Activation of Adenosine A 1 and A 2A Receptors Modulates Dopamine D 2 Receptor-Induced Responses in Stably Transfected Human Neuroblastoma Cells *²Hassan Salim, ‡Sergi Ferre ´, ²Abderrahim Dalal, §Robert A. Peterfreund, ‡Kjell Fuxe, *Jean-Didier Vincent, and *Pierre-Marie Lledo *CNRS, Institut Alfred Fessard, Gif-sur-Yvette, France; ² De ´partement de Biologie, Faculte ´ des Sciences, Universite ´ Cadi Ayyad, Marrakech, Morocco; ‡Department of Neuroscience, Division of Cellular and Molecular Neurochemistry, Karolinska Institute, Stockholm, Sweden; Department of Neurochemistry, I.I.B.B., C.S.I.C., Barcelona, Spain; and §Department of Anesthesia and Critical Care Clinics 3, Massachusetts General Hospital, Boston, Massachusetts, U.S.A. Abstract: Adenosine can influence dopaminergic neuro- transmission in the basal ganglia via postsynaptic inter- action between adenosine A 2A and dopamine D 2 recep- tors. We have used a human neuroblastoma cell line (SH-SY5Y) that was found to express constitutively mod- erate levels of adenosine A 1 and A 2A receptors (100 fmol/mg of protein) to investigate the interactions of A 2A /D 2 receptors, at a cellular level. After transfection with human D 2L receptor cDNA, SH-SY5Y cells ex- pressed between 500 and 1,100 fmol of D 2 receptors/mg of protein. In membrane preparations, stimulation of adenosine A 2A receptors decreased the affinity of dopa- mine D 2 receptors for dopamine. In intact cells, the cal- cium concentration elevation induced by KCl treatment was moderate, and dopamine had no effect on either resting intracellular free Ca 2+ concentration ([Ca 2+ ] i ) or KCl-induced responses. In contrast, pretreatment with adenosine deaminase for 2 days dramatically increased the elevation of [Ca 2+ ] i evoked by KCl, which then was totally reversed by dopamine. The effects induced by 48-h adenosine inactivation were mimicked by applica- tion of adenosine A 1 antagonists and could not be further reversed by acute activation of either A 1 or A 2A receptors. Acute application of the selective A 2 receptor agonist CGS-21680 counteracted the D 2 receptor-induced [Ca 2+ ] i responses. The present study shows that SH- SY5Y cells are endowed with functional adenosine A 2A and A 1 receptors and that A 2A receptors exert an antag- onistic acute effect on dopamine D 2 receptor-mediated functions. In contrast, A 1 receptors induce a tonic mod- ulatory role on these dopamine functions. Key Words: Intracellular calcium—Adenosine receptors—Dopamine receptors—Stable transfection—Basal ganglia—Adeno- sine deaminase. J. Neurochem. 74, 432– 439 (2000). The purine nucleoside adenosine has been proposed to act as a neuromodulator in the CNS (Fredholm et al., 1993). Adenosine exerts its effects on neuronal activity via four G protein-coupled receptors: A 1 ,A 2A ,A 2B , and A 3 (for review, see Fredholm et al., 1994). Although adenosine A 1 and A 2B receptors are widely distributed in the brain, A 2A receptor distribution is restricted to do- pamine-innervated regions such as the striatum, nucleus accumbens, and olfactory tubercule (Alexander and Red- dington, 1989; Dixon et al., 1996; Ferre ´ et al., 1997). From these anatomical data, it was proposed that dopa- mine and adenosine receptors may interact in multiple brain functions (for review, see Ferre ´ et al., 1997). In agreement with this hypothesis, large numbers of studies have demonstrated that adenosine can interact with do- paminergic neurotransmission in the basal ganglia. It was suggested that adenosine receptor agonists inhibit, whereas antagonists potentiate, the function of dopamine in this region (Ferre ´ et al., 1992). Dopamine receptors are subdivided into D 1 -like (D 1 and D 5 ) and D 2 -like (D 2S ,D 2L ,D 3 , and D 4 ) receptors (for reviews, see Gingrich and Caron, 1993; Cardinaud et al., 1998; Missale et al., 1998). In striatopallidal neurons, the antagonistic adenosine– dopamine interaction seems to be mediated by postsynaptic A 2A receptors colocalized with D 2 dopamine receptors (Schiffmann et al., 1991; Ferre ´ et al., 1993a). Behavioral studies have shown that A 2A receptor stimulation inhibits D 2 -mediated activation in mice and that D 2 receptor stimulation inhibits A 2A - Received August 11, 1999; revised manuscript received September 20, 1999; accepted September 20, 1999. Address correspondence and reprint requests to Dr. P.-M. Lledo at CNRS, Institut Alfred Fessard, Avenue de la Terrasse, 91198 Gif-sur- Yvette Cedex, France. E-mail: lledo@iaf.cnrs-gif.fr Abbreviations used: ADA, adenosine deaminase; [Ca 2+ ] i , intracel- lular free Ca 2+ concentration; CGS-21680, 2-p-(2-carboxyethyl)phen- ethylamino-5'-N-ethylcarboxyamidoadenosine; CPT, 8-cyclopentyl- 1,3-dimethylxanthine; DPCPX, 1,3-dipropyl-8-cyclopentylxanthine; R- PIA, N 6 -(R)-(2-phenylisopropyl)adenosine. 432 Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 2000 International Society for Neurochemistry