Epsilon PKC interacts with mitochondrial ATP-dependent potassium channel during ischemic preconditioning in organotypic slice cultures

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Abstract

Background: Mitochondrial ATP-dependent potassium (mtK+ATP) channel activation has been reported to mediate cardiac ischemic preconditioning (IPC) via generating reactive oxygen species (ROS). The role of the mtK+ATP channel in IPC induction in the brain is still not well defined. Previously we demonstrated that IPC-induced neuronal survival in the hippocampal CA1 required epsilon protein kinase C (?PKC) activation (1, 2). Thus, the goal of the present study was to define whether mtK+ATP channel activation is required during the triggering phase of IPC. Further, we hypothesize that ?PKC-mediated neuroprotection requires mtK+ATP channel phosphorylation. Methods: Hippocampal slices were obtained from 9-11 days old Sprague Dawley rats and cultured for 14-15 days. Slices exposed to oxygen/glucose deprivation (OGD) for 40 min ('test' ischemia) were used as the ischemia group. Slices exposed to OGD for 15 min (or a pharmacological agent), 48 h prior to 'test' ischemia were used as the IPC/pharmacological preconditioned (PPC) groups. Propidium Iodide (PI) fluorescence images were obtained using a SPOT CCD camera and were digitized using SPOT advanced software. Percentage of relative optical intensity was used as an index of cell death. We confirmed the presence of mtK+ATP channel specific subunits (Kir 6.1 and Kir 6.2) in hippocampal mitochondria by Western blotting. Immunoprecipitation was carried out to study phosphorylation of Kir 6.2 in different experimental conditions. Results are expressed, as mean ± SEM. Statistical significance was determined with an ANOVA test followed by a Bonferroni's post-hoc test. Results: Opening of mtK+ATP channel (diazoxide, 50 μM) 48 h prior to ischemia protected the hippocampal CA1 and mimicked neuroprotection mediated by IPC/PPC with the εPKC agonist. PI fluorescence level of ischemic-, IPC-, and mtK+ATP channel agonist (diazoxide) treated groups were 50.6 ± 5.2 (n = 6); 28.5 ± 4.9 (n = 4); 16.8 ± 7.6 % (50 mM; n = 8; p < 0.001, compared to ischemia) respectively. Blockade of the mtK+ATP channel using 5-hydroxydecanoic acid (HD) abolished IPC/εPKC mediated neuroprotection. PI fluorescence values of HD-treated and IPC groups were 58.13 ± 15.9 % (n =12) and 29.7 ± 15.7 % (n =8), respectively (p < 0.001). Similarly, PI fluorescence values of εPKC agonist and εPKC agonist plus HD-treated groups were 33.5 ± 6.7 % (n = 5), and 54.3 ± 3.3 % (n = 5), respectively. Finally, we determined if ischemic and εPKC agonist-mediated PPC leads to phosphorylation of mtK+ATP channel specific subunits Kir 6.2. The levels of Kir 6.2 phosphorylation were higher by 79 (n = 3, p<0.01) and 104% (n = 3, p<0.01) in IPC and PPC groups, respectively, when compared with mitochondria from sham treated slices (n = 3). Inhibition of εPKC activation after IPC prevented Kir 6.2 phosphorylation, suggesting that εPKC-mediated Kir 6.2 phosphorylation following IPC. Conclusion: The signal transduction pathway that ensues following IPC/εPKC agonist-mediated PPC requires opening of the mtK+ATP channel.

Original languageEnglish (US)
Pages (from-to)BP8-01U
JournalJournal of Cerebral Blood Flow and Metabolism
Volume27
Issue numberSUPPL. 1
StatePublished - Nov 13 2007

ASJC Scopus subject areas

  • Neurology
  • Clinical Neurology
  • Cardiology and Cardiovascular Medicine

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