Magni P, Barnea A
Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas 75235-9032.
Endocrinology. 1992 Feb;130(2):976-84. doi: 10.1210/endo.130.2.1370798.
Using fetal brain cells in culture, we have previously shown that activation of the cAMP pathway by forskolin induces the production and secretion of neuropeptide Y (NPY). In this study we wished to ascertain 1) if activation of the protein kinase C pathway induces NPY production and/or secretion and if there is synergism between the pathways, and 2) the role of protein/RNA synthesis and influx of extracellular calcium. Aggregates, formed from dissociated cells obtained from the hypothalamus-olfactory tubercle area of 17-day-old rat fetuses, were cultured in serum-free medium for 12 days. The NPY content of aggregates incubated for 24 h with solvent (control) was 4.4 ng/flask, and the medium content was 7.6 ng. Forskolin (10 microM) or phorbol 12-myristate 13-acetate (PMA; 20 nM) marginally affected aggregate content, but each increased medium content 2- to 3-fold; forskolin and PMA were additive. When cycloheximide (75 microM) was included along with forskolin, PMA, or forskolin plus PMA for a period of 10 h, the increase in NPY medium content was abolished. Actinomycin D (Act-D; 5 micrograms/ml) inhibited the response to each secretagogue in a time-dependent manner. When Act-D was included along with forskolin, PMA, or forskolin plus PMA for a total period of 12 or 24 h, the 12 h increase in content was not affected, whereas the 24 h increase was abolished. When the presence of Act-D was limited to 0-24, 6-24, or 12-24 h, and forskolin plus PMA were included for the entire 24-h period, the increase in NPY content was inhibited by 94%, 57%, and 12%, respectively. Verapamil (100 microM) totally inhibited the 24 h response to forskolin and partially (40-50%) inhibited the response to PMA or forskolin plus PMA. In none of these conditions was the inhibition of the increase in medium NPY content accompanied by an increase in aggregate content, nor was the NPY content of aggregate/medium of control cultures affected.(ABSTRACT TRUNCATED AT 400 WORDS)
我们之前利用培养的胎儿脑细胞发现，福司可林激活环磷酸腺苷（cAMP）途径可诱导神经肽Y（NPY）的产生和分泌。在本研究中，我们希望确定：1）蛋白激酶C途径的激活是否会诱导NPY的产生和/或分泌，以及这些途径之间是否存在协同作用；2）蛋白质/RNA合成以及细胞外钙内流的作用。从17日龄大鼠胎儿下丘脑 - 嗅结节区域分离的细胞形成的聚集体，在无血清培养基中培养12天。用溶剂（对照）孵育24小时的聚集体中NPY含量为4.4纳克/瓶，培养基中含量为7.6纳克。福司可林（10微摩尔）或佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA；20纳摩尔）对聚集体含量影响不大，但每种都使培养基中含量增加2至3倍；福司可林和PMA具有相加作用。当与福司可林、PMA或福司可林加PMA一起加入放线菌酮（75微摩尔）10小时，NPY培养基中含量的增加被消除。放线菌素D（Act - D；5微克/毫升）以时间依赖性方式抑制对每种促分泌剂的反应。当与福司可林、PMA或福司可林加PMA一起加入Act - D共12或24小时时，12小时时聚集体中含量增加不受影响，而24小时时增加被消除。当Act - D的存在时间限制在0 - 24、6 - 24或12 - 24小时，且在整个24小时期间加入福司可林加PMA时，NPY含量的增加分别被抑制94%、57%和12%。维拉帕米（100微摩尔）完全抑制对福司可林的24小时反应，并部分（40 - 50%）抑制对PMA或福司可林加PMA的反应。在这些情况下，培养基中NPY含量增加的抑制均未伴随聚集体中含量的增加，对照培养物的聚集体/培养基中的NPY含量也未受影响。（摘要截短至400字）
