Discrimination between changes in glucocorticoid receptor expression and activation in rat brain using western blot analysis

These studies investigated autoregulation of glucocorticoid receptor (GR) p rotein expression and activation in rat brain using western blot methodolog y. By comparing GR immunoblot reactivity present in various tissue subcellu lar preparations we were able to discriminate between corticosterone-induce d changes in GR activation or GR protein expression. Our cytosolic tissue p reparation yielded a similar pattern of treatment effects on relative GR as measured by receptor binding assay or western blot. In both cases, short-t erm adrenalectomy (18 h) produced no change in cytosolic GR. On the other h and, long-term adrenalectomy (3-14 days) resulted in a large increase in cy tosolic CR, whereas acute (1-2 h) treatment with high dose corticosterone p roduced a large decrease in cytosolic GR. Western blot measurement of GR le vels in a nuclear extract or whole-cell extract from the same brains indica ted that acute corticosterone treatment produced a large increase in nuclea r GR and no change in whole-cell GR. Thus, all of the decrease in cytosolic GR observed after acute corticosterone treatment could be accounted for by receptor redistribution to the nuclear tissue fraction as opposed to rapid receptor protein downregulation. Long-term treatment of rats with adrenale ctomy or high dose corticosterone produced a large increase and decrease, r espectively, in whole-cell GR, indicating genuine changes in receptor prote in expression. These studies indicate that in vivo regulation of GR protein expression in the rat brain can be studied using western blot analysis of a whole-cell tissue preparation. This procedure has an important advantage over receptor binding studies in that GR protein expression can be measured in adrenal-intact rats. These studies also support the validity of using c ytosolic receptor binding assays to estimate relative changes in GR occupat ion/activation when appropriate comparison groups are included. (C) 2000 El sevier Science B.V. All rights reserved.