Protein kinase C-dependent growth hormone releasing peptides stimulate cyclic adenosine 3',5'-monophosphate production by human pituitary somatotropinomas expressing gsp oncogenes: evidence for crosstalk between transduction pathways.

The effects of the synthetic GH-releasing peptides, GHRP-2 and GHRP-6, on phosphatidylinositol (PI) hydrolysis and cAMP production have been examined in human pituitary somatotropinomas with and without adenylyl cyclase-activating gsp oncogenes. Both peptides dose-dependently stimulated the rate of PI hydrolysis and GH secretion by cell cultures of both types of somatotropinoma. GHRP-2 was considerably more potent than GHRP-6. The effects on GH secretion were reduced or abolished by phloretin, an inhibitor of protein kinase C, and W7, an inhibitor of calmodulin. However, antagonism of the GHRH-receptor and of protein kinase A with (N-Ac-Tyr1,D-Arg2)GRF-(1-29)-NH2 and Rp-adenosine-3',5'-cyclic monophosphothioate, respectively, did not alter the stimulatory effects of GHRP-2 and GHRP-6 on GH secretion. The effect of GHRP-2 and/or GHRP-6 on cAMP production was studied in 15 tumors, seven of which possessed constitutive adenylyl cyclase activity as evidenced by presence of gsp oncogenes. Both peptides stimulated cAMP production in the latter but not former types of tumor. Moreover, GHRP-2 and GHRP-6 potentiated the stimulation of cAMP production induced by GHRH and pituitary adenylate cyclase-activating polypeptide in tumors without gsp oncogenes. These results demonstrate that GHRP-2 and GHRP-6 exert identical effects on human pituitary somatotropinomas, except for differences in potency. Additionally, under conditions of adenylyl cyclase activity above basal levels (i.e. through stimulation of G2-protein coupled receptors or because of gsp oncogene expression), cAMP production can be increased even further by GHRP, providing evidence for cross-talk between the PI and adenylyl cyclase transduction systems in pituitary cells.

Ghrelin.

BACKGROUND: The gastrointestinal peptide hormone ghrelin was discovered in 1999 as the endogenous ligand of the growth hormone secretagogue receptor. Increasing evidence supports more complicated and nuanced roles for the hormone, which go beyond the regulation of systemic energy metabolism. SCOPE OF REVIEW: In this review, we discuss the diverse biological functions of ghrelin, the regulation of its secretion, and address questions that still remain 15 years after its discovery. MAJOR CONCLUSIONS: In recent years, ghrelin has been found to have a plethora of central and peripheral actions in distinct areas including learning and memory, gut motility and gastric acid secretion, sleep/wake rhythm, reward seeking behavior, taste sensation and glucose metabolism.

Parallel studies of His-DTrp-Ala-Trp-DPhe-Lys-NH2 and human pancreatic growth hormone-releasing factor-44-NH2 in rat primary pituitary cell monolayer culture.

His-DTrp-Ala-Trp-DPhe-Lys-NH2 (GH-RP-6) is a synthetic hexapeptide that specifically releases GH both in vivo and in vitro in pituitary incubates. In this study, for the first time, GH-RP-6 was studied in primary pituitary cell monolayer culture. Parallel studies were performed with human pancreatic GH-releasing factor-44 (hpGRF-44). Culture conditions optimal for GH-RP-6 were not optimal for hpGRF-44. Both peptides released GH in a dose- and time-dependent manner. In this assay system, the ED50 for GH-RP-6 was 9 nM, and the ED50 for hp-GRF-44 was 1.6 nM. Calcium-blocking agents inhibited the GH responses of both peptides as well as basal GH release. Pretreatment with GH-RP-6 decreased the subsequent response to both GH-RP-6 and hpGRF-44. hpGRF-44 down regulated itself but not GH-RP-6. Rat sera potentiated the GH response of hpGRF-44 but not that of GH-RP-6. GH-RP-6 and hpGRF-44 GH responses were additive. These results suggest that GH-RP-6 and hpGRF-44 stimulate GH release via different somatotroph receptors.

On the in vitro and in vivo activity of a new synthetic hexapeptide that acts on the pituitary to specifically release growth hormone.

His-DTrp-Ala-Trp-DPhe-LysNH2, [His1,Lys6] GHRP, is a new synthetic hexapeptide which specifically elicits a dosage-related release of GH in vitro and in vivo without a concomitant release of LH, FSH, TSH, or PRL and, in limited in vivo studies, insulin or glucagon. Our results indicate that this small peptide has the attributes of a hypophysiotropic hormone. In vitro the minimum and maximum active dosages ranged from 1-10 ng/ml in the pituitary incubate assay. It was active in rats, monkeys, lambs, calves, and under special experimental conditions chicks, indicating its lack of species dependency. It was active when administered iv, sc, or ip to rats. After iv injection, GH levels rose within 2 min, peaked at +10-20 min, and by 2 h usually had returned to normal. It was not possible to directly compare the potencies of [His1,Lys6]GHRP, and the GH-releasing factors GHRF-44 and GHRF-40 after a single sc injection in rats because the time course of the GH response of these peptides was different. The GH response of [His1,Lys6]GHRP was longer in duration than either of these larger peptides. Both SRIF-14 and SRIF-28 inhibited the GH response of the hexapeptide; however, SRIF-28 was about four times more active than SRIF-14 in vitro and 7.5 times more active in vivo. When this small peptide was administered sc once or twice daily to immature rats for 9 or 25 days, the BW gain increased above the control. At the end of the weight gain studies the pituitary remained fully responsive to the peptide. Thus, [His1,Lys6] GHRP may be a valuable peptide for investigating the function of the pituitary somatotrophs and, in addition, it has the potential for increasing BW gain of a variety of normal animals by inducing GH release via a direct pituitary site of action.

Variables determining the growth hormone response of His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 in the rat.

Previous studies of His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GH-RP-6) have shown this synthetic hexapeptide to be a potent and specific stimulator of GH secretion both in vivo and in vitro. In this study the variables determining the in vivo responses were examined in the rat. The magnitude of the GH response to sc GH-RP-6 was dependent on the age and sex of the rat. Animals less than 15 days of age had much larger responses than did rats 21 days and older. At 10 days of age the male rat had a larger GH response than the female. At 21 days of age, bis(4-methyl 1-homo-piperazinyl-thiocarbonyl) disulfide (Fla-63)-pretreated females had larger responses than did Fla-63-pretreated males. In the Fla-63-pretreated adult rat, sc GH-RP-6 stimulated GH release in the female but not in the male. In the 10-day-old male, the ED50 for sc GH-RP-6 was 0.4 micrograms, and the maximal serum GH response was 800 ng/ml. In the 21-day-old female Fla-63-pretreated rat, the ED50 for sc GH-RP-6 was 3.0 micrograms, and the maximal GH response was 200 ng/ml. In the 21-day-old female pentobarbital-anesthetized rat, iv GH-RP-6 had an ED50 of 0.5 micrograms and a maximal serum GH response of 2500 ng/ml. A marked dose- and time-dependent decrease of subsequent GH-RP-6 responses occurred after a single sc GH-RP-6 injection. Decreases in pituitary GH or increases in somatostatin secretion would not explain this decreased response because the GH response of MRZ 2549, an opiate agonist, was unchanged by GH-RP-6 pretreatment. In contrast to the acute effect of GH-RP-6, chronic daily injections of GH-RP-6 resulted in an enhancement of the GH-RP-6 response.

Dose-response characteristics of various peptides with growth hormone-releasing activity in the unanesthetized male rat.

The characterization of GH-releasing peptides in vivo has been complicated by the effects of endogenous hypothalamic regulation of GH secretion. We describe a model to minimize endogenous hypothalamic interference by pretreating adult male rats with iv diethyldithiocarbamate and antisomatostatin serum. This pretreatment regimen established stable, detectable basal levels of plasma GH and eliminated spontaneous GH pulses for 12 h. Repeated pulsatile administration of 400 ng/kg iv rat hypothalamic GH-releasing factor (rGRF) produced consistent GH responses. Linear, nearly identical, dose responses (from 300-5000 ng/kg) were observed with rGRF and human pancreatic GH-releasing factor (GRF44) with ED50 values of 1059.3 and 1116.9 ng/kg, respectively. We also investigated a synthetic hexapeptide, His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP), which was previously reported to have potent GH-releasing activity. In contrast to either rGRF or GRF44, repeated administration of the same dose of GHRP did not produce consistent GH responses. The first bolus of GHRP produced a larger GH pulse than the second (P less than 0.01), followed by increasing GH responses from injections 2 to 7. GHRP was about 2 log orders less potent than either rGRF or GRF44 on a molar basis. The disparity between the native peptides and GHRP suggests that the synthetic peptide may act to release GH through a different mechanism(s). In summary, these data indicate that the diethyldithiocarbamate/anti-somatostatin serum-treated animal may be a useful model for investigating the pituitary actions of GH-releasing peptides.

The effects of growth hormone (GH)-releasing peptides on GH secretion in perifused pituitary cells of adult male rats.

The present study investigated the GH secretory activities of two distinctively different peptides: human pancreatic GH-releasing factor 44 (GRF-44) and a synthetic peptide His-DTrp-Ala-Trp-DPhe-Lys-NH2 (GHRP). GH secretion was studied in perifused dispersed anterior pituitary cells from male rats 24 or 48 h postdispersion. GRF-44 was 60 times more potent than GHRP and elicited linear increases in GH secretion between 0.3 and 30 ng (0.06-6 pmol), whereas the GHRP dose range was 3-300 ng (3.44-344 pmol). Hourly pulses of GHRP (30 ng) and GRF-44 (12.5 ng) stimulated consistent GH responses. An apparent priming effect was observed with GRF-44 at a dose of 3 ng. Continuous infusion of either peptide resulted in desensitization, with GH secretion being monophasic during GHRP infusion and biphasic during GRF-44 infusion. These results demonstrate that GRF-44 and GHRP, two peptides that have striking differences in their structure and size, stimulate in vitro GH secretion with remarkable similarity. However, the differences in potency, slope of the dose response, and pattern of GH secretion during continuous GRF-44 or GHRP infusion suggest that each stimulates in vitro GH secretion by slightly different mechanisms. GHRP or similar synthetic peptides may be useful probes to study GH secretory mechanisms.

Conformational energy studies and in vitro and in vivo activity data on growth hormone-releasing peptides.

A series of growth hormone-releasing peptides have been designed and tested for both in vitro and in vivo activity. In vitro activity at 1-10 ng/ml was obtained for the pentapeptide, His-DTrp-Ala-Trp-DPhe-NH2 (I) and the hexapeptide, His-DTrp-Ala-Trp-DPhe-Lys-NH2 (II). These peptides, as well as others to be described, are active in releasing GH in vivo at low microgram dosages. In this manuscript, the conformational properties and in vitro and in vivo activity of a series of small peptides are reported. Results of the biological studies are reported in an accompanying paper.

On the actions of the growth hormone-releasing hexapeptide, GHRP.

GH-releasing peptide (His-DTrp-Ala-Trp-DPhe-Lys-NH2 or GHRP) releases GH by a unique and complementary dual site of action on the hypothalamus and pituitary. These effects are mediated via non-GH-releasing hormone (non-GHRH) and nonopiate receptors in rats. Select types of opiates are known to release GH by a hypothalamic site of action, and thus, the dermorphin heptapeptide and benzomorphan opiate agonist 2549 used in this study presumably act on the hypothalamus to release GH. Neither dermorphin nor 2549 released GH or augmented the GH responses of GHRP or GHRH in vitro by a direct pituitary action, while GHRH antiserum inhibited the GH response of both dermorphin and 2549 in vivo. Evidence indicates that these opiates and GHRP administered together synergistically release GH, demonstrating the independent action(s) of GHRP and the opiates. Present data indicate that one of the major differences in the actions of dermorphin, 2549, and GHRP is the inhibition of somatostatin (SRIF) release by the opiates but not by GHRP. Although the actions of dermorphin, 2549, and GHRP on GH release are GHRH dependent, release of endogenous GHRH does not explain how GH is released synergistically by the combination of these peptides. It is proposed that dermorphin/2549 synergistically release GH with GHRP or GHRH because these opiates inhibit SRIF release. Since the GHRP plus GHRH synergistic GH release was not explained by inhibition of SRIF or stimulation of GHRH, an alternative mechanism is proposed to explain how GHRP synergistically release GH in combination with GHRH. The complementary, rather dramatic synergistic interaction of GHRP, GHRH, and dermorphin or GHRP, GHRH, and 2549 in releasing GH again strongly supports the independent actions of these compounds.

On the inhibitory effects of luteinizing hormone-releasing hormone analogs.

A detailed study of the activity of LHRH analog antagonists has been made in four assay systems which measure inhibition of the action of LHRH on isolated rat pituitaries in vitro, inhibition of the release of the LH induced by LHRH in vivo in adult male rats and adult male chimpanzees, and inhibition of spontaneous ovulation in cycling female rats. Only a partial correlation was observed between the in vitro and in vivo assays. Currently, the most potent LHRH analog antagonists in the present study were based on a 1,2,3,6-tetra-substituted LHRH sequence. The analogs [D less than Glu1,DPhe2,DTrp3,DTrp6]-LHRH, Ac-[Pro1,DPhe2,DTrp3,DTrp6]LHRH and [(Glu-Pro)1, dphe2,DTrp3,DTrp6]LHRH completely inhibited spontaneous ovulation in cycling rats at a dosage of 200 microgram/rat, sc. The most potent inhibitors of ovulation were always very potent in vitro, but other analogs having identical in vitro activities had little or no antiovulatory activity even at substantially higher dosages. The analogs inhibited the action of LHRH in the rat more easily than in the chimpanzee. Twelve of 13 analogs at the analog to LHRH ratio of 100:1 significantly inhibited the LH response, while only 5 of 9 of these same analogs inhibited the LH response in the chimpanzee at the analog to LHRH ratio of 333:1. Only 1 of 8 analogs at a high dosage inhibited the binding of labeled LH to the gonadal LH receptor in vitro. The inability of the less polar (cyclopentane carboxylic acid) analogs to inhibit ovulation could be explained, at least partially, in terms of impaired absorption sc. Although the cyclopentane carboxylic acid analogs effectively inhibited the action of LHRH in vitro and, when given iv in vivo, they were not effective in blocking the LHRH-stimulated LH response in adult male rats when given sc, which is the mode of administration of the antiovulatory assay, suggesting the importance of the route of administration.

Design, synthesis, and biological activity of peptides which release growth hormone in vitro.

Experimental observations showed that the analogs [D-Trp2]- and [D-Phe2]methionine enkephalin amide were weakly active in releasing GH from rat pituitary in vitro. These observations were used to design more active GH-releasing factors. Conformational energy calculations were carried out, and energetically favored conformations of these polypeptides were found. Structural similarities as well as structural differences between active and inactive analogs were examined, and new sequences were predicted. Progressively more active analogs were designed, then synthesized, and tested. This cycle of steps was repeated, each time using structural and chemical concepts as design guides, until a series of very active analogs resulted. The most active analog to date, Tyr-D-Trp-Ala-Trp-D-Phe-NH2, was shown to release GH in vitro at 10-30 ng/ml medium, which is approximately 10(3) times more active than the two starting enkephalin-based analogs. From the structure-activity data, a mechanism for binding at the receptors is formulated, and a comparison is made between the structural relationships of the GH-releasing peptide analogs and the GH inhibitor, somatostatin.

Oral estradiol administration modulates continuous intravenous growth hormone (GH)-releasing peptide-2-driven GH secretion in postmenopausal women.

Exactly how estradiol (E2) regulates the human GH-insulin-like growth factor I axis is not known. Here, we explore the impact of oral E2 supplementation on the stimulatory actions of a potent and specific synthetic GH-releasing peptide (GHRP), GHRP-2. To this end, we studied 10 healthy postmenopausal women following the administration of placebo or 17beta-estradiol (1 mg twice daily orally) for 7-12 days in a prospectively randomized, double-blind, within-subject crossover design. To drive GH secretion via the GHRP-receptor/ effector pathway, we infused GHRP-2 (1 microg/kg x h) or saline continuously iv for 24 h. Deconvolution analysis was used to quantitate the separate basal and pulsatile modes of GH secretion based on 24-h serum GH concentrations profiles collected at 10-min intervals and assayed by chemiluminescence. As complementary (nonpulsatile) measures, we used the approximate entropy (ApEn) statistic and cosine regression to define feedback-dependent and circadian-related changes, respectively. E2 administration amplified the mass of GH secreted per burst by 1.9-fold over placebo, 24-h GHRP-2 infusion by 7.0-fold, and, the two agonists together by 8.8-fold (P < 10(-14)). Intravenous GHRP-2 infusion augmented the basal (nonpulsatile) rate of GH secretion by 4.4-fold (P < 10(-4)). E2 treatment had no effect alone, but doubled the stimulatory effect of GHRP-2, on basal GH secretion. Neither E2 nor GHRP-2 influenced 24-h GH pulse frequency, interburst interval, half-life or pulse duration. Combined E2 and GHRP-2 elevated the ApEn of GH secretory profiles significantly above control, thereby indicating a marked alteration of within-axis feedback control (P = 0.00033). Dual stimulation with E2 and GHRP-2 also synergistically increased the amplitude (by 11-fold, P < 10(-11)) and the mesor (by 10-fold, P < 10(-10)) of the 24-h GH rhythm. Infusion of GHRP-2 advanced the GH acrophase (time of daily maximum of GH release) by 8.75 h, whereas combined treatment with E2 and GHRP-2 normalized the acrophase. Cross-correlation analysis showed that GHRP-2 infusion (but not E2 administration) significantly synchronized paired 24-h serum GH concentration profiles (P < 10(-3)). In summary, short-term oral E2 replacement in post-menopausal women strongly modulates the actions of a synthetic hexapeptide GH secretagogue on three quantifiable modes of GH secretion [i.e. 1) basal (nonpulsatile) GH release; 2) feedback-dependent ApEn; and 3) the mesor, amplitude and timing of the 24-h GH rhythm]. Moreover, a continuous GHRP-2 stimulus also synchronizes inter diem GH secretory patterns. The present pharmacological study, thus, offers a further framework for exploring the nature of the interactions of E2 with the GHRP-receptor/effector pathway in the aging and/or gonadoprival human.

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 i.v. infusion of GHRH and GHRP-2 each at a rate of 1 microg/kg x h on d 10 of randomly ordered placebo or 17beta-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 microg/liter (placebo) and 7.7 +/- 0.89 microg/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) microg/liter x 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 x 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.

Pharmacokinetics and pharmacodynamics of growth hormone-releasing peptide-2: a phase I study in children.

Administration of GH-releasing peptide-2 (GHRP-2) represents a potential mode of therapy for children of short stature with inadequate secretion of GH. Requisite information to determine the dosing route and frequency for GHRP-2 consists of the pharmacokinetics (PK) and pharmacodynamics (PD) for this compound, neither of which have been previously evaluated in children. The purpose of this study was to characterize the PK and PD of GHRP-2 in children with short stature. Ten prepubertal children (nine boys and one girl; 7.7 +/- 2.4 yr old) received a single 1 microg/kg i.v. dose of GHRP-2 over 1 min, followed by repeated (n = 9) blood sampling over 2 h. GHRP-2 and GH were quantitated by specific RIA methods. PK parameters were calculated from curve fitting of GHRP-2 and GH vs. time data. Posttreatment plasma GH concentrations (normalized for pretreatment values) were used as the effect measurement. PD parameters were generated using the sigmoid Emax model. Disposition of GHRP-2 best fit a biexponential function. GHRP-2 PK parameters (mean +/- SD) were: alpha = 13.4 +/- 9.7 h(-1), beta = 1.3 +/- 0.3 h(-1), t(1/2beta) = 0.55 +/- 0.14 h, AUC(0-infinity) = 2.02 +/- 1.37 ng/mL x h, Cmax = 7.4 +/- 3.8 ng/mL, plasma clearance = 0.66 +/- 0.32 L/h x kg, and apparent volume of distribution = 0.32 +/- 0.14 L/kg. PK parameters for GH were: appearance rate constant = 5.9 +/- 3.1 h(-1), elimination t(1/2) = 0.37 +/- 0.15 h, lag time = 0.05 +/- 0.01 h, Cmax = 50.7 +/- 17.2 ng/mL, Tmax = 0.42 +/- 0.16 h, and AUC(0-infinity) = 47.9 +/- 26.1 ng/mL x h. PD parameters for GHRP-2 were: Ke0 = 1.13 +/- 0.94 h(-1), gamma = 13.15 +/- 9.44, E0 = 6.63 +/- 4.86 ng/mL (GH), Emax = 67.5 +/- 23.5 ng/mL (GH), and EC50 = 1.09 +/- 0.59 ng/mL. We concluded that 1) GHRP-2 produced a predictable and significant (i.e. compared to pretreatment values) increase in plasma GH concentrations; 2) the PK-PD link model enabled quantitative assessment of GHRP-2 modulation of serum GH levels; and 3) definition of the EC50 for GHRP-2 will enable PD and PK evaluations of extravascular dosing regimens for children.

The growth hormone-releasing activity of a synthetic hexapeptide in normal men and short statured children after oral administration.

It has been shown that iv GH-releasing peptide (GHRP; His-DTrp-Ala-Trp-DPhe-Lys-NH2) specifically releases GH in man. Because of the clinical value of having an orally active peptide to release GH, five normal men were given GHRP orally at dosages of 100 and 300 micrograms/kg. At the 300 micrograms/kg dosage, the GH rise was detectable at 30 min, peaked between 60-75 min, and gradually declined to the baseline level between 150-180 min. Peak GH levels rose 63- and 202-fold above the baseline after administration of 100 and 300 micrograms/kg GHRP, respectively. Nine short stature children with varying degrees of GH deficiency were also included in this study. All children had short stature, slow growth, and delayed bone age and were being treated with biosynthetic human GH. The studies were performed after stopping GH treatment for 2-3 weeks. The oral GHRP dose administered to all of the children was 300 micrograms/kg, because this dosage was found to consistently increase GH levels in adults. The magnitude and pattern of the GH response to oral GHRP in four of the nine children were essentially the same as those in the five normal men. In the other five children, the GH responses were low but still measurable in three and undetectable in two of the children. Serum immunoreactive GHRP (irGHRP) levels were measured before and at 15- to 30-min intervals 3-5 h after oral as well as iv bolus GHRP administration. For comparison of serum irGHRP and GH levels, results were included after iv bolus administration of 0.1, 0.3, and 1.0 micrograms/kg GHRP to normal men. Since 300 micrograms/kg oral GHRP released about the same amount of GH as 1 microgram/kg, iv, in normal men, it was calculated that oral GHRP has about 0.3% the activity of iv GHRP. After iv GHRP, the peak serum irGHRP levels were immediate and proportional to the dosage, and the disappearance rate decreased exponentially. Between 100-240 min, the mean serum irGHRP level was essentially the same and remained slightly elevated. After administration of 300 micrograms/kg GHRP orally to normal men, serum irGHRP was measurable within 15 min, the peak serum irGHRP level coincided with the GH rise at 60 min, and serum irGHRP levels fell more slowly than serum GH levels. The serum half-life was 20 min, and the distribution volume was 2.5 L after both oral and iv administration.(ABSTRACT TRUNCATED AT 400 WORDS)

Tripartite neuroendocrine activation of the human growth hormone (GH) axis in women by continuous 24-hour GH-releasing peptide infusion: pulsatile, entropic, and nyctohemeral mechanisms.

Despite the discovery of potent GH-releasing peptides (GHRPs) more than 15 yr ago and the recent cloning of human, rat, and pig GHRP receptors in the hypothalamus and pituitary gland, the neuroregulatory mechanisms of action of GHRP agonists on the human hypothalamo-somatotroph unit are not well delineated. To gain such clinical insights, we evaluated the ultradian (pulsatile), entropic (pattern orderliness), and nyctohemeral GH secretory responses during continuous 24-h i.v. infusion of saline vs. the most potent clinically available hexapeptide, GHRP-2 (1 microg/kg x h) in estrogen-unreplaced (mean serum estradiol, 12 +/- 2.4 pg/mL) postmenopausal women (n = 7) in a paired, randomized design. Blood was sampled every 10 min for 24 h during infusions and was assayed by ultrasensitive GH chemiluminescence assay. Pulsatile GH secretion was quantitated by deconvolution analysis, orderliness of GH release patterns by the approximate entropy statistic, and 24-h GH rhythmicity by cosinor analysis. Statistical analysis revealed that GHRP-2 elicited a 7.7-fold increase in (24-h) mean serum (+/-SEM) GH concentrations, viz. from 0.32 +/- 0.042 (saline) to 2.4 +/- 0.34 microg/L (GHRP-2; P = 0.0006). This occurred via markedly stimulated pulsatile GH release, namely a 7.1-fold augmentation of GH secretory burst mass: 0.87 +/- 0.18 (control) vs. 6.3 +/- 1.3 microg/L (GHRP-2; P = 0.0038). Enhanced GH pulse mass reflected a commensurate 10-fold (P = 0.023) rise in GH secretory burst amplitude [maximal GH secretory rate (micrograms per L/min) attained within a secretory pulse] with no prolongation in event duration. GH burst frequency, interpulse interval, and calculated GH half-life were all invariant of GHRP-2 treatment. Concurrently, as detected in the ultrasensitive GH assay, GHRP-2 augmented deconvolution-estimated interpulse (basal) GH secretion by 4.5-fold (P = 0.025). The approximate entropy of 24-h serum GH concentration profiles rose significantly during GHRP-2 infusion; i.e. from 0.592 +/- 0.073 (saline) to 0.824 +/- 0.074 (GHRP-2; P = 0.0011), signifying more irregular or disorderly GH release patterns during secretagogue stimulation. Cosinor analysis of 24-h GH rhythms disclosed a significantly earlier (daytime) acrophase at 2138 h (+/- 140 min) during GHRP-2 stimulation vs. 0457 h (+/-42 min) during saline infusion (P = 0.013). Concomitantly, the cosinor amplitude rose 6-fold (P = 0.018), and the mesor (cosine mean) rose 5-fold (P = 0.003). Fasting (0800 h) plasma insulin-like growth factor (IGF-I) concentrations rose by -11 +/- 12 microg/L during saline infusion and by 102 +/- 18 microg/L during GHRP-2 infusion (P = 0.0036). GHRP-2 infusion did not modify (24-h pooled) serum LH, FSH, or TSH concentrations and minimally increased serum (pooled) daily PRL (6.8 +/- 0.83 vs. 12 +/- 1.2 microg/L; P < 0.05) and cortisol (5.3 +/- 0.59 to 7.0 +/- 0.74; P < 0.05) concentrations. In summary, 24-h constant iv GHRP-2 infusion in the gonadoprival female neurophysiologically activates the GH-IGF-I axis by potentiating GH secretory burst mass and amplitude by 7- to 10-fold and augmenting the basal (nonpulsatile) GH secretion by 4.5-fold. GHRP-2 action is highly selective, as it does not alter GH secretory burst frequency, interpulse interval, event duration, or GH half-life. GHRP-2 effectively elevates IGF-I concentrations, unleashes greater disorderliness of GH release patterns, and heightens the 24-h rhythmicity of GH secretion. These tripartite features of GHRP-2's action in estrogen-withdrawn (postmenopausal) women also characterize normal human puberty and/or sex steroid regulation of the GH-IGF-I axis. However, how or whether GHRP-2 interacts further with sex hormone modulation of GH neurosecretory control in older women and men is not yet known.

Acute growth hormone (GH) response to GH-releasing hexapeptide in humans is independent of endogenous GH-releasing hormone.

The synthetic GH-releasing hexapeptide (GHRP: His-DTrp-Ala-Trp-DPhe-Lys-NH2) releases GH in man by an undetermined mechanism. To investigate whether acute GH response to GHRP is mediated by endogenous GHRH, we examined the effect of GHRP on GH release during pituitary desensitization to GHRH induced by short-term GHRH infusion. In five healthy men on six occasions, we infused saline (sal) or 1 microgram/kg.h GHRH-44 for 6 h. After 4 h, a bolus of sal, GHRH-44 1 microgram/kg body weight, or GHRP 1 microgram/kg body weight was given iv. GH concentration, measured by RIA, was analyzed by mean area under the curve (AUC) of GH released over the 2 h immediately after bolus injection. Infusion of GHRH had a biphasic effect on GH release; plasma GH increased to 12.7 +/- 3.3 micrograms/L within the first hour, with subsequent decrease to 2.9 +/- 0.3 micrograms/L during the last 2 h of infusion. GH AUC (hours 4-6 of infusion) microgram/L.2 h [table: see text] GH response to bolus GHRH was abolished by GHRH infusion, whereas GH response to GHRP persisted under the same conditions. Thus, we conclude that acute GH response to GHRP in humans is not mediated by endogenous GHRH.

E2 supplementation selectively relieves GH's autonegative feedback on GH-releasing peptide-2-stimulated GH secretion.

Female gender confers resistance to GH autonegative feedback in the adult rat, thereby suggesting gonadal or estrogenic modulation of autoregulation of the somatotropic axis. Here we test the clinical hypothesis that short-term E2 replacement in ovariprival women reduces GH's repression of spontaneous, GHRH-, and GH-releasing peptide (GHRP)-stimulated GH secretion. To this end, we appraised GH autoinhibition in nine healthy postmenopausal volunteers during a prospective, randomly ordered supplementation with placebo vs. E [1 mg micronized 17 beta-E2 orally twice daily for 6-23 d]. The GH autofeedback paradigm consisted of a 6-min pulsed i.v. infusion of recombinant human GH (10 microg/kg square-wave injection) or saline (control) followed by i.v. bolus GHRH (1 microg/kg), GHRP-2 (1 microg/kg), or saline 2 h later. Blood was sampled every 10 min and serum GH concentrations were measured by chemiluminescence. Poststimulus GH release was quantitated by multiparameter deconvolution analysis using published biexponential kinetics and by the incremental peak serum GH concentration response (maximal poststimulus value minus prepeak nadir). Outcomes were analyzed on the logarithmic scale by mixed-effects ANOVA at a multiple-comparison type I error rate of 0.05. E2 supplementation increased the (mean +/- SEM) serum E2 concentration from 43 +/- 1.8 (control) to 121 +/- 4 pg/ml (E2) (158 +/- 6.6 to 440 +/- 15 pmol/liter; P < 0.001), lowered the 0800 h (preinfusion) serum IGF-I concentration from 127 +/- 7.7 to 73 +/- 3.6 microg/liter (P < 0.01), and amplified spontaneous pulsatile GH production from 7.5 +/- 1.1 to 13 +/- 2.3 microg/liter per 6 h (P = 0.020). In the absence of exogenously imposed GH autofeedback, E2 replacement enhanced the stimulatory effect of GHRP-2 on incremental peak GH release by 1.58-fold [95% confidence interval, 1.2- to 2.1-fold] (P = 0.0034) but did not alter the action of GHRH (0.83-fold [0.62- to 1.1-fold]). In the E2-deficient state, bolus GH infusion significantly inhibited subsequent spontaneous, GHRH-, and GHRP-induced incremental peak GH responses by, respectively, 33% (1-55%; P = 0.044 vs. saline), 79% (68-86%; P < 0.0001), and 54% (32-69%; P = 0.0002). E2 repletion failed to influence GH autofeedback on either spontaneous or GHRH-stimulated incremental peak GH output. In contrast, E2 replenishment augmented the GHRP-2-stimulated incremental peak GH response in the face of GH autoinhibition by 1.7-fold (1.2- to 2.5-fold; P = 0.009). Mechanistically, the latter effect of E2 mirrored its enhancement of GH-repressed/GHRP-2-stimulated GH secretory pulse mass, which rose by 1.5-fold (0.95- to 2.5-fold over placebo; P = 0.078). In summary, the present clinical investigation documents the ability of short-term oral E2 supplementation in postmenopausal women to selectively rescue GHRP-2 (but not spontaneous or GHRH)-stimulated GH secretion from autonegative feedback. The secretagogue specificity of E's relief of GH autoinhibition suggests that this sex steroid may enhance activity of the hypothalamopituitary GHRP-receptor/effector pathway.

Leptin levels in protracted critical illness: effects of growth hormone-secretagogues and thyrotropin-releasing hormone.

Prolonged critical illness is characterized by feeding-resistant wasting of protein, whereas reesterification, instead of oxidation of fatty acids, allows fat stores to accrue and associate with a low-activity status of the somatotropic and thyrotropic axis, which seems to be partly of hypothalamic origin. To further unravel this paradoxical metabolic condition, and in search of potential therapeutic strategies, we measured serum concentrations of leptin; studied the relationship with body mass index, insulin, cortisol, thyroid hormones, and somatomedins; and documented the effects of hypothalamic releasing factors, in particular, GH-secretagogues and TRH. Twenty adults, critically ill for several weeks and supported with normocaloric, continuously administered parenteral and/or enteral feeding, were studied for 45 h. They had been randomized to receive one of three combinations of peptide infusions, in random order: TRH (one day) and placebo (other day); TRH + GH-releasing peptide (GHRP)-2 and GHRP-2; TRH + GHRH + GHRP-2 and GHRH + GHRP-2. Peptide infusions were started after a 1-microgram/kg bolus at 0900 h and infused (1 microgram/kg.h) until 0600 h the next morning. Serum concentrations of leptin, insulin, cortisol, T4, T3, insulin-like growth factor (IGF)-I, IGF-binding protein-3 and the acid-labile subunit (ALS) were measured at 0900 h, 2100 h, and 0600 h on each of the 2 study days. Baseline leptin levels (mean +/- SEM: 12.4 +/- 2.1 micrograms/L) were independent of body mass index (25 +/- 1 kg/m2), insulin (18.6 +/- 2.9 microIU/mL), cortisol (504 +/- 43 mmol/L), and thyroid hormones (T4: 63 +/- 5 nmol/L, T3: 0.72 +/- 0.08 nmol/L) but correlated positively with circulating levels of IGF-I [86 +/- 6 micrograms/L, determination coefficient (R2) = 0.25] and ALS (7.2 +/- 0.6 mg/L, R2 = 0.32). Infusion of placebo or TRH had no effect on leptin. In contrast, GH-secretagogues elevated leptin levels within 12 h. Infusion of GHRP-2 alone induced a maximal leptin increase of +87% after 24 h, whereas GHRH + GHRP-2 elevated leptin by up to +157% after 24 h. The increase in leptin within 12 h was related (R2 = 0.58) to the substantial rise in insulin. After 45 h, and having reached a plateau, leptin was related to the increased IGF-I (R2 = 0.37). In conclusion, circulating leptin levels during protracted critical illness were linked to the activity state of the GH/IGF-I axis. Stimulating the GH/IGF-I axis with GH-secretagogues increased leptin levels within 12 h. Because leptin may stimulate oxidation of fatty acids, and because GH, IGF-I, and insulin have a protein-sparing effect, GH-secretagogue administration may be expected to result in increased utilization of fat as preferential substrate and to restore protein content in vital tissues and, consequently, has potential as a strategy to reverse the paradoxical metabolic condition of protracted critical illness.

Diagnostic studies with intravenous and intranasal growth hormone-releasing peptide-2 in children of short stature.

GH secretion is primarily regulated by the hypothalamic-releasing hormones GHRH and somatostatin. Additionally, several neurotransmitters act at the hypothalamus and pituitary to modulate GH release. The agents commonly used in clinical practice to diagnose GH deficiency, such as arginine, insulin and L-dopa, act through the neural GH network. Many children with a poor GH response to conventional agents have a significant serum GH response to iv GHRH. GH-releasing peptides (GHRPs) are synthetic peptides that like GHRH act directly on pituitary somatotrophs to stimulate GH release. GHRP-2, an investigational drug, is one of the most potent members of the GHRP family. It has been shown to be effective in adults via the oral and intranasal as well as the iv route of administration. In this study, GH responses to GHRP-2 were compared with GH responses to other provocative agents in children of short stature. GHRP-2 was administered iv or intranasally to children with short stature. In the same subjects, GHRP-2 was administered iv in combination with GHRH. Twenty-four children undergoing evaluation for GH deficiency received at least one conventional agent (arginine, L-dopa/exercise, insulin) in addition to iv GHRH and GHRP-2. The GH responses to GHRH or GHRP-2 were similar in each child, and both were equally reliable predictors of pituitary reserve. The conventional agents used in GH testing were less likely to predict the capacity of the pituitary to release GH than were either GHRH or GHRP-2. There was no correlation between maximal GH response to standard tests with GH responses to GHRH or GHRP-2. A subset of the group of 21 children who had a robust response to iv GHRP-2 were later administered GHRH+GHRP-2 simultaneously. The GH response to GHRH+GHRP-2 was synergistic in this group of 12 children, similar to previously reported observations in adults of normal stature. Fifteen of the 21 children who had a robust response to the iv GH-releasing factors also received intranasal GHRP-2. All 15 of these children had a significant GH response to intranasal GHRP-2 over a dose range of 5-20 micrograms/kg per dose. The mean peak GH response to 15 micrograms/kg was 31.3 micrograms/L. The intranasal preparation was well tolerated.(ABSTRACT TRUNCATED AT 400 WORDS)