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

1 Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio 45267-0576; and 2 Sucampo Pharmaceuticals, Inc., Bethesda, Maryland 20814 Submitted 26 November 2003 ; accepted in final form 21 June 2004 ABSTRACT 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 EC 50 of 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 EC 50 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. cystic fibrosis transmembrane regulator; intestinal chloride channels; chloride channel opener Address for reprint requests and other correspondence: J. Cuppoletti, Dept. of Molecular and Cellular Physiology, College of Medicine, Univ. of Cincinnati, PO Box 670576, Cincinnati, OH 45267-0576 (E-mail: John.Cuppoletti{at}uc.edu )