GHRP-6 Research Articles

Scavenger Receptor CD36 Mediates Inhibition of Cholesterol Synthesis via Activation of the LKB1-AMPK Pathway and Insig1/Insig2 Expression in Hepatocytes

Scavenger receptor CD36 is known to play a central role in regulating lipid metabolism, in part, through the uptake of oxidized LDL in macrophages and its ability to internalize long chain fatty acids in adipocytes. Here, we demonstrate that CD36 activity impacts cholesterol synthesis in hepatocytes. Using hexarelin, a growth hormone-releasing peptide that interacts with CD36, we found a rapid phosphorylation of LKB1, which led to subsequent AMPK phosphorylation in treated HepG2 cells. HMG-CoA reductase, which catalyzes the rate-limiting step in cholesterol synthesis, was phosphorylated and inactivated following hexarelin treatment. This was accompanied with a significant degradation of HMG-CoA reductase by the ubiquitin-proteasome pathway in response to CD36 activation and an enhanced recruitment of escort proteins Insig-1 and Insig-2. Key enzymes involved in cholesterol biosynthesis and under SREBP-2 control remained downregulated upon long-term hexarelin treatment despite sterol depletion. Increased expression of Insig-1 and Insig-2 in HepG2 cells was dependent on nuclear receptor PPARγ activation by hexarelin, providing a role for PPARγ in upregulating Insig expression. This study provides a mechanistic basis by which CD36 modulates HMG-CoA reductase function and Insig expression through activation of PPARγ, thereby supporting a novel role of scavenger receptor CD36 to regulate cholesterol synthesis in hepatocytes.

Establishment of a Gastric Cell-Based Assay System for Exploring Inhibitors of Octanoylated Ghrelin Production

Ghrelin, a gastric hormone, is a growth hormone-releasing peptide. Its serine-3 acylation with octanoic acid is essential for its orexigenic activity, and therefore, inhibition of the acylation of ghrelin may help in decreasing appetite and preventing obesity. This study aimed to establish a human gastric cell-based assay system to evaluate candidate inhibitors of octanoylated ghrelin production. In human gastric carcinoma AGS cells, obligatory factors for the posttranslational modification of ghrelin, such as certain prohormone convertases responsible for processing of proghrelin to the mature ghrelin and the enzyme-catalyzing acyl-modification of ghrelin, were well expressed, but ghrelin was expressed at low levels. Accordingly, we transfected a ghrelin-expressing vector into AGS cells and isolated a stable ghrelin-expressing cell line (AGS-GHRL8). AGS-GHRL8 cells secreted octanoylated ghrelin in accordance with the concentrations of octanoic acid in the culture medium. Given that ingested heptanoic acid is used for the acyl-modification of ghrelin, we evaluated whether heptanoic acid inhibits production of octanoylated ghrelin in AGS-GHRL8 cells. Butyric acid was used as a control. Indeed, heptanoic acid predictably decreased the secretion of octanoylated ghrelin, whereas butyric acid did not. The AGS-GHRL8 line established in this study will facilitate the screening of inhibitors of octanoylated ghrelin production.

Differential effects of GH and GH-releasing peptide-6 on astrocytes

GH and GH secretagogues (GHSs) are involved in many cellular activities such as stimulation of mitosis, proliferation and differentiation. As astrocytes are involved in developmental and protective functions, our aim was to analyse the effects of GH and GH-releasing hexapeptide on astrocyte proliferation and differentiation in the hypothalamus and hippocampus. Treatment of adult male Wistar rats with GH (i.v., 100 μg/day) for 1 week increased the levels of glial fibrillary acidic protein (GFAP) and decreased the levels of vimentin in the hypothalamus and hippocampus. These changes were not accompanied by increased proliferation. By contrast, GH-releasing hexapeptide (i.v., 150 μg/day) did not affect GFAP levels but increased proliferation in the areas studied. To further study the intracellular mechanisms involved in these effects, we treated C6 astrocytoma cells with GH or GH-releasing hexapeptide and the phosphatidylinositol 3'-kinase (PI3K) inhibitor, LY294002, and observed that the presence of this inhibitor reverted the increase in GFAP levels induced by GH and the proliferation induced by GH-releasing hexapeptide. We conclude that although GH-releasing hexapeptide is a GHS, it may exert GH-independent effects centrally on astrocytes when administered i.v., although the effects of both substances appear to be mediated by the PI3K/Akt pathway.

Differential pulsatile secretagogue control of GH secretion in healthy men

Pulsatile growth hormone (GH) secretion putatively reflects integrated regulation by GH-releasing hormone (GHRH), somatostatin (SST), and GH-releasing peptide (GHRP). GHRH and SST secretion is itself pulsatile. However, how GHRH and SST pulses act along with GHRP to jointly determine pulsatile GH secretion is unclear. Moreover, how testosterone (T) modulates such interactions is unknown. These queries were assessed in a prospectively randomized, placebo-controlled double-blind cohort comprising 26 healthy older men randomized to testosterone (T) vs. placebo supplementation. Pulses of GHRH, SST, or saline were infused intravenously at 90-min intervals for 13 h, along with either continuous saline or ghrelin analog (GHRP-2). The train of pulses was followed by a triple stimulus (combined l-arginine, GHRH, and GHRP-2) to estimate near-maximal GH secretion over a final 3 h. Testosterone vs. placebo supplementation doubled pulsatile GH secretion during GHRH pulses combined with continuous saline (GHRH/saline) (P < 0.01). Pulsatile GH secretion correlated positively with T concentrations (270-1,170 ng/dl) in the 26 men during saline pulses/saline (P = 0.015, R(2) = 0.24), GHRH pulses/saline (P = 0.020, R(2) = 0.22), and combined GHRH pulses/GHRP-2 (P = 0.016, R(2) = 0.25) infusions. Basal nonpulsatile GH secretion correlated with T during saline pulses/GHRP-2 drive (P = 0.020, R(2) = 0.16). By regression analysis, pulsatile GH secretion varied negatively with body mass index (BMI) during saline/GHRP-2 infusion (P = 0.001, R(2) = 0.36), as well as after the triple stimulus preceded by GHRH/GHRP-2 (P = 0.013, R(2) = 0.23). Mean (10-h) GH concentrations under GHRP-2 were predicted jointly by estradiol (positively) and BMI (negatively) (P < 0.001, R(2) = 0.520). These data indicate that estradiol, T, and BMI control pulsatile secretagogue-specific GH-regulatory mechanisms in older men.

The effects of coadministration of ghrelin agonist (GHRP-2) and GH on TNF-α, IL-6, and iNOS genes expression induced by LPS in mouse liver

Growth hormone (GH)-releasing peptide-2 (GHRP-2), a ghrelin receptor agonist, has been reported to bear an anti-inflammatory effect. The aim of this study is to assess the impact of GHRP-2 and GH on the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS) genes induced by lipopolysaccharide (LPS) in mouse liver tissues. Thirty-five male NMRI mice (25 ± 5 g) were used. Mice were divided into five groups (n = 7). GHRP-2 (100 μg/kg), GH (25 μg/kg), and (GHRP-2 + GH) were injected through the mice tail vein 30 min before the injection of LPS. Then, inflammation was induced by intraperitoneal injection of LPS (5 mg/kg) while the control animals received sterile saline. Changes in the levels of expression of TNF-α, IL-6, and iNOS genes in the mice liver induced by LPS injection for 2 h were studied by a semiquantitative RT-polymerase chain reaction method. Administration of LPS increased hepatic TNF-α, IL-6, and iNOS mRNAs. The results showed that intravenous administration of GHRP-2and GH significantly reduced the elevated TNF-α, IL-6, and iNOS mRNA levels 2 h after the injection. In contrast, injection of GHRP-2 prior to injection of LPS reduced IL-6. Injection of GH reduced the expression of iNOS in liver tissues. Coadministration of two drugs had a positive effect on the reduction of TNF-α, IL-6, and iNOS expression.

Epidermal growth factor and growth hormone-releasing peptide-6: Combined therapeutic approach in experimental stroke

Stroke is the second cause of mortality worldwide, with a high incidence of disability in survivors. Promising candidate drugs have failed in stroke trials. Combined therapies are attractive strategies that simultaneously target different points of stroke pathophysiology. The aim of this work is to determine whether the combined effects of epidermal growth factor (EGF) and growth hormone-releasing peptide-6 (GHRP6) can attenuate clinical signs and pathology in an experimental stroke model. Brain global ischemia was generated in Mongolian gerbils by 15 minutes of carotid occlusion. After reperfusion, EGF, GHRP6 or EGF+GHRP6 were intraperitoneally administered. Clinical manifestations were monitored daily. Three days after reperfusion, animals were anesthetized and perfused with an ink solution. The anatomy of the Circle of Willis was characterized. Infarct volume and neuronal density were analyzed. EGF+GHRP6 co-administration reduced clinical manifestations and infarct volume and preserved neuronal density. No correlation was observed between the grade of anastomosis of the Circle of Willis and clinical manifestations in the animals receiving EGF+GHRP6, as opposed to the vehicle-treated gerbils. Co-treatment with EGF and GHRP6 affects both the clinical and pathological outcomes in a global brain ischemia model, suggesting a suitable therapeutic approach for the acute management of stroke.

The arginine and GHRP-2 tests as alternatives to the insulin tolerance test for the diagnosis of adult GH deficiency in Japanese patients: A comparison

The arginine + GHRH test has been established as an alternative to the insulin tolerance test (ITT) for the diagnosis of adult GH deficiency (AGHD). However, the glucagon, arginine, and GH releasing peptide-2 (GHRP-2) test are recommended as alternatives in Japan. The objective of this study was to evaluate the arginine and GHRP-2 tests as alternatives to the ITT for the diagnosis of AGHD in a Japanese population. Three stimulation tests (ITT, arginine test, and GHRP-2 test) were conducted in 71 pre-operative adult patients with pituitary tumors (age, 18-65 years). The peak GH responses to each test were examined. The peak GH responses were significantly lower with the ARG test (median 4.43 μg/L) (p < 0.0001) than with the ITT (median 9.38 μg/L), and the peak GH responses with the GHRP-2 test (median 28.88 μg/L) were higher (p < 0.0001). However, among the AGHD patients, there was no significant difference between the peak GH responses to the ARG test and the ITT. The sensitivities and specificities of the ARG / GHRP-2 tests compared to the ITT for the diagnosis of severe AGHD (peak GH responses to ITT ≤ 1.8 μg/L) were 93.8% / 81.3% and 85.5% / 94.5%, respectively. The arginine and GHRP-2 stimulation tests are acceptable alternatives to the ITT for the diagnosis of AGHD in Japanese patients. The method and criterion for the diagnosis of AGHD should be reconsidered and adjusted to each population.

Decreased Serum Ghrelin Levels in Patients with Acute Myocardial Infarction

Ghrelin is a novel growth hormone-releasing peptide isolated from the stomach and possesses various cardioprotective effects, including energy balance improvement and regulation of autonomic nervous system activity. We investigated the changes in serum ghrelin levels and its association with cardiac function and myocardial infarct size in patients with acute myocardial infarction (AMI). Forty-seven consecutive patients were divided into the following 4 groups: 16 patients with AMI, 12 patients with unstable angina pectoris (UAP), 13 patients with stable angina pectoris (SAP), and 6 control patients. Serum levels were measured with the ELISA kit. Compared to the control (72 ± 26 fmol/mL), SAP (69 ± 47 fmol/mL), and UAP (72 ± 31 fmol/mL) groups, serum ghrelin levels on admission were significantly lower in the AMI group (27 ± 12 fmol/mL, P < 0.01). After admission, the serum ghrelin level gradually increased (30 ± 15 fmol/mL on day 2 and 39 ± 18 fmol/mL on day 7) and became significantly higher on day 14 (49 ± 28 fmol/mL, P < 0.01), compared to the level on admission. In patients with AMI, the ratio of day 14 to admission serum ghrelin levels, an index of AMI-related acute changes in ghrelin, correlated positively with peak creatine phosphokinase levels (R = 0.72, P < 0.01) and the double products (R = 0.60, P < 0.01) and inversely with left ventricular ejection fraction (R = -0.53, P < 0.05). In conclusion, serum ghrelin levels are significantly decreased in association with myocardial infarct size and cardiac function.

Metabolism of Growth Hormone Releasing Peptides

New, potentially performance enhancing compounds have frequently been introduced to licit and illicit markets and rapidly distributed via worldwide operating Internet platforms. Developing fast analytical strategies to follow these new trends is one the most challenging issues for modern doping control analysis. Even if reference compounds for the active drugs are readily obtained, their unknown metabolism complicates effective testing strategies. Recently, a new class of small C-terminally amidated peptides comprising four to seven amino acid residues received considerable attention of sports drug testing authorities due to their ability to stimulate growth hormone release from the pituitary. The most promising candidates are the growth hormone releasing peptide (GHRP)-1, -2, -4, -5, -6, hexarelin, alexamorelin, and ipamorelin. With the exemption of GHRP-2, the entity of these peptides represents nonapproved pharmaceuticals; however, via Internet providers, all compounds are readily available. To date, only limited information on the metabolism of these substances is available and merely one metabolite for GHRP-2 is established. Therefore, a comprehensive in vivo (po and iv administration in rats) and in vitro (with human serum and recombinant amidase) study was performed in order to generate information on urinary metabolites potentially useful for routine doping controls. The urine samples from the in vivo experiments were purified by mixed-mode cation-exchange solid-phase extraction and analyzed by ultrahigh-performance liquid chromatography (UHPLC) separation followed by high-resolution/high-accuracy mass spectrometry. Combining the high resolution power of a benchtop Orbitrap mass analyzer for the first metabolite screening and the speed of a quadrupole/time-of-flight (Q-TOF) instrument for identification, urinary metabolites were screened by means of a sensitive full scan analysis and subsequently confirmed by high-accuracy product ion scan experiments. Two deuterium-labeled internal standards (triply deuterated GHRP-4 and GHRP-2 metabolite) were used to optimize the extraction and analysis procedure. Overall, 28 metabolites (at least three for each GHRP) were identified from the in vivo samples and main metabolites were confirmed by the human in vitro model. All identified metabolites were formed due to exopeptidase- (amino- or carboxy-), amidase-, or endopeptidase activity.

Pharmacokinetic study of Growth Hormone-Releasing Peptide 6 (GHRP-6) in nine male healthy volunteers

GHRP-6 is a growth hormone secretagogue that also enhances tissue viability in different organs. In the present work, we studied the pharmacokinetics of this short therapeutic hexapeptide (His-(D-Trp)-Ala-Trp-(D-Phe)-Lys-NH2, MW=872.44Da) in nine male healthy volunteers after a single intravenous bolus administration of 100, 200 and 400μg/kg of body weight. GHRP-6 was quantified in human plasma by a specific LC–MS method, previously developed and validated following FDA guidelines, using 13C3Ala-GHRP-6 as internal standard (Gil et al., 2012, J. Pharm. Biomed. Anal. 60, 19–25). The Lower Limit of Quantification (5ng/mL) was reached in all subjects at 12h post-administration, which was sufficient for modeling a pharmacokinetic profile including over 85% of the Area under the Curve (AUC). Disposition of GHRP-6 best fitted a bi-exponential function with R2 higher than 0.99, according to a mathematic modeling and confirmed by an Akaike index (AIC) lower than that of the corresponding one-compartment model for all subjects. Averaging all three dose levels, the distribution and elimination half-life of GHRP-6 were 7.6±1.9min and 2.5±1.1h, respectively. These values are coherent with existing data for other drugs whose disposition also fits this model. Dose dependence analysis revealed a noticeable trend for AUC to increase proportionally with administered dose. Atypical GHRP-6 concentration spikes were observed during the elimination phase in four out of the nine subjects studied.

Determination of prohibited, small peptides in urine for sports drug testing by means of nano-liquid chromatography/benchtop quadrupole orbitrap tandem-mass spectrometry

In the present study, a screening assay was developed comprising 11 prohibited peptides (<1.5kDa) that are sufficiently purified from urine using weak cation exchange with subsequent determination of all substances by means of nanoUHPLC separation coupled to high resolution tandem mass spectrometry. These peptides included Gonadorelin (LH-RH), Desmopressin and 9 growth hormone releasing peptides (GHRP-1, -2, -4, -5, -6, Hexarelin, Alexamorelin, Ipamorelin and a GHRP-2 metabolite); however, the procedure is expandable to further target analytes or metabolites. The method was validated with a main focus on qualitative result interpretation considering the parameters specificity, linearity (0–500pg/mL), recovery (45–95%), precision (<20% at 100pg/mL), limits of detection (2–10pg/mL), robustnesss and ion suppression. The proof-of-principle was shown by analysing excretion study urine samples for LHRH, Desmopressin and GHRP-2.

Azapeptide Analogues of the Growth Hormone Releasing Peptide 6 as Cluster of Differentiation 36 Receptor Ligands with Reduced Affinity for the Growth Hormone Secretagogue Receptor 1a

The synthetic hexapeptide growth hormone releasing peptide-6 (GHRP-6) exhibits dual affinity for the growth hormone secretagogue receptor 1a (GHS-R1a) and the cluster of differentiation 36 (CD36) receptor. Azapeptide GHRP-6 analogues have been synthesized, exhibiting micromolar affinity to the CD36 receptor with reduced affinity toward the GHS-R1a. A combinatorial split-and-mix approach furnished aza-GHRP-6 leads, which were further examined by alanine scanning. Incorporation of an aza-amino acid residue respectively at the D-Trp(2), Ala(3), or Trp(4) position gave aza-GHRP-6 analogues with reduced affinity toward the GHS-R1a by at least a factor of 100 and in certain cases retained affinity for the CD36 receptor. In the latter cases, the D-Trp(2) residue proved important for CD36 receptor affinity; however, His(1) could be replaced by Ala(1) without considerable loss of binding. In a microvascular sprouting assay using a choroid explant, [azaTyr(4)]-GHRP-6 (15), [Ala(1), azaPhe(2)]-GHRP-6 (16), and [azaLeu(3), Ala(6)]-GHRP-6 (33) all exhibited antiangiogenic activity.

Ghrelin Prevents Incidence of Malignant Arrhythmia after Acute Myocardial Infarction through Vagal Afferent Nerves

Ghrelin is a GH-releasing peptide mainly excreted from the stomach. Ghrelin administration has been shown to inhibit cardiac sympathetic nerve activity (CSNA), reduce malignant arrhythmia, and improve prognosis after acute myocardial infarction (MI). We therefore investigated the effects and potential mechanisms of the action of endogenous ghrelin on survival rate and CSNA after MI by using ghrelin-knockout (KO) mice. MI was induced by left coronary artery ligation in 46 KO mice and 41 wild-type mice. On the first day, malignant arrhythmia-induced mortality was observed within 30 min of the ligation and had an incidence of 2.4% in wild-type and 17.4% in KO mice (P < 0.05). We next evaluated CSNA by spectral analysis of heart rate variability. CSNA, represented by the low frequency/high frequency ratio, was higher in KO mice at baseline (2.18 ± 0.43 vs. 0.98 ± 0.09; P < 0.05), and especially after MI (25.5 ± 11.8 vs. 1.4 ± 0.3; P < 0.05), than in wild-type mice. Ghrelin (150 μg/kg, s.c.) 15 min before ligation suppressed the activation of CSNA and reduced mortality in KO mice. Further, this effect of ghrelin was inhibited by methylatropine bromide (1 mg/kg, i.p.) or by perineural treatment of both cervical vagal trunks with capsaicin (a specific afferent neurotoxin). Our data demonstrated that both exogenous and endogenous ghrelin suppressed CSNA, prevented the incidence of malignant arrhythmia, and improved the prognosis after acute MI. These effects are likely to be via the vagal afferent nerves.

Growth hormone response to growth hormone-releasing peptide-2 in growth hormone-deficient Little mice

OBJECTIVE:To investigate a possible direct, growth hormone-releasing, hormone-independent action of a growth hormone secretagogue, GHRP-2, in pituitary somatotroph cells in the presence of inactive growth hormone-releasing hormone receptors.MATERIALS AND METHODS:The responses of serum growth hormone to acutely injected growth hormone-releasing P-2 in lit/lit mice, which represent a model of GH deficiency arising from mutated growth hormone-releasing hormone-receptors, were compared to those observed in the heterozygous (lit/+) littermates and wild-type (+/+) C57BL/6J mice.RESULTS:After the administration of 10 mcg of growth hormone-releasing P-2 to lit/lit mice, a growth hormone release of 9.3±1.5 ng/ml was observed compared with 1.04±1.15 ng/ml in controls (p<0.001). In comparison, an intermediate growth hormone release of 34.5±9.7 ng/ml and a higher growth hormone release of 163±46 ng/ml were induced in the lit/+ mice and wild-type mice, respectively. Thus, GHRP-2 stimulated growth hormone in the lit/lit mice, and the release of growth hormone in vivo may be only partially dependent on growth hormone-releasing hormone. Additionally, the plasma leptin and ghrelin levels were evaluated in the lit/lit mice under basal and stimulated conditions.CONCLUSIONS:Here, we have demonstrated that lit/lit mice, which harbor a germline mutation in the Growth hormone-releasing hormone gene, maintain a limited but statistically significant growth hormone elevation after exogenous stimulation with GHRP-2. The present data probably reflect a direct, growth hormone-independent effect on Growth hormone S (ghrelin) stimulation in the remaining pituitary somatotrophs of little mice that is mediated by growth hormone S-R 1a.

Interaction between PEO–PPO–PEO Copolymers and a Hexapeptide in Aqueous Solutions

Interaction between PEO-PPO-PEO copolymers and a hexapeptide, growth hormone releasing peptide-6 (GHRP-6), was investigated by NMR to study the potential use of the copolymers in peptide drug delivery. (1)H NMR and nuclear Overhauser effect spectroscopy (NOESY) measurements determined that PO methyl protons interacted with methyl protons of the Ala moiety, aromatic protons of the Trp moiety, and some of the Phe aromatic protons. The Lys moiety and part of the Phe moiety entered the hydrophilic EO environment via hydrogen bonding. PEO-PPO-PEO copolymers and the peptide formed a complex in 1:1 stoichiometry. Binding constants between copolymers and GHRP-6 were determined, and it was indicated that the copolymers containing more EO and PO units will lead to greater affinity with the peptide. Isothermal titration calorimetry (ITC) measurements confirmed the results of NMR experiments. This study indicates that PEO-PPO-PEO copolymers have great potential in delivering peptide drugs.

Protective effect of GHRP-6 and estrogen supplementation against some cardiometabolic risk factors in ovariectomized rats

This study was aimed to assess the effect of both the estrogen and growth hormone releasing peptide-6 (GHRP-6), on the cardiovascular and metabolic diseases in ovariectomized (OVX) rats. Four groups of adult female rats were used: sham operated, OVX, OVX plus estrogen supplemented, and OVX plus GHRP-6 supplemented. After 8 weeks, blood samples were collected from the retro-orbital plexus and total cholesterol (TC), triglycerides (TGs), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), glucose and insulin in serum were estimated. The ovariectomy resulted in significant increase of body weight gain, food intake, fasting serum glucose, insulin, insulin resistance, TC, and LDL-C as well as in significant decrease of serum TGs and HDL-C levels. Estrogen supplementation to OVX rats reversed the effect of OVX on all the above mentioned parameters. However, GHRP-6 supplemented to OVX rats failed to produce significant change in both the body weight gain and food intake, but reversed the effect of OVX on fasting serum glucose, insulin, insulin resistance, and the assessed lipid fractions. The decrease in ovarian hormones produced by OVX resulted in arising of several risk factors related to carbohydrate metabolism and cardiovascular system. It appeared that such negative effects of OVX can be reversed by estrogen or GHRP-6 supplementation. However, the effect of GHRP-6 on improving dyslipidemia after OVX was more potent than that of estrogen, while the effect of estrogen on improving carbohydrate metabolism after OVX was more potent than that of GHRP-6.

Gastric mucosal damage in water immersion stress: Mechanism and prevention with GHRP-6

To investigate the mechanism of gastric mucosal demage induced by water immersion restraint stress (WRS) and its prevention by growth hormone releasing peptide-6 (GHRP-6). Male Wistar rats were subjected to conscious or unconscious (anesthetized) WRS, simple restraint (SR), free swimming (FS), non-water fluid immersion, immersion without water contact, or rats were placed in a cage surrounded by sand. To explore the sensitivity structures that influence the stress reaction besides skin stimuli, a group the rats had their eyes occluded. Cervical bilateral trunk vagotomy or atropine injection was performed in some rats to assess the parasympathetic role in mucosal damage. Gastric mucosal lesions, acid output and heart rate variability were measured. Plasma renin, endothelin-1 and thromboxane B2 and gastric heat shock protein 70 were also assayed. GHRP-6 was injected [intraperitoneal (IP) or intracerebroventricular (ICV)] 2 h before the onset of stress to observe its potential prevention of the mucosal lesion. WRS for 6 h induced serious gastric mucosal lesion [lesion area, WRS 81.8 ± 6.4 mm² vs normal control 0.0 ± 0.0 mm², P < 0.01], decreased the heart rate, and increased the heart rate variability and gastric acid secretion, suggesting an increase in vagal nerve-carrying stimuli. The mucosal injury was inversely correlated with water temperature (lesion area, WRS at 35 °C 56.4 ± 5.2 mm² vs WRS at 23 °C 81.8 ± 6.4 mm², P < 0.01) and was consciousness-dependent. The injury could not be prevented by eye occlusion, but could be prevented by avoiding contact of the rat body with the water by dressing it in an impermeable plastic suit. When water was replaced by vegetable oil or liquid paraffin, there were gastric lesions in the same grade of water immersion. When rat were placed in a cage surrounded by sand, there were no gastric lesions. All these data point to a remarkable importance of cutenuous information transmitted to the high neural center that by vagal nerves reaching the gastric mucosa. FS alone also induced serious gastric injury, but SR could not induce gastric injury. Bilateral vagotomy or atropine prevented the WRS-induced mucosal lesion, indicating that increased outflow from the vagal center is a decisive factor in WRS-induced gastric injury. The mucosal lesions were prevented by prior injection of GHRP-6 via IP did, but not via ICV, suggesting that the protection is peripheral, although a sudden injection is not equivalent to a physiological release and uptake, which eventually may affect the vagal center. From the central nervous system, vagal nerves carry the cutaneous stimuli brought about by the immersion restraint, an experimental model for inducing acute gastric erosions. GHRP-6 prevents the occurrence of these lesions.

Pharmacological Characteristics of KP-102 (GHRP-2), a Potent Growth Hormone-Releasing Peptide

KP-102 (D-alanyl-3-(2-naphthyl)-D-alanyl-L-alanyl-L-tryptophyl-D-phenylalanyl-L-lysinamide dihydrochloride, growth hormone-releasing peptide-2, GHRP-2, pralmorelin, CAS 158861-67-7), is a potent synthetic growth hormone (GH) secretagogue. In the present study, the pharmacological characteristics of the GH-releasing property of KP-102 were investigated by means of in vivo and in vitro experiments. In conscious rats, the GH-releasing activity of KP-102 was more potent than that of exogenously injected GH-releasing hormone (GHRH). Under pentobarbital anesthesia in which endogenous somatostatin secretion is known to be decreased, KP-102 and GHRH, both showed an almost equivalent GH-releasing potency, which was also similar to that of KP-102 in conscious rats. Besides, KP-102 showed GH-releasing activity in conscious dogs as well, while GHRH failed to increase serum GH levels in conscious dogs. These findings suggest that the GH-releasing activity of KP-102 was less sensitive to GH suppression by endogenous somatostatin as compared with that of GHRH. The GH-releasing activity of KP-102 was completely absent in hypophysectomized rats, but present in median eminence-lesioned rats in which secreted GH amounts were significantly less than those normal rats, indicating necessity of the median eminence (endogenous GHRH) to exert the full activity of KP-102 in GH stimulation. KP-102 directly stimulated GH secretion from cultured rat anterior pituitary cells, although the GH-releasing potency of KP-102 was significantly weaker than that of GHRH in vitro. In conscious rats, KP-102 stimulated the secretion of both adrenocorticotrophic hormone (ACTH) and corticosterone, but not of prolactin. Three weeks administration of KP-102 showed growth-accelerating effect, a slight increase of body weight and wet weight of some organs in both normal and monosodium glutamate (MSG)-treated rats. These results suggest that KP-102 showed specific GH-releasing activity apart from slight ACTH secretion, and that the GH-releasing activity was stable in comparison with that of exogenously injected GHRH.

General Pharmacology of KP-102 (GHRP-2), a Potent Growth Hormone-Releasing Peptide

The general pharmacological effects of the hexapeptide KP-102 (D-alanyl-3-(2-naphthyl)-D-alanyl-L-alanyl-L-tryptophyl-D-phenylalanyl-L-lysinamide dihydrochloride, growth hormone-releasing peptide-2, GHRP-2, pralmorelin, CAS 158861-67-7), which potently promotes growth hormone (GH) release by acting at both hypothalamic and pituitary sites, were evaluated in various animal experimental models. The administration of KP-102 showed no obvious effect at a pharmacological dose on the central nervous system. KP-102 had no significant effect on the autonomic nervous system and smooth muscle except a slight and transient increase in spontaneous motility of isolated rabbit ileum and contraction of isolated guinea pig ileum at high doses. There was negligible effect on the respiratory and cardiovascular systems, digestive system, renal function and blood system after KP-102 treatment. These results suggest that KP-102 has no serious general pharmacological effects at dose levels showing GH-releasing activity in the experimental animals. Therefore, it is concluded that KP-102 will be a useful drug for the diagnosis of serious GH deficiency and for treatment of short stature.

Identification of Ghrelin Receptor Blocker, D-[Lys3] GHRP-6 as a CXCR4 Receptor Antagonist

[D-Lys3]-Growth Hormone Releasing Peptide-6 (DLS) is widely utilized in vivo and in vitro as a selective ghrelin receptor (GHS-R) antagonist. Unexpectedly, we identified that DLS also has the ability to block CXCL12 binding and activity through CXCR4 on T cells and peripheral blood mononuclear cells (PBMCs). Moreover, as CXCR4 has been shown to act as a major co-receptor for HIV-1 entry into CD4 positive host cells, we have also found that DLS partially blocks CXCR4-mediated HIV-1 entry and propagation in activated human PBMCs. These data demonstrate that DLS is not the specific and selective antagonist as thought for GHS-R1a and appears to have additional effects on the CXCR4 chemokine receptor. Our findings also suggest that structural analogues that mimic DLS binding properties may also have properties of blocking HIV infectivity, CXCR4 dependent cancer cell migration and attenuating chemokine-mediated immune cell trafficking in inflammatory disorders.