title: An optimized approach to study endocannabinoid signaling: evidence against constitutive activity of rat brain adenosine A1 and cannabinoid CB1 receptors.
abstract: 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]GTPgammaS 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. Unlike lipophilic A1 receptor antagonists (potency order DPCPX>>N-0840 approximately cirsimarin>caffeine), 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. SR141716 and AM251, at concentrations fully effective in reversing CB1-mediated responses (10-6 m), did not reduce basal G-protein activity, indicating that CB1 receptors are not constitutively active in these preparations.4 At higher concentrations (1-2.5 x 10-5 m), both antagonists reduced basal G-protein activity in control and ADA-treated membranes, but had no effect when A1 receptor signaling was blocked with DPCPX. Moreover, the CB1 antagonists right-shifted A1 agonist dose-response curves without affecting maximal responses, suggesting competitive mode of antagonist action. The CB1 antagonists did not affect muscarinic acetylcholine or GABAB receptor signaling. When further optimizing G-protein activation assay for the labile endocannabinoid 2-arachidonoylglycerol (2-AG), we show, by using HPLC, that pretreatment of cerebellar membranes with methyl arachidonoyl fluorophosphonate (MAFP) fully prevented enzymatic degradation of 2-AG and concomitantly enhanced the potency of 2-AG. In contrast to previous claims, MAFP exhibited no antagonist activity at the CB1 receptor.6 The findings establish an optimized method with improved signal-to-noise ratio to assess endocannabinoid-dependent G-protein activity in brain membranes, under assay conditions where basal adenosinergic tone and enzymatic degradation of 2-AG are fully eliminated.
authors: Department of Physiology, University of Kuopio, PO Box 1627, Kuopio FIN-70211, Finland.
authors: Juha R
authors: JR
authors: Savinainen
authors: Susanna M
authors: SM
authors: Saario
authors: Riku
authors: R
authors: Niemi
authors: Tomi
authors: T
authors: Järvinen
authors: Jarmo T
authors: JT
authors: Laitinen
mesh_terms: D058916:Adenosine A1 Receptor Antagonists; D000243:Adenosine Deaminase / Q000494:pharmacology; D000818:Animals; D001095:Arachidonic Acids / Q000494:pharmacology; D002531:Cerebellum / Q000187:drug effects / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D063065:Organophosphonates / Q000494:pharmacology; D010880:Piperidines / Q000008:administration & dosage / Q000494:pharmacology; D011720:Pyrazoles / Q000008:administration & dosage / Q000494:pharmacology; D011869:Radioligand Assay; D051381:Rats; D017208:Rats, Wistar; D043682:Receptor, Adenosine A1 / Q000378:metabolism*; D043884:Receptor, Cannabinoid, CB1 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000077285:Rimonabant; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: D058916:Adenosine A1 Receptor Antagonists; D001095:Arachidonic Acids; D063065:Organophosphonates; D010880:Piperidines; D011720:Pyrazoles; D043682:Receptor, Adenosine A1; D043884:Receptor, Cannabinoid, CB1; C104810:methyl arachidonylfluorophosphonate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C103505:AM 251; D000243:Adenosine Deaminase; D000077285:Rimonabant