[Studies on the glucocorticoid receptor in human peripheral lymphocytes. I. Fundamental investigation on the method of measurement of the glucocorticoid receptor in whole cell and nucleus].
There have been several reports about the glucocorticoid receptor (GC-R) in human peripheral lymphocytes (HPL). However, little has been investigated about the validity of the assay method of GC-R in HPL. such as the effect of receptor occupancy by intrinsic cortisol or administered GC. In the present study, it was clarified that receptor-bound prednisolone or cortisol was almost completely dissociated from the receptor by washing lymphocytes three times with 30 min equilibration at 37 degrees C between each centrifugation. The GC receptor was measured by whole cell binding assay using [3H] dexamethasone as a ligand. The receptor number and dissociation constant (Kd) of lymphocytes, which were preincubated with prednisolone (10(-7) M) or cortisol (10(-6) M) at 37 degrees C for 1 h, were equal to those lymphocytes preincubated without GC. These data indicated that the receptor number and Kd of HPL can be determined without the effects of receptor occupancy by this assay procedure. The GC and GC-R complex in the nucleus are quite important for the action of GC. However, there have been few reports about the assay of nuclear GC-R in HPL. In this study, it was elucidated that nuclear receptor-bound [3H] dexamethasone could be measured by centrifugation (2000 g, 10 min) of the nuclei of lymphocytes after being lysed in 1.5 mM MgCl2 (0 degree C). By these assay procedures, the receptor number in whole lymphocytes of normal HPL (n = 22) was determined to be 10.48 +/- 2.40 (M +/- SD) fmoles/10(6) cells, and Kd was 5.8 +/- 1.24 nM, while the receptor number and Kd in the nuclei were 5.68 +/- 1.53 and 6.29 +/- 1.49, respectively.
D000293:Adolescent; D000328:Adult; D002467:Cell Nucleus / Q000378:metabolism; D003907:Dexamethasone / Q000378:metabolism; D005260:Female; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism*; D008297:Male; D008722:Methods; D011965:Receptors, Glucocorticoid / Q000032:analysis*
D011965:Receptors, Glucocorticoid; D003907:Dexamethasone