Thyrotropin-releasing hormone and a homologous peptide in the male rat reproductive system

TRH and a TRH-like peptide have been shown to occur throughout the male rat reproductive system by TRH radioimmunoassay, SP-Sephadex C25 cation exchange chromatography, high pressure liquid chromatography and parallel line analysis. The total concentration of TRH and TRH-like peptide was highest in the prostate followed by the testis, cauda epididymis and seminal vesicles. Dilution curves for extracts of prostate, testis and seminal vesicles were parallel with TRH while the corresponding curve for epididymis was nonparallel.     

Characterization of neutral TRH-like peptides in mammary gland, mammary tumors and milk

Three pyroglutamylpeptide amides, pGlu-Glu-Pro amide, pGlu-Phe-Pro amide and pGlu-Gln-Pro amide, with similar structures to thyrotropin-releasing hormone (TRH), have been identified previously in the male reproductive system. We report here that rat and human mammary gland contain neutral TRH-immunoreactive peptides which are not retained on cation or anion exchange chromatography and that similar peptides occur in the milk of rat, cow, ewe and sow. The TRH-like peptides in lyophilized milk from the cow were purified by gel exclusion chromatography, mini-column cation exchange chromatography and reversed phase high performance liquid chromatography (HPLC) and the chromatographed peptides were located by TRH radioimmunoassay (RIA). In each chromatographic system the major TRH-immunoreactive peptide from cow milk exhibited identical behavior to pGlu-Phe-Pro amide; in addition there were two minor TRH-immunoreactive components. The possible physiological role of the TRH-like peptides in the mammary gland is discussed. In a series of patients with breast carcinoma, mammary tumor tissue was shown to contain approximately four times more TRH-like peptide than normal mammary tissue from the same patient, raising the possibility that the TRH-like peptides may be implicated in tumor development.     

Identification of the TRH-like peptides pGlu-Glu-Pro amide and pGlu-Phe-Pro amide in rat thyroid: regulation by thyroid status.

Rat thyroid contains thyrotropin-releasing hormone (TRH) and TRH-like peptides which react with TRH antisera. We have identified the TRH-like peptides in the thyroid and examined whether their levels are influenced by thyroid status. The peptides were extracted from the thyroid glands of five hyperthyroid rats and purified by ion-exchange chromatography on SP-Sephadex C25 and reversed-phase high performance liquid chromatography. The principal TRH-immunoreactive component exhibited the same retention on HPLC as synthetic pGlu-Glu-Pro amide and a secondary component corresponded to synthetic pGlu-Phe-Pro amide. In agreement with these assignments the main peptide was shown to be acidic when chromatographed on DEAE-Sephadex A25 and the second peptide neutral. The levels of TRH and TRH-like peptides in the thyroid were investigated in hyper-, hypo- and euthyroid rats. Hyperthyroidism was induced by chronic subcutaneous administration of triiodothyronine (T3) and hypothyroidism was produced by addition of propylthiouracil (PTU) to the drinking water. The amounts of the peptides were determined by radioimmunoassay with a TRH-antiserum, carried out after extraction from the tissues and purification by ion exchange chromatography. The mean concentration of TRH-like peptides in the thyroids of the hyperthyroid rats was 95.5+/-25.5 pmol/g, the mean concentration in the hypothyroid rats was 11.7+/-3.4 pmol/g, and in the euthyroid rats 17.6+/-3.2 pmol/g. The concentrations of TRH were less influenced by thyroid status: the values in hyper-, hypo- and euthyroid rats were 47.5+/-9.4, 42.1+/-6.3, and 17.2+/-1.6 pmol/g respectively. The results show that the levels of the TRH-like peptides in rat thyroid are highly sensitive to thyroid status, suggesting a possible involvement in thyroid regulation.     

TRH-like peptides in prostate gland and other tissues

This minireview is aimed to recapitulate the occurrence of TRH-like peptides in the prostate gland and other tissues and to discuss their known functions in the organism. The hypothalamic thyrotropin-releasing hormone (TRH) was the first chemically defined hypophyseotropic hormone with the primary structure pGLU-HIS-PRO.NH2. However, the presence of extrahypothalamic TRH-immunoreactive peptides was reported in peripheral tissues including the gastrointestinal tract, placenta, neural tissues, male reproductive system and certain endocrine tissues. It was supposed that this TRH immunoreactivity can partially originate from TRH-homologous peptides and that these peptides have significant cross-reactions with the antibody specific against authentic TRH. This assumption was confirmed by the identification of prostatic TRH immunoreactivity as pyroGLU-GLU-PRO.NH2 using fast atom bombardment mass spectrometry and gas phase sequence analysis. TRH-like peptides are characterized by substitution of the basic amino acid histidine (related to authentic TRH) for neutral or acidic amino acids, such as glutamic acid, phenylalanine, glutamine or tyrosine. The physiological role of TRH-like peptides in peripheral tissues is not precisely known, but they possess a C-terminal amide group which is characteristic for many biologically active peptides. The occurrence of these peptides in the male reproductive system can influence male fertility. They are also closely related to circulating thyroid and steroid hormones. There might be an important connection of TRH-like peptides to the prostatic local autocrine/paracrine network mediated by extrahypothalamic TRH immunoreactivity corresponding to TRH-like peptides and extrapituitary thyrotropin (TSH) immunoreactivity also found in the prostatic tissue. A similar system of intraepithelial lymphocyte hormonal regulation due to the local paracrine network of TRH/TSH has been described in the gastrointestinal tract. The local network of TRH-like peptides/TSH may be involved in possible regulation of prostatic growth.     

Effects of dopamine on the release of thyrotropin-releasing hormone from the rat retina in vitro.

The effects of dopamine on the release of thyrotropin-releasing hormone (TRH) from the rat retina in vitro were studied. The rat retina was incubated in the medium 199 (pH 7.4) with 1.0 mg/ml of bacitracin and 100 micrograms/ml of ascorbic acid. The amount of TRH release into the medium was measured by radioimmunoassay. The TRH release from the rat retina was inhibited significantly in a dose-related manner with the addition of dopamine, but not with pimozide. The inhibitory effects of dopamine on TRH release from the rat retina were blocked with an addition of pimozide to the medium. The elution profile of methanol-extracted rat retina on sephadex G-10 was identical to that of synthetic TRH. From these findings it is concluded that the dopaminergic system inhibits TRH release from the rat retina in vitro.     

Detection of TRH extended peptides in rat hypothalamus using an antibody raised to pGluHisProGlyLys

An antibody was raised to the synthetic pentapeptide pGluHisProGlyLys which, in radioimmunoassay (RIA), could detect the pentapeptide at a level of 10 fmole per tube and exhibited <0.5 per cent cross reactivity with a series of related peptides. The RIA was used to demonstrate the presence of C-terminally extended forms of thyrotropin releasing hormone (TRH) in rat hypothalamus. After extraction, the endogenous peptides were resolved by gel exclusion chromatography and TRH-extended peptides were revealed by trypsin digestion to release the pentapeptide. The TRH extended peptides occurred in substantial quantity, approximately 11 pmoles/g, indicating that only partial processing of the gene duplicated prohormone takes place.     

Effects of acetylcholine on thyrotropin-releasing hormone release from the rat caecum in vitro

Effects of acetylcholine on the release of thyrotropin-releasing hormone (TRH) from the rat caecum in vitro were studied. The rat caecum was incubated in medium 199 with 1.0 mg/ml of bacitracin and 100 micrograms/ml of ascorbic acid (pH 7.4) (medium). The amount of TRH release into the medium was measured by radioimmunoassay. The immunoreactive TRH (ir-TRH) release from the rat caecum was enhanced significantly in a dose-related manner with the addition of acetylcholine, but not changed with atropine. The stimulatory effect of acetylcholine on ir-TRH release from the rat caecum was blocked with an addition of atropine. Elution profile of acid-methanol-extracted rat caecum on Sephadex G-10 was identical to that of synthetic TRH. The findings suggest that the cholinergic system stimulates TRH release from the rat caecum in vitro.     

Rapid modulation of TRH and TRH-like peptide release in rat brain and peripheral tissues by ghrelin and 3-TRP-ghrelin

Ghrelin is not only a modulator of feeding and energy expenditure but also regulates reproductive functions, CNS development and mood. Obesity and major depression are growing public health concerns which may derive, in part, from dysregulation of ghrelin feedback at brain regions regulating feeding and mood. We and others have previously reported that thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH(2)) and TRH-like peptides (pGlu-X-Pro-NH(2), where "X" can be any amino acid residue) have neuroprotective, antidepressant, anti-epileptic, analeptic, anti-ataxic, and anorectic properties. For this reason male Sprague-Dawley rats were injected ip with 0.1mg/kg rat ghrelin or 0.9mg/kg 3-Trp-rat ghrelin. Twelve brain regions: cerebellum, medulla oblongata, anterior cingulate, posterior cingulate, frontal cortex, nucleus accumbens, hypothalamus, entorhinal cortex, hippocampus, striatum, amygdala, piriform cortex and 5 peripheral tissues (adrenals, testes, epididymis, pancreas and prostate) were analyzed. Rapid and profound decreases in TRH and TRH-like peptide levels (increased release) occurred throughout brain and peripheral tissues following ip ghrelin. Because ghrelin is rapidly deacylated in vivo we also studied 3-Trp-ghrelin which cannot be deacylated. Significant increases in TRH and TRH-like peptide levels following 3-Trp-ghrelin, relative to those after ghrelin were observed in all brain regions except posterior cingulate and all peripheral tissues except prostate and testis. The rapid stimulation of TRH and TRH-like peptide release by ghrelin in contrast with the inhibition of such release by 3-Trp-TRH is consistent with TRH and TRH-like peptides modulating the downstream effects of both ghrelin and unacylated ghrelin.     

Dopamine inhibits thyrotropin-releasing hormone release from rat adrenal gland in vitro

The effects of dopamine on the release of thyrotropin-releasing hormone (TRH) from the rat adrenal gland were studied in vitro. The rat adrenal glands were incubated in medium 199 with 1.0 mg/ml of bacitracin and 100 micrograms/ml of ascorbic acid (pH 7.4) (medium) for 20 min. The amount of TRH release into the medium was measured by radioimmunoassay. The immunoreactive TRH (ir-TRH) release from the rat adrenal gland was inhibited significantly in a dose-related manner with the addition of dopamine and enhanced with the addition of pimozide or domperidone to the medium. Dopamine's effects on ir-TRH release from the adrenal gland were blocked with the addition of pimozide or domperidone. The elution profile of methanol-extracted rat adrenal gland was identical to that of synthetic TRH. The findings suggest that the dopaminergic system inhibits TRH release from the rat adrenal gland.     

TRH-related peptides in the rabbit prostate complex during development.

The novel peptide, pyroglutamylglutamylprolineamide (pGlu-Glu-ProNH2), has recently been isolated and characterized from the rabbit prostate complex. The tripeptide is present in high concentrations in the prostate complex and semen, together with a 40-50 residue polypeptide which contains a TRH-immunoreactive fragment at its C-terminus. The present study investigates changes in the levels of these TRH-related peptides in rabbits aged 11 weeks, 4 months, 7 months, 13 months and 2 years. For each age group the peptides were extracted from the prostate complex, separated by gel exclusion chromatography, and located by TRH radioimmunoassay. The TRH-immunoreactive fragment was released from the polypeptide by trypsin digestion prior to radioimmunoassay. Very low concentrations of TRH-immunoreactive peptides were present at 11 weeks of age, but considerable levels of both peptides were found in all the other age groups. Anion exchange chromatography, under conditions which resolve TRH and pGlu-Glu-ProNH2, showed that the majority of the low molecular weight TRH immunoreactivity co-eluted with synthetic pGlu-Glu-ProNH2. The remaining TRH immunoreactivity, which had not bound to the anion resin, also failed to bind to a cation exchange column at pH 2.0, indicating that it was not authentic TRH. Dissection of the prostate complex into its four constitutive regions (vesicular gland, coagulating gland, prostate and bulbourethral gland) followed by extraction, chromatography and TRH radioimmunoassay of each region showed that the TRH-related peptides were located in the prostate.     

The computer modelling of human TRH receptor, TRH and TRH-like peptides

The aim of this work was to verify the possibility of interactions between the human TRH receptor (an integral membrane protein which belongs to family 1 of G-protein coupled receptors) and TRH-like peptides presented in the prostate gland. These peptides are characterized by substitution of basic amino acid histidine (related to authentic TRH) for neutral or acidic amino acid, such as glutamic acid, phenylalanine, glutamine or tyrosine. The physiological function of TRH-like peptides in peripheral tissues is not precisely known. However, according to our recent experiments, we assume the existence of a local hormonal network formed by TRH-like peptides and TSH in the prostate gland. The network can be associated with circulating thyroid and steroid hormones, and may represent a new regulatory mechanism influencing the proliferative ability of prostatic tissue. A similar network of authentic TRH and TSH was already found in the gastrointestinal tract. The experimentally determined 3D-structures of human TRH receptor (hTRHr) and TRH-like peptides are not available. From this point of view we used de novo modeling procedures of G-protein coupled receptors on an automated protein modeling server used at the Glaxo Wellcome Experimental Research (Geneva, Switzerland). 3D-structures of TRH-like peptides were determined with a computer program CORINA (written by the team of J. Gasteiger, Computer-Chemie-Centrum and Institute for Organic Chemistry, University of Erlangen-Nurenberg, Germany). The generated PDB files with 3D-coordinates were visualized with Swiss-Pdb Viewer Release 3.51 (Glaxo Wellcome). From recent results it is evident that polar amino acids belonging to the extracellular terminus of hTRHr transmembrane regions can participate in interactions between TRH and hTRHr. There is no direct evidence that TRH-like peptides interact with the presented hTRHr model. On the contrary, with respect to the similar 3D-shape and the identity of terminal amino acids, it appears that these interactions are highly probable as well as the nearly 100 % cross-reactions between TRH or TRH-like peptides and antibody specific against authentic TRH. Closed terminal amino acids (pyroglutamic acid and proline-amide) of TRH or TRH-like peptides are important for these interactions. Desamido-TRH or glutamyl metabolites will be repelled by the negative potential of ASP195 (E: D93) and GLU298 (G: E137).     

Effects of histamine and related compounds on the release of immunoreactive thyrotropin-releasing hormone from the rat stomach in vitro

Effects of histamine and related compounds on the release of immunoreactive thyrotropin-releasing hormone (ir-TRH) from the rat stomach in vitro were studied. The rat stomach was incubated in medium 199 with 1.0 mg/ml of bacitracin and 100 micrograms/ml of ascorbic acid (pH 7.4) for 20 min. The amount of TRH release into the medium was measured by radioimmunoassay. The ir-TRH release from the rat stomach was enhanced significantly in a dose-related manner with the addition of histamine and inhibited with the addition of famotidine, but not with mepyramine. The stimulatory effect of histamine on ir-TRH release from the stomach was partially blocked with the addition of famotidine, but not with mepyramine. The elution profile of acid-methanol-extracted rat stomach on Sephadex G-10 was identical to that of synthetic TRH. These findings suggest that histamine stimulated ir-TRH release from the rat stomach in vitro, and that histamine's effects may be mediated via a H2-receptor.     

Effects of opioid peptides on thyrotropin-releasing hormone release from the rat caecum in vitro

Effects of opioid peptides (beta-endorphin, dynorphin (1-13). alpha-neoendorphin, beta-neoendorphin, leucine-enkephalin, methionine-enkephalin) on the release of thyrotropin-releasing hormone (TRH) from the rat caecum were studied in vitro. The rat caecum was incubated in medium 199 with 1.0 mg/ml of bacitracin (pH 7.4) (medium). The amount of TRH release from the rat caecum into the medium was measured by radioimmunoassay. The immunoreactive TRH (ir-TRH) release from the rat caecum was inhibited significantly in a dose-related manner with the addition of opioid peptides. The inhibitory effects of opioid peptides on ir-TRH release from the rat caecum were blocked with an addition of naloxone. The elution profile of acid-methanol-extracts of rat caecum on Sephadex G-10 was identical to that of synthetic TRH. The findings suggest that opioid peptides inhibit TRH release from the rat caecum in vitro.     

Regional distribution of in vitro release of thyrotropin releasing hormone in rat brain

To increase our knowledge of the TRH functions in brain and the processes of TRH compartmentalization and release, we studied the in vitro release of endogenous TRH in different brain areas. We also determined the correlation between TRH levels and release under both basal and stimulated conditions. TRH concentration was measured in tissues and media by specific radioimmunoassay. TRH-like material detected in olfactory bulb and hypothalamic incubates (basal or K+ stimulated) were shown to be chromatographically identical to synthetic TRH. Different brain regions showed high variability in the basal release of TRH (1-20% of tissue content). This suggests the existence of different pools. The response to depolarizing stimulus (56 mM K+) was significant only in the following regions: median eminence, total hypothalamus, preoptic area, nucleus accumbens-lateral septum, amygdala, mesencephalon, medulla oblongata and the cervical region of the spinal cord. These regions have been shown to contain a high number of receptors, a high concentration of TRH nerve endings and are susceptible to TRH effects. These results support the hypothesis that TRH functions as neuromodulator in these areas.     

Valproate and copper accelerate TRH-like peptide synthesis in male rat pancreas and reproductive tissues

Treatment with valproate (Valp) facilitates the synthesis of TRH-like peptides (pGlu-X-Pro-NH(2)) in rat brain where "X" can be any amino acid residue. Because high levels of TRH-like peptides occur in the pancreas and pGlu-Glu-Pro-NH(2) (Glu-TRH) has been shown to be a fertilization promoting peptide, we hypothesized that these peptides mediate some of the metabolic and reproductive side effects of Valp. Male WKY rats were treated with Valp acutely (AC), chronically (CHR) or chronically followed by a 2 day withdrawal (WD). AC, CHR and WD treatments significantly altered TRH and/or TRH-like peptide levels in pancreas and reproductive tissues. Glu-TRH was the predominant TRH-like peptide in epididymis, consistent with its fertilization promoting activity. Glu-TRH levels in the epididymis increased 3-fold with AC Valp. Phe-TRH, the most abundant TRH-like peptide in the pancreas, increased 4-fold with AC Valp. Phe-TRH inhibits both basal and TRH-stimulated insulin release. Large dense core vesicles (LDCV's) contain a copper-dependent enzyme responsible for the post-translational processing of precursors of TRH and TRH-like peptides. Copper (500 microM) increased the in vitro C-terminal amidation of TRH-like peptides by 8- and 4-fold during 24 degrees C incubation of homogenates of pancreas and testis, respectively. Valp (7 microM) accelerated 3-fold the processing of TRH and TRH-like peptide precursors in pancreatic LDCV's incubated at 24 degrees C. We conclude that copper, an essential cofactor for TRH and TRH-like peptide biosynthesis that is chelated by Valp, mediates some of the metabolic and reproductive effects of Valp treatment via acceleration of intravesicular synthesis and altered release of these peptides.     

The TRH-related peptide pyroglutamyglutamylprolinamide is present in human semen

We have recently identified a novel peptide in the rabbit prostate complex which cross-reacts with an antibody to thyrotrophin-releasing hormone (TRH) and has the structure pGlu-Glu-ProNH2. In the present study, high concentrations of a TRH-related tripeptide and also a polypeptide (10-12 kDa) containing a TRH-immunoreactive peptide at its C-terminus were detected in human semen. The low molecular mass TRH-like peptide and the immunoreactive fragment from the polypeptide were isolated from human semen and shown to have identical structures. Amino acid analysis suggested compositions Glx2, Pro1, and after mild acid hydrolysis, the same sequence, Glu-Glu-Pro, was established for the two peptides. Fast atom bombardment (FAB) mass spectrometry yielded a pseudomolecular ion (M + H)+ of 355.38 which was identical to that of the synthetic peptide pGlu-Glu-ProNH2. The data demonstrate that human semen contains the TRH-like peptide pyroglutamylglutamylprolinamide and also a polypeptide terminating in the sequence Gln-Glu-ProNH2.     

Thyrotropin-releasing hormone and a homologous peptide in the reproductive system of the female rat and pig

TRH and a TRH homologous peptide have been shown to occur throughout the female rat and pig reproductive systems by TRH radioimmunoassay, SP-Sephadex C-25 cation exchange chromatography, and parallel line analysis of the assays. The total amount of TRH and TRH homologous peptide immunoreactivity was highest in the oviducts followed by the ovary and then uterus. The concentration of TRH immunoreactivity in all reproductive organs of the rat fell gradually from one month of age. TRH and the TRH homologous peptide were not parallel on serial dilution and measurement in the same TRH radioimmunoassay. The rapid degradation of TRH by pig follicular fluid may explain the higher measured concentration of TRH homologous peptide compared to TRH not only in pig follicular fluid but also in the pig ovary as a whole.     

Effects of calcium hopantenate on the release of thyrotropin-releasing hormone from the rat adrenal gland in vitro

The effects of calcium hopantenate (HOPA), a GABA agonist, on the release of thyrotropin-releasing hormone (TRH) from the rat adrenal gland were studied in vitro. The adrenal glands were incubated in medium 199 with 1.0 mg/ml of bacitracin (pH 7.4) (medium) for 20 min. The amount of TRH release into the medium was measured by radioimmunoassay. The TRH release from the rat adrenal gland was inhibited significantly in a dose-related manner with the addition of HOPA to the medium. HOPA's effects on TRH release from the adrenal gland were blocked with the addition of bicuculline, a GABA receptor inhibitor. The elution profile of methanol-extracted rat adrenal gland TRH was identical to that for synthetic TRH. The findings suggest that HOPA inhibits TRH release from the rat adrenal gland, and that its effects are mediated via the GABA receptor.     

Three TRH-Like Molecules Are Released from Rat Hypothalamus In Vitro

TRH-like immunoreactivity distinct from TRH is present in various tissues and fluids. In order to determine whether TRH-like molecules are secreted by the hypothalamus, we analyzed tissues and media from hypothalamic slices incubated in Krebs Ringer bicarbonate. Media from basal or high KCl conditions contained 3 TRH-like molecules evidenced by reverse phase high performance liquid chromatography followed by TRH radioimmunoassay. Peak I corresponded to authentic TRH (73% of total immunoreactivity) and peaks II and III had a higher retention time. These additional TRH-like forms were neither detected in hypothalamic tissue nor in tissue or medium from olfactory bulb. Gel filtration analysis of hypothalamic media revealed only one TRH-like peak eluting as TRH, suggesting that the molecular weights of peaks II and III are similar to that of TRH. Peak II retention time was similar to that of pglu-phe-proNH₂. We analysed if they could be produced by post secretory metabolism of TRH. Incubation of hypothalamic slices with [³H-Pro]-TRH did not produce radioactive species comigrating with peaks II or III. However, it induced rapid degradation to [³H-Pro]-his-prodiketopiperazine ([³H]-HPDKP). Inhibitor profile suggested that pyroglutamyl aminopeptidase II, but not pyroglutamyl aminopeptidase I, is responsible for [³H]-HPDKP production. These data are consistent with the hypothesis that pyroglutamyl aminopeptidase II is the main aminopeptidase degrading TRH in hypothalamic extracellular fluid. Furthermore, we suggest that the hypothalamus releases additional TRH-like molecules, one of them possibly pglu-phe-proNH₂, which may participate in control of adenohypophyseal secretions.