The present proposal continues our efforts aimed at understanding the expression, regulation and function of the Beta-Endorphin/ACTH precursor, proopiomelanocortin (POMC) in pituitary and in brain. This precursor gives rise to several biologically important substances including the stress hormone ACTH and the potent opioid analgesic, Beta-Endorphin (BetaE). In the past several years, we have examined the effects of various challenges, particularly stress, on pituitary POMC and have achieved some understanding of the cellular control and regulation of this precursor. We have described the interplay between pre-translational and post-translational mechanisms in maintaining the homeostasis of POMC cells in the anterior and intermediate lobes of that gland. As these mechanisms exhibit different time dependencies, they can lead to the production of unique combinations of peptide end-products as a function of the regulatory history of the POMC cell. This endows the system with a previously unforeseen level of plasticity. We have also begun to examine the pituitary POMC cells in the context of the overall circuit which controls their responsiveness to stress, the limbic-hypothalamo-pituitary adrenal axis. Finally, we have shown that the brain POMC systems shares common regulatory mechanisms with the pituitary, as revealed by mRNA changes following stress, and biosynthetic changes following chronic morphine administration. However, the neuronal regulation of these systems in the context of complex circuits and unknown inputs remains to be fully explored. This proposal builds toward a better understanding of the brain POMC systems (arcuate and NTS) by ascending from a molecular level of discourse, to a more cellular level and finally to the level of brain circuitry. At the molecular level, we shall examine the structural determinants of POMC, which lead to its highly ordered tissue-specific processing, using gene transfer and site-directed mutagenesis. At the cellular level, we shall study, in anterior pituitary cells, the consequences of activation by secretagogues and inhibition by glucocorticoids on various putative transacting factors which lead to changes in POMC transcription and levels of expression. At the integrative level, we shall describe the brain elements of the circuit which controls anterior lobe POMC, and study the coordinate regulation, at the mRNA and peptide level, of the stress- responsive hypothalamic secretagogues, CRH and vasopressin. This knowledge base will serve to guide us in our studies of brain BetaE systems at a cellular level, and in an anatomical context. We shall attempt to understand the dynamics of these systems in biosynthetic terms (mRNA levels, peptide forms, processing enzymes, releasability), starting with stress as a challenge which also engages brain POMC. We shall extend these studies to other challenges, particularly to studying the effects of analgesic electrical stimulation, and chronic administration of opiate agonists. It is hoped that this multipronged approach can yield valuable information on the role of Beta-Endorphin and related peptides in mechanisms of stress, pain and drug addiction.
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