Receptors for thyrotropin-releasing hormone, thyroid-stimulating hormone, and thyroid hormones in the macaque uterus: effects of long-term sex hormone treatment.

OBJECTIVE: Thyroid gland dysfunction is associated with menstrual cycle disturbances, infertility, and increased risk of miscarriage, but the mechanisms are poorly understood. However, little is known about the regulation of these receptors in the uterus. The aim of this study was to determine the effects of long-term treatment with steroid hormones on the expression, distribution, and regulation of the receptors for thyrotropin-releasing hormone (TRHR) and thyroid-stimulating hormone (TSHR), thyroid hormone receptor α1/α2 (THRα1/α2), and THRß1 in the uterus of surgically menopausal monkeys. METHODS: Eighty-eight cynomolgus macaques were ovariectomized and treated orally with conjugated equine estrogens (CEE; n = 20), a combination of CEE and medroxyprogesterone acetate (MPA; n = 20), or tibolone (n = 28) for 2 years. The control group (OvxC; n = 20) received no treatment. Immunohistochemistry was used to evaluate the protein expression and distribution of the receptors in luminal epithelium, glands, stroma, and myometrium of the uterus. RESULTS: Immunostaining of TRHR, TSHR, and THRs was detected in all uterine compartments. Epithelial immunostaining of TRHR was down-regulated in the CEE + MPA group, whereas in stroma, both TRHR and TSHR were increased by CEE + MPA treatment as compared with OvxC. TRHR immunoreactivity was up-regulated, but THRα and THRß were down-regulated, in the myometrium of the CEE and CEE + MPA groups. The thyroid-stimulating hormone level was higher in the CEE and tibolone groups as compared with OvxC, but the level of free thyroxin did not differ between groups. CONCLUSIONS: All receptors involved in thyroid hormone function are expressed in monkey uterus, and they are all regulated by long-term steroid hormone treatment. These findings suggest that there is a possibility of direct actions of thyroid hormones, thyroid-stimulating hormone and thyrotropin-releasing hormone on uterine function.

High levels of thyrotropin-releasing hormone receptors activate programmed cell death in human pancreatic precursors.

OBJECTIVES: Thyrotropin-releasing hormone (TRH) is expressed in rodent and human adult pancreata and in mouse pancreas during embryonic development. However, expression of TRH receptors (TRHRs) in the pancreas is controversial. We sought to provide evidence that the TRH/TRHR system might play a role in fetal development. METHODS: We used quantitative reverse transcription-polymerase chain reaction to measure TRH and TRHR messenger RNA (mRNA). To study the effects of TRHR expression in a pancreatic progenitor population, we expressed TRHRs in human islet-derived precursor cells (hIPCs) by infection with adenoviral vector AdCMVmTRHR. Thyrotropin-releasing hormone receptor signaling was measured as inositol phosphate production and intracellular calcium transients. Thyrotropin-releasing hormone receptor expression was measured by [H]methyl-TRH binding. Apoptosis was monitored by release of cytochrome c from mitochondria. RESULTS: We show that TRH mRNA is expressed in human fetal and adult pancreata, and that TRHR mRNA is expressed in fetal human pancreas but not in adult human pancreas. Thyrotropin-releasing hormone receptors expressed in hIPCs were shown to signal normally. Most importantly, TRH treatment for several days stimulated apoptosis in hIPCs expressing approximately 400,000 TRHRs per cell. CONCLUSIONS: These findings suggest a possible role for TRH/TRHR signaling in pancreatic precursors to promote programmed cell death, a normal constituent of morphogenesis during embryonic development in humans.

Increasing plasma thyroxine levels during late embryogenesis and hatching in the chicken are not caused by an increased sensitivity of the thyrotropes to hypothalamic stimulation.

The hatching process in the chicken is accompanied by dramatic changes in plasma thyroid hormones. The cause of these changes, though crucial for hatching and the onset of endothermy, is not known. One hypothesis is that the pituitary gland becomes more sensitive to hypothalamic stimulation during this period. We have tested whether the responsiveness of the thyrotropes to hypothalamic stimuli changes throughout the last week of embryonic development and hatching by studying the mRNA expression of receptors involved in the control of the secretory activity of this cell type. We used a real-time PCR set-up to quantify whole pituitary mRNA expression of the beta subunit of thyrotrophin (TSH-beta), type 1 thyrotrophin-releasing hormone receptor (TRH-R1), corticotrophin-releasing hormone receptors (CRH-R1 and CRH-R2) and somatostatin subtype receptor 2 (SSTR2) on every day of the last week of embryonic development, including the day of hatch and the first day of posthatch life. The thyrotrope-specific expression was investigated by a combination of in situ hybridization with immunohistochemistry at selected ages. Although TSH-beta mRNA levels increased towards day 19 of incubation (E19), the expression of CRH-R2 and TRH-R1 mRNA by the thyrotropes tended to decrease during this period, suggesting a lower sensitivity of the thyrotropes to the stimulatory factors CRH and TRH. CRH-R1, which is not involved in the control of TSH secretion, increased steadily throughout the period tested. The expression of SSTR2 mRNA by the thyrotropes was low during embryonic development and increased just before hatching. We have concluded that the sensitivity of the pituitary thyrotropes to hypothalamic stimulation decreases throughout the last week of embryonic development, so that the higher expression of TSH-beta mRNA around E16-E19, and hence the increasing plasma thyroxine level, is unlikely to be the result of an increased stimulation by either TRH or CRH.

Effects of TRH on heteromeric rat erg1a/1b K+ channels are dominated by the rerg1b subunit.

The erg1a (HERG) K+ channel subunit and its N-terminal splice variant erg1b are coexpressed in several tissues and both isoforms have been shown to form heteromultimeric erg channels in heart and brain. The reduction of erg1a current by thyrotropin-releasing hormone (TRH) is well studied, but no comparable data exist for erg1b. Since TRH and TRH receptors are widely expressed in the brain, we have now studied the different TRH effects on the biophysical properties of homomeric rat erg1b as well as heteromeric rat erg1a/1b channels. The erg channels were overexpressed in the clonal somatomammotroph pituitary cell line GH3/B6, which contains TRH receptors and endogenous erg channels. Compared to rerg1a, homomeric rerg1b channels exhibited not only faster deactivation kinetics, but also considerably less steady-state inactivation, and half-maximal activation occurred at about 10 mV more positive potentials. Coexpression of both isoforms resulted in erg currents with intermediate properties concerning the deactivation kinetics, whereas rerg1a dominated the voltage dependence of activation and rerg1b strongly influenced steady-state inactivation. Application of TRH induced a reduction of maximal erg conductance for all tested erg1 currents without effects on the voltage dependence of steady-state inactivation. Nevertheless, homomeric rerg1b channels significantly differed in their response to TRH from rerg1a channels. The TRH-induced shift in the activation curve to more positive potentials, the dramatic slowing of activation and the acceleration of deactivation typical for rerg1a modulation were absent in rerg1b channels. Surprisingly, most effects of TRH on heteromeric rerg1 channels were dominated by the rerg1b subunit.

Involvement of thyrotropin-releasing hormone receptor, somatostatin receptor subtype 2 and corticotropin-releasing hormone receptor type 1 in the control of chicken thyrotropin secretion.

Thyrotropin or thyroid-stimulating hormone (TSH) secretion in the chicken is controlled by several hypothalamic hormones. It is stimulated by thyrotropin-releasing hormone (TRH) and corticotropin-releasing hormone (CRH), whereas somatostatin (SRIH) exerts an inhibitory effect. In order to determine the mechanism by which these hypothalamic hormones modulate chicken TSH release, we examined the cellular localization of TRH receptors (TRH-R), CRH receptors type 1 (CRH-R1) and somatostatin subtype 2 receptors (SSTR2) in the chicken pars distalis by in situ hybridization (ISH), combined with immunological staining of thyrotropes. We show that thyrotropes express TRH-Rs and SSTR2s, allowing a direct action of TRH and SRIH at the level of the thyrotropes. CRH-R1 expression is virtually confined to corticotropes, suggesting that CRH-induced adrenocorticotropin release is the result of a direct stimulation of corticotropes, whereas CRH-stimulated TSH release is not directly mediated by the known chicken CRH-R1. Possibly CRH-induced TSH secretion is mediated by a yet unknown type of CRH-R in the chicken. Alternatively, a pro-opiomelanocortin (POMC)-derived peptide, secreted by the corticotropes following CRH stimulation, could act as an activator of TSH secretion in a paracrine way.

Analysis of thyrotropin-releasing hormone-signaling components in pituitary adenomas of patients with acromegaly.

In many acromegalic patients the paradoxical release of GH in response to TRH has been well documented, but the mechanisms underlying this phenomenon are not understood. It has been suggested that aberrant GH secretion may result from TRH endogenously synthesized by the adenoma. In 32 adenomas from acromegalic patients, TRH-like immunoreactivity (TRH-LI) was measured using 2 well characterized antisera. TRH-LI was not detectable in 10 samples, and in 19 samples, TRH-LI was measured only by the less specific antibody. With the TRH-specific antibody, TRH-LI was identified only in 3 samples, 2 of which contained exceedingly high concentrations (40 and 96 pg/mg tissue). In the latter 2 samples, prepro-TRH messenger ribonucleic acid was identified by Northern blot analysis, but not in the control tissue sample of a patient without pituitary disease and also not in the other adenomas analyzed by this technique. Transcripts of the TRH receptor were almost undetectable in all adenomas analyzed. For the TRH-degrading ectoenzyme, a potential regulator of TRH signals at adenohypophyseal target sites, transcripts were significantly expressed only in the TRH-producing adenomas. We conclude that the TRH-signaling elements examined are, in general, not directly involved in the mechanisms causing paradoxical GH secretion in acromegalic patients.

Distribution of thyrotropin releasing hormone receptor type 2 in rats: an immunohistochemical study.

OBJECTIVE: To investigate the organ distribution of thyrotropin releasing hormone receptor (TRHR) type 2 in rats by immunohistochemical method. METHODS: TRHR type 2 was identified immunohistochemically in the rat tissues using specific anti-TRHR antiserum raised in New Zealand white rabbits immunized with a conjugate of synthetic TRHR type 2 (5-23) with bovine serum albumin. Immunohistochemical analysis was performed by avidin-biotin complex method. RESULTS: TRHR type 2 immunoreactivity was visualized in the central nervous system, anterior pituitary, gastric mucosa, Auerbach's and Meissner's nervous branch of the stomach, small intestine and colon, retina amd testis. Significant stain was detected in neural perikarya, axons and dendrites. When using antiserum preincubated with synthetic TRHR type 2(5-23) or anterior pituitary homogenates, no significant stain of anterior pituitary was detected. CONCLUSIONS: These findings suggest that TRHR type 2 is widely distributed and that the method used is valuable in studying the distribution of TRHR type 2 in rats.

Receptors for thyrotropin-releasing hormone on rat lactotropes and thyrotropes.

Primary cultures of rat pituitary cells were stained with an antibody to the native thyrotropin-releasing hormone (TRH) receptor and with a bioactive, fluorescent analogue of TRH, Rhod-TRH. Rhod-TRH specifically stained 86% of lactotropes and 21% of nonlactotropes from primary pituitary cell cultures. Lactotropes and thyrotropes accounted for 90% of cells that stained with Rhod-TRH, but there were occasional lactotropes and thyrotropes that did not show detectable staining with antireceptor antibodies or with Rhod-TRH. The intensity of staining was generally higher in the GH3 line of tumor cells than in normal pituicytes, and 100% of the tumor cells stained with Rhod-TRH. To determine whether the TRH receptor undergoes ligand-directed endocytosis in normal cells, TRH receptor immunocytochemistry was performed before and after TRH binding. TRH receptors were localized on the surface of cells prior to TRH exposure, and Rhod-TRH fluorescence was confined to the plasma membrane when TRH binding was performed at 0 degrees C, where endocytosis is blocked. When cells were incubated with TRH at 37 degrees C, receptors were found in intracellular vesicles in both lactotropes and thyrotropes, and Rhod-TRH was rapidly internalized into endosomes at elevated temperatures. Internalization of Rhod-TRH was inhibited by hypertonic sucrose, indicating that it occurs through clathrin-coated pits. These findings show that some of the heterogeneity in the secretory and calcium responses of pituicytes to TRH occurs at the level of the TRH receptor.

Human TRH receptor messenger ribonucleic acid levels in normal and adenomatous pituitary: analysis by the competitive reverse transcription polymerase chain reaction method.

OBJECTIVE: Little is known about the mechanism of diversity in in-vivo hormonal responsiveness to TRH in patients with functional pituitary adenomas. In order to clarify the relation between the responsiveness to TRH and TRH receptor messenger ribonucleic acid (mRNA) expression, we attempted to measure TRH receptor mRNA levels in human pituitary adenoma tissues by competitive reverse transcription polymerase chain reaction (RT-PCR) method. PATIENTS: Pituitary tissue samples were obtained at autopsy from 5 patients without pituitary disease. Pituitary adenoma tissue samples were obtained at surgery from 18 patients with pituitary adenoma (4 non-functioning, 8 prolactinoma, 4 acromegaly, 1 Cushing's disease and 1 FSH producing adenoma). METHODS: Partial TRH receptor cDNA from a human GH producing adenoma cDNA library was amplified by PCR under low stringency conditions using primers encoding the transmembrane domains III and VI of pituitary TRH receptor cDNA. The partial sequence of the amplified cDNA determined by a dideoxy-chain termination method was identical to the corresponding sequence of human TRH receptor cDNA. A competitor was generated by deleting the inner 111 bp from the amplified TRH receptor cDNA and subcloning. RNA extracted from human pituitary was reverse transcribed and co-amplified with competitor by PCR under higher stringency conditions. The TRH receptor mRNA levels, expressed as the relative intensity against the amplified levels of competitor, were compared among various pituitary tissues. RESULTS: The relative TRH receptor mRNA levels of pituitary tissues in patients without pituitary disease were detectable and variable (M +/- SD) (0.370 +/- 0.231, n = 5), and slightly but not significantly lower than those in patients with pituitary tumours (0.598 +/- 0.265, n = 18). In patients with prolactinoma, the relative levels of TRH receptor mRNA were quite variable (0.02-1.170, 0.604 +/- 0.358, n = 8) and not correlated with PRL responsiveness to TRH (responder 0.457; non-responder 0.340-0.950). In patients with acromegaly, TRH receptor mRNA was detectable not only in the paradoxical GH responder to TRH (0.718) but also in the non-responder (0.758 and 0.765). In one patient with Cushing's disease, a relatively low level of TRH receptor mRNA could be detected (0.415). In the patient with a FSH producing tumour whose plasma FSH did not respond to TRH, a small amount of TRH receptor mRNA was detectable (0.447). CONCLUSIONS: In patients with functioning pituitary adenomas, hormonal responsiveness to TRH in vivo might not be assessable by TRH receptor mRNA levels in the adenoma cells.

Production and characterization of polyclonal anti-idiotypic anti-TRH antibodies: application to the study of pituitary TRH receptor.

cDNA encoding the thyrotropin-releasing hormone receptor (TRH-R) was recently cloned in rat pituitary prolactin cells and in mouse thyrotropes. The molecular weights of the protein sequences obtained are 46.6 and 44.5 kD. However, TRH-R has not yet been purified to homogeneity and specific anti-TRH-R antibody could not yet be obtained by classical biochemical methods. We thus attempted to obtain antibodies specific for TRH-R using an anti-idiotypic approach. Rabbits of the same allotype were immunized using Igs (Ab1) extracted from rabbit polyclonal anti-TRH immune serum. Anti-idiotypic rabbit polyclonal anti-anti-TRH antibodies (Ab2) were obtained, as shown by their ability to inhibit the formation of TRH-anti-TRH complexes in a radioimmunoassay system. One of them, the polyclonal Ab2 R38/B12, was tested for its ability to recognize the TRH-R in rat pituitary, tumor-derived, GH3/B6 prolactin-secreting cells. Immunoreactive material was immunocytochemically detected in fixed and saponin-permeabilized GH3/B6 cells. The immunostaining was localized at the plasma membrane and on intracellular structures. It was not observed using non-anti-TRH Ab2 and was abolished in the presence of excess TRH. Furthermore, binding of [125I]R38/B12 on fixed and saponin-permeabilized GH3/B6 cells was partially inhibited by excess TRH. By immunoblot analyses of Triton X-114 cell extracts performed under reducing or nonreducing conditions, the polyclonal R38/B12 Igs revealed two main protein species of approximately 98 and approximately 76 kD as well as several proteins < or = 46 kD. In the presence of excess TRH, the approximately 98- and approximately 42-kD bands were abolished, whereas the intensity of the other bands was faintly attenuated only. The approximately 98-kD protein was also revealed in a two-dimensional PAGE analysis. Nevertheless, the effects of R38/B12 Igs on [3H]TRH binding by GH3/B6 cells and on basal or TRH-induced prolactin secretion were not markedly different from those elicited by control Ab2. These data suggest that we have characterized Ab2 antibodies which recognize a molecular entity that might be related to the TRH-R in GH3B6 cells.