Effect of Tamoxifen on Carbachol-triggered Intracellular Calcium Responses in Chicken Granulosa Cells

Abstract

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 Ca²⁺-responsive fluorescent dye fura-2. Resting [Ca²⁺]_i was 96 ± 5 nm (n = 20), and CCh (1 nm) 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 µm) did not by itself affect [Ca²⁺]_i but pretreating granulosa cells with tamoxifen (10 µm) 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 µm), but not by the structurally related antiestrogens nafoxidine (10 µm) or clomiphene citrate (10 µm). The tamoxifen effect on the CCh-triggered [Ca²⁺]_i response was not mediated through estrogen receptors since pretreating granulosa cells with 17β-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 Ca²⁺-free medium. Tamoxifen did not affect [Ca²⁺]_i surges triggered by 17β-estradiol (10⁻⁶ m) or dimethyl sulfoxide (1%) which mobilize Ca²⁺ from internal stores. Pretreating granulosa cells with tamoxifen (10 µm) or 4-hydroxytamoxifen (10 µm) 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.