Adenosine A 2A Receptors Regulate the Extracellular Accumulation of Excitatory Amino Acids upon Metabolic Dysfunction in Chick Cultured Retinal Cells ANA CRISTINA REGO a , PAULA AGOSTINHO a , JOANA MELO a , RODRIGO A. CUNHA b AND CATARINA R. OLIVEIRA a * a Center for Neurosciences of Coimbra and Faculty of Medicine, University of Coimbra, 3004-504 Coimbra, Portugal and b Department of Chemistry and Biochemistry, Faculty of Sciences, University of Lisbon, 1749-028 Lisbon, Portugal (Received Cleveland 10 August 1999 and accepted in revised form 9 December 1999) The role of endogenous extracellular adenosine as a tonic modulator of the extracellular accumulation of excitatory amino acids (glutamate and aspartate) caused by metabolic inhibition was investigated in cultured retinal cells. The selective adenosine A 2A receptor antagonist, 4-[2-[7-amino-2-(2-furyl)(1,2,4)- triazin-5-ylamino]-ethyl]phenol (ZM241385) (50 nM), increased the release of glutamate (three- to four- fold) and of aspartate (nearly two-fold) upon iodoacetic acid-induced glycolysis inhibition, in the presence or in the absence of Ca 2 . Blockade of tonic activation of A 2A receptors by ZM241385 also increased (nearly two-fold) the ischemia-induced release of glutamate and aspartate. Furthermore, another selective A 2A receptor antagonist, 5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo[4,3-e]-1,2,4- triazolo[1,5-c]pyrimidine (SCH58261), also increased the release of aspartate and glutamate by about two-fold in cells submitted to glycolysis inhibition. In contrast, the selective adenosine A 1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (100 nM), did not signi®cantly modify the extracellular accumulation of either glutamate or aspartate caused by inducers of chemical ischemia or glycolytic inhibitors. Inhibition of glycolysis also increased (about three-fold) the extracellular accumulation of GABA, which was virtually unchanged by ZM241385. Furthermore, the GABA A receptor antagonist, bicuculline (10 mM), only increased (nearly two-fold) the iodoacetic acid-induced Ca 2 -dependent release of glutamate, whereas the GABA B receptor antagonist, 3-aminopropyl(diethox- ymethyl) phosphinic acid, CGP35348 (100 mM), was devoid of effects on the extracellular accumulation of glutamate and aspartate. These results show that endogenous extracellular adenosine, which rises under conditions of inhibited glycolysis, tonically inhibits the extracellular accumulation of excitatory amino acid through the activation of A 2A , but not A 1 , adenosine receptors, and this effect is independent of GABA A and GABA B functions in the cultured retinal cells. # 2000 Academic Press Key words: aspartate; A 1 adenosine receptors; A 2A adenosine receptors; adenosine; glutamate; metabolic stress; retinal cells. 1. Introduction Extracellular adenosine modulates the function of neuronal cells by acting on G protein-coupled adeno- sine receptors, named A 1 ,A 2A ,A 2B and A 3 receptors (Fredholm et al., 1994). Adenosine decreases neur- onal excitability through activation of A 1 receptors and may also enhance neuronal excitability through activation of high af®nity A 2A receptors (Cunha et al., 1994). The role of low af®nity facilitatory A 2B receptors. for which there is a lack of selective pharmacological tools, and the role of the low af®nity A 3 receptors are not yet clearly understood. The levels of extracellular adenosine increase upon electrical stimulation of brain preparations (e.g. Cunha et al., 1996) and increase even further during graded hypoxia (Fowler, 1993a), glycolysis inhibition (Rego, Santos and Oliveira, 1997) or ischemia (Phillis, Walter and Simpson, 1991; Pedata et al., 1993). Extracellular adenosine ful®lls two parallel roles to modulate cellular excitability (Cunha, 1997): (i) as a homeostatic modulator, a function common to all types of eucaryotic cells, and (ii) as a neuromodulator at the synaptic level, a role which can be part of the homeostatic role of adenosine, but which can also occur independently of changes in neuronal metabolic status (Mitchell, Lupica and Dunwiddie, 1993). It is now established that tonic neuromodulation by endogenous adenosine is a balance between inhibit- ory A 1 and facilitatory A 2A adenosine receptors (Brown et al., 1990; Cunha et al., 1994; Correia-de- Sa Â, Timo Âteo and Ribeiro, 1996). In contrast, whereas the homeostatic role of tonic activation of inhibitory A 1 receptors as a neuroprotective mechanism is well understood (e.g. Rudolphi et al., 1992), the homeo- static role of facilitatory A 2A receptors is not well established. Exp. Eye Res. (2000) 70, 577±587 doi:10.1006/exer.1999.0815, available online at http://www.idealibrary.com on 0014-4835/00/05057711 $35.00/0 # 2000 Academic Press * Address correspondence to: Catarina Resende de Oliveira, Center for Neurosciences of Coimbra, Faculty of Medicine, University of Coimbra, 3004-504 Coimbra, Portugal.