Growth Hormone-Releasing Factor Production by Fetal Rat Cerebrocortical and Hypothalamic Cells in Primary Culture*

Abstract

Recent data from our laboratory and others have shown radioimmunoassayable GRF (IR-GRF) in the rat brain cortex. In the present study the ontogenesis of immunoreactive rat(r) GRF (IR-GRF) in long term dissociated fetal rat cerebrocortical and hypothalamic cell cultures and the regulation of its secretion by potassium depolarization and calcium channel-blocking agents were investigated. The chromatographic profiles of IR-rGRF from cell extracts were determined and compared with those from in vivo cerebrocortical and hypothalamic tissues. Mechanically dispersed cultured telencephalic and diencephalic cells from 17- to 21-day-old fetal rats showed a progressive increase in IR-rGRF, reaching maximum values (media plus cells) between 775-1020 (pg/mg protein) in hypothalamic cells on days 10-20 and between 450 and 950 pg/mg protein in cortical cells on days 25-30. IR-rGRF from acidic extracts of cells and adult cortical and hypothalamic tissues adsorbed onto octadecylsilyl-silica columns corresponds primarily to rGRF-(1-43)OH on HPLC. In gel filtration chromatography, almost all IR-rGRF from cultured cerebrocortical cells and fetal and adult cortical tissues coeluted with rGRF-(1-43)OH. IR-rGRF from cultured hypothalamic cells showed an additional component with a higher mol wt eluting in the void volume. To determine the influence of membrane depolarization of rGRF release, potassium (K+) concentrations in the medium were increased to 30 and 56 mM, inducing a marked increase in medium rGRF concentrations. Verapamil, a Ca2+ channel blocker (20 microns) reverses the effect of 56 mM potassium depolarization, suggesting that it is at least partially dependent upon Ca2+ transport. These data indicate that fetal rat cerebrocortical and hypothalamic cells in culture produce and release rGRF in response to depolarizing agents. The presence of rGRF in cortical tissue suggests that there are other physiological roles besides those implicated in the stimulation of GH secretion.

Insights into a role of GH secretagogues in reversing the age-related decline in the GH/IGF-I axis

“…This inhibition was completely abolished with the blockade of SS with antiserum, thus indicating that the inhibitory effect was exerted by SS. This inhibition could be due to the fact that SS secretion by fetal hypothalamic cells in this in vitro model is 10 times greater than that of GHRH (11,14).…”

“…After 5 h of incubation, defined medium was replaced by Neurobasal medium. Experiments were performed after 15 days in vitro, which is when we have observed that high levels of SS secretion and the peak of GHRH secretion occur (11,14). Medium was changed every 3-4 days.…”

“…IR-GHRH and IR-SS were measured in hypothalamic tissue extracts by previously described RIAs with specific GHRH and SS antisera raised in our laboratory as previously described (14,15). The tissue peptide content was expressed as a function of the total protein determined by the Bradford method.…”

“…Free-floating sections were collected in cryoprotectant (30% glycerol, 30% ethyleneglycol in 0.1 M phosphate buffer) and stored at Ϫ80°C until being processed. Specific polyclonal antisera raised in rabbits against rat GHRH and SS were used in this study (14,15). All antisera were diluted in PBS containing 0.2% Triton X-100 (Sigma).…”

“…ronment, we established a model of coculture of pituitary and fetal hypothalamic cells, where interactions between the two cellular populations and certain treatments could be studied. To this end, hypothalamic cells from 17-day-old embryos were cultured for 15 days, the time at which we had observed the peak of GHRH and high levels of SS secretion (11,14).…”