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Aniracetam Attenuates Apoptosis of Astrocytes Subjected to Simulated Ischemia In Vitro
NeuroToxicology 23 (2002) 385–395
Aniracetam Attenuates Apoptosis of Astrocytes Subjected to Simulated Ischemia In Vitro Boz˙ena Gabryel1,*, Jakub Adamczyk2, Małgorzata Huzarska3, Anna Pudełko1, Henryk I. Trzeciak1 1
Department of Pharmacology, Silesian Medical University, 18 Medyko´w Street, PL 40752 Katowice, Poland Department and Clinic of Internal Diseases and Physical Medicine, Center of Laser Diagnostics and Therapy, Silesian Medical University, 15 Batory Street, PL 41902 Bytom, Poland 3 Department of Clinical Pharmacology, Silesian Medical University, 18 Medyko´w Street, PL 40752 Katowice, Poland 2
Received 11 March 2002; accepted 10 June 2002
Abstract The aim of the present study was to establish whether aniracetam is capable of protecting cultured rat astrocytes against ischemic injury. Treatment of the cultures with aniracetam (1, 10 and 100 mM) during 24 h ischemia simulated in vitro significantly decreased the number of apoptotic cells. The antiapoptotic effects of the drug were confirmed by the increase of intracellular ATP and phosphocreatine (PCr) levels and the inhibition of the caspase-3 activity. Aniracetam also attenuated cellular oxidative stress by decreased production of reactive oxygen species (ROS). These effects were associated with the decrease in levels of c-fos and c-jun mRNA in primary astrocyte cultures exposed to 24 h ischemia. When cultured astrocytes were incubated during 24 h simulated ischemia with wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor or PD98059, a mitogen-activated protein (MAP)/extracellular signal regulated kinase (ERK) (MEK) inhibitor, the cell apoptosis was accelerated. This effect was antagonized by adding 100 mM aniracetam to the culture medium. These findings suggest that the protective effect of aniracetam is mediated by PI 3-kinase and MEK pathways in the downstream mechanisms. # 2002 Elsevier Science Inc. All rights reserved.
Keywords: Aniracetam; Astrocytes; Ischemia; Apoptosis
INTRODUCTION Aniracetam (1-anisoyl-2-pyrrolidinone) is a potent modulator of AMPA (a-amino-3-hydroxy-5-methyl4-izoxazolepropionate) receptors. The modulation mechanism is based on inhibiting within the receptor the desensitization of the GluR2 subunit which is characterized by the low level of Ca2þ permeability (Tsuzuki et al., 1992). Aniracetam is being clinically used for the treatment of emotional disturbances, sleep disorders, cognition impairments and behavioral abnormalities in patients with cerebrovascular diseases, progressive supranuclear palsy and Parkinson’s and Alzheimer’s diseases (Otomo et al., 1991). A * Corresponding author. Tel.: þ48-32-252-3835; fax: þ48-32-252-3835. E-mail address: [email protected] (B. Gabryel).
number of studies, have shown beneficial effect of aniracetam on cognitive functions both in naive animals (Martin et al., 1992) and under conditions of cognitive deficits induced by impairment of the cholinergic system functions (Bartolini et al., 1992), electroshock application (Cumin et al., 1982) or ischemia (DeNoble et al., 1986). Recently, Shirane and Nakamura (2000) found that N-anisoyl-g-aminobutyric acid (N-anisoyl-GABA), one of the active in vivo metabolites of aniracetam modulates group II mGlu receptors, leading to enhance acetylcholine release in the prefrontal cortex of stroke-prone spontaneously hypertensive rats. However, the detailed molecular mechanisms of action of aniracetam are still unknown. A decrease in expression of the GluR2 subunit of the AMPA receptor is observed in brain ischemia (Gu et al., 1996). This may lead to the increase in the sensitivity
0161-813X/02/$ – see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 6 1 - 8 1 3 X ( 0 2 ) 0 0 0 8 4 - 0
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and number of the AMPA receptors permeable for Ca2þ which in turn may results in cell damage and changes connected with late phase of neurodegeneration (Gu et al., 1996). This phenomenon seems particularly important in the context of co-existence of apoptotic death of both neurons and astrocytes as a result of ischemic brain damage (Petito et al., 1998). The role of degenerated astrocytes in the pathomechanism of brain ischemia is not clear. These cells may be impaired in their ability to take up and inactivate excess excitatory amino acids, causing excitotoxic stress, neuronal cell death, oligodendrocyte death and further gliosis. They may also fail to regulate extracellular Kþ and Ca2þ concentrations and pH (Aschner, 1998; Gabryel and Trzeciak, 2001). A detailed understanding of the cellular pathways that are elicited in response to ischemia-death promoting stimuli can provide potential targets for therapeutic intervention. The aim of paper was to investigate the influence of aniracetam, a potentially neuroprotecting drug on astrocytes from rat cerebral cortex in primary cell culture during in vitro simulated ischemia. The effect of aniracetam on astrocyte apoptosis by determining the amount of apoptotic nuclei, intracellular highenergy phosphate (ATP and phosphocreatine (PCr)) levels, caspase-3 activity and the expression of immediate early genes (c-fos and c-jun) was examined. Moreover, because reactive oxygen species (ROS) production is implicated in ischemia-mediated cellular processes, this factor was investigated in astrocytes in an in vitro ischemia model.
MATERIALS AND METHODS Materials Chemicals and materials were obtained from the following sources: aniracetam (Hoffmann-La Roche, Switzerland), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), phosphate buffered saline (PBS), antibiotic–antimycotic mixture, TRIzol (all from GIBCO Laboratories, USA), DAKO PAP kit system glial fibrillary acidic protein (GFAP) test (DAKO Co., Denmark), 20 ,70 -dichlorofluorescein diacetate (DCF-DA) (Molecular Probes Inc., USA), caspase-3 colorimetric assay (R&D System, UK), ATP monitoring kit (Labsystems, Finland), creatine kinase (Boehringen Mannheim, Germany), adenosine 50 pentaphosphate, ADP, poly-D-lysine, Hoechst 33342, ethylenediaminetetraacetic acid disodium salt
(EDTA 2 Na), wortmannin, 20 -amino-30 -methoxyflavone (PD98059) and other chemicals all from Sigma (USA). Cell Culture Astrocytes were isolated from 1-day-old Wistar rat pups and cultured essentially according to the method of Hertz et al. (1985). Before the material was plated onto the plastic dishes, the cells were counted in a Coulter Z1 counter (Coulter Counter, UK). Cells destined for bioluminescent and fluorescent studies were plated at a density 3 105 cell per dish (35 mm in diameter). For 20 ,70 -dichlorofluorescein (DCF), fluorescence measurements cells were sieved at 1 104 per well on 96-well black plates. The cells destined for determination of caspase-3 activity and RT-PCR were sieved onto plastic dishes of 100 mm in diameter at the density of 1 106 per dish. For Hoechst 33342 staining, astrocytes were grown on coverslips covered with poly-D-lysine (100 mg/ml). The culture medium initially contained 20% of FBS and after 4 days was replaced with medium containing 10% FBS. The whole volume of culture medium was changed twice a week. In order to remove contaminating non-astroglial cells, confluent cultures were shaken overnight on a rotary platform at 200 rpm (37 8C) (McCarthy and DeVellis, 1980). Cell culture medium was replaced after overnight shaking. To identify astrocytes, cultures were stained immunocytochemically for GFAP. Analysis of the cultures has shown that 90–95% of cells were GFAP-positive. All experiments were performed on 21-day cultures. Experimental Procedure Normoxic astrocytes were cultured in DMEM medium with 5.5 mM glucose and 10% FBS. In order to mimic the in vivo ischemia, cultures were placed for 24 h in 37 8C in 3% O2/5% CO2/92% N2 (CO2 incubator, Heraeus, Germany) in DMEM without glucose and serum. Osmolarity of the medium was measured and adjusted to 319 mOsm with mannitol. Cells were treated with aniracetam (1, 10 and 100 mM) for 24 h in normoxia and for 24 h of simulated ischemia. Aniracetam was dissolved in dimethyl sulphoxide (DMSO) at an initial concentration of 10 mM. Further dilutions were performed in the appropriate medium. Kinase inhibitor: PD98059 was prepared as DMSO stocks at 50 mM and added to the culture medium at final concentration 50 mM; wortmannin was added to the culture medium at 0.1 mM.
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Assessment of Apoptosis Cell nuclei staining by Hoechst 33342 enables visualization of fragmented and condensed DNA, a characteristic feature of apoptosis. After washing with PBS astrocytes cultured on coverslips were fixed for 10 min with a 4% paraformaldehyde at room temperature. Subsequently, after being washed twice with PBS the samples were dehydrated first in 70% ethanol and then in absolute ethanol. The samples were kept in 20 8C until they were stained with Hoechst 33342 (5 mg/ml in PBS) for 5 min at room temperature. Then the cells were washed again with PBS. Cell nuclei analysis was conducted with the fluorescence imaging MiraCal Pro III workstation (Life Science Resources Ltd., UK) comprising of inverted microscope Eclipse TE200 (Nikon, Japan) (ex/em 340/510 nm). A 20 objective was used. The number of apoptotic nuclei was determined on at least six randomly selected areas from three coverslips of every experimental group, each containing approximately 200 cells. The results were expressed as a percentage of apoptotic cells relative to the total number of cells. Measurement of ATP and PCr Concentrations The ATP and PCr concentrations were determined by high-specific firefly luciferin-luciferase bioluminescence assay kit using a fluoroscan ascent plate reader (Labsystems, Finland). The extraction of ATP and PCr was performed according to procedure described by Fitzpatrick et al. (1988). The conversion of PCr into ATP catalyzed by creatine kinase was performed according to the method of Lowry and Passonneau (1972). ATP concentrations were measured by bioluminescence quantification according to the method of Lust et al. (1981). Briefly, 100 ml samples of disrupted cells were added to equal volumes of ice-cold 5% TCA solution with 2 mM EDTA, vortexed rapidly for 5 s and incubated for 30 min at room temperature. Then the samples were 20-fold diluted with Tris–acetate buffer pH 7.75 and 10 ml solution of known ATP concentration was added to each well as internal standard and the total amount of ATP was determined. The results were calculated as nM of ATP or PCr per mg of protein (nM/ mg). Measurement of Caspase-3 Activity Enzymatic activity of caspase-3 was determined by caspase-3 colorimetric assay (R&D System, UK),
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according to the manufacturer’s instructions. This assay is based on the release of the fluorochrome pnitroaniline (p-NA) combined with a caspase-3 specific peptide substrate (DEVD–p-NA). Peptide cleavage through an active caspase-3 releases chromophor pNA, which can be quantified using colorimetric plate reader (405 nm). Briefly, cultured astocytes were lysed and cell extracts were centrifuged to eliminate cellular debris. Aliquots (20 ml of the cell extracts) were incubated for 2 h at 37 8C in the presence of the substrate. The level of caspase-3 activity, proportional to the color reaction intensity was expressed as a percentage of control. Cellular Oxidative Stress [20 ,70 -Dichlorofluorescein (DCF) Fluorescence] Cellular oxidative stress was determined on the basis of ROS-mediated conversion on 20 ,70 -DCF-DA into fluorescent DCF (Mattson et al., 1995). Cultured astrocytes were loaded with 100 mM DCF-DA by incubating for 50 min. Cells were washed three times with HBSS and DCF fluorescence was quantified using fluoroscan microplate reader (Labsystems, Finland). The dye was excited at 485 nm, and emission was filtered using a 538 nm barrier filter. ROS production was expressed as a percentage of control cells. RT-PCR RT-PCR analysis was used to determine expression of c-fos and c-jun genes in cultured rat astrocytes exposed to 24 ischemia and treated with 1, 10 or 100 mM aniracetam. Total RNA was isolated from astrocytes cultured on 100 mm culture cell dishes by Chomczynski extraction method (Chomczynski and Sacchi, 1987) with the TRIzol reagent. Precipitated and purified RNA was dissolved in sterile nuclease-free water (Promega, USA) and stored at 70 8C before use. Isolated RNA was quantified by determining absorbance at 260 nm (one absorbance unit at 260 nm equals 42 mg/ ml RNA). The reverse transcription reaction was performed using 100 ng of total RNA per 10 ml RT reaction volume (10 mM Tris–HCl buffer, 2.5 Thermus thermophilus (Tth) polymerase (Promega)) for 30 min at 60 8C. The total PCR mixture (50 ml) contained chelating buffer (0.75 mM EGTA, 100 mM KCl, 0.05% Tween, 5% glycerol), 2.5 mM Mg2þ, 10 mM dNTP and 1.0 U Thermus aquaticus (Taq) polymerase (Promega, USA). Amplification was carried out for 30 cycles for c-fos, c-jun and b-actin primers with 45 s denaturation at
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94 8C, 45 s annealing at 57 8C and 1 min extension 72 8C. The last extension step at 72 8C was prolonged for 7 min. The primers used in the PCR analysis correspond to the exon 1 DNA sequences of the rat c-fos gene, 50 -CTT GAA GAC GAG AAG TCT GCG30 for the upstream primer and 50 -GGT CAT TGA GAA GAG GCA GG-30 for the downstream primer (Simi et al., 2000). The primers for c-jun corresponded to the following DNA sequences in exon 1 of the rat c-jun gene: 50 -AAC AGA TCC CGG TGC AGC AC-30 for the upstream, and 50 -CCA CCT GTT CCC TGA GCA TG-30 for the downstream primer (Simi et al., 2000). The b-actin primers corresponded to the following sequences of the rat b-actin gene 50 -CCT GCG TCT GGA CCT GGC TG-30 and 50 -CTC AGG AGG AGC AAT GAT CT-30 for the upstream and the downstream primer, respectively (Fang et al., 1998). RT and PCR were performed in BIOMETRA UNOII Termoblock thermocycler (Germany). The 10 ml of amplified PCR products were analyzed on a 2% LMPagarose gel (Promega, USA) stained by ethidium-bromide in 1% TAE buffer and analyzed by densitometry. The results were quantified using Image Pro Plus software and expressed in average pixel intensity. All the results were normalized with the expression of the b-actin housekeeping gene. Experiments were repeated three times and the values of relative optical density were subjected to statistical analysis. Protein Determination
decreased the amount of apoptotic nuclei. Exposing astrocytes to 100 mM aniracetam during 24 h ischemia period turned out to be the most effective method in the prevention of apoptosis induced by simulated ischemia. To investigate the role of phosphatidylinositol 3kinase (PI 3-kinase) and mitogen-activated protein (MAP) kinase (MEK) in antiapoptic effect of aniracetam, we examined the effects of the phosphoinositide 3-kinase inhibitor wortmannin (0.1 mM) and MEK inhibitor PD98059 (50 mM). When the cells were treated with 0.1 mM wortmannin and 50 mM PD98059 during ischemia, 86 7:3 and 38:7 6% of the cells died, respectively. Simultaneous use of 100 mM aniracetam and wortmannin or PD98059 decreased the number of apoptotic nuclei to 3:9 þ 1:3 and 11:8 þ 2:9%, respectively, which indicates the drug’s strong influence on PI 3-kinase and MEK pathways (Fig. 1). In the cultures of astrocytes exposed to ischemia in the presence wortmannin (Fig. 2A) or PD98059 (Fig. 2B), a lot of fragmented and pyknotic nuclei occurred. Treatment of astrocytes with 100 mM aniracetam and wortmannin (Fig. 2C) or PD98059 (Fig. 2D) during ischemia prevented the process of apoptosis. Influence of Aniracetam on Intracellular ATP and PCr Concentrations Increased ATP and PCr levels in cells treated with 1, 10 or 100 mM aniracetam was observed in normoxia. The exposure of astrocytes to 24 h simulated ischemia
Protein content in astrocytes was measured according to the method of Lowry et al. (1951). Statistical Analysis Data were analyzed using one-way analysis of variance (ANOVA) followed by the Student–Newman– Keul’s test. A P-value of 0.05 was considered as statistically significant. All data were expressed as mean S:D:
RESULTS Influence of Aniracetam on Apoptosis (Hoechst 33342 Staining) The 24 h simulated ischemia significantly increased the number of apoptotic nuclei as compared to the normoxic conditions. The presence of aniracetam in the culture medium in these conditions significantly
Fig. 1. Effect of aniracetam (ANI) on astrocyte apoptosis induced by simulated ischemia. The results are shown as a percentage relation of the apoptotic nuclei to the total amount of nuclei in the field and are a mean S:D: of the six randomly selected areas from three culture dishes; (*) P < 0:05 vs. normoxia; (#) P < 0:05 vs. ischemia.
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Fig. 2. Effect of aniracetam on astrocyte apoptosis induced by simulated ischemia and PD98059 or wortmannin exposure. Cells were incubated with 100 nM wortmannin (A) and 50 mM PD98059 (C) in the absence or presence of 100 mM aniracetam (B, D, respectively) for 24 h ischemia. Then, the cells were fixed and stained with the Hoechst 33342 dye. Microphotographs were taken with the use of fluorescent microscope and 20 objective. Thin arrows point at apoptotic nuclei and thick arrows point at live nuclei.
caused a significant reduction in the amount of both high-energy phosphates. In this experimental option aniracetam produced significant increase of the intracellular ATP concentration. The significant decrease in value of these parameters were observed in ischemic conditions after treatment with 0.1 mM wortmannin. This effect was antagonized by aniracetam. Administration of 50 mM PD98059 did not affect the ATP level in ‘‘ischemic’’ astrocytes, but the protective effect of aniracetam in comparison to the normoxia control was observed. At the same time no changes were seen in intracellular concentrations of PCr when the cells were exposed during simulated ischemia to PD98059 and aniracetam (Figs. 3 and 4).
Effect of Aniracetam on ROS Production
Influence of Aniracetam on Caspase-3 Activity
Effect of Aniracetam on c-fos and c-jun Expressions
Treatment with 1 mM aniracetam did not change the enzyme proteolytic activity in comparison with simulated ischemia. However, simultaneous exposure of astrocytes to ischemia and 10 or 100 mM aniracetam substantially decreased the caspase-3 activity. After 100 mM aniracetam administration for 24 h ischemia a similar activity of the enzyme to that in normoxia was observed. (Fig. 5).
Fig. 6. shows the results of the influence of aniracetam on cellular oxidative stress. A substantial decrease of DCF fluorescence in astrocytes in normoxic conditions stimulated by aniracetam indicated a significant decrease in ROS production (Fig. 6A). A 24 h simulated ischemia intensified, while administration of aniracetam during this period attenuated the conversion of DCF. An additional exposition of astrocytes in these conditions to PD98059 increased ROS production in comparison to the normoxia control. However, treatment with 100 mM aniracetam together with kinase inhibitors decreased the DCF fluorescence intensity during 24 h ischemia (Fig. 6B).
The exposure of astrocyte cultures to 24 h simulated ischemia caused an increase both genes expression, the c-jun expression in examined cultures was weaker than c-fos expression. A treatment with 1 mM aniracetam was without effect on the expression of cfos and c-jun genes. However, a 24 h treatment with 10 and 100 mM aniracetam markedly attenuated
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Fig. 3. Effect of aniracetam (ANI) on intracellular ATP concentration in cultured rat astrocytes exposed to normoxia (A) and simulated ischemia (B). The results are shown as nM/ mg protein. Each value is mean obtained form eight dishes S:D: (n ¼ 8); (*) P < 0:05 vs. normoxia; (#) P < 0:05 vs. ischemia.
Fig. 4. Effect of aniracetam (ANI) on intracellular PCr concentration in cultured rat astrocytes exposed to normoxia (A) and simulated ischemia (B). The results are shown as nM/mg protein. Each value is mean obtained form eight dishes S:D: (n ¼ 8); (*) P < 0:05 vs. normoxia; (#) P < 0:05 vs. ischemia.
ischemia-induced expression c-fos and c-jun in astrocytes (Fig. 7A,B,C).
DISCUSSION We examined the protective effects of aniracetam against simulated ischemia-induced apoptosis in cultured rat astrocytes. This experimental model may contribute to clarification of the mechanisms of drugs that ameliorate ischemia-induced brain dysfunction (Takuma et al., 2000). The glial cells that interact with neurons play important roles in ischemic brain injury (Petito et al., 1998). Astrocytes produce several different antiapoptic growth factors (e.g. bFGF, NGF, BDNF), while microglia produce neurotoxic substances such as nitric oxide and excitotoxins (Schwartz and Nishiyama, 1994; Mattson et al., 2000).
Fig. 5. Effect of aniracetam (ANI) on caspase-3 activity in cultured rat astrocytes exposed to simulated ischemia. The results are shown as a percentages of the control value in the normoxia conditions. Each value is mean of eight dishes S:D: (n ¼ 8); (*) P < 0:05 vs. normoxia; (#) P < 0:05 vs. ischemia.
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Fig. 6. Effect of aniracetam (ANI) on DCF fluorescence (as measure of ROS production) in cultured rat astrocytes exposed to normoxia (A) and simulated ischemia (B). For each experimental group fluorescence intensity was determined. The results are shown as a percentage relation of the control value in the normoxia conditions. Each value is mean activity obtained form 12 wells S:D: (n ¼ 12); (*) P < 0:05 vs. normoxia; (#) P < 0:05 vs. ischemia.
Astrocyte apoptosis in vitro is induced by several signals, e.g. HIV-1 infection (He et al., 1997), S100 b (Hu and Van Eldik, 1996), staurosporine (Mangura and Dawson, 1998), Ca2þ deprivation (Takuma et al., 1999; Chiesa et al., 1998) and hypoxia (Gabryel et al., 2001). The factors determining the apoptotic cell death are the change in mitochondrial membrane permeability as a result of the occurrence of mitochondrial permeability transition pore (MTP) (Zoratti and Szabo’, 1995) and intracellular level of ATP (Leist et al., 1997; Eguchi et al., 1997). The opening of MTP is accompanied by inhibition of oxidative phosphorylation and activation of the mitochondrial ATP-ase which increases the consumption of ATP (Nieminen et al., 1994; Cai and Jones, 1998). When glycolysis or the use of energy stores accumulated in PCr are able to partly compensate the ATP deficiency, the apoptosis program is
Fig. 7. Effect of aniracetam (ANI) on c-fos and c-jun expression in cultured rat astrocytes exposed to 24 h hypoxia. (A) The cDNA fragments obtained after RT-PCR using primers specific for c-fos, c-jun and b-actin were visualized under UV after electrophoresis in the presence of EtBr; lane 1: control cultures (normoxia), lane 2: astrocytes exposed to 24 h simulated ischemia (ischemia), lane 3: 1 mM aniracetam, lane 4: 10 mM aniracetam, lane 5: 100 mM aniracetam. (B) The c-fos and (C) c-jun expressions after 24 h ischemia as measured by relative optical density of bands. The signals from c-fos and c-jun cDNA fragments were normalized to b-actin at different experimental conditions. Data represent mean S:D: (*) P < 0:05 vs. normoxia; (#) P < 0:05 vs. ischemia.
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initiated. The energy stored in ATP is necessarily used in major apoptotic pathways leading from cytochrome c (Li et al., 1997), apoptosis-inducing factor (AIF) and death receptors to caspases activity (Susin et al., 1999). Likewise, morphological alternations of the cell nuclei, featuring for apoptosis, are the result of the signal transduction from cytoplasm to the nuclei through active transport requiring ATP hydrolysis (Melchior and Gerace, 1995; Pante and Aebi, 1996). In the cultures subjected to 24 h simulated ischemia we observed a significant increase in the number of apoptotic astrocytes in comparison to the control. Simultaneous exposure of the cells during ischemia to increasing concentrations of aniracetam (1, 10, 100 mM) prevented apoptosis. To investigate the role of PI 3-kinase and MAP kinase (MEK) in antiapoptotic effect of aniracetam, the effects of the PI 3-kinase inhibitor wortmannin (0.1 mM) and MEK inhibitor PD98059 (50 mM) were examined. The addition of both substances into the culture medium of the cells exposed to 24 h ischemia caused a significant increase of the amount of apoptotic cells. At the same time it has been observed that administration of kinase inhibitors with 100 mM aniracetam prevents the apoptosis completely and the amount of apoptotic cells was similar to the amount in normoxia conditions. These results indicate a distinct protective action of aniracetam on astrocytes damaged by simulated ischemia. With ischemia, analytic measures of high-energy compounds (ATP and PCr) in rapidly frozen decapitated brain showed an initial partial loss of PCr followed by a simultaneous loss of both PCr and ATP (Siesjo¨ and Zwetnow, 1970). Since the restoration by cells ability to produce high-energy compounds enables regeneration of the ischemic damages we assessed the influence of aniracetam on intracellular amount of ATP and PCr in normoxic and ischemic conditions. In both situations we have demonstrated a significant increase of the ATP amount. Aniracetam showed weaker influence on the PCr than on ATP in astrocytes damaged by simulated ischemia. Both used kinase inhibitors caused a significant decrease of the intracellular ATP, while the use of aniracetam in this conditions reversed the effect. PD98059 or aniracetam together with PD98059 did not change the intracellular concentration of PCr. On the other hand, exposure the cells to aniracetam and wortmannin restored the PCr level to the value similar to normoxia conditions. PCr level was significantly decreased when only wortmannin was added. The drug prevented ATP lose probably through partial use of PCr as an energy store which also enabled maintenance of mitochondria capacity.
Caspase-3 activity is one of the fundamental events of the so-called apoptosis execution phase (Gorman et al., 1998). This enzyme decomposes various proteins, i.e. cell cycle regulatory proteins, poly(ADPrybose)polymerase and activates other caspase family members, such as caspase-2, caspase-6 and caspase-7 (Nicholson, 1999). There is same evidence that phosphorylation or other covalent post-translational modification (other than proteolytic activation) participates in caspase regulation. The serine/threonine kinase Akt/ PKB is activated by PI 3-kinase and opposes apoptosis by several mechanisms including catalytic inactivation of caspase-9 (Yuan et al., 1993). This suggests a particularly important regulatory role for caspase-9 in apoptosis, since there is currently no evidence for similar Akt-mediated inactivation of other caspases. Inhibition of caspase-9, together with cytochrome c and Apaf-1 form apoptosom disables activation of caspase-3. We demonstrated significant increase of the apoptotic nuclei after adding wortmannin as a PI 3-kinase inhibitor as well as aniracetam ability to reverse apoptotic effect of wortmannin. Therefore, the inhibiting influence of aniracetam on caspase-3 activity which we observed may be result of the drug’s involvement in the PI 3-kinase/Akt pathway. Caspases also disrupt activation of the transcription factor NFkB, which normally activates antiapoptotic gene expression, including members of IAP family (Wang et al., 1998). In normal conditions, NFkB is present in cytoplasm as a inactive complex, composed with a p50/p65 heterodimer bound to an inhibitory subunit IkB. Activation of NFkB is induced by phosphorylation of IkB, which leads to the dissociation of this complex. Subsequently, IkB is degraded, and the p50/p65 heterodimer translocated from cytoplasm into nucleus, where may induce expression of several genes (Thanos and Maniatis, 1985). Caspases cleave the inhibitor of NFkB, IkBa, producing a C-terminal fragment that is resistant to proteosomal degradation and stable inhibits NFkB, sensitizing cells to apoptosis (Reuther and Baldwin, 1999). Tissue injury including cerebral ischemia is a potent stimulus for activation of NFkB (Kolesnick and Golde, 1994; Liu et al., 1994). Among others, NFkB constitutes an important component of transcriptional regulation of antioxidant enzyme—manganese superoxide dismutace (MnSOD) (Mattson et al., 1997). Antioxidant effect of aniracetam was shown by the fluorescent method where we observed a reduced occurrence of ROS as a decrease in DCF fluorescence. Moreover, we have also observed a significant decrease of the stimulating influence of wortmannin and PD98059 on forming
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ROS with the simultaneous use of aniracetam (Fig. 6). MAP kinase (MEK 1/2) whose inhibitor is PD98059 (Dudley et al., 1995) takes part in the process of IkB phosphorylation (Lee et al., 1997). The MEK signal pathway is proposed to play a role in cell survival (Yujiri et al., 1998), it is possible that the MAP signal pathways may be target for the drugs to ameliorate ischemia/reperfusion injury. Our results indirectly indicate this pathway’s participation in the protective effect exerted by aniracetam in ischemia. From studies showing that AMPA receptor agonists stimulate Ca2þ-dependent phosphorylation of CREB (cAMP response element-binding protein), a common glutamate receptor-mediated signal transduction pathway involving Ras and MAP kinase has emerged in different glial cell types (Pende et al., 1997). Mitogen-activated protein kinases (MAPKs) are well-known intracellular signaling cascades involved in gene regulation. Activation of the MAPKs results in the phosphorylation and activation of transcription factors binding to the corresponding response elements in their promoters, which results in a subsequent gene activation. Therefore, another goal of this study was to analyze the expression of the genes c-jun and c-fos known to be activated by MAPKs and implicated in apoptosis (Treisman, 1996). Both fos and jun family genes have been studied in models of experimental ischemia, mostly in rodents (Feuerstain et al., 1997; Lu et al., 1997; Yang et al., 1997). Despite the fact that cfos and c-jun belong to the s.c. immediate early genes family, many articles present assessment of their expression in vitro 24 h or even 48 h after occurrence of the damaging factor, e.g. calcium deprivation or treatment with ceramide (Chiesa et al., 1998; Willaime et al., 2001). We assessed the intensification of the c-fos and c-jun genes expression after the 24 h exposition of the astrocyte cell culture to ischemia and treatment with aniracetam in order to, together with other parameters measured in ‘‘one point of time’’, assess the level of apoptosis in cells exposed to damaging factor and treated pharmacologically. Using semiquantitive RT-PCR we observed the increase in amount of mRNA of both genes in astrocytes exposed to 24 h simulated ischemia. Aniracetam caused decrease of the c-fos and c-jun expressions proportionately to the concentration used (Fig. 7). A glioprotective action of aniracetam against simulated ischemia was seen after addition of a high concentration of aniracetam (100 mM), but not after a low concentration (1 mM). But, in some experiments in vitro on neuronal cultures the 100 mM concentration of aniracetam was added to the medium (Copani et al.,
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1992; Pizzi et al., 1993; Kolta et al., 1998). Moreover, in several electrophysiological studies the concentrations 300–1000 mM in different model systems were used (Kapus et al., 2000; Buldakova et al., 2000). It should be noted that, our data for the first time demonstrate that aniracetam exerts protective action against apoptosis induced by simulated in vitro ischemia in primary culture of astrocytes. It cannot be excluded that the final outcome of the action of aniracetam might be dependent on the metabolic differences between astrocytes and neurons (Gabryel and Trzeciak, 2001). Summing up, the observed glioprotective effects of aniracetam are the result of this drug interference with a number of factors determining cell apoptosis and their viability. Our study shows that antiapoptotic affect of aniracetam is connected with its stimulating influence on the intracellular amount of high-energy phosphates, decreased caspase-3 activity, ROS production and c-fos and c-jun genes expression. We demonstrated that aniracetam completely reverses the apoptotic effect of MEK inhibitor PD98059 and PI 3-kinase inhibitor wortmannin. The results suggest that protective effect of aniracetam is mediated both through MEK kinase and phosphoinisitide 3-kinase signal pathways. It seems that studies bring the hypothesis of the new mechanisms of aniracetam antiischemic activity indicating their potential protective influence towards astrocytes in ischemia conditions.
ACKNOWLEDGEMENTS The work was supported by grant of State Committee for Scientific Research 4 PO5D 00516 and grant from Silesian Medical University.
REFERENCES Aschner M. Astrocytic functions and physiological reactions to injury: the potential to induce and/or exacerbate neuronal dysfunction—a forum position paper. Neurotoxicology 1998; 19:7–18. Bartolini L, Risaliti R, Pepeu G. Effect of scopolamine and nootropic drugs on rewarded alternation in a T-maze. Pharmacol Biochem Behav 1992;43:1161–4. Buldakova SL, Bolshakov KV, Tikhonov DB, Magazanik LG. Ca2þdependent desensitization of AMPA receptors. NeuroReport 2000;11:2937–41. Cai J, Jones DP. Superoxide in apoptosis. Mitochondrial generation triggered by cytochrome c loss. J Biol Chem 1998;273:11401–4. Chiesa R, Angeretti N, Del Bo R, Lucca E, Munna E, Forloni G. Extracellular calcium deprivation in astrocytes: regulation of
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mRNA expression and apoptosis. J Neurochem 1998;70:1474– 83. Chomczynski P, Sacchi N. Single step method of RNA isolation by acid guanidinum thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156–9. Copani A, Genazzani AA, Aleppo G, Casabona G, Canonico PL, Scapagnini U, Nicoletti F. Noortropic drugs positively modulate a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-sensitive glutamate receptors in neuronal cultures. J Neurochem 1992;58:1199–204. Cumin R, Bandle EF, Gamzu E, Haefely WE. Effects of the novel compound aniracetam (Ro 13-5057) upon impaired learning and memory in rodents. Psychopharmacology (Berl) 1982;78:104– 11. DeNoble VJ, Repeti SJ, Gelpke LW, Wood LM, Keim KL. Vinpocetine: nootropic effects on scopolamine-induced and hypoxia-induced retrieval deficits of a step-through passive avoidance response in rats. Pharmacol Biochem Behav 1986; 24:1123–8. Dudley DT, Pang L, Decker SJ, Bridges AJ, Saltiel AR. Synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci USA 1995;92:7686–9. Eguchi Y, Shimizu S, Tsujimoto Y. Intracellular ATP levels determine cell fated by apoptosis or necrosis. Cancer Res 1997; 57:1835–40. Fang C, Lindros KO, Badger TM, Ronis MJ, Ingelman-Sundberg M. Zonated expression of cytokines in the rat liver: effect of chronic ethanol and the cytochrome P450 2E1 inhibitor, chlomethiazole. Hepatology 1998;27:1304–10. Feuerstain GZ, Wang X, Barone FC. Inflammatory gene expression in cerebral ischemia and trauma. Ann N Y Acad Sci 1997;825:179–93. Fitzpatrick A, Cooper AJL, Hertz L. Effects of ammonia and bmethylene-DL-asparate on the oxidation of glucose and pyruvate by neurons and astrocytes in primary culture. J Neurochem 1988;51:1197–203. Gabryel B, Trzeciak HI. Role of astrocytes in pathogenesis of ischemic brain injury. Neurotoxicity Res 2001;3:205–21. Gabryel B, Adamek M, Trzeciak HI. Does trimetazidine exert cytoprotective activity on astrocytes subjected to hypoxia in vitro? Neurotoxicology 2001;22:455–65. Gorman AM, Orrenius S, Cecatelli S. Apoptosis in neuronal cells: role of caspases. NeuroReport 1998;9:49–55. Gu JG, Albuquerque C, Lee CJ, Macdermott AB. Synaptic strengthening through activation of Ca2þ-permeable AMPA receptors. Nature 1996;381:793–6. He J, DeCastro JM, Vandenbark GR, Busciglio J, Gabuzda D. Astrocyte apoptosis induced by HIV-1 transactivation of the ckit protooncogene. Proc Natl Acad Sci USA 1997;94:3954–9. Hertz L, Juurlink BHJ, Szuchet S. Cell cultures. In: Lajtha A, editor. Handbook of neurochemistry, vol. 8. New York: Plenum Press, 1985. p. 603–61. Hu J, Van Eldik LJ. S 100 b induces apoptotic cell death in cultured astrocytes via a nitric oxide-dependent pathway. Biochim Biophys Acta 1996;1313:239–45. Kapus G, Bodi I, Pataki A, Gueritaud J-P, Szekely JI, Tarnawa I. Differential modulation of the GYKI 53784-induced inhibition of AMPA currents by various AMPA-positive modulators in cerebellar Purkinje cells. Eur J Pharmacol 2000;397:43–7. Kolesnick R, Golde DW. The sphingomyelin pathway in tumor necrosis factor and interleukin-1 signaling. Cell 1994;77:325–8.
Kolta A, Lynch G, Ambros-Ingerson J. Effects of aniracetam after LTP induction are suggestive of interactions on the kinetics of the AMPA receptor channel. Brain Res 1998;788:269–86. Lee FS, Hagler J, Chen ZJ, Maniatis T. Activation of the IkB alpha kinase complex by MEKK1, a kinase of the JNK pathway. Cell 1997;88:213–22. Leist M, Single B, Castoldi AF, Kuhlne S, Nicotera P. Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis. J Exp Med 1997; 185:1481–6. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptosis protease cascade. Cell 1997;91:479–89. Liu T, Clark RK, Mc Donnell PC, Young PR, White MS, Barone FC, Feuerstein GZ. Tumor necrosis factor-a expression in ischemic neurons. Stroke 1994;25:1481–8. Lowry OH, Passonneau JV. A flexible system of enzymatic analysis. New York: Academic Press, 1972. p. 120–6. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193:265–75. Lu XC, Tortella FC, Ved HS, Garcia GE, Dave JR. Neuroprotective role of c-fos antisense oligonucleotide: in vitro and in vivo studies. NeuroReport 1997;8:2925–9. Lust WD, Feussner GK, Barbehenn EK, Passoneau JV. The enzymatic measurement of adenine nucleotides and p-creatine in picomole amounts. Anal Biochem 1981;110:258–66. Mangura D, Dawson G. Programmed cell death in cortical chick embryo astrocytes is associated with activation of protein kinase PK60 and ceramide formation. J Neurochem 1998;70: 130–8. Martin JR, Cumin R, Aschwanden W, Moreau JL, Jenck F, Haefely WE. Aniracetam improves radial maze performance in rats. NeuroReport 1992;3:81–3. Mattson MP, Barger SW, Begley JG, Mark RJ. Calcium, free radicals, and excitotoxic neuronal death in primary cell culture. Methods Cell Biol 1995;46:187–215. Mattson MP, Goodman Y, Luo H, Fu W, Furukawa K. Activation of NF-kB protects hippocampal neurons against oxidative stress-induced apoptosis: evidence for induction of manganese superoxide dismutase and suppression of peroxynitrite production and protein tyrosine nitration. J Neurosci Res 1997; 49:681–97. Mattson MP, Culmsee C, Yu ZF. Apoptotic and antiapoptotic mechanisms in stroke. Cell Tissue Res 2000;301:173–87. McCarthy KD, DeVellis J. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol 1980;85:890–902. Melchior F, Gerace L. Mechanisms of nuclear protein import. Curr Opin Cell Biol 1995;7:310–8. Nicholson DW. Caspase structure, proteolytic substrates, and function during apoptotic cell death. Cell Death Differ 1999; 6:1028–42. Nieminen AL, Saylor AK, Herman B, Lemasters JJ. ATP depletion rather than mitochondrial depolarization mediates hepatocyte killing after metabolic inhibition. Am J Physiol 1994;267:67–74. Otomo E, Hirai S, Terashi A, Hasegawa Y, Tazaki Y, Araki G, Itoh E, Nishimura T, Furukawa T. Clinical usefulness of aniracetam for psychiatric symptoms in patients with cerebrovascular
B. Gabryel et al. / NeuroToxicology 23 (2002) 385–395 disorders: placebo controlled double-blind trial. J Clin Exp Med 1991;156:143–87. Pante N, Aebi U. Toward the molecular dissection of protein import into nuclei. Curr Opin Cell Biol 1996;8:397–406. Pende M, Fischer TL, Simpson PB, Russell JT, Blenis J, Gallo V. Neurotransmitter and growth factor-induced cAMP response element binding protein phosphorylation in glial cell progenitors: role of calcium ions, protein kinase C, and mitogen-activated protein kinase/ribosomal S6 kinase pathway. J Neurosci 1997;15:1291–301. Petito CK, Olarte JP, Roberts B, Nowak TS, Pulsinelli WA. Selective glial vulnerability following transient global ischemia in rat brain. J Neuropathol Exp Neurol 1998;57:231–8. Reuther JY, Baldwin AS. Apoptosis promotes a caspase induced amino-terminal truncation of IkBa that function as a stable inhibitor of NFkB. J Biol Chem 1999;274:20664–206670. Pizzi M, Fallacara C, Arrighi V, Memo M, Spano P. Attenuation of excitatory amino acid toxicity by metabotropic glutamate receptor agonists and aniracetam in primary cultures of cerebellar granule cells. J Neurochem 1993;61:683–9. Schwartz JP, Nishiyama N. Neurotrophic factor gene expression in astrocytes during development and following injury. Brain Res Bull 1994;35:403–7. Shirane M, Nakamura K. Group II metabotropic glutamate receptors are a common target of N-anisoyl-GABA and 1S, 3RACPD in enhancing ACh release in the prefrontal cortex of freely moving SHRSP. Neurpharmacology 2000;39:866–72. Siesjo¨ B, Zwetnow N. The effect of hypovolemic hypotension on extra- and intracellular acid-base parameters and energy metabolites in the rat brain. Acta Physiol Scand 1970;79:114– 24. Simi A, Ingelman-Sundberg M, Tindberg N. Neuroprotective agent chlomethiazole attenuates c-fos, c-jun and AP-1 activation through inhibition of p38 MAP kinase. J Cereb Blood Flow Metab 2000;20:1077–88. Susin SA, Lorenzo HK, Zamzani N, Marzo I, Snow BE, Brothers GM, Mangion J, Jacotot E, Constantini P, Loeffler M, Larochette N, Goodlett DR, Aebersold R, Siderovski DP, Penninger JM, Kroemer G. Molecular characterization of
395
mitochondrial apoptosis-inducing factor. Nature 1999;397:441– 6. Takuma K, Lee E, Kidawara M, Mori K, Kimura Y, Baba A, Matsuda T. Apoptosis in Ca2þ reperfusion injury of cultured astrocytes: roles of reactive oxygen species and NF-kB activation. Eur J Neurosci 1999;11:4204–12. Takuma K, Fujita T, Kimura Y, Tanabe M, Yamamuro A, Lee E, Mori K, Koyama Y, Baba A, Matsuda T. T-588 inhibits astrocyte apoptosis via mitogen-activated protein kinase signal pathway. Eur J Pharmacol 2000;399:1–8. Thanos D, Maniatis T. NF-kB, a lesson in family values. Cell 1985;80:529–32. Treisman R. Regulation of transcription by MAP kinase cascades. Curr Opin Cell Biol 1996;8:205–15. Tsuzuki K, Takeuchi T, Ozawa S. Agonist-dependent and subunitdependent potentiation of glutamate receptors by nootropic drug aniracetam. Mol Brain Res 1992;16:105–10. Wang CY, Mayo MW, Korneluk RG, Goeddel DV, Baldwin AS. NFkB antiapoptosis: Induction of TRAF1 and TRAF2 and cIAP1 and c-IAP2 to suppress caspase-8 activation. Science 1998;281:1680–3. Willaime S, Vanhoutle P, Caboche J, Lemaigre-Dubreuil Y, Mariani J, Brugg B. Ceramide-induced apoptosis in cortical neurons is mediated by an increase in p38 phosphorylation and not by the decrease in ERK phosphorylation. Eur J Neurosci 2001;13:2037–46. Yang DD, Kuan CY, Whitmarsh AJ, Rincon M, Zheng TS, Davis RJ, Rakic P, Flavell RA. Absence of excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene. Nature 1997;389:865–70. Yuan J, Shaham S, Ledoux S, Ellis HM, Horvitz HR. The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interlekin-1b-converting enzyme. Cell 1993;75: 641–52. Yujiri T, Sather S, Fanger GR, Johnson GL. Role of MEKK1 in cell survival and activation of JNK and ERK pathways defined by targeted gene disruption. Science 1998;282:1911–4. Zoratti M, Szabo’ I. The mitochondrial permeability transition. Biochim Biophys Acta 1995;1241:139–76.
Aniracetam Attenuates Apoptosis of Astrocytes Subjected to Simulated Ischemia In Vitro Boz˙ena Gabryel1,*, Jakub Adamczyk2, Małgorzata Huzarska3, Anna Pudełko1, Henryk I. Trzeciak1 1
Department of Pharmacology, Silesian Medical University, 18 Medyko´w Street, PL 40752 Katowice, Poland Department and Clinic of Internal Diseases and Physical Medicine, Center of Laser Diagnostics and Therapy, Silesian Medical University, 15 Batory Street, PL 41902 Bytom, Poland 3 Department of Clinical Pharmacology, Silesian Medical University, 18 Medyko´w Street, PL 40752 Katowice, Poland 2
Received 11 March 2002; accepted 10 June 2002
Abstract The aim of the present study was to establish whether aniracetam is capable of protecting cultured rat astrocytes against ischemic injury. Treatment of the cultures with aniracetam (1, 10 and 100 mM) during 24 h ischemia simulated in vitro significantly decreased the number of apoptotic cells. The antiapoptotic effects of the drug were confirmed by the increase of intracellular ATP and phosphocreatine (PCr) levels and the inhibition of the caspase-3 activity. Aniracetam also attenuated cellular oxidative stress by decreased production of reactive oxygen species (ROS). These effects were associated with the decrease in levels of c-fos and c-jun mRNA in primary astrocyte cultures exposed to 24 h ischemia. When cultured astrocytes were incubated during 24 h simulated ischemia with wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor or PD98059, a mitogen-activated protein (MAP)/extracellular signal regulated kinase (ERK) (MEK) inhibitor, the cell apoptosis was accelerated. This effect was antagonized by adding 100 mM aniracetam to the culture medium. These findings suggest that the protective effect of aniracetam is mediated by PI 3-kinase and MEK pathways in the downstream mechanisms. # 2002 Elsevier Science Inc. All rights reserved.
Keywords: Aniracetam; Astrocytes; Ischemia; Apoptosis
INTRODUCTION Aniracetam (1-anisoyl-2-pyrrolidinone) is a potent modulator of AMPA (a-amino-3-hydroxy-5-methyl4-izoxazolepropionate) receptors. The modulation mechanism is based on inhibiting within the receptor the desensitization of the GluR2 subunit which is characterized by the low level of Ca2þ permeability (Tsuzuki et al., 1992). Aniracetam is being clinically used for the treatment of emotional disturbances, sleep disorders, cognition impairments and behavioral abnormalities in patients with cerebrovascular diseases, progressive supranuclear palsy and Parkinson’s and Alzheimer’s diseases (Otomo et al., 1991). A * Corresponding author. Tel.: þ48-32-252-3835; fax: þ48-32-252-3835. E-mail address: [email protected] (B. Gabryel).
number of studies, have shown beneficial effect of aniracetam on cognitive functions both in naive animals (Martin et al., 1992) and under conditions of cognitive deficits induced by impairment of the cholinergic system functions (Bartolini et al., 1992), electroshock application (Cumin et al., 1982) or ischemia (DeNoble et al., 1986). Recently, Shirane and Nakamura (2000) found that N-anisoyl-g-aminobutyric acid (N-anisoyl-GABA), one of the active in vivo metabolites of aniracetam modulates group II mGlu receptors, leading to enhance acetylcholine release in the prefrontal cortex of stroke-prone spontaneously hypertensive rats. However, the detailed molecular mechanisms of action of aniracetam are still unknown. A decrease in expression of the GluR2 subunit of the AMPA receptor is observed in brain ischemia (Gu et al., 1996). This may lead to the increase in the sensitivity
0161-813X/02/$ – see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 6 1 - 8 1 3 X ( 0 2 ) 0 0 0 8 4 - 0
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and number of the AMPA receptors permeable for Ca2þ which in turn may results in cell damage and changes connected with late phase of neurodegeneration (Gu et al., 1996). This phenomenon seems particularly important in the context of co-existence of apoptotic death of both neurons and astrocytes as a result of ischemic brain damage (Petito et al., 1998). The role of degenerated astrocytes in the pathomechanism of brain ischemia is not clear. These cells may be impaired in their ability to take up and inactivate excess excitatory amino acids, causing excitotoxic stress, neuronal cell death, oligodendrocyte death and further gliosis. They may also fail to regulate extracellular Kþ and Ca2þ concentrations and pH (Aschner, 1998; Gabryel and Trzeciak, 2001). A detailed understanding of the cellular pathways that are elicited in response to ischemia-death promoting stimuli can provide potential targets for therapeutic intervention. The aim of paper was to investigate the influence of aniracetam, a potentially neuroprotecting drug on astrocytes from rat cerebral cortex in primary cell culture during in vitro simulated ischemia. The effect of aniracetam on astrocyte apoptosis by determining the amount of apoptotic nuclei, intracellular highenergy phosphate (ATP and phosphocreatine (PCr)) levels, caspase-3 activity and the expression of immediate early genes (c-fos and c-jun) was examined. Moreover, because reactive oxygen species (ROS) production is implicated in ischemia-mediated cellular processes, this factor was investigated in astrocytes in an in vitro ischemia model.
MATERIALS AND METHODS Materials Chemicals and materials were obtained from the following sources: aniracetam (Hoffmann-La Roche, Switzerland), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), phosphate buffered saline (PBS), antibiotic–antimycotic mixture, TRIzol (all from GIBCO Laboratories, USA), DAKO PAP kit system glial fibrillary acidic protein (GFAP) test (DAKO Co., Denmark), 20 ,70 -dichlorofluorescein diacetate (DCF-DA) (Molecular Probes Inc., USA), caspase-3 colorimetric assay (R&D System, UK), ATP monitoring kit (Labsystems, Finland), creatine kinase (Boehringen Mannheim, Germany), adenosine 50 pentaphosphate, ADP, poly-D-lysine, Hoechst 33342, ethylenediaminetetraacetic acid disodium salt
(EDTA 2 Na), wortmannin, 20 -amino-30 -methoxyflavone (PD98059) and other chemicals all from Sigma (USA). Cell Culture Astrocytes were isolated from 1-day-old Wistar rat pups and cultured essentially according to the method of Hertz et al. (1985). Before the material was plated onto the plastic dishes, the cells were counted in a Coulter Z1 counter (Coulter Counter, UK). Cells destined for bioluminescent and fluorescent studies were plated at a density 3 105 cell per dish (35 mm in diameter). For 20 ,70 -dichlorofluorescein (DCF), fluorescence measurements cells were sieved at 1 104 per well on 96-well black plates. The cells destined for determination of caspase-3 activity and RT-PCR were sieved onto plastic dishes of 100 mm in diameter at the density of 1 106 per dish. For Hoechst 33342 staining, astrocytes were grown on coverslips covered with poly-D-lysine (100 mg/ml). The culture medium initially contained 20% of FBS and after 4 days was replaced with medium containing 10% FBS. The whole volume of culture medium was changed twice a week. In order to remove contaminating non-astroglial cells, confluent cultures were shaken overnight on a rotary platform at 200 rpm (37 8C) (McCarthy and DeVellis, 1980). Cell culture medium was replaced after overnight shaking. To identify astrocytes, cultures were stained immunocytochemically for GFAP. Analysis of the cultures has shown that 90–95% of cells were GFAP-positive. All experiments were performed on 21-day cultures. Experimental Procedure Normoxic astrocytes were cultured in DMEM medium with 5.5 mM glucose and 10% FBS. In order to mimic the in vivo ischemia, cultures were placed for 24 h in 37 8C in 3% O2/5% CO2/92% N2 (CO2 incubator, Heraeus, Germany) in DMEM without glucose and serum. Osmolarity of the medium was measured and adjusted to 319 mOsm with mannitol. Cells were treated with aniracetam (1, 10 and 100 mM) for 24 h in normoxia and for 24 h of simulated ischemia. Aniracetam was dissolved in dimethyl sulphoxide (DMSO) at an initial concentration of 10 mM. Further dilutions were performed in the appropriate medium. Kinase inhibitor: PD98059 was prepared as DMSO stocks at 50 mM and added to the culture medium at final concentration 50 mM; wortmannin was added to the culture medium at 0.1 mM.
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Assessment of Apoptosis Cell nuclei staining by Hoechst 33342 enables visualization of fragmented and condensed DNA, a characteristic feature of apoptosis. After washing with PBS astrocytes cultured on coverslips were fixed for 10 min with a 4% paraformaldehyde at room temperature. Subsequently, after being washed twice with PBS the samples were dehydrated first in 70% ethanol and then in absolute ethanol. The samples were kept in 20 8C until they were stained with Hoechst 33342 (5 mg/ml in PBS) for 5 min at room temperature. Then the cells were washed again with PBS. Cell nuclei analysis was conducted with the fluorescence imaging MiraCal Pro III workstation (Life Science Resources Ltd., UK) comprising of inverted microscope Eclipse TE200 (Nikon, Japan) (ex/em 340/510 nm). A 20 objective was used. The number of apoptotic nuclei was determined on at least six randomly selected areas from three coverslips of every experimental group, each containing approximately 200 cells. The results were expressed as a percentage of apoptotic cells relative to the total number of cells. Measurement of ATP and PCr Concentrations The ATP and PCr concentrations were determined by high-specific firefly luciferin-luciferase bioluminescence assay kit using a fluoroscan ascent plate reader (Labsystems, Finland). The extraction of ATP and PCr was performed according to procedure described by Fitzpatrick et al. (1988). The conversion of PCr into ATP catalyzed by creatine kinase was performed according to the method of Lowry and Passonneau (1972). ATP concentrations were measured by bioluminescence quantification according to the method of Lust et al. (1981). Briefly, 100 ml samples of disrupted cells were added to equal volumes of ice-cold 5% TCA solution with 2 mM EDTA, vortexed rapidly for 5 s and incubated for 30 min at room temperature. Then the samples were 20-fold diluted with Tris–acetate buffer pH 7.75 and 10 ml solution of known ATP concentration was added to each well as internal standard and the total amount of ATP was determined. The results were calculated as nM of ATP or PCr per mg of protein (nM/ mg). Measurement of Caspase-3 Activity Enzymatic activity of caspase-3 was determined by caspase-3 colorimetric assay (R&D System, UK),
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according to the manufacturer’s instructions. This assay is based on the release of the fluorochrome pnitroaniline (p-NA) combined with a caspase-3 specific peptide substrate (DEVD–p-NA). Peptide cleavage through an active caspase-3 releases chromophor pNA, which can be quantified using colorimetric plate reader (405 nm). Briefly, cultured astocytes were lysed and cell extracts were centrifuged to eliminate cellular debris. Aliquots (20 ml of the cell extracts) were incubated for 2 h at 37 8C in the presence of the substrate. The level of caspase-3 activity, proportional to the color reaction intensity was expressed as a percentage of control. Cellular Oxidative Stress [20 ,70 -Dichlorofluorescein (DCF) Fluorescence] Cellular oxidative stress was determined on the basis of ROS-mediated conversion on 20 ,70 -DCF-DA into fluorescent DCF (Mattson et al., 1995). Cultured astrocytes were loaded with 100 mM DCF-DA by incubating for 50 min. Cells were washed three times with HBSS and DCF fluorescence was quantified using fluoroscan microplate reader (Labsystems, Finland). The dye was excited at 485 nm, and emission was filtered using a 538 nm barrier filter. ROS production was expressed as a percentage of control cells. RT-PCR RT-PCR analysis was used to determine expression of c-fos and c-jun genes in cultured rat astrocytes exposed to 24 ischemia and treated with 1, 10 or 100 mM aniracetam. Total RNA was isolated from astrocytes cultured on 100 mm culture cell dishes by Chomczynski extraction method (Chomczynski and Sacchi, 1987) with the TRIzol reagent. Precipitated and purified RNA was dissolved in sterile nuclease-free water (Promega, USA) and stored at 70 8C before use. Isolated RNA was quantified by determining absorbance at 260 nm (one absorbance unit at 260 nm equals 42 mg/ ml RNA). The reverse transcription reaction was performed using 100 ng of total RNA per 10 ml RT reaction volume (10 mM Tris–HCl buffer, 2.5 Thermus thermophilus (Tth) polymerase (Promega)) for 30 min at 60 8C. The total PCR mixture (50 ml) contained chelating buffer (0.75 mM EGTA, 100 mM KCl, 0.05% Tween, 5% glycerol), 2.5 mM Mg2þ, 10 mM dNTP and 1.0 U Thermus aquaticus (Taq) polymerase (Promega, USA). Amplification was carried out for 30 cycles for c-fos, c-jun and b-actin primers with 45 s denaturation at
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94 8C, 45 s annealing at 57 8C and 1 min extension 72 8C. The last extension step at 72 8C was prolonged for 7 min. The primers used in the PCR analysis correspond to the exon 1 DNA sequences of the rat c-fos gene, 50 -CTT GAA GAC GAG AAG TCT GCG30 for the upstream primer and 50 -GGT CAT TGA GAA GAG GCA GG-30 for the downstream primer (Simi et al., 2000). The primers for c-jun corresponded to the following DNA sequences in exon 1 of the rat c-jun gene: 50 -AAC AGA TCC CGG TGC AGC AC-30 for the upstream, and 50 -CCA CCT GTT CCC TGA GCA TG-30 for the downstream primer (Simi et al., 2000). The b-actin primers corresponded to the following sequences of the rat b-actin gene 50 -CCT GCG TCT GGA CCT GGC TG-30 and 50 -CTC AGG AGG AGC AAT GAT CT-30 for the upstream and the downstream primer, respectively (Fang et al., 1998). RT and PCR were performed in BIOMETRA UNOII Termoblock thermocycler (Germany). The 10 ml of amplified PCR products were analyzed on a 2% LMPagarose gel (Promega, USA) stained by ethidium-bromide in 1% TAE buffer and analyzed by densitometry. The results were quantified using Image Pro Plus software and expressed in average pixel intensity. All the results were normalized with the expression of the b-actin housekeeping gene. Experiments were repeated three times and the values of relative optical density were subjected to statistical analysis. Protein Determination
decreased the amount of apoptotic nuclei. Exposing astrocytes to 100 mM aniracetam during 24 h ischemia period turned out to be the most effective method in the prevention of apoptosis induced by simulated ischemia. To investigate the role of phosphatidylinositol 3kinase (PI 3-kinase) and mitogen-activated protein (MAP) kinase (MEK) in antiapoptic effect of aniracetam, we examined the effects of the phosphoinositide 3-kinase inhibitor wortmannin (0.1 mM) and MEK inhibitor PD98059 (50 mM). When the cells were treated with 0.1 mM wortmannin and 50 mM PD98059 during ischemia, 86 7:3 and 38:7 6% of the cells died, respectively. Simultaneous use of 100 mM aniracetam and wortmannin or PD98059 decreased the number of apoptotic nuclei to 3:9 þ 1:3 and 11:8 þ 2:9%, respectively, which indicates the drug’s strong influence on PI 3-kinase and MEK pathways (Fig. 1). In the cultures of astrocytes exposed to ischemia in the presence wortmannin (Fig. 2A) or PD98059 (Fig. 2B), a lot of fragmented and pyknotic nuclei occurred. Treatment of astrocytes with 100 mM aniracetam and wortmannin (Fig. 2C) or PD98059 (Fig. 2D) during ischemia prevented the process of apoptosis. Influence of Aniracetam on Intracellular ATP and PCr Concentrations Increased ATP and PCr levels in cells treated with 1, 10 or 100 mM aniracetam was observed in normoxia. The exposure of astrocytes to 24 h simulated ischemia
Protein content in astrocytes was measured according to the method of Lowry et al. (1951). Statistical Analysis Data were analyzed using one-way analysis of variance (ANOVA) followed by the Student–Newman– Keul’s test. A P-value of 0.05 was considered as statistically significant. All data were expressed as mean S:D:
RESULTS Influence of Aniracetam on Apoptosis (Hoechst 33342 Staining) The 24 h simulated ischemia significantly increased the number of apoptotic nuclei as compared to the normoxic conditions. The presence of aniracetam in the culture medium in these conditions significantly
Fig. 1. Effect of aniracetam (ANI) on astrocyte apoptosis induced by simulated ischemia. The results are shown as a percentage relation of the apoptotic nuclei to the total amount of nuclei in the field and are a mean S:D: of the six randomly selected areas from three culture dishes; (*) P < 0:05 vs. normoxia; (#) P < 0:05 vs. ischemia.
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Fig. 2. Effect of aniracetam on astrocyte apoptosis induced by simulated ischemia and PD98059 or wortmannin exposure. Cells were incubated with 100 nM wortmannin (A) and 50 mM PD98059 (C) in the absence or presence of 100 mM aniracetam (B, D, respectively) for 24 h ischemia. Then, the cells were fixed and stained with the Hoechst 33342 dye. Microphotographs were taken with the use of fluorescent microscope and 20 objective. Thin arrows point at apoptotic nuclei and thick arrows point at live nuclei.
caused a significant reduction in the amount of both high-energy phosphates. In this experimental option aniracetam produced significant increase of the intracellular ATP concentration. The significant decrease in value of these parameters were observed in ischemic conditions after treatment with 0.1 mM wortmannin. This effect was antagonized by aniracetam. Administration of 50 mM PD98059 did not affect the ATP level in ‘‘ischemic’’ astrocytes, but the protective effect of aniracetam in comparison to the normoxia control was observed. At the same time no changes were seen in intracellular concentrations of PCr when the cells were exposed during simulated ischemia to PD98059 and aniracetam (Figs. 3 and 4).
Effect of Aniracetam on ROS Production
Influence of Aniracetam on Caspase-3 Activity
Effect of Aniracetam on c-fos and c-jun Expressions
Treatment with 1 mM aniracetam did not change the enzyme proteolytic activity in comparison with simulated ischemia. However, simultaneous exposure of astrocytes to ischemia and 10 or 100 mM aniracetam substantially decreased the caspase-3 activity. After 100 mM aniracetam administration for 24 h ischemia a similar activity of the enzyme to that in normoxia was observed. (Fig. 5).
Fig. 6. shows the results of the influence of aniracetam on cellular oxidative stress. A substantial decrease of DCF fluorescence in astrocytes in normoxic conditions stimulated by aniracetam indicated a significant decrease in ROS production (Fig. 6A). A 24 h simulated ischemia intensified, while administration of aniracetam during this period attenuated the conversion of DCF. An additional exposition of astrocytes in these conditions to PD98059 increased ROS production in comparison to the normoxia control. However, treatment with 100 mM aniracetam together with kinase inhibitors decreased the DCF fluorescence intensity during 24 h ischemia (Fig. 6B).
The exposure of astrocyte cultures to 24 h simulated ischemia caused an increase both genes expression, the c-jun expression in examined cultures was weaker than c-fos expression. A treatment with 1 mM aniracetam was without effect on the expression of cfos and c-jun genes. However, a 24 h treatment with 10 and 100 mM aniracetam markedly attenuated
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Fig. 3. Effect of aniracetam (ANI) on intracellular ATP concentration in cultured rat astrocytes exposed to normoxia (A) and simulated ischemia (B). The results are shown as nM/ mg protein. Each value is mean obtained form eight dishes S:D: (n ¼ 8); (*) P < 0:05 vs. normoxia; (#) P < 0:05 vs. ischemia.
Fig. 4. Effect of aniracetam (ANI) on intracellular PCr concentration in cultured rat astrocytes exposed to normoxia (A) and simulated ischemia (B). The results are shown as nM/mg protein. Each value is mean obtained form eight dishes S:D: (n ¼ 8); (*) P < 0:05 vs. normoxia; (#) P < 0:05 vs. ischemia.
ischemia-induced expression c-fos and c-jun in astrocytes (Fig. 7A,B,C).
DISCUSSION We examined the protective effects of aniracetam against simulated ischemia-induced apoptosis in cultured rat astrocytes. This experimental model may contribute to clarification of the mechanisms of drugs that ameliorate ischemia-induced brain dysfunction (Takuma et al., 2000). The glial cells that interact with neurons play important roles in ischemic brain injury (Petito et al., 1998). Astrocytes produce several different antiapoptic growth factors (e.g. bFGF, NGF, BDNF), while microglia produce neurotoxic substances such as nitric oxide and excitotoxins (Schwartz and Nishiyama, 1994; Mattson et al., 2000).
Fig. 5. Effect of aniracetam (ANI) on caspase-3 activity in cultured rat astrocytes exposed to simulated ischemia. The results are shown as a percentages of the control value in the normoxia conditions. Each value is mean of eight dishes S:D: (n ¼ 8); (*) P < 0:05 vs. normoxia; (#) P < 0:05 vs. ischemia.
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Fig. 6. Effect of aniracetam (ANI) on DCF fluorescence (as measure of ROS production) in cultured rat astrocytes exposed to normoxia (A) and simulated ischemia (B). For each experimental group fluorescence intensity was determined. The results are shown as a percentage relation of the control value in the normoxia conditions. Each value is mean activity obtained form 12 wells S:D: (n ¼ 12); (*) P < 0:05 vs. normoxia; (#) P < 0:05 vs. ischemia.
Astrocyte apoptosis in vitro is induced by several signals, e.g. HIV-1 infection (He et al., 1997), S100 b (Hu and Van Eldik, 1996), staurosporine (Mangura and Dawson, 1998), Ca2þ deprivation (Takuma et al., 1999; Chiesa et al., 1998) and hypoxia (Gabryel et al., 2001). The factors determining the apoptotic cell death are the change in mitochondrial membrane permeability as a result of the occurrence of mitochondrial permeability transition pore (MTP) (Zoratti and Szabo’, 1995) and intracellular level of ATP (Leist et al., 1997; Eguchi et al., 1997). The opening of MTP is accompanied by inhibition of oxidative phosphorylation and activation of the mitochondrial ATP-ase which increases the consumption of ATP (Nieminen et al., 1994; Cai and Jones, 1998). When glycolysis or the use of energy stores accumulated in PCr are able to partly compensate the ATP deficiency, the apoptosis program is
Fig. 7. Effect of aniracetam (ANI) on c-fos and c-jun expression in cultured rat astrocytes exposed to 24 h hypoxia. (A) The cDNA fragments obtained after RT-PCR using primers specific for c-fos, c-jun and b-actin were visualized under UV after electrophoresis in the presence of EtBr; lane 1: control cultures (normoxia), lane 2: astrocytes exposed to 24 h simulated ischemia (ischemia), lane 3: 1 mM aniracetam, lane 4: 10 mM aniracetam, lane 5: 100 mM aniracetam. (B) The c-fos and (C) c-jun expressions after 24 h ischemia as measured by relative optical density of bands. The signals from c-fos and c-jun cDNA fragments were normalized to b-actin at different experimental conditions. Data represent mean S:D: (*) P < 0:05 vs. normoxia; (#) P < 0:05 vs. ischemia.
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initiated. The energy stored in ATP is necessarily used in major apoptotic pathways leading from cytochrome c (Li et al., 1997), apoptosis-inducing factor (AIF) and death receptors to caspases activity (Susin et al., 1999). Likewise, morphological alternations of the cell nuclei, featuring for apoptosis, are the result of the signal transduction from cytoplasm to the nuclei through active transport requiring ATP hydrolysis (Melchior and Gerace, 1995; Pante and Aebi, 1996). In the cultures subjected to 24 h simulated ischemia we observed a significant increase in the number of apoptotic astrocytes in comparison to the control. Simultaneous exposure of the cells during ischemia to increasing concentrations of aniracetam (1, 10, 100 mM) prevented apoptosis. To investigate the role of PI 3-kinase and MAP kinase (MEK) in antiapoptotic effect of aniracetam, the effects of the PI 3-kinase inhibitor wortmannin (0.1 mM) and MEK inhibitor PD98059 (50 mM) were examined. The addition of both substances into the culture medium of the cells exposed to 24 h ischemia caused a significant increase of the amount of apoptotic cells. At the same time it has been observed that administration of kinase inhibitors with 100 mM aniracetam prevents the apoptosis completely and the amount of apoptotic cells was similar to the amount in normoxia conditions. These results indicate a distinct protective action of aniracetam on astrocytes damaged by simulated ischemia. With ischemia, analytic measures of high-energy compounds (ATP and PCr) in rapidly frozen decapitated brain showed an initial partial loss of PCr followed by a simultaneous loss of both PCr and ATP (Siesjo¨ and Zwetnow, 1970). Since the restoration by cells ability to produce high-energy compounds enables regeneration of the ischemic damages we assessed the influence of aniracetam on intracellular amount of ATP and PCr in normoxic and ischemic conditions. In both situations we have demonstrated a significant increase of the ATP amount. Aniracetam showed weaker influence on the PCr than on ATP in astrocytes damaged by simulated ischemia. Both used kinase inhibitors caused a significant decrease of the intracellular ATP, while the use of aniracetam in this conditions reversed the effect. PD98059 or aniracetam together with PD98059 did not change the intracellular concentration of PCr. On the other hand, exposure the cells to aniracetam and wortmannin restored the PCr level to the value similar to normoxia conditions. PCr level was significantly decreased when only wortmannin was added. The drug prevented ATP lose probably through partial use of PCr as an energy store which also enabled maintenance of mitochondria capacity.
Caspase-3 activity is one of the fundamental events of the so-called apoptosis execution phase (Gorman et al., 1998). This enzyme decomposes various proteins, i.e. cell cycle regulatory proteins, poly(ADPrybose)polymerase and activates other caspase family members, such as caspase-2, caspase-6 and caspase-7 (Nicholson, 1999). There is same evidence that phosphorylation or other covalent post-translational modification (other than proteolytic activation) participates in caspase regulation. The serine/threonine kinase Akt/ PKB is activated by PI 3-kinase and opposes apoptosis by several mechanisms including catalytic inactivation of caspase-9 (Yuan et al., 1993). This suggests a particularly important regulatory role for caspase-9 in apoptosis, since there is currently no evidence for similar Akt-mediated inactivation of other caspases. Inhibition of caspase-9, together with cytochrome c and Apaf-1 form apoptosom disables activation of caspase-3. We demonstrated significant increase of the apoptotic nuclei after adding wortmannin as a PI 3-kinase inhibitor as well as aniracetam ability to reverse apoptotic effect of wortmannin. Therefore, the inhibiting influence of aniracetam on caspase-3 activity which we observed may be result of the drug’s involvement in the PI 3-kinase/Akt pathway. Caspases also disrupt activation of the transcription factor NFkB, which normally activates antiapoptotic gene expression, including members of IAP family (Wang et al., 1998). In normal conditions, NFkB is present in cytoplasm as a inactive complex, composed with a p50/p65 heterodimer bound to an inhibitory subunit IkB. Activation of NFkB is induced by phosphorylation of IkB, which leads to the dissociation of this complex. Subsequently, IkB is degraded, and the p50/p65 heterodimer translocated from cytoplasm into nucleus, where may induce expression of several genes (Thanos and Maniatis, 1985). Caspases cleave the inhibitor of NFkB, IkBa, producing a C-terminal fragment that is resistant to proteosomal degradation and stable inhibits NFkB, sensitizing cells to apoptosis (Reuther and Baldwin, 1999). Tissue injury including cerebral ischemia is a potent stimulus for activation of NFkB (Kolesnick and Golde, 1994; Liu et al., 1994). Among others, NFkB constitutes an important component of transcriptional regulation of antioxidant enzyme—manganese superoxide dismutace (MnSOD) (Mattson et al., 1997). Antioxidant effect of aniracetam was shown by the fluorescent method where we observed a reduced occurrence of ROS as a decrease in DCF fluorescence. Moreover, we have also observed a significant decrease of the stimulating influence of wortmannin and PD98059 on forming
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ROS with the simultaneous use of aniracetam (Fig. 6). MAP kinase (MEK 1/2) whose inhibitor is PD98059 (Dudley et al., 1995) takes part in the process of IkB phosphorylation (Lee et al., 1997). The MEK signal pathway is proposed to play a role in cell survival (Yujiri et al., 1998), it is possible that the MAP signal pathways may be target for the drugs to ameliorate ischemia/reperfusion injury. Our results indirectly indicate this pathway’s participation in the protective effect exerted by aniracetam in ischemia. From studies showing that AMPA receptor agonists stimulate Ca2þ-dependent phosphorylation of CREB (cAMP response element-binding protein), a common glutamate receptor-mediated signal transduction pathway involving Ras and MAP kinase has emerged in different glial cell types (Pende et al., 1997). Mitogen-activated protein kinases (MAPKs) are well-known intracellular signaling cascades involved in gene regulation. Activation of the MAPKs results in the phosphorylation and activation of transcription factors binding to the corresponding response elements in their promoters, which results in a subsequent gene activation. Therefore, another goal of this study was to analyze the expression of the genes c-jun and c-fos known to be activated by MAPKs and implicated in apoptosis (Treisman, 1996). Both fos and jun family genes have been studied in models of experimental ischemia, mostly in rodents (Feuerstain et al., 1997; Lu et al., 1997; Yang et al., 1997). Despite the fact that cfos and c-jun belong to the s.c. immediate early genes family, many articles present assessment of their expression in vitro 24 h or even 48 h after occurrence of the damaging factor, e.g. calcium deprivation or treatment with ceramide (Chiesa et al., 1998; Willaime et al., 2001). We assessed the intensification of the c-fos and c-jun genes expression after the 24 h exposition of the astrocyte cell culture to ischemia and treatment with aniracetam in order to, together with other parameters measured in ‘‘one point of time’’, assess the level of apoptosis in cells exposed to damaging factor and treated pharmacologically. Using semiquantitive RT-PCR we observed the increase in amount of mRNA of both genes in astrocytes exposed to 24 h simulated ischemia. Aniracetam caused decrease of the c-fos and c-jun expressions proportionately to the concentration used (Fig. 7). A glioprotective action of aniracetam against simulated ischemia was seen after addition of a high concentration of aniracetam (100 mM), but not after a low concentration (1 mM). But, in some experiments in vitro on neuronal cultures the 100 mM concentration of aniracetam was added to the medium (Copani et al.,
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1992; Pizzi et al., 1993; Kolta et al., 1998). Moreover, in several electrophysiological studies the concentrations 300–1000 mM in different model systems were used (Kapus et al., 2000; Buldakova et al., 2000). It should be noted that, our data for the first time demonstrate that aniracetam exerts protective action against apoptosis induced by simulated in vitro ischemia in primary culture of astrocytes. It cannot be excluded that the final outcome of the action of aniracetam might be dependent on the metabolic differences between astrocytes and neurons (Gabryel and Trzeciak, 2001). Summing up, the observed glioprotective effects of aniracetam are the result of this drug interference with a number of factors determining cell apoptosis and their viability. Our study shows that antiapoptotic affect of aniracetam is connected with its stimulating influence on the intracellular amount of high-energy phosphates, decreased caspase-3 activity, ROS production and c-fos and c-jun genes expression. We demonstrated that aniracetam completely reverses the apoptotic effect of MEK inhibitor PD98059 and PI 3-kinase inhibitor wortmannin. The results suggest that protective effect of aniracetam is mediated both through MEK kinase and phosphoinisitide 3-kinase signal pathways. It seems that studies bring the hypothesis of the new mechanisms of aniracetam antiischemic activity indicating their potential protective influence towards astrocytes in ischemia conditions.
ACKNOWLEDGEMENTS The work was supported by grant of State Committee for Scientific Research 4 PO5D 00516 and grant from Silesian Medical University.
REFERENCES Aschner M. Astrocytic functions and physiological reactions to injury: the potential to induce and/or exacerbate neuronal dysfunction—a forum position paper. Neurotoxicology 1998; 19:7–18. Bartolini L, Risaliti R, Pepeu G. Effect of scopolamine and nootropic drugs on rewarded alternation in a T-maze. Pharmacol Biochem Behav 1992;43:1161–4. Buldakova SL, Bolshakov KV, Tikhonov DB, Magazanik LG. Ca2þdependent desensitization of AMPA receptors. NeuroReport 2000;11:2937–41. Cai J, Jones DP. Superoxide in apoptosis. Mitochondrial generation triggered by cytochrome c loss. J Biol Chem 1998;273:11401–4. Chiesa R, Angeretti N, Del Bo R, Lucca E, Munna E, Forloni G. Extracellular calcium deprivation in astrocytes: regulation of
394
B. Gabryel et al. / NeuroToxicology 23 (2002) 385–395
mRNA expression and apoptosis. J Neurochem 1998;70:1474– 83. Chomczynski P, Sacchi N. Single step method of RNA isolation by acid guanidinum thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156–9. Copani A, Genazzani AA, Aleppo G, Casabona G, Canonico PL, Scapagnini U, Nicoletti F. Noortropic drugs positively modulate a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-sensitive glutamate receptors in neuronal cultures. J Neurochem 1992;58:1199–204. Cumin R, Bandle EF, Gamzu E, Haefely WE. Effects of the novel compound aniracetam (Ro 13-5057) upon impaired learning and memory in rodents. Psychopharmacology (Berl) 1982;78:104– 11. DeNoble VJ, Repeti SJ, Gelpke LW, Wood LM, Keim KL. Vinpocetine: nootropic effects on scopolamine-induced and hypoxia-induced retrieval deficits of a step-through passive avoidance response in rats. Pharmacol Biochem Behav 1986; 24:1123–8. Dudley DT, Pang L, Decker SJ, Bridges AJ, Saltiel AR. Synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci USA 1995;92:7686–9. Eguchi Y, Shimizu S, Tsujimoto Y. Intracellular ATP levels determine cell fated by apoptosis or necrosis. Cancer Res 1997; 57:1835–40. Fang C, Lindros KO, Badger TM, Ronis MJ, Ingelman-Sundberg M. Zonated expression of cytokines in the rat liver: effect of chronic ethanol and the cytochrome P450 2E1 inhibitor, chlomethiazole. Hepatology 1998;27:1304–10. Feuerstain GZ, Wang X, Barone FC. Inflammatory gene expression in cerebral ischemia and trauma. Ann N Y Acad Sci 1997;825:179–93. Fitzpatrick A, Cooper AJL, Hertz L. Effects of ammonia and bmethylene-DL-asparate on the oxidation of glucose and pyruvate by neurons and astrocytes in primary culture. J Neurochem 1988;51:1197–203. Gabryel B, Trzeciak HI. Role of astrocytes in pathogenesis of ischemic brain injury. Neurotoxicity Res 2001;3:205–21. Gabryel B, Adamek M, Trzeciak HI. Does trimetazidine exert cytoprotective activity on astrocytes subjected to hypoxia in vitro? Neurotoxicology 2001;22:455–65. Gorman AM, Orrenius S, Cecatelli S. Apoptosis in neuronal cells: role of caspases. NeuroReport 1998;9:49–55. Gu JG, Albuquerque C, Lee CJ, Macdermott AB. Synaptic strengthening through activation of Ca2þ-permeable AMPA receptors. Nature 1996;381:793–6. He J, DeCastro JM, Vandenbark GR, Busciglio J, Gabuzda D. Astrocyte apoptosis induced by HIV-1 transactivation of the ckit protooncogene. Proc Natl Acad Sci USA 1997;94:3954–9. Hertz L, Juurlink BHJ, Szuchet S. Cell cultures. In: Lajtha A, editor. Handbook of neurochemistry, vol. 8. New York: Plenum Press, 1985. p. 603–61. Hu J, Van Eldik LJ. S 100 b induces apoptotic cell death in cultured astrocytes via a nitric oxide-dependent pathway. Biochim Biophys Acta 1996;1313:239–45. Kapus G, Bodi I, Pataki A, Gueritaud J-P, Szekely JI, Tarnawa I. Differential modulation of the GYKI 53784-induced inhibition of AMPA currents by various AMPA-positive modulators in cerebellar Purkinje cells. Eur J Pharmacol 2000;397:43–7. Kolesnick R, Golde DW. The sphingomyelin pathway in tumor necrosis factor and interleukin-1 signaling. Cell 1994;77:325–8.
Kolta A, Lynch G, Ambros-Ingerson J. Effects of aniracetam after LTP induction are suggestive of interactions on the kinetics of the AMPA receptor channel. Brain Res 1998;788:269–86. Lee FS, Hagler J, Chen ZJ, Maniatis T. Activation of the IkB alpha kinase complex by MEKK1, a kinase of the JNK pathway. Cell 1997;88:213–22. Leist M, Single B, Castoldi AF, Kuhlne S, Nicotera P. Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis. J Exp Med 1997; 185:1481–6. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptosis protease cascade. Cell 1997;91:479–89. Liu T, Clark RK, Mc Donnell PC, Young PR, White MS, Barone FC, Feuerstein GZ. Tumor necrosis factor-a expression in ischemic neurons. Stroke 1994;25:1481–8. Lowry OH, Passonneau JV. A flexible system of enzymatic analysis. New York: Academic Press, 1972. p. 120–6. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193:265–75. Lu XC, Tortella FC, Ved HS, Garcia GE, Dave JR. Neuroprotective role of c-fos antisense oligonucleotide: in vitro and in vivo studies. NeuroReport 1997;8:2925–9. Lust WD, Feussner GK, Barbehenn EK, Passoneau JV. The enzymatic measurement of adenine nucleotides and p-creatine in picomole amounts. Anal Biochem 1981;110:258–66. Mangura D, Dawson G. Programmed cell death in cortical chick embryo astrocytes is associated with activation of protein kinase PK60 and ceramide formation. J Neurochem 1998;70: 130–8. Martin JR, Cumin R, Aschwanden W, Moreau JL, Jenck F, Haefely WE. Aniracetam improves radial maze performance in rats. NeuroReport 1992;3:81–3. Mattson MP, Barger SW, Begley JG, Mark RJ. Calcium, free radicals, and excitotoxic neuronal death in primary cell culture. Methods Cell Biol 1995;46:187–215. Mattson MP, Goodman Y, Luo H, Fu W, Furukawa K. Activation of NF-kB protects hippocampal neurons against oxidative stress-induced apoptosis: evidence for induction of manganese superoxide dismutase and suppression of peroxynitrite production and protein tyrosine nitration. J Neurosci Res 1997; 49:681–97. Mattson MP, Culmsee C, Yu ZF. Apoptotic and antiapoptotic mechanisms in stroke. Cell Tissue Res 2000;301:173–87. McCarthy KD, DeVellis J. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol 1980;85:890–902. Melchior F, Gerace L. Mechanisms of nuclear protein import. Curr Opin Cell Biol 1995;7:310–8. Nicholson DW. Caspase structure, proteolytic substrates, and function during apoptotic cell death. Cell Death Differ 1999; 6:1028–42. Nieminen AL, Saylor AK, Herman B, Lemasters JJ. ATP depletion rather than mitochondrial depolarization mediates hepatocyte killing after metabolic inhibition. Am J Physiol 1994;267:67–74. Otomo E, Hirai S, Terashi A, Hasegawa Y, Tazaki Y, Araki G, Itoh E, Nishimura T, Furukawa T. Clinical usefulness of aniracetam for psychiatric symptoms in patients with cerebrovascular
B. Gabryel et al. / NeuroToxicology 23 (2002) 385–395 disorders: placebo controlled double-blind trial. J Clin Exp Med 1991;156:143–87. Pante N, Aebi U. Toward the molecular dissection of protein import into nuclei. Curr Opin Cell Biol 1996;8:397–406. Pende M, Fischer TL, Simpson PB, Russell JT, Blenis J, Gallo V. Neurotransmitter and growth factor-induced cAMP response element binding protein phosphorylation in glial cell progenitors: role of calcium ions, protein kinase C, and mitogen-activated protein kinase/ribosomal S6 kinase pathway. J Neurosci 1997;15:1291–301. Petito CK, Olarte JP, Roberts B, Nowak TS, Pulsinelli WA. Selective glial vulnerability following transient global ischemia in rat brain. J Neuropathol Exp Neurol 1998;57:231–8. Reuther JY, Baldwin AS. Apoptosis promotes a caspase induced amino-terminal truncation of IkBa that function as a stable inhibitor of NFkB. J Biol Chem 1999;274:20664–206670. Pizzi M, Fallacara C, Arrighi V, Memo M, Spano P. Attenuation of excitatory amino acid toxicity by metabotropic glutamate receptor agonists and aniracetam in primary cultures of cerebellar granule cells. J Neurochem 1993;61:683–9. Schwartz JP, Nishiyama N. Neurotrophic factor gene expression in astrocytes during development and following injury. Brain Res Bull 1994;35:403–7. Shirane M, Nakamura K. Group II metabotropic glutamate receptors are a common target of N-anisoyl-GABA and 1S, 3RACPD in enhancing ACh release in the prefrontal cortex of freely moving SHRSP. Neurpharmacology 2000;39:866–72. Siesjo¨ B, Zwetnow N. The effect of hypovolemic hypotension on extra- and intracellular acid-base parameters and energy metabolites in the rat brain. Acta Physiol Scand 1970;79:114– 24. Simi A, Ingelman-Sundberg M, Tindberg N. Neuroprotective agent chlomethiazole attenuates c-fos, c-jun and AP-1 activation through inhibition of p38 MAP kinase. J Cereb Blood Flow Metab 2000;20:1077–88. Susin SA, Lorenzo HK, Zamzani N, Marzo I, Snow BE, Brothers GM, Mangion J, Jacotot E, Constantini P, Loeffler M, Larochette N, Goodlett DR, Aebersold R, Siderovski DP, Penninger JM, Kroemer G. Molecular characterization of
395
mitochondrial apoptosis-inducing factor. Nature 1999;397:441– 6. Takuma K, Lee E, Kidawara M, Mori K, Kimura Y, Baba A, Matsuda T. Apoptosis in Ca2þ reperfusion injury of cultured astrocytes: roles of reactive oxygen species and NF-kB activation. Eur J Neurosci 1999;11:4204–12. Takuma K, Fujita T, Kimura Y, Tanabe M, Yamamuro A, Lee E, Mori K, Koyama Y, Baba A, Matsuda T. T-588 inhibits astrocyte apoptosis via mitogen-activated protein kinase signal pathway. Eur J Pharmacol 2000;399:1–8. Thanos D, Maniatis T. NF-kB, a lesson in family values. Cell 1985;80:529–32. Treisman R. Regulation of transcription by MAP kinase cascades. Curr Opin Cell Biol 1996;8:205–15. Tsuzuki K, Takeuchi T, Ozawa S. Agonist-dependent and subunitdependent potentiation of glutamate receptors by nootropic drug aniracetam. Mol Brain Res 1992;16:105–10. Wang CY, Mayo MW, Korneluk RG, Goeddel DV, Baldwin AS. NFkB antiapoptosis: Induction of TRAF1 and TRAF2 and cIAP1 and c-IAP2 to suppress caspase-8 activation. Science 1998;281:1680–3. Willaime S, Vanhoutle P, Caboche J, Lemaigre-Dubreuil Y, Mariani J, Brugg B. Ceramide-induced apoptosis in cortical neurons is mediated by an increase in p38 phosphorylation and not by the decrease in ERK phosphorylation. Eur J Neurosci 2001;13:2037–46. Yang DD, Kuan CY, Whitmarsh AJ, Rincon M, Zheng TS, Davis RJ, Rakic P, Flavell RA. Absence of excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene. Nature 1997;389:865–70. Yuan J, Shaham S, Ledoux S, Ellis HM, Horvitz HR. The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interlekin-1b-converting enzyme. Cell 1993;75: 641–52. Yujiri T, Sather S, Fanger GR, Johnson GL. Role of MEKK1 in cell survival and activation of JNK and ERK pathways defined by targeted gene disruption. Science 1998;282:1911–4. Zoratti M, Szabo’ I. The mitochondrial permeability transition. Biochim Biophys Acta 1995;1241:139–76.