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.     

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.     

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.     

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.     

Rapid modulation of TRH-like peptides in rat brain by thyroid hormones

Recent identification of membrane receptors for T4, T3, 3,5-T2, and 3-iodothyronamine that mediate rapid physiologic effects of thyroid hormones suggested that such receptors may supplement the regulation of TRH and TRH-like peptides by nuclear T3 receptors. For this reason 200 g male Sprague-Dawley rats received daily i.p. injections of PTU or T4. Levels of TRH and TRH-like peptides were measured 0, 2 h or 1, 2, 3, or 4 days later. Rapid increases or decreases in TRH and TRH-like peptide levels were observed in response to PTU and T4 treatments in various brain regions involved in mood regulation. Significant effects were measured within 2 h of T4 injection. Nuclear T3 receptor-mediated changes in gene expression altering translation, post-translational processing and constitutive release of peptides require more than 2 h. We conclude that non-genomic mechanisms may contribute to the psychiatric effects of thyroid disease and thyroid hormone adjuvant treatment for major depression.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Glucocorticoid regulation of enkephalins in cultured rat adrenal medulla

The effect of dexamethasone on enkephalin-containing (EC) peptide levels and preproenkephalin mRNA levels was determined in adrenal medullary explants (glands) from sham and hypophysectomized (hypox) rats. Culture for 4 days in serum-free medium without dexamethasone resulted in a 13- and 4-fold increase in EC peptide levels in sham and hypox glands, respectively. The addition of dexamethasone (10(-5) M) produced a 20- to 26-fold increase in EC peptides in sham and hypox glands. In serum free medium, hypox glands showed a concentration dependent increase in EC peptides with the ED50 for dexamethasone equal to 5.7 x 10(-7) M. Since the glucocorticoid antagonist RU486 partially blocked the rise in EC peptides in sham glands, it appears that the increase in EC peptides in sham glands in the absence of dexamethasone is a result of a higher concentration of endogenous corticosterone in sham compared to hypox glands. Dexamethasone resulted in a 6-fold increase in preproenkephalin mRNA in hypox glands cultured for 2 days. This increase was approximately proportional to the increase in EC peptides seen at 4 days. In serum free medium progesterone, testosterone, and deoxycorticosterone failed to increase EC peptides in hypox glands. These results indicate that glucocorticoid treatment is required for maximal proenkephalin gene expression and EC peptide biosynthesis in cultured glands.     

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).     

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.     

Immunocytochemical location of thyrotropin-releasing hormone (TRH) in the B-cells of adult hypothyroid rat pancreas

Thyrotropin-releasing hormone (TRH) is present in small quantities in the rat adult pancreas. As hypothyroidism increases dramatically the pancreatic content of this peptide, this model was used to localize TRH in the gland by immunocytochemistry. Immunocytochemical staining of semithin (0.5–1.0 μm) and thin (golden) sections was performed as well as antibody and method controls to check the specificity of the immunoperoxidase staining. At the light microscope level, a very faint TRH-like immunoreactivity was apparent in the pancreas of normal untreated animals. In hypothyroid rats, a strong TRH immunostaining was observed in the central portion of the islets of Langerhans. On the contrary, in previously hypothyroid rats made euthyroid, no TRH-like immunoreactivity was found. Serial sections alternately labelled with TRH and insulin antisera revealed the simultaneous occurrence of both immunoreactivities. In addition, the TRH immunoreactive cells were distinct from glucagon- or somatostatin-containing cells. At the electron microscope level, immunoreactive TRH was found over the secretory granules of insulin-containing cells. Hypothyroid animals offer therefore a suitable model for the study of TRH in the pancreas.     

Glucocorticoids stimulate the production of preprocalcitonin-derived secretory peptides by a rat medullary thyroid carcinoma cell line

A rat medullary thyroid carcinoma cell line, CA-77, has been established as a model system for investigating calcitonin biosynthesis and secretion. Growth of this cell line in serum-free defined medium provided suitable conditions for studying steroid hormone effects on the production of calcitonin and related peptides. After exposure for 5 days to a variety of steroids, only dexamethasone and corticosterone increased cellular content of calcitonin and a second secretory peptide (CCAP) derived from the same mRNA translation product as calcitonin. Glucocorticoids had no effect on cellular somatostatin, another secretory product of these cells. Increasing doses of dexamethasone progressively elevated cellular calcitonin and CCAP, with a maximal effect at 10(-8) M; 10(-9) M and lower doses were ineffective. On a molar basis, corticosterone was approximately 50-fold less potent than the synthetic glucocorticoid. An increase in cellular calcitonin content was observed only after 48 h of glucocorticoid treatment; a maximum increase (13-fold) occurred after 7 days. Glucocorticoids also increased basal calcitonin secretion. Similar effects were observed for cellular and secreted CCAP. Withdrawal of dexamethasone after 4 days of treatment lowered cellular calcitonin toward the level of control cultures. Dexamethasone pretreatment potentiated the acute secretory response to calcium for both calcitonin and CCAP, while no such enhancement was noted for calcium stimulation of somatostatin secretion. We conclude that the glucocorticoids specifically stimulate the production and secretion of calcitonin and CCAP, two secretory peptides derived from preprocalcitonin.     

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 (TRH) precursor processing. Characterization of mature TRH and non-TRH peptides synthesized by transfected mammalian cells

Prepro-thyrotropin-releasing hormone (TRH) contains five TRH progenitor sequences and at least six other potential peptides (Lechan, R. M., Wu, P., Jackson, I. M. D., Wolf, H., Cooperman, S., Mandel, G., and Goodman, R. H. (1986a) Science 231, 159-161). Previous studies using radioimmunoassays developed against discrete regions of prepro-TRH have demonstrated that several of the potential peptides are present in rat brain and pancreas (Wu, P., Lechan, R. M., and Jackson, I. M. D. (1987) Endocrinology 121, 108-115; Wu, P. and Jackson, I. M. D. (1988a) Brain Res. 456, 22-28; Wu, P., and Jackson, I. M. D. (1988b) Regul. Pept. 22, 347-360). However, the low level of peptides present in intact tissues has made isolation of the peptides difficult. CA77 cells, a medullary thyroid carcinoma cell line, also express prepro-TRH and display processing similar to that found in tissues. However, peptide content in this tumor cell line is enhanced only 3-fold compared with normal tissues (Sevarino, K. A., Wu, P., Jackson, I. M. D., Roos, B. A., Mandel, G., and Goodman, R. H. (1988) J. Biol. Chem. 263, 620-623). To achieve higher levels of expression for facilitating peptide sequencing studies and to see if alternate processing of prepro-TRH could be detected in different cell types, we transfected into 3T3, GH4, AtT20, and RIN 5F cells a cDNA vector under control of the cytomegalovirus immediate-early promoter. 3T3 and GH4 cells failed to process prepro-TRH beyond cleavage of the signal sequence. Both AtT20 and RIN 5F cells efficiently cleaved the precursor at dibasic sites to generate mature TRH and the non-TRH peptides previously identified in vivo. Peptide content was up to 30 times greater than in hypothalamic extracts and 10 times greater than in CA77 cells. Secretion experiments with transfected AtT20 cells demonstrated that both mature TRH and the non-TRH peptides were secreted via a regulated secretory pathway similar to that utilized by endogenously synthesized peptides. We isolated several of the non-TRH peptides synthesized by transfected AtT20 cells and characterized these peptides by sequential Edman degradation. These studies identified the signal sequence cleavage site and determined that the non-TRH peptides are generated by cleavage at the dibasic sites flanking the five TRH progenitor sequences. Further, we determined that processing occurs at the Arg51-Arg52 site located in the amino-terminal portion of the precursor, the only dibasic site not flanking a TRH progenitor sequence.