Department of Pathology, Immunology, and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, Florida 32610-00275, USA.
Am J Physiol Gastrointest Liver Physiol. 2009 Nov;297(5):G918-29. doi: 10.1152/ajpgi.00251.2009.
The purpose of this investigation was to quantitate the contribution of the anion exchanger PAT-1 (putative anion transporter-1), encoded by SLC26A6, to oxalate transport in a model intestinal epithelium and to discern some characteristics of this exchanger expressed in its native environment. Control (Con) Caco-2 BBe1 monolayers, 6-8 days postseeding, were compared with those transfected with a small interfering RNA targeted to SLC26A6 (A6KD). Radiotracer and Ussing chamber techniques were used to determine the transepithelial unidirectional fluxes of Ox(2-), Cl(-), and SO(4)(2-) whereas fluorometric/BCECF measurements of intracellular pH were used to assess HCO(3)(-) exchange. PAT-1 was functionally targeted to the apical membrane, and SLC26A6 knockdown reduced PAT-1 protein (>60%) and mRNA (>75%) expression in A6KD. No net flux of Ox(2-), Cl(-), or SO(4)(2-) was detected in Con or A6KD monolayers, yet the unidirectional fluxes in A6KD were reduced 50, 35, and 15%, respectively. Cl(-)-dependent HCO(3)(-) efflux from A6KD was reduced 50% compared with Con. The difference between Con and A6KD properties represents that mediated solely by PAT-1, and by this approach we found that PAT-1-mediated oxalate influx and efflux are inhibited equally by mucosal DIDS (EC(50) approximately 5 microM) and that mucosal Cl(-) inhibits oxalate uptake with an EC(50) < 20 mM. Transepithelial Cl(-) gradients supported large, DIDS-sensitive net absorptive or secretory fluxes of oxalate in a direction opposite that of the imposed Cl(-) gradient. The overall symmetry of PAT-1-mediated oxalate exchange suggests that vectorial oxalate transport observed in vivo is principally dependent on the magnitude and direction of counterion gradients.
这项研究的目的是定量分析阴离子交换器 PAT-1（假定的阴离子转运蛋白-1）在模型肠上皮细胞中对草酸盐转运的贡献，并辨别其在天然环境中表达的一些特性。将 6-8 天传代后的对照（Con）Caco-2 BBe1 单层与靶向 SLC26A6 的小干扰 RNA 转染的单层（A6KD）进行比较。放射性示踪和 Ussing 室技术用于测定 Ox（2-）、Cl（-）和 SO（4）（2-）的跨上皮单向通量，而荧光/BCECF 测量细胞内 pH 用于评估 HCO（3）（-）交换。PAT-1 被功能性地靶向到顶膜，并且 SLC26A6 的敲低降低了 A6KD 中的 PAT-1 蛋白（>60%）和 mRNA（>75%）表达。在 Con 或 A6KD 单层中未检测到 Ox（2-）、Cl（-）或 SO（4）（2-）的净通量，但 A6KD 的单向通量分别降低了 50%、35%和 15%。与 Con 相比，A6KD 中的 Cl（-）依赖性 HCO（3）（-）外排减少了 50%。Con 和 A6KD 之间的差异代表仅由 PAT-1 介导的差异，通过这种方法，我们发现 PAT-1 介导的草酸盐内流和外流均被粘膜 DIDS 同等抑制（EC（50）约 5 microM），并且粘膜 Cl（-）抑制草酸盐摄取，EC（50）<20 mM。跨上皮 Cl（-）梯度支持草酸盐的大、DIDS 敏感的净吸收或分泌通量，方向与施加的 Cl（-）梯度相反。PAT-1 介导的草酸盐交换的整体对称性表明，体内观察到的载体草酸盐转运主要取决于抗衡离子梯度的大小和方向。
