Different Regulation of Human δ-Opioid Receptors by SNC-80 [(+)-4-[(αR)-α-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide] and Endogenous Enkephalins

Abstract

Among the different mechanisms underlying opioid tolerance, receptor desensitization would represent a major cellular adaptation process in which the role of receptor internalization is still a matter of debate. In the present study, we examined desensitization of the human ␦-opioid receptor (hDOR) produced by endogenous opioid peptides Leu-enkephalin (Tyr-Gly-Gly-PheLeu) and Met-enkephalin (Tyr-Gly-Gly-Phe-Met), and the contribution of internalization in this process. Results obtained with natural peptides were compared with those produced by a synthetic opioid agonist, SNC-80. After a 30-min treatment, we observed a different regulation of hDOR between agonists. SNC-80 produced a stronger and faster desensitization and was associated with a loss of opioid binding sites by 50%. SNC-80 also caused a marked hDOR down-regulation by 30% as observed by Western blot. Immunocytochemistry revealed that SNC-80 induced a complete redistribution of hDOR from cell surface into intracellular compartments, whereas a partial internalization was visualized upon enkephalin exposure. In constrast, a stronger hDOR recycling and resensitization were measured after enkephalin treatment compared with SNC-80. These data strongly suggested a differential sorting of the internalized receptors caused by enkephalins and SNC-80 that was further confirmed by chloroquine as a lysosomal degradation blocker and monensin as a recycling endosome inhibitor. Finally, by preventing hDOR internalization with 0.5 M sucrose, we demonstrated that hDOR internalization contributes partially to desensitization. In conclusion, hDOR desensitization depends both on its internalization and its sorting either to the recycling pathway or to lysosomes.The three opioid receptors, namely, ␦-, -, and -opioid receptors, are regulated not only by endogenous opioid peptides but also by numerous pharmacologically active drugs, particularly those used in the clinical management of pain (Reisine and Pasternak, 1996). These receptors belong to the superfamily of seven-transmembrane domain G protein-coupled receptors (GPCRs) and are subjected to the regulatory mechanisms described for the ␤2-adrenergic receptor, the prototypic GPCR. Similarly to many GPCRs, a sustained or repeated activation of opioid receptors by agonist results in a reduction of responsiveness known as desensitization that is postulated to underlie opioid tolerance in vivo (Loh et al., 1988). Recently, by using ␤-arrestin 2 knockout mice, Bohn et al. (2000) clearly established a direct link between opioid receptor desensitization and tolerance. Chronic treatment of these transgenic animals promoted neither tolerance to morphine-induced analgesia nor -opioid receptor desensitization evaluated using [35 S]guanosine 5Ј-O-(3-thio)triphosphate binding. Desensitization could result from uncoupling between opioid receptors and G proteins, whereas contribution of a decrease of opioid receptor number is less clear in this process. For example, a prolonged treatment (24 h) of the R1.1 cell line by (trans)...

The selective delta opioid agonist SNC80 enhances amphetamine-mediated efflux of dopamine from rat striatum

“…Alternatively, tolerance may be occurring since the behavioral effects of SNC80 to promote locomotor activity alone, as well as enhance amphetamine-mediated locomotor activity are subject to profound tolerance (Jutkiewicz et al, 2008). The lack of effectiveness of SNC80 after a 60 min perfusion in vitro would agree with the rapid development of desensitization and/or downregulation of the delta opioid receptor (Lecoq et al, 2004).…”

Differential effects of opioid agonists on G protein expression in CHO cells expressing cloned human opioid receptors

“…Because of the role that receptor internalization plays in opioid receptor desensitization and opioid tolerance, studies of biased agonism, with the end-point of receptor internalization, is a subject of contemporary interest. Opioid agonists, for example, differ in their ability to induce receptor internalization of μ (Koch et al, 2005), δ (Lecoq et al, 2004) and κ (Liu-Chen, 2004) opioid receptors. A striking example of this is the recently described μ agonist HERK which, unlike DAMGO and other internalizing μ opioid agonists, does not recruit β-arrestin and promote μ receptor internalization, even in cells that over express β-arrestin and the GPCR kinase, GRK2, (Groer et al, 2007).…”

“…Photolabeling of Gα-subunits with azidoanilido-[α-32 P]GTP showed that constitutively active μ opioid receptors activate individual G proteins differently than those stimulated by DAMGO (Tyr-D-Ala-Gly-N-MePhe-Gly-ol) (Liu et al, 2001) and that peptide and non-peptide μ agonists induce different patterns of μ receptor phosphorylation (Chakrabarti et al, 1998). Moreover, opioid agonists differ in their ability to induce receptor internalization of μ (Koch et al, 2005), δ (Lecoq et al, 2004) and κ (Liu-Chen, 2004) opioid receptors. In this regard, recent studies showed that herkinorin (HERK) ((2S,4aR,6aR,7R,9S,10aS,10bR)-9-(benzoyloxy)-2-(3-furanyl) dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naphtho-[2,1-c]pyran-7-carboxylic acid methyl ester) is a non-nitrogenous neoclerodane diterpene fully efficacious μ agonist (Harding et al, 2005), that, unlike DAMGO, does not promote β-arrestin recruitment and μ receptor internalization, even in cells that over express β-arrestin and the GPCR kinase, GRK2, (Groer et al, 2007).…”

Adenosine A1 receptor agonist N6-cyclohexyl-adenosine induced phosphorylation of delta opioid receptor and desensitization of its signaling

“…After internalization, receptor could either recycle from endosomes to the plasma membrane and reduce desensitization, or degraded in lysosomes to decrease functional receptors on cell membrane and enhance desensitization [23] . In this study, prolonged CHA treatment did not induce DOR down-regulation, which suggests that receptor down-regulation is not a necessary consequence of phosphorylation.…”

“…However, it is [9,13] , and heterologous desensitization was suggested to correlate with heterologous receptor phosphorylation [20] . DOR undergoes down-regulation after chronic selective agonist exposure, for different agonists the magnitude and time-course of receptor down-regulation varied [21][22][23] , nevertheless, all recent studies favor the idea that reduction of active opioid receptors from the cell surface would potentiate their desensitization.…”