Oestradiol stimulates prolactin secretion in women through oestrogen receptors

OBJECTIVE: To examine the effects of clomiphene and raloxifene on basal and GnRH-induced prolactin (PRL) secretion in postmenopausal women. DESIGN: Postmenopausal women participated in two experimental procedures a month apart. In one experiment they received raloxifene (180 mg/day) (R-Exp) and in the other clomiphene (150 mg/day) (Cl-Exp). In Group 1, the women (n = 8) received raloxifene or clomiphene for 30 days plus oestradiol via skin patches (100 microg/24 h) for the last 10 days. In Group 2, the women (n = 8) received oestradiol for 30 days plus raloxifene (R-Exp) or clomiphene (Cl-Exp) for the last 10 days. The pituitary response to GnRH (100 microg i.v.) was investigated in all women on days 0, 10, 20 and 30 of each experiment. PATIENTS: The study included 16 healthy postmenopausal volunteer women aged 56-60 years. MEASUREMENTS: Basal levels of PRL and the area under the curve (AUC) of DeltaPRL response to GnRH were calculated. RESULTS: In Group 1, basal levels of PRL and the area under the curve (AUC) of PRL response to GnRH did not change significantly in both experiments. In Group 2, during both experiments basal levels of PRL and the AUC of PRL increased significantly on days 10 (P < 0.05) and 20 (P < 0.05) as compared to day 0 and then they decreased significantly on day 30 as compared to day 20 (P < 0.05). CONCLUSIONS: Our study demonstrates for the first time that raloxifene and clomiphene affect the secretion of PRL in postmenopausal women in a similar manner. It is suggested that oestradiol stimulates the secretion of PRL in women by acting through oestrogen receptors.     

Relaxin stimulates prolactin secretion from anterior pituitary cells

The anterior pituitary has recently been implicated as a relaxin target issue because of the cAMP elevation after relaxin treatment. We attempted to correlate this finding with an endocrine response to relaxin in rats. Anterior pituitary cells were enzymatically dispersed and subjected to the reverse hemolytic plaque assay. PRL secretion was significantly stimulated 1.31-fold by human relaxin at the lowest concentration studied (30 pM) and maximally stimulated 1.65-fold at 0.3 nM relaxin. Antibodies directed against relaxin inhibited this effect, as did the PRL inhibitory hormone, dopamine. In contrast to the response of PRL cells, there was no effect or a slight inhibition of LH release after incubation with relaxin. In conclusion, we propose that one of the pituitary cell types responsive to relaxin in culture is the PRL-secreting mammotroph.     

Activation of GABA B receptors in the anterior pituitary inhibits prolactin and luteinizing hormone secretion.

Previous work from our laboratory showed that baclofen could lower serum prolactin (PRL) levels acting at the central nervous system. The present experiments were designed to evaluate whether the gamma-aminobutyric acid B agonist was also effective in inhibiting hormone release at the pituitary level. In monolayer cultures of adenohypophyseal dispersed cells, baclofen inhibited basal PRL secretion after 1 or 2 h of incubation. This inhibition was significantly abolished by three antagonists: phaclofen, 3-aminopropyl-phosphonic acid and 4-aminobutylphosphonic acid. Furthermore, baclofen inhibited the thyrotropin-releasing hormone-induced PRL release in a concentration-dependent manner. With regard to gonadotropin secretion, baclofen was unable to modify basal luteinizing hormone (LH) secretion, but significantly inhibited the LH-releasing hormone-induced LH release. These results show that baclofen, in addition to its central neuroendocrine effects, inhibits pituitary hormone secretion, under basal and/or stimulated conditions, by direct action at the pituitary level.     

Kallidin stimulates prolactin secretion from individual rat anterior pituitary cells

Kinins are localized within the adenohypophysis where they have been shown to stimulate the release of pituitary hormones. In the present study we have investigated the effect of [Lys]-bradykinin (kallidin) on prolactin secretion at the single cell level from cultured male rat anterior pituitary cells. This was assessed by use of a reverse haemolytic plaque assay which permits quantitative evaluation of the proportion of all pituitary cells which are secreting prolactin, and the amount of prolactin secreted per lactotroph (plaque area). The rate of plaque development was used as an index of the rate of hormone secretion in time-course studies. Kallidin induced a dose-dependent increase in both the percentage of plaque-forming cells and the median plaque area during the first 2 and 3 h of incubation respectively. The threshold concentration of kallidin was 10 nmol/l. After 4 h of kallidin stimulation there was no difference between treated and control monolayers with respect to median plaque area and the total secretion index. Although recruitment of additional cells into the secretory pool cannot be excluded, this seems unlikely since at 3 and 4 h little or no difference was observed in the number of plaque-forming cells. The data suggest that initially kallidin accelerated the rate of prolactin secretion primarily by inducing an increase in the number of cells secreting prolactin, and subsequently by increasing the amount of hormone secreted per lactotroph. The results presented here are consistent with the proposed role of the kallikrein-kinin system in the paracrine or autocrine control of prolactin release from the pituitary gland.     

Bradykinin stimulates phosphoinositide metabolism and prolactin secretion in rat anterior pituitary cells

Bradykinin stimulated prolactin secretion from monolayer cultures of rat anterior pituitary cells, the stimulation being greater from the cells of male rats. This stimulated secretion was accompanied by a rise in total inositol phosphate accumulation, suggesting that the action of bradykinin is mediated by phosphoinositide hydrolysis. The increase in inositol phosphate accumulation was biphasic; a further sharp rise occurred when the concentration of bradykinin exceeded 1 mumol/l. This may indicate that bradykinin acts on other cell types in the pituitary gland. Bradykinin had no effect on growth hormone secretion from cells of normal pituitary glands, or on prolactin secretion and phosphoinositide metabolism in GH3 rat pituitary tumour cells. Bradykinin receptor antagonists (both B1 and B2) had no effect on either bradykinin-stimulated inositol phosphate accumulation or prolactin secretion. Kallikreins, the enzymes responsible for the generation of kinins, are known to be present in the adenohypophysis. Therefore, the results presented here would suggest that kinins may have a role as paracrine agents in the pituitary gland.     

Thyrotropin-releasing hormone rapidly stimulates a biphasic secretion of prolactin and growth hormone in GH4C1 rat pituitary tumor cells

The characteristics of TRH-induced acute PRL and GH secretion were studied in GH4C1 cells, a clonal rat anterior pituitary tumor cell line which secretes PRL and GH. The experiments were carried out both in a flow system in which microcarrier (Cytodex)-attached cells were perifused at a constant rate and in a conventional static culture system. In both systems, cells responded to TRH in a qualitatively similar manner. TRH significantly stimulated PRL and GH secretion within 5 sec without a detectable lag period. The secretion rate was highest during the initial 1 min, declined sharply thereafter despite the continuous presence of TRH, and plateaued at a lower level. The maximum dose of TRH caused 250-700% of basal secretion during the early period (approximately 8 min; first phase) and about 150% of basal secretion thereafter (second phase). The sustained lower secretion (second phase) was maintained as long as cells were exposed to TRH (up to 2.5 h), and the secretion rate returned to the basal level within 30 min of removal of TRH from the medium. The half-maximal doses for the first and second phase secretion were 2-3 and 0.5-1 nM, respectively, in both the perifusion and static culture systems. Over a 2-day period, TRH stimulated PRL synthesis and inhibited GH synthesis. The dose-response curves for these long term effects on hormone synthesis were similar to the dose-response curves for the first phase of release. [N3-methyl-His2]TRH gave similar results, but was more potent than TRH. [N3-methyl-His2]TRH stimulated first phase release with an ED50 of 0.4-0.8 nM, second phase release with an ED50 of 0.1-0.2 nM, and hormone synthesis with an ED50 of 0.7-0.8 nM. Preincubation of the cells with Ca+2-free medium significantly depressed both first and second phase secretion. Preexposure of the cells to cycloheximide (10 micrograms/ml) had little effect on the first phase of secretion, but reduced second phase secretion. The acute effects of TRH on GH and PRL were identical, except that the secretory response tended to be greater for PRL. We conclude that 1) TRH causes hormone secretion very rapidly in a biphasic manner; 2) the first phase of secretion consists primarily of the release of stored hormone, whereas the second phase includes the release of newly synthesized hormone; 3) the dose-response curve of second phase secretion is shifted to the left compared with that of first phase secretion; and 4) both phases of secretion are at least partially dependent on extracellular Ca+2.     

Bombesin stimulates prolactin and growth hormone release by pituitary cells in culture

Bombesin (BBS) has been previously shown to stimulate the secretion of PRL and GH in steroid-primed rats. To determine whether these effects were mediated by the central nervous system or were due to direct action on the pituitary gland, we studied the interaction of BBS with GH4C1 cells, a clonal strain of rat pituitary cells which synthesizes and secretes PRL and GH. The addition of 100 nM BBS to GH4C1 cells for 60 min increased PRL release to 140 +/- 3% of the control value (mean +/- SE) and GH release to 133 +/- 5% of the control value. Stimulation of hormone secretion was observed within 15 min of treatment with 100 nM BBS and continued for at least 2 h. Half-maximal stimulation of PRL release occurred with 0.5 nM BBS, and a maximal effect was observed with 10 nM peptide. The BBS analogs ranatensin, litorin, and [Tyr4]BBS, each at a concentration of 100 nM, caused the same stimulation of PRL release as maximal concentrations of BBS itself. BBS stimulated hormone release selectively in two of five different clonal pituitary cell strains examined. Pretreatment of GH4C1 cells with 1 nM estradiol and/or 100 nM insulin resulted in more powerful stimulation of PRL release by both TRH and BBS. When epidermal growth factor and vasoactive intestinal peptide were added simultaneously with BBS, PRL release was greater than in the presence of either peptide alone. In contrast, the stimulatory effects of TRH and BBS were not additive. Somatostatin inhibited both basal and stimulated PRL release. Thus, low concentrations of BBS can directly stimulate PRL and GH release by a clonal pituitary cell strain in culture. These results suggest that BBS may stimulate PRL and GH secretion in vivo by direct action on the pituitary gland.     

Functional melanocortin-2 receptors are expressed by mouse aorta-derived mesenchymal progenitor cells

A local melanocortin system is active during tissue injury and inflammation. Thus far this system has been described as autocrine in nature where local production of pro-opiomelanocortin (POMC) peptides by leukocytes feeds back on melanocortin receptor (MC-R) expressing immune cells to quell inflammatory cytokine production. Here we present evidence that POMC peptides may generate extracellular matrix (ECM) changes by inducing matrix production by cells of the mesenchymal lineage through activation of the MC2-R. Using immunoblot, we determined that mouse aorta-derived mesenchymal progenitor cells express both MC2-R and MC3-R. These progenitors respond to treatment with ACTH by increasing collagen matrix synthesis as assessed by picrosirius red stain and (3)H-proline incorporation. ACTH also induces transient increases in intracellular calcium ([Ca(2+)](i)) as assessed using the fluorescent Ca(2+) indicator, fura-2. The ACTH-induced changes in [Ca(2+)](i) are consistent with MC2-R signaling and consist of both an intracellular release and an extracellular influx of Ca(2+). Both mouse aortic mesenchymal progenitors and mouse macrophage cells express POMC and the prohormone convertase 1/3 (PC1/3) indicating they have the potential to contribute to the local production of POMC peptides. These data demonstrate functional MC2-R expression in mouse aorta-derived mesenchymal progenitors and implicate both macrophage and mesenchymal cells as relevant sources of local POMC peptides.     

Alpha-melanocyte-stimulating hormone stimulation of aldosterone secretion in hypophysectomized rats

Intact and hypophysectomized male rats on low and high sodium diets were treated with sc infused ACTH and alpha MSH, and the levels of aldosterone and corticosterone were determined in truncal blood. Plasma aldosterone levels were lower in hypophysectomized animals on the low sodium diet than in intact animals on the low sodium diet. Alpha MSH (8 micrograms/day) restored plasma levels of aldosterone to normal in hypophysectomized rats on the low sodium diet. ACTH (6 micrograms/day) did not cause significant changes in plasma levels of aldosterone in hypophysectomized animals. ACTH restored plasma corticosterone to normal in hypophysectomized rats, whereas alpha MSH had no effect on corticosterone. Alpha MSH did not increase plasma aldosterone levels in intact rats. These data suggest that alpha MSH may be important in the regulation of aldosterone secretion in the rat and that zona glomerulosa responsiveness to alpha MSH in vivo is increased by hypophysectomy. The mechanisms of alpha MSH action on glomerulosa cells are different from those of ACTH.     

Caffeine stimulates growth hormone secretion by cultured rat pituitary cells

To study the effect of caffeine on growth hormone secretion a culture system of dispersed rat anterior pituitary cells was employed. The cells were incubated overnight in medium 199 containing 10(-5) to 10(-1) M caffeine. The medium was then collected and assayed for rat growth hormone content. A dose dependent stimulatory effect of caffeine on growth hormone secretion into the culture medium was observed. It is concluded that caffeine, like other xanthine phosphodiesterase inhibitors stimulates growth hormone secretion by a direct effect on pituitary cells.     

Dermorphin stimulates prolactin secretion in the rat

The effect of dermorphin, a new opioid peptide originally isolated from amphibian skin, on the release of prolactin (Prl) was studied in vivo and in vitro. In vivo experiments: subcutaneous administrations of different doses of dermorphin ranging from 0.1 to 5 mg/kg body weight to normal male rats induce a statistically significant, dose-related increase in serum Prl levels. Pretreatment with the specific opioid antagonist, naloxone (2 mg/kg i.p.) completely prevents the rise in serum Prl, induced by 2 mg/kg of dermorphin. In normal male rats, the intraventricular injection of 0.25 micrograms/kg of dermorphin is not able to induce any significant changes in serum Prl levels 10 min after injection. Serum Prl levels show a significant enhancement 30 min after the administration of this dose of dermorphin, and return to control values at 60 min. On the contrary, 1 microgram/kg of dermorphin significantly elevates Prl concentrations 10 min after injection, leaving serum Prl levels unchanged 30 and 60 min after the administration. Naloxone (25 and 100 micrograms/kg) alone does not substantially modify serum Prl concentrations at any time interval considered. Treatment with either dose of naloxone performed together with either 0.25 or 1 microgram/kg of dermorphin completely counteracts the stimulatory effect of the peptide at all time intervals in which dermorphin was active when given alone. In orchidectomized (3 weeks) rats, the intraventricular administration of dermorphin at the dose of 0.25 micrograms/kg appears effective in enhancing Prl levels only 30 min after treatment. No statistically significant modifications are observed at 10 and 60 min with this dose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leptin stimulates pituitary prolactin release through an extracellular signal-regulated kinase-dependent pathway

Leptin was initially identified as a regulator of appetite and weight control centers in the hypothalamus, but appears to be involved in a number of physiological processes. This study was carried out to examine the possible role of leptin in regulating prolactin (PRL) release using the teleost pituitary model system. This advantageous system allows isolation of a nearly pure population of lactotropes in their natural, in situ aggregated state. The rostral pars distalis were dissected from tilapia pituitaries and exposed to varying concentrations of leptin (0, 1, 10, 100 nM) for 1 h. Release of PRL was stimulated by leptin in a potent and concentration-dependent manner. A time-course experiment showed that the strongest response in PRL release with leptin occurs within the first hour (approximately sixfold), and stimulation was sustained after 16 h (approximately twofold). Many of the actions of leptin are mediated by the activation of extracellular signal-regulated kinase (ERK1/2) but nothing is known about the cellular mechanisms by which leptin might regulate PRL secretion in vertebrates. We therefore tested whether ERK1/2 might be involved in the leptin PRL response and found that the ERK inhibitor, PD98059, hindered leptin-induced PRL release. We further analyzed leptin response by quantifying tyrosine and threonine phosphorylation of ERK1/2 using western blots. One hour incubation with leptin induced a concentration-dependent increase in phosphorylated, and thus active, ERK1/2. Our data show that leptin is a powerful stimulator of in vitro PRL release and that its actions occur in part through stimulation of ERK1/2.     

Progesterone stimulates luteinizing hormone secretion by acting directly on the pituitary.

To determine if progesterone (P) does affect gonadotropin secretion by acting directly on the pituitary, six women with hypothalamic gonadotropin deficiency were studied. They were treated with 17 beta-estradiol (E2; 2 mg/day, orally) to induce P receptors and maintain constant plasma E2 levels during two 15-day periods separated by 1 month. GnRH was administered iv at a dose of 10 microgram/pulse every 90 min during the last 5 days of E2 treatment. Either P (400 mg/day) or a placebo was administered intravaginally in a cross-over randomized design during the 5 days of pulsatile GnRH therapy. A baseline study of pulsatile LH secretion was performed, with sampling performed every 10 min for 8 h. The sampling was then repeated on day 15 of each study period at the end of pulsatile GnRH administration. Plasma levels of E2 and P were measured every day during the 5 days of either GnRH and P or GnRH and placebo treatment. In the six patients, the observed apulsatile pattern of LH during the baseline study confirmed the diagnosis of complete gonadotropin deficiency. Plasma E2 levels were not significantly different at the time of each pulse analysis (288 +/- 61 vs. 252 +/- 77 pmol/L). The plasma P level achieved with the vaginal pessaries was 22 +/- 5 nmol/L. P treatment resulted in all cases in a significant increase in the mean plasma LH level (5.2 +/- 0.9 vs. 3.6 +/- 0.7 IU/L after GnRH plus placebo; P less than 0.001). Furthermore, LH pulse amplitude was significantly increased by P compared to placebo (3.1 +/- 0.3 vs. 1.4 +/- 0.1 IU/L, respectively; P less than 0.01). Mean plasma FSH levels were significantly increased by GnRH regardless of whether P or placebo was present. In conclusion, these data indicate that a short exposure to physiological levels of P in the range of early luteal phase levels has a stimulatory effect on LH secretion by acting directly at the pituitary level.     

Alpha-melanocyte-stimulating hormone stimulates sodium excretion in the salt gland of the duck.

Salt glands of ducks were induced to secrete sodium through the ingestion of salt water. In salt-adapted animals the administration of melanocyte-stimulating hormone (MSH) produced a rise in the sodium excreted by the salt gland, an effect which was not mimicked by adrenocorticotropin. Studies in vitro using incubations of gland slices and radioactive sodium ion showed that MSH increased sodium efflux, indicating that it acted directly upon the gland. We have previously observed that MSH has no effect on the pigmentary system of the duck. It is proposed that in the evolutionary process this hormone has acquired new target tissues in these birds.     

Estradiol-17 beta stimulates basal and thyrotropin releasing hormone induced prolactin secretion by bovine pituitary cells in primary culture.

A series of experiments were conducted which demonstrate that estradiol-17β directly affects bovine pituitary cells in primary culture causing an increase in basal and thyrotropin releasing hormone (TRH)-induced prolactin secretion. Prolactin release by pituitary cells incubated with TRH at concentrations of 0.001, 0.01, 0.1 and 1 ng/ml increased linearly with increasing log concentrations. Exposure of pituitary cells to 5, 50 or 500 ng/ml estradiol for 4 h did not affect basal or TRH-induced prolactin release. However, when the period of exposure to estradiol was prolonged to 6, 12, or 24 h, 0.5, 5 or 50 ng estradiol/ml medium caused pituitary cells to release more prolactin and there was more total prolactin in the system (medium +cell content) than for comparable controls. These increases were linearly related to increasing log concentrations of estradiol used. To determine the chronic effect of estradiol on prolactin secretion, pituitary cells were incubated with estradiol-17β for 11 days during which medium was collected at 24 h intervals beginning on day 3. On day 3, prolactin accumulation in medium of control cultures averaged 2.5 ng/ml, and decreased gradually reaching relatively low levels by day 11 (100 ng/ml). Although prolactin secretion decreased during the culture period, stimulatory effects of estradiol were evident throughout. In addition, these cells still released prolactin in response to TRH (1 ng/ml) on day 11 and magnitude of TRH-induced prolactin release increased with increasing concentrations of estradiol-17β. We conclude that estradiol will increase basal and TRH-induced prolactin release by bovine lactotrophs. These results are consistent with the view that the increase in estradiol that occurs at the end of pregnancy in cattle, may participate in the prolactin surge that occurs at parturition in this species.     

Mainly mu-opiate receptors are involved in luteinizing hormone and prolactin secretion

We evaluated plasma PRL and LH concentrations in the rat after the administration of drugs that exert a specificity directed mainly, although not absolutely, toward the mu-, delta-, or kappa-opiate receptors, in order to investigate the role of different receptors and thus the respective endogenous ligands in the modulation of the release of these anterior pituitary hormones. LH concentrations were evaluated in prepuberal female rats, in adult male rats, and in ovariectomized, estradiol benzoate-treated rats. PRL concentrations were evaluated in suckling rats, in ovariectomized, estradiol benzoate-treated rats, and in ether-stressed rats. The delta-antagonist ICI 154129 never affected PRL or LH concentrations, whereas both the mu- and kappa-antagonists, naloxone and MR 1452, respectively, seemed to be effective. However, when graded doses of the two classes of antagonists were tested, the mu-antagonist appeared to be effective on both hormones at doses that were one tenth of those of the kappa-antagonist. In conclusion, the mu-receptor seems to be the most profoundly involved in the regulation of PRL and LH secretion.     

Epidermal growth factor stimulates secretion of rat pituitary luteinizing hormone in vitro

The effects of epidermal growth factor (EGF) on pituitary luteinizing hormone (LH) release and on the releases induced by oestradiol (E2) and LH-releasing hormone (LRH) were examined in a sequential double chamber perifusion system. In this system the mediobasal hypothalami (MBH) and/or pituitaries excised from normally cycling female rats in dioestrus were perifused with test media. Perifusion with EGF at 1 ng/ml for 30 min induced significant release (80-100% increase, P less than 0.05) of LH from hypothalamo-pituitary pairs, but not from the pituitary alone. Perifusion of the pituitary alone with medium containing 1 ng/ml EGF, resulted in significant release of LH (70-140% increase, P less than 0.05) after administration of 10(-7) M E2, but did not significantly influence LH release in response to 20 ng/ml LRH. These findings suggest that EGF may be involved in the regulation of pituitary gonadotrophin secretion by a direct effect on the hypothalamus and indirectly by increasing the pituitary responsiveness to E2.     

Acetylcholine stimulates alpha-melanocyte-stimulating hormone release from frog pituitary melanotrophs through activation of muscarinic and nicotinic receptors

The release of alpha MSH from the pars intermedia of amphibians is regulated by multiple factors, including classical neurotransmitters and neuropeptides. In this study we have examined the possible involvement of acetylcholine (ACh) in the regulation of alpha MSH secretion from the pars intermedia of the frog (Rana ridibunda) using the perifusion technique. When intact neurointermediate lobes (NIL) were exposed to graded doses of ACh (3 X 10(-7) to 3 X 10(-4) M), a dose-dependent stimulation of alpha MSH release was observed. Repeated administration of ACh (10(-4) M) induced reproducible responses of NIL without any desensitization phenomenon. ACh was also capable of stimulating alpha MSH release from dispersed intermediate lobe cells, indicating that the neurotransmitter exerts its effect by acting directly on frog melanotrophs. Using the monoclonal antibody M-35 against calf muscarinic receptors we have visualized, by the immunofluorescence technique, the presence of muscarinic receptor-like immunoreactivity in the frog pars intermedia. The stimulatory action of ACh was mimicked by both nicotine and muscarine (10(-5) M each). Nicotine-induced stimulation of alpha MSH release was partially abolished by alpha-bungarotoxin (10(-6) M) and hexamethonium (10(-4) M). The stimulatory effect of muscarine was suppressed by atropine and the M1-muscarinic antagonist pirenzepine (10(-5) M), but not by the M2-muscarinic antagonist gallamine. We have investigated the effect of ACh during administration of specific nicotinic and muscarinic antagonists. While hexomethonium or atropine could block only part of the stimulatory effect of ACh, concomitant administration of these antagonists totally abolished the response of NIL to ACh. Finally, the stimulatory effect of ACh was not impaired during prolonged administration of the beta-adrenergic antagonist propranolol. These data show that ACh stimulates in vitro alpha MSH secretion by frog NIL. Our results also indicate that amphibian pars intermedia cells possess two types of cholinergic receptors, an M1-muscarinic receptor sensitive to pirenzepine and nicotinic receptors sensitive to hexamethonium and alpha-bungarotoxin.     

Melatonin stimulates growth hormone secretion through pathways other than the growth hormone-releasing hormone

OBJECTIVE There is evidence that melatonin plays a role in the regulation of GH secretion. The aim of this study was to investigate the neuroendocrine mechanisms by which melatonin modulates GH secretion. Thus we assessed the effect of oral melatonin on the GH responses to GHRH administration and compared the effects of melatonin with those of pyridostigmine, a cholinergic agonist drug which is likely to suppress hypothalamic somatostatin release. DESIGN The study consisted of four protocols carried out during the afternoon hours. Study 1: oral melatonin (10 mg) or placebo were administered 60 minutes prior to GHRH (100 μg i.v. bolus). Study 2: GHRH (100 μg i.v. bolus) or placebo were administered at 0 minutes; oral melatonin or placebo were given at 60 minutes and were followed by a second GHRH stimulus (100 μg i.v. bolus) at 120 minutes. Study 3: placebo; oral melatonin (10 mg); oral pyridostigmine (120 mg); melatonin (10 mg) plus pyridostigmine (120 mg) were administered on separate occasions. Study 4: placebo; oral melatonin (10 mg); oral pyridostigmine (120 mg); melatonin (10 mg) plus pyridostigmine (120 mg) were administered on separate occasions 60 minutes prior to a submaximal dose (3 μg i.v. bolus) of GHRH. SUBJECTS Four groups of eight normal male subjects, ages 22–35 years, were randomly assigned to each protocol. MEASUREMENTS Growth hormone was measured by RIA at 15-minute intervals. RESULTS Oral melatonin administration had a weak stimulatory effect on GH basal levels. Prior melatonin administration approximately doubled the GH release induced by supramaximal (100 μg) or submaximal (3 μg) doses of GHRH. Melatonin administration restored the GH response to a second GHRH challenge, given 120 minutes after a first GHRH i.v. bolus. The GH releasing effects of pyridostigmine, either alone or followed by GHRH, were greater than those of melatonin. However, the simultaneous administration of melatonin and pyridostigmine was not followed by any further enhancement of GH release, either in the absence or in the presence of exogenous GHRH. CONCLUSIONS Our data indicate that oral administration of melatonin to normal human males increases basal GH release and GH responsiveness to GHRH through the same pathways as pyridostigmine. Therefore it is likely that melatonin plays this facilitatory role at the hypothalamic level by inhibiting endogenous somatostatin release, although with a lower potency than pyridostigmine. The physiological role of melatonin in GH neuroregulation remains to be established.