544 Uroguanylin-GUCY2C Brain Gut Axis Regulating Feeding Behavior Is Mediated by Nodose Ganglia HCN2 Ion Channel

Abstract

Opiates differ in their ability to trigger μOR endocytosis and to promote physiological tolerance, which involve changes at the receptor levels and downstream of the receptor. The aims of this study were to test whether agonists with different internalizing abilities activate MAPK, a μOR downstream signaling, in enteric neurons, and the effect of chronic morphine treatment on opioid-induced activation of MAPK. Cultured rat myenteric neurons were incubated with 1 μM DAMGO, a potent μOR-internalizing agonist or 10 μM morphine, a poor μOR-internalizing agonist for 0'-30' at 37oC. MAPK or extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation was determined byWestern Blot using membranes from cell lysates and the phospho-p44/p42 MAPK and total ERK1/2 antibodies. Specificity controls included pre-incubation with the opioid receptor antagonist, naloxone (10 μM), tetrodotoxin (TTX; 10μM) to block neurotransmitter release or the MAPK inhibitor, U0126 (10 μM). To determine whether opioid-induced MAPK requires receptor internalization and the role of the intracellular trafficking protein, dynamin, myenteric neurons were treated with hypertonic sucrose solution (0.5 M) to block endocytosis, with the dynamin inhibitor, dynasore (10μM) prior to opioid stimulation, or transfected with a wild-type dynamin or a mutated-dynamin using lentiviral vectors. There was a significant increase in MAPK phosphorylation in neurons exposed to DAMGO at 5' and 10', which returned to control levels at 20', while morphine did not induce MAPK activation. DAMGO-induced MAPK phosphorylation was blocked by naloxone and MAPK inhibitor U0126, and by sucrose, dynasore and mutated-dynamin transfection. TTX did not prevent MAPK phosphorylation. To determine the effect of chronic μOR activation on opioid-induced MAPK phosphorylation, cells were treated with 10 μM morphine for 4 days before exposure to DAMGO or morphine. Both DAMGO and morphine acute stimulation induced MAPK activation in enteric neurons chronically treated with morphine, with a peak at 5' and 10' returning to control levels at 20' as in normal cells. Stimulation with DAMGObut not morphine induces μOR internalization in Naive myenteric neurons, whereas both DAMGO and morphine stimulation trigger μOR internalization in neurons chronically treated with morphine. This study shows that opioid stimulation induces a rapid and transient activation of MAPK/ERK in enteric neurons, which is ligand dependent and mediated by direct interaction of the ligand with μOR. Opioid stimulation of MAPK/ERK pathway in enteric neurons requires μOR internalization and is dynamin-dependent. This suggests that receptor endocytosis regulates MAPK, which in turn could trigger feedback signaling affecting cellular response and adaptations, mechanisms underlying the development of chronic opiate adverse effects.