NONGENOMIC STIMULATION OF ESTROGEN

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DESCRIPTION (Adapted from applicant's abstract): The long-term objectives are to understand the non-genomic actions of E upon the CNS, an issue of relevance since steroid hormones are known to affect a variety of brain functions. We propose, on the grounds of our preliminary work and the work of others, that this steroid can influence cell activity not only by actions on the classical nuclear receptors but also by actions on the plasmalemma of neurons or glia cells through a specific membrane estrogen receptor (mER) leading to changes in intracellular messengers responsible for a particular neurochemical activation. The central hypothesis of this revised project postulates that the rapid stimulation of E upon DA release from rat striatal tissue depends on an interaction between a particular chemical group of the steroid and a specific binding domain in the mER of the corpus striatum (CS) cells. This, depending on the estrous cycle, leads to changes in intracellular messengers ultimately responsible for DA release. The specific aims are: (1) to establish that the rapid effect of E upon in vitro DA release from rat striatal fragments is a membrane mediated event taking place during a particular phase of the rat estrous cycle; (2) to establish that E binds stereospecifically and with high affinity to sites in the plasmalemma fraction from the rat CS and that the specific binding sites for E correspond to mERs and not to "acceptor" molecules; (3) to isolate, purify and chemically characterize the mER from the rat CS and generate polyclonal and monoclonal antibodies against this protein; and (4) to initiate cloning studies to isolate a cDNA clone for E-6-125-IBSA through screening a rat brain lambda ZAP-II library for selective phage plaques producing proteins with affinity for E-6-IBSA. To address these goals, I will take advantage of novel ligands to examine steroid membrane interactions, the so called steroid-BSA complexes. The hormone is covalently bound to bovine serum albumin (BSA), a protein that can either be radioiodinated (for use as a ligand in binding studies) or coupled to an agarose matrix (for use as an affinity chromatography column to isolate and purify the receptor). The work will lead to a better understanding of the role of steroids at the neuronal membrane level and the characterization of a membrane receptor for E may lead to new and useful pharmacological drugs.