Figure 1.
μOR immunoreactivity in naïve enteric neurons (A–C) and in neurons chronically treated with morphine (D–F).
μOR immunoreactivity is at the cell surface in unstimulated and morphine-stimulated neurons (A, C arrows), and it is in the cytosol following stimulation with DAMGO (B) in naïve enteric neurons. μOR immunoreactivity is at the cell surface in unstimulated neurons (D, arrows), but in the cytosol following DAMGO or morphine stimulation (E, F) in chronic enteric neurons.
Figure 2.
Opioid-induced MAPK activation in naïve (A) and chronically treated (B) enteric neurons.
DAMGO (1 µM, black bars) induced a transient MAPK/ERK1/2 activation in naïve (A) and chronic (B) neurons at 5 and 10 minutes, whereas morphine (grey bars) induced MAPK/ERK1/2 activation only in chronic (B) neurons. **p<0.01 compared to controls (white bars). N = 4–7 experiments in triplicate. Representative gels of pERK1/2 and tERK are shown at the bottom of each graph. tERK was used to verify that the treatment did not affect the total level of this protein and to confirm equal gel loading.
Figure 3.
Characterization of opioid-induced MAPK pathway in enteric neurons.
DAMGO-induced MAPK activation in naive (A, C, E) and DAMGO- and morphine-activation of MAPK in chronic (B, D, F) enteric neurons. Naloxone (A, B), MEK1/2 inhibitor, U0126 (C, D) and hypertonic sucrose (E, F) prevented opioid induced MAPK activation in naïve (A, C, E) and chronic (B, D, F) neurons. **p<0.01 significantly different from control. N = 4–7 experiments in triplicate per group.
Figure 4.
Role of dynamin on MAPK activation in enteric neurons.
DAMGO-induced MAPK in naïve neurons is inhibited by dynasore, a dynamin inhibitor (A) and it is not observed in neurons transfected with mutated K44E dynamin (B). Both DAMGO- and morphine-induced MAPK activation in chronic neurons is blocked by dynasore (D) and is not detected in neurons transfected with mutated dynamin (E). C shows the increased expression of dynamin (dyn) immunoreactivity in enteric neurons transfected with wild type (WT) or mutated dynamin, confirming effectiveness of neuronal transfection. **p<0.01 compared to controls; n = 5–7 performed in triplicate per group. Representative immunoblots of dynamin immunoreactivities are shown at the bottom of histogram in C. GAPDH served as housekeeping protein to verify that the same amount of proteins was loaded on each gel.
Figure 5.
Desensitization of μOR signaling in enteric neurons.
A: Single exposure to DAMGO (1 µM, 5 min) induced significant MAPK activation in naïve enteric neurons, whereas a second exposure to the same DAMGO dose following 2 hours DAMGO pretreatment abolished DAMGO-mediated MAPK response, indicating desensitization. B: Single exposure to DAMGO (1 µM) or morphine (10 µM) activated MAPK in chronic neurons. A second exposure to the same dose of DAMGO or morphine following 2 hours DAMGO or morphine pretreatment induced the same effect in chronic neurons as single exposures, indicating suppression of desensitization. (** p<0.01 vs. control in A and B). C and D: DAMGO and morphine inhibit forskolin-stimulated cAMP in naïve (C) and chronic (D) enteric neurons. This effect was not observed in naïve enteric neurons (C) with a second opioid stimulation following a prior 2 hour exposure, indicative of desensitization. D: Note the over 2 fold increase in cAMP in unstimulated chronic neurons (cAMP superactivation or “overshooting”) vs. naïve control; DAMGO and morphine inhibition of cAMP was not prevented by 2 hours DAMGO or morphine pretreatment in chronic neurons, indicating suppression of desensitization. **p<0.01 vs. controls. N = 5–7 experiments performed in duplicate per group.
Figure 6.
Effect of opioids on CREB phosphorylation in enteric neurons.
Naïve (A) and chronic (B) neurons were stimulated with 1 µM DAMGO, 10 µM morphine or medium (control) for 0–20 minutes. DAMGO and morphine induced a significant, transient CREB activation in chronic, but not naïve enteric neurons. CREB phosphorylation in chronic neurons was blocked by the MEK1/2 inhibitor (U0126) treatment. *p<0.05 and **p<0.01 vs. control; n = 4–7 experiments in triplicate per group. Representative gels of pCREB and CREB are shown at the bottom of the figure. Total CREB was used to verify that the treatment did not affect the total level of this protein and to confirm equal gel loading.
Figure 7.
Effect of MEK1/2 inhibitor (U0126) on GI transit delay induced by chronic morphine in rats.
Chronic morphine (MORPH) significantly delayed GI transit (reduction of geometric center) (**p<0.01 vs. control). MEK1/2 inhibitor alone (U0126) did not affect transit. Administration of MEK1/2 inhibitor together with morphine (MORPH+ U0126) reversed the delay in GI transit (**p<0.01 vs. MORPH). N = 5 per group.