The mechanisms underlying narcotic addiction, abstinence and withdrawal are largely unknown. One possible clue to a better understanding of these mechanisms is the recent suggestion that intake of opiods may affect the production, release or activity of the endogenous opiod, beta-endorphin. The major site of production of beta-endorphin is the pituitary. The aim of this proposal is to better understand how synthesis, processing and release of beta-endorphin is regulated in the anterior and intermedate lobes of the pituitary and the hypothalamus and to determine if and how exogenous opioids affect these processes. Primary cell cultures from rat and human pituitary and rat hypothalamus will be used as model systems for these studies. Earlier studies have shown that ACTH and beta-lipotropin are synthesized from a common precursor protein in the pituitary gland. Pulse label and pulse chase techniques of protein labeling will be used to study synthesis and processing of the common precursor to beta-endorphin and ACTH in primary cultures. Release of the ACTH/endorphin peptides will be investigated by prelabeling cells with radioactive amino acids and then purifying and quantifying radioactive hormones released into the culture medium by specific immunoprecipitation and SDS gel electrophoresis techniques. We will then determine the effects of hypothalamic extracts, glucocorticoids, catecholamines, and opioids on synthesis and release of labeled ACTH/endorphin peptides. If we find an effect of opioids on these processes, binding studies will be performed with 3H-labeled opioids in order to detect opioid receptors in corticotrophic cells and membrane preparations from these cells.