Evidence for Irreversible, Actinomycin D-sensitive, and Temperature-sensitive Steps following Binding of Cortisol to Glucocorticoid Receptors and Preceding Effects on Glucose Metabolism in Rat Thymus Cells

Following cortisol binding to the specific glucocorticoid receptors of thymus cells at 37° (a process which takes about 7 min), there is a 5- to 10-min lag period before a cortisol effect can be observed on glucose 6-phosphate accumulation after a glucose pulse. Evidence is presented that during this period the signal initiated by cortisol binding traverses an irreversible, an actinomycin D-sensitive, and a temperature-sensitive step. The irreversible step is shown by the fact that removal of cortisol from the glucocorticoid receptors (by displacement with cortexolone, a metabolically inactive glucocorticoid competitor, or by washing) well before any metabolic effect has appeared does not prevent the subsequent appearance of the cortisol effect. The actinomycin D-sensitive step is shown by the fact that, whereas addition of actinomycin D to thymus cells together with cortisol prevents the cortisol effect from developing subsequently, addition of actinomycin D 5 min after cortisol does not prevent the cortisol effect. To produce these actions, actinomycin D must be used at 100 µg per ml. This same concentration is necessary to inhibit RNA synthesis rapidly in thymus cells by about 80%. The temperature-sensitive step is shown by the fact that the duration of the lag period preceding the appearance of a cortisol effect increases markedly at temperatures below 37° to more than 120 min at 20°. The duration of the lag period at 37° can be shortened if cells are first incubated with cortisol at 20°. The temperature dependence of the irreversible step is such that it cannot be identical with, but must precede, the temperature-dependent step. The actinomycin D-sensitive step may or may not be identical with one of the other two steps.