Thyroid hormone effects on pituitary ACTH have not been well established. Adult male Sprague-Dawley rats were rendered hypo- and hyperthyroid while undergoing treatment with 6-Propylthiouracil (PTU) and L-Thyroxine (T4). At the time of decapitation, plasma values for T4 (micrograms/100 ml) were 3.9 +/- 0.4 in the control, 17.3 +/- 2.2 in the T4 and less than 2 in the PTU treated group; plasma T3 and TSH confirmed hyper- and hypothyroidism in the T4 and PTU treated groups respectively. Plasma immunoassayable ACTH and corticosterone were significantly increased in hyperthyroid and decreased in the PTU treated animals. Pituitaries were removed and incubated in DMEM. After 3 h incubation, ACTH content and secretion to the medium were significantly lower in the PTU group. As expected, pituitary TSH content and secretion were decreased in the T4 treated animals. These data indicate that thyroid hormones influence pituitary-adrenal function by increasing ACTH secretion and consequently corticosterone production.

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.

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.

Thyroid hormone (TH) action on somatostatin (SRIF) secretion and synthesis by fetal rat brain cells in culture was studied. Cortical and hypothalamic brain cells were maintained as monolayer cultures for 7-10 days. T3, T4, and [3H] phenylalanine [( 3H]Phe) (40 microCi/plate) were added simultaneously for 48 h. Alternately, cultures were pulse labeled with [3H] Phe for only the last 3 h, after being exposed to TH for 45 h. 3H-Labeled SRIF-like material [( 3H]IR-SRIF) was purified by immunoaffinity chromatography and further characterized by gel filtration in Bio-Gel P-10. Total protein synthesis was determined by the incorporation of [3H]Phe into trichloroacetic acid precipitable proteins. Forty eight-hour T3 treatment had a biphasic effect on secretion of IR-SRIF by both cortical and hypothalamic cells. In cortical cells, low doses of T3 (10(-11) M) significantly increased (P less than 0.01) and high T3 doses (10(-7) M) significantly decreased (P less than 0.05) total IR-SRIF (nanograms per plate); control: 2 +/- 0.25; T3 (10(-11) M): 3 +/- 0.3; T3 (10(-7) M): 1.3 +/- 0.1. Similarly, T4 had a significant stimulatory action at 10(-9) M, being inhibitory at 10(-7) M (picograms/plate); control: 290 +/- 20 T4 (10(-9) M): 510 +/- 40; T4 (10(-7) M): 201 +/- 10. When [3H]Phe was added during the 48 h of the experiment, [3H]IR-SRIF synthesis in response to T3 by cortical cells significantly increased after exposure to 10(-11) M (P less than 0.05) and decreased with 10(-7) M (P less than 0.05). When [3H]Phe was added for only the last 3 h or incubation with T3, the action was inhibitory at both 10(-11) M and 10(-7) M. Trichloroacetic acid precipitable material decreased in a dose response manner between T3, 10(-11) M and 10(-7) M. These findings suggest that at this time of brain development, SRIF synthesis by cortical and hypothalamic cells is affected by TH.

The effects of hypothyroidism duration on several factors implicated in GH secretion control were studied in the male rat at different maturity stages, ranging from the peripuberal period to adulthood. Thyroid ablation was performed on 22-day-old Wistar male rats maintained on a low iodine diet (T group). Age-paired controls (C group) were fed with the same diet, supplemented with potassium iodide. Subgroups of T and C animals (aged 32, 42, 52, 82 and 112 days) were studied 10, 20, 30, 60 and 90 days after surgery. After pentobarbital anesthesia, jugular blood was withdrawn before and 5 min after an intravenous TRH stimulus, for GH assay. Hypothalamic and pituitary tissues were obtained in order to measure GH, immunoreactive somatostatin (IR-SRIF) and growth hormone-releasing factor (IR-GRF). Growth rate and serum testosterone confirmed that C rats reached sexual maturity by day 30 of the study. Mean ± SE serum GH (ng/ml) increased (p < 0.05) in C animals from day 10 (38.5 ± 5) to day 30 (67.4 ± 7.3), with no significant variations thereafter. The same time sequence pattern was observed in pituitary GH concentrations. In T rats, both serum and pituitary GH decreased progressively from day 10 to 90, being significantly lower than in C at all times of the study. No GH response to TRH could be found in C groups. In contrast, GH increased significantly (p < 0.05) in T animals after TRH at days 20 and 30. Hypothalamic IR-SRIF concentrations (ng/mg protein) increased (p < 0.05) to adult levels in C rats between days 10 (15.7 ± 3) and 30 (26.3 ± 2.8), remaining unchanged thereafter. T animals showed the same pattern up to day 60. On day 90, IR-SRIF decreased abruptly in T (9.7 ± 2.9) as compared to both normal peers (30.8 ± 7.2; p < 0.01) and 60-day T rats (24.3 ± 5.1; p < 0.05). Hypothalamic IR-GRF (ng/mg protein) also increased (p < 0.05) between day 10 (2.9 ± 0.3) and 30 (4.3 ± 0.7) in C animals. This puberal rise did not occur in 30-day Trats, whose IR-GRF was lower (2.6 ± 0.3; p < 0.01) than in C. It can thus be concluded that a decrease in pituitary GH availability is not the only factor implicated in the abnormal GH secretion found in hypothyroidism. An enhanced GH response to TRH was found to coexist with a 94% reduction in pituitary GH content on days 20 and 30 of hypothyroidism. In the normal rat, hypothalamic IR-SRIF and IR-GRF content increased to adult values in the puberal period. The influence of hypothyroidism on the hypothalamic IR-SRIF content is closely related to its duration, occurring only in long-term thyroid deprivation. Hypothyroidism also prevents the puberal rise of IR-GRF, but does not affect the concomitant increase of IR-SRIF. The blunting of the puberal rise in IR-GRF occurs despite the normal testosterone increase in hypothyroid rats, suggesting that the diminished concentration of thyroid hormone impairs the testosterone-mediated IR-GRF rise.