Volume 140, Issue 2, Pages 618-626 (February 2011) Morphine Induces μ Opioid Receptor Endocytosis in Guinea Pig Enteric Neurons Following Prolonged Receptor Activation  Simona Patierno, Laura Anselmi, Ingrid Jaramillo, David Scott, Rachel Garcia, Catia Sternini  Gastroenterology  Volume 140, Issue 2, Pages 618-626 (February 2011) DOI: 10.1053/j.gastro.2010.11.005 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 μOR immunoreactivity in enteric neurons from animals chronically treated with (A–C) saline (naive) or (D–F) morphine (tolerant). μOR immunoreactivity is predominantly at the cell surface (arrows) of enteric neurons from (A) unstimulated and (B) morphine-stimulated tissue from naive animals. (D) A low level of μOR internalization is observed in unstimulated enteric neurons from animals chronically treated with morphine. μOR internalization is observed in DAMGO-stimulated neurons from (C) saline-treated animals as well as in (E) morphine-stimulated and (F) DAMGO-stimulated neurons from animals chronically treated with morphine. (A, B, and D) Arrows point to μOR immunoreactivity on the cell surface, (C, E, and F) arrows point to examples of μOR immunoreactivity in endosomes inside the cytoplasm. Calibration bars: (A and D) 4 μm; (B, C, E and F) 5 μm. Gastroenterology 2011 140, 618-626DOI: (10.1053/j.gastro.2010.11.005) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Levels of μOR immunoreactivity in the cytoplasm of enteric neurons from animals treated with saline (white bars; naive) and animals chronically exposed to morphine (black bars; tolerant) incubated with medium (Krebs; unstimulated), morphine, or DAMGO. Fluorescence density values are expressed as the mean ± standard error of the mean of measures taken from 10 neurons per experiment (70–80 neurons from 7–8 experiments). **P < .001 vs unstimulated (Krebs) neurons and morphine-stimulated naive neurons. Gastroenterology 2011 140, 618-626DOI: (10.1053/j.gastro.2010.11.005) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 μOR immunoreactivity in neurons from animals chronically treated with morphine that were pretreated in organotypic cultures with TTX and then fixed (A) immediately or (B) after exposure to Krebs, (C) morphine, or (D) DAMGO. μOR is internalized in (C) morphine- and (D) DAMGO-stimulated neurons, but not in (A) control and (B) Krebs treated neurons, indicating that blockade of endogenous transmitter release with TTX does not prevent μOR internalization in vitro. Calibration bars: (A and B) 6 μm; (C and D) 5 μm. Graph on the right shows the quantification of μOR internalization in control (corresponding to panel A), exposure to Krebs solution (corresponding to panel B), and exposure to either (C) morphine or (D) DAMGO in enteric neurons from tolerant animals pretreated with TTX to block transmitter release. **P < .01 compared with control and Krebs. Gastroenterology 2011 140, 618-626DOI: (10.1053/j.gastro.2010.11.005) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 (A–C) Specificity of μOR endocytosis in response to ligand stimulation in tolerant animals. (A) μOR is predominantly at the cell surface in unstimulated neurons, and it remains at the plasma membrane in neurons from tolerant animals, which were treated with the selective μOR antagonist d-Phe-Cys-Tyr-d-Trp-Orn-Yhr-NH2 before exposure to either (B) morphine or (C) DAMGO. (D and E) Effect of morphine on μOR internalization in tolerant neurons in the (D) absence or (E) presence of the dynamin inhibitor, dynasore. (D) μOR internalized in a neuron from a tolerant animal stimulated with morphine. (E) μOR is predominantly at the cell surface in a neuron of the ileum from a tolerant animal stimulated with morphine in the presence of the dynamin inhibitor, dynasore. (D) Arrows point to endosomes, and (E) to the cell membrane. Calibration bars: (A) 4 μm; (B) 6 μm; (C) 5 μm; and (D and E) 10 μm. Gastroenterology 2011 140, 618-626DOI: (10.1053/j.gastro.2010.11.005) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 Quantification of dynamin 2 (dyn) and β-arrestin 1 (β-arr) immunoreactivity in LMMPs of the guinea pig distal ileum from animals treated with saline (white bars; naive) and animals chronically treated with morphine (black bars; chronic) measured by Western blot. Results represent the percentage increase of dynamin or β-arrestin compared with tissue from naive animals. ***P < .001 compared with control (saline). Representative immunoblots are shown on top of the histograms. Gastroenterology 2011 140, 618-626DOI: (10.1053/j.gastro.2010.11.005) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 Distribution of dynamin 2 immunoreactivity in subcellular fractionation of LMMPs of distal ileum from saline-treated (white bars) animals and animals chronically treated with morphine (black bars). *P < .05 and **P < .01 compared with control (saline). Values are expressed as the percentage of dynamin immunoreactivity in control (saline). Gastroenterology 2011 140, 618-626DOI: (10.1053/j.gastro.2010.11.005) Copyright © 2011 AGA Institute Terms and Conditions

Figure 7 Morphine inhibition of electrically induced twitch contractions of LMMPs from guinea pigs chronically treated with saline (open circles) or chronic morphine (black circles). Morphine (1 nmol/L–1 μmol/L) induces a dose-dependent inhibition of muscle twitch. In animals chronically treated with morphine, the muscle twitch inhibitory curve induced by morphine was shifted to the right in a parallel manner without reduction of the maximum response, indicative of tolerance. Gastroenterology 2011 140, 618-626DOI: (10.1053/j.gastro.2010.11.005) Copyright © 2011 AGA Institute Terms and Conditions