Identification of N-arachidonoyl dopamine as a highly biased ligand at cannabinoid CB1 receptors

Background and Purpose N-arachidonyl dopamine (NADA) has been identified as a putative endocannabinoid, but there is little information about which signalling pathways it activates. The purpose of this study was to identify the signalling pathways activated by NADA in vitro. Experimental Approach Human or rat cannabinoid CB₁ receptors were expressed in AtT20, CHO or HEK 293 cells. NADA displacement of radiolabelled cannabinoids, and CB₁ receptor mediated activation of K channels or ERK phosphorylation, release of intracellular calcium ([Ca]_i) and modulation of adenylyl cyclase were measured in addition to NADA effects on CB₁ receptor trafficking. Key Results At concentrations up to 30 μM, NADA failed to activate any signalling pathways via CB₁ receptors, with the exception of mobilization of [Ca]_i. The elevations of [Ca]_i were insensitive to pertussis toxin, and reduced or abolished by blockers of G_q/₁₁-dependent processes including U73122, thapsigargin and a peptide antagonist of G_q/₁₁ activation. Prolonged NADA incubation produced modest loss of cell surface CB₁ receptors. The prototypical cannabinoid agonist CP55940 signalled as expected in all assays. Conclusions and Implications NADA is an ineffective agonist at most canonical cannabinoid receptor signalling pathways, but did promote mobilization of [Ca]_i via G_q-dependent processes and some CB₁ receptor trafficking. This signalling profile is distinct from that of any known cannabinoid, and suggests that NADA may have a unique spectrum of effects in vivo. Our results also indicate that it may be possible to identify highly biased CB₁ receptor ligands displaying a subset of the pharmacological or therapeutic effects usually attributed to CB₁ ligands.