Recently, the endogenous peptide ligand for an opioid-like g protein-coupled receptor (called LC 132 or ORL-10 has been identified and named orphanin FQ [OFQ] or nociceptin. The cDNA has been cloned and initial observations suggest that OFQ is contained in a larger precursor molecule as ae the enkephalins, endorphins and dynorphins. There appears to be several other putative peptides contained in the OFQ precursor, all bounded by paired-basic amino acids, a motif shared by many prohomones. We hypothesize that several other biologically active peptides are derived from the OFQ precursor and that the release of these peptides, including OFQ are regulated by dopamine and morphine. In order to test these hypotheses we will: 1) Determine the processing of prepro(PP)- OFQ-derived peptides in the rodent hypothalamus. These studies will characterize pp-OFQ-derived peptide liberated from the precusor and provide evidence that the peptides other than OFQ that are predicted from the cDNA are produced in hypothalamic neurons. 2) characterize the regulation of OFQ in monolayer cultures of hypothalamic neurons. Monolayer cultures of rat hypothalamic neurons will be incubated in the presence of ppiate receptor agonists or dopamine and the culture medium assayed for OFQ immunoreactivity. We will also continue to develop radioimmunoassays (RIAs) for each of the other neuropeptides potentially derived from pp-OfQ and use these assays to monitor the regulation of their secretion by dopamine and drugs of abuse. 3) Determien of glucocorticoids regulate pp-OFQ mRNA and peptide levels in vivo and ni vitro. We hypothesisze that OFQ-derived peptides are majormodulators of the stress response. We will use transgenic mie that have negligible circulating glucosteroids and subject them to acute and chronic restraint-stress. Hypothalamic OFQ peptide levlels will be measured by RIA and OFQ mRNA levels using molecular billogical methods. These studies will reveal whether OFQ levels are regulated by glucosteroids in the intact animal and thus potentially responsive to stress paradigms.
