The potent effects of morphine, a plant alkaloid, have long led to the assumption that the brain produces endogenous opioids. Several peptides with opioid properties and varying degrees of selectivity for the delta and kappa receptors have been discovers, whereas endogenous ligands with a clear selectivity for the mu receptor have remained elusive. We have recently isolated from brain two novel peptides, endomorphin-1 and -2, that have high affinity and selectivity for the mu opiate receptor (nature 386:499, 1997). We proposed to characterize the neurobiology of these newly discovered peptides. To determine whether they are produced endogenously from precursor proteins, we will clone and sequence the prohormone precursor(s) of these peptides and analyze the upstream region to identify key regulatory elements. Antibodies directed against each of the peptides have been generated and will be used to determine the distribution of the peptides within the nervous system, a crucial step toward understanding physiological functions of these peptides. Once the sequence of the gene(s) for the peptide is identified, riboprobes for in situ hybridization will be made and used to localize cells expressings the endomorphin genes. The possibility of a unique cell signaling profile will be tested with GTP gamma-S binding to measure capacity to activate G-proteins. Finally, the potential for abuse for the peptides will be tested in behavioral studies of opiate dependence and place preference. We take as our formal hypothesis that endomorphin-1 and -2 are endogenous, selective ligands for the mu receptor.
Our specific aims are: 1. Isolate and sequence the gene(s) for endomorphin-1 and -2. 2. Demonstrate cellular localization of endomorphin protein and mRNA. 3. Determine the cellular signaling profile of the endomorphins. 4. Determine tolerance, dependence, and rewarding properties of endomorphins.
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