Betamethasone receptor levels following betamethasone administration.

Betamethasone (BM) receptor cncentrations in the liver cytosol from adrenalectomized rats treated with 0. 2 and 2 mg BM were measured and correlated with BM concentrations in the serum and liver. The BM levels in the serum, liver cytosol and nuclei changed in a parallel fashion. One hr after BM administration, serum BM reached a peak, then decreased gradually and was undetectable at 24-48 hr. The peak levels of BM in the serum, liver cytosol and nuclei from 0. 2 mg BM treated rats were 880. 0±96. 0 ng/ml, 32. 5±8. 1 ng/mg protein and 9. 6±2. 4 ng/mg DNA or 12. 1 ±2. 6 ng/g wet liver. Those from 2 mg BM treated rats were 1540. 0±942. 0 ng/ml, 47. 4±38. 8 ng/mg protein and 14. 2 ± 3. 7 ng/mg DNA or 16. 6 ±5. 6 ng/g wet liver. In the liver cytosol, there are two types of binding sites for BM, one with high affinity (Kd=6. 0x10-9 mol/liter) and low capacity (6. 6x10-1s mol/mg protein or 13. 0±5. 2 ng/g wet liver) and one with low affinity (Kd>10-7 mol/liter) and high capacity (>10-11 mol/mg protein). The peak levels of BM in the liver nuclei from 0. 2 and 2 mg BM treated rats were close tO the binding capacity of high affinity binding site in cytosol (12. 1 and 16. 6 vs. 13. 0 ng/g wet liver). [SHJBM binding to the liver cytosol from both 0. 2 and 2 mg BM treated rats was lost completely at 1 to 6 hr, and recovered at 24 hr in the 0. 2 mg BM treated rats and at 48 hr in the 2 mg BM treated rats. Thus, the cytosol and nuclear levels of BM are reciprocally related to [SH] BM binding capacity in the liver cytosol, and the fall of [SH] BM binding to the cytosol is accompanied by the appearence of BM in the nuclei. Therefore, our in vivo study suggested that almost all of high affinity receptor bound BM is transferred rapidly to nuclei and remains there in the presence of a sufficient amount of BM in cytoplasm, and thereafter the receptor is released from nuclei to cytoplasm. 599 INTRODUCTION after administration of two doses of BM to rats, with cytosol BM receptor in the liver. The mechanism of action of glucocorticoid hormones has been studied extensively at the molecular level1• 2• 8>. However, the bioavailability of administered synthetic glucocorticoid and its relationship to their own receptors still remain unclear. The purpose of this study was to correlate liver cytosol and nuclear levels of betamethasone (BM) at various time intervals MATERIALS AND METHOD Isotopes and chemicals 1, 2, 4(n)-[3HJBM (32 Ci/m,mol) was obtained from the Radiochemical Centre Amersham (Buckinghamshire, U. K.). Prednisolone and BM were gifts of Shionogi Co. (Osaka, Japan). DNA, cytochrome C, hen egg albumin and 600 K. Tanaka et al. aldolase were obtained from Baehringer Mannheim GmnH (Mannheim, West Germany). Corticosterone, bovine serum albumin, bovine' serum gammaglobulin and tr~zffi:a base, wer~ purchased from Sigma Chemical C0. ·(St. Louis, U.S. A.). Other chemicals were purchased from Katayama Chemical Co. (Osaka, Japan). Animals and preparation of cytosol and nucleus Male Wistar rats weighing 200-240 g were used throughout these experiments 3-4 days after adrenalectomy. They were maintained on Oriental brand foods and drinking water supplemented 0. 4% saline ad lib. A water suspension of 0. 2 or 2 mg of BM was administered to these rats through a gastric tube. Before and 1, 3, 6, 24, 48 hr after BM administration, tats w~re sacrificed, blood was obtained from abdominal aorta, and the liver was removed after perfusion with 50 ml of cold 0. 9 % saline via the portal vein. The liver was minced with scissors, homogenized in an equal volume of 0. 25 M sucrose, 3 mM MgCl2, 50 mM Tris-HCl buffer, pH 7. 4, and centrifuged at 105, 000 G for 60 min at 4°C. The supernatant was termed cytosol and its protein concentration was determined by the method of Lowry using bovine serum albumin as a standard15>. To prepare the nuclear fraction, the liver was homogenized in four volumes of the same buffer and centrifuged at 700 G for 20 min. The pellet was suspended in 2. 5 M sucrose, 3 mM MgCl2, · 50 mM Tris-HCl buffer, pH 7. 4 and recentrifuged at 40, 000 G for 20 min. The Pellet was resuspended in 0. 25 M sucrose, 50 mM TrisHCl buffer, filtered through several layers of gauze and centrifuged at 700 G for 5 min. The final nuclear fraction was washed three times. The DNA concentration in the nuclear fraction was determined according to the Burton's method?>. BM radioimmunoassay An appropriate volume of the serum and liver cytosol, or the liver nucleus was extracted with 4 ml of dichloromethane by shaking for one min. These extracts or standards containing 10 pg-10 ng of BM dissolved in ethanol were pipetted into assay tubes. The tubes were then dried under an air stream at 45°C. Antiserum '(final . dilution 1 : 5, 000) and [3HJBM (10, 000 cpm) dissolved in 0. 1% gammaglobulin in saline were added to each tube in a total· volume of 1 ml. After incubation at 4°C for 16 hr, 0. 2 ml of the 0. 5 % dextran coated charcoal (DCC) was add~d to each' tube and the tubes were centrifuged at 3, 000 rpm for 15 min. The supernatarits were. decanted into counting vials which contained 10 ml of Bray's solution and the radioactivity was measured with a liquid schintillation spectrometer. The radioimmunoassay dose-response curves and the sample values were analyzed by the logit-log method presented by Rodbard, Bridson & Rayfort19>. The validity of this·· radioimmunoassay method has been described elsewhere17, 15>. Gel chromatography Aliquots of liver cytosol were incubated with [ 3H] BM in the absence or presence of a 100 fold excess of non radioactive BM at 4° C for 3 hr, and then applied to a column of Sephacryl S-300 (2. 5 x 80 cm) equilibrated with 50 mM Tris-HCl buffer, pH 7. 4. The column was eluted with the same buffer and 8 ml aliquot of the eluate was collected, 1 ml of which was then pipetted into the counting vials which contained 10 ml of Bray's solution. The radioactivity was measured by a liquid schintillation spectrometer. For the evaluation of BM molecules in the cytosol, the liver cytosol from rats 1 hr after 2 mg BM administration was applied to the same column and eluted with 50 mM Tris-HCl buffer, pH 7. 4. One ml of 8 ml each fraction was mixed with 4 ml of dfohloromethane, and BM or BM like immunoactivity both in aqeous and dichloromethane phase was measured by radioimmunoassay. Analysis of BM receptor Standards containing 5-500 nM of BM or other glucocorticoids in ethanol were pipetted into the assay tubes. After evaporation under an air stream at 45° C, 3. 5 nM of [HJBM, liver cytosol and 50 mM Tris-HCl buffer, pH 7. 4 were added to the tubes in a total volume of 0. 5 ml. Following the incubation at 4°C for 3 hr, 0. 5 ml of 1% DCC was placed in each tube. The tubes were centrifuged at 3, 000 rpm for 15 min and the radioactivity in the supernatant was determined. The inhibition curve was obtained by plotting the percent binding against the logarithm of the dose. The binding capacity and dissociation constant (Kd) of BM receptor were determined from Scatchard plot22>. The receptor content of the liver cytosol after Betamethasone Receptor 601 BM administration was determined in · DCC treated cytosol according to the method mentioned above.