Synthesis and secretion of vasoactive intestinal peptide by rat fetal cerebral cortical and hypothalamic cells in culture.
To establish the neurosecretory activity of brain vasoactive intestinal peptide (VIP)ergic neurons and to characterize the molecular forms of secretion of these cells, fetal cerebrocortical and hypothalamic cells were grown in primary cultures for periods up to 4 weeks, their regulation by depolarization and calcium and sodium channel active agents was studied, and the chromatographic patterns of cell and medium VIP were determined. Mechanically dispersed cultured fetal telencephalic and diencephalic cells showed a progressive increase in immunoreactive VIP in both cells and media, reaching maximum values between 110-290 pg/mg protein.culture plate on days 15-20. Immunoreactive VIP in both cells and media corresponded almost exclusively to VIP-28 on exclusion chromatography and HPLC, and was identical to the form extracted from whole fetal brain. This indicates that both the stored and secreted forms of VIP are the mature 28-amino acid-containing peptide. To determine the influence of membrane depolarization on VIP release, the potassium concentration in the medium was increased to 30 and 56 mM, inducing a marked increase in medium VIP concentrations. The effects of K+ were dependent on Ca2+ transport, since release was blocked by the addition of verapamil, a Ca2+ channel blocker (20 microM). VIP release was stimulated by Na+ channel activation using the drug veratridine (100 microM); this effect was blocked by tetrodoxin. The influence of GH-releasing factor (GRF) on VIP release was studied by adding GRF (10(-9)-10(-7) M), alone or in the presence of anti-rGRF immunoglobulins (antirat GRF immunoglobulin G) to the incubation medium. Rat GRF stimulated VIP release and the simultaneous addition of antirat GRF immunoglobulin G blocked this effect. These findings confirm that VIP regulation by brain cells corresponds to the well defined patterns of membrane activation observed in other neuronal systems. They, furthermore, demonstrate that VIP release is under GRF influence.
Lorenzo
M J
MJ
Servicio de Endocrinologia, Hospital Ramón y Cajal, Madrid, Spain.
Sánchez-Franco
F
F
de los Frailes
M T
MT
Reichlin
S
S
Férnandez
G
G
Cacicedo
L
L
D000818:Animals; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D002850:Chromatography, Gel; D004594:Electrophysiology; D005333:Fetus / Q000378:metabolism; D013007:Growth Hormone-Releasing Hormone / Q000494:pharmacology; D007031:Hypothalamus / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D014660:Vasoactive Intestinal Peptide / Q000096:biosynthesis* / Q000378:metabolism; D014701:Veratridine / Q000494:pharmacology
D014660:Vasoactive Intestinal Peptide; D014701:Veratridine; D013007:Growth Hormone-Releasing Hormone; D011188:Potassium