However, whether postendocytotic trafficking

of MORs can be modulated by DORs remains to be examined. Furthermore, if DORs and MORs were colocalized in sensory afferent fibers, it would be interesting to learn more explore the physical interaction and functional correlation between these two types of opioid receptors in vivo. The aim of the present study was to investigate the postendocytotic process of the MOR/DOR complex after agonist stimulation and its correlation with the DOR-mediated negative regulation of MOR-mediated spinal analgesia. We found that the activation of DORs in the MOR/DOR complex could target MORs into the postendocytotic degradation pathway, resulting in MOR desensitization. Furthermore, morphine analgesia could be facilitated by disrupting the MOR/DOR interaction with an interfering peptide that corresponds to the first transmembrane domain (TM1)

of MOR fused with the TAT peptide, which is the cell membrane transduction domain of the human immunodeficiency virus and used as a cell-penetrating vector to deliver small cargos or large molecules (Schwarze et al., 1999). Therefore, physical disassociation of MORs from DORs could be a strategy to enhance MOR-mediated analgesia. To assess whether MOR trafficking could be modulated Alisertib cell line by activation of DORs, we examined the distribution and translocation of MORs and DORs Tryptophan synthase in human embryonic kidney 293 (HEK293) cells that were cotransfected with plasmids expressing MOR with an N-terminal hemagglutinin (HA) tag (HA-MOR) and DOR with an N-terminal Myc tag (Myc-DOR). Because HA and Myc were tagged at the N termini of MOR and DOR, respectively, and exposed to the extracellular space following insertion of the receptors into the plasma membrane, HA-MOR and Myc-DOR on the cell surface of living cells could be prelabeled using rabbit anti-HA and mouse anti-Myc antibodies. Under control conditions, the prelabeled DORs and MORs were mainly present on the surface of the double-transfected HEK293 cells (Figure 1A).

Interestingly, after a 30 min treatment with the selective DOR agonists deltorphin (Delt) I, Delt II, or (+)-4-[(αR)-α-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80) (1 μM), the prelabeled DORs and MORs were cointernalized and colocalized in the same vesicular structures (Figure 1A). When DAMGO (1 μM), a selective MOR agonist, was applied for 30 min, the cointernalization of prelabeled MORs and DORs was also observed in the double-transfected HEK293 cells (Figure 1A). The reaction induced by Delt I or DAMGO could be abolished using the DOR antagonist naltrindole (NTI) or the MOR antagonists naloxone and D-Phe-Cys-Tyr-D-Trp-Om-Thr-Pen-Thr-NH2 (CTOP) (Figure 1A), indicating that the receptor cointernalization is induced in a receptor-specific manner.