Isolation and sequencing of cDNAs for splice variants of growth hormone-releasing hormone receptors from human cancers

The proliferation of various tumors is inhibited by the antagonists of growth hormone-releasing hormone (GHRH) in vitro and in vivo, but the receptors mediating the effects of GHRH antagonists have not been identified so far. Using an approach based on PCR, we detected two major splice variants (SVs) of mRNA for human GHRH receptor (GHRH-R) in human cancer cell lines, including LNCaP prostatic, MiaPaCa-2 pancreatic, MDA-MB-468 breast, OV-1063 ovarian, and H-69 small-cell lung carcinomas. In addition, high-affinity, low-capacity binding sites for GHRH antagonists were found on the membranes of cancer cell lines such as MiaPaCa-2 that are negative for the vasoactive intestinal peptide/pituitary adenylate cyclase-activating polypeptide receptor (VPAC-R) or lines such as LNCaP that are positive for VPAC-R. Sequence analysis of cDNAs revealed that the first three exons in SV1 and SV2 are replaced by a fragment of retained intron 3 having a new putative in-frame start codon. The rest of the coding region of SV1 is identical to that of human pituitary GHRH-R, whereas in SV2 exon 7 is spliced out, resulting in a 1-nt upstream frameshift, which leads to a premature stop codon in exon 8. The intronic sequence may encode a distinct 25-aa fragment of the N-terminal extracellular domain, which could serve as a proposed signal peptide. The continuation of the deduced protein sequence coded by exons 4–13 in SV1 is identical to that of pituitary GHRH-R. SV2 may encode a GHRH-R isoform truncated after the second transmembrane domain. Thus SVs of GHRH-Rs have now been identified in human extrapituitary cells. The findings support the view that distinct receptors are expressed on human cancer cells, which may mediate the antiproliferative effect of GHRH antagonists.

Luteinizing hormone-releasing hormone (LH-RH) antagonist Cetrorelix down-regulates the mRNA expression of pituitary receptors for LH-RH by counteracting the stimulatory effect of endogenous LH-RH

The mechanisms through which LH-RH antagonists suppress gonadotroph functions and LH-RH receptor (LH-RH-R) production are incompletely understood. To elucidate these mechanisms, we investigated the effects of Cetrorelix on the mRNA expression of pituitary LH-RH-R and luteinizing hormone (LH) secretion in three experimental systems with different pituitary LH-RH environments. Ovariectomy induced 3.61-fold and 6.34-fold increases in the mRNA expression of pituitary LH-RH-R in rats after 11 and 21 days, respectively. After (5 h) a single injection of 100 μg Cetrorelix, no significant decrease occurred in the mRNA levels of pituitary LH-RH-R in ovariectomized (OVX) rats with high pituitary exposure to LH-RH, but there was a significant 23.2% reduction in cycling rats with normal hypophysial LH-RH environment. Prolonged treatment for 10 days with a Cetrorelix depot formulation releasing 100 μg/day decreased the concentration of mRNA for pituitary LH-RH-R by 72.6% in OVX rats, but only by 32.9% in normal rats. The decline in serum LH was 98.7% in OVX rats and 63.2% in normal rats, resulting in a minimal 0.1–0.2 ng/ml LH concentration in both groups. A continuous exposure of pituitary cells to 100 nM Cetrorelix in the superfusion system, which is devoid of LH-RH, did not cause any significant changes in LH-RH-R mRNA level. These studies demonstrate that prolonged exposure to Cetrorelix in vivo, but not in vitro, down-regulates the mRNA expression of the pituitary receptors for LH-RH. Our findings indicate that LH-RH antagonists exert their inhibitory effects on the gene expression of pituitary LH-RH-R by counteracting the stimulatory effect of endogenous LH-RH.

Long-term, progressive hippocampal cell loss and dysfunction induced by early-life administration of corticotropin-releasing hormone reproduce the effects of early-life stress

Stress early in postnatal life may result in long-term memory deficits and selective loss of hippocampal neurons. The mechanisms involved are poorly understood, but they may involve molecules and processes in the immature limbic system that are activated by stressful challenges. We report that administration of corticotropin-releasing hormone (CRH), the key limbic stress modulator, to the brains of immature rats reproduced the consequences of early-life stress, reducing memory functions throughout life. These deficits were associated with progressive loss of hippocampal CA3 neurons and chronic up-regulation of hippocampal CRH expression. Importantly, they did not require the presence of stress levels of glucocorticoids. These findings indicate a critical role for CRH in the mechanisms underlying the long-term effects of early-life stress on hippocampal integrity and function.

Cytotoxic analogs of luteinizing hormone-releasing hormone containing doxorubicin or 2-pyrrolinodoxorubicin, a derivative 500-1000 times more potent.

Doxorubicin (DOX) and its daunosamine-modified derivative, 2-pyrrolino-DOX, which is 500-1000 times more active than DOX, were incorporated into agonistic and antagonistic analogs of luteinizing hormone-releasing hormone (LH-RH). The conjugation of DOX with LH-RH analogs was performed by using N-(9-fluorenylmethoxycarbonyl)-DOX-14-O-hemiglutarate, a dicarboxylic acid ester derivative of DOX. Coupling this derivative covalently to the epsilon-amino group of the D-Lys side chain of agonist [D-Lys6]LH-RH or antagonistic analog AC-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys-Leu-Arg-Pro-D-Ala-NH 2 [where Nal(2) = 3-(2-naphthyl)alanine, Pal(3) = 3-(3-pyridyl)alanine, and Phe(4CI) = 4-chlorophenylalanine] was followed by the removal of the 9-fluorenylmethoxycarbonyl protective group to yield cytotoxic derivatives of LH-RH analogs containing DOX. From these DOX containing LH-RH hybrids, intensely potent analogs with daunosamine-modified derivatives of DOX can be readily formed. Thus, cytotoxic LH-RH agonist containing DOX (AN-152) can be converted in a 66% yield by a reaction with a 30-fold excess of 4-iodobutyraldehyde in N,N-dimethylformamide into a derivative having 2-pyrrolino-DOX (AN-207). Hybrid molecules AN-152 and AN-207 fully preserve the cytotoxic activity of their radicals, DOX or 2-pyrrolino-DOX, respectively, in vitro, and also retain the high binding affinity of the peptide hormone portion of the conjugates to rat pituitary receptors for LH-RH. These highly potent cytotoxic analogs of LH-RH were designed as targeted anti-cancer agents for the treatment of various tumors that possess receptors for the carrier peptide. Initial in vivo studies show that the hybrid molecules are much less toxic than the respective cytotoxic radicals incorporated and significantly more active in inhibiting tumor growth.

Transcription of the human corticotropin-releasing hormone gene in NPLC cells is correlated with Z-DNA formation.

The intron of the corticotropin-releasing hormone (corticoliberin; CRH) gene contains a sequence of over 100 bp of alternating purine/pyrimidine residues. We have used binding of a Z-DNA-specific antibody in metabolically active, permeabilized nuclei to study the formation of Z-DNA in this sequence at various levels of transcription. In the NPLC human primary liver carcinoma cell line, activation of cAMP-dependent pathways increased the level of transcription, while adding glucocorticoids inhibited transcription of the CRH gene. These cells respond in a manner similar to hypothalamic cells. Z-DNA formation in this sequence was detected at the basal level of transcription, as well as after stimulation with forskolin. Inhibition of transcription by dexamethasone abolished Z-DNA formation. Z-DNA formation in the WC gene (c-myc) was affected differently in the same experiment. Thus, changes in Z-DNA formation in the CRH gene are gene specific and are linked to the transcription of the gene.

Down-regulation of pituitary receptors for luteinizing hormone-releasing hormone (LH-RH) in rats by LH-RH antagonist Cetrorelix.

Antagonists of luteinizing hormone-releasing hormone (LH-RH), unlike the LH-RH agonists, suppress gonadotropins and sex steroid secretion immediately after administration, without initial stimulatory effects. [Ac-D-Nal(2)1,D-Ph(4Cl)2,D-Pal(3)3,D-Cit6,D-Ala10]LH-R H (SB-75; Cetrorelix) is a modern, potent antagonistic analog of LH-RH. In this study, the binding characteristics of receptors for LH-RH in membrane fractions from rat anterior pituitaries were investigated after a single injection of Cetrorelix at a dose of 100 microg per rat. To determine whether the treatment with Cetrorelix can affect the concentration of measurable LH-RH binding sites, we applied an in vitro method to desaturate LH-RH receptors by chaotropic agents such as manganous chloride (MnCl2) and ammonium thiocyanate (NH4SCN). Our results show that the percentages of occupied LH-RH receptors at 1, 3, and 6 h after administration of Cetrorelix were approximately 28%, 14%, and 10%, respectively, of total receptors. At later time intervals, we could not detect occupied LH-RH binding sites. Ligand competition assays, following in vitro desaturation, demonstrated that rat pituitary LH-RH receptors were significantly (P < 0.01) down-regulated for at least 72 h after administration of Cetrorelix. The lowest receptor concentration was found 3-6 h after Cetrorelix treatment and a recovery in receptor number began within approximately 24 h. The down-regulation of LH-RH binding sites induced by Cetrorelix was accompanied by serum LH and testosterone suppression. Higher LH-RH receptor concentrations coincided with elevated serum hormone levels at later time intervals. Our results indicate that administration of LH-RH antagonist Cetrorelix produces a marked down-regulation of pituitary receptors for LH-RH and not merely an occupancy of binding sites.

Synthesis and biological evaluation of superactive agonists of growth hormone-releasing hormone.

Analogs of the 29 amino acid sequence of human growth hormone-releasing hormone (hGH-RH) with agmatine (Agm) in position 29, desaminotyrosine (Dat) in position 1, norleucine (Nle) in position 27, and L-alpha-aminobutyric acid (Abu) in position 15 have been synthesized, and their biological activity was evaluated. Some peptides contained one or two residues of ornithine (Orn) instead of Lys in positions 12 and 21 and additional replacements in positions 8 and 28. All analogs were found to be more potent than hGH-RH-(1-29)-NH2 in the superfused rat pituitary cell system. In tests in vivo in rats after subcutaneous administration, the analogs JI-22, [Dat1, Orn12,21, Abu15, Nle27, Agm29]hGH-RH-(1-29); JI-34, [Dat1, Orn12,21,Abu15,Nle27, Asp28, Agm29]hGH-RH-(1-29); JI-36, [Dat1, Thr8, Orn12,21, Abu15,Nle27,Asp28,Agm29]hGH-RH-(1-29); and JI-38, [Dat1,Gln8, Orn12,21,Abu15,Nle27,Asp28,Agm29]hGH-RH-(1 -29) displayed a potency 44.6,80.9,95.8, and 71.4 times greater, respectively, than that of hGH-RH-(1-29)-NH2 at 15 min and 217.1, 89.7, 87.9, and 116.8 times greater at 30 min. After intravenous administration, JI-22, JI-36, and JI-38 were 3.2-3.8 times more potent than hGH-RH-(1-29)-NH2 at 5 min and 6.1-8.5 times more active at 15 min. All analogs were found to have higher binding affinities for GH-RH receptors on rat pituitary cells than hGH-RH-(1-29)-NH2. Because of high activity and greater stability, these analogs could be considered for therapy of patients with growth hormone deficiency.

Electrical and synaptic properties of embryonic luteinizing hormone-releasing hormone neurons in explant cultures.

Voltage- and ligand-activated channels in embryonic neurons containing luteinizing hormone-releasing hormone (LHRH) were studied by patch-pipette, whole-cell current and voltage clamp techniques. LHRH neurons were maintained in explant cultures derived from olfactory pit regions of embryonic mice. Cells were marked intracellularly with Lucifer yellow following recording. Sixty-two cells were unequivocally identified as LHRH neurons by Lucifer yellow and LHRH immunocytochemistry. The cultured LHRH neurons had resting potentials around -50 mV, exhibited spontaneous discharges generated by intrinsic and/or synaptic activities and contained a time-dependent inward rectifier (Iir). Voltage clamp analysis of ionic currents in the LHRH neuron soma revealed a tetrodotoxin-sensitive Na+ current (INa) and two major types of K+ currents, a transient current (IA), a delayed rectifier current (IK) and low- and high-voltage-activated Ca2+ currents. Spontaneous depolarizing synaptic potentials and depolarizations induced by direct application of gamma-aminobutyrate were both inhibited by picrotoxin or bicuculline, demonstrating the presence of functional gamma-aminobutyrate type A synapses on these neurons. Responses to glutamate were found in LHRH neurons in older cultures. Thus, embryonic LHRH neurons not yet positioned in their postnatal environment in the forebrain contained a highly differentiated repertoire of voltage- and ligand-gated channels.

Ethanol inhibits luteinizing hormone-releasing hormone (LHRH) secretion by blocking the response of LHRH neuronal terminals to nitric oxide.

It has previously been shown that alcohol can suppress reproduction in humans, monkeys, and small rodents by inhibiting release of luteinizing hormone (LH). The principal action is via suppression of the release of LH-releasing hormone (LHRH) both in vivo and in vitro. The present experiments were designed to determine the mechanism by which alcohol inhibits LHRH release. Previous research has indicated that the release of LHRH is controlled by nitric oxide (NO). The proposed pathway is via norepinephrine-induced release of NO from NOergic neurons, which then activates LHRH release. In the present experiments, we further evaluated the details of this mechanism in male rats by incubating medial basal hypothalamic (MBH) explants in vitro and examining the release of NO, prostaglandin E2 (PGE2), conversion of arachidonic acid to prostanoids, and production of cGMP. The results have provided further support for our theory of LHRH control. Norepinephrine increased the release of NO as measured by conversion of [14C]arginine to [14C]citrulline, and this increase was blocked by the alpha 1 receptor blocker prazosin. Furthermore, the release of LHRH induced by nitroprusside (NP), a donor of NO, is related to the activation of soluble guanylate cyclase by NO since NP increased cGMP release from MBHs and cGMP also released LHRH. Ethanol had no effect on the production of NO by MBH explants or the increased release of NO induced by norepinephrine. Therefore, it does not act at that step in the pathway. Ethanol also failed to affect the increase in cGMP induced by NP. On the other hand, as might be expected from previous experiments indicating that LHRH release was brought about by PGE2, NP increased the conversion of [14C]arachidonic acid to its metabolites, particularly PGE2. Ethanol completely blocked the release of LHRH induced by NP and the increase in PGE2 induced by NP. Therefore, the results support the theory that norepinephrine acts to stimulate NO release from NOergic neurons. This NO diffuses to the LHRH terminals where it activates guanylate cyclase, leading to an increase in cGMP. At the same time, it also activates cyclooxygenase. The increase in cGMP increases intracellular free calcium, activating phospholipase A2 to provide arachidonic acid, the substrate for conversion by the activated cyclooxygenase to PGE2, which then activates the release of LHRH. Since alcohol inhibits the conversion of labeled arachidonic acid to PGE2, it must act either directly to inhibit cyclooxygenase or perhaps it may act by blocking the increase in intracellular free calcium induced by cGMP, which is crucial for activation of of both phospholipase A2 and cyclooxygenase.

Nitric oxide inhibits hypothalamic luteinizing hormone-releasing hormone release by releasing gamma-aminobutyric acid.

Nitric oxide synthase (NOS)-containing neurons, termed NOergic neurons, occur in various regions of the hypothalamus, including the median eminence-arcuate region, which plays an important role in controlling the release of luteinzing hormone-releasing hormone (LHRH). We examined the effect of NO on release of gamma-aminobutyric acid (GABA) from medial basal hypothalamic (MBH) explants incubated in vitro. Sodium nitroprusside (NP) (300 microM), a spontaneous releaser of NO, doubled the release of GABA. This release was significantly reduced by incubation of the tissue with hemoglobin, a scavenger of NO, whereas hemoglobin alone had no effect on the basal release of GABA. Elevation of the potassium concentration (40 mM) in the medium increased GABA release 15-fold; this release was further augmented by NP. Hemoglobin blocked the increase in GABA release induced by NP but had no effect on potassium-induced release, suggesting that the latter is not related to NO. As in the case of hemoglobin, NG-monomethyl-L-arginine (NMMA), a competitive inhibitor of NOS, had no effect on basal release of GABA, which indicates again that NO is not significant to basal GABA release. However, NMMA markedly inhibited the release of GABA induced by high potassium, which indicates that NO plays a role in potassium-induced release of GABA. In conditions in which the release of GABA was substantially augmented, there was a reduction in GABA tissue stores as well, suggesting that synthesis of GABA in these conditions did not keep up with release of the amine. Although NO released GABA, there was no effect of the released GABA on NO production, for incubation of MBH explants with GABA had no effect on NO release as measured by [14C]citrulline production. To determine whether GABA had any effect on the release of LHRH from these MBH explants, GABA was incubated with the tissue and the effect on LHRH release was determined. GABA (10(-5) or 10(-6) M) induced a 70% decrease in the release of LHRH, indicating that in the male rat GABA inhibits the release of this hypothalamic peptide. This inhibition in LHRH release induced by GABA was blocked by NMMA (300 microM), which indicates that GABA converts the stimulatory effect of NO on LHRH release into an inhibitory one, presumably via GABA receptors, which activate chloride channels that hyperpolarize the cell. Previous results have indicated that norepinephrine stimulates release of NO from the NOergic neurons, which then stimulates the release of LHRH. The current results indicate that the NO released also induces release of GABA, which then inhibits further LHRH release. Thus, in vivo the norepinephrinergic-driven pulses of LHRH release may be terminated by GABA released from GABAergic neurons via NO.

Effect of naloxone-precipitated morphine withdrawal on c-fos expression in rat corticotropin-releasing hormone neurons in the paraventricular hypothalamus and extended amygdala

Morphine withdrawal is characterized by physical symptoms and a negative affective state. The 41 amino acid polypeptide corticotropin-releasing, hormone (CRH) is hypothesized to mediate, in part, both the negative affective state and the physical withdrawal syndrome. Here, by means of dual-immunohistochemical methodology, we examined the co-expression of the c-Fos protein and CRH following naloxone-precipitated morphine withdrawal. Rats were treated with slow-release morphine 50 mg/kg (subcutaneous, s.c.) or vehicle every 48 It for 5 days, then withdrawn with naloxone 5 mg/kg (s.c.) or saline 48 h after the final morphine injection. Two hours after withdrawal rats were perfused transcardially and their brains were removed and processed for immunohistochemistry. We found that naloxone-precipitated withdrawal of morphine-dependent rats increased c-Fos immunoreactivity (IR) in CRH positive neurons in the paraventricular hypothalamus. Withdrawal of morphine-dependent rats also increased c-Fos-IR in the central amygdala and bed nucleus of the stria terminalis. however these were in CRH negative neurons. (C) 2004 Published by Elsevier Ireland Ltd.

Testosterone secretion and pharmacological spermatozoal recovery in the cane toad (Bufo marinus)

The cane toad (Bufo marinus) was used as a model to study male anuran reproductive endocrinology and to develop a protocol for non-invasive sperm recovery. Circulating testosterone concentrations in 6-hourly samples did not vary significantly (P < 0.05) over a 24 h period although there was a tendency (P = 0.06) for testosterone to be elevated at 19:00 h relative to other times of the day, which may be related to the nocturnal activity pattern of this species. Testosterone secretion after intraperitoneal (IP) injection of either a GnRH agonist (5 mu g IP) or hCG (1000 IU) was also examined. While the GnRH agonist did not produce a significant increase above basal plasma testosterone (0.29, 95% C.I. of 0.05-1.10 ng/ml), injection of hCG resulted in an increase (P < 0.01) of plasma testosterone with peak concentrations at approximately 120 min (4.17, 95% C.I. of 2.69-7.44 ng/ml) after injection. Non-invasive pharmaceutical sperm recovery was attempted following IP injection of graded doses of GnRH agonist, hCG or FSH. Urine was collected at 3, 6 and 12 h after treatment to assess sperm quality and quantity. The optimal protocol for sperm recovery in cane toads was injection of either 1000 or 2000 IU hCG; there was no significant difference in the quality of the spermic urine samples obtained using either dose of hCG or with respect to collection time. The findings indicated that hCG can be used to assess testicular steroidogenic status and also to induce sperm recovery in the cane toad. The hCG protocols developed in this study will have application in studies on the reproductive biology of rare and endangered male anurans. (c) 2005 Elsevier B.V. All rights reserved.

Testicular germ cell tumors exhibit evidence of hormone dependence

The aim of this investigation was to test the hypothesis that testicular germ cell tumors (TGCTs) are hormone-dependent cancers. Human TGCT cells were implanted in the left testis of male severe combined immunodeficient mice receiving either no treatment or hormone manipulation treatment [blockade of gonadotropin-releasing hormone secretion and/or signaling using leuprolide or leuprolide plus exogenous testosterone]. Real-time RT-PCR analysis was used to determine the expression profiles of hormone pathway-associated genes. Tumor burden was significantly smaller in mice receiving both leuprolide and testosterone. Real-time RTPCR analysis of follicle-stimulating hormone (FSH) receptor, luteinizing hormone (LH) receptor and P450 aromatase revealed changes in expression in normal testis tissue related to presence of xenograft tumors and manipulation of hormone levels but a complete absence of expression of these genes in tumor cells themselves. This was confirmed in human specimens of TGCT. Reduced TGCT growth in vivo was associated with significant downregulation of LH receptor and P450 aromatase expression in normal testes. In conclusion, manipulation of hormone levels influenced the growth of TGCT in vivo, while the presence of xenografted tumors influenced the expression of hormone-related genes in otherwise untreated animals. Human TGCTs, both in the animal model and in clinical specimens, appear not to express receptors for FSH or LH. Similarly, expression of the P450 aromatase gene is absent in TGCTs. Impaired estrogen synthesis and/or signaling may be at least partly responsible for inhibition of TGCT growth in the animal model. (c) 2005 Wiley-Liss, Inc.