TRANSLATIONAL PHYSIOLOGY SPI-0211 activates T84 cell chloride transport and recombinant human ClC-2 chloride currents

Cuppoletti, John, Danuta H. Malinowska, Kirti P. Tewari, Qiu-ju Li, Ann M. Sherry, Myra L. Patchen, and Ryuji Ueno. SPI-0211 activates T84 cell chloride transport and recombinant human ClC-2 chloride currents. Am J Physiol Cell Physiol 287: C1173– C1183, 2004. First published June 22, 2004; doi:10.1152/ajpcell. 00528.2003.—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 18 nM measured by short-circuit current (Isc) 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 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.