Glucose deprivation and chemical hypoxia: neuroprotection by P2 receptor antagonists

Neurochemistry International 38 (2001) 189 – 197 www.elsevier.com/locate/neuint

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Glucose deprivation and chemical hypoxia: neuroprotection by P2 receptor antagonists Fabio Cavaliere a,c, Nadia D’Ambrosi a,c, Maria Teresa Ciotti b, Giorgio Mancino d, Giuseppe Sancesario a,c, Giorgio Bernardi a,c, Cinzia Volonte´ a,b,* a Fondazione Santa Lucia, Via Ardeatina 306, 00179 Rome, Italy Institute of Neurobiology, CNR, Viale Marx 15, 00137 Rome, Italy c Uni6ersita` Tor Vergata, Facolta` di Medicina, Dipartimento di Neuroscienze, Via di Tor Vergata 135, 00133 Rome, Italy d Centro di Ricerca Scientifica, Ospedale S. Pietro, Fatebenefratelli, Rome, Italy b

Received 11 June 2000; accepted 21 July 2000

Abstract In this work we investigate cell survival after glucose deprivation and/or chemical hypoxia and we analyse the neuroprotective properties of selected antagonists of P2 ATP receptors. We find that in rat cerebellar granule neurones, the antagonist basilen blue prevents neuronal death under hypoglycaemia. Basilen blue acts through a wide temporal range and it retains its efficacy under chemically induced hypoxic conditions, in the presence of the respiratory inhibitors of mitochondria electron transport chain complexes II (3-nitropropionic acid) and III (antimycin A). In spite of the presence of these compounds, basilen blue maintains normal intracellular ATP levels. It furthermore prevents neuronal death caused by agents blocking the mitochondrial calcium uptake (ruthenium red) or discharging the mitochondrial membrane potential (carbonyl cyanide m-chlorophenylhydrazone). Inhibition of poly (ADP-ribose) polymerase, modulation of the enzyme GAPDH and mitochondrial transport of mono-carboxylic acids are not conceivable targets for the action of basilen blue. Survival is sustained by basilen blue also in CNS primary cultures from hippocampus and in PNS sympathetic-like neurones. Partial neuroprotection is furthermore provided by three additional P2 receptor antagonists: suramin, pyridoxal-phosphate-6-azophenyl-2%,4%-disulphonic acid 4-sodium and 4,4%-diisothiocyanatostilbene2,2%disulphonic acid. Our data suggest the exploitation of selected P2 receptor antagonists as potential neuroprotective agents. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Cerebellar granule neurones; Hippocampal neurones; PC12 cells; ATP; Energy metabolism; Basilen blue

1. Introduction

* Corresponding author. Tel.: +39-06-51501557; fax: + 39-0651501556. E-mail address: [email protected] (C. Volonte´). Abbre6iations: BB, basilen blue; BME, Eagle’s basal medium; BZM, Benzamide; 4-CIN, a-cyano-4-hydroxycinnamic acid; CCCP, carbonyl cyanide m-chlorophenylhydrazone; CGN, cerebellar granule neurones; DIV, days in vitro; EBSS, Earle’s Balanced Salt Solution; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MTT, 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; NMDA, Nmethyl-D-aspartic acid; PARP, poly (ADP-ribose) polymerase; RuR, Ruthenium red.

The pathogenesis of brain damage under conditions of impaired energy metabolism continues to be of primary interest, since much still needs to be understood about the events affecting cellular metabolic injury in the brain. ATP is fundamental not only in the intracellular environment as energy molecule, but also in the extracellular milieu as neuromodulator, neurotransmitter and growth factor (Neary et al., 1996; Fredholm et al., 1996; Burnstock, 1997; Ralevic and Burnstock, 1998; Rathbone et al., 1999; Vizi and Sperlagh, 1999; Weight et al., 1999; Boeynaems et al., 2000; Vizi, 2000). Therefore, P1/P2 (adenosine/ATP) receptor occupancy

0197-0186/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 1 9 7 - 0 1 8 6 ( 0 0 ) 0 0 0 8 8 - 7

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may contribute, alone or in combination, to a neurodegenerative process. Not surprisingly, ATP and adenosine are already known to participate in brain responses to trauma and ischemia and are involved in the etiopathology of chronic neurodegenerative diseases. Whereas the neuroprotective properties of adenosine analogues active at the A1 receptor subtype have been known for several years (Ongini et al., 1997; Fredholm, 1997; Doolette, 1997), the function of other receptor subtypes (A2A and A3) is still controversial. In a similar way, the role of P2 receptors (P2X ionotropic and P2Y metabotropic) in the modulation of ischemic or traumatic brain damage still awaits further characterisation. Recent data would suggest synergism between P2 receptor modulation and glutamate-induced cytotoxicity or apoptotic cell death (Franceschi et al., 1996; Volonte´ and Merlo, 1996, 1997; Merlo and Volonte´, 1996; Volonte´ et al., 1999) and induction of cyclooxygenase 2 (Brambilla et al., 1999), an enzyme associated to inflammatory events in both acute and chronic neurodegenerative diseases. Because of their homogeneity and abundance in the mammalian CNS, cerebellar granule neurones (CGN) are some of the most suitable primary cell systems for analysing the molecular mechanisms of neuronal injury. Transient oxygen-glucose deprivation is already known to induce in these neurones both excitotoxic necrosis as well as apoptosis (Gwag et al., 1995; Budd and Nichols, 1996; Sagara and Schubert, 1998; Castilho et al., 1999). In this regard, we have investigated the involvement of P2 ATP receptors and precisely the functional properties and mechanism of action of selected P2 antagonists. Among these, the sulfonic derivative of anthraquinone basilen blue (BB) (Burnstock and Warland, 1987), an antagonist not discriminating between P2X and P2Y receptors, has been tested as a prototypic molecule.

ated hippocampal or cortical cells were plated on polyL-lysine-coated dishes and maintained in culture at 37°C with 5% CO2, in Eagle’s basal medium (BME), supplemented with 5 mM KCl, 2 mM glutamine, 0.1 mg/ml gentamycin, 10% heat inactivated foetal calf serum. At 2 DIV the cultures were supplemented with 10 mM cytosine arabinoside and were kept for 8–12 days.

2.2. Hypoglycaemia studies Hypoglycaemic conditions were obtained by maintaining the cells in glucose-free buffer (Earle’s Balanced Salt Solution, EBSS) (116 mM NaCl, 25 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, 26 mM NaHCO3, 0.6 mM NaH2PO4, pH 7.4). For control conditions, EBSS was supplemented with 1 mg/ml glucose. BB (Sigma, MIItaly) or other agents were added at different concentrations and for different times, as indicated. After treatment, cells were returned to the previously saved culture medium and cell survival was evaluated 20 h later, by direct count of intact nuclei (Volonte´ et al., 1994) or by propidium iodide incorporation.

2.3. Determination of ATP content Intracellular or extracellular ATP content was determined with the use of an ATP bioluminescent assay kit. CGN at 8 DIV were maintained for different times in EBSS buffer supplemented with glucose or in hypoglycaemic conditions, in the absence or presence of 100 mM BB. After lysing the cells in lysing buffer (Firezyme Diagnostic Technologies Limit, Windsor, N.S., Canada) total intracellular ATP content was monitored. An aliquot (100 ml, 105 cells/sample) was diluted with 100 ml of appropriate dilution buffer, Luciferin– luciferase solution (100 ml) was added directly in the test chamber of the luminometer and light emission was recorded.

2. Experimental procedures

2.4. Protein quantification 2.1. Primary cell cultures Cerebellar granule cultures from Wistar 8-day-old rat cerebellum were prepared as previously described (Levi et al., 1989) and seeded on poly-L-lysine-coated plastic dishes in Eagle’s basal medium (BME) (Gibco BRL, MI-Italy), supplemented with 25 mM KCl, 2 mM glutamine, 0.1 mg/ml gentamycin, 10% heat inactivated foetal calf serum (Gibco BRL, MI-Italy). At 1 DIV the cultures were supplemented with 10 mM cytosine arabinoside and were kept for 7 – 9 days, without replacing the culture medium. Rat hippocampal or cortical cultures from embryonic day 18 were prepared following the method described by Toselli and Taglietti, (1992), and Keilhoff and Erdo¨, (1991), respectively. Dissoci-

Protein concentrations were determined by the method of Bradford, (1976), with bovine serum albumin as standard.

3. Results

3.1. Neuroprotecti6e action of P2 receptor antagonists against glucose depri6ation In order to study their susceptibility to glucose starvation, CGN at 8 DIV were switched from culture medium to hypoglycaemic conditions (glucose-free physiological extracellular buffer) in a humidified atmo-

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sphere of 5% CO2 and 95% air at 37°C. Cells were then returned to the previously saved culture medium and survival was assessed the day after, by direct count of intact viable nuclei. Under these conditions, cell death increases linearly with maximal effect (75 – 95%) obtained in 3 h (Fig. 1A). When inspected by phase-contrast microscopy immediately after hypoglycaemia, granule neurones reveal loss of brightness, progressively damaged cell bodies and increasingly fragmented branching processes (not shown). The P2 receptor antagonist basilen blue (BB) fully sustains healthy morphological appearance (not shown) and survival of CGN, in a time- (Fig. 1A, Table 1) and dose-dependent manner (Fig. 1B). Pre-treatment for about 24 h with BB (Table 1, second horizontal bar) prevents cell death by about 50%. Immediately after 3 h of glucose starvation, post-treatment with BB for 1 h (Table 1 5th horizontal bar) or longer (Table 1 7th horizontal bar) maintains  80% of neuronal survival. BB-treatment could be delayed for 30 min after the beginning (Table 1 3rd bar) or the end (Table 1 6th bar) of the hypoglycaemic insult and still provide remarkable neuroprotection (respectively 85 and 60%).

Fig. 1. Basilen blue prevents hypoglycaemia-induced cell death in cerebellar granule neurones: time-course and dose–response effects. Cerebellar granule neurones at 8 DIV (days in vitro) were maintained under hypoglycaemic conditions for different times (0–180 min), in the absence (Control) or presence of 100 mM basilen blue (A). In (B), cultures were maintained under hypoglycaemic conditions for 3 h, in the simultaneous presence of different concentrations of basilen blue. Cell survival was assessed 20 h later, by direct count of intact nuclei. Counts represent means9 SEM (n=6) and 100% cell survival is referred to cultures maintained in the presence of 1 mg/ml glucose.

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As shown in Table 2, we find that, among additional ligands targeting P2 receptors, suramin (an antagonist of both P2X and P2Y receptors) (Hoyle et al., 1990) and the P2X antagonist PPADS (pyridoxal-phosphate6-azophenyl-2%,4%-disulphonic acid 4-sodium) (Ziganshin et al., 1994) protect respectively  67 or 36% of neurones from cell death. The antagonist DIDS (4,4%diisothiocyanatostilbene-2, 2%disulphonic acid) (Soltoff et al., 1993) is only  40% effective in sustaining survival. Ox-ATP (ATP-2%, 3%-dialdehyde) (an irreversible inhibitor of P2X7 receptor) (Murgia et al., 1992), KN62 (1 - (N,O - bis[5 - Isoquinolinesulphonyl] - N - methyl - L tyrosyl)-4-phenylpiperazine) (Humphreys et al., 1998) and P2Y antagonist PIT (2,2%-pyridylisatogen) (Spedding et al., 1975) fail under this regard. Cibacron blue, the other commercially available isomer of sulfonic derivatives of antraquinone, is instead effective, suggesting that the exact position of the sulfonic group on the A-ring (which distinguishes it from BB) is not crucial for survival (Table 2).

3.2. Neuroprotecti6e action of BB against energy metabolism failure: potential mechanisms of action The action of BB is only partially inhibited (40–50%) by cycloheximide (10 mg/ml during the hypoglycaemic insult), actinomycin D (1 mg/ml) or anisomycin (1 mg/ ml), therefore suggesting that transcription-dependent and transcription-independent events are both responsible for the survival effect. During ischemic insults, conservation of energy metabolism is a key feature to sustain neuronal survival. We find that activities of reduction of MTT into formazan, occurring in healthy cells with NADH or NADPH as co-substrates (Mosmann, 1983), are totally inhibited when tested after 3 h of glucose starvation (data not shown). In 30 min post-treatment, only cultures previously maintained with BB are instead capable of full enzymatic recovery (data not shown). Furthermore, we show that glucose starvation reduces intracellular content of ATP to  1/8 of control values, whereas ATP is still normally produced in neurones maintained in the simultaneous presence of BB (Table 3, hypoglycaemia). BB maintains efficacy in preserving intracellular ATP under selective, chemically induced, hypoxic conditions, that is the presence of inhibitors of mitochondria electron transport chain complexes II (Table 3 3-nitropopionic acid, acting on succinateubiquinone reductase) and III (Table 3, antimycin, acting on ubiquinone–cytochrome-c reductase). Consistently, both with (Table 3) or without extracellular glucose (data not shown), 100 mM BB offsets cell death induced only by 3-nitropropionic acid or by antimycin A. Neither neuronal survival, nor ATP levels are instead significantly sustained by BB when cell death is

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Table 1 Basilen blue prevents neuronal cell death evoked by glucose deprivationa

a Cerebellar granule neurons were maintained under hypoglycaemic conditions for 3 h (red box) in the absence or presence of 100 mM BB (blue horizontal bars) added at and for different times. Red horizontal bars represent glucose starvation in the absence of BB. Cell survival was assessed 24 h after the hypoglycaemic insult, by direct count of intact nuclei. Data are expressed as % of neuronal survival and represent means 9 SEM (n=8).

evoked by sodium azide (targeting cytochrome-c oxidase or complex IV), rotenone (inhibitor of NADH– ubiquinone reductase, complex I) or oligomycin (inhibitor of ATP synthase, complex V) (Table 3). BB neither rescues mortality caused by inhibition of the rate-limiting enzyme of glycolysis GAPDH with iodoacetate (Fig. 2A), or by inhibition of the transport of mono-carboxylic acids through the mitochondria with a-cyano-4-hydroxycinnamic acid (Fig. 2A). The mitochondrial Ca2 + entry and the highly negative potential of the inner mitochondrial membrane (DCm) are instead known to indirectly control some of the previous activities. Ca2 + uptake by mitochondria can be blocked by ruthenium red and it also stops if mitochondrial membrane potential is discharged by application of protonophores, such as carbonyl cyanide m-chlorophenylhydrazone (CCCP) (Babcock and Hille, 1998). In CGN, these compounds cause dramatic cell death, which can be effectively impeded by BB (Fig. 3). A toxic insult may not only reduce ATP synthesis, but also increase ATP hydrolysis by cells, particularly via activation of the energy-consuming and NAD-depleting enzyme poly (ADP-ribose) polymerase (PARP), activated by DNA strand breaks (Zhang et al., 1994). During hypoglycaemic conditions, we find that PARP inhibitor benzamide does not reduce cell death. Therefore, PARP modulation does not seem to account for ATP depletion (data not shown) and glucose-starvation-dependent neuronal loss or consequent BB-sustained neuroprotection (Fig. 2B).

3.3. Neuroprotection in different neuronal populations. We have extended our studies to additional neuronal cultures. As with CGN, hippocampal primary neurones maintained under hypoglycaemic conditions reveal by phase-contrast microscopy loss of brightness, progres-

bTable 2 Selected P2 receptor antagonists sustaim neuronal survival under hypoglycaemic conditionsa Time (h)

Antagonist (mM)

Cell Survival %

0 3 ’’ ’’ ’’ ’’ ’’ ’’ ’’ ’’

– – 100 mM BB 100 mM CibB 100 mM SUR 100 mM DIDS 60 mM PPADS 200 mM ox-ATP 10 mM PIT 10 mM KN62

10095 1593 80 94 82 9 2 6792 4195 36 9 2 5 91 5 9l 5 92

a Cerebellar granule cultures at 8 DIV were maintained under hypoglycaernic conditions for 3 h, in the presence of various P2 receptor antagonists (BB, Basilen Blue; CibB, Cibacron Blue; SUF, Suramine; DIDS, 4,4%-diisothiocyanatostilbene-2, 2% disulphonic acid; PPADS, pyridoxal-phosphate-6-azophenyl-2%, 4%-disulphonic acid 4sodium; ox-ATP, ATP-2%, 3%-dialdehyde; PIT, 2,2%-pyridylisatogen; KN62, 1-(N,O-bis[5-Isoquinolinesulphonyll-N-methyl-L-tyrosyl)-4phenylpiperazine). Cells were returned to the previously saved culture conditioned medium and survival was assessed 20 h later, by direct count of intact nuclei. Counts represent means 9 SEM (n =6).

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Table 3 Basilen blue maintains elevated intracellular ATP levels and sustains neuronal survival in the presence of mitochondrial electron transport chain complexes II and III inhibitorsa

a Cerebellar granule neurons were maintained for 3 h under hypoglycaemic conditions, or for 2 h in the presence of 1 mg/ml glucose and rotenone (1 mM), or 3-nitropropionic acid (1 mM), or antimycin A (3 ng/ml), or sodium azide (10 mM), or oligomycin (3 ng/ml), with or without 100 mM BB. Intracellular ATP content was measured 2 h after the insult and cell survival was assessed 20 h later, by direct count of intact nuclei. Data represent means 9SEM (n=8).

sively damaged cell bodies and fragmented branching processes (data not shown). Remarkably, in the presence of BB (100 mM), glucose starvation-induced cell death is totally prevented (Fig. 4) and neurones maintain healthy-looking cell bodies and neurites (data not shown). BB is still effective (85 – 95% protection) with the sympathetic-like NGF-differentiated PC12 cells (a cellular model for the peripheral nervous system) (Fig. 4), but not with cortical neurones in primary cultures (data not shown).

4. Discussion Glucose deprivation, mitochondrial dysfunction or severe hypoglycaemia cause functional disarrangement of the brain and interruption of its spontaneous electrical activity. Under these conditions, massive amounts of purines are discharged in the extracellular space. This is due to pathologically increased release of ATP-containing presynaptic vesicles, outflow of cytoplasmic purines from hypoxic cells as a

consequence of membrane permeability loss, degradation of nucleic acids from dying cells. Brain cells are hence exposed to high concentrations of purines and this has raised the hypothesis that they may play a key role in brain damage and/or repair. Our main new experimental findings show that different antagonists of P2 ATP receptor are able to rescue various neuronal populations from several metabolic hazards. Among suramin, PPADS and DIDS, the most potent BB drastically increases neuronal tolerance either to glucose-free conditions and/or to selected forms of mitochondrial dysfunction. This occurs in primary cultures from cerebellum, hippocampus, in sympathetic-like neurones, but not in primary cultures from cerebral cortex and thus it would indicate selectivity of action, correlated to a possible differential distribution among P2 receptor subclasses in CNS and PNS neurones (Burnstock, 1997). The effectiveness of the various and structurally different P2 antagonists supports the potential involvement of P2 receptors in the mechanisms of neuronal death/survival. Our hypothesis is moreover reinforced by: (a) the

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correspondence between the doses of BB which prevent cell death after hypoglycaemia in CGN (this work) and those which inhibit the extracellular binding of ATP to these same neurones (Volonte´ and Merlo, 1996; Merlo

Fig. 2. Basilen blue does not sustain neuronal survival through PARP, GAPDH or transport of mono-carboxylic acids. In (A), cerebellar granule neurones at 8 DIV (days in vitro) were incubated for 1 h with 1 mM iodoacetate (iodoac, inhibitor of GAPDH) in the presence of extracellular glucose, or with 10 mM a-cyano-4-hydroxycinnamic acid (4-CIN, inhibiting the transport of mono-carboxylic acids through the mitochondria) and returned to the previously saved conditioned medium. In (B), sister cultures were maintained under hypoglycaemic conditions for 3 h, in the presence of the PARP inhibitor benzamide up to 1 mM. Survival was assessed 20 h later as described and counts represent means 9SEM (n=6).

Fig. 3. Basilen blue prevents neuronal death evoked by ruthenium red and CCCP. Cerebellar granule neurones at 8 DIV (days in vitro) were maintained for 1.5 h in EBSS buffer supplement with 1 mg/ml glucose and in the presence of ruthenium red (100 mM) or CCCP (carbonyl cyanide m-chlorophenylhydrazone) (10 mM), with or without 100 mM basilen blue. Cells were returned to the saved culture conditioned medium and survival was assessed 20 h later, as described. Data represent means 9SEM (n= 6).

Fig. 4. Basilen blue prevents hypoglycaemia-induced cell death in different primary neuronal cultures. Hippocampal, cortical primary cultures at 8 DIV (days in vitro) or NGF-differentiated (2 weeks) PC12 cells were maintained respectively for 3 or 5 h under hypoglycaemic conditions, in the simultaneous absence or presence of basilen blue 50 mM (hippocampal, cortical neurones) or basilen blue 20 mM (PC12 cells). Cell survival was then assessed as described in the legend of Fig. 1. Data represent means 9SEM (n = 6).

and Volonte´, 1996); (b) the direct toxic effect exhibited by extracellular ATP in CGN (Amadio et al., manuscript in preparation). Considering that complex biological events such as survival/cell death work through multiple receptor/effector systems, we do not exclude that conjoint and/or alternative signalling pathways might still be involved. Nevertheless, the contribution of extracellular ATP-utilising enzymes, such as ecto-ATPases and ectokinase, is excluded by the experimental conditions adopted and by previously obtained results (Volonte´ and Merlo, 1996; Merlo and Volonte´, 1996; D’Ambrosi et al., 2000). Investigating some features of neuroprotection in CGN (Volonte´ and Merlo, 1996; Volonte´ et al., 1999; Cavaliere et al., 2000), we have also shown here that conservation of intracellular ATP levels (Frieberg et al., 1998; Lemaster et al., 1998; Patel et al., 1998) is necessary for maintaining neuronal survival under conditions of hypoglycaemia and/or chemical hypoxia. BB can enforce such conservation despite absence of extracellular glucose, acute inhibition of cellular overall MTT-reducing activities, without targeting the rate limiting glycolytic enzyme GAPDH or the transport of mono-carboxylic acids to the mitochon-

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dria. Moreover, it is known that intracellular energy imbalance can result in energy depletion also through increased ATP consumption. Nevertheless, supraphysiological activation of PARP (a process which exhausts the intracellular NAD+ pool) is unlikely to play a crucial role in hypoglycaemia-induced cell death and BB-evoked neuroprotection of CGN. Similar result was also confirmed by inducing apoptotic cell death of CGN by peroxynitrite and nitric oxide donors (Leist et al., 1997). The increased ATP demand that follows a lack in extracellular glucose often induces overactivity of the mitochondrial ATP synthase (complex V). From our results we evince that this enzyme is not a target of BB’s action in the safeguard of normal intracellular ATP levels. On the contrary, a neuroprotective action of BB can be definitely exerted on the mitochondrial electron transport chain complexes II and III, but not I and IV. The increased ATP demand following deficiency of extracellular glucose induces drain in the proton gradient, depolarisation of the mitochondrial membrane, overflow of cellular Ca2 + . Our data show that BB efficiently halts the detrimental effects on neuronal survival induced by blocking mitochondrial calcium uptake (with ruthenium red) or discharging mitochondrial membrane potential (with the protonophore CCCP). Whereas all these mechanisms contribute to explain how BB ‘‘locks’’ the mitochondria into a poised metabolic status, further studies are needed to establish the sequence of events between extracellular antagonistic action of BB on P2 receptors (People and Li, 1998) and intracellular modulation of those pathways functional to survival (Cavaliere et al., 2000). On this regard, we should not forget that pathologic mechanisms extend beyond simple mitochondrial enzyme inhibition and several neurodegenerative diseases unravel interrelationships between mitochondrial insufficiency and excitotoxic mechanisms (Choi, 1993; Beal, 1995; Gwag et al., 1995; Budd and Nichols, 1996; Sagara and Schubert, 1998; Castilho et al., 1999). The same specificity of mitochondrial inhibitors extends to differential effects on neurotransmitter systems, ionic current responses and free radical activity (Matsuoka et al., 1997; Zhang et al., 1996; Nakai et al., 1999; Sperlagh et al., 1998). Such combined secondary effects might therefore contribute to explain the selective and specific action of BB in preventing chemically induced hypoxia. In conclusion, the biological solution chosen by a cell to bypass a situation of metabolic hazard is certainly multifactorial (neurotransmitter and receptor cross talk, ion flux modulation, mitochondrial function, signal transduction activity, second messenger generation, gene expression). Not by chance, the modulation of P2 receptors by BB seems to be a multifac-

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torial strategy, with a wide range of actions and several distinct direct or indirect biological targets (Volonte´ et al., 1999; D’Ambrosi et al., 2000; Cavaliere et al., 2000). This would explain why BB maintains considerable efficacy with respect to several different neuronal populations and against various causes of death, including excitotoxicity (Volonte´ and Merlo, 1996; Cavaliere et al., 2000), apoptosis (Volonte´ et al., 1999; Cavaliere et al., 2000), glucose starvation and selected mitochondrial dysfunction. Such features might propound a possible clinical exploitation of P2 receptor ligands, not excluding BB, as novel therapeutic agents to slow or halt neurodegenerative diseases sustained by deregulation of energy metabolism.

Acknowledgements The research presented was supported by C.N.R. Bilateral Research Project c9700728, by Progetto Finalizzato Ministero della Sanita` Ref 98.85 and by Cofinanziamento MURST 99 ‘‘Purinoceptors and Neuroprotection’’.

References Babcock, D.F., Hille, B., 1998. Mitochondrial oversight of cellular Ca2 + signaling. Curr. Opinion Neurobiol. 8, 398 – 404. Beal, M.F., 1995. Aging, energy and oxidative stress in neurodegenerative diseases. Ann. Neurol. 42, 646 – 654. Boeynaems, J.M., Communi, C., Savi, P., Herbert, J.M., 2000. P2Y receptors: in the middle of the road. Trends in Pharmacology 21, 1 – 3. Bradford, M., 1976. A rapid sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal. Biochem. 72, 248 – 252. Brambilla, R., Burnstock, G., Bonazzi, A., Ceruti, S., Cattabeni, F., Abbracchio, M.P., 1999. Cyclo-oxygenase-2 mediates P2Y receptor-induced reactive astrogliosis. Br. J. Pharmac. 126 (3), 563– 567. Budd, S.L., Nichols, D.G., 1996. Mitochondria, calcium regulation, and acute glutamate excitotoxicity in cultured granule cells. J. Neurochem. 67, 2282 – 2291. Burnstock, G., 1997. The past, present and future of purine nucleotides as signalling molecules. Neuropharmacology 36, 11,427– 11,439. Burnstock, G., Warland, J.J.I., 1987. P2-purinoceptors of two subtypes in the rabbit mesenteric artery: reactive blue 2 selectively inhibits responses mediated via the P2Y- but not P2X-purinoceptor. Br. J. Pharmac. 90, 383 – 391. Castilho, R.F., Ward, M.W., Nichols, D.G., 1999. Oxidative stress, mitochondrial function and acute glutamate excitotoxicity in cultured cerebellar granule cells. J. Neurochem. 72, 1394–1401. Cavaliere, F., D’Ambrosi, N., Sancesario, G., Bernardi, G., Volonte´, C., 2000. Hypoglycaemia-induced cell death: features of neuroprotection by the P2 receptor antagonist basilen blue. Neurochem. Int. (in press).

196

F. Ca6aliere et al. / Neurochemistry International 38 (2001) 189–197

Choi, D., 1993. NMDA receptors and AMPA/Kainate receptors mediate parallel injury in cerebral cortical cultures subjected to oxygen-glucose deprivation. Prog. Brain Res. 96, 137–143. D’Ambrosi, N., Cavaliere, F., Merlo, D., Milazzo, L., Mercanti, D., Volonte´, C., 2000. Antagonists of P2 receptors prevent NGF-dependent neuritogenesis in PC12 cells. Neuropharmacology 39, 1083 – 1094. Doolette, D.J., 1997. Mechanism of adenosine accumulation in the hippocampal slice during energy deprivation. Neurochem. Int. 30, 211 – 223. Franceschi, C., Abbracchio, M.P., Barbieri, D., Ceruti, S., Ferrari, D., Iliou, J.P., Rounds, S., Schubert, P., Schulze-Lohoff, E., Rassendren, F.A., Staub, M., Volonte´, C., Wakade, A.R., Burnstock, G., 1996. Purines and cell death. Drug Dev. Res. 139, 442 – 449. Fredholm, B.B., 1997. Adenosine and neuroprotection. In: Green, A.R., Cross, A.J. (Eds.), Neuroprotective Agents and Cerebral Ischemia. San Diego, Academic Press, pp. 259–280. Fredholm, B.B., Abbracchio, M.P., Burnstock, G., Daly, J.W., Harden, K., Jacobson, K.A., Leff, P., Williams, M., 1996. Nomenclature and classification of purinoceptors. Pharmac. Rev. 46, 143 – 156. Frieberg, H., Ferrand-Drake, M., Bengtsson, F., Halestrap, A.P., Wieloch, T., 1998. Cyclosporin A, but not FK 506, protects Mitochondria and neurons against hypoglycemic damage and implicates the mitochondrial permeability transition in cell death. J. Neurosci. 18, 5151–5159. Gwag, B.J., Lobner, D., Koh, J.Y., Wie, M.B., Choi, D.W., 1995. Blockade of glutamate receptors unmasks neuronal apoptosis after oxygen-glucose deprivation in vitro. Neuroscience 68, 615 – 619. Hoyle, C.H., Knight, C.E., Burnstock, G., 1990. Suramin antagonises responses to P2 purinoceptor agonists and purinergic nerve stimulation in the guinea- pig urinary bladder and taenia coli. Br. J. Pharmac. 99, 617–621. Humphreys, B.D., Virginio, C., Surprenant, A., Rice, J., Dubyak, G.R., 1998. Isoquinolines as antagonists of the P2X7 nucleotide receptor: high selectivity for the human versus the ratreceptor homologues. Molec. Pharmac. 54, 22–32. Keilhoff, G., Erdo¨, S.L., 1991. Parallel development of excitotoxic vulnerability to NMDA and kainate in dispersed cultures of rat cerebral cortex. Neuroscience. 43, 35–40. Leist, M., Fava, E., Montecucco, C., Nicotera, P., 1997. Peroxynitrite and nitric oxide donors induce neuronal apoptosis by eliciting autocrine excitotoxicity. Eur. J. Neurosci. 9, 1488–1498. Lemaster, J.J., Nieminen, A.L., Qian, T., Trost, L.C., Elmore, S.P., Nishimura, Y., Crowe, R.A., Cascio, W.E., Bradham, C.A., Brenner, D.A., Herman, B., 1998. The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy. Biochim. Biophys. Acta 1366, 177 – 196. Levi, G., Aloisi, F., Ciotti, M.T., Thangnipon, W., Kingsbury, A., Bala`zs, R., 1989. Preparation of 98% pure cerebellar granule cell cultures. In: Shahar, A., deVellis, J., Vernadakis, A., Haber, B. (Eds.), A Dissection and Tissue Culture Manual of the Nervous System. Liss, New York, pp. 211–214. Matsuoka, Y., Kitamura, Y., Fukunaga, R., Shimohama, S., Nabeshima, T., Tooyama, I., Kimura, H., Taniguchi, T., 1997. In vivo hypoxia-induced neuronal damage in dentate gyrus of rat hippocampus: changes in NMDA receptor and the effect of MK-801. Neurochem. Int. 30, 533–542. Merlo, D., Volonte´, C., 1996. Binding and functions of extracellular ATP in cultured cerebellar granule neurons. Biochem. Biophys. Res. Commun. 225, 907–914. Mosmann, 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assay. J. Immunol. Methods 65, 55–63. .

Murgia, M., Hanau, S., Pizzo, P., Rippa, M., Di Virgilio, F., 1992. Oxidized ATP, an irreversible inhibitor of the macrophage purinergi P2z receptor. J. Biol. Chem. 268, 8199 – 8203. Nakai, T., Milusheva, E., Baranyi, M., Uchihashi, Y., Satoh, T., Vizi, E.S., 1999. Excessive release of [3H] noradrenalin and glutamate in response to stimulation of ischemic conditions in rat spinal cord slice preparation: effect NMDA and AMPA receptor antagonists. Eur. J. Pharmac. 366, 143 – 150. Neary, J.T., Rathbone, M., Cattabeni, F., Abbracchio, M.P., Burnstock, G., 1996. Trophic actions of extracellular nucleotides and nucleosides on glial and neuronal cells. Trends Neurosci. 19, 13 – 18. Ongini, E., Adami, M., Ferri, C., Bertorelli, R., 1997. Adenosine A2A receptors and neuroprotection. Ann. N.Y. Acad. Sci. 825, 30 – 48. Patel, A.J., Lauritzen, I., Lazdunski, M., Honore, E., 1998. Disruption of mitochondrial respiration inhibits volume-regulated anion channels and provokes neuronal cell swelling. J. Neurosci. 18, 3117 – 3123. People, R.W., Li, C., 1998. Inhibition of NMDA-gated ion channels by the P2 purinoceptor antagonists suramin and reactive blue 2 in mouse hippocampal neurones. Br. J. Pharmac. 124, 400–408. Ralevic, V., Burnstock, G., 1998. Receptor for purines and pyrimidines. Pharmac. Rev. 50, 413 – 492. Rathbone, M.P., Middlemiss, P.J., Gysberg, J.W., Craig, A., Herman, M.A.R., Reed, J.K., Ciccarelli, R., Di Iorio, P., Caciagli, F., 1999. Trophic effects of purines in neurons and glial cells. Prog. Neurobiol. 59, 663 – 690. Sagara, Y., Schubert, D., 1998. The activation of metabotropic glutamate receptors protects nerve cells from oxidative stress. J. Neurosci. 18, 6662 – 6671. Soltoff, S.P., McMillian, M.K., Talamo, B.R., Cantley, L.C., 1993. Blockade of ATP binding site of P2 purinoceptors in rat parotid acinar cells by iso thiocyanate compounds. Biochem. Biophys. Res. Commun. 45, 1936 – 1940. Spedding, M., Sweetman, A.J., Weetman, D.F., 1975. Antagonism of adenosine 5%-triphosphate-induced relaxation by 2-2%pyridylisatogen in the taenia of guinea-pig caecum. Br. J. Pharmac. 53, 575 – 583. Sperlagh, B., Sershen, H., Lajtha, A., Vizi, E.S., 1998. Co-release of endogenous ATP and [3H] noradrenaline from rat hypothalamic slices: origin and modulation by a2-adrenoceptors. Neuroscience 82, 511 – 520. Toselli, M., Taglietti, V., 1992. Kinetic and pharmacological properties of high and low-threshold calcium channels in primary cultures of rat hippocampal neurons. Pflu¨gers Arch. 421, 59–66. Vizi, E.S., 2000. Role of high-affinity receptors and membrane transporters in nonsynaptic communication and drug action in the central nervous system. Pharmac. Rev. 52, 63 – 89. Vizi, E.S., Sperlagh, B., 1999. Receptor- and carrier-mediated release of ATP of postsynaptic origin: cascade transmission. Prog. Brain Res. 120, 159 – 169. Volonte´, C., Ciotti, M.T., Battistini, L., 1994. Development of a method for measuring cell number: application to CNS primary neuronal cultures. Cytometry 17, 274 – 276. Volonte´, C., Merlo, D., 1996. Selected P2 purinoceptor modulators prevent glutamate-evoked cytotoxicity in cultured cerebellar granule neurons. J. Neurosci. Res. 45, 183 – 193. Volonte´, C., Merlo, D., 1997. Biological effects of P2 purinoceptor modulators in cultured primary cerebellar granule neurons. In: Teelken, A.W., Korf, J. (Eds.), Neurochemistry, vol. 60. Plenum, New York, pp. 357 – 360. Volonte´, C., Ciotti, M.T., D’Ambrosi, N., Lockhart, B., Spedding, M., 1999. Neuroprotective effects of modulators of P2 receptors in primary culture of CNS neurons. Neuropharmacology 38, 1335 – 1342.

F. Ca6aliere et al. / Neurochemistry International 38 (2001) 189–197 Weight, F.F., Li, C., Peoples, R.W., 1999. Alcohol action on membrane ion channels gated by extracellular ATP (P2X receptors). Neurochem. Int. 35, 143 –152. Zhang, J., Dawson, V.L., Dawson, T.M., Snyder, S.H., 1994. Nitric oxide activation of poly(ADP-ribose) synthetase in neurotoxicity. Science 263, 687 – 689.

.

197

Zhang, Y.X., Yamashita, H., Ohshita, T., Sawamoto, N., Nakamura, S., 1996. ATP induces release of newly synthesized dopamine in the rat striatum. Neurochem. Int. 28, 395 – 400. Ziganshin, A.U., Hoyle, C.H.V., Lambrecht, G., Mutschler, E., Ba¨umert, H.G., Burnstock, G., 1994. Selective antagonism by PPADS at P2X-purinoceptors in rabbit isolated blood vessels. Br. J. Pharmacol. 111, 923 – 929.