Search

Immunohistochemistry and confocal microscopy were used to investigate mu opioid receptor (muOR) internalization in enteric neurons of the guinea-pig ileum following abdominal surgery. The following surgical procedures were performed under halothane or isofluorane anesthesia: a) midline abdominal skin incision, b) laparotomy or c) laparotomy with intestinal manipulation. Gastrointestinal transit was evaluated by using a non-absorbable marker and measuring fecal pellet output. In neurons from normal and control (anesthesia alone) animals, muOR was predominantly at the cell surface. muOR endocytosis following skin incision was not significantly different from controls (21.2+/-3.5% vs. 13.7+/-2.1%, mean+/-S.E.M.), whereas it was significantly increased by laparotomy (46.5+/-6.1%; P<0.01 vs. controls) or laparotomy plus intestinal manipulation (40.5+/-6.1%; P<0.01 vs. controls) 30 min following surgery compared with controls. muOR endocytosis remained elevated at 4 h (38.6+/-1.2%; P<0.01 vs. controls), whereas it was similar to controls at 6 and 12 h (17.5+/-5.8% and 11.2+/-3.0%). muOR endocytosis occurred in cholinergic and nitrergic neurons. Gastrointestinal transit was significantly delayed by laparotomy or laparotomy plus intestinal manipulation (12.8+/-1.2 and 13.8+/-0.6 h vs. 7.0+/-0.5 in controls; P<0.01), but was not significantly changed by skin incision (8.2+/-0.6 h). The findings of the present study support the concept that the noxious stimulation caused by abdominal surgery induces release of endogenous opioids thus resulting in muOR endocytosis in neurochemically distinct enteric neurons. muOR internalization can serve as indirect evidence of opioid release and as a means to visualize neuronal pathways activated by opioids.

Immunohistochemistry and confocal microscopy were used to investigate endocytosis and recycling of the native mu opioid receptor (muOR) in enteric neurons. Isolated segments of the guinea-pig ileum were exposed to increasing concentrations of muOR agonists at 4 degrees C to allow ligand binding and warming to 37 degrees C for 0 min (baseline) to 6 h in ligand-free medium to allow receptor internalization and recycling. The endogenous ligand, [Met]enkephalin, and [D-Ala(2),MePhe(4),Gly-ol(5)] enkephalin (DAMGO), an opioid analog, and the alkaloids, etorphine and fentanyl, induced rapid internalization of muOR immunoreactivity in enteric neurons, whereas morphine did not. muOR internalization was prevented by muOR antagonists. Basal levels of muOR immunoreactivity in the cytoplasm were 10.52+/-2.05%. DAMGO (1 nM-100 microM) induced a concentration-dependent increase of muOR immunofluorescence density in the cytoplasm to a maximum of 84.37+/-2.26%. Translocation of muOR immunoreactivity in the cytoplasm was detected at 2 min, reached the maximum at 15-30 min, remained at similar levels for 2 h, began decreasing at 4 h, and was at baseline values at 6 h. A second exposure to DAMGO (100 nM) following recovery of internalized muOR immunoreactivity at the cell surface induced a translocation of muOR immunoreactivity in the cytoplasm comparable to the one observed following the first exposure (46.89+/-3.11% versus 43.31+/-3.80%). muOR internalization was prevented by hyperosmolar sucrose, phenylarsine oxide or potassium depletion, which inhibit clathrin-mediated endocytosis. muOR recycling was prevented by pre-treatment with bafilomycin A1, an acidotropic agent that inhibits endosomal acidification, but not by the protein synthesis inhibitor, cycloheximide. This study shows that native muOR in enteric neurons undergoes ligand-selective endocytosis, which is primarily clathrin-mediated, and recycles following endosomal acidification. Following recycling, muOR is activated and internalized by DAMGO indicating that recycled receptors are functional.