Discrimination between changes in glucocorticoid receptor expression and activation in rat brain using western blot analysis.
These studies investigated autoregulation of glucocorticoid receptor (GR) protein expression and activation in rat brain using western blot methodology. By comparing GR immunoblot reactivity present in various tissue subcellular preparations we were able to discriminate between corticosterone-induced changes in GR activation or GR protein expression. Our cytosolic tissue preparation yielded a similar pattern of treatment effects on relative GR as measured by receptor binding assay or western blot. In both cases, short-term adrenalectomy (18 h) produced no change in cytosolic GR. On the other hand, long-term adrenalectomy (3-14 days) resulted in a large increase in cytosolic GR, whereas acute (1-2 h) treatment with high dose corticosterone produced a large decrease in cytosolic GR. Western blot measurement of GR levels in a nuclear extract or whole-cell extract from the same brains indicated that acute corticosterone treatment produced a large increase in nuclear 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 adrenalectomy or high dose corticosterone produced a large increase and decrease, respectively, in whole-cell GR, indicating genuine changes in receptor protein 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 cytosolic receptor binding assays to estimate relative changes in GR occupation/activation when appropriate comparison groups are included.
Spencer
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Department of Psychology, Campus Box 345, University of Colorado, Boulder, CO 80309, USA. spencer@psych.colorado.edu
Kalman
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Cotter
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Deak
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D000315:Adrenalectomy / Q000009:adverse effects; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D002457:Cell Extracts / Q000032:analysis; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D003348:Cortisone / Q000378:metabolism / Q000494:pharmacology; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004334:Drug Administration Schedule; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008297:Male; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D011965:Receptors, Glucocorticoid / Q000187:drug effects* / Q000378:metabolism*
D002457:Cell Extracts; D011965:Receptors, Glucocorticoid; D003348:Cortisone