Role of vasoactive intestinal polypeptide (VIP) in regulating the pituitary function in man

Intramuscular injection of synthetic VIP (200 micrograms) resulted in a rapid increase in plasma prolactin (PRL) concentrations in normal women, which was accompanied by the 4- to 7-fold increase in plasma VIP levels. Mean (+/- SE) peak values of plasma PRL obtained 15 min after the injection of VIP were higher than those of saline control (28.1 +/- 6.7 ng/ml vs. 11.4 +/- 1.6 ng/ml, p less than 0.05). Plasma growth hormone (GH) and cortisol levels were not affected by VIP in normal subjects. VIP injection raised plasma PRL levels (greater than 120% of the basal value) in all of 5 patients with prolactinoma. In 3 of 8 acromegalic patients, plasma GH was increased (greater than 150% of the basal value) by VIP injection. In the in vitro experiments, VIP (10(-8), 10(-7) and 10(-6) M) stimulated PRL release in a dose-related manner from the superfused pituitary adenoma cells obtained from two patients with prolactinoma. VIP-induced GH release from the superfused pituitary adenoma cells was also shown in 5 out of 6 acromegalic patients. VIP concentrations in the CSF were increased in most patients with hyperprolactinemia and a few cases with acromegaly. These findings indicate that VIP may play a role in regulating PRL secretion in man and may affect GH secretion from pituitary adenoma in acromegaly.     

Novel prolactin related mRNAs in rat pituitary cells

From cytoplasm of rat pituitary GH4C1 tumour cells, anti prolactin anti-serum precipitates a polypeptide with apparent molecular weight of 75.000 in addition to prolactin. In vitro translation of size fractionated RNA shows that a 82.000 molecular weight PRL-like polypeptide is encoded by a mRNA larger than the 1 kb prolactin mRNA. Northern blot analysis shows that a rat prolactin cDNA probe hybridize to a 3.2 kb RNA and a 1.5 kb RNA in addition to the 1 kb PRL mRNA. The 82.000 molecular weight translation product and the 3.2 kb mRNA is also detected in rat anterior pituitary cytoplasm. We conclude that at least one high molecular weight mRNA which code for a prolactin-like polypeptide, is present in normal rat anterior pituitary gland and in GH4C1 cells.     

Tumor necrosis factor-alpha affects growth hormone secretion by a direct pituitary interaction.

Administration of 50, 250, and 1,250 ng/kg iv of recombinant bovine tumor necrosis factor-alpha (RBTNF) did not affect basal plasma concentrations of growth hormone (GH) or thyroid-stimulating hormone in male calves. However, when administered 30 min before challenge with 1 microgram/kg iv of thyrotropin-releasing hormone (TRH), 250 ng/kg of RBTNF increased the subsequent incremental GH response. At 1,250 ng/kg of RBTNF, GH response to TRH was significantly blunted. For each dose of RBTNF administered, the incremental change in plasma thyroid-stimulating hormone following TRH was not significantly different from control. To examine direct effects of RBTNF on pituitary function, fresh bovine pituitaries were sliced into 1-mm cubes and incubated with 0 or 10(-8), 10(-9), or 10(-10) M RBTNF. Additional cultures were treated with 10(-8) or 10(-9) M GH-releasing factor or 10(-8) M TRH and 0 or 10(-8) M RBTNF. Media GH increased in cultures with 10(-10) M RBTNF and declined linearly as RBTNF concentration increased. RBTNF blocked GH release from GH-releasing factor- and TRH-challenged pituitary slices. Membranes prepared from homogenized bovine pituitaries had specific saturable binding characteristics for monomeric 125I-RBTNF. Membranes treated with 4 M MgCl2 for 10 min and washed free of Mg2+ produced Scatchard plots fit to a two-site model (high affinity site Kd = 6.6 nM), while Scatchards of non-Mg(2+)-treated membranes fit a single site (Kd = 8.9 nM). Polyacrylamide gel electrophoresis separation of 125I-RBTNF cross-linked pituitary membranes showed specific binding of monomeric 125I-RBTNF to protein components ranging in molecular weight from 19,000 to 77,000. The data suggest that RBTNF has modulatory effects on the regulation of GH secretion acting directly at the pituitary through specific receptors.     

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.     

The effects of testosterone and estrogen on the pituitary growth hormone response to growth hormone-releasing factor

The effects of testosterone and estrogen on the pituitary growth hormone response to hypothalamic growth hormone-releasing factor (GRF) were evaluated in vivo using male and female rats and in vitro using a pituitary cell monolayer culture system. In vivo the increase in plasma growth hormone (GH) concentration in response to a 500 ng/kg dose of GRF was similar in gonadectomized male and female rats. Pretreatment of intact and gonadectomized male rats with testosterone caused significant enhancement of the pituitary GH response to GRF, whereas pretreatment of gonadectomized female rats with 17 beta-estradiol did not alter the response. The GH response to GRF was not different between prepubertal (i.e., 30-day-old) male and female rats. However, following puberty (i.e., by 60 days of age), the response in male rats was significantly greater than that observed in female rats. The in vitro preincubation of anterior pituitary cells with either testosterone or 17 beta-estradiol did not cause any shift in the dose-response curve between GRF and GH. These results demonstrated that androgens play an active role in modulating the pituitary response to GRF in vivo.     

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.     

Heterogeneity of immunoreactive prolactin in the rat brain

Three immunoreactive prolactin proteins (24 Kd, 16 Kd, and 12 Kd) were identified in the rat brain using sodium dodecyl sulphate polyacrylamide gel electrophoresis, and western blot analyses. In male and female brains, the primary prolactin protein has a molecular weight of 24 Kd which is similar to that of pituitary prolactin. Two additional proteins with apparent molecular weights of 16 Kd and 12 Kd were also identified and were found in greater concentrations in the brain than in the pituitary, and were more predominant in the female brain. In addition, brain extracts proteolytically modify the 24K dalton PRL resulting in the formation of two fragments with apparent molecular weights of 16 and 8 Kd. These data indicate that the prolactin identified in the rat brain is similar to pituitary prolactin, and suggests, that like other PRL target tissues the brain may have the capacity to proteolytically modify prolactin.     

Growth hormone, thyrotropin and prolactin responses to simultaneous administration of human growth hormone-releasing factor and thyrotropin releasing hormone in the bovine

The effects of intravenous injection of synthetic human pancreatic growth hormone-releasing factor-44-NH2 (hpGRF-44) and synthetic thyrotropin releasing hormone (TRH), or hpGRF-44 in combination with TRH on growth hormone (GH), thyrotropin (TSH), and prolactin (PRL) release in dairy female calves (6- and 12-month-old) were studied. When 0.25 microgram of hpGRF-44 per kg of body weight (bw) was injected in combination with TRH (1.0 microgram per kg of bw), the mean plasma GH concentration of the 12-month-old calves rose to a maximum level of 191.5 ng/ml (P less than 0.001) at 15 min from the value of 6.8 ng/ml before injection at 0 min. The maximum level was 3.1 and 6.1 times as high as the peak values obtained after injection of hpGRF-44 (0.25 microgram per kg of bw) and TRH (1.0 microgram per kg of bw), respectively (P less than 0.001). The area under the GH response curve for the 12-month-old calves for 3 hr after injection of hpGRF-44 in combination with TRH was 2.5 times as large as the sum of the areas obtained by hpGRF-44 and TRH injections. In contrast, the mean plasma GH level was unchanged in saline injected calves. The magnitudes of the first and the second plasma GH responses in the 6-month-old calves to two consecutive injections of hpGRF-44 in combination with TRH at a 3-hr interval were very similar. The peak values of plasma GH in the calves after hpGRF-44 injection were 2-4 times as high as those after TRH injection.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Isolation and characterization of rat-mouse somatic cell hybrids secreting growth hormone and prolactin

Interspecific somatic cell hybrid clones have been isolated and characterized in order to study growth hormone (GH) and prolactin (PRL) gene expression. Rat pituitary tumor cells (GH3, 69 chromosomes) secreting rat GH and PRL were grown for 48 h together with nonhormone secreting, aminopterin-sensitive murine fibroblast cells (LMTK-, 55 chromosomes) and fused using polyethylene glycol. Resultant heterokaryons were selected in hypoxanthine-aminopterin-thymidine (HAT) medium and cloned. Five clones produced rat GH and PRL. Hormone-producing hybrids morphologically resembled the mouse parent fibroblast. Hybrids grew in monolayers and contained 80-142 chromosomes, and marker chromosomes for both rat (small submetacentric) and mouse (bi-armed and large true metacentric) were identified. The interspecific nature of the hybrids was further confirmed by the presence of both rat and mouse adenosine deaminase and superoxide dismutase isozymes. Using specific antisera and indirect immunoperoxidase staining, both hybrid clones and GH3 rat parental cells stained positively for rat GH and PRL, while the murine fibroblast parental cells were negative. Hormone production by the hybrids has been sustained for over twenty subcultures; secretion rates were initially 150 ng PRL and 321 ng GH/10(6) cells/24 h and are currently 100 ng PRL and 90 ng GH/10(6) cells/24 h. Parental GH3 rat cells secreted 720 ng PRL and 660 ng GH/10(6) cells/24 h. Exposure of hybrids to KCl (50 mM) resulted in acute stimulation of rat PRL, but not rat GH release, and long-term incubation with thyrotropin-releasing hormone (TRH, 80 nM) stimulated PRL secretion. Hormone-dependent modulation of PRL secretion was transferred to the hybrid cell thus enabling the model to be used in studying regulation of PRL gene expression.     

Growth hormone and prolactin secretion in hypophysial stalk-transected pigs as affected by growth hormone and prolactin-releasing and inhibiting factors.

Control of growth hormone (GH) and prolactin (PRL) release was investigated in hypophysial stalk-transected (HST) and stalk-intact pigs by determining the effects of analogs of GH-releasing factors (GHRF), somatostatin (SRIF), arginine, thyrotropin-releasing hormone, alpha-methyl-rho-tyrosine, and haloperidol. HST and control gilts were challenged with intravenous injections of human pancreatic GHRF(1-40)OH, thyrotropin-releasing hormone, and analogs of rat hypothalamic GHRF. HST animals remained acutely responsive to GHRF by releasing 2-fold greater quantities of GH than seen in controls. This occurred in spite of a 38% reduction in pituitary gland weight and a 32 and 55% decrease in GH concentration and total content. During SRIF infusion, GH remained at similar basal concentrations in HST and control gilts, but increased immediately after stopping SRIF infusion only in the controls. Releasable pituitary GH appears to accumulate during SRIF infusion. GHRF given during SRIF infusion caused a 2-fold greater release of GH than seen in animals receiving only GHRF. Arginine increased (P less than 0.05) GH release in controls, but not in HST gilts, which suggests that it acts through the central nervous system. Basal PRL concentrations were greater (P less than 0.05) in HST gilts than in control gilts. TRH acutely elevated circulating PRL (P less than 0.001) in HST gilts, suggesting that it acts directly on the pituitary gland. Haloperidol, a dopamine receptor antagonist, increased circulating PRL in controls but not in HST animals. alpha-Methyl-rho-tyrosine did not consistently increase circulating PRL, however, suggesting that it did not sufficiently alter turnover rate of the tyrosine hydroxylase pool. The results indicate that the isolated pituitary after HST remains acutely responsive to hypothalamic releasing and inhibiting factors for both GH and PRL release in the pig.     

Effects of homologous ghrelins on the growth hormone/insulin-like growth factor-I axis in the tilapia, Oreochromis mossambicus

Ghrelin is a gut-brain peptide synthesized mainly in the oxyntic mucosal cells of the stomach, and has potent growth hormone (GH)-releasing and orexigenic activities. Recently, two forms of ghrelin, ghrelin-C8 and -C10, were identified in the Mozambique tilapia (Oreochromis mossambicus). The present study describes in vitro and in vivo effects of these endogenous ghrelins on the GH/insulin-like growth factor-I (IGF-I) axis. Ghrelin-C8 (100 nM) stimulated GH release from primary cultures of pituitary cells after 4 and 8 h of incubation, whereas no effect was seen on prolactin (PRL) release. Stimulatory effects of ghrelin-C8 and -C10 (100 nM) on GH release during 6 h of incubation were blocked by pre-incubation with GHS receptor antagonist, [D-Lys(3)]-GHRP-6 (10 microM). Intraperitoneal injection of ghrelin-C8 (1 ng/g body weight) and -C10 (0.1 and 1 ng/g body weight) significantly increased plasma GH levels after 5 h. Significant increases were observed also in hepatic expression of IGF-I and GH receptor (GHR) mRNA following injections of both forms of ghrelin (0.1 and 1 ng/g body weight), although there was no effect on plasma levels of IGF-I. In the next experiment, both forms of ghrelin (1 ng/g body weight) significantly increased plasma IGF-I levels 10 h after the injection. No significant effect of either ghrelin was observed on plasma PRL levels. Both forms of GHS receptor (GHSR-1a and -1b) were found in the pituitary, clearly indicating that tilapia ghrelins stimulate primarily GH release through the GHS receptor. Stimulation of hepatic expression of IGF-I and GHR suggests metabolic roles of ghrelin in tilapia.     

Effect of estradiol & testosterone on the pituitary prolactin levels in developing male & female rats

Testosterone priopionate (TP) or estradiol-17 beta (E2) were injected into male and female rats from day of birth to 15 days of age to determine the effect of these steroids on the pituitary prolactin (PRL) level in developing rats. Animals were autopsied on Days 5, 7, 10, 14, 17, 22, 25, 30, 37, 45, 52, and 60 and pituitary PRL estimated by radioimmunoassay. Neonatal administration of TP or E2 markedly increased PRL content in male rats. The peak of PRL was advanced to Days 14 and 23, respectively, in E2- and TP-treated groups. The content of pituitary PRL declined sharply and values increased again by Day 52 in TP-treated rats and Day 37 in E2-treated rats. In the female rat both the steroids advanced the time of PRL peak. Peaks were observed on Days 22 and 30, respectively. Although PRL content declined following these peaks, values remained at a significantly higher level than normal. These results suggest that mechanisms controlling the PRL secretion are functional during the neonatal period. It is also suggested that TP acts to increase PRL levels by 1st being converted to E2.     

Possible involvement of endogenous opioid peptides in prolactin secretion induced by alpha 2-adrenergic stimulation in rats

Noradrenergic mechanisms have a stimulatory role in regulating prolactin (PRL) secretion in the rat. We investigated the mechanism by which the alpha 2-adrenergic system stimulates PRL release in urethane-anesthetized male rats. Intracerebroventricular injection of norepinephrine (2 micrograms/rat) or epinephrine (100 ng and 1 microgram/rat) caused an increase in plasma PRL levels. The PRL increase induced by epinephrine was much greater than that by norepinephrine. Intracerebroventricular injection of phentolamine (1 microgram/rat), an alpha-antagonist, blunted the plasma PRL increase induced by epinephrine (100 ng intracerebroventricularly). Plasma PRL levels were increased by intravenous injection of alpha 2-agonists, clonidine (15 micrograms/100 g of body wt), and xylazine (200 micrograms/100 g of body wt). Plasma PRL increase induced by clonidine or xylazine was suppressed by intravenous injection of naloxone (125 micrograms/100 g of body wt), an opiate antagonist. These findings suggest that alpha 2-adrenergic mechanisms stimulate pituitary PRL secretion, at least partly, by activating endogenous opioid peptides in the rat.     

Discrepancy between prolactin (PRL) messenger ribonucleic acid and PRL content in rat fetal pituitary cells: possible role of dopamine

Data are controversial concerning the time when PRL-synthesizing cells are detected for the first time in the rat pituitary. Using a very sensitive immunocytochemical technique, we could visualize only a few PRL cells before day 10 after birth. At that time, pituitary PRL was still 200 times less abundant than in the adult (on a tissue weight basis) whereas PRL mRNA per mg total RNA was only 80 times lower than in the adult. However, by in situ hybridization, we could demonstrate the presence of PRL mRNA in cells from fetal day 18 on. We have also followed the expression of GH gene in rat pituitary cells during development. In contrast to results obtained with PRL cells, quantitative analysis of cDNA probe hybridization to GH mRNA correlated well with measurements of immunostained cells. We found that PRL was released in the blood from fetal day 19 onwards. Thus, at that time PRL is synthesized and secreted but not stored. We therefore measured brain dopamine levels, and the data support the idea that the rise in dopamine levels after birth contributes to PRL storage. We confirmed in vitro that newborn pituitary cells can store PRL when cultured in the presence of dopamine.     

Effect of growth hormone-releasing factor on plasma growth hormone, prolactin and somatomedin C in hypopituitary and short normal children

We studied the effect of a single intravenous bolus of 0.5 microgram/kg of growth hormone-releasing factor (GRF) on plasma GH, prolactin (PRL) and somatomedin C (SMC) in 12 short normal children and 24 patients with severe GH deficiency (GHD), i.e. GH less than 5 ng/ml after insulin and glucagon tolerance tests. GRF elicited an increase in plasma GH in both short normal and GHD children. The mean GH peak was lower in the GHD than in the short normal children (8.2 +/- 2.5 vs. 39.2 +/- 5.1 ng/ml, p less than 0.001). In the GHD patients (but not in the short normals) there was a negative correlation between bone age and peak GH after GRF (r = -0.58, p less than 0.005); GH peaks within the normal range were seen in 5 out of 8 GHD children with a bone age less than 5 years. In the short normal children, GRF had no effect on plasma PRL, which decreased continuously between 8.30 and 11 a.m. (from 206 +/- 22 to 86 +/- 10 microU/ml, p less than 0.005), a reflection of its circadian rhythm. In the majority of the GHD patients, PRL levels were higher than in the short normal children but had the same circadian rhythm, except that a slight increase in PRL was observed 15 min after GRF; this increase in PRL was seen both in children with isolated GHD and in those with multiple hormone deficiencies; it did occur in some GHD patients who had no GH response to GRF. Serum SMC did not change 24 h after GRF in the short normal children. We conclude that: (1) in short normal children: (a) the mean GH response to a single intravenous bolus of 0.5 microgram/kg of GRF is similar to that reported in young adults and (b) GRF has no effect on PRL secretion; (2) in GHD patients: (a) normal GH responses to GRF are seen in patients with a bone age less than 5 years and establish the integrity of the somatotrophs in those cases; (b) the GH responsiveness to GRF decreases with age, which probably reflects the duration of endogenous GRF deficiency, and (c) although the PRL response to GRF is heterogeneous, it does in some patients provide additional evidence of responsive pituitary tissue.     

Growth hormone response to human pancreatic growth hormone releasing factor in cattle

The effects of a growth hormone releasing factor, human pancreatic growth hormone releasing factor-44 (hpGRF-44), on growth hormone (GH) secretion in calves, heifers and cows were studied. A single intravenous (iv) injection of 0.1, 0.25, 0.5 or 1.0 microgram of synthetic hpGRF-44 per kg of body weight (bw) in calves significantly elevated the circulating GH level within 2-5 min, while no increase in plasma GH was observed in saline injected control calves. The plasma GH level increased proportionally to the log dose of hpGRF-44, and reached a peak at 5-10 min (p less than 0.01). Subcutaneous injection of hpGRF-44 also elevated the plasma GH level, but the peak value at 15 min was 37% of that of iv injection (p less than 0.05). Intravenous injection of 0.25 microgram of hpGRF-44 per kg of bw to female calves, heifers, and cows significantly elevated mean the GH levels from 8.5, 2.3, and 1.6 ng/ml at 0 time to peak values of 97, 26, and 11.6 ng/ml, respectively (p less than 0.01). The plasma GH response and basal level in calves were significantly higher than those of heifers or cows (p less than 0.025). The plasma GH response to hpGRF-44 as well as the basal level decreased with advancing age. The plasma GH response to hpGRF-44 and basal GH in male calves were significantly greater than those in female calves (p less than 0.001). These results indicate that synthetic hpGRF-44 is a potent secretogogue for bovine GH, and suggest its usefulness in the assessment of GH secretion and reserve in cattle.