Activation of the luteinizing hormone-releasing hormone (LHRH) receptor by its agonist triggers multiple signaling pathways and an array of responses. The immediate event is an explosive exocytosis of LH and involves postranslational protein modifications. Delayed responses to LHRH are manifest within one to several hours and, in some ways, prepare the gonadotrope for subsequent LHRH challenges. LHRH self-priming is an example of a delayed response; it provides for LHRH signal amplification and as such is a critical component of the preovulatory LH surge. LHRH self-potentiation shares strikingly similar characteristics with the augmentation of LHRH-stimulated LH secretion by either cAMP or acute progesterone exposure (neither of which can act acutely as a LH secretagogue). Our observations have led us to hypothesize that a signaling pathway stimulated by the LHRH receptor leads to ligand- independent activation of the progesterone receptor and that this mechanism is part of the normal, physiological operation of the gonadotrope. The goal of the project is the delineation of the LHRH self-priming pathway and its intersection with the progesterone receptor. The specific aims are: 1) To assess the transcription activation function of the progesterone receptor in response to stimulation at various points in the post-LHRH receptor pathway in primary cultured gonadotropes and in a gonadotrope cell line. The target of the experimental manipulations will be the PR normally expressed in the gonadotrope. 2) To establish a single cell model of gonadotropin secretion that uses changes in membrane capacitance as a real- time electrophysiological measure of exocytosis. This single cell model in combination with microinjection will be used to define the required role for cAMP-dependent protein kinase in LHRH self-priming. Specific targets will be a PKA pathway and a PKC pathway and their intersection with the PR. Cell models to achieve these aims will be cultured rat anterior pituitary cells, gonadotrope-enriched cultures, and a mouse betaT202 clonal gonadotrope cell line.