Molecular cloning and characterization of two putative appetite regulators in winter flounder (Pleuronectes americanus): preprothyrotropin-releasing hormone (TRH) and preproorexin (OX).

cDNAs encoding for preproTRH and preproorexin were cloned in winter flounder, a species that undergoes a period of natural fasting during the winter. For both peptides, the deduced amino acid structure of the hormone precursor shows 30-70% similarities with their homologs in other fish species. RT-PCR studies show that these peptides are present not only in the brain, but also in several peripheral tissues, including gastrointestinal tract and testes. Fasting induced increases in both preproorexin and preproTRH expressions in the hypothalamus, but did not affect their expression levels in the telencephalon/preoptic area. In addition, the mRNA expressions of both preproorexin and preproTRH were higher in the winter than in the summer in both hypothalamus and telencephalon/preoptic area. Our results suggest that orexin and thyrotropin-releasing hormone (TRH) might have a role in the seasonal regulation of food intake in winter flounder.

Quantification of thyrotropin-releasing hormone by liquid chromatography-electrospray mass spectrometry.

Thyrotropin-releasing hormone (TRH) is involved in a wide range of biological responses. It has a central role in the endocrine system and regulates several neurobiological activities. In the present study, a rapid, sensitive and selective liquid chromatography-mass spectrometry method for the identification and quantification of TRH has been developed. The methodology takes advantage of the specificity of the selected-ion monitoring acquisition mode with a limit of detection of 1 fmol. Furthermore, the MS/MS fragmentation pattern of TRH has been investigated to develop a selected reaction monitoring (SRM) method that allows the detection of a specific b2 product ion at m/z 249.1, corresponding to the N-terminus dipeptide pyroglutamic acid-histidine. The method has been tested on rat hypothalami to evaluate its suitability for the detection within very complex biological samples.

Structure-activity studies with high-affinity inhibitors of pyroglutamyl-peptidase II.

Inhibitors of PPII (pyroglutamyl-peptidase II) (EC 3.4.19.6) have potential applications as investigative and therapeutic agents. The rational design of inhibitors is hindered, however, by the lack of an experimental structure for PPII. Previous studies have demonstrated that replacement of histidine in TRH (thyrotropin-releasing hormone) with asparagine produces a competitive PPII inhibitor (Ki 17.5 microM). To gain further insight into which functional groups are significant for inhibitory activity, we investigated the effects on inhibition of structural modifications to Glp-Asn-ProNH2 (pyroglutamyl-asparaginyl-prolineamide). Synthesis and kinetic analysis of a diverse series of carboxamide and C-terminally extended Glp-Asn-ProNH2 analogues were undertaken. Extensive quantitative structure-activity relationships were generated, which indicated that key functionalities in the basic molecular structure of the inhibitors combine in a unique way to cause PPII inhibition. Data from kinetic and molecular modelling studies suggest that hydrogen bonding between the asparagine side chain and PPII may provide a basis for the inhibitory properties of the asparagine-containing peptides. Prolineamide appeared to be important for interaction with the S2' subsite, but some modifications were tolerated. Extension of Glp-Asn-ProNH2 with hydrophobic amino acids at the C-terminus led to a novel set of PPII inhibitors active in vitro at nanomolar concentrations. Such inhibitors were shown to enhance recovery of TRH released from rat brain slices. Glp-Asn-Pro-Tyr-Trp-Trp-7-amido-4-methylcoumarin displayed a Ki of 1 nM, making it the most potent competitive PPII inhibitor described to date. PPII inhibitors with this level of potency should find application in exploring the biological functions of TRH and PPII, and potentially provide a basis for development of novel therapeutics.

Neuropharmacodynamic evaluation of the centrally active thyrotropin-releasing hormone analogue [Leu2]TRH and its chemical brain-targeting system.

The centrally active thyrotropin-releasing hormone (TRH) analogue pGlu-Leu-Pro-NH(2) ([Leu(2)]TRH) showed a significant increase in the extracellular acetylcholine concentration during its perfusion to the hippocampus in rats, and this effect was manifested upon the delivery of the analogue in much smaller quantities compared to TRH when measured by in vivo intracranial microdialysis. The neuropharmacodynamic efficacy of [Leu(2)]TRH upon intravenous administration was augmented by the use of a brain-targeting derivative in which the progenitor sequence of the mature peptide was embedded in a molecular architecture that promoted enhanced brain delivery, retention and in situ generation of the pharmacologically active molecule. Compared to the unmodified peptide, the targeting system significantly improved the cumulative effect of the treatment on extracellular acetylcholine levels in rats.

Conformationally restricted TRH analogues: constraining the pyroglutamate region.

A modified synthetic route has been developed so that the steric size of constraints added to the pyroglutamate region of TRH (pGluHisProNH(2)) can be varied. Both an analogue with a smaller ethylene bridge and a larger, more flexible propane bridge in this region have been synthesized. These analogues were synthesized in order to probe why the initial incorporation of an ethane bridge into this region of the molecule had led to an analogue with a binding constant and potency three times lower than that of an directly analogous unconstrained analogue. The data for both analogues indicated that the fall off in activity caused by the ethane bridge in the initial analogue was not caused by the size of the bridge.

Hydropathy profiles of predicted thyrotropin-releasing hormone precursors are highly conserved despite low similarity of primary structures.

Two types of cDNAs encoding thyrotropin-releasing hormone (TRH) precursors (TRH-A and TRH-B) were amplified from hypothalamic mRNA of sockeye salmon by reverse transcriptase-polymerase chain reaction (RT-PCR). The amplification was achieved using two primers which correspond to TRH progenitor sequence (Lys/Arg-Arg-Gln-His-Pro-Gly-Lys/Arg-Arg). A full length cDNA encoding TRH-A was obtained by 5'- and 3'-RACE methods. It has a length of 1324 base pairs (bp) that contains sequences of 5' and 3' untranslated regions and an open reading frame of 259 codons. The sockeye salmon TRH-A deduced from the nucleotide sequence tandemly contains 8 copies of TRH progenitor sequences. Another cDNA which encodes a part of TRH-B consists of 242 bp, and the sequence homology between TRH-A and -B cDNAs is 90%. The result of Southern blot analysis of sockeye and masu salmon genomic DNAs supported the evidence that there are at least two TRH genes in the salmonid. A RT-PCR analysis of TRH gene expression in various tissues of sockeye salmon showed that strong expression was observed only in the brain. The primary structure of the sockeye salmon TRH-A shares low similarity to those of human, rat and Xenopus TRH precursors (35, 27 and 44%, respectively). However, their hydropathy profiles were almost the same with each other. The profile of sockeye salmon TRH-A showed the presence of two discrete hydrophobic regions, one in the N-terminal region which corresponds to the signal peptide and the other in the C-terminal region. All of the repetitive TRH progenitor sequences are included in three hydrophilic regions easily recognizable. The present results thus suggest that the three-dimensional structures of TRH precursors are highly conserved, although the primary structures of TRH precursors have diverged through the evolutionary pathway of vertebrates.

In vitro and in vivo evaluation of thyrotrophin releasing hormone release from copoly(dl-lactic/glycolic acid) microspheres.

In vitro and in vivo release of thyrotrophin releasing hormone (TRH) from copoly(dl-lactic/glycolic acid) (PLGA) microspheres were evaluated. Factors affecting the TRH release from the microspheres were examined to clarify the release mechanisms by changing the medium composition in the in vitro release test. The hydrolysis rate of PLGA, the matrix-forming substance in the microspheres, was faster in acidic medium than in neutral medium. The release rate of TRH from the PLGA microspheres increased with the increase in the degradation rate of PLGA. A decrease in an osmolarity of the medium also caused an increase in the TRH release rate even though no significant change in PLGA degradation was observed. The effect of osmolarity appears to be characteristic of water-soluble drug-containing microspheres composed of hydrophobic polymer. The release rate of TRH from PLGA microspheres was largely affected by the medium composition in the in vitro release test. A proper choice of medium was found to be important for the estimation of in vivo release. The in vivo release rate of TRH from the PLGA microspheres following administration to rats correlated with the in vitro release in pH 7, 1/30 M buffer.

[Molecular cloning and posttranslational processing of human preprothyrotropin-releasing hormone].

Human PreproTRH gene and its hypothalamic cDNA were cloned. The human preproTRH protein contains six copies of TRH progenitor sequence flanked by paired basic amino acids. Radioimmunoassays with specific antibodies raised against five human preproTRH cryptic peptides and chromatographic analyses identified that the posttranslational processing of preproTRH in hypothalami and placentae could be different. Moreover, transfection assays using human preproTRH promoter-luciferase chimeric-plasmids demonstrated that human preproTRH promoter activities were regulated negatively by T3 in GH3 cells. Cloning of the human preproTRH gene would facilitate the elucidation of the molecular mechanism of actions of TRH and TRH cryptic peptides in human hypothalamo-pituitary-thyroid axis and central nervous system.

A cryptic peptide from the preprothyrotropin-releasing hormone precursor stimulates thyrotropin gene expression.

The precursor peptide of TRH (prepro-TRH) contains five copes of pro-TRH linked by other peptide sequences. These peptides are coprocessed with TRH in the median eminence of the hypothalamus and released into the portal circulation, rendering this family of peptides available to act at the level of the anterior pituitary. Therefore, we tested the potential bioactivity of one cryptic peptide, prepro-TRH amino acids 160-169 [prepro-TRH-(160-169)], in a TRH-responsive pituitary cell line (GH3). In a heterologous TSH expression assay, we found that prepro-TRH-(160-169) stimulated TSH beta gene promoter activity in a time- and dose-dependent manner; moreover, the effect of prepro-TRH-(160-169) was more rapid and of greater magnitude than that of TRH on TSH beta-directed chloramphenicol acetyltransferase synthesis. In the same cells, we found that prepro-TRH-(160-169) stimulated PRL synthesis and secretion, but the effect was similar in magnitude and duration to that of TRH. The effect of prepro-TRH-(160-169) appears to be additive to that of TRH, suggesting that prepro-TRH-(160-169) may act through a mechanism separate from that of TRH. Thus, prepro-TRH-(160-169) has potent endocrinological effects at the level of the genome.

Testosterone increases TRH biosynthesis in epididymis but not heart of zinc-deficient rats.

Enzymes responsible for the posttranslational processing of precursor proteins to form alpha-amidated peptide hormones require the availability of several cofactors, including zinc, copper, and ascorbic acid. For this reason, we studied the effects of 6 weeks of a zinc-deficient diet (ZD1; 1 microgram zinc per g diet), pair-feeding (PF), and marginal zinc deficiency (ZD6; 6 micrograms zinc per g diet) compared to a control diet (36 micrograms/g zinc) on the conversion of prepro-TRH to TRH in epididymides, testes, prostate, pancreas, and heart of young adult, male Sprague-Dawley rats. In the epididymis, severe zinc deficiency (ZD1 diet) reduced TRH and TRH-like peptides to undetectable levels. In ZD6 animals, TRH was selectively inhibited 80%, while pair-feeding increased all of these peptide levels compared to controls. A similar effect of zinc deficiency on the TRH precursor peptides was observed. A quantitative loss of TRH from the testes of ZD1 was also observed. Zinc deficiency results in a substantial reduction in body weight and testosterone production in male rats. Exogenous testosterone (T) supplementation of ZD1 rats resulted in a selective increase in the TRH concentration of the epididymis but not of the heart. The change in steady-state levels of TRH precursor peptides in the hearts of the ZD1+T rats was consistent with a reduction in the activity of the zinc-dependent carboxypeptidase H enzyme. We conclude that severe zinc deficiency inhibits TRH biosynthesis in reproductive tissues of the male rat due to the combined effects of hypogonadism and inhibition of the zinc-dependent carboxypeptidase H.

Thyrotropin-releasing hormone (TRH) is the major prolactin-releasing factor in the bullfrog hypothalamus.

A substance exhibiting potent activity in stimulating the release of prolactin from bullfrog (Rana catesbeiana) pituitary in vitro was isolated from an acid extract of bullfrog hypothalami by gel-filtration chromatography (Sephadex G-15), ion-exchange chromatography (Mono-S HR 5/5), and reverse-phase high-performance liquid chromatography (TSK-gel ODS-120T). Its amino acid composition was similar to that of synthetic thyrotropin-releasing hormone (TRH). Radioimmunoassay confirmed that the substance had TRH immunoreactivity. Moreover, it exhibited the same chromatographic behavior as that of synthetic TRH. These results clearly indicate that the isolated hypothalamic substance is TRH, and that it is the major prolactin-releasing factor present in the bullfrog hypothalamus.

Isolation and amino acid sequence of the TRH-potentiating peptide from bovine hypothalamus.

A neuropeptide termed TRH-potentiating peptide, which potentiates TRH-evoked thyrotropin secretion by antehypophysis in vitro, was isolated from an acetonic powder of bovine hypothalamus. The peptide was purified to homogeneity by a 3-step protocol involving molecular sieve filtration, ion-exchange chromatography and reverse phase high performance liquid chromatography. The complete amino acid sequence of the decapeptide was determined as Ser-Phe-Pro-Trp-Met-Glu-Ser-Asp-Val-Thr by automated Edman degradation with a solid-phase sequencer. Bovine TRH-potentiating peptide is structurally identical to Ps4, a decapeptide which was deduced from the cDNA encoding the rat TRH precursor. This study provides for the first time a direct chemical evidence for the existence of non-TRH peptides originating from posttranslational processing of the TRH precursor in vivo.

Interactions between adrenergic systems, anaesthetic and TRH analogue induced analeptic effects on VBT transmission.

Previous behavioural and electrophysiological studies have indicated an antinarcotic action of thyrotropin-releasing hormone (TRH) and its analogues in antagonizing the action of CNS depressant drugs, including baclofen and a variety of anesthetics. While beta-adrenergic receptors are implicated in the level of anaesthesia/arousal, whether the analeptic action of TRH involves adrenergic systems for its expression is uncertain. The object of the present experiments, therefore, was to examine interactions between adrenergic systems and the anti-anaesthetic effects of TRH analogue CG3703. It was found that pretreatment with the beta-block (+/-)propranolol did not abolish or reduce the ability of CG3703 to antagonize urethane-induced depression of VBT transmission. These results suggest therefore, that beta-adrenergic systems are unlikely to be involved in the anti-anaesthetic effect of the tripeptide.