The effect of thyroid hormone and glucocorticoids on carp growth hormone-releasing factor (GRF)-induced growth hormone (GH) release in rainbow trout (Oncorhynchus mykiss).

1. The effect of thyroid hormone and glucocorticoids on carp growth hormone-releasing factor (GRF)-induced growth hormone (GH) secretion was studied on rainbow trout using a dispersed pituitary cell culture system. 2. A combined administration of lower doses (0.01 microM) of 3,5,3'-triiodo-L-thyronine (T3) and dexamethasone (Dex) significantly increased spontaneous as well as carp GRF-induced GH release. 3. Lower doses of Dex alone had no effect, and T3 had a marginal effect on GH release. Higher doses of either Dex or T3 potentially reduced GH release. 4. This study indicates an important role of thyroid hormone and/or glucocorticoids in the hypothalamic regulation of GH secretion in fish.     

Characterization of functional receptors for somatostatin in rat pituitary cells in culture.

GH4C1 cells are a clonal strain of rat pituitary tumor cells which synthesize and secrete prolactin and growth hormone. Somatostatin, a hypothalamic tetradecapeptide, inhibits the release of growth hormone and, under certain circumstances, also prolactin from normal pituitary cells. We have prepared [125I-Tyr1]somatostatin (approximately 2200 C1/mmol) and have shown that this ligand binds to a limited number of high affinity sites on GH4C1 cells. Half-maximal binding of somatostatin occurred at a concentration of 6 x 10(-10) M. A maximum of 0.11 pmol of [125I-Tyr1]somatostatin was bound per mg of cell protein, equivalent to 13,000 receptor sites per cell. The rate constant for binding (kon) was 8 x 10(7) M(-1) min(-1). The rate constant for dissociation (koff) was determined by direct measurement to be 0.02 min(-1) both in the presence and absence of excess nonradioactive somatostatin. Binding of [125I-Tyr1]somatostatin was not inhibited by 10(-7) M thyrotropin-releasing hormones. Substance P, neurotensin, luteinizing hormone-releasing hormone, calcitonin, adrenocorticotropin, or insulin. Of seven nonpituitary cell lines tested, none had specific receptors for somatostatin. Somatostatin was shown to inhibit prolactin and growth hormone production by CH4C1 cells. The dose-response characteristics for binding and the biological actions of somatostatin were essentially coincident. Furthermore, among several clonal pituitary cell strains tested, only those which had receptors for somatostatin showed a biological response to the hormone. We conclude that the characterized somatostatin receptor is necessary for the biological actions of somatostatin on GH4C1 cells.     

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.     

Anatomy of the Hypophysiotropic Somatostatinergic and Growth Hormone-releasing Hormone System Minireview

The central control of growth hormone (GH) secretion from the pituitary gland is ultimately achieved by the interaction between two hypothalamic neurohormones, somatostatin which inhibits and growth hormone-releasing hormone (GHRH) which stimulates GH release. The regulation of the somatostatin and GHRH release from the hypothalamus is regulated by a range of other neuropeptides, neurotransmitters, neurohormones. In this mini review we attempt to provide a short summary covering the anatomy and chemical characteristics of the various cell populations regulating GH secretion as a tribute to Miklós Palkovits who pioneered the field of functional neuroanatomy of hypothalamic networks.Special Issue Dedicated to Miklós Palkovits.     

Neuroendocrine Regulation of Growth Hormone Secretion in Teleost Fishes with Emphasis on the Involvement of Gonadal Sex Steroids

In teleost fishes, growth hormone (GH) appears to play an important regulatory role in several, apparently disparate, physiological events, including reproduction, osmotic or ionic regulation, metabolism, growth and development. GH secretion is regulated by hypothalamic neuroendocrine factors that either act directly on the somatotrophic cells in the pituitary gland, or modulate the secretion or activity of other neuroendocrine factors. In addition, the degree of the neuroendocrine influence on GH release is influenced by the nutritional and reproductive state of the fish; moreover, there appear to be marked species differences in some aspects of this neuroendocrine-physiological condition relationship among fish species. Thus, the neuroendocrine control of GH secretion in fishes is complex, and still poorly understood. The neuropeptides, gonadotrophin-releasing hormone, growth hormone-releasing hormone, thyrotrophin-releasing hormone, neuropeptide Y, serotonin and pituitary adenylate cyclase-activating polypeptide have all been demonstrated to stimulate GH in fish, as has the glutamate agonist, N-methyl-d,l-aspartate. Conversely, somatostatin has a potent inhibitory action on GH release in goldfish and carp, but is less effective in salmon and trout species.This review examines the interactive nature of the neuroendocrine control of GH secretion in fishes, and the manner in which gonadal steroids, directly or indirectly, modulate GH secretion and/or the release, or the activity, of the neuroendocrine factors.     

Actions of releasing factors on isolated secretory granules mediated by calcium.

Synthetic TRH, crude hypothalamic extract and partially purified prolactin releasing factor stimulated prolactin and growth hormone release from isolated secretory granules. Somatostatin and partially purified prolactin release-inhibiting factor inhibited release of both hormones. Calcium promoted hormone release from granules; its releasing action was potentiated by TRH and ionophore A23187 but reduced by somatostatin.     

Stimulatory effect of thyrotrophin-releasing hormone (TRH) on the release of luteinizing hormone (LH) from rat anterior pituitary cells in vitro

The effect of thyrotrophin-releasing hormone (TRH, 10(-7) M) on luteinizing hormone (LH) release from rat anterior pituitary cells was examined using organ and primary cell culture. The addition of TRH to the culture medium resulted in a slightly enhanced release of LH from the cultured pituitary tissues. However, the amount of LH release stimulated by TRH was not greater than that produced by luteinizing hormone-releasing hormone (LH-RH, 10(-7) M). Actinomycin D (2 X 10(-5) M) and cycloheximide (10(-4) M) had an inhibitory effect on the action of TRH on LH release. The inability of TRH to elicit gonadotrophin release from the anterior pituitary glands in vivo may partly be due to physiological inhibition of its action by other hypothalamic factor(s).     

Peptidase inactivation of hypothalamic releasing hormones.

With the structural characterization of the hypothalamic hormones, luteinizing hormone-releasing hormone (LH-RH), thyrotrophin-releasing (TRH), melanocyte-stimulating hormone release-inhibiting hormine (MIH), and growth hormone release-inhibiting hormone, (GH-RIH or somatostatin), it has been possible to investigate their enzymic inactivation by peptidases which are present at various sites in the body. Enzymes may play an important part in the control of polypeptide hormone levels and the peptidases acting on these four hypothalamic hormones may regulate the amount of TRH, LH-RH, MIH and somatostatin released from the hypothalamus, or their action at the level of the pituitary and their removal from the circulation. By studying the peptidase enzymes, further information may be obtained on the physiological mechanisms controlling the secretion and actions of hypothalamic hormones, as well as on the design of analogues with increased or competitive activity.     

Synaptic communication between somatostatinergic axons and growth hormone-releasing factor (GRF) synthesizing neurons in the arcuate nucleus of the rat

Growth hormone (GH) production of the anterior pituitary gland is controlled by inhibiting and releasing hormones that are synthesized in the diencephalon. In order to elucidate the possible interrelationships between somatostatin and growth hormone-releasing factor (GRF) synthesizing neurons at the hypothalamic level, immunocytochemical double labelling studies were performed on sections containing the arcuate nucleus (ARC) of the rat. Somatostatin producing neurons were located in the dorsomedial part of the ARC, while somatostatin immunoreactive (IR) axons were found in the ventro-lateral part of the nucleus, an area containing GRF-synthesizing cells. The use of the dual antigen localization technique revealed the approach and juxtaposition of somatostatin containing axons to dendrites and cell bodies of GRF-synthesizing neurons. At the light microscopic level, several somatostatinergic axon varicosities were clustered around single GRF-synthesizing cells. Ultrastructural analysis of the ventro-lateral part of the ARC showed that (i), somatostatinergic axons established synaptic connections (ii), GRF-producing neurons received axons terminals on their somata and dendrites and (iii), somatostatin-IR axons formed asymmetric synaptic specializations with both dendrites and somata of GRF-synthesizing neurons. These morphological findings indicate that the hormone production and release of hypophysiotrophic GRF-IR neurons can be influenced by the central somatostatin system via direct synaptic mechanisms. The data support the concept, that the interaction of inhibiting and releasing hormones, which determines responses of the pituitary target cells, may take place also at the hypothalamic level.     

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 effect of d-fenfluramine on anterior pituitary hormone release in the rat: in vivo and in vitro studies

Administration of d-fenfluramine, a serotonin-releasing drug, to male rats induced a dose-dependent increase in both serum prolactin and corticosterone concentrations. Serum growth hormone levels increased, but not significantly, at a dose of 1.25 mg/kg i.p. and decreased significantly at higher doses. When rats were pretreated with the serotonin uptake inhibitor fluoxetine (10 mg/kg i.p.) 30 min prior to injection of d-fenfluramine (5 mg/kg i.p.), the serum prolactin response to d-fenfluramine was partially inhibited, whereas the growth hormone response was not significantly modified. Fluoxetine pretreatment increased the serum corticosterone to the same level as did d-fenfluramine. d-Fenfluramine's effect on prolactin and growth hormone release was further tested in a hypothalamic-pituitary in vitro system. The addition of d-fenfluramine (5-500 ng/mL) for 30 min to rat hypothalami resulted in an enhancement of prolactin and growth hormone-releasing activities. These were expressed as the ability of the media in which the hypothalami had been incubated to stimulate prolactin and growth hormone release by cultured pituitary cells. The data suggest that the effect of d-fenfluramine on prolactin secretion is exerted through the hypothalamus and is probably mediated, at least partially, by a serotoninergic mechanism. The mechanism of d-fenfluramine's effect on corticosterone and growth hormone release needs further evaluation.     

In vitro release of growth hormone-releasing factor (GRF) from the hypothalamus: somatostatin inhibits GRF release.

The manner of release of growth hormone-releasing factor (GRF) from the rat hypothalamus was studied in a perifusion system using a highly sensitive radioimmunoassay for rat GRF. The recovery of GRF in this system was 50-60%. The release of GRF from the rat hypothalamic blocks was almost stable for 20-240 min after the start of the perifusion and was stimulated by depolarization induced by high K+ concentration. The release of GRF was inhibited by somatostatin at concentrations of 10(-11) to 10(-8) M with maximum inhibition to 52.5% of the basal release at a concentration of 10(-9) M. These results suggest that this system is useful in studying the regulatory mechanism of GRF release and that, in addition to its action on the pituitary, somatostatin appears to act at the level of the hypothalamus in inhibiting GRF release in the regulation of GH secretion.     

The glycopeptide moiety of vasopressin-neurophysin precursor is neurohypophysial prolactin releasing factor

All of the classically-described hypothalamic, hypophysiotropic factors that regulate anterior pituitary hormone secretion have now been isolated and identified except for prolactin releasing factor. We report here that the 39-amino acid glycopeptide comprising the carboxyterminus of the neurohypophysial vasopressin-neurophysin precursor stimulates prolactin release from cultured pituitary cells as potently as does thyrotropin releasing hormone but has no effect on the secretion of other pituitary hormones. Furthermore, antisera to the glycopeptide administered to lactating rats attenuated suckling-induced prolactin secretion. Thus, this glycopeptide appears to be the neurohypophysial prolactin releasing factor.     

Growth hormone-releasing factor releases ACTH from an AtT-20 mouse pituitary tumor cell line but not from normal pituitary cells

Corticotropin-releasing factor (CRF) and both human pancreatic growth hormone-releasing factor (hp-GRF) and rat hypothalamic GRF (rh-GRF) stimulated ACTH release from neoplastic AtT-20 mouse pituitary tumor cells in a dose-dependent fashion, with CRF inducing a 10-fold increase and GRF a maximal increment of approximately one-half that of CRF. Neither rh-GRF nor hp-GRF induced ACTH release in normal anterior pituitary cells. Pretreatment with either dexamethasone or somatostatin prior to the addition of rh-GRF inhibited the increase in ACTH release. Both ovine CRF and rh-GRF stimulated adenosine 3,5-monophosphate production in AtT-20 cells. The weak but clearly discernible effect of GRF on ACTH release from AtT-20 cells may be due to an abnormality in the AtT-20 cell receptor.     

Effects of TRH and somatostatin on releases of prolactin and growth hormone in vitro by the pituitary of Poecilia latipinna

Summary Pituitary glands from a teleost fish were incubated in the presence of the synthetic hypophysiotropic peptides, thyrotrophin-releasing hormone and somatostatin, in two media of different osmotic pressure.The effects on prolactin and growth hormone cells were detected by electron-microscopic morphometry with the aid of an image analyser. Thyrotrophin-releasing hormone caused changes in prolactin cell ultrastructure consistent with stimulated hormone release and, in the low osmotic pressure medium, appeared to increase synthetic activity. There was no effect on growth hormone cells. After somatostatin treatment, both synthesis and release in prolactin cells appeared to be inhibited, and there was an obvious inhibition of synthesis and release in growth hormone cells. The response of both cell types to somatostatin did not appear to be dependent on the osmotic pressure of the medium.     

Somatostatin-14 neurons in the ovine hypothalamus: colocalization with estrogen receptor alpha and somatostatin-28(1-12) immunoreactivity,and activation in response to estradiol

Pituitary gland growth hormone (GH) secretion is influenced by two hypothalamic neuropeptides: growth hormone-releasing hormone (GHRH) and somatostatin. Recent data also suggest that estrogen modulates GH release, particularly at the time of the preovulatory luteinizing hormone surge, when a coincident surge of GH is observed in sheep. The GHRH neurons do not possess estrogen receptor alpha (ERalpha), suggesting that estrogen does not act directly on GHRH neurons. Similarly, few somatotropes express ERalpha, suggesting a weak pituitary effect of estradiol on GH. It was hypothesized, therefore, that estradiol may affect somatostatin neurons to modulate GH release from the pituitary. Using immunocytochemical approaches, the present study revealed that although somatostatin neurons were located in several hypothalamic sites, only those in the arcuate nucleus (13% +/- 2%) and ventromedial nucleus (VMN; 29% +/- 1%) expressed ERalpha. In addition, we found that all neurons immunoreactive for somatostatin-14 were also immunoreactive for somatostatin-28(1-12). To determine whether increased GH secretion in response to estradiol is through modulation of GHRH and/or somatostatin neuronal activity, a final study investigated whether c-fos expression increased in somatostatin- and GHRH-immunoreactive cells at the time of the estradiol-induced LH surge in intact anestrous ewes. Estradiol significantly (P < 0.05) increased the percentage of GHRH (estradiol, 75% +/- 3%; no estradiol, 19% +/- 2%) neurons expressing c-fos in the hypothalamus. The percentage of somatostatin-immunoreactive neurons coexpressing c-fos in the estradiol-treated animals was significantly (P < 0.05) higher (periventricular, 44% +/- 3%; arcuate, 72% +/- 5%; VMN, 81% +/- 5%) than in the control animals (periventricular, 22% +/- 1%; arcuate, 29% +/- 3%; VMN, 31% +/- 3%). The present study suggests that estradiol modulates the activity of GHRH and somatostatin neurons but that this effect is most likely mediated through an indirect interneuronal pathway.     

ACTH release in pituitary cell cultures. Effect of neurogenic peptides and neurotransmitter substances on ACTH release induced by hypothalamic corticotropin releasing factor (CRF).

The effects of various neurogenic peptides and neurotransmitter substances on the release of ACTH induced by hypothalamic corticotropin releasing factor (HY-CRF) were investigated using monolayer cultured anterior pituitary cells. Test substances were given in combination with 0.05-0.1 hypothalamic extract (HE)/ml, because HE evoked a significant ACTH release and a linear dose response relationship was demonstrated sequentially between 0.0165 HE/ml and 0.5 HE/ml. Relative high doses of lysine-vasopressin showed a slight additive effect on the release of ACTH induced by 0.1 HE/ml. Leu-enkephalin, dopamine, prostaglandin E1 and E2 slightly reduced the release of ACTH induced by HY-CRF, but the inhibitory effect of these substances were not dose-related. Other tested substances including luteinizing hormone releasing hormone, thyrotropin releasing hormone, somatostatin, melanocyte stimulating hormone release inhibiting factor, beta-endorphin, neurotensin, substance P, vasoactive intestinal polypeptide, angiotensin II, norepinephrine, serotonin, acetylcholine, histamine and gamma-amino butyric acid showed neither agonistic nor antagonistic effect on the release of ACTH induced by HY-CRF. These results indicate that the release of ACTH is controlled specifically by HY-CRF and corticosterone, and modified slightly by some other substances such as vasopressin and prostaglandins, and that the effect of most other neurogenic peptides and neurotransmitter substances is negligible or non-physiological at the pituitary level.     

Regulation of hormone secretion in primary cell cultures of human somatotropinomas]

The effects of somatostatin and thyroliberin (thyrotropin-releasing hormone; TRH) on growth hormone (GH) and prolactin (PRL) secretion were studied in short-term (0.5-3h) or long-term (21-24h) incubations using monolayer cell cultures of somatotropin obtained from surgical material of patients with acromegaly. High sensitivity of both GH and PRL release to inhibitory action of somatostatin (10(-11) M) was established. We could not reveal the unambiguous influence of TRH on somatotropic function in the in vivo and in vitro conditions, as compared to the action of this tripeptide on PRL secretion. The results obtained permit us to propose that cell cultures of pituitary adenomata represent adequate and convenient models for studying the pathogenesis of tumor processes in the pituitary gland and for the development of new procedures of pharmacotherapy.     

Synaptic communication between somatostatinergic axons and growth hormone-releasing factor (GRF) synthesizing neurons in the arcuate nucleus of the rat

Summary Growth hormone (GH) production of the anterior pituitary gland is controlled by inhibiting and releasing hormones that are synthesized in the diencephalon. In order to elucidate the possible interrelationships between somatostatin and growth hormone-releasing factor (GRF) synthesizing neurons at the hypothalamic level, immunocytochemical double labelling studies were performed on sections containing the arcuate nucleus (ARC) of the rat. Somatostatin producing neurons were located in the dorsomedial part of the ARC, while somatostatin immunoreactive (IR) axons were found in the ventro-lateral part of the nucleus, an area containing GRF-synthesizing cells. The use of the dual antigen localization technique revealed the approach and juxtaposition of somatostatin containing axons to dendrites and cell bodies of GRF-synthesizing neurons. At the light microscopic level, several somatostatinergic axon varicosities were clustered around single GRF-synthesizing cells. Ultrastructural analysis of the ventrolateral part of the ARC showed that (i), somatostatinergic axons established synaptic connections (ii), GRF-producing neurons received axons terminals on their somata and dendrites and (iii), somatostatin-IR axons formed asymmetric synaptic specializations with both dendrites and somata of GRF-synthesizing neurons.These morphological findings indicate that the hormone preduction and release of hypophysiotrophic GRF-IR neurons can be influenced by the central somatostatin system via direct synaptic mechanisms. The data support the concent that the interaction of inhibiting and releasing hormones, which determines responses of the pituitary target cells, may take place also at the hypothalamic level.Supported by grants from the National Institute of Health (NIH NS 19266), the National Science Foundation (INT 8703030, 8602688), the Hungarian Academy of Sciences (OTKA 104) and the National Foundation of Technical Development (OKKFT Tt 286/1986)