Pituitary Thyrotropin-releasing Hormone Research Articles

IGSF1 Deficiency Leads to Reduced TSH Production Independent of Alterations in Thyroid Hormone Action in Male Mice.

Loss of function mutations in IGSF1/Igsf1 cause central hypothyroidism. Igsf1 knockout mice have reduced pituitary thyrotropin-releasing hormone receptor, Trhr, expression, perhaps contributing to the phenotype. Because thyroid hormones negatively regulate Trhr, we hypothesized that IGSF1 might affect thyroid hormone availability in pituitary thyrotropes. Consistent with this idea, IGSF1 coimmunoprecipitated with the thyroid hormone transporter monocarboxylate transporter 8 (MCT8) in transfected cells. This association was impaired with IGSF1 bearing patient-derived mutations. Wild-type IGSF1 did not, however, alter MCT8-mediated thyroid hormone import into heterologous cells. IGSF1 and MCT8 are both expressed in the apical membrane of the choroid plexus. However, MCT8 protein levels and localization in the choroid plexus were unaltered in Igsf1 knockout mice, ruling out a necessary chaperone function for IGSF1. MCT8 expression was low in the pituitary and was similarly unaffected in Igsf1 knockouts. We next assessed whether IGSF1 affects thyroid hormone transport or action, by MCT8 or otherwise, in vivo. To this end, we treated hypothyroid wild-type and Igsf1 knockout mice with exogenous thyroid hormones. T4 and T3 inhibited TSH release and regulated pituitary and forebrain gene expression similarly in both genotypes. Interestingly, pituitary TSH beta subunit (Tshb) expression was consistently reduced in Igsf1 knockouts relative to wild-type regardless of experimental condition, whereas Trhr was more variably affected. Although IGSF1 and MCT8 can interact in heterologous cells, the physiological relevance of their association is not clear. Nevertheless, the results suggest that IGSF1 loss can impair TSH production independently of alterations in TRHR levels or thyroid hormone action.

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.

Hypothalamic–pituitary thyroid axis alterations in female mice with deletion of the neuromedin B receptor gene

Neuromedin B, a peptide highly expressed at the pituitary, has been shown to act as autocrine/paracrine inhibitor of thyrotropin (TSH) release. Here we studied the thyroid axis of adult female mice lacking neuromedin B receptor (NBR-KO), compared to wild type (WT) littermates. They exhibited slight increase in serum TSH (18%), with normal pituitary expression of mRNA coding for α-glycoprotein subunit (Cga), but reduced TSH β-subunit mRNA (Tshb, 41%), lower intra-pituitary TSH content (24%) and increased thyroid hormone transporter MCT-8 (Slc16a2, 44%) and thyroid hormone receptor β mRNA expression (Thrb, 39%). NBR-KO mice exhibited normal thyroxine (T4) and reduced triiodothyronine (T3) (30%), with no alterations in the intra-thyroidal content of T4 and T3 or thyroid morphological changes. Hypothalamic thyrotropin-releasing hormone (TRH) mRNA (Trh) was increased (68%), concomitant with a reduction in type 2 deiodinase mRNA (Dio2, 30%) and no changes in MCT-8 and thyroid hormone receptor mRNA expression. NBR-KO mice exhibited a 56% higher increase in serum TSH in response to an acute single intraperitoneal injection of TRH concomitant with a non-significant increase in pituitary TRH receptor (Trhr) mRNA at basal state. The phenotype of female NBR-KO mice at the hypothalamus–pituitary axis revealed alterations in pituitary and hypothalamic gene expression, associated with reduced serum T3, and higher TSH response to TRH, with apparently normal thyroid morphology and hormonal production. Thus, results confirm that neuromedin B pathways are importantly involved in secretory pathways of TSH and revealed its participation in the in vivo regulation of gene expression of TSH β-subunit and pituitary MCT8 and Thrb and hypothalamic TRH and type 2 deiodinase.

Loss-of-function mutations in IGSF1 cause an X-linked syndrome of central hypothyroidism and testicular enlargement.

Congenital central hypothyroidism occurs either in isolation or in conjunction with other pituitary hormone deficits. Using exome and candidate gene sequencing, we identified eight distinct mutations and two deletions in IGSF1 in males from eleven unrelated families with central hypothyroidism, testicular enlargement, and variably low prolactin concentrations. IGSF1 is a membrane glycoprotein highly expressed in the anterior pituitary gland and the identified mutations impair its trafficking to the cell surface in heterologous cells. Igsf1-deficient male mice show diminished pituitary and serum thyroid-stimulating hormone (TSH) concentrations, reduced pituitary thyrotropin-releasing hormone (TRH) receptor expression, decreased triiodothyronine concentrations, and increased body mass. Collectively, our observations delineate a novel X-linked disorder in which loss-of-function mutations in IGSF1 cause central hypothyroidism, likely secondary to an associated impairment in pituitary TRH signaling.

Desensitization, Trafficking, and Resensitization of the Pituitary Thyrotropin-Releasing Hormone Receptor

The pituitary receptor for thyrotropin-releasing hormone (TRH) is a calcium-mobilizing G protein-coupled receptor (GPCR) that signals through Gq/11, elevating calcium, and activating protein kinase C. TRH receptor signaling is quickly desensitized as a consequence of receptor phosphorylation, arrestin binding, and internalization. Following activation, TRH receptors are phosphorylated at multiple Ser/Thr residues in the cytoplasmic tail. Phosphorylation catalyzed by GPCR kinase 2 (GRK2) takes place rapidly, reaching a maximum within seconds. Arrestins bind to two phosphorylated regions, but only arrestin bound to the proximal region causes desensitization and internalization. Phosphorylation at Thr365 is critical for these responses. TRH receptors internalize in clathrin-coated vesicles with bound arrestin. Following endocytosis, vesicles containing phosphorylated TRH receptors soon merge with rab5-positive vesicles. Over approximately 20 min these form larger endosomes rich in rab4 and rab5, early sorting endosomes. After TRH is removed from the medium, dephosphorylated receptors start to accumulate in rab4-positive, rab5-negative recycling endosomes. The mechanisms responsible for sorting dephosphorylated receptors to recycling endosomes are unknown. TRH receptors from internal pools help repopulate the plasma membrane. Dephosphorylation of TRH receptors begins when TRH is removed from the medium regardless of receptor localization, although dephosphorylation is fastest when the receptor is on the plasma membrane. Protein phosphatase 1 is involved in dephosphorylation but the details of how the enzyme is targeted to the receptor remain obscure. It is likely that future studies will identify biased ligands for the TRH receptor, novel arrestin-dependent signaling pathways, mechanisms responsible for targeting kinases and phosphatases to the receptor, and principles governing receptor trafficking.

Thyrotropin-releasing hormone and oestrogen differentially regulate prolactin and prolactin receptor expression in female human skin and hair follicles in vitro

Human skin and scalp hair follicles are both a nonclassical target and an extrapituitary source of prolactin (PRL), which is a potent hair growth modulator. However, how the expression of PRL and PRL receptor (PRLR) is regulated in human skin is unknown. To investigate whether two key stimulators of pituitary PRL secretion, thyrotropin-releasing hormone (TRH) and oestrogen, also regulate cutaneous PRL and PRLR expression. Female scalp skin and/or microdissected hair follicles were treated for 6 days in serum-free organ culture with oestrogen (100 nmol L(-1)), TRH (1-10 ng mL(-1), 3-30 nm) or vehicle control. Quantitative immunohistomorphometry of skin and hair follicle sections was complemented with quantitative polymerase chain reaction for PRL and PRLR in cultured hair follicles and/or female human outer root sheath (ORS) keratinocytes. Oestrogen treatment significantly upregulated PRL and PRLR immunoreactivity in selected skin and hair follicle compartments, at the gene and protein level (P < 0.05). TRH significantly increased PRL immunoreactivity and transcription in hair follicles (P < 0.05); however, while it also increased PRLR transcription in hair follicles, it downregulated PRLR immunoreactivity in the hair follicle ORS (P < 0.05). Our pilot study shows that two key endocrine controls of pituitary PRL secretion, oestrogen and TRH, also regulate PRL and PRLR expression in human skin. This provides novel insights into the regulation of extrapituitary PRL and PRLR expression, and invites exploration of oestrogen and TRH as novel therapeutic agents in the management of skin and hair diseases characterized by aberrant PRLR-mediated signalling.

Interaction between salsolinol (SAL) and thyrotropin-releasing hormone (TRH) or dopamine (DA) on the secretion of prolactin in ruminants

We have recently demonstrated that salsolinol (SAL), a dopamine (DA)-derived compound, is present in the posterior pituitary gland and is able to stimulate the release of prolactin (PRL) in ruminants. The aim of the present study was to clarify the effect that the interaction of SAL with thyrotropin-releasing hormone (TRH) or DA has on the secretion of PRL in ruminants. A single intravenous (i.v.) injection of SAL (5 mg/kg body weight (b.w.)), TRH (1 μg/kg b.w.), and SAL plus TRH significantly stimulated the release of PRL in goats ( P < 0.05). The cumulative response curve (area under the curve: AUC) during 120 min was 1.53 and 1.47 times greater after the injection of SAL plus TRH than either SAL or TRH alone, respectively ( P < 0.05). A single i.v. injection of sulpiride (a DA receptor antagonist, 0.1 mg/kg b.w.), sulpiride plus SAL (5 mg/kg b.w.), and sulpiride plus TRH (1 μg/kg b.w.) significantly stimulated the release of PRL in goats ( P < 0.05). The AUC of PRL during 120 min was 2.12 and 1.78 times greater after the injection of sulpiride plus TRH than either sulpiride alone or sulpiride plus SAL, respectively ( P < 0.05). In cultured bovine anterior pituitary (AP) cells, SAL (10 −6 M), TRH (10 −8 M), and SAL plus TRH significantly increased the release of PRL ( P < 0.05), but the additive effect of SAL and TRH detected in vivo was not observed in vitro. In contrast, DA (10 −6 M) inhibited the TRH-, as well as SAL-induced PRL release in vitro. All together, these results clearly show that SAL can stimulate the release of PRL in ruminants. Furthermore, they also demonstrate that the additive effect of SAL and TRH on the release of PRL detected in vivo may not be mediated at the level of the AP, but that DA can overcome their releasing activity both in vivo and in vitro, confirming the dominant role of DA in the inhibitory regulation of PRL secretion in ruminants.

Thyrotropin-releasing hormone concentrations in different regions of the chicken brain and pituitary: an ontogenetic study

The regional distribution of thyrotropin-releasing hormone (TRH) was studied in the chicken brain. The hypothalamus and the brain stem contained the highest concentration of TRH. Lower amounts were present in the telencephalon, the optic lobes and the cerebellum. Within the hypothalamus, TRH was most abundant in the median eminence. Other important TRH sites were the nucleus paraventricularis magnocellularis, nucleus periventricularis hypothalami, nucleus ventromedialis hypothalami, nucleus dorsomedialis hypothalami and nucleus preopticus periventricularis. On the 14th day of embryonic development (E14), TRH was mostly found in the brain stem. Towards hatching, TRH concentrations increased gradually in both the hypothalamic area and the brain stem. TRH concentrations in the telencephalon, optic lobes and cerebellum remained low. Pituitaries from E14 to E16 chickens were characterized by a high TRH concentration, whereas hypophyseal TRH concentrations dropped towards hatching. Our results support the hypothesis that TRH exerts both endocrine and neurocrine actions in the chicken. On the other hand, high pituitary TRH concentrations were present when hypothalamic concentrations were low and vice versa. Therefore, the chicken pituitary may function as an important source of TRH during early in ovo development at least until the moment hypothalamic control develops.

Cloning and Characterization of a cDNA Encoding a Novel Subtype of Rat Thyrotropin-releasing Hormone Receptor

A cDNA encoding a thyrotropin-releasing hormone (TRH) receptor expressed in the pituitary was previously cloned (De La Pena, P., Delgado, L. M., Del Camino, D., and Barros, F. (1992) Biochem. J. 284, 891-899; De La Pena, P., Delgado, L. M., Del Camino, D., and Barros, F. (1992) J. Biol. Chem. 267, 25703-25708; Duthie, S. M., Taylor, P. L., Anderson, J., Cook, J., and Eidne, K. A. (1993) Mol. Cell Endocrinol. 95, R11-R15). We now describe the isolation of a rat cDNA encoding a novel subtype of TRH receptor (termed TRHR2) displaying an overall homology of 50% to the pituitary TRH receptor. Introduction of TRHR2 cDNA in HEK-293 cells resulted in expression of high affinity TRH binding with a different pharmacological profile than the pituitary TRH receptor. De novo expressed receptors were functional and resulted in stimulation of calcium transient as assessed by fluorometric imaging plate reader analysis. The message for TRHR2 was exclusive to central nervous system tissues as judged by Northern blot analysis. Studies of the expression of TRHR-2 message by in situ hybridization revealed a pattern of expression remarkably distinct (present in spinothalamic tract, spinal cord dorsal horn) from that of the pituitary TRH receptor (present in hypothalamus, and ventral horn of the spinal cord, anterior pituitary). Therefore, we have identified a novel, pharmacologically distinct receptor for thyrotropin-releasing hormone that appears to be more restricted to the central nervous system particularly to the sensory neurons of spinothalamic tract and spinal cord dorsal horn, which may account for the sensory antinociceptive actions of TRH.

Ectopic expression of thyrotropin releasing hormone (TRH) receptors in liver modulates organ function to regulate blood glucose by TRH

Maintenance of blood glucose by the liver is normally initiated by extracellular regulatory molecules such as glucagon and vasopressin triggering specific hepatocyte receptors to activate the cAMP or phosphoinositide signal transduction pathways, respectively. We now show that the normal ligand-receptor regulators of blood glucose in the liver can be bypassed using an adenovirus vector expressing the mouse pituitary thyrotropin releasing hormone receptor (TRHR) cDNA ectopically in rat liver in vivo. The ectopically expressed TRHR links to the phosphoinositide pathway, providing a means to regulate liver function with TRH, an extracellular ligand that does not normally affect hepatic function. Administration of TRH to these animals activates the phosphoinositide pathway, resulting in a sustained rise in blood glucose. It should be possible to use this general strategy to modulate the differentiated functions of target organs in a wide variety of pathologic states.

Posttranscriptional up-regulation of thyrotropin-releasing hormone (TRH) receptor messenger ribonucleic acid by TRH in COS-1 cells transfected with mouse pituitary TRH receptor complementary deoxyribonucleic acid.

We found previously that the level of endogenous TRH receptor (TRH-R) mRNA in pituitary (GH3) cells and the level of mouse TRH-R mRNA in GH3 cells stably transfected with mouse pituitary TRH-R cDNA are down-regulated by TRH. This down-regulation is caused by TRH stimulation of TRH-R mRNA degradation via a mechanism that appears to involve protein kinase-C. In this report we study regulation of TRH-R mRNA in monkey kidney (COS-1) cells transiently transfected with mouse pituitary TRH-R cDNA. In transfected COS-1 cells, TRH and phorbol 12-myristate 13-acetate (PMA) caused increases in the level of TRH-R mRNA. In contrast, TRH caused only a small transient increase in the level of the mRNA for the neomycin resistance gene, which was cotransfected with TRH-R, and did not affect the level of the mRNA for glyceraldehyde phosphate dehydrogenase, an endogenous gene. The increases in TRH-R mRNA caused by TRH and PMA were inhibited to similar extents by H-7 (1-[5-isoquinolinesulfonyl]2-methyl piperazine dihydrochloride), an inhibitor of protein kinases. The effect of TRH was observed in cells transfected with expression vectors in which TRH-R cDNA was controlled by cytomegalovirus or Rous sarcoma virus promoters. There was no effect of TRH or PMA on the rate of transcription of the transfected TRH-R cDNA. In contrast, TRH caused the rate of degradation of TRH-R mRNA to decrease from 8.0% to 5.1%/h. Hence, TRH, most likely via a protein kinase-C-mediated mechanism, up-regulates TRH-R mRNA levels in transfected COS-1 cells by decreasing the rate of TRH-R mRNA degradation. Since TRH and PMA down-regulate TRH-R mRNA in GH3 cells, posttranscriptional regulation of TRH-R mRNA is a cell-type specific process.

Posttranscriptional up-regulation of thyrotropin-releasing hormone (TRH) receptor messenger ribonucleic acid by TRH in COS-1 cells transfected with mouse pituitary TRH receptor complementary deoxyribonucleic acid

Posttranscriptional up-regulation of thyrotropin-releasing hormone (TRH) receptor messenger ribonucleic acid by TRH in COS-1 cells transfected with mouse pituitary TRH receptor complementary deoxyribonucleic acid

Inverse relationship between CSF TRH concentrations and the TSH response to TRH in abstinent alcohol-dependent patients.

The authors performed the thyrotropin-releasing hormone (TRH) stimulation test and measured CSF concentrations of TRH in 13 abstinent alcohol-dependent subjects. They found an inverse correlation between the thyrotropin (TSH) response to TRH and endogenous CSF TRH concentrations. This finding supports the hypothesis that as the concentration of CSF TRH increases, anterior pituitary TRH receptor density decreases, resulting in a blunted TSH response to TRH stimulation.

Dose-response studies with thyrotropin-releasing hormone (TRH) in abstinent male alcoholics: evidence for selective thyrotroph dysfunction?

A reduced thyrotropin (TSH) response to thyrotropin-releasing hormone (TRH) has been reported in subjects with a history of alcoholism whereas prolactin (PRL) responses have generally been normal. One hypothesis proposed to explain the reduced TSH response is down-regulation of pituitary TRH receptors. If this is correct, PRL response should also be diminished. To account for the different dose-response characteristics of TSH/PRL we have given four dosages of TRH (25, 100, 500 and 800 micrograms) to eight noncirrhotic, male alcoholics abstinent from ethanol a minimum of 28 days and to seven male control subjects. Across the TRH dose range the alcoholic subjects exhibited reduced basal TSH (p = .01) and a reduced TSH response (p = .0023) but no differences in basal and stimulated PRL levels. Alcoholic subjects had higher basal T4, T3 and FT4I values than did control subjects but covarying for T4, T3 and FT4I did not change the significance of either TSH or PRL findings. No significant differences in estradiol, estrone, testosterone, cortisol or glucose were noted between groups. The present study confirms the observation of a lower TSH response to TRH in abstinent alcoholics and indicates that the lower response cannot be overcome by increasing TRH dosage. The similar PRL response between groups suggests normal lactotroph function in noncirrhotic abstinent alcoholics and argues against the pituitary TRH receptor down-regulation hypothesis.

Expression cloning of a cDNA encoding the mouse pituitary thyrotropin-releasing hormone receptor.

Thyrotropin-releasing hormone (TRH) is an important extracellular regulatory molecule that functions as a releasing factor in the anterior pituitary gland and as a neurotransmitter/neuromodulator in the central and peripheral nervous systems. Binding sites for TRH are present in these tissues, but the TRH receptor (TRH-R) has not been purified from any source. Using Xenopus laevis oocytes in an expression cloning strategy, we have isolated a cDNA clone that encodes the mouse pituitary TRH-R. This conclusion is based on the following evidence. Injection of sense RNA transcribed in vitro from this cDNA into Xenopus oocytes leads to expression of cell-surface receptors that bind TRH and the competitive antagonist chlordiazepoxide with appropriate affinities and that elicit electrophysiological responses to TRH with the appropriate concentration dependency. Antisense RNA inhibits the TRH response in Xenopus oocytes injected with RNA isolated from normal rat anterior pituitary glands. Finally, transfection of COS-1 cells with this cDNA leads to expression of receptors that bind TRH and chlordiazepoxide with appropriate affinities and that transduce TRH stimulation of inositol phosphate formation. The 3.8-kilobase mouse TRH-R cDNA encodes a protein of 393 amino acids that shows similarities to other guanine nucleotide-binding regulatory protein-coupled receptors.

A novel substance P binding site in rat brain regions modulates TRH receptor binding

Binding sites for thyrotropin-releasing hormone (TRH) were labelled with [3H](2-Me-His3)TRH ([3H]MeTRH) on membranes from rat brain regions at 0 degrees C for 5 h. Amygdaloid membranes bound [3H]MeTRH with high-affinity (Kd = 3.1 +/- 0.5 nM (n = 4)). Five TRH analogs competed for this binding with the same rank order and with affinities that matched the pharmacological specificity of pituitary TRH receptors. Substance P (SP) and its C-terminal fragments reduced amygdaloid TRH receptor binding in a concentration dependent manner (IC50 for SP = 65 microM). The rank order of potency of SP analogs at inhibiting TRH receptor binding was: SP greater than nonapeptide (3-11) greater than hexapeptide (6-11) greater than heptapeptide (5-11) greater than pentapeptide (7-11). However, other tachykinins were inactive in this system. SP was a potent inhibitor of [3H]MeTRH binding in hippocampus greater than spinal cord greater than retina greater than n. accumbens greater than hypothalamus greater than amygdaloid greater than olfactory bulb greater than or equal to pituitary greater than pons/medulla in parallel assays. In amygdaloid membranes SP (50 microM) reduced the apparent maximum receptor density by 39% (p less than 0.01) without altering the binding affinity, and 100 microM SP induced a biphasic dissociation of [3H]MeTRH with kinetics faster than those induced by both TRH (10 microM) and serotonin (100 microM). In contrast, other neuropeptides such as neurotensin, proctolin, angiotensin II, bombesin and luteinizing hormone releasing hormone did not significantly inhibit [3H]MeTRH binding to amygdaloid membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyrotropic activity of estradiol in the chick embryo develops without increase in pituitary thyrotropin releasing hormone binding.

The influence of 17β-estradiol (E2) injections on plasma thyroid hormonesand growth hormone (GH) and on pituitary thyrotropin-releasing hormone(TRH) binding was studied in 19-day-old chick embryos. The E2 injectionraised plasma levels of thyroxine (T4) and triiodothyronine (T3) withoutincreases in the peripheral 5'-monodeiodination (5'-D) and the T3 to T4 ratio.The injection had no significant effect on plasma GH concentrations. PituitaryTRH binding was not affected by E2-treatment, suggesting that as contrastedwith findings in mammals, changes in pituitary TRH receptor levels may not beresponsible for the thyrotropic action of E2 in the chick embryo.

Pituitary Thyrotropin-Releasing Hormone (TRH) Receptors: Effects of TRH, Drugs Mimicking TRH Action, and Chlordiazepoxide

Binding of TRH to specific cell surface receptors on clonal GH4C1 cells is followed within 10 min by receptor sequestration and over 24 h by receptor down-regulation. These experiments were designed to determine if TRH-activated second messenger systems are responsible for changes in receptor localization or number. BAY K8644 and A23187, which increase intracellular calcium, alone or together with 12-O-tetradecanoyl phorbol acetate (TPA), which activates protein kinase C, did not appear to internalize TRH receptors. Drug treatment did not alter the rate of [3H]MeTRH association or internalization, determined by resistance to an acid/salt wash, or the amount of [3H]MeTRH able to bind at 0 C, where only surface receptors are accessible. TPA (0-100 nM) alone or in combination with BAY K8644 or A23187, also failed to change receptor number or affinity after 48 h when TRH caused a 75% decrease in the density of specific binding sites. Chlordiazepoxide has been reported antagonize TRH binding and TRH-induced phospholipid breakdown. Chlordiazepoxide shifted the dose-response curves for TRH stimulation of PRL release and synthesis to the right, and did not change PRL release alone. The affinity of receptors for chlordiazepoxide was not affected by a nonhydrolyzable analog of GTP whereas affinity for TRH was decreased; these properties are consistent with the classification of chlordiazepoxide as a competitive antagonist. Several experiments tested whether chlordiazepoxide would cause receptor internalization and down-regulation. Chlordiazepoxide did not appear to internalize TRH receptors, because TRH-binding sites became available rapidly and at the same rate after they had been saturated with chlordiazepoxide at 0 or 37 C.(ABSTRACT TRUNCATED AT 250 WORDS)

Pituitary Thyrotropin-Releasing Hormone Receptors: Local Anesthetic Effects on Binding and Responses

Pharmacological agents are widely used to probe the mechanism of action of TRH. A number of these drugs behave as local anesthetics at high concentrations. The effect of local anesthetics on the binding of [3H]Me-TRH to specific receptors was studied using the GH4C1 line of rat pituitary tumor cells. [3H]Me-TRH binding was inhibited by classical local anesthetics with the order of potency (IC50 values): dibucaine (0.37 mM) greater than tetracaine (1.2 mM) greater than lidocaine (3.3 mM) greater than procaine and benzocaine (greater than 10 mM). IC50 values for other drugs with local anesthetic properties that inhibited [3H]Me-TRH were: 100 microM trifluoperazine, 100 microM imipramine, 170 microM chlorpromazine, 300 microM verapamil, and 700 microM propranolol. Inhibition by tetracaine and verapamil increased as the pH was raised from 6 to 8.5, indicating that the free base form of the amine drugs was the inhibitory species, and the local anesthetic effect was greater at 37 C than at 24 C or 0 C. [3H]Me-TRH binding to receptors in isolated membranes was inhibited to the same extent as binding to receptors on intact cells. Local anesthetics were 3- to 20-fold less potent at inhibiting [3H]Me-TRH to digitonin-solubilized receptors than binding to intact cells. In contrast, the potency of chlordiazepoxide, a putative TRH antagonist, to inhibit [3H]Me-TRH binding was equal using cells and solubilized receptors (IC50 = 10 microM). Local anesthetics inhibited TRH-stimulated PRL release and also inhibited basal PRL secretion and secretion stimulated by two nonhormonal secretagogues, (Bu)2cAMP and a phorbol ester.(ABSTRACT TRUNCATED AT 250 WORDS)

Pituitary TRH Receptors

Disclosed for the first time is the isolation, sequence, and expression cloning of a cDNA encoding for pituitary thyrotropin-releasing hormone receptor, as well as the amino acid sequence for the receptor per se.