An optimized approach to study endocannabinoid signaling: evidence against constitutive activity of rat brain adenosine A1 and cannabinoid CB1 receptors

1 At nanomolar concentrations, SR141716 and AM251 act as specific and selective antagonists of the cannabinoid CB1 receptor. In the micromolar range, these compounds were shown to inhibit basal G-protein activity, and this is often interpreted to implicate constitutive activity of the CB1 receptors in native tissue. We show here, using [35S]GTPgS binding techniques, that micromolar concentrations of SR141716 and AM251 inhibit basal G-protein activity in rat cerebellar membranes, but only in conditions where tonic adenosine A1 receptor signaling is not eliminated. 2 Unlike lipophilic A1 receptor antagonists (potency order DPCPXbN-0840 Ecirsimarin4caf-feine), adenosine deaminase (ADA) was not fully capable in eliminating basal A1 receptor-dependent G-protein activity. Importantly, all antagonists reduced basal signal to the same extent (20%), and the response evoked by the inverse agonist DPCPX was not reversed by the neutral antagonist N-0840. These data indicate that rat brain A1 receptors are not constitutively active, but that an ADA-resistant adenosine pool is responsible for tonic A1 receptor activity in brain membranes. 3 SR141716 and AM251, at concentrations fully effective in reversing CB1-mediated responses (106M), did not reduce basal G-protein activity, indicating that CB1 receptors are not constitutively active in these preparations