Alteration in Binding of Dexamethasone to Glucocorticoid Receptors in RegeneratingRat Liverafter Partial Hepatectomy1

Animalsand Treatments. Male albinoDonryurats, weighing 100 to 120 g (supplied by Kitayama Rabes Corp., Kyoto, Japan), were given laboratory chow and water ad !ibitum and were adapted to constant laboratory conditions (17, 18). Rats weighing 190 to 220 g were used for experiments. When specified, the rats were adrenalectomized 7 days before 70% hepatectomy. At the beginning of the experiment, the rats were subjected to 70% hepatectomy by the procedure of Higgins and Anderson (1 5) or to a sham operation (27). The animals were sacrificed at the times after partial hepatectomy or sham operation indicated in the charts. Preparation of Cytoplasmic Extracts. Animalswere killed by decapitation, and their livers were perfused with 30 ml of ice-cold 50 mM Tnis-HCI buffer (pH 7.55) containing 250 mM sucrose, 25 mM KCI, 3 m@i MgCI2,and 1 mM mercaptoethanol (1 ). The cytoplasmic supemnatant fraction (cytosol) was pre pared from a homogenate of the tissue by centrifugation at 105,000 x g for 1 hr at 2°as described previously (17, 18). Dexamethasone Binding Assay. The binding of [3Hjdexa methasone (28 Ci/mmol; The Radiochemical Centre, Amen sham, England) to cytosol receptors of admenalectomized rat liver was assayed as described by Beato and Feigelson (1), with the modifications reported previously (1 7, 18). Specific binding was estimated by subtracting the binding of 5 x 10_8 M [3H]dexamethasone plus 1000-fold excess of unlabeled ste. moidfrom that of 5 x 10_8 M [3H]dexamethasone (18). Radio activity was measured as described elsewhere (17). The method of Giannopoulos (1 2) was used for measurement of the binding of the hormone to cytosol receptors of the liver of nonadnenalectomized rats with the modification that samples of 0.2 ml of cytosol were incubated for 20 hr at 0°with 5 x 10_@M [3H]dexamethasone with or without 1000-fold excess of unlabeled glucocorticoid. Apparent equilibrium constants (Kd's) for dexamethasone were estimated by plotting the bind ing data according to the method of Scatchard (26). Protein was estimated with bovine serum albumin as a stan dard (14).