Evaluation of hypothalamic-pituitary function in a combination of tests with four hypothalamic releasing hormones and L-dopa in normal subjects and in patients with hypothalamic and/or pituitary disorders

Hypothalamic-pituitary function was evaluated in a combination of tests with four hypothalamic releasing hormones (4RHs) and L-dopa in normal subjects and in patients with hypothalamic and/or pituitary disorders. Plasma concentrations of anterior pituitary hormones (GH, ACTH, TSH, PRL, LH and FSH) were measured before and after simultaneous iv administration of GHRH, CRH, TRH and LHRH. In addition, changes in the plasma levels of GHRH and GH were investigated before and after oral administration of L-dopa. Normal subjects showed appreciable responses to both tests. In five patients with hypothalamic disorders, the response of plasma anterior pituitary hormones varied, but plasma GHRH and GH did not respond to L-dopa. Patients with idiopathic and postpartum hypopituitarism showed low response to 4RHs or none at all, but L-dopa evoked a normal GHRH response in 2 of the 4 cases having no GH response. In the patients with hypopituitarism due to resection of a pituitary tumor, the response of anterior pituitary hormones to 4RHs was low, and L-dopa administration induced a normal GHRH and low GH response in 5 out of the 7 cases. After 4RHs administration, the patients with ACTH deficiency syndrome showed different patterns of impaired ACTH secretion, and isolated, combined or limited ACTH reserve. Seven patients with anorexia nervosa showed exaggerated GH, delayed TSH and FSH, low ACTH and LH, that is, normal PRL response to 4RHs, but no response of plasma GHRH or GH to L-dopa, suggesting the presence of hypothalamic dysfunction. These results indicate that the combination of the 4RHs test and L-dopa test is a simple and useful means for evaluating hypothalamic-pituitary function by measuring the response of plasma GHRH and six anterior pituitary hormones in the patients with endocrine disorders.     

Insights into a role of GH secretagogues in reversing the age-related decline in the GH/IGF-I axis

Growth hormone (GH) secretion and serum insulin-like growth factor-I (IGF-I) decline with aging. This study addresses the role played by the hypothalamic regulators in the aging GH decline and investigates the mechanisms through which growth hormone secretagogues (GHS) activate GH secretion in the aging rats. Two groups of male Wistar rats were studied: young-adult (3 mo) and old (24 mo). Hypothalamic growth hormone-releasing hormone (GHRH) mRNA and immunoreactive (IR) GHRH dramatically decreased (P < 0.01 and P < 0.001) in the old rats, as did median eminence IR-GHRH. Decreases of hypothalamic IR-somatostatin (SS; P < 0.001) and SS mRNA (P < 0.01), and median eminence IR-SS were found in old rats as were GHS receptor and IGF-I mRNA (P < 0.01 and P < 0.05). Hypothalamic IGF-I receptor mRNA and protein were unmodified. Both young and old pituitary cells, cultured alone or cocultured with fetal hypothalamic cells, responded to ghrelin. Only in the presence of fetal hypothalamic cells did ghrelin elevate the age-related decrease of GH secretion to within normal adult range. In old rats, growth hormone-releasing peptide-6 returned the levels of GH and IGF-I secretion and liver IGF-I mRNA, and partially restored the lower pituitary IR-GH and GH mRNA levels to those of young untreated rats. These results suggest that the aging GH decline may result from decreased GHRH function rather than from increased SS action. The reduction of hypothalamic GHS-R gene expression might impair the action of ghrelin on GH release. The role of IGF-I is not altered. The aging GH/IGF-I axis decline could be rejuvenated by GHS treatment.     

Growth hormone-releasing hormone facilitates hypoglycemia-induced release of cortisol

Early sleep in humans is characterized by a distinct suppression of pituitary-adrenal activity coinciding with enhanced activity of the somatotropic axis. Here, we tested in awake humans the hypothesis of an inhibiting influence of hypothalamic growth hormone-releasing hormone (GHRH) on pituitary-adrenal activity. For this purpose, pituitary-adrenal activity was stimulated in 10 men through a standard insulin-hypoglycemia-test (IHT) and in another 10 men through combined administration of CRH/vasopressin. Stimulation was performed in each man on three conditions following pretreatment with Placebo and GHRH administered intravenously (50 microg) or intranasally (300 microg) 1 h before. GH, ACTH and cortisol as well as blood pressure and heart rate were measured repeatedly. Contrary to expectations, pretreatment with GHRH did not suppress but enhanced secretion of cortisol upon insulin-induced hypoglycemia regardless of the route of GHRH pretreatment (p<0.05). In contrast, GHRH did not facilitate cortisol release after stimulation with CRH/vasopressin. Changes in ACTH remained inconsistent. Plasma levels of GH increased significantly after i.v. GHRH application, but remained unchanged after the intranasal administration. Blood pressure and heart rate were not influenced by the treatments. Results indicate facilitating effects of GHRH mediated at a suprapituitary (i.e. hypothalamic) level as suggested by restriction of the effect to the hypoglycemia-induced cortisol release with no effects after pituitary stimulation with CRH/vasopressin.     

Vasopressin pressor receptor-mediated activation of HPA axis by acute ethanol stress in rats

The plasma arginine vasopressin (AVP), ACTH, and corticosterone levels and the hypothalamic corticotropin-releasing hormone (CRH) content were measured after oral administration of 1 ml of 75% ethanol to rats, a model known to induce acute gastric erosions and stress. Elevated plasma AVP, ACTH, and corticosterone levels were detected 1 h after ethanol administration. Treatment with the vasopressin pressor (V(1)) receptor antagonist [d(CH(2))(5)Tyr(Me)-AVP] before ethanol administration significantly reduced the ACTH and corticosterone level increases. A higher hypothalamic CRH content was measured at 30 or 60 min after ethanol administration. V(1) receptor antagonist injection, 5 min before ethanol administration, inhibited the rise in hypothalamic CRH content. The protein synthesis blocker cycloheximide prevented the hypothalamic CRH content elevation after stress. The AVP-, CRH-, and AVP + CRH-induced in vitro ACTH release in normal anterior pituitary tissue cultures was also prevented by pretreatment with the V(1) receptor antagonist. The results support the hypothesis that stress-induced AVP may not only act directly on the ACTH producing anterior pituitary cells but also indirectly at the hypothalamic level via the synthesis and release of CRH.     

Interleukin-1 potentiates basal and AVP-stimulated ACTH secretion in vitro--the role of CRH pre-incubation.

The acute-phase cytokine interleukin-1 (IL-1) is known to activate the hypothalamic pituitary adrenal axis, primarily via corticotropin releasing hormone (CRH). The aim of this study was to determine whether IL-1beta could directly stimulate ACTH secretion from perifused equine anterior pituitary cells, and whether CRH pre-incubation affected corticotroph responsiveness. Isolated equine anterior pituitary cells were pre-incubated with media containing 10 nM CRH or vehicle for 20 hours before being loaded onto columns and perifused with 0.02 nM CRH and 100 nM cortisol. Columns were given a 5-minute pulse of arginine vasopressin (AVP, 10 nM), perifused for 4 hours with 0 (control) or 1 nM IL-1beta, then given a further 5-minute pulse of AVP (10nM). ACTH was measured in 5 minute fractions. In the setting of CRH pre-incubation, cells perifused with IL-1beta for 4 hours showed increased basal ACTH secretion compared to control (114 +/- 6 pM vs. 86 +/- 4 pM [means +/- S.E.M.], p < 0.001) and a significantly greater ACTH response to the final AVP pulse (240 +/- 32% vs. 96 +/- 30%, p = 0.009, expressed as % of ACTH response to the initial AVP pulse). The potentiation of AVP-stimulated ACTH release by IL-1 was not observed in cells pre-incubated with vehicle alone. In conclusion, IL-1 increases ACTH release in equine corticotroph cells pre-incubated with CRH and potentiates responsivity to AVP.     

Corticotrophin-releasing factors in the hypothalamus of the developing fetal sheep.

Corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) are secreted from the hypothalamic median eminence to elicit the secretion of ACTH from the pituitary corticotrophs. During fetal development there is progressive maturation of the hypothalamic-pituitary-adrenal axis, manifest as increasing plasma ACTH and cortisol concentrations, which in species such as sheep culminates in the onset of birth. However, the precise nature of the hypothalamic signal controlling fetal pituitary ACTH secretion remains poorly understood. To investigate the ontogeny of this hypothalamic signal, the present study examined immunoreactive and bioactive ACTH-releasing factors in the developing fetal sheep hypothalamus. Immunoreactive CRH and AVP were measured by radioimmunoassay in extracts of hypothalami taken at day 70, day 100, and day 130 gestation (term = 145 days). There was a progressive and significant (P < 0.01) increase in hypothalamic CRH and AVP concentrations which was particularly marked between d100 and d130 gestation. AVP was always present in higher concentrations that CRH, although this difference was significantly reduced by day 130 gestation as the result of a large increase in the content of CRH relative to AVP. Sephadex G50 chromatography revealed that immunoreactive CRH and AVP in hypothalamic extracts existed as single molecular forms corresponding to synthetic peptides at each gestational age. In addition, these immunoreactive forms of CRH and AVP possessed significant ACTH-releasing bioactivity as measured in primary cultures of adult sheep anterior pituitary cells. Furthermore, significant bioactivity was present in high and low molecular weight fractions eluted after chromatography which did not contain any CRH or AVP immunoreactivity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth hormone releasing hormone receptors on thymocytes and splenocytes from rats.

In the present study, we determined that rat mononuclear leukocytes possess specific receptors for growth hormone releasing hormone (GHRH). The results show that the binding of 125I-labeled GHRH to spleen and thymic cells was saturable and of a high affinity, approximately 3.5 and 2.5 nM for thymus and spleen cells, respectively. The Scatchard analysis revealed a binding capacity of approximately 54 and 35 fmol per 10(6) cells on thymus and spleen, respectively. The binding of GHRH was not competed by 10(-6) M growth hormone, corticotropin releasing factor, substance P or luteinizing hormone releasing hormone and vasointestinal peptide (VIP). Partial characterization of the receptor was accomplished by crosslinking 125I-labeled GHRH to thymus cells with disuccinimidyl suberate and polyacrylamide gel electrophoresis. Autoradiography of dried gels showed two major components in leukocytes and pituitary cells at approximately 42 and 27 kDa which could be diminished by unlabeled GHRH. The treatment of leukocytes with GHRH (10 nM) rapidly increased the intracellular free calcium concentration from a basal level of 70 +/- 20 nM to a plateau value of 150 +/- 20 nM in 6 min after stimulation. The functional activity of GHRH receptors was studied further by measuring lymphocyte proliferative responses and the increase in the level of cytoplasmic GH RNA. The presence of GHRH alone resulted in a dose-dependent increase in thymidine and uridine incorporation and a dose-dependent increase in the levels of GH RNA in the cytoplasm. Taken together, the results show that lymphocytes contain specific receptors for GHRH that are coupled to important biological responses and further support the concept of bidirectional communication between the immune and neuroendocrine tissues.     

Influence of membrane ion channel in pituitary somatotrophs by hypothalamic regulators

The secretion of growth hormone (GH) from somatotrophs located within the anterior pituitary gland is stimulated by endogenous hypothalamic growth hormone-releasing hormone (GHRH) and the GH secretagogue (GHS) ghrelin, and inhibited by somatotropin-releasing inhibitory factor (SRIF, also known as somatostatin). These factors bind to specific G-protein-coupled receptors on the cell membrane, and directly or indirectly modify the properties of ion channels and second messenger systems. Ultimately this results in a change in intracellular free Ca(2+) concentration ([Ca(2+)](i)) and the secretion of GH. Somatotrophs possess a variety of ion channels on their membranes, and modification of these ion channels, especially Ca(2+), K(+), and Na(+) channels, is tightly linked to intracellular Ca(2+) levels and therefore hormone secretion. Various issues regarding receptor distribution, role of ion channels, alteration of membrane potential, and involvement of intracellular signaling system in the control of GH secretion are discussed in this review. In particular, this work will focus on ion channels and [Ca(2+)](i) in somatotrophs.     

睡眠中间断低氧对大鼠下丘脑-垂体-肾上腺轴和生长激素的影响

目的:探讨睡眠中间断低氧对大鼠下丘脑-垂体-肾上腺轴和生长激素水平的影响.方法:大鼠分别给予吸入空气,持续低氧和间断低氧气体,在1 d,3 d,7 d和30 d后测定下丘脑促肾上腺皮质激素释放激素(CRH)和生长激素释放激素(GHRH)mRNA水平,并测定30d后血浆CRH,GHRH,促肾上腺皮质激素(ACTH)和皮质酮水平,分析其间的变化关系.结果:与对照组比较,在低氧后1 d,3 d,7 d后大鼠下丘脑CRH mRNA升高,GHRH mRNA降低,在30 d后,间断低氧组下丘脑CRH mRNA升高,GHRH mRNA降低,而持续低氧组则接近正常.间断低氧30 d后,血浆CRH、ACTH,皮质酮均升高,GHRH降低,而生长激素没有明显变化.结论:大鼠睡眠中慢性间断低氧可以引起下丘脑-垂体-肾上腺轴激素水平升高,反馈调节紊乱,可引起GHRH分泌抑制.     

Growth hormone secretion: molecular and cellular mechanisms and in vivo approaches

Growth hormone (GH) release is under the direct control of hypothalamic releasing hormones, some being also produced peripherally. The role of these hypothalamic factors has been understood by in vitro studies together with such in vivo approaches as stalk sectioning. Secretion of GH is stimulated by GH-releasing hormone (GHRH) and ghrelin (acting via the GH secretagogue [GHS] receptor [GHSR]), and inhibited by somatostatin (SRIF). Other peptides/proteins influence GH secretion, at least in some species. The cellular mechanism by which the releasing hormones affect GH secretion from the somatotrope requires specific signal transduction systems (cAMP and/or calcium influx and/or mobilization of intracellular calcium) and/ or tyrosine kinase(s) and/or nitric oxide (NO)/cGMP. At the subcellular level, GH release (at least in response to GHS) is accomplished by the following. The GH-containing secretory granules are moved close to the cell surface. There is then transient fusion of the secretory granules with the fusion pores in the multiple secretory pits in the somatotrope cell surface.     

Insulin hypoglycemia test and releasing hormone (corticotropin-releasing hormone and growth hormone-releasing hormone) stimulation in patients with pituitary failure of different origin

To investigate the efficacy of endocrine evaluation in diagnosing and localizing the cause of anterior pituitary failure, 17 patients with suprasellar space-occupying lesions, 4 patients with intrasellar tumors, 8 patients with no detectable anatomical lesion, 1 patient with posttraumatic failure and 1 patient with septooptical dysplasia were investigated. Endocrine evaluation consisted of measuring adrenocorticotropic hormone (ACTH), cortisol, and growth hormone (GH) levels during insulin hypoglycemia test (IHT) and after administration of corticotropin-releasing hormone (CRH) and growth hormone-releasing hormone (GRH). In addition, basal prolactin levels, gonadal and thyroid function were evaluated. The results showed that 4 of 17 patients with suprasellar tumors had normal ACTH and GH responses during IHT and after releasing hormone (RH) administration. Five of these patients had a normal ACTH or cortisol rise but no GH response during IHT. All 5 had a normal ACTH and 3 had normal GH rise after RH. Seven patients with suprasellar tumors had no ACTH or GH response during IHT, but all had an ACTH response to CRH. Only 3 of this group had a GH response to GRH. There was one exception of a patient who showed a GH and ACTH rise during IHT but only a blunted ACTH and no GH rise after RH administration. Four patients with pituitary failure and no demonstrable lesion had an ACTH rise after CRH but no GH rise after GRH, whereas in 3 patients with isolated ACTH deficiency no ACTH rise after CRH was seen. In 4 patients with nonsecreting pituitary tumors normal ACTH responses to IHT and CRH were seen, whereas GH rose during IHT only in 1 patient.(ABSTRACT TRUNCATED AT 250 WORDS)     

CRH and lysine-vasopressin stimulation tests in the diagnosis of hypoadrenalism secondary to hypothalamic or pituitary disorders

Ten patients with secondary hypoadrenalism have been tested with corticotropin releasing hormone (CRH) and lysine-vasopressin (LVP). One patient had isolated ACTH deficiency; 9 had deficiency of other pituitary hormones attributable to a primary pituitary disease in 3 and to an hypothalamic disorder in 6. After CRH administration, a definite increase in plasma ACTH was observed in all 6 patients with hypothalamic disorder. No response was elicited in the 3 patients with pituitary disease and in the patient with isolated ACTH deficiency. In the responsive patients. ACTH showed a delayed and prolonged pattern of response. Lysine-vasopressin administration produced an increase in plasma ACTH in 4 of the 6 hypothalamic patients and no response in those with pituitary disease and in the patient with isolated ACTH deficiency. These findings suggest that CRH represents a reliable test in differentiating hypothalamic from pituitary adrenal failure; LVP appeared a less sensitive diagnostic test.     

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.     

Influence of chronic treatment with the growth hormone secretagogue Ipamorelin,in young female rats: somatotroph response in vitro

Growth hormone (GH) is secreted in the anterior pituitary gland by the somatotroph cells. Secretion is regulated by growth hormone releasing hormone (GHRH) and somatostatin. Morever, GH secretagogues (GHS) can exert a considerable effect on GH secretion. In order to determine the effects of chronic treatment with the GHS Ipamorelin on the composition of the somatotroph cell population and on somatotroph GH content, an in vitro analysis was performed of the percentage of somatotroph cells (% of total), the ratio of different GH cell types (strongly/weakly-staining) and individual GH content, in pituitary cell cultures obtained from young female rats receiving Ipamorelin over 21 days (Ipamorelin group) and the effects were compared with those of GHRH (GHRH group) or saline (saline group). The ultrastructure of somatotroph cells did not change, but the volume density of secretion granules was increased (P<0.05) by previous in vivo Ipamorelin or GHRH treatment. In 3-day basal pituitary cell monolayer cultures, the percentage of somatotroph cells showed no modifications between groups, nor was there any change in the ratio of strongly/weakly immunostaining GH cells. In the Ipamorelin group alone, in vitro treatment with Ipamorelin (10(-8) M), or GHRP 6 (10(-8) M), or GHRH (10(-8) M) for 4 hours, increased the percentage of somatotroph cells, without modifying the ratio of strongly/weakly immunostained GH cells. Basal intracellular GH content in somatotroph cells over 4 hours was lower in the Ipamorelin group and the GHRH group than in the saline group. Only in the Ipamorelin group did Ipamorelin (10(-8) M), GHRP 6 (10(-8) M) and GHRH (10(-8) M) prompt increased intracellular GH content. These data suggest that, at least in the young female rat, the GHS Ipamorelin is able to exert a dynamic control effect on the somatotroph population and on GH hormone content.     

Effects of long-term treatment with growth hormone-releasing peptide-2 in the GHRH knockout mouse

Growth hormone (GH) secretagogues (GHS) stimulate GH secretion in vivo in humans and in animals. They act on the ghrelin receptor, expressed in both the hypothalamus and the pituitary. It is unknown whether GHSs act predominantly by increasing the release of hypothalamic GH-releasing hormone (GHRH) or by acting directly on the somatotroph cells. We studied whether a potent GHS could stimulate growth in the absence of endogenous GHRH. To this end, we used GHRH knockout (GHRH-KO) mice. These animals have proportionate dwarfism due to severe GH deficiency (GHD) and pituitary hypoplasia due to reduced somatotroph cell mass. We treated male GHRH-KO mice for 6 wk (from week 1 to week 7 of age) with GH-releasing peptide-2 (GHRP-2, 10 microg s.c. twice a day). Chronic treatment with GHRP-2 failed to stimulate somatotroph cell proliferation and GH secretion and to promote longitudinal growth. GHRP-2-treated mice showed an increase in total body weight compared with placebo-treated animals, due to worsening of the body composition alterations typical of GHD animals. These data demonstrate that GHRP-2 failed to reverse the severe GHD caused by lack of GHRH.     

Deterministic construct of amplifying actions of ghrelin on pulsatile growth hormone secretion

Ghrelin is a native ligand for the growth hormone secretagogue (GHS) receptor that stimulates pulsatile GH secretion markedly. At present, no formal construct exists to unify ensemble effects of ghrelin, GH-releasing hormone (GHRH), somatostatin (SRIF), and GH feedback. To model such interactions, we have assumed that ghrelin can stimulate pituitary GH secretion directly, antagonize inhibition of pituitary GH release by SRIF, oppose suppression of GHRH neurons in the arcuate nucleus (ArC) by SRIF, and induce GHRH secretion from ArC. The dynamics of such connectivity yield self-renewable GH pulse patterns mirroring those in the adult male and female rat and explicate the following key experimental observations. 1) Constant GHS infusion stimulates pulsatile GH secretion. 2) GHS and GHRH display synergy in vivo. 3) A systemic pulse of GHS stimulates GH secretion in the female rat at any time and in the male more during a spontaneous peak than during a trough. 4) Transgenetic silencing of the neuronal GHS receptor blunts GH pulses in the female. 5) Intracerebroventricular administration of GHS induces GH secretion. The minimal construct of GHS-GHRH-SRIF-GH interactions should aid in integrating physiological data, testing regulatory hypotheses, and forecasting innovative experiments.     

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.     

Increased growth hormone responses to growth hormone releasing hormone and thyrotropin releasing hormone in patients with metastatic testicular cancer

In 16 patients with metastatic testicular cancer and 10 age matched male control subjects growth hormone (GH) responses to growth hormone releasing hormone (GHRH; 1 microgram/kg body weight iv.) and thyrotropin releasing hormone (TRH; 200 micrograms iv.) were measured. Basal GH levels and GH levels following stimulation with GHRH or TRH were significantly increased in cancer patients compared to control subjects. 9 patients with testicular cancer were studied both in the stage of metastatic disease and after they had reached a complete remission. In complete remission GH responses to GHRH tended to decrease but the differences did not reach statistical significance. Our data suggest an alteration of hypothalamic and/or pituitary regulation of GH secretion in patients with metastatic testicular cancer.