Thyrotropin-releasing Hormone Binding Research Articles

In vitro biochemical characterization and autoradiographic distribution of 3H-thyrotropin-releasing hormone binding sites in rat brain sections.

In the present study, we describe the biochemical characteristics and the autoradiographic distribution of thyrotropin-releasing hormone (TRH) receptors in the rat central nervous system (CNS) after in vitro incubation of brain slices with 3H-TRH. Scatchard analysis showed that, in the range of concentrations tested (0.7-35 nM), 3H-TRH bound to a single-class of receptors with a dissociation constant of 6 nM and a number of binding sites of 20 fmol/mg protein. Increasing concentrations of unlabeled TRH produced a dose-dependent inhibition of 3H-TRH binding. The only analogue as potent as TRH to displace 3H-TRH binding was 3-Me-TRH, whereas 1-Me-TRH or TRH-free acid as well as pGlu-His, pGlu-Pro-NH2 or His-Pro-diketopiperazine were ineffective. Neither Luteinizing hormone-releasing hormone (LHRH), neurotensin, somatostatin, D-Ala-Met-enkephalin nor VIP showed any significant affinity for TRH binding sites. Autoradiograms obtained by apposition of LKB 3H-Ultrofilm showed that the highest concentrations of 3H-TRH binding sites were found in the ventral dentate gyrus of the hippocampal formation, the lateral amygdaloid nucleus, the nucleus accumbens, and the thalamic paraventricular nucleus. The biochemical characterization of 3H-TRH binding in brain sections is in good agreement with previous reports on membrane preparations and the autoradiographic localization of the binding sites provides anatomical support for the effects of TRH in the CNS.

Polypeptide hormone regulation of gene expression. Thyrotropin-releasing hormone rapidly stimulates both transcription of the prolactin gene and the phosphorylation of a specific nuclear protein.

The tripeptide hypothalamic releasing factor, thyrotropin-releasing hormone (TRH), rapidly increases prolactin gene transcription 7- to 12-fold in the GH4 rat pituitary cell line. The maximal rates of transcription are achieved within minutes and begin to attenuate within 1 h following addition of TRH. This transcriptional response appears to account for the observed effects of TRH on the accumulation of prolactin mRNA and the stimulation of prolactin biosynthesis. The rapid transcriptional effects of TRH include a large number of polymerase II-catalyzed transcription units. Addition of TRH to GH4 cell cultures also rapidly induces the phosphorylation of a 23,000-dalton chromatin-associated basic protein, distinct from known high mobility group or histone proteins and referred to as basic regulated phosphoprotein. The time course of TRH-stimulated prolactin transcription and basic regulated phosphoprotein phosphorylation demonstrates that the signal generated by TRH binding to its plasma membrane receptor rapidly reaches the cell nucleus.

Benzodiazepines compete for thyrotropin-releasing hormone receptor binding: Micromolar potency in rat pituitary, retina and amygdala

In screening neuroactive compounds for their possible interaction with rat thyrotropin-releasing hormone (TRH) receptors, some benzodiazepines were found to compete relatively potently for specific [ 3H](3-Me-His 2)TRH ([ 3H]MeTRH) binding. The profile of activity (chlordiazepoxide > diazepam > flurazepam > lurazepam > flunitrazepam) was almost identical for washed pituitary, retina and amygdala preparations. Corresponding K i values for the latter region were (μM): 0.33, 3.24, 17.4 for the 3 most active inhibitors. Other (47) neuroactive substances exhibited little activity on [ 3H]MeTRH binding. All benzodiazepines tested were competitive inhibitors in the three tissues. These data support previous evidence for a close similarity of TRH receptors in the pituitary gland and central nervous system, and invoke the possibility of TRH receptor-mediated effects for some benzodiazepines.

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)

Barbiturate competition for TRH receptors in mouse brain: Neuromodulation of anesthesia

In vitro thyrotropin releasing hormone (TRH) radioligand binding assays were performed using purified presynaptic and postsynaptic membranes derived from various regions of mouse brain. These studies revealed the pattern of central distribution of specific TRH binding sites. The highest concentrations of both types of membrane receptors were localized in the limbic forebrain. The brain stem contained a high density of only presynaptic receptors, and the cerebral cortex contained a moderate-high level of only postsynaptic receptors. Barbiturate analogues effectively competed for all forebrain and brain stem, but not cortical, TRH receptors, thus implicating these specific receptors in the neuromodulation of barbiturate anesthesia. The results of in vivo radioligand binding assays for [ 3H] TRH disposition after central infusions concomitant with barbiturate vs. saline challenges further support this viewpoint.

Chronic thyrotropin releasing hormone (TRH) administration on TRH receptors and muscarinic cholinergic receptors in CNS

Intraperitoneal injections of thyrotropin releasing hormone (TRH, 6 mg/kg/day) to rats for 14 days resulted in a significant loss (20–25%) of TRH binding sites in the hippocampus, hypothalamus and cerebral cortex. The TRH-mediated loss was reversible and reflected a decrease in binding sites but no change in affinity. On the other hand, this TRH treatment did not affect the specific brain binding of [ 3H]QNB. The results suggest that TRH participates in regulating CNS-TRH receptors.

Characteristics of thyrotropin releasing hormone (TRH) receptors in rat brain

Characteristics of TRH-receptors were studied in the rat central nervous system (CNS). Ion species, pH and temperature importantly influenced TRH-receptor binding. Subcellular distribution of TRH-receptor binding revealed that synaptic membranes had the greatest percentage of total sites. Scatchard analysis suggested that the rat CNS had two distinct TRH binding sites with apparent dissociation constants (K d) of 5×10 −9 M and 13×10 −8 M. Receptor activity is sensitive to trypsin and phospholipase A digestion, suggesting that protein and phospholipid moieties are essential for the binding of [ 3H]TRH. Thiol reagents reduced the binding activity of the receptor, suggesting that an intrachain disulfide bond may form an important constituent of the binding site for TRH. The TRH-receptor in the rat brain was successfully solubilized with non-ionic detergent Triton X-100. On gel chromatography with Sepharose 6B column, the solubilized TRH-receptor molecule eluted at the fraction corresponding to an apparent molecular weight of 300,000 daltons, with Stokes' radius of 5.8 nm. The regional distribution of TRH-receptor binding was examined to clarify the site of TRH action. The highest level of binding was in the hypothalamus, cerebral cortex and hippocampus, indicating that TRH affects the CNS function mainly through the limbic system, cerebral cortex and hypothalamus. Moreover, tricyclic anti-depressants and Li + decreased the binding of [ 3H]TRH. These findings suggest that endogenous TRH and TRH receptor may play the role of a neurotransmission modulator in the brain to control emotional and mental functions.

Purification and characterization of plasma membrane fractions from cultured pituitary cells

Highly purified plasma membrane fractions have been prepared from GH 3 pituitary cells grown in suspension cultures. These membrane fractions have been obtained by differential and sucrose gradient centrifugation and were characterized in terms of their lipid content, marker enzyme analysis and the binding of 3H-labelled thyrotropin-releasing hormone (TRH) to its receptor. Alkaline phosphatase and 5′-nucleotidase activities were enriched 12- to 15-fold in the plasma membrane fraction with somewhat greater enrichment (28-fold) of the specific binding component for [ 3H]TRH, with a specific activity of 2286 fmol [ 3H]TRH bound per mg protein. A single class of binding sites for TRH was observed with an apparent dissociation constant of 18 nM, a value similar to that observed for intact cells. No detectable TRH binding to the nuclear fraction was observed that could not be ascribed to residual plasma membrane contamination. By electron microscopy, these fragments appeared to be sealed vesicles with an average diameter of approximately 1800 Å. The binding of 125I-labelled wheat germ agglutinin was used as a marker for plasma membrane purification. In addition to specific binding to this membrane fraction, specific binding was also observed in the nuclear fraction. Studies with fluorescein-labelled wheat germ agglutinin revealed that, in fixed cells, fluorescence was restricted to the plasma membrane. However, if the cells were treated with Triton before labelling, most of the fluorescence was then associated with the cell nucleus. Hence, the use of wheat germ agglutinin binding as a specific plasma membrane marker must be reevaluated.

Guanine nucleotides modulate trh-receptor binding in sheep anterior pituitary

Binding of thyrotropin-releasing hormone (TRH, thyroliberin) to receptors in chilled membrane resuspensions from sheep anterior pituitary glands is reduced in affinity almost 2-fold by micromolar concentrations of guanosine 5'-triphosphate (GTP) and the hydrolysis-resistant analog guanyl-5'-yl imidodiphosphate (GppNHp) added to tissue during a 10-min preincubation at 37°C. Guanosine 5'-diphosphate (GDP) produced a similar effect at 1 mM, while GMP, ATP, ADP and AMP appeared relatively inactive. The number of TRH-binding sites was not affected by the nucleotide treatments. These results are consistent with reports of guanine nucleotide effects on other receptor types and with evidence suggesting an adenylate cyclase mechanism for some of TRH's effects in the anterior pituitary.

Cyclo (His-Pro): A selective inhibitor of rat prolactin secretion [formula omitted

Cyclo (His-Pro) (10 ng/ml), inhibits KCl (59 mM) or thyrotropin-releasing hormone (10 ng/ml) stimulated, but not basal, release of prolactin from rat hemipituitaries in vitro . However, cyclo (His-Pro) has no effect on the basal or stimulated release of thyrotropin and growth hormone. Cyclo (His-Pro) does not inhibit the binding of thyrotropin-releasing hormone to pituitary membrane suggesting that cyclo (His-Pro) inhibition of prolactin release is not mediated via the pituitary TRH-receptor.

Thyrotropin releasing hormone: Apparent receptor binding in retina

Thyrotropin releasing hormone (TRH) binds with high affinity to sites on sheep retinal membranes. These sites closely resemble sheep pituitary receptors for TRH, as determined by direct comparison of binding in the two tissues. Specific [ 3H]TRH binding in sheep pituitary and retina was measured relative to blanks containing 1 μ m [3-Me-His 2]TRH, an analog more selective and 10-fold more potent than TRH. Incubations were at 0°C; bound radioactivity was separated by filtration. High affinity retinal TRH binding sites resembled pituitary receptors in (1) equilibrium dissociation constant (about 20–40 n m), (2) rate constant for association (about 1–2 μ m −1 min −1), (3) rate constant for dissociation (about 0·07 min −1), (4) effectiveness of 16 TRH analogs, ranging over six orders of magnitude in potency, in competing for binding. Major differences were (1) the retinal binding sites were present in only one quarter the concentration of pituitary receptors (about 5 pmol/g wet weight vs. 20 pmol/g wet weight) and (2) the pharmacological properties of retinal binding sites were partially obscured by the simultaneous presence of low affinity binding sites, absent in pituitary, with which 6 analogs appeared to compete more potently than for high affinity sites. These findings support the possibility that TRH or a closely related substance acts as a peptide neurotransmitter or neuromodulator in the retina.

Hydrocortisone increases the number of receptors for thyrotropin-releasing hormone on pituitary cells in culture

Hydrocortisone (cortisol) increased the binding of thyrotropin-releasing hormone (TRH) to specific membrane receptors in 4 clonal strains of rat pituitary cells. At the highest effective cortisol concentration (3–5 × 10 −6 M), the increase was observed within 6–8 hr and became maximal (140 to 160% of control binding) by 18–24 hr. Half-maximum stimulation occurred in serum-containing medium at 9 × 10 −8 M cortisol, and a significant increase in TRH binding was seen at 3 × 10 −8 M. Equilibrium binding studies showed that enhanced TRH binding was explained by an increase in receptor number with no change in affinity. Similar effects were seen with Dexamethasone, but no increase in TRH binding was noted when testosterone, methyltestosterone, progesterone, estradiol or the antiestrogen Lilly 88571 were added to the culture medium. Cortisol treatment did not cause the appearance of specific TRH binding sites in cell strains previously shown to lack receptors for the tripeptide (F 4C 1, GH 12C 1 and R 5 cells). When added cortisol was removed from medium, receptor number decayed to control values with a T 1 2 of about 30 hr. Previous studies have shown that TRH receptors in GH-cells can be down-modulated by TRH and thyroid hormones; the present findings demonstrate that glucocorticoid hormones can increase the number of TRH receptors in GH-cells.

Inhibition of degradation and measurement of immunoreactive thyrotropin-releasing hormone in rat blood and plasma.

A radioimmunoassay (RIA) for thyrotropin-releasing hormone (TRH) is described. The cross-reactivity of the antiserum was tested with 26 analogs of TRH, 5 amino acids, and LH-releasing hormone. Use of this RIA revealed that inactivation of TRH by rat blood was prevented if the blood was frozen and thawed prior to its incubation with TRH. This procedure did not interfere with the binding of TRH to its antibody. The degradation of TRH by blood or plasma was inhibited by 2,3-dimercaptopropanol (BAL) or benzamidine, but these compounds nonspecifically inhibited the binding of [125I]TRH to anti-TRH. At 37 C, 50% of the synthetic TRH added to rat blood was degraded within seconds, whereas at 1C, 60-65% was recovered after 90 min. When blood was frozen and thawed prior to its incubation with TRH at 1C, essentially all of the hormone was recovered after a 90-min incubation period. In contrast, incubation of TRH with frozen and thawed blood at 37 C resulted in a rapid loss of TRH. BAL (10 mM) or bensamidine (100 mM) afforded complete protection for TRH for at least 30 min at 1 C. At 37 C, protection was incomplete. Exposure of rats to cold (2C) resulted in a significant increase in serum TSH levels, but TRH was undetectable (less than3 pg) in 100 mul of blood regardless of whether the blood contained BAL (10 mM) or benzamidine (100 mM), or was frozen quickly and thawed before RIA. However, when 5-8 ml of trunk blood was extracted with methanol, 8-11 pg/ml of TRH was found, and the TRH levels were slightly but significantly elevated after cold exposure.

Binding of thyrotropin releasing hormone and prolactin release by synthronized GH3 rat pituitary cell line

GH3 cells were synchronized by growing them in a low serum concentration (1%). They were thereafter put back in normal medium (17.5% serum) (time 0 of synchronization). Four parameters were then examined every two hours for up to 40 hours : rate of [ 3H] thymidine incorporation, cell number, binding of [ 3H] Thyrotropin Releasing Hormone (TRH) after a 30 min exposure, and prolactin (PRL) content of culture medium and cell extract. The rate of thymidine incorporation presented a 10–20 fold increase in S phase, beginning on 12–16 hours and lasting at 26 hours. The cell population was doubled at 28 hours. [ 3H] TRH binding to attached cells was observed throughout the cell cycle, but presented a significant increase (40–80%) during the S phase. In contrast, the % increase of PRL release in response to TRH was optimum (300% of control) in G 1 phase. Variations of the PRL cell content as well as of the PRL spontaneous release ability of the cell do not account for the variations of TRH responsiveness. The discrepancy between the two parameters of the TRH-GH3 cells interaction strongly suggest a morphological or functional heterogeneity of the TRH-binding sites.

Thyrotropin releasing hormone (TRH): Apparent receptor binding in rat brain membranes

Thyrotropin releasing hormone (TRH) binds to membranes of rat brain tissue via high- and low-affinity binding components. The high-affinity binding of TRH to brain membranes resembles binding to pituitary membranes in terms of its affinity and specificity for most peptides. In equilibrium studies, the affinity and specificity for most peptides. In equilibrium studies, the dissociation constant for high-affinity binding to brain membranes is about 50 dissociation constant for high-affinity binding to brain membranes is about 50nM, which is about the same as for aat pituitary membranes, while low-affinity binding to brain membranes has a dissociation constant of about 5 muM. Liver membranes display low-affinity binding for TRH with a dissociation constant similar to the low-affinity binding component of brain membranes. No high-affinity binding can be detected with liver membranes. Negligible saturable binding to TRH can be detected with membranes of any tissues examined other than liver, pituitary and brain...

Intrinsic tryptophan fluorescence of membranes of GH 3 pituitary cells: Quenching by thyrotropin-releasing hormone

The intrinsic tryptophan fluorescence of membranes prepared from the GH 3 strain of hormone-producing pituitary cells was monitored by spectrofluorometry. Membranes of GH 3 cells have specific receptors which bind thyrotropin-releasing hormone (TRH). When TRH binds to GH 3 membranes there is quenching of tryptophan fluorescence. The kinetics of the change in fluorescence of GH 3 membranes and of TRH binding are similar. In addition, the concentration of TRH required to produce a half-maximum change in fluorescence is 10 nM, and that required for half-maximum binding of TRH to receptors is 11 nM. Inactive TRH analogs which do not bind to TRH receptors likewise do not alter GH 3 membrane fluorescence, and a pituitary cell strain which lacks TRH receptors does not change membrane fluorescence on incubation with TRH. We conclude that the TRH-receptor interaction in GH 3 membranes is associated with a change in membrane conformation that is readily measured by differential spectrofluorometry.

Receptor for Thyrotropin-releasing Hormone in Plasma Membranes of Bovine Anterior Pituitary Gland: ROLE OF LIPIDS

Abstract The receptor for thyrotropin-releasing hormone has been located in the plasma membranes of bovine anterior pituitary gland. Treatment of membranes with phospholipase A, phospholipase C, or digitonin or addition of lysophosphatidylcholine diminishes or completely destroys the ability of membranes to bind 3H-labeled thyrotropin-releasing hormone. Phospholipase D was without effect. These reductions in thyrotropin-releasing hormone binding capacity stem from a decrease in the affinity for thyrotropin-releasing hormone of the membrane-located receptor. The effects of phospholipase A were completely Ca2+-dependent. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine are the four major phospholipids of adenohypophyseal cell plasma membranes. When added in excess of 1 mg per ml to intact plasma membranes, sonicated suspensions of these four phospholipids present either singly or in combination result in an inhibition of thyrotropin-releasing hormone binding. If plasma membranes are used with a concentration of phospholipase A or C to give a reduction in thyrotropin-releasing hormone binding of at least 50%, partial recovery of binding activity is achieved on phospholipid addition. Inhibition of thyrotropin-releasing hormone binding by phospholipid addition to intact membranes is mediated by a decrease in the Vmax of the reaction. These data show membrane phospholipids to be intimately involved in the receptor for thyrotropin-releasing hormone.

Binding of thyrotropin releasing hormone by thyrotropin-secreting cells

In order to localize and identify receptor structures, the binding of radiolabeled thyrotropin releasing hormone (TRH) to thyrotropin (TSH)-secreting cells from rat and bovine anterior pituitaries and from a mouse TSH-secreting tumor was studied in vitro . The binding of TRH to rat anterior pituitaries increased linearly with the log of TRH concentration in the incubation medium. Plasma membranes were the only subcellular fractions isolated after incubation from bovine anterior pituitary and the TSH tumor which bound detectable quantities of TRH.

Receptors for Thyrotropin-releasing Hormone in Prolactin-producing Rat Pituitary Cells in Culture

Abstract A hypothalamic tripeptide, thyrotropin-releasing hormone (TRH), causes an increase in the rate of synthesis of prolactin by GH3 cells, a clonal strain of rat pituitary cells grown in culture. This cell system has been used to study the initial interaction between TRH and prolactin-producing cells. Tritiated TRH binds to a limited number of sites on GH3 cells, but TRH does not bind to control cell strains which do not respond biologically to the neurohormone. Half-maximum binding of TRH to GH3 cells occurs at a TRH concentration of 11 nm, while a half-maximum biological response to TRH, an increase in prolactin production, is obtained at a concentration of 2 nm. A maximum of 1.1 pmoles of TRH is bound per mg of GH3 cell protein, equivalent to 130,000 TRH receptors per cell. Initially the binding of TRH to GH3 cells is reversible, but it becomes irreversible over the ensuing 24-hour period. Binding of TRH to receptors has been studied in GH3 cell homogenates using a filter assay technique. At 0°, the dissociation constant of the TRH-GH3 receptor complex is 25 nm, and there is 0.75 pmole of TRH receptors per mg of GH3 cell protein. The halftime for the dissociation of the TRH-GH3 receptor complex is 2 to 3 hours. The binding of TRH to the GH3 receptor has a pH optimum of 7.5, does not require the presence of divalent cations, and is inhibited by high concentrations of calcium ion. We conclude that the action of TRH on GH3 cells involves an initial binding of the tripeptide to specific receptors.

Binding of thyrotropin-releasing hormone to plasma membranes of bovine anterior pituitary gland (hormone receptor-adenylate cyclase-equilibrium constant-( 3 H)thyrotropin).

An assay for the binding of [(3)H]thyrotropin-releasing hormone ([(3)H]TRH) is described. Plasma membranes isolated from bovine anterior pituitary gland bind about 600 femtomoles of this hormone per mg of protein, as compared to 15 femtomoles per mg of protein in the total adenohypophyseal homogenate (40-fold purification). The equilibrium constant of membrane receptor-[(3)H]TRH binding at 0 degrees C is 4.3 x 10(7) L.M(-1), or a half-maximal binding of this hormone at 23 nM. The binding is time-dependent; addition of unlabeled hormone induces dissociation of the receptor-[(3)H]TRH complex with a half-life of 14 min. The binding of TRH is not altered by 10 muM melanocyte-stimulating hormone-release inhibiting hormone, lysine-vasopressin, adrenocorticotropin, growth hormone, prolactin, luteinizing hormone, insulin, glucagon, L-thyroxine, or L-triiodothyronine. K(+) and Mg(++) increase formation of the receptor-TRH complex at optimal concentrations of 5-25 mM and 0.5-2.5 mM, respectively, with inhibition at higher concentrations. Ca(++) inhibits binding of TRH at all concentrations tested.