Our aim is to determine to what extent heterogeneity in responsiveness of individual pituitary LH secreting cells to hypothalamic gonadotropin-releasing hormone (GnRH) is responsible for differences in pituitary responsiveness to GnRH in different physiological states, and to identify the factors involved in determining the responsiveness of individual gonadotropes to GnRH. To accomplish this aim, we have developed a method, the reverse hemolytic plaque assay, that permits detection and measurement of LH secretion from individual gonadotropes. In the reverse hemolytic plaque assay, LH secreting cells in a mixed pituitary cell culture form microscopically identifiable plaques (zones of hemolysis around secreting cells) when incubated as a monolayer with Staphlococcal protein-A-coupled ovine erythrocytes in the presence of LH antiserum and complement. The presence of a plaque surrounding a cell idenfifies it as a gonadotrope and the size of the plaque is related to the amount of LH released from the cell. GnRH causes an increase in both the size and number of plaques in a dose dependent manner. The number of plaque forming cells seen with maximal doses of GnRH corresponds well with the number of gonadotropes in the same cultures determined by immunocytochemistry (ICC). By combining the plaque assay with ICC or autoradiography (using radiolabeled GnRH as a secretagogue) we can correlate the amount of LH secreted by individual cells with GnRH receptor binding or LH content. By employing quantitative ICC, autoradiography or fluorescence with the plaque assay for LH secretion I will test the following specific aims: 1) to study LH secretion by gonadotropes obtained from animals at various physiological states and their heterogeneity in response to GnRH, 2) to determine the contribution of GnRH binding to this responsiveness on individual gonadotropes, 3) to examine the relationship between LH content (ICC) and LH secretion and 4) to investigate the role of intracellular [Ca++[ (Fura-2 fluorescence) in the functional heterogeneity of gonadotropes. The scientific disciplines involved are cell biology, biochemistry, physiology, anatomy and immunology. The findings of these studies will increase our understanding of the role of receptors in the cell function and should contribute to our understanding of receptor related diseases.
