Investigations into the potential clinical applications of growth hormone (GH) has increased over the past 10 years, following the availability of human recombinant GH (hGH). Although GH replacement had proven to be effective and well-tolerated in GH-deficient children, its use was not widespread until the mid-1980s. Until then, supply had been limited because GH was obtained by extraction from pituitary glands of human cadavers. The purity of the extracted GH became an issue because of an association with outbreaks of Jacob-Creutzfeldt disease, a fatal neurodegenerative disorder. However, the manufacture of recombinant hGH in the 1980s prompted investigators to evaluate uses of GH beyond the treatment of GH-deficient children. As a result, investigators were encouraged to learn more about the basic mechanisms controlling the episodic nature of GH release and about the function of GH at the cellular level. In this introductory chapter, I first review the regulatory role of growth hormone releasing-hormone (GHRH), somatostatin (sst or SRIF), and the GH-secre-tagogue receptor (GHS-R), referring to relevant chapters in this volume. I then review the content of the remaining chapters. This will provide a brief summary of the current molecular understanding of the GH receptor and its potential role in the central nervous system (CNS) followed by an overview of the clinical aspects of GH deficiency, indications for its replacement, and associated benefits of direct and indirect GH replacement.
From: Human Growth Hormone: Research and Clinical Practice Edited by: R. G. Smith and M. O. Thorner © Humana Press Inc., Totowa, NJ
THE ROLES OF GHRH, SST, AND GHS-R IN REGULATING GH RELEASE
Like many hormones, GH is released episodically and is regulated by tightly controlled feedback pathways. This tightly controlled process provides a mechanism that has presumably been optimized biologically for hormone-receptor interactions resulting in activation, inactivation, and reactivation of signal transduction cascades. The frequency of GH pulses appears to be conserved in all mammalian species, occurring at approx 3-h intervals. How this frequency is regulated is a topic that continues to hold great fascination for scientists. Historically, GH secretion was considered to be regulated by a positive/negative feedback loop controlled by the two hypothalamic hormones, GHRH and sst. GHRH is released from arcuate neurons into the median eminence and transported through the portal vessels to the pituitary gland, where it stimulates GH release from somatotrophs. Negative feedback is thought to be mediated by GH stimulating the release of sst from hypothalamic neurons. sst acts to inhibit GH release from the pituitary gland.
The discovery of the GH-releasing peptides (GHRPs) by Bowers was a very important episode in defining the characteristics of the regulation of GH secretion, because the GHRPs do not act directly through the GHRH or sst receptors (Chapter 2). Based on Bowers' discovery, a series of nonpeptide mimetics were designed to allow optimization of oral bioavailability and other pharmacokinetic properties, resulting in the development of MK-0677 (Chapter 3). A single oral dose of MK-0677 amplified GH pulses for 24 h (1). To distinguish the GHRPs and their mimetics from the natural hormone GHRH, the synthetic molecules were termed "GH-secretagogues" (GHS).
Early studies had suggested that GHRPs and their mimetics elicited their effects on GH release through a pathway distinct from that of GHRH (2). Characterization of the receptor was frustrated by the very low abundance of binding sites in the pituitary gland. However, by incorporating 35S into the MK-0677 molecule, a high specific activity radioligand was synthesized (3). Using this ligand, Pong et al. showed that MK-0677 bound with high affinity (Kd = 200 pM) to the plasma membrane fraction of pituitary and hypothalamic tissues (2,4). The concentration of binding sites was remarkably low (2 fmole and 6 fmole/mg protein in pituitary and hypothalamic membranes, respectively). 35S-MK-0677 binding was competitively inhibited by L-692,429, MK-0677, and GHRPs, but not by GHRH or sst; thus, this new receptor is selective for this specific class of synthetic growth hormone secretagogues (GHS). High affinity binding was inhibited noncompetitively by GTP-y-S, suggesting that the receptor was G-protein coupled (2,4).
The identification of the first of the optimized drug candidates, MK-0677, led to the cloning and molecular characterization of a new orphan G-protein coupled receptor for the GH-secretagogues, GHS-R. Activation of the GHS-R by synthetic ligands initiates and amplifies pulsatile GH release in animals, including humans (5). This new receptor was cloned using a strategy that exploited the observation that ligands for this receptor activated IP3-induced release of intracellular Ca2+ (Chapter 4). Based on this property, the receptor was expression-cloned from a pituitary cDNA library using Xenopus oocytes (Chapter 4). The predicted amino-acid sequence of the receptor (GHS-R) was consistent with biochemical studies that predicted it belonged to the G-protein coupled receptor family. Based on knowledge derived from the structure of other G-protein coupled receptors, the X-ray structure of MK-0677 and energy calculations derived from nuclear magnetic resonance (NMR) studies with MK-0677, a three-dimensional model for the ligand-receptor complex was proposed (6,7). A series of experiments were designed to test the validity of the model. The orientation of the receptor in the cell membrane was examined using antibodies generated to peptide sequences that, according to the model, were in the extracellular and intracellular loops. Immunofluorescence studies on intact and permeablized cells expressing GHS-R confirmed the orientation of the extracellular and extracellular loops (6).
The binding pocket occupied by MK-0677 was characterized using selected site-directed mutagenesis based on a computer-generated, space-filling model (Fig. 1) for the receptor-ligand complex (7). The consequences of mutating each amino acid were evaluated by measuring the ability of different ligands to activate the mutated receptors (6). The model illustrated in Fig. 2 suggests that E124 in helix 3 serves as a counter-ion to the basic N in the MK-0677 side chain, and this was confirmed by site-directed mutagenesis. Docking the structurally distinct agonists L-692,585 and GHRP-6 into the pocket also supported this hypothesis (6).
In situ hybridization studies using selective nonoverlapping radiolabeled oligonucleotides showed that GHS-R was expressed in the pituitary gland and brain (8). In the rat pituitary gland, expression of GHS-R was confined to the anterior lobe (8). This observation was consistent with experiments showing that GHRP-6 and its mimetics selectively activate somatotrophs and somatomammotrophs, and that a fluorescently tagged analog of MK-0677 selectively binds to GH-producing cells (2,5). Based on a combination of RNAse protection assays and in situ hybridization to different tissues, it appears that GHS-R expression is confined to the anterior pituitary gland and CNS (8).
In the brain, the GHS-R is widely expressed (8). Intriguingly, although we anticipated expression in those nuclei that play a role in the control of GH release, the receptor is also expressed in areas of the brain that affect mood, cognition, memory, learning, feeding, and sleep. The GHS-R mRNA is expressed in multiple hypothalamic nuclei including anteroventral preoptic nucleus, anterior hypothalamic area, suprachiasmatic nucleus, lateroanterior hypothalamic nucleus, supraoptic nucleus, ventromedial hypothalamic nucleus, arcuate nucleus, paraventricular nucleus, and tuberomamillary nucleus (8). Expression is also found in the dentate gyrus of the hippocampal formation, the CA2 and CA3 areas of the hippocampus, the pars compacta of the substantia nigra, ventral tegmental area, dorsal and medial raphae nuclei, and Edinger-Westphal nucleus (8). The functional significance of expression of the GHS-R in areas of the brain other than those involved in GH release is critical for our understanding of the complete physiological significance of the GHS-R. Evidence for activation of this receptor in the CNS by GHRPs and mimetics is presented in Chapter 5.
Interaction of GHS-R, GHRH, and SST Hypothalamic Neurons
The key role of the hypothalamic hormone GHRH in stimulating GH release from the pituitary gland is well-established (9). However, because its biological half-life is short, a series of analogs have been synthesized in attempts to develop compounds with
increased potency and improved pharmacokinetics for use in the clinic (Chapter 6). An alternative clinical approach is to identify a compound that induces the release of GHRH from arcuate neurons in the hypothalamus. The localization of GHS-R in the arcuate nucleus implicated the GHS-R ligands as regulators of GHRH release. Intravenous administration of the ligands results in stimulation of c-fos and electrical activity in arcuate neurons that project to the median eminence (Chapter 5; ). In situ hybridization studies to monkey and rat brains showed that GHS-R is expressed in
arcuate neurons, suggesting that the GHS-R ligands stimulate these neurons directly (5). A recent study using immunohistological techniques showed that the GHS-R is expressed in GHRH neurons (11). Furthermore, studies in sheep have shown that GHRH is released into the portal vessels immediately following treatment with a GHS-R hexapeptide ligand (12). Collectively, these data are consistent with GHS-R ligands acting directly on arcuate neurons to stimulate the release of GHRH. Activation of these neurons by MK-0677 can be inhibited by sst or pretreatment with GH (13). These important studies link the action of the GHS-R ligands with the two known endogenous regulators of GH-release GHRH and somatostatin.
GH self-entrains the ultradian rhythm of episodic GH release (14,15). Presumably, the negative feedback phase is either mediated directly by GH, or indirectly by causing the release of sst. Ligand binding to GHS-R results in functional antagonism of sst by depolarization of target cell membranes (16). Hence, if sst is involved in sustaining the rhythm of GH pulsatility, it follows that the rhythm can be interrupted by ligands that activate the GHS-R. Indeed, this is precisely what is observed in animals. Administration of GHS-R ligands immediately stimulate GH release and, as a consequence, the GH ultradian rhythm is reset (16,17). These observations support the suggestion that sst plays a critical role in the control of the pulse frequency of GH release (18,19-25).
It is reasonable to speculate that feedback control is regulated through GHRH containing arcuate neurons. Negative feedback could be regulated directly by GH or indirectly by GH causing the release of sst from hypothalamic neurons. To determine whether sst is involved, we generated transgenic mice in which the somatostatin receptor subtype-2 (sstr2) gene was selectively inactivated. Of the five sst subtypes (Chapter 7), we selected sstr2 because this particular subtype had already been implicated in the regulation of GH release (26). Subsequent studies with more selective nonpeptide sstr2 agonists support these earlier studies (27,28). The sstr2 was inactivated by homologous recombination in mouse embryonic stem cells (13). Mice homozygous for the deleted sstr2 allele appeared normal and healthy and were indistinguishable in appearance from sstr2 intact animals. The central effects of GH and MK-0677 were evaluated using induction of Fos immunoactivity to monitor activation of hypothalamic neurons (13). In parallel, wildtype and sstr2 null mice were treated with GH or placebo 10 min prior to injection with MK-0677 or vehicle. Thirty minutes later, the mice were sacrificed and brain sections isolated to measure Fos expression by immunohistochemistry. In both wild-type and sstr2, knockout mice treatment with GH caused expression of Fos in the periventricular nucleus, but not in the arcuate nucleus. In wild-type mice, pretreatment with mouse GH completely prevented MK-0677 activation of Fos in arcuate neurons. By contrast, in sstr2 null mice, pretreatment with GH failed to prevent activation of Fos in arcuate neurons. These results are consistent with GH-mediated negative feedback on GHRH neurons being regulated through sst and specifically sstr2. However, more work is needed before concluding that GH induced sst release is sufficient to explain entrainment of the 3 h pulses of GH.
The results summarized above with the sstr2 -/- and wild-type mice suggest that GH pulsatility is regulated at the hypothalamic level through GH receptors on periventricular neurons. Activation of these neurons results in the release of sst to suppress the activity of GHRH neurons in the arcuate nucleus. This interpretation is supported by electro-physiology experiments where stimulation of periventricular neurons results in inhibition of arcuate neurons (29,30). However, histological evidence for the projection of periventricular neurons to the arcuate nucleus is lacking at this time. An alternative explanation is that the negative feedback signal is mediated from periventricular to the arcuate nucleus indirectly through the basal lateral amygdala (BLA); electrical stimulation of BLA neurons also inhibits activity of arcuate neurons (29,30). The amplitude of GH pulses is apparently regulated through the GH receptor. When GH-receptor antisense RNA is administered to rats by intracerebral ventricular injection, the amplitude of GH pulses increases (31). Whether the effect is mediated indirectly by sst at the hypothalamic or pituitary level is not clear; however, the GH receptor apparently plays a pivotal role in the regulation.
Figure 3 illustrates a model of GH/sst mediated feedback regulation through arcuate neurons that express GHRH, GHS-R, and sstr2. Although not illustrated in Fig. 3, sst
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