epsilonPKC phosphorylates the mitochondrial K(+) (ATP) channel during induction of ischemic preconditioning in the rat hippocampus.
Neuroprotection against cerebral ischemia conferred by ischemic preconditioning (IPC) requires translocation of epsilon protein kinase C (epsilonPKC). A major goal in our laboratory is to define the cellular targets by which epsilonPKC confers protection. We tested the hypothesis that epsilonPKC targets the mitochondrial K(+)(ATP) channel (mtK(+)(ATP)) after IPC. Our results demonstrated a rapid translocation of epsilonPKC to rat hippocampal mitochondria after IPC. Because in other tissues epsilonPKC targets mtK(+)(ATP) channels, but its presence in brain mitochondria is controversial, we determined the presence of the K(+)(ATP) channel-specific subunits (Kir6.1 and Kir6.2) in mitochondria isolated from rat hippocampus. Next, we determined whether mtK(+)(ATP) channels play a role in the IPC induction. In hippocampal organotypic slice cultures, IPC and lethal ischemia were induced by oxygen-glucose deprivation. Subsequent cell death in the CA1 region was quantified using propidium iodide staining. Treatment with the K(+)(ATP) channel openers diazoxide or pinacidil 48 h prior to lethal ischemia protected hippocampal CA1 neurons, mimicking the induction of neuroprotection conferred by either IPC or epsilonPKC agonist-induced preconditioning. Blockade of mtK(+)(ATP) channels using 5-hydroxydecanoic acid abolished the neuroprotection due to either IPC or epsilonPKC preconditioning. Both ischemic and epsilonPKC agonist-mediated preconditioning resulted in phosphorylation of the mtK(+)(ATP) channel subunit Kir6.2. After IPC, selective inhibition of epsilonPKC activation prevented Kir6.2 phosphorylation, consistent with Kir6.2 as a phosphorylation target of epsilonPKC or its downstream effectors. Our results support the hypothesis that the brain mtK(+)(ATP) channel is an important target of IPC and the signal transduction pathways initiated by epsilonPKC.
Raval
Ami P
AP
Cerebral Vascular Disease Research Center, Department of Neurology and Neuroscience Program, University of Miami Leonard M Miller School of Medicine, Miami, FL 33101, USA. araval@med.miami.edu
Dave
Kunjan R
KR
DeFazio
R Anthony
RA
Perez-Pinzon
Miguel A
MA
D000818:Animals; D000831:Animals, Newborn; D000959:Antihypertensive Agents / Q000494:pharmacology; D003981:Diazoxide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology* / Q000502:physiology; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D007511:Ischemia / Q000378:metabolism; D019194:Ischemic Preconditioning / Q000379:methods*; D054086:KATP Channels / Q000378:metabolism*; D008297:Male; D008928:Mitochondria / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000187:drug effects; D008954:Models, Biological; D009474:Neurons / Q000502:physiology / Q000648:ultrastructure*; D026902:Potassium Channel Blockers / Q000494:pharmacology; D051744:Protein Kinase C-epsilon / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
D000959:Antihypertensive Agents; D004791:Enzyme Inhibitors; D054086:KATP Channels; D026902:Potassium Channel Blockers; D051744:Protein Kinase C-epsilon; D003981:Diazoxide