Pharmacological characterization of AR-M1000390 at human delta opioid receptors

We investigated the pharmacological properties of a newly synthesised delta agonist AR-M1000390, derived from SNC-80 ((+)-4-[(alpha R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethyl-benzamide), in the neuroblastoma cell line SK-N-BE expressing only human delta-opioid receptors. Binding and functional experiments showed a weak affinity (K(i) = 106 +/- 34 nM) correlated with a weak potency (EC(50) = 111 +/- 31 nM) to inhibit the forskolin-stimulated cAMP accumulation. Sustained activation of opioid receptors in the presence of the maximal inhibitory concentration of AR-M1000390 produced a rapid and strong desensitization. In order to examine the contribution of internalization and down-regulation in the desensitization processes, binding and functional experiments were conducted in the presence or in the absence of hypertonic sucrose solution to block clathrin-dependent opioid receptor endocytosis. We observed both the inability of AR-M1000390 to down-regulate opioid receptors and the absence of any effect of sucrose on desensitization. The lack of delta-opioid receptor internalization by AR-M1000390 was further corroborated by confocal microscopy using antibodies directed either against the endogenous delta-opioid receptors or the FLAG-tagged delta-opioid receptors stably expressed in the SK-N-BE cells. These data suggest that uncoupling rather than internalization is responsible for delta-opioid receptors desensitization by AR-M1000390.