Author:

Keywords:

Animals, Binding Sites, Cells, Cultured, Female, In Vitro, Pituitary Gland, Pituitary Hormone-Releasing Hormones, Rats, Science & Technology, Life Sciences & Biomedicine, Pharmacology & Pharmacy, In Vitro Techniques

Abstract:

Cell type-enriched subpopulations can be obtained by unit gravity sedimentation of dispersed pituitary cells. Using pituitary cells of 14-day-old female rats, it is possible to obtain a highly enriched population of gonadotrophs. There was a good correlation between the amount of binding of 125I [D-Ala6-des-Gly10-ethylamide] GnRH to 2 x 105 cells and the proportional number of gonadotrophs in the different gradient fractions, suggesting that specific binding only occurs on GnRH-responsive cells. The number of binding sites per gonadotroph increased from top to bottom of the gradient. As the unit gravity sedimentation technique separates pituitary cells mainly according to size, it is clear that large gonadotrophs had considerably more binding sites then the other gonadotrophs. This finding correlates well with our previous observations that the responsiveness to GnRH of these gradient-separated gonadotrophs in culture increases with the size of the cells. The affinity constants were the same in all gradient fractions. It is concluded that the specific binding sites of 125I [D-Ala6-des-Gly10-ethylamide] GnRH in cultured pituitary cells are binding sites with the characteristics of functional GnRH receptors. The present assay system seems to be a suitable and reliable in vitro system for studying the in vivo characteristics of the GnRH receptors.