A new, nongenomic estrogen action: the rapid release of intracellular calcium

We have investigated the effects of steroids on the intracellular calcium ion concentration [Ca2+]i in chicken granulosa cells obtained from the two largest preovulatory follicles of laying hens. [Ca2+]i was measured in cells loaded with the Ca(2+)-responsive fluorescent dye fura-2. The resting [Ca2+]i in these cells was 100 +/- 5 nM. There was an immediate (i.e. less than 5 sec) 4- to 8-fold increase in [Ca2+]i in all of the 76 cells examined after the addition of 10(-7) M estradiol-17 bdta. Estradiol-17 beta was effective between 10(-10)-10(-6) M. Estradiol-17 alpha, estrone, and estriol (10(-8)-10(-6) M) were as effective as estradiol-17 beta, but the progestins, pregnenolone, and progesterone, and the androgens, testosterone, androstenedione, or 5 alpha-dihydrotestosterone were ineffective at concentrations up to 10(-5) M. The prompt estradiol-17 beta-induced [Ca2+]i spike was not affected by incubating the cells in Ca(2+)-free medium containing 2 mM EGTA or by pretreating them with the Ca2+ channel blockers lanthanum (1 mM), cobalt (5 mM), methoxyverapamil (D600; 50 microM), or nifedipine (20 microM). The estrogen-triggered [Ca2+]i surge was also not affected by pretreating the cells with the conventional estrogen receptor antagonist tamoxifen (10(-5) M), or the RNA and protein synthesis inhibitors actinomycin D (1 microgram/ml) and cycloheximide (1 microgram/ml), but was abolished by pretreating the cells with inhibitors of inositol phospholipid hydrolysis, neomycin (1.5 mM) and U-73,122 (2.5 microM). The closely related, but inactive, compound U-73,343 (1 microM) did not affect the estrogen-triggered [Ca2+]i surge. Estradiol-17 beta (10(-7) M), but not progesterone (10(-5) M), also triggered a large [Ca2+]i surge in pig granulosa cells, which, like the [Ca2+]i surge in chicken granulosa cells, was almost immediate, transient, and unaffected by incubation in Ca(2+)-free medium or pretreatment with methoxyverapamil (D600; 50 microM), lanthanum (1 mM), or tamoxifen (10(-5)M). However, granulosa cells from immature rats primed with diethylstilbestrol or PMSG did not respond to estradiol-17 beta, even at concentrations as high as 10(-5) M, although they promptly generated a [Ca2+]i transient upon exposure to LHRH (10(-5) M). These results suggest that estrogens almost instantaneously trigger the release of Ca2+ from intracellular stores which may be mediated through phosphoinositide breakdown. The striking rapidity of this estrogen-induced internal Ca2+ mobilization is consistent with the activation of a cell surface receptor which is different from the conventional slowly acting, gene-stimulating nuclear estrogen receptor.