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 δ-opioid receptor (hDOR) produced by endogenous opioid peptides Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) 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 [(+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide). 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.
Footnotes
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DOI: 10.1124/jpet.103.063958.
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ABBREVIATIONS: GPCR, G protein-coupled receptor; MOR, μ-opioid receptor; DPDPE, [d-Pen2,d-Pen5]-enkephalin; deltorphin I, Tyr-d-Ala-Phe-Asp-Val-Val-Gly-NH2; hDOR, human δ-opioid receptor; Leu-enkephalin, Tyr-Gly-Gly-Phe-Leu; Met-enkephalin, Tyr-Gly-Gly-Phe-Met; SNC-80, (+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; IBMX, 3-isobutyl-1-methylxanthine; FSK, forskolin; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; mDOR, mouse δ-opioid receptor; ANOVA, analysis of variance; NTI, naltrindole; RB3020, 2-(3-[(1-amino-ethyl)-hydroxy-phosphinyl]-2-biphenyl-4-ylmethyl-propionylamino)-propionic acid; RB101, N-{(R,S)-2-benzyl-3[(S)(2-amino-4-methylthio)butyl dithio]-1-oxopropyl}-l-phenylalanine benzyl ester.
- Received December 8, 2003.
- Accepted April 12, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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