Western blotting for detection of glucocorticoid receptors in the brain and pituitary gland from adrenal intact pigs

Abstract

The objectives of the experiments presented here were to develop a method for measuring total glucocorticoid receptors (GRs), in adrenal intact pigs, and to describe the distribution of this receptor in the pituitary gland and selected brain regions. Six male pigs, 14 days of age, were euthanized and the pituitary gland, hypothalamus, prefrontal cortex, and hippocampus were prepared for western blotting using a traditional soluble cytosolic preparation or a whole cell extract preparation. Samples were electrophoresed and immunoblotted using the anti-human GR antibodies #57 and #51. The #57 antibody labeled a doublet at 95 and 87 kDa whereas the #51 GR antibody labeled a single band at 87 kDa. Affinity labeling of GR with [ 3H] dexamethasone mesylate revealed a single band at 87 kDa. Using the soluble cytosolic preparation GR concentrations were highest in the pituitary gland followed by the hypothalamus ( P<0.0001). The amount of GR in the hippocampus and prefrontal cortex was equivalent but lesser ( P<0.0001) than that found in the pituitary gland or hypothalamus. With the whole cell extract tissue preparation, GR concentrations were highest ( P<0.05) in the prefrontal cortex and hippocampus with lower ( P<0.05) levels detected in the hypothalamus. Taken together, these results indicated that the 87-kDa band was the functional GR and that regional distribution of GRs is specific to the type of tissue preparation used. In addition, we have described a procedure (from whole cell extracts) for measuring total GRs in neural and nonneural tissues from adrenal intact pigs.