In vivo autoregulation of rat adenohypophyseal thyrotropin-releasing hormone receptor

The possible mechanism of attenuation of thyrotropin response to exogenous thyrotropin-releasing hormone $\text{in vivo}$ after repeated administrations of the releasing hormone has been studied. To this end, the effect of prolonged hormone treatment on the binding of hormone to its receptor in the anterior pituitary gland has been evaluated. The data show that prolonged hormonal treatment resulted in a reduction in the number (B_max) but not the binding affinity (K_D) of the receptor. The effect was reversible and depended on the duration of treatment. This phenomenon of down regulation or the decrease in the receptor number was found not to be due to either the metabolism of releasing hormone or its ability to activate pituitary-thyroid-axis.     

Effect of thyrotropin-releasing hormone on rat myometrium

The effect of TRH in vitro was observed on electromyograms and isometric tension changes in the uterine horn isolated from the rat. TRH induced transient prolongation of the duration of spike bursts in the electromyogram and an increased tension in contraction of diestrous uterine horns. No distinct response to TRH was elicited in preparations from rats during other estrous stages. TRH produced a contraction associated with a burst of spike potentials in the quiescent horn from the estrogen-primed ovariectomized rat. Priming with progesterone was not a prerequisite for responsiveness to TRH. In a medium with a high Ca concentration, diestrous uteri were quiescent but a transient contraction associated with a burst of spike potentials was induced by TRH. In a Ca-free medium, TRH failed to elicit any response in the diestrous uterus but acetylcholine induced a contraction without associated spike potentials. It appears that TRH stimulates Ca-influx into the uterine muscle in which responsiveness is dependent on estrogen priming.     

Regulated dimerization of the thyrotropin-releasing hormone receptor affects receptor trafficking but not signaling

To investigate the function of dimerization of the TRH receptor, a controlled dimerization system was developed. A variant FK506 binding protein (FKBP) domain was fused to the receptor C terminus and dimerization induced by incubating cells with dimeric FKBP ligand, AP20187. The TRH receptor-fusion bound hormone and signaled normally. Addition of dimerizer to cells expressing the receptor-FKBP fusion dramatically increased the fraction of receptor running as dimer on SDS-PAGE. AP20187 caused dimerization in a time- and concentration-dependent manner, acting within 1 min. Dimerizer had no effect on TRH receptors lacking the FKBP domain, and its effects were blocked by excess monomeric FKBP ligand. AP20187-induced dimerization did not cause receptor phosphorylation, inositol phosphate production, or ERK1/2 activation, and dimerizer did not alter signaling by TRH. Induced dimerization did, however, alter TRH receptor trafficking. TRH promoted greater receptor internalization in cells treated with AP20187 but not monomeric ligand, based on loss of surface binding sites and immunostaining. Dimerization increased the rate of internalization of TRH receptors and decreased the apparent rate of receptor recycling. AP20187 enhanced the small amount of TRH-induced receptor internalization when the receptor-FKBP fusion protein was expressed in cells lacking beta-arrestins. The results show that controlled dimerization of the TRH receptor potentiates hormone-induced receptor trafficking.     

Modulation of dopamine receptors by thyrotropin-releasing hormone in the rat brain

Nanomolar concentration of thyrotropin-releasing hormone (TRH) in vitro caused a significant reduction of [3H]apomorphine binding sites (70% of the control) in the rat striatum and the limbic forebrain. [3H]Spiperone binding was not affected by TRH. On the other hand, dopamine and apomorphine displaced [3H]TRH binding partially, suggesting the presence of a TRH receptor subpopulation that has a high affinity for dopamine agonist. Most of the neuroleptics displaced [3H]TRH binding dose-dependently in the micromolar range. (-)-Sulpiride had no affinity to TRH receptors. These findings suggest that one of the important roles of TRH as a neuromodulator is to modulate receptors for classical neurotransmitters, and this receptor-receptor interaction may be of importance in explaining the well known stimulating effects of TRH on the dopaminergic system.     

Brain thyrotropin-releasing hormone matures independently of the hypothalamus in the rat

To evaluate the relationship of the extrahypothalamic brain thyrotropin-releasing hormone (TRH) to its hypothalamic counterpart, we studied the maturation of hypothalamic and extrahypothalamic TRH in the rat. The absolute increase of TRH in the whole brain and the extrahypothalamus reached adult levels at 7 days of age, whereas the hypothalamic TRH concentrations did not differ from the adult levels at 23 days. Moreover, the TRH concentrations at 7 days were greater than the adult levels in the striatum, hippocampus, pons-medulla and cerebellum, and similar to the adult levels in the midbrain and cortex. These data indicate the developmental divergency of hypothalamic and extrahypothalamic TRH, implying that the maturation of extrahypothalamic TRH is independent of the hypothalamus. The present study suggests that extrahypothalamic TRH may play a neurophysiological role in the central nervous system at an early infantile age, at which hypothalamic TRH is not ripe for its endocrinological action.     

Regulation by thyrotropin-releasing hormone (TRH) of TRH receptor mRNA degradation in rat pituitary GH3 cells.

In rat pituitary GH3 cells, thyrotropin-releasing hormone (TRH) down-regulates TRH receptor (TRH-R) mRNA (Fujimoto, J., Straub, R.E., and Gershengorn, M.C. (1991) Mol. Endocrinol. 5, 1527-1532), at least in part, by stimulating its degradation (Fujimoto, J., Narayanan, C.S., Benjamin, J.E., Heinflink, M., and Gershengorn, M.C. (1992) Endocrinology 130, 1879-1884). Here we show that TRH regulates RNase activity in GH3 cells and that specific mRNA sequences are needed for in vivo regulation of TRH-R mRNA by TRH. TRH affected RNase activity in a biphasic manner with rapid stimulation (by 10 min) followed by a decrease to a rate slower than in control lysates within 6 h. This time course paralleled the effects of TRH on degradation of TRH-R mRNA in vivo. The regulated RNase activity was in a polysome-free fraction of the lysates and was not specific for TRH-R RNA. A truncated form of TRH-R RNA that was missing the entire 3'-untranslated region (TRHR-R5) was more stable than full-length TRH-R RNA (TRHR-WT). In contrast to TRHR-WT mRNA, TRHR-R5 mRNA and TRHR-D9 mRNA, which was missing the 143 nucleotides 5' of the poly(A) tail, were not down-regulated by TRH in stably transfected GH3 cells as their rates of degradation were not increased. These data show that TRH regulates RNase activity in GH3 cells, that the 3'-untranslated region bestows decreased stability on TRH-R mRNA and that the 3' end of the mRNA is necessary for regulation by TRH of TRH-R mRNA degradation. We present an hypothesis that explains specific regulation of TRH-R mRNA degradation by TRH in GH3 pituitary cells.     

Evidence for thyrotropin-releasing hormone (TRH) biosynthesis in rat prostate

TRH occurs in very high concentration in rat prostate. A species specific protein with repetitive -Gln-His-Pro-Gly- sequences, which are flanked on the N- and C-terminus by paired basic residues, has been shown to be the source of TRH in frog skin and rat hypothalamus. Following cleavage by trypsin-like enzymes, the peptide fragments with N-terminal Gln spontaneously cyclize to pGlu while Gly within the C-terminally extended peptides serves as the -NH2 donor for the alpha-amidation of the proline residue. Because this last step in the biosynthesis of TRH is rate limiting for pGlu-His-Pro-Gly, we have combined several chromatographic and radioimmunoassay techniques to identify this TRH precursor in rat prostate.     

Dopamine inhibits thyrotropin-releasing hormone release from rat adrenal gland in vitro

The effects of dopamine on the release of thyrotropin-releasing hormone (TRH) from the rat adrenal gland were studied in vitro. The rat adrenal glands were incubated in medium 199 with 1.0 mg/ml of bacitracin and 100 micrograms/ml of ascorbic acid (pH 7.4) (medium) for 20 min. The amount of TRH release into the medium was measured by radioimmunoassay. The immunoreactive TRH (ir-TRH) release from the rat adrenal gland was inhibited significantly in a dose-related manner with the addition of dopamine and enhanced with the addition of pimozide or domperidone to the medium. Dopamine's effects on ir-TRH release from the adrenal gland were blocked with the addition of pimozide or domperidone. The elution profile of methanol-extracted rat adrenal gland was identical to that of synthetic TRH. The findings suggest that the dopaminergic system inhibits TRH release from the rat adrenal gland.     

Purification and characterization of a thyrotropin-releasing hormone deamidase from rat brain.

This report describes the purification of a rat brain thyrotropin-releasing hormone (TRH) deamidating enzyme to apparent homogeneity. Criteria for purity include sodium dodecyl sulfate and disc gel electrophoresis, as well as isoelectric focusing (pI = 4.5). Enzyme purification was facilitated by development of a rapid and sensitive continuous assay using the substrate L-pyroglutamyl-Nim-benzylhistidyl-L-prolyl-beta-naphthylamide, which, upon hydrolysis of the naphthylamide, results in the appearance of the fluorescent product, beta-naphthylamine (beta NA). With this substrate the homogeneous enzyme had a specific activity of 14.5 mumol of beta NA min-1 mg-1. The only peptide product formed was shown to be L-pyroglutamyl-Nim-benzylhistidyl-L-proline. Hydrolysis of [L-prolyl-2,3-3H]TRH was shown to yield L-pyro-glutamyl-L-histidyl-L-proline as the only radiolabeled product. Characterization of the brain deamidase by gel filtration chromatography and sodium dodecyl sulfate gel electrophoresis indicated that the enzyme consists of a single polypeptide chain having molecular weights of 70,000 and 73,500, respectively. Rat brain TRH deamidase has an apparent Km of 34 micron, and a pH optimum between 7 and 8 using L-pyroglutamyl-Nim-benzylhistidyl-L-prolyl-beta-naphthylamide as a substrate. With this substrate, TRH was shown to be a competitive inhibitor with an apparent Ki of 120 +/- 20 micron.     

Biosynthesis of thyrotropin-releasing hormone by a rat medullary thyroid carcinoma cell line

Prepro-thyrotropin-releasing hormone (TRH) messenger RNA was detected in the rat medullary thyroid carcinoma cell line CA77. The RNA of 1.6 kilobases comigrated with that found in rat hypothalamus. Using three radioimmunoassays specific for pro-TRH-derived peptides, we demonstrated that CA77 cells synthesize high levels of immunoreactive TRH and all of the other pro-TRH-derived peptides identified in hypothalamic tissue. The relative levels of the pro-TRH-derived peptides also indicate that CA77 cells process the TRH precursor in a manner similar to hypothalamic tissue. CA77 cells provide a promising model system for further studies of prepro-TRH gene regulation and post-translational maturation.     

The molecular mechanism of EGF receptor activation in pancreatic beta-cells by thyrotropin-releasing hormone

Thyrotropin-releasing hormone (TRH) and its receptor subtype TRH receptor-1 (TRHR1) are found in pancreatic beta-cells, and it has been shown that TRH might have potential for autocrine/paracrine regulation through the TRHR1 receptor. In this paper, TRHR1 is studied to find whether it can initiate multiple signal transduction pathways to activate the epidermal growth factor (EGF) receptor in pancreatic beta-cells. By initiating TRHR1 G protein-coupled receptor (GPCR) and dissociated alphabetagamma-complex, TRH (200 nM) activates tyrosine residues at Tyr845 (a known target for Src) and Tyr1068 in the EGF receptor complex of an immortalized mouse beta-cell line, betaTC-6. Through manipulating the activation of Src, PKC, and heparin-binding EGF-like growth factor (HB-EGF), with corresponding individual inhibitors and activators, multiple signal transduction pathways linking TRH to EGF receptors in betaTC-6 cell line have been revealed. The pathways include the activation of Src kinase and the release of HB-EGF as a consequence of matrix metalloproteinase (MMP)-3 activation. Alternatively, TRH inhibited PKC activity by reducing the EGF receptor serine/threonine phosphorylation, thereby enhancing tyrosine phosphorylation. TRH receptor activation of Src may have a central role in mediating the effects of TRH on the EGF receptor. The activation of the EGF receptor by TRH in multiple circumstances may have important implications for pancreatic beta-cell biology.     

Effects of subconvulsive and repeated electroconvulsive shock on thyrotropin-releasing hormone in rat brain

Male Sprague-Dawley rats were given a single electroconvulsive shock (ECS) on alternate days and sacrificed 48 hrs after 1, 3, or 5 seizures. The content of TRH in hippocampus, pyriform cortex and amygdala was increased 2.5-fold, 5.4-fold and 4.3-fold respectively, 48 hrs. after 3 alternate-day electroconvulsive shocks (ECS) and remained unchanged after 2 additional shocks. Pyriform cortex exhibited a significant intermediate increase (1.7-fold) after only 1 ECS. In a second study, rats were sacrificed 48 hrs after a series of 5 alternate-day ECS vs. subconvulsive shocks (SCS). SCS had no significant effect in these same regions, but was seen to alter TRH in striatum. These results provide an interesting parallel to several aspects of clinical electroconvulsive treatment (ECT) of depression. Together with other findings, these data suggest also, that endogenous TRH may play a role in the modulation of convulsive seizures.     

Effect of electroconvulsive shock on the content of thyrotropin-releasing hormone in rat brain

Five grand-mal seizures were electrically induced in rats on alternate days. Forty-eight hours following the last seizure, TRH was quantitated in extracts of anterior cortex, hippocampus, striatum, thalamus plus midbrain, and hypothalamus. When compared to sham treated controls, TRH was found to be elevated 5-fold in the hippocampus and 2-fold in the striatum with no changes observed in the remaining regions. Since the time chosen for analysis excludes acute post-ictal effects, these results draw attention to a prolonged alteration of TRH levels in specific brain regions in an animal model of electroconvulsive treatment.     

Characterization and subcellular distribution of specific thyrotropin-releasing hormone binding sites in rat cerebellum

The specific binding of thyrotropin-releasing hormone (TRH) by 30,000g pellet fraction was ubiquitously distributed throughout various rat brain regions including cerebellum. Although the cerebellum had the lowest apparent density of specific TRH binding sites found in any of the brain regions studied, it represented a single class of high afinity receptor (K _D=37.73±4.88 nM,B _max=156.0±5.7 fmol/mg protein,n=4). Furthermore, the cerebellar synaptic plasma membrane fractions were richly endowed with TRH-binding, two other membrane fractions (light-synaptic plasma membrane and microsomal) exhibited high TRH-binding whereas nuclear, mitochondrial or myelin fractions were devoid of significant binding activity. These data show for the first time the existence of specific TRH-binding in cerebellum, and thus suggest that TRH may modulate cerebellar synaptic functions by acting through a specific high affinity-receptor.