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.     

Regulation of TRH and TRH-related peptides in rat brain by thyroid and steroid hormones.

To investigate the possibility that TRH (pGlu-His-Pro-NH(2)) and EEP (pGlu-Glu-Pro-NH(2)) contribute to the behavioral and mood changes attending hypothyroidism, hyperthyroidism and hypogonadism, we have treated young, adult, male Sprague-Dawley rats (5/group, 250 g bw at time of sacrifice) for one week with either daily ip injections of saline, 5 microg T(4), 3 mg PTU or castration. Immunoreactivity for TRH (TRH-IR), TRH-Gly (pGlu-His-Pro-Gly, a TRH precursor), EEP and Ps4 (prepro-TRH-derived TRH-enhancing peptide) was measured in 8 brain regions by RIA. Castration reduced the Ps4-IR levels in hippocampus by 80%. High pressure liquid chromatography revealed that in many brain regions EEP-IR and TRH-IR consisted of a mixture of TRH and other TRH-like peptides including EEP, Val(2)-TRH, Tyr(2)-TRH, Leu(2)-TRH and Phe(2)-TRH. Transition from the hyperthyroid to the hypothyroid state increased the Val(2)-TRH and Tyr(2)-TRH levels in the accumbens by 10-fold and 15-fold, respectively, and the corresponding ratios for the pyriform cortex increased 9-fold and 12-fold, respectively. Hypothyroidism and castration reduced the levels of TRH and the majority of other TRH-like peptides in the entorhinal cortex. This is the first report that thyroid and steroid hormones alter the levels of TRH, prepro-TRH-derived peptides, and a newly discovered array of TRH-like neuropeptides in limbic brain regions.     

Valproate modulates TRH receptor, TRH and TRH-like peptide levels in rat brain

We have tested our hypothesis that alterations in the levels of TRH receptors, and the synthesis and release of tripeptide TRH, and other neurotropic TRH-like peptides mediate some of the mood stabilizing effects of valproate (Valp). We have directly compared the effect of 1 week of feeding two major mood stabilizers, Valp and lithium chloride (LiCl) on TRH binding in limbic and extra-limbic regions of male WKY rats. Valp increased TRH receptor levels in nucleus accumbens and frontal cortex. Li increased TRH receptor binding in amygdala, posterior cortex and cerebellum. The acute, chronic and withdrawal effects of Valp on brain levels of TRH (pGlu-His-Pro-NH2, His-TRH) and five other TRH-like peptides, Glu-TRH, Val-TRH, Tyr-TRH, Leu-TRH and Phe-TRH were measured by combined HPLC and RIA. Acute treatment increased TRH and TRH-like peptide levels within most brain regions, most strikingly in pyriform cortex. The fold increases (in parentheses) were: Val-TRH (58), Phe-TRH (54), Tyr-TRH (25), TRH (9), Glu-TRH (4) and Leu-TRH (3). We conclude that the mood stabilizing effects of Valp may be due, at least in part, to its ability to alter TRH and TRH-like peptide, and TRH receptor levels in the limbic system and other brain regions implicated in mood regulation and behavior.     

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).     

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.     

TRH-like peptides

TRH-like peptides are characterized by substitution of basic amino acid histidine (related to authentic TRH) with neutral or acidic amino acid, like glutamic acid, phenylalanine, glutamine, tyrosine, leucin, valin, aspartic acid and asparagine. The presence of extrahypothalamic TRH-like peptides was reported in peripheral tissues including gastrointestinal tract, placenta, neural tissues, male reproductive system and certain endocrine tissues. Work deals with the biological function of TRH-like peptides in different parts of organisms where various mechanisms may serve for realisation of biological function of TRH-like peptides as negative feedback to the pituitary exerted by the TRH-like peptides, the role of pEEPam such as fertilization-promoting peptide, the mechanism influencing the proliferative ability of prostatic tissues, the neuroprotective and antidepressant function of TRH-like peptides in brain and the regulation of thyroid status by TRH-like peptides.     

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.     

Ketamine modulates TRH and TRH-like peptide turnover in brain and peripheral tissues of male rats

Major depression is the largest single healthcare burden with treatments of slow onset and often limited efficacy. Ketamine, a NMDA antagonist used extensively as a pediatric and veterinary anesthetic, has recently been shown to be a rapid acting antidepressant, making it a potential lifesaver for suicidal patients. Side effects and risk of abuse limit the chronic use of ketamine. More complete understanding of the neurobiochemical mechanisms of ketamine should lead to safer alternatives. Some of the physiological and pharmacological actions of ketamine are consistent with increased synthesis and release of TRH (pGlu-His-Pro-NH₂), and TRH-like peptides (pGlu-X-Pro-NH₂) where “X” can be any amino acid residue. Moreover, TRH-like peptides are themselves potential therapeutic agents for the treatment of major depression, anxiety, bipolar disorder, epilepsy, Alzheimer's and Parkinson's diseases. For these reasons, male Sprague–Dawley rats were anesthetized with 162 mg/kg ip ketamine and then infused intranasally with 20 μl of sterile saline containing either 0 or 5 mg/ml Glu-TRH. One, 2 or 4 h later, the brain levels of TRH and TRH-like peptides were measured in various brain regions and peripheral tissues. At 1 h in brain following ketamine only, the levels of TRH and TRH-like peptides were significantly increased in 52 instances (due to increased biosynthesis and/or decreased release) or decreased in five instances. These changes, listed by brain region in order of decreasing number of significant increases (↑) and/or decreases (↓), were: hypothalamus (9↑); piriform cortex (8↑); entorhinal cortex (7↑); nucleus accumbens (7↑); posterior cingulate (5↑); striatum (4↑); frontal cortex (2↑,3↓); amygdala (3↑); medulla oblongata (1↑,2↓); cerebellum (2↑); hippocampus (2↑); anterior cingulate (2↑). The corresponding changes in peripheral tissues were: adrenals (8↑); epididymis (4↑); testis (1↑,3↓); pancreas (1↑); prostate (1↑). We conclude that TRH and TRH-like peptides may be downstream mediators of the rapid antidepressant actions of ketamine.     

Dietary thyrotropin-releasing hormone stimulates growth rate and increases the insulin: glucagon molar ratio of broiler chickens

The effects of thyroid manipulation on growth, feed efficiency, and plasma hormone levels were determined in rapidly growing chickens. Beginning at 3 weeks of age, eight broiler cockerels were provided with control feed (CF) or feed containing either 1 ppm of triiodothyronine (T3), 1 ppm of thyroxine (T4), 0.3% propylthiouracil (PTU), or 5 ppm of thyrotropin-releasing hormone (TRH) for 3 weeks. Blood samples were taken at 4, 5, and 6 weeks for determination of plasma levels of growth hormone, insulin-like growth factor, T3, T4, insulin, glucagon, glucose, and nonesterified fatty acids. Dietary TRH increased (P less than 0.05) the growth rate of chickens by 14% when compared with the CF group. Plasma growth hormone levels were reduced (P less than 0.05) 65% by dietary T3 and 33% by treatment with either T4 or TRH when compared with the CF group. Plasma insulin-like growth factor levels were 16% lower (P less than 0.05) in PTU-fed birds than the other treatment groups. Plasma T3 levels were elevated (P less than 0.05) 3-fold by dietary T3 and 38% by TRH whereas plasma T3 in the PTU group was 38% below the average of CF birds. Plasma T4 levels were increased (P less than 0.05) by 12-fold in T4-fed birds, decreased 48% in TRH-fed birds, and nondetectable in birds treated with either T3 or PTU. Compared with the other treatments, dietary PTU increased (P less than 0.01) plasma insulin levels 4.3-fold whereas TRH provided a 2.7-fold increase in plasma insulin. Plasma glucagon levels were 26% higher (P less than 0.05) in T3-fed birds than those fed either T4 or PTU. These observations indicate that thyroid activity plays an important role in regulating secretion of GH and the pancreatic hormones. Furthermore, our study demonstrates the potential use of TRH as an orally active growth promoter for poultry.     

Rapid modulation of TRH and TRH-like peptide release in rat brain and peripheral tissues by prazosin

Hyperresponsiveness to norepinephrine contributes to post-traumatic stress disorder (PTSD). Prazosin, a brain-active blocker of α₁-adrenoceptors, originally used for the treatment of hypertension, has been reported to alleviate trauma nightmares, sleep disturbance and improve global clinical status in war veterans with PTSD. Thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH₂) may play a role in the pathophysiology and treatment of neuropsychiatric disorders such as major depression, and PTSD (an anxiety disorder). To investigate whether TRH or TRH-like peptides (pGlu-X-Pro-NH₂, where “X” can be any amino acid residue) participate in the therapeutic effects of prazosin, male rats were injected with prazosin and these peptides then measured in brain and endocrine tissues. Prazosin stimulated TRH and TRH-like peptide release in those tissues with high α₁-adrenoceptor levels suggesting that these peptides may play a role in the therapeutic effects of prazosin.     

Thyrotropin-releasing hormone receptor expression in thyroid follicular cells: a new paracrine role of C-cells?

Thyrotropin-releasing hormone (TRH) synthesized in the hypothalamus has the capability of inducing the release of thyroid-stimulating hormone (TSH) from the anterior pituitary, which in turn stimulates the production of thyroid hormones in the thyroid gland. Immunoreactivity for TRH and TRH-like peptides has been found in some tissues outside the nervous system, including thyroid. It has been demonstrated that thyroid C-cells express authentic TRH, affecting thyroid hormone secretion by follicular cells. Therefore, C-cells could have a paracrine role in thyroid homeostasis. If this hypothesis is true, follicular cells should express TRH receptors (TRH-Rs) for the paracrine modulation carried out by C-cells. In order to elucidate whether or not C-cell TRH production could act over follicular cells modulating thyroid function, we studied TRH-Rs expression in PC C13 follicular cells from rat thyroid, by means of immunofluorescence technique and RT-PCR analysis. We also investigated the possibility that C-cells present TRH-Rs for the autocrine control of its own TRH production. Our results showed consistent expression for both receptors, TRH-R1 and TRH-R2, in 6-23 C-cells, and only for TRH-R2 in PC C13 follicular cells. Our data provide new evidence for a novel intrathyroidal regulatory pathway of thyroid hormone secretion via paracrine/autocrine TRH signaling.     

Effects of thyroid state alterations in ovo on the plasma levels of thyroid hormones and on the populations of fibers in the plantaris muscle of male and female chickens.

Propylthiouracil (PTU), thyroxine (T4) or thyreoliberin (TRH) were injected in ovo to modify the thyroid state of chicken embryos. Significant sexual differences were observed in the effects of these treatments on the plasma concentrations of thyroid hormones and on plantaris muscle characteristics (DNA, RNA, populations of muscle fibers) in 3- and 35-day old male and female chickens. The T4 plasma concentration is lower in control males; it is decreased in PTU treated females and in the T4 treated females at 35 days. The T3 plasma concentration is lowered at 3 days in all treated chickens and also at 35 days in the TRH treated animals. The slow (STnO) and the fast (FTOG) fibers of the plantaris are always more numerous in males. In controls, the number of FTOG fibers remains steady between 3 and 35 days; at the same time, the number of STnO fibers rises in males only. Both PTU and T4 treatments increase the number of the FTOG and the STnO fibers respectively before and after the 3rd day. TRH treatment increases the number of STnO fibers at 3 and 35 days in males, but reduces it at 3 days in females. Thus changes in the number of FTOG fibers can be induced during in ovo myogenesis, whereas the number of STnO fibers may increase after hatching.     

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.     

Cocaine regulates TRH-related peptides in rat brain

Cocaine administration has previously been reported to alter the levels of prepro-TRH mRNA and TRH (pGlu-His-Pro-NH₂) in the limbic system of rats (J. Neurochem. 60 (1993) 1151). We have now demonstrated that a previously unrecognized family of TRH-like peptides is involved in the actions of cocaine. We treated young adult male Sprague-Dawley rats (five per group, 250 g body weight at sacrifice) for 2 weeks with either twice daily injections of saline (control group), twice daily injections of 15 mg/kg cocaine until sacrifice (chronic group), single injection of 15 mg/kg cocaine 2 h prior to sacrifice (acute group) or chronic cocaine injections replaced by saline injections 72 h prior to sacrifice (withdrawal group (WD)). Twelve different brain regions were dissected and immunoreactivity for TRH (TRH-IR), EEP (pGlu-Glu-Pro-NH₂; EEP-IR) and related peptides were measured by radioimmunoassay (RIA). High pressure liquid chromatography (HPLC) revealed that in many brain regions EEP-IR and TRH-IR consisted of a mixture of TRH, and other TRH-like peptides including EEP, pGlu-Val-Pro-NH₂ (Val²-TRH), pGlu-Tyr-Pro-NH₂ (Tyr²-TRH), pGlu-Leu-Pro-NH₂ (Leu²-TRH), and pGlu-Phe-Pro-NH₂ (Phe²-TRH). Following i.p. injection, these TRH-like peptides readily crossed the blood–brain barrier but cleared very slowly from brain tissues.

Acute cocaine produced a 4.1-fold increase in Val²-TRH level in medulla while Val²-TRH and Tyr²-TRH, increased 6.2- and 2.9-fold, respectively in pyriform cortex PYR. TRH and Leu²-TRH, decreased 47 and 93%, respectively in the nucleus accumbens (AM) while other EEP-IR peaks decreased 50–100% consistent with the significant decrease in total EEP-IR in the AMs following acute cocaine treatment. Because 2 h is too short a time to alter levels of neuropeptides via changes in the rate of biosynthesis, the acute cocaine-induced elevation or reduction in TRH and related peptides is most likely due to suppression or stimulation, respectively, of the corresponding peptide secretion rate. Because TRH and TRH-like peptides have antidepressant, analeptic and euphorigenic properties, we conclude that these endogenous substances are potential mediators of both the cocaine “high” and withdrawal symptoms.     

Dexamethasone stimulates thyrotropin-releasing hormone production in a C cell line

The hypothalamic peptide hormone TRH is also found in other tissues, including the thyroid. While TRH may be regulated by T3 in the hypothalamus, other regulators of TRH have not been identified and the regulation of TRH in nonhypothalamic tissues is unknown. We recently demonstrated the biosynthesis of TRH in the CA77 neoplastic thyroidal C cell line. We studied the regulation of TRH by dexamethasone in this cell line because glucocorticoids have been postulated to inhibit TSH secretion by decreasing TRH in the hypothalamus. Furthermore, TRH in the thyroid inhibits thyroid hormone release. Thus by regulating thyroidal TRH, glucocorticoids could also directly affect thyroid hormone secretion. Treatment of CA77 cells for 4 days with dexamethasone produced dose-dependent increases in both TRH mRNA and cellular and secreted TRH. Increases in TRH mRNA and peptide levels could be seen with 10(-9) M dexamethasone. A 4.8-fold increase in TRH mRNA and a 4-fold increase in secreted peptide were seen with 10(-7) M dexamethasone. Dexamethasone treatment did not increase beta-actin mRNA levels or cell growth. These results suggest that glucocorticoids may be physiological regulators of TRH in normal C cells. In addition to their inhibitory effects on TSH, glucocorticoids may decrease thyroid hormone levels by increasing thyroidal TRH. Since the glucocorticoid effects on C cell TRH are the converse of what is expected for hypothalamic TRH, glucocorticoid effects in these two tissues may be mediated by different regulators.     

Increase of IGF I binding to erythrocyte receptors during the TRH-test: correlation between IGF I binding and thyroid hormone values

The effects of TRH on insulin-like growth factor I receptors were investigated on erythrocytes from 7 GH-deficient children having plasma GH levels less than 10 ng/ml during two provocation tests. Intravenous injection of synthetic TRH (0.2 mg/m2) was followed by a marked increase of IGF I binding on erythrocytes, from 3.9% +/- 0.3% to 5.9% +/- 0.3% (P less than 0.005) after 1 hour and 7.3% +/- 0.4% (P less than 0.005) after 2 hours. The IGF I binding variations were due to an increase in both the receptor affinity and the number of sites. The levels of plasma GH, IGF I, T3, T4, free T4, TSH and prolactin having been determined during the TRH test at 0, 1 hour, and 2 hours after the injection, the increase in the IGF I binding to erythrocytes at the same time correlated with the rise of thyroid hormones: triiodothyronine T3 (P less than 0.001) and thyroxine T4 (P less than 0.005) and not with the level of the other hormones. These findings suggest that thyroid hormones play a role in the regulation of insulin-like growth factor I receptors.     

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.     

Helodermin, but not cholecystokinin, somatostatin, or thyrotropin releasing hormone, acutely increases thyroid blood flow in the rat

In the present study, we investigated whether peptides located within the thyroid gland, but not directly found in nerve fibers associated with blood vessels, might influence thyroid blood flow. Specifically, we evaluated the effects of helodermin, cholecystokinin (CCK), somatostatin (SRIF) and thyrotropin releasing hormone (TRH) given systemically on thyroid blood flow and circulating thyroid hormone levels. Blood flows in the thyroid and six other organs were measured in male rats using 141Ce-labeled microspheres. Circulating thyrotropin (TSH) and thyroid hormone levels were monitored by RIA. Helodermin (10(-10) mol/100 g BW, i.v. over 4 min) markedly elevated thyroid blood flow (52 +/- 6 vs. 10 +/- 2 ml/min.g in vehicle-infused rats; n = 5). Blood flows to the salivary gland, pancreas, lacrimal gland and stomach (but not adrenal and kidney) were also increased during helodermin infusions. CCK, SRIF, and TRH were without effect on blood flows to the thyroid and other organs even though these peptides were tested at higher molar doses than helodermin. Helodermin, CCK, or SRIF did not affect thyroid hormone or plasma calcium levels. As expected however, plasma TSH and T3 levels were increased at 20 min and 2 h, respectively, following TRH infusions. Since helodermin shares sequence homology with VIP, we next compared the relative effects of these two peptides on thyroid and other organ blood flows. VIP (10(-11) mol/100 g BW, i.v.) was more potent in increasing blood flows to the thyroid, salivary gland, and pancreas than an equimolar dose of helodermin. This study shows that while helodermin, like VIP, has the ability to increase thyroid and other organ blood flows, it appears to be a less potent vasodilator.     

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.