SPI-0211 activates T84 cell chloride transport and recombinant human ClC-2 chloride currents
SPI-0211 activates T84 cell chloride transport and recombinant human ClC-2 chloride currents
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DOI：
10.1152/ajpcell.00528.2003
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发表时间：
2004-11-01
影响因子：
5.5
通讯作者：
Ueno, R
中科院分区：
文献类型：
--
作者：
Cuppoletti, J;Malinowska, DH;Ueno, R
本研究的目的是确定SPI-0211（鲁比前列酮）的作用机制，SPI-0211是一种新型双环脂肪酸，正在开发用于治疗肠功能障碍。RT-PCR、北方印迹分析和原位杂交显示成年兔肠中含有ClC-2的mRNA。在可渗透支持物上生长至汇合的T84细胞显示在顶膜中表达ClC-2通道蛋白。SPI-0211增加了T84细胞顶膜的产电Cl-转运，在用制霉菌素透化基底外侧膜后，通过短路电流（I-sc）测量的EC 50类似于18 nM。还使用用重组人ClC-2或重组人囊性纤维化跨膜调节因子（CFTR）稳定转染的人胚肾（HEK）-293细胞系，通过全细胞膜片钳测量SPI-0211对Cl-电流的影响。在这些研究中，SPI-0211以浓度依赖性方式激活ClC-2 Cl-电流，EC 50类似于17 nM，并且在未转染的HEK-293细胞中没有影响。相比之下，SPI-0211对CFTR转染的HEK-293细胞中测量的CFTR Cl-通道电流没有影响。SPI-0211对ClC-2的激活不依赖于PKA。总之，这些研究表明SPI-0211是ClC-2 Cl-通道的有效激活剂，并表明SPI-0211给药后ClC-2 Cl-通道在肠道Cl-转运中的生理相关作用。
The purpose of this study was to determine the mechanism of action of SPI-0211 (lubiprostone), a novel bicyclic fatty acid in development for the treatment of bowel dysfunction. Adult rabbit intestine was shown to contain mRNA for ClC-2 using RT-PCR, Northern blot analysis, and in situ hybridization. T84 cells grown to confluence on permeable supports were shown to express ClC-2 channel protein in the apical membrane. SPI-0211 increased electrogenic Cl- transport across the apical membrane of T84 cells, with an EC50 of similar to 18 nM measured by short-circuit current (I-sc) after permeabilization of the basolateral membrane with nystatin. SPI-0211 effects on Cl- currents were also measured by whole cell patch clamp using the human embryonic kidney (HEK)-293 cell line stably transfected with either recombinant human ClC-2 or recombinant human cystic fibrosis transmembrane regulator ( CFTR). In these studies, SPI-0211 activated ClC-2 Cl- currents in a concentration- dependent manner, with an EC50 of similar to 17 nM, and had no effect in nontransfected HEK-293 cells. In contrast, SPI-0211 had no effect on CFTR Cl- channel currents measured in CFTR-transfected HEK-293 cells. Activation of ClC-2 by SPI-0211 was independent of PKA. Together, these studies demonstrate that SPI-0211 is a potent activator of ClC-2 Cl- channels and suggest a physiologically relevant role for ClC-2 Cl- channels in intestinal Cl- transport after SPI-0211 administration.