Human GHRH Research Articles

Rescue of Isolated GH Deficiency Type II (IGHD II) via Pharmacologic Modulation of GH-1 Splicing.

Isolated GH deficiency (IGHD) type II, the autosomal dominant form of GHD, is mainly caused by mutations that affect splicing of GH-1. When misspliced RNA is translated, it produces a toxic 17.5-kDa GH isoform that reduces the accumulation and secretion of wild-type-human GH (wt-hGH). Usually, isolated GHD type II patients are treated with daily injections of recombinant human GH in order to maintain normal growth. However, this type of replacement therapy does not prevent toxic effects of the 17.5-kDa GH isoform on the pituitary gland, which can eventually lead to other hormonal deficiencies. Here, we tested the possibility to restore the constitutive splicing pattern of GH-1 by using butyrate, a drug that mainly acts as histone deacetylase inhibitor. To this aim, wt-hGH and/or different hGH-splice site mutants (GH-IVS3+2, GH-IVS3+6, and GH-ISE+28) were transfected in rat pituitary cells expressing human GHRH receptor (GHRHR) (GC-GHRHR). Upon butyrate treatment, GC-GHRHR cells coexpressing wt-hGH and each of the mutants displayed increased GH transcript level, intracellular GH content, and GH secretion when compared with the corresponding untreated condition. The effect of butyrate was most likely mediated by the alternative splicing factor/splicing factor 2. Overexpression of alternative ASF/SF2 in the same experimental setting, indeed, promoted the amount of full-length transcripts thus increasing synthesis and secretion of the 22-kDa GH isoform. In conclusion, our results support the hypothesis that modulation of GH-1 splicing pattern to increase the 22-kDa GH isoform levels can be clinically beneficial and hence a crucial challenge in GHD research.

Growth Hormone (GH)-Releasing Hormone Increases the Expression of the Dominant-Negative GH Isoform in Cases of Isolated GH Deficiency due to GH Splice-Site Mutations

An autosomal dominant form of isolated GH deficiency (IGHD II) can result from heterozygous splice site mutations that weaken recognition of exon 3 leading to aberrant splicing of GH-1 transcripts and production of a dominant-negative 17.5-kDa GH isoform. Previous studies suggested that the extent of missplicing varies with different mutations and the level of GH expression and/or secretion. To study this, wt-hGH and/or different hGH-splice site mutants (GH-IVS+2, GH-IVS+6, GH-ISE+28) were transfected in rat pituitary cells expressing human GHRH receptor (GC-GHRHR). Upon GHRH stimulation, GC-GHRHR cells coexpressing wt-hGH and each of the mutants displayed reduced hGH secretion and intracellular GH content when compared with cells expressing only wt-hGH, confirming the dominant-negative effect of 17.5-kDa isoform on the secretion of 22-kDa GH. Furthermore, increased amount of 17.5-kDa isoform produced after GHRH stimulation in cells expressing GH-splice site mutants reduced production of endogenous rat GH, which was not observed after GHRH-induced increase in wt-hGH. In conclusion, our results support the hypothesis that after GHRH stimulation, the severity of IGHD II depends on the position of splice site mutation leading to the production of increasing amounts of 17.5-kDa protein, which reduces the storage and secretion of wt-GH in the most severely affected cases. Due to the absence of GH and IGF-I-negative feedback in IGHD II, a chronic up-regulation of GHRH would lead to an increased stimulatory drive to somatotrophs to produce more 17.5-kDa GH from the severest mutant alleles, thereby accelerating autodestruction of somatotrophs in a vicious cycle.

Identification of two novel chicken GHRH receptor splice variants: implications for the roles of aspartate 56 in the receptor activation and direct ligand–receptor interaction

In this study, two novel GHRHR receptor splice variants, named chicken GHRHR-v1 (cGHRHR-v1) and cGHRHR-v2 respectively, were identified from chicken pituitary using RT-PCR assay. cGHRHR-v1 is characterized by an N-terminal deletion of 36 amino acid residues, including an aspartate at position 56 (Asp(56)) conserved in G protein-coupled receptor B-I subfamily. cGHRHR-v2 is a carboxyl-terminal truncated receptor variant with four putative transmembrane domains, which arose from alternative use of a splice acceptor site on intron 8. Using the pGL3-CRE-luciferase reporter system, the functionality of the two variants was examined in Chinese hamster ovary cells. cGHRHR-v1 was shown to be capable of transmitting signal upon agonist stimulation, but cGHRHR-v2 could not. Both GHRH and pituitary adenylate cyclase-activating peptide (PACAP) could activate cGHRHR-v1 at high dosages (GHRH >/=10(-8) M; PACAP >/=10(-6) M) and GHRH was much more potent than PACAP, suggesting that cGHRHR-v1 is a functional membrane-spanning receptor with an impairment in high-affinity ligand binding, rather than in receptor activation and ligand-binding specificity. This finding also points out the possibility that Asp(56) is not a critical determinant for receptor activation and direct ligand-receptor interaction. To substantiate this hypothesis, using site-directed mutagenesis, two receptor mutants with replacement of Asp(56) by Ala or Gly were generated. Expectedly, chicken or human GHRH could still activate both receptor mutants with reduced potencies (about 2- to 14-fold less potent). Taken together, our findings not only suggest that cGHRHR variants may play a role in controlling normal pituitary functions, but also support that Asp(56) is nonessential for receptor activation and direct ligand-receptor interaction.

Endocrine and orexigenic actions of growth hormone secretagogues in rainbow trout (Oncorhynchus mykiss)

The effects of growth hormone secretagogues (GHSs) on the teleost somatotropic axis are poorly understood, particularly with respect to insulin-like growth factor-I (IGF-I) and the IGF-binding proteins (IGFBPs). To assess the endocrine and orexigenic responses of rainbow trout (Oncorhynchus mykiss) to GHS treatment, animals were injected with human GHRH(1-29)-amide, KP-102 or rat ghrelin at 0, 1 or 10 pmol/g body mass. Feed intake was tested at 2 and 5 h post-injection and plasma levels of growth hormone (GH), IGF-I and the IGFBPs were determined at 3, 6 and 12 h post-injection. Feed intake was significantly elevated by all of the GHSs tested at both post-injection time points. All GHSs elevated plasma GH levels in a time-dependent manner. Plasma IGF-I levels were elevated by all GHSs at 3 h post-injection, whereas those animals treated with KP-102 and ghrelin exhibited depressions at 6 h. Four IGFBPs were identified in the plasma by western blotting. Levels of the 20 kDa IGFBP decreased over the sampling time. Levels of the 32 kDa IGFBP were significantly depressed by all GHSs tested. Levels of the 42 kDa IGFBP were significantly elevated by all GHSs tested. Plasma levels of the 50 kDa IGFBP was decreased in some treatment groups at 3 h, but elevated by 6 h in the ghrelin-treated groups and elevated in all treatment groups by 12 h post-injection. The endocrine and orexigenic responses demonstrate that GHSs influence the teleost neuroendocrine system beyond short-term actions (<3 h post-injection) on GH release and the responses of the IGFBPs to GHS treatment support this notion and clarify their identification as functional homologues to mammalian IGFBPs.

Cloning of the Chicken Pituitary Receptor for Growth Hormone-Releasing Hormone

Details of the regulation of GH in birds are unclear. In this report, a receptor was cloned from chicken pituitary cDNA with 61% amino acid sequence identity to the human pituitary GHRH receptor. Phylogenies inferred from sequence alignments support that this is the chicken counterpart of the GHRH receptor known in mammals. Northern blotting shows that this receptor message is expressed in chicken pituitary, with lesser amounts seen in hypothalamus and brain but not in liver. The recombinant chicken receptor binds human GHRH with high affinity and specificity and signals cAMP accumulation. Surprisingly, available peptides synthesized to the published sequence for chicken GHRH-like peptide (cGHRH-LP) were inactive at this receptor. To address this we recloned the cDNA for this cGHRH-LP from chicken hypothalami. The revised sequence encodes lysine at position 21, which is consistent with all reported GHRH sequences from other species but different from the originally published chicken sequence. When this revised cGHRH-LP sequence was synthesized, it had improved but still weak potency at the cloned receptor. Consistent with the activity at the cloned receptor, human GHRH was potent when assayed in live chickens or on chicken pituitary membranes, but cGHRH-LP was not. We conclude that we have cloned a putative GHRH receptor that is homologous to mammalian GHRH receptors and functionally expressed in chicken pituitary, but that the identity of the endogenous ligand remains unclear. The chicken GHRH receptor cloned in this study can serve as a tool to identify its ligand and to clarify the evolutionary development of the regulation of GH.

Effect of recombinant human GH and GHRH on plasma metabolite levels in rainbow trout (Oncorhynchus mykiss)

The aim of the present work was to study metabolic changes in rainbow trout treated with preparations of human recombinants of growth hormone (rhGH) and GH-releasing hormone (rhGHRH), by analysing time course variations in plasma metabolite levels. Trouts were given the hormones by intraperitoneal injection in a single dose (rhGH: 0.5microg/g b.w.; rhGHRH: 0.06microg/g b.w.) or as a weekly injection over a period of 4 weeks. The concentrations of glucose, lactate, free fatty acids and glycerol were determined in plasma immediately before injection and 1, 3, 6, 12, 24, 48 and 168 hours post-injection. Results indicate that glucose and lactate levels increase, while slight variations in free fatty acids was observed after the injection of rhGH and rhGHRH.

Inhibitory effects of antagonistic analogs of GHRH on GH3 pituitary cells overexpressing the human GHRH receptor.

GH3 rat pituitary tumor cells produce GH and prolactin (PRL), but lack the GHRH receptor (GHRH-R). We expressed human GHRH-R (hGHRH-R) in GH3 cells using recombinant adenoviral vectors and studied the effects of GHRH antagonists. The mRNA expression of the GHRH-R gene in the cells was demonstrated by RT-PCR. An exposure of the GH3 cells infected with hGHRH-R to 10(-10), 10(-9) and 10(-8) m hGHRH for 1 or 2 h in culture caused a dose-dependent elevation of the intracellular cAMP concentration and the cAMP efflux. Exposure to hGHRH also elicited dose-dependent increases in GH and PRL secretion from these cells. Neither the uninfected nor the antisense hGHRH-R-infected control cells exhibited cAMP, GH and PRL responses to GHRH stimulation. GHRH antagonists JV-1-38 and jv-1-36 applied at 3x10(-8) m for 3 h, together with 10(-9) m GHRH, significantly inhibited the GHRH-stimulated cAMP efflux from the hGHRH-R-infected cells by 36 and 80% respectively. The more potent antagonist JV-1-36 also decreased the intracellular cAMP levels in these cells by 55%. Exposure to JV-1-36 for 1 h nullified the stimulatory effect of GHRH on GH secretion and significantly inhibited it by 64 and 77% after 2 and 3 h respectively. In a superfusion system, GHRH at 10(-10), 10(-9) and 10(-8) m concentrations induced prompt and dose-related high cAMP responses and smaller increases in the spontaneous GH secretion of the hGHRH-R-infected cells. Antagonists JV-1-36 and JV-1-38 applied at 3x10(-8) m for 15 min, together with 10(-9) m GHRH, inhibited the GHRH-stimulated cAMP response by 59 and 35% respectively. This work demonstrates that GHRH antagonists can effectively inhibit the actions of GHRH on the hGHRH-R. Our results support the view that this class of compounds would be active clinically.

Impact of Estradiol Supplementation on Dual Peptidyl Drive of GH Secretion in Postmenopausal Women

As an indirect probe of estrogen-regulated hypothalamic somatostatin restraint, the present study monitors the ability of short-term oral E2 supplementation to modulate GH secretion during combined continuous stimulation by recombinant human GHRH [GHRH-(1–44)-amide] and the potent and selective synthetic GH-releasing peptide, GHRP-2. According to a simplified tripeptidyl model of GH neuroregulation, the effects of estrogen in this dual secretagogue paradigm should mirror alterations in endogenous somatostatinergic signaling. To this end, seven healthy postmenopausal women underwent frequent (10-min) blood sampling for 24 h during simultaneous iv infusion of GHRH and GHRP-2 each at a rate of 1 μg/kg·h on d 10 of randomly ordered placebo or 17β-estradiol (E2) (1 mg orally twice daily) replacement. Serum GH concentrations (n = 280/subject) were assayed by chemiluminescence. The resultant GH time series was evaluated by deconvolution analysis, the approximate entropy statistic, and cosine regression to quantitate pulsatile, entropic (feedback-sensitive), and 24-h rhythmic GH release, respectively. Statistical comparisons revealed that E2 repletion increased the mean (±sem) serum E2 concentration to 222 ± 26 pg/ml from 16 ± 1.7 pg/ml during placebo (P < 0.001) and suppressed the serum LH by 48% (P = 0.0033), serum FSH by 64% (P < 0.001), and serum IGF-I by 44% (P = 0.021). Double peptidyl secretagogue stimulation elevated mean 24-h serum GH concentrations to 8.1 ± 1.0 μg/liter (placebo) and 7.7 ± 0.89 μg/liter (E2; P = NS) and evoked prominently pulsatile patterns of GH secretion. No primary measure of pulsatile or basal GH release was altered by the disparate sex steroid milieu, i.e. GH secretory burst amplitudes of 0.62 ± 0.93 (placebo) and 0.72 ± 0.16 (E2) μg/liter·min, GH pulse frequencies of 27 ± 1.8 (placebo) and 23 ± 1.9 (E2) events/24 h, GH half-lives of 12 ± 0.74 (placebo) and 15 ± 4.5 (E2) min, and basal (nonpulsatile) GH secretion 70 ± 22 (placebo) and 57 ± 18 (E2) ng/liter·min. The approximate entropy (ApEn) of serial GH release [1.297 ± 0.061 (placebo) and 1.323 ± 0.06 (E2)] and the mesor (cosine mean), amplitude, and acrophase (time of the maximum) of 24-h rhythmic GH secretion were likewise invariant of estrogen supplementation. Estimated statistical power exceeded 90% for detecting significant (P < 0.05) within-subject changes exceeding 30–50% in the mean serum GH concentration, GH ApEn, or GH mesor. In contrast, ApEn analysis of the evolution of successive GH secretory burst-mass values over 24 h disclosed that E2 replacement disrupts the serial regularity of pulsatile GH output (elevates the ApEn ratio) during combined GHRH/GHRP-2 stimulation (P = 0.004). In summary, short-term elevation of serum E2 concentrations in postmenopausal individuals into the midfollicular phase range observed in young women does not significantly alter 24-h basal, pulsatile, entropic, or nyctohemeral GH secretion monitored under continuous combined drive by GHRH and GHRP-2. As E2 repletion without enforced GHRH/GHRP-2 stimulation augments each of the foregoing regulated facets of GH release, we infer that one or both of the infused peptidyl secretagogues may itself participate in E2’s short-term amplification of GH secretion in postmenopausal individuals. Estrogen’s disruption of the orderliness of sequential GH pulse-mass values during fixed GHRH/GHRP-2 feedforward would be consistent with a subtle reduction in the release and/or actions of hypothalamic somatostatin or an (unexpected) direct pituitary action of the sex steroid. Whether comparable dynamics mediate the effects of endogenous estrogen on the GH axis in premenopausal women or pubertal girls is not known.

The effect of GHRH on somatotrope hyperplasia and tumor formation in the presence and absence of GH signaling.

Excessive GHRH stimulation leads to somatotrope hyperplasia and, ultimately, pituitary adenoma formation in the metallothionein promoter-driven human GHRH (hGHRH) transgenic mouse. This pituitary phenotype is similar to that observed in humans with ectopic production of GHRH. In both mice and man, GHRH hyperstimulation also results in dramatic increases in circulating GH and IGF-I. To determine whether GH/IGF-I modulates the development and growth rate of GHRH-induced pituitary tumors, pituitary growth and histology were evaluated in mice generated from cross-breeding metallothionein promoter-driven hGHRH transgenic mice with GH receptor binding protein (GHR) gene disrupted mice (GHR(-/-)). Expression of the hGHRH transgene in 2-month-old GHR intact (GHR(+)) mice resulted in the doubling of pituitary weight that was largely attributed to an increase in the number of GH-immunopositive cells. Pituitary weight of GHR(+) hGHRH mice did not significantly change between 2 and 6 months of age, whereas at 12 months, weights increased up to 100-fold those of GHR(+) pituitaries, and 70% of the glands contained grossly visible adenomas. All adenomas stained positively for GH, whereas some showed scattered PRL staining. Pituitaries of GHR(-/-) mice were half the size of those of GHR(+) mice. Although reduced in size, the histological features of GHR(-/-) mouse pituitaries were suggestive of somatotrope hyperplasia. Despite evidence of somatotrope hyperplasia, pituitaries from GHR(-/-) mice as old as 28 months of age were similar in size to those of 2-month-old mice and did not show signs of adenoma formation. Expression of the hGHRH transgene in GHR(-/-) mice did not significantly increase pituitary size between 2 and 6 months of age. However, at 12 months the majority of GHR(-/-), hGHRH pituitaries developed adenomas with mean pituitary weight and histological features similar to those of GHR(+), hGHRH mice. These observations demonstrate that intact GH signaling is not required for GHRH tumor formation. Although the majority of GHR(+), hGHRH and GHR(-/-), hGHRH pituitaries developed tumors by 12 months of age, a small subset remained morphologically indistinct from those at 2 months of age. These observations taken together with the fact that overt tumor formation is preceded by a static pituitary growth phase between 2 and 6 months, indicates that protective mechanisms are in place to maintain pituitary mass despite hGHRH hyperstimulation.

A two-sample method for assessing growth hormone response to growth hormone-releasing hormone challenge: use as a predictor of gain in beef bulls.

In two experiments, Black Angus bulls were challenged at weaning with GHRH analog and evaluated for their GH response to determine whether GH response can predict subsequent growth characteristics. The GH response was determined by measuring GH in blood serum collected 0 and 10 min after GHRH injection (Exp. 1: 1.5 microg/100 kg BW human GHRH, n = 34; Exp. 2: 1.5 and 4.5 microg/100 kg BW bovine GHRH [treatments LGHRH and HGHRH, respectively] administered 3 h after a 4.5 microg/100 kg BW "clearance dose" of GHRH, n = 38]. In Exp. 1, GH response did not predict growth or carcass measurements. In Exp. 2, GH response to LGHRH was positively related to ADG (R2 = .18; P = .007) during a 112-d controlled feeding trial. In addition, there was a tendency for bulls with a greater GH response to HGHRH to exhibit greater ADG than animals with a low response. However, GH response to GHRH was not related to changes in hip height (HH) or carcass ultrasound measurements at d 112 of the growth performance trial. Response of GH to repeated GHRH challenges was consistent within animal over time (r = .47; P = .003). The use of a clearance dose 3 h prior to GHRH challenge improved the relationship between GH response and ADG. Results of this study suggest that GH response to GHRH challenge is a useful tool for identifying beef bulls with superior growth potential.

Predicting bull growth performance and carcass composition from growth hormone response to growth hormone-releasing hormone.

Development of practical, physiologically based methods that provide an early, yet accurate, evaluation of a bull's genetic merit could benefit the beef industry. The use of GH response to a single, acute dose of GHRH was evaluated as a predictor of future growth performance and carcass characteristics of weanling bulls. Fifty-six Angus bulls averaging 229 d (SD = 27) of age were administered three doses i.v. (0, 1.5, and 4.5 microg/100 kg BW) of human GHRH (1-29) analog in a Latin square design balanced for residual effects. Blood samples were collected via jugular catheter at -60, -45, -30, -15, 0, 5, 10, 15, 30, 45, 60, 90 and 120 min relative to GHRH injection. Serum concentrations of GH were plotted over time. Response to GHRH was calculated as the area under the GH response curve (AUC-GH) using the trapezoidal approximation. Relationships between AUC-GH, weaning weight adjusted to 205 d of age (205-d WW), and direct weaning weight EPD (WWEPD) versus age-adjusted BW (BWadj), ADG, and carcass measurements from a 140-d growth performance test were evaluated using simple linear regression. A positive correlation between AUC-GH and ADG and an inverse relationship between AUC-GH and carcass fat were observed. The present study provides evidence that AUC-GH is a better predictor of future growth performance in beef bulls than 205-d WW or WWEPD values. Thus, GH response to GHRH is associated with subsequent growth and may be a useful tool for sire selection in beef production.

Pathophysiology of the neuroregulation of growth hormone secretion in experimental animals and the human.

During the last decade, the GH axis has become the compelling focus of remarkably active and broad-ranging basic and clinical research. Molecular and genetic models, the discovery of human GHRH and its receptor, the cloning of the GHRP receptor, and the clinical availability of recombinant GH and IGF-I have allowed surprisingly rapid advances in our knowledge of the neuroregulation of the GH-IGF-I axis in many pathophysiological contexts. The complexity of the GHRH/somatostatin-GH-IGF-I axis thus commends itself to more formalized modeling (154, 155), since the multivalent feedback-control activities are difficult to assimilate fully on an intuitive scale. Understanding the dynamic neuroendocrine mechanisms that direct the pulsatile secretion of this fundamental growth-promoting and metabolic hormone remains a critical goal, the realization of which is challenged by the exponentially accumulating matrix of experimental and clinical data in this arena. To the above end, we review here the pathophysiology of the GHRH somatostatin-GH-IGF-I feedback axis consisting of corresponding key neurotransmitters, neuromodulators, and metabolic effectors, and their cloned receptors and signaling pathways. We propose that this system is best viewed as a multivalent feedback network that is exquisitely sensitive to an array of neuroregulators and environmental stressors and genetic restraints. Feedback and feedforward mechanisms acting within the intact somatotropic axis mediate homeostatic control throughout the human lifetime and are disrupted in disease. Novel effectors of the GH axis, such as GHRPs, also offer promise as investigative probes and possible therapeutic agents. Further understanding of the mechanisms of GH neuroregulation will likely allow development of progressively more specific molecular and clinical tools for the diagnosis and treatment of various conditions in which GH secretion is regulated abnormally. Thus, we predict that unexpected and enriching insights in the domain of the neuroendocrine pathophysiology of the GH axis are likely be achieved in the succeeding decades of basic and clinical research.

Tissue-specific molecular heterogeneity of human growth hormone-releasing hormone receptor protein

A site-directed anti-peptide antibody (anti-hGHRHRc18) was generated against the cytoplasmic tail of human GHRH receptor. The dissociation constant ( K d ) and the antibody binding site (AbT) of anti-hGHRHRc18 were 2.5 nmol/l and 0.54 nmol/l, respectively. In an immunoblotting experiment, affinity-purified anti-hGHRHRc18 specifically recognized a single 50-kDa protein in human pituitary. In a screening of the expression of GHRH receptor protein in extra-pituitary tissues, only human kidney showed a single 52-kDa protein. Our results suggest that the GHRH receptor protein exhibits tissue-specific molecular heterogeneity.

Hypothalamic control of growth hormone (GH) secretion in type I diabetic men: effect of the combined administration of GH-releasing hormone and hexarelin, a novel GHRP-6 analog.

Insulin dependent (type I) diabetic patients show abnormal growth hormone (GH) secretion. Hexarelin is an analog of GHRP-6 which releases GH in part via somatostatin inhibition. The aim of our study was to evaluate the effects of hexarelin and GHRH, administered either alone or in combination, on GH secretion in 10 type I diabetic and 7 normal men. All the subjects were administered: 1) human GHRH (1-29) NH2 100 micrograms i.v. bolus at 0 min; 2) hexarelin 100 micrograms i.v. bolus at 0 min; 3) hexarelin 100 micrograms + hGHRH 100 micrograms i.v. bolus at 0 min. In type I diabetic patients significantly greater GH responses to GHRH and hexarelin have been observed with normal subjects. Hexarelin caused a significantly (p < 0.05) greater GH response as compared to GHRH in both diabetic and control subjects. After the administration of hexarelin+GHRH, a significant increase in both GH absolute and peak levels as compared to hexarelin or GHRH alone was found in all the subjects. However, the GH responses to the combined stimuli were not significantly different in diabetics as compared to normals; moreover, the interaction of GHRH and hexarelin was synergistic in controls and additive in diabetics. We hypothesize that a reduction in the hypothalamic somatostatin inhibitory tone combined with increased pituitary GH production may be responsible for the pattern of the GH responses to hexarelin and GHRH observed in our type I diabetic patients.

Effect of intravenous or intracerebroventricular injections of His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 on GH release in conscious, freely moving male rats.

The present study was designed to examine the effects of intracerebroventricular injection of His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP) on GH secretion in freely moving conscious male rats and to determine whether the central action of GHRP is mediated by increased GHRH and/or somatostatin (SRIF) release. A significant and dose-related suppression of natural fluctuations in plasma GH occurred immediately after intracerebroventricular injection of GHRP at the doses of 1, 3 and 10 mu g/rat and was sustained for more than 1 h. An intracerebroventricular injection of GHRP also suppressed plasma GH rises after an intravenous injection of [D-Ala2, Nle27] human GHRH (1-28)agmatine (hGHRH analog), but the GH suppression by intracerebroventricular GHRP was significantly blunted in rats treated with antirat SRIF gamma-globulin (SRIF-ab). In addition, the suppression by intracerebroventricular GHRP of hGHRH-analog-induced GH release was observed even in rats treated with anti-rat GHRH goat gamma-globulin (GHRH-ab) which does not cross-react with hGHRH analog, and again its suppression was significantly attenuated by simultaneous treatment with SRIF-ab and GHRH-ab. Furthermore, intracerebroventricularly injected GHRP was effective enough to inhibit the central-nervous-system (endogenous GHRH)-driven natural GH-secretory surges in SRIF-ab-treated rats. When 10 mu g/kg GHRP was injected intravenously every 1 h during a 7-hour observation period in control rats without antibody treatment, plasma GH levels were increased with the peak 10 min after each injection during the surge period of the GH-secretory rhythm, but the plasma GH response to GHRP was frequently absent during its trough period. In addition, in SRIF-ab-treated rats, basal GH levels as well as plasma GH peaks after GHRP injections were markedly elevated and the obvious plasma GH rises after GHRP could be observed even during the trough period. In GHRH-ab-treated rats, basal GH levels were lower, and the plasma GH response to GHRP was significantly attenuated as compared with those in control rats. Plasma GH peaks after GHRP injections in rats simultaneously treated with SRIF-ab and GHRH-ab were higher than those in rats given GHRH-ab alone, but its difference in GH response was obviously smaller than that observed between SRIF-ab-treated and control rats, suggesting a crucial role of endogenous GHRH in GHRP-induced GH release. To further clarify the interaction of GHRH and GHRP in GH release, hGHRH analog was injected intravenously alone or simultaneously with GHRP in rats treated with SRIF-ab and GHRH-ab. The peak GH values after simultaneous injection of hGHRH analog and GHRP were significantly higher than the sum of GH peaks after each injection of hGHRH analog and GHRP. In conclusion, an intracerebroventricular injection of GHRP suppresses GH secretion by a central mechanism which is likely to include increased SRIF release, reduced GHRH release or both. We have confirmed that intravenous injection of GHRP can stimulate GH release by itself in an immunologically nullified condition of circulating endogenous GHRH and SRIF in rats, and that endogenous SRIF modestly suppresses the GH-stimulating effect of intravenously injected GHRP while both endogenous and exogenous GHRH markedly enhance GH release by intravenously injected GHRP in a synergistic manner.

Effects of methimazole treatment on growth hormone (GH) response to GH-releasing hormone in patients with hyperthyroidism

In vitro studies have demonstrated that thyroid hormones can enhance basal and stimulated growth hormone secretion by cultured pituitary cells. However, both in man and in the rat the effects of high thyroid hormone levels on GH secretion are unclear. The aim of our study was to test the GH response to human GHRH in hyperthyroid patients and to evaluate the effects on GH secretion of short- and long-term pharmacological decrease of circulating thyroid hormones. We examined 10 hyperthyroid patients with recent diagnosis of Graves' disease. Twelve healthy volunteers served as controls. All subjects received a bolus iv injection of GHRH(1-29)NH2, 100 micrograms. Hyperthyroid patients underwent a GHRH test one and three months after starting antithyroid therapy with methimazole, 10 mg/day po. GH levels at 15, 30, 45, 60 min and GH peak after stimulus were significantly lower in hyperthyroid patients than in normal subjects. The GH peak was also delayed in hyperthyroid patients. After one month of methimazole therapy, most of the hyperthyroid patients had thyroid hormone levels in the normal range, but they did not show significant changes in GH levels after GHRH, and the GH peak was again delayed. After three months of therapy with methimazole, the hyperthyroid patients did not show a further significant decrease in serum thyroid hormone levels. However, mean GH levels from 15 to 60 min were significantly increased compared with the control study. The GH peak after GHRH was also earlier than in the pre-treatment study.(ABSTRACT TRUNCATED AT 250 WORDS)

Pyridostigmine Blocks the Inhibitory Effect of Glucocorticoids on Growth Hormone-Releasing Hormone Stimulated Growth Hormone Secretion in Normal Man

Glucocorticoids have been shown to inhibit GH secretion in normal man when acutely and chronically administered in pharmacological amounts. Pyridostigmine (PD), an acetylcholinesterase inhibitor, is able to elicit GH secretion when administered alone and to enhance the GH response to GHRH in normal subjects probably via a decrease in the hypothalamic release of somatostatin. The aim of the present study was to investigate the influence of glucocorticoids on the GH response to PD administered either alone or in combination with GHRH in normal adult subjects. Six healthy adult volunteers underwent six experimental protocols. They received 1) human (h) GHRH(1-29)NH2, 100 micrograms injected as an iv bolus; 2) cortisone acetate, 50 mg administered orally (po) 60 min before an hGHRH iv bolus injection; 3) PD, 120 mg administered po, 60 min before an hGHRH iv bolus injection; 4) PD and cortisone acetate, administered po 60 min before an hGHRH iv bolus injection; 5) PD, administered po 60 min before a saline iv bolus injection; 6) PD and cortisone acetate administered po 60 min before a saline iv bolus injection. Mean GH levels, peak GH levels, and GH area under the curves (AUCs) were significantly lower after GHRH + cortisone as compared to GHRH alone. However, these parameters were not significantly different after PD + GHRH + cortisone when compared to PD + GHRH and after PD + cortisone when compared to PD alone. We conclude that acute administration of pharmacological amounts of glucocorticoids cannot inhibit the GH response to PD alone or in combination with GHRH. Thus, we hypothesize that the inhibitory action of glucocorticoids on the GH response to GHRH in man may be mediated by an enhancement of either somatostatin release by the hypothalamus or somatostatin action on the pituitary.

Neuropeptides influencing in vitro pituitary hormone secretion in hatchling turtles

AbstractStatic culture techniques were used to examine the effects of neuropeptides on thyrotropin (TSH), growth hormone (GH), and luteinizing hormone (LH) secretion by turtle pituitaries (Pseudemys scripta). Pituitaries were exposed to 2.5–2,500 nM thyrotropin‐releasing hormone (TRH), ovine corticotropin‐releasing hormone (oCRH), sauvagine (SV), carp urotensin I (UI), rat growth hormone‐releasing hormone (rGHRH), human GHRH (hGHRH), carp GHRH‐like immunoreactive peptide (cGHRH‐LIP), vasoactive intestinal peptide (VIP), arginine vasotocin (AVT), salmon gonadotropin‐releasing hormone (sGnRH) or chicken II GnRH (cGnRH). Medium concentrations of TSH, GH, and LH were estimated by RIA. Significant dose responses in TSH secretion were observed with TRH, oCRH, rGHRH, and hGHRH; VIP was also a potent stimulator of TSH, but a dose response was not observed. The two CRH‐like molecules, SV and UI, also stimulated TSH at the single dose tested (25 nM). All these neuropeptides had similar potencies. In one test in which PRL was measured, it was stimulated by 25 nM TRH but not by an equivalent dose of the CRH‐like neuropeptides. GH secretion was elevated by TRH, rGHRH, hGHRH, and VIP. oCRH at the highest dose (250 nM) stimulated GH, but SV and UI had no effect on GH at the single dose tested (25 nM). Neither AVT, cGHRH‐LIP, nor the two GnRH molecules had any effect on TSH or GH secretion. Data obtained by bioassay of TSH in representative samples were consistent with results of RIA.In contrast to the diversity of neuropeptides affecting pituitary TSH and GH, only GnRH stimulated significant and marked increases in LH secretion. AVT produced a small increase in LH output at the higher doses tested (250 and 2,500 nM), and all other neuropeptides were inactive at all doses. Taken together with other comparative data, these results suggest that in ectothermal vertebrates, TSH and GH responses are generally not as specific for neuropeptides as they are in normal mammals but may resemble mammals in certain abnormal states. However, there is not a clear phylogenetic pattern in which neuropeptides may be stimulatory to TSH or GH. The gonadotropes in both ectotherms and mammals appear to be relatively specific for molecules of the GnRH family.

Maternal Modulation of Growth Hormone Secretion in the Neonatal Rat. I. Involvement of Milk Factors*

The present experiments investigated the time course of maternal modulation of GH secretion and examined the possible role of milk in the regulation of GH secretion in neonatal rats. Serum GH concentrations in neonatal rats were high at birth and declined over time postpartum. Separation of rat pups from their mothers decreased, while a subsequent period of suckling increased, serum GH levels in rat pups on postpartum days 1-14, but not on day 20. The water-soluble fraction (infranatant) of rat milk contained immunoreactive (ir) rat GH-releasing hormone (rGHRH)-like material (725.06 +/- 81.29 pg/ml), ir-somatostatin-like activity (1.64 +/- 0.2 ng/ml), and irGH (4.79 +/- 0.73 ng/ml). The concentrations of these hormones tended to decrease with time postpartum and were positively correlated with each other (r = 0.70; P less than or equal to 0.0001). IrPRL was also present in the infranatant (148.44 +/- 14.55 ng/ml), but levels were not correlated with the other hormones detected. Milk infranatant stimulated GH secretion from perifused neonatal rat pituitary glands in vitro. Milk infranatant also stimulated GH secretion in vivo when administered sc or intragastrically to 2- or 8-day-old rat pups. The GH-releasing effect was not due to gastric distension or nonspecific nutritive components, as neither 0.9% saline nor nutrients (5% glucose and 10% BSA) increased serum GH levels. The presence of high concentrations of irGHRH in rat milk infranatant and the strong correlation between the irGHRH concentrations of milk samples and the in vitro GH response (r = 0.71; P less than or equal to 0.005) suggested that this peptide is a major candidate for producing the in vitro and in vivo GH-stimulating activity in rat milk. However, the minimally effective concentration of synthetic rGHRH required to stimulate GH release in the superfusion system was between 1-10 nM, which exceeds milk irGHRH levels by 100- to 1000-fold. Moreover, in vivo administration of synthetic rGHRH (sc or intragastrically) was unable to increase serum GH concentrations in 2-day-old pups, although a large dose (100 ng/g) of human GHRH sc was effective. These findings indicate that rat milk may be an important maternal factor that modulates GH secretion and, consequently, growth during the neonatal period. Rat milk has GH-releasing activity both in vivo and in vitro in neonatal rats, but the GH-releasing activities of milk are probably only minimally due to its rGHRH content.

Bioactivity of growth hormone releasing hormone (1–29) analogues after SC injection in man

The bioactivity of growth hormone releasing hormone 1-29 [GHRH(1-29)NH2] has been compared with that of an agonist analogue [Ac-D-Tyr1,D-Ala2]-GHRH(1-29)NH2, in normal male volunteers. Using a submaximal dose of 3 micrograms/kg administered subcutaneously, peak growth hormone (GH) response and area under the GH curve were similar for the native and agonist analogue. In addition, no significant differences were found in peak GHRH(1-29) immunoreactivity, area under the GHRH(1-29) curves or plasma disappearance rates of the two peptides. The results suggest that, in keeping with the relative activities of other "superactive" analogues tested so far, the greatly enhanced activity of [Ac-D-Tyr1,D-Ala2]-GHRH(1-29)NH2 observed in the rat is not found in humans. It is possible that this species difference is due to differences in the interaction of GHRH peptides with the rat and the human somatotroph GHRH receptor.