title: Platelet-activating factor-induced chloride channel activation is associated with intracellular acidosis and apoptosis of intestinal epithelial cells.
abstract: Platelet-activating factor (PAF) is a phospholipid inter- and intracellular mediator implicated in intestinal injury primarily via induction of an inflammatory cascade. We find that PAF also has direct pathological effects on intestinal epithelial cells (IEC). PAF induces Cl(-) channel activation, which is associated with intracellular acidosis and apoptosis. Using the rat small IEC line IEC-6, electrophysiological experiments demonstrated that PAF induces Cl(-) channel activation. This PAF-activated Cl(-) current was inhibited by Ca(2+) chelation and a calcium calmodulin kinase II inhibitor, suggesting PAF activation of a Ca(2+)-activated Cl(-) channel. To determine the pathological 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 Ca(2+) 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 construct to enhance H(+) extruding ability and prevent intracellular acidosis. As ClC-3 is a known intestinal Cl(-) channel dependent on both Ca(2+) and calcium calmodulin kinase II phosphorylation, we generated ClC-3 knockdown cells using short hairpin RNA. 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 Ca(2+)-dependent Cl(-) channel ClC-3, resulting in apoptosis of IEC.
authors.affiliation: Department of Pediatrics, University of Chicago, Chicago, IL 60637, USA. eclaud@peds.bsd.uchicago.edu
authors.forename: Erika C
authors.initials: EC
authors.lastname: Claud
authors.forename: Jing
authors.initials: J
authors.lastname: Lu
authors.forename: Xue Qing
authors.initials: XQ
authors.lastname: Wang
authors.forename: Mark
authors.initials: M
authors.lastname: Abe
authors.forename: Elaine O
authors.initials: EO
authors.lastname: Petrof
authors.forename: Jun
authors.initials: J
authors.lastname: Sun
authors.forename: Deborah J
authors.initials: DJ
authors.lastname: Nelson
authors.forename: Jeremy
authors.initials: J
authors.lastname: Marks
authors.forename: Tamas
authors.initials: T
authors.lastname: Jilling
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000138:Acidosis / Q000139:chemically induced / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology; D065101:Chloride Channel Agonists; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004594:Electrophysiology; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D016257:Fura-2 / Q000737:chemistry; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007422:Intestines / Q000166:cytology; D010455:Peptides / Q000494:pharmacology; D010972:Platelet Activating Factor / Q000494:pharmacology*; D047428:Protein Kinase Inhibitors; D034622:RNA Interference; D051381:Rats; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics
chemical_list: C503464:CaMKII inhibitor AIP; D002614:Chelating Agents; D065101:Chloride Channel Agonists; D018118:Chloride Channels; C115376:ClC-3 channel; D005456:Fluorescent Dyes; D010455:Peptides; D010972:Platelet Activating Factor; D047428:Protein Kinase Inhibitors; C452850:Slc9a1 protein, rat; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers; D004533:Egtazic Acid; D053148:Caspase 3; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium; D016257:Fura-2