Regulation of Secretory Ion Channels in Colonic Crypts

描述(由申请人提供)：本项目的中心目标是确定调节哺乳动物结肠上皮中活性Cl-和K+分泌的Cl-和K+通道的机制，因为这些通道是刺激这些不同的离子分泌模式的关键因素；粘膜分泌反应是激活和抑制途径之间的平衡。分泌性腹泻可由隐窝上皮细胞的病理生理刺激引起，隐窝上皮细胞在细胞内cAMP增加时主动分泌Cl-和K+。炎症性肠病和囊性纤维化等情况也会通过对这些分泌上皮细胞的作用而导致离子和水运输的改变。尤其是对抑制物通路的不适当抑制，可能是溃疡性结肠炎患者分泌性腹泻的一个来源。工作模型是，结肠隐窝上皮的柱状细胞对Cl-和K+的分泌过程活跃，因此这些细胞是许多胃肠道疾病的功能障碍中心。使用膜片钳电生理技术记录膜离子电流，检测参与Cl-和K+分泌的离子通道。来自隔离隐窝的记录允许研究顶端和基底外侧膜通道以及控制离子通道活动的第二信使。荧光探针将被用来监测细胞内钙离子和pH作为控制离子通道的第二信使的参与。其具体目的是确定参与液体分泌的基底膜K+通道、氯离子基侧膜通道和顶膜K+和Cl-通道的调节机制。长期目标是确定生理刺激过程中对肠上皮细胞多种分泌功能的调节机制。这些不同的离子通道控制模式是分泌性腹泻、溃疡性结肠炎和囊性纤维化等分泌功能过度和受损的基础。前列腺素刺激分泌和炎症反应之间的密切关系为开发针对所涉及的前列腺素受体亚型的不同药物提供了机会。支持每种分泌模式的离子通道类型的差异也可以被用来开发进一步的药物，用于选择性的临床干预，而不会产生有害的副作用。因此，该项目将导致对结肠液分泌的调节控制的更深入的了解，并可能为具有危及生命的分泌活动的患者进行治疗干预。

DESCRIPTION (provided by applicant): The central objective of this project is to determine the mechanisms regulating Cl- and K+ channels involved in active Cl- and K+ secretion across the mammalian colonic epithelium, since these channels are key elements in stimulating these distinct modes of ion secretion; Mucosal secretory responses are a balance between activator and repressor pathways. Secretory diarrhea can result from a pathophysiologic stimulation of crypt epithelial cells, which actively secrete Cl- and K+ in response to increased intracellular cAMP. Conditions such as inflammatory bowel disease and cystic fibrosis also lead to altered ion and water transport via actions at these secretory epithelial cells. Inappropriate inhibitions of repressor pathways, in particular, are a likely source of secretory diarrhea for patients with ulcerative colitis. The working model is that active secretory processes for Cl- and K+ occur in columnar cells of the colonic crypt epithelium, such that these cells are a locus of dysfunction in many gastrointestinal diseases. Ion channels involved in Cl- and K+ secretion will be examined using patch-clamp electrophysiologic techniques to record membrane ionic currents. Recordings from isolated crypts allow study of both apical and basolateral membrane channels as well as the second messengers controlling ion channel activity. Fluorescent probes will be used to monitor involvement of cell Ca++ and pH as second messengers controlling ion channels. The specific aims are to determine regulatory mechanisms for the basolateral membrane K+ channels, the Cl- basolateral membrane channels and the apical membrane K+ and Cl- channels involved in fluid secretion. The long-term objective is to determine the regulatory mechanisms acting on the multiple secretory functions of intestinal epithelial cells during physiologic stimulation. These distinct modes of ion channel control are the basis for excessive and impaired secretory functions that occur in secretory diarrhea, ulcerative colitis and cystic fibrosis. The close relationship between prostaglandin stimulation of secretion and inflammatory responses provides an opportunity to develop pharmaceutical agents that differentially target the prostanoid receptor subtypes involved. Differences in the ion channel types supporting each mode of secretion also can be exploited to develop further agents for selective clinical intervention, without producing deleterious side effects. Thus, this project will lead to greater insight into the regulatory control of colonic fluid secretion and likely to therapeutic interventions for patients with life-threatening secretory activity.