SPI-0211 activates T84 cell chloride transport and recombinant human ClC-2 chloride currents.
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 approximately 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 approximately 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.
Cuppoletti
John
J
Department of Molecular and Cellular Physiology, College of Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0576, USA. John.Cuppoletti@uc.edu
Malinowska
Danuta H
DH
Tewari
Kirti P
KP
Li
Qiu-Ju
QJ
Sherry
Ann M
AM
Patchen
Myra L
ML
Ueno
Ryuji
R
D000818:Animals; D015152:Blotting, Northern; D000090007:CLC-2 Chloride Channels; D018118:Chloride Channels / Q000187:drug effects / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004305:Dose-Response Relationship, Drug; D005227:Fatty Acids / Q000494:pharmacology*; D006801:Humans; D017403:In Situ Hybridization; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000187:drug effects; D017136:Ion Transport / Q000187:drug effects; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D018613:Microscopy, Confocal; D018408:Patch-Clamp Techniques; D012333:RNA, Messenger / Q000032:analysis; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction
C505032:CFTR protein, human; D000090007:CLC-2 Chloride Channels; D018118:Chloride Channels; D002712:Chlorides; D005227:Fatty Acids; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator