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（由申请方提供）：腹泻和实验诱导的液体 分泌几乎总是与富含HCO 3的血浆样溶液有关。在 相反，在体外模型中很少观察到HCO 3分泌，但有优雅的 研究了25年的活性氯分泌机制。模型 结肠HCO 3分泌不足。我们已经建立了方法来执行 分离的大鼠和小鼠结肠隐窝的微灌注（分泌的位点 环腺苷酸依赖性和钙依赖性激动剂诱导HCO 3 分泌物cAMP刺激HCO 3分泌的研究揭示了 HCO 3分泌和活性Cl分泌之间的关系，提示顶端HCO 3 运动是通过阴离子通道，而不是C1-HCO 3交换，HCO 3不是 产生的内源性，但从细胞外来源和它的运动， 基底外侧膜（BLM）是Na-K-2Cl偶联的结果， 共转运（NKCCI）和Cl-HCO 3交换或Na-HCO 3共转运（NBC）。我们 建议在正常大鼠和CFTR敲除大鼠中进行隐窝微灌注研究， NKCC 1基因敲除小鼠（及其对照同窝仔），以建立机制 HCO 3的分泌用NKCC 1敲除小鼠进行的初步研究支持 NKCC 1和Cl-HCO 3交换的耦合作为BLM吸收的机制。贴片 钳研究将表征HCO 3电导的阴离子通道中， 隐窝顶膜。虽然Cl-穿过基底外侧膜的运动 （除了通过NKCCl）长期以来一直被怀疑，以前的研究已经 未能识别载体介导的过程，例如，Cl-阴离子交换使用 蛋白酶抑制剂制备BLM囊泡，我们最近已经确定了一个 Cl-HCO 3交换并计划其表征实验。我们相信AE 2 编码这种Cl-HCO 3交换，并克隆了一个全长cDNA，具有90 与胃AE 2的同源性百分比并提出其在HEK 293细胞中的表达。我们 也有证据表明远端结肠中的NBC（即，cNBC）与 可能代表一种新的上皮细胞NBC。我们提出 全面的运输、电生理和分子研究， 表征隐窝HCO 3分泌。