Ligand-induced receptor endocytosis is one of the key steps regulating receptor-mediated signal transduction. An understanding of opioid receptor (uOR) trafficking is of importance, since uOR mediates many opiate effects, including analgesia, tolerance, respiratory depression and impairment of intestinal transit. The major findings during the previous funding period were that: 1) Ligand-induced uOR endocytosis reduces the neurogenic response of intestinal neuromuscular preparations in which spare uOR receptors have been inactivated, and 2) uOR endocytosis occurs in enteric neurons in response to endogenously released opioids. The hypothesis of the present application is that agonist-induced uOR endocytosis serves as a regulatory mechanism to attenuate neuronal responsiveness. The long-term goals of this program are to elucidate uOR trafficking mechanisms and effects, and the role of uOR in intestinal motility. These studies will use both transfected cells and enteric neurons in organotypic and primary cell cultures. Guinea pigs and mice will serve as animal models. Specific Aim 1 will investigate a) the effect of ligand-induced uOR endocytosis on the nerve-mediated response (cholinergic contraction and non-adrenergic/non-cholinergic relaxation evoked by field stimulation) of longitudinal muscle-myenteric plexus preparations from chronically treated and untreated guinea pigs, and b) the effect of ligands that differ in their ability to induce receptor endocytosis acutely on the cellular distribution of uOR in chronically treated neurons. Specific Aim 2 will examine a) the role of B arrestins, dynamin and rab GTPase in agonist-induced endocytosis and trafficking of uOR in transfected cells, and b) the cellular distribution and expression of B arrestins, dynamin and rab GTPase in enteric neurons following chronic opiate treatment. Specific Aim 3 will use uOR endocytosis to visualize the enteric neuronal circuits activated by endogenous opioids released in response to visceral noxious stimuli and test the hypothesis that opioid release induced by visceral noxious stimuli is mediated by NMDA receptors, which are glutamate, ion-gated receptors. Specific Aim 4 will a) determine the effect of ablation of enteric uORs by using the ribosome-inactivating protein saporin conjugated to dermorphin (a uOR agonist that induces receptor internalization) on intestinal motility; and b) examine the effect of visceral noxious stimuli on intestinal transit in uOR knockout (-/-) and wild type (+1+) mice. These studies will further our understanding of uOR function in the gastrointestinal tract.