The nongenomic effects of physiological and environmental estrogens on dopamine transporter function

Neurological diseases and neuropsychiatric disorders that vary depending on female life stages suggest that sex hormones may influence the function of neurotransmitter regulatory machinery such as the dopamine transporter (DAT). In this dissertation we tested the rapid nongenomic effects of several physiological estrogens [estradiol (E2) estrone (E1), and estriol (E3)] and xenoestrogens (XEs), such as organochlorine pesticides [dieldrin, endosulfan, and o¡¯,p¡¯-dichlorodiphenylethylene (DDE)], plastics manufacturing by-products/detergents (nonylphenol, bisphenol A), and the synthetic estrogen DES for effects on DAT function. We characterized membrane estrogen receptors (ERs) and their responses using a nontransfected continuous rat cell line (PC12). We showed that PC12 cells express membrane ER¥á, ER©¬, GPR30, and the DAT, and that a two day NGF-©¬ treatment increased the levels of DAT and ER¥á. Low concentrations (10-14-10-8M) of E2 administered for 9 minutes caused DA efflux, which was not dependent on extracellular Ca2+-mediated exocytotic release from vesicular monoamine transporter vesicles (VMATs). XE concentrations ranging from 10-14-10-8M caused dose-response patterns distinct from that caused by E2. The rapidity and the mimicking of this response by a cell membrane-impermeable E2-dendrimer conjugate both suggest a non-genomic, membrane-initiated response mechanism. Using receptor-selective agonist/antagonists and siRNA knockdowns for ER¥á, ER¥â, and GPR30, we determined that E2-mediated dopamine efflux at 9 minutes is mediated by ER¥á, with inhibitor contributions from ER©¬ and GPR30. Using kinase inhibitors we showed that E2-mediated dopamine efflux is also dependent on protein kinase C and MEK activation, but not on PI3K or protein kinase A. In plasma membrane-enriched fractions there were ligand-independent associations of ER¥á and ER©¬ (but not GPR30) with the DAT. Conditions which caused efflux (a 9 min 10-9M E2 treatment) also caused trafficking of ER¥á to, and ER©¬ away, from the plasma membrane. In contrast, E1 and E3 inhibited efflux while causing DAT to leave the membrane. We used kinase activity screens to develop signaling profiles for E2 and BPA; XEs causing dopamine efflux have distinct kinase activation profiles, which could contribute to their abilities to disregulate endocrine functions. Mechanisms that we described in these studies could explain how both physiological and environmental contaminant estrogens influence DAT-dependent diseases.