Influence of chemical pollution on brain adenosine receptors of Mugil cephalus
Introduction
The purine nucleoside adenosine, acting via specific cell surface receptors, modulates several neuronal functions in the mammalian central nervous system (London and Wolff, 1977; Daly, 1983; Dunwiddie, 1985; Richardson et al., 1987; Fredholm and Dunwiddie, 1988). Adenosine receptors have been classified into A1, A2a, A2b and A3 subtypes based on the pharmacological profile for agonist and antagonist ligands and their effects on intracellular cAMP accumulation (Van Calker et al., 1979; Palmer and Stiles, 1995; Ongini and Fredholm, 1996; Linden, 1994). All these receptor subtypes have the structural features of G-protein coupled receptors. Whereas A2a and A2b receptors activate adenylyl cyclase, A1 and A3 receptors are negatively coupled to adenylyl cyclase and also modulate other effector systems (Van Calker et al., 1979; Linden, 1994). In mammalian brain, A2a receptors are mainly localized in the striatum, while A1 and A2b 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 A3 adenosine receptor subtype has a relatively low expression in the mammalian central nervous system (Linden, 1994). A1, A2a, and A2b receptors are blocked by xanthines, while the A3 receptor appears to be functionally insensitive to these compounds (Linden, 1994). The pharmacological characteristics of mammalian A1 adenosine receptors have been widely investigated using selective radioligands, such as the agonists, [3H]-CHA (Bruns et al., 1980), and [3H]-R-PIA (Schwabe and Trost, 1980), or the antagonist [3H]-DPCPX (Lohse et al., 1987). Mammalian brain A2a adenosine receptors have been characterized using either the non-selective agonist, [3H]-NECA (Bruns et al., 1986), or the selective agonist, [3H]-CGS21680 (Jarvis and William, 1989; Hutchison et al., 1989).
The presence of A1 adenosine receptors is also detectable in brain of lower vertebrates including marine teleosts, but not in invertebrate nervous system (Siebenaller and Murray, 1986). Lucchi et al. (1992) have demonstrated the presence of A1 adenosine receptors in brain membranes of the freshwater teleost Carassius auratus (goldfish). Since adenosine can be considered as the ‘prototype neuromodulator’, the evolutionary development of A1 adenosine receptors may be related to neuromodulation of an increasingly complex nervous system. In teleost brain, the presence and physiological role of other adenosine receptor subtypes have not been investigated yet.
In a previous paper, we have defined and characterized a low-affinity binding site for [3H]-CHA in brain of the saltwater teleost, Mullus surmuletus (Giannaccini et al., 1996). This adenosine binding site shows some pharmacological properties of mammalian A1 adenosine receptors.
The aim of the present study was to characterize brain adenosine receptors of the teleost Mugil cephalus and to evaluate the effects of chemical pollution on receptor affinity for the agonist ligand.
Section snippets
Material
[3H]-CHA (specific activity 31.2 Ci/mmol) was obtained from NEN (Boston, MA). Adenosine deaminase, R-PIA and CHA were purchased from Boehringer-Mannheim (Mannheim, Germany). CGS21680, CPCA and DPMA were from Research Biochemicals International (Natik, MA). Other agents and reagents were obtained from commercial sources.
Results
Saturation experiments were carried out using brain membranes obtained from the two fish groups, A and B. Fig. 1A shows a saturation experiment which was carried out using brain membranes prepared from fish of Group A (control). Specific binding was saturable, whereas non-specific binding increased linearly with the concentration of the radiolabeled ligand (data not shown). Analysis of data by a non-linear curve-fitting computerized program (EBDA/LIGAND) revealed that the best fit observed was
Discussion
In this work we have investigated adenosine receptors sites in whole brain membranes of the saltwater fish, Mugil cephalus, using the A1 receptor selective agonist, [3H]-CHA.
The presence of A1 adenosine receptors in the brain of lower vertebrates, such as fresh and salt water teleost fish, has been indicated by radioligand studies (Siebenaller and Murray, 1986; Lucchi et al., 1992). In mammalian species based on biochemical effects, pharmacological properties and cDNA structures, four adenosine
Acknowledgements
We wish to thank Professor R. Packham for his helpful assistance in the language correction of the manuscript.
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