Glucocorticoid Receptor in Circulating Mononuclear Leukocytes

Abstract

Binding of [³H]dexamethasone ([³H]Dex) and [³H]cortisol to normal female goat mononuclear leukocytes (MNL) was studied at 4 and 20 C. Dissociation studies of [³H]Dex and [³H]cortisol from MNL after 3 h of incubation at 20 C indicated the presence of at least two components; a small pool with a dissociation rate constant (k_Ofr) of 15 ± 8 x 10^-3 min^-1 for [³H]Dex and 35 ± 11 x 10^-3 min^-1 for [³H]cortisol, and a large capacity pool with a kOff of 1.0 ± 0.1 X 10^-3 min^-1 for [³H]Dex and 11 ± 2 X 10^-3 min^-1 for [³H]cortisol. The calculated k_off for the slow component was indistinguishable from the k_Ofr for nuclei isolated from cells that had been preincubated with [³H]Dex or [³H]cortisol. Thus, for the intact cells the slow component probably represents the dissociation of an activated steroid-receptor complex transformed by a temperature-dependent process at 20 C. The rapid phase may represent the dissociation of an inactive steroid-receptor complex in the cytosol at 20 C. The K_d obtained from equilibrium Scatchard analysis (Dex, 0.27 ± 0.02 nM; cortisol, 4.5 ± 0.1 nM) was in satisfactory agreement with the ratio of k_Ofr to association rate from kinetic studies. The dissociation of [³H]Dex and [³H]cortisol from MNL at 4 C can be described by a single exponential decay. The ratio of koff tp association rate at 4 C corresponds to the K_dS obtained using equilibrium Scatchard analysis (at 15 h) for either intact cells (Dex, 2.0 ± 0.3 nM; cortisol, 8.0 ± 0.9 nM) or cytosols (Dex, 1.4 ± 0.4 nM; cortisol, 8.5 ± 1.9 nM). The higher affinity of Dex to the intact cell at 20 C may explain the previously noted discrepancy between the increased biological potency of Dex compared to that of cortisol and the modest increase in binding affinity of Dex to cytosol receptors.