Simultaneous measurement of hormone release and secretagogue binding by individual pituitary cells.

Abstract

The quantitative relationship between receptor binding and hormone secretion at the single-cell level was investigated in the present study by combining a reverse hemolytic plaque assay for measurement of luteinizing hormone (LH) secretion from individual pituitary cells with an autoradiographic assay of 125I-labeled gonadotropin-releasing hormone (GnRH) agonist binding to the same cells. In the plaque assay, LH secretion induces complement-mediated lysis of the LH-antibody-coated erythrocytes around the gonadotropes, resulting in areas of lysis (plaques). LH release from individual gonadotropes was quantified by comparing radioimmunoassayable LH release to hemolytic area in similarly treated cohort groups of cells; plaque area was linearly related to the amount of LH secreted. Receptor autoradiography was performed using 125I-labeled GnRH-A (a superagonist analog of GnRH) both as the ligand and as the stimulant for LH release in the plaque assay; the developed silver grains appearing over cells in the center of plaques were measured microscopically. The grains appeared to represent specific and high-affinity receptors for GnRH because no pituitary cells other than gonadotropes bound the labeled ligand and grain development was progressively inhibited by coincubation with increasing doses of unlabeled GnRH-A. Despite high correlations between mean grain number and mean plaque area in dose-response curves, the correlation coefficients for these parameters were low (range 0.02-0.38) in the individual cells comprising these groups. We conclude that GnRH receptor number for any individual gonadotrope is a weak determinant of the amount of LH it can secrete; nevertheless, full occupancy of all its GnRH receptors is required for any gonadotrope to reach its full LH-secretory capacity. Apparently the levels of other factors comprising the steps along the secretory pathway determine the secretory capacity of an individual cell.