Effects of Progesterone on Intracellular Ca²⁺ Levels of Immortalized Hypothalamic Neurons (GT1-7): Fluorescence Imaging Study

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The effects of progesterone on intracellular Ca²⁺ levels have been examined in immortalized hypothalamic neurons (GT1-7) with fluorescence microscopy. The effects observed were classified into two types: 1) the rapid effects and 2) the slow effects. To observe the rapid effects of progesterone, we added these reagents to cells at 10 μM. Upon stimulation with progesterone, the increase in intracellular Ca²⁺ level was observed at around 40% of cells within two minutes. The progesterone-induced Ca²⁺ signals were also observed with membrane-impermeable progesterone conjugated to BSA. These results suggest that progesterone stimulates Ca²⁺ signals in a nongenomic way by acting on a target in the plasma membrane. Next, to observe the slow effects, cells were incubated with progesterone at 100 nM for 72 hours. The incubation for 72 hours significantly increased the intracellular Ca²⁺ level and stimulated the spontaneous fluctuations. Further, the 72h incubation significantly suppressed GABA-induced Ca²⁺ signals. We have also measured effects of tributyltin, a possible analogue of steroid hormones, on intracellular Ca²⁺ levels in GT1-7 cells. The effects were similar to those of progesterone, implying that tributyltin might mimic the progesterone action on intracellular Ca²⁺ in GT1-7 cells.