Thyrotropin-releasing Hormone Receptor Expression Research Articles

Optical and pharmacological manipulation of hypoglossal motor nucleus identifies differential effects of taltirelin on sleeping tonic motor activity and responsiveness

Pharyngeal muscle activity and responsiveness are key pathophysiological traits in human obstructive sleep apnea (OSA) and strong contributors to improvements with pharmacotherapy. The thyrotropin-releasing hormone (TRH) analog taltirelin is of high pre-clinical interest given its neuronal-stimulant properties, minimal endocrine activity, tongue muscle activation following microperfusion into the hypoglossal motor nucleus (HMN) or systemic delivery, and high TRH receptor expression at the HMN compared to rest of the brain. Here we test the hypothesis that taltirelin increases HMN activity and/or responsivity to excitatory stimuli applied across sleep–wake states in-vivo. To target hypoglossal motoneurons with simultaneous pharmacological and optical stimuli we used customized “opto-dialysis” probes and chronically implanted them in mice expressing a light sensitive cation channel exclusively on cholinergic neurons (ChAT–ChR2, n = 12) and wild-type mice lacking the opsin (n = 10). Both optical stimuli applied across a range of powers (P < 0.001) and microperfusion of taltirelin into the HMN (P < 0.020) increased tongue motor activity in sleeping ChAT–ChR2 mice. Notably, taltirelin increased tonic background tongue motor activity (P < 0.001) but not responsivity to excitatory optical stimuli across sleep–wake states (P > 0.098). This differential effect on tonic motor activity versus responsivity informs human studies of the potential beneficial effects of taltirelin on pharyngeal motor control and OSA pharmacotherapy.

Paternal developmental thyrotoxicosis disrupts neonatal leptin leading to increased adiposity and altered physiology of the melanocortin system.

The genetic code does not fully explain individual variability and inheritance of susceptibility to endocrine conditions, suggesting the contribution of epigenetic factors acting across generations. We used a mouse model of developmental thyrotoxicosis (Dio3-/- mouse) to analyze endocrine outcomes in the adult offspring of Dio3-/- males using standard methods for body composition, and baseline and fasting hormonal and gene expression determinations in serum and tissues of relevance to the control of energy balance. Compared to controls, adult females with an exposed father (EF females) exhibited higher body weight and fat mass, but not lean mass, a phenotype that was much milder in EF males. After fasting, both EF females and males exhibited a more pronounced decrease in body weight than controls. EF females also showed markedly elevated serum leptin, increased white adipose tissue mRNA expression of leptin and mesoderm-specific transcript but decreased expression of type 2 deiodinase. EF females exhibited decreased serum ghrelin, which showed more pronounced post-fasting changes in EF females than in control females. EF female hypothalami also revealed significant decreases in the expression of pro-opiomelanocortin, agouti-related peptide, neuropeptide Y and melanocortin receptor 4. These markers also showed larger changes in response to fasting in EF females than in control females. Adult EF females showed no abnormalities in serum thyroid hormones, but pituitary expression of thyrotropin-releasing hormone receptor 1 and thyroid gland expression of thyroid-stimulating hormone receptor, thyroid peroxidase and iodotyrosine deiodinase were increased at baseline and showed differential regulation after fasting, with no increase in Trhr1 expression and more pronounced reductions in Tshr, Tpo and Iyd. In EF males, these abnormalities were generally milder. In addition, postnatal day 14 (P14) serum leptin was markedly reduced in EF pups. A paternal excess of thyroid hormone during development modifies the endocrine programming and energy balance in the offspring in a sexually dimorphic manner, with baseline and dynamic range alterations in the leptin-melanocortin system and thyroid gland, and consequences for adiposity phenotypes. We conclude that thyroid hormone overexposure may have important implications for the non-genetic, inherited etiology of endocrine and metabolic pathologies.

Interaction between the hypothalamo-pituitary-adrenal and thyroid axes during immobilization stress.

The location of corticotropin-releasing hormone receptor 2 (CRH-R2) on thyrotropes within the avian anterior pituitary (APit) and its activation by different stressors indicate a possible communication between hypothalamo-pituitary-adrenal (HPA) and thyroid (HPT) axes. Therefore, an experiment was designed to 1) compare the timing of major components of the HPT axis to those of the HPA axis; 2) address whether stressors activating the HPA axis may simultaneously upregulate components of the HPT axis. Blood, brain, and APit were sampled from chicks prior to stress (control) and 15, 30, 60, 90, and 120min following immobilization (IM) stress. The nucleus of the hippocampal commissure (NHpC) and paraventricular nucleus (PVN) were cryo-dissected from brains for RT-qPCR. Gene expression of thyrotropin-releasing hormone (TRH) and its receptors (TRH-R1 and TRH-R3), urocortin3 (UCN3), deiodinase 2 (D2), and the second type of corticotropin-releasing hormone (CRH2) within the NHpC and PVN was measured. Additionally, gene expression of TRH receptors, thyroid stimulating hormone subunit beta (TSHβ), and D2 was determined in the APit and corticosterone assayed in blood. In brains, a significant upregulation in examined genes occurred at different times of IM. Specifically, UCN3 and CRH2 which have a high affinity to CRH-R2 showed a rapid increase in their mRNA levels that were accompanied by an early upregulation of TRHR1 in the NHpC. In the APit, a significant increase in gene expression of TSHβ and TRH receptors was observed. Therefore, results supported concurrent activation of major brain and APit genes associated with the HPA and HPT axes following IM. The initial neural gene expression originating within the NHpC resulted in the increase of TSHβ mRNA in the APit. Specifically, the rapid upregulation of UCN3 in the NHpC appeared responsible for the early activation of TSHβ in the APit. While sustaining TSHβ activation appeared to be due to both CRH2 and TRH. Therefore, data indicate that CRH-producing neurons and corticotropes as well as CRH- and TRH-producing neurons and thyrotropes are activated to produce the necessary energy required to maintain homeostasis in birds undergoing stress. Overall, data support the inclusion of the NHpC in the classical avian HPA axis and for the first time show the concurrent activation of the HPA axis and components of the HPT axis following a psychogenic stressor.

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.

GATA2 Mediates Thyrotropin-Releasing Hormone-Induced Transcriptional Activation of the Thyrotropin β Gene

Thyrotropin-releasing hormone (TRH) activates not only the secretion of thyrotropin (TSH) but also the transcription of TSHβ and α-glycoprotein (αGSU) subunit genes. TSHβ expression is maintained by two transcription factors, Pit1 and GATA2, and is negatively regulated by thyroid hormone (T3). Our prior studies suggest that the main activator of the TSHβ gene is GATA2, not Pit1 or unliganded T3 receptor (TR). In previous studies on the mechanism of TRH-induced activation of the TSHβ gene, the involvements of Pit1 and TR have been investigated, but the role of GATA2 has not been clarified. Using kidney-derived CV1 cells and pituitary-derived GH3 and TαT1 cells, we demonstrate here that TRH signaling enhances GATA2-dependent activation of the TSHβ promoter and that TRH-induced activity is abolished by amino acid substitution in the GATA2-Zn finger domain or mutation of GATA-responsive element in the TSHβ gene. In CV1 cells transfected with TRH receptor expression plasmid, GATA2-dependent transactivation of αGSU and endothelin-1 promoters was enhanced by TRH. In the gel shift assay, TRH signal potentiated the DNA-binding capacity of GATA2. While inhibition by T3 is dominant over TRH-induced activation, unliganded TR or the putative negative T3-responsive element are not required for TRH-induced stimulation. Studies using GH3 cells showed that TRH-induced activity of the TSHβ promoter depends on protein kinase C but not the mitogen-activated protein kinase, suggesting that the signaling pathway is different from that in the prolactin gene. These results indicate that GATA2 is the principal mediator of the TRH signaling pathway in TSHβ expression.

High Levels of Thyrotropin-Releasing Hormone Receptors Activate Programmed Cell Death in Human Pancreatic Precursors

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. 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. 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. 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.

Expression of thyrotropin-releasing hormone receptors in rat testis and their role in isolated Leydig cells

Thyrotropin-releasing hormone (TRH) was initially discovered as a neuropeptide synthesized in the hypothalamus. Receptors for this hormone include TRH-receptor-1 (TRH-R1) and -2 (TRH-R2). Previous studies have shown that TRH-R1 and TRH-R2 are localized exclusively in adult Leydig cells (ALCs). We have investigated TRH-R1 and TRH-R2 expression in the testes of postnatal 8-, 14-, 21- 35-, 60-, and 90-day-old rats and in ethane dimethane sulfonate (EDS)-treated adult rats by using Western blotting, immunohistochemistry, and immunofluorescence. The effects of TRH on testosterone secretion of primary cultured ALCs from 90-day-old rats and DNA synthesis in Leydig cells from 21-day-old rats have also been examined. Western blotting and immunohistochemistry demonstrated that TRH-R1 and TRH-R2 were expressed in fetal Leydig cells (in 8-day-old rats) and in all stages of adult-type Leydig cells during development. Immunofluorescence double-staining revealed that newly regenerated Leydig cells in post-EDS 21-day rats expressed TRH-R1 and TRH-R2 on their first reappearance. Incubation with various doses of TRH affected testosterone secretion of primary cultured ALCs. Low concentrations of TRH (0.001, 0.01, and 0.1 ng/ml) inhibited basal and human chorionic gonadotrophin (hCG)-stimulated testosterone secretion of isolated ALCs, whereas relatively high doses of TRH (1 and 10 ng/ml) increased hCG-stimulated testosterone secretion. As detected by a 5-bromo-2'-deoxyuridine incorporation test, the DNA synthesis of Leydig cells from 21-day-old rats was promoted by low TRH concentrations. Thus, we have clarified the effect of TRH on testicular function: TRH might regulate the development of Leydig cells before maturation and the secretion of testosterone after maturation.

Amygdala kindling differentially regulates the expression of the elements involved in TRH transmission

Subthreshold electrical stimulation of the amygdala (kindling) activates neuronal pathways increasing the expression of several neuropeptides including thyrotropin releasing-hormone (TRH). Partial kindling enhances TRH expression and the activity or its inactivating ectoenzyme; once kindling is established (stage V), TRH and its mRNA levels are further increased but TRH-binding and pyroglutamyl aminopeptidase II (PPII) activity decreased in epileptogenic areas. To determine whether variations in TRH receptor binding or PPII activity are due to regulation of their synthesis, mRNA levels of TRH receptors (R1, R2) and PPII were semi-quantified by RT-PCR in amygdala, frontal cortex and hippocampus of kindled rats sacrificed at stage II or V. Increased mRNA levels of PPII were found at stage II in amygdala and frontal cortex, and of pro-TRH and TRH-R2, in amygdala and hippocampus. At stage V, pro-TRH mRNA levels increased and those of PPII, decreased in the three regions; TRH-R2 mRNA levels diminished in amygdala and frontal cortex and of TRH-R1 only in amygdala. In situ hybridization analyses revealed, at stage II, enhanced TRH-R1 mRNA levels in dentate gyrus and amygdala while decreased in piriform cortex; those of TRH-R2 increased in amygdala, CA2, dentate gyrus, piriform cortex, thalamus and subiculum and of PPII, in CAs and piriform cortex. In contrast, at stage V decreased expression of TRH-R1 occurred in amygdala, CA2/3, dentate gyrus and piriform cortex; of TRH-R2 in CA2, thalamus and piriform cortex, and of PPII in CA2, and amygdala. The magnitude of changes differed between ipsi and contralateral side. These results support a trans-synaptic modulation of all elements involved in TRH transmission in conditions that stimulate the activity of TRHergic neurons. They show that reported changes in PPII activity or TRH-binding caused by kindling relate to regulation of the expression of TRH receptors and degrading enzyme.

Thyrotropin-releasing hormone receptor expression in thyroid follicular cells: a new paracrine role of C-cells?

Thyrotropin-releasing hormone (TRH) synthesized in the hypothalamus has the capability of inducing the release of thyroid-stimulating hormone (TSH) from the anterior pituitary, which in turn stimulates the production of thyroid hormones in the thyroid gland. Immunoreactivity for TRH and TRH-like peptides has been found in some tissues outside the nervous system, including thyroid. It has been demonstrated that thyroid C-cells express authentic TRH, affecting thyroid hormone secretion by follicular cells. Therefore, C-cells could have a paracrine role in thyroid homeostasis. If this hypothesis is true, follicular cells should express TRH receptors (TRH-Rs) for the paracrine modulation carried out by C-cells. In order to elucidate whether or not C-cell TRH production could act over follicular cells modulating thyroid function, we studied TRH-Rs expression in PC C13 follicular cells from rat thyroid, by means of immunofluorescence technique and RT-PCR analysis. We also investigated the possibility that C-cells present TRH-Rs for the autocrine control of its own TRH production. Our results showed consistent expression for both receptors, TRH-R1 and TRH-R2, in 6-23 C-cells, and only for TRH-R2 in PC C13 follicular cells. Our data provide new evidence for a novel intrathyroidal regulatory pathway of thyroid hormone secretion via paracrine/autocrine TRH signaling.

Expression of thyrotropin-releasing hormone receptor in immortalized beta-cell lines and rat pancreas

Thyrotropin-releasing hormone (TRH), a hypothalamic tripeptide, is expressed in pancreatic islets at peak levels during the late gestation and early neonate period. TRH increases insulin production in cultured beta-cells, suggesting that it might play a role in regulating pancreatic beta-cell function. However, there is limited information on TRH receptor expression in the pancreas. The aim of the present study was to explore the distribution of the TRH receptor in the pancreas and its function in pancreatic beta-cells. TRH receptor type 1 (TRHR1) gene expression was detected by RT-PCR and verified by Northern blotting and immunoblotting in the beta-cell lines, INS-1 and betaTC-6, and the rat pancreatic organ. The absence of TRH receptor type 2 expression in the tissue and cells indicated the tissue specificity of TRH receptor expression in the pancreas. The TRHR1 signals (detected by in situ hybridization) were distributed not only in islets but also in the surrounding areas of the pancreatic ductal and vasal epithelia. The apparent dissociation constant value for the affinity of [(3)H]3-methyl-histidine TRH (MeTRH) is 4.19 in INS-1 and 3.09 nM in betaTC-6. In addition, TRH induced epidermal growth factor (EGF) receptor phosphorylation with a half-maximum concentration of approximately 50 nM, whereas the high affinity analogue of TRH, MeTRH, was 1 nM. This suggested that the affinity of TRH ligands for the TRH receptor influences the activation of EGF receptor phosphorylation in betaTC-6 cells. Our observations suggested that the biological role of TRH in pancreatic beta-cells is via the activation of TRHR1. Further research is required to identify the role of TRHR1 in the pancreas aside from the islets.

Lithium modulates expression of TRH receptors and TRH-related peptides in rat brain

Lithium is an established mood stabilizer and neuroprotective agent frequently used in the treatment of bipolar disorder and as an adjuvant in drug-resistant unipolar depression. The mechanisms underlying both the therapeutic efficacy of lithium and the exacerbation of symptoms following rapid withdrawal are not understood. From previous studies showing antidepressant and neuroprotective activities of thyrotropin releasing hormone (TRH) and TRH-related neuropeptides we hypothesized that lithium may have substantial effects on the expression and secretion of these peptides and/or their receptors in various rat brain regions involved in the regulation of mood. Chronic lithium effect on TRH receptor binding studies: The effect of 1 and 2 weeks of dietary lithium on [ 3H]3-Me-His-TRH binding to plasma membranes of nucleus accumbens, amygdala and pituitary of young adult male Wistar and the endogenously ‘depressed’ Wistar Kyoto (WKY) rats was measured by the method of Burt and Taylor [Burt, D.R., Taylor, R.L., Endocrinology 106 (1980) 1416–1423]. Acute, chronic and withdrawal effect of lithium on TRH and TRH-like peptide levels in young, adult male Sprague–Dawley rats: Rats were divided into four lithium treatment groups. Control animals received a standard laboratory rodent chow. The acute group received a single i.p. injection of 1.5 milli-equivalents of LiCl 2 h prior to killing. The chronic and withdrawal groups received standard rodent chow containing 1.7 g/kg LiCl for 2 weeks. Withdrawal rats were returned to standard chow 48 h prior to killing while the chronic animals continued on the LiCl diet. TRH, TRH-Gly (pGlu-His-Pro-Gly, a TRH precursor), EEP (pGlu-Glu-Pro-NH 2, a TRH-like peptide with antidepressant activity) and Ps4 (a prepro-TRH-derived TRH-enhancing decapeptide) immunoreactivity (IR) were measured in 13 brain regions. The remaining samples were pooled and fractionated by high-pressure liquid chromatography followed by EEP radioimmunoassay. Chronic lithium treatment increased [ 3H]3Me-TRH binding in the nucleus accumbens and amygdala about two-fold in both Wistar and WKY rats but no change was observed in pituitary binding. The most widespread changes in TRH and TRH-related peptide levels were observed in the withdrawal group compared to the controls. The direction of change for the total IR was consistent for all TRH-IR and TRH-related peptide-IR within a given tissue. For example, withdrawal increased all peptide levels in the pyriform cortex and striatum but decreased these levels in the anterior cingulate and lateral cerebellum. Both acute injection and chronic treatment with LiCl decreased TRH and TRH-related peptide levels in the entorhinal cortex. Acute injection and withdrawal both increased EEP-IR in striatum by more than two-fold. The acute effects are most likely due to changes in the release of these peptides since 2 h is not sufficient time for alterations in peptide biosynthesis. Chronic treatment increased levels of pGlu-Phe-Pro-NH 2 levels in hippocampus, pGlu-Leu-Pro-NH 2, and peak ‘2’ in septum by more than four-fold. The present results are consistent with a component role for TRH and related peptides in the mood-altering effects of lithium administration and withdrawal frequently observed during treatment for depression and bipolar disorder.

Changes of Thyrotropin-Releasing Hormone (Trh) Levels in Brain Regions and Pituitary During Induced Metamorphosis of Ambystoma Mexicanum

Axolotls (Ambystoma mexicanum) are neotenic amphibians: they keep larval features during their entire lifetime. However, metamorphosis can be induced by injection of thyroid hormones or thyrotropin (TSH). The role played by thyrotropin-releasing hormone (TRH) in amphibian metamorphosis is not clear, since TRH treatment never succeeded in inducing metamorphosis in amphibian larvae. We studied the changes of TRH and plasma thyroid hormone levels in axolotls during thyroxine (T4)-induced metamorphosis. TRH levels in skin, hypothalamus, pituitary and extrahypothalamic region of the brain as well as plasma T4 and triiodothyronine (T3) levels were measured by radioimmunoassay (RIA). Preliminary data on the expression of TRH receptors in the pituitary of a neotenic and a metamorphosed animal were obtained by in situ hybridisation with an antisense oligonucleotide probe, based on nucleotides 1011 to 1060 of chicken TRH receptor mRNA. In general, TRH RIA showed that hypothalamus, extrahypothalamic region and pituitary contained high TRH levels. During metamorphosis, TRH levels decreased in hypothalamus (and extrahypothalamic region) and increased in pituitary; postmetamorphic values did not differ from neotenic controls. TRH levels in skin did not change significantly. During metamorphosis, plasma thyroid hormone levels were high, followed by a decrease to premetamorphic values after metamorphosis. Preliminary in situ hybridisation results suggested that the expression of TRH receptors is low in neotenic animals and increases during T4-induced metamorphosis.

Expression of thyrotropin-releasing hormone receptor 2 (TRH-R2) in the central nervous system of rats

The distribution of the recently discovered thyrotropin-releasing hormone (TRH) receptor subtype TRH-R2 was studied in rat brain, pituitary, and spinal cord by in situ hybridization histochemistry and compared with the distribution patterns of the other elements of TRH signaling, namely TRH, TRH-R1, and the TRH-degrading ectoenzyme (TRH-DE). In contrast to the very restricted mRNA expression of TRH-R1 in the central nervous system, TRH-R2 mRNA was widely distributed with highest transcript levels throughout the thalamus, in the cerebral and cerebellar cortex, medial habenulae, medial geniculate nucleus, pontine nuclei, and throughout the reticular formation. In accordance with the well-known endocrine function of TRH, TRH-R1 is found predominantly expressed in hypothalamic regions. Expression of TRH-R1 in various brainstem nuclei and spinal cord motoneurons seems to be associated with the described effects of TRH on the vegetative and autonomic system as well as on the somatomotor system. Furthermore, the fully complementary expression of both receptor subtypes, even in regions where transcripts for both receptors were found (e.g., medial septum, lateral hypothalamus superior colliculi, substantia nigra, etc.), indicates that in discrete neuroanatomical pathways the two receptors serve highly specific functions for the transmission of TRH signals. Together with TRH-DE, the putative terminator of TRH actions that shows in various, but not all, brain areas, an overlapping mRNA distribution pattern with both receptors, the distribution of TRH-R2 mRNA seems to provide the anatomical basis for the described effects of TRH on higher cognitive functions as well as its effect on arousal, locomotor activity, and pain perception.

Desensitization of Inositol 1,4,5-Trisphosphate/Ca2+-induced Cl− Currents by Prolonged Activation of G Proteins in Xenopus Oocytes

Expression of G protein alpha subunits of the Gq family with various G protein-coupled receptors induces activation of an inositol 1,4, 5-trisphosphate (IP3)/Ca2+-mediated Cl- conductance in Xenopus oocytes. Our present data show that two members of this family, the human Galpha16 subunit and the murine homologue Galpha15, can induce both activation and inhibition of these agonist-induced currents. Although extremely low amounts (10-50 pg) of injected Galpha16 subunit cRNA cause modest ( approximately 2-fold) enhancement of ligand-induced Cl- currents in oocytes co-injected with thyrotropin-releasing hormone (TRH) receptor cRNA 48 h postinjection, larger Galpha16 and Galpha15 cRNA injections cause >10-fold inhibition of TRH or 5HT2c receptor responses. The inhibition is analyzed in this study. The inhibited currents are recovered if various Gbetagamma subunit combinations are also expressed with the Galpha subunits. The constitutively active mutant, Galpha16Q212L, also causes a strong attenuation of the ligand-induced Cl- currents, but this inhibition is not recovered by co-expression of Gbetagamma subunits. These results indicate that the free Galpha subunit is responsible for the inhibitory signal. Although expression of TRH receptor alone produces maximum responses approximately 48 h after injection, co-expression of TRH receptor with Galpha16 results in enhanced responses 6-12 h postinjection, followed by complete attenuation at 36 h. Furthermore, injection of Galpha16 cRNA alone at comparable levels gives rise to spontaneous Cl- currents within 6-12 h postinjection, suggesting that the early spontaneous activation underlies the later suppression. Expression of other G protein alpha subunits of the Gq family, at cRNA levels considerably higher than effective for Galpha16, produces both analogous spontaneous Cl- currents and, later, inhibition of ligand-induced Cl- currents. Experiments with direct injection of IP3 and of Ca2+ suggest that this inhibition is consistent with the down-regulation of IP3 receptors. These data indicate that both enhancement and inhibition of signaling through G protein-coupled receptors can be mediated by the expression level and/or activity of an individual G protein.

Thyrotropin-releasing hormone (TRH) receptor number determines the size of the TRH-responsive phosphoinositide pool. Demonstration using controlled expression of TRH receptors by adenovirus mediated gene transfer.

We use an adenovirus vector, AdCMVmTRHR, to express thyrotropin-releasing hormone (TRH) receptors (TRH-Rs) to determine whether the size of the hormone-responsive phosphoinositide pool in mammalian cells is directly related to receptor number. Infection of HeLa cells with increasing numbers of AdCMVmTRHR caused time-dependent graded expression of TRH-Rs. Measurement of cytoplasmic free Ca2+ in individual cells permitted quantitation of the fraction of cells responsive to TRH. Infection with 100 AdCMVmTRHR particles/cell or more led to TRH responsiveness in > or = 90% of HeLa cells. Measurement of prelabeled phosphoinositides hydrolyzed during prolonged TRH stimulation assesses the size of the TRH-responsive pool. In cells infected with AdCMVmTRHR for 24 h, the size of the TRH-responsive phosphoinositide pool increased with increasing TRH-R expression. The TRH-responsive pool also increased with time after infection as the number of TRH-Rs increased. Similar observations were made in GHY and KB cells. These data confirm our previous suggestion (Cubitt, A. B., Geras-Raaka, E., and Gershengorn, M. C. (1990) Biochem. J. 271, 331-336) that there are hormone-responsive and -unresponsive pools of cellular phosphoinositides and that the maximal size of the TRH-responsive pool is directly related to the number of TRH-Rs.