This proposal examines the requirement for arachidonic acid release in hormone secretion. It will evaluate whether arachidonic acid release is a necessary, second messenger step leading to anterior pituitary hormone secretion. Our preliminary data indicate that arachidonic acid release correlates with prolactin secretion. The hypothesis is the hypothalamic releasing factors induce release of arachidonic acid as a necessary step in the stimulation of pituitary hormone secretion. This hypothesis will be examined first by determining whether arachidoic acid release and the rate of hormone secretion always correlate. For this purpose cells secreting pituitary hormones will be cultured in vitro and incubated with 14C arachidonc acid to incorporate it into membrane phospholipids. A homogeneous cell population will be employed. Secretion will be measured as hormone released into the media, and arachidonic acid release will be quantified by 3 methods. High pressure liquid chromatography (HPLC) will be used to isolate arachidonic acid and TLC for phospholipids before their quantification by LSC. Releasing factors, other secretagogues or phospholipase A2 inhibitors will be used to alter the rate of hormone secretion. The main hypothesis will be tested secondly by investigating the ability of arachidonic acid metabolites to stimulate hormone secretion. Our data indicate that exogenous arachidonic acid stimulates prolactin release. The ability of exogenous arachidonic acid to stimulate pituitary hormone release will be further characterized e.g. dose, time course, interaction with other modulators of secretion such as dopamine, estrogen glucocortcoids. Additionally the metabolism of exogenous C14 arachidonic acid or releasing factor liberated arachidonic acid will be investigated. Metabolites of the lipooxygenase or P450 pathway will be HPLC fractionated and identified by comparison to standard or by mass fragmentography. Additionally this study will characterize the biochemical step leading to arachidonic acid release. These will include the relationship between phosphotidyl inositol turnover, hydrolysis of other phospholipids, calcium mobilization and their effect on arachidonic acid release. This proposal examines a new uncharacterized step by which releasing factors control hormone secretion.
