Colonic Crypt HCO3 Secretion

结肠隐窝 HCO3 分泌

• 批准号: 6623648
• 负责人: HENRY J BINDER
• 金额: $ 35.47万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-15 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6623648
• 关键词: acidity /alkalinity; apical membrane; basolateral membrane; bicarbonates; calcium ion; cell line; chloride channels; colon; cyclic AMP; cyclic GMP; gastrointestinal absorption /transport; gene targeting; genetically modified animals; intestinal villi; ion transport; laboratory mouse; laboratory rat; membrane channels; membrane transport proteins; protease inhibitor; protein isoforms; secretion; voltage /patch clamp

DESCRIPTION (provided by applicant): Diarrhea and experimentally-induced fluid secretion is almost always associated with HCO3-rich, plasma-like solution. In contrast, in vitro models rarely observe HCO3 secretion but have elegantly dissected the mechanisms of active Cl secretion for twenty-five years. A model of colonic HCO3 secretion is lacking. We have established methods to perform microperfusion of isolated rat and mice colonic crypts (the site for secretory processes) in which cyclic AMP-dependent and Ca-dependent agonists induce HCO3 secretion. Studies of cAMP-stimulated HCO3 secretion reveal a close association between HCO3 secretion and active Cl secretion with suggestion that apical HCO3 movement is via an anion channel, not C1-HCO3 exchange and that HCO3 is not generated endogenously, but from extracellular sources and its movement across the basolateral membrane (BLM) is a result of either the coupling of Na-K-2Cl co-transport (NKCCI) and Cl-HCO3 exchange or Na-HCO3 co-transport (NBC). We propose crypt microperfusion studies in normal rats and in CFTR knockout and NKCC1 knockout mice (and their control littermates) to establish the mechanism of HCO3 secretion. Initial studies with NKCCl knockout mice support the coupling of NKCC1 and Cl-HCO3 exchange as the mechanism of BLM uptake. Patch clamp studies will characterize the HCO3 conductance of anion channels in the crypt apical membrane. Although Cl movement across the basolateral membrane (other than that via NKCCl) has long been suspected, previous studies have failed to identify a carrier-mediated process, e.g., Cl-anion exchange. Using protease inhibitors to prepare BLM vesicles, we have recently identified a Cl-HCO3 exchange and plan experiments for its characterization. We believe AE2 encodes this Cl-HCO3 exchange and have cloned a full-length cDNA with 90 percent homology to gastric AE2 and propose its expression in HEK293 cells. We also have evidence that NBC in distal colon (i.e., cNBC) differs substantially from other NBCs and may represent a novel epithelial cell NBC. We propose comprehensive transport, electrophysiological and molecular studies to characterize crypt HCO3 secretion.

描述（由申请人提供）：腹泻和实验引起的液体 分泌几乎总是与富含 HCO3 的血浆样溶液相关。在 相比之下，体外模型很少观察到 HCO3 的分泌，但已经优雅地观察到 剖析了活性氯离子分泌机制二十五年。一个模型 结肠 HCO3 分泌不足。我们已经制定了执行方法 离体大鼠和小鼠结肠隐窝（分泌部位）的微灌注 过程），其中环 AMP 依赖性和 Ca 依赖性激动剂诱导 HCO3 分泌。对 cAMP 刺激的 HCO3 分泌的研究揭示了密切的关联 HCO3 分泌和活性 Cl 分泌之间的关系，表明顶端 HCO3 运动是通过阴离子通道进行的，而不是 C1-HCO3 交换，并且 HCO3 不是 内源性产生，但来自细胞外来源及其跨细胞运动 基底外侧膜 (BLM) 是 Na-K-2Cl 偶联的结果 共转运 (NKCCI) 和 Cl-HCO3 交换或 Na-HCO3 共转运 (NBC)。我们 提出在正常大鼠和 CFTR 敲除中进行隐窝微灌注研究 NKCC1 敲除小鼠（及其对照同窝小鼠）建立机制 HCO3 的分泌。 NKCCl 敲除小鼠的初步研究支持 NKCC1 和 Cl-HCO3 交换的耦合作为 BLM 摄取的机制。修补 钳位研究将表征阴离子通道的 HCO3 电导 隐窝顶膜。尽管 Cl 穿过基底外侧膜 （除了通过 NKCCl 之外）长期以来一直被怀疑，之前的研究已经 未能识别载体介导的过程，例如 Cl-阴离子交换。使用 蛋白酶抑制剂来制备 BLM 囊泡，我们最近发现了一种 Cl-HCO3 交换并计划实验以表征其特性。我们相信AE2 编码此 Cl-HCO3 交换，并克隆了全长 cDNA，具有 90 与胃 AE2 的同源性百分比，并提出其在 HEK293 细胞中的表达。我们 也有证据表明远端结肠中的 NBC（即 CNBC）存在显着差异 与其他 NBC 不同，可能代表一种新型上皮细胞 NBC。我们建议 综合运输、电生理学和分子研究 表征隐窝 HCO3 分泌。