P38/TRHr-Dependent Regulation of TPO in Thyroid Cells Contributes to the Hypothyroidism of Triclosan-Treated Rats.

BACKGROUND/AIMS: Triclosan, as an antimicrobial agent and a potential endocrine disruptor, has been used extensively in diverse products, resulting in widespread human exposure. In recent years, studies suggest that triclosan could disturb thyroid functions and decline thyroid hormones (THs). METHODS: To verify our hypothesis that the MAPK pathway may function significantly in triclosan-induced hypothyroidism, Sprague-Dawley rats were gavaged with triclosan for 31 consecutive days; Nthy-ori 3-1 cells were treated with triclosan in the presence/absence of NAC, inhibitors (SB203580 and SB202474), or TRHr siRNA. Tissues and/or cells were analyzed by several techniques including transmission electron microscopy, confocal laser scanning microscopy, gene silencing, western blot, and real-time PCR. RESULTS: Triclosan led to histopathologic changes in the thyroid and decreases in triiodothyronine (T3) and thyroxine (T4). Triclosan stimulated ROS production and oxidative stress occurrence, thereby activating the p38 pathway in vivo and in vitro. Thyrotropin releasing hormone receptor (TRHr) was induced when the p38 pathway was activated, and was suppressed when that pathway was inhibited. Moreover, thyroid peroxidase (TPO) was restrained and modulated by the p38/TRHr pathway after triclosan treatment. Furthermore, deiodinase 3 (D3) and hepatic enzymes (Ugt2b1, CYP1a1, CYP1a2, CYP2b1, CYP3a1, and Sult1e1) were also induced by triclosan. CONCLUSION: Taken together, p38/TRHr-dependent regulation of TPO in thyroid cells contributes to the hypothyroidism of triclosan-treated rats.

TRH receptor mobility in the plasma membrane is strongly affected by agonist binding and by interaction with some cognate signaling proteins.

OBJECTIVES: Extensive research has been dedicated to elucidating the mechanisms of signal transduction through different G protein-coupled receptors (GPCRs). However, relatively little is known about the regulation of receptor movement within the cell membrane upon ligand binding. In this study we focused our attention on the thyrotropin-releasing hormone (TRH) receptor that typically couples to Gq/11 proteins. METHODS: We monitored receptor diffusion in the plasma membrane of HEK293 cells stably expressing yellow fluorescent protein (YFP)-tagged TRH receptor (TRHR-YFP) by fluorescence recovery after photobleaching (FRAP). RESULTS: FRAP analysis indicated that the lateral movement of the TRH receptor was markedly reduced upon TRH binding as the value of its diffusion coefficient fell down by 55%. This effect was prevented by the addition of the TRH receptor antagonist midazolam. We also found that siRNA-mediated knockdown of Gq/11α, Gß, ß-arrestin2 and phospholipase Cß1, but not of Giα1, ß-arrestin1 or G protein-coupled receptor kinase 2, resulted in a significant decrease in the rate of TRHR-YFP diffusion, indicating the involvement of the former proteins in the regulation of TRH receptor behavior. The observed partial reduction of the TRHR-YFP mobile fraction caused by down-regulation of Giα1 and ß-arrestin1 suggests that these proteins may also play distinct roles in THR receptor-mediated signaling. CONCLUSION: These results demonstrate for the first time that not only agonist binding but also abundance of some signaling proteins may strongly affect TRH receptor dynamics in the plasma membrane.

Excitation of locus coeruleus noradrenergic neurons by thyrotropin-releasing hormone.

Locus coeruleus (LC) noradrenergic neurons are implicated in a variety of functions including the regulation of vigilance and the modulation of sensory processing. Thyrotropin-releasing hormone (TRH) is an endogenous neuropeptide that induces a variety of behavioural changes including arousal and antinociception. In the present study, we explored whether the activity of LC noradrenergic neurons is modulated by TRH. Using current-clamp recording from isolated rat LC neurons, we found that TRH increased the firing rate of spontaneous action potentials. The TRH action was mimicked by TRH analogues including taltirelin and TRH-gly. In voltage-clamp recording at a holding potential of 50 mV, TRH produced an inward current associated with a decrease in the membrane K+ conductance. This current was inhibited by the TRH receptor antagonist chlordiazepoxide. Following inhibition of the pH-sensitive K+ conductance by extracellular acidification, the TRH response was fully inhibited. The TRH-induced current was also inhibited by the phospholipase C (PLC) inhibitor U-73122, but not by the protein kinase C inhibitor chelerythrine nor by chelation of intracellular Ca2+ by BAPTA. The recovery from the facilitatory action of TRH on the spike frequency was markedly inhibited by a high concentration of wortmannin. These results suggest that TRH activates LC noradrenergic neurons by decreasing an acid-sensitive K+ conductance via PLC-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate. The present findings demonstrate that TRH activates LC neurons and characterize the underlying signalling mechanisms. The action of TRH on LC neurons may influence a variety of CNS functions related to the noradrenergic system which include arousal and analgesia.

Excitation of histaminergic tuberomamillary neurons by thyrotropin-releasing hormone.

The histaminergic tuberomamillary nucleus (TMN) controls arousal and attention, and the firing of TMN neurons is state-dependent, active during waking, silent during sleep. Thyrotropin-releasing hormone (TRH) promotes arousal and combats sleepiness associated with narcolepsy. Single-cell reverse-transcription-PCR demonstrated variable expression of the two known TRH receptors in the majority of TMN neurons. TRH increased the firing rate of most (ca 70%) TMN neurons. This excitation was abolished by the TRH receptor antagonist chlordiazepoxide (CDZ; 50 mum). In the presence of tetrodotoxin (TTX), TRH depolarized TMN neurons without obvious change of their input resistance. This effect reversed at the potential typical for nonselective cation channels. The potassium channel blockers barium and cesium did not influence the TRH-induced depolarization. TRH effects were antagonized by inhibitors of the Na(+)/Ca(2+) exchanger, KB-R7943 and benzamil. The frequency of GABAergic spontaneous IPSCs was either increased (TTX-insensitive) or decreased [TTX-sensitive spontaneous IPSCs (sIPSCs)] by TRH, indicating a heterogeneous modulation of GABAergic inputs by TRH. Facilitation but not depression of sIPSC frequency by TRH was missing in the presence of the kappa-opioid receptor antagonist nor-binaltorphimine. Montirelin (TRH analog, 1 mg/kg, i.p.) induced waking in wild-type mice but not in histidine decarboxylase knock-out mice lacking histamine. Inhibition of histamine synthesis by (S)-alpha-fluoromethylhistidine blocked the arousal effect of montirelin in wild-type mice. We conclude that direct receptor-mediated excitation of rodent TMN neurons by TRH demands activation of nonselective cation channels as well as electrogenic Na(+)/Ca(2+) exchange. Our findings indicate a key role of the brain histamine system in TRH-induced arousal.

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.

1-(Phenyl)isoquinoline carboxamides: a novel class of subtype selective inhibitors of thyrotropin-releasing hormone (TRH) receptors.

We report the synthesis of and binding to the two subtypes of mouse thyrotropin-releasing hormone (TRH) receptors, TRH-R1 and TRH-R2, of several 1-(phenyl)isoquinoline carboxamide analogues. These analogues showed a degree of selectivity for binding at TRH-R2. These are the first ligands reported that show selective binding to these receptors.

Design, synthesis, and biological evaluation of novel, centrally-acting thyrotropin-releasing hormone analogues.

Novel, metabolically stable and centrally acting TRH analogues with substituted pyridinium moieties replacing the [His(2)] residue of the endogenous peptide were prepared by solid-phase Zincke reaction. The 1,4-dihydropyridine prodrugs of these analogues obtained after reducing the pyridinium moiety were able to reach the brain and maintain a sustained concentration of the charged, degradation-resistant analogues formed after enzymatic oxidation of the prodrug, as manifested by the analeptic action measured in mice. Among the four analogues reported, compound 2a showed the highest potency and longest duration of action in reducing the pentobarbital-induced sleeping time compared to the parent TRH. No binding to the endocrine TRH-receptor was measured for 2a; thus, this compound emerged as a potent, centrally acting TRH analogue.

Overexpression of the G protein G11alpha prevents desensitization of Ca2+ response to thyrotropin-releasing hormone.

Doubly transfected human embryonal kidney cells (clone E2M11 of the HEK 293 cell line) expressing both thyrotropin-releasing hormone (TRH) receptors and G11alpha protein in high amounts were used to analyze the desensitization phenomenon of the Ca2+-mobilizing pathway. Quite unexpectedly, we did not observe any significant desensitization of the [Ca2+]i response to TRH in these cells after repeated or prolonged incubation with the hormone (up to 5 h). Under the same conditions, the TRH-induced [Ca2+]i response was completely desensitized in the parent cell line (293-E2 cels) expressing TRH receptors alone. In both cell lines, inositol phosphate response was desensitized after TRH exposure, although basal levels of inositol phospates in TRH-pretreated cells were much higher than in "naive" TRH-unexposed cells. These data suggest a significant role of the G protein G11alpha in desensitization of the Ca2+-mobilizing pathway occuring after repeated or long-term exposure of target cells to TRH-receptor agonists.

Inverse agonist abolishes desensitization of a constitutively active mutant of thyrotropin-releasing hormone receptor: role of cellular calcium and protein kinase C.

1. C335Stop is a constitutively active mutant of the TRH receptor (TRH-R). To investigate the mechanism of the decreased responsiveness of C335Stop TRH-R, we studied cellular Ca2+ concentrations ([Ca2+]i) in AtT20 cells stably transfected with C335Stop TRH-R cDNA, or Ca2+-activated chloride currents in Xenopus laevis oocytes expressing this mutant receptor after injection of cRNA. The competitive TRH-R binding antagonist, chlorodiazepoxide (CDE), was used as an inverse agonist to study the contribution of constitutive activity to desensitization. 2. Acute treatment with CDE resulted in a rapid (within minutes) decrease in [Ca2+]i and an increase in the response amplitude to TRH with no measurable change in receptor density. Conversely, removal of chronically administered CDE caused a rapid increase in [Ca2+]i and a decrease in TRH response amplitude. 3. CDE abolished heterologous desensitization induced by C335Stop TRH-R on muscarinic m1-receptor (ml-R) co-expressed in Xenopus oocytes. 4. Chelation of extracellular calcium with EGTA caused a rapid decrease in [Ca2+]i and a concomitant increase in the response to TRH in AtT20 cells expressing C335Stop TRH-Rs. 5. Chelerythrine, a specific inhibitor of protein kinase C (PKC), reversed the heterologous desensitization of the response to acetylcholine (ACh). The phosphoserine/phosphothreonine phosphatase inhibitor, okadaic acid, abolished the effect of chelerythrine. 6. Down-regulation of PKC by chronic exposure to phorbol 12-myristate 13-acetate (PMA) or acute inhibition with chelerythrine caused a partial resensitization of the response to TRH. 7. Western analysis indicated that the alpha subtype of protein kinase C was down-regulated in cells expressing C335Stop TRH-Rs. Following a 5 min exposure to PMA, the residual alphaPKC translocated to the particular fraction. 8. We propose that cells expressing the constitutively active mutant TRH-R rapidly desensitize their response, utilizing a mechanism mediated by an increase in [Ca2+]i and PKC.

Signal transduction, desensitization, and recovery of responses to thyrotropin-releasing hormone after inhibition of receptor internalization.

Three independent methods were used to block internalization of the TRH receptor: cells were infected with vaccinia virus encoding a dominant negative dynamin, incubated in hypertonic sucrose, or stably transfected with a receptor lacking the C-terminal tail. Internalization was blocked in all three paradigms as judged by microscopy using a fluorescently labeled TRH agonist and biochemically. The initial inositol trisphosphate (IP3) and Ca2+ responses to TRH were normal when internalization was inhibited. The IP3 increase was sustained rather than transient, however, in cells expressing the truncated TRH receptor, implying that the C-terminal tail of the receptor may be important for uncoupling from phospholipase C. After withdrawal of TRH, cells were refractory to TRH until both ligand dissociation and resensitization of the receptor had occurred. When surface-bound TRH was removed by a mild acid wash, which did not impair receptor function, neither wild-type nor truncated receptors were able to generate full IP3 responses for about 10 min. The rate of recovery was not altered by blocking internalization. Recovery of intracellular Ca2+ responses also depended on the rate of Ca2+ pool refilling. In summary, in the continued presence of TRH, phospholipase C activity declines quickly due to receptor uncoupling; this desensitization does not take place for the truncated receptor. After TRH is withdrawn, cells are refractory to TRH. Before cells can respond, TRH must dissociate and a resensitization step, which takes place on the plasma membrane and does not require the C-terminal tail of the receptor, must occur.

Results of a combined dexamethasone suppression/thyrotropin-releasing hormone stimulation test in healthy horses and horses suspected to have a pars intermedia pituitary adenoma.

OBJECTIVE: To evaluate results of a combined dexamethasone suppression/thyrotropin-releasing hormone (TRH) stimulation test in horses suspected clinically to have a pars intermedia pituitary adenoma (PIPA). DESIGN: Case-control study. ANIMALS: 7 healthy adult horses and 5 horses suspected to have a PIPA. PROCEDURE: A baseline blood sample was collected, and dexamethasone (40 micrograms/kg [18 micrograms/lb] of body weight, IV) was administered; a second blood sample was collected 3 hours later, and TRH (1.1 mg, IV) was administered; serial blood samples were collected 15, 30, 45, 60, and 90 minutes and 21 hours after TRH administration (24 hours after dexamethasone injection). Cortisol concentration was determined for all blood samples. RESULTS: Baseline cortisol concentration was significantly lower in horses suspected to have a PIPA than in healthy horses. Cortisol concentration was suppressed by dexamethasone in both groups; however, after TRH administration, cortisol concentration returned to baseline values in horses suspected to have a PIPA, but not in healthy horses. Concentration was still less than the baseline value 24 hours after dexamethasone administration in healthy horses. CLINICAL IMPLICATIONS: The combined dexamethasone suppression/TRH stimulation test may be a useful diagnostic test in horses suspected to have a PIPA. For clinical application, collection of a blood sample 30 minutes after TRH administration is recommended.

Intracisternal antisense oligonucleotides to TRH receptor abolish TRH-evoked gastric motor excitation.

Thyrotropin-releasing hormone (TRH) from the nucleus raphe obscurus (nROb) innervates the dorsal vagal complex (DVC) and activates gastric motor function. Assessment of the importance of TRH has been hampered by the lack of TRH receptor antagonists. To overcome this, rats were given intracisternal antisense oligonucleotides against the first 18 bases of TRH receptor mRNA, mismatch oligonucleotides, or saline. Rats were anesthetized, and L-glutamate (15 nmol), TRH (1 and 10 pmol), and saline were microinjected into the DVC and nROb while gastric motor function was monitored. Intracisternal TRH mRNA antisense oligonucleotides abolished the gastric excitatory affects of microinjection of TRH, but not L-glutamate, into the DVC, and the response to TRH recovered after 2 wk of no antisense treatment. Chemical stimulation of the nROb increased intragastric pressure in saline- and mismatch- but not antisense-treated animals. These studies demonstrate that intracisternal TRH receptor antisense oligonucleotides produce a selective and reversible "knockdown" of responsiveness to exogenous TRH in the DVC, as well as to excitation of an endogenous TRH pathway controlling gastric function. It also provides a new tool for assessment of TRH pathways in hindbrain control of gastric function.

Antisense to thyrotropin releasing hormone receptor reduces arterial blood pressure in spontaneously hypertensive rats.

We report in the present study the effect of intrathecal treatment with antisense oligonucleotides complementary to thyrotropin releasing hormone (TRH) receptor mRNA on the pressor response to intrathecal administration of TRH and on resting arterial blood pressure in Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). In 16-week-old male WKY rats, 18-base phosphodiester antisense or mismatch oligonucleotides to TRH receptor mRNA (100 micrograms per day) were injected intrathecally for 3 days. Twenty-four hours after the last injection, the magnitude of the pressor response to intrathecal TRH (10 micrograms) was significantly smaller in the antisense-treated group (n = 7) compared with mismatch-treated controls (n = 7) (change in mean arterial pressure, +20.3 +/- 3.0 versus +32.6 +/- 2.5 mm Hg, P < .01). No differences were observed in the pressor responses to injection of N-methyl-D-aspartic acid. Resting arterial blood pressure was unaffected by antisense treatment in WKY rats. In separate experiments, 16-week-old male SHR were treated with antisense (n = 7) or mismatch (n = 6) oligonucleotides for 3 days. Mean resting arterial blood pressure was significantly reduced by treatment with antisense oligonucleotides (from 157 +/- 4.8 to 119 +/- 8.8 mm Hg, P < .01), but no significant changes were observed in mismatch-treated animals. Our results suggest that the expression of TRH receptors in spinal sympathetic preganglionic neurons can be selectively reduced by intrathecal treatment with antisense oligonucleotides and that TRH projections to sympathetic preganglionic neurons play an important role in the elevation of arterial blood pressure in SHR.

Caffeine inhibits the binding of thyrotropin-releasing hormone in GH4C1 pituitary cells.

Caffeine can modulate intracellular Ca2+ concentration ([Ca2+]i) by triggering the mobilization of Ca2+ from intracellular Ca2+ stores. In the present study we show that in Fura 2 loaded GH4C1 cells, caffeine inhibited, in a dose-dependent manner, the Ca2+ response induced by a submaximally effective dose (3 nM) of thyrotropin-releasing hormone (TRH). We also show that caffeine decreased the specific binding of [3H]TRH. Equilibrium binding studies with [3H]TRH and Scatchard analysis of the binding data showed that caffeine increased the dissociation constant (Kd) from 8 +/- 1 nM to 26 +/- 3 nM, while the maximum amount of [3H]TRH bound to the cells was increased by 32%. Thus, caffeine inhibited the TRH-evoked increase in [Ca2+]i by inhibiting the binding of TRH to its receptor.