Ligand-induced trafficking of G protein-coupled receptors and their interaction with associated proteins are key steps regulating receptor-mediated signal transduction and cellular responsiveness. ? opioid receptor (?OR) mediates the analgesic effects of opiates, potent drugs used for pain control, and their side effects, including opioid bowel syndrome (profound constipation and abdominal pain), and tolerance. We previously showed that a) ?OR endocytoses in enteric neurons in response to endogenously opioids and to most opiates, but not to morphine, b) chronic ?OR activation confers morphine the ability to trigger ?OR endocytosis and induces over expression and translocation of dynamin, an intracellular protein that is important for receptor internalization, c) the magnitude of ?OR endocytosis induced by high efficacy opiates does not change in a condition of opioid tolerance, d) activation of ?OR with an internalizing agonist stimulates mitogen-activated protein kinase (MAPK) phosphorylation in cultured myenteric neurons. This application will test the hypothesis that prolonged ?OR activation induces alterations in the intracellular machinery that regulates agonist-induced ?OR trafficking and post-receptor signaling in enteric neurons, which are possible mechanisms underlying the development of tolerance. Specific Aim 1 will establish which intracellular proteins regulate ?OR trafficking following acute and chronic activation in vitro using neuronal transfection of native cells expressing ?OR and correlate their expression with receptor endocytosis and state of gut tolerance in vivo. Specific Aim 2 will determine the effects of acute and chronic ?OR stimulation on MAPK activation in enteric neurons, the effects of MAPK inhibitors on agonist- induced ?OR desensitization and internalization, and whether chronic opiate treatment resulting in a state of tolerance in the gut induces sustained MAPK activation in intact segments of the ileum. These studies will provide a better knowledge of opiate-induced adaptations in enteric neurons naturally expressing ?OR, which will increase our understanding of opioid bowel syndrome, a condition affecting patients receiving chronic opiates for pain, and tolerance, which hamper the use of these drugs.