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，都以上皮细胞分泌C1的失调为特征。该项目最初确定了氨离子(NH4+，通常存在于结肠腔中的高浓度)可能是一种通过对K+通道的作用而影响C1-分泌的新型内源性调节因子，并开始确定NH4+与Cl-分泌所需的基侧膜K+转运体之间的相互作用。基于已经完成的工作，目前的应用考虑K+通道调节改变如何影响各种肠道疾病状态。初步数据表明，氨源性氧化剂一氯胺可能通过增强钙依赖的K+通道而导致结肠炎的腹泻。实验还表明，二十二碳六烯酸(DHA，鱼油的一种成分)可以增强钙依赖的K+通道，这一发现特别令人感兴趣，因为DHA开始作为治疗CF的临床评估。初步研究结果表明，肌动蛋白细胞骨架在功能上改变了钙依赖的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。本文提出了三套研究方案。首先，将在培养的上皮细胞和人的结肠粘膜标本中进一步研究氨对结肠上皮运输的影响，并关注NH4+与基侧Na+-K+-2Cl-共转运体、Na+_K+ATPase和K+通道的相互作用。其次，将以培养的上皮细胞为模型系统，探讨cAMP和NH_2Cl对基底外侧钙依赖的K~+通道的增强作用，目的是确定这些看似不同的刺激物对K~+通道增强的共同机制。将探索治疗调节基底外侧K+通道的可能性，特别是检测4r二十二碳六烯酸(DHA)是否能增加T84细胞和人结肠的钙依赖的Cl-分泌，如果是的话，确定其作用机制。最后，这些研究将明确F-肌动蛋白的化学操作对钙依赖的K+通道调节的影响，并扩展初步的发现，即K+通道调节影响肌动蛋白调节的上皮修复过程。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.