Thyrotropin-releasing Hormone Antibody Research Articles

TRH/TRH-R1 Receptor Signaling in the Brain Medulla as a Pathway of Vagally Mediated Gut Responses During the Cephalic Phase

Pavlov's seminal findings in the early twentieth century showed that the sight, smell or taste of food in dogs with chronic esophagostomy induces a vagal-dependent gastric acid secretion. These observations established the concept of the cephalic phase of digestion. Compelling experimental evidence in rats indicates that the three amino acid peptide thyrotropin-releasing hormone (TRH) expressed in the brainstem plays a key role in the vagal stimulation of gastric function. Neurons in the dorsal motor nucleus of the vagus (DMN) expressed TRH receptor subtype (TRH-R1) and received efferent input from TRH containing fibers arising from TRH synthesizing neurons in the raphe pallidus, raphe obscurus, and the parapyramidal regions. TRH microinjected into the DMN or intracisternally excites the firing of DMN neurons and stimulates efferent activity in the gastric branch of the vagus nerve and gastric myenteric cholinergic neurons. At the functional level, this results in a vagally-mediated and atropine-sensitive stimulation of gastric epithelial and endocrine cells secreting acid, pepsin, serotonin, histamine and ghrelin, and enteric neurons leading to increased gastric motility and emptying. Importantly, the blockade of TRH or TRH-R1 in the brainstem by pretreatment into the cisterna magna or the DMN with TRH antibody or TRH-R1 oligodeoxynucleotide antisense respectively abolishes the stimulation of gastric acid induced by sham-feeding. The gastric response to TRH injected into the DMN is potentiated by serotonin and the proTRH flanking peptide, Ps4 and suppressed by a number of brainstem peptides and cytokines activated during stress or immune response and inhibiting food intake and gastric acid secretion. These convergent data strongly support a physiological involvement of TRH signaling pathway in the brainstem to stimulate vagal activity and identified TRH-TRH-R1 system as a major effector in the dorsal vagal complex to drive the vagally mediated gut response triggered by the cephalic phase.

CREB/TRH pathway in the central nervous system regulates energy expenditure in response to deprivation of an essential amino acid.

In the central nervous system (CNS), thyrotropin-releasing hormone (TRH) has an important role in regulating energy balance. We previously showed that dietary deprivation of leucine in mice increases energy expenditure through CNS-dependent regulation. However, the involvement of central TRH in this regulation has not been reported. Male C57J/B6 mice were maintained on a control or leucine-deficient diet for 7 days. Leucine-deprived mice were either third intracerebroventricular (i.c.v.) injected with a TRH antibody followed by intraperitoneal (i.p.) injection of triiodothyronine (T3) or i.c.v. administrated with an adenovirus of shCREB (cAMP-response element binding protein) followed by i.c.v. injection of TRH. Food intake and body weight were monitored daily. Oxygen consumption, physical activity and rectal temperature were assessed after the treatment. After being killed, the hypothalamus and the brown adipose tissue were collected and the expression of related genes and proteins related was analyzed. In other experiments, control or leucine-deficient medium incubated primary cultured neurons were either infected with adenovirus-mediated short hairpin RNA targeting extracellular signal-regulated kinases 1 and 2 (Ad-shERK1/2) or transfected with plasmid-overexpressing protein phosphatase 1 regulatory subunit 3C (PPP1R3C). I.c.v. administration of anti-TRH antibodies significantly reduced leucine deprivation-stimulated energy expenditure. Furthermore, the effects of i.c.v. TRH antibodies were reversed by i.p. injection of T3 during leucine deprivation. Moreover, i.c.v. injection of Ad-shCREB (adenovirus-mediated short hairpin RNA targeting CREB) significantly suppressed leucine deprivation-stimulated energy expenditure via modulation of TRH expression. Lastly, TRH expression was regulated by CREB, which was phosphorylated by ERK1/2 and dephosphorylated by PPP1R3C-containing protein Ser/Thr phosphatase type 1 (PP1) under leucine deprivation in vitro. Our data indicate a novel role for TRH in regulating energy expenditure via T3 during leucine deprivation. Furthermore, our findings reveal that TRH expression is activated by CREB, which is phosphorylated by ERK1/2 and dephosphorylated by PPP1R3C-containing PP1. Collectively, our studies provide novel insights into the regulation of energy homeostasis by the CNS in response to an essential amino-acid deprivation.

Activation of raphe pallidus neurons increases insulin through medullary thyrotropin-releasing hormone (TRH)-vagal pathways.

Pancreatic insulin secretion is regulated by the vagus nerve. Medullary thyrotropin-releasing hormone (TRH) containing projections from the raphe pallidus (Rpa) neurons innervate vagal preganglionic motor neurons in the dorsal vagal complex (DVC) and are involved in vagal regulation of gastric functions. To investigate whether chemical stimulation of Rpa neurons influences circulating insulin levels through brain medullary TRH-vagal pathways. In fasted, pentobarbital-anesthetized rats, kainic acid (10 ng/50 nL) was microinjected into the Rpa, and serum insulin levels were measured. Gastric acid secretion was monitored as a control of vagally mediated visceral response. Chemical stimulation of Rpa neuronal cell bodies significantly increased serum insulin levels. Values before and at 30, 60, and 90 minutes after the microinjection of kainic acid were 0.34 +/- 0.02, 0.54 +/- 0.06, 0.60 +/- 0.06, and 0.99 +/- 0.13 ng/mL, respectively. In the same rats, gastric acid secretion was stimulated (basal, 2.3 +/- 0.6, versus 26.1 +/- 8.6 micromol/15 min at 30 minutes). Microinjections outside of the Rpa had no effect. The Rpa stimulation-induced increase in serum insulin could be mimicked by DVC microinjection of TRH analog, completely prevented by bilateral cervical vagotomy, and significantly reduced by bilateral microinjection of TRH antibody into the DVC. Chemical activation of Rpa neurons increases pancreatic insulin release through medullary TRH and vagal-mediated pathways.

PROSTATE-THYROID AXIS: PROSTATIC TRH IS ONE OF THE STIMULATORS OF THYROID HORMONE

Ventral prostatectomy decreased serum thyroid hormones and histology of the thyroid gland indicate that hypothyroid condition. Co-culture of thyroid gland and ventral prostate stimulates thyroid hormone secretion. In the present study we report prostatic thyrotropin releasing hormone (TRH) is the stimulating factor of thyroid hormone secretion. Mature rat (90 days old) ventral prostate, anterior pituitary and thyroid glands were co-cultured in vitro with or without TRH antibody to assess the direct influence of prostatic TRH on thyroid hormone secretion. Total thyroxine (T4) and triiodothyronine (T3) were increased significantly in the culture media of ventral prostate, anterior pituitary and thyroid gland when compared with thyroid gland plus anterior pituitary culture media. However, media T4 and T3 concentration decreased significantly in thyroid gland alone; also in thyroid gland plus ventral prostate, thyroid gland plus anterior pituitary and thyroid gland plus anterior pituitary plus ventral prostate were co-cultured with TRH antibody (Ab) in a dose dependent manner. The results suggest that ventral prostatic TRH is one of the stimulating factors of thyroid hormone secretion under these in vitro conditions.

Medullary thyrotropin-releasing hormone mediates vagal-dependent adaptive gastric protection induced by mild acid in rats

Background & Aims : Adaptive gastric protection is dependent on vagal pathways in rats. It is hypothesized that medullary thyrotropin-releasing hormone (TRH), known to regulate vagal function, is part of the brain mechanisms mediating adaptive gastric protection. Methods : Urethane-anesthetized rats were pretreated with either acute bilateral subdiaphragmatic vagotomy, sham operation, or intracisternal injection of purified control, TRH, or peptide YY antibody. Gastric lesions were assessed 75 minutes after orogastric administration of 1 mL of either vehicle or 0.35N HCI followed 15 minutes later by 0.6N or 1.0N HCI. Results : Injection of 0.6N and 1.0N HCI induced gastric lesions covering 23.1% ± 2.7% and 37.8% ± 3.3% of the corpus mucosa, respectively. Pretreatment with 0.35N HCI resulted in 67.3% and 50.5% reductions in gastric lesions induced by 0.6N and 1.0N HCI, respectively. Subdiaphragmatic vagotomy or intracisternal injection of TRH antibody increased gastric lesions induced by 0.6N HCI to 32.2% ± 2.2% and 42.9% ± 5.6%, respectively, and completely abolished the protective effect of 0.35N HCI pretreatment. Control or peptide YY antibody injected intracisternally did not alter the gastric protection induced by mild acid. Conclusions : These results indicate that medullary TRH plays a role in the vagally mediated adaptive gastric protection induced by mild acid.

Medullary TRH is involved in gastric protection induced by low dose of kainic acid into the raphe pallidus.

The gastroprotective effect of kainic acid microinjected into the raphe pallidus (Rpa) at a dose subthreshold to increase acid secretion was investigated in urethan-anesthetized rats. Kainic acid (25 pg/30 nl) microinjected into the Rpa inhibited by 65.8% gastric damage induced by intragastric ethanol (60%). No protection was observed when kainic acid was injected outside of the Rpa. The cytoprotective effect was completely abolished by thyrotropin-releasing hormone (TRH) antibody microinjected bilaterally (1.3 micrograms/site) into the dorsal motor nucleus of the vagus (DMN), indomethacin (5 mg/kg ip), and atropine (0.3 mg/kg sc). Microinjection of TRH antibody outside of the DMN or of control antibody into the DMN did not modify the protective action induced by kainic acid into the Rpa. The TRH antibody microinjected alone into the DMN did not alter the severity of the ethanol-induced gastric lesions. These data indicate that excitation of Rpa neurons by a low dose of kainic acid results in cytoprotection against ethanol lesions. Furthermore, this cytoprotection occurs as a result of TRH action in the DMN and activation of muscarinic and prostaglandin pathways.

Intracisternal injection of TRH antibody blocks gastric emptying stimulated by 2-deoxy- d-glucose in rats

We evaluated the effect of 2-deoxy- d-glucose (2-DG) on gastric emptying of a non nutrient solution in conscious rats using a Phenol red method. Intravenous injection of 2-deoxy- d-glucose dose-dependently increased the rate of gastric emptying. This stimulatory action of 2-DG was abolished by subdiaphragmatic vagotomy. Intracisternal injection of thyrotropin-releasing hormone (TRH) antibody blocked intracisternal TRH and intravenous 2-DG-induced enhancement of gastric empyting but not the stimulation of gastric emptying induced by intracisternal pancreatic polypeptide. The TRH antibody injected intraperitoneally had no effect. These results suggest that endogenous TRH in the brain is involved in vagal-dependent stimulation of gastric emptying by 2-DG.

New hapten-protein conjugation method using N-( m-aminobenzoyloxy) succinimide as a two-level heterobifunctional agent: thyrotropin-releasing hormone as a model peptide without free amino or carboxyl groups

The use of a two-level heterobifunctional agent N-( m-aminobenzoyloxy)succinimide ( m-ABS) allowed us to develop a new method for preparing hapten-protein conjugates. This was demonstrated by a conjugation between thyrotropin-releasing hormone (TRH) and bovine or human serum albumin (BSA or HSA). The conjugation is based on the principle that the succinimidyl ester group of m-ABS immediately acts on an ε-amino group of lysine residues of carrier protein BSA (or HSA) and a m-aminobenzoyl group incorporated into the protein is then activated by diazotization to a functional m-diazobenzoyl group ( m-DB) acting on a histidyl group of TRH. The TRH-BSA containing about 3.5 mol of TRH per BSA molecule, elicited the production of TRH antibody in rabbits. A new type of enzyme-linked immunosorbent assay (ELISA) for TRH was developed using the antiserum, the solid-phase antigen TRH-HSA and the commercially available horseradish peroxidase-labeled goat anti-rabbit IgG/Fab′ as a marker, revealing that the ELISA was monospecific to the hormone and measured as low as 50 pg of the hormone reproducibly. Also, using the antiserum by the indirect immunoperoxidase method the distribution of immunoreactive TRH in the rat brain was demonstrated in neurons of the paraventricular nucleus and neuronal processes of the median eminence. These results strongly suggested that the use of m-ABS provided a simple and efficient new method for preparing immunogens not only for the previously reported haptens with a primary amino group(s) (J. Immunol. Methods 134 (1990) 227), but also for haptens with an imidazole, phenolic, or indole group(s) in the molecule.

Raphe pallidus stimulation increases gastric contractility via TRH projections to the dorsal vagal complex in rats

The role of thyrotropin releasing hormone (TRH) in the dorsal vagal complex (DVC) in mediating the enhanced gastric contractility induced by glutamate (100 pmol) microinjected into the raphe pallidus (Rpa) was investigated in urethane-anesthetized rats acutely implanted with miniature strain gauge force transducers on the corpus of the stomach. Glutamate-induced stimulation of gastric contractility was dose-dependently inhibited by bilateral microinjection into the DVC of TRH antibody (0.17, 0.85 or 1.7 μg/100 nl/site) but not by vehicle. TRH antibody microinjected into the dorsal medullary reticular field had no effect. These data indicate that activation of Rpa neurons by glutamate increases gastric motor function through TRH release in the DVC.

TRH in dorsal vagal complex mediates acid response to excitation of raphe pallidus neurons in rats.

The role of thyrotropin-releasing hormone (TRH) in the dorsal vagal complex (DVC) in the acid response to excitation of raphe pallidus neurons was investigated in urethan-anesthetized rats with gastric fistula. Kainic acid (0.19 microgram/30 nl) microinjected into the raphe pallidus stimulated gastric acid secretion. The response was prevented by vagotomy. A specific polyclonal TRH antibody, 8964, was raised and characterized (50% inhibitory dose for TRH was 80 pg/ml at an antibody final dilution of 1:10(5)). The TRH antibody injected intracisternally blocked the acid response to intracisternal TRH, but not that of the TRH analogue RX-77368. The TRH antibody (0.33, 0.65, or 1.3 micrograms.100 nl-1.site-1) microinjected bilaterally into the DVC prevented dose dependently by 31, 60, and 76%, respectively, the increase in acid secretion induced by kainic acid injected into the raphe pallidus. The TRH antibody (1.3 microgram/site) microinjected into medullary sites outside of the DVC had no effect. These data indicate that excitation of raphe pallidus neurons induces a vagal-dependent stimulation of gastric acid secretion that is mediated by endogenous TRH in the DVC. TRH neurons in the raphe pallidus projecting to the DVC may have a physiological relevance in the vagal regulation of gastric function.

Isolation and identification of two neutral thyrotropin releasing hormone-like peptides, pyroglutamylphenylalanineproline amide and pyroglutamylglutamineproline amide, from human seminal fluid.

Two tripeptide amides with stuctures similar to thyrotropin releasing hormone were isolated from human seminal fluid and their amino acid sequences determined. The peptides were purified by gel exclusion from Sephadex G50 and were detected by radioimmunoassay with thyrotropin releasing hormone antibody; in addition, N-terminally extended forms were demonstrated by radioimmunoassay after trypsin digestion. Further purification of the tripeptides was by chromatography on SP-Sephadex C25 and by high performance liquid chromatography on C18 Microbondapak using an HCl/acetonitrile gradient. After exclusion from mini-columns of SP-Sephadex C25 and DEAE-Sephadex A25, two neutral peptides were obtained in homogeneous form by high performance liquid chromatography with an HCl/methanol gradient. Amino acid analysis gave the following compositions: Glu, 0.74, Phe, 1.0, Pro, 1.0; and Glu, 1.72, Pro, 1.0. Both peptides possessed a blocked N terminus, but after opening the pyroglutamyl ring the sequences Glu-Phe-Pro and Glu-Glx-Pro were demonstrated. The chromatographic properties of the endogenous peptides were identical to the properties of the corresponding synthetic peptides. The structure of pGlu-Phe-Pro (where p-indicates pyro-) amide was confirmed by fast atom bombardment mass spectrometry. The presence in human semen of three structurally related peptides, pGlu-Phe-Pro amide, pGlu-Gln-Pro amide, and the previously reported pGlu-Glu-Pro amide (Cockle, S. M., Aitken, A., Beg, F., and Smyth, D. G. (1989) J. Biol. Chem. 264, 7788-7791), suggests that this series of peptides may have evolved to fulfil complementary biological roles.

Enzyme immunoassay of thyrotropin releasing hormone (TRH).

A sensitive and specific double-antibody enzyme immunoassay (EIA) for a thyrotropin releasing hormone (TRH)-like immunoreactive substance has been developed. In order to synthesize TRH-labeled beta-D-galactosidase (beta-gal), a newly devised TRH derivative, pGlu-His-Pro-NH-(CH2)6-NH2 (TRH-Hex), was employed. TRH-Hex was linked to beta-gal by the N-(epsilon-maleimidocaproyloxy) succinimide coupling procedure. For competitive reactions, the TRH antibody was incubated with standard TRH and TRH-Hex-beta-gal (delayed addition). Free and antibody-bound enzyme hapten were separated by using an anti-rabbit immunoglobulin G coated immunoplate. Activity of the enzyme on the plate was fluorometrically determined. The present immunoassay allows detection of 0.8 to 100 pmol/well of TRH.

Role of thyrotropin-releasing hormone in thyroid-stimulating hormone and growth hormone regulation during postnatal maturation in female Wistar rats.

The role of endogenous thyrotropin-releasing hormone (TRH) in the control of pituitary thyroid-stimulating hormone (TSH) and growth hormone (GH) secretion was studied during postnatal maturation in female Wistar rats. Half of the sucklings in each litter was treated intraperitoneally with either specific rabbit antiserum against TRH or normal rabbit serum (0.1-0.3 ml according to age). All animals were decapitated after 2 h. The presence of anti-TRH activity was checked as a binding of labelled TRH with plasma of the experimental animals. Immunoneutralization of endogenous TRH resulted in a decrease of plasma TSH in 3- to 15-day-old female pups as compared to control littermates. No effect of TRH antibody injection was seen at the ages of 1, 21, 30 and 70 days despite the presence of excess antibody in the plasma. A profound effect of TRH antibody on plasma TSH was seen again at the age of 100 days. Plasma GH in the same animals exhibited a paradoxical increase after TRH immunoneutralization at the age of 5 and 8 days, a decrease was found at the age of 21 days. It was concluded that hypothalamic TRH control of TSH secretion matures early in Wistar rats. Hypothalamic secretion of TRH at the ages of 1, 21, 30, and 70 days is low and(or) its role in TSH regulation is masked by other regulating factors. TRH may play a dual role in the regulation of GH secretion during the postnatal period.