Ontogenetic development of the inhibition of growth hormone release by somatostatin in the rat: in-vivo and in-vitro (perifusion) study

An in-vitro study of GH secretion by rat fetal and neonatal pituitary glands was conducted using a perifusion system. After a 2 h period the GH content of the effluent was constant. Theophylline, thyrotrophin releasing hormone (TRH) and rat stalk median emience extract (SME) were effective stimuli of GH release from the pituitary glands of the 19.5-day-old fetuses. Somatostatin, added to the medium (10 microgram/ml), had no inhibitory effect on GH release (basal or stimulated by either theophylline or SME) before day 4 after birth. After postnatal day 5, somatostatin always inhibited GH secretion. These findings were consistent with the results of experiments in vivo. In rats tested within 4 days of birth, sodium pentobarbitone-stimulated plasma GH levels were not reduced by somatostatin; on day 4 and thereafter somatostatin depressed the response to pentobarbitone injection. These results indicate a postnatal maturation of the regulation of GH release by the hypothalamo-hypophysial system in the rat.     

Growth hormone (GH) autofeedback action in the neonatal rat: involvement of GH-releasing hormone and somatostatin

It is known that in adult rats, GH by itself and by promoting secretion of the somatomedins acts at the level of the hypothalamus to trigger release of somatostatin and decrease output of GH-releasing hormone (GHRH), thereby inhibiting further secretion of GH. To assess whether these mechanisms are already operative in the early postnatal period, we have evaluated the effect of short-term administration of GH in 10-day-old rats. Twice-daily s.c. administration of 25 micrograms human GH/rat, from days 5 to 9 of life, significantly reduced pituitary content of GH, decreased hypothalamic levels of GHRH mRNA and abolished the in-vivo GH response to a challenge dose of GHRH (20 ng/100 g body weight, s.c.). GHRH (20 ng/100 g body weight, twice daily, s.c.) given concomitantly with the GH treatment, completely counteracted the inhibitory effect of the latter on pituitary content of GH and restored to normal the in-vivo GH response to the GHRH challenge. These data indicate that impaired secretion of GHRH is involved in the inhibitory effect elicited by GH treatment in infant rats. However, concomitant involvement of hypothalamic somatostatin as a result of GH treatment cannot be ruled out. In fact, pituitaries from rats pretreated with GH responded in the same manner as pituitaries from control rats to the GHRH challenge in vitro.     

Normal and growth hormone (GH)-secreting adenomatous human pituitaries release somatostatin and GH-releasing hormone

Neuropeptides such as vasoactive intestinal peptide, LHRH, or TRH have been found in rat pituitary tissue and could act via paracrine or autocrine actions in this tissue. In this study we investigated whether normal human pituitary tissue and GH-secreting human pituitary adenomas could release somatostatin (SRIH) and GHRH. Fragments from three human pituitaries and dispersed cells from six GH-secreting adenomas (four adenomas were studied for GHRH release and five for SRIH release) were perifused using a Krebs-Ringer culture medium, and the perifusion medium was collected every 2 min (1 mL/fraction for 5 h). GH, GHRH, and SRIH were measured by RIA under basal conditions and in the presence of 10(-6) mol/L TRH or SRIH. Both normal pituitaries and GH-secreting pituitary adenomas released SRIH and GHRH. SRIH release commenced 90-180 min after initiation of the perifusion, at which time GH secretion had decreased significantly. TRH stimulated SRIH release from normal pituitary tissue and inhibited SRIH release from adenoma tissue. GHRH was present at the start of the perifusion, but rapidly disappeared. However, SRIH stimulated GHRH release from normal pituitary tissue, but not from adenoma tissue. Significant amounts of GHRH and SRIH were released during the experiments, suggesting their local synthesis. These results indicate that pituitary cells can release hypothalamic peptides. The liberation of these neuropeptides is regulated, and moreover, their regulation differs between normal and adenomatous pituitaries.     

Maturation of the inhibitory response of growth hormone secretion to ether stress in postnatal rat

To study the maturation of inhibitory influences on growth hormone (GH) secretion the effect of ether stress on plasma GH levels was studied during postnatal ontogenesis in female rats. Ether stress did not affect plasma GH levels in 1-day-old pups. A distinct decrease of plasma GH was found in 3- and 9-day-old pups, and the response was prevented by treatment of 3-day-old animals with somatostatin antiserum. No effect of ether stress on plasma GH was noted in 12-, 15-, 18- and 21-day-old rats. Treatment of intact 12-day-old pups with the somatostatin antiserum increased plasma GH level under basal conditions. The inhibitory effect of ether stress on plasma GH was noted again at the age 30 days and in adult animals. It is concluded that the hypothalamus of 3-day-old rats is able to release enough somatostatin to inhibit GH secretion after stress. At the period 12-18 days a phase of pituitary refractoriness was noted: ether stress as well as TRH injection (our previous observation) fail to affect plasma GH in female pups, probably due to high somatostatin secretion under basal conditions and (or) low capacity of pituitary to release GH. It is suggested that regulation of GH secretion is not mature until after the 21st day of life.     

Neuroendocrine regulation of thyrotropin release in cultured human pituitary cells

The regulation of TSH release in man was investigated using cell cultures derived from human pituitaries obtained within 24 h of accidental death. TRH stimulated TSH release in a dose-dependent manner. The ED50 was 2.9 +/- 0.6 (+/- SEM) nmol/L, similar to that reported for rat pituitary cell cultures. The release of TSH was calcium dependent, since the calcium channel antagonist verapamil inhibited TRH-stimulated TSH release, and the calcium ionophore A23187 stimulated TSH release. 12-O-Tetradecanoyl-phorbol-13-acetate stimulated TSH secretion, while dibuytryl cAMP had no effect. Epinephrine and serotonin stimulated TSH release, and dopamine and somatostatin inhibited TRH-stimulated TSH release. These findings have directly demonstrated that the regulation of TSH secretion by hypothalamic neuropeptides and biogenic amines in the human pituitary is similar to that in the rat. The development of a tissue culture system to study thyrotrophs from postmortem human pituitaries provides the means for detailed studies of the regulation of TSH secretion in man.     

Altered responsiveness to hypophysiotrophic hormones of perifused rat pituitary tumours.

Since growth hormone (GH) and prolactin (Prl) secretion by human pituitary tumours is often influenced by the hypophysiotrophic hormones thyrotrophin-releasing hormone (TRH) and somatostatin (SRIF), we have examined the responses of several transplantable rat pituitary tumours to these substances in a perifusion apparatus. The MStT/W15 tumour did not alter its secretion of GH and Prl in response to TRH, SRIF, or a partially purified porcine hypothalamic extract containing GH-releasing activity; normal rat pituitaries show clear responses to each of these substances. Theophylline and dibutyryl cyclic AMP each provoked increased GH and Prl release from the tumour. A second specimen of the MStT/W15 tumour and a specimen of the MStT/W5 tumour behaved in a manner identical to the original MStT/W15, showing no response to TRH or SRIF, but releasing both GH and Prl when theophylline or dibutyryl cyclic AMP was given. The MtT/F4 tumour increased its secretion of GH in response to TRH, 10 mug/ml, and theophylline, but no effect was seen with lower concentrations of TRH or with SRIF; Prl secretion by the F4 tumour was increased by theophylline, but TRH and SRIF had no effect. The autonomy demonstrated in these experimental tumours may be due to a loss of specific hypophysiotrophic hormone receptors or of secretory activating mechanisms.     

gamma-Aminobutyric acid stimulates pituitary growth hormone secretion in the neonatal rat. A superfusion study

The putative inhibitory neurotransmitter gamma-aminobutyric acid (GABA) elicited a dose-dependent increase in GH secretion from the pituitary of newborn rats. GH secretion increased within 3 min after GABA administration with a peak response at 5-6 min. The lowest effective dose of the GABA agonist muscimol was about 10 times smaller than that of GABA. The GABA effect was antagonized by picrotoxin and bicuculline, suggesting that GABA acts at GABA-A type receptors. The pituitary responsiveness to GABA gradually decreased during the second and third postnatal weeks. If the neonatal pituitaries were continuously exposed to GABA for 3 h GH secretion rapidly increased to a maximum within the first 10 min and then gradually decreased to a less elevated level by 1 h and remained at this level for the next 2 h. After 3 h of GABA exposure muscimol had no effect on GH secretion but human pancreatic GH-releasing factor stimulated it, indicating receptor desensitization during prolonged GABA administration. The significance of GABAergic regulation of GH secretion in the neonate is emphasized by the finding that simultaneous administration of picrotoxin diminished the GH releasing activity of the hypothalamic extract of 2-day-old rats by more than 60%. These results indicate that in the postnatal period the regulation of GH secretion differs from that of the adult animal and GABA might play an important role in the maintenance of the high GH secretion during the first days of life.     

Growth hormone regulation in primary fetal and neonatal rat pituitary cell cultures: the role of thyroid hormone

GH is first detectable in the fetal rat pituitary between gestational days 18 and 19. The reasons for the GH surge soon after birth and subsequent postnatal decline to adult levels remain unclear. We therefore determined whether GH gene regulation in the developing pituitary could be distinguished from adult rat somatotroph function. In primary cultures of fetal and neonatal rat pituitary cells, GH secretion was detected by the 20th gestational day. These cells were stimulated by GH-releasing hormone (GHRH), but not by T3 or the morphogen retinoic acid. The stimulatory effect of T3 (0.25 mM) on GH secretion was detected only on the 2nd neonatal day and was similar to that seen in mature rat pituitary cell cultures. GHRH (10 nM) treatment for 24 h caused a 5-fold induction of GH secretion in pituitary cells derived from 2-, 5-, and 12-day-old neonatal rats. The presence or absence of T3 in the culture medium did not alter the response to GHRH. In contrast, only 2-fold induction of GH was observed in adult male pituitary cells during the same time course. Insulin-like growth factor-I (IGF-I; 6.5 nM), the peripheral target hormone for GH, resulted in a modest (20%) attenuation of GH secretion from pituitary cells derived from 20-day-old fetuses. IGF-I, however, produced a 70% reduction in GH levels in adult male pituitary cells grown under similar conditions. The effects of IGF-I on adult pituitary cells grown in T3-depleted medium were blunted. Addition of T3 partially restored the responsiveness of these cells to IGF-I. The results suggest that the high circulating GH levels in the fetal and neonatal rat may be secondary to relative insensitivity of the immature somatotroph to the inhibitory actions of IGF-I in addition to enhanced responsiveness to GHRH compared with the adult rat pituitary. Relative thyroid hormone deficiency in the immature rat may be contributory to this early transient state of pituitary IGF-I resistance.     

Neuropeptides of anterior pituitary origin

Several neuropeptides classically associated with the hypothalamus have been found in the anterior pituitary. The question arises whether they are locally synthesized and if they play a paracrine or autocrine role on pituitary hormone secretion. Using normal and tumoral human pituitaries we found neuropeptides (TRH, SRIH, GHRH) and dopamine in variable quantities according to the nature of the tissue. They were all present in normal pituitaries, while stimulatory hormones (TRH and GHRH) were predominantly found in tumoral tissue, implying an imbalance of pathophysiological importance between the stimulatory and inhibitory control of hypophyseal hormones (PRL and GH) in pituitary adenomas. Both normal and tumoral pituitaries released TRH, SRIH and GHRH in large amounts suggesting their local synthesis. The in situ synthesis was demonstrated for SRIH by the evidence of SRIH mRNA, the detection of SRIH immunoreactivity in peculiar cells and the presence of SRIH precursor. The possible role of these pituitary neuropeptides was suggested for instance by the negative correlation found in vitro between SRIH and GH secretions. Moreover neuropeptides could interact on each other. Indeed DA stimulated TRH release while PRL secretion decreased at the same time. Pulses of TRH had differential effects on SRIH release according to the nature of the tissue as TRH inhibited SRIH release from adenoma while it stimulated SRIH release from normal pituitary. Concerning the effects of SRIH and GHRH on GH secretion, there was an endogenous regulatory pattern comparable to that described in rat portal blood vessels. Pulses of GHRH induced GH secretion only when endogenous SRIH release was not stimulated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Age-related changes in prolactin and growth hormone release from pituitary glands in vitro

The basal release of prolactin from cockerel anterior pituitary glands in vitro declined between 1 and 7 weeks of age, to a level less than that released by pituitary glands from 18 week old (adult) cockerels and hens. Basal growth hormone (GH) release increased between 1 and 7 weeks of age but had declined in adults to a level similar to that released from 4 weeks old cockerels. The responsiveness of the pituitary gland to hypothalamic stimulation, using hypothalami from 8 week old broiler fowl, was also age-related. Prolactin release was considerably higher from pituitaries of 1 week old cockerels compared to the other age groups. Stimulation of GH release by the hypothalamus was higher from pituitaries of both 1 and 7 week old cockerels compared to the other groups of birds. The increase in release of prolactin following incubation with thyrotrophin releasing hormone (TRH) declined between 1 and 7 weeks, but increased slightly in adult birds, whereas the increase in release of GH following TRH was higher from pituitaries of both 1 and 7 week old cockerels. Hypothalamic prolactin (Prl) releasing activity, measured as the ability of the hypothalamus to stimulate hormone release from 8 week old broiler fowl anterior pituitary glands, declined with the age of the donor cockerels. The hypothalami from adult hens secreted significantly more Prl releasing activity than did adult cockerel hypothalami. The secretion of GH releasing activity decreased markedly with the age of the donor bird. These results suggest that maturational patterns of hormone secretion in fowl are partly due to changes in autonomous hormone release, to changing patterns of hypothalamic activity and to differences in pituitary responsiveness to provocative stimuli.     

Inhibition of hypothalamic somatostatin release by beta-adrenergic antagonists

A number of in vivo studies suggest that hypothalamic somatostatin (SRIF) tone is stimulated by the beta-adrenergic system. Employing dispersed adult male rat hypothalamic cells, we studied the effects of beta-adrenergic antagonists on the release of hypothalamic SRIF. Propranolol, at concentrations of 1-100 microM, had no detectable effect on basal SRIF release, but caused dose-dependent inhibition of SRIF release stimulated by ouabain. Two other beta-adrenergic antagonists, labetolol and metoprolol, also caused inhibition of ouabain-stimulated SRIF release. The alpha 2-agonist clonidine was without effect on SRIF release under basal or stimulated conditions. GH secretion from monolayers of dispersed rat anterior pituitary cells was also examined. Propranolol (1-100 microM) had no significant effect on basal GH secretion or GH secretion stimulated by rat GRF. In conclusion, 1) beta-adrenergic antagonists caused inhibition of stimulated SRIF release; 2) clonidine had no detectable effect on SRIF release; and 3) propranolol did not affect GH secretion in vitro. These findings support the hypothesis that beta-adrenergic antagonists augment GH responsivity by inhibiting hypothalamic SRIF release.     

Influence of endogenous somatostatin on growth hormone and thyrotropin secretion in neonatal rats

When gestating rats were injected iv with an antiserum to somatostatin (SRIF-AS) during the last week of gestation, serum GH levels in fetuses and 6-h-old newborn rats were not significantly different from controls. Similarly, 2 h after the ip administration of SRIF-AS, no change in serum GH concentration was observed in 2-h-old rats. However, under the same conditions, a significant increase in serum GH was observed in 24-h-old rats and in 2- to 60-day-old rats. The injection of SRIF-AS neither changed basal serum TSH levels during the neonatal development nor in the adult stage. A significant increase in TRH-induced TSH release was observed after the third postnatal day. It is concluded that endogenous SRIF plays a physiological role in GH release by 24 h of age in the rat and that the fall in GH secretion that normally occurs during the first week of life is due to the development of inhibitory mechanisms mediated by hypothalamus SRIF. Additionally the results suggest that the influence of SRIF upon TSH secretion is present before that of TRH.     

Inhibition of adrenocorticotropin secretion by somatostatin in pituitary cells in culture

AtT20/D16v is a clonal strain of mouse pituitary tumor cells which synthesizes and secretes ACTH. Somatostatin, a hypothalamic tetradecapeptide, has been shown to inhibit the release of PRL, GH, and TSH from the pituitary gland. We have characterized specific binding sites for somatostatin on AtT20/D16v cells and demonstrate that somatostatin inhibits stimulated ACTH release by these cells. Equilibrium binding studies with [125I]Tyr1]somatostatin showed the presence of a single class of noninteracting binding sites on AtT20/D16v cells. Half-maximal binding of somatostatin occurred at 1.7 X 10(-9) M, and there were 26,300 binding sites/cell. The binding of [125I]Tyr1]somatostatin was not significantly inhibited by the hypothalamic peptides TRH, LHRH, and substance P. Somatostatin had no consistent effect on basal ACTH secretion by AtT20/D16v cells, but it inhibited ACTH secretion stimulated with either 50 mM KCl or a hypothalamic extract. Half-maximal inhibition occurred with 4 X 10(-10) M somatostatin. TRH, LHRH, and substance P at concentrations of 10(-7) M were without effect. Somatostatin had no effect on either basal or stimulated hormone secretion by GH12C1 or F4C1 cells, two cell strains which lack specific somatostatin-binding sites. A critical concentration of extracellular calcium was required for the stimulation of ACTH secretion in AtT20/D16v cells. No response to 50 mM KCl occurred in the presence of EGTA or cobalt. Increased extracellular calcium overcame the inhibition of stimulated hormone secretion by EGTA, cobalt, and somatostatin. Therefore, we conclude that the inhibition of stimulated ACTH secretion by somatostatin involves the interaction of the peptide with specific binding sites on AtT20/D16v cells and the inhibition of stimulus-elicited calcium influx.     

Galanin secretion from anterior pituitary cells in vitro is regulated by dopamine, somatostatin, and thyrotropin-releasing hormone.

Lactotrophs, somatotrophs, and thyrotrophs have been shown to contain immunoreactive galanin. Furthermore, estrogen stimulates galanin mRNA and peptide levels in the rat anterior pituitary, particularly within lactotrophs. To determine whether galanin is released from the anterior pituitary in a regulated manner, we used cultured pituitary cells from male and ovariectomized Fischer 344 rats implanted with estrogen-containing capsules. Anterior pituitary cells (5 x 10(5) cells/well) were challenged (0.5-3 h) with hypothalamic factors known to regulate anterior pituitary hormone secretion, and medium galanin levels were measured by RIA. In female pituitary cells, galanin secretion was inhibited by dopamine (10 and 100 nM) and stimulated by TRH (20 and 100 nM). Although galanin release was significantly lower in male pituitary cells, dopamine and TRH inhibited and stimulated galanin secretion, respectively. Medium galanin levels were also significantly reduced by somatostatin (5 nM) in both female and male cells. The pattern of PRL release in response to dopamine, TRH, and somatostatin was similar to that observed for galanin, regardless of the sex of the pituitary donor. Although galanin has been localized in somatotrophs, 5 nM GH-releasing hormone (GRF) failed to alter galanin release in male as well as female pituitary cells; GH secretion was significantly increased by GRF. LHRH (5 nM) and CRF (5 nM) failed to alter galanin release in vitro. We conclude that in estrogen-exposed pituitary cells obtained from male and ovariectomized Fischer 344 rats: 1) galanin secretion is inhibited by dopamine and somatostatin, and stimulated by TRH; 2) GRF, LHRH, and CRF do not regulate galanin release in these cells; and 3) the profile of the regulated pathway for galanin release suggests that the primary location of galanin is the lactotroph, probably within secretory granules.     

Insulin, glucagon, somatostatin, and thyrotropin-releasing hormone content and secretion by perifused fetal rat islets during culture

In the neonatal period of the rat, pancreatic thyrotropin-releasing hormone content decreases and the sensitivity of insulin secretion to glucose increases. In adult rat islets, TRH inhibits glucose-induced insulin release. The aim of this study was to investigate whether a high TRH content and release can be part of the explanation for the functional immaturity of neonatal islets. For that purpose, we have measured the tissue content and the secretion of immunoreactive insulin, glucagon, somatostatin and TRH in islets from 21.5-day-old rat fetuses cultured for up to one week. Insulin, glucagon and somatostatin content increased during one week of culture in the presence of 11.1 mmol/l glucose. The TRH content decreased during culture, but did not equal adult values. Insulin, glucagon and somatostatin responses to glucose were present after one week of culture. Glucose had no effect on TRH release in cultured fetal islets, but inhibited TRH release in adult islets. We conclude that glucose can stimulate insulin secretion without inhibiting TRH release, but that a decrease in islet TRH content and a sensitization of TRH secretion to glucose may be important in the full maturation of fetal pancreatic islets.     

Acute effects of hypothalamic ablation on plasma thyrotropin and prolactin concentrations in the suckling rat: evidence that early postnatal pituitary-thyroid regulation is independent of hypothalamic control.

Hypothalamic ablation was performed at various periods postnatally in animals previously administered propylthiouracil to raise plasma TSH concentrations. There was no significant change in plasma Tsh up to 8 h after hypothalamic ablation in pups 1--4 days old, whereas hypophysectomy of such pups produced a 60% fall in plasma TSH within 4 h. By the 5th postnatal day, hypothalamic ablation produced a 30% fall in plasma TSH within 4 h (P less than 0.05). By the 12th postnatal day and thereafter, the fall in plasma TSH after hypothalamic ablation was not significantly different from that seen in adults, except in 30-day-old rats in which there was a lesser effect of hypothalamic ablation on plasma TSH (P less than 0.01 in comparison to 23-day-old and adult groups). The greatest effect of hypothalamic ablation on plasma TSH was in 45-day-old animals (P less than 0.01 in comparison to adults). No significant change was produced in plasma PRL within 4 h postoperatively at any age. Our data indicate that regulation of TSH secretion in the rat is independent of hypothalamic control until after the 5th postnatal day and is fully developed by day 12. This corresponds temporally with the postnatal rise of plasma TSH, T4, and T3 and hypothalamic TRH to adult concentrations and indicates maturation of the hypothalamic regulation of TSH secretion.     

In vivo studies with growth hormone (GH)-releasing factor and clonidine in rat pups: ontogenetic development of their effect on GH release and synthesis

The demonstration that GH-releasing factor (GRF) stimulates GH synthesis and release in rat pups prompted studies to evaluate the effects on the same indices of clonidine (CLO), an alpha 2-adrenoceptor and potent GH secretagogue, purported to act in adult rats via GRF release. Our first aim was to ascertain whether CLO elicits GH release in rat pups via GRF, and if this is the case, to evaluate the ontogenetic development in 1- to 10-day-old pups of the GH response to acute CLO or GRF administration and, finally, the effects of short term CLO or GRF treatment on plasma and pituitary GH concentrations and on the GH response to an acute challenge with the homologous secretagogue. CLO (15 micrograms/100 g BW, sc) induced a clearcut GH rise in 10-day-old rats but not in pups pretreated with a specific anti-GRF serum. Moreover, unlike GRF (10(-8) M), CLO (10(-6) to 10(-5) M) did not stimulate GH release in vitro from anterior pituitaries of 10-day-old rats. In 1-day-old rats, neither CLO (15 micrograms/100 g BW, sc) nor GRF (20 ng/100 g BW, sc) stimulated GH release, whereas significant GH stimulation was elicited by GRF, but not CLO, in 5-day-old rats and by both secretagogues in 10-day-old rats. Short term treatment with CLO (15 micrograms/100 g BW, sc, twice daily) or GRF (20 ng/100 g BW, sc, twice daily) on postnatal days 1 through 5 did not modify either plasma or pituitary GH concentrations 14 h after the last drug administration, but did so when either secretagogue was administered on postnatal days 5 through 9. Finally, an acute challenge with GRF, but not with CLO, induced a further rise in the already elevated plasma GH levels of pups pretreated from postnatal day 5 through 9, but neither secretagogue did so in pups pretreated from postnatal days 1 to 5. Viewed together, these data indicate that in infant rats CLO releases GH via GRF release and that the somatotropes respond earlier to GRF (5 days) than the GRF-secreting structures do to alpha 2-adrenergic stimulation (10 days). Both GRF and CLO stimulate GH synthesis when administered repeatedly. However, whereas repeated GRF treatment has a priming effect on the somatotropes, CLO does not, probably because of down-regulation of hypothalamic alpha 2-adrenoceptors.     

Normal and adenomatous human pituitaries secrete thyrotropin-releasing hormone in vitro: modulation by dopamine, haloperidol, and somatostatin

TRH is present in human normal pituitaries and in pituitary adenomas. In this study we demonstrated that the same tissues can release TRH in vitro. Fragments from seven normal pituitaries (10-15 mg/syringe) and dispersed cells from eight prolactinomas, four GH-secreting and two nonsecreting adenomas (1-3 x 10(6) cells/syringe) were perifused using a Krebs-Ringer culture medium. After 1 h of equilibration the perifusion medium was collected every 2 min (1 mL/fraction) for 3 h. TRH, PRL, and GH were measured by RIA under basal conditions and in the presence of 10(-10) to 10(-6) mol/L dopamine (DA), alone or concomitant with haloperidol, or in the presence of 10(-10) or 10(-6) mol/L somatostatin. Both normal pituitary fragments and pituitary adenomatous cells (from all types of adenomas studied) spontaneously released TRH in vitro. TRH was detected in the perifusion medium either immediately after the end of the equilibration period or 30-60 min later. The molecular identity of TRH was assessed by high pressure liquid chromatography. There was no difference in the profile and the rate of TRH secretion between normal and tumoral tissues, and no correlation was found between the level of TRH release and that of PRL or GH secretion. DA stimulated TRH release from normal pituitaries and from PRL- and GH-secreting adenomas at doses as low as 10(-10) mol/L. A concomitant decrease in PRL and GH release was observed from adenomatous cells and in one case of normal tissue. Haloperidol (10(-7) mol/L) antagonized the effect of 10(-8) mol/L DA on both TRH and PRL secretion in normal pituitary and in prolactinomas. DA had no effect on TRH release from two nonsecreting tumors. The amounts of TRH released during 1 h of perifusion were 60-1640 pg/2 mg wet wt tissue in normal pituitaries and 54-2174 pg/10(6) cells in adenomas; these values were very high compared to those precedently reported within the tissues. These results indicate that pituitary cells can release TRH in vitro and suggest that TRH might be synthesized in situ. We suggest that TRH could act on pituitary hormone secretion and/or cell proliferation via a paracrine and/or an autocrine mechanism.     

Differential responsiveness of the somatotroph to growth hormone-releasing factor during early neonatal development in the rat

The effects of human pancreatic GH-releasing factor-40 (hpGRF-40; 0.01-100 nM) and (Bu)2cAMP (0.015-1.5 mM) on GH release from primary monolayer cultures of pituitary cells were evaluated in rats of three age groups: postnatal days 2 and 12, and young adult males (3-4 months). Both hpGRF-40 and (Bu)2cAMP elicited a dose-related increase in GH release in cell cultures from each age group. However, the magnitude of the fractional increase over basal release was markedly age dependent. hpGRF-40-stimulated GH release (expressed as a percentage of control values) was greater in cultured cells of 2-day-old than of 12-day-old rats, which was, in turn, significantly greater than in cells of adult rats (P less than 0.001). Maximum hpGRF-40-stimulated GH release was 1058 +/- 50% of control values (+/- SE) in 2-day-old, 617 +/- 21% of control values in 12-day-old, and 405 +/- 6% of control values in adult pituitary cell cultures. The slopes of the dose-response curves differed significantly among the three age groups (P less than 0.001) and varied inversely with increasing age. GH release induced by (Bu)2cAMP was similarly age dependent; maximal stimulated release was 1073 +/- 20%, 414 +/- 4%, and 259 +/- 7% of control values in cultured cells of 2-day-old, 12-day-old, and adult rats, respectively (P less than 0.001 for age effect at each dose). As with hpGRF-40, the slopes of the dose-response curves for (Bu)2cAMP decreased with advancing age (P less than 0.001). Intracellular GH storage during culture, basal release of GH, and serum GH were also age dependent. Pooled serum GH was consistently elevated in 2-day-old rats (139 +/- 2 ng ml-1), became lower and more variable in 12-day-old rats (62 +/- 14 ng ml-1), and was even more variable in adult male rats (79 +/- 23 ng ml-1), owing to random sampling during spontaneous secretory pulses. These results indicate that the stimulatory effects of GRF and (Bu)2cAMP on GH secretion from cultured rat pituitaries vary with age; pituitary cells of newborn rats are relatively more sensitive to these secretagogues than those of adult rats. This increased responsiveness of the neonatal somatotroph to GRF may contribute to the elevation of the plasma GH concentration which is characteristic of the perinatal period in the rat.