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 l251-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 1251-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 (t) no pituitary cells other than gonadotropes bound the labeled ligand and (ii) 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 doseresponse 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.The quantitative relationship between receptor binding and hormone secretion has been difficult to establish at the single-cell level because of the presence of "spare receptors" on cells and because of the absence of methods permitting measurement of secretion by individual cells. In the present study, we combined a reverse hemolytic plaque assay for measurement of luteinizing hormone (LH) secretion from individual pituitary cells (1, 2) with an autoradiographic assay of 1251I-labeled gonadotropin-releasing hormone (GnRH) agonist binding to the same cells under conditions in which the spare receptors had been removed by trypsin treatment.The hypothalamic decapeptide GnRH is the key neural regulator of the reproductive process of mammals (3, 4). It is synthesized by hypothalamic neurons and reaches the anterior lobe of the pituitary gland after secretion into the hypothalamohypophyseal portal circulation, the blood vessels comprising a direct link between the hypothalamus and pituitary gland. There, GnRH stimulates the release of LH from the gonadotropes (comprising about 5% of the...

Proestrous Surge of Gonadotropin-Releasing Hormone Secretion Inhibits Apoptosis of Anterior Pituitary Cells in Cycling Female Rats

“…Our finding that Bax is expressed predominantly in the gonadotrope combined with the fact that the gonadotrope is the primary pituitary target of GnRH [38, 39]support the hypothesis that GnRH inhibits apoptosis of pituitary gonadotropes by suppressing Bax expression. Because GnRH acts on the gonadotrope to stimulate gonadotropin secretion as well as cell proliferation [40, 41], the concurrent stimulation of cell proliferation and cell survival of gonadotropes at estrus may be accounted for by GnRH, a multifunctional hormone that regulates all events of cell proliferation, apoptotic cell death, and hormone secretion.…”

Variation in levels of luteinizing hormone and reproductive photoresponsiveness in a population of white-footed mice (Peromyscus leucopus)

“…The gonadal sex steroids are part of a negative feedback pathway inhibiting LH and FSH release at least partially via inhibition of GnRH release (6,12,23,28,37). Previous work has suggested that enhanced sensitivity of GnRH neurons to the sex steroid negative feedback occurring specifically in response to the short photoperiods of winter may be one cause of winter suppression of fertility in seasonally reproducing mammals (14,25,32).…”

Glucose-inducible hypertrophy and suppression of anion efflux in rat beta cells

“…This assay permits assessment of the activity of individual hormone-secreting cells (Smith & Neill 1987). Beta cells were exposed to glucose (1·4-16·5 mM) plus insulin antibody for 2 h, then guinea pig complement for 30 min.…”