Estradiol modulates acetylcholine-induced Ca2+ signals in LHRH-releasing GT1-7 cells through a membrane binding site.

Abstract

Estrogen regulation of the female reproductive axis involves the rapid inhibition (< 30 min) of luteinizing hormone-releasing hormone (LHRH) secretion from hypothalamic neurons. This fast time-course suggests interactions with potential plasma membrane binding sites that could result in short-term effects on LHRH neurons. Because LHRH release is calcium dependent, we have studied the acute effects of 17beta-estradiol (E2) and estradiol-peroxidase (E-HRP) on the elevations of intracellular calcium ([Ca2+]i) induced by acetylcholine (ACh) in LHRH-producing GT1-7 cells. Exposure to ACh (1-100 micro m) induced transient increases of [Ca2+]i, whereas pretreatment with E2 or E-HRP (10 nm) for 2 min reduced this response by 50-60%. The effect was specific for E2 as neither 17alpha-estradiol (1 micro m) nor the synthetic antiestrogens ICI182 780 (1 micro m) or tamoxifen (1 micro m) elicited any change on the ACh-induced Ca2+ signal. Both the latency of the effect and the response to the membrane impermeant conjugate suggested a membrane-mediated mechanism. Such membrane binding sites for E2 in GT1-7 cells were demonstrated by visualizing the binding of E-HRP and estradiol-BSA-fluorescein isothiocyanate (E-BSA-FITC) conjugates. Competition studies showed that E-HRP binding was blocked by preincubation with E2, but not with 17alpha-E2, ICI182 780, tamoxifen or progesterone, indicating that the plasma membrane binding site is highly specific for E2 and exhibits a pharmacological profile different from classical estrogen receptors. We conclude that ACh-induced increase in [Ca2+]i in GT1-7 cells is modulated acutely by physiological E2 concentrations in a manner which is compatible with the existence of an estrogen-specific membrane binding site.