Gonadotropin-releasing hormone (GnRH) analogs: relationship between their structure, proteolytic inactivation and pharmacokinetics in rats

There are two types of superactive agonists of gonadotropin-releasing hormone (GnRHa-I: (D-amino acid)6-GnRH and GnRHa-II: (D-amino acid)6-(desGly)10-GnRH- ethylamide) the high hormonal activity of which is understood to be due to their higher receptor affinity and their higher proteolytic stability as compared with the native GnRH sequence. Using the soluble fractions of various rat tissues in studies on the inactivation of GnRH peptides, we confirmed the higher proteolytic resistance of GnRHa-II, but not of D-Phe6-GnRH (GnRHa-I) and of another analog, D-Trp3-D-Phe6-GnRH, as compared with GnRH. The exact behaviour of the peptides during degradation was found to be dependent on the peptide concentrations used, showing the importance of using conditions as near to the physiological ones a possible. Towards the membrane fractions, however, the order of degradability was found to be GnRH much greater than D-Phe6-GnRH much greater than D-Trp3-D-Phe6-GnRH. The pharmacokinetic consequences of the different proteolytic degradabilities of the GnRH peptides, observed in rats, were a moderate increase in the biological half-life of D-Phe6-GnRH by 2.5-fold, as compared with GnRH, and a small increase in half-life of D-Trp3-D-Phe6-GnRH by 1.4-fold when compared with D-Phe6-GnRH. Whereas no intact GnRH was recovered in rat urine, small amounts of D-Phe6-GnRH (about 1% of dose) and high amounts of D-Trp3-D-Phe6-GnRH (25.5%) were excreted into urine. Combining the biochemical and pharmacokinetic data, it is concluded that proteolytic stability of GnRH analogs in pharmacological terms means stability towards membrane enzymes (pharmacologically-related stability) and that designing analogs with further increased proteolytic stability will be of only limited consequences with respect to their biological half-lives, the glomerular filtration rate of the kidney becoming the determining factor in the peptide clearance.     

A peptidomics strategy to elucidate the proteolytic pathways that inactivate peptide hormones

Proteolysis plays a key role in regulating the levels and activity of peptide hormones. Characterization of the proteolytic pathways that cleave peptide hormones is of basic interest and can, in some cases, spur the development of novel therapeutics. The lack, however, of an efficient approach to identify endogenous fragments of peptide hormones has hindered the elucidation of these proteolytic pathways. Here, we apply a mass spectrometry (MS) based peptidomics approach to characterize the intestinal fragments of peptide histidine isoleucine (PHI), a hormone that promotes glucose-stimulated insulin secretion (GSIS). Our approach reveals a proteolytic pathway in the intestine that truncates PHI at its C-terminus to produce a PHI fragment that is inactive in a GSIS assay, a result that provides a potential mechanism of PHI regulation in vivo. Differences between these in vivo peptidomics studies and in vitro lysate experiments, which showed N- and C-terminal processing of PHI, underscore the effectiveness of this approach to discover physiologically relevant proteolytic pathways. Moreover, integrating this peptidomics approach with bioassays (i.e., GSIS) provides a general strategy to reveal proteolytic pathways that may regulate the activity of peptide hormones.     

Combined expression of different hormone genes in single cells of normal rat and mouse pituitary

Cells displaying combined expression of different pituitary hormone genes (further referred to as 'multi-hormone mRNA cells') were identified in normal rat and mouse pituitary by single cell RT-PCR. These cells do not seem to produce or store all the respective hormones the mRNAs encode for. The cells are already developed at day 16 of embryonic life (E16) in the mouse. Different peptides, such as gamma3-melanocyte-stimulating hormone (gamma3-MSH) and gonadotropin-releasing hormone (GnRH), affect different subsets of these cells. In culture, estrogen and GnRH increase the number of 'multi-hormone mRNA cells' that contain prolactin (PRL) mRNA or mRNA of the alpha-subunit of the glycoprotein hormones (alpha-GSU) but not the number of 'multi-hormone mRNA cells' not containing PRL or alpha-GSU mRNA. 'Multi-hormone mRNA cells' may function as 'reserve cells' in which a particular hormone mRNA may be translated under a particular physiological condition demanding a rapid increase of that hormone.     

Interactions of gonadotropin-releasing hormone (GnRH) and gonadotropin-inhibitory hormone (GnIH) in birds and mammals

Gonadotropin-releasing hormone (GnRH) regulates secretion of both of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone. Thus, it is a key hormone for vertebrate reproduction. GnRH was considered to be unusual among hypothalamic neuropeptides in that it appeared to have no direct antagonist, although some neurochemicals and peripheral hormones (opiates, GABA, gonadal steroids, inhibin) can modulate gonadotropin release to a degree. Five years ago, a vertebrate hypothalamic neuropeptide that inhibited pituitary gonadotropin release in a dose-dependent manner was discovered in quail by Tsutsui et al. (2000. Biochem Biophys Res Commun 275:661-667). We now know that this inhibitory peptide, named gonadotropin-inhibitory hormone, or GnIH, is a regulator of gonadotropin release in vitro and in vivo. Its discovery has opened the door to an entirely new line of research within the realm of reproductive biology. In our collaborative studies, we have begun to elucidate the manner in which GnIH interacts with GnRH to time release of gonadotropins and thus time reproductive activity in birds and mammals. This paper reviews the distribution of GnIH in songbirds relative to GnRHs, and our findings on its modes of action in vitro and in vivo, based on laboratory and field studies. These data are simultaneously compared with our findings in mammals, highlighting how the use of different model species within different vertebrate classes can be a useful approach to identify the conserved actions of this novel neuropeptide, along with its potential importance to vertebrate reproduction.     

Early pituitary follicle stimulating hormone response to gonadotrophin releasing hormone in ewes

The object of our experiments was to characterize the response of plasma follicle stimulating hormone (FSH) within minutes of an i.v. injection of high or low doses of gonadotrophin releasing hormone (GnRH), especially in relation to contemporary changes in luteinizing hormone (LH) concentrations. In the deep anoestrous period (June), three intact ewes and two ovariectomized ewes were injected with 1 mug synthetic GnRH followed 2 h later by a second identical injection. A week later, the same regimen was repeated with the same sheep but with 50 mug GnRH after an interval of 5 h 20 min. Blood samples were collected every 15 sec for 15 min after each injection (early release), then at longer intervals (main release) till the next treatment, followed by sampling for a further 6-h period after the second treatment. FSH was released as soon as the second minute after GnRH injection in all ewes. The mean pituitary FSH response, during this early release, in intact and ovariectomized ewes was similar after either 1 or 50 mug GnRH. However, the main release was less pronounced in the ovariectomized sheep and was not stimulated after the second treatment in all sheep. Three other ewes were injected with 40 mug GnRH and sampled every 15 sec for seven, 6-min periods during the period of release to compare FSH and LH secretion. The profiles reflected a similarity in sensitivity and responsiveness to GnRH, especially soon after GnRH injection. Increases in both hormones were formed by several grouped associated spikes. It is suggested that a readily releasable pool of FSH exists in the ewe. There are probably differences in the mechanisms of synthesis and/or release between pituitary FSH and LH.     

Three forms of gonadotropin-releasing hormone,including a novel form,in a basal salmonid,Coregonus clupeaformis

Multiple forms of GnRH within individual brains may have different functions. However, some vertebrates such as salmonids continue to reproduce even though they have lost or do not express 1 of the 3 forms of GnRH found in most other teleosts. We examined a basal salmonid, lake whitefish, to determine the mechanism by which a reduction in the number of GnRH forms occurs. We identified for the first time 3 distinct GnRHs in a salmonid. One form is novel and is designated whitefish GnRH. The primary structure is pGlu-His-Trp-Ser-Tyr-Gly-Met-Asn-Pro-Gly-NH(2). HPLC and RIA were used for purification followed by Edman degradation for sequence determination. Mass spectroscopy was used to confirm the sequence and amidation of the peptide. The other 2 forms, salmon GnRH and chicken GnRH-II, are identical to the 2 forms found in salmon, which evolved later than whitefish. Synthetic whitefish GnRH is biologically active, as it increased mRNA expression of growth hormone and the alpha-subunit for LH and thyroid-stimulating hormone in dispersed fish pituitary cells. Our data support the hypothesis that the ancestral salmonid had a third GnRH form when the genome doubled (tetraploidization), but the third form was lost later in some salmonids due to chromosomal rearrangements. We suggest that the salmon GnRH form compensated for the loss of the third form.     

Design and synthesis of a gonadotropin-releasing hormone (GnRH) analogue, [Tyr(OMe)5,d-Glu6,Aze9]GnRH: Receptor binding, gonadotropin release and ovulation studies

Summary Gonadotropin-releasing hormone (GnRH) stimulates the release and synthesis of gonadotropin hormones (GtH) and is the key regulator of reproduction. The present study was carried out to design a potent GnRH analogue containing Tyr(OMe) at position 5 and ad-amino acid at position 6. This was based on a previous study in which [Tyr(OMe)⁵]GnRH was shown to have reduced potency compared to GnRH. A novel GnRH peptide containing Tyr(OMe)⁵ andd-Glu⁶ in combination with other substitutions at positions 9 and 10 was synthesized in the present study and tested for binding to the rat pituitary as well as potency in terms of gonadotropin (GtH) release in the goldfish pituitary and ovulation in sea bass. The results demonstrate that the replacement of the glycine residue at position 6 with ad-Glu in combination with the substitution of proline at position 9 with azetidine (Aze) increased the binding and biological activity of [Tyr(OMe)⁵]GnRH. The observed increased potency is likely to be related to the improved resistance to degradation. The present findings may lead to the development of a more potent GnRH agonist for inducing ovulation in fish.     

Internalization and recycling of receptor-bound gonadotropin-releasing hormone agonist in pituitary gonadotropes

The fate of cell surface gonadotropin-releasing hormone (GnRH) receptors on pituitary cells was studied utilizing lysosomotropic agents and monensin. Labeling of pituitary cells with a photoreactive GnRH derivative, [azidobenzoyl-D-Lys6]GnRH, revealed a specific band of Mr = 60,000. When photoaffinity-labeled cells were exposed to trypsin immediately after completion of the binding, the radioactivity incorporated into the Mr = 60,000 band decreased, with a concomitant appearance of a proteolytic fragment (Mr = 45,000). This fragment reflects cell surface receptors. Following GnRH binding, the hormone-receptor complexes underwent internalization, partial degradation, and recycling. The process of hormone-receptor complex degradation was substantially prevented by lysosomotropic agents, such as chloroquine and methylamine, or the proton ionophore, monensin. Chloroquine and monensin, however, did not affect receptor recycling, since the tryptic fragment of Mr = 45,000 was evident after treatment with these agents. This suggests that recycling of GnRH receptors in gonadotropes occurs whether or not the internal environment is acidic. Based on these findings, we propose a model describing the intracellular pathway of GnRH receptors.     

Novel localization of NMDA receptors within neuroendocrine gonadotropin-releasing hormone terminals

About 1000 hypothalamic neurons synthesize and release gonadotropin-releasing hormone (GnRH), the master molecule of reproduction in all mammals. At the level of the median eminence at the base of the brain, where GnRH and other hypothalamic releasing hormones are secreted into the capillary system leading to the anterior pituitary gland, there is non-synaptic regulation of neurohormone release by a number of central neurotransmitters. For example, glutamate, the major excitatory amino acid in the brain, directly regulates GnRH release from nerve terminals via NMDA receptors (NMDARs). Moreover, the effects of glutamate action on GnRH secretion are potentiated by estrogens, and this relates to the physiologic control of ovulation by the hypothalamus. We sought to determine the ultrastructural relationship between GnRH neuroterminals and NMDARs, and this regulation by estradiol. Using immunofluorescent confocal microscopy, postembedding immunogold electron microscopy, fractionation, and Western blotting, we demonstrated: (i) GnRH is localized in large dense-core vesicles of neurosecretory profiles/terminals, (ii) the NMDAR1 subunit is found primarily on large dense-core vesicles of neurosecretory profiles/terminals, (iii) there is extensive colocalization of GnRH and NMDAR1 on the same vesicles, and (iv) estradiol modestly but significantly alters the distribution of NMDAR1 in GnRH neuroterminals by increasing expression of NMDAR1 on large dense-core vesicles. Western blots of fractionated median eminence support the presence of NMDAR1 in subcellular fractions containing large dense-core vesicles. These data are the first to show the presence of the NMDAR on neuroendocrine secretory vesicles, its co-expression with GnRH, and its regulation by estradiol. The results provide a novel anatomical site for the NMDAR and may represent a new mechanism for the regulation of GnRH release.     

Interaction of fluorescent gonadotropin-releasing hormone with receptors in cultured pituitary cells

A fluorescent derivative of the gonadotropin-releasing hormone (GnRH) agonist analog, [D-Lys6]GnRH, was synthesized for receptor studies and shown to be biologically active. The rhodamine-derivatized peptide (Rh-GnRH) retained 40% of the receptor binding activity of [D-Lys6]GnRH, and 50% of the luteinizing hormone-releasing activity assayed in cultured pituitary cells. The fluorescent analog was employed to visualize the distribution of GnRH receptors in cultured pituitary cells, using the technique of video-intensified fluorescence microscopy. The binding of Rh-GnRH was confined to the large gonadotrophs which comprised 15% of the cell population. The specificity of the binding was shown by the absence of significant fluorescence in the presence of a 100-fold excess of [D-Lys6]GnRH, or when Rh-GnRH was incubated with choriocarcinoma, neuroblastoma, or 3T3 cell lines devoid of GnRH receptors. The interaction of Rh-GnRH with living pituitary cells was characterized by an initial diffuse distribution, followed by the formation of polar aggregates that later appeared to be internalized. These observations emphasize the value of fluorescent derivatives of GnRH for elucidating the course of the interaction with specific receptors on pituitary gonadotrophs. The initial results indicate that GnRH-receptor complexes undergo aggregation during stimulation of luteinizing hormone release, and are later internalized for subsequent degradation and/ or intracellular actions.     

Gonadotropin-releasing hormone: GnRH receptor signaling in extrapituitary tissues

Gonadotropin-releasing hormone (GnRH) has historically been known as a pituitary hormone; however, in the past few years, interest has been raised in locally produced, extrapituitary GnRH. GnRH receptor (GnRHR) was found to be expressed in normal human reproductive tissues (e.g. breast, endometrium, ovary, and prostate) and tumors derived from these tissues. Numerous studies have provided evidence for a role of GnRH in cell proliferation. More recently, we and others have reported a novel role for GnRH in other aspects of tumor progression, such as metastasis and angiogenesis. The multiple actions of GnRH could be linked to the divergence of signaling pathways that are activated by GnRHR. Recent observations also demonstrate cross-talk between GnRHR and growth factor receptors. Intriguingly, the classical G(alphaq)-11-phospholipase C signal transduction pathway, known to function in pituitary gonadotropes, is not involved in GnRH actions at nonpituitary targets. Herein, we review the key findings on the role of GnRH in the control of tumor growth, progression, and dissemination. The emerging role of GnRHR in actin cytoskeleton remodeling (small Rho GTPases), expression and/or activity of adhesion molecules (integrins), proteolytic enzymes (matrix metalloproteinases) and angiogenic factors is explored. The signal transduction mechanisms of GnRHR in mediating these activities is described. Finally, we discuss how a common GnRHR may mediate different, even opposite, responses to GnRH in the same tissue/cell type and whether an additional receptor(s) for GnRH exists.     

Agonist-induced regulation of pituitary receptors for gonadotropin-releasing hormone. Dissociation of receptor recruitment from hormone release in cultured gonadotrophs

The regulation of receptors for gonadotropin-releasing hormone (GnRH) by the homologous decapeptide ligand was analyzed in cultured rat anterior pituitary cells. Assay of GnRH receptors in both intact and disrupted cells showed that GnRH binding to gonadotrophs was rapidly followed by dose-dependent loss of sites that was maximal within 1 h. This early loss of GnRH receptors was not dependent on protein synthesis, and was attributable to ligand-induced processing of the peptide binding sites. No loss of GnRH sites was observed after receptor occupancy by a GnRH antagonist, or after target cell activation by exposure to a depolarizing concentration of KCl to stimulate luteinizing hormone release. After their initial down-regulation, GnRH receptors returned to normal and subsequently increased in concentration after 6 h of incubation. The delayed phase of receptor up-regulation was prevented by treatment with cycloheximide or actinomycin D and was calcium-dependent, being induced by 50 mM KCl and by low concentrations of the calcium ionophore, A23187. Conversely, calcium antagonists such as verapamil and MgCl2 impaired the agonist-induced increase of GnRH receptor sites. These findings have demonstrated that pituitary GnRH receptors undergo two distinct phases of regulation after interaction with the homologous ligand. The initial phase of agonist-dependent receptor loss is followed by a postsecretory phase of receptor recruitment that is dependent on protein synthesis. The expression of GnRH receptors can be completely dissociated from gonadotropin secretion, indicating that fusion of luteinizing hormone secretory granules with the plasma membrane is not a major pathway for transport of GnRH receptors to the cell surface in cultured gonadotrophs. Such changes in cell surface GnRH receptors during activation by the peptide agonist are relevant to the alterations in gonadotroph sensitivity that occur in vivo during physiological regulation of the pituitary gland by GnRH.     

Gonadotropin-releasing hormone II: a multi-purpose neuropeptide

Close to 30 forms of gonadotropin releasing hormone (GnRH) and at least five GnRH receptors have been identified in a wide variety of vertebrates and some invertebrates. One form, now called GnRH II, has the broadest distribution and the most ancient and conserved phylogeny. The distribution of the neurons that produce this peptide are completely nonoverlapping with any other GnRH forms. Fibers that project from these neurons overlap with GnRH I cells and/or fibers in a few regions, but are primarily divergent. The musk shrew (Suncus murinus) continues to be the most tractable mammalian species to use for studies of the function of GnRH II. The brain of the musk shrew has two GnRH genes (I and II), two GnRH receptors (types-1 and -2), and two different behaviors can be influenced by central infusion of GnRH II, but not by GnRH I; receptivity and feeding. Here, we summarize research on the musk shrew relative to the behavioral functions of GnRH II. First, female musk shrews are continually sexually receptive by virtue of their lack of an ovarian and/or behavioral estrus cycle. This feature of their reproductive ecology may be related to their semi-tropical distribution and their breeding season is highly dependent on changes in the availability of food. When food is not abundant, females stop mating, but brief bouts of feeding reinstate reproductive behavior. Likewise, intake of food is related to GnRH II mRNA and peptide content in the brain; after mild food restriction both decline. When GnRH II is infused centrally, at times when its content is low, it can both enhance receptivity and inhibit food intake. Simultaneous administration of a type-1 antagonist does not change the effect of GnRH II and use of an analog (135-18) that is a specific GnRH II agonist as well as a type-1 antagonist has the same effect as the endogenous GnRH II peptide. We propose that GnRH II plays a critical role by orchestrating the coordination of reproduction with the availability of nutritional support for these activities. Humans are bombarded with copious nutritional opportunities and at present obesity is a larger threat to health in many parts of the world than is under nutrition. It is our hope that understanding neuropeptides such as GnRH II that regulate food intake can ultimately lead to products that may curb appetite and thus decrease obesity and related risks to health.     

Primary structure of two forms of gonadotropin-releasing hormone from brains of the American alligator (Alligator mississippiensis)

Two forms of gonadotropin-releasing hormone (GnRH) have been purified from brains of the American alligator, Alligator mississippiensis, using reverse-phase high-pressure liquid chromatography (HPLC). The concentration of total GnRH was 8.8 ng/g of frozen brain tissue or 21.1 ng per brain. The amino acid sequence of each form of GnRH was determined using automated Edman degradation. The presence of the N-terminal pGlu residue was established by digestion studies with bovine pyroglutamyl aminopeptidase and coelution with synthetic forms of the native peptide. The primary structure of alligator GnRH I is pGlu-His-Trp-Ser-Tyr-Gly-Leu-Gln-Pro-Gly-NH2 and alligator GnRH II is pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2.     

Gonadotropin-releasing hormone release by superfused hypothalami in response to norepinephrine

We have been studying the release of gonadotropin-releasing hormone (GnRH) from adult male rat medial basal hypothalamus (MBH) utilizing a continuous flow superfusion system. This model system allows for direct application of modifying substances into the superfusion chambers and for continuous collection of effluent for radioimmunoassay of GnRH levels. Gonadotropin-releasing hormone is rapidly released in response to specific chemical stimuli. As demonstrated by others, pulses of KCl or prostaglandin E2 (PGE2) result in sharp peaks of GnRH release followed by rapid return to baseline. Forty millimolar KCl increases [GnRH] 3- to 4-fold, consistent with a membrane-associated secretory process for GnRH release. A 50-micrograms bolus of PGE2 results in a 2-fold rise in GnRH. Norepinephrine stimulates the release of GnRH in a log-linear dose-dependent manner in the range of 10(-9) to 10(-5) M norepinephrine (NE). At 10(-11) M, NE does not increase GnRH release above baseline, whereas at 10(-9) M NE GnRH release is increased 2-fold. The alpha-receptor blocker phentolamine significantly inhibits the NE-induced rise in GnRH. Propranolol, a beta-adrenergic receptor blocker, does not inhibit the GnRH response to NE. This study demonstrates a direct, dose-dependent, alpha-mediated stimulatory effect of NE on GnRH release from superfused male rat MBH, and establishes the potential of this system for the investigation of the GnRH response to other aminergic agents and their extraneural modifiers, including steroid hormones.     

Tryptic digestion of peptides corresponding to modified fragments of human growth hormone-releasing hormone

The objective of this study was to examine the degradation of short peptides corresponding to modified fragments of human growth hormone-releasing hormone by trypsin. Six analogues of pentapeptide 9-13 of human growth hormone-releasing hormone containing homoarginine, ornithine, glutamic acid, glycine, leucine or phenylalanine residue in position 11, two analogues of hexapeptide 8-13 of human growth hormone-releasing hormone and two analogues of heptapeptide 7-13 of human growth hormone-releasing hormone containing homoarginine or glycine residue in position 11 were obtained. The peptides were subjected to digestion by trypsin and the course of reaction was monitored using HPLC. It was found that the rate of hydrolysis of the Lys(12)-Val(13) peptide bond depends on the amino-acid residue preceding Lys(12). The extension of the peptide chain towards the N-terminus by introduction of consecutive amino-acid residues corresponding to the human growth hormone-releasing hormone sequence accelerates the hydrolysis process. These results may be of assistance in designing new analogues of human growth hormone-releasing hormone, more resistant to the activity of proteolytic enzymes.     

Evolutionary aspects of gonadotropin-releasing hormone and its receptor

Summary 1. Gonadotropin-releasing hormone (GnRH) was originally isolated as a hypothalamic peptide hormone that regulates the reproductive system by stimulating the release of gonadotropins from the anterior pituitary. However, during evolution the peptide was subject to gene duplication and structural changes, and multiple molecular forms have evolved.2. Eight variants of GnRH are known, and at least two different forms are expressed in species from all vertebrate classes: chicken GnRH II and a second, unique, GnRH isoform.3. The peptide has been recruited during evolution for diverse regulatory functions: as a neurotransmitter in the central and sympathetic nervous systems, as a paracrine regulator in the gonads and placenta, and as an autocrine regulator in tumor cells.4. Evidence suggests that in most species the early-evolved and highly conserved chicken GnRH II has a neurotransmitter function, while the second form, which varies across classes, has a physiologic role in regulating gonadotropin release.5. We review here evolutionary aspects of the family of GnRH peptides and their receptors.