Estrogen inhibition of norepinephrine responsiveness is initiated at the plasma membrane of GnRH-producing GT1-7 cells.
The modulatory action of estradiol (E2) on the GnRH network can be exerted indirectly on presynaptic neurons or directly on estrogen receptors (ERs) located within GnRH hypothalamic neurons. Using the GnRH-producing GT1-7 cell line, we have investigated whether E2 is able to modify the response of these cells to norepinephrine (NE) stimulation. A 48-h exposure of GT1-7 cells to 10 nM E2 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 E2 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 membrane-binding sites for E2. Binding of E-BSA coupled to FITC was blocked by preincubation of cells with either E2, antiestrogen ICI 182 780, or tamoxifen. Moreover, fluorescence staining of non-permeabilized cells with antibodies against receptors alpha and beta 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 ERalpha 4,4',4''-(4-propyl-[1H]pyrazole-1,3,5-triyl)tris-phenol (PPT) and ERbeta 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) were used. Since PPT, but not DPN, reproduced the effect of E2, it is suggested that estrogen-induced modulatory action on NE responsiveness was mediated by the ERalpha isoform. Taken together, these results indicate that E2 modulates the adrenergic sensitivity of GT1-7 cells by a mechanism compatible with the activation of membrane-associated ERs.
Morales
Araceli
A
Laboratory of Cellular Neurobiology, Department of Physiology, University of La Laguna, La Laguna, Spain. mamoral@ull.es
Gonzalez
Miriam
M
Marin
Raquel
R
Diaz
Mario
M
Alonso
Rafael
R
D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D004958:Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000494:pharmacology; D047629:Estrogen Receptor beta / Q000378:metabolism*; D004967:Estrogens / Q000378:metabolism / Q000494:pharmacology*; D004966:Estrogens, Conjugated (USP) / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000494:pharmacology; D000077267:Fulvestrant; D007987:Gonadotropin-Releasing Hormone / Q000096:biosynthesis*; D006801:Humans; D007031:Hypothalamus / Q000378:metabolism*; D009243:NAD / Q000494:pharmacology; D009474:Neurons / Q000378:metabolism*; D009638:Norepinephrine / Q000378:metabolism*; D010636:Phenols; D011485:Protein Binding; D011720:Pyrazoles / Q000494:pharmacology; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013629:Tamoxifen / Q000494:pharmacology
D004965:Estrogen Antagonists; D047629:Estrogen Receptor beta; D004967:Estrogens; D004966:Estrogens, Conjugated (USP); D010636:Phenols; D011720:Pyrazoles; C043089:estradiol 6-O-carboxymethyloxime-bovine serum albumin-fluorescein isothiocyanate; C043090:estradiol-bovine serum albumin; D013629:Tamoxifen; C486184:4,4',4''-(4-propyl-((1)H)-pyrazole-1,3,5-triyl) tris-phenol; D009243:NAD; D000077267:Fulvestrant; D012710:Serum Albumin, Bovine; D007987:Gonadotropin-Releasing Hormone; D004958:Estradiol; D000242:Cyclic AMP; D016650:Fluorescein-5-isothiocyanate; D009638:Norepinephrine