Growth hormone releasing hormone-sensitive adenylate cyclase activity in growth hormone-producing pituitary adenoma: correlation to the response of plasma growth hormone to growth hormone releasing hormone in patients with acromegaly

The correlation between response of plasma GH to GHRH and the GHRH-induced stimulation of the intracellular adenylate cyclase (AC) activity in pituitary adenoma cell membranes in acromegalic patients was investigated. Each peak plasma GH level after iv administration of GHRH ranged from 1.1 to 13.8 times the basal level in 13 acromegalic patients. On the other hand, the maximal stimulation of intracellular AC activity (cAMP production) induced by GHRH varied from 1.4 to 6.4 times the control level in each GH-producing pituitary adenoma cell membrane. A significant positive correlation (r = 0.89, P less than 0.005) between plasma GH response to GHRH and intracellular cAMP production stimulated by GHRH was observed in nine of the acromegalic patients. In contrast, the response of plasma GH to GHRH was significantly blunted, despite a fairly large production of intracellular cAMP stimulated by GHRH, in the other four acromegalic patients. These results suggest that GHRH-induced GH release from GH-producing pituitary adenomas of patients with acromegaly may be regulated not only by GHRH receptor-adenylate cyclase system but also modified by several other factors including somatostatin and Sm-C.     

Gender governs the relationship between exercise intensity and growth hormone release in young adults.

We previously reported that in young adult males growth hormone (GH) release is related to exercise intensity in a linear dose-response manner (Pritzlaff et al. J Appl Physiol 87: 498-504, 1999). To investigate the effects of gender and exercise intensity on GH release, eight women (24.3 +/- 1.3 yr, 171 +/- 3.2 cm height, 63.6 +/- 8.7 kg weight) were each tested on six randomly ordered occasions [1 control condition (C), 5 exercise conditions (Ex)]. Serum GH concentrations were measured in samples obtained at 10-min intervals between 0700 and 0900 (baseline) and 0900 and 1300 (Ex + recovery or C). Integrated GH concentrations (IGHC) were calculated by trapezoidal reconstruction. During Ex, subjects exercised for 30 min (0900-0930) at one of the following intensities [normalized to the lactate threshold (LT)]: 25 and 75% of the difference between LT and rest, at LT, and at 25 and 75% of the difference between LT and peak O2 uptake. No differences were observed among conditions for baseline IGHC. To determine whether total (Ex + recovery) IGHC changed with increasing exercise intensity, slopes associated with individual linear regression models were subjected to a Wilcoxon signed-rank test. To test for gender differences, data in women were compared with the previously published data in men. A Wilcoxon ranked-sums two-tailed test was used to analyze the slopes and intercepts from the regression models. Total IGHC increased linearly with increasing exercise intensity. The slope and intercept values for the relationship between total IGHC and exercise intensity were greater in women than in men. Deconvolution analysis (0700-1300 h) revealed that, regardless of gender, increasing exercise intensity resulted in a linear increase in the mass of GH secreted per pulse and summed GH production rate, with no changes in GH secretory pulse frequency or apparent half-life of elimination. Exercise reduced the half-duration of GH secretory burst in men but not in women. Gender comparisons revealed that women had greater basal (nonpulsatile) GH secretion across all conditions, more frequent GH secretory pulses, a greater GH secretory pulse amplitude, a greater production rate, and a trend for a greater mass of GH secreted per pulse than men. We conclude that, in young adults, the GH secretory response to exercise is related to exercise intensity in a linear dose-response pattern. For each incremental increase in exercise intensity, the fractional stimulation of GH secretion is greater in women than in men.     

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.     

Ghrelin Stimulation of Growth Hormone-Releasing Hormone Neurons Is Direct in the Arcuate Nucleus

Background

Ghrelin targets the arcuate nucleus, from where growth hormone releasing hormone (GHRH) neurones trigger GH secretion. This hypothalamic nucleus also contains neuropeptide Y (NPY) neurons which play a master role in the effect of ghrelin on feeding. Interestingly, connections between NPY and GHRH neurons have been reported, leading to the hypothesis that the GH axis and the feeding circuits might be co-regulated by ghrelin.

Principal Findings

Here, we show that ghrelin stimulates the firing rate of identified GHRH neurons, in transgenic GHRH-GFP mice. This stimulation is prevented by growth hormone secretagogue receptor-1 antagonism as well as by U-73122, a phospholipase C inhibitor and by calcium channels blockers. The effect of ghrelin does not require synaptic transmission, as it is not antagonized by γ-aminobutyric acid, glutamate and NPY receptor antagonists. In addition, this hypothalamic effect of ghrelin is independent of somatostatin, the inhibitor of the GH axis, since it is also found in somatostatin knockout mice. Indeed, ghrelin does not modify synaptic currents of GHRH neurons. However, ghrelin exerts a strong and direct depolarizing effect on GHRH neurons, which supports their increased firing rate.

Conclusion

Thus, GHRH neurons are a specific target for ghrelin within the brain, and not activated secondary to altered activity in feeding circuits. These results support the view that ghrelin related therapeutic approaches could be directed separately towards GH deficiency or feeding disorders.     

Synaptic interaction between ghrelin- and ghrelin-containing neurons in the rat hypothalamus

Synaptic relationships between ghrelin-like immunoreactive axon terminals and other neurons in the hypothalamic arcuate nucleus (ARC) were studied using immunostaining methods at the light and electron microscope levels. Many ghrelin-like immunoreactive axon terminals were found to be in apposition to ghrelin-like immunoreactive neurons at the light microscopic level. At the electron microscopic level, ghrelin-like immunoreactive axon terminals were found to make synapses on ghrelin-like immunoreactive cell bodies and dendrites in the ARC. While the axo-dendritic synapses between ghrelin- and ghrelin-like immunoreactive neurons were mostly the asymmetric type, the axo-somatic synapses were both asymmetric and symmetric type of synapses. Ghrelin at 10(-10) M increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) in the neurons isolated from the ARC, some of which were immunocytochemically identified as ghrelin-positive. Ghrelin at 10(-10) M also increased [Ca(2+)](i) in 12% of ghrelin-like immunoreactive neurons in the ARC. These findings suggest that ghrelin serves as a transmitter and/or modulator that stimulates [Ca(2+)](i) signaling in ghrelin neurons of the ARC, which may participate in the orexigenic action of ghrelin. Our data suggests a possibility of existing a novel circuit implicating regulation of feeding and/or energy metabolism.     

Impact of acute exercise intensity on pulsatile growth hormone release in men.

To investigate the effects of exercise intensity on growth hormone (GH) release, 10 male subjects were tested on 6 randomly ordered occasions [1 control condition (C), 5 exercise conditions (Ex)]. Serum GH concentrations were measured in samples obtained at 10-min intervals between 0700 and 0900 (baseline) and 0900 and 1300 (exercise+ recovery). Integrated GH concentrations (IGHC) were calculated by trapezoidal reconstruction. During Ex subjects exercised for 30 min (0900-0930) at one of the following intensities [normalized to the lactate threshold (LT)]: 25 and 75% of the difference between LT and rest (0.25LT and 0.75LT, respectively), at LT, and at 25 and 75% of the difference between LT and peak (1.25LT and 1.75LT, respectively). No differences were observed among conditions for baseline IGHC. Exercise+recovery IGHC (mean +/- SE: C = 250 +/- 60; 0.25LT = 203 +/- 69; 0.75LT = 448 +/- 125; LT = 452 +/- 119; 1.25LT = 512 +/- 121; 1.75LT = 713 +/- 115 microg x l(-1) x min(-1)) increased linearly with increasing exercise intensity (P < 0.05). Deconvolution analysis revealed that increasing exercise intensity resulted in a linear increase in the mass of GH secreted per pulse and GH production rate [production rate increased from 16. 5 +/- 4.5 (C) to 32.1 +/- 5.2 microg x distribution volume(-1) x min(-1) (1.75LT), P < 0.05], with no changes in GH pulse frequency or half-life of elimination. We conclude that the GH secretory response to exercise is related to exercise intensity in a linear dose-response pattern in young men.     

The role of sexual steroids in the modulation of growth hormone (GH) secretion in humans

Sex steroids contribute to modulate GH secretion in man. However, both the exact locus and mechanism by which their actions are exerted still remain not clearly understood. We undertook a number of studies designed to ascertain: (1) whether or not sudden or chronic changes in circulating gonadal steroids may affect GH secretion in normal adults; and (2) the reason(s) for gender-related dimorphic pattern of GH release. The pituitary reserve of GH, as evaluated by means of a GHRH challenge, was similar in women with anorexia nervosa and in normally menstruating women. Estrogenic receptor blockade with tamoxifen (TMX) did not significantly change GHRH-induced GH response in these normal women. Therefore, acute or chronic hypoestrogenism apparently had no important effects at level of somatotrophs. In another group of normal women we tested the possibility that changes in circulating estrogens might induce changes in the hypothalamic-somatotroph rhythm (HSR). GHRH challenges were performed throughout a menstrual cycle, and again after having achieved functional ovarian blockade with a GnRH agonist treatment. Short-term ovarian blockade did not significantly affect the parameters of GH response to GHRH, although it was accompanied by an increase in the number of women in a refractory HSR phase at testing. This suggested a low potentiating effect on the basic pattern of somatostatin (SS) release occurring as a consequence of the decrease in circulating estrogens. In normal men, neither the GH response to GHRH nor the HSR were affected by functional testicular blockade (after GnRH agonist treatment). However, the administration of testosterone enanthate (250 mg) to another group of men increased both the GHRH-induced GH release and the number of subjects in a spontaneous secretory HSR phase at testing; these were reversed by estrogenic receptor blockade with TMS. In another group of normal men, the fraction of GH secreted in pulses (FGHP) during a nocturnal sampling period was significantly decreased by testicular blockade. Other parameters of GH secretion,such as the number of GH pulses and their mean amplitude (A), and the mean plasma GH concentration (MCGH), showed a slight, although not significant, decrease following the lack of androgens. The administration of testosterone enanthate (500 mg) reversed these parameters to values similar to those in the basal study. Interestingly, when tamoxifen was given after testosterone enanthate, A, MCGH and FGHP increased to values significantly higher than in any other experimental condition in that study.(ABSTRACT TRUNCATED AT 400 WORDS)     

PYY(3-36) induces Fos in the arcuate nucleus and in both catecholaminergic and non-catecholaminergic neurons in the nucleus tractus solitarius of rats

Peptide YY (3-36) [PYY(3-36)] inhibits feeding in rodents, nonhuman primates and humans, yet the neural circuits underlying this action remain to be determined. Here we assessed whether PYY(3-36) inhibits feeding by activating neurons in forebrain and hindbrain sites containing Y2 receptors and linked to control of food intake, or in hindbrain sites immediately downstream of vagal afferent neurons. Rats received an anorexigenic dose of PYY(3-36), and the number of neurons expressing Fos, an indicator of neuronal activation, was determined in anterior hypothalamus (AH), arcuate nucleus (ARC), dorsomedial hypothalamus (DMH), lateral hypothalamus (LH), ventromedial hypothalamus (VMH), central nucleus of the amygdala (CeA), area postrema (AP), and caudal medial nucleus tractus solitarius (cmNTS), commissural NTS (cNTS), and gelatinosus NTS (gNTS). Expression of tyrosine hydroxylase (TH), an indicator of catecholamine synthesis, was also measured in the cmNTS. PYY(3-36) increased Fos in ARC, cmNTS, gNTS and AP. Approximately 10% of Fos+ neurons in the cmNTS were TH+. These results suggest that PYY(3-36) inhibits feeding through direct activation of ARC neurons, and direct and/or indirect activation via vagal afferent nerves of cmNTS, gNTS and AP, including some catecholaminergic neurons in the cmNTS.     

Galanin-like peptide and ghrelin increase cytosolic Ca2+ in neurons containing growth hormone-releasing hormone in the arcuate nucleus

Galanin-like peptide (GALP), discovered in the porcine hypothalamus, is expressed predominantly in the arcuate nucleus (ARC), a feeding-controlling center. Intracerebroventricular injection of GALP has been shown to stimulate food intake in the rats. However, the mechanisms underlying the orexigenic effect of GALP are unknown. The present study aimed to determine the target neurons of GALP in the ARC. We investigated the effects of GALP on cytosolic free Ca2+ concentration ([Ca2+]i) in the neurons isolated from the rat ARC, followed by neurochemical identification of these neurons by immunocytochemistry using antisera against growth hormone-releasing hormone (GHRH), neuropeptide Y (NPY) and proopiomelanocortin (POMC), the peptides localized in the ARC. GALP at 10(-10) M increased [Ca2+]i in 11% of single neurons of the ARC, while ghrelin, an orexigenic and GH-releasing peptide, at 10(-10) M increased [Ca2+]i in 35% of the ARC neurons. Some of these GALP- and/or ghrelin-responsive neurons were proved to contain GHRH. In contrast, NPY- and POMC-containing neurons did not respond to GALP. These results indicate that GALP directly targets GHRH neurons, but not NPY and POMC neurons, and that ghrelin directly targets GHRH neurons in the ARC. The former action may be involved in the orexigenic effect of GALP and the latter in the GH-releasing and/or orexigenic effects ghrelin.     

Mutual priming effects of GHRH and arginine on GH secretion: informative procedure for evaluating GH secretory dynamics

To establish a single and reliable test for evaluating GH secretion, we examined successive GH provocation by two agents with different modes of action: GHRH and arginine (Arg). In 4 normal subjects, a bolus injection of 50 micrograms of GHRH followed by 0.5 g/kg Arg infusion after 90 min evoked two GH peaks and the priming of the GHRH potentiated Arg-induced GH peak by 88% of that by Arg alone. In contrast, Arg pretreatment suppressed the GHRH-induced GH peak to a level of 15%. This inhibitory effect of Arg priming was not recovered by an increase in the GHRH dose (100 micrograms) or by prolongation of the GHRH injection period to 180 min. During Arg infusion, plasma somatostatin (SRIH) was significantly reduced and there was a linear correlation between Arg-induced GH peaks and basal TSH levels. This suggests that GH release by Arg is mediated by suppression of hypothalamic SRIH. One subject showed a blunted GH peak in response to GHRH but a normal peak in response to Arg repeatedly, suggesting an endogenous hypertonicity of SRIH. In 4 other normal subjects, the effect of endogenous GH fluctuation on the GHRH-Arg test was examined in the morning, afternoon and evening. The GH secretory profile was fairly consistent in individuals, but in 2 of them, GH response to GHRH was exaggerated in the evening and Arg-unresponsiveness ensued. This potentiation of GH release appears to be due to an increase in endogenous GHRH secretion or a decrease in SRIH tone. The GHRH-Arg test is therefore able to evaluate GH secretory dynamics through two major mechanisms, GHRH stimulation and SRIH inhibition in a single procedure, reducing the incidence of false negative GH response to Arg.     

Peripheral estrogen receptor-alpha selectively modulates the waveform of GH secretory bursts in healthy women

Estradiol (E(2)) drives growth hormone (GH) secretion via estrogen receptors (ER) located in the hypothalamus and pituitary gland. ERalpha is expressed in GH releasing hormone (GHRH) neurons and GH-secreting cells (somatotropes). Moreover, estrogen regulates receptors for somatostatin, GHR peptide (GHRP, ghrelin), and GH itself, while potentiating signaling by IGF-I. Given this complex network, one cannot a priori predict the selective roles of hypothalamic compared with pituitary ER pathways. To make such a distinction, we introduce an investigative model comprising 1) specific ERalpha blockade with a pure antiestrogen, fulvestrant, that does not penetrate the blood-brain barrier; 2) graded transdermal E(2) administration, which doubles GH concentrations in postmenopausal women; 3) stimulation of fasting GH secretion by pairs of GHRH, GHRP-2 (a ghrelin analog), and l-arginine (to putatively limit somatostatin outflow); and 4) implementation of a flexible waveform deconvolution model to estimate the shape of secretory bursts independently of their size. The combined strategy unveiled that 1) E(2) prolongs GH secretory bursts via fulvestrant-antagonizable mechanisms; 2) fulvestrant extends GHRH/GHRP-2-stimulated secretory bursts; 3) l-arginine/GHRP-2 stimulation lengthens GH secretory bursts whether or not E(2) is present; 4) E(2) limits the capability of l-arginine/GHRP-2 to expand GH secretory bursts, and fulvestrant does not inhibit this effect; and 5) E(2) and/or fulvestrant do not alter the time evolution of l-arginine/GHRH-induced GH secretory bursts. The collective data indicate that peripheral ERalpha-dependent mechanisms determine the shape (waveform) of in vivo GH secretory bursts and that such mechanisms operate with secretagogue selectivity.     

Neuronal interactions between galanin-like-peptide- and orexin- or melanin-concentrating hormone-containing neurons

Galanin-like peptide (GALP) is a novel orexigenic neuropeptide that is recently isolated from the porcine hypothalamus. GALP-containing neurons predominantly locate in the hypothalamic arcuate nucleus (ARC). The expression of GALP mRNA within the ARC is increased after the administration of leptin. GALP-containing neurons express leptin receptor and contain alpha-melanocyte-stimulating hormone. We have recently reported that neuropeptide Y (NPY)- and orexin-containing axon terminals are in close apposition with GALP-containing neurons in the ARC. In addition, GALP-containing neurons express orexin-1 receptor (OX1-R). Thus, GALP may function under the influence of leptin and orexin. However, the target neurons of GALP have not yet been clarified. To clarify the neuronal interaction between GALP-containing and other feeding regulating neurons, double-immunostaining method using antibodies against GALP- and orexin- or melanin-concentrating hormone (MCH) was performed in the rat lateral hypothalamus (LH). GALP-immunoreactive fibers appeared to project to the LH around the fornix. They were also found from the rostral to the caudal part of the ARC, paraventricular nucleus (PVH), stria terminalis (BST), medial preoptic area (MPA), and lateral septal nucleus (LSV). Moreover, GALP-like immunoreactive nerve fibers were directly contacted with orexin- and melanin-concentrating hormone (MCH)-like immunoreactive neurons in the LH. Our findings strongly suggest that GALP-containing neurons interact with orexin- and/or MCH-containing neurons in the lateral hypothalamus and that it participates in the regulation of feeding behavior in harmony with other feeding-regulating neurons in the hypothalamus.     

Selective alteration at the growth-hormone- releasing-hormone nerve terminals during aging in GHRH-green fluorescent protein mice

Growth hormone (GH) secretion decreases spontaneously during lifespan, and the resulting GH deficiency participates in aging-related morbidity. This deficiency appears to involve a defect in the activity of hypothalamic GH-releasing hormone (GHRH) neurons. Here, we investigated this hypothesis, as well as the underlying mechanisms, in identified GHRH neurons from adult ( approximately 13 weeks old) and aged ( approximately 100 weeks old) transgenic GHRH-green fluorescent protein mice, using morphological, biochemical and electrophysiological methods. Surprisingly, the spontaneous action potential frequency was similar in adult and aged GHRH neurons studied in brain slices. This was explained by a lack of change in the intrinsic excitability, and simultaneous increases in both stimulatory glutamatergic- and inhibitory GABAergic-synaptic currents of aged GHRH neurons. Aging did not decrease GHRH and enhanced green fluorescent protein contents, GHRH neuronal number or GHRH-fibre distribution, but we found a striking enlargement of GHRH-positive axons, suggesting neuropeptide accumulation. Unlike in adults, autophagic vacuoles were evident in aged GHRH-axonal profiles using electron microscopy. Thus, GHRH neurons are involved in aging of the GH axis. Aging had a subtle effect at the nerve terminal level in GHRH neurons, contrasting with the view that neuronal aging is accompanied by more widespread damage.     

Validation of urinary cortisol as an indicator of hypothalamic-pituitary-adrenal function in the bearded emperor tamarin (Saguinus imperator subgrisescens)

The use of cortisol levels as a measure of stress is often complicated by the use of invasive techniques that may increase hypothalamic-pituitary-adrenal (HPA) axis activity during sample collection. The goal of this study was to collect samples noninvasively and validate an enzyme-immunoassay (EIA) for the measurement of cortisol in urine to quantify HPA axis activity in the bearded emperor tamarin (Saguinus imperator subgrisescens). Urine samples were collected from trained subjects between 0700 and 0730 hr during a 1-month period, and were pooled for immunological validation. We validated the assay immunologically by demonstrating specificity, accuracy, precision, and sensitivity. For biological validation of the assay, we showed that levels of urinary cortisol (in samples collected between 0700 and 1700 hr) varied significantly across the day. Cortisol concentration was lowest at 0700 hr, increased to a mid-morning peak (0900 hr), and declined across the remainder of the day in a typical mammalian circadian pattern. We thus demonstrate that urinary cortisol can be used to quantify HPA activity in S.i. subgrisescens.     

Effect of feeding regimen on diurnal variation of breathing movements in late-gestation fetal sheep

There is a diurnal variation in the mean incidence and amplitude of fetal breathing movements (FBMs) in sheep after approximately 120 days gestation. To determine whether this variation is caused by diurnal fluctuations in plasma glucose or prostaglandin (PG) concentrations, we studied two groups of pregnant sheep fed either once daily at 1100 h or every 2 h for 24 h. Maternal and fetal blood samples were taken every 2 h from 0900 to 0900 h the next day for assay of plasma glucose and PGE2 and PGF2 alpha concentrations. FBMs were recorded throughout the 24 h. The mean fetal plasma glucose concentrations of the once-daily and multifed groups were not different, but there was a significant difference between the two groups in the 24-h pattern of fetal glucose concentrations. In the once-daily fed group, plasma glucose concentrations reached a peak 8 h after maternal feeding and then declined, whereas in the multifed group, fetal plasma glucose concentrations reached a plateau and were constant from 1300 to 0900 h the next day. Fetal plasma PGE2 and PGF2 alpha concentrations did not show a significant change with time of day in either group. There was a significant diurnal variation in the incidence and amplitude of FBMs in each of the two feeding groups, and the 24-h pattern of FBMs did not differ significantly between groups. Therefore it would appear that the diurnal variation of FBMs is not a consequence of the maternal feeding regimen or diurnal changes in plasma glucose or PG concentrations.