Neuronal preconditioning with the antianginal ... - Wiley Online Library

Journal of Neurochemistry, 2007, 102, 595–608

doi:10.1111/j.1471-4159.2007.04501.x

Neuronal preconditioning with the antianginal drug, bepridil Tama´s Ga´spa´r,* Be´la Kis,* James A. Snipes,* Ga´bor Lenzse´r,*, Keita Mayanagi,* Ferenc Barià and David W. Busija* *Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, North Carolina, USA Institute of Clinical Experimental Research and Human Physiology, Semmelweis University, Budapest, Hungary àDepartment of Physiology, Faculty of Medicine, University of Szeged, Szeged, Hungary

Abstract It has recently been shown that the antianginal drug bepridil (BEP) activates mitochondrial ATP-sensitive potassium (mitoKATP) channels and thus confers cardioprotection. Our aim was to investigate whether BEP could induce preconditioning in cultured rat cortical neurons. Although BEP depolarized isolated and in situ mitochondria and increased reactive oxygen species generation, no acute protection was observed. However, a 3-day BEP-treatment elicited dosedependent delayed neuroprotection against 180 min of oxygen–glucose deprivation (cell viability: untreated, 52.5 ± 0.85%; BEP 1 lmol/L, 59.6 ± 1.53%*; BEP 2.5 lmol/L, 71.9 ± 1.23%*; BEP 5 lmol/L, 95.3 ± 0.89%*; mean ± SEM; *p < 0.05 vs. untreated) and 60 min of glutamate excitotoxicity (200 lmol/L; cell viability: untreated, 54.1 ± 0.69%; BEP 1 lmol/L, 61.2 ± 1.19%*; BEP 2.5 lmol/L, 78.1 ± 1.67%*;

Preconditioning (PC) was first reported in 1986 as a protection of the heart against otherwise lethal insults induced by a preceding sublethal stimulus (Murry et al. 1986). The first PC studies were conducted using a brief period of ischemia as an initiating event (Schurr et al. 1986; Kitagawa et al. 1990). Later paradigms such as chemical and pharmacological PC were reported to induce comparable tolerance to ischemic PC (Riepe et al. 1997; Kis et al. 2003). Subsequent studies have been performed on different organs and cell types, and PC has been demonstrated to protect not only the heart but also other organs such as the brain, liver, and skeletal muscle. Bepridil (1-isobutoxy-2-pyrrolidino-3-N-benzylanilinopropane; BEP) is a synthetic compound with antianginal, antiand proarrhythmic, and mild antihypertensive properties and is used in the treatment of chronic stable angina pectoris (Hollingshead et al. 1992). This drug has negative inotropic, dromotropic, and chronotropic effects on the heart and has been reported to affect a large variety of ion channels. In fact,

BEP 5 lmol/L, 91.2 ± 1.20%*; mean ± SEM; *p < 0.05 vs. untreated), and inhibited the reactive oxygen species surge upon glutamate exposure. The protection was antagonized with co-application of the superoxide dismutase mimetic M40401, but not with reduced glutathione, catalase, or with the mitoKATP blocker 5-hydroxydecanoate. Furthermore, BEP treatment resulted in increased levels of phosphorylated protein kinase C, manganese-dependent superoxide dismutase, glutathione peroxidase, and Bcl-2. Our results indicate that BEP induces delayed neuronal preconditioning which is dependent on superoxide generation but perhaps not on direct mitoKATP activation. Keywords: Bcl-2, mitochondria, neuronal culture, neuroprotection, protein kinase C, reactive oxygen species. J. Neurochem. (2007) 102, 595–608.

Received August 25, 2006; revised manuscript received November 20, 2006; accepted December 13, 2006. Address correspondence and reprint requests to Tama´s Ga´spa´r, Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Medical Center Boulevard, Winston-Salem, NC 271571010, USA. E-mail: [email protected] Abbreviations used: 5HD, 5-hydroxydecanoate; [Ca2+]i, intracellular free calcium level; BEP, bepridil; di-8-ANEPPS, 4-{2-[6-(dioctylamino)2-naphthalenyl]ethenyl}-1-(3-sulfopropyl)-pyridinium; EBSS, Earle’s balanced salt solution; Glib, glibenclamide; GPx, glutathione peroxidase; GSH, reduced glutathione; HEt, hydroethidine; MIB, mitochondrial isolation buffer; mitoKATP, mitochondrial ATP-sensitive potassium channel; MnSOD, manganese-dependent superoxide dismutase; NCX, Na+/Ca2+ exchanger; NMDA, N-methyl-D-aspartate; NR1, NMDAreceptor subunit 1; OGD, oxygen–glucose deprivation; PBS, phosphatebuffered saline; PC, preconditioning; PKC, protein kinase C; ROS, reactive oxygen species; SD, Sprague–Dawley; SNP, sodium nitroprusside; SOD, superoxide dismutase; TMRE, tetramethylrhodamine ethyl ester.

2007 The Authors Journal Compilation 2007 International Society for Neurochemistry, J. Neurochem. (2007) 102, 595–608

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596 T. Ga´spa´r et al.

BEP inhibits fast Na+ channels, L- and T-type Ca2+ channels, and different types of K+ channels (Gill et al. 1992). It has also been shown to block the Na+/Ca2+ exchanger (NCX) system (Kaczorowski et al. 1988) and N-methyl-D-aspartate (NMDA) type glutamate receptors (Sobolevsky et al. 1997). The drug penetrates the cell membrane, acts as a calmodulin antagonist (Gill et al. 1992), and inhibits oxidative phosphorylation (Younes et al. 1977; Matlib 1985). A recent study has reported that BEP pre-treatment protects the heart against ischemia/ reperfusion injury via activation of mitochondrial ATPsensitive potassium (mitoKATP) channels (Sato et al. 2006). Very little is known, however, concerning neuroprotective effects of BEP. In one study, BEP was shown to increase infarct volume when administered 3–22 h following permanent middle cerebral artery occlusion in the rat (Pignataro et al. 2004). In a second study, the neuroprotective effect of the reverse mode NCX activator sodium nitroprusside (SNP) against chemical hypoxia in vitro was reversed by BEP (Amoroso et al. 2000). Contrary to this limited evidence, numerous studies including our own (Domoki et al. 1999; Kis et al. 2003, 2004; Busija et al. 2004; Farkas et al. 2004, 2006; Lenzser et al. 2005; Mayanagi et al. 2007) have shown that activation of mitoKATP channels is neuroprotective in vivo and in vitro against a wide variety of lethal stimuli. Therefore, additional studies are needed to determine whether BEP activates mitoKATP channels and protects neurons against anoxic and chemical stress. The purpose of this in vitro study was to examine whether BEP can induce neuronal PC and to reveal its mechanism of action. We investigated the effect of BEP on mitochondrial membrane potential, reactive oxygen species (ROS) generation, and calcium homeostasis using rat cortical neuronal cultures and isolated rat brain mitochondria.

Materials and methods Materials Cell culture plastics were purchased from Becton-Dickinson (San Jose, CA, USA). Dulbecco’s modified Eagle’s medium, neurobasal medium, B27 supplement, 2-mercaptoethanol, and horse serum were obtained from Gibco BRL (Grand Island, NY, USA), and sucrose from Fisher Scientific (Fair Lawn, NJ, USA). Percoll was purchased from Amersham Biosciences (Uppsala, Sweden), and M40401 from Metaphore Pharmaceuticals (St Louis, MO, USA). CellTiter 96 AQueous One Solution assay and SV Total RNA Isolation kit were procured from Promega (Madison, WI, USA). Hydroethidine (HEt), Fluo-4 AM, Pluronic F-127, tetramethylrhodamine ethyl ester (TMRE), 4-{2-[6-(dioctylamino)-2-naphthalenyl]ethenyl}-1-(3-sulfopropyl)-pyridinium (di-8-ANEPPS), and Amplex Red catalase assay kit were purchased from Molecular Probes (Eugene, OR, USA). Superoxide dismutase (SOD) assay kit was purchased from Fluka (Buchs, Switzerland) and glutathione peroxidase (GPx) assay kit from Chayman Chemical Company (Ann Arbor, MI, USA). Taqman gene expression assay for catalase

(assay identification number: RN00560930_m1) was procured from Applied Biosystems (Foster City, CA, USA). Antibodies were obtained from the following sources: anti-glial fibrillary acidic protein antibody from Chemicon (Temecula, CA, USA); antimicrotubule-associated protein-2 antibody, monoclonal anti-manganese-dependent superoxide dismutase (MnSOD), and monoclonal anti-Bcl-2 antibodies from Becton-Dickinson; polyclonal antiphospho-protein kinase C (PKC) (pan) antibody from Cell Signaling Technology (Danvers, MA, USA); polyclonal anti-catalase antibody from Calbiochem (San Diego, CA, USA); polyclonal antibody to GPx 1 from Abcam (Cambridge, MA, USA); monoclonal antiNMDA-receptor subunit 1 (NR1) antibody from Upstate (Lake Placid, NY, USA); and anti-rabbit IgG and anti-mouse IgG from Jackson Immuno-Research (West Grove, PA, USA). All other chemicals were from Sigma (St Louis, MO, USA). Primary rat cortical neuronal culture All animals were maintained and used in compliance with the principles set forth by the Animal Care and Use Committee of Wake Forest University Health Sciences. Timed pregnant Sprague– Dawley (SD) rats were obtained from Harlan (Indianapolis, IN, USA). As described previously, primary rat cortical neurons were isolated from E18 SD fetuses (Kis et al. 2003). For confocal microscopic analysis, the cells were plated onto poly-L-lysine coated glass coverslips at a density of 2 · 105 cells/cm2. For the other experiments, 106 cells/cm2 were seeded onto poly-D-lysine coated plates or dishes in plating medium consisting of 60% Dulbecco’s modified Eagle’s medium, 20% Ham’s F-12 nutrient mixture, 20% horse serum, and L-glutamine (0.5 mmol/L). The cultures were maintained in a humidified 5% CO2 incubator at 37C within normoxic conditions. After cell attachment, the plating medium was replaced with Neurobasal medium supplemented with B27 (2%), L-glutamine (0.5 mmol/L), 2-mercaptoethanol (55 lmol/L), and KCl (25 mmol/L). Positive immunostaining for microtubule-associated protein-2 and negative immunostaining for glial fibrillary acidic protein verified that the cultures consisted of more than 99% of neurons on day 7 in vitro. Experiments were carried out in 7 to 9-day-old cultures. During this period, neurons expressed NMDA, a-amino-3-hydroxy-5-methylisoxazole-4-propionate, and kainate receptors. In addition, they were vulnerable to glutamate cytotoxicity and glucose deprivation (Mattson et al. 1991, 1993). Treatment with BEP To induce delayed PC, 7-day-old neuronal cultures were treated with different doses of BEP (1, 2.5, 5, and 10 lmol/L) in the regular culture medium once a day for three consecutive days. In other experiments, the cells were co-treated once a day for 3 days with 5 lmol/L BEP and with either the SOD-mimetic M40401 (50 lmol/ L), catalase (200 U/mL), reduced glutathione (GSH; 200 lmol/L), the reverse mode NCX activator SNP (300 lmol/L), the K+ channel blocker glibenclamide (Glib; 5 lmol/L), or with the putatively selective mitoKATP channel antagonist 5-hydroxydecanoic acid (5HD; 1 mmol/L). The selected doses of these compounds did not induce neurotoxicity during preliminary experiments. Oxygen–glucose deprivation Neurons in 96-well plates were exposed to oxygen–glucose deprivation (OGD) for 180 min at 37C 24 h after the last


Neuronal preconditioning with bepridil 597

treatment, using a protocol described previously (Goldberg and Choi 1993; Kis et al. 2003). Briefly, the cells were rinsed three times with phosphate-buffered saline (PBS) and then the cultures were placed in a ShelLab Bactron Anaerobic Chamber (Sheldon Manufacturing Inc., Cornelius, OR, USA) filled with a humidified anaerobic gas mixture (5% CO2, 5% H2, and 90% N2), and the medium was replaced with deoxygenated glucose-free Earle’s balanced salt solution (EBSS). Control cell cultures were incubated in glucose-containing (5.5 mmol/L) EBSS in a regular 5% CO2 cell culture incubator for 180 min. OGD was terminated by replacing the glucose-free EBSS with the regular culture medium and thereafter the cultures were maintained in the regular 5% CO2 incubator within normoxic conditions. Glutamate excitotoxicity Cell cultures in 96-well plates were washed three times with PBS 24 h after the last treatment, and then were exposed to glutamate (200 lmol/L) in the regular culture medium for 60 min at 37C in the 5% CO2 incubator. Afterward the cells were rinsed and returned to the 5% CO2 incubator in the regular culture medium. Quantification of neuronal survival Cell viability in neuronal cultures was determined 24 h after the neurotoxic insults with a FLUOstar OPTIMA microplate reader (BMG Labtech GmbH, Offenburg, Germany) using the tetrazolium-based CellTiter 96 AQueous One Solution assay (kabs = 492 nm), as described previously (Kis et al. 2003). Comparisons were made to sister cultures exposed to the same neurotoxic stimulus on the same day, and cell viability was expressed as a percentage of the corresponding control culture (untreated and not exposed to the lethal insult): % viabilitySAMPLE ¼ ðabsorbanceSAMPLE absorbanceBACKGROUND Þ 100=ðabsorbanceCONTROL absorbanceBACKGROUND Þ: Isolation of rat brain mitochondria Brain mitochondria were isolated using a discontinuous Percoll gradient as described previously, with some modifications (Sims 1990; Rajapakse et al. 2001). All the instruments and buffers were kept ice-cold during the procedure. Male SD rats (280–320 g; Harlan) were over-anesthetized with isoflurane and decapitated. The brain, without the cerebellum, was removed and weighed and was then homogenized in mitochondrial isolation buffer [MIB; sucrose, 250 mmol/L; K+-EDTA, 0.5 mmol/L; Tris–HCl (pH 7.4), 10 mmol/L; and bovine serum albumin, 1%] using Dounce homogenizers and glass pestles (Kontes Glass Co., Vineland, NJ, USA). The homogenate was centrifuged for 3 min at 500 g, and then the supernatant was collected and resuspended in equal amounts of 24% Percoll in MIB. This suspension was layered onto a discontinuous Percoll gradient (24% and 40% in MIB). The gradient was centrifuged for 5 min at 28 000 g and the layer between the 24% and 40% Percoll suspensions containing the purified mitochondria was collected. The preparation was washed in MIB and centrifuged for 12 min at 15 000 g. For confocal microscopic analysis, the pellet was transferred into cell culture dishes with poly-D-lysine coated glass bottom and was centrifuged for 5 min at 500 g. For fluorescent plate reader measurements, protein content was determined, and then equal amounts of mitochondria were transferred in MIB into black-walled 96-well plates.

Determination of mitochondrial membrane potential The changes of mitochondrial membrane potential (DYm) were analyzed using the DYm-sensitive dye TMRE. Neuronal cultures and isolated mitochondria were loaded in the dark with TMRE (0.5 lmol/ L) at 37C in a 5% CO2 incubator for 20 min, and then were rinsed three times. Experiments were performed in glucose-containing (1 mg/mL) PBS (neurons) or MIB (mitochondria) at 37C. To measure immediate changes, confocal images of cellular TMRE-fluorescence (kex = 543 nm and kem > 560 nm) were acquired using a laser-scanning microscope (LSM 510; Zeiss, Jena, Germany) with a 63X water immersion objective (Zeiss). Fluorescent images were recorded every 20 s for 10 min after the application of vehicle or BEP (1, 5, and 10 lmol/L). In some experiments, 5HD (1, 2, and 5 mmol/L) was also co-applied with the highest dose (10 lmol/L) of BEP. The average pixel intensity of individual cell bodies and mitochondria was determined using the software supplied by the manufacturer (Zeiss). To analyze delayed effects on DYm, neuronal cultures were treated with BEP (5 lmol/L) and 5HD (1, 2, and 5 mmol/L) once a day for 3 days, then 24 h after the last treatment, TMREfluorescence in each well was measured using the same microplate reader used for viability measurements (kex = 510 nm and kem = 590 nm). In both cases, data were expressed as a percentage of the starting intensity of the untreated control culture: %TMRE-fluorescenceSAMPLE ¼ ðTMRE-fluorescenceSAMPLE TMRE-fluorescenceBACKGROUND Þ 100=ðTMRE-fluorescenceCONTROL TMRE-fluorescenceBACKGROUND Þ: Measurement of plasma membrane potential Plasma membrane potential was monitored using the voltagesensitive dye di-8-ANEPPS. Neuronal cultures were loaded with 1 lmol/L di-8-ANEPPS in the dark for 30 min at 37C, and then were washed three times with PBS. Confocal images of cellular di8-ANEPPS fluorescence were acquired with the same laser-scanning microscope used for mitochondrial membrane potential measurements. Fluorescent images (kex = 488 nm, kem1 > 650 nm, and kem2 = 500–550 nm) of randomly selected fields were recorded every 20 s for 10 min. The tested compounds (BEP, 10 lmol/L and NS1619, 20 lmol/L) were administered after 60 s. The average pixel intensity of individual cell bodies was determined using the software supplied by the manufacturer (Zeiss). Ratio of emissions was calculated and data were expressed as a percentage of the starting ratio of the corresponding control culture using the following equations: di-8-ANEPPS-fluorescent ratioSAMPLE ¼ (di-8-ANEPPS-fluorescenceSAMPLE at kem1 Þ =(di-8-ANEPPS-fluorescenceSAMPLE at kem2 Þ and %di-8-ANEPPS-fluorescent ratioSAMPLE ¼ (di-8-ANEPPS-fluorescent ratioSAMPLE di-8-ANEPPS-fluorescent ratioBACKGROUND Þ 100/(di-8-ANEPPS-fluorescent ratioCONTROL di-8-ANEPPS-fluorescent ratioBACKGROUND Þ:



Analysis of ROS formation Reactive oxygen species generation was assessed in 96-well plates with HEt using the same microplate reader used for the viability measurements (kex = 510 and kem = 590 nm). Cultured neurons and isolated mitochondria were washed then loaded with HEt (5 lmol/L) in glucose-containing (1 mg/mL) PBS (neurons) or MIB (mitochondria) 1 min before the assay. HEt-fluorescence in each well was measured at 37C every minute for 30 min after the application of the tested compounds or the vehicle. Data were expressed as a percentage of the starting intensity of the untreated control: %HEt-fluorescenceSAMPLE ¼ ðHEt-fluorescenceSAMPLE HEt-fluorescenceBACKGROUND Þ 100=ðHEt-fluorescenceCONTROL HEt-fluorescenceBACKGROUND Þ: Monitoring intracellular free calcium levels Changes of intracellular free calcium level ([Ca2+]i) were monitored using the Ca-indicator dye Fluo-4 AM in glucose-containing (1 mg/ mL) PBS. Neuronal cultures were loaded with 2 lmol/L Fluo-4 AM and 1 lmol/L Pluronic F-127 in PBS in the dark for 60 min at room temperature (22C), and then were washed three times. Glutamate (200 lmol/L) or NMDA (100 lmol/L) was applied into the medium and confocal images of cellular Fluo-4 AM fluorescence (kex = 488 nm and kem = 520 nm) were acquired using the same confocal microscopy system used for TMRE-measurements. Images were recorded every 20 s for 10 min and the average pixel intensity of individual cell bodies was determined using the software supplied by the manufacturer (Zeiss). Data were expressed as a percentage of the starting intensity of the untreated control culture: ð% Fluo-4fluorescenceSAMPLE ¼ ðFluo-4fluorescenceSAMPLE Fluo-4fluorescenceBACKGROUND Þ 100=ðFluo-4fluorescenceCONTROL Fluo-4fluorescenceBACKGROUNDÞ: Western blotting for phosphorylated PKC, MnSOD, catalase, GPx, Bcl-2, and NR1 Cultured cells were harvested by scraping in ice-cold Nonidet P-40 lysis buffer supplemented with proteinase inhibitors (aprotinin, 1 lg/mL; phenylmethylsulfonyl fluoride, 50 lg/mL; and leupeptin; 1 lg/mL), and a phosphatase inhibitor cocktail (EDTA, 1 mmol/L; sodium o-vanadate, 1 mmol/L; benzamidine, 10 lg/mL; sodium pyrophosphate, 1 mmol/L; and sodium fluoride; 1 mmol/L). For each sample, equal amounts of protein were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a polyvinylidine difluoride sheet (Polyscreen PVDF; Perkin Elmer Life Sciences, Boston, MA, USA). Membranes were incubated in a blocking buffer (Tris-buffered saline, 0.1% Tween 20, and 5% skimmed milk powder) for 1 h at room temperature (22C) followed by incubation with polyclonal anti-phospho-PKC (pan) (1 : 3000), monoclonal anti-MnSOD (1 : 3000), polyclonal anti-catalase (1 : 4000), polyclonal anti-GPx 1 (1 : 2000), monoclonal anti-Bcl-2 (1 : 500), and monoclonal anti-NR1 (1 : 500) antibodies overnight at 4C. The membranes were then washed three times in Tris-buffered saline with 0.1% Tween 20 and incubated for 1 h in the blocking

buffer with anti-rabbit IgG (1 : 50 000) or anti-mouse IgG (1 : 5000) conjugated to horseradish peroxidase. The final reaction products were visualized using enhanced chemiluminescence (SuperSignal West Pico; Pierce, Rockford, IL, USA) and recorded on X-ray film. Enzyme activity assay for MnSOD Manganese-dependent superoxide dismutase activity was measured using the tetrazolium-based SOD assay kit as directed by the manufacturer. Neurons on 96-well plates were pre-treated with BEP (5 lmol/L) for 1, 2, or 3 days, and then 24 h after the last treatment the cells were incubated with NaCN (5 mmol/L) for 10 min to inhibit copper–zinc SOD activity. Subsequently, the plates were rinsed three times with PBS and enzyme activity was measured with the same microplate reader that was used for the viability measurements (kabs = 460 nm). Data were expressed as a percentage of the activity of the untreated control culture using the following equations: SOD activitySAMPLE ¼ f½ðabsorbancePOSITIVE CONTROL absorbanceNEGATIVE CONTROL Þ ðabsorbanceSAMPLE absorbanceBACKGROUND Þ=ðabsorbancePOSITIVE CONTROL absorbanceNEGATIVE CONTROL Þg 100 and %SOD activitySAMPLE ¼ SOD activitySAMPLE 100=SOD activityCONTROL : Catalase assay Catalase activity was assessed with the Amplex Red catalase activity assay kit. Neurons on 96-well plates were pre-treated with BEP (5 lmol/L) for 1, 2, or 3 days, then 24 h after the last treatment the plates were rinsed three times with PBS, and enzyme activity was measured with the same microplate reader that was used for other fluorescent measurements (kex = 555 nm and kem = 590 nm). A standard curve was generated for each measurement to calculate catalase activity of wells. Data were expressed as a percentage of the activity of the untreated control culture using the following equation: % catalase activitySAMPLE ¼ catalase activitySAMPLE 100=catalase activityCONTROL : GPx assay Glutathione peroxidase activity was measured with the Gpx assay kit as directed by the manufacturer. The assay measures GPx activity indirectly by the oxidation of NADPH to NADP+. Neurons on 96well plates were pre-treated with BEP (5 lmol/L) for 1, 2, or 3 days, then 24 h after last treatment the plates were rinsed three times with PBS, and changes in absorbance at 350 nm were measured every minute for 10 min with the same microplate reader as that used for the viability measurements. Data were expressed as a percentage of the activity of the untreated control culture using the following equations: GPx activitySAMPLE ¼ ½ðchange of absorbanceSAMPLE min change of absorbanceBACKGROUND minÞ=0:00373lmol=L ð0:19mL=0:02mLÞ dilution (where 0.00373 lmol/L is the adjusted NADPH extinction coefficient, 0.02 mL is the sample volume, and 0.19 mL is the final volume) and



% GPx activitySAMPLE ¼ GPx activitySAMPLE 100=GPx activityCONTROL : Real time RT-PCR for catalase After pre-treatment for 1, 2, or 3 days with BEP (5 lmol/L), RNA was isolated from neurons using the SV Total RNA Isolation kit as directed by the manufacturer. The RNA was incubated with DNase to eliminate any residual DNA that would amplify during the polymerase chain reaction. Real time quantitative RT-PCR experiments were carried out in the ABI/ Prism 7000 Sequence Detection System (Applied Biosystems). From each sample, 25 pg total RNA was reverse transcribed and amplified using QuantiTect Probe RT-PCR Kit (Qiagen, Valencia, CA, USA) and a cycle program as follows: 30 min at 50C,

Fig. 1 Bepridil (BEP) causes mitochondrial depolarization. The mitochondrial membrane potential was measured using tetramethylrhodamine ethyl ester (TMRE). Isolated rat brain mitochondria (a) and cultured rat cortical neurons (b) were subjected to different doses of BEP (1, 5, and 10 lmol/L) and the intensity of TMRE-fluorescence was recorded using confocal microscopy. Another set of cortical neurons (c) were treated with the putative selective mitochondrial ATP-sensitive potassium (mitoKATP) channel antagonist 5-hydroxydecanoic acid (5HD; 1, 2, and 5 mmol/L) 5 min before the application of BEP (10 lmol/L). Fluorescent intensity of the starting value was

15 min at 95C, and then 40 cycles each at 94C for 15 s and 60C for 60 s. All amplifications were conducted with the predeveloped Taqman gene expression assay for catalase. All reactions were performed in triplicate with b-actin serving as an internal control. Relative quantification of the mRNA expression levels of target genes was calculated using the 2)DDCT method (Schmittgen et al. 2000). Statistical analysis Statistical analysis was performed with SigmaStat (SPSS, Chicago, IL, USA). Data are presented as means ± SEM. Differences between groups were assessed by one-way analysis of variance followed by Tukey comparison tests. A value of p < 0.05 was considered to be statistically significant.

considered 100%. *First significant difference (p < 0.05) compared with untreated control. Data are expressed as mean ± SEM; cells from at least two individual cultures (n = 55–176); mitochondria from at least four animals (n = 16–32). Three-day co-treatment with 5HD (1, 2, and 5 mmol/L) did not affect the mitochondrial depolarizing effect of BEP (5 lmol/L) measured with TMRE 24 h after the last treatment using a fluorescent microplate reader (d). #Significant difference (p < 0.05) compared with untreated control. Data are expressed as mean ± SEM; cells from at least two individual cultures (n = 16–32).



Results

BEP depolarized mitochondria but did not affect plasma membrane potential Application of BEP resulted in the reduction of mitochondrial membrane potential, measured as a decrease in the intensity of TMRE-fluorescence, in both isolated brain mitochondria (Fig. 1a) and cultured cortical neurons (Fig. 1b). This effect was dose-dependent: the highest concentration of BEP (10 lmol/L) caused the most robust depolarization, resulting in the total loss of mitochondrial membrane potential in isolated mitochondria. The depolarizing effect of BEP on mitochondria in situ was weaker and could not be inhibited with 5HD (Figs 1c and d), but was maintained for 3 days (Fig. 1d). On the other hand, BEP (10 lmol/L) had no acute effect on the plasma membrane potential of cultured neurons, while the calcium-activated potassium channel opener NS1619 (20 lmol/L) induced hyperpolarization shown as an increase in the di-8-ANEPPS-fluorescence ratio (Fig. 2).

cells and isolated mitochondria. Administration of BEP (10 lmol/L) induced a significant elevation of ROS generation in isolated mitochondria (Fig. 3a). A similar increase in ROS production was seen in neuronal cultures after BEP application (Fig. 3b). This effect was completely inhibited with the SOD-mimetic M40401. BEP-induced delayed PC against OGD and glutamate excitotoxicity One-day treatment with BEP did not induce neuroprotection (data not shown); therefore, a once-daily treatment protocol for 3 days was used to induce delayed PC. Preliminary data showed that 1, 6, and 24 h daily treatments for 3 days

BEP application increased ROS production The ethidium signal increased modestly over time in the control groups because of the basal ROS formation by the

Fig. 2 Bepridil (BEP) does not affect the plasma membrane potential. Plasma membrane potential was assessed with the voltage-sensitive dye, 4-{2-[6-(dioctylamino)-2-naphthalenyl]ethenyl}-1-(3-sulfopropyl)pyridinium (di-8-ANEPPS). Neuronal cultures were challenged with either BEP (10 lmol/L) or the calcium-activated potassium channel opener NS1619 (20 lmol/L) and the intensity of di-8-ANEPPS fluorescence was recorded using confocal microscopy. The ratio of emissions (kem1 > 650 nm and kem2 = 500–550 nm) was calculated and the fluorescent intensity ratio of the starting value was considered 100%. While NS1619 hyperpolarized the cells, BEP had no acute effect on the plasma membrane potential. *First significant difference (p < 0.05) compared with untreated control. Data are expressed as mean ± SEM; cells from at least two individual cultures (n = 74–105).

Fig. 3 Bepridil (BEP) induces reactive oxygen species generation. Free radical production in isolated mitochondria (a) and cultured neurons (b) was measured with a fluorescent microplate reader using hydroethidine (HEt) after exposure to vehicle (control) or BEP (10 lmol/L). A set of neurons in panel (b) was treated with the SODmimetic M40401 (50 lmol/L) 2 min before the application of BEP. Fluorescent intensity of the starting value was regarded as 100%. *First significant difference (p < 0.05) compared with untreated control. Values represent mean ± SEM; cells from at least two individual cultures (n = 68–105); mitochondria from at least four animals (n = 14–32).



induced increasing tolerance against both OGD (viability: untreated, 48.8 ± 0.73%; BEP 5 lmol/L for 1 h daily, 81.3 ± 0.57%*; BEP 5 lmol/L for 6 h daily, 90.5 ± 0.61%*; BEP 5 lmol/L for 24 h daily, 92.8 ± 1.47%*; data shown as mean ± SEM, n = 32–64; *p < 0.05 compared with the untreated group) and glutamate excitotoxicity (viability: untreated, 51.2 ± 0.75%; BEP 5 lmol/L for 1 h daily, 72.7 ± 0.83%*; BEP 5 lmol/L for 6 h daily, 83.5 ± 0.87%*; BEP 5 lmol/L for 24 h daily, 85.8 ± 1.36%*; data shown as mean ± SEM, n = 32–64; *p < 0.05 compared with the untreated group) with the best protection after the longest daily treatment. Therefore, the 24 h protocol was chosen for the further experiments. The 3-day treatment with concentrations of BEP below 10 lmol/L had no toxic effect on the survival of quiescent cells, whereas doses above 10 lmol/L destroyed neurons (cell viability: untreated, 100 ± 0.85%; BEP 1 lmol/L, 97.1 ± 0.94%; BEP 2.5 lmol/L, 98.6 ± 0.76%; BEP 5 lmol/L 104.6 ± 0.58%; BEP 10 lmol/L, 102.5 ± 2.99%; BEP 20 lmol/L, 40.9 ± 0.54%*; data shown as mean ± SEM, n = 32–64; *p < 0.05 compared with untreated control). The 10 lmol/L dose seemed to be the threshold concentration in this context: in some experiments, it had no toxic effect, while in others it caused mild but significant cell death. A dose of 20 lmol/L, however, proved always to be toxic. In our experiments, therefore, we used 1, 2.5, 5, and 10 lmol/L of BEP to induce delayed PC. Three-day treatment with BEP induced a dosedependent protection against 180 min of OGD, with the best result in the 5 lmol/L BEP group, whereas cell survival in the 10 lmol/L BEP group was somewhat worse (Fig. 4a). Exposure to glutamate (200 lmol/L) for 60 min destroyed almost 50% of untreated neurons. Treatment with BEP, however, dose-dependently protected neurons against glutamate excitotoxicity resulting in almost 100% cell survival in the 10 lmol/L BEP group (Fig. 4b). Acute PC experiments were also performed [treatment with BEP (1–50 lmol/ L) for 15–60 min, followed by either OGD or glutamate excitotoxicity 30 min later], but no protection could be found (data not shown). 5HD and Glib did not block the neuroprotection When neuronal cultures were co-treated with BEP (5 lmol/ L) and with either 5HD (1 mmol/L; Fig. 5a) or Glib (5 lmol/ L; Fig. 5b) for 3 days, the viability of the cells was not altered within basal conditions. When exposed to OGD (180 min), none of the K+ channel blockers diminished the protection induced by BEP. M40401 antagonized the neuroprotective effect of BEP Co-treatment for 3 days with BEP (5 lmol/L) and with either the SOD-mimetic M40401 (50 lmol/L), catalase (200 U/mL), or GSH (200 lmol/L) did not influence the survival of neurons under control conditions (Fig. 5c). However, when the cells were exposed to OGD for

Fig. 4 Bepridil (BEP) induces delayed preconditioning in neurons. Cultured cortical neurons were treated with different doses of BEP once a day for 3 days, then 24 h after the last treatment the cells were exposed to either oxygen and glucose deprivation (OGD) for 180 min (a) or glutamate (200 lmol/L) for 60 min (b). BEP induced a dosedependent protection against both insults. The viability of cell cultures exposed to the toxic insults (gray bars) was expressed as percent of the viability of control cultures which were not treated with BEP and were not exposed to any lethal stimuli (open bars). *Significant difference (p < 0.05) compared with untreated control. #Significant difference (p < 0.05) compared with untreated cultures which were exposed to either OGD or glutamate. Data are expressed as mean ± SEM; cells from at least two individual cultures (n = 16–32).

180 min, M40401 completely abolished the neuroprotective effect of BEP resulting in pronounced cell death, whereas catalase and GSH had no effect on the protection (Fig. 5d). The reverse mode NCX activator SNP (300 lmol/L) did not influence the survival of quiescent neurons and did not antagonize the protection induced by 3-day treatment with BEP (Fig. 5e). PC with BEP prevented ROS generation during glutamate exposure In neurons pre-treated with BEP (5 lmol/L) for 3 days prior to measurement, the moderately increasing baseline curve of



Fig. 5 Superoxide anion generation but not the activation of the mitochondrial ATP-sensitive K+ (mitoKATP) or the inhibition of the Na+/ Ca2+ exchanger (NCX) is needed to induce the preconditioned state. Neuronal cultures were co-treated with bepridil (BEP) (5 lmol/L) and either 5-hydroxydecanoate (5HD) (1 mmol/L) (a), glibenclamide (Glib; 5 lmol/L) (b), the SOD-mimetic M40401 (50 lmol/L), catalase (200 U/ mL), and GSH (200 lmol/L) (c and d), or the reverse mode NCX activator sodium nitroprusside (SNP; 300 lmol/L) (e) once a day for 3 days, then were exposed to oxygen–glucose deprivation (OGD) (180 min) 24 h after the last treatment. The K+ channel inhibitors did not affect the neuroprotection induced by BEP. The tested antioxi-

dants had no influence on the viability of resting cells (c). When exposed to OGD (d), M40401 completely blocked the neuroprotective effect of BEP, while the hydrogen peroxide scavengers were ineffective. SNP had no effect on neuronal survival. The viability of cell cultures that were treated with any of the drugs or were exposed to OGD (gray bars) was expressed as percent of the viability of control cultures which were not treated with the drugs and were not exposed to OGD (open bars). *Significant difference (p < 0.05) compared with control. #Significant difference (p < 0.05) compared with untreated cultures that were exposed to OGD. Data are expressed as mean ± SEM; cells from at least two individual cultures (n = 16–32).



untreated, the translational level (2)DDCT-values: 99.3 ± 2.57%; BEP 5 lmol/L for 24 h, 129.0 ± 4.07%*; BEP 5 lmol/L for 48 h, 189.5 ± 2.87%*; BEP 5 lmol/L for 72 h, 216.3 ± 6.52%*; data shown as mean ± SEM, n = 5; *p < 0.05 compared with untreated control).

Fig. 6 Preconditioning with bepridil (BEP) inhibits excessive reactive oxygen species (ROS) generation in neurons upon exposure to glutamate. ROS production in cultured cortical neurons was assessed with a fluorescent microplate reader using hydroethidine (HEt) after exposure to vehicle (control) or glutamate (200 lmol/L). Two groups of cells were treated with BEP (5 lmol/L), and a third group with BEP (5 lmol/L) and M40401 (50 lmol/L) once a day for 3 days before the experiment. Fluorescent intensity at the starting point was regarded as 100%. The basal ROS generation of the BEP-treated group did not differ from that of the control group. BEP pre-treatment blocked the ROS surge during glutamate exposure. M40401 counteracted this effect, resulting in similar increase in ROS production as in the untreated control group. *First significant difference (p < 0.05) compared with untreated control. #First significant difference (p < 0.05) compared with untreated, glutamate-stimulated group. Data are expressed as mean ± SEM; cells from at least two individual cultures (n = 16– 32).

HEt-fluorescence did not differ from that of untreated control cells. Exposing control cells to glutamate (200 lmol/L) resulted in a significant increase in the HEt-signal, suggesting an elevation in ROS generation, whereas no change in the fluorescent intensity of BEP-treated cells was found upon glutamate challenge. After co-treatment with M40401 (50 lmol/L) and BEP (5 lmol/L), glutamate exposure induced an increase in ROS production similar to that seen in control cultures (Fig. 6). BEP-treatment induced PKC activation and increased the expression and activity of cytoprotective proteins Western blot analysis revealed augmented expression of phosphorylated PKC after 12 h of incubation with 5 lmol/L BEP (Fig. 7a). Three-day treatment with BEP (5 lmol/L) resulted in elevated protein levels of MnSOD (Fig. 7b), GPx (Fig. 7c), catalase (Fig. 7d), and Bcl-2 (Fig. 7e) with the highest increases after the longest period of treatment. A parallel increase was detected in the enzymatic activity of MnSOD and GPx, whereas the activity of catalase did not change (Table 1). Therefore, real time RT-PCR was performed for catalase mRNA, which showed an increase at the transcriptional level similar to that seen in the western blot at

PC with BEP reduced the Ca2+ surge upon exposure to glutamate and NMDA In quiescent cells, the intensity of Fluo-4 fluorescence did not change over time. Exposure of control neurons to glutamate (200 lmol/L) resulted in an immediate eightfold increase in the signal. The increase of fluorescent intensity was much less in neurons incubated with BEP (5 lmol/L) for 3 days. Ten minutes later, at the end of the measurement, however, no significant difference between the stimulated signals was observed (Fig. 8a). Similar to glutamate, a challenge with NMDA (100 lmol/L) caused a significantly higher peak value in the [Ca2+]i of control neurons. Additionally, neurons preconditioned with BEP eliminated the excess of [Ca2+]i after NMDA much faster than control cells (Fig. 8b). BEP treatment did not alter the expression of NR1 (Fig. 8c). Discussion

The main findings of the present study are: (i) BEP elicits ROS generation and mitochondrial depolarization in both neurons and isolated brain mitochondria; (ii) BEP induces delayed but not immediate PC in rat cortical neuronal cultures; (iii) neuroprotective effects of BEP are diminished by co-treatment with the SOD-mimetic M40401 but not by hydrogen peroxide scavengers, K+ channel blockers, or by an NCX activator; (iv) PC with BEP prevents the ROS surge and reduces Ca2+-influx upon exposure to glutamate; (v) BEP treatment induces sequential activation of PKC, augmented levels of antioxidants, and Bcl-2. We conclude that delayed PC by BEP is due to ROS/PKC signaling events, which lead to enhanced levels of proteins, which limit injury to cells by reducing oxidative stress upon exposure to potentially lethal stimuli. We are unaware of other studies concerning BEP and delayed PC. Bepridil has been used in the treatment of chronic stable angina pectoris (Hollingshead et al. 1992). Recent studies have shown that BEP has diverse pharmacological effects on both cell membrane and intracellular targets (Younes et al. 1977; Matlib 1985; Kaczorowski et al. 1988; Gill et al. 1992; Sobolevsky et al. 1997; Chen and Jan 2001; Watanabe and Kimura 2001). While in previous neuroprotective studies BEP was administered during or after the toxic stimulus and was shown to increase cell death in vitro and infarct volume in vivo (Amoroso et al. 2000; Tortiglione et al. 2002; Pignataro et al. 2004); a recent paper has reported that pretreatment with BEP induces acute PC in the heart via activation of mitoKATP channels (Sato et al. 2006). However,



Fig. 7 Bepridil (BEP) treatment induces the overexpression of cytoprotective proteins. Neuronal cultures were treated with BEP (5 lmol/ L) for 1, 3, 6, 12, and 24 h, then proteins were extracted and subjected to western blot analysis for phosphorylated PKC (a). BEP induced the overexpression of phosphorylated PKC after 12 h of treatment. For manganese-dependent superoxide dismutase (MnSOD) (b), glutathione peroxidase (GPx) (c), catalase (d), and Bcl-2 (e), neuronal cultures were treated with BEP (5 lmol/L) once a day for 1–3 days, then proteins were extracted and subjected to western blot analysis. BEP

treatment induced a significant elevation in the protein levels of MnSOD, GPx, and catalase after 2 days and Bcl-2 after 1 day. The intensity of bands was normalized to that of b-actin, and the normalized level of the examined protein in the untreated control group (open bar) was considered 100%. Representative western blots are shown below the graphs. *Significant difference (p < 0.05) compared with untreated control. Data are expressed as mean ± SEM; cells from at least two individual cultures (n = 4).

whether pre-treatment with BEP can provide immediate or delayed PC in the central nervous system has not previously been investigated.

In our experiments, application of BEP did not change the plasma membrane potential of cultured neurons, but depolarized mitochondria and increased ROS levels in both



Table 1 Activity of antioxidants after different periods of treatment with BEP Untreated control (%)

BEP 24 h (%)

BEP 48 h (%)

BEP 72 h (%)

MnSOD 100.0 ± 11.4 140.6 ± 11.6 189.8 ± 15.3* 201.9 ± 16.5* Catalase 100.0 ± 5.89 104.5 ± 0.48 101.5 ± 0.91 102.5 ± 1.07 GPx 100.0 ± 8.47 109.7 ± 8.14 119.5 ± 6.99 137.4 ± 8.05* Cultured neurons in 96-well plates were treated with BEP (5 lmol/L) for 1, 2, or 3 days, then the enzyme activity of manganese-dependent superoxide dismutase (MnSOD), catalase, and glutathione peroxidase (GPx) was measured using commercially available kits. Whereas BEP-treatment induced increased activity of MnSOD and GPx, no change in the activity of catalase was observed. The activity of the tested enzyme in the untreated control group was regarded as 100%. *Significant difference (p < 0.05) compared with the activity of untreated control. Data are expressed as mean ± SEM; cells from at least two individual cultures (n = 14–32). BEP, bepridil.

isolated mitochondria and cultured neurons, and these effects were similar in magnitude and timing to previous findings using the prototypical mitoKATP channel opener diazoxide (Kis et al. 2003). Reduced membrane potential was maintained for more than 3 days, however, unlike results with diazoxide, this effect could not be antagonized with the putatively selective mitoKATP channel antagonist, 5HD, suggesting the involvement of non-mitoKATP channeldependent mechanisms. We hypothesize that both the increased ROS levels and the depolarization of mitochondria may be due to inhibitory effects of BEP on the mitochondrial electron transport chain (Younes et al. 1977; Matlib 1985) rather than to the opening of the mitoKATP channel. Similar results were reported on mitochondrial membrane potential after BEP-application by Storozhevykh et al. (1996). These authors speculated that the collapse of mitochondrial membrane potential resulted from the inhibitory action of the compound on respiration and oxidative phosphorylation. Despite these acute effects, BEP did not induce early PC in cultured neurons. The reason why immediate PC with BEP could be observed in the heart but not in neuronal cultures is unclear, but may be due to differences in the cellular physiology and/or experimental conditions; nevertheless, a similar phenomenon has been shown following diazoxide treatment (Kis et al. 2003). In contrast to the results with immediate PC, we found BEP to be an effective inducer of delayed PC. Thus, treatment of neurons with BEP for 3 days caused a time- and dose-dependent protection against OGD and glutamate excitotoxicity. Similar to the findings concerning mitochondrial membrane potential, 5HD and the non-selective K+ channel blocker, Glib, failed to block delayed PC. Addition-

Fig. 8 Preconditioning with bepridil (BEP) reduces intracellular calcium load upon stimulation with either glutamate or NMDA. Intracellular free calcium ([Ca2+]i) levels in cultured neurons were measured by a confocal microscope using Fluo-4 AM during exposure to either glutamate (200 lmol/L) (a) or NMDA (100 lmol/L) (b). To induce preconditioning, two groups of neurons were treated with BEP (5 lmol/L) once a day for 3 days prior to measurement. Fluorescent intensity of the untreated control at the starting point was regarded as 100%. *Significant difference (p < 0.05) compared with untreated control. #First significant difference (p < 0.05) compared with untreated control. Data are expressed as mean ± SEM; cells from at least two individual cultures (n = 40–203). (c) Representative western blot of NMDA receptor subunit 1 (NR1). Neuronal cultures were treated with BEP (5 lmol/L) once a day for 1–3 days, then proteins were extracted and subjected to western blot analysis. No significant change in the protein level of NR1 was found.

ally, the lack of effect of SNP indicates that inhibition of NCX signaling is not involved in the induction of PC either. Free radicals are known to play an important role in the initiation of PC. ROS can act as second messengers and directly activate protein kinases (Oldenburg et al. 2003; Otani 2004). Furthermore, eliminating ROS during the PC phase abolishes the protection, including delayed PC with



diazoxide (Ravati et al. 2001; Kis et al. 2003; Nagy et al. 2004; Nie et al. 2006). As our experiments showed that BEP augmented ROS generation in both neuronal cells and isolated mitochondria, we examined whether free radicals produced during the early phase of BEP treatment have any role in the initiation of neuroprotection. Therefore, we coincubated neuronal cultures with BEP and the SOD-mimetic M40401, catalase, or GSH for 3 days, and we found that M40401 but not the hydrogen peroxide scavengers eliminated the protection, suggesting that the early increase in superoxide generation after BEP application is essential for the preconditioned state. Additionally, we found elevated levels of phosphorylated PKC 12 h after the initiation of BEP-treatment. Free radicals can directly activate PKC (Cohen et al. 2000), which in turn promotes the activation of transcription factors such as nuclear factor-jB, mitogenactivated protein kinases, antioxidants, and other elements of the neuroprotective pathways (for a review see Otani 2004). Decreased ROS availability and attenuated Ca2+ influx are key elements of the cytoprotective effect of PC (Wang et al. 2001; Ozcan et al. 2002; Arthur et al. 2004; Danielisova et al. 2005). In agreement with these authors, we have also found that increased neuronal viability against glutamate administration was associated with reduced ROS levels and Ca2+ influx in BEP-treated cells. One possible reason for reduced Ca2+ influx could be the direct inhibitory effect of BEP on the NCX system. Indeed, Czyz et al. (2002) reported that under hypoglycemic conditions, intracellular Ca2+ levels upon NMDA receptor activation largely depend on the reverse mode operation of the NCX. Nevertheless, our Ca2+ measurements were performed under normoglycemic conditions, where the majority of Ca2+ – as reported by these authors – entered the cell directly via the NMDA ion channel complex. Furthermore, BEP was thoroughly washed out before the experiments, thus, the NCX system seems to have no direct role in the observed changes of [Ca2+]i. As we did not find any alterations in the protein level of the NR1 subunit of the NMDA receptor, we hypothesize that the observed effects on intracellular Ca2+ levels are due to receptor modulation by ROS or other causes rather than down-regulation of the NMDA receptor. To identify further factors leading to PC, we examined the expression of MnSOD, catalase, GPx, and Bcl-2 and found that the levels of these proteins increased with PC. MnSOD is a scavenger of superoxide anion and has been shown to be inducible by nuclear factor-jB (Mattson et al. 1997), and increases in protein level and/or activity of MnSOD have been reported to be protective in different paradigms (Keller et al. 1998; Ravati et al. 2001; Danielisova et al. 2005). While catalase is mainly found in peroxisomes, the glutathione system is also present in mitochondria; both are key antioxidants that decompose the hydrogen peroxide produced by SODs such as MnSOD. The enzyme activity of MnSOD

and GPx increased parallel with their protein levels. Both catalase mRNA and protein showed robust elevation, while activity did not change. Therefore, we speculate that some of the detected protein is enzymatically inactive. Bcl-2 is a major anti-apoptotic protein and exerts many positive effects on mitochondria. It maintains the open configuration of the voltage-dependent anion channel in the outer mitochondrial membrane and promotes metabolic passage between the outer and inner mitochondrial membranes (Gottlieb et al. 2000; Vander Heiden et al. 2001). This effect appears to be particularly important in promoting rapid restoration of ATP production and transport to the cytosol during recovery from anoxia. Additionally, Bcl-2 has been shown to preserve mitochondrial membrane potential during stress (Vander Heiden et al. 2001; Juhaszova et al. 2004). Furthermore, Bcl-2 has been suggested to possess direct ROS scavenging capacity; its overexpression is also known to protect cultured neurons against glutamate excitotoxicity, and the protection is accompanied with the inhibition of glutamate-induced superoxide accumulation (Lawrence et al. 1996; Howard et al. 2002). We found increased expression of Bcl-2 after 24 h of incubation with BEP and a further increase after 48 and 72 h of treatment. Bcl-2 overexpression was also accompanied with reduced ROS levels and increased neuronal survival upon glutamate excitotoxicity. Consequently, Bcl-2 seems to be another key component in the neuroprotective effect of BEP. Our results present the first evidence that the antianginal drug BEP is a neuroprotective agent in vitro. We propose that BEP-induced increases in ROS levels initiate a chain of events that include PKC and transcription factor activation and increased levels and/or activity of MnSOD, catalase, GPx, and Bcl-2, resulting in attenuated oxidative stress during a subsequent toxic insult. In contrast to the heart, the direct involvement of mitoKATP channels in the mechanism of the delayed PC with BEP is unlikely. Acknowledgements The authors gratefully thank Nancy Busija M.A. for critical reading of the manuscript. This work was supported by the National Institutes of Health Grants (HL-077731, HL-030260, DK-062372, and HL-065380), Y. F. Wu Research and Education Fund, WFUSM Venture Fund, and K. G. Phillips Fund for the Prevention and Treatment of Heart Disease, and WFUSM Interim Funding for DW Busija and partly by the Hungarian Science Fund (OTKA T046531) for F Bari.

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