Biochemical and pharmacological characterization of adenosine A1 receptors in eel (Anguilla anguilla) brain

Abstract

N6-cyclohexyl[3H]adenosine ([3H]CHA) was used to label adenosine A1 receptors in membranes prepared from male and female eel whole brain. The A1 receptor agonist [3H]CHA bound saturably, reversibly and with high affinity (Kd = 0.91 ± 0.12 nM; Bmax = 120.36 ± 5.2 fmol mg−1 protein). In equilibrium competition experiments, the adenosine agonists and antagonists all displaced [3H]CHA from high-affinity binding sites with the rank order of potency in displacing, characteristics of an A1 adenosine receptor. Mg2+ dramatically increased the affinity of [3H]CHA without modifying the maximal binding capacity. The specific binding was inhibited by guanosine 5′-triphosphate (Ki = 2.54 ± 0.98 μM). The [3H]CHA binding sites are ubiquitously distributed with a maximum in cerebellum and a minimum in olfactory bulb. No difference was observed between male and female brain. In eel brain, synaptosomes (P2), stimulation of adenosine 3′,5′-monophosphate (cyclic AMP) accumulation with 10−5 M forskolin was markedly reduced (45.5%) by treatment with the adenosine A1 receptor agonist CHA (10−4 M), and the reduction was reversed in presence of the selective A1 receptor antagonist 8-cyclopentyltheophylline (10−5 M). In superfused eel cerebellar synaptosomes, K+ stimulated the release of adenosine in a partially Ca2+-dependent manner. The findings, taken together, suggest the hypothesis that adenosine A1 receptors present in eel brain could modulate synaptic transmission, as A1 receptors do in other vertebrates.