Sex-dependent and -independent regulation of thyrotropin-releasing hormone expression in the hypothalamic dorsomedial nucleus by negative energy balance, exercise, and chronic stress.

The dorsomedial nucleus of the hypothalamus (DMH) is part of the brain circuits that modulate organism responses to the circadian cycle, energy balance, and psychological stress. A large group of thyrotropin-releasing hormone (Trh) neurons is localized in the DMH; they comprise about one third of the DMH neurons that project to the lateral hypothalamus area (LH). We tested their response to various paradigms. In male Wistar rats, food restriction during adulthood, or chronic variable stress (CVS) during adolescence down-regulated adult DMH Trh mRNA levels compared to those in sedentary animals fed ad libitum; two weeks of voluntary wheel running during adulthood enhanced DMH Trh mRNA levels compared to pair-fed rats. Except for their magnitude, female responses to exercise were like those in male rats; in contrast, in female rats CVS did not change DMH Trh mRNA levels. A very strong negative correlation between DMH Trh mRNA levels and serum corticosterone concentration in rats of either sex was lost in CVS rats. CVS canceled the response to food restriction, but not that to exercise in either sex. TRH receptor 1 (Trhr) cells were numerous along the rostro-caudal extent of the medial LH. In either sex, fasting during adulthood reduced DMH Trh mRNA levels, and increased LH Trhr mRNA levels, suggesting fasting may inhibit the activity of TRHDMH->LH neurons. Thus, in Wistar rats DMH Trh mRNA levels are regulated by negative energy balance, exercise and chronic variable stress through sex-dependent and -independent pathways.

Neonatal exposure to bisphenol A alters the hypothalamic-pituitary-thyroid axis in female rats.

Bisphenol A (BPA) is a component of polycarbonate plastics, epoxy resins and polystyrene found in many common products. Several reports revealed potent in vivo and in vitro effects. In this study we analyzed the effects of the exposure to BPA in the hypothalamic-pituitary-thyroid axis in female rats, both in vivo and in vitro. Female Sprague-Dawley rats were injected sc from postnatal day 1 (PND1) to PND10 with BPA: 500 µg 50 µl-1 oil (B500), or 50 µg 50 µl-1 (B50), or 5 µg 50 µl-1 (B5). Controls were injected with 50 µl vehicle during the same period. Neonatal exposure to BPA did not modify TSH levels in PND13 females, but it increased them in adults in estrus. Serum T4 was lower in B5 and B500 with regards to Control, whereas no difference was seen in T3. No significant differences were observed in TRH, TSHß and TRH receptor expression between groups. TSH release from PPC obtained from adults in estrus was also higher in B50 with regard to Control. In vitro 24 h pre-treatment with BPA or E2 increased basal TSH as well as prolactin release. On the other hand, both BPA and E2 lowered the response to TRH. The results presented here show that the neonatal exposure to BPA alters the hypothalamic pituitary-thyroid axis in adult rats in estrus, possibly with effects on the pituitary and thyroid. They also show that BPA alters TSH release from rat PPC through direct actions on the pituitary.

Tanycytes control the hormonal output of the hypothalamic-pituitary-thyroid axis.

The hypothalamic-pituitary-thyroid (HPT) axis maintains circulating thyroid hormone levels in a narrow physiological range. As axons containing thyrotropin-releasing hormone (TRH) terminate on hypothalamic tanycytes, these specialized glial cells have been suggested to influence the activity of the HPT axis, but their exact role remained enigmatic. Here, we demonstrate that stimulation of the TRH receptor 1 increases intracellular calcium in tanycytes of the median eminence via Gαq/11 proteins. Activation of Gαq/11 pathways increases the size of tanycyte endfeet that shield pituitary vessels and induces the activity of the TRH-degrading ectoenzyme. Both mechanisms may limit the TRH release to the pituitary. Indeed, blocking TRH signaling in tanycytes by deleting Gαq/11 proteins in vivo enhances the response of the HPT axis to the chemogenetic activation of TRH neurons. In conclusion, we identify new TRH- and Gαq/11-dependent mechanisms in the median eminence by which tanycytes control the activity of the HPT axis.The hypothalamic-pituitary-thyroid (HPT) axis regulates a wide range of physiological processes. Here the authors show that hypothalamic tanycytes play a role in the homeostatic regulation of the HPT axis; activation of TRH signaling in tanycytes elevates their intracellular Ca2+ via Gαq/11 pathway, ultimately resulting in reduced TRH release into the pituitary vessels.

TRH Action Is Impaired in Pituitaries of Male IGSF1-Deficient Mice.

Loss-of-function mutations in the X-linked immunoglobulin superfamily, member 1 (IGSF1) gene cause central hypothyroidism. IGSF1 is a transmembrane glycoprotein of unknown function expressed in thyrotropin (TSH)-producing thyrotrope cells of the anterior pituitary gland. The protein is cotranslationally cleaved, with only its C-terminal domain (CTD) being trafficked to the plasma membrane. Most intragenic IGSF1 mutations in humans map to the CTD. In this study, we used CRISPR-Cas9 to introduce a loss-of-function mutation into the IGSF1-CTD in mice. The modified allele encodes a truncated protein that fails to traffic to the plasma membrane. Under standard laboratory conditions, Igsf1-deficient males exhibit normal serum TSH levels as well as normal numbers of TSH-expressing thyrotropes. However, pituitary expression of the TSH subunit genes and TSH protein content are reduced, as is expression of the receptor for thyrotropin-releasing hormone (TRH). When challenged with exogenous TRH, Igsf1-deficient males release TSH, but to a significantly lesser extent than do their wild-type littermates. The mice show similarly attenuated TSH secretion when rendered profoundly hypothyroid with a low iodine diet supplemented with propylthiouracil. Collectively, these results indicate that impairments in pituitary TRH receptor expression and/or downstream signaling underlie central hypothyroidism in IGSF1 deficiency syndrome.

Hypothalamo­hypophysial system in rats with autotransplantation of the adrenal cortex.

Patients with bilateral pheochromocytoma often require an adrenalectomy. Autotransplantation of the adrenal cortex is an alternative therapy that could potentially be performed instead of receiving glucocorticoid replacement following adrenalectomy. Adrenal cortex autotransplantation aims to avoid the side effects of long­term steroid treatment and adrenal insufficiency. Although the function of the hypothalamo­hypophysial system is critical for patients who have undergone adrenal cortex autotransplantation, the details of that system, with the exception of adrenocorticotropic hormone in the subjects with adrenal autotransplantation, have been overlooked for a long time. To clarify the precise effect of adrenal autotransplantation on the pituitary gland and hypothalamus, the current study examined the gene expression of hormones produced from the hypothalamus and pituitary gland. Bilateral adrenalectomy and adrenal autotransplantation were performed in 8 to 9­week­old male rats. The hypothalamus and pituitary tissues were collected at 4 weeks after surgery. Transcriptional regulation of hypothalamic and pituitary hormones was subsequently examined by reverse transcription­quantitative polymerase chain reaction. Proopiomelanocortin, glycoprotein hormone α polypeptide, and thyroid stimulating hormone ß were significantly elevated in the pituitary gland of autotransplanted rats when compared with sham­operated rats. In addition, there were significant differences in the levels of corticotropin releasing hormone receptor 1 (Crhr1), Crhr2, nuclear receptor subfamily 3 group C member 1 and thyrotropin releasing hormone receptor between the sham­operated rats and autotransplanted rats in the pituitary gland. In the hypothalamus, corticotropin releasing hormone and urocortin 2 mRNA was significantly upregulated in autotransplanted rats compared with sham­operated rats. The authors identified significant alterations in the function of not only the hypothalamus­pituitary­adrenal axis, but also the adenohypophysis thyrotropes in autotransplanted rats. In the future, it will be important to examine other tissues affected by glucocorticoids following adrenal cortex autotransplantation.

Di2-ethylhexyl phthalate disrupts thyroid hormone homeostasis through activating the Ras/Akt/TRHr pathway and inducing hepatic enzymes.

Di(2-ethylhexyl) phthalate (DEHP), as a widespread environmental pollutant and an endocrine disruptor, can disturb the homeostasis of thyroid hormones (THs). In order to elucidate roles of the MAPK and PI3K/Akt pathways and hepatic enzymes in thyroid-disrupting effects of DEHP, Sprague-Dawley rats were dosed with DEHP by gavage for 30 consecutive days; Nthy-ori 3-1 cells were treated with DEHP with NAC, k-Ras siRNA or inhibitors (U0126 and wortmannin). Results showed that DEHP led to histopathologic changes in rat thyroid and liver, such as the decrease in thyroid follicular cavity diameter, hepatocyte edema. Triiodothyronine (T3), thyroxine (T4) and thyrotropin releasing hormone (TRH) were reduced. DEHP caused ROS production, oxidative stress and k-Ras upregulation, thereby activating the ERK and Akt pathways in vivo and in vitro. Moreover, TRH receptor (TRHr) level was elevated after the activation of the Akt pathway and was downregulated after the inhibition of the Akt pathway. However, TRHr was not modulated by the ERK pathway. Additionally, hepatic enzymes, including Ugt1a1, CYP2b1, Sult1e1, and Sult2b1, were significantly induced after DEHP exposure. Taken together, DEHP can perturb TH homeostasis and reduce TH levels. The activated Ras/Akt/TRHr pathway and induced hepatic enzymes play vital roles in thyroid-disrupting effects of DEHP.

The PI3K/Akt and ERK pathways elevate thyroid hormone receptor ß1 and TRH receptor to decrease thyroid hormones after exposure to PCB153 and p,p'-DDE.

PCBs and DDT cause the disturbance of thyroid hormone (TH) homeostasis in humans and animals. To test the hypothesis that the PI3K/Akt and MAPK pathways would play significant roles in TH imbalance caused by PCBs and DDT, Sprague-Dawley rats were dosed with PCB153 and p,p'-DDE intraperitoneally for 5 consecutive days, and human thyroid follicular epithelial (Nthy-ori 3-1 cell line) were treated with PCB153 and p,p'-DDE for different time. Results showed that serum total thyroxine (TT4), free thyroxine (FT4), total triiodothyronine (TT3) and thyroid stimulating hormone (TSH) were decreased, whereas serum free triiodothyronine (FT3) and thyrotropin releasing hormone (TRH) were not changed. The PI3K/Akt and ERK pathways were activated in vivo and in vitro after the treatment with PCB153 and p,p'-DDE. Moreover, TH receptor ß1 (TRß1) was elevated after the activation of the PI3K/Akt pathway and was depressed after the inhibition of the PI3K/Akt pathway; TRH receptor (TRHr) was increased after the activation of the ERK pathway and was decreased after the inhibition of the ERK pathway. Though TH receptor α1 (TRα1) level was increased in the hypothalamus, TRα1 and TSHr were not influenced by the status of signaling pathways in in vitro study. Taken together, after exposure to PCB153 and p,p'-DDE, activated PI3K/Akt and ERK pathways disrupt the hypothalamic-pituitary-thyroid (HPT) axis via TRß1 and TRHr and then decrease TH levels, and that would be a potential mechanism by which PCBs and DDT disturb TH homeostasis.

Absence of TRH receptor 1 in male mice affects gastric ghrelin production.

TRH not only functions as a thyrotropin releasing hormone but also acts as a neuropeptide in central circuits regulating food intake and energy expenditure. As one suggested mode of action, TRH expressed in the caudal brainstem influences vagal activity by activating TRH receptor 1 (TRH-R1). In order to evaluate the impact of a diminished medullary TRH signaling on ghrelin metabolism, we analyzed metabolic changes of TRH-R1 knockout (R1ko) mice in response to 24 hours of food deprivation. Because R1ko mice are hypothyroid, we also studied eu- and hypothyroid wild-type (wt) animals and R1ko mice rendered euthyroid by thyroid hormone treatment. Independent of their thyroidal state, R1ko mice displayed a higher body weight loss than wt animals and a delayed reduction in locomotor activity upon fasting. Ghrelin transcript levels in the stomach as well as total ghrelin levels in the circulation were equally high in fasted wt and R1ko mice. In contrast, only wt mice responded to fasting with a rise in ghrelin-O-acyltransferase mRNA expression and consequently an increase in serum levels of acylated ghrelin. Together, our data suggest that an up-regulation of medullary TRH expression and subsequently enhanced activation of TRH-R1 in the vagal system represents a critical step in the stimulation of ghrelin-O-acyltransferase expression upon starvation that in turn is important for adjusting the circulating levels of acylated ghrelin to the fasting condition.

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

BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

NR4A1 (Nur77) mediates thyrotropin-releasing hormone-induced stimulation of transcription of the thyrotropin ß gene: analysis of TRH knockout mice.

Thyrotropin-releasing hormone (TRH) is a major stimulator of thyrotropin-stimulating hormone (TSH) synthesis in the anterior pituitary, though precisely how TRH stimulates the TSHß gene remains unclear. Analysis of TRH-deficient mice differing in thyroid hormone status demonstrated that TRH was critical for the basal activity and responsiveness to thyroid hormone of the TSHß gene. cDNA microarray and K-means cluster analyses with pituitaries from wild-type mice, TRH-deficient mice and TRH-deficient mice with thyroid hormone replacement revealed that the largest and most consistent decrease in expression in the absence of TRH and on supplementation with thyroid hormone was shown by the TSHß gene, and the NR4A1 gene belonged to the same cluster as and showed a similar expression profile to the TSHß gene. Immunohistochemical analysis demonstrated that NR4A1 was expressed not only in ACTH- and FSH- producing cells but also in thyrotrophs and the expression was remarkably reduced in TRH-deficient pituitary. Furthermore, experiments in vitro demonstrated that incubation with TRH in GH4C1 cells increased the endogenous NR4A1 mRNA level by approximately 50-fold within one hour, and this stimulation was inhibited by inhibitors for PKC and ERK1/2. Western blot analysis confirmed that TRH increased NR4A1 expression within 2 h. A series of deletions of the promoter demonstrated that the region between bp -138 and +37 of the TSHß gene was responsible for the TRH-induced stimulation, and Chip analysis revealed that NR4A1 was recruited to this region. Conversely, knockdown of NR4A1 by siRNA led to a significant reduction in TRH-induced TSHß promoter activity. Furthermore, TRH stimulated NR4A1 promoter activity through the TRH receptor. These findings demonstrated that 1) TRH is a highly specific regulator of the TSHß gene, and 2) TRH mediated induction of the TSHß gene, at least in part by sequential stimulation of the NR4A1-TSHß genes through a PKC and ERK1/2 pathway.

Constitutively active thyrotropin and thyrotropin-releasing hormone receptors and their inverse agonists.

Receptors for thyrotropin-releasing hormone (TRH) and thyrotropin (thyroid-stimulating hormone-TSH) are important regulators of the function of the TSH-producing cells of the anterior pituitary gland and the thyroid gland, respectively, and thereby play a central role in thyroid hormone homeostasis. Although the roles of TRH- and TSH-stimulated signaling in these endocrine glands are well understood, these receptors are expressed in other sites and their roles in these extraglandular tissues are less well known. Moreover, one of the two subtypes of TRH receptors (TRH-R2) and the single TSH receptor (TSHR) exhibit constitutive signaling activity and the roles of constitutive signaling by these receptors are poorly understood. One approach to studying constitutive signaling is to use inverse agonists. In this chapter, we will describe the experimental procedures used to measure constitutive signaling by TRH-R2 and TSHR and the effects of their specific inverse agonists.

Myrtucommulones F-I, phloroglucinols with thyrotropin-releasing hormone receptor-2 binding affinity from the seeds of Corymbia scabrida.

High-throughput screening of a plant and marine invertebrate extract library to find natural products with rat thyrotropin-releasing hormone (TRH) receptor-2 binding affinity led to the isolation of four new, myrtucommulones F-I (3-6), and two known, myrtucommulones A (1) and D (2), active acylphloroglucinols from the seeds of the Queensland tree Corymbia scabrida. Their structures were assigned from interpretation of 2D NMR and high-resolution ESIMS data. The relative configuration of the stereogenic centers for all six compounds was deduced from ROESY correlations. This is the first time that myrtucommulone A (1) has been isolated as a single pure compound. The structure of myrtucommulone D (2) has been revised. Myrtucommulones A, D, and F-I showed rat TRH receptor-2 binding affinity with IC50 values of 39, 11, 16, 24, 31, and 16 microM, respectively.

The thyrotropin-releasing hormone secretory system in the hypothalamus of the Siberian hamster in long and short photoperiods.

Thyrotropin-releasing hormone (TRH) is not only essential for the regulation of the pituitary-thyroid axis, but also exerts complementary effects on energy metabolism within the brain. We hypothesised that increased activity of the TRH secretory system may contribute to seasonal adaptations in the Siberian hamster whereby food intake is decreased in winter, and catabolism of fat stores is increased to support thermogenesis. We determined the distribution of TRH producing neurones and TRH-R1 receptor expressing cells in the hypothalamus, and investigated whether photoperiod regulated this system. TRH-immunoreactive (ir) cell somata and preproTRH mRNA expression were found to be widely distributed throughout the medial hypothalamus, with particular clusters in the paraventricular nucleus, the medial preoptic area and periventricular nucleus, and in the dorsomedial hypothalamus extending into the lateral hypothalamic area. A partial sequence encoding TRH-R1 was cloned from hamster hypothalamic cDNA and used to generate a riboprobe for in situ hybridisation studies. TRH-R1 mRNA expressing cells were abundant throughout the hypothalamus, corresponding to the widespread presence of TRH-ir fibres. Photoperiod did not affect the expression of preproTRH mRNA in any region, and the only significant change in TRH-R1 expression was in the dorsomedial posterior arcuate region. This wide distribution of TRH-producing and receptive cells in the hypothalamus is consistent with its hypothesised neuromodulatory roles in the short-term homeostatic control of appetite, thermoregulation and energy expenditure, but the lack of photoperiodic change in TRH mRNA expression does not support the hypothesis that changes in this system underlie long-term seasonal changes in body weight.

A virtual screen for diverse ligands: discovery of selective G protein-coupled receptor antagonists.

Virtual screening has become a major focus of bioactive small molecule lead identification, and reports of agonists and antagonists discovered via virtual methods are becoming more frequent. G protein-coupled receptors (GPCRs) are the one class of protein targets for which success with this approach has been limited. This is likely due to the paucity of detailed experimental information describing GPCR structure and the intrinsic function-associated structural flexibility of GPCRs which present major challenges in the application of receptor-based virtual screening. Here we describe an in silico methodology that diminishes the effects of structural uncertainty, allowing for more inclusive representation of a potential docking interaction with exogenous ligands. Using this approach, we screened one million compounds from a virtual database, and a diverse subgroup of 100 compounds was selected, leading to experimental identification of five structurally diverse antagonists of the thyrotropin-releasing hormone receptors (TRH-R1 and TRH-R2). The chirality of the most potent chemotype was demonstrated to be important in its binding affinity to TRH receptors; the most potent stereoisomer was noted to have a 13-fold selectivity for TRH-R1 over TRH-R2. A comprehensive mutational analysis of key amino acid residues that form the putative binding pocket of TRH receptors further verified the binding modality of these small molecule antagonists. The described virtual screening approach may prove applicable in the search for novel small molecule agonists and antagonists of other GPCRs.

Hypothalamic neuronal histamine mediates the thyrotropin-releasing hormone-induced suppression of food intake.

We examined the involvement of thyrotropin-releasing hormone (TRH) and TRH type 1 and 2 receptors (TRH-R1 and TRH-R2, respectively) in the regulation of hypothalamic neuronal histamine. Infusion of 100 nmol TRH into the rat third cerebroventricle (3vt) significantly decreased food intake (p < 0.05) compared to controls infused with phosphate- buffered saline. This TRH-induced suppression of food intake was attenuated partially in histamine-depleted rats pre-treated with alpha-fluoromethylhistidine (a specific suicide inhibitor of histidine decarboxylase) and in mice with targeted disruption of histamine H1 receptors. Infusion of TRH into the 3vt increased histamine turnover as assessed by pargyline-induced accumulation of tele-methylhistamine (t-MH, a major metabolite of neuronal histamine in the brain) in the tuberomammillary nucleus (TMN), the paraventricular nucleus, and the ventromedial hypothalamic nucleus in rats. In addition, TRH-induced decrease of food intake and increase of histamine turnover were in a dose-dependent manner. Microinfusion of TRH into the TMN increased t-MH content, histidine decarboxylase (HDC) activity and expression of HDC mRNA in the TMN. Immunohistochemical analysis revealed that TRH-R2, but not TRH-R1, was expressed within the cell bodies of histaminergic neurons in the TMN of rats. These results indicate that hypothalamic neuronal histamine mediates the TRH-induced suppression of feeding behavior.

Thyrotropin-releasing hormone induced thermogenesis in Syrian hamsters: site of action and receptor subtype.

Early work in our laboratory has revealed the important role played by thyrotropin-releasing hormone (TRH) in the arousal from hibernation in Syrian hamsters. In the present study, we investigated the thermogenic mechanism of TRH in Syrian hamsters. Six to 10 female Syrian hamsters were used in the respective experiments. Intracerebroventricular (icv) injection of TRH elevated the intrascapular brown adipose tissue (IBAT) temperature (T(IBAT)) and rectal temperature (T rec) in Syrian hamsters. Thermogenic response of icv TRH was suppressed by bilateral denervation of the sympathetic nerve. Icv injection of TRH increased the norepinephrin (NE) turnover rate in IBAT without affecting the total serum triiodothyronine (T3) level. Moreover, TRH microinjections into the dorsomedial hypothalamus (DMH), preoptic area (PO), anterior hypothalamus (AH) and ventromedial hypothalamus (VMH) induced T(IBAT) and T(rec) increases. However, neither T(IBAT) nor T rec was affected by similar TRH administrations into the lateral hypothalamus and posterior hypothalamus. Interestingly, although TRH-induced hyperthermia was suppressed by pretreatment of anti-TRH-R1 antibodies, no changes were induced by anti-TRH-R2 antibodies. These results suggest that the sites of action of TRH associated with thermogenesis are probably localized in the DMH, PO, AH and VMH. In addition, TRH-induced thermogenesis is probably elicited by facilitation of the sympathetic nerve system via the central TRH-R1 irrelevant of T3.

Conformationally restricted TRH analogues: constraining the pyroglutamate region.

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

The coumarin osthol attenuates the binding of thyrotropin-releasing hormone in rat pituitary GH4C1 cells.

The influence of two plant coumarins, osthol and xanthotoxin, on intracellular Ca2+ ([Ca2+]i) transients evoked by TRH were studied in clonal rat pituitary GH4C1 cells. Osthol, but not xanthotoxin, decreased the TRH-induced transient increase in [Ca2+]i in Fluo-3 loaded cells incubated in Ca(2+)-free buffer. Binding experiments with [3H]TRH showed that osthol decreased the binding of TRH to its receptor, whereas the affinity of the receptor for TRH increased. This resulted in a decreased TRH-evoked production of IP3 in cells treated with osthol, and a decreased mobilization of sequestered calcium. Osthol did not inhibit the release of calcium evoked by exogenous IP3 in permeabilized cells. Furthermore, osthol decreased the uptake of 45Ca2+ in response to high K+. Xanthotoxin had no effects in these experiments. The results show that osthol modulates TRH-evoked responses by interacting with the TRH receptor.

Expression of thyrotropin-releasing hormone receptor 2 (TRH-R2) in the central nervous system of rats.

The distribution of the recently discovered thyrotropin-releasing hormone (TRH) receptor subtype TRH-R2 was studied in rat brain, pituitary, and spinal cord by in situ hybridization histochemistry and compared with the distribution patterns of the other elements of TRH signaling, namely TRH, TRH-R1, and the TRH-degrading ectoenzyme (TRH-DE). In contrast to the very restricted mRNA expression of TRH-R1 in the central nervous system, TRH-R2 mRNA was widely distributed with highest transcript levels throughout the thalamus, in the cerebral and cerebellar cortex, medial habenulae, medial geniculate nucleus, pontine nuclei, and throughout the reticular formation. In accordance with the well-known endocrine function of TRH, TRH-R1 is found predominantly expressed in hypothalamic regions. Expression of TRH-R1 in various brainstem nuclei and spinal cord motoneurons seems to be associated with the described effects of TRH on the vegetative and autonomic system as well as on the somatomotor system. Furthermore, the fully complementary expression of both receptor subtypes, even in regions where transcripts for both receptors were found (e.g., medial septum, lateral hypothalamus superior colliculi, substantia nigra, etc.), indicates that in discrete neuroanatomical pathways the two receptors serve highly specific functions for the transmission of TRH signals. Together with TRH-DE, the putative terminator of TRH actions that shows in various, but not all, brain areas, an overlapping mRNA distribution pattern with both receptors, the distribution of TRH-R2 mRNA seems to provide the anatomical basis for the described effects of TRH on higher cognitive functions as well as its effect on arousal, locomotor activity, and pain perception.

A novel transcript for the thyrotropin-releasing hormone receptor in human pituitary and pituitary tumors.

We measured the amounts of TRH receptor (TRHR) messenger ribonucleic acid (mRNA) in human normal pituitary and pituitary tumors and found a novel transcript of the TRHR gene. Competitive PCR revealed expression of the TRHR mRNA in all pituitary adenomas examined, and its level was variable and similar to that in the normal pituitary. When the C-terminal region was amplified by PCR, an additional short product was observed. Cloning and sequence analysis of this short fragment revealed that the deleted sequence corresponded exactly to the 5'-sequence of exon 3, indicating alternative splicing of the TRHR mRNA. This alternative splicing resulted in a frame shift, yielding a C-terminal truncated protein (HTRHR2) on translation. Expression analysis of HTRHR2 in Chinese hamster ovary cells showed no significant binding to [3HIMeTRH or response of intracellular calcium to TRH administration. However, the mRNA ratio of HTRHR2 vs. the wild type (HTRHR1) was significantly different among pituitary tumors. The highest ratio was observed in prolactinomas (30%), and almost no detectable expression was found in GH-producing tumors. These findings indicate that this novel transcript of the human TRH receptor gene is produced in a tumor-specific manner and may be a useful parameter for evaluation of individual pituitary tumors.