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 (Kd) of 5 X 10(09) M and 13 X 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.     

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)     

Effects of Chronic Chlorpromazine Treatment on Peripheral Benzodiazepine Binding Sites in Heart, Kidney, and Cerebral Cortex of Rats

Daily intraperitoneal administration to rats of 5 mg/kg of chlorpromazine (CPZ) for 21 days induced a significant up-regulation (51%) of peripheral benzodiazepine binding sites (PBSs) in cerebral cortex and a down-regulation of PBSs in the heart (25%) and kidney (14%), whereas no alteration in [3H]flunitrazepam binding in cerebral cortex was observed. [3H]PK 11195 binding to cerebral cortex returned to normal following 5 days of CPZ withdrawal, whereas the density of PBSs in the heart and kidney remained reduced. The affinity of PBSs for the ligand [3H]PK 11195 in the cerebral cortex and heart was not affected by the drug treatment or withdrawal. The CPZ-induced alterations in PBSs may be relevant to the effects of the drug on CNS and/or peripheral organs.     

Chronic nicotine administration does not increase nicotinic receptors labeled by [125I]epibatidine in adrenal gland,superior cervical ganglia,pineal or retina

Neuronal nicotinic acetylcholine receptors (nAChRs) were measured in CNS and peripheral tissues following continuous exposure to saline or nicotine hydrogen tartrate (3.3 or 10 mg/kg/day) for 14 days via osmotic pumps. Initially, binding of [3H](-)nicotine, [3H]cytisine and [3H]epibatidine to nAChRs was compared to determine the suitability of each for these kinds of studies. The predominant nAChR labeled by agonists in the cerebral cortex is an alpha 4 beta 2 subtype, whereas the predominant nicotinic receptors in the adrenal gland, superior cervical ganglia and pineal gland contain an alpha 3 subunit, and they do not bind either [3H](-)nicotine or [3H]cytisine with high affinity. In retina some nAChRs bind all three ligands with high affinity, and others appear to bind only [3H]epibatidine. Thus, only [3H]epibatidine had high enough affinity to be useful for measuring the nAChRs in all of the tissues. The receptors from nicotine-treated rats were then measured using [125I]epibatidine, which has binding characteristics very similar to [3H]epibatidine. Treatment with the two doses of nicotine hydrogen tartrate increased binding sites in the cerebral cortex by 40% and 70%, respectively. In contrast, no significant changes in the density of receptor binding sites were found in the adrenal gland, superior cervical ganglia, pineal gland or retina. These data indicate that chronic administration of nicotine even at high doses does not increase all nicotinic receptor subtypes, and that receptors containing alpha 3 subunits may be particularly resistant to this nicotine-induced change.     

Loss of [3H]kainate and of NMDA-displaceable [3H]glutamate binding sites in brain in thiamine deficiency: Results of a quantitative autoradiographic study

Previous studies suggest that alterations of brain glutamate synthesis and release occur in experimental thiamine deficiency. In order to assess the integrity of post-synaptic glutamatergic receptors in thiamine deficiency, binding sites for [³H]glutamate (displaced by NMDA), [³H]-kainate, and [³H]quisqualate (AMPA sites) were evaluated using Quantitative Receptor Autoradiography in rat brain following 14 days of treatment with the central thiamine antagonist pyrithiamine. Compared to pair-fed controls, brains of symptomatic thiamine-deficient animals contained significantly fewer NMDA-displaceable binding sites in cerebral cortex, medial septum and hippocampus. It has been suggested that NMDA-receptor mediated glutamate excitotoxicity plays a role in the pathogenesis of neuronal loss in thiamine deficiency. If such is the case, the selective loss of NMDA binding sites in cerebral cortex and hippocampus offers a possible explanation for the relative nonvulnerability of these brain regions to pyrithiamine-induced thiamine deficiency. [³H]quisqualate (AMPA) binding sites were unchanged in all brain regions of pyrithiamine-treated rats whereas [³H]kainate sites were significantly reduced in density in medial and lateral thalamus. The decline in these binding sites may be due to neuronal loss in pyrithiamine-induced thiamine deficiency. Alterations of glutamatergic synaptic function involving both NMDA and kainate receptor subclasses could contribute to the pathogenesis of neurological dysfunction in Wernicke's Encephalopathy in humans.     

In Vivo Partial Inactivation of Dopamine D1 Receptors Induces Hypersensitivity of Cortical Dopamine-Sensitive Adenylate Cyclase: Permissive Role of α1-Adrenergic Receptors

As shown by autoradiography, peripheral injections of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) induced a dose-dependent decrease of [3H]SCH 23390 and [3H]prazosin high-affinity binding sites in the rat prefrontal cortex. EEDQ showed similar efficacy in inactivating cortical and striatal dopamine (DA) D1 receptors, whereas prazosin-sensitive alpha 1-adrenergic receptors were more sensitive to the action of the alkylating agent, as for all doses of EEDQ tested (from 0.8 to 3 mg/kg, i.p.), the decrease in cortical [3H]SCH 23390 binding was less pronounced than that of [3H]prazosin. The effects of EEDQ on [3H]SCH 23390 binding and DA-sensitive adenylate cyclase activity were then simultaneously compared in individual rats. In the striatum, whatever the dose of EEDQ used, the decrease of DA-sensitive adenylate cyclase activity was always lower than that of D1 binding sites, suggesting the occurrence of a large proportion of spare D1 receptors. In the prefrontal cortex, a significant increase in DA-sensitive adenylate cyclase activity was observed in rats treated with a low dose of EEDQ (0.8 mg/kg), this effect being associated with a slight reduction in [3H]SCH 23390 binding sites (-20%). Parallel decreases in the enzyme activity and D1 binding sites were observed with higher doses. The EEDQ-induced supersensitivity of DA-sensitive adenylate cyclase did not occur in rats in which the decrease in [3H]prazosin binding sites was higher than 35%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of thyroid status on the characteristics of alpha 1-, alpha 2-, beta, imipramine and GABA receptors in the rat brain.

The effects of a chronic treatment with L-triiodothyronine (T3; 100 mg/rat/day s.c. for 7 days) or with propylthiouracil (PTU; 50 mg/rat/day for 35 days by stomach tube) on the characteristics of alpha 1, alpha 2, beta, imipramine and GABA binding sites in different brain areas of the adult rat have been studied. T3-treatment caused an increase in the number of [3H]dihydroalprenolol and a decrease in the number of [3H]muscimol binding sites in the cerebral cortex. PTU-treatment caused a decrease in the number of [3H]prazosin, [3H]yohimbine and [3H]dihydroalprenolol binding sites in the cerebral cortex, while the number of [3H]imipramine binding sites was reduced in the cerebral cortex and hypothalamus, and increased in the hippocampus. Affinity constants were never modified. Concurrent experiments showed that the "in vitro" addition of T3 and PTU did not influence the binding of any of the ligands employed to control rat brain membranes. The present data further support the view that neurotransmission in the CNS is influenced by the thyroid status.     

[3H]8-Hydroxy-2-(Di-n-Propylamino)Tetralin Binding to Pre- and Postsynaptic 5-Hydroxytryptamine Sites in Various Regions of the Rat Brain

The specific binding of [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([ 3H]8-OH-DPAT) to 5-hydroxytryptamine (5-HT)-related sites was investigated in several regions of the rat brain. Marked differences were observed in the characteristics of binding to membranes from hippocampus, striatum, and cerebral cortex. Hippocampal sites exhibited the highest affinity (KD approximately 2 nM) followed by the cerebral cortex (KD approximately 6 nM) and the striatum (KD approximately 10 nM). Ascorbic acid inhibited specific [3H]8-OH-DPAT binding in all three regions but millimolar concentrations of Ca2+, Mg2+, and Mn2+ enhanced specific binding to hippocampal membranes, whereas only Mn2+ increased it in the cerebral cortex and all three cations inhibited specific binding to striatal membranes. Guanine nucleotides (0.1 mM GDP, GTP) inhibited binding to hippocampal and cortical membranes only. As intracerebral 5,7-dihydroxytryptamine markedly decreased [3H]8-OH-DPAT binding sites in the striatum, but not in the hippocampus, the striatal sites appear to be on serotoninergic afferent fibers. In contrast, in the hippocampus the sites appear to be on postsynaptic 5-HT target cells, as local injection of kainic acid decreased their density. Both types of sites appear to be present in the cerebral cortex. The postsynaptic hippocampal [3H]8-OH-DPAT binding sites are probably identical to the 5-HT1A subsites, but the relationship between the presynaptic binding sites and the presynaptic autoreceptors controlling 5-HT release deserves further investigation.     

Solubilization of Serotonin1a and Serotonin1b Binding Sites from Bovine Brain

Serotonin1 (5-hydroxytryptamine1, 5-HT1) binding sites have been solubilized from bovine brain cortex using a mixture of 0.1% Nonidet P-40 and 0.3% digitonin in a low-salt buffer containing 0.1% ascorbic acid. The affinity of [3H]5-HT for the soluble cortical binding sites (2.1 nM) is identical to its affinity at membrane-bound binding sites (2.1 nM). [3H]8-Hydroxy-2-(di-n-propylamino)tetralin ([3H]DPAT), a selective 5-HT1a radioligand, also binds to soluble cortical binding sites with high affinity (1.8 nM) comparable with its affinity in the crude membranes (1.7 nM). A significant correlation exists in the rank order potency of serotonergic agents for [3H]5-HT binding and for [3H]DPAT binding to crude and soluble membranes. The density of [3H]DPAT binding sites relative to the [3H]5-HT sites in the solubilized cortical membranes (35%) corresponds well with the proportion of 5-HT1a sites in the crude membranes determined by spiperone displacement (33%), suggesting that both the 5-HT1a and 5-HT1b binding sites have been cosolubilized. [3H]5-HT binding in the soluble preparations was inhibited by GTP, suggesting that a receptor complex may have been solubilized. [3H]Spiperone-specific binding was not detectable in this preparation, suggesting that 5-HT2 sites were not cosolubilized.     

Electroconvulsive Shock (ECS) and the Adenosine Neuromodulatory System: Effect of Single and Repeated ECS on the Adenosine A1 and A2 Receptors, Adenylate Cyclase, and the Adenosine Uptake Site

The effect of a single electroconvulsive shock (ECS) (30 min and 24 h after treatment) and repeated ECS (10 once-daily) on the adenosine neuromodulatory system was investigated in rat cerebral cortex, cerebellum, hippocampus, and striatum. The present study examined the adenosine A1 receptor using N6-[3H]cyclohexyladenosine ([3H]CHA), the A2 receptor using 5'-N-[3H]ethylcarboxyamidoadenosine ([ 3H]NECA), adenylate cyclase using [3H]forskolin, and the adenosine uptake site using [3H]nitrobenzylthioinosine ([3H]NBI). At 30 min after a single ECS, the Bmax of the [3H]NBI binding in striatum was increased by 20%, which is in good agreement with the well-known postictal adenosine release. The Bmax of [3H]forskolin binding in striatum and cerebellum was increased by 60 and 20%, respectively. In contrast to earlier reported changes following chemically induced seizures, [3H]CHA binding was not altered postictally. At 24 h after a single ECS, there were no changes for any ligand in any brain region. Following repeated ECS, there was a 20% increase of [3H]CHA binding sites in cerebral cortex, which lasted for at least 14 days after the last ECS. [3H]Forskolin binding in hippocampus and striatum was 20% lowered 24 h after 10 once-daily ECS but had already returned to control levels 48 h after the last treatment. Evidence is provided that the upregulated adenosine A1 receptors are coupled to guanine nucleotide binding proteins and, furthermore, that this upregulation is not paralleled by an increase in adenylate cyclase activity as labeled by [3H]forskolin.     

Polychlorinated Convulsant Insecticides Potentiate the Protective Effect of NaCl Against Heat Inactivation of [3H]Flunitrazepam Binding Sites

Six polychlorinated convulsant insecticides that potently inhibit t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to rat brain membranes also potentiate the protective effect of NaCl (200 mM) against heat inactivation of [3H]flunitrazepam binding sites on the same membranes. Similar effects were obtained with all "cage" convulsants tested. The rank order of potencies as heat protection potentiators was similar to the rank order of potencies as inhibitors of [35S]TBPS binding (alpha-endosulfan greater than endrin greater than dieldrin greater than toxaphene greater than lindane). alpha-Endosulfan and endrin are more potent in both respects than any previously reported picrotoxin-like (cage) convulsant, but are much less toxic to mammals. The greatly reduced toxicities of alpha-endosulfan and endrin in mammals may reflect partial gamma-aminobutyric acid (GABA)-neutral properties of these compounds. Time courses of heat inactivation of [3H]flunitrazepam binding sites in the presence of 200 mM NaCl plus saturating concentrations of endrin or picrotoxin revealed monophasic components constituting about 88% of the binding sites, suggesting that virtually all [3H]flunitrazepam binding sites are coupled to picrotoxin binding sites in the GABA/benzodiazepine/picrotoxin receptor complex. Protection against heat inactivation constitutes a useful tool for characterizing the various allosterically linked binding sites in neurotransmitter receptor complexes.     

Electroconvulsive Shock Increases α1b-but Not α1a-Adrenoceptor Binding Sites in Rat Cerebral Cortex

Repeated administration of electroconvulsive shock (ECS) increases [3H]prazosin binding to alpha 1-adrenoceptors in rat cerebral cortex. In contrast, [3H]WB4101 binding in cortex has been reported to be unchanged after ECS. [3H]Prazosin labels two alpha 1-adrenoceptor subtypes, termed alpha 1a and alpha 1b, whereas [3H]WB4101 labels the alpha 1a subtype preferentially. The purpose of this study was to determine whether ECS increases one or both alpha 1-adrenoceptor subtypes in rat cerebral cortex. We found that treatment of rats with ECS once daily for 10-12 days increased [3H]prazosin binding in cortex by about 25% but did not significantly alter [3H]WB4101 binding to alpha 1-adrenoceptors. Measurement of alpha 1a and alpha 1b receptors by competition analysis of the selective alpha 1a antagonist 5-methylurapidil against [3H]prazosin and measurement of [3H]prazosin binding in homogenates preincubated with chlorethylclonidine, which alkylates alpha 1b binding sites, also indicated that the ECS-induced increase in alpha 1-adrenoceptors is confined to the alpha 1b subtype. In contrast to its effect on [3H]prazosin binding, ECS did not increase phosphoinositide hydrolysis as measured by [3H]inositol 1-phosphate accumulation in slices of rat cerebral cortex stimulated by either norepinephrine or phenylephrine. The failure of ECS to increase [3H]inositol 1-phosphate accumulation stimulated by phenylephrine, which is a partial agonist for this response, suggests that spare receptors do not account for the apparent absence of effect of ECS on alpha 1-adrenoceptor-mediated phosphoinositide hydrolysis.     

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.     

Effect of estradiol on expression and activation of Akt protein in rat hypothalamus exposed to chronic [D-Met2, Pro5]-enkephalinamide treatment

Adult ovariectomized rats were implanted with [D-Met2, Pro5]-enkephalinamide (ENK)-containing osmotic minipumps. Two hours prior to sacrifice, some animals were treated with estradiol-17beta (E2) at a dose 10 microg/100 g bodyweight (BW). Expression and activation of Akt proteins, nuclear [3H]estradiol binding, and the expression of estrogen receptor alpha (ERalpha) and beta (ERbeta) and of progesterone receptor (PR) were investigated. Estradiol increased the level of activated Akt protein (pAkt473) in the hypothalamus by 52 +/- 11% in comparison to the vehicle-treated controls. No such effect of E2 was observed 24 and 48 h after ENK implantation. This effect of ENK was abolished by concomitant treatment with naloxone. Time-dependent changes in nuclear [3H]estradiol binding and the expression of estrogen and progesterone receptors were also detected in the hypothalamus of ENK-implanted and E2-treated rats. At 24-48 h following ENK implantation, expression of ERalpha and high affinity [3H]estradiol binding decreased. At this time point, the PR level was also reduced, while the ERbeta level was augmented. In conclusion, these results suggest that the stimulatory effects of E2 on the expression and activation of Akt protein and the expression of ERalpha and PR are negatively regulated in rat hypothalamus exposed to chronic ENK treatment.     

Allosteric Modulation of Flunitrazepam Binding to Rat Brain Benzodiazepine Receptors by Methyl β-Carboline-3-Carboxylate

The inhibition of flunitrazepam (FNP) binding to rat brain benzodiazepine (BZ) receptors by methyl beta-carboline-3-carboxylate (MCC) was studied. Biphasic dissociation was observed for [3H]FNP and [3H]MCC in cerebral cortex, cerebellum, and hippocampus, although the dissociation of [3H]MCC was much faster. The dissociation rate of [3H]FNP was increased by MCC in the cerebellum, but was not altered in cerebral cortex or hippocampus. [3H]FNP binding stimulated by gamma-aminobutyric acid was enhanced in the presence of MCC in all three regions examined. These results indicate that MCC exerts these effects by interacting with allosteric sites that are different from the FNP recognition sites on the BZ receptors.     

Enhancement of γ-Aminobutyric Acid Binding by the Anxiolytic β-Carbolines ZK 93423 and ZK 91296

The effects of two anxiolytic beta-carboline derivatives, ZK 93423 and ZK 91296, on the binding of gamma-[3H]aminobutyric acid ([3H]GABA) to brain membrane preparations from rat cerebral cortex were examined. ZK 93423 concentration-dependently enhanced the specific binding of [3H]GABA, with a maximal increase of 45% above control at a 50 microM concentration. A less pronounced increase was induced by diazepam and by the partial agonist ZK 91296. Scatchard plot analysis revealed that the effect of ZK 93423 was due to an increase in the total number of high- and low-affinity GABA binding sites. The action of ZK 93423 was mediated by benzodiazepine recognition sites since it was blocked by the benzodiazepine antagonists Ro 15-1788 and ZK 93426 at concentrations that failed to modify [3H]GABA binding on their own. Moreover the stimulatory effect of ZK 93423 on [3H]GABA binding was also blocked by the beta-carboline inverse agonist ethyl beta-carboline-3-carboxylate. These results are consistent with the view that ZK 93423 and ZK 91296, similarly to benzodiazepines, exert their pharmacological effects by enhancing the GABAergic transmission at the level of the GABA/benzodiazepine receptor complex.     

Presynaptic Nicotinic Cholinergic Receptors Labeled by [3H]Acetylcholine on Catecholamine and Serotonin Axons in Brain

Nicotinic cholinergic receptor binding sites labeled by [3H]acetylcholine were measured in the cerebral cortices, thalami, striata, and hypothalami of rats lesioned by intraventricular injection of either 6-hydroxydopamine or 5, 7-dihydroxytryptamine. In addition, [3H]acetylcholine binding sites were measured in the cerebral cortices of rats lesioned by injection of ibotenic acid into the nucleus basalis magnocellularis. [3H]Acetylcholine binding was significantly decreased in the striata and hypothalami of both 6-hydroxydopamine- and 5,7-dihydroxytryptamine-lesioned rats. There was no change in binding in the cortex or thalamus by either lesion. Ibotenic acid lesions of the nucleus basalis magnocellularis, which projects cholinergic axons to the cortex, did not alter [3H]acetylcholine binding. These results provide evidence for a presynaptic location of nicotinic cholinergic binding sites on catecholamine and serotonin axons in the striatum and hypothalamus.     

Characterization of the Binding of [3H]Ro 5-4864, a Convulsant Benzodiazepine, to Guinea Pig Brain

The density of high affinity binding sites for [3H]4'-chlorodiazepam [( 3H]Ro 5-4864) in guinea pig cerebral cortex is significantly higher (3.8-fold) than the density reported in the rat, and is nearly equal to the density of binding sites for other [3H]benzodiazepines (e.g., diazepam, flunitrazepam). The density of these [3H]Ro 5-4864 binding sites was generally higher in guinea pig brain than in rat brain, with the exception of olfactory bulb. Both the subcellular distribution and pharmacologic profile of these sites in guinea pig brain appears qualitatively similar to observations previously reported in the rat. The high density of binding sites for [3H]Ro 5-4864, coupled with the potency of this compound as a convulsant in the guinea pig, suggest this species will be a valuable model for elucidating putative pharmacologic and physiologic functions of these sites in brain.     

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.     

Increased Binding of [3H]Muscimol and [3H]Flunitrazepam in the Rat Brain Under Hypoxia

We examined the effects of in vivo hypoxia (10% O2/90% N2) on the gamma-aminobutyric acid (GABA)/benzodiazepine receptors and on glutamic acid decarboxylase (GAD) activity in the rat brain. Male Wistar rats were exposed to a mixture of 10% O2 and 90% N2 in a chamber for various periods (3, 6, 12, and 24 h). The control rats were exposed to room air. The brain regions examined were the cerebral cortex, striatum, hippocampus, and cerebellum. GABA and benzodiazepine receptors were assessed using [3H]muscimol and [3H]flunitrazepam, respectively. Compared with control values, GAD activity was decreased significantly following a 6-h exposure to hypoxia in all four regions studied. On the other hand, the numbers of both [3H]muscimol and [3H]flunitrazepam binding sites were increased significantly. The increase in receptor number tended to return to control values after 24 h. Treatment of the membrane preparations with 0.05% Triton X-100 eliminated the increase in the binding capacity. These results may represent an up-regulation of postsynaptically located GABA/benzodiazepine receptors corresponding to the impaired presynaptic activity under hypoxia.