Mineralocorticoids (e.g., aldosterone, ALDO) and glucocorticoids (e.g., corticosterone, CORT) have traditionally been defined in terms of their peripheral actions on electrolyte and carbohydrate metabolism; however it is now known that they can exert a number of important behavioral and other actions in the brain. Most of these actions are mediated by genomic modulations of various neurochemicals following he interactions of ALDO and CORT with their intracellular Type I and Type II receptors. The relatively low affinity of Type II receptors for CORT, and ALDO, suggests an interaction with this receptor mainly during conditions of heightened secretion or exogenous steroid administration. Since Type I receptors display an equivalently high affinity for CORT and ALDO, both hormones have the capacity to compete for binding, and in doing so, may elicit different genomic actions. Before these steroid-receptor complexes can elicit this response, they must first undergo a temperature-dependent transformation to the nuclear/DNA binding form known as activation. One of the main goals of this grant is to compare the underlying biochemical causes and consequences of ALDO- vs. CORT-Type I receptor activation in mouse brain. For example, what are the changes in size, shape, density, subunit structure and in surface charge, hydrophobicity and DNA affinity of ALDO- vs. CORT-Type I receptor complexes resulting from and/or producing activation? Are there intermediate steps in this process, and if so, what are the kinetics and energy requirements for the final sequence of transformation? Does the rate and energy requirements of ALDO- vs CORT-Type I receptor association and dissociation changes as a consequence of activation? What role, if any, does RNA and RNase play in this process? What role, if any, do sulfhydryl groups on the Type I receptor play in the reversible binding of ALDO, CORT AND DNA? What are the pH, dose- and time-dependent actions of molybdate on these processes? Lastly, we will attempt to purify unoccupied, occupied-unactivated and occupied-activated Type I receptors from rat hippocampal brain samples. This work will involve the combined use of various biochemical differences between the receptors and a novel selective presaturation treatment prior to steroid affinity resin chromatography. Following this purification, we will be able to study a number of new questions including the possible roles or receptor phosphorylation/dephosphorylation. We could also try to generate monoclonal antibodies against these purified receptors for use in additional biochemical and immunohistochemical localization studies.
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