The BK channel opener NS1619 induces neuronal preconditioning in vitro

Tamas Gaspar, F. Domoki, Keita Mayanagi, James A. Snipes, F. Bari, David W. Busija

Research output: Contribution to journalArticle

Abstract

Background and aims. Targeting the large conductance Ca activated potassium (BK) channels is a novel approach to induce preconditioning (PC). NS1619 (NS) is a selective opener of the BK channel and was shown to protect the heart against an otherwise lethal ischemic insult. Recently, the BK channel has been reported to be present in the inner mitochondrial membrane of rat neurons, but its role in tolerance induction in the central nervous system has not yet been explored. The aim of our in vitro study was to examine whether NS can induce PC and protect rat cortical neuronal cultures against toxic stimuli. We also investigated its effect on reactive oxygen species (ROS) production, plasma membrane potential, and expression and activity of cytoprotective proteins. Methods. Cortical neurons of 18-day old Sprague-Dawley rat fetuses were cultured in B27 supplemented Neurobasal medium. One week after plating, a 6-hour daily treatment with different doses of NS (10 ? 100 μM) for 3 days was introduced. Eighteen hours after the last treatment, neuronal cultures were exposed to 180 min of oxygen and glucose deprivation (OGD), 6 hour of exogenous hydrogen peroxide toxicity (80 μM), or 60 min of glutamate excitotoxicity (200 μM). Cell viability was evaluated with CellTiter 96 AQueous One Solution assay. ROS generation, plasma and mitochondrial membrane potentials were monitored using hydroethidine, di-8-ANEPPS, and tetramethylrhodamine ethyl ester, respectively. The expression of manganese dependent superoxide dismutase (MnSOD), cupper and zinc dependent superoxide dismutase (CuZnSOD), glutathione peroxidase (GPx), and catalase was examined using Western blotting. Enzyme activity of antioxidants was assessed with a microplate reader using commercially available kits. Results. Application of NS increased ROS generation, depolarized mitochondria, and hyperpolarized the cell membrane of neurons. Three-day treatment with NS dose-dependently protected the cell cultures against OGD, H2O2, and glutamate, although higher concentrations of the compound were needed to elicit PC than previously reported in the heart. The highest tolerance was observed against H2O2 where a dose of 40 μM provided complete protection (viability: control, 100.0±1.97%; untreated, 50.1±2.81%*; NS 40 μM, 93.2±3.25%#; *p

Original languageEnglish
JournalJournal of Cerebral Blood Flow and Metabolism
Volume27
Issue numberSUPPL. 1
Publication statusPublished - Nov 13 2007

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Large-Conductance Calcium-Activated Potassium Channels
Reactive Oxygen Species
Cell Membrane
Neurons
Superoxide Dismutase
Glutamic Acid
Oxygen
Glucose
Mitochondrial Membrane Potential
Poisons
Potassium Channels
Mitochondrial Membranes
Glutathione Peroxidase
Membrane Potentials
Catalase
Hydrogen Peroxide
Sprague Dawley Rats
In Vitro Techniques
NS 1619
Zinc

ASJC Scopus subject areas

  • Endocrinology
  • Neuroscience(all)
  • Endocrinology, Diabetes and Metabolism

Cite this

The BK channel opener NS1619 induces neuronal preconditioning in vitro. / Gaspar, Tamas; Domoki, F.; Mayanagi, Keita; Snipes, James A.; Bari, F.; Busija, David W.

In: Journal of Cerebral Blood Flow and Metabolism, Vol. 27, No. SUPPL. 1, 13.11.2007.

Research output: Contribution to journalArticle

Gaspar, Tamas ; Domoki, F. ; Mayanagi, Keita ; Snipes, James A. ; Bari, F. ; Busija, David W. / The BK channel opener NS1619 induces neuronal preconditioning in vitro. In: Journal of Cerebral Blood Flow and Metabolism. 2007 ; Vol. 27, No. SUPPL. 1.
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T1 - The BK channel opener NS1619 induces neuronal preconditioning in vitro

AU - Gaspar, Tamas

AU - Domoki, F.

AU - Mayanagi, Keita

AU - Snipes, James A.

AU - Bari, F.

AU - Busija, David W.

PY - 2007/11/13

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N2 - Background and aims. Targeting the large conductance Ca activated potassium (BK) channels is a novel approach to induce preconditioning (PC). NS1619 (NS) is a selective opener of the BK channel and was shown to protect the heart against an otherwise lethal ischemic insult. Recently, the BK channel has been reported to be present in the inner mitochondrial membrane of rat neurons, but its role in tolerance induction in the central nervous system has not yet been explored. The aim of our in vitro study was to examine whether NS can induce PC and protect rat cortical neuronal cultures against toxic stimuli. We also investigated its effect on reactive oxygen species (ROS) production, plasma membrane potential, and expression and activity of cytoprotective proteins. Methods. Cortical neurons of 18-day old Sprague-Dawley rat fetuses were cultured in B27 supplemented Neurobasal medium. One week after plating, a 6-hour daily treatment with different doses of NS (10 ? 100 μM) for 3 days was introduced. Eighteen hours after the last treatment, neuronal cultures were exposed to 180 min of oxygen and glucose deprivation (OGD), 6 hour of exogenous hydrogen peroxide toxicity (80 μM), or 60 min of glutamate excitotoxicity (200 μM). Cell viability was evaluated with CellTiter 96 AQueous One Solution assay. ROS generation, plasma and mitochondrial membrane potentials were monitored using hydroethidine, di-8-ANEPPS, and tetramethylrhodamine ethyl ester, respectively. The expression of manganese dependent superoxide dismutase (MnSOD), cupper and zinc dependent superoxide dismutase (CuZnSOD), glutathione peroxidase (GPx), and catalase was examined using Western blotting. Enzyme activity of antioxidants was assessed with a microplate reader using commercially available kits. Results. Application of NS increased ROS generation, depolarized mitochondria, and hyperpolarized the cell membrane of neurons. Three-day treatment with NS dose-dependently protected the cell cultures against OGD, H2O2, and glutamate, although higher concentrations of the compound were needed to elicit PC than previously reported in the heart. The highest tolerance was observed against H2O2 where a dose of 40 μM provided complete protection (viability: control, 100.0±1.97%; untreated, 50.1±2.81%*; NS 40 μM, 93.2±3.25%#; *p

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