Growth hormone receptors in cultured adipocytes: a model to study receptor regulation.
Acutely isolated rat adipocytes have been maintained in primary culture for several days and the effects of culture on the kinetics of 125I-human growth hormone (hGH) binding to adipocytes have been determined. A marked increase (500-1000%) in specific binding of 125I-hGH was observed over the first 3 days of culture--acutely isolated adipocytes (5.5 +/- 1.4%, mean +/- SE, n = 47) compared to 3-day cultured adipocytes (48 +/- 7%, mean +/- SE, n = 8). Specific binding of 125I-hGH to both acutely isolated and cultured adipocytes was dependent on incubation time and temperature (equilibrium being reached in 1 h at 37 degrees C and 2 h at 22 degrees C). Binding was reversible (t1/2 approximately 1.5 h). Scatchard analysis revealed linear plots and showed that the increase in binding during culture was due to an increase in the number of receptors per cell (approximately 20 000 to approximately 170 000) with little or no change in binding affinity (Ka approximately 1 X 10(9) M-1). Cycloheximide inhibited the increase in binding sites during culture suggesting a requirement for de novo protein synthesis. Addition of unlabelled hGH to the culture medium resulted in a marked down-regulation of the GH receptor by 2 days. The GH-induced decrease in receptor number was to due to receptor occupancy by exogenously added GH. The studies to date indicate that the cultured rat adipocyte should provide a useful model for a comprehensive study of the cellular mechanisms and dynamics of GH receptor regulation.