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Opposite effects of cyclohexyladenosine and theophylline on hypoxic damage in cultured neurons
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Neuroscience Letters 175 (1994) 114-116
N[UROSCIENC[ LETTERS
Opposite effects of cyclohexyladenosine and theophylline on hypoxic damage in cultured neurons Jean-Luc Daval*, Fr6d6ric Nicolas I N S E R M U.272, BP 3069, Universit~ de Nancy I, 24-30 rue Lionnois, 54013 Nancy, France Received 5 April 1994; Revised version received 11 May 1994; Accepted 16 May 1994
Abstract
To study the central effects of adenosine on hypoxia, the influence of treatment by the A 1 receptor agonist cyclohexyladenosine (1 gM) or by the antagonist theophylline (10/.tM) was tested on cell damage in a model of neuronal culture. Whereas theophylline enhanced cell injury induced by 8 h hypoxia, cyclohexyladenosinedecreased lactate dehydrogenase leakage, abolished the transient increase in 2-D-deoxyglucosetransport and improved cell morphology. Such actions might involve regulation of excitatory amino acid release and maintenance of calcium homeostasis. Key words." Cultured neuron; Hypoxia; Lactate dehydrogenase efflux;Deoxyglucosetransport; Cyclohexyladenosine;TheophyUine
Numerous cellular processes are known, or have been hypothesized to be involved in the pathophysiology of brain hypoxia. Among them, the release of excitatory amino acids, mainly glutamate and aspartate, as well as calcium influx and overload have been shown to play a key role in hypoxia-ischemia neuronal damage [8,15]. The role of adenosine as a neuromodulator in the central nervous system has been well documented [7,16]. Via the activation of its specific A~ receptors, the nucleoside inhibits neuronal calcium influx [1] and counteracts the presynaptic release of the excitotoxic neurotransmitters [4]. Therefore, adenosine appears as an endogenous neuroprotective agent, and numerous studies have provided evidence for a beneficial effect of adenosine analogues, e.g. cyclohexyladenosine (CHA) or L-phenyl-isopropyladenosine (L-PIA), in brain ischemia, whereas blockade of adenosine receptors by antagonists such as theophylline or caffeine exacerbates ischemic brain injury [14]. In addition to its neuromodulatory effects, the ubiquitous properties of adenosine (and its stable analogues), including cardiovascular depression, vasodilatory actions as well as hypothermia, may also contribute to the cerebral protection induced by adenosine in vivo. In
* Corresponding author. Fax: (33) 83.32.43.40. 0304-3940/94/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(94)00398-T
order to alleviate these side effects, the influence of the adenosine A~ receptor agonist CHA and the antagonist theophylline was studied on hypoxic damage in a model of primary cultures of cortical and hippocampal neurons. Neuronal cell cultures were obtained from 14-day-old rat embryo cortex and hippocampus according to Daval et al. [6]. Dispersed cells were plated on poly-lysine-precoated 35 mm dishes containing a mixture of Dulbeccomodified Eagle's and Ham-F12 media (50:50) with 5% inactivated foetal calf serum. After 24 h, neurons were grown for 5 additional days under standard normoxic conditions (95% air/5% CO2) in a chemically-defined serum-free medium. Cells were submitted to transient hypoxia by incubating the culture dishes for 8 h in a humidified chamber flushed with 95% Nil5% CO2, while control cultures were maintained under normoxic conditions, as previously described [6]. Pharmacological treatments were done just before the hypoxic insult by adding either CHA (dissolved in 2% ethanol, final concentration 1/IM in 0.02% ethanol) or theophylline (final concentration 10/IM) to the culture medium. Concentrations of both drugs were chosen in a preliminary study in order to exert an action on hypoxic cell injury, while not altering cell morphology and viability per se within the 3 following days. Cells
J.-L. Daval, E NicolaslNeuroscience Letters 175 (1994) 114-116
were studied either 24 or 72 h after hypoxia by the assessment of cell survival and morphology, lactate dehydrogenase (LDH) efflux [9], whereas the functional activity of the cells was estimated by measuring the specific uptake of [3H]2-deoxy-D-glucose ([3H]2DG, 969.4 GBq/ mmol, NEN, Boston, USA) during a 30 min incubation of the cells at 37°C in the presence of 2 ml of a solution of Krebs-Ringer saline buffered with HEPES (pH 7.40) and containing 1 mM [3H]2DG [5]. Cells were finally washed twice by 2 ml 0.9% NaC1 warmed up to 37°C, treated by 1 ml NaOH (1 M), and then scrapped off for protein measurement according to Bradford [2]. After 6 days in vitro, cultures exhibited a very high percent of living neuronal cells interconnected by a dense fiber network, as previously reported [11]. Whatever the pharmacological treatment of the cells was, no cell damage could be observed by microscopic observation 24 h after the hypoxic insult which induced a 78% reduction of the partial pressure of oxygen in the culture medium which decreased from 139.2 + 0.2 to 31.7 + 3.3 mmHg. By contrast, signs of cell degeneration appeared 3 days after hypoxia, concomitantly with a 22% decrease in protein levels (Table 2). Whereas CHA and theophylline themselves had no incidence on cell morphology, the treatment of the cells by the adenosine agonist improved cell morphology 3 days post-hypoxia, leading to a non significant 9% reduction of the protein concentration. Conversely, theophylline treatment tended to aggravate the deleterious influence of hypoxia on neuron survival and morphology, as reflected by increased Trypan blue penetration into the cells, the presence of larger amounts of cellular debris in culture dishes, and a 37% reduction of the protein concentrations in the cultures. Cell exposure to hypoxia for 8 h led to an important efflux of L D H to the extracellular medium which increased with time (Tables 1 and 2). While not inducing significant LDH leakage when not associated to hypoxia, CHA reduced hypoxia-related amounts of LDH Table 1 Changes in LDH efflux, 2DG specific transport and protein contents in cultured neurons 24 hours after hypoxia L D H efflux 2DG transport units/liter nmol/mg prot.
proteins pg/dish
Controls 1 # M CHA 10 # M Theophylline
1.3 -+ 0.2 1.6 _+ 0.2 1.7 _+ 0.3
91.6 -+ 3.9 88.5 _+ 4.6 111.2 _+ 3.7
203.2 _+ 8.3 211.0 _+4.1 198.6 _+ 4.9
Hypoxia Hypoxia + CHA Hypoxia + Theophylline
4.6 _+ 0.7** 2.4 _+ 0.4* 6.5 ± 1.0"*
121.2 _+4.2** 199.4 _+ 5.4 92.6 _+4.0 °0 207.7 _+ 9.1 152.3 +_ 3.61 **° 198.2 -+ 8.2
Data are reported as mean values (_+S.D.) obtained from 4 separate experiments using 6 or 7 culture dishes for each experimental condition. Statistically differences with the appropriate pharmacologically-treated control: *P < 0.05, **P < 0.01. Statistically significant differences with hypoxia-values: °P<0.05, °°P <0.01 (Bonferroni t-test for multiple comparisons).
115
Table 2 Changes in L D H efflux, 2DG specific transport and protein contents in cultured neurons 72 hours after hypoxia L D H efflux 2DG transport proteins units/liter nmol/mg prot. #g/dish Controls 1 / t M CHA 10 # M Theophylline
1.5 _+0.1 2.2 _+ 0.5 3.4 _+ 0.6
90.2 _+ 3.6 92.8 _+ 6.3 98.4 _+ 5.1
Hypoxia 6.3 _+ 0.5 ** 6 4 . 0 + 3 . 5 * * Hypoxia + CHA 2.3 _+ 0.3 °0 89.7_+4.7 °° Hypoxia + Theophylline 8.0 _+ 0.7 **° 52.1 _+ 4.4 **0
170.7 _+ 11.2 168.6 ± 10.7 159.6 _+ 9.5 133.6_+12.5 * 154.5_+ 14.1 101.6 +_ 16.8 *
Data are reported as mean values (+ S.D.) obtained from 4 separate experiments using 6 or 7 culture dishes for each experimental condition. Statistically significant differences with the appropriate pharmacologically-treated control: *P < 0.05, **P< 0.01. Statistically significant differences with hypoxia-values: °P <0.05, °°P <0.01 (Bonferroni t-test for multiple comparisons).
detected in the culture medium of the cells at the 2 timepoints studied. Theophylline treatment increased slightly LDH release in cells exposed to normoxia, and more dramatically following cell exposure to hypoxia. As shown in Tables 1 and 2, hypoxia enhanced transiently 2DG-specific transport which increased 24 h after hypoxia, and then decreased below normal values in the following days. While it did not change 2DG transport under normoxic conditions, CHA was able to abolish hypoxia-induced alterations of [3H]2DG transport in neuronal cells. Theophylline alone, at the concentration used, increased only moderately 2DG uptake by the cells in vitro. However, the adenosine antagonist enhanced the effects of hypoxia on 2DG transport. Since conditions that closely mimic those which occur in vivo can be produced easily in vitro by manipulating the extracellular environment, cell cultures are useful tools for studying the cellular mechanisms involved in hypoxia- or ischemia-induced cell death. Also, cultures from the central nervous system are of interest to counteract peripheral effects of substances which are known to have protective properties in vivo. In addition, neuron-enriched cultures from cortex and hippocampus, as used in the present study, appear as the most appropriate model, since it has been shown that (i) glia can protect neurons against both hypoxia [17] and excitatory amino acid-induced insult [10], and (ii) cortex and hippocampus are particularly vulnerable to reduced oxygen supply [3]. This study shows that an adenosine analogue which interacts preferentially with A~ receptors, and at pharmacologically-relevant concentration, is able to reduce hypoxic injury in a model of primary culture of neurons isolated from rat cortex and hippocampus. Indeed, CHA treatment appears to limit LDH efflux, a good marker of cell injury, and to abolish the transient increase in 2DG uptake which might reflect, at least partly, excitatory amino acid-induced cell stimulation that corresponds to the first step of a deleterious cascade of cellular
116
J.-L. Daval, E Nicolas l Neuroscience Letters 175 (1994) 114-116
events. Conversely, the non-specific receptor antagonist theophylline increases cell d a m a g e following hypoxia in vitro. Such data are in g o o d agreement with those obtained f r o m in vivo studies. In the brain, adenosine depresses the presynaptic release o f glutamate. Accordingly, it has been shown that in rat hippocampal synaptosomes, adenosine and its stable analogues decreased the K ÷ or the kainate-evoked release o f glutamate and aspartate, whereas glutamate was able to stimulate adenosine release f r o m the nerve terminals [13]. Also, adenosine reduces postsynaptic m e m b r a n e depolarization by acting on K ÷ and CI- conductances. At both pre- and postsynaptic levels, adenosine action is relevant for the maintenance o f calcium homeostasis [1,7,16]. All these central effects are certainly effective to limit and/or prevent pathophysiological processes triggered by hypoxia or ischemia, and might a c c o u n t for our observations. A l t h o u g h A 1 receptor-mediated n e u r o m o d u l a t o r y actions o f adenosine are o f special importance, the mechanisms underlying the anti-hypoxic effects o f the nucleoside m a y also involve the participation o f A2 receptors [12,14]. In this respect, C H A at the concentration used in the present study ( 1 / t M ) m a y also act, although to a lesser extent, at A2 receptors, leading to the assumption that the activation o f the two categories o f adenosine sites could be responsible for the protection observed. Therefore, further studies using highly selective agonists and antagonists o f b o t h types o f adenosine receptors would provide new insights to the neuroprotective role o f adenosine derivatives. The authors wish to thank V. Koziel for her excellent technical assistance. [1] Andinr, P., Involvement of adenosine in ischemic and postischemic calcium regulation, Mol. Chem. Neuropathol., 18 (1983) 3549. [2] Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248-254.
[3] Brierley, J.B., Cerebral hypoxia. In W. Blackwood and J.A. Corsellis (Eds.), Greenfield's Neuropathology, Edward Arnold, London, 1976, pp.41-85. [4] Corradetti, R., Lo Conte, F., Moroni, F., Passani, M.B. and Pepeu, G., Adenosine decreases aspartate and glutamate release from rat hippocampal slices, Eur. J. Pharmacol., 104 (1984) 19-26. [5] Daval, J.L., Anglard, P., Gerard, M.J., Vincendon, G. and Louis, J.C., Regulation of deoxyglucose uptake by adrenocorticotropic hormone in cultured neurons, J. Cell. Physiol., 124 (1985) 7580. [6] Daval, J.L., Koziel, V. and Nicolas, F., Functional changes in cultured neurons following transient asphyxia, NeuroReport, 2 (1991) 97-100. [7] Daval, J.L., Nehlig, A. and Nicolas, F., Physiological and pharmacological properties of adenosine: therapeutic implications, Life Sci., 49 (1991) 1435-1453. [8] J6rgensen, M.B. and Diemer, N.H., Selective neuronal loss after cerebral ischemia in the rat: possible role of transmitter glutamate, Acta Neurol. Scand., 66 (1982) 536-546. [9] Koh, J.Y. and Choi, D.W., Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay, J. Neurosci. Methods, 20 (1987) 8390. [10] Mattson, M.P. and Rychlik, B., Glia protects hippocampal neurons against excitatory amino acid-induced degeneration: involvement of fibroblast growth factor, Int. J. Dev. Neurosci., 8 (1990) 399415. [11] Nicolas, F., Oillet, J., Koziel, V. and Daval, J.L., Characterization of adenosine receptors in a model of cultured neurons from rat forebrain, Neurochem. Res., 19 (1994) 507-515. [12] Phillis, J.W., The selective adenosine A: receptor agonist, CGS 21680, is a potent depressant of cerebral cortical neuronal activity, Brain Res., 509 (1990) 328-330. [13] Poli, A., Lucchi, R., Vibio, M. and Barnabei, O., Adenosine and glutamate modulate each other's release from rat hippocampal synaptosomes, J. Neurochem., 57 (1991) 298-306. [14] Rudolphi, K.A., Schubert, P., Parkinson, F.E. and Fredholm, B.B., Adenosine and brain ischemia, Cerebrovasc. Brain Metab. Rev., 4 (1992) 346-369. [15] Siesj6, B.K. and Bengtsson, F., Calcium fluxes, calcium antagonists, and calcium related pathology in brain ischemia, hypoglycemia, and spreading depression: a unifying hypothesis, J. Cereb. Blood Flow Metab., 9 (1989) 127-140. [16] Snyder, S.H., Adenosine as a neuromodulator, Annu. Rev. Neurosci., 8 (1985) 103-124. [17] Vibulsreth, S., Hefti, F., Ginsberg, M.D., Dietrich, W.D. and Busto, R., Astrocytes protect cultured neurons from degeneration induced by anoxia, Brain Res., 422 (1987) 303-311.
Neuroscience Letters 175 (1994) 114-116
N[UROSCIENC[ LETTERS
Opposite effects of cyclohexyladenosine and theophylline on hypoxic damage in cultured neurons Jean-Luc Daval*, Fr6d6ric Nicolas I N S E R M U.272, BP 3069, Universit~ de Nancy I, 24-30 rue Lionnois, 54013 Nancy, France Received 5 April 1994; Revised version received 11 May 1994; Accepted 16 May 1994
Abstract
To study the central effects of adenosine on hypoxia, the influence of treatment by the A 1 receptor agonist cyclohexyladenosine (1 gM) or by the antagonist theophylline (10/.tM) was tested on cell damage in a model of neuronal culture. Whereas theophylline enhanced cell injury induced by 8 h hypoxia, cyclohexyladenosinedecreased lactate dehydrogenase leakage, abolished the transient increase in 2-D-deoxyglucosetransport and improved cell morphology. Such actions might involve regulation of excitatory amino acid release and maintenance of calcium homeostasis. Key words." Cultured neuron; Hypoxia; Lactate dehydrogenase efflux;Deoxyglucosetransport; Cyclohexyladenosine;TheophyUine
Numerous cellular processes are known, or have been hypothesized to be involved in the pathophysiology of brain hypoxia. Among them, the release of excitatory amino acids, mainly glutamate and aspartate, as well as calcium influx and overload have been shown to play a key role in hypoxia-ischemia neuronal damage [8,15]. The role of adenosine as a neuromodulator in the central nervous system has been well documented [7,16]. Via the activation of its specific A~ receptors, the nucleoside inhibits neuronal calcium influx [1] and counteracts the presynaptic release of the excitotoxic neurotransmitters [4]. Therefore, adenosine appears as an endogenous neuroprotective agent, and numerous studies have provided evidence for a beneficial effect of adenosine analogues, e.g. cyclohexyladenosine (CHA) or L-phenyl-isopropyladenosine (L-PIA), in brain ischemia, whereas blockade of adenosine receptors by antagonists such as theophylline or caffeine exacerbates ischemic brain injury [14]. In addition to its neuromodulatory effects, the ubiquitous properties of adenosine (and its stable analogues), including cardiovascular depression, vasodilatory actions as well as hypothermia, may also contribute to the cerebral protection induced by adenosine in vivo. In
* Corresponding author. Fax: (33) 83.32.43.40. 0304-3940/94/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(94)00398-T
order to alleviate these side effects, the influence of the adenosine A~ receptor agonist CHA and the antagonist theophylline was studied on hypoxic damage in a model of primary cultures of cortical and hippocampal neurons. Neuronal cell cultures were obtained from 14-day-old rat embryo cortex and hippocampus according to Daval et al. [6]. Dispersed cells were plated on poly-lysine-precoated 35 mm dishes containing a mixture of Dulbeccomodified Eagle's and Ham-F12 media (50:50) with 5% inactivated foetal calf serum. After 24 h, neurons were grown for 5 additional days under standard normoxic conditions (95% air/5% CO2) in a chemically-defined serum-free medium. Cells were submitted to transient hypoxia by incubating the culture dishes for 8 h in a humidified chamber flushed with 95% Nil5% CO2, while control cultures were maintained under normoxic conditions, as previously described [6]. Pharmacological treatments were done just before the hypoxic insult by adding either CHA (dissolved in 2% ethanol, final concentration 1/IM in 0.02% ethanol) or theophylline (final concentration 10/IM) to the culture medium. Concentrations of both drugs were chosen in a preliminary study in order to exert an action on hypoxic cell injury, while not altering cell morphology and viability per se within the 3 following days. Cells
J.-L. Daval, E NicolaslNeuroscience Letters 175 (1994) 114-116
were studied either 24 or 72 h after hypoxia by the assessment of cell survival and morphology, lactate dehydrogenase (LDH) efflux [9], whereas the functional activity of the cells was estimated by measuring the specific uptake of [3H]2-deoxy-D-glucose ([3H]2DG, 969.4 GBq/ mmol, NEN, Boston, USA) during a 30 min incubation of the cells at 37°C in the presence of 2 ml of a solution of Krebs-Ringer saline buffered with HEPES (pH 7.40) and containing 1 mM [3H]2DG [5]. Cells were finally washed twice by 2 ml 0.9% NaC1 warmed up to 37°C, treated by 1 ml NaOH (1 M), and then scrapped off for protein measurement according to Bradford [2]. After 6 days in vitro, cultures exhibited a very high percent of living neuronal cells interconnected by a dense fiber network, as previously reported [11]. Whatever the pharmacological treatment of the cells was, no cell damage could be observed by microscopic observation 24 h after the hypoxic insult which induced a 78% reduction of the partial pressure of oxygen in the culture medium which decreased from 139.2 + 0.2 to 31.7 + 3.3 mmHg. By contrast, signs of cell degeneration appeared 3 days after hypoxia, concomitantly with a 22% decrease in protein levels (Table 2). Whereas CHA and theophylline themselves had no incidence on cell morphology, the treatment of the cells by the adenosine agonist improved cell morphology 3 days post-hypoxia, leading to a non significant 9% reduction of the protein concentration. Conversely, theophylline treatment tended to aggravate the deleterious influence of hypoxia on neuron survival and morphology, as reflected by increased Trypan blue penetration into the cells, the presence of larger amounts of cellular debris in culture dishes, and a 37% reduction of the protein concentrations in the cultures. Cell exposure to hypoxia for 8 h led to an important efflux of L D H to the extracellular medium which increased with time (Tables 1 and 2). While not inducing significant LDH leakage when not associated to hypoxia, CHA reduced hypoxia-related amounts of LDH Table 1 Changes in LDH efflux, 2DG specific transport and protein contents in cultured neurons 24 hours after hypoxia L D H efflux 2DG transport units/liter nmol/mg prot.
proteins pg/dish
Controls 1 # M CHA 10 # M Theophylline
1.3 -+ 0.2 1.6 _+ 0.2 1.7 _+ 0.3
91.6 -+ 3.9 88.5 _+ 4.6 111.2 _+ 3.7
203.2 _+ 8.3 211.0 _+4.1 198.6 _+ 4.9
Hypoxia Hypoxia + CHA Hypoxia + Theophylline
4.6 _+ 0.7** 2.4 _+ 0.4* 6.5 ± 1.0"*
121.2 _+4.2** 199.4 _+ 5.4 92.6 _+4.0 °0 207.7 _+ 9.1 152.3 +_ 3.61 **° 198.2 -+ 8.2
Data are reported as mean values (_+S.D.) obtained from 4 separate experiments using 6 or 7 culture dishes for each experimental condition. Statistically differences with the appropriate pharmacologically-treated control: *P < 0.05, **P < 0.01. Statistically significant differences with hypoxia-values: °P<0.05, °°P <0.01 (Bonferroni t-test for multiple comparisons).
115
Table 2 Changes in L D H efflux, 2DG specific transport and protein contents in cultured neurons 72 hours after hypoxia L D H efflux 2DG transport proteins units/liter nmol/mg prot. #g/dish Controls 1 / t M CHA 10 # M Theophylline
1.5 _+0.1 2.2 _+ 0.5 3.4 _+ 0.6
90.2 _+ 3.6 92.8 _+ 6.3 98.4 _+ 5.1
Hypoxia 6.3 _+ 0.5 ** 6 4 . 0 + 3 . 5 * * Hypoxia + CHA 2.3 _+ 0.3 °0 89.7_+4.7 °° Hypoxia + Theophylline 8.0 _+ 0.7 **° 52.1 _+ 4.4 **0
170.7 _+ 11.2 168.6 ± 10.7 159.6 _+ 9.5 133.6_+12.5 * 154.5_+ 14.1 101.6 +_ 16.8 *
Data are reported as mean values (+ S.D.) obtained from 4 separate experiments using 6 or 7 culture dishes for each experimental condition. Statistically significant differences with the appropriate pharmacologically-treated control: *P < 0.05, **P< 0.01. Statistically significant differences with hypoxia-values: °P <0.05, °°P <0.01 (Bonferroni t-test for multiple comparisons).
detected in the culture medium of the cells at the 2 timepoints studied. Theophylline treatment increased slightly LDH release in cells exposed to normoxia, and more dramatically following cell exposure to hypoxia. As shown in Tables 1 and 2, hypoxia enhanced transiently 2DG-specific transport which increased 24 h after hypoxia, and then decreased below normal values in the following days. While it did not change 2DG transport under normoxic conditions, CHA was able to abolish hypoxia-induced alterations of [3H]2DG transport in neuronal cells. Theophylline alone, at the concentration used, increased only moderately 2DG uptake by the cells in vitro. However, the adenosine antagonist enhanced the effects of hypoxia on 2DG transport. Since conditions that closely mimic those which occur in vivo can be produced easily in vitro by manipulating the extracellular environment, cell cultures are useful tools for studying the cellular mechanisms involved in hypoxia- or ischemia-induced cell death. Also, cultures from the central nervous system are of interest to counteract peripheral effects of substances which are known to have protective properties in vivo. In addition, neuron-enriched cultures from cortex and hippocampus, as used in the present study, appear as the most appropriate model, since it has been shown that (i) glia can protect neurons against both hypoxia [17] and excitatory amino acid-induced insult [10], and (ii) cortex and hippocampus are particularly vulnerable to reduced oxygen supply [3]. This study shows that an adenosine analogue which interacts preferentially with A~ receptors, and at pharmacologically-relevant concentration, is able to reduce hypoxic injury in a model of primary culture of neurons isolated from rat cortex and hippocampus. Indeed, CHA treatment appears to limit LDH efflux, a good marker of cell injury, and to abolish the transient increase in 2DG uptake which might reflect, at least partly, excitatory amino acid-induced cell stimulation that corresponds to the first step of a deleterious cascade of cellular
116
J.-L. Daval, E Nicolas l Neuroscience Letters 175 (1994) 114-116
events. Conversely, the non-specific receptor antagonist theophylline increases cell d a m a g e following hypoxia in vitro. Such data are in g o o d agreement with those obtained f r o m in vivo studies. In the brain, adenosine depresses the presynaptic release o f glutamate. Accordingly, it has been shown that in rat hippocampal synaptosomes, adenosine and its stable analogues decreased the K ÷ or the kainate-evoked release o f glutamate and aspartate, whereas glutamate was able to stimulate adenosine release f r o m the nerve terminals [13]. Also, adenosine reduces postsynaptic m e m b r a n e depolarization by acting on K ÷ and CI- conductances. At both pre- and postsynaptic levels, adenosine action is relevant for the maintenance o f calcium homeostasis [1,7,16]. All these central effects are certainly effective to limit and/or prevent pathophysiological processes triggered by hypoxia or ischemia, and might a c c o u n t for our observations. A l t h o u g h A 1 receptor-mediated n e u r o m o d u l a t o r y actions o f adenosine are o f special importance, the mechanisms underlying the anti-hypoxic effects o f the nucleoside m a y also involve the participation o f A2 receptors [12,14]. In this respect, C H A at the concentration used in the present study ( 1 / t M ) m a y also act, although to a lesser extent, at A2 receptors, leading to the assumption that the activation o f the two categories o f adenosine sites could be responsible for the protection observed. Therefore, further studies using highly selective agonists and antagonists o f b o t h types o f adenosine receptors would provide new insights to the neuroprotective role o f adenosine derivatives. The authors wish to thank V. Koziel for her excellent technical assistance. [1] Andinr, P., Involvement of adenosine in ischemic and postischemic calcium regulation, Mol. Chem. Neuropathol., 18 (1983) 3549. [2] Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248-254.
[3] Brierley, J.B., Cerebral hypoxia. In W. Blackwood and J.A. Corsellis (Eds.), Greenfield's Neuropathology, Edward Arnold, London, 1976, pp.41-85. [4] Corradetti, R., Lo Conte, F., Moroni, F., Passani, M.B. and Pepeu, G., Adenosine decreases aspartate and glutamate release from rat hippocampal slices, Eur. J. Pharmacol., 104 (1984) 19-26. [5] Daval, J.L., Anglard, P., Gerard, M.J., Vincendon, G. and Louis, J.C., Regulation of deoxyglucose uptake by adrenocorticotropic hormone in cultured neurons, J. Cell. Physiol., 124 (1985) 7580. [6] Daval, J.L., Koziel, V. and Nicolas, F., Functional changes in cultured neurons following transient asphyxia, NeuroReport, 2 (1991) 97-100. [7] Daval, J.L., Nehlig, A. and Nicolas, F., Physiological and pharmacological properties of adenosine: therapeutic implications, Life Sci., 49 (1991) 1435-1453. [8] J6rgensen, M.B. and Diemer, N.H., Selective neuronal loss after cerebral ischemia in the rat: possible role of transmitter glutamate, Acta Neurol. Scand., 66 (1982) 536-546. [9] Koh, J.Y. and Choi, D.W., Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay, J. Neurosci. Methods, 20 (1987) 8390. [10] Mattson, M.P. and Rychlik, B., Glia protects hippocampal neurons against excitatory amino acid-induced degeneration: involvement of fibroblast growth factor, Int. J. Dev. Neurosci., 8 (1990) 399415. [11] Nicolas, F., Oillet, J., Koziel, V. and Daval, J.L., Characterization of adenosine receptors in a model of cultured neurons from rat forebrain, Neurochem. Res., 19 (1994) 507-515. [12] Phillis, J.W., The selective adenosine A: receptor agonist, CGS 21680, is a potent depressant of cerebral cortical neuronal activity, Brain Res., 509 (1990) 328-330. [13] Poli, A., Lucchi, R., Vibio, M. and Barnabei, O., Adenosine and glutamate modulate each other's release from rat hippocampal synaptosomes, J. Neurochem., 57 (1991) 298-306. [14] Rudolphi, K.A., Schubert, P., Parkinson, F.E. and Fredholm, B.B., Adenosine and brain ischemia, Cerebrovasc. Brain Metab. Rev., 4 (1992) 346-369. [15] Siesj6, B.K. and Bengtsson, F., Calcium fluxes, calcium antagonists, and calcium related pathology in brain ischemia, hypoglycemia, and spreading depression: a unifying hypothesis, J. Cereb. Blood Flow Metab., 9 (1989) 127-140. [16] Snyder, S.H., Adenosine as a neuromodulator, Annu. Rev. Neurosci., 8 (1985) 103-124. [17] Vibulsreth, S., Hefti, F., Ginsberg, M.D., Dietrich, W.D. and Busto, R., Astrocytes protect cultured neurons from degeneration induced by anoxia, Brain Res., 422 (1987) 303-311.