ACTH RELEASE BY DYN A--ROLE OF NMDA RECEPTORS

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DESCRIPTION (Applicant's Abstract): The long term objective of this proposal is to provide an understanding of the cellular mechanisms underlying the non-opioid stimulation of anterior pituitary corticotrophs by dynorphin (Dyn) to release ACTH. The specific aims are: 1) to determine if Dyn acts on anterior pituitary cells, in the absence of hypothalamic releasing factors, to release ACTRh, via non-opioid mechanisms. This will be accomplished by determing ACTH secretion from mouse anterior pituitary tumor cells, AtT-20, in response to Dyn and other mu-, delta-, and kappaid-opioid receptor peptides, and determing the sensitivity of those repsonses to naloxone. 2) to determine the role of NMDA receptors (NMDAR) and Ca2+ flux in Dyn-stimulation of ACTH secretion from AtT-20 cells. Functional and binding studies, as well as Western Blots will be used to determine the presence of NMDAR on pituitary cells. The zeta1 NMDAR subunit will be co-transfected with epsilon1, epsilon2, epsilon3, epsilon4 both transiently, into COS-1 cells, and stably into HEK 293 cells, to make the following combinations of NMDA subunits: Zeta1 epsilon1, zeta1 epsilon2, zeta1 epsilon3, zeta1-epsilon4. These constructs will be characterized for binding by NMDAR ligand and Dyn, and changes in intracellular Ca2+ upon stimulation. 3) to determine at the molecular and structural level the binding site of Dyn on the NMDAR, chimeric NMDAR subunits will be constructed, employing the conservative segement exchange approach, which involves the exchange of selected chemically similar residues on the extracellular domain of one NMDAR subunit with chemically similar residues on another NMDAR subunit. These chimeras will be analyzed for binding and change of intracellular Ca2+ levels in response to Dyn and NMDAR ligands. An understanding of the regulatory control of Dyn in ACTH secretion, and its mechanism of interaction with NMDAR is important since the actions of Dyn at excitatory amino acid and receptors may be therapeutically useful in the treatment of neuroendocrine disorders of alternatively may have the potential for producing adverse effects on neuroendocrine function. In addition, Dyn has been shown to inhibit the development of tolerance to opiates, restore spinal/supraspinal synergism in morphine tolerant mice, and to eliminate the development of sensitization to cocaine. The mechanism of these actions are not known but they are all, at least in part, non-opioid. Thus, the studies may shed new light into the mechanisms behind the role of Dyn for treatment of cocaine and opioid abuse.