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MECHANISM OF CELLULAR REGULATION OF BICARBONATE TRANSPORTER FUNCTION : AN APPROACH WITH RUMINANT PAROTID TOWARD ITS UNDERSTANDING

碳酸氢盐转运蛋白功能的细胞调节机制：反刍动物腮腺的理解方法

基本信息

批准号：
16590160
负责人：
ISHIKAWA Toru
金额：
$ 2.3万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16590160/
关键词：
electrogenic sodium-bicarbonate cotransporter bicarbonate secretion bovine parotid patch-clamp

项目摘要

Although the electrogenic Na^+- HCO_<3^-> cotransporter (NBCe) is believed to play a crucial role in vectorial HCO_<3^-> transport by mediating a basolateral HCO_<3^-> influx or efflux pathway in various epithelia, its transport properties and cellular regulation under native conditions have not been well understood. The main aim of the present project was to identify and characterize native NBCe currents in acinar cells (BPA cells) acutely isolated from bovine parotid, which is known to produce an enormous amount of HCO_<3^-> - rich fluid. Using the whole-cell patch-clamp technique, we could first demonstrate that BPA cells indeed express a membrane current, which is dependent on the presence of both extracellular Na^+ and HCO_<3^->, is blocked by extracellular DIDS, and has an apparent coupling ratio of 1 Na^+ to 2 HCO_<3^->. Using molecular biological techniques, we could also show that BPA cells express transcripts of NBCe1-B, and that electrophysiological and pharmacological properties of the native cotransporter current are similar to those of recombinant current of NBCe1-B expressed in HEK293 cells. Futhermore, we could provide evidence that the cotransporter activity likely contributes to the resting membrane potential of BPA cells. The results together not only provide the first electrophysiological characterization of native NBCe currents in a ruminant parotid, but also form a first step toward understanding cellular regulation mechanism of the NBCe in a native HCO_<3^-> transporting epithelium.

虽然生电性Na^+- HCO_3^-转运蛋白（NBCe）被认为在各种上皮细胞中通过介导基底外侧HCO_3 ^->内流或外流途径在载体HCO_3^->转运中起关键作用，但其转运特性及其在天然条件下的细胞调控尚不清楚。牛腮腺腺泡细胞（BPA细胞）产生大量富含HCO_（3^-）的液体，本研究的主要目的是鉴定和表征BPA细胞的NBCe电流。利用全细胞膜片钳技术，我们可以首先证明BPA细胞确实表达膜电流，该电流依赖于细胞外Na^+和HCO_3^-的存在，并被细胞外DIDS阻断，其表观偶联比为1 Na^+：2 HCO_3^-。利用分子生物学技术，我们还可以表明BPA细胞表达NBCe 1-B的转录本，并且天然协同转运蛋白电流的电生理学和药理学性质与HEK 293细胞中表达的NBCe 1-B的重组电流的电生理学和药理学性质相似。因此，我们可以提供证据表明协同转运蛋白活性可能有助于BPA细胞的静息膜电位。这些结果不仅首次提供了反刍动物腮腺中天然NBCe电流的电生理特征，而且为理解天然HCO_3^-转运上皮中NBCe的细胞调节机制迈出了第一步。