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 μM) 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 μM) 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 μg/ 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 μM) 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. In summary, 1) forskolin and PMA induce an increase in NPY content and secretion by a process requiring both RNA and protein synthesis; 2) there is no evidence for synergism between the two agents; 3) the mRNA species essential for the induced increase in NPY content and secretion (including NPY mRNA itself) exhibit an apparent overall half-life greater than 12 h; and 4) a major fraction of the culture content of NPY is secreted, and this occurs independently of influx of extracellular calcium via a verapamil-sensitive process; the latter, however, is required for forskolin and a partial one for PMA induction of the increase in NPY content. These results are consistent with the cAMP and protein kinase C pathways regulating three major events in the cultured NPY neuron: transcription, translation, and secretion.

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 a transcriptional and post-transcriptional level / P. Magni, A. Barnea. - In: ENDOCRINOLOGY. - ISSN 0013-7227. - 130:2(1992), pp. 976-984. [10.1210/endo.130.2.1370798]

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 a transcriptional and post-transcriptional level

P. Magni
Primo
;
1992

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 μM) 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 μM) 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 μg/ 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 μM) 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. In summary, 1) forskolin and PMA induce an increase in NPY content and secretion by a process requiring both RNA and protein synthesis; 2) there is no evidence for synergism between the two agents; 3) the mRNA species essential for the induced increase in NPY content and secretion (including NPY mRNA itself) exhibit an apparent overall half-life greater than 12 h; and 4) a major fraction of the culture content of NPY is secreted, and this occurs independently of influx of extracellular calcium via a verapamil-sensitive process; the latter, however, is required for forskolin and a partial one for PMA induction of the increase in NPY content. These results are consistent with the cAMP and protein kinase C pathways regulating three major events in the cultured NPY neuron: transcription, translation, and secretion.
somatostatin-like immunoreactivity; vasoactive intestinal peptide; central nervous-system; rat-brain; cyclic-amp; biochemical differentiation; paraventricular nucles; tyrosine-hydroxylase; adenylate-cyclase; dopamine neurons
Settore MED/05 - Patologia Clinica
Settore MED/04 - Patologia Generale
Settore MED/13 - Endocrinologia
Settore MED/46 - Scienze Tecniche di Medicina di Laboratorio
1992
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