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

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 17β‐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 µm) 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 17α‐estradiol (1 µm) nor the synthetic antiestrogens ICI182 780 (1 µm) or tamoxifen (1 µm) 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 17α‐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.