A possible new signal trunsduction mechanism of anterior pituitary cells

研究了一种新型钙磷脂结合蛋白armexin v在脑垂体前叶功能中的生理重要性。我们合成了重组大鼠膜联蛋白V，并通过对磷脂膜的亲和力和反相高效液相色谱法对其进行了纯化。将膜联蛋白V应用于大鼠垂体前叶细胞原代培养，可显著增加LH释放，抑制催乳素释放。这些不同的影响是由不同的机制驱动的。由于抗膜联蛋白V本身增加催乳素的释放，而吲哚美辛逆转了这一作用，膜联蛋白V被认为是通过抑制磷脂酶A2和减少原代培养中前列腺素的合成来抑制催乳素的释放。前列腺素在体外被证明能刺激催乳素的释放。另一方面，膜联蛋白V对LH分泌的刺激作用是促性腺激素所特有的。通过半定量RT-PCR，膜联蛋白V也被证明可以刺激LH β亚基的合成。因此，膜联蛋白V被证明可以促进LH分泌的整个过程，即从合成到释放。由于我们通过免疫细胞化学观察到了膜联蛋白V在促性腺激素上的分布，因此我们检测了促性腺激素本身是否能合成膜联蛋白V。为此，我们利用促性腺激素克隆细胞系alpha - 3-1检测了膜联蛋白V mRNA的表达，发现该细胞系能够合成膜联蛋白V，结果表明垂体促性腺激素能够合成膜联蛋白V。膜联蛋白V作为促性腺激素信号转导因子，促进促性腺激素LH的合成和释放，同时作为旁分泌因子，抑制催乳素细胞功能。

Physiological importance of a novel calcium-phospholipid binding protein, armexin V.in functions of the anterior pituitary gland was investigated. We synthesized recombinant rat annexin V, and purified it by the affinity to phospholipid membranes and the reverse phase HPLC.When annexin V was applied to the primary culture of rat anterior pituitary cells, it dramatically increased LH release but inhibited prolactin release. These diverse effects were driven by different mechanisms. As anti annexin V itself augmented prolactin release and Indomethacin reversed this effect, annexin V was thought to inhibit prolactin release by inhibiting phospholipase A2 and reducing prostaglandin synthesis in the primary culture. Prostaglandins are shown to stimulate prolactin release in vitro. On the other hand, the stimulating effect of annexin V on LH secretion is specific to gonadotropes. By using semi-quantitative RT-PCR, annexin V was also shown to stimulate LH beta subunit synthesis. Hence, annexin V was shown to enhance the all process of LH secretion, namely from synthesis to release. As we had observed annexin V distributed on gonadotropes by immunocytochemistry, we examined whether gonadoiropes themselves could synthesize annexin V.For this purpose, we utilized clonal cell line of the gonadotropes, alphaT3-1, to examine the expression of annexin V mRNA and found this cell line synthesizes annexin V.This result suggests the pituitary gonadotropes synthesize annexin V.Getting together, annexin V is suggested to function as a factor of the signal transduction to enhance the synthesis and the release of LH at the gonadotropes, and it is supposed to work simultaneously as a paracrine factor to inhibit prolactin cell function.