In this project, the PI proposes a series of experiments to determine whether dynorphin and related peptides, acting through a mechanism independent of the protein-coupled opioid receptors, synergize with glucose to stimulate insulin secretion from beta cells of the pancreatic islets of Langerhans. The following hypothesis will be tested. Dynorphin sensitizes beta cells to glucose-induced insulin secretion by activating (or prolonging the activation of) N-methyl-D-aspartate (NMDA)-selective excitatory ionotropic glutamate receptors. Dynorphin A is a member of the family of opioid peptides that appear to act primarily as neuromodulators by interacting with G protein-coupled receptors (GPCRs); mu and kappa and NMDA receptors are members of a class of ligand-gated ion channels that when activated increase the permeability of the cell surface membrane to Ca2+ (and Na+ and K+) and thereby elevate cytoplasmic free Ca 2+ concentration. Elevations in cytoplasmic free C2+ will in turn sensitize the cell to stimulation by glucose and couple stimulation to insulin secretion. The Specific Aims that will be pursued are: 1) To determine whether dynorphin and related peptides synergize with glucose stimulation of insulin secretion by signaling via NMDA receptors. The PI will employ a mouse insulinoma cell line, MIN6 to study binding and signaling characteristics of endogenously expressed NMDA receptors in insulin- secreting cells and compare those findings with observations made in human embryonic kidney cells (HEK 293 cells) and monkey kidney COS-1 cells expressing NMDA receptors comprised of specific subunits by gene transfer. 2) To determine which subunits form dynorphin-binding NMDA receptors so as to begin to delineate the domain(s) on the subunit(s) that directly bind dynorphin and related peptides. The experiments involving expression of NMDA receptor subunits will be performed in transfected HEK 293 cells and COS-1 cells in which the receptors can be expressed to high levels. 3) To determine the pharmacophore within the dynorphin peptide. That is, to determine the smallest peptide that retains the NMDA receptor-binding and insulin secretagogue characteristics of Dyn A(1-17). These experiments will be performed in MIN6, HEK 293 and COS-1 cells. If the dynorphin-NMDA receptor-calcium pathway were shown to sensitize beta cells to glucose-induced insulin secretion, a long-term goal of this research will be to develop nonpeptidic, orally active drugs that can be used to treat diabetes mellitus in humans.