Thyrotropin-releasing Hormone Receptor Binding Research Articles

Contransmitters: differential effects of serotonin (5-HT)-depleting drugs on levels of 5-HT and TRH and their receptors in rat brain and spinal cord

Following codepletion of endogenous serotonin (5-HT, >90%) and thyrotropin-releasing hormone (TRH, 66%) by neonatal treatment with the serotonergic neurotoxin, 5,7-dihydroxytryptamine (DHT), a 33% ( n = 12, P < 0.01) increase in specific TRH receptor binding was observed in adult rat spinal cord (SC) homogenates. A 20–21% increase in TRH receptors was also observed in the medulla/pons (MP) ( n = 12, P < 0.05) and midbrain (MB) ( n = 12, P < 0.02), but no changes were detected in 6 rostral brain regions. The depletion of 5-HT after DHT-treatment was also accompanied by a 34–42% increase in 5-HT 1 binding in the SC, MP and MB. Eadle-Hofstee analysis revealed that the changes in TRH receptor levels observed after DHT-lesions were due to an increase in receptor number rather than any significant changes in receptor affinity. Chronic treatment of adult rats with the 5-HT-depleting drugs, p-chlorophenylalanine (PCPA) and reserpine, produced a 90–97% decrease in 5-HT in the SC, MP and MB and elevated 5-HT 1 binding in any of these tissues. In conclusion, these results have provided further support for the coexistence of 5-HT and TRH in the MP and SC and revealed possible new areas of such colocalization in the MB. Furthermore, these data have demonstrated that only DHT-treatment, as apposed to PCPA or reserpine, can produce long-lasting codepletion of 5-HT and TRH with simultaneous compensatory up-regulation of their receptor systems in the SC and other caudal tissues.

Inhibition of the binding and the behavioral effects of thyrotropin-releasing hormone (TRH) by the triazolobenzodiazepines.

The abilities of 4 triazolobenzodiazepines, adinazolam, alprazolam, estazolam and triazolam, to inhibit thyrotropin-releasing hormone (TRH) receptor binding and to antagonize the narcoleptic effects of TRH were examined. The IC50 values for inhibition of 3H-3-methyl-His-2-TRH (MeTRH) binding ranged from 19 microM to 477 microM, and the Hill coefficient from 0.53 to 0.98. Similar ranges of values were obtained from benzodiazepines of other structural classes. Thus, the inhibition of TRH receptor binding by the triazolobenzodiazepines is similar to that produced by other types of benzodiazepines. Furthermore, the triazolobenzodiazepine, alprazolam, antagonized the narcoleptic effect of TRH. However, this action is not necessarily linked to its inhibition of TRH receptor binding since alprazolam also inhibited the narcoleptic effect of amphetamine.

Simultaneous modulation of TRH and opioid receptor binding in rat spinal cord after chronic naloxone administration

[ 3H]MeTRH binds to crude membranes prepared from the spinal cord with high affinity, K = 3.13 (± 0.29) × 10 8 L/M. After chronic treatment with naloxone (2 mg-kg/h), the density of spinal opioid (μ) receptors increased 100% at the same time as the following changes in MeTRH binding were observed: the affinity of [ 3H]MeTRH decreased 58% (K = 1.32 (± 0.17) × 10 8 L/M; p < 0.005), and binding capacity increased 71%, from 28 (± 4) × 10 −12 M/L or 56 fmol/mg protein to 48 (± 9) × 10 −12 M/L or 96 fmol/mg (p < 0.05). The relative potencies of TRH and MeTRH in competition for the binding of [ 3H]MeTRH changed from a high IC 50 ratio of 10 in control rats to a low IC 50 ratio of about 3 only after naloxone treatment. The results of this study show for the first time that the opiate antagonist-induced upregulation of opioid receptors is associated with modulation in binding of TRH receptors.

Regulation of TRH receptor binding sites by cyclic AMP-dependent protein kinase in GH 3 cells

Regulation of TRH receptor binding sites by cyclic AMP-dependent protein kinase in GH 3 cells

Receptor-stimulated system mediated interactions of neuropeptides in GH3 cells.

The interaction between two neuropeptides, VIP and TRH, was studied. The TRH receptor binding ability was examined using intact GH3 cells and its membrane fraction. The TRH binding ability decreased when intact cells were preincubated with VIP, forskolin or db-cAMP, but not when the membrane fraction was treated with these agents. The binding was reduced only when the membrane fraction was treated with catalytic subunit of cAMP dependent protein kinase (A-kinase). These results indicate that the binding ability of TRH receptors, which are linked to inositol phospholipid metabolism, is suppressed when A-kinase increases due to activation of the adenylate cyclase or its dependent system. This, in turn, suggests the presence of a communication via a cytoplasmic factor (probably A-kinase) between the two principal second messenger systems in the cell.

Guanine nucleotide regulation of receptor binding of thyrotropin-releasing hormone (TRH) in rat brain regions, retina and pituitary

Guanine nucleotides inhibited the specific binding of [ 3H](3-Me-His 2)thyrotropin-releasing hormone ([ 3H]MeTRH) to receptors for TRH in washed homogenates of rat anterior pituitary gland in a dose-related manner. The order of potency (at 100 and 500 μM final) was Gpp(NH)p (a stable analog of GTP) >GTP> >GDP> > >cGMP (with the adenine nucleotides being inactive) in the pituitary and various brain regions. Gpp(NH)p at 1 mM caused 17–35% inhibition of [ 3H]MeTRH binding to different tissues including the pituitary, hypothalamus, retina and nucleus accumbens. A statistically significant nucleotide effect was not observed, however, in the olfactory bulb and medulla/pons membranes. Gpp(NH)p (1 mM) increased the dissociation constants for [ 3H]MeTRH binding by 1.9- to 2.4-fold in the pituitary, n. accumbens and retinal preparations without altering the apparent binding capacity. These data suggest that TRH receptor binding can be allosterically regulated by guanine nucleotides and provide further evidence for the existence of guanine nucleotide binding protein(s) coupled to the TRH receptor.

Changes in brain thyrotropin-releasing hormone (TRH) of El mice.

The present study showed that DN-1417 had a dose-dependent anticonvulsant activity on El mouse seizure. This finding is consistent with other reports using the kindling model of epilepsy. Since both the El mouse and kindling preparations have been regarded as complex partial seizure with secondary generalization, endogenous brain TRH, as well as exogenous TRH, may act as an anticonvulsant substance to such a seizure type of epilepsy. Moreover, this study showed IR-TRH of the El mouse changed significantly in the striatum or hippocampus genetically or postictally without a change in the TRH receptor binding. A transient decrease in hippocampal IR-TRH after convulsion shown in this study may suggest an increased release of TRH during and after the seizure. Further studies are required to clarify the relationship between a change in the brain TRH system and seizure susceptibility in the El mouse.

Regulation of thyrotropin-releasing hormone receptor binding and phospholipase C activation by a single GTP-binding protein.

In a crude membrane preparation of rat 7315c cells, GTP was found to enhance thyrotropin-releasing hormone- (TRH) stimulated inositol triphosphate (IP3) formation with a potency of 0.97 +/- 0.1 microM. TRH stimulation of IP3 formation was inhibited by high GDP concentrations. Neither nucleotide had any effect in the absence of TRH. 5'-Guanosine gamma-thiotriphosphate (GTP gamma S) stimulated IP3 formation in the absence of TRH; the apparent affinity of GTP gamma S was 0.16 +/- 0.05 microM. GTP blocked GTP gamma S stimulation of IP3 formation in a concentration-dependent manner. The apparent affinity of GTP for the site of action shared by GTP gamma S was calculated to be 0.98 +/- 0.3 microM. TRH was able to reverse inhibition of GTP gamma S-stimulated IP3 formation by GTP but could not reverse inhibition by GDP. A lag in the rate of IP3 formation in response to GTP gamma S was abolished by addition of TRH. These data support the proposal that activation of the TRH receptor enhances turnover of guanine nucleotides at the binding protein coupling the receptor to phospholipase C. In addition, GTP gamma S diminished high affinity [3H]Me-TRH binding. The potency of GTP gamma S at decreasing [3H]Me-TRH binding was 0.092 +/- 0.03 microM. GTP gamma S (0.1 microM) decreased the affinity of the TRH receptor for [3H]Me-TRH from 2 to 100 nM. Maximally effective concentrations of GTP gamma S, Gpp(NH)p, GTP, and GDP decreased specific [3H]Me-TRH binding by 80%. Pretreatment of cells with pertussis toxin (30 ng/ml for 24 h) failed to affect TRH receptor affinity or the potency or efficacy of GTP gamma S in diminishing [3H]Me-TRH binding, supporting the identification of Gp (a GTP-binding protein associated with phospholipase C and Ca2+-mobilizing receptors) as distinct from Gi (an inhibitory GTP-binding protein). In contrast to its lack of effect on TRH receptor binding, 3-h pertussis toxin treatment decreased agonist affinity of the mu-opiate receptor and abolished the ability of GTP gamma S to shift the affinity of the mu-opiate receptor for its agonist. The affinities calculated for GTP, GDP, GTP gamma S, and Gpp (NH)p for the G-protein regulating receptor affinity and IP3 formation are nearly identical for each guanine nucleotide tested, suggesting the same G-protein regulates both activities.

Long-term increase in striatal thyrotropin-releasing hormone receptor binding caused by amygdaloid kindling.

Our previous finding that intracerebroventricular (i.c.v.) administration of both thyrotropin-releasing hormone (TRH) and its analogue, gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-prolinamide citrate (DN-1417), suppressed seizure development of amygdaloid (AM) kindling and kindled AM seizures leads to a new hypothesis that endogenous TRH may be an antiepileptic substance in the brain. In this study, we examined postictal chronological changes in both immunoreactive TRH (IR-TRH) and TRH receptor binding activity in discrete brain regions of AM-kindled rats to study the relationship of the brain TRH system to kindling-induced seizure susceptibility. AM-kindled rats were decapitated 30 min, 24 h, 48 h, 7 days, and 21 days after the last kindled convulsion. IR-TRH increased markedly in the AM/pyriform cortex and hippocampus 24 and 48 h after the last convulsion, and returned to the control (unstimulated, sham-operated) value within 3 weeks after the convulsions ended. In contrast, a significant increase in the striatal TRH binding sites was evident 24 h after the cessation of convulsions which lasted 21 days. A lasting change in the striatal TRH neural system may be related to kindling-induced seizure susceptibility.

Benzodiazepines inhibit thyrotropin (TSH)-releasing hormone-induced TSH and growth hormone release from perifused rat pituitaries.

The perifusion technique was used to investigate the action of diazepam (DZ), a benzodiazepine molecule known to compete for TRH receptor binding in rat pituitary, on TRH-induced TSH and GH release. The release kinetics for the two hormones from quartered pituitaries were measured in response to a 6-min pulse of TRH (10 nM), without or with DZ addition for a period of 30 min before and during the TRH pulse, plus an additional 15-min period. The dynamic patterns of TSH and GH release in response to TRH were characterized by a rapid increase in hormone release, declining slowly over the next 20 min. The rate of release represented 2.98 +/- 0.02 (+/- SE) and 1.75 +/- 0.06 times the corresponding basal level for TSH and GH, respectively, when evaluated over the first 15 min of the response to TRH. Addition of increasing doses of DZ suppressed the stimulatory effect of TRH in a dose-related manner, with an ID50 of 3 nM for both TSH and GH. The maximal effect of DZ was obtained with a concentration of 10 nM for both hormones. Ro 15-1788 (100 nM), a selective antagonist of the central type of benzodiazepine-binding sites (added to the perifusion system 30 min before DZ and then during the whole period of DZ perifusion), completely abolished (P less than 0.01) the inhibitory effect of DZ (10 nM) on the TRH-induced TSH and GH responses. When added alone before the TRH pulse, Ro 15-1788 had no effect on the TSH response to TRH. In contrast, PK 11,195 (100 nM), a selective antagonist of the nonneuronal benzodiazepine-binding sites, was unable to abolish the inhibitory action of DZ on TRH-stimulated TSH release. In addition, the effects of four other types of benzodiazepine (flurazepam, chlordiazepoxide, midazolam, and medazepam), all tested at a 10-nM concentration, corroborated these findings. Furthermore, DZ inhibition of the TSH response was nullified by picrotoxin (1 microM), but not by bicuculline (1 microM), two gamma-aminobutyric acid antagonists that had no effect, by themselves, on this response. For comparison, the effect of DZ (10 nM) was also tested on the release of GH in response to human GH-releasing factor-(1-44)-NH2 (10 nM) and was found to be ineffective.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of immobilization stress on neuropeptides and their receptors in rat central nervous system

The effect of immobilization stress (IM-stress) on the concentration and the receptor binding of substance P (SP), methionine-enkephalin (ME) and thyrotropin-releasing hormone (TRH) was determined in eight brain regions and the spinal cord. The concentration of SP was decreased in the septum, striatum and hippocampus, and SP receptor binding was decreased in the septum, amygdala + pyriform cortex and hypothalamus. Scatchard analysis indicated that the decrease in the SP binding is mainly due to the decrease in the number of receptors. The concentration of ME was not changed, but ME receptor binding was decreased in the septum. The concentration of TRH was decreased in the frontal cortex, septum, amygdala + pyriform cortex and pons + medulla oblongata, but increased in the spinal cord. TRH receptor binding was decreased in the septum, amygdala + pyriform cortex and hypothalamus. Scatchard analysis indicated that the decrease in TRH binding is due to the decrease in the number of receptors. These results show that IM-stress affects the neuropeptide receptor as well as neuropeptide concentration, and that the septum is a very important region under IM-stress.

Change in brain thyrotropin-releasing hormone (TRH) mechanism of amygdaloid kindled rats.

We reported previously that DN-1417, a potent analog of thyrotropin-releasing hormone (TRH), suppressed both the progression of amygdaloid (AM) kindling and AM kindled seizure. To study a functional role of the cerebral TRH mechanism in AM kindling, immunoreactive TRH (IR-TRH) and specific TRH receptor binding were examined in the rat brains kindled from the left AM. The IR-TRH concentration elevated significantly in the amygdala plus piriform cortex and the hippocampus 24 and 48 hours after the AM kindled convulsion. Such an elevation of IR-TRH was not found 7 days after the last convulsion, indicating that the elevation of IR-TRH was a transient change seen after the AM kindled convulsion. By contrast, the specific TRH receptor binding in the striatum increased 48 hours, 7 and 21 days after the AM kindled convulsion. This indicates that the increase of the specific TRH binding in the striatum was a long-lasting change. The present study suggests that the change in the striatal TRH receptors may be associated with a long-lasting seizure susceptibility of AM kindled rats.

Experimental spinal cord injury: effects of trauma or ischemia on TRH and muscarinic receptors.

Traumatic spinal cord injury in rats and ischemic spinal cord injury in rabbits were associated with time-dependent, localized decreases in thyrotropin-releasing hormone (TRH) and muscarinic receptor binding. Changes were not evident in the first 24 to 48 hours, consistent with the hypothesis that secondary alterations in spinal cord may occur relatively late after injury. TRH receptor binding, but not muscarinic receptor binding, recovered by 3 weeks following trauma. Since TRH and acetylcholine may serve as excitatory neurotransmitters within the spinal cord, such changes could contribute to the functional neurologic impairment that follows injury.

Involvement of thyrotropin-releasing hormone (TRH) neural system of the brain in pentylenetetrazol-induced seizures

In order to study the relationship between pentylenetetrazol (PTZ)-induced seizures and the thyrotropin-releasing hormone (TRH) neural system, immunoreactive TRH (IR-TRH) and TRH receptor binding activity were determined in discrete regions of the rat brain before as well as 40 s (immediately before seizures), 150 s (during seizures) and 24 h after an intraperitoneal injection of PTZ (75 mg/kg). IR-TRH markedly increased in the septum 40 and 150 s after the injection, and also in the hippocampus and the thalamus-midbrain region 40 and 150 s after the injection, respectively. However, no significant changes were observed in the TRH receptor binding before, during or after the seizures, suggesting that the increased IR-TRH was not released into the synaptic cleft. This speculation was supported by the dose-dependent inhibition of PTZ-induced generalized seizures by the pre-treatment with TRH or its analogue DN-1417 into the cerebral ventricle.

Ibotenic acid decreases thyrotropin-releasing hormone receptor binding in the rat amygdala

Thyrotropin-releasing hormone (TRH) receptor densities in the amygdala were examined with quantitative autoradiography in rats treated with the cellular neurotoxin ibotenic acid (IBO). Microinjections of IBO (10 μg) into the right basolateral amygdaloid nucleus (BsA) reduced the concentration of TRH receptors in this nucleus by over 50%, when compared to the contralateral BsA and to vehicle-injected control rats. IBO lesions left amygdaloid terminals intact, as shown by normal levels of presynaptic choline uptake sites. Our results strongly suggest that TRH receptors in the amygdala are predominantly located on cell bodies.

Seizures and thyrotropin-releasing hormone (TRH) neural system in the rat brain.

In order to study the relationship between seizures and the thyrotropin-releasing hormone (TRH) neural system, immunoreactive TRH (IR-TRH) and TRH receptor binding activity were determined by pentylenetetrazol (PTZ)-induced seizures and amygdaloid (AM) kindling. IR-TRH markedly increased in the septum 40 and 150 seconds after the PTZ injection. A significant increase in the IR-TRH concentrations was also noted in the hippocampus and thalamus/midbrain 40 and 150 seconds after the PTZ injection, respectively. However, no significant changes were observed in the TRH receptor binding before, during or after the PTZ-induced seizures. In addition, a lasting change in the striatal TRH receptors after AM kindling as well as a transient IR-TRH increase in the limbic structures were seen 48 hours after Am-kindled convulsions. TRH and its analog (DN-1417) inhibited PTZ-induced generalized seizures dose-dependently. These findings indicate the involvement of the TRH neural system in seizure mechanisms, and suggest that endogenous TRH may be an antiepileptic substance in the brain.

Up-regulation of thyrotropin-releasing hormone (TRH) receptors in rat spinal cord after codepletion of serotonin and TRH.

Immunohistochemical evidence indicates the coexistence of serotonin and TRH in many raphe neurons. We examined the biochemical changes in TRH receptors after destruction of the serotonergic pathways by 5,7-dihydroxytryptamine (5,7-DHT). 2 weeks after an intracerebroventricular injection of 5,7-DHT, rats were killed, and specific brain regions were dissected on ice. Serotonin levels in the CNS of lesioned rats was reduced by 50-85% in all regions, with the highest reduction in the spinal cord and hippocampus. Immunoreactive TRH was reduced in the spinal cord by 70%, but other brain regions contained normal levels of TRH. TRH receptor binding was increased by 40% in the spinal cord of lesioned rats, but appeared unchanged in rostral brain regions in which no decrease in TRH content was detected. Scatchard plots of TRH receptor binding in the spinal cord indicated that the increased binding after 5,7-DHT administration reflected an increased receptor number. These findings suggest that biochemical up-regulation of TRH receptors occurs in the spinal cord following depletion of TRH.

Characterization and autoradiographic localization of TRH receptors in sections of rat brain

Receptors for thyrotropin-releasing hormone (TRH) in rat brain have been localized autoradiographically by exposing tritium-sensitive film to sections labeled with [ 3H]3-Me-His 2-TRH. Greatest grain density was found over certain nuclei of the amygdala, with considerable density over several other forebrain areas. Properties of TRH receptor binding in frozen sections closely resembled those of receptors in fresh membrane fragments.

Micromolar substance P reduces spinal receptor binding for thyrotropin-releasing hormone — possible relevance to neuropeptide coexistence?

Receptors for thyrotropin-releasing hormone (TRH) on membranes of rat spinal cord (SC) were labeled with [ 3H](3-Me-His 2)TRH ([ 3H]MeTRH) (dissociation constant = 3.6 ± 0.8 (6) nM). Substance P (SP) caused a concentration-dependent inhibition of TRH receptor binding in the micromolar range (43 ± 4 (6)% at 50 μM). Scatchard analyses of competition data revealed that 50 μM SP reduced TRH receptor number (40–66%, P < 0.05) with little or no effect on affinity. SP appeared more potent in reducing [ 3H]MeTRH binding to ventral cord membranes than those of dorsal or whole SC. A number of SP analogs also reduced TRH receptor binding in a dose-related manner but with different potencies. In contrast, various amino acids and serotonin (250 μM) produced little or no inhibition of [ 3H]MeTRH binding, and cholecystokinin, Leu- and Met-enkephalins, angiotensin II, LH-RH, bombesin and somatostatin were also markedly less potent than SP. Although it is unclear whether spinal TRH receptors are ever exposed to micromolar concentrations of SP in vivo, the reported colocalization of these neuropeptides in raphe efferents to the SC suggests that our findings may be of physiological relevance.

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.