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Adenosine enhances glial glutamate efflux via A2a adenosine receptors
Life Sciences 68 (2001) 1343–1350
Adenosine enhances glial glutamate efflux via A2a adenosine receptors X.X. Li, T. Nomura, H. Aihara, T. Nishizaki* Department of Physiology, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan Received 22 May 2000; accepted 16 August 2000
Abstract The present study investigated the effect of adenosine on glial glutamate efflux. Adenosine (from 1 nM to 100 mM) enhanced the release from cultured rat glial cells in a bell-shaped dose-responsive manner for the hippocampus and in a dose-dependent manner for the superior colliculus, and a similar increase was obtained with the A2a adenosine receptor agonist, 2-p-(2-carboxyethyl) phenethylamino59-N-ethylcarboxamidoadenosine hydrochloride (CGS21680), but not with the A1 adenosine receptor agonist, N6-cyclohexyladenosine (CHA). Adenosine and CGS21680 also enhanced glutamate efflux from Xenopus oocytes injected with the poly (A)1 mRNAs derived from cultured glial cells for the hippocampus and the superior colliculus together with and without the A2a adenosine receptor mRNA, but instead such increase was not found in oocytes expressing A2a adenosine receptors alone. The results of the present study thus suggest that adenosine enhances glutamate efflux from glial cells via A2a adenosine receptors, and this may represent a mechanism underlying the facilitatory action of adenosine on hippocampal and superior colliculus neurotransmissions. © 2001 Elsevier Science Inc. All rights reserved. Keywords: A2a adenosine receptor; Glutamate efflux; Glial cells
Introduction Adenosine is well-recognized to serve as a neuromodulator in the peripheral and central nervous systems [1]. Adenosine receptors (P1 purinoceptors) are classified on the basis of their differential selectivity for adenosine analogs and the A1 (A1a, A1b, A3), A2 (A2a, A2b), and A4 subtypes have been cloned [2–4]. It is shown that adenosine exerts its excitatory (# 1 mM) and inhibitory ($ 10 mM) bilateral actions on hippocampal neurotransmission [5], and its dose-
* Corresponding author: Tel.: 181-78-382-5363; fax: 181-78-382-5379. E-mail address: [email protected]. (T. Nishizaki) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 1 0 3 6 -5
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dependent excitatory action on superior colliculus neurotransmission [6]. The inhibition appears to be mediated by A1 adenosine receptors, involving an enhancement in the postsynaptic K1 conductances [7] and a decrease in the release of the neurotransmitters, e.g., noradrenaline in the hippocampus and cerebral cortex [8], dopamine and/or acetylcholine in the rat striatum [9.10], and glutamate in the goldfish cerebellum [11]. In contrast, the excitation may be mediated by A2a adenosine receptors [12], involving an enhancement in the release of neurotransmitters from presynaptic terminals, e.g., acetylcholine in the rat striatum [10,13]. Previous studies show that adenosine increases glutamate released from electrically stimulated slices of the guinea pig hippocampus [14] and superior colliculus [15], suggesting that adenosine facilitates neurotransmissions by stimulating glutamate release from neurons. The increase, however, is also obtained with slices still under non-excitable conditions [15]. Then, we hypothesized that adenosine might be involved in the glial glutamate efflux. To prove this hypothesis, we assayed glutamate released from either cultured glial cells of the rat hippocampus and superior colliculus, or Xenopus oocytes injecting with the poly (A)1 mRNAs derived from cultured glial cells for each region together with and without the A2a adenosine receptor mRNAs. The results of the present study suggest that adenosine stimulates glutamate efflux from glial cells via A2a adenosine receptors. Methods Primary cultures of glial cells Glial cells from the neonatal rat brain on day 1 were cultured through described previously methods [16]. Briefly, the hippocampus and the superior colliculus were removed from the brain under ether anesthesia. Tissues were incubated in 0.25% trypsin in Ca21-, Mg21-free saline for a few min at room temperature and then mechanically dissociated by triturating with a Pasteur pipette. The dissociated cells were plated on collagen-coated cover-slips and grown in Eagle’s minimum essential medium containing 2 mM glutamine, double concentrations of the other amino acids, quadruple concentrations of the vitamines, 7 mM glucose, 50 IU/ml penicillin, 50 mg/ml streptomycin and 15% fetal bovine serum at 378C in a humidified atmosphere of 95% air and 5% CO2. The first cultured cells were split at a rate of 1:3 one week after plating, and 1 week later cells were used for experiments. We confirmed that the cells are astrocytes; in a whole-cell patch-clamp configuration, voltage-dependent Na1 currents, a specific marker for neurons, were never evoked; and in the immunohistochemistry, the cells were reactive to a mouse monoclonal antibody against human glial fibrillary acidic protein (GFAP). Preparation of mRNAs and translation in Xenopus oocytes mRNAs were extracted from cultured glial cells of the rat hippocampus and superior colliculus using a guanidinium thiocyanate/caesium chloride method, and purified chromatographically on oligo (dT)-cellulose columns [17]. mRNAs coding for the dog A2a adenosine receptor were synthesized by in vitro transcription [2]. Xenopus oocytes were manually separated from the ovary, and injected with the poly (A)1 mRNAs derived from cultured glial cells for each region together with and without the A2a adenosine receptor mRNAs. The
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oocytes were incubated in Barth’s solution (in mM, 88 NaCl, 1 KCl, 2.4 NaHCO3, 0.82 MgSO4, 0.33 Ca(NO2)2, 0.41 CaCl2, and 7.5 Tris, pH 7.6) at 188C for 5–7 days. Assay of glutamate Cultured cells were washed twice with extracellular solution (in mM; 145 NaCl, 5 KCl, 2.4 CaCl2, 10 HEPES, pH 7.4). Subsequently, cells were incubated with an extracellular solution (500 ml) in the presence and absence of adenosine, N6-cyclohexyladenosine (CHA) (RBI, USA), or 2-p-(2-carboxyethyl) phenethylamino-59-N-ethylcarboxamidoadenosine hydrochloride (CGS21680) (RBI, USA) for 30 min. In the oocyte expression systems, 100 mM glutamate was added to Barth’s solution one day before experiments. Oocytes were washed twice with frog Ringer’s solution (in mM; 115 NaCl, 2 KCl, 1.8 CaCl2, and 5 HEPES, pH 7.0) and incubated in 200 ml of the solution containing adenosine, CHA, or CGS21680 for 30 min at room temperature (20–228C). The medium collected was filtered with millipore filters (0.45 mm) and glutamate concentrations were determined with using a high performance liquid chromatography. Results An increase in the glutamate efflux from cultured glial cells induced by adenosine Adenosine increased glutamate efflux from hippocampal glial cells in a bell-shaped doseresponsive manner at concentrations ranged from 1 nM to 100 mM, with a maximum at 10 nM, the corresponding value (7.6 nmol/mg protein/30 min) being 15 times of control (nontreatment with adenosine) (Fig. 1A). A similar increase was obtained with the selective A2a adenosine receptor agonist, CGS21680 (1 mM), while the selective A1 adenosine receptor agonist, CHA (10 mM), had no effect (Fig. 1A), suggesting that adenosine stimulates glutamate efflux from glial cells via A2a adenosine receptors. For glial cells from the superior colliculus, adenosine enhanced glutamate efflux in a dose-dependent manner at concentrations ranged from 1 nM to 100 mM, reaching 9.4 nmol/mg protein/30 min (Fig. 1B). CGS21680 (1 mM), but not CHA (10 mM), also enhanced glutamate efflux from superior colliculus glial cells (Fig. 1B), further supporting the idea that A2a adenosine receptors are involved in the glial glutamate efflux. An increase in the glutamate efflux from oocytes expressing glial components derived from the hippocampus and the superior colliculus together with and without A2a adenosine receptors As is the case in cultured glial cells, adenosine increased glutamate efflux from oocytes injected with the poly (A)1 mRNA derived from cultured glial cells for the hippocampus and the superior colliculus (Fig. 2A,B). The increase was reinforced by co-expressing A2a adenosine receptors, reaching 4.5 and 1.7 times of control (non-treatment with adenosine) for the hippocampus, and 7.3 and 9.8 times of control for the superior colliculus, at 10 nM and 100 mM of adenosine, respectively (Fig. 2A,B). Likewise, CGS21680 (1 mM) caused 2.8- and 6.6fold increase in the glutamate efflux for the hippocampus and the superior colliculus, respectively, but CHA (10 mM) had no effect (Fig. 2A,B). In oocytes expressing A2a adenosine re-
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Fig. 1. Adenosine-induced glutamate efflux from cultured glial cells. Adenosine at concentrations as indicated, CHA (10 mM), or CGS21680 (1 mM) was applied to cultured glial cells from the hippocampus (A) and superior colliculus (B), and glutamate released in extracellular medium was assayed. Each value represents the mean glutamate concentration of 5–7 independent experiments and the SEM is indicated by the bars. * P,0.1, ** P,0.01, ANOVA with Fisher’s least-significance difference test.
ceptors alone, however, such increase was not found (Fig. 2C). It is strongly suggested from these results that adenosine stimulates glutamate efflux via A2a adenosine receptors, but that unknown factors or components expressed in glial cells are required for the adenosine action. Discussion Adenosine is shown to induce a facilitation of neurotransmission in the guinea pig hippocampus (# 1 mM) [5] and superior colliculus (. 1 nM) [6]. One thought that the facilitatory action is caused by increasing glutamate from presynaptic terminals, based upon the data that adenosine increased glutamate released from electrically stimulated slices from the guinea pig hippocampus [14] and superior colliculus [15]. Adenosine, however, increased glutamate released from slices also under non-excitable conditions [15], suggesting
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Fig. 2. Adenosine-induced glutamate efflux from oocytes. Oocytes were injected with the poly (A)1 mRNAs derived from cultured glial cells; for the hippocampus together with (A, right panel) and without (A, left panel) the A2a adenosine receptor mRNA; or for the superior colliculus together with (B, right panel) and without (B, left panel) the A2a adenosine receptor mRNA. In C, A2a adenosine receptors alone were expressed in oocytes. Oocytes were incubated in the presence and absence of adenosine (0.01 and 100 mM), CHA (10 mM), or CGS21680 (1 mM), and glutamate released in extracellular medium was assayed. Each value represents the mean glutamate concentration of 8–10 independent experiments and the SEM is indicated by the bars. * P,0.1, ** P,0.01, ANOVA with Fisher’s least-significance difference test.
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that the facilitatory action of adenosine is not caused simply by an increase in the glutamate release from neurons. This, taken together with the result of our preliminary study that adenosine had no effect on the rate of spontaneous miniature excitatory postsynaptic currents in cultured solitary neurons from the rat hippocampus (unpublished data), raises the possibility that adenosine might facilitate neurotransmission in part by stimulating glial glutamate release. In support of this idea, the results of the present study showed that adenosine actually enhances glutamate efflux from cultured glial cells. Interestingly, adenosine dose-responsive relations in the release, i.e., bell-shaped curve for the hippocampus and sigmoid curve for the superior colliculus, are in agreement with the relations in the facilitation of neurotransmissions for each region [5,6]. Then, one would wonder why the dose-dependency of responses to adenosine in the two regions is quite different. Possible explanation is that such difference might be due to the difference either in the amount of adenosine deaminase between the regions, i.e., much higher in the superior colliculus than in the hippocampus, or in the distribution of the relevant receptors, or in the glial subtypes, although glial cells used here are astrocytes. The question is as to which adenosine receptor mediates glial glutamate efflux. In cultured glial cells, the A2a adenosine receptor agonist, CGS21680, but not the A1 adenosine receptor agonist, CHA, increased glutamate efflux in a fashion that mimics the adenosine action. This, in the light of the previous study showing that the facilitatory action of adenosine on hippocampal neurotransmission is likely mediated by A2a adenosine receptors [12], suggests strongly that adenosine might enhance glial glutamate efflux via A2a adenosine receptors. In the Xenopus oocyte expression systems, adenosine or CGS21680 increased glutamate efflux from oocytes co-expressing A2a adenosine receptors and glial components from the hippocampus or the superior colliculus, but such increase was not obtained with oocytes expressing A2a adenosine receptors alone. This may account for glial unknown factors or components as a target of A2a adenosine receptor signal in the glutamate efflux. A2a adenosine receptors are linked to Gs-protein stimulating adenylylcyclase [7], and therefore, the glial efflux observed here may be regulated via a cAMP-dependent protein kinase pathway. Lines of evidence have pointed to major two pathways in the glutamate release; Ca21dependent vesicular release from presynaptic terminals [18, 19] and Ca21-independent, nonvesicular release from neuronal cytoplasmic pools and glial cells, such as reverse transport of glutamate by glutamate transporters [20–23]. Then, one would discuss whether adenosine releases glutamate through reverse transport by glutamate transporters or not. It is recognized that glutamate transporters run backwards under extracellular high K1 concentrations associated with ischemia and anoxia [24]. Adenosine indeed enhances glutamate release from rat cerebral cortex [25] or cultured chick retinal cells after ischemic insult [26], suggesting the implication of glutamate transporters in adenosine-induced glutamate release. However, this is unlikely, since in the present study, an increase in the glutamate efflux induced by adenosine was found under normal conditions. Adenosine, thus, appears to stimulate glutamate efflux from glial cells via an undescribed pathway. To address this pathway, we are carrying out further experiments. In conclusion, the results presented here suggest that adenosine enhances glial glutamate efflux via A2a adenosine receptors, at least in part responsible for the facilitatory action on neurotransmissions, providing a new communication between neurons and glial cells.
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Acknowledgments We are grateful to Dr. G. Vassart (Université Libre de Bruxelles) for providing us with the A2a adenosine receptor cDNA clone. References 1. Stiles GL. Adenosine receptors: structure, functions, and regulation. Trends in Pharmacological Sciences 1986; 12 (12): 486–90. 2. Libert F, Parmentier M, Lefort A, Dinsart C, Van Sande J, Maenhaut C, Simons M-J, Dumont JE, Vassart G. Selective amplification and cloning of four new members of the G protein-coupled receptor family. Science 1989 (4904); 244: 569–72. 3. Mahan LC, Mcvittie LD, Smyk-Randall EM, Nakata H, Monsma FJJr, Gerfen CR, Sibley DR. Cloning and expression of an A1 adenosine receptor from rat brain. Molecular Pharmacology 1991; 40 (1): 1–7. 4. Tucker AL, Linden J. Cloned receptors and cardiovascular responses to adenosine. Cardiovascular Research 1993; 27 (1): 62–7. 5. Nishimura S, Mohri M, Okada Y, Mori M. Excitatory and inhibitory effects of adenosine on the neurotransmission in the hippocampal slices of guinea pig. Brain Research 1990; 525 (1): 165–9. 6. Okada Y, Nishimura S, Miyamoto T. Excitatory effect of adenosine on neurotransmission in the slices of superior colliculus and hippocampus of guinea pig. Neuroscience Letters 1990; 120 (2): 205–8. 7. Stiles GL. Adenosine receptors. The Journal of Biological Chemistry 1992; 267 (10): 6451–4. 8. von Kügelgen I. Purinoceptors modulating the release of noradrenaline. Journal of Autonomic Pharmacology 1994; 14 (1): 11–2. 9. Jin S, Johansson B, Fredholm BB. Effects of adenosine A1 and A2 receptor activation on electrically evoked dopamine and acetylcholine release from rat striatal slices. The Journal of Pharmacology and Experimental Therapeutics 1993; 267 (2): 801–8. 10. Kirkpatrick KA, Richardson PJ. Adenosine receptor-mediated modulation of acetylcholine release from rat striatal synaptosomes. British Journal of Pharmacology 1993; 110 (3): 949–54. 11. Lucchi R, Poli A, Traversa U, Barnabei O. Functional adenosine receptors in goldfish brain: regional distribution and inhibition of K1-evoked glutamate release from cerebellar slices. Neuroscience 193; 58 (2): 237–43. 12. Sebastião AM, Ribeiro JA. Evidence for the presence of excitatory A2 adenosine receptors in the rat hippocampus. Neuroscience Letters 1992; 138 (1): 41–4. 13. Kirk IP, Richardson PJ. Adenosine A2 receptor-mediated modulation of striatal [3H]GABA and [3H]acetylcholine release. Journal of Neurochemistry 1994; 62 (3): 960–6. 14. Okada Y, Sakurai T, Mori M. Excitatory effect of adenosine on neurotransmission is due to increase of transmitter release in the hippocampal slices. Neuroscience Letters 1992; 142 (2): 233–236. 15. Hirai H, Okada Y. Adenosine facilitates glutamate release in a protein kinase-dependent manner in superior colliculus slices. European Journal of Pharmacology 1994; 256 (1): 65–71. 16. Ikeuchi Y, Nishizaki T, Matsuoka T. Lysophosphatidylcholine inhibits NMDA-induced currents by mechanism independent of phospholipase A2-mediated protein kinase C activation in hippocampal glial cells. Biochemical and Biophysical Research Communications 1995; 217 (3): 811–6. 17. Sumikawa K. Parker I, Miledi R. Effects of tuncamycin on the expression of functional neurotransmitter receptors and voltage-operated channels in Xenopus oocytes. Molecular Brain Research 1988; 4 (3): 191–9. 18. Kauppinen RA, McMahon HT, Nicholls DG. Ca21-dependent and Ca21-independent glutamate release, energy status and cytosolic free Ca21 concentration in isolated nerve terminals. Neuroscience 1988; 27 (1): 175–82. 19. Wahl F, Obrenovitch TP, Hardy AM, Plotkine M, Boulu R, Symon L. Extracellular glutamate during focal cerebral ischaemia in rats: time course and calcium dependency. Journal of Neurochemistry 1994; 63 (3): 1003–11. 20. Attwell D, Barbour B, Szatkowski M. Nonvascular release of neurotransmitter. Neuron 1993; 11 (3): 401–7.
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21. Nicholls DG, Attwell D. The release and uptake of expiatory amino acids. Trends in Pharmacological Science 1990; 11 (11): 462–8. 22. Szatkowski M, Attwell D. Triggering and execution of neuronal death in brain ischaemia: two phases of glutamate release by different mechanisms. Trends in Neuroscience 1996; 17 (9): 359–64. 23. Takahashi M, Billups B, Rossi D, Sarantis M, Hamann M, Attwell, D. The role of glutamate transporters in glutamate homeostasis in the brain. Journal of Experimental Biology 1997; 200 (2): 401–9. 24. Levy LM, Warr O, Attwell D. Stoichiometry of the glial glutamate transporter GLT-1 expressed inducibly in a Chinese hamster ovary cell line selected for low endogenous Na1-dependent glutamate uptake. The Journal of Neuroscience 1998; 18 (23): 9620–8. 25. Simpson RE, O’Regan MH, Perkins LM, Phillis JW. Excitatory transmitter amino acid release from the ischemic rat cerebral cortex. Journal of Neurochemistry 1992; 58 (5): 1683–90. 26. Rego AC, Santos MS, Oliveira CR. Adenosine triphosphate degradation produces after oxidative stress and metabolic dysfunction in cultured retinal cells. Journal of Neurochemistry 1997; 69 (3): 1228–35.
Adenosine enhances glial glutamate efflux via A2a adenosine receptors X.X. Li, T. Nomura, H. Aihara, T. Nishizaki* Department of Physiology, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan Received 22 May 2000; accepted 16 August 2000
Abstract The present study investigated the effect of adenosine on glial glutamate efflux. Adenosine (from 1 nM to 100 mM) enhanced the release from cultured rat glial cells in a bell-shaped dose-responsive manner for the hippocampus and in a dose-dependent manner for the superior colliculus, and a similar increase was obtained with the A2a adenosine receptor agonist, 2-p-(2-carboxyethyl) phenethylamino59-N-ethylcarboxamidoadenosine hydrochloride (CGS21680), but not with the A1 adenosine receptor agonist, N6-cyclohexyladenosine (CHA). Adenosine and CGS21680 also enhanced glutamate efflux from Xenopus oocytes injected with the poly (A)1 mRNAs derived from cultured glial cells for the hippocampus and the superior colliculus together with and without the A2a adenosine receptor mRNA, but instead such increase was not found in oocytes expressing A2a adenosine receptors alone. The results of the present study thus suggest that adenosine enhances glutamate efflux from glial cells via A2a adenosine receptors, and this may represent a mechanism underlying the facilitatory action of adenosine on hippocampal and superior colliculus neurotransmissions. © 2001 Elsevier Science Inc. All rights reserved. Keywords: A2a adenosine receptor; Glutamate efflux; Glial cells
Introduction Adenosine is well-recognized to serve as a neuromodulator in the peripheral and central nervous systems [1]. Adenosine receptors (P1 purinoceptors) are classified on the basis of their differential selectivity for adenosine analogs and the A1 (A1a, A1b, A3), A2 (A2a, A2b), and A4 subtypes have been cloned [2–4]. It is shown that adenosine exerts its excitatory (# 1 mM) and inhibitory ($ 10 mM) bilateral actions on hippocampal neurotransmission [5], and its dose-
* Corresponding author: Tel.: 181-78-382-5363; fax: 181-78-382-5379. E-mail address: [email protected]. (T. Nishizaki) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 1 0 3 6 -5
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dependent excitatory action on superior colliculus neurotransmission [6]. The inhibition appears to be mediated by A1 adenosine receptors, involving an enhancement in the postsynaptic K1 conductances [7] and a decrease in the release of the neurotransmitters, e.g., noradrenaline in the hippocampus and cerebral cortex [8], dopamine and/or acetylcholine in the rat striatum [9.10], and glutamate in the goldfish cerebellum [11]. In contrast, the excitation may be mediated by A2a adenosine receptors [12], involving an enhancement in the release of neurotransmitters from presynaptic terminals, e.g., acetylcholine in the rat striatum [10,13]. Previous studies show that adenosine increases glutamate released from electrically stimulated slices of the guinea pig hippocampus [14] and superior colliculus [15], suggesting that adenosine facilitates neurotransmissions by stimulating glutamate release from neurons. The increase, however, is also obtained with slices still under non-excitable conditions [15]. Then, we hypothesized that adenosine might be involved in the glial glutamate efflux. To prove this hypothesis, we assayed glutamate released from either cultured glial cells of the rat hippocampus and superior colliculus, or Xenopus oocytes injecting with the poly (A)1 mRNAs derived from cultured glial cells for each region together with and without the A2a adenosine receptor mRNAs. The results of the present study suggest that adenosine stimulates glutamate efflux from glial cells via A2a adenosine receptors. Methods Primary cultures of glial cells Glial cells from the neonatal rat brain on day 1 were cultured through described previously methods [16]. Briefly, the hippocampus and the superior colliculus were removed from the brain under ether anesthesia. Tissues were incubated in 0.25% trypsin in Ca21-, Mg21-free saline for a few min at room temperature and then mechanically dissociated by triturating with a Pasteur pipette. The dissociated cells were plated on collagen-coated cover-slips and grown in Eagle’s minimum essential medium containing 2 mM glutamine, double concentrations of the other amino acids, quadruple concentrations of the vitamines, 7 mM glucose, 50 IU/ml penicillin, 50 mg/ml streptomycin and 15% fetal bovine serum at 378C in a humidified atmosphere of 95% air and 5% CO2. The first cultured cells were split at a rate of 1:3 one week after plating, and 1 week later cells were used for experiments. We confirmed that the cells are astrocytes; in a whole-cell patch-clamp configuration, voltage-dependent Na1 currents, a specific marker for neurons, were never evoked; and in the immunohistochemistry, the cells were reactive to a mouse monoclonal antibody against human glial fibrillary acidic protein (GFAP). Preparation of mRNAs and translation in Xenopus oocytes mRNAs were extracted from cultured glial cells of the rat hippocampus and superior colliculus using a guanidinium thiocyanate/caesium chloride method, and purified chromatographically on oligo (dT)-cellulose columns [17]. mRNAs coding for the dog A2a adenosine receptor were synthesized by in vitro transcription [2]. Xenopus oocytes were manually separated from the ovary, and injected with the poly (A)1 mRNAs derived from cultured glial cells for each region together with and without the A2a adenosine receptor mRNAs. The
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oocytes were incubated in Barth’s solution (in mM, 88 NaCl, 1 KCl, 2.4 NaHCO3, 0.82 MgSO4, 0.33 Ca(NO2)2, 0.41 CaCl2, and 7.5 Tris, pH 7.6) at 188C for 5–7 days. Assay of glutamate Cultured cells were washed twice with extracellular solution (in mM; 145 NaCl, 5 KCl, 2.4 CaCl2, 10 HEPES, pH 7.4). Subsequently, cells were incubated with an extracellular solution (500 ml) in the presence and absence of adenosine, N6-cyclohexyladenosine (CHA) (RBI, USA), or 2-p-(2-carboxyethyl) phenethylamino-59-N-ethylcarboxamidoadenosine hydrochloride (CGS21680) (RBI, USA) for 30 min. In the oocyte expression systems, 100 mM glutamate was added to Barth’s solution one day before experiments. Oocytes were washed twice with frog Ringer’s solution (in mM; 115 NaCl, 2 KCl, 1.8 CaCl2, and 5 HEPES, pH 7.0) and incubated in 200 ml of the solution containing adenosine, CHA, or CGS21680 for 30 min at room temperature (20–228C). The medium collected was filtered with millipore filters (0.45 mm) and glutamate concentrations were determined with using a high performance liquid chromatography. Results An increase in the glutamate efflux from cultured glial cells induced by adenosine Adenosine increased glutamate efflux from hippocampal glial cells in a bell-shaped doseresponsive manner at concentrations ranged from 1 nM to 100 mM, with a maximum at 10 nM, the corresponding value (7.6 nmol/mg protein/30 min) being 15 times of control (nontreatment with adenosine) (Fig. 1A). A similar increase was obtained with the selective A2a adenosine receptor agonist, CGS21680 (1 mM), while the selective A1 adenosine receptor agonist, CHA (10 mM), had no effect (Fig. 1A), suggesting that adenosine stimulates glutamate efflux from glial cells via A2a adenosine receptors. For glial cells from the superior colliculus, adenosine enhanced glutamate efflux in a dose-dependent manner at concentrations ranged from 1 nM to 100 mM, reaching 9.4 nmol/mg protein/30 min (Fig. 1B). CGS21680 (1 mM), but not CHA (10 mM), also enhanced glutamate efflux from superior colliculus glial cells (Fig. 1B), further supporting the idea that A2a adenosine receptors are involved in the glial glutamate efflux. An increase in the glutamate efflux from oocytes expressing glial components derived from the hippocampus and the superior colliculus together with and without A2a adenosine receptors As is the case in cultured glial cells, adenosine increased glutamate efflux from oocytes injected with the poly (A)1 mRNA derived from cultured glial cells for the hippocampus and the superior colliculus (Fig. 2A,B). The increase was reinforced by co-expressing A2a adenosine receptors, reaching 4.5 and 1.7 times of control (non-treatment with adenosine) for the hippocampus, and 7.3 and 9.8 times of control for the superior colliculus, at 10 nM and 100 mM of adenosine, respectively (Fig. 2A,B). Likewise, CGS21680 (1 mM) caused 2.8- and 6.6fold increase in the glutamate efflux for the hippocampus and the superior colliculus, respectively, but CHA (10 mM) had no effect (Fig. 2A,B). In oocytes expressing A2a adenosine re-
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Fig. 1. Adenosine-induced glutamate efflux from cultured glial cells. Adenosine at concentrations as indicated, CHA (10 mM), or CGS21680 (1 mM) was applied to cultured glial cells from the hippocampus (A) and superior colliculus (B), and glutamate released in extracellular medium was assayed. Each value represents the mean glutamate concentration of 5–7 independent experiments and the SEM is indicated by the bars. * P,0.1, ** P,0.01, ANOVA with Fisher’s least-significance difference test.
ceptors alone, however, such increase was not found (Fig. 2C). It is strongly suggested from these results that adenosine stimulates glutamate efflux via A2a adenosine receptors, but that unknown factors or components expressed in glial cells are required for the adenosine action. Discussion Adenosine is shown to induce a facilitation of neurotransmission in the guinea pig hippocampus (# 1 mM) [5] and superior colliculus (. 1 nM) [6]. One thought that the facilitatory action is caused by increasing glutamate from presynaptic terminals, based upon the data that adenosine increased glutamate released from electrically stimulated slices from the guinea pig hippocampus [14] and superior colliculus [15]. Adenosine, however, increased glutamate released from slices also under non-excitable conditions [15], suggesting
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Fig. 2. Adenosine-induced glutamate efflux from oocytes. Oocytes were injected with the poly (A)1 mRNAs derived from cultured glial cells; for the hippocampus together with (A, right panel) and without (A, left panel) the A2a adenosine receptor mRNA; or for the superior colliculus together with (B, right panel) and without (B, left panel) the A2a adenosine receptor mRNA. In C, A2a adenosine receptors alone were expressed in oocytes. Oocytes were incubated in the presence and absence of adenosine (0.01 and 100 mM), CHA (10 mM), or CGS21680 (1 mM), and glutamate released in extracellular medium was assayed. Each value represents the mean glutamate concentration of 8–10 independent experiments and the SEM is indicated by the bars. * P,0.1, ** P,0.01, ANOVA with Fisher’s least-significance difference test.
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that the facilitatory action of adenosine is not caused simply by an increase in the glutamate release from neurons. This, taken together with the result of our preliminary study that adenosine had no effect on the rate of spontaneous miniature excitatory postsynaptic currents in cultured solitary neurons from the rat hippocampus (unpublished data), raises the possibility that adenosine might facilitate neurotransmission in part by stimulating glial glutamate release. In support of this idea, the results of the present study showed that adenosine actually enhances glutamate efflux from cultured glial cells. Interestingly, adenosine dose-responsive relations in the release, i.e., bell-shaped curve for the hippocampus and sigmoid curve for the superior colliculus, are in agreement with the relations in the facilitation of neurotransmissions for each region [5,6]. Then, one would wonder why the dose-dependency of responses to adenosine in the two regions is quite different. Possible explanation is that such difference might be due to the difference either in the amount of adenosine deaminase between the regions, i.e., much higher in the superior colliculus than in the hippocampus, or in the distribution of the relevant receptors, or in the glial subtypes, although glial cells used here are astrocytes. The question is as to which adenosine receptor mediates glial glutamate efflux. In cultured glial cells, the A2a adenosine receptor agonist, CGS21680, but not the A1 adenosine receptor agonist, CHA, increased glutamate efflux in a fashion that mimics the adenosine action. This, in the light of the previous study showing that the facilitatory action of adenosine on hippocampal neurotransmission is likely mediated by A2a adenosine receptors [12], suggests strongly that adenosine might enhance glial glutamate efflux via A2a adenosine receptors. In the Xenopus oocyte expression systems, adenosine or CGS21680 increased glutamate efflux from oocytes co-expressing A2a adenosine receptors and glial components from the hippocampus or the superior colliculus, but such increase was not obtained with oocytes expressing A2a adenosine receptors alone. This may account for glial unknown factors or components as a target of A2a adenosine receptor signal in the glutamate efflux. A2a adenosine receptors are linked to Gs-protein stimulating adenylylcyclase [7], and therefore, the glial efflux observed here may be regulated via a cAMP-dependent protein kinase pathway. Lines of evidence have pointed to major two pathways in the glutamate release; Ca21dependent vesicular release from presynaptic terminals [18, 19] and Ca21-independent, nonvesicular release from neuronal cytoplasmic pools and glial cells, such as reverse transport of glutamate by glutamate transporters [20–23]. Then, one would discuss whether adenosine releases glutamate through reverse transport by glutamate transporters or not. It is recognized that glutamate transporters run backwards under extracellular high K1 concentrations associated with ischemia and anoxia [24]. Adenosine indeed enhances glutamate release from rat cerebral cortex [25] or cultured chick retinal cells after ischemic insult [26], suggesting the implication of glutamate transporters in adenosine-induced glutamate release. However, this is unlikely, since in the present study, an increase in the glutamate efflux induced by adenosine was found under normal conditions. Adenosine, thus, appears to stimulate glutamate efflux from glial cells via an undescribed pathway. To address this pathway, we are carrying out further experiments. In conclusion, the results presented here suggest that adenosine enhances glial glutamate efflux via A2a adenosine receptors, at least in part responsible for the facilitatory action on neurotransmissions, providing a new communication between neurons and glial cells.
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