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  • Title: Forskolin and phorbol ester stimulation of neuropeptide Y (NPY) production and secretion by aggregating fetal brain cells in culture: evidence for regulation of NPY biosynthesis at transcriptional and posttranscriptional levels.
    Author: Magni P, Barnea A.
    Journal: Endocrinology; 1992 Feb; 130(2):976-84. PubMed ID: 1370798.
    Abstract:
    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)
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