Does Trimetazidine Exert Cytoprotective Activity on Astrocytes Subjected to Hypoxia In Vitro?

NeuroToxicology 22 (2001) 455±465

Does Trimetazidine Exert Cytoprotective Activity on Astrocytes Subjected to Hypoxia In Vitro? Bozena Gabryel*, Mariusz Adamek, Henryk I. Trzeciak Department of Pharmacology, Silesian Medical University, 18 MedykoÂw Street, PL-40752 Katowice, Poland Received 16 March 2001; accepted 16 May 2001

Abstract The aim of the present study was to establish whether trimetazidine (TMZ) is capable of protecting astrocytes against hypoxic injury. Using the model of astrocyte cell culture we tried to observe the cells treated with TMZ before, during and after hypoxia simulated in vitro. Cell viability was determined by Live/Dead (viability/cytotoxicity) Assay Kit and MTT conversion test. Apoptotic cell death was distinguished by a method using ¯uorescence microscopy with Hoechst 33342. The effect of the drug on the DNA synthesis was evaluated by measuring the incorporation of [3 H]thymidine into DNA of astrocytes. TMZ stimulates the proliferation of astrocytes most signi®cant one when the astrocytes are exposed to the drug in normoxia, hypoxia and/or re-oxygenation. Adding TMZ into cultures during re-oxygenation and hypoxia/ re-oxygenation signi®cantly decreases the number of dead and apoptotic cells. Our experiment has proved that TMZ exerts the most signi®cantly cytoprotective effect on astrocytes in vitro when added during hypoxia and/or reoxygenation. We may conclude that the protective effect of TMZ depends on the sequence of drug adding and hypoxia/ re-oxygenation onset. # 2001 Elsevier Science Inc. All rights reserved.

Keywords: Trimetazidine; Astrocytes; Hypoxia; Apoptosis

INTRODUCTION Disturbances in oxygen and glucose delivery to the brain caused by stroke initiate a series of biochemical changes leading to swelling, leakage of intracellular material and functional brain damage (SiesjoÈ, 1985). Studies on the pathomechanism of brain ischemia have mainly focused on neurones and, accordingly, therapeutic strategies have been designed to counteract neuronal dysfunction. However, recent data indicate that a decrease in neuronal survival during and after ischemia is also associated with astrocytic dysfunction (Aschner, 1998; Gabryel and Trzeciak, 2001). Perineuronal glial cells react to brain damage resulting from, among others, hypoxia by transforming themselves into so-called reactive astrocytes. They are

*

Corresponding author. E-mail address: [email protected] (B. Gabryel).

characterised by stellate morphology, increased glial ®brillary acidic protein (GFAP) expression, increased number of mitochondria and increased antioxidant activity, the last of which results in an increased ability of astrocytes to protect neurons against the damaging effect of free radicals (Petito, 1986; Liu et al., 1993; Takizawa et al., 1994). Astrocytes show a greater than neurons resistance to stress caused by ischemia/reperfusion (Petito et al., 1990). Factors that determine astroglial resistance to ischemic injury include: the presence of glycogen stores, rapid glutathione turnover, considerable antioxidant enzyme activity and high expression of the anti-apoptotic gene bcl-2 (Sagara et al., 1993; Vanella et al., 1989). Prolonged hypoxia however, causes disturbances in the astrocyte functions, such as, loss of cell volume control resulting in swelling of brain parenchyma and blood vessels and consequently leading to termination of brain circulation (Hossmann, 1985). Thus, counteracting the metabolic imbalance in astrocytes during ischemia and

0161-813X/01/$ ± see front matter # 2001 Elsevier Science Inc. All rights reserved. PII: S 0 1 6 1 - 8 1 3 X ( 0 1 ) 0 0 0 4 1 - 9

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reperfusion provide a new option of the pharmacological intervention in the prevention of brain damage. Trimetazidine (TMZ, 1-(2,3,4-trimethoxy-benzyl) piperazine dihydrochloride) was found effective in preventing changes caused by ischemia/reperfusion in cardiomiocytes (Morin et al., 1998). TMZ is used in the treatment of angina pectoris, during coronary angioplasty and in coronary artery by-pass graft surgery (Levy, 1995; Detry et al., 1994; Fantini et al., 1994). The mechanism of TMZ activity con®rming its effectiveness as an anti-ischemic drug was studied on the cell level mainly on cardiomiocytes and erythrocytes (Harpey et al., 1989; Guarnieri and Muscari, 1993; Maridonneau-Parini and Harpey, 1985). There is a still growing evidence suggesting the potential use of TMZ in anoxia and/or ischemia of CNS. TMZ penetrates blood±brain barrier at therapeutic concentration in serum equal to 0.15 mM/l (Ancerewicz et al., 1998). Its content in brain has been established to reach 70% of the concentration in heart muscle (Naito et al., 1972). In an experimental model mimicking CNS ischemia and reperfusion in vivo Smirnov et al. (1998a,b, 1999) have demonstrated TMZ to increase the levels of high-energy phosphates having at the same time an antioxidant activity. So far no experiments have been conducted concerning the possible protective in¯uence of the drug on the glial cells. Astrocytes show similar resistance against ischemic damage both: in vivo and in vitro. For this particular reason, primary astrocyte culture obtained from rat cerebral cortex provides a valuable experimental model to study biochemical, physiological and pharmacological properties of astrocytes in normal and pathological conditions, including hypoxia (Yu et al., 1989). The aim of the present study was to assess the ability of TMZ to protect astrocytes in culture against hypoxic injury. Using the model the effectiveness of TMZ added before, during and after hypoxia simulated in vitro was compared. MATERIALS AND METHODS Materials Chemicals and materials were obtained from the following sources: trimetazidine (Vastarel1, Biopharma, France), Dulbecco's modi®ed Eagle's medium (DMEM), foetal bovine serum (FBS), phosphate buffered saline (PBS), antibiotic±antimycotic mixture (all from GIBCO Laboratories, USA), DAKO PAP kit system glial ®brillary acidic protein (GFAP) test,

(DAKO Co., Denmark), Live/Dead Kit (Molecular Probes Inc., USA), poly-D-lysine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolinum bromide (MTT); Hoechst 33342, etylenediaminetetraacetic acid disodium salt (EDTA 2 Na), trichloracetic acid (TCA), trypan blue solution (0.4%) and other chemicals, all from Sigma, USA, [methyl-3 H]thymidine (2.00 Ci/ mM) (Dupont, NEN, USA), plastic tissue culture petri dishes (Becton Dickinson, USA). Primary Culture of Rat Astrocytes Astrocytes were isolated and cultured essentially according to the method of Hertz et al. (1985). Brie¯y, hemispheres from 1-day-old Wistar rat pups were removed aseptically from skulls, freed of the meninges, minced and mechanically disrupted by vortexing in DMEM containing penicillin (100 U/ml) and streptomycin (100 mg/ml). This suspension was ®ltered through sterile nylon screening cloth with pore sizes 70 mm (®rst sieving) and 10 mm (second sieving). Subsequently, cultures were incubated at 378C in 95% air and 5% CO2 with relative humidity 95% (CO2-incubator, Kebo-Assab, Sweden). Before the material was plated onto the plastic dishes the cells were counted in a Coulter Z1 counter (Coulter Counter, UK). Cells destined for isotopic study were plated at a density 3  105 cells per dish (35 mm in diameter). Cultures destined for MTT conversion measurements were conducted on 96-well dishes onto which the cells were sieved at 1  104 cells per well. Astrocytes for ¯uorescent studies (Live/Dead Kit and Hoechst 33342 staining) were grown on coverslips covered with polyD-lysine (100 mg/ml). The culture medium initially contained 20% of FBS. Then, the culture medium was changed after 4 days of seeding and replaced with medium containing 10% FBS twice a week. To identify astrocytes, cultures were stained immunocytochemically for GFAP. Analysis of the cultures has shown that 90±95% of the cells were GFAP-positive. All experiments were performed on 21-day cultures. Experimental Set-up Cell cultures were divided into experimental groups according to the scheme shown in Fig. 1. Control astrocytes (normoxia) 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 8 h in 378C in 3% O2/5% CO2/92% N2 (CO2 incubator, Heraeus, Germany) in DMEM without glucose and serum. Osmolarity (Osm) of the medium was measured

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457

Fig. 1. Cell culture treatment protocol. For details see materials and methods section.

and adjusted to 319 Osm with mannitol. Some astrocyte cultures were exposed for 8 h only to hypoxia. Cells were treated with TMZ according to six distinct protocols: (i) Group I, for 8 h in normoxia; (ii) Group II, only during 24 h in normoxia prior to 8 h of hypoxia; (iii) Group III, both, during 24 h of normoxia and subsequent 8 h of hypoxia; (iv) Group IV, only during 8 h of hypoxia; (v) Group V, only during 24 h of re-oxygenation; and (vi) Group VI, both, during 8 h of hypoxia and 24 h of re-oxygenation. Treatment of astrocytes with TMZ before hypoxia or 24 h re-oxygenation was in a medium containing glucose and serum, at 378C, in 95% air/5% CO2. Cell Death Cell death was determined by a ¯uorescent method with the use of a Live/Dead Kit (Molecular Probes Inc., USA) containing calcein/AM and ethidium homodimer-1 (EthD-1). Calcein/AM is absorbed by living cells and subsequently cleaved by cytosolic esterases into a green ¯uorescent (excitation/emission (ex/em) 495/530 nm) product, whereas EthD-1 is known to enter only the cell with compromised cell membrane permeability and after being attached to nucleic acids yields red ¯uorescence (ex/em 495/635 nm). Astrocytes plated on coverslips were treated with 10 mM TMZ. As soon as the experiments was terminated the cells were incubated with 2 mM of calcein/AM and 4 mM of EthD-1 for 30 min at 378C. The number of dead and live cells was determined on at least six

randomly selected areas from three coverslips of every experimental group, each containing approximately 200 cells. Cells were examined by ¯uorescence imaging MiraCal Pro III workstation (Life Science Resources Ltd., UK) comprising of inverted microscope Eclipse TE200 (Nicon, Japan) and high-resolution cooled CCD camera (Photonic Science Ltd., UK). The results were expressed as a percentage of dead cells relative to the total number of cells. Assessment of Apoptosis Cell nuclei staining by Hoechst 33342 enables determination of the DNA condensate occurrence, a characteristic feature of apoptosis. Astrocyte cultured on coverslips were treated with TMZ at the concentration 10 mM. After being washed with PBS, they were ®xed for 10 min with a 4% paraformaldehyde in room temperature. Then, after being washed twice with PBS the samples were dehydrated ®rst in 70% ethanol and then in absolute ethanol. The samples were kept in 208C, 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 above-described imaging system using the excitation/ emission wavelength of 340/510 nm, respectively. 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

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results were expressed as a percentage of apoptotic cells relative to the total number of cells. Mitochondrial Function Mitochondrial function of astrocytes treated with TMZ at 0.1, 1, 10 and 100 mM was evaluated with MTT conversion method (Mosman, 1983). The cells ability for MTT conversion by active mitochondrial dehydrogenases proves the integrity and activity of mitochondria, which might, in turn, indicate cell viability. MTT (®nal concentration, 0.25 mg/ml) was added to the medium 3 h before the scheduled end of the experiment and then the cultures were incubated

at 378C in proper conditions. At the end of experiment, after being washed twice with PBS, the cells were lysed in 100 ml dimethyl sulphoxide which enabled the release of the blue reaction product, formasan. Absorbency at the wavelength of 570 nm was read on a microplate reader, a results were expressed as a percentage of absorbency measured in control cells. [3H]Thymidine Incorporation The amount of incorporated [3 H]thymidine was measured according to the method of Neary et al. (1994) with slight modi®cations. In brief, astrocytes treated with TMZ (at 0.1, 1, 10 and 100 mM) in the

Fig. 2. Live/dead measurement in the astrocyte cultures treated with trimetazidine (1 mM). The microphotographs show experimental groups of astrocyte cultures treated with trimetazidine according to the scheme presented in Fig. 1. Live and dead cells were determined by the Live/Dead Kit. Microphotographs were taken with the use of fluorescent microscope with a 20 objective. Open-spiked arrows point at live cells stained with calceine, closed-spiked arrows point at nuclei of dead cells stained with EthD-1.

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above-mentioned experimental versions were incubated with [3 H]thymidine (20 Ci/mM) 0.5 mCi/ dish (2 ml) for 18 h in DMEM containing 0.5% FBS. At the end of incubation, cells were washed twice with 1 ml of ice-cold PBS. Then, cells were scraped from the dishes with a plastic policeman (Costar Co., USA) and centrifuged for 10 min (2000 rpm/min). About 1 ml of 10% TCA was added to the sediment and samples were incubated at 48C for 30 min. Then samples were centrifuged again for 10 min (2000 rpm/min). About 1 ml of 0.1 N NaOH was then added to each tube and samples were lysed overnight at 48C. Next day, the samples were neutralised by 10% formic acid and transferred to scintillation vials, each with 8 ml of Bray's scintillating solution. Radioactivity was determined in a Beckman LS 6000 IC counter (Beckman Instruments Inc., USA). The results were calculated in dpm/mg of protein and expressed as percentage of control. Statistical Analysis Data were analysed using one-way analysis of variance (ANOVA) followed by the Newman±Keuls test. Results at P < 0:05 were considered as statistically signi®cant. All data were expressed as mean  S:E:M.

459

RESULTS Effect of TMZ on Cell Death The number of dead astrocytes in cell cultures treated with TMZ was determined by ¯uorescent staining with the use of the Live/Dead Kit. Nuclei of cells undergoing necrosis stain red whereas calcein identi®es viable cells, giving at the same time an accurate insight into their morphology. Fig. 2 shows microphotographs of cells stained with calceine representative for particular experimental groups. Cells of the control group are stained with calcein almost regularly (Fig. 2; K). Small cells with intensive ¯uorescence appear in the astrocytes culture exposed to hypoxia (Fig. 2; H). Staining with calcein showed that the exposure of astrocytes to TMZ in normoxia conditions and before and/or during hypoxia signi®cantly changes morphology of the cells and ¯uorescence intensity (Fig. 2; Groups I±VI). The most signi®cant morphological changes in comparison with the control group were observed in Groups V and VI where astrocytes were treated with the drug during re-oxygenation and hypoxia/ re-oxygenation period. The percentage of dead cells stained with EthD-1 in comparison with the cells stained with calcein in

Fig. 3. Influence of trimetazidine (1 mM) on astrocyte death induced by hypoxia. The results are shown as a percentage relation of dead cells to the total amount of cells in the field and are a mean  S:E:M: of six randomly selected areas from three culture dishes. At least 1000 cells were counted in each experiment. Data were analysed with the use one-way ANOVA in order to compare mean value among different groups. Newman±Keuls test was performed in order to compare effects of trimetazidine treatment.  P < 0:05 vs. control (normoxia); # P < 0:05 vs. hypoxia.

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particular groups is presented in Fig. 3. Hypoxia causes a signi®cant increase of dead astrocytes number in the cultures (4692  850/cm2) in comparison to control (1279  294/cm2). Adding TMZ into cultures in normoxia (Group I), before (Group II) and during reoxygenation and hypoxia/re-oxygenation decreases the number of dead cells. Influence of TMZ on Apoptosis (Hoechst 33342 Staining) In order to determine the in¯uence of TMZ on the process of apoptosis the astrocyte cell nuclei were visualised by DNA-speci®c ¯uorescent dye Hoechst 33342. Fig. 4 shows microphotographs representative

for particular experimental groups. Cells from the control group (normoxia) had big, regular nuclei and few, characteristic for apoptosis, nuclei with condensed chromatin (Fig. 4; K). In the culture of astrocytes exposed to hypoxia there occurred many more picnotic nuclei and bright apoptotic bodies resulting from their fragmentation (Fig. 4; H). Exposure of astrocytes to TMZ in normoxic conditions before or during hypoxia did not appear to prevent apoptosis (Fig. 4; Groups I± V). In group VI, where astrocytes were treated with the drug both during hypoxia and re-oxygenation, big, regular nuclei with dispersed chromatin were observed, and the number of apoptotic nuclei was similar to that in normoxia group. Quantitative analysis of the pictures con®rmed that treating astrocytes with TMZ

Fig. 4. Influence of trimetazidine (1 mM) on astrocyte apoptosis induced by hypoxia. Cell nuclei were stained with the Hoechst 33342 dye. Microphotographs show experimental groups of astrocyte cultures treated with trimetazidine according to scheme presented in Fig. 1. Apoptotic nuclei were differentiated by virtue of fragmentation and of the level of the DNA condensation. Microphotographs were taken with the use of fluorescent microscope with the 20 objective. Open-spiked point at apoptotic cell nuclei and the closed-spiked arrows point at live cell nuclei.

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461

Fig. 5. Influence of trimetazidine (1 mM) on astrocyte apoptosis induced by hypoxia. 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:E:M: of the six randomly selected areas from three culture dishes. Data were analysed with the use of one-way ANOVA in order to compare mean values among different groups. Newman±Keuls test was performed in order to compare effects of trimetazidine treatment. P < 0:05 vs. control (normoxia); #P < 0:05 vs. hypoxia.

during 8 h hypoxia and 24 h re-oxygenation was the most effective in preventing hypoxia-induced apoptosis. Microscopic pictures were analysed in order to determine the percentage of apoptotic cells in particular groups. The results are presented in Fig. 5. Effect of TMZ on Mitochondrial Function Mitochondrial function was measured by the MTT reduction method, which may also serve as a general indicator of cell viability. The cleavage of tetrazoline ring in MTT takes place mainly with the participation of the mitochondrial succinate dehydrogenase and depends on the activity of the respiratory chain and the state of the redox mitochondria (Mosman, 1983; Shearman et al., 1995). Table 1 presents the results of the in¯uence of TMZ on MTT conversion into formasan in the cultures. A signi®cant increase of MTT reduction in astrocytes was observed in hypoxia conditions, which indicates a signi®cant intensi®cation in mitochondrial activity and cell reactivity. TMZ added to the cell culture for 8 h does not in¯uence the mitochondrial function (Group I). Yet, the exposure of astrocytes to the drug both 24 h prior to and during hypoxia signi®cantly intensi®es MTT reduction (Groups II±IV). Intensi®cation of mitochondrial function was much weaker when

the cells were treated with TMZ during re-oxygenation and hypoxia/re-oxygenation (Groups Vand VI) and the parameters measured were similar to the control group. Influence of TMZ on [3H]Thymidine Incorporation [3 H]thymidine incorporation into astrocytes treated with TMZ was studied as an indicator of DNA synthesis and astrocytic proliferation. It has been observed that 8 h hypoxia slowed [3 H]thymidine incorporation (16389  853 dpm/mg protein) in comparison with the cells maintained in normoxia condition (40746 1678 dpm/mg protein). Exposure of astrocytes to TMZ (0.1±100 mM) increased [3 H]thymidine incorporation into the DNA of astrocytes in all experimental protocols but magnitude of the increase varied between groups and depended on whether the drug was added before or after hypoxia (Table 2). The increase was most signi®cant when TMZ was added into the medium in normoxic conditions (Group I), during hypoxia (Group IV) and hypoxia/re-oxygenation (Groups Vand VI). The weakest mitogenic effect of TMZ was observed when the drug was added into the culture before the episode of hypoxia regardless of whether during the episode the drug was (Group III) or was not (Group II) present in the medium.

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Table 1 Effect of trimetazidine on mitochondrial function of astrocytes measured by MTT conversion assaya

Table 2 Effect of trimetazidine on cultured rat astrocytesa

Treatment

Percentage of control

Treatment

Control Hypoxia

100.0  3.7 190.9  14.4*

Control Hypoxia (TMZ (mM))

100.0  4.2 40.2  5.2*

Group I (TMZ (mM)) 0.1 1 10 100

117.9 100.3 107.7 87.8

   

5.0*,# 12.5# 16.6# 5.1#

Group I (TMZ (mM)) 0.1 1 10 100

667.7 884.6 939.3 557.2

   

17.8*,# 11.8*,# 14.1*,# 15.7*,#

Group II (TMZ (mM)) 0.1 1 10 100

228.7 232.2 209.7 226.5

   

12.5*,# 8.8*,# 9.5*,# 12.4*,#

Group II (TMZ (mM)) 0.1 1 10 100

267.5 296.1 130.2 150.2

   

6.9*,# 6.8*,# 3.6*,# 4.5*,#

Group III (TMZ (mM)) 0.1 1 10 100

190.7 206.0 191.9 210.9

   

13.2*,# 9.2*,# 9.5*,# 9.6*,#

Group III (TMZ (mM)) 0.1 1 10 100

271.9 167.1 132.4 102.0

   

4.8*,# 5.2*,# 6.2*,# 6.5#

Group IV (TMZ (mM)) 0.1 1 10 100

171.9 170.7 170.6 180.8

   

6.4* 7.7* 9.6* 13.8*

Group IV (TMZ (mM)) 0.1 1 10 100

825.3 1025.9 594.4 297.1

   

8.7*,# 14.4*,# 14.7*,# 11.6*,#

Group V (TMZ (mM)) 0.1 1 10 100

121.2 170.5 142.6 105.5

   

14.5# 31.5* 10.3*,# 1.8#

Group V (TMZ (mM)) 0.1 1 10 100

784.5 660.4 630.8 798.7

   

3.4*,# 6.4*,# 3.5*,# 3.6*,#

Group VI (TMZ (mM)) 0.1 1 10 100

99.0 139.2 107.5 145.3

   

9.6# 11.1*,# 12.6# 16.3*,#

Group VI (TMZ (mM)) 0.1 1 10 100

501.7 820.3 760.1 768.4

   

13.9*,# 2.1*,# 5.6*,# 4.2*,#

3

H-thymidine incorporation into Percentage of control

a Cells incubated with trimetazidine in the conditions shown in Fig. 1. MTT conversion was determined as described in materials and methods section. The results are expressed as percentages of the control value in the normoxia conditions. Data are mean  S:E:M: (n ˆ 8); *P < 0:01 vs. control (normoxia); #P < 0:01 vs. hypoxia.

a Cells were incubated with trimetazidine in the conditions shown in Fig. 1. [3 H]thymidine incorporation was determined as described in materials and methods section. The results are expressed as percentages of the control value in the normoxia conditions. Each value is mean of eight dishes  S:E:M: (n ˆ 8); *P < 0:01 vs. control (normoxia); #P < 0:01 vs. hypoxia.

DISCUSSION

microscopy, have shown that hypoxia-induced cell death possesses features characteristic of both necrosis and apoptosis. Also in our experiment, conducted with Live/Dead Kit and Hoechst 33342 we observed a signi®cant increase in the number of both necrotic and apoptotic cells in the astrocytes culture subjected to hypoxia in comparison with the control. On the other hand, hypoxia signi®cantly intensi®ed mitochondrial activity of the surviving cells which was proved by the MTT conversion method. This is consistent with increase metabolic activity of astrocytes that survive

Simultaneous determination of the number of dead and apoptotic cells, mitochondrial activity and DNA synthesis intensity enables evaluation of the in¯uence of hypoxia on the type of cell death, metabolic activity of the surviving cells and their proliferation. Shimizu et al. (1996) in their studies of morphological features of the PC12 and 7316 rat hepatoma cell line subjected to hypoxia, using both electron and confocal microscopy as well as non-confocal ¯uorescent

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stressing conditions, as previously observed by others (Cookson et al., 1995; Norenberg, 1994; Petito, 1986). It is currently believed that mitochondria are the intracellular organelles which constitute a kind of switch between necrosis and apoptosis. The factor determining the type of cell death is the change in their membrane permeability as a result of the induction of mitochondrial permeability transition pore (MTP) (Haworth and Hunter, 1979; Zoratti and Szabo', 1995). Data collected through experiments show that the induction of MTP may initiate the process of cell death observed in pathological states, i.e. ischemia/ reperfusion (Wilson, 1997). The fact that cell death is controlled by mitochondrial factors is of major importance for development of cytoprotective drugs (Kroemer et al., 1998). Experiments on peripheral tissues have shown that TMZ is a drug which ®rst of all in¯uences the mitochondrial enzymatic systems, control mechanisms of their activity and the permeability of ions through mitochondrial membranes. Guarnieri and Muscari (1993) showed that TMZ improves mitochondrial function during ischemic damage. Also, Salducci et al. (1996) observed restoration of adenosine 50 -triphosphate (ATP) synthesis by TMZ in isolated mitochondria previously overloaded with Ca2‡ or exposed to cyclosporine A (CsA). Lavanchy et al. (1987) proved that TMZ prevents a decrease of ATP and phosphocreatine (PCr) in myocardium ®bres after experimental ischemia. The present study is the ®rst to show that TMZ is effective in countering apoptotic or necrotic death of astrocytes. In the experiment we conducted, TMZ improved the mitochondrial function of astrocytes subjected to hypoxia without changing the parameter in oxygen conditions. We have also observed, a intriguing interrelation between the intensi®cation of the MTT conversion and the measured amount of dead and apoptotic cells depending on whether TMZ was present in the medium before or after hypoxic episode. The exposure of astrocytes to the drug before hypoxia, regardless of whether it was subsequently present in the medium or not during hypoxia signi®cantly intensi®es the MTT reduction decreasing the amount of dead cells but not of the apoptotic ones. The phenomenon was accompanied by a reduced intensity of cell proliferation represented by the measured amount of incorporated [3 H]thymidine. Different changes were observed when the drug was added during hypoxia and/ or re-oxygenation. In such case TMZ intensi®ed MTT conversion less signi®cantly decreasing the number of dead cell, whereas of apoptotic cells present in the culture was close to the control.

463

Morin et al. (1998) with the use of [3 H]TMZ, demonstrated the presence of two classes of speci®c TMZ binding sites on mitochondria (i) high af®nity site (K d ˆ 1 mM) amounts to 4% of the total binding sites and is located on the outer mitochondrial membrane; whereas (ii) low af®nity site appears to be con®ned to the inner membrane. The authors suggested that these sites might be engaged in the process of MTP closing in mitochondria. Studies of functional signi®cance of the TMZ-binding sites allowed to advance a hypothesis that TMZ ability to close MTP is connected by characteristic physicochemical features. TMZ, as amphiphilic cation is cable of binding itself to the membranes. TMZ ability to close MTP was also ascribed to its ability to in¯uence the mitochondrial membrane potential (Broekemeier and Pfeiffer, 1995). Cation compounds such as sphingosine, tri¯uoperazine, spermine or two-charged cations such as TMZ make the membrane potential more positive and, as a result, prevent the opening of MTP (Lapidus and Sokolove, 1994; Pereira et al., 1992). The results of many studies prove that the opening of MTP may be the initial stage of apoptosis and hence, compounds preventing its occurrence would also decrease cell death. This is indirectly con®rmed by our results: a signi®cant decrease of both necrotic and apoptotic cells in astrocyte cultures exposed to hypoxia and then treated with TMZ. In our study, regardless of the option considered, we observed a signi®cant in¯uence of TMZ on the amount of [3 H]thymidine incorporated into the astrocyte DNA. The result prove that TMZ is a potent stimulator of astrocyte proliferation. The key enzyme taking part in the regulation of the DNA synthesis is protein kinase C (PKC) (Aroor and Baker, 1997). It has been shown that the increase of PKC activity may both stimulate and prevent mitogenesis depending on the cell type and mitogen (Huang et al., 1989). Many PKC isoenzymes are involved in the process of DNA synthesis. Therefore, signi®cance of PKC isoenzymes activated by phorbol esters was examined in situ on various types of cells including astrocytes (Abe and Saito, 1997). It has been proved that the PKC, an isoenzyme is an apoptotic inhibitor and this particular effect is caused by the prevention of ceramide engagement in the apoptotic process. Thus, what should perhaps be considered is the possible common mitogenic and antiapoptotic effect of TMZ caused by its possible in¯uence on the PKC activity in astrocytes. What also seems necessary in the future is the determination of the GFAP immunoreactivity as a con®rmation of the occurrence of reactive astrocytes.

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What seems to be of particularly interest is the nonhomogenous in¯uence of TMZ on the astrocyte morphology. Increase in the amount of radiolabelled incorporated nucleotide was accompanied by signi®cant changes in the morphological image of both the whole cells and the nuclei in comparison with the control. Administration of TMZ before hypoxia and the astrocytes exposure to TMZ solely during hypoxia caused increased condensation of cytoplasm in the perinuclear area and the occurrence of thin processes (Fig. 2; Groups II±IV). On the other hand, astrocytes treated with TMZ after hypoxia are ¯at and not branching. It should be stressed, however, that the in¯uence of the increase in cAMP cellular concentration on the astrocyte branching is generally recognised (Goldman and Abramson, 1990; Ramakers and Moolenaar, 1998). Therefore, in the future, in¯uence of TMZ on cAMP level and/or the activity of protein kinase A (PKA) will be studied. The study would be of particular importance with reference to a possible in¯uence of TMZ on the survival of astrocytes during hypoxia as a result of the PKA-stimulated breakdown of the intracellular glycogen stores. In summary, our experiment showed that TMZ is capable of protecting astrocytes against hypoxia insult. The effect appeared most signi®cant when the drug was added during hypoxia and/or re-oxygenation. Yet, it is currently impossible to precisely determine the mechanism by which the drug functions, although it cannot be ruled out that the protective effect of TMZ results partly from the drug antioxidant properties. ACKNOWLEDGEMENTS The authors are deeply indebted Prof. Jan Albrecht for his critical reading of the manuscript. The work was supported by grant of State Committee for Scienti®c Research 4 PO5D 00516. REFERENCES Abe K, Saito H. Developmental changes in cyclic AMPstimulated stellation of cultured rat cortical astrocytes. Jpn J Pharmacol 1997;75:433±8. Ancerewicz J, Migliavacca E, Carrupt PA, Testa B, Bree F, Zini R, Tillement JP, Labidalle S, Guyot D, Chauvet-Monges AM, Crevat A, Le Ridat A. Structure±property relationships of trimetazidine derivates and model compounds as potential antioxidants. Free Radic Biol Med 1998;25:113±20. Aroor AR, Baker RC. Negative and positive regulation of astrocyte DNA synthesis by ethanol. J Neurosci Res 1997;50:1010±7.

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