e{open}PKC phosphorylates the mitochondrial K+ATP channel during induction of ischemic preconditioning in the rat hippocampus

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Abstract

Neuroprotection against cerebral ischemia conferred by ischemic preconditioning (IPC) requires translocation of epsilon protein kinase C (εPKC). A major goal in our laboratory is to define the cellular targets by which εPKC confers protection. We tested the hypothesis that εPKC targets the mitochondrial K+ATP channel (mtK+ATP) after IPC. Our results demonstrated a rapid translocation of εPKC to rat hippocampal mitochondria after IPC. Because in other tissues εPKC 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 εPKC agonist-induced preconditioning. Blockade of mtK+ATP channels using 5-hydroxydecanoic acid abolished the neuroprotection due to either IPC or εPKC preconditioning. Both ischemic and εPKC agonist-mediated preconditioning resulted in phosphorylation of the mtK+ATP channel subunit Kir6.2. After IPC, selective inhibition of εPKC activation prevented Kir6.2 phosphorylation, consistent with Kir6.2 as a phosphorylation target of εPKC 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 εPKC.

Original languageEnglish
Pages (from-to)345-353
Number of pages9
JournalBrain Research
Volume1184
Issue number1
DOIs
StatePublished - Dec 12 2007

Fingerprint

Ischemic Preconditioning
Protein Kinase C
Hippocampus
Mitochondria
Phosphorylation
Ischemia
Adenosine Triphosphate
mitochondrial K(ATP) channel
Pinacidil
Protein Kinase C-epsilon
Diazoxide
Propidium
Brain
Brain Ischemia
Signal Transduction
Cell Death
Staining and Labeling
Oxygen
Neurons
Glucose

Keywords

  • Cell death
  • Diazoxide
  • Ischemic tolerance
  • Organotypic slice culture
  • Protein kinase C
  • Signal transduction

ASJC Scopus subject areas

  • Neuroscience(all)
  • Clinical Neurology
  • Developmental Biology
  • Molecular Biology

Cite this

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title = "e{open}PKC phosphorylates the mitochondrial K+ATP channel during induction of ischemic preconditioning in the rat hippocampus",
abstract = "Neuroprotection against cerebral ischemia conferred by ischemic preconditioning (IPC) requires translocation of epsilon protein kinase C (εPKC). A major goal in our laboratory is to define the cellular targets by which εPKC confers protection. We tested the hypothesis that εPKC targets the mitochondrial K+ATP channel (mtK+ATP) after IPC. Our results demonstrated a rapid translocation of εPKC to rat hippocampal mitochondria after IPC. Because in other tissues εPKC 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 εPKC agonist-induced preconditioning. Blockade of mtK+ATP channels using 5-hydroxydecanoic acid abolished the neuroprotection due to either IPC or εPKC preconditioning. Both ischemic and εPKC agonist-mediated preconditioning resulted in phosphorylation of the mtK+ATP channel subunit Kir6.2. After IPC, selective inhibition of εPKC activation prevented Kir6.2 phosphorylation, consistent with Kir6.2 as a phosphorylation target of εPKC 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 εPKC.",
keywords = "Cell death, Diazoxide, Ischemic tolerance, Organotypic slice culture, Protein kinase C, Signal transduction",
author = "Ami Raval and Dave, {Kunjan R} and DeFazio, {R. Anthony} and Miguel Perez-Pinzon",
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T1 - e{open}PKC phosphorylates the mitochondrial K+ATP channel during induction of ischemic preconditioning in the rat hippocampus

AU - Raval, Ami

AU - Dave, Kunjan R

AU - DeFazio, R. Anthony

AU - Perez-Pinzon, Miguel

PY - 2007/12/12

Y1 - 2007/12/12

N2 - Neuroprotection against cerebral ischemia conferred by ischemic preconditioning (IPC) requires translocation of epsilon protein kinase C (εPKC). A major goal in our laboratory is to define the cellular targets by which εPKC confers protection. We tested the hypothesis that εPKC targets the mitochondrial K+ATP channel (mtK+ATP) after IPC. Our results demonstrated a rapid translocation of εPKC to rat hippocampal mitochondria after IPC. Because in other tissues εPKC 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 εPKC agonist-induced preconditioning. Blockade of mtK+ATP channels using 5-hydroxydecanoic acid abolished the neuroprotection due to either IPC or εPKC preconditioning. Both ischemic and εPKC agonist-mediated preconditioning resulted in phosphorylation of the mtK+ATP channel subunit Kir6.2. After IPC, selective inhibition of εPKC activation prevented Kir6.2 phosphorylation, consistent with Kir6.2 as a phosphorylation target of εPKC 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 εPKC.

AB - Neuroprotection against cerebral ischemia conferred by ischemic preconditioning (IPC) requires translocation of epsilon protein kinase C (εPKC). A major goal in our laboratory is to define the cellular targets by which εPKC confers protection. We tested the hypothesis that εPKC targets the mitochondrial K+ATP channel (mtK+ATP) after IPC. Our results demonstrated a rapid translocation of εPKC to rat hippocampal mitochondria after IPC. Because in other tissues εPKC 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 εPKC agonist-induced preconditioning. Blockade of mtK+ATP channels using 5-hydroxydecanoic acid abolished the neuroprotection due to either IPC or εPKC preconditioning. Both ischemic and εPKC agonist-mediated preconditioning resulted in phosphorylation of the mtK+ATP channel subunit Kir6.2. After IPC, selective inhibition of εPKC activation prevented Kir6.2 phosphorylation, consistent with Kir6.2 as a phosphorylation target of εPKC 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 εPKC.

KW - Cell death

KW - Diazoxide

KW - Ischemic tolerance

KW - Organotypic slice culture

KW - Protein kinase C

KW - Signal transduction

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