Figure 5

Na ₂ S induces MOR activation and phosphorylation, the recruitment of β arrestin and MOR internalization. Both DAMGO (1 μM) and Na₂S (50 μM) induce MOR activation (panel A and B respectively). Treating SKNMCs with both DAMGO and Na₂S results in MOR phosphorylation that is time-dependent. DAMGO induces MOR phosphorylation at Ser(377) that is maximal at 30 minutes and, similarly, H₂S induces MOR phosphorylation that peaks at 3-6 minutes and persists until 30 minutes (panel C). The total DAMGO-induced and H₂S-induced MOR phosphorylation is unchanged within the duration of the experiment (panel D). Co-immunoprecipitation experiments demonstrate that DAMGO induces the rapid complex between MOR and β arrestin with the peak at 5-15 minutes and, similarly, H₂S induces the co-immunoprecipitation of MOR and β arrestin that peaked at 30 minutes (panel E), indicating that H₂S induces the interaction between β arrestin and MOR. At the cell membrane fractioning experiments, DAMGO (1 μM) causes the disappearance of MOR from the plasma membrane fraction at 5 minutes and this effect is maximal at 60 minutes. At the same time there is a progressive increment of MOR presence in the cytoplasmatic fraction (panel F). After Na₂S (50 μM), MOR disappears from the plasma membrane fraction at 30 minutes with the maximal effect at 60 minutes and, in contemporary, it passes into the cytoplasmatic fraction (panel G). At the confocal microscopy SKNMCs exhibit MOR immunoreactivity predominantly localized at the cell surface in nonstimulated condition (panel H) and it translocates to cytoplasm after activation with DAMGO (panel I), which is known to induce MOR internalization. Na₂S induces a massive translocation of MOR from plasma membrane into the cytoplasm in most neurons (panel L). Data are representative of at least 3 experiments. *p < 0.05 vs control.