Evidence that pH induced activation of the rat hepatic glucocorticoid-receptor complex is irreversible

Abstract

The possible reversibility of pH induced activation of the glucocorticoid-receptor complex was studied. Generally, this was accomplished by activating rat liver cytosol at pH 8.5 (15°C, 30 min), and then returning it to pH 6.5 for a second incubation (15°C, 30 min). Activation was quantitated by measuring the binding of [ 3H]triamcinolone acetonide ([ 3H]TA)-receptor complexes to DNA-cellulose. When cytosol was incubated at pH 6.5, only 4.1% of the [ 3H]TA-receptor complexes bound to DNA-cellulose. However, 39.2% of the complexes bound when the cytosol was pH activated. When pH activation was followed by a second incubation at pH 6.5, 47.0% of the steroid-receptor complexes bound. Thus, according to the DNA-cellulose binding assay, pH induced activation was irreversible. In order to visualize both activated and unactivated [ 3H]TA-receptor complexes during this process, diethylaminoethyl (DEAE)-cellulose chromatography was performed. When cytosol was incubated at pH 6.5, only 19.6% of the [ 3H]TA-receptor complexes were eluted in the activated form from DEAE-cellulose. However, 67.5% of the complexes were eluted in the activated form when cytosol was pH activated. When pH activation was followed by a second incubation at pH 6.5, 74.9% of the steroid-receptor complexes were eluted in the activated form. Thus, DEAE-cellulose chromatography also showed that pH induced activation was irreversible. This is the first known report that the combination of DNA-cellulose binding and DEAE-cellulose chromatography have been used to study pH induced activation of the glucocorticoid-receptor complex. By these criteria, we conclude that in vitro pH induced activation is irreversible.