Effect of temperature on glucocorticoid uptake and glucocorticoidreceptor translocation in rat thymocytes

The temperature dependence of uptake of ³H]dexamethasone by rat thymocytes in suspension and of the intracellular distribution of the bound hormone was studied as a function of time of incubation. The transport of ³H]dexamethasone was found to obey a simple solubility-diffusion mechanism. The permeability coefficient for glucocorticoid transport corresponded to values reported for other nonelectrolytes of a similar size through biological membranes. At temperatures ranging from 0 to 42 °C, the permeability coefficient increased with temperature and no maximum was observed. However, the maximum cellular uptake of the hormone varied depending on the temperature and time of incubation. Maximal uptake of ³H]dexamethasone was observed at 30 min when the reaction mixture was incubated at 30 °C; when incubated at 20 °C, maximum uptake of ³H]dexamethasone was observed at 3 h. These data were interpreted to mean that there was competition between two temperature-dependent processes, namely steroid transport and inactivation of intracellular binding sites. Intracellular hormone was observed to bind to specific sites as well as to nonspecific, presumably membranal sites. Two independent methods, one of which is based on a linear plot of uptake versus extracellular hormone concentration, gave similar values for the amount of specifically bound hormone, estimated to be 3300 molecules per cell. The binding results are in accord with the sequence of events previously proposed for the interaction of glucocorticoids with thymocytes. These events include nonspecific uptake, specific cytoplasmic binding, a highly temperature-dependent translocation into the nucleus, intranuclear binding, as well as receptor inactivation and regeneration. The amount of intracellular bound hormone and its distribution between the cytoplasmic and nuclear fractions showed no equilibrium or steady-state phenomenon throughout extended periods of incubation up to 28 h. The experiments verified kinetic equations which predicted maximum nuclear binding of the hormone at a given time, followed by an appreciable and progressive reduction in the binding of the hormone to cytoplasmic and nuclear fractions.