In¯uence of chemical pollution on brain adenosine receptors of Mugil cephalus G. Giannaccini, L. Betti, M. Dini, P. Giannoni, M. Gori, M.R. Mazzoni, A. Lucacchini* Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie dell'Universita Á di Pisa, Via Bonnano 6, 56100, Pisa, Italy Received 18 February 1998; accepted 8 July 1998 Abstract The aim of this study was to investigate adenosine receptor sites in whole brain membranes of the saltwater teleost ®sh, Mugil cephalus, using the A 1 receptor selective agonist, [ 3 H]- N 6 -cyclohexyladenosine ([ 3 H]-CHA). Speci®c binding was saturable and reversible. A single class of high-anity binding sites was identify, showing K d and B max values of 1.430.26 nM and 69.55.4 fmol/mg protein, respectively. In competition experiments, the following adenosine analogues, CHA, (2-[ p-(2-carboxyethyl)-phe- nylethylamino]-5 0 -N-ethylcarboxamidoadenosine) (CGS21680), 5 0 -(N-cyclopropyl)carboxamidoadenosina (CPCA) and N 6 -[2-(3,5- dimethoxyphenyl)-2-(2methylphenyl)-ethyl]adenosine (DPMA), displaced [ 3 H]-CHA speci®cally bound to brain membranes, revealing that the high-anity binding sites have some pharmacological properties of mammalian A 1 adenosine receptors. Fur- thermore, our ®ndings indicate that in captivity chemical pollution determines a decrease of the K d value corresponding to a 5-fold increase in anity, whereas it does not induce any variation of the receptor density. # 1998 Elsevier Science Ltd. All rights reserved. Keywords: Adenosine receptors; Brain; Chemical pollution; Fish; Mugil cephalus Abbreviations: ADA, adenosine deaminase; CGS21680, (2-[p-(2-carboxyethyl)-phenylethylamino]-5 0 -N-ethylcarboxamidoadenosine); CHA, N 6 - cyclohexyladenosine; R-PIA, N 6 -R-phenylisopropyladenosine; CPCA; 5 0 -(N-cyclopropyl)carboxamidoadenosina; DPMA, N 6 -[2-(3,5-dimethox- yphenyl)-2-(2methylphenyl)-ethyl]adenosine 1. Introduction The purine nucleoside adenosine, acting via speci®c cell surface receptors, modulates several neuronal func- tions in the mammalian central nervous system (London and Wol, 1977; Daly, 1983; Dunwiddie, 1985; Richardson et al., 1987; Fredholm and Dunwiddie, 1988). Adenosine receptors have been classi®ed into A 1 ,A 2a ,A 2b and A 3 subtypes based on the pharmaco- logical pro®le for agonist and antagonist ligands and their eects on intracellular cAMP accumulation (Van Calker et al., 1979; Palmer and Stiles, 1995; Ongini and Fredholm, 1996; Linden, 1994). All these receptor sub- types have the structural features of G-protein coupled receptors. Whereas A 2a and A 2b receptors activate ade- nylyl cyclase, A 1 and A 3 receptors are negatively cou- pled to adenylyl cyclase and also modulate other eector systems (Van Calker et al., 1979; Linden, 1994). In mammalian brain, A 2a receptors are mainly localized in the striatum, while A 1 and A 2b receptors show a more ubiquitous distribution (Bridges and Fredholm, 1988; Jarvis and William, 1989; Parkinson and Fredholm, 1990). Adenosine modulates the neuronal activity both in physiological and pathological conditions mainly through the interaction with these receptor subtypes. The A 3 adenosine receptor subtype has a relatively low expression in the mammalian central nervous system (Linden, 1994). A 1 ,A 2a , and A 2b receptors are blocked by xanthines, while the A 3 receptor appears to be func- tionally insensitive to these compounds (Linden, 1994). The pharmacological characteristics of mammalian A 1 adenosine receptors have been widely investigated using selective radioligands, such as the agonists, [ 3 H]-CHA (Bruns et al., 1980), and [ 3 H]-R-PIA (Schwabe and Trost, 1980), or the antagonist [ 3 H]-DPCPX (Lohse et al., 1987). Mammalian brain A 2a adenosine receptors have been characterized using either the non-selective 0269-7491/98/$Ðsee front matter # 1998 Elsevier Science Ltd. All rights reserved. PII: S0269-7491(98)00131-6 ENVIRONMENTAL POLLUTION Environmental Pollution 103 (1998) 25±30 * Corresponding author. Tel.: +39-50-24092; fax: +39-50-40517; e-mail: lucas@farm.unipi.it.