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Peptidomimetic Regulation of Growth Hormone Secretion

Merck Research Laboratories, Rahway, New Jersey 07065

	Abstract

Top Abstract I. Introduction II. Identification of... III. Molecular Design by... IV. Characterization of the... V. Signal Transduction Pathway VI. Cloning the GH... VII. Action of the... VIII. Peptidomimetic GH... IX. Regulation of Pulsatile... X. Concluding Comments References

 

I. Introduction

II. Identification of Peptidomimetic GH Secretagogues

A. Mechanism of action of GHRH, GHRP-6, and somatostatin

B. In vitro assays

III. Molecular Design by Medicinal Chemistry

A. Benzolactams and L-692,429

B. Spiroindanes and MK-0677

C. Isonipecotic acid peptidomimetics

IV. Characterization of the MK-0677 Receptor

A. Pituitary gland

B. Hypothalamus

V. Signal Transduction Pathway

VI. Cloning the GH Secretagogue Receptor

A. Chromosomal localization

VII. Action of the Peptidomimetic GH Secretagogues in the Central Nervous System

VIII. Peptidomimetic GH Secretagogues in Vivo

A. Animal models

B. Clinical studies in humans

IX. Regulation of Pulsatile GH Release

A. GHRH and somatostatin

B. The role of GHS-R

X. Concluding Comments

	I. Introduction

Top Abstract I. Introduction II. Identification of... III. Molecular Design by... IV. Characterization of the... V. Signal Transduction Pathway VI. Cloning the GH... VII. Action of the... VIII. Peptidomimetic GH... IX. Regulation of Pulsatile... X. Concluding Comments References

 
HORMONES are generally released episodically, yet to our knowledge no attempt has been made to design drugs for treating hormone deficiencies that imitate or amplify the endogenous oscillators governing pulsatile hormone release. For example, although GH is normally secreted in a pulsatile manner with peaks occurring approximately every 3 h, GH deficiency is treated by injecting recombinant GH either once daily or every 3 days. Clearly, this dosing regimen does not result in a physiological GH profile and urges the development of a more natural approach for the treatment of GH deficiency. For convenience to the patient, a pathway was sought that could be activated by a drug given orally once daily. By design, conventional peptides were excluded, because the molecular template had to be readily manipulable for optimization of oral absorption and pharmacokinetics. A receptor that controlled GH release by interaction with an endogenous small molecule was unknown, necessitating a heterodox approach to drug discovery. This reverse pharmacology involved: 1) establishing assays for the functional endpoint, stimulation of episodic GH release; 2) understanding at the cellular level how known peptides controlled GH release so that new specific targets of intervention could be selected; 3) identifying selective GH secretagogues of appropriate structure for optimization of pharmacokinetic properties; 4) characterizing and cloning the receptor for these ligands; and 5) identifying the receptor’s natural ligand. This review provides a summary of points 1–4, focusing on the discovery and properties of new GH secretagogues. These molecules were subsequently shown to be peptide mimetics of the GH-releasing peptides (GHRPs) first identified by the pioneering work of Bowers, Momany, and colleagues (1, 2, 3); for a comprehensive review of GHRPs the reader is referred to Refs. 4–9. The natural ligand for the receptor activated by GHRPs and its peptidomimetics remains elusive, but now that the receptor has been cloned and distributed, we anticipate its ligand will soon be discovered.

	II. Identification of Peptidomimetic GH Secretagogues

Top Abstract I. Introduction II. Identification of... III. Molecular Design by... IV. Characterization of the... V. Signal Transduction Pathway VI. Cloning the GH... VII. Action of the... VIII. Peptidomimetic GH... IX. Regulation of Pulsatile... X. Concluding Comments References

 
A. Mechanism of action of GHRH, GHRP-6, and somatostatin
The objective was to somehow amplify the GH-secretory pathway with a small orally active molecule. This required a detailed knowledge of the mechanisms governing GH release at the cellular biochemistry level as well as understanding the interplay between the central nervous system and the anterior pituitary gland. Two known hypothalamic hormones, GHRH and somatostatin, are key regulators of GH release from somatotrophs in the pituitary gland; GHRH stimulates GH secretion, and somatostatin is inhibitory (10, 11, 12, 13). In addition to GHRH, a synthetic hexapeptide, His-D-Trp-Ala-Trp-D-Phe-Lys-NH₂ (GHRP-6), first described by Bowers’ group, is also a potent GH secretagogue (1, 2). The GHRPs were first described in 1981 (3), before GHRH (14, 15), but until recently their receptor had not been defined (16). The obvious approach was to identify nonpeptide mimetics of either GHRH or GHRP-6; however, in contrast to antagonists, the probability of finding nonpeptide agonists of peptides was considered to be very low. The only precedent was morphine and its congeners, which are nonpeptide mimetics of the opioid peptides. Therefore, from a medicinal chemistry perspective, a more reasonable approach to amplify GH release with a small molecule was to design a nonpeptide antagonist of somatostatin. However, the potential for extrapituitary effects of a nonselective somatostatin antagonist made somatostatin a less than ideal target; moreover, before the cloning of somatostatin receptor subtypes, screening for selectivity was not an option.

In spite of the historical difficulties of identifying peptidomimetics, GHRH and GHRP-6 were excellent drug targets. A priori, a peptidomimetic of GHRH seemed the most obvious choice because it had been studied extensively. However, structure-activity relationships indicated that the size of the molecule could not be reduced below 29 amino acids without a significant loss in activity (17). The peptide GHRP-6 was of ideal size, but because its receptor had not been identified, and cell lines responsive to GHRP-6 were unknown, high volume screening for a peptidomimetic was impractical. Based on these considerations, investigators modified the structure of GHRP-6 and identified more potent peptides (4, 5, 6, 18). For example, activity was enhanced by replacing D-Trp2 by D-2-(2-napthyl)alanine and His by D-alanine to furnish GHRP-2 (D-Ala-D-2 Nal-Ala-Trp-D-Phe-Lys-NH₂) (5). However, the peptides still had low oral bioavailability.

The development of the GHRPs had primarily emerged from in vivo studies and before attempting to identify peptidomimetics, it was essential to understand the cellular mechanisms involved in the action of GHRP-6. In 1989, the signal transduction pathway activated by GHRP-6 was reported (19). It was established that GHRP-6 acted directly on somatotrophs to cause GH release and to potentiate the effects of GHRH. In contrast to GHRH, which increases cAMP in somatotrophs, GHRP-6 alone had no effect on intracellular cAMP, but when combined with GHRH, the hexapeptide amplified the effects of GHRH on cAMP production (19). GHRP-6 was subsequently shown to activate L-type Ca²⁺ channels, to depolarize the plasma membrane of somatotrophs by inhibiting K⁺ channels, and behave as a functional antagonist of somatostatin (8, 20, 21, 22). In contrast to GHRH, which stimulates GH release through the kinase A pathway, GHRP-6 apparently transduced its signal through protein kinase C (23). Phloretin, an inhibitor of protein kinase C, inhibited GHRP-6-stimulated GH release (23). Also, prolonged exposure of pituitary cells to phorbol esters before GHRP-6 treatment markedly attenuated the action of GHRP-6 without affecting GH release induced by GHRH (23). Subsequent studies showed that GHRP-6 stimulated IP₃ turnover, activated protein kinase C, and caused the release of intracellular stores of Ca²⁺ (24, 25, 26). Collectively, these data provided evidence that GHRP-6 acted through a receptor distinct from that of the GHRH receptor and were consistent with the notion that the GHRP-6 receptor was G-protein coupled.

To further evaluate differences between GHRH and GHRP-6 receptors, the kinetics of desensitization and resensitization of pituitary cells exposed to GHRP-6 and GHRH (27) were compared. These studies clearly showed that pituitary cells were desensitized very rapidly by GHRP-6 compared with GHRH, and that complete resensitization required interruption of exposure to GHRP-6 for at least 1 h (27). Having established the kinetics of desensitization, tachyphylaxis was deliberately induced to GHRH and GHRP-6 by prolonged continuous perifusion of each secretagogue. The cells were then challenged with GHRH and GHRP-6 to test for stimulation of GH secretion. Cross-desensitization was not evident, confirming that discrete receptors for GHRH and GHRP-6 were involved in the GH release pathways (27). The rapid rate of tachyphylaxis observed with GHRP-6 explained why some early studies failed to demonstrate an effect on GH release. In contrast to studies with the longer acting GHRH, where increases in GH in the medium of cultured pituitary cells are generally measured over 2–3 h, GHRP-6 culture medium must be sampled within 10–15 min of treatment for optimal results.

B. In vitro assays
Primary cultures of rat pituitary cells were used to screen for small molecules that selectively caused GH release. Because it was impractical to efficiently screen thousands of compounds in a primary cell assay, the structural classes to be assayed were rationally selected based on the key structural features of GHRP-6. However, to maximize the probability of finding a small molecule GH secretagogue, assay conditions were chosen that would identify compounds acting on GHRP-6, GHRH, or ion channel pathways (28). Selectivity for activation of somatotrophs, rather than mammotrophs or corticotrophs, was determined by assaying the pituitary cell culture medium for PRL and ACTH as well as GH.

The mechanism through which each active compound stimulated GH release was ascertained. For example, each compound was evaluated to determine whether it caused increases in cAMP or whether the protein kinase C pathway was involved in signal transduction. The actives were also assayed in the presence of specific GHRH and GHRP-6 antagonists to determine whether they were GHRH or GHRP-6 mimetics (29). Their effects on Ca²⁺, K⁺, and Na⁺ channels and on membrane potential were also investigated. Finally, after identification of somatotrophs with the reverse hemolytic plaque assay (30), electrophysiology and Ca²⁺ imaging studies were applied to more carefully determine effects on the target cell (29). Having established the signal transduction pathway involved, pituitary cells were treated with each active compound in combination with GHRH or GHRP-6 to test for amplification of the respective pathways.

	III. Molecular Design by Medicinal Chemistry

Top Abstract I. Introduction II. Identification of... III. Molecular Design by... IV. Characterization of the... V. Signal Transduction Pathway VI. Cloning the GH... VII. Action of the... VIII. Peptidomimetic GH... IX. Regulation of Pulsatile... X. Concluding Comments References

 
A. Benzolactams and L-692,429
Based on the structure of GHRP-6, and the knowledge that benzodiazepine and related templates mimic small peptides, approximately 120 compounds were selected for their ability to stimulate GH release. A benzolactam 1 (Fig. 1) with an EC₅₀ of 3 µM for GH release in the rat pituitary cell assay was identified (29, 31). Replacement of the carboxylic acid moiety with a tetrazole and resolution of the enantiomers led to the discovery of L-692,429 (Fig. 1, compound 2), a nonpeptide GH secretagogue with an EC₅₀ of 60 nM (29, 31, 32, 33). L-692,429 was the first example of a potent nonpeptide GH secretagogue (29). L-692,428, the S enantiomer of L-692,429, was inactive, demonstrating the stereoselectivity of the response (Fig. 2). Using fluorescent ratio imaging, L-692,429 was shown to increase free intracellular Ca²⁺ in somatotrophs (Fig. 3); L-692,428 was ineffective (29). A structurally related nonpeptide antagonist of L-692,429 was identified and used in conjunction with peptide antagonists of GHRP-6 and GHRH to define the receptor target of L-692,429 (29). L-692,429 synergized with GHRH but, in the presence of a maximally effective concentration of GHRP-6, exhibited no additive effect on stimulating GH release from rat pituitary cells. The activity of L-692,429 was blocked by both peptide and nonpeptide antagonists of GHRP-6, but not by an antagonist of GHRH (29). These results were consistent with the notion that L-692,429 was a peptidomimetic of GHRP-6.

View larger version (17K): [in this window] [in a new window]   Figure 1. Benzolactam GH secretagogues

View larger version (12K): [in this window] [in a new window]   Figure 2. Stereoselectivity of a nonpeptide GH secretagogue. A comparison of the effects of L-692,429 (R-enantiomer), •, and L-692,428 (S-enantiomer), , on inducing GH release from cultured rat pituitary cells (29). [Reprinted with permission from R. G. Smith et al.: Science 260:1640–1643 (29). © 1993 American Association for the Advancement of Science.]

View larger version (70K): [in this window] [in a new window]   Figure 3. Fluorescent ratio imaging showing the effect of L-692,429 on cytoplasmic free Ca2+ in a rat somatotroph. Images of a somatotroph at 340 nm and 380 nm are shown as a function of time after addition of L-692,429. The concentration of L-692,429 selected was 33-fold the EC50 for GH release, and the free intracellular Ca2+ increased from approximately 100 to 780 nM (29). [Reprinted with permission from R. G. Smith et al.: Science 260:1640–1643 (29). © 1993 American Association for the Advancement of Science.]

Attempts were made to improve the efficacy of L-692,429. A comparison of the biological activity of a series of six and eight-member lactam analogs of L-692,429 showed that the seven-member ring was preferred (31). Substitution with heterocyclic analogs of the benzolactam nucleus resulted in diminished activity (37). Continued exploration of structures related to L-692,429, focusing on refining structure-activity relationships in the amino acid side chain, revealed that the basic amine was an essential pharmacophore for GH-releasing activity (38). A systematic investigation of this dimethyl-ß-alanine side chain led to the identification of L-692,585, a 2-(R)-hydroxypropyl analog (Fig. 1, compound 3), which was 20-fold more potent than L-692,429 (38, 39). Comparison of the binding data for L-692,585 [inhibition constant (K_i) = 0.8 nM] vs. L-692,429 (K_i = 63 nM) strongly suggested that the 2-hydroxypropyl moiety in L-692,585 makes an additional binding interaction with the GH secretagogue receptor. Although L-692,585 had much improved potency, and subsequent studies in beagle dogs showed it had highly reproducible oral activity, its oral bioavailability was unacceptably low (4%) for clinical development. Replacement of the central phenyl ring of the biphenyl moiety in L-692,429 and L-692,585 was evaluated. A cyclohexenyl analog of L-692,585 showed similar activity to its parent, showing that the aromaticity of the central ring was not critical for bioactivity and suggested that this ring may serve to orient the benzolactam and phenyltetrazole pharmacophore (40). However, this structural change provided no improvement in oral bioavailability.

To investigate replacements for the 2'-tetrazole moiety of L-692,429, a variety of 2'-carboxamides and 2'-biphenyl analogs were evaluated. A 2'-carboxamide and N-2-hyroxypropyl tetrazole were found to have similar potency to the acidic tetrazole; however, N-alkyl tetrazoles, sulfonamides, and acyl sulfonamides were generally less potent replacements (41, 42). The primary and secondary carboxamides were potent GH secretagogues, and L-700,653 (Fig. 1, compound 4) had improved oral bioavailability in dogs and in swine (42, 43). However, in spite of excellent potency, selectivity, and tolerability in animals, the relatively low bioavailability remained an issue with the benzolactam structural class; hence a different structural lead was sought (44, 45, 46).

B. Spiroindanes and MK-0677
A new structural class of GH secretagogues was discovered by screening compounds from a project to prepare derivatized privileged structures for broad testing in receptor assays. The term "privileged structures" refers to structural units that are found on a recurring basis in receptor ligands. Their recognition and derivatization have been proposed as a useful way to prepare receptor agonists and antagonists (47). In the current instance, the successful strategy was to derivatize a spiroindanylpiperidine with capped amino acids. This piperidine derivative was considered a privileged structure since it was present in - (48) and oxytocin receptor ligands (49) and, in fact, was also present in a camphor sulfonamide lead 1 (L-368, 112, Fig. 4), which showed weak GH secretagogue activity (50). Thus, it could be inferred that the spiroindanylpiperidine would be an appropriate core from which to elaborate ligands for the putative GH secretagogue receptor. Its derivatization afforded L-252,564 (Fig. 4, compound 2) whose activity as a secretagogue (EC₅₀ = 50 nM) was remarkable since it was an unseparated mixture of four diastereoisomers. The other components of compound 2 are tryptophan and a quinuclidinylurea. To account for the high activity of this lead, it was noted that the quinuclidene group was also present in an unpublished Merck GH secretagogue lead, and tryptophan is a key amino acid in the GHRP-6 structure (50).

View larger version (17K): [in this window] [in a new window]   Figure 4. Spiroindane GH secretagogues

Potency enhancement was achieved by the introduction of a carbonyl or hydroxyl substituent at the indane benzylic position. The oral activity of these compounds was disappointing; however, it was restored by the introduction of a methanesulfonamide group in this position. In addition, replacement of D-Trp by O-benzyl-D-serine further improved oral bioavailability. The resultant compound 4 (L-163,191) was active in the rat pituitary cell assay (EC₅₀ = 1.3 nM) and was a specific GH secretagogue when counterscreened in more than 50 in vitro assays (50). These included oxytocin, enkephalin, cholinergic, adrenergic, serotonin, neurokinin, and galanin receptors. Its pharmacokinetic properties in rats included an oral bioavailability of between 6–22% (52), and in beagle dogs its bioavailability was greater than 60% with a terminal half-life of between 5–6 h (50, 52). In beagles, after oral administration of 1 mg/kg, GH was elevated for more than 6 h (50, 53). Insulin-like growth factor I (IGF-I) levels were also increased significantly. Most importantly, L-163,191 was the first GH secretagogue in its class demonstrated to provide a sustained increase in IGF-I levels for up to 24 h after a single oral dose (53, 54). Based on these properties, L-163,191 entered safety studies and then clinical studies as MK-0677. A series of analogs was also prepared (55, 56) to evaluate more extensively interactions with the MK-0677 receptor using site-directed mutagenesis studies.

C. Isonipecotic acid peptidomimetics
The Genentech group used a multidisciplinary approach in their discovery of a new series of small molecule GH secretagogues (Fig. 5). Toward the goal of determining the topographical requirements for the GH-releasing activity of the GHRPs, G-7203 (EC₅₀ = 0.43 ± 0.11 nM), a cyclic analog of the linear hexapeptide GHRP-2, was developed (57, 58). Nuclear magnetic resonance studies showed that G-7203 was structured in water. Furthermore, the D-2-Nal-Ala-Trp-D-Phe fragment adopts a compact conformation with nested hairpin turns initiated at D-Lys1 and Ala3. Other less active cyclic GHRP-2 analogs did not readily adopt this conformation, suggesting that a precise arrangement of the three aromatic side chains was crucial for GH-releasing activity.

View larger version (20K): [in this window] [in a new window]   Figure 5. GHRP-2 and Genentech GH secretagogues

	IV. Characterization of the MK-0677 Receptor

Top Abstract I. Introduction II. Identification of... III. Molecular Design by... IV. Characterization of the... V. Signal Transduction Pathway VI. Cloning the GH... VII. Action of the... VIII. Peptidomimetic GH... IX. Regulation of Pulsatile... X. Concluding Comments References

 
A. Pituitary gland
Based on binding studies using [³H]naloxone as a ligand, it was clear that although GHRP-6 and its analogs were derived from met-enkephalin, neither GHRP-6 nor the peptidomimetics bind to opiate receptors. Moreover, their GH-releasing activity was not blocked by coadministration of naloxone (34). Extensive evaluation of L-692,429 and MK-0677 showed that they lacked activity on different opiate receptor subtypes and in more than 50 different receptor assays (29, 50, 59). Attempts to define a receptor in the pituitary gland using radiolabeled GHRP-6 failed to show a correlation between high-affinity, low-capacity binding and biological activity of both peptide and peptidomimetic GH secretagogues (60).

To characterize the MK-0677 receptor, high-specific activity radiolabeled MK-0677 (800–1100 Ci/mmol) was synthesized by substituting ³⁵S for ³²S in the molecule (60, 61). [³⁵S]MK-0677 bound with high affinity [dissociation constant (K_d) = 140 pM] and limited capacity (B_max = 6.4 fmol/mg protein) to porcine pituitary membranes (Fig. 6). This concentration of binding sites in pig pituitary is remarkably low, but in rat pituitary membranes the concentration is even lower (2 fmol/mg protein). [³⁵S]MK-0677 binding was displaced by L-692,429, L-692,585, and by the peptide GH secretagogue GHRP-6, but not by GHRH or somatostatin (60). The K_i values in the binding assay correlated with the EC₅₀ values for stimulating GH release in the rat pituitary cell assay (Table 1). Consistent with [³⁵S]MK-0677 binding to a G protein-coupled receptor, binding was dependent upon Mg²⁺ (5 mM) and displaced by GTP--S (10 nM) but not by ATP--S (59, 60). Remarkably, competition binding studies with L-692,429 and GHRP-6 showed that these two structurally distinct molecules were competitive inhibitors of MK-0677 binding (59). By contrast, as anticipated for a G protein-coupled receptor, GTP--S was an allosteric inhibitor (59, 60). To further explore the specificity of [³⁵S]MK-0677 binding, met-enkephalin, GnRH, TRH, galanin, gastrin releasing peptide, substance P, MSH, isoproterenol, dopamine, bromocriptine, propanolol, and clonidene were tested in the binding assay at a concentration of 1 µM; none competed for [³⁵S]MK-0677 binding (60).

View larger version (22K): [in this window] [in a new window]   Figure 6. Scatchard analysis of [35S]MK-0677 binding to pig pituitary membrane showing a single class of high-affinity binding sites (60). [Reproduced with permission from S.-S. Pong et al.: Mol Endocrinol 10:57–61, 1996 (60). © The Endocrine Society.]

To determine whether the binding to pituitary membranes was localized to somatotrophs, a biotinylated analog of MK-0677, L-164,683, was prepared as a suitable ligand for immunofluorescence studies (63). L-164,683 was an excellent competitor for [³⁵S]MK-0677 binding (IC₅₀ = 0.2 nM) and stimulated GH release with an EC₅₀ of 2.5 nM. Primary cultures of rat pituitary cells were treated with L-164,683 for 3 min at 37 C and treated with avidin-Texas Red. GH-containing cells were labeled with fluorescein-conjugated goat anti-rabbit IgG. Dual fluorophor labeling for GH and the MK-0677 receptor by confocal microscopy showed that about half of the GH-containing cells also expressed the MK-0677 receptor (Fig. 7). L-164,683 binding was confined to cells that contained GH (63). While only about half of the GH-containing cells showed binding of the MK-0677 analog, based on limits of sensitivity of the assay, this proportion should be considered a low estimate. More quantitative estimates have been attempted by localization using MK-0677 receptor antibodies, and preliminary results suggest that all GH-containing cells in the pituitary gland express the MK-0677 receptor (R. G. Smith, A. D. Howard, S. D. Feighner, and J. W. Woods, unpublished results).

View larger version (34K): [in this window] [in a new window]   Figure 7. Dual fluorophore localization of MK-0677-binding sites and GH in rat pituitary primary cultures (63). Left panel, Identification of rat GH with a rabbit anti-rat GH antibody and fluorescein-conjugated goat anti-rabbit antibodies. Right panel, Identification of GH secretagogue-binding sites on the same cells with biotinylated MK-0677 (L-164,683; 1 µM; 3 min 37 C) and avidin Texas red. Cells were first reacted with L-164,683 (15 min 4 C) and avidin, after which the cells were fixed and permeabilized and treated for GH detection. Note that approximately 50% somatotrophs are labeled for MK-0677-binding sites, and all cells labeled with the MK-0677 derivative contain GH.

	V. Signal Transduction Pathway

Top Abstract I. Introduction II. Identification of... III. Molecular Design by... IV. Characterization of the... V. Signal Transduction Pathway VI. Cloning the GH... VII. Action of the... VIII. Peptidomimetic GH... IX. Regulation of Pulsatile... X. Concluding Comments References

 
MK-0677, in common with L-692,429 but in contrast to GHRH, does not increase intracellular cAMP levels in pituitary cells (50, 59). However, in combination with GHRH, MK-0677 amplifies the GHRH-induced increase in cAMP and potentiates GH release (59). At doses of MK-0677 that maximally stimulated GH release in vitro, additional stimulation is not observed in the presence of maximally stimulating concentrations of L-692,429. Similar observations were made when GHRP-6 was substituted for L-692,429 indicating, in agreement with binding studies, that GHRP-6 and these peptidomimetics all mediate their effects through the same receptor. The synergistic effects of the GH secretagogues on the GHRH pathway were mimicked by phorbol myristic acetate. Preincubation of rat pituitary cells with phorbol myristic acetate for 24 h, before treatment with MK-0677, profoundly attenuated the stimulation of GH release by MK-0677 (50). These results, and the cation and nucleotide dependence of MK-0677 binding, suggested that MK-0677 interacts with a G-protein coupled receptor that activates phospholipase C (59, 60). Consistent with activation of this pathway were the earlier observations that other ligands for this receptor, such as L-692,429 and GHRP-6, increase IP₃ turnover (65), increase free intracellular Ca²⁺ through an IP₃ pathway, and cause translocation of protein kinase C (24, 25, 26, 65).

The role of L-type Ca²⁺ channels in the transduction pathway was confirmed by fluorescence ratio imaging in somatotrophs after treatment with either L-692,429 or MK-0677 (29, 59). Nifedipine and -agatoxin IIIA, but not conotoxin, were shown to block increases in intracellular Ca²⁺, consistent with activation of L-type Ca²⁺ channels (66, 67, 68). Electrophysiology studies showed that the GHRP-6 peptidomimetics blocked K⁺ currents in somatotrophs, resulting in depolarization and electrical spiking to enhance Ca²⁺ entry through voltage gate channels (21, 69, 70). Modulation of these channels was evident when perforated patch clamp or on-cell single-channel recording techniques were used, but not when the cells were dialyzed using the whole cell voltage clamp configuration, suggesting that a soluble second messenger was involved. The depolarizing effects were confirmed using the membrane-sensitive dye bisoxanol (29). Depolarizing agents such as the potassium channel blockers tetraethylammonium and 4-amino pyridine and the sodium channel agonist veratridine had no additive effects on GH secretion induced by the peptidomimetics; rather, they mimicked the peptidomimetics by amplifying the effects of GHRH (8). The fact that the peptidomimetics cause depolarization explains their functional antagonism of somatostatin, since somatostatin hyperpolarizes somatotrophs by increasing potassium conductance (71). The magnitude of GH release caused by depolarizing agents alone is less than that induced by the peptidomimetics, emphasizing the importance of other aspects of the signal transduction pathway. The MK-0677 receptor via phospholipase C activates Ca²⁺ and inhibits K⁺ channels to facilitate GH secretion (Fig. 8A). The IP₃-mediated redistribution of intracellular Ca²⁺ alone does not explain all the effects of these secretagogues because somatostatin does not prevent the Ca²⁺ redistribution but does inhibit GH release (30); rather, it is speculated that IP₃ facilitates GH release by synchronizing docking of GH-secretory granules to the plasma membrane (72, 73).

View larger version (70K): [in this window] [in a new window]   Figure 8. A, Signal transduction pathway activated by ligands that interact with the MK-0677 receptor. B, GHRH and MK-0677 act on discrete receptors and transduce their signal through different pathways.

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	VI. Cloning the GH Secretagogue Receptor

Top Abstract I. Introduction II. Identification of... III. Molecular Design by... IV. Characterization of the... V. Signal Transduction Pathway VI. Cloning the GH... VII. Action of the... VIII. Peptidomimetic GH... IX. Regulation of Pulsatile... X. Concluding Comments References

 
Having established from binding data and biochemical studies that MK-0677 probably binds to a G protein-coupled receptor that signals through phospholipase C, an expression-cloning strategy was developed for the MK-0677 receptor by microinjecting size-fractionated poly A⁺ RNA from pig pituitaries into Xenopus oocytes (16). Expression of the RNA from one fraction resulted in an MK-0677-induced calcium activated chloride current, but the assay reproducibility was low. A more robust cloning strategy was developed, which coupled receptor expression in Xenopus oocytes to a functional endpoint that measured MK-0677-induced Ca²⁺ release (16). The Ca²⁺-sensitive bioluminescent reporter protein aequorin was used successfully as a signal for positive coupling, but responses to MK-0677 were still weak and variable. It was speculated that expression of a specific G protein essential for receptor coupling might be limiting; therefore, cRNAs encoding a series of G proteins (G₁₁, G_q, G₁₆, G₁₃, Gi₁, Gi₃, G_o) were individually coinjected into Xenopus oocytes together with pituitary gland poly A⁺ RNA and aequorin cRNA. Only G₁₁ provided a highly reproducible Ca²⁺-mediated luminescence signal in response to MK-0677 (16). This robust expression system was used to screen pools of cRNA from a pig pituitary gland cDNA library for an MK-0677-inducible signal. Approximately 2 x 10⁶ individual cDNAs from a pig pituitary library were screened in pools of 10,000. Stepwise fractionation of a single positive pool resulted in the isolation of a single cDNA clone that conferred both MK-0677-activated aequorin luminescence and an inward chloride current in Xenopus oocytes. Interestingly, supplementation with G₁₁ was unnecessary when cRNA pool complexity dropped below 50 clones.

The nucleotide sequence of the full-length swine MK-0677 receptor cDNA-1a predicted a protein of 366 amino acids with seven transmembrane (7-TM)-spanning domains, three intra- and extracellular loops, and a G protein-coupled receptor triplet signature sequence (16). The human receptor was subsequently cloned, and its predicted topology is represented in Fig. 9. Genomic analysis by Southern blotting was consistent with a single highly conserved gene in human, chimpanzee, bovine, rat, and mouse (16). Sequence alignments showed that the swine MK-0677 receptor was 93% identical and 98% similar at the amino acid level to the human receptor (Fig. 10). Additional cDNAs clones were obtained from pig and human libraries that encode a shorter form of the MK-0677 receptor (16). Receptor 1b cDNA encodes a polypeptide of 289 amino acids that lacks transmembrane domains 6 and 7 of the 1a receptor (16). This truncated receptor is identical to the 1a receptor from the translation initiation codon to Leu-265 beyond which the cDNA is fused to a short contiguous reading frame of 24 amino acids followed by a translation stop codon. This 24-amino acid sequence is highly conserved in both the pig and human MK-0677 receptor genes (16). A similarly truncated mRNA has been reported for the neuropeptide Y1 (NPY1) receptor (78). Inspection of the amino acid sequence of the human 1a receptor revealed a G protein-coupled receptor signature [ERY142], a series of potential N-glycosylation sites, protein kinase C, casein kinase II phosphorylation sites, a cAMP/cGMP-dependent phosphorylation site [346–349 RKLS], myristoylation sites, and an amidation site (235–238 IGRK).

View larger version (45K): [in this window] [in a new window]   Figure 9. Predicted membrane topology and amino acid sequence of the human type 1a GHS-R. TM denotes transmembrane domains 1–7.

View larger version (73K): [in this window] [in a new window]   Figure 10. Predicted amino acid sequences for the human and swine type 1a and 1b GHS-R. Identities are highlighted in the boxed amino acid sequences (16). Conserved cysteine residues and the GPC-R signature sequence (ERY142) are shaded gray. N-Linked glycosylation sites and potential phosphorylation sites are highlighted with arrowheads and asterisks, respectively. The transmembrane (TM) domains are overlined and numbered sequentially. The numbers on the left and right refer to the sequentially numbered amino acid residues (1–366 for the type 1a and 1–289 for the type 1b receptors). [Reprinted with permission from A. D. Howard et al.: Science 273:974–977 (16). © 1996 American Association for the Advancement of Science.]

View larger version (20K): [in this window] [in a new window]   Figure 11. Competition binding assay showing displacement of [35S]MK-0677 (.24 nM) from COS cell membranes isolated from cells transiently expressing GHS-R1a. [Reprinted with permission from A. D. Howard et al.: Science273:974–977 (16). © American Association for the Advancement of Science.]

View larger version (38K): [in this window] [in a new window]   Figure 12. Dendrogram of GHS-R and other G protein-coupled receptors. Database searches (Genbank 92, EMBL 43, Swiss-Prot 31, PIR 45, dEST (Gbest 92), Prosite 12), sequence alignments, and analysis of the GHS-R nucleotide and protein sequences were carried out using the GCG Sequence Analysis Software Package (Madison, WI; pileup, peptide structure and motif programs), FASTA and BLAST search programs, the PC/Gene software suite from Intelligenetics (San Francisco, CA; protein analysis programs) and Lasergene software (DNA Star, Madison, WI). The amino acid sequence of representative members (51 sequences) for all known classes (Families I-IV and pheromone) of G protein-coupled receptors were used to construct the dendrogram using the clustal method (PAM-250; gap and length penalty = 10). The length of each pair of branches represents the distance between sequence pairs. The scale below the tree measures the distance between sequences. Units indicate the percent of substitution events. All receptor sequences aligned to human GHS-R type Ia and type 1b cDNAs were human unless otherwise noted, and accession numbers are for the SwissProt database, GenBank database (designated with "G"), or PIR database ("P"): 5HT-2A, serotonin, P28223; RDC-1, orphan receptor, A39714; somatostatinR4, somatostatin, A47457; V1a, vasopressin, A53046; V1b, vasopressin, A55089; A2b, adenosine, P29275; M1, muscarinic; P11229; APJ, orphan, P11229; C5a, chemotactic, P21730; CASR, extracellular calcium-sensing; P41180; CB1, cannabinoid, P21554; CRF, corticotropin-releasing factor, P34998; FSHR, FSH, P23945; CC-BR, gastrin/cholecystokinin type B, P32239; GRFR, GH-releasing, Q02643; GnRH, gonadotropin-releasing, P30968; CALR, calcitonin, X69920 (G); GlucR, glucagon, U03469 (G); OR-, opioid, U10504 (G); Mel-1a, melatonin, U14108 (G); SecR, secretin, U20178 (G); PTHCaR, parathyroid cell calcium-sensing, U20759 (G); Br1, bradykinin, U22346 (G); 1AD, alpha-1 adrenergic, L31772 (G); GalR, galanin, L34339 (G); H1R, histamine, D14436 (G); TRHR, TRH, D16845 (G); MetGlu5a, metabotropic glutamate, JC2132 (P); 1AD, alpha-1 adrenergic, JC2331 (P); Br2, bradykinin, JH0712 (P); NK-1, substance P, P25103; NM-BR, neuromedin-B, P28336; NT-R, neurotensin, P30989; NPY1R, neuropeptide Y, P25929; OR-µ, opioid, P35372; RHO-fish, rhodopsin, P35356; EP-1, prostaglandin E1, P34995; PTH-R, PTH, Q03431; OR-, opioid-rat, S39015 (P); ORL-1, opioid-orphanin FQ, S43087 (P); ET-B, endothelin, S44866 (G); D2, dopamine, S62137 (G); STE2-yeast, pheromone factor, P06842; SSTR2, SSTR3, SSTR5, SSTR2A-mouse, SSTR2B, SSTR1, somatostatin, P30874, P32745, P35346, P30875, P30934, P30872, respectively; PACAP-R, pituitary adenylate cyclase-activating peptide, D17516 (G); TR-R, thrombin, P25116; TSH-R1, TSH, S49816 (G).

A. Chromosomal localization
The human GHS-R was mapped by fluorescence in situhybridization to band 3Q26.2 (82). Genes whose deficiencies affect GH release do not map to this region. Interestingly, however, this location is close to the map position reported for the Brachmann-de-Lange Syndrome, a pre- and postnatal growth deficiency (83, 84, 85). Mapping of the Brachmann-de-Lange Syndrome is based on chromosome duplication and translocation mutants, which always include region 3q26 (interval 3q26.31–q27.3). Given the close proximity between the GHS-R gene and the presumed Brachmann-de-Lange location, it will be important to determine whether these subjects respond to MK-0677 treatment and whether they have alterations in the gene encoding the GHS-R.

	VII. Action of the Peptidomimetic GH Secretagogues in the Central Nervous System

Top Abstract I. Introduction II. Identification of... III. Molecular Design by... IV. Characterization of the... V. Signal Transduction Pathway VI. Cloning the GH... VII. Action of the... VIII. Peptidomimetic GH... IX. Regulation of Pulsatile... X. Concluding Comments References

 
The central effects of GHRP-6 and the peptidomimetics are inferred by the demonstration in sheep and guinea pigs that the doses required to cause GH release are at least 10- to 100-fold lower when injected into the third ventricle than when the compounds are administered peripherally (86, 87). Intravenous administration of GHRP-6 and the peptidomimetics L-692,429, L-692,585, and MK-0677 into conscious rats and mice increased c-fos expression in the arcuate nucleus at concentrations consistent with their relative biological activites in releasing GH (86, 88, 89, 90). In situ hybridization histochemistry studies with little (lit/lit) mice and dwarf (dw/dw) mice treated with MK-0677 (Fig. 13) also showed pronounced increases in c-fos expression (89). Lit/lit mice have a reduced capacity to release GH because they lack a functional GHRH receptor (74, 91, 92, 93), and dw/dw mice lack GH (94, 95). Therefore, activation of fos by GHRP-6 and the peptidomimetics cannot be explained by indirect effects on GHRH receptors or through GH (86, 88, 89, 96), but rather as a direct consequence of a central action of GHS-R ligands. In rats treated with GHRP-6, approximately 25% of neurons showing an increase in c-fos activity express GHRH mRNA, and 51% express NPY mRNA (97). The activation of GHRH-containing neurons suggests that GHRP-6 and the peptidomimetic secretagogues stimulate the release of GHRH. Indeed, increases in GHRH have been measured in hypothalamic-pituitary portal vessels of sheep after systemic treatment with GHRPs (98, 99). The finding that GHRP-6 also activates NPY-containing neurons potentially explains the increased feeding behavior seen in rats treated with GHRP-6 (100) and the observed increases in corticosteroid and ACTH release (101, 102). Clearly, activation of Fos immunoactivity or c-fos mRNA expression should not be considered a direct and sole marker since other neurons in the brain that do not express Fos may be activated, and of course those expressing Fos might be activated indirectly as a result of derepression.

View larger version (87K): [in this window] [in a new window]   Figure 13. Activation of c-fos gene expression in the central nervous system of control and dw/dw mice after intraperitoneal MK-0677 administration (89). Wild type C57 BL/6J mice and dw/dw mice were injected intraperitoneally with either PBS or MK-0677 (50 mg/kg) and killed 40 min later. In situ hybridizations for c-fos mRNA were performed on coronal brain sections. Images have been artificially colored: blue represents background; white e represents the most intense signals (23).

In situ hybridization studies using a cDNA probe selective for GHS-R 1a to sections of rhesus monkey and rat brains demonstrate that the receptor is expressed in the arcuate nucleus (16). Localization of expression in this area of the hypothalamus is consistent with electrophysiology experiments and c-fos expression, suggesting that these molecules act on GHRH-containing arcuate neurons (97). More complete localization studies have been completed in rat brain and pituitary gland and show that GHS-R is expressed in the anterior pituitary gland and in regions of the brain outside those generally considered to be involved in GH release (Fig. 14 and 105 . Indeed, expression of GHS-R is seen in the anterior hypothalamus, suprachiasmatic nucleus, supraoptic nucleus, ventromedial hypothalamus, arcuate nucleus, dentate gyrus, tuberomamillary nucleus, pars compacta of substantia nigra, the ventral tegmental area, dorsal raphe nuclei, and median raphe nuclei (105).

View larger version (112K): [in this window] [in a new window]   Figure 14. In situ hybridization of GHS-R mRNA in rat brain (105). 33P-Labeled rat GHS-R oligonucleotide probes were used for in situ hybridization of coronal rat brain sections. Signals were highly specific and could be displaced by 100-fold molar excess of cold probe. Abbreviations are as follows. Top left panel: AHA, anterior hypothalamic area; Sch, suprachiasmatic nucleus; SO, supraoptic nucleus. Top right panel: ARC, arcuate nucleus; VMH, ventromedial hypothalamus. Bottom left panel: DG, dentate gyrus; CA2/CA3, CA2 and CA3 regions; TM, tuberomammillary nucleus. Bottom right panel: SNC, pars compacta of substantia nigra; VTA, ventral tegmental area. [Reprinted from X.-M. Guan et al.: Mol Brain Res 48:23–29, 1997, with kind permission from Elsevier Science-NL, Sara Burgerhartstraat 25, 1055 KV Amsterdam, The Netherlands.]

	VIII. Peptidomimetic GH Secretagogues in Vivo

Top Abstract I. Introduction II. Identification of... III. Molecular Design by... IV. Characterization of the... V. Signal Transduction Pathway VI. Cloning the GH... VII. Action of the... VIII. Peptidomimetic GH... IX. Regulation of Pulsatile... X. Concluding Comments References

 
A. Animal models
Experiments with cultured pituitary cells demonstrated that in vitro desensitization to the peptidomimetic GH secretagogues occurred rapidly. However, animal studies with a compound having a relatively long half-life suggested that the effects on GH release were sustained (43). In guinea pigs, when GH was monitored at 10-min intervals during a constant infusion of L-692,585, a sustained amplification of episodic GH release was observed, similar to that seen with GHRP-6 (7, 87). Intriguingly, L-692,585 initiated GH pulsatility, suggesting that this class of compounds has the capacity to reset the ultradian rhythm of GH release (87).

The action of the peptidomimetic GH secretagogues, L-692,429 and L-692,585 (5–100 µg/kg), on GH dynamics was evaluated in a crossover design with four male and four female beagle dogs (110, 111). Peak GH levels were recorded within 5–15 min after dosing, and L-692,585, consistent with its in vitro potency, was effective at approximately 1/20th of the dose compared with L-692,429 (111). No sex-related difference was detected and, apart from small transient increases in ACTH and cortisol, both compounds were very selective at stimulating GH release (89, 103). In a more extensive chronic study in which L-692,585 was given once daily for up to 14 days, desensitization to repeated dosing was not observed. Increases in IGF-I were evident 6 h after dosing, but the increase was transient and IGF-I levels returned to baseline within 24 h. PRL, insulin, and T₄ levels were unaltered over the course of the study (111).

Pharmacokinetic oral bioavailability measurements had established that MK-0677 was a viable candidate for once-daily oral dosing (50, 52). To determine whether the effects on GH release could be sustained during repeated oral treatment, dogs were treated with MK-0677 (1 mg/kg) for 4 days. On days 1 and 4, blood was collected at 15-min intervals for 8 h and assayed for GH. MK-0677 treatment resulted in sustained amplification of the pulsatile profile of GH. However, because the magnitude of GH release caused by MK-0677 decreased markedly by the fourth day (59), a second study of 14 days duration was implemented to determine whether complete desensitization would occur during more prolonged treatment. Although reduced amplitude of the GH response to MK-0677 was again observed by day 4, treatment for up to 14 days did not result in further attenuation. A sustained increase in serum IGF-I levels accompanied the reduced amplitude of GH release (54). Interestingly, a similar study with the shorter acting secretagogue L-692,585 gave neither a reduced GH response nor a sustained increase in IGF-I levels during repeated daily treatment (111). When dogs were dosed with MK-0677 on alternate days for up to 9 days, reduced GH responses to repeated dosing was not evident. This particular dosing regimen allowed IGF-I to return to basal levels before dogs received the next dose of MK-0677. Based on these observations it was speculated that tachyphylaxis might be associated with increases in IGF-I rather than desensitization of the GHS-R. To address this possibility, dogs were treated with MK-0677 on day 1; on days 2 and 3 the dogs were dosed with porcine GH; and on day 4 the dogs were treated with MK-0677. At the time of treatment on day 4, GH concentrations had returned to basal levels but IGF-I was still elevated. The GH response to MK-0677 on day 4 was markedly attenuated compared with day 1. Thus reduced responsiveness to chronic MK-0677 treatment is not necessarily due to desensitization of the GHS-R but might be explained by reduced responsiveness of the GH/GHRH axis caused by a sustained increase in IGF-I (54). Perhaps chronically, IGF-I feeds back on arcuate neurons to reduce the expression of both GHRH and the GHS-R. The demonstration that the magnitude of the stimulatory effect of MK-0677 on GH release is limited by negative feedback is very attractive clinically because it prevents hyperstimulation of the GH/IGF-I axis.

Like GHRP-6, L-692,429 and L-692,585 also cause transient increases in ACTH and cortisol (110, 111). The effect on ACTH is probably mediated by action of the secretagogues on the hypothalamus, because in cultured pituitary cells the peptidomimetics do not significantly increase ACTH release. MK-0677 also increased cortisol levels when given acutely; however, during chronic administration the stimulatory effect on cortisol became insignificant (53, 54). When MK-0677 was given chronically to dogs on alternate days, just as observed with GH, the cortisol response was not attenuated (54). These results suggest that the GH and cortisol responses are linked and perhaps similarly attenuated during repeated daily treatment through an IGF-I-mediated pathway.

Interesting anabolic activities were reported for the Genentech compound G-7039. Body weight gain in 150-day-old female Sprague Dawley rats was significantly greater when G-7039 was administered by subcutaneous minipump twice daily for 14 days as compared with the body weight gain after subcutaneous minipump infusion of G-7039 for 14 days (58). The implication is that optimal growth requires intermittent administration of these secretagogues rather than continuous treatment. It remains to be tested whether this applies to species other than rats.

B. Clinical studies in humans
GH treatment may prove to be beneficial to a variety of subjects other than GH-deficient children. Most individuals over the age of 60 yr might be considered GH deficient according to young adult standards (112, 113, 114), and it has been suggested that this relative deficiency is responsible for decreases in bone and muscle mass and increased adiposity during aging (115). Since GH increases bone turnover, GH in combination with an inhibitor of bone resorption may have great benefit in severe osteoporosis. GH deficiency is associated with lipid profiles that favor the likelihood of atherosclerosis and mortality due to cardiovascular disease (116, 117, 118, 119). Depressed subjects present a significant decrease in 24-h GH production (120). Whether the decreased amplitude of the GH pulses reported in major depressive illness is due to changes in the activity of neurotransmitters or is related to GH itself remains to be established.

Perhaps the greatest potential for GH replacement is in the frail elderly population. Although pulsatile GH secretion declines during aging, rodent studies show that the pituitary gland continues to synthesize GH and remains responsive to GHRH (121, 122). The advantage of increasing GH in the elderly is suggested by the recent demonstration that in a population of healthy men over 60 yr old, once-daily GH treatment over a 3-month period increased lean body mass, muscle mass, and thigh strength measured by isokinetic dynamometry (123). Therefore, identification of a compound that rejuvenates the GH/IGF-I axis provides a way to optimally evaluate the potential clinical benefits of reversing GH deficiency in the musculoskeletally impaired elderly (59, 124). The GHRPs and the peptidomimetic GH secretagogues provide a more physiological approach to GH replacement; however, based on our understanding of the mechanisms involved, subjects must have an intact hypothalamic/pituitary axis and produce normal GH. Consequently, in certain instances, treatment with recombinant GH is the only option.

1. L-692,429.
A series of studies demonstrated that the GHRPs showed reproducible GH-releasing activity in humans (18, 125, 126, 127, 128, 129, 130, 131, 132). The first nonpeptide GH secretagogue discovered, L-692,429, was shown to be a mimetic of GHRP-6 (29). Based on small animal studies, L-692,429 had improved oral bioavailability (4%) compared with the GHRPs. Although its bioavailability was considered inadequate for a clinical development candidate, L-692,429 was selected to test the concept that this new class of compounds would stimulate GH release in humans. Its efficacy, tolerability, and selectivity in clinically relevant target groups were determined. Healthy normal volunteers and the elderly tolerated L-692,429 well. Dose-dependent increases in GH were observed that were accompanied by small, but significant, transient increases in cortisol (133, 134). Elderly subjects were somewhat less responsive than young adults as were subjects chronically treated with glucocorticoids (134, 135). However, this deficit was overcome by administering a 3-fold higher dose. L-692,429 also stimulated GH release in obese subjects (136).

Constant infusion of L-692,429 in healthy elderly adults enhanced pulsatile GH secretion similar to that observed with GHRP-6 (128, 137). Infusion of 0.05 -0.1 mg/kg·h for 12–24 h increased pulsatile GH concentrations in older adults (60–80 yr old) in a dose-dependent manner (137). The number of GH-secretory pulses and the half-life of GH disappearance were not markedly affected by L-692,429 infusion; rather, the pulse amplitude was increased. Remarkably, the profile and GH levels resulting from L-692,429 infusion into the elderly approximated that of young adults (137). These results indicated that the peptidomimetic GH secretagogues had excellent potential for treatment of GH deficiency in humans. Since constant infusion provided amplification of a physiological profile, a molecule having high oral bioavailability and appropriate half-life for once-daily treatment was sought.

2. MK-0677.
Based on studies in rodents and beagle dogs, MK-0677 was identified as having appropriate properties for a once-daily oral drug capable of sustaining amplification of pulsatile GH release (54, 59). Studies in humans showed MK-0677 to be well tolerated, and once-daily oral administration was sufficient to increase serum concentrations of both GH and IGF-I. Seventeen male and 15 female healthy elderly volunteers (64–81 yr old) were treated orally once daily with MK-0677 at doses of 2, 10, and 25 mg in a randomized, double-blind, placebo-controlled trial. Serum was collected every 20 min for 24 h at baseline and after each of two 14-day treatment periods the serum was analyzed for GH, cortisol, and PRL. The assessment of effects on pulsatile GH release was evaluated by three independent algorithms. MK-0677 increased GH concentrations dose dependently (138, 139). Serum IGF-I concentrations were also significantly increased at both the 10- and 25-mg doses while the 2-mg dose was ineffective. At the 25-mg dose the mean 24-h GH concentration increased by 97 ± 23% due to enhancement of preexisting pulsatile GH secretion. Similarly, in these elderly subjects, serum IGF-I levels increased from 141 ± 21 µg/liter into the normal range for young adults (219 ± 21 µg/liter). These increases were sustained for at least 28 days (265 ± 29 µg/liter). Most importantly, serum cortisol, diurnal cortisol pattern, and urinary free cortisol remained in the normal range and were all unchanged compared with the placebo group (138, 139). Also, thyroid hormones were not significantly affected by MK-0677. Small increases in PRL from pretreatment levels were observed, but the concentrations remained in the normal range. After 4 weeks, MK-0677 treatment was associated with a significant increase in fasting glucose (5.4 ± 0.3 to 6.8 ± 0.4 mmol/liter) and insulin concentrations. The increases in glucose were correlated with body mass index, suggesting that impaired glucose tolerance might be an issue in subjects with predisposing risk factors. However, it is likely that insulin sensitivity would improve over time coincident with anticipated improvements in body composition. Consistent with a GH increase, IGF-II and IGF binding protein-3 (IGFBP-3) increased, and IGFBP-1 and IGFBP-2 levels decreased. Figure 15A illustrates the sustained increases in pulsatile GH release observed in elderly men and women during repeated daily dosing with MK-0677. The GH and IGF-I levels attained are similar to those of healthy young adults. Cortisol levels (Fig. 15B) are unchanged, demonstrating the selectivity of this peptidomimetic GH secretagogue (139).

View larger version (29K): [in this window] [in a new window]   Figure 15. A, Rejuvenation of the GH axis in elderly men and women treated for 14 days once daily with MK-0677 showing a clear increase in amplitude of pulsatile GH release. Mean (±SE) serum GH concentrations after 2 weeks of treatment with placebo (; n = 10), 10 mg/day MK-0677 (•; n = 12) and 25 mg/day MK-0677 (; n = 10). Evening treatment time (between 2200 and 2300 h) is indicated by the arrow. B, Effect of oral 25 mg/day MK-0677 administered to 10 elderly men and women once daily for 14 days on serum cortisol. Serum cortisol at baseline () and on day 14 (•). Blood samples were collected every 20 min. [Reproduced with permission from I. M. Chapman et al.: J Clin Endocrinol Metab81:4249–4257, 1996 (139). © The Endocrine Society.]

	IX. Regulation of Pulsatile GH Release

Top Abstract I. Introduction II. Identification of... III. Molecular Design by... IV. Characterization of the... V. Signal Transduction Pathway VI. Cloning the GH... VII. Action of the... VIII. Peptidomimetic GH... IX. Regulation of Pulsatile... X. Concluding Comments References

The experimental evidence points to GH periodicity being self-entraining. For example, when GH is given exogenously at intervals of 3 h, approximately in phase with endogenous GH pulses, the exogenous and endogenous GH peaks become entrained (146). If the exogenous pulses are repeated more frequently (every 90 min), the regular endogenous pulsatility disappears (146). Entrainment of the 3-h pulses can be accomplished also by administering a GHRH analog (147) or L-692,585 instead of GH at 3-h intervals (7, 87). These regular episodic GH peaks at 3- to 3.5-h intervals are best explained by GH-induced feedback rather than by changes in environmental factors or ultradian rhythms, because in male rats, irrespective of light-dark cycle, the time between GH peaks is unaffected (148). GH does not inhibit GH release from primary cultures of rat pituitary cells (149), and introduction of GH into the third ventricle inhibits endogenous GH release (150); therefore, GH-negative feedback is probably mediated at the hypothalamic level, perhaps by retrograde transport through the portal vessels (151, 152). Persuasive evidence for hypothalamic GH-mediated negative feedback has been elegantly illustrated by showing that increased pulsatility follows administration of antisense GH receptor mRNA into the brain (153).

Pulsatile GH release appears to be related to reduced GHRH secretion and increased somatostatin secretion during GH troughs (154, 155, 156, 157, 158). Experiments using GHRH antibodies and GHRH antagonists show that GH pulses are dependent on the release of GHRH (159, 160). Hypothalamic/pituitary stalk disconnection results in loss of normal GH pulsatility in sheep (161), suggesting that pulsatility is controlled centrally by changes in GHRH, somatostatin, or some other hypothalamic factor(s). When GHRH is constantly infused into humans, pulsatile GH release is amplified and sustained. Thus, changes in GHRH alone do not necessarily explain GH pulsatility (128, 137, 162) and implicate an important role for somatostatin. Increases in somatostatin suppress endogenous GH release (146, 163, 164), whereas a decline in somatostatin facilitates the release of GHRH from the hypothalamus and GH release from somatotrophs (165, 166).

The strict adherence to a 3-h cycle of GH pulsatility is likely governed by the pharmacodynamics of GH on somatostatin tone. The rationalization that desensitization to somatostatin explains the 3-h cycle is untenable because continuous infusion of a somatostatin agonist, MK-678, prevents pulsatile GH secretion for at least 12 h (K. Cheng and R. G. Smith, unpublished observations). Studies demonstrating that endogenous GH pulsatility is sustained when somatostatin is neutralized by infusion of somatostatin antibodies (154) is not inconsistent with the notion of a central role for somatostatin since these antibodies may not be capable of readily accessing and thereby influencing the appropriate somatostatin neurons. Whether GH feeds back directly on somatostatin-containing neurons or whether the effect is indirectly mediated by release of another mediator, such as NPY, from arcuate neurons is currently being elucidated (167).

An important question is whether pulsatility is also controlled at the level of the pituitary gland. If so, by monitoring hypothalamic secretions, it should be possible to correlate peaks in GHRH and troughs in somatostatin with GH peaks in peripheral blood. When GHRH and somatostatin were monitored in the hypothalamic-pituitary portal vessels of conscious sheep and pigs to test this possibility, the results were far from satisfying. In one study the occurrence of a simultaneous GH and GHRH peak associated with a fall in somatostatin occurred 48% of the time. A GH peak in the absence of a change in GHRH but accompanied by a fall in somatostatin was observed 18.5% of the time (168). Another study gave a 62% match of GH and GHRH peaks (P < 0.02) without a significant correlation with somatostatin troughs (169). Recently, using a transorbital approach, Drisko et al. (170) measured somatostatin and GHRH in the portal vessels of pigs and observed a closer correlation of GH peaks with somatostatin troughs (63%) than with GHRH peaks (33%) (J. E. Drisko and G. J. Hickey, unpublished observations). The experimental findings are difficult to interpret conclusively for various reasons. Limited sensitivities of the GHRH and somatostatin assays do not allow quantification at 0.5–1 min sampling intervals. Ideally, such frequent sampling is important because perifusion experiments show that pituitary cells respond to somatostatin and GHRH within 1 min (27). The problem is exacerbated because the half-lives of these hypothalamic hormones are relatively short. Furthermore, the concentrations of GHRH and somatostatin in portal blood are very low, and based on in vitro generated GHRH and somatostatin dose-response curves for modulating GH release, small changes in GHRH and somatostatin concentrations are likely to cause more profound increases in GH. Therefore, because small dynamic changes in somatostatin and GHRH are difficult to accurately and reproducibly quantitate in portal blood, the precise roles of these hypothalamic hormones in controlling GH pulsatility at the level of the pituitary gland remain obscure.

B. The role of GHS-R
The sites of action of the peptidomimetic GH secretagogues are illustrated in Fig. 16. GHS-Rs have been identified in the pituitary and hypothalamus, and modification of electrical activity of arcuate neurons has been demonstrated. Since ligands for the GHS-R behave as functional antagonists of somatostatin and amplifiers of GHRH activity, it is predictable that the GHS-R ligands would amplify GH secretion in vivo. Less obvious is how they sustain GH pulsatility. Although sustained exposure to MK-0677, L-692,429, L-692,585, and GHRP-6 amplifies pulsatile GH release for at least 24 h (59, 87, 128, 137, 139), when pituitary cells are exposed to these secretagogues in vitro the cells become refractory to repeated stimulation within minutes (27, 171). The paradox between the in vivo and in vitro results must be explained by other factors that modulate GHS-R function in vivo.

View larger version (19K): [in this window] [in a new window]   Figure 16. Sites of action of the peptidomimetic GH secretagogues. The ligands for the GHS-R act directly on somatotrophs to cause GH secretion, amplify the affects of GHRH, and behave as functional antagonists of somatostatin. They also act in the arcuate nucleus to increase the activity of GHRH-containing neurons.

The in vitro studies described above suggest that, pharmacologically, GH pulsatility can be controlled at the level of the pituitary. However, there is considerable evidence that, physiologically, regulation is exerted at the level of GHRH neurons in the arcuate nucleus. Electrophysiology and c-fos activation studies on hypothalamic neurons (86, 88, 97) suggest that ligands for the GHS-R regulate pulsatility, at least in part, by inducing release of GHRH from arcuate neurons. Consistent with this prediction, in conscious sheep, GHRPs have been shown to increase GHRH and the frequency of GHRH pulsing in the hypothalamic-portal vessels without affecting somatostatin levels (98, 99). However, experiments with the peptidomimetics have given inconsistent results. Although we consistently observed elevations in GH in peripheral blood after treatment of sheep with L-692,585, only on rare occasions were we able to measure significant increases in GHRH in portal blood (59). Similar frustrating results were obtained in pigs and rhesus monkeys (J. E. Drisko, G. J. Hickey, H. G. Spies, and R. G. Smith, unpublished results). Although these findings might indicate the peptidomimetics act solely on the anterior pituitary gland, this conclusion does not explain why L-692,585 causes GH release and activates arcuate neurons at lower doses when injected into the third ventricle compared with the intravenous route (7, 86, 87). The lack of a perfect correlation between acute GHRH and GH release in response to L-692,585 might simply be explained by variability among animals in transporting L-692,585 across the blood-brain barrier. Clearly, rapid brain penetration is essential to synchronize hypothalamic and pituitary events. Indeed, after intravenous injection of L-692,585, recordings from electrodes placed on arcuate neurons of rats showed a 5- to 10-min lag period before excitation was detected (86, 88, 90). Therefore, while stimulation of GHRH release by the peptidomimetics has not been causally linked to increased GH secretion, reproducible, more intensive investigations are needed before a relationship can be ruled out.

Bowers (4) has argued that the increased activity of the GHS-R ligands in vivo compared with in vitro cannot simply be explained by stimulation of GHRH release and inhibition of somatostatin tone. These conclusions are based on observations that in the presence of maximum dosages of GHRH, or when animals were pretreated with somatostatin antibodies, synergism in response to GHRPs was still observed. Additional supporting evidence for Bowers’ view is that the magnitude of the increases in cAMP measured in rat pituitary gland after administration of GHRP and GHRH did not correlate with the level of GH release (4). With GHRP, the increase in cAMP was low and the increase in GH release was high, whereas GHRH provided a large increase in both cAMP and GH. Since cAMP increases GH secretion, if GHRP caused GHRH release, a closer correlation would have been anticipated. Another study revealed the concentrations of GHRP, GHRH, and GHRP + GHRH in vitro and in vivo that inhibited the activity of somatostatin on GH release by 50% (4). While the effects of GHRP and GHRH were found to attenuate the effects of somatostatin equally in vitro and in vivo, the combined effects of GHRP + GHRH were additive in vitro but synergistic in vivo. Bowers concluded from these studies that GHRPs cause the release of an unknown hypothalamic factor (U-factor) that results in a synergistic effect on GH release (4). However, administration of GHRPs and somatostatin exogenously to animals is complicated by how well these compounds penetrate the brain and activate specific hypothalamic neurons. Furthermore, the magnitude of GH release regulated by GHS-R ligands, GHRH, somatostatin, and cAMP is not a quantitative linear relationship. Hence, while Bowers’ hypothesis is very appealing, the need for a U-factor to explain the synergistic effects of the GHS-R ligands awaits confirmation.

Our working model is that, physiologically, GH self-entrains its pulsatility through the coupling of three biological oscillators: somatostatin, GHRH, and the natural ligand for the GHS-R. However, when a GHS-R ligand is administered exogenously, it acts dominantly to antagonize somatostatin and stimulate GHRH release. This results in synergy between GHRH and the GHS-R ligand to cause increased GH release from somatotrophs. GH then feeds back on the hypothalamus to entrain a new cycle by increasing somatostatin tone on GHRH-containing neurons, thus inhibiting GHRH and GH release. Hence, administration of a GHS-R ligand is capable of resetting the coupled oscillators.

It is tempting to speculate on the mechanism of the interplay between somatostatin and the GHS-R. As discussed above, somatostatin appears to prevent desensitization of the GHS-R. However, until somatostatin falls to a critical concentration, the GHS-R ligand cannot overcome the inhibitory effects of somatostatin on GH release because somatostatin receptors are present in excess over GHS-R (60). The inhibitory effects of somatostatin on GH release and maintenance of GHS-R in an active state probably occurs through key phosphorylation/dephosphorylation reactions since somatostatin causes translocation of a tyrosine phosphatase and activation of protein phosphatase-2A (178, 179, 180, 181). MK-0677 activates protein kinase C, perhaps causing tyrosine phosphorylation of a K⁺ channel, as described for the m1 muscarinic receptor (182). This would explain the observed depolarization of the somatotroph membrane and stimulation of GH release. The phosphorylated K⁺ channel is a potential substrate for the tyrosine phosphatase activated by somatostatin. Dephosphorylation of the channel would cause hyperpolarization and inhibit GH release. The serine/threonine phosphorylation sites on the GHS-R are likely involved in receptor desensitization, and we speculate that somatostatin-induced protein phosphatase-2A regulates the phosphorylated state of phosphorylated GHS-R. In spite of the fact that a proportion of phosphorylated GHS-Rs may be reactivated by somatostatin, GH release will still be tightly controlled because the somatotrophs remain hyperpolarized until somatostatin tone is reduced (16). Thus, GH could self-entrain its pulsatility indirectly through a somatostatin feedback loop that acts in a concentration-dependent manner on the GHS-R and K⁺ channels.

	X. Concluding Comments

Top Abstract I. Introduction II. Identification of... III. Molecular Design by... IV. Characterization of the... V. Signal Transduction Pathway VI. Cloning the GH... VII. Action of the... VIII. Peptidomimetic GH... IX. Regulation of Pulsatile... X. Concluding Comments References

 
In summary, a class of orally active small molecules have been described that are mimetics of the synthetic peptide GH secretagogue GHRP-6. The receptor for these molecules has been cloned and shown to be distinct from that of the GHRH receptor. This new receptor is G protein coupled and does not obviously fall into a known family of G protein-coupled receptors. However, the GHS-R is highly conserved across species, suggesting that an unidentified hormone for this receptor plays an important physiological role in the control of pulsatile GH release. The importance of the GHS-R pathway might be exemplified in GH-deficient children because the majority of children respond to ligands that bind to the GHS-R (125). Perhaps these children lack the natural ligand for the receptor. If this speculation is true, individuals lacking the GHS-R or its natural ligand would have growth-retarded phenotypes because of reduced amplitude of episodic GH release. Similarly, the attenuation of GH pulsatility during aging, which can be corrected by administration of MK-0677, is suggestive of reduced production of the endogenous ligand. Based on the localization of GHS-R expression in the brain, a decline in the ligand concentration might be associated with age-related deficits in cognitive function. Indeed, expression of the GHS-R in specific regions of the brain not implicated in GH release implies a much broader role for the natural ligand in human physiology.

	Acknowledgments

 
The authors greatly acknowledge the patience, good humor, and expert assistance of Ms. Laura Peterson in preparing this manuscript.

	Footnotes

 
Address reprint requests to: Roy G. Smith, Ph.D., Merck Research Laboratories, P.O. Box 2000, RY80K Rahway, New Jersey 07065.

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Top Abstract I. Introduction II. Identification of... III. Molecular Design by... IV. Characterization of the... V. Signal Transduction Pathway VI. Cloning the GH... VII. Action of the... VIII. Peptidomimetic GH... IX. Regulation of Pulsatile... X. Concluding Comments References

 

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				G.-L. Wang, X. Shi, E. Salisbury, Y. Sun, J. H. Albrecht, R. G. Smith, and N. A. Timchenko Growth Hormone Corrects Proliferation and Transcription of Phosphoenolpyruvate Carboxykinase in Livers of Old Mice via Elimination of CCAAT/Enhancer-binding Protein {alpha}-Brm Complex J. Biol. Chem., January 12, 2007; 282(2): 1468 - 1478. [Abstract] [Full Text] [PDF]

				J. O. L. Jorgensen A simple twist of science: the convoluted tale of ghrelin continues. J. Clin. Endocrinol. Metab., September 1, 2006; 91(9): 3279 - 3280. [Full Text] [PDF]

				A. Mano-Otagiri, T. Nemoto, A. Sekino, N. Yamauchi, Y. Shuto, H. Sugihara, S. Oikawa, and T. Shibasaki Growth Hormone-Releasing Hormone (GHRH) Neurons in the Arcuate Nucleus (Arc) of the Hypothalamus Are Decreased in Transgenic Rats Whose Expression of Ghrelin Receptor Is Attenuated: Evidence that Ghrelin Receptor Is Involved in the Up-Regulation of GHRH Expression in the Arc Endocrinology, September 1, 2006; 147(9): 4093 - 4103. [Abstract] [Full Text] [PDF]

				B. Holst, M. Lang, E. Brandt, A. Bach, A. Howard, T. M. Frimurer, A. Beck-Sickinger, and T. W. Schwartz Ghrelin Receptor Inverse Agonists: Identification of an Active Peptide Core and Its Interaction Epitopes on the Receptor Mol. Pharmacol., September 1, 2006; 70(3): 936 - 946. [Abstract] [Full Text] [PDF]

				H D. Falls, B. D Dayton, D. G Fry, C. A Ogiela, V. G Schaefer, S. Brodjian, R. M Reilly, C. A Collins, and W. Kaszubska Characterization of ghrelin receptor activity in a rat pituitary cell line RC-4B/C. J. Mol. Endocrinol., August 1, 2006; 37(1): 51 - 62. [Abstract] [Full Text] [PDF]

				H. Jiang, L. Betancourt, and R. G. Smith Ghrelin Amplifies Dopamine Signaling by Cross Talk Involving Formation of Growth Hormone Secretagogue Receptor/Dopamine Receptor Subtype 1 Heterodimers Mol. Endocrinol., August 1, 2006; 20(8): 1772 - 1785. [Abstract] [Full Text] [PDF]

				G. Segal-Lieberman, H. Rubinfeld, M. Glick, N. Kronfeld-Schor, and I. Shimon Melanin-concentrating hormone stimulates human growth hormone secretion: a novel effect of MCH on the hypothalamic-pituitary axis Am J Physiol Endocrinol Metab, May 1, 2006; 290(5): E982 - E988. [Abstract] [Full Text] [PDF]

				J. D. Veldhuis, J. N. Roemmich, E. J. Richmond, and C. Y. Bowers Somatotropic and Gonadotropic Axes Linkages in Infancy, Childhood, and the Puberty-Adult Transition Endocr. Rev., April 1, 2006; 27(2): 101 - 140. [Abstract] [Full Text] [PDF]

				J. V. Zhang, P.-G. Ren, O. Avsian-Kretchmer, C.-W. Luo, R. Rauch, C. Klein, and A. J. W. Hsueh Obestatin, a Peptide Encoded by the Ghrelin Gene, Opposes Ghrelin's Effects on Food Intake Science, November 11, 2005; 310(5750): 996 - 999. [Abstract] [Full Text] [PDF]

				N. Wettschureck and S. Offermanns Mammalian G Proteins and Their Cell Type Specific Functions Physiol Rev, October 1, 2005; 85(4): 1159 - 1204. [Abstract] [Full Text] [PDF]

				B. Holst, E. Brandt, A. Bach, A. Heding, and T. W. Schwartz Nonpeptide and Peptide Growth Hormone Secretagogues Act Both as Ghrelin Receptor Agonist and as Positive or Negative Allosteric Modulators of Ghrelin Signaling Mol. Endocrinol., September 1, 2005; 19(9): 2400 - 2411. [Abstract] [Full Text] [PDF]

				Y. Nishi, H. Hiejima, H. Mifune, T. Sato, K. Kangawa, and M. Kojima Developmental Changes in the Pattern of Ghrelin's Acyl Modification and the Levels of Acyl-Modified Ghrelins in Murine Stomach Endocrinology, June 1, 2005; 146(6): 2709 - 2715. [Abstract] [Full Text] [PDF]

				R. G. Smith Development of Growth Hormone Secretagogues Endocr. Rev., May 1, 2005; 26(3): 346 - 360. [Abstract] [Full Text] [PDF]

				J. M. Garcia, M. Garcia-Touza, R. A. Hijazi, G. Taffet, D. Epner, D. Mann, R. G. Smith, G. R. Cunningham, and M. Marcelli Active Ghrelin Levels and Active to Total Ghrelin Ratio in Cancer-Induced Cachexia J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 2920 - 2926. [Abstract] [Full Text] [PDF]

				M. Kojima and K. Kangawa Ghrelin: Structure and Function Physiol Rev, April 1, 2005; 85(2): 495 - 522. [Abstract] [Full Text] [PDF]

				R. G. Smith, L. Betancourt, and Y. Sun Molecular Endocrinology and Physiology of the Aging Central Nervous System Endocr. Rev., April 1, 2005; 26(2): 203 - 250. [Abstract] [Full Text] [PDF]

				N. Wettschureck, A. Moers, B. Wallenwein, A. F. Parlow, C. Maser-Gluth, and S. Offermanns Loss of Gq/11 Family G Proteins in the Nervous System Causes Pituitary Somatotroph Hypoplasia and Dwarfism in Mice Mol. Cell. Biol., March 1, 2005; 25(5): 1942 - 1948. [Abstract] [Full Text] [PDF]

				C. Gauna, F. M. Meyler, J. A. M. J. L. Janssen, P. J. D. Delhanty, T. Abribat, P. van Koetsveld, L. J. Hofland, F. Broglio, E. Ghigo, and A. J. van der Lely Administration of Acylated Ghrelin Reduces Insulin Sensitivity, Whereas the Combination of Acylated Plus Unacylated Ghrelin Strongly Improves Insulin Sensitivity J. Clin. Endocrinol. Metab., October 1, 2004; 89(10): 5035 - 5042. [Abstract] [Full Text] [PDF]

				A. J. van der Lely, M. Tschop, M. L. Heiman, and E. Ghigo Biological, Physiological, Pathophysiological, and Pharmacological Aspects of Ghrelin Endocr. Rev., June 1, 2004; 25(3): 426 - 457. [Abstract] [Full Text] [PDF]

				F. Broglio, C. Gottero, P. Van Koetsveld, F. Prodam, S. Destefanis, A. Benso, C. Gauna, L. Hofland, E. Arvat, A. J. van der Lely, et al. Acetylcholine Regulates Ghrelin Secretion in Humans J. Clin. Endocrinol. Metab., May 1, 2004; 89(5): 2429 - 2433. [Abstract] [Full Text] [PDF]

				Y. Sun, P. Wang, H. Zheng, and R. G. Smith Ghrelin stimulation of growth hormone release and appetite is mediated through the growth hormone secretagogue receptor PNAS, March 30, 2004; 101(13): 4679 - 4684. [Abstract] [Full Text] [PDF]

				A. INUI, A. ASAKAWA, C. Y. BOWERS, G. MANTOVANI, A. LAVIANO, M. M. MEGUID, and M. FUJIMIYA Ghrelin, appetite, and gastric motility: the emerging role of the stomach as an endocrine organ FASEB J, March 1, 2004; 18(3): 439 - 456. [Abstract] [Full Text] [PDF]

				J. P. Camina, M. C. Carreira, S. El Messari, C. Llorens-Cortes, R. G. Smith, and F. F. Casanueva Desensitization and Endocytosis Mechanisms of Ghrelin-Activated Growth Hormone Secretagogue Receptor 1a Endocrinology, February 1, 2004; 145(2): 930 - 940. [Abstract] [Full Text] [PDF]

				N. M. Thompson, D. A. S. Gill, R. Davies, N. Loveridge, P. A. Houston, I. C. A. F. Robinson, and T. Wells Ghrelin and Des-Octanoyl Ghrelin Promote Adipogenesis Directly in Vivo by a Mechanism Independent of the Type 1a Growth Hormone Secretagogue Receptor Endocrinology, January 1, 2004; 145(1): 234 - 242. [Abstract] [Full Text] [PDF]

				M. Iwase, H. Kanazawa, Y. Kato, T. Nishizawa, F. Somura, R. Ishiki, K. Nagata, K. Hashimoto, K. Takagi, H. Izawa, et al. Growth hormone-releasing peptide can improve left ventricular dysfunction and attenuate dilation in dilated cardiomyopathic hamsters Cardiovasc Res, January 1, 2004; 61(1): 30 - 38. [Abstract] [Full Text] [PDF]

				Y. Sun, S. Ahmed, and R. G. Smith Deletion of Ghrelin Impairs neither Growth nor Appetite Mol. Cell. Biol., November 15, 2003; 23(22): 7973 - 7981. [Abstract] [Full Text] [PDF]

				F. Tassone, F. Broglio, S. Destefanis, S. Rovere, A. Benso, C. Gottero, F. Prodam, R. Rossetto, C. Gauna, A. J. van der Lely, et al. Neuroendocrine and Metabolic Effects of Acute Ghrelin Administration in Human Obesity J. Clin. Endocrinol. Metab., November 1, 2003; 88(11): 5478 - 5483. [Abstract] [Full Text] [PDF]

				B. Holst, A. Cygankiewicz, T. H. Jensen, M. Ankersen, and T. W. Schwartz High Constitutive Signaling of the Ghrelin Receptor--Identification of a Potent Inverse Agonist Mol. Endocrinol., November 1, 2003; 17(11): 2201 - 2210. [Abstract] [Full Text] [PDF]

				J. E. Caminos, R. Nogueiras, M. Blanco, L. M. Seoane, S. Bravo, C. V. Alvarez, T. Garcia-Caballero, F. F. Casanueva, and C. Dieguez Cellular Distribution and Regulation of Ghrelin Messenger Ribonucleic Acid in the Rat Pituitary Gland Endocrinology, November 1, 2003; 144(11): 5089 - 5097. [Abstract] [Full Text] [PDF]

				F. Broglio, C. Gottero, A. Benso, F. Prodam, S. Destefanis, C. Gauna, M. Maccario, R. Deghenghi, A. J. van der Lely, and E. Ghigo Effects of Ghrelin on the Insulin and Glycemic Responses to Glucose, Arginine, or Free Fatty Acids Load in Humans J. Clin. Endocrinol. Metab., September 1, 2003; 88(9): 4268 - 4272. [Abstract] [Full Text] [PDF]

				K. Kawamura, N. Sato, J. Fukuda, H. Kodama, J. Kumagai, H. Tanikawa, A. Nakamura, Y. Honda, T. Sato, and T. Tanaka Ghrelin Inhibits the Development of Mouse Preimplantation Embryos in Vitro Endocrinology, June 1, 2003; 144(6): 2623 - 2633. [Abstract] [Full Text] [PDF]

				M.L. Barreiro, J.S. Suominen, F. Gaytan, L. Pinilla, L.K. Chopin, F.F. Casanueva, C. Dieguez, E. Aguilar, J. Toppari, and M. Tena-Sempere Developmental, Stage-Specific, and Hormonally Regulated Expression of Growth Hormone Secretagogue Receptor Messenger RNA in Rat Testis Biol Reprod, May 1, 2003; 68(5): 1631 - 1640. [Abstract] [Full Text] [PDF]

				F. Broglio, A. Benso, C. Castiglioni, C. Gottero, F. Prodam, S. Destefanis, C. Gauna, A. J. van der Lely, R. Deghenghi, M. Bo, et al. The Endocrine Response to Ghrelin as a Function of Gender in Humans in Young and Elderly Subjects J. Clin. Endocrinol. Metab., April 1, 2003; 88(4): 1537 - 1542. [Abstract] [Full Text] [PDF]

				J. E. Caminos, M. Tena-Sempere, F. Gaytan, J. E. Sanchez-Criado, M. L. Barreiro, R. Nogueiras, F. F. Casanueva, E. Aguilar, and C. Dieguez Expression of Ghrelin in the Cyclic and Pregnant Rat Ovary Endocrinology, April 1, 2003; 144(4): 1594 - 1602. [Abstract] [Full Text] [PDF]

				G. S. Tannenbaum, J. Epelbaum, and C. Y. Bowers Interrelationship between the Novel Peptide Ghrelin and Somatostatin/Growth Hormone-Releasing Hormone in Regulation of Pulsatile Growth Hormone Secretion Endocrinology, March 1, 2003; 144(3): 967 - 974. [Abstract] [Full Text] [PDF]

				F. Gaytan, M. L. Barreiro, L. K. Chopin, A. C. Herington, C. Morales, L. Pinilla, F. F. Casanueva, E. Aguilar, C. Dieguez, and M. Tena-Sempere Immunolocalization of Ghrelin and Its Functional Receptor, the Type 1a Growth Hormone Secretagogue Receptor, in the Cyclic Human Ovary J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 879 - 887. [Abstract] [Full Text] [PDF]

				L. M. Seoane, M. Lopez, S. Tovar, F. F. Casanueva, R. Senaris, and C. Dieguez Agouti-Related Peptide, Neuropeptide Y, and Somatostatin-Producing Neurons Are Targets for Ghrelin Actions in the Rat Hypothalamus Endocrinology, February 1, 2003; 144(2): 544 - 551. [Abstract] [Full Text] [PDF]

				H. Hosoda, M. Kojima, T. Mizushima, S. Shimizu, and K. Kangawa Structural Divergence of Human Ghrelin. IDENTIFICATION OF MULTIPLE GHRELIN-DERIVED MOLECULES PRODUCED BY POST-TRANSLATIONAL PROCESSING J. Biol. Chem., January 3, 2003; 278(1): 64 - 70. [Abstract] [Full Text] [PDF]

				H. Hosoda, M. Kojima, and K. Kangawa Ghrelin and the Regulation of Food Intake and Energy Balance Mol. Interv., December 1, 2002; 2(8): 494 - 503. [Abstract] [Full Text]

				S. R. Cunha and K. E. Mayo Ghrelin and Growth Hormone (GH) Secretagogues Potentiate GH-Releasing Hormone (GHRH)-Induced Cyclic Adenosine 3',5'-Monophosphate Production in Cells Expressing Transfected GHRH and GH Secretagogue Receptors Endocrinology, December 1, 2002; 143(12): 4570 - 4582. [Abstract] [Full Text] [PDF]

				F. Broglio, E. Arvat, A. Benso, C. Gottero, F. Prodam, S. Grottoli, M. Papotti, G. Muccioli, A. J. van der Lely, R. Deghenghi, et al. Endocrine Activities of Cortistatin-14 and Its Interaction with GHRH and Ghrelin in Humans J. Clin. Endocrinol. Metab., August 1, 2002; 87(8): 3783 - 3790. [Abstract] [Full Text] [PDF]

				V. Bodart, M. Febbraio, A. Demers, N. McNicoll, P. Pohankova, A. Perreault, T. Sejlitz, E. Escher, R.L. Silverstein, D. Lamontagne, et al. CD36 Mediates the Cardiovascular Action of Growth Hormone-Releasing Peptides in the Heart Circ. Res., May 3, 2002; 90(8): 844 - 849. [Abstract] [Full Text] [PDF]

				S. Ishizaki, T. Murase, Y. Sugimura, S. Kakiya, H. Yokoi, K. Tachikawa, H. Arima, Y. Miura, and Y. Oiso Role of Ghrelin in the Regulation of Vasopressin Release in Conscious Rats Endocrinology, May 1, 2002; 143(5): 1589 - 1593. [Abstract] [Full Text] [PDF]

				J. L. Ramirez, F. Gracia-Navarro, S. Garcia-Navarro, R. Torronteras, M. M. Malagon, and J. P. Castano Somatostatin Stimulates GH Secretion in Two Porcine Somatotrope Subpopulations through a cAMP-Dependent Pathway Endocrinology, March 1, 2002; 143(3): 889 - 897. [Abstract] [Full Text] [PDF]

				M. Volante, E. AllIa, P. Gugliotta, A. Funaro, F. Broglio, R. Deghenghi, G. Muccioli, E. Ghigo, and M. Papotti Expression of Ghrelin and of the GH Secretagogue Receptor by Pancreatic Islet Cells and Related Endocrine Tumors J. Clin. Endocrinol. Metab., March 1, 2002; 87(3): 1300 - 1308. [Abstract] [Full Text] [PDF]

				L. S. Farhy, M. Straume, M. L. Johnson, B. Kovatchev, and J. D. Veldhuis Unequal autonegative feedback by GH models the sexual dimorphism in GH secretory dynamics Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2002; 282(3): R753 - R764. [Abstract] [Full Text] [PDF]

				C. Ghe, P. Cassoni, F. Catapano, T. Marrocco, R. Deghenghi, E. Ghigo, G. Muccioli, and M. Papotti The Antiproliferative Effect of Synthetic Peptidyl GH Secretagogues in Human CALU-1 Lung Carcinoma Cells Endocrinology, February 1, 2002; 143(2): 484 - 491. [Abstract] [Full Text] [PDF]

				M. Baratta, R. Saleri, G. L. Mainardi, D. Valle, A. Giustina, and C. Tamanini Leptin Regulates GH Gene Expression and Secretion and Nitric Oxide Production in Pig Pituitary Cells Endocrinology, February 1, 2002; 143(2): 551 - 557. [Abstract] [Full Text] [PDF]

				C. B. Lawrence, A. C. Snape, F. M.-H. Baudoin, and S. M. Luckman Acute Central Ghrelin and GH Secretagogues Induce Feeding and Activate Brain Appetite Centers Endocrinology, January 1, 2002; 143(1): 155 - 162. [Abstract] [Full Text] [PDF]

				K. Wallenius, K. Sjogren, X.-D. Peng, S. Park, V. Wallenius, J.-L. Liu, M. Umaerus, H. Wennbo, O. Isaksson, L. Frohman, et al. Liver-Derived IGF-I Regulates GH Secretion at the Pituitary Level in Mice Endocrinology, November 1, 2001; 142(11): 4762 - 4770. [Abstract] [Full Text] [PDF]

				J. Kamegai, H. Tamura, T. Shimizu, S. Ishii, H. Sugihara, and I. Wakabayashi Chronic Central Infusion of Ghrelin Increases Hypothalamic Neuropeptide Y and Agouti-Related Protein mRNA Levels and Body Weight in Rats Diabetes, November 1, 2001; 50(11): 2438 - 2443. [Abstract] [Full Text] [PDF]

				M. Papotti, P. Cassoni, M. Volante, R. Deghenghi, G. Muccioli, and E. Ghigo Ghrelin-Producing Endocrine Tumors of the Stomach and Intestine J. Clin. Endocrinol. Metab., October 1, 2001; 86(10): 5052 - 5059. [Abstract] [Full Text] [PDF]

				T. L. Horvath, S. Diano, P. Sotonyi, M. Heiman, and M. Tschop Minireview: Ghrelin and the Regulation of Energy Balance--A Hypothalamic Perspective Endocrinology, October 1, 2001; 142(10): 4163 - 4169. [Abstract] [Full Text] [PDF]

				A. Garcia, C. V. Alvarez, R. G. Smith, and C. Dieguez Regulation of PIT-1 Expression By Ghrelin and GHRP-6 Through the GH Secretagogue Receptor Mol. Endocrinol., September 1, 2001; 15(9): 1484 - 1495. [Abstract] [Full Text] [PDF]

				J. L. Clasey, A. Weltman, J. Patrie, J. Y. Weltman, S. Pezzoli, C. Bouchard, M. O. Thorner, and M. L. Hartman Abdominal Visceral Fat and Fasting Insulin Are Important Predictors of 24-Hour GH Release Independent of Age, Gender, and Other Physiological Factors J. Clin. Endocrinol. Metab., August 1, 2001; 86(8): 3845 - 3852. [Abstract] [Full Text] [PDF]

				M. J. Bray, T. M. Vick, N. Shah, S. M. Anderson, L. W. Rice, A. Iranmanesh, W. S. Evans, and J. D. Veldhuis Short-Term Estradiol Replacement in Postmenopausal Women Selectively Mutes Somatostatin's Dose-Dependent Inhibition of Fasting Growth Hormone Secretion J. Clin. Endocrinol. Metab., July 1, 2001; 86(7): 3143 - 3149. [Abstract] [Full Text] [PDF]

				L. S. Farhy, M. Straume, M. L. Johnson, B. Kovatchev, and J. D. Veldhuis A construct of interactive feedback control of the GH axis in the male Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2001; 281(1): R38 - R51. [Abstract] [Full Text] [PDF]

				U. Pagotto, G. Marsicano, F. Fezza, M. Theodoropoulou, Y. Grubler, J. Stalla, T. Arzberger, A. Milone, M. Losa, V. Di Marzo, et al. Normal Human Pituitary Gland and Pituitary Adenomas Express Cannabinoid Receptor Type 1 and Synthesize Endogenous Cannabinoids: First Evidence for a Direct Role of Cannabinoids on Hormone Modulation at the Human Pituitary Level J. Clin. Endocrinol. Metab., June 1, 2001; 86(6): 2687 - 2696. [Abstract] [Full Text] [PDF]

				N. Nagaya, M. Kojima, M. Uematsu, M. Yamagishi, H. Hosoda, H. Oya, Y. Hayashi, and K. Kangawa Hemodynamic and hormonal effects of human ghrelin in healthy volunteers Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2001; 280(5): R1483 - R1487. [Abstract] [Full Text] [PDF]

				P. Cassoni, M. Papotti, C. Ghè, F. Catapano, A. Sapino, A. Graziani, R. Deghenghi, T. Reissmann, E. Ghigo, and G. Muccioli Identification, Characterization, and Biological Activity of Specific Receptors for Natural (Ghrelin) and Synthetic Growth Hormone Secretagogues and Analogs in Human Breast Carcinomas and Cell Lines J. Clin. Endocrinol. Metab., April 1, 2001; 86(4): 1738 - 1745. [Abstract] [Full Text]

				E. Arvat, M. Maccario, L. Di Vito, F. Broglio, A. Benso, C. Gottero, M. Papotti, G. Muccioli, C. Dieguez, F. F. Casanueva, et al. Endocrine Activities of Ghrelin, a Natural Growth Hormone Secretagogue (GHS), in Humans: Comparison and Interactions with Hexarelin, a Nonnatural Peptidyl GHS, and GH-Releasing Hormone J. Clin. Endocrinol. Metab., March 1, 2001; 86(3): 1169 - 1174. [Abstract] [Full Text]

				M. Korbonits, S. A. Bustin, M. Kojima, S. Jordan, E. F. Adams, D. G. Lowe, K. Kangawa, and A. B. Grossman The Expression of the Growth Hormone Secretagogue Receptor Ligand Ghrelin in Normal and Abnormal Human Pituitary and Other Neuroendocrine Tumors J. Clin. Endocrinol. Metab., February 1, 2001; 86(2): 881 - 887. [Abstract] [Full Text]

				R. Salvatori, X. Fan, J. A. Phillips III, R. Espigares-Martin, I. Martin de Lara, K. L. Freeman, L. Plotnick, A. Al-Ashwal, and M. A. Levine Three New Mutations in the Gene for the Growth Hormone (GH)-Releasing Hormone Receptor in Familial Isolated GH Deficiency Type IB J. Clin. Endocrinol. Metab., January 1, 2001; 86(1): 273 - 279. [Abstract] [Full Text]

				L. Gianotti, M. Maccario, F. Lanfranco, J. Ramunni, L. Di Vito, S. Grottoli, E. E. Muller, E. Ghigo, and E. Arvat Arginine Counteracts the Inhibitory Effect of Recombinant Human Insulin-Like Growth Factor I on the Somatotroph Responsiveness to Growth Hormone-Releasing Hormone in Humans J. Clin. Endocrinol. Metab., October 1, 2000; 85(10): 3604 - 3608. [Abstract] [Full Text]

				M. Papotti, C. Ghè, P. Cassoni, F. Catapano, R. Deghenghi, E. Ghigo, and G. Muccioli Growth Hormone Secretagogue Binding Sites in Peripheral Human Tissues J. Clin. Endocrinol. Metab., October 1, 2000; 85(10): 3803 - 3807. [Abstract] [Full Text]

				R. Nass, J. Gilrain, S. Anderson, B. Gaylinn, A. Dalkin, R. Day, M. Peruggia, and M. O. Thorner High Plasma Growth Hormone (GH) Levels Inhibit Expression of GH Secretagogue Receptor Messenger Ribonucleic Acid Levels in the Rat Pituitary Endocrinology, June 1, 2000; 141(6): 2084 - 2089. [Abstract] [Full Text] [PDF]

				M. Roumi, S. Marleau, P. du Souich, T. Maggi, R. Deghenghi, and H. Ong Kinetics and Disposition of Hexarelin, A Peptidic Growth Hormone Secretagogue, in Rats Drug Metab. Dispos., January 1, 2000; 28(1): 44 - 50. [Abstract] [Full Text]

				O. C. Palyha, S. D. Feighner, C. P. Tan, K. K. McKee, D. L. Hreniuk, Y.-D. Gao, K. D. Schleim, L. Yang, G. J. Morriello, R. Nargund, et al. Ligand Activation Domain of Human Orphan Growth Hormone (GH) Secretagogue Receptor (GHS-R) Conserved from Pufferfish to Humans Mol. Endocrinol., January 1, 2000; 14(1): 160 - 169. [Abstract] [Full Text]

				V. Bodart, J. F. Bouchard, N. McNicoll, E. Escher, P. Carriere, E. Ghigo, T. Sejlitz, M. G. Sirois, D. Lamontagne, and H. Ong Identification and Characterization of a New Growth Hormone-Releasing Peptide Receptor in the Heart Circ. Res., October 29, 1999; 85(9): 796 - 802. [Abstract] [Full Text] [PDF]

				S. D. Feighner, C. P. Tan, K. K. McKee, O. C. Palyha, D. L. Hreniuk, S. Pong, C. P. Austin, D. Figueroa, D. MacNeil, M. A. Cascieri, et al. Receptor for Motilin Identified in the Human Gastrointestinal System Science, June 25, 1999; 284(5423): 2184 - 2188. [Abstract] [Full Text]

				J. L. Ramírez, J. P. Castaño, R. Torronteras, A. J. Martínez-Fuentes, L. S. Frawley, S. García-Navarro, and F. Gracia-Navarro Growth Hormone (GH)-Releasing Factor Differentially Activates Cyclic Adenosine 3',5'-Monophosphate- and Inositol Phosphate-Dependent Pathways to Stimulate GH Release in Two Porcine Somatotrope Subpopulations Endocrinology, April 1, 1999; 140(4): 1752 - 1759. [Abstract] [Full Text]

				E. E. Muller, V. Locatelli, and D. Cocchi Neuroendocrine Control of Growth Hormone Secretion Physiol Rev, April 1, 1999; 79(2): 511 - 607. [Abstract] [Full Text] [PDF]

				J. Mendlewicz, P. Linkowski, M. Kerkhofs, R. Leproult, G. Copinschi, and E. Van Cauter Genetic Control of 24-Hour Growth Hormone Secretion in Man: A Twin Study J. Clin. Endocrinol. Metab., March 1, 1999; 84(3): 856 - 862. [Abstract] [Full Text]

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