Estrogen inhibition of norepinephrine responsiveness is initiated at the plasma membrane of GnRH-producing GT1-7 cells

The modulatory action of estradiol (E,) on the GnRH network can be exerted indirectly on presynaptic neurons or directly on estrogen receptors (ER's) located within GnRH hypothalamic neurons. Using the GnPH-producing GT1-7 cell line, we have investigated whether E-2 is able to modify the response of these cells to norepinephrine (NE) stimulation. A 48-h exposure of GT1-7 cells to 10 nM E, reduced NE-induced cAMP accumulation. However, 15-min exposure was enough to induce this inhibitory action, provided that a hormone-free period of 48 h after steroid treatment was allowed. Furthermore, this effect was mimicked by E, coupled to (E-BSA), indicating that it may be exerted through a membrane-mediated mechanism. In addition, competition experiments using E-BSA coupled to fluorescein isothiocyanate (FITC) revealed the presence of cell in membrane-binding ding sites for E2. Binding of E-BSA coupled to FITC was blocked by preincubation of cells with either E,, antiestrogen ICI 182 780, or tamoxifen. Moreover, fluorescence staining of non-permeabilized cells with antibodies against receptors a and 0 confirmed the presence of both receptor subtypes at the cell membrane. To determine the nature of the ER involved in this response, specific agonists for ER-alpha 4,4,4-(4-propyl-['H]pyrizole-1,3,5triyl)tris-phenol (PPT) and ER beta 2,3-bis(4-hydroxyphenyJ)propionitrile (DPN) were used. Since PPT, but not DPN, reproduced the effect of E,, it is suggested that estrogen-induced modulatory action on NE responsiveness was mediated by the EP alpha isoform. Taken together, these results indicate that E, modulates the adrenergic sensitivity of GT1-7 cells by a mechanism compatible with the activation of membrane-associated ERs.