Huerta-Ocampo Icnelia, Fiordelisio Tatiana, Díaz Nestor, Navarro Nilda, Castilla Alejandra, Cárabez Alfonso, Aguilar Manuel B, Morales Teresa, Hernández-Cruz Arturo, Mena Flavio
Departamentos de Neurobiología Celular y Molecular, Instituto de Neurobiología, Campus UNAM, Juriquilla-Querétaro, México.
Neuroendocrinology. 2007;85(1):1-15. doi: 10.1159/000100507. Epub 2007 Mar 5.
This study demonstrates that conditioned media (CM) from the anterior pituitary gland (AP) of lactating rats contains soluble factors that promote in vitro prolactin (PRL) release from the pituitary glands of male rats. CM-induced PRL release was confirmed by polyacrylamide gel electrophoresis, ELISA and bioassay. In cultured AP cells challenged with CM, increased intracellular staining with the dye FM1-43 was observed, suggesting vesicular PRL release and subsequent endocytosis. The percentage and hormone content of PRL-containing cells but not of growth hormone-containing cells increased in cultured male AP cells when exposed to CM. When the release of PRL, prelabeled with [3H] leucine for 30 min to 24 h was examined, no stimulatory effect of CM was observed, suggesting that released PRL originates from hormone synthesized more than 24 h earlier. Accordingly, the PRL content of mature granules from male pituitary tissues decreased after CM treatment. These findings were confirmed by electron microscopy immunogold PRL labeling. Treatment with inhibitors of protein synthesis or vesicle trafficking between the endoplasmic reticulum and the Golgi complex did not prevent the stimulatory effect of CM on PRL release. However, blockage of traffic to the plasma membrane completely abolished the effect of CM. These results suggest that CM from the AP of lactators contains soluble factor(s) capable of inducing rapid vesicular release of PRL in the male AP, which originates from preformed, mature granules by mechanisms independent of protein synthesis.
本研究表明,哺乳期大鼠垂体前叶(AP)的条件培养基(CM)含有可促进雄性大鼠垂体体外释放催乳素(PRL)的可溶性因子。通过聚丙烯酰胺凝胶电泳、酶联免疫吸附测定(ELISA)和生物测定法证实了CM诱导的PRL释放。在用CM刺激的培养AP细胞中,观察到用染料FM1-43进行的细胞内染色增加,提示囊泡性PRL释放及随后的内吞作用。当培养的雄性AP细胞暴露于CM时,含PRL细胞的百分比和激素含量增加,而含生长激素细胞的百分比和激素含量未增加。当检测预先用[3H]亮氨酸标记30分钟至24小时的PRL释放时,未观察到CM的刺激作用,这表明释放的PRL源自24小时前合成的激素。因此,CM处理后雄性垂体组织成熟颗粒中的PRL含量降低。这些发现通过电子显微镜免疫金PRL标记得到证实。用蛋白质合成抑制剂或内质网与高尔基体复合体之间的囊泡运输抑制剂处理,并未阻止CM对PRL释放的刺激作用。然而,阻断向质膜的运输则完全消除了CM的作用。这些结果表明,哺乳期大鼠AP的CM含有能够诱导雄性AP中PRL快速囊泡释放的可溶性因子,其通过独立于蛋白质合成的机制源自预先形成的成熟颗粒。