Stimulatory action of prolactin on gonadotropin secretion in vitro

The action of prolactin (PRL) on the secretion of gonadotropin was investigated by means of a cell culture system of rat anterior pituitary gland. Anterior pituitary glands were removed from Wistar male rats, enzymatically digested and cultured. Luteinizing hormone (LH) release into medium was increased by adding PRL dose-dependently in the range between 10 ng/ml and 1 microgram/ml. This effect of PRL was further augmented by the presence of either gonadotropin-releasing hormone or estradiol. The intracellular LH concentration was also increased by PRL. PRL also caused an increase in follicle-stimulating hormone release into medium dose-dependently. In conclusion, PRL was shown to stimulate the secretion of gonadotropin at the pituitary level, thus suggesting a paracrine mode of PRL action in the anterior pituitary gland.     

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.     

Hypocretin/orexin suppresses corticotroph responsiveness in vitro

The hypocretin/orexins (Hcrts/ORXs) are peptides produced in neurons in the lateral hypothalamic area that project to neuroendocrine centers in the hypothalamus. Hcrt/ORX receptors are present in the hypothalamus and anterior pituitary gland. We examined the possibility that the Hcrts/ORXs, which we have demonstrated previously to act in the brain to stimulate sympathetic function, could alter stress hormone secretion by a direct pituitary action. In vitro studies revealed a dose-related inhibitory effect of the Hcrts/ORXs on corticotropin-releasing hormone-stimulated ACTH secretion that appeared to be mediated via the orexin-1 receptor and to be expressed at doses (threshold dose 1 nM orexin A) similar to the affinity constant for the receptor. The effect was not due to abrogation of the cAMP response of the corticotroph to corticotropin-releasing hormone and was not pertussis toxin sensitive, suggesting a non-G(i)-mediated mechanism. Instead, a G(q)-mediated signaling mechanism was indicated by the ability of protein kinase C blockade with calphostin C to reverse the inhibitory action of orexin A. Orexin A and orexin B did not significantly alter basal ACTH secretion in vitro and did not alter basal or releasing factor-stimulated secretion of luteinizing hormone, prolactin, thyroid-stimulating hormone or growth hormone from cells harvested from male or random-cycle female donors. Our data suggest a direct, pituitary action of the Hcrts/ORXs to modulate the endocrine response to stress and identify the potential cellular mechanism of a unique biological action of the peptides in the anterior pituitary gland.     

荷包牡丹碱对垂体前叶组织块释放促肾上腺皮质激素的刺激效应

本实验利用垂体组织块离体灌流技术,观察到－氨基丁酸Ａ受体拮抗剂荷包牡丹碱对切除双侧肾上腺９６ｈ后的大鼠垂体前叶ＡＣＴＨ的分泌具有强烈的刺激作用。但同样浓度的荷包牡丹碱对分离的垂体前叶细胞的ＡＣＴＨ分泌无影响。提示肾上腺切除后,－氨基丁酸在垂体前叶直接或通过间接途径抑制ＡＣＴＨ分泌。     

Growth hormone secretion of the neonatal rat pituitaries is stimulated by gamma-aminobutyric acid in vitro

Growth hormone secretion from pituitaries of neonatal rats was stimulated by gamma-aminobutyric acid (GABA) and the GABA agonist muscimol $\text{in vitro}$. This response to GABA was absent after the 9th postnatal day. The stimulation of growth hormone secretion by GABA was antagonized by bicuculline-methiodide and by picrotoxin. Diazepam stimulated while baclophen had no effect on growth hormone secretion. This stimulatory GABA effect might be related to a certain developmental stage of the pituitary GABA receptors or to the lack of hypothalamic regulatory influence(s) in the newborn.     

Aminergic regulation of neuroendocrinological functions: theoretical background and some clinical examples

Remarkable progress has been made during recent years in the central regulation of the hypothalamic releasing and inhibiting factors and the respective anterior pituitary hormones. There are two nearly universal inhibitory organizations: short tuberoinfundibular dopamine (TIDA) neurons and somatostatinergic system originating from the periventricular hypothalamus and terminating to the median eminence. It is now known that e.g. dopamine, noradrenaline and acetylcholine enhance while 5-hydroxytryptamine and GABA inhibit somatostatin secretion. These transmitters are also involved in the regulation of all releasing factors and pituitary hormones. Clinical applications have been developed based on the regulation of prolactin and growth hormone. Inhibitory TIDA neurons are undoubtedly the major determinants of prolactin secretion. Hyperprolactinaemia is one of the most common endocrinological side-effects of the drugs antagonizing dopaminergic transmission. Expectedly, dopaminergic drugs (bromocryptine, lergotrile, piribedil, dopamine and levodopa) are quite effective in reducing high prolactin levels regardless of the reason. The secretion of growth hormone is predominantly under dual dopaminergic control: hypothalamic stimulation and pituitary inhibition. The former masters the function of the normal gland, while the peripheral inhibitory component takes over in acromegalic gland. Hence dopaminergic drugs are able to reduce elevated growth hormone levels in 30-50% of the acromegalic patients. In normal man, dopamine agonists increase growth hormone levels. An analogous situation can be seen in Cushing's disease regarding ACTH secretion.     

The met-enkephalin analog FK 33-824 directly inhibits ACTH release by the rat pituitary gland in vitro

Systematic administration of the enkephalin analog FK 33-824 was previously shown to stimulate PRL secretion and to inhibit ACTH secretion in man. Naloxone prevented the effect on PRL release, but not on ACTH release. In this study, the direct action of this analog on hormone release by rat anterior pituitary lobes $\text{in}$$\text{vitro}$ were investigated. 1 uM FK 33-824 inhibited basal ACTH secretion by anterior pituitary glands in vitro, while 0.1 uM and 1 uM attenuated the lysine vasopressin stimulated ACTH release. Naloxone did not reverse the inhibitory action of the analog on ACTH release. β-Endorphin (0.01 - 1 uM) did not directly affect ACTH release. Basal and dopamine-induced inhibition of PRL release by anterior pituitary glands was neither influenced by FK 33-824 (0.1 and 1 uM), nor by β-endorphin (0.1 and 1 uM) with or without bacitracin. This study shows that the long-acting met-enkephalin analog FK 33-824 differentially affects PRL and ACTH secretion by the pituitary gland. It seems to stimulate PRL release at a suprapituitary site and this action probably involves u opiate receptors, because naloxone prevents these stimulatory effects. The inhibitory effect of FK 33-824 on ACTH release, however, is mediated via a direct effect at the pituitary level, which does not involve u receptors, as naloxone did not prevent this effect. In this respect, its action differs from that of β-endorphin, which does not directly affect ACTH release by the anterior pituitary gland.     

葆包牡丹碱对垂体前叶组织块释放促肾上腺皮质激素的刺激效应

本实验利用垂体组织块离体灌流技术,观察到γ－氨基丁酸Ａ受体拮抗剂荷包牡丹笃切除双侧肾上腺９６ｈ后的大鼠垂体前叶ＡＣＴＨ的分泌具有强烈的刺激作用。但同样浓度的荷包牡丹笃分离的垂体前叶细胞的ＡＣＴＨ分泌无影响,提示肾上腺切除后,γ－氨基丁酸在垂体前叶直接或通过间接途径抑制ＡＣＴＨ分泌。     

Tachykinins and the control of prolactin secretion

Tachykinins are present in the pituitary gland and in brain areas involved in the control of the secretion of pituitary hormones. Tachykinins have been demonstrated to stimulate prolactin release acting directly on the anterior pituitary gland. These peptides have also been revealed to be able to act at the hypothalamic level, interacting with neurotransmitters and neuropeptides that have the potential to affect prolactin secretion. Tachykinins seem to act by stimulating or inhibiting the release of the factors that affect prolactin secretion. Among them, tachykinins have been demonstrated to stimulate oxytocin and vasopressin release, which in turn results in prolactin release. Tachykinins also potentiated the response to vasoactive intestinal peptide (VIP) and reinforced the action of glutamate, which in turn result in prolactin release. They have also been shown to interact with serotonin, a neurotransmitter involved in the control of prolactin secretion. In addition, tachykinins have been shown to inhibit GABA release, a neurotransmitter with prolactin-release inhibiting effect. This inhibition may result in an increased prolactin secretion by removal of the GABA inhibition. On the other hand, tachykinins have also been shown to stimulate dopamine release by the hypothalamus, an action that results in an inhibition of prolactin release. Dopamine is a well known inhibitor of prolactin secretion. In conclusion, although tachykinins have been shown to have a predominantly stimulatory effect on prolactin secretion, especially at the pituitary level, under some circumstances they may also exert an inhibitory influence on prolactin release, by stimulating dopamine release at the hypothalamic level.     

Effects of GABA(A) receptor modulation on the expression of GnRH gene and GnRH receptor (GnRH-R) gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland of follicular-phase ewes

The effect of prolonged, intermittent infusion of GABA(A) receptor agonist (muscimol) or GABA(A) receptor antagonist (bicuculline) into the third cerebral ventricle on the expression of GnRH gene and GnRH-R gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland was examined in follicular-phase ewes by real-time PCR. The activation or inhibition of GABA(A) receptors in the hypothalamus decreased or increased the expression of GnRH and GnRH-R genes and LH secretion, respectively. The present results indicate that the GABAergic system in the hypothalamus of follicular-phase ewes may suppress, via hypothalamic GABA(A) receptors, the expression of GnRH and GnRH-R genes in this structure. The decrease or increase of GnRH-R mRNA in the anterior pituitary gland and LH secretion in the muscimol- or bicuculline-treated ewes, respectively, is probably a consequence of parallel changes in the release of GnRH from the hypothalamus activating GnRH-R gene expression. It is suggested that GABA acting through the GABA(A) receptor mechanism on the expression of GnRH gene and GnRH-R gene in the hypothalamus may be involved in two processes: the biosynthesis of GnRH and the release of this neurohormone in the hypothalamus.     

Effect of THIP and SL 76002, two clinically experimented GABA-mimetic compounds, on anterior pituitary GABA receptors and prolactin secretion in the rat

In the present study, the ability of three direct GABA agonists, muscimol, THIP and SL 76002 to displace 3H-GABA binding from anterior pituitary and medio-basal hypothalamus membranes was evaluated. Further, the effect of both THIP and SL 76002 on baseline prolactin levels or after stimulation of hormone release with haloperidol has been also studied. Either muscimol, THIP or SL 76002 have shown to posses 7-, 7- and 3-fold higher affinity, respectively, for the central nervous system than for the anterior pituitary 3H-GABA binding sites. Moreover, THIP and SL 76002 have demonstrated to be respectively, 25- and 1000- fold less potent than muscimol in inhibiting 3H- GABA binding at the level of the anterior pituitary and about 25- and 2700- fold less potent at the level of the medio-basal hypothalamus. Under basal conditions, either THIP or SL 76002 were ineffective to reduce prolactin release. However, after stimulation of prolactin secretion through blockade of the dopaminergic neurotransmission with haloperidol (0.1 mg/kg), both THIP (10 mg/kg) and SL 76002 (200 mg/kg) significantly counteracted the neuroleptic-induced prolactin rise with a potency which is in line with their ability to inhibit 3H-GABA binding in the anterior pituitary. The present results indicate that both compounds inhibit prolactin release under specific experimental situations probably through a GABAergic mechanism. In view of the endocrine effects of these GABA-mimetic compounds, the possibility arises for an application of these type of drugs in clinical neuroendocrinology.     

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.     

电场刺激对离体温育垂体前叶促肾上腺皮质激素分泌的影响

本实验应用离体温育大鼠垂体前叶组织块结合电场刺激及放射免疫测定方法,观察了垂体前叶内的神经纤维兴奋对促肾上腺皮质激素（ＡＣＴＨ）分泌的影响以及其它因素的作用。结果表明,一定参数的电场刺激使温育的大鼠四分之一垂体前叶组织块ＡＣＴＨ分泌增加,能被河豚毒素（ＴＴＸ）和藜芦碱部分阻断,此效应也可被地塞米松显著抑制,而同一参数的电场刺激与促肾上腺皮质激素释放激素（ＣＲＨ）诱发的ＡＣＴＨ分泌没有相互作用。经典神经递质受体阻断剂阿托品、心得安、酚妥拉明对电场刺激诱发的ＡＣＴＨ分泌没有显著影响;ＧＡＥＡ＿Ａ受体拮抗剂荷包牡丹碱明显促进此效应。该作用不受Ｐ物质拮抗剂ｓｐａｎｔｉｄｅ的影响,而降钙素基因相关肽（ＣＧＲＰ）受体拮抗剂ＣＧＲＰ片段８－３７能部分阻断这一作用。上述结果提示电场刺激所诱发的ＡＣＴＨ分泌可能部分由垂体前叶内ＣＧＲＰ能神经纤维介导。     

垂体前叶内神经纤维可能参与ACTH分泌的调节

我们建立了垂体组织块短时温育并施加电场刺激的离体实验体系,运用此方法并结合放射免疫测定激素含量,观察了大鼠垂体前叶内神经纤维对促肾上腺皮质激素（ＡＣＴＨ）释放的影响。结果表明,电场刺激能够促使垂体前叶ＡＣＴＨ释放显著增加,刺激参数为强度３０ｍＡ,波宽０．５ｍｓ,频率１０Ｈｚ。这个效应可为温育液中加入河豚毒素（ＴＴＸ）和藜芦碱所阻断,但ＴＴＸ不能阻断精氨酸加压素（ＡＶＰ）诱发的ＡＣＴＨ分泌。同样参数的电场刺激对分散培养的大鼠垂体前叶细胞ＡＣＴＨ的分泌没有显著作用。以上结果说明,我们所用参数的电场刺激产生的效应是兴奋了垂体前叶内的神经纤维,而非直接刺激腺细胞所致。上述结果提示：垂体前叶激素分泌的调节除了传统的体液途径之外,还可能存在直接的神经控制。     

Stimulation of prolactin release in rats by GABA.

Infusion of GABA into the lateral ventricle of intact female rats on the morning of proestrus and in ovariectomized rats significantly stimulated PRL release. This response apparently is not mediated through a direct action on the pituitary since injection of GABA into hypophysectomized rats with a pituitary transplant under the kindney capsule did not alter serum prolactin levels. These observations suggest that GABA may have a role in regulating prolactin secretion.     

The role of amino acid neurotransmitters in the regulation of pituitary gonadotropin release in fish.

Both glutamate and gamma-aminobutyric acid (GABA) are involved in pituitary hormone release in fish. Glutamate serves 2 purposes, both as a neurotransmitter and as a precursor for GABA synthesis. Glutamate can be catabolized to GABA by the actions of 2 distinct but related enzymes, glutamate decarboxylase 65 (GAD65) and GAD67. They derive from 2 different genes that likely arose from an early gene duplication prior to the emergence of teleosts more than 400 million years ago. There is good evidence for the involvement of GABA in luteinizing hormone (LH) release in fish. The mechanism of GABA action to stimulate LH release appears to be a combination of effects on GnRH release, potentiation of gonadotropin hormone-releasing hormone (GnRH) action, and in some cases directly at the LH cell. These actions appear to be dependent on such factors as sex or sex steroid levels, and there may also be species differences. Nevertheless, the stimulatory effects of GABA on LH are present in at least 4 fish species. In contrast, convincing data for the inhibitory effects of GABA on LH release have only been observed in 1 fish species. The sites and mechanisms of action of amino acid neurotransmitters on LH release have yet to be fully characterized. Both 130N-methyl-D-aspartic acid (NMDA) and S-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptors are likely to have important roles. We suggest that it is a receptor similar to the GABA(A) type which mediates the effects of GABA on LH release in fish, at least partially acting on the GnRH neuron, but likely directly acting at the gonadotroph as well. GABA may also be involved in regulating the release of other pituitary hormones in fish, namely follicle stimulating hormone (FSH = GTH-I), prolactin, and growth hormone. Based on the findings described in this review, a working model for the involvement of glutamate and GABA in the regulation of LH release in teleost fish is proposed.     

Perifusion model system to culture bovine hypothalamic slices in series with dispersed anterior pituitary cells

Summary Dispersed bovine anterior pituitary cells were incubated either in static or perifusion cultures to assess basal growth hormone release as well as stimulatory and inhibitory effects of growth hormone-releasing hormone and somatostatin, respectively, on growth hormone release. Total concentrations of growth hormones over a 12-hour incubation period were fivefold greater in perifused than in static cultures (2034 ± 160 vs. 387 ± 33 ng/12 h). A dose-dependent increase in growth hormone secretion in response to challenge with growth hormone-releasing hormone (10⁻¹² to 10⁻⁸ M) for 1 h was observed in both static and perifusion cultures; however, perifused cells were more responsive to the same concentration of neuropeptide than those in static culture. Concentrations of somatostatin (10⁻¹² to 10⁻⁸ M) for 1 h did not inhibit basal growth hormone secretion in either static or perifusion cultures. To establish model, slices of the hypothalamus, immediately adjacent to the sagittal midline, were perifused in series with anterior pituitary cells, and media effluent was assayed for growth hormone concentrations. Release of growth hormone was pulsatile and seemed to mimic the episodic pattern of bovine secretion. Hypothalamic slices were placed in one chamber of the perifusion system, and basal secretion of growth hormone-releasing hormone and somatostatin was pulsatile in media effluent. Tissue viability of hypothalamic slices and anterior pituitary cells was evaluated by KCl depolarization. Tissues were viable for at least 120 h. Thus, this hypothalamo-pituitary dual chamber perifusion system is a valid in vitro model to study regulation of growth hormone secretion.     

Mode of action and functional significance of avian gonadotropin-inhibitory hormone (GnIH): a review

Neuropeptide control of gonadotropin secretion at the level of the anterior pituitary gland is primarily through the stimulatory action of the hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH). However, a hypothalamic neuropeptide acting at the level of the pituitary to negatively regulate gonadotropin secretion has, until recently, remained unknown in any vertebrate. In 2000, we discovered a novel hypothalamic neuropeptide inhibiting gonadotropin release at the level of the pituitary in quail and termed it gonadotropin-inhibitory hormone (GnIH). A gonadotropin-inhibitory system is an intriguing concept and provides us with an unprecedented opportunity to study the regulation of avian reproduction from an entirely novel standpoint. To elucidate the mode of action of GnIH, we further identified the receptor for GnIH and characterized its expression and binding activity in quail. The identified GnIH receptor possessed seven transmembrane domains and specifically bound to GnIH in a concentration-dependent manner. The expression of GnIH receptor was found in the pituitary and several brain regions including the hypothalamus. These results suggest that GnIH acts directly on the pituitary via GnIH receptor to inhibit gonadotropin release. GnIH may also act on the hypothalamus to inhibit GnRH release. To understand the functional significance of GnIH in avian reproduction, we also investigated the mechanism that regulates GnIH expression. Interestingly, melatonin induced dose-dependently GnIH expression and melatonin receptor (Mel(1c)) was expressed in GnIH neurons. Thus melatonin appears to act directly on GnIH neurons via its receptor to induce GnIH expression. Based on these studies, GnIH is likely an important neuropeptide for the regulation of avian reproduction.     

Rat atrial natriuretic factor suppresses proopiomelanocortin-derived peptides secretion from both anterior and intermediate lobe cells and growth hormone release from anterior lobe cells of rat pituitary in vitro

Synthetic rat atrial natriuretic factor (ANF) was found to attenuate, in a dose-dependent manner, basal and corticotropin-releasing factor-induced secretion of proopiomelanocortin-derived peptides from cultured anterior and intermediate lobe cells of rat pituitary. ANF was also found to suppress basal and growth hormone-releasing factor-stimulated secretion of growth hormone from anterior lobe cells of rat pituitary. These results, together with reports of the existence of ANF-positive neurons in the hypothalamus and ANF-positive fibers in the median eminence, suggest that hypothalamic ANF is probably involved in the regulation of pituitary hormone secretion, especially that of proopiomelanocortin-derived peptides and growth hormone.     

Changes in growth hormone (GH) messenger RNA (GH mRNA) expression in the rat anterior pituitary after single interferon (IFN) alpha administration

INTRODUCTION: Interferon alpha (IFN-alpha) is a cytokine with pleiotropic effects which, via different pathways, influences the secretion of certain cytokines and hormones. Growth hormone (GH) secreted from the pituitary has physiological effects on various target tissues. The question is how IFN-alpha administered in various types of disease influences GH secretion. This study investigated the acute effect of IFN-alpha on GH mRNA expression in the rat anterior pituitary. OBJECTIVE: The aim of the study was to measure the cellular expression of GH mRNA by in situ hybridisation in the anterior pituitary after a single administration of IFN-alpha. MATERIAL AND METHODS: Rats were administered an intraperitoneal injection of IFN-alpha or saline. The rat pituitaries were taken 2 and 4 hours after IFN/saline administration and kept frozen until in situ hybridisation histochemistry. A 31-base(35)S-labelled oligonucleotide probe complementary to part of the exonic mRNA sequence coding for GH mRNA was used. All control and experimental sections were hybridised in the same hybridisation reaction. RESULTS: Acute administration of interferon alpha increased GH mRNA expression in the anterior pituitary in the 4-hour group in comparison with the control group, and there was no difference between the control group and the 2-hour rats. CONCLUSION: A single IFN-alpha administration was found to exert an influence on anterior pituitary GH mRNA expression. These observations may pave the way for presenting a possible new action of IFN-alpha.