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Departments of Physiology (T.S., D.D.B.) and Medicine (A.N.E.H., M.H.), University of Toronto and Division of Cellular and Molecular Biology (M.H., D.D.B.), Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario, Canada M5S 1A8
Address all correspondence and requests for reprints to: Denise D. Belsham, Ph.D., Department of Physiology, University of Toronto, Medical Sciences Building 3247A, 1 King’s College Circle, Toronto, Ontario, Canada M5S 1A8. E-mail: d.belsham{at}utoronto.ca.
Steroid hormones induce rapid membrane receptor-mediated effects that appear to be separate from long-term genomic events. The membrane receptor-mediated effects of androgens on GT1-7 GnRH-secreting neurons were examined. We observed androgen binding activity with a cell-impermeable BSA-conjugated testosterone [testosterone 3-(O-carboxymethyl)oxime (T-3-BSA)] and were able to detect a 110-kDa protein recognized by the androgen receptor (AR) monoclonal MA1–150 antibody in the plasma membrane fraction of the GT1-7 cells by Western analysis. Further, a transfected green fluorescent protein-tagged AR translocates and colocalizes to the plasma membrane of the GT1-7 neuron. Treatment with 10 nM 5
-dihydrotestosterone (DHT) inhibits forskolin-stimulated accumulation of cAMP, through a pertussis toxin-sensitive G protein, but has no effect on basal cAMP levels. The inhibition of forskolin-stimulated cAMP accumulation by DHT was blocked by hydroxyflutamide, a specific inhibitor of the nuclear AR. DHT, testosterone (T), and T-3-BSA, all caused significant elevations in intracellular calcium concentrations ([Ca2+]i). T-3-BSA stimulates GnRH secretion 2-fold in the GT1-7 neuron, as did DHT or T. Interestingly GnRH mRNA levels were down-regulated by DHT and T as has been reported, but not by treatment with T-3-BSA or testosterone 17ß-hemisuccinate BSA. These studies indicate that androgen can differentially regulate GnRH secretion and gene expression through specific membrane-mediated or nuclear mechanisms.
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