Agonists of growth hormone-releasing hormone (GHRH) inhibit human experimental cancers in vivo by down-regulating receptors for GHRH.

The effects of the growth hormone-releasing hormone (GHRH) agonist MR409 on various human cancer cells were investigated. In H446 small cell lung cancer (SCLC) and HCC827 and H460 (non-SCLC) cells, MR409 promoted cell viability, reduced cell apoptosis, and induced the production of cellular cAMP in vitro. Western blot analyses showed that treatment of cancer cells with MR409 up-regulated the expression of cyclins D1 and D2 and cyclin-dependent kinases 4 and 6, down-regulated p27kip1, and significantly increased the expression of the pituitary-type GHRH receptor (pGHRH-R) and its splice-variant (SV1). Hence, in vitro MR409 exerts agonistic action on lung cancer cells in contrast to GHRH antagonists. However, in vivo, MR409 inhibited growth of lung cancers xenografted into nude mice. MR409 given s.c. at 5 µg/day for 4 to 8 weeks significantly suppressed growth of HCC827, H460, and H446 tumors by 48.2%, 48.7%, and 65.6%, respectively. This inhibition of tumor growth by MR409 was accompanied by the down-regulation of the expression of pGHRH-R and SV1 in the pituitary gland and tumors. Tumor inhibitory effects of MR409 in vivo were also observed in other human cancers, including gastric, pancreatic, urothelial, prostatic, mammary, and colorectal. This inhibition of tumor growth parallel to the down-regulation of GHRH-Rs is similar and comparable to the suppression of sex hormone-dependent cancers after the down-regulation of receptors for luteinizing hormone-releasing hormone (LHRH) by LHRH agonists. Further oncological investigations with GHRH agonists are needed to elucidate the underlying mechanisms.

Anti-tumor effects of peptide analogs targeting neuropeptide hormone receptors on mouse pheochromocytoma cells.

Pheochromocytoma is a rare but potentially lethal chromaffin cell tumor with currently no effective treatment. Peptide hormone receptors are frequently overexpressed on endocrine tumor cells and can be specifically targeted by various anti-tumor peptide analogs. The present study carried out on mouse pheochromocytoma cells (MPCs) and a more aggressive mouse tumor tissue-derived (MTT) cell line revealed that these cells are characterized by pronounced expression of the somatostatin receptor 2 (sst2), growth hormone-releasing hormone (GHRH) receptor and the luteinizing hormone-releasing hormone (LHRH) receptor. We further demonstrated significant anti-tumor effects mediated by cytotoxic somatostatin analogs, AN-162 and AN-238, by LHRH antagonist, Cetrorelix, by the cytotoxic LHRH analog, AN-152, and by recently developed GHRH antagonist, MIA-602, on MPC and for AN-152 and MIA-602 on MTT cells. Studies of novel anti-tumor compounds on these mouse cell lines serve as an important basis for mouse models of metastatic pheochromocytoma, which we are currently establishing.

Identification of two novel chicken GHRH receptor splice variants: implications for the roles of aspartate 56 in the receptor activation and direct ligand receptor interaction.

In this study, two novel GHRHR receptor splice variants, named chicken GHRHR-v1 (cGHRHR-v1) and cGHRHR-v2 respectively, were identified from chicken pituitary using RT-PCR assay. cGHRHR-v1 is characterized by an N-terminal deletion of 36 amino acid residues, including an aspartate at position 56 (Asp(56)) conserved in G protein-coupled receptor B-I subfamily. cGHRHR-v2 is a carboxyl-terminal truncated receptor variant with four putative transmembrane domains, which arose from alternative use of a splice acceptor site on intron 8. Using the pGL3-CRE-luciferase reporter system, the functionality of the two variants was examined in Chinese hamster ovary cells. cGHRHR-v1 was shown to be capable of transmitting signal upon agonist stimulation, but cGHRHR-v2 could not. Both GHRH and pituitary adenylate cyclase-activating peptide (PACAP) could activate cGHRHR-v1 at high dosages (GHRH >/=10(-8) M; PACAP >/=10(-6) M) and GHRH was much more potent than PACAP, suggesting that cGHRHR-v1 is a functional membrane-spanning receptor with an impairment in high-affinity ligand binding, rather than in receptor activation and ligand-binding specificity. This finding also points out the possibility that Asp(56) is not a critical determinant for receptor activation and direct ligand-receptor interaction. To substantiate this hypothesis, using site-directed mutagenesis, two receptor mutants with replacement of Asp(56) by Ala or Gly were generated. Expectedly, chicken or human GHRH could still activate both receptor mutants with reduced potencies (about 2- to 14-fold less potent). Taken together, our findings not only suggest that cGHRHR variants may play a role in controlling normal pituitary functions, but also support that Asp(56) is nonessential for receptor activation and direct ligand-receptor interaction.

Evidence that ghrelin is as potent as growth hormone (GH)-releasing hormone (GHRH) in releasing GH from primary pituitary cell cultures of a nonhuman primate (Papio anubis), acting through intracellular signaling pathways distinct from GHRH.

Ghrelin is more effective than GHRH in stimulating GH release in normal adult humans and monkeys in vivo. This robust effect of ghrelin has been largely attributed to regulation of hypothalamic input, whereas the direct effect of ghrelin on pituitary GH release has been minimized by the observation that ghrelin has only a modest impact on GH release, compared with GHRH, in cultures prepared from human fetal pituitaries and GH-producing adenomas, as well as pituitaries from nonprimate species. However, comparable in vitro studies have not been performed to test the direct effect of ghrelin on normal adult primates. Therefore, in the present study, primary pituitary cell cultures from female baboons (Papio anubis) were used as a model system to test the direct effects of ghrelin on primate somatotrope function. In this model, both ghrelin and GHRH increased GH release in a dose-dependent fashion. Surprisingly, at maximal concentrations (10 nM), both ghrelin and GHRH elicited a robust increase in GH release (4 and 24 h, respectively), and both up-regulated GH secretagogue-receptor and GHRH-receptor mRNA levels (24 h). Combined treatment with ghrelin and GHRH resulted in an additive effect on GH release, suggesting that distinct intracellular signaling pathways are activated by each ligand, as confirmed by the use of specific inhibitors of intracellular signaling. Together, these results present the first evidence that a direct effect of ghrelin on somatotrope function may play a major role in stimulating GH release in primates.

Lipopeptide antagonists of growth hormone-releasing hormone with improved antitumor activities.

Antagonists of growth hormone-releasing hormone (GHRH) synthesized previously inhibit proliferation of various human cancers, but derivatisation with fatty acids could enhance their clinical efficacy. We synthesized a series of antagonists of GHRH(1-29)NH(2) acylated at the N terminus with monocarboxylic or alpha,omega-dicarboxylic acids containing six to sixteen carbon atoms. These peptides are analogs of prior potent antagonists JV-1-36, JV-1-38, and JV-1-65 with phenylacetyl group at their N terminus. Several new analogs, including MZ-J-7-46 and MZ-J-7-30, more effectively inhibited GHRH-induced GH release in vitro in a superfused rat pituitary system than their parent compound JV-1-36 and had increased binding affinities to rat pituitary GHRH receptors, but they showed weaker inhibition of GH release in vivo than JV-1-36. All antagonists acylated with fatty acids containing 8-14 carbon atoms inhibited the proliferation of MiaPaCa-2 human pancreatic cancer cells in vitro better than JV-1-36 or JV-1-65. GHRH antagonist MZ-J-7-114 (5 mug/day) significantly suppressed the growth of PC-3 human androgen-independent prostate cancers xenografted into nude mice and reduced serum IGF-I levels, whereas antagonist JV-1-38 had no effect at the dose of 10 mug/day. GHRH antagonists including MZ-J-7-46 and MZ-J-7-114 acylated with octanoic acid and MZ-J-7-30 and MZ-J-7-110 acylated with 1,12-dodecanedicarboxylic acid represent relevant improvements over earlier antagonists. These and previous results suggest that this class of GHRH antagonists might be effective in the treatment of various cancers.

Changes in expression of hypothalamic releasing hormone receptors in individual rat anterior pituitary cells during maturation, puberty and senescence.

Anterior pituitary (AP) is formed by five different cell types, each one producing a different AP hormone whose secretion is regulated by a specific hypothalamic-releasing hormone (HRH). On the other hand, a significant number of AP cells express multiple HRH receptors (multiresponsive cells). Plastic changes in expression of HRH receptors in individual AP cells are involved in critical endocrine events. Here we have characterized the changes in functional responses to CRH, LHRH, TRH, and GHRH in individual AP cells throughout the whole life span of the rat. To this end, calcium responses to the HRHs were followed by single-cell imaging in freshly dispersed AP cells prepared from rats of different ages (0-540 postnatal days). Three different cell pools were identified: 1) monoresponsive cells, holding a single class of HRH receptor; 2) multiresponsive cells; and 3) nonresponsive cells. The relative abundance of each pool changed with age. Nonresponsive cells were abundant at birth, multiresponsive cells were abundant at puberty, and monoresponsive cells dominated at senescence. The relative abundance of each HRH receptor changed largely with age but not gender. In addition, the contribution of monoresponsive and multiresponsive cells to responses to each HRH changed very much with age. Thus, the anterior pituitary shows large changes in cell populations typed by functional responses to HRHs during maturation, puberty, and senescence.

Receptor-controlled phosphorylation of alpha 1 soluble guanylyl cyclase enhances nitric oxide-dependent cyclic guanosine 5'-monophosphate production in pituitary cells.

It is generally accepted that G protein-coupled receptors stimulate soluble guanylyl cyclase (sGC)-mediated cGMP production indirectly, by increasing nitric oxide (NO) synthase activity in a calcium- and kinase-dependent manner. Here we show that normal and GH(3) immortalized pituitary cells expressed alpha(1)beta(1)-sGC heterodimer. Activation of adenylyl cyclase by GHRH, pituitary adenylate cyclase-activating polypeptide, vasoactive intestinal peptide, and forskolin increased NO and cGMP levels, and basal and stimulated cGMP production was abolished by inhibition of NO synthase activity. However, activators of adenylyl cyclase were found to enhance this NO-dependent cGMP production even when NO was held constant at basal levels. Receptor-activated cGMP production was mimicked by expression of a constitutive active protein kinase A and was accompanied with phosphorylation of native and recombinant alpha(1)-sGC subunit. Addition of a protein kinase A inhibitor, overexpression of a dominant negative mutant of regulatory protein kinase A subunit, and substitution of Ser(107)-Ser(108) N-terminal residues of alpha(1)-subunit with alanine abolished adenylyl cyclase-dependent cGMP production without affecting basal and NO donor-stimulated cGMP production. These results indicate that phosphorylation of alpha(1)-subunit by protein kinase A enlarges the NO-dependent sGC activity, most likely by stabilizing the NO/alpha(1)beta(1) complex. This is the major pathway by which adenylyl cyclase-coupled receptors stimulate cGMP production.

Growth hormone-releasing hormone and pituitary somatotrope proliferation.

Growth hormone-releasing hormone (GHRH) is a hypothalamic hormone that is essential for normal expansion of the somatotrope lineage during pituitary development. Decreased GHRH secretion and/or action leads to impairment of this process and somatotrope hypoplasia in both humans and experimental animals. Excessive GHRH secretion and/or action result in dysregulated somatotrope proliferation, leading to hyperplasia and neoplastic transformation. Our understanding of the molecular and morphologic bases for these effects from both animal and clinical studies has greatly increased during the past decade. However, many features of the cellular pathways remain to be defined, including the interaction of other genes in the multistep process of somatotrope tumorigenesis.

Growth hormone-releasing hormone stimulates mitogen-activated protein kinase.

GH-releasing hormone (GHRH) can induce proliferation of somatotroph cells. The pathway involving adenylyl cyclase/cAMP/protein kinase A pathway in its target cells seems to be important for this action, or at least it is deregulated in some somatotroph pituitary adenomas. We studied in this work whether GHRH can also stimulate mitogen-activated protein (MAP) kinase. GHRH can activate MAP kinase both in pituitary cells and in a cell line overexpressing the GHRH receptor. Although both protein kinase A and protein kinase C could activate MAP kinase in the CHO cell line studied, neither protein kinase A nor protein kinase C appears to be required for GHRH activation of MAP kinase in this system. However, sequestration of the betagamma-subunits of the G protein coupled to the receptor inhibits MAP kinase activation mediated by GHRH. This pathway also involves p21ras and a phosphatidylinositol 3-kinase, probably phosphatidylinositol 3-kinase-gamma. Despite the involvement of p21ras, the protein kinase Raf-1 is not hyperphosphorylated in response to GHRH, contrary to what usually occurs when the Ras-Raf-MAP kinase pathway is activated. In summary, this work describes for the first time the activation of MAP kinase by GHRH and outlines a path for this activation that is different from the cAMP-dependent mechanism that has been traditionally described as mediating the mitogenic actions of GHRH.

Effect of insulin-like growth factor-I on growth hormone-releasing factor receptor expression in primary rat anterior pituitary cell culture.

We examined the effect of insulin-like growth factor-I (IGF-I) on GH-releasing factor (GRF) receptor expression in the primary rat anterior pituitary cell culture. The levels of GRF receptor mRNA were dose-dependently reduced by IGF-I treatment for 24 h. To clarify whether altered levels of GRF receptor mRNA contribute to GRF receptor concentrations, we examined the GH response to GRF in vitro. There was no difference in basal GH secretion between control and IGF-I pretreated cells, while GRF-stimulated GH secretion in cells pretreated with IGF-I for 24 h was significantly lower than that in control cells. Moreover, specific [125I] Tyr10-human GRF binding to pituitary cells was reduced significantly by IGF-I treatment. These results suggest that IGF-I acts directly on the pituitary and participates in the regulation of GRF receptor expression.

Growth hormone-releasing factor mobilizes cytosolic free calcium through different mechanisms in two somatotrope subpopulations from porcine pituitary.

Porcine somatotropes can be separated by Percoll density gradient centrifugation into low (LD) and high density (HD) subpopulations that differ ultrastructurally and functionally. Here, we report the effects of growth hormone-releasing factor (GRF) on the cytosolic free calcium concentration ([Ca2+]i) of single LD and HD somatotropes. Resting [Ca2+]i in LD somatotropes was 2-fold higher than in HD cells. GRF induced [Ca2+]i increases in a similar percentage of somatotropes from both subsets. However, amplitude and kinetics of the responses were markedly different. In all responsive LD somatotropes, GRF evoked a rapid initial peak followed by a sustained plateau (plateau-type response). Blockade of extracellular Ca2+ entry by 3 mM EDTA, 2 mM CoCl2, or 100 microM verapamil completely abolished the plateau phase without affecting the initial Ca2+ spike. Conversely, only the plateau phase was preserved in thapsigargin (TG)-treated LD cells. The vast majority of GRF-responsive HD somatotropes exhibited a transient [Ca2+]i peak that returned gradually to baseline (transient-type response). This response was completely blocked by removal of extracellular Ca2+, whereas TG treatment had no effect. Taken together, our results indicate that the response of LD somatotropes to GRF depends on mobilization of Ca2+ of both extra- and intracellular origin, whereas that of HD somatotropes seems to be exclusively dependent on extracellular Ca2+ entry through L-type voltage sensitive Ca2+ channels (VSCC). These findings are the first to demonstrate a differential effect of GRF on Ca2+ mobilization in two somatotrope subpopulations, and suggest the existence of differences in the GRF receptor(s) expressed in each subpopulation and/or in the intracellular signalling pathways activated upon GRF binding.

Serotonergic mechanisms involved in calcitonin potentiation of kappa-opioid receptor-mediated effects on adrenal secretion.

Calcitonin can selectively modulate the effects of opioids on the rat hypothalamic-pituitary-adrenal axis and increase the release of corticosterone induced by a kappa-opioid receptor agonist. Considerable evidence supports the involvement of opioid and serotonergic systems in the analgesic effect of calcitonin. In this study, the involvement of hypothalamic serotonergic pathways in the calcitonin potentiation of the effect of (trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl]-benzeneacetamide methane sulphonate (U-50,488H) on the secretion of corticosterone was examined. The correlation between the calcitonin-induced potentiation of the pituitary adrenal response to U-50,488H and changes in serotonin turnover was evaluated. Our results show that the increase in the release of corticosterone induced by treatment with calcitonin + U-50,488H was not evident when the turnover of serotonin was decreased by inhibition of its synthesis with m-hydroxybenzylhydrazine (NSD 1015) or by blockade of its metabolism with trans-2-phenylcyclopropylamine (tranylcypromine). Although other factors can not be discarded, from the present data it can be suggested that the serotonergic system plays an important role in the interaction calcitonin-kappa-opioid receptor agonist in the hypothalamic-pituitary-adrenal axis.

Gene expression of the receptor for growth-hormone-releasing hormone is physiologically regulated by glucocorticoids and estrogen.

We investigated the effects of glucocorticoids and estrogen on the gene expression of growth hormone (GH) and the receptor for growth-hormone-releasing hormone (GHRH) by measuring the mRNA levels of GH and GHRH receptor in pituitary tissues of Sprague-Dawley rats using Northern blot hybridization and specific cDNA probes. Male rats, 6 weeks of age, were either adrenalectomized (or sham-operated) or treated with varying doses of dexamethasone (40, 200, 500 or 1,000 micrograms/kg/day, i.p.) for 3 days. Female rats, 4 weeks of age, were oophorectomized or sham-operated, and treated with 17 beta-estradiol benzoate 25 micrograms/kg/day (or vehicle) s.c. for 5 days starting 10 days after oophorectomy. Adrenalectomy was associated with a reduction in weight gain and decreased GHRH receptor mRNA levels (p < 0.05 and p < 0.0001 versus sham-operated, respectively). Dexamethasone treatment, however, was associated with a dose-dependent reduction in weight gain (p < 0.0001) but dose-dependent increases in GHRH receptor mRNA and GH mRNA levels (p < 0.0001 and p < 0.05, respectively). In the female rats, weight gain was increased by oophorectomy (p < 0.005 vs. sham-operated) and decreased by estrogen treatment (p < 0.05 vs. vehicle-treated). Pituitary GHRH receptor mRNA levels were also increased by oophorectomy (p < 0.05) and decreased by estrogen (p < 0.005). GH mRNA levels were unchanged by oophorectomy but decreased after estrogen treatment (p < 0.05). In conclusion, our findings suggest that endogenous glucocorticoids and estrogen are physiological regulators of pituitary GHRH receptor gene expression. Glucocorticoids and estrogen also regulate GH secretion via effects on GH gene expression. Changes in GHRH receptor and GH mRNA levels cannot explain the growth retardation in dexamethasone-treated rats.

Effects of aging on pituitary growth hormone-releasing factor receptor binding sites: in vitro mimicry by guanyl nucleotides and reducing agents.

We have investigated the effect of 5'-guanylylimidodiphosphate (Gpp(NH)p) and two disulfide bond reducing agents, reduced glutathione (GSH) and dithiothreitol (DTT), on the modulation of [125I-Tyr10]hGRF(1-44)NH2 binding to GRF receptor binding sites, in pituitaries of young and aging rats. In pituitaries from 2-month-old rats, Gpp(NH)p (0.1-1.0 mM), GSH and DTT (1-50 mM) exhibited a partial but concentration-dependent inhibitory effect on GRF specific binding. These effects were associated with a conversion of the high affinity GRF binding sites to lower affinity sites and to a reduction of the apparent number of total binding sites (high and low). No potentiation of these effects was observed when Gpp(NH)p (1 mM) and DTT (1 mM) were combined. In pituitaries from 14-month-old rats, Gpp(NH)p (1 mM) was capable of modulating GRF binding parameters in a similar fashion to that in pituitaries from 2-month-old rats. In pituitaries from 18-month-old rats, the high affinity GRF binding sites were already blunted and neither Gpp(NH)p nor Gpp(NH)p plus DTT significantly altered GRF binding parameters. In addition, in 20-month-old rats, the affinity of hGRF(1-29)NH2 and that of the full antagonist N alpha-Ac-[D-Arg2,Ala15]rGRF(1-29)NH2 were respectively decreased 9.3- and 9.9-fold. Our results suggest that in aging, alterations of GRF receptor binding sites could involve disulfide bond reduction or other structural modifications leading to conformational changes, similar to those induced by GSH or DTT. Such structural changes may prevent an efficient coupling of the GRF receptor with its ligands and G-protein, leading to a loss of somatotroph responsiveness.

Characterization of growth hormone-releasing hormone (GHRH) binding to cloned porcine GHRH receptor.

To study structure-activity relationships of growth hormone-releasing hormone (GHRH), a competitive binding assay was developed using cloned porcine adenopituitary GHRH receptors expressed in human kidney 293 cells. Specific binding of [His1, 125I-Tyr10,Nle27]hGHRH(1-32)-NH2 increased linearly with protein concentration (10-45 micrograms protein/ tube). Binding reached equilibrium after 90 min at 30 degrees C and remained constant for at least 240 min. Binding was reversible to one class of high-affinity sites (Kd = 1.04 +/- 0.19 nM, Bmax = 3.9 +/- 0.53 pmol/mg protein). Binding was selective with a rank order of affinity (IC50) for porcine GHRH (2.8 +/- 0.51 nM), rat GHRH (3.1 +/- 0.69 nM), [N-Ac-Tyr1, D-Arg2]hGHRH(3-29)-NH2 (3.9 +/- 0.58 nM), and [D-Thr7]GHRH(1-29)-NH2 (189.7 +/- 14.3 nM), consistent with their binding to a GHRH receptor. Nonhydrolyzable guanine nucleotides inhibited binding. These data describe a selective and reliable method for a competitive GHRH binding assay that for the first time utilizes rapid filtration to terminate the binding assay.

Identification of receptor-binding pharmacophores of growth-hormone-releasing factor in rat adenopituitary.

Previous structure-activity studies on growth-hormone-releasing factor (GRF) have mainly been carried out in pituitary cell culture assays. In such systems, the molecular features necessary to increase GRF receptor affinity cannot be fully distinguished from those that improve proteolytic resistance. To assess the affinity of GRF analogues, we have recently characterized [125I-Tyr10]hGRF(1-44)NH2 binding to rat adenopituitary, developing a reliable binding assay in which GRF-carboxamide-related peptides are stable. In the present study, we have determined the binding affinity of two series of analogues in which the entire sequence of hGRF(1-29)NH2 was scanned with D-amino acid and alanine substitutions. To further document their potency, we have evaluated the ability of representative candidates of each series to stimulate cAMP production. In the first series, a D-amino acid substitution at Ala4, Ile5, Phe6, Thr7, Val13, Gln16, Leu17, Ala19, Arg20 and Ile26 decreased drastically the binding affinity of hGRF(1-29)NH2 while it induced a smaller decrease at Tyr1, Asp3, Ser9, Tyr10, Arg11, Lys12, Leu14, Ala15, Ser18, Lys21, Leu22, Leu23, Gln24, Met27 and Ser28. Interestingly, a D-substitution in position 8 generated an analogue exhibiting a significantly greater binding affinity than hGRF(1-29)NH2, while it had no influence on hGRF(1-29)NH2 affinity at Ala2, Asp25 and Arg29. Adenylate cyclase activities of [D-Tyr1], [D-Tyr10] and [D-Arg20]hGRF(1-29)NH2 correlate with their binding affinity. In the second series, the largest decrease of binding affinity was observed with an alanine substitution at Tyr1, Asp3, Ile5, Phe6, Tyr10, Arg11, Lys12, Leu14, Leu17, Arg20 and Lys21.(ABSTRACT TRUNCATED AT 250 WORDS)