Effect of tamoxifen on carbachol-triggered intracellular calcium responses in chicken granulosa cells.

The effect of the nonsteroidal antiestrogen tamoxifen on carbachol (CCh)-triggered intracellular Ca2+ surges was determined in granulosa cells from the two largest preovulatory follicles of laying hens. The intracellular calcium ion concentration ([Ca2+]i) was measured in cells loaded with the Ca(2+)-responsive fluorescent dye fura-2. Resting [Ca2+]i was 96 +/- 5 nM (n = 20), and CCh (1 mM) triggered a large initial [Ca2+]i spike to 600-800 nM, due to the mobilization of Ca2+ from internal stores. Following the spike, the [Ca2+]i dropped to a lower, suprabasal level with super-imposed oscillations, which depended on Ca2+ influx, and returned to the resting level by 2 to 4 min. Tamoxifen (10 microM) did not by itself affect [Ca2+]i but pretreating granulosa cells with tamoxifen (10 microM) prolonged the CCh-triggered [Ca2+]i surge and oscillations by as much as 10 to 30 min. Pretreatment with much higher concentrations of tamoxifen (e.g., 0.5 mM) also had no effect by themselves, but caused a prolonged rise in [Ca2+]i following CCh (1 mM) stimulation. The effect of tamoxifen on CCh-triggered [Ca2+]i responses was mimicked by the tamoxifen metabolite 4-hydroxytamoxifen (10 microM), but not by the structurally related antiestrogens nafoxidine (10 microM) or clomiphene citrate (10 microM). The tamoxifen effect on the CCh-triggered [Ca2+]i response was not mediated through estrogen receptors since pretreating granulosa cells with 17 beta-estradiol (10(-6) M) did not mimic the tamoxifen response. The effect of tamoxifen was inhibited by pretreating granulosa cells with the Ca2+ channel blocker, lanthanum (1 mM), or by incubating the cells in Ca(2+)-free medium. Tamoxifen did not affect [Ca2+]i surges triggered by 17 beta-estradiol (10(-6) M) or dimethyl sulfoxide (1%) which mobilize Ca2+ from internal stores. Pretreating granulosa cells with tamoxifen (10 microM) or 4-hydroxytamoxifen (10 microM) before inducing Ca2+ influx through voltage-dependent Ca2+ channels by depolarizing the cells with 45 mM external K+, caused a prolonged rise of [Ca2+]i, with oscillations, similar to the CCh response. These studies demonstrate that tamoxifen affects the activation of chicken granulosa cell Ca2+ channels by CCh or by raising the external K+ concentration, resulting in a prolongation of the sustained [Ca2+]i elevation and oscillations, which result from the influx of extracellular Ca2+. These observations suggest that tamoxifen interacts with open Ca2+ channels in chicken granulosa cells and keeps them open for prolonged periods of time.