Internalization and degradation of human chorionic gonadotropin in ovine luteal cells: effects of inhibition of protein synthesis

Abstract

Studies were undertaken to investigate the possibility that receptors for LH in monolayer cultures of enzymatically dissociated ovine luteal cells are recycled. Cultured cells (3-10 X 10(5) total steroidogenic cells/dish) were incubated with or without cycloheximide (CHX; 10(-4) M) and 2 X 10(6) cpm [125I]iodo-hCG in the presence or absence of 30 micrograms nonradioactively labeled hCG for 0, 12, 24, 36, or 48 h. At each time point, the amounts of radioactivity bound to the cells (bound [125I]iodo-hCG), located intracellularly (intracellular [125I]iodo-hCG), and degraded and returned to the medium as [125I]monoiodotyrosine (degraded [125I]iodo-hCG) were determined. The number of receptors for LH was determined by Scatchard analysis. Cell viability was also monitored by: 1) trypan blue dye exclusion, 2) the ability of the cells to synthesize protein, and 3) basal and hCG-stimulated secretion of progesterone. More than 90% of the cells remained viable after 48 h of culture, and CHX had no effect on cell viability. Protein synthesis in CHX-treated cells was inhibited by more than 90%. Basal and hCG-stimulated secretion of progesterone were also inhibited by CHX. Treatment with CHX increased the amounts of membrane-bound and internalized [125I]iodo-hCG and decreased the amounts of [125I]iodo-hCG that were degraded. When the quantities of radioactivity in these three fractions (plasma membrane-bound, internalized, and degraded) were added together to obtain a value for the total amount of [125I]iodo-hCG that had been bound to receptor during the 48-h time course (total receptor-associated [125I]iodo-hCG), the value for control cells was not significantly different from the value for CHX-treated cells. Furthermore, the total receptor-associated [125I]iodo-hCG was approximately 2-fold greater than the amount of [125I]iodo-hCG required to saturate receptors at time zero. These data indicate that synthesis of new receptors is not required for the continued binding, internalization, and degradation of [125I]iodo-hCG. Further, the data are compatible with the hypothesis that receptors for LH are recycled or that a portion of the total receptor population is in an unavailable form when the cells are intact but are available for binding after homogenization of the cells.