Regulation of Secretory Ion Channels in Colonic Crypts

描述（由申请人提供）：该项目的中心目标是确定调节Cl-和K+通道的机制，这些通道参与哺乳动物结肠上皮中Cl-和K+的活性分泌，因为这些通道是刺激这些不同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增加的情况下积极分泌Cl-和K+。炎症性肠病和囊性纤维化等疾病也会通过这些分泌性上皮细胞的作用导致离子和水运输的改变。特别是抑制通路的不适当抑制，是溃疡性结肠炎患者分泌性腹泻的可能来源。工作模型是Cl-和K+的活跃分泌过程发生在结肠隐窝上皮的柱状细胞中，因此这些细胞是许多胃肠道疾病的功能障碍位点。参与Cl-和K+分泌的离子通道将使用膜片钳电生理技术来记录膜离子电流。来自孤立隐窝的记录允许研究根尖和基底外侧膜通道以及控制离子通道活性的第二信使。荧光探针将用于监测细胞钙离子和pH作为控制离子通道的第二信使的参与。具体目的是确定基底外侧膜K+通道、基底外侧膜Cl-通道和根尖膜K+和Cl-通道参与液体分泌的调控机制。长期目标是确定在生理刺激下肠上皮细胞多种分泌功能的调控机制。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中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前列腺素受体亚型的药物提供了机会。支持每种分泌模式的离子通道类型的差异也可以用于开发选择性临床干预的进一步药物，而不会产生有害的副作用。因此，该项目将导致更深入了解结肠液体分泌的调节控制，并可能对危及生命的分泌活动患者进行治疗干预。

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.