Colonic Crypt HCO3 Secretion

描述(申请人提供)：腹泻和实验诱导的液体 分泌物几乎总是与富含HCO3的血浆样溶液有关。在……里面 相比之下，体外模型很少观察到HCO3的分泌，但有优雅的 剖析了25年来活动性氯气分泌的机制。一位模特 结肠HCO3分泌缺乏。我们已经建立了执行任务的方法 大鼠和小鼠离体肠隐窝(分泌部位)的微灌流 过程)，其中环AMP依赖和钙依赖激动剂诱导HCO3 分泌物。对cAMP刺激的HCO3分泌的研究表明 HCO3分泌与活性氯分泌的关系提示心尖HCO3 运动是通过阴离子通道，而不是C1-HCO3交换，HCO3不是 由内源产生，但来自细胞外来源，并通过 基侧膜(BLM)是Na-K-2Cl偶联的结果 共转运(NKCCI)和氯-HCO3交换或Na-HCO3共转运(NBC)。我们 建议对正常大鼠和CFTR基因敲除大鼠进行隐窝微灌注研究 NKCC1基因敲除小鼠(及其对照仔鼠)机制的建立 HCO3分泌物。对NKCC1基因敲除小鼠的初步研究支持 NKCC1和Cl-HCO3交换的偶联是BLM摄取的机制。补片 钳位研究将表征阴离子通道的HCO3电导 隐窝顶膜。尽管氯在基底外侧膜上的移动 (不是通过NKCCl)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，氯-阴离子交换。vbl.使用 蛋白水解酶抑制剂制备博莱姆囊泡，我们最近发现了一种 CL-HCO3交换，并为其表征计划实验。我们相信AE2 编码了这个氯-HCO3交换，并克隆了一个全长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)有很大不同 可能代表了一种新的上皮细胞NBC。我们建议 综合运输、电生理和分子研究 确定隐窝HCO3分泌物的特征。

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.