Major metabolic pathways and hormone production in unstimulated monolayer cultures of the rat anterior pituitary

Summary In cell cultures derived from anterior pituitary glands of rats, enzyme activities of cell homogenates and hormone (GH, PRL, LH, and FSH) content of the culture media were measured. Sex differences in enzyme activities representing major metabolic pathways (citrate cycles, pentose cycles, and glycolysis) were demonstrated both in freshly dispersed cells and in 8-day-old cultures; in cultures of both sexes enzyme activities increased during cultivation. In cultures derived from female rats, cell protein doubled by the 12th day and remained constant for up to the 24th day in culture, whereas enzyme activities showed changes suggesting that cell metabolism shifted to anaerobic glycolysis during cultivation. In the culture media the presence of four pituitary hormones was demonstrated for as long as 3 weeks of cultivation with variable secretion dynamics; the release of gonadotropic hormones diminished gradually whereas that of GH remained constant and PRL levels increased with time. These results indicate that under strictly defined culture conditions pituitary cells may function in spite of profound metabolic changes.     

Inhibitory effects of cysteamine on neuroendocrine function

The action of cysteamine on anterior pituitary hormone secretion was studied in vivo using conscious, freely moving male rats and in vitro using anterior pituitary cells in monolayer culture. Administration of 500 micrograms cysteamine into the lateral cerebral ventricles of normal rats caused the complete inhibition of pulsatile GH secretion for a minimum of 6 h. This treatment also significantly decreased plasma concentrations of LH for at least 6 h in orchiectomized rat, TSH in short-term (0.5 month) thyroidectomized rats, and PRL in long-term (6 months) thyroidectomized rats. The in vivo stimulation of GH, LH, TSH and PRL with their respective releasing hormones 60 min after administration of cysteamine was not different from the response observed in rats pretreated with saline except for PRL where cysteamine pretreatment significantly inhibited the expected PRL increase. In vitro, 1 mM cysteamine decreased basal and TRH stimulated PRL release while not affecting basal or stimulated GH, LH, TSH and ACTH secretion. These data demonstrate the dramatic and wide-ranging effects of cysteamine on anterior pituitary hormone secretion. This action appears to be mediated through hypothalamic pathways for GH, LH and TSH and through a pituitary pathway for PRL.     

Placental lactogen not growth hormone and prolactin regulates secretion of progesterone in vitro by the 40-45 day ovine corpus luteum of pregnancy

The study was designed to compare the direct effect of three prolactin-like hormones on steroidogenesis of ovine luteal cells collected at day 40-45 of pregnancy. 100 ng/ml of ovine placental lactogen or 100 ng/ml of ovine growth hormone or 100 ng/ml of ovine prolactin were added to the media of luteal cell cultures. After 48 h incubation, all cultures were terminated and the media were frozen until further steroid analysis. To determine to what extent growth hormone (GH), prolactin (PRL) and lactogen (PL) regulate the activity of 3 beta-HSD, an enzyme involved in progesterone synthesis, the classical steroidal competitive inhibitor of 3 beta-HSD trilostane, was investigated for its effects on basal and GH-, PRL-, and PL-stimulated progesterone biosynthesis since there is a possibility that the luteotropic effect of these hormones are mediated via 3 beta-HSD. oPL resulted in an increase of progesterone secretion in a statistically significant manner, while GH or PRL had no effect on progesterone secretion. A decrease in progesterone secretion as an effect of 100 mM trilostane was observed in all culture types. An explanation for the luteotropic effect of PL and the lack of this effect for GH is that the GH receptor associates with a different molecule within the ovarian tissue and forms a heterodimeric receptor for PL, and the possibility that physiological effects of native oPL may be mediated through its binding to specific PL receptors, which have low affinities for oGH and oPRL.     

Catecholaminergic control of plasma growth hormone and thyrotropin levels in hypophysectomized rats bearing ectopic pituitary transplants

Transplantation of the anterior pituitary to an ectopic site leads to stimulation of PRL secretion and suppression of the release of other adenohypophyseal hormones. We have previously reported that precursors and blockers of catecholamine synthesis can affect PRL release from the ectopic pituitary. In the present study we have measured the effects of L-3,4-dihydroxyphenylalanine (DOPA), DL-threo-dihydroxyphenylserine (DOPS), alpha-methyl-para-tyrosine (alpha-mpt) and diethyldithiocarbamate (ddc) on plasma growth hormone (GH) and thyrotropin (TSH) levels in hypophysectomized rats with pituitary transplants under the renal capsule. In these animals, peripheral plasma GH levels were elevated by a precursor (DOPA) and reduced by a blocker (alpha-mpt) of catecholamine synthesis. Plasma TSH levels were increased by a precursor (DOPS) and reduced by a blocker (ddc) of norepinephrine synthesis. We suspect that GH and TSH present in the circulation of pituitary-grafted animals were derived, in part, from the ectopic pituitary tissue and suggest that the small but detectable secretion of hormones other than PRL in this animal model is under the control of endogenous catecholamines.     

Prolactin and growth hormone synthesis: effects of perphenazine, alpha-methyltyrosine and estrogen in different thyroid states.

The effects of thyroid hormones on prolactin (PRL) and growth hormone (GH) synthesis by the rat anterior pituitary gland were assessed in vitro. A marked reduction (84-87%) in the rate of H3-leucine incorporation into GH was evident 2-4 weeks after thyroidectomy, while incorporation into PRL was 52-71% less than that measured in glands from intact rats. A single injection of T4 (200 mug/kg) administered to thyroidectomized (THX) rats 48 hr before sacrifice significantly increased incorporation into both pituitary hormones, although the stimulation of GH synthesis was much more dramatic. Perphenazine, alpha-methyltyrosine and estrogen enhanced the rate of PRL synthesis in intact rats. Thyroid ablation did not affect the response to perphenazine, but significantly increased the response to alpha-methyltyrosine and estrogen. On the other hand, administration of T4 to THX rats receiving perphenazine, alpha-methyltyrosine or estrogen diminished the stimulatory influence of these treatments on PRL synthesis. Perphenazine, alpha-methyltyrosine and estrogen had no effect on the rate of GH synthesis in THX rats, nor did they alter the ability of T4 to restore GH synthesis in these animals. These results indicate that GH synthesis in the rat is dependent upon thyroid hormones and support the concept that these hormones exert their stimulatory effect directly on pituitary somatotrophs. Pituitary lactotrophs, however, appear to retain much of their capacity to synthesize PRL under conditions of thyroid deficiency. The changes in pituitary PRL levels and synthesis rate induced by thyroid ablation might reflect differences in the number rather than the activity of these cells.     

Choice of extraction procedure for estimation of anterior pituitary hormone content

Searching for the best procedure for simultaneous estimation of the anterior pituitary hormones, extraction efficiencies of various media, additives such as urea and triton X-100, and physical treatments such as freezing-thawing (F-T) and sonication, were examined by measuring prolactin (PRL), growth hormone (GH), lutropin (LH), follitropin (FSH), and thyrotropin (TSH) in the extracts. Ethanolic media (60% EtOH) gave high yields of PRL at neutral to alkaline pH, but poor extraction of GH accompanied by a marked loss of its immunoreactivity during storage. Ethanolic media also gave a poor yield of LH even at high pH. Aqueous media like PBS at various pH, 0.1 M acetic acid and distilled water were considerably effective in the extraction of GH, LH, FSH and TSH if they were coupled with F-T and sonication. However, high yields of PRL could not be obtained with these aqueous media even with F-T and sonication. Hartree's 40% EtOH-6% ammonium acetate, pH 5.1, solubilized considerable amounts of glycoprotein hormones, but yielded almost no GH and only a small amount of PRL. The addition of triton X-100 to PBS (pH 7) at 0.1% resulted in the maximum extraction of glycoprotein hormones with homogenization and F-T, but further sonication was necessary for GH and PRL. When the anterior pituitaries were homogenized and frozen-thawed in PBS (pH 7) containing 1 M urea, yields of PRL, GH, LH, FSH, and TSH were maximum, and sonication did not cause any additional extraction, indicating that this procedure, i.e. homogenization and F-T in 1 M urea-PBS, would be the best for the simultaneous estimation of these anterior pituitary hormones.     

Inversely related evolution of growth hormone and prolactin secretions in long-term tissue cultures of human pituitary adenomas from acromegalic patients

Summary Pituitary tumoral tissue from 20 acromegalic patients was cultured for up to 120 d in a medium containing 5 nM cortisol. In all cultures, growth hormone (GH) release decreased. At the beginning of the culture, prolactin (PRL) was detected in 18 adenomas, varying from 0.5 to 1000 ng per flask per day. Thereafter, in 10 cases PRL secretion increased from 3 to 50 times the basal level, most frequently after a lapse of 9 to 30 d. PRL secretion remained low in three cases, undetectable in one case only. When added at 350 nM, cortisol increased GH secretion up to 20-fold and simultaneously decreased PRL secretion by as much as 10% of the basal level. Withdrawing cortisol reversed the situation. Immunocytochemical studies of the tumor at surgery showed, besides GH immunoreactive (IR) cells, PRL-IR cells (from rare cells to 10% of total cells) in 15 adenomas, correlating with the first days of culture PRL levels. In cultured explants, mitoses were never found. In 5 nM cortisol medium, the number of GH-IR cells decreased and PRL-IR cells increased or appeared. With 350 nM cortisol, the number of GH-IR cells increased, and PRL-IR cells were scarce or absent. Immunoreactivities for GH and PRL were found in different cells. Care was taken to exclude cultures containing normal pituitary tissue, and because no mitoses were found, these results suggest that most somatotropic adenomas can reversibly shift their secretion from GH to PRL in culture. This capacity to secrete PRL, hidden or low in vivo, is revealed by the favorable low cortisol conditions present in vitro.     

45Ca2+ uptake and peptide hormones secretion in the presence of excess Mg2+ content

Studies have been performed on the relationship between PRL and GH production and the 45Ca2+ influx in high magnesium content in vitro. The obtained data show that an elevated magnesium concentration in Krebs-Ringer solution is capable of inhibiting some hormonal function of the pituitary gland. It has been found, that PRL and GH released into the media in normal KRB solution revealed nearly two times higher concentration than in the presence of high Mg2+. Instead the cellular iPRL and iGH did not show any significant differences in control and in treated cultures. The incorporation of 4.5-3H-leucine into the prolactin and growth hormone demonstrate a significant decrease in the presence of high Mg2+ indicating that the ion is able to inhibit the secretion of newly synthesized PRL an GH. High concentration of Mg2+ abolished either the stimulation effect of releasing hormones on calcium uptake.     

Adenohypophysial cell types in the lamprey pituitary: current state of the art

Adenohypophysial cell types in the pituitary of adult sea lampreys, Petromyzon marinus, was localized by means of immunocytochemical and lectin cytochemical techniques. At least four types of adenohypophysial hormone cells are present in the pituitary of adult sea lampreys. The first type of cell is ACTH-like and occupies most parts of the rostral pars distalis (RPD), but a few scattered ACTH-like cells are also present in the proximal pars distalis (PPD). The second type of cell is MSH-like and occupies the whole pars intermedia. The third type of cell is GH/PRL-like and occupies the dorsal half of the PPD. These GH/PRL-like cells were initially detected by heterologous immunocytochemistry using antibodies to salmon GH, salmon PRL and blue shark GH, after hydrated autoclave pretreatment of sections. Later, by use of an antiserum raised against a synthetic peptide corresponding to the partial sequence of lamprey GH/PRL, the same cells as those containing GH/PRL-like immunoreactivity were stained positively. Similarity of the topographic distributions between lamprey GH/PRL-like cells and gnathostome fish GH cells in the pituitary suggests that GH/PRL-like cells in the lamprey may be GH cells. The last type of cell is GTH-like and occupies the ventral half of the PPD. Although GTH has not yet been isolated from the lamprey pituitary, our immunocytochemical data suggest that GTH-like material in the sea lamprey pituitary is more closely related to mammalian-like LH, rather than to FSH or TSH. These four types of adenohypophysial cells occupy most parts of the lamprey adenohypophysis and indeed there is little room for TSH or PRL cells. Thus, the present study further suggests that GH and LH-like GTH are ancestral forms of GH/PRL/SL family and glycoprotein hormones, respectively.     

Studies of anterior pituitary-grafted rats: II. Normal growth hormone secretion

Of the various animal models used to study chronic hyperprolactinemia, the otherwise intact rat implanted with extra anterior pituitary glands (AP) under the kidney capsule is assumed to be normal except for excess circulating prolactin (PRL). Since the ectopic glands contain numerous somatotropes in addition to abundant and active lactotropes, it was important to assess growth hormone (GH) secretion as well in this model of hyperprolactinemia. The structural and functional similarities of PRL and GH are such that it is necessary to demonstrate that metabolic abnormalities noted in AP-implanted rats are due to hyperprolactinemia and not to altered GH secretion. AP-implanted female rats have significantly higher resting serum PRL concentrations when compared to sham-operated control rats, but baseline serum GH levels are similar in normal and pituitary-grafted rats. Suppression of GH by insulin and clonidine is comparable in AP-implanted and control rats. The intrasellar pituitary GH concentration is also similar (ca. 20 μg/mg wet weight) in hyperprolactinemic and normal rats. We conclude that GH secretion is normal in the non-hypophysectomized AP-implanted rat, in contrast to the hypophysectomized AP-implanted rat model which has been reported to have diminished GH secretion. Despite the presence of recognizable somatotropes, the ectopic anterior pituitary does not appear to secrete significant amounts of GH, making the intact rat bearing multiple pituitary grafts an excellent model of chronic hyperprolactinemia.     

Secretory activity of lactotrophs and its regulation by hypothalamic hormones in primary cultures of pituitary cells of rats of different ages]

Basal prolactin (PRL) secretion and the responses of lactotrophs to thyroliberin, dopamine and somatostatin were studied in the experiments employing primary monolayer cultures of pituitary cells obtained from developing rats of different ages. High responsiveness of PRL-secreting cells to the action of hypothalamic hormones was observed in the group of neonatal rats, although basal PRL release was about two orders lower in pituitary cultures of neonatal rats as compared to the cultures of immature, pubertal and adult animals. The investigation performed could reveal quantitative, but not qualitative differences in the reactions of lactotrophs of various age groups. It is concluded that postnatal development in the rat is coupled with significant changes of basal PRL release and to a lesser extent, with changes of lactotroph responsiveness to hypothalamic hormones.     

Effect of experimentally induced hyperprolactinemia on growth hormone secretion

In order to study the existence of possible interrelation-ships between prolactin (PRL) and growth hormone (GH) secretions, adult male rats bearing an anterior pituitary graft under the kidney capsule since day 90 of life and their sham-operated controls were submitted to a single i.p. administration of L-dopa (50 mg/kg weight) or saline 30 days after the operation. Plasma PRL and GH levels were measured by using specific RIA methods. Dopamine (DA) and norepinephrine (NE) contents in the hypothalamus and in the in situ anterior pituitary gland were measured by using a specific radioenzymatic assay. An increase in plasma PRL levels and a decrease in plasma GH levels were shown in grafted rats. Hypothalamic contents of DA and NE were increased in these animals, while the anterior pituitary content of DA was not modified as compared to controls. The administration of a single injection of L-dopa led to decreases of plasma PRL and GH levels in both grafted and control rats, but while marked increases in hypothalamic and anterior pituitary contents of DA were shown in both groups, the hypothalamic content of NE was only increased in control animals. These data suggest that PRL and GH secretions were closely related. Dopamine could be mediating the action of PRL on GH, while NE would be less involved.     

Studies on S-adenosylmethionine:protein-carboxyl methyltransferase in the hypothalamo-neurohypophysial complex in organ culture.

The level of protein methylase II (S-adenosylmethionine: protein-carboxyl-methyltransferase, EC.2.1.1.24) activity in the hypothalamo-neurohypophysial complex in organ culture was studied during the period of 21 days. 1. The endogenous enzyme activity, which is a measure of both enzyme and substrate protein levels, in hypothalamus is maximum at the 7th day of culture showing 100% increase, compared to the activity at 0 day. Exogenous enzyme activity in hypothalamus which is a measure of enzyme level only, did not show a peak at 7th day. Thus, this increase is a measure of enzyme level only, did not show a peak at 7th day. Thus, this increase of the activity indicates that newly synthesized neurophysin served as endogenous substrate protein. 2. Endogenous enzyme activity of cytosol fraction from anterior pituitary gland gradually increased during the culture period reaching 3-fold increase at 12th day of culture, while the exogenous activity remained unchanged. This specific increase of endogenous enzyme activity also indicates that $\text{in vivo}$ newly synthesized anterior pituitary peptide hormones serve as substrate.     

Growth hormone and prolactin actions on osmoregulation and energy metabolism of gilthead sea bream (Sparus auratus)

The gilthead sea bream (Sparus auratus) is an euryhaline fish where prolactin (PRL) and growth hormone (GH) play a role in the adaptation to different environmental salinities. To find out the role of these pituitary hormones in osmoregulation and energy metabolism, fish were implanted with slow release implants of ovine GH (oGH, 5 microg g(-1) body mass) or ovine prolactin (oPRL, 5 microg g(-1) body mass), and sampled 7 days after the start of the treatment. GH increased branchial Na(+),K(+)-ATPase activity and decreased sodium levels in line with its predicted hypoosmoregulatory action. GH had metabolic effects as indicated by lowered plasma protein and lactate levels, while glucose, triglycerides and plasma cortisol levels were not affected. Also, GH changed liver glucose and lipid metabolism, stimulated branchial and renal glucose metabolism and glycolytic activity, and enhanced glycogenolysis in brain. PRL induced hypernatremia. Furthermore, this hormone decreased liver lipid oxidation potential, and increased glucose availability in kidney and brain. Both hormones have opposite osmoregulatory effects and different metabolic effects. These metabolic changes may support a role for both hormones in the control of energy metabolism in fish that could be related to the metabolic changes occurring during osmotic acclimation.     

Relation of endogenous opioid peptides and morphine to neuroendocrine functions.

Morphine and the endogenous opioid peptides (EOP) exert similar effects on the neuroendocrine system. When adminstered acutely, they stimulate growth hormone (GH), prolactin (PRL), and adrenocorticotropin (ACTH) release, and inhibit release of luteinizing hormone (LH), follicle stimulating hormone (FSH),and thyrotropin (TSH). Recent studies indicate that the EOP probably have a physiological role in regulating pituitary hormone secretion. Thus injection of naloxone (opiate antagonist) alone in rats resulted in a rapid fall in serum concentrations of GH and PRL, and a rise in serum LH and FSH, suggesting that the EOP help maintain basal secretion of these hormones. Prior administration of naloxone or naltrexon inhibited stress-induced PRL release, and elevated serum LH in castrated male rats to greater than normal castrate levels. Studies on the mechanisms of action of the EOP and morphine on hormone secretion indicate that they have no direct effect on the pituitary, but act via the hypothalamus. There is no evidence that the EOP or morphine alter the action of the hypothalamic hypophysiotropic hormones on pituitary hormone secretion; they probably act via hypothalamic neurotransmitters to influence release of the hypothalamic hormones into the pituitary portal vessels. Preliminary observations indicate that they may increase serotonin and decrease dopamine metabolism in the hypothalamus, which could account for practically all of their effects on pituitary hormone secretion.