Solubilization of receptors for thyrotropin-releasing hormone from GH4C1 rat pituitary cells: demonstration of guanyl nucleotide sensitivity

TRH receptors have been solubilized from GH4C1 cells using the plant glycoside digitonin. Solubilized receptors retain the principal binding characteristics exhibited by the TRH receptor in intact pituitary cells and their membranes. The binding of the methylhistidyl derivative of TRH [( 3H]MeTRH) attained equilibrium within 2-3 h at 4 C, and it was reversible, dissociating with a t1/2 of 7 h. Analysis of [3H]MeTRH binding to soluble receptors at 4 C yielded a dissociation constant (Kd) of 3.8 nM and a total binding capacity (Bmax) of 3.9 pmol/mg protein. Peptides known to interact with non-TRH receptors on GH cells failed to interfere with the binding of [3H]MeTRH, indicating that the TRH binding was specific. Chlordiazepoxide, a competitive antagonist for TRH action in GH cells, inhibited TRH binding to soluble receptors with an IC50 of 11 microM. When [3H]MeTRH was bound to membranes and the membrane proteins were then solubilized, we found enhanced dissociation of the prebound [3H]MeTRH from its solubilized receptor by guanyl nucleotides. Maximal enhancement of [3H]MeTRH dissociation by 10 microM GTP gamma S occurred within about 45 min at 22 C. GTP gamma S, GTP, GDP beta S, and GDP were all effectors of [3H]MeTRH dissociation, exhibiting EC50s in the range of 14-450 nM. The rank order of potency of the tested nucleotides was GTP gamma S greater than GTP congruent to GDP beta S greater than GDP much greater than ATP gamma S greater than GMP. We conclude that TRH receptors have been solubilized from GH cells with digitonin and retain the binding characteristics of TRH receptors in intact pituitary cells. Furthermore, prebinding [3H]MeTRH to GH4C1 cell membranes results in the solubilization of a complex in which the TRH receptor is linked functionally to a GTP binding protein.     

Lack of Usefulness of DN-1417 for Characterization of a CNS Receptor for Thyrotropin-Releasing Hormone

CNS receptors for thyrotropin-releasing hormone (TRH) and its analogs are likely to mediate the experimentally and clinically observed net excitatory effect of these peptides on lower motor neurons. Previous findings suggest that several types of TRH receptors with distinct TRH analog specificities may be present in rat CNS. In particular, based on competition isotherm assays with unlabeled analog gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-prolineamide (DN-1417). Funatsu et al. claim the existence of a limbic forebrain site that binds this peptide and TRH with high affinity but that does not bind [3-methyl-histidyl2]-TRH (MeTRH). Using saturation and competition isotherm experiments, we have examined the binding of [3H]TRH and [3H]DN-1417 in three regions of rat CNS: pyriform cortex/amygdala, limbic forebrain, and lumbosacral spinal cord. In all three regions, saturation assays with [3H]TRH (0.4-100 nM) resolved only a single, saturable receptor with high affinity (KD = 12-14 nM) for TRH; in no case could more than one saturable site be identified. When [3H]DN-1417 was substituted as the assay ligand, no high-affinity binding component for this analog could be detected in the three regions. Competition curves for the binding of unlabeled DN-1417 to limbic forebrain and lumbosacral spinal cord ([3H]TRH as assay ligand) were monophasic (not biphasic like those of Funatsu et al.) and indicative of low-affinity binding of DN-1417 in these regions (Ki values = 2-3 microM; in agreement with values obtained in similar assays with [3H]MeTRH).(ABSTRACT TRUNCATED AT 250 WORDS)     

Proliferation of thyrotropin releasing hormone receptors in specific brain regions during the development of hypertension in spontaneously hypertensive rats

The binding of [3H] [3-MeHis2] thyrotropin releasing hormone [( 3H]MeTRH) to brain membranes prepared from 8 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. [3H]MeTRH bound specifically to rat brain membranes at a single high affinity site. The density (Bmax value) of [3H]MeTRH binding sites was significantly greater (28%) in SHR rats compared to WKY rats. The apparent dissociation constants (Kd values) for the binding of [3H]MeTRH in SHR and WKY rats did not differ. Binding in the various brain regions revealed that the density of [3H]MeTRH was highest in the hypothalamus followed in decreasing order by pons + medulla, midbrain, cortex and striatum. The binding of [3H]MeTRH was approximately 25% greater in cortex, hypothalamus and striatum of SHR rats in comparison to WKY rats. The binding in pons + medulla, midbrain and pituitary of SHR and WKY rats did not differ. To assess the significance of increased binding sites for [3H]MeTRH in some brain regions of SHR rats, the binding studies were carried out during normotensive and hypertensive stages of postnatal age in the two strains. In 3 and 4 week old SHR rats there was neither an increase in blood pressure nor any increase in [3H]MeTRH binding in the hypothalamus and striatum as compared to age matched WKY rats. With the development of elevated blood pressure at 6 weeks, an increase in [3H]MeTRH binding in the hypothalamus and striatum of SHR rats in comparison to the tissues from WKY rats was observed. The results provide, for the first time, evidence for a parallel increase in the density of brain TRH receptors with elevation of blood pressure, and suggest that brain TRH receptors may play an important role in the pathophysiology of hypertension.     

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)     

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°C for 5 h. Amygdaloid membranes bound [³H]MeTRH with high-affinity (K _d=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 M). The rank order of potency of SP analogs at inhibiting TRH receptor binding was: SP>nonapeptide (3–11)>hexapeptide (6–11)>heptapeptide (5–11)>pentapeptide (7–11). However, other tachykinins were inactive in this system. SP was a potent inhibitor of [³H]MeTRH binding in hippocampus> spinal cord>retina>n. accumbens>hypothalamus>amygdaloid>olfactory bulb pituitary>pons/medulla in parallel assays. In amygdaloid membranes SP (50 M) reduced the apparent maximum receptor density by 39% (p<0.01) without altering the binding affinity, and 100 M SP induced a biphasic dissociation of [³H]MeTRH with kinetics faster than those induced by both TRH (10 M) and serotonin (100 M). In contrast, other neuropeptides such as neurotensin, proctolin, angiotensin II, bombesin and luteinizing hormone releasing hormone did not significantly inhibit [³H]MeTRH binding to amydaloid membranes. Thus, the SP site with low affinity in the rat brain is not like any of the previously described tachykinin/neurokinin binding sites but resembles the site found on neuroblastoma cells (108CC15) and on adrenal chromaffin cells that modulate cation permeability and nicotinic receptors respectively. The physiological role of these atypical SP sites in the rat brain remains to be determined.A preliminary account of these studies has been presented to the British Pharmacological Society (9).     

Analog specificity of the thyrotropin-releasing hormone receptor in the central nervous system: possible clinical implications

TRH has rapid-onset (30 sec), slow-offset (1-12 days) clinical benefit in patients with amyotrophic lateral sclerosis and other motor neuron disorders. This benefit is probably receptor-mediated and may have at least 2 components. To obtain a better understanding of the various responses to TRH of the spinal lower motor neurons (LMNs) in patients, and possibly to help guide selection of additional therapeutic agents, we utilized rat CNS (spinal-cord and brain membranes) to analyze the ability of certain molecules to inhibit specific binding of [3H]methyl TRH [( 3H]MeTRH) to the TRH receptor. We found: a) lack of high-affinity binding of the TRH-analog DN-1417 by spinal-cord and brain TRH receptor, despite its known strong TRH-like action physiologically on LMNs; b) lack of high-affinity binding of the TRH-product cyclo(His-Pro) by spinal-cord and brain TRH receptor despite its having some strong TRH-like physiologic actions on the CNS; and c) lack of any identifiable high-affinity receptor for cyclo(His-Pro) in spinal cord and brain. From these data we hypothesize that the acute transmitter-like action of DN-1417, TRH, and possibly other TRH-analogs and products on LMNs is via a non-TRH receptor, such as an amine or amino acid neurotransmitter receptor, e.g. a 5-hydroxytryptamine receptor. We further postulate that the CNS TRH-receptor may modulate a trophic-like influence of TRH on LMNs.     

Thyrotropin-releasing hormone binding to the mouse pituitary receptor does not involve ionic interactions. A model for neutral peptide binding to G protein-coupled receptors.

Thyrotropin-releasing hormone, TRH (< Glu-His-Proamide), and [N tau-Me-His]TRH (MeTRH) are present as neutral and positively charged forms at physiologic pH, and it was possible that they bind to the TRH receptor (TRH-R) as charged (protonated) species. Binding affinities of TRH and MeTRH to endogenous rat TRH-Rs and to transfected wild type mouse TRH-Rs decreased below pH 7.1. Half-maximal decreases in binding occurred at the approximate pK alpha values of these ligands. Asp to Ala mutations in extracellular loop 1, TM-4, and TM-5 did not decrease binding affinity, but an Asp to Ala mutation in TM-2 caused the affinity to decrease 8-fold. The pH dependences of binding of MeTRH, however, were similar in wild type and all mutant receptors and were consistent with the protonated form of MeTRH binding less well. Thus, the binding of TRH to its receptor does not involve ionic interactions and may be a prototype for binding of neutral peptide ligands to G protein-coupled receptors.     

Modulation of Receptors for Thyrotropin-Releasing Hormone by Benzodiazepines: Brain Regional Differences

The benzodiazepines (BZDs) chlordiazepoxide (CDE), diazepam (DZM), and flurazepam (FLM) inhibited receptor binding for thyrotropin-releasing hormone (TRH) with low micromolar potency. In contrast, numerous other categories of drugs were previously shown to be inactive. Scatchard analysis of competition data suggested that the BZDs reduced TRH receptor affinity, consistent with competitive inhibition. Receptors from amygdala, retina, and pituitary appeared more sensitive to inhibition by BZDs than those from hypothalamus, hippocampus, spinal cord, or cerebellum. The latter four regions also gave shallower inhibition curves. CDE revealed an apparently biphasic dissociation of [3-Me-His2]TRH([3H]MeTRH) from amygdala membranes at 4 degrees C, with kinetics similar to those with TRH. These results suggest that TRH receptors in the brain are heterogeneous and that certain BZDs in high therapeutic concentrations may exert central effects through actions at TRH receptors or coupled proteins.     

Sulfhydryl Groups in Receptor Binding of Thyrotropin-Releasing Hormone to Rat Amygdala

Abstract: Binding of [³H]-[3-Me-His²]thyrotropin-releasing hormone ([³H]MeTRH) to TRH receptors in rat amygdala was decreased by sulfhydryl reagents in a time-, temperature-, and concentration-dependent manner. A pronounced reduction in receptor density, with little or no change in binding affinity, was apparent following disulfide bond reduction by dithiothreitol (DTT), alkylation of thiol groups by N -ethylmaleimide (NEM), and their oxidation by 5,5'-dithiobis (2-nitrobenzoic acid). Heavy metals (Cd²⁺, Hg²⁺), which complex with reactive -SH residues, also potently inhibited binding. The pharmacological specificity of residual [³H]MeTRH binding in chemically modified amygdala membranes was the same as that in control preparations. Sequential exposure to thiol reagents, in the presence or absence of cations, revealed possible additive effects. Pretreatment of membranes with TRH (10^--⁸-^-10^--⁶ M ), and its continued presence during modification, afforded protection against DTT and NEM. These results indicate the possible importance of thiol groups in the maintenance of TRH receptor conformation.     

Analogs of thyrotropin-releasing hormone (TRH): Receptor affinities in brains,spinal cords,and pituitaries of different species

[³H](3-Me-His²) thyrotropin-releasing hormone ([³H]MeTRH) bound to TRH receptors in rodent, rabbit and dog brain and spinal cord (SC), and in rat, sheep, bovine and dog anterior pituitary (PIT) glands, with high affinity (dissociation constants, K_ds=5–9 nM; n=3–4) but to different densities of these sites (B _max range 6–145 fmol/mg protein) (rabbit SC>sheep PITG.pig brain>dog brain>rat brain>bovine and dog PIT). Various TRH analogs competitively inhibited [³H]MeTRH binding in these tissues with a similar rank order of potency: MeTRH>TRH> CG3703RX77368MK-771>TRH Glycinamide>Glu¹-TRHCG3509NVal²-TRH>>>TRH free acid>>>and cyclo-His-Pro, indicating a pharmacological similarity of CNS and pituitary TRH receptors. While most TRH analogs displaced [³H]MeTRH binding with a similar potency in the different species, TRH exhibited a 2-fold lower affinity in the rat and G.pig brain than in other tissues of other species. Similarly, CG3703 was 2.4–4.5 times more active in the rabbit brain than in the rodent and dog brain, and also more potent in the rabbit brain as compared to the sheep PIT. However, MK-771 and RX77368 had a similar affinity for the brain TRH receptors in the different species but RX77368 was 2-fold more active in the SC preparations and 3–4-fold less active in the sheep PIT when compared to the brain homogenates. RX 77368 exhibited the highest affinity for the dog PIT TRH receptor. In contrast, MK-771 showed a similar affinity for the brain, SC and PIT TRH receptor apart from in the rat PIT where it had the highest affinity. Similarly, TRH glycinamide was more active in the dog brain than rodent and rabbit brain. These data suggest that while the rank order of potency of TRH analogs is similar in the species examined, certain analogs appear to be more potent in certain tissues of some species than in others. In addition, the current results have shown that CG3703 is almost equipotent with RX77368 and MK-771 in most species but is substantially more active than its related analog, CG3509 in the brain, SC and PIT. Taken together, these observations may have some relevance to the future clinical applications of these metabolically stabilized TRH analogs.     

Intracisternal injection of TRH precursor, TRH-glycine, stimulates gastric acid secretion in rats

Intracisternal injection of thyrotropin-releasing hormone (TRH)-Gly (pGlu-His-Pro-Gly) produced a dose-dependent (1-100 micrograms) stimulation of gastric acid secretion in urethane-anesthetized rats implanted acutely with a gastric fistula. The peak response occurred 20-30 min after intracisternal injection and lasted for more than 2 h. Intravenous injection of TRH-Gly (100 micrograms) did not modify gastric acid secretion. Following intracisternal injection of TRH-Gly, a peak elevation of both TRH-Gly and TRH levels is observed in the cerebrospinal fluid (CSF) within 15 min. Thereafter, TRH values are returned to basal levels at 75 min after the injection, whereas TRH-Gly concentrations remain significantly elevated throughout the 2-h period of measurement. Compartmental analysis revealed that CSF conversion of TRH-Gly to TRH was only 0.0072%/min. Medullary coronal sections containing the dorsal vagal complex and the raphé nucleus revealed increased content of TRH-Gly, but not TRH, 40 min after administration of TRH-Gly at an intracisternal dose effective in stimulating gastric acid secretion (100 micrograms). In addition, TRH but not TRH-Gly (10(-7)-10(-5) M) displaced [3H]MeTRH binding from rat medullary blocks containing the dorsal vagal complex. These data suggest that the intracisternal TRH-Gly-induced stimulation of gastric acid secretion is not related to its conversion to TRH in the CSF, or direct activation of TRH receptors in the medulla. The acid secretory response of TRH-Gly may be due to the formation of TRH at the active brain sites, or alternatively to activation of its own specific receptors.     

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.     

Signal transduction and hormone-dependent internalization of the thyrotropin-releasing hormone receptor in cells lacking Gq and G11.

The thyrotropin-releasing hormone (TRH) receptor was expressed in embryonic fibroblasts from mice lacking the alpha subunits of Gq and G11 (Fq/11 cells) to determine whether G protein coupling is necessary for agonist-dependent receptor internalization. Neither TRH nor agonists acting on endogenous receptors increased intracellular calcium unless the cells were co-transfected with the alpha subunit of Gq. In contrast, temperature-dependent internalization of [3H]MeTRH in Fq/11 cells was the same whether Gqalpha was expressed or not. A rhodamine-labeled TRH analog and fluorescein-labeled transferrin co-localized in endocytic vesicles in Fq/11 cells, indicating that endocytosis took place via the normal clathrin pathway. Cotransfection with beta-arrestin or V53D beta-arrestin increased TRH-dependent receptor sequestration. Fq/11 cells were co-transfected with the TRH receptor and a green fluorescent protein (GFP)-beta-arrestin conjugate. GFP-beta-arrestin was uniformly distributed in the cytoplasm of untreated cells and quickly translocated to the periphery of the cells when TRH was added. A truncated TRH receptor that lacks potential phosphorylation sites in the cytoplasmic carboxyl terminus signaled but did not internalize or cause membrane localization of GFP-beta-arrestin. These results prove that calcium signaling by the TRH receptor requires coupling to a G protein in the Gq family, but TRH-dependent binding of beta-arrestin and sequestration do not.     

Dopaminergic Receptors in Bovine Retina and Their Interaction with Thyrotropin-Releasing Hormone

A superfusion technique was employed to study the release of [3H]dopamine from isolated bovine retina. Only K+-stimulated release was observed from both light- and dark-adapted retina; release by other stimuli was from dark-adapted retina only. Light-evoked release of [3H]dopamine from dark-adapted retina was blocked by thyrotropin-releasing hormone (TRH), which has previously been identified as a retinal neuropeptide. TRH itself released small amounts of [3H]dopamine from dark-adapted retina. These results are interpreted as indicating that TRH acts as a modulator of dopaminergic activity in retina through the agency of presynaptic autoreceptors. Evidence of the existence of a feedback inhibition system, probably mediated by dopaminergic autoreceptors, was found by the inclusion of sulpiride, a dopaminergic D2 receptor antagonist in the perfusate, which, in a stereoselective manner, enhanced spontaneous and light-evoked release of [3H]dopamine. On the other hand, dopamine (1 microM) reduced these effects. TRH did not affect the high-affinity uptake system for dopamine in retina; this, then, could not account for the effects on release. Radioligand binding showed a specific, saturable high-affinity binding system for [3H]TRH, with an apparent KD of 2.2 nM and a Bmax of 23 fmol/mg protein in bovine retinal membranes. Displacement experiments showed that specific [3H]TRH binding was displaced in the nanomolar range by spiperone and in the micromolar range by dopamine, whereas L-(--)-sulpiride was virtually inactive in displacing [3H]TRH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta-arrestin mediates desensitization and internalization but does not affect dephosphorylation of the thyrotropin-releasing hormone receptor

The G protein-coupled thyrotropin-releasing hormone (TRH) receptor is phosphorylated and binds to beta-arrestin after agonist exposure. To define the importance of receptor phosphorylation and beta-arrestin binding in desensitization, and to determine whether beta-arrestin binding and receptor endocytosis are required for receptor dephosphorylation, we expressed TRH receptors in fibroblasts from mice lacking beta-arrestin-1 and/or beta-arrestin-2. Apparent affinity for [(3)H]MeTRH was increased 8-fold in cells expressing beta-arrestins, including a beta-arrestin mutant that did not permit receptor internalization. TRH caused extensive receptor endocytosis in the presence of beta-arrestins, but receptors remained primarily on the plasma membrane without beta-arrestin. beta-Arrestins strongly inhibited inositol 1,4,5-trisphosphate production within 10 s. At 30 min, endogenous beta-arrestins reduced TRH-stimulated inositol phosphate production by 48% (beta-arrestin-1), 71% (beta-arrestin-2), and 84% (beta-arrestins-1 and -2). In contrast, receptor phosphorylation, detected by the mobility shift of deglycosylated receptor, was unaffected by beta-arrestins. Receptors were fully phosphorylated within 15 s of TRH addition. Receptor dephosphorylation was identical with or without beta-arrestins and almost complete 20 min after TRH withdrawal. Blocking endocytosis with hypertonic sucrose did not alter the rate of receptor phosphorylation or dephosphorylation. Expressing receptors in cells lacking Galpha(q) and Galpha(11) or inhibiting protein kinase C pharmacologically did not prevent receptor phosphorylation or dephosphorylation. Overexpression of dominant negative G protein-coupled receptor kinase-2 (GRK2), however, retarded receptor phosphorylation. Receptor activation caused translocation of endogenous GRK2 to the plasma membrane. The results show conclusively that receptor dephosphorylation can take place on the plasma membrane and that beta-arrestin binding is critical for desensitization and internalization.     

Thyrotropin releasing hormone (TRH) binding sites in the adult human brain: localization and characterization

In the current study, we found evidence for the existence of binding sites for TRH in synaptic membrane preparations of several regions of the postmortem adult human brain. High levels of specific binding (fmol [3H]Me-TRH/mg protein/2 hr) were found in limbic structures: amygdala (7.1 +/- 0.6, Mean +/- SE), hippocampus (2.8 +/- 0.3), and temporal cortex (2.4 +/- 0.8). Intermediate levels of binding were found in the hypothalamus and nucleus accumbens whereas binding was low to undetectable in frontal and occipital cortex, cerebellum, pons, medulla and corpus striatum. Binding of the radioligand was linear over protein concentrations of 0.05-1.5 mg, and greater than 6 hr of incubation was required to achieve maximal binding. In the amygdala, binding was inhibited in the presence of TRH and Me-TRH but not in the presence of up to 1 microM concentrations of cyclo (His-Pro), TRH-OH, pGlu-His or peptides unrelated to TRH. Pretreatment of amygdala synaptic membranes with detergents, proteases or phospholipases disrupted [3H]Me-TRH binding; pretreatment with DNase or collagenase had no effect on binding. Saturation and association/dissociation analyses of the binding of [3H]Me-TRH to purified amygdala synaptic membranes revealed the presence of a high affinity (KD = 2.0 nM), low capacity (Bmax = 180 +/- 16 fmoles/mg protein) binding site. These results demonstrate that a highly specific membrane associated receptor for TRH is present in the adult human brain. The specific role that this receptor plays in brain function remains to be elucidated.     

Evidence for tight coupling of thyrotropin-releasing hormone receptors to stimulated inositol trisphosphate formation in rat pituitary cells

The effect of decreasing the concentration of receptors for thyrotropin-releasing hormone (TRH) on the surface of cloned rat pituitary (GH3) cells on TRH-stimulated inositol trisphosphate (Ins-P3) formation was investigated. Incubation of cells with dibutyryl cAMP (Bt2cAMP) for 16 h caused a decrease in [3H] TRH binding to intact cells to a minimum level 37 +/- 9.1% of control. Scatchard analysis of the concentration dependency of [3H]TRH binding showed that the effect of Bt2cAMP was to lower the receptor concentration without affecting its affinity for TRH. Similar decreases in [3H]TRH binding were found in cells incubated with 8-bromo-cAMP, cholera toxin, and sodium butyrate and, as shown previously, with TRH. In cells incubated with 1 mM Bt2cAMP for 16 h, but not for 1 h, the maximum TRH-induced increase in Ins-P3 was inhibited to 25 +/- 3.2% of that in control cells. Inhibition of TRH-induced Ins-P3 formation was also observed in cells treated with 8-bromo-cAMP, cholera toxin, and sodium butyrate for 16 h, and with TRH for 48 h. Inhibition of TRH-induced Ins-P3 formation and lowering of TRH receptor concentration caused by Bt2cAMP occurred in parallel with increasing doses of Bt2cAMP; at 16 h of exposure, half-maximal effects occurred with 0.3 mM Bt2cAMP. The concentration dependency of TRH-induced Ins-P3 formation was the same in control and Bt2cAMP-treated cells; half-maximal effects occurred with 10 nM TRH. These data demonstrate that decreases in TRH receptor concentration caused by several agents that act via different mechanisms are associated with reduced stimulation of Ins-P3 formation and suggest that the TRH receptor is tightly coupled to stimulation of hydrolysis of phosphatidylinositol 4,5-bisphosphate by a phospholipase C.     

Brain receptor binding characteristics and pharmacokinetics of JTP-2942, a novel thyrotropin-releasing hormone (TRH) analogue

JTP-2942 competed with [3H]-Me-TRH for the binding sites in rat brain in vitro, and its inhibitory effect was approximately 17 times less potent than TRH, as shown by Ki values of 673 and 39.7 nM, respectively. Both JTP-2942 and TRH significantly increased apparent dissociation constant (Kd values) for brain [3H]-Me-TRH binding. Intravenous injection of JTP-2942 (0.3-3 mg/kg) and TRH (3 and 10 mg/kg) produced a significant reduction of [3H]-Me-TRH binding sites (Bmax values) in rat brain. Although the decrease by TRH was maximal 10 min after the injection and declined rapidly with time, the decrease by JTP-2942 (1 and 3 mg/kg) tended to be maximal at 30 min later and it lasted until 120 min. The intravenous injection of JTP-2942 was at least 3 times more potent than that of TRH in decreasing Bmax values for brain [3H]-Me-TRH binding. Plasma concentration of JTP-2942 (0.3-3 mg/kg) after intravenous injection in rats rose with the increase of dose, and it peaked immediately after the injection, thereafter decreasing with t1/2 of 19.3-29.9 min. It is concluded that JTP-2942, compared to TRH, may exert fairly potent and sustained occupation of brain TRH receptors under in vivo condition. Thus, JTP-2942 could be clinically useful for the treatment of CNS disorders.     

A receptor-like binding macromolecule for 1 alpha, 25-dihydroxycholecalciferol in cultured mouse bone cells.

1alpha, 25-Dihydroxycholecalciferol (1,25-(OH)2D3), the active form of vitamin D, like other steroid hormones, initiates its action by binding to cytoplasmic receptors in target cells. Although the 1,25-(OH)2D3 receptor has been well studied in intestine, little information beyond sucrose gradient analyses is presently available from mammalian bone. We, therefore, employed primary cultures of mouse calvarial cells to characterize the mammalian receptor in bone. A hypertonic molybdate-containing buffer was found to protect receptor binding. On hypertonic sucrose gradients, the 1,25-(OH)2-[3H]D3 binder sedimented at 3.2 S. Scatchard analysis of specific 1,25-(OH)2[3H]D3 binding sites at 0 degrees C yielded an apparent Kd of 0.26 nM and an Nmax of 75 fmol/mg of cytosol protein. Competitive binding experiments revealed the receptor to prefer 1,25-(OH)2D3 greater than 25-(OH)-D3 = 1 alpha-(OH)-D3 greater than 24R,25-(OH)2D3; vitamin D3, dihydrotachysterol, sex steroids, and glucocorticoids exhibited negligible binding. As shown in other systems, the receptor could be distinguished from a 25-(OH)-[3H]D3 binder which sedimented at approximately 6 S. In summary, cultured mouse calvarial cells possess a macromolecule with receptor-like properties. This system appears to be an ideal model for the investigation of 1,25-(OH)2D3 receptor binding and action in mammalian bone.     

Sphingosine inhibits thyrotropin-releasing hormone binding to pituitary cells by a mechanism independent of protein kinase C

Sphingosine inhibited [3H]methylhistidine-thyrotropin-releasing hormone (MeTRH) binding to intact GH3 cells and to GH3 membranes. This inhibition was dependent on the concentration of sphingosine and on the ratio of sphingosine to cell number (or membrane protein) and was partly reversed by washing. In intact cells, the IC50 was 63 microM (1.8 X 10(6) cells/ml; 2 nM MeTRH), and 100 microM sphingosine was found, by Scatchard analysis, to increase the apparent dissociation constant (Kd) from 1.1 +/- 0.3 to 6.5 +/- 2.3 nM and to decrease the maximal binding capacity (Bmax) to 41 +/- 9.5% of control. Kinetic analysis showed that the major effect of sphingosine on Kd was due to a marked decrease in the apparent association rate constant for MeTRH from 2.5 +/- 0.4 X 10(5) M-1 s-1 to 0.10 +/- 0.015 X 10(5) M-1 s-1. At 100 microM, sterylamine was as effective as sphingosine in inhibiting MeTRH binding, whereas sphinganine was less effective, and psychosine and steroylsphingosine were without effect. The following observations show that sphingosine inhibition of MeTRH binding did not involve protein kinase C. The IC50 for sphingosine inhibition of MeTRH binding was the same in GH3 cells that had been incubated with 1 microM phorbol 12-myristate 13-acetate for 16 h, to "down-regulate" protein kinase C, as in control cells. Sphingosine inhibited MeTRH binding to membranes isolated from GH3 cells that contain very little protein kinase C activity. In GH3 membranes, 100 microM sphingosine increased the Kd for MeTRH from 3.4 +/- 0.1 to 13 +/- 3.1 nM but did not significantly decrease Bmax (12 +/- 5.0% of control, p greater than 0.05). And, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, an inhibitor of protein kinase C, failed to decrease MeTRH binding to intact GH3 cells or to membranes, and did not interfere with the effects of sphingosine. These data show that sphingosine and its analogs have complex actions to inhibit MeTRH binding to GH3 cells, at least some of which are independent of protein kinase C, and thereby demonstrate that sphingolipids cannot be used as specific inhibitors of protein kinase C.