In this study, we assessed whether β-endorphin could affect the trafficking properties of MORs using immunocytochemical methods in BHK cells with confocal microscope. Confocal imaging of the BHK cells expressing Venus-fused MOR with β-arrestin-2 revealed that the yellow fluorescence was largely confined to the plasma membrane ( and ). In both the presence and absence of 100 nM β-endorphin, at which concentration there did not cause any internalization of MORs (), cells expressing MORs treated with 10 μM morphine () showed little internalization of MORs, while the cells treated with 100 nM fentanyl (, and ) and 10 μM oxycodone () showed robust internalization of the receptor. These findings were consistent with previous reports that fentanyl and etorphine caused partial internalization, while morphine failed to induce detectable MOR endocytosis (Koch et al., 2005
). We next investigated the resensitization properties of MORs after the washing-out of agonists. In the absence of β-endorphin, internalized MOR returned to the plasma membrane from 90 min after the washing-out of fentanyl (). However, in the presence of β-endorphin, the internalized MOR induced by fentanyl remained in the cytosolic fraction at 3–6 h after the washing-out of β-endorphin and fentanyl (). However, in both the presence and absence of β-endorphin, the internalized MOR induced by oxycodone returned to the plasma membrane after the washing-out of agonist in a time-dependent manner (). We performed quantitative analysis of the agonist-induced internalization of MORs after the washing-out of each agonist shown in Materials and Methods. At 30 min after the washing-out of agonists, cells treated with fentanyl or oxycodone showed robust internalization of MORs (fentanyl: 79.0 ± 5.14%, β-endorphin fentanyl: 80.2 ± 3.7%, oxycodone: 70.5 ± 7.09%, β-endorphin oxycodone: 70.7 ± 5.35%), which was not seen in morphine-treated cells (morphine: 19.67 ± 3.93%, β-endorphin morphine: 21.5 ± 4.76%; ). However, while there was no difference in the degree of oxycodone-induced MOR internalization between the presence and absence of β-endorphin 3 h after washing-out (oxycodone: 23.17 ± 5.12%, β-endorphin oxycodone: 30.5 ± 4.72%), in fentanyl-treated cells, β-endorphin caused the prolonged internalization of MORs and fluorescence was stayed in the cytosolic fraction (fentanyl: 27.67 ± 5.47%, β-endorphin fentanyl: 76.5 ± 6.02%; ).
Fig. 1 Confocal imaging of agonist-induced internalization of MORs in BHK cells expressing Venus-fused MORs. The cells were incubated in the absence (A, C, E, and G) or presence (B, D, F, and H) of 100 nM β-endorphin (β-END) for 30 min at 37°C (more ...)
Fig. 2 Confocal imaging of agonist-induced internalization of MORs in BHK cells expressing Venus-fused MORs. Typical cells where most of MOR-Venus intensity was at the plasma membranes, [A, control cells (Control)] or in the cytosolic fraction [B, 100 nM fentanyl-stimurated (more ...)
Fig. 3 Confocal imaging of resensitization of MORs in BHK cells expressing Venus-fused MORs. Cells were incubated with 100 nM fentanyl (A–H) or 10 μM oxycodone (I–P) in the absence (A-D and I-L) or presence (E-H and M-P) of β-endorphin, (more ...)
It has been widely accepted that receptor desensitization, internalization and trafficking appear to play a key role in the development of opioid tolerance (Claing et al., 2002
; Gainetdinov et al., 2004
). The initial process in these events is the phosphorylation of intracellular domains of MOR. Phosphorylated MORs are mostly internalized via clathrin-coated pits into early endosomes and subsequently dephosphorylated by intracellular protein phosphatases. The dephosphorylated MORs might either be recycled to the plasma membrane or transported to lysosomes for degradation. A growing body (Smalheiser and Lugli) of evidence suggests that among diverse serine/threonine (Thr) residues of the intracellular domain of MOR, the phosphorylation of Ser 375 in the mouse MOR is essential for the internalization of MORs (Schulz et al., 2004
). In a previous study, we found that repeated treatment with fentanyl, but not morphine, resulted in an increase in the levels of phosphorylated-MOR (Ser 375) associated with the enhanced inactivation of protein phosphatase 2A and a reduction in Rab4-dependent MOR resensitization in the spinal cord of mice that showed inflammatory pain (Imai et al., 2006
). However, several lines of evidence indicate that, in response to pain stimulus, endogenous β-endorphin is released within some brain regions (Zubieta et al., 2001
). We previously reported that β-endorphin released in the ventral tegmental area is a key factor in regulating the dysfunction of MOR to negatively modulate opioid reward under a neuropathic pain-like state (Niikura et al., 2008
). Taken together, although further studies are still needed, these findings support the idea that inhibition of the resensitization system of MOR following chronic treatment with fentanyl in the presence of β-endorphin may be associated with antihyperalgesic tolerance to fentanyl under a chronic pain-like state.
In conclusion, we demonstrated here that unlikely morphine, either fentanyl or oxycodone induced a robust MOR internalization and, in turn, its resensitization. In the presence of β-endorphin, the internalized MOR induced by fentanyl, but not oxycodone, remained within the cytosolic fraction even after washing out. These findings strongly support that idea that fentanyl has different pharmacological profile form that of morphine or oxycodone.