Rapid, high temperature exchange assay for the hepatic glucocorticoid receptor

Liver cytosol from adrenalectomized rats was prebound for 2 hr at 4 degrees C with unlabeled 10(-5) M corticosterone. After treatment of cytosol with dextran-coated charcoal to remove free steroid, samples were incubated at 15-25 degrees C in the presence of 10 mM molybdate plus 5 mM dithiothreitol (followed by a 60 min incubation at 4 degrees C). Essentially, complete exchange of [3H] dexamethasone for receptor-bound unlabeled steroid was observed after 120 min at 15 degrees C, and near complete (80-95%) exchange occurred within 60 min at 25 degrees C using these conditions. However in control, 5 mM dithiothreitol (alone) and 10 mM molybdate (alone) treated samples, less than 50% exchange was found. Using a similar protocol, only partial exchange was found in brain and kidney cytosols, suggesting at least partial specificity for the hepatic system. We have used this rapid, high temperature exchange assay to study the regulation of hepatic cytoplasmic glucocorticoid receptors under some experimental conditions.