title: Glycogen synthase kinase 3 inhibition slows mitochondrial adenine nucleotide transport and regulates voltage-dependent anion channel phosphorylation.
abstract: Inhibition of glycogen synthase kinase (GSK)-3 reduces ischemia/reperfusion injury by mechanisms that involve the mitochondria. The goal of this study was to explore possible molecular targets and mechanistic basis of this cardioprotective effect. In perfused rat hearts, treatment with GSK inhibitors before ischemia significantly improved recovery of function. To assess the effect of GSK inhibitors on mitochondrial function under ischemic conditions, mitochondria were isolated from rat hearts perfused with GSK inhibitors and were treated with uncoupler or cyanide or were made anoxic. GSK inhibition slowed ATP consumption under these conditions, which could be attributable to inhibition of ATP entry into the mitochondria through the voltage-dependent anion channel (VDAC) and/or adenine nucleotide transporter (ANT) or to inhibition of the F(1)F(0)-ATPase. To determine the site of the inhibitory effect on ATP consumption, we measured the conversion of ADP to AMP by adenylate kinase located in the intermembrane space. This assay requires adenine nucleotide transport across the outer but not the inner mitochondrial membrane, and we found that GSK inhibitors slow AMP production similar to their effect on ATP consumption. This suggests that GSK inhibitors are acting on outer mitochondrial membrane transport. In sonicated mitochondria, GSK inhibition had no effect on ATP consumption or AMP production. In intact mitochondria, cyclosporin A had no effect, indicating that ATP consumption is not caused by opening of the mitochondrial permeability transition pore. Because GSK is a kinase, we assessed whether protein phosphorylation might be involved. Therefore, we performed Western blot and 1D/2D gel phosphorylation site analysis using phos-tag staining to indicate proteins that had decreased phosphorylation in hearts treated with GSK inhibitors. Liquid chromatographic-mass spectrometric analysis revealed 1 of these proteins to be VDAC2. Taken together, we found that GSK-mediated signaling modulates transport through the outer membrane of the mitochondria. Both proteomics and adenine nucleotide transport data suggest that GSK regulates VDAC and that VDAC may be an important regulatory site in ischemia/reperfusion injury.
authors.affiliation: Department of Pathology, Johns Hopkins University, Baltimore, MD 21205, USA.
authors.forename: Samarjit
authors.initials: S
authors.lastname: Das
authors.forename: Renee
authors.initials: R
authors.lastname: Wong
authors.forename: Nishadi
authors.initials: N
authors.lastname: Rajapakse
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Murphy
authors.forename: Charles
authors.initials: C
authors.lastname: Steenbergen
mesh_terms: D000227:Adenine Nucleotides / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000627:Aminophenols / Q000494:pharmacology; D000818:Animals; D002316:Cardiotonic Agents / Q000494:pharmacology*; D004734:Energy Metabolism / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D007211:Indoles / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008301:Maleimides / Q000494:pharmacology; D008929:Mitochondria, Heart / Q000187:drug effects* / Q000201:enzymology; D000226:Mitochondrial ADP, ATP Translocases / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000187:drug effects* / Q000378:metabolism; D000083162:Mitochondrial Permeability Transition Pore; D009200:Myocardial Contraction / Q000187:drug effects; D015428:Myocardial Reperfusion Injury / Q000201:enzymology / Q000517:prevention & control; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D016277:Ventricular Function, Left / Q000187:drug effects; D050996:Voltage-Dependent Anion Channel 2 / Q000187:drug effects / Q000378:metabolism; D050994:Voltage-Dependent Anion Channels / Q000187:drug effects* / Q000378:metabolism
chemical_list: C417520:3-(3-chloro-4-hydroxyphenylamino)-4-(4-nitrophenyl)-1H-pyrrole-2,5-dione; D000227:Adenine Nucleotides; D000627:Aminophenols; D002316:Cardiotonic Agents; D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D019253:Proto-Oncogene Proteins c-bcl-2; C417521:SB 216763; C494767:Vdac2 protein, rat; D050996:Voltage-Dependent Anion Channel 2; D050994:Voltage-Dependent Anion Channels; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000226:Mitochondrial ADP, ATP Translocases; D000071679:Glycogen Synthase Kinase 3 beta; D051057:Proto-Oncogene Proteins c-akt; D038362:Glycogen Synthase Kinase 3; D006180:Proton-Translocating ATPases
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