Phosphorylation of the endogenous thyrotropin-releasing hormone receptor in pituitary GH3 cells and pituitary tissue revealed by phosphosite-specific antibodies.

To study phosphorylation of the endogenous type I thyrotropin-releasing hormone receptor in the anterior pituitary, we generated phosphosite-specific polyclonal antibodies. The major phosphorylation site of receptor endogenously expressed in pituitary GH3 cells was Thr(365) in the receptor tail; distal sites were more phosphorylated in some heterologous models. beta-Arrestin 2 reduced thyrotropin-releasing hormone (TRH)-stimulated inositol phosphate production and accelerated internalization of the wild type receptor but not receptor mutants where the critical phosphosites were mutated to Ala. Phosphorylation peaked within seconds and was maximal at 100 nm TRH. Based on dominant negative kinase and small interfering RNA approaches, phosphorylation was mediated primarily by G protein-coupled receptor kinase 2. Phosphorylated receptor, visualized by immunofluorescence microscopy, was initially at the plasma membrane, and over 5-30 min it moved to intracellular vesicles in GH3 cells. Dephosphorylation was rapid (t((1/2)) approximately 1 min) if agonist was removed while receptor was at the surface. Dephosphorylation was slower (t((1/2)) approximately 4 min) if agonist was withdrawn after receptor had internalized. After agonist removal and dephosphorylation, a second pulse of agonist caused extensive rephosphorylation, particularly if most receptor was still on the plasma membrane. Phosphorylated receptor staining was visible in prolactin- and thyrotropin-producing cells in rat pituitary tissue from untreated rats and much stronger in tissue from animals injected with TRH. Our results show that the TRH receptor can rapidly cycle between a phosphorylated and nonphosphorylated state in response to changing agonist concentrations and that phosphorylation can be used as an indicator of receptor activity in vivo.

Production and characterization of an antiserum which recognizes the native receptor for thyrotropin-releasing hormone.

Despite attempts in several laboratories, it has been difficult to prepare antiserum to the thyrotropin-releasing hormone receptor (TRHR). We have prepared a polyclonal anti-rat TRHR antiserum by immunization of rabbits with a synthetic peptide corresponding to the C-terminus of the TRHR. The specificity of the antiserum was assessed by enzyme-linked immunosorbent assay. The affinity-purified antibody recognized a major broad band at 50-60 kDa and a minor broad band at 100-120 kDa in Western blot analysis of membrane proteins from TRHR-transfected, but not control, HEK293t cells. Binding to both bands was abolished by preincubation with the immunizing peptide but not control peptide. The approach was repeated with rat pituitary F4C1 cells, which lack endogenous TRHRs; membranes from F4C1 cells transfected with TRHR cDNA, but not control cells, showed specific binding by Western blot. Using laser confocal microscopy, the TRHR was visualized on the plasma membrane of transfected, but not control, F4C1 cells. Similar confocal findings were observed in TRHR-transfected HEK293t cells. Within 5 min after TRH addition, the TRHR signal translocated from the plasma membrane to the cytoplasm of F4C1 cells transfected with TRHR cDNA. Ten minutes after TRH addition, the TRHR signal formed aggregates in the cytoplasm. Thirty minutes after TRH treatment, both cytoplasmic and plasma membrane localizations were observed, suggesting recycling of some TRHRs back to the plasma membrane. These observations are consistent with our previous findings using an epitope-tagged TRHR. In conclusion, we have prepared an antiserum that recognizes the native TRHR by Western blot analysis and confocal microscopy.

Thyrotropin-releasing hormone and its receptor in glia.

The presence of thyrotropin-releasing hormone (Thyroliberin, TRH) and its receptor (TRH-R) in frozen coronal sections of the adult rat spinal cord and neonatal rat astroglial cultures was investigated by means of immunocytochemistry and Western blot using polyclonal antibodies generated against the hormone and monoclonal antibodies originated against discrete sequences of the type 1 rat TRH receptor (TRH-R1). TRH-R1 and TRH are present both in astroglial cells from adult rats and in cultured cells from newborn animals. The localization of TRH and TRH-R1 in nonneuronal cells in the central nervous system may reflect that some of the neurotrophic actions of TRH upon the central nervous system are mediated by glial cells.

Expression analysis of the thyrotropin-releasing hormone receptor (TRHR) in the immune system using agonist anti-TRHR monoclonal antibodies.

Monoclonal anti-rat thyrotropin-releasing hormone (TRH) receptor (TRHR)-specific antibodies (mAb) were generated by immunization with synthetic peptides of rat TRHR partial amino acid sequences; one (TRHR01) was directed against a sequence (84-98) in the extracellular portion of the rat TRHR reported to be constant among different species, including man, and the second (TRHR02) recognizes the C-terminal region sequence 399-412. In lysates from GH4C1 cells, a clonal rat pituitary cell line, both mAb recognize the TRHR in Western blot analysis, and TRHR02 immunoprecipitates the TRHR. Incubation of GH4C1 cells with the mAb causes a fluorescence shift in fluorescence-activated cell sorting analysis. The cells were stained specifically by both mAb using immunocytochemical techniques. Furthermore, TRHR01 is agonistic in its ability to trigger Ca2+ flux, and desensitizes the TRH receptor. We tested for TRHR in several rat organs and found expression in lymphoid tissues. TRHR01 recognizes the human TRHR, and analysis of human peripheral blood lymphocyte and tonsil-derived leukocyte populations showed receptor expression in non-activated and phytohemagglutinin-activated T and B cells.

Outcome of treatment of hyperthyroidism.

This is a retrospective study designed to evaluate the initial response to carbimazole in patients with Graves' disease (GD), possible determinants of that response, the frequency of occurrence of adverse effects during treatment with carbimazole and the frequency of transient and permanent hypothyroidism after treatment with 131I in patients with GD and multinodular goiter (MNG). Data were collected from patients who first presented with GD or MNG at the Department of Endocrinology of the Royal Infirmary of Edinburgh between 1 January 1993 and 31 August 1996. Patients were divided into three groups: patients with GD treated with a daily dose of 40 mg carbimazole, patients with GD treated with a single dose of 400 MBq 1311, and patients with MNG treated with the same dose of 131I. Of the patients younger than 30 years, 50% remained biochemically hyperthyroid after 4-6 weeks of treatment with carbimazole, compared to 14% of patients over 30. Other determinants of the response to carbimazole expressed as the fall in thyroid hormone levels after 4-6 weeks were: pretreatment levels of FT4, T3, TRAb and the 4 h 131I uptake, patients with the higher levels responding significantly better to carbimazole. Adverse effects were reported in 11.5% of patients. Of the patients with GD treated with 1311, 62.6% became hypothyroid, transient hypothyroidism occurred in only 2.4% of these cases. The main predictors of development of hypothyroidism were positive titres of antithyroid peroxidase antibodies (AbTPO) and antithyroglobulin antibodies (AbTg), with positive predictive values of 79.5 and 91.6 respectively. None of the patients with MNG became hypothyroid after treatment with 131I, a response significantly different from patients with GD. In conclusion, GD younger patients might benefit from higher initial doses of carbimazole. In patients with positive titres of AbTPO and AbTg, lower doses of 1311 might prevent hypothyroidism. Transient hypothyroidism was underestimated in this study. No permanent thyroxin replacement therapy should be started within the first six months after 131I treatment.

Intraluminal thyrotropin-releasing hormone affects gastric somatostatin and acid secretion through its specific receptor in rats.

BACKGROUND: Although thyrotropin-releasing hormone (TRH) is present in the gastric mucosa and juice, the pathophysiologic significance of TRH is poorly understood at the peripheral level of the stomach. In the present study we analyzed the TRH-induced secretion profiles of somatostatin, histamine, and acid in the rat stomach. METHODS: The effects of intraluminal perfusion of TRH on somatostatin, histamine, and acid and the influence of the specific anti-TRH receptor antibody were investigated by using the rat gastric intraluminal perfusion model. RESULTS: Intraluminal TRH caused an immediate decrease in somatostatin secretion in a dose-dependent manner, and this change occurred preceding an increase in acid secretion. In contrast, this treatment did not yield any significant changes in histamine contents in the effluent. Pretreatment of the gastric lumen with the anti-TRH receptor antibody caused a complete inhibition of TRH-induced changes in somatostatin and acid secretion. CONCLUSIONS: These findings suggest that intraluminal TRH affects somatostatin and acid secretion in a paracrine manner via its specific receptor in the rat stomach.

Thyrotropin-releasing hormone and somatostatin inhibit each others release in vitro in the rat retina.

The effect of thyrotropin-releasing hormone (TRH), somatostatin (SS) or octreotide, an analogue of SS, on release of TRH or SS from the rat retina was studied in vitro. The retina was incubated in medium 199 (pH 7.4) with 1.0 mg/ml of bacitracin (medium) for 20 min. The amount of TRH or SS release into the medium was measured by individual radioimmunoassays. The TRH release from the rat retina was inhibited significantly in a dose-related manner by the addition of SS or octreotide. The SS release from the retina was inhibited by TRH, and the inhibitory effect of TRH on SS release from the rat retina was blocked by the addition of anti-TRH receptor antiserum immunoglobulin fraction. The findings suggest an interaction between TRH and SS in the rat retina by which the addition of one inhibits the release of the other.