Submitted on July 16, 2007
Accepted on March 11, 2008
Platelet-activating Factor induced chloride channel activation is associated with intracellular acidosis and apoptosis of intestinal epithelial cells
Erika C Claud1*, Jing Lu2, Xue Qing Wang3, Mark Abe1, Elaine O Petrof4, Jun Sun5, Deborah J Nelson3, Jeremy D Marks1, and Tamas Jilling6
1 Pediatrics, University of Chicago, Chicago, Illinois, United States
2 Pediatrics, ENH Research Institute Northwestern University, Evanston, Illinois, United States
3 Neurobiology, Pharmacology and Physiology, University of Chicago, Chicago, Illinois, United States
4 Medicine, University of Chicago, Chicago, Illinois, United States
5 Pathology, University of Chicago, Chicago, Illinois, United States
6 Pediatrics, ENH Research Institute, Northwestern University, Evanston, Illinois, United States
* To whom correspondence should be addressed. E-mail: eclaud{at}peds.bsd.uchicago.edu.
Platelet activating factor (PAF) is a phospholipid inter and intra-cellular mediator implicated in intestinal injury primarily via induction of an inflammatory cascade. We find that PAF also has direct pathologic effects on intestinal epithelial cells. PAF induces Cl- channel activation which is associated with intracellular acidosis and apoptosis. Using the rat small intestinal epithelial cell line IEC-6, electrophysiologic experiments demonstrated that PAF induces Cl- channel activation. This PAF activated Cl- current was inhibited by Ca2+ chelation and a calcium calmodulin Kinase II inhibitor suggesting PAF activation of a Ca2+ activated Cl- channel. To determine the pathologic consequences of Cl- channel activation, microfluorimetry experiments were performed which revealed PAF induced intracellular acidosis which is also inhibited by the Cl- channel inhibitor 4, 4' diisothiocyanostilbene-2, 2' disulfonic acid and Ca2+ chelation. PAF-induced intracellular acidosis is associated with caspase 3 activation and DNA fragmentation. PAF-induced caspase activation was abolished in cells transfected with a pH compensatory Na/H exchanger (NHE1) construct to enhance H+ extruding ability and prevent intracellular acidosis. As ClC-3 is a known intestinal Cl- channel dependent on both Ca2+ and calcium calmodulin Kinase II phosphorylation, we generated ClC-3 knockdown cells using shRNA. PAF induced Cl- current, acidosis and apoptosis were all significantly decreased in ClC-3 knockdown cells. Our data suggest a novel mechanism of PAF-induced injury by which PAF induces intracellular acidosis via activation of the Ca2+ dependent Cl- channel ClC-3 resulting in apoptosis of intestinal epithelial cells.
