道疾病状态。初步数据表明，氨衍生的氧化剂单氯胺（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）可以增加Ca+2依赖性的K+通道，随着DHA开始作为CF治疗的临床评估，这一发现特别有趣。初步发现表明，肌动蛋白细胞骨架可以在功能上改变Ca2+依赖性的K+通道，相反，这些K+通道可以调节细胞功能，如涉及肌动蛋白重塑的上皮修复。提出了三套研究方案。首先，氨对结肠上皮运输的影响将在培养的上皮细胞和人类结肠粘膜制剂中进一步表征，重点关注NH4+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。其次，将使用培养的上皮细胞作为模型系统，探索cAMP和NH2Cl对基底侧Ca+2依赖性K+通道的增强作用，目的是确定这些看似不同的刺激对K+通道增强作用的共同机制。将探讨基底外侧K +通道治疗性调节的潜力，特别是检查DHA是否可以增加T84细胞和人结肠中Ca2+依赖性Cl-分泌，如果是这样，确定其作用机制。最后，这些研究将确定F-actin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，并加强了它们作为新药设计靶点的潜力。

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.