The effects of cell membrane alteration on glucocorticoid uptake by the AtT-20/D-1 target cell

Abstract

The glucocorticoid-sensitive AtT-20/D-1 cell line was used to study cellular uptake of glucocorticoids. A previous observation that glucocorticoid uptake by these cells was temperature dependent had prompted us to postulate that glucocorticoids entered the cell by a temperature-sensitive transport process located in the cell membrane. Attempts were then made to perturb the membrane mechanism. In some of these experiments, intact cells were treated with neuraminidase or pronase. The release of sialic acid in the case of neuraminidase treatmentand of sialic acid and cell surface peptides in the case of pronase treatment demonstrated that the enzymes were effective. Approx. 60% of total cellular sialic acid was released by a 15 min incubation with 20 μg/ml neuraminidase at 25°C. The treated cells appeared to be viable, in that they continued to produce corticotropin at a normal rate, yet intact cell glucocorticoid binding at both 4 and 25°C was only 20–30% of that of untreated cells. Treatment with pronase also caused steroid uptake at 4 and 25°C to be reduced, although the extent of reduction was less than that seen following neuraminidase treatment. In other experiments, the effect of exposure of AtT-20/D-1 cells to ethanol or dimethyl sulfoxide was determined. The solvent concentrations used (0.5–10%) did not alter cell viability significantly, and the ability of the cytosol receptor to bind steroid in a cell-free preparation was unimpaired. However, incubation of intact cells with 10% (v/v) dimethyl sulfoxide or ethanol resulted in an 80–90% decrease in steroid uptake at 25°C. We conclude that steroid uptake by the intact cell can be perturbed by treatments which do not affect the cytosol receptor or alter cell viability. These results support the postulate that glucocorticoids enter the AtT-20/D-1 cell by a specific membrane-associated mechanism.