Glucocorticoid Transport Research Articles

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.

Evidence for glucocorticoid transport into AtT-20/D-1 cells

Glucocorticoid uptake by AtT-20/D-1 mouse pituitary adenocarcinoma cells grown in tissue culture was examined. The binding of triamcinolone acetonide, a potent synthetic glucocorticoid, by intact cells and by cell cytosol was studied at both 4 and 25 degrees. Specific binding of [3H]triamcinolone acetonide by intact cells was markedly different from cell-free cytosol binding at 4 degrees. Intact cells bound a relatively small amount of labeled steroid within 2 min, after which no further binding was observed. In contrast, the receptor in a cell-free cytosol preparation was capable of binding steroid progressively at 4 degrees, indicating that the limited binding by intact cells was not a consequence of receptor characteristics. At 25 degrees, uptake by intact cells and cytosol was nearly identical and appeared to be limited only by the binding kinetics of the cytosol receptor. Estradiol-17 beta, a nonglucocorticoid steroid, was not bound by the AtT-20/D-1 cell at 4 degrees. Triamcinolone was not bound significantly at 4 or 25 degrees by an adrenal carcinoma cell that does not appear to be a glucocorticoid target cell. An Arrhenius plot of cell steroid uptake vs. the reciprocal of absolute temperature revealed an abrupt change in slope at 16 degrees, which is compatible with the temperature-dependent mechanism involved in glucocortidoid uptake being associated with lipid constituents of the cell membrane. These data suggest that glucocorticoid uptake by this target cell involves a mechanism of specific, temperature-dependent transport through the cell membrane.

Evidence for glucocorticoid transport through the target cell membrane

Summary Treatment of intact cells from the glucocorticoid-responsive AtT-20/D-1 cell line with neuraminidase or phospholipase A 2 caused a reduction in the ability of these cells to incorporate labeled glucocorticoids. Similar treatments with proteolytic enzymes had no effect on steroid uptake. In contrast, cell-free binding to the cytosol receptor was not affected by neuraminidase or phospholipase A 2 treatment but was abolished by treatment with proteolytic enzymes indicating that the effect on cell uptake was not secondary to an effect on the cytosol receptor. These data suggest that steroid entry into the AtT-20/D-1 cell is mediated by components of the cell membrane which are sensitive to the loss of sialic acid groups or disruption of membrane architecture.