Neuronal preconditioning with the antianginal drug, bepridil

Tamas Gaspar, Bela Kis, James A. Snipes, Gabor Lenzser, Keita Mayanagi, Ferenc Bari, David W. Busija

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Abstract

Background and aims. Bepridil (BEP) is an antianginal drug with a large variety of cardiovascular effects. While a recent study has demonstrated that BEP activates mitochondrial ATP sensitive potassium (mitoKATP) channels and protects the heart against ischemia, the effects of this drug on the central nervous system are not entirely known. The aim of our in vitro study was to investigate whether BEP can protect rat cortical neuronal cultures against toxic stimuli. We also examined the effect of BEP on reactive oxygen species (ROS) generation, plasma membrane and mitochondrial membrane potentials, mitoKATP channels, and on the expression and activity of cytoprotective proteins. Methods. Cortical neurons of 18-day old Sprague-Dawley rat fetuses were cultured in B27 supplemented Neurobasal medium. Preconditioning was carried out by treating the cells with different doses of BEP for 3 days, once a day. Twenty-four hours after the last treatment, neuronal cultures were exposed to either 180 min of oxygen and glucose deprivation (OGD) or 60 min of glutamate excitotoxicity (200 μM). Cell viability was evaluated with CellTiter 96 AQueous One Solution assay. ROS production was monitored with a fluorescent microplate reader using hydroethidine, plasma membrane and mitochondrial membrane potentials with confocal microscopy using di-8-ANEPPS and tetramethylrhodamine ethyl ester, respectively. The expression of phosphorylated protein kinase C (PKC), manganese dependent superoxide dismutase (MnSOD), glutathione peroxidase (GPx), catalase, and Bcl2 was examined using Western blotting. Enzyme activity of antioxidants was assessed with a microplate reader using commercially available kits. Results. As an immediate effect, application of BEP did not change plasma membrane potential, induced an increase in ROS generation, and dose-dependently depolarized mitochondria, but no acute protection could be found. Three-day treatment with BEP, however, provided dose-dependent neuroprotection against both OGD (cell viability: untreated, 54.7±0.67%; BEP 1μM, 66.4±1.45%*; BEP 2.5 μM, 77.3±1.19%*; BEP 5 μM, 97.8±0.94%*; BEP 10 μM, 84.7±1.67%*; p<0.05 vs. untreated) and glutamate excitotoxicity (cell viability: untreated, 54.1±0.69%; BEP 1 μM, 61.2±1.19% *; BEP 2.5 μM, 78.1±1.67%*; BEP 5 μM, 91.2±1.20% *; BEP 10 μM, 94.6±2.12%* ; p<0.05 vs. untreated), and inhibited the ROS surge upon exposure to glutamate. The neuroprotective effect of BEP could be antagonized with the SOD mimetic M40401 (cell viability after OGD for 180 min: untreated, 36.7±0.59%; BEP 5 μM, 82.0±1.23% *; BEP 5 μM + M40401 50 μM, 20.5±2.70%*#; *p<0.05 vs. untreated, #p<0.05 vs. BEP 5 μM), but not with catalase, reduced glutathione, or the putative mitoKATP channel blocker, 5-hydroxydecanoate. BEP treatment resulted in elevated protein levels of PKC, MnSOD, GPx, catalase, and Bcl-2 and increased enzymatic activity of MnSOD and GPx. Conclusion. Our results indicate that BEP induces late preconditioning in isolated rat neuronal cultures conferring neuroprotection which is dependent on superoxide generation and a sequential activation of PKC and cytoprotective proteins but perhaps not on direct mitoKATP activation.

Original languageEnglish
Pages (from-to)BP10-02H
JournalJournal of Cerebral Blood Flow and Metabolism
Volume27
Issue numberSUPPL. 1
Publication statusPublished - Nov 13 2007

ASJC Scopus subject areas

  • Neurology
  • Clinical Neurology
  • Cardiology and Cardiovascular Medicine

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  • Cite this

    Gaspar, T., Kis, B., Snipes, J. A., Lenzser, G., Mayanagi, K., Bari, F., & Busija, D. W. (2007). Neuronal preconditioning with the antianginal drug, bepridil. Journal of Cerebral Blood Flow and Metabolism, 27(SUPPL. 1), BP10-02H.