Colonic Crypt HCO3 Secretion

结肠隐窝 HCO3 分泌

基本信息

批准号：
7060943
负责人：
HENRY J BINDER
金额：
$ 37.85万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-05-15 至 2008-06-30
项目状态：
已结题

项目摘要

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）：腹泻和实验诱导的液体分泌几乎总是与富含HCO3的类似血浆的溶液有关。在对比，体外模型很少观察到HCO3分泌，但优雅解剖了二十五年的主动CL分泌的机制。模型缺乏结肠HCO3分泌。我们已经建立了执行的方法分离的大鼠和小鼠结肠隐窝的微灌注（分泌的位置过程）在其中依赖于AMP的循环AMP诱导HCO3 分泌。 cAMP刺激的HCO3分泌的研究表明了密切的关联在HCO3分泌和主动CL分泌之间，建议顶端HCO3 运动是通过阴离子通道，而不是C1-HCO3交换，而HCO3不是内源性产生，但是从细胞外来源及其跨过的运动基底外侧膜（BLM）是Na-K-2cl耦合的结果共同传输（NKCCI）和CL-HCO3 Exchange或NA-HCO3 Co-Transport（NBC）。我们在正常大鼠和CFTR敲除和 NKCC1敲除小鼠（及其对照同窝仔）建立机制 HCO3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。修补夹具研究将表征阴离子通道的HCO3电导地穴顶膜。尽管CL横穿基底外侧膜（除了通过NKCCL以外）长期以来一直怀疑，先前的研究已经无法识别载体介导的过程，例如Cl-anion Exchange。使用蛋白酶抑制剂准备BLM囊泡，我们最近确定了 CL-HCO3交换和计划实验的表征。我们相信AE2 编码此CL-HCO3交换，并用90克隆了全长cDNA 与胃AE2同源性百分比同源，并提出其在HEK293细胞中的表达。我们还有证据表明远端结肠（即CNBC）中的NBC有很大不同来自其他NBC，可能代表一种新型的上皮细胞NBC。我们建议综合运输，电生理和分子研究 crypt hco3分泌的特征。