Regional variations in binding capacities at mu-, delta- and kappa-opioid sites in membrane suspensions from rabbit brain

The highest maximum binding capacities at the mu-sites of rabbit brain are in the striatum, with intermediate levels in the diencephalon, mesencephalon, cerebellum and cortex and low levels in the pons-medulla and hippocampus. For the delta-site the highest maximum binding capacity is also in the striatum; there are almost equally low levels in the other brain regions. At the kappa-sites the maximum binding capacities are highest in the diencephalon; there are intermediate levels in the cortex and striatum, and low levels in the mesencephalon, cerebellum, hippocampus and pons-medulla. The KD values lack reproducibility; there are no regional variations at the kappa-site as estimated with [3H](-)-bremazocine, but the possibility cannot be excluded that there are regional variations in the KD values for [3H][D-Ala2,MePhe4,Gly-ol5]enkephalin at the mu-site or for [3H][D-Ala2,D-Leu5]enkephalin at the delta-site. It may be important to use saturation analysis in future investigations of the distributions of the binding sites.     

Chronic administration of methamphetamine increases the mRNA expression of diazepam binding inhibitor in rat brain

Anxiety is one of the common features of withdrawal syndrome caused by abuse-inducing drugs such as methamphetamine (MAP). The neural pathways associated with anxiety are established within the network sustained by diencephalon, cerebral cortex, cerebellum and hippocampus. Diazepam binding inhibitor (DBI), a peptide consisting of 87 amino acids, serves as an inverse agonist for the type A receptor of the gamma-aminobutyric acid (GABAA receptor) with endogenous anxiogenic potential. We examined the effect of chronic administration of MAP on the mRNA expression of DBI and DBI-related proteins, such as alpha 2 subunit of GABAA receptor (GABA-α2), peripheral-type benzodiazepine receptor (PBR), and pituitary adenylate cyclase-activating polypeptide (PACAP) in seven regions (diencephalon, cerebral cortex, cerebellum, striatum, hippocampus, midbrain, and pons-medulla) of the rat brain. The mRNA expression of DBI increased significantly in all areas of the brain, especially diencephalon, after chronic administration of MAP. The mRNA expression of PBR, GABA-α2 and PACAP increased significantly in all areas of the brain, especially cerebral cortex, after chronic administration of MAP. These results suggest that anxiety is associated with the mRNA expression of DBI as well as DBI-related genes.     

Kinetic studies of [3H]-N-methylscopolamine binding to muscarinic receptors in the rat central nervous system: evidence for the existence of three classes of binding sites

We compared the binding of [N-methyl-3H]scopolamine methyl chloride [( 3H]NMS) and pirenzepine to muscarinic receptors in four regions of the rat central nervous system (cortex, hippocampus, striatum, and cerebellum) and in rat heart. Equilibrium binding studies suggested the existence of three classes of receptors: A, receptors with high affinity for pirenzepine and [3H] NMS (in cortex, hippocampus, and striatum); B, receptors with intermediate affinity for pirenzepine and high affinity for [3H]NMS (in the same brain regions); and C, receptors with low affinity for pirenzepine and [3H]NMS (in cerebellum and heart). Dissociation kinetic studies indicated that the receptor types A, B, and C had different koff values allowing, therefore, a separate study of their binding properties. We observed that: [3H]NMS recognized muscarinic receptors A, B, and C with the following order of potency: B greater than A much greater than C; and pirenzepine recognized these receptors with a different order of potency: A much greater than B greater than C. Thus, dissociation kinetics provide a useful tool to identify muscarinic receptor types.     

Regionally different N-methyl-D-aspartate receptors distinguished by ligand binding and quantitative autoradiography of [3H]-CGP 39653 in rat brain.

1. Binding of D,L-(E)-2-amino-4-[3H]-propyl-5-phosphono-3-pentenoic acid ([3H]-CGP 39653), a high affinity, selective antagonist at the glutamate site of the N-methyl-D-aspartate (NMDA) receptor, was investigated in rat brain by means of receptor binding and quantitative autoradiography techniques. 2. [3H]-CGP 39653 interacted with striatal and cerebellar membranes in a saturable manner and to a single binding site, with KD values of 15.5 nM and 10.0 nM and receptor binding densities (Bmax values) of 3.1 and 0.5 pmol mg-1 protein, respectively. These KD values were not significantly different from that previously reported in the cerebral cortex (10.7 nM). 3. Displacement analyses of [3H]-CGP 39653 in striatum and cerebellum, performed with L-glutamic acid, 3-((+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) and glycine showed a pharmacological profile similar to that reported in the cerebral cortex. L-Glutamic acid and CPP produced complete displacement of specific binding with Ki values not significantly different from the cerebral cortex. Glycine inhibited [3H]CGP 39653 binding with shallow, biphasic curves, characterized by a high and a low affinity component. Furthermore, glycine discriminated between these regions (P < 0.005, one-way ANOVA), since the apparent Ki of the high affinity component of the glycine inhibition curve (KiH) was significantly lower (Fisher's protected LSD) in the striatum than the cortex (33 nM and 104 nM, respectively). 4. Regional binding of [3H]-CGP 39653 to horizontal sections of rat brain revealed a heterogeneous distribution of binding sites, similar to that reported for other radiolabelled antagonists at the NMDA site (D-2-[3H]-amino-5-phosphonopentanoic acid ([3H]-D-AP5) and [3H]-CPP). High values of binding were detected in the hippocampal formation, cerebral cortex and thalamus, with low levels in striatum and cerebellum. 5. [3H]-CGP 39653 binding was inhibited by increasing concentrations of L-glutamic acid, CPP and glycine. L-Glutamic acid and CPP completely displaced specific binding in all regions tested, with similar IC50 values throughout. Similarly, glycine was able to inhibit the binding in all areas considered: 10 microM and 1 mM glycine reduced the binding to 80% and 65% of control (average between areas) respectively. The percentage of specific [3H]-CGP 39653 binding inhibited by 1 mM glycine varied among regions (P < 0.05, two-ways ANOVA). Multiple comparison, performed by Fisher's protected LSD method, showed that the inhibition was lower in striatum (72% of control), with respect to cortex (66% of control) and hippocampal formation (58% of control). 6. The inhibitory action of 10 microM glycine was reversed by 100 microM 7-chloro-kynurenic acid (7-CKA), a competitive antagonist of the glycine site of the NMDA receptor channel complex, in all areas tested. Moreover, reversal by 7-CKA was not the same in all regions (P < 0.05, two-ways ANOVA). In fact, in the presence of 10 microM glycine and 100 microM 7-KCA, specific [3H]-CGP 39653 binding in the striatum was 131% of control, which was significantly greater (Fisher's protected LSD) than binding in the hippocampus and the thalamus (104% and 112% of control, respectively). 7. These results demonstrate that [3H]-CGP 39653 binding can be inhibited by glycine in rat brain regions containing NMDA receptors; moreover, they suggest the existence of regionally distinct NMDA receptor subtypes with a different allosteric mechanism of [3H]-CGP 39653 binding modulation through the associated glycine site.     

Theophylline-induced upregulation of A1-adenosine receptors associated with reduced sensitivity to convulsants

Chronic administration of theophylline (50 mg/kg twice daily for 14 consecutive days) significantly increased the specific binding of [3H]CHA in membranes of the cerebral cortex and cerebellum of the rat, but not in membranes derived from the hippocampus or diencephalon. To characterize further the upregulation of A1 = adenosine receptors induced by theophylline, saturation analysis with [3H]CHA was performed in membranes of the cerebral cortex and cerebellum. In both saline- and theophylline-treated cortical membranes the binding isotherms for [3H]CHA could be resolved into receptor affinity states having respectively high (KH) and low (KL) affinity for [3H]CHA. The high and low affinity dissociation constants obtained from theophylline-exposed membranes of the cerebral cortex were 1.14 nM and 25.2 nM and did not differ significantly from the corresponding values in saline-treated animals. Chronic exposure to theophylline did, however, produce significant increases in the densities of both the high and low affinity forms of A1-adenosine receptors in the cerebral cortex. Qualitatively and quantitatively similar results were observed in cerebellar membranes. These results suggest that chronic exposure to theophylline increases the density of A1-receptor in the cerebral cortex and cerebellum with no concomitant changes in the ability of [3H]CHA to distinguish separate agonist affinity states of the receptor. The physiological significance of theophylline-induced upregulation was assessed by determining seizure thresholds for convulsants in rats treated chronically with saline or theophylline.(ABSTRACT TRUNCATED AT 250 WORDS)     

'GABA shift' in vivo: enhancement of benzodiazepine binding in vivo by modulation of endogenous GABA

The enhancement of benzodiazepine binding by gamma-aminobutyric acid (GABA) and its analogues has been described in detail in brain membrane preparations, but results in in vivo preparations such as tissue slices or animals treated with GABA modulators are conflicting. This 'GABA shift' in vitro has been reported for compounds with agonist effects at the benzodiazepine receptor but not for antagonists. We examined the effects of modulators of endogenous GABA on benzodiazepine receptor binding in vivo as determined by specific uptake of the benzodiazepine antagonist [3H]Ro 15-1788. Enhancement of radioligand uptake was observed in cortex, hypothalamus, hippocampus and pons-medulla 4 h after treatment with aminooxyacetic acid (AOAA), in cortex, cerebellum, hypothalamus, hippocampus and pons-medulla 0.5 h after treatment with valproic acid, and in cortex, cerebellum, hypothalamus and hippocampus 6 h after treatment with gamma-vinyl-GABA. GABA concentrations were increased at each of these points, as were synaptosomal GABA concentrations in prior studies. In contrast, no changes in radioligand uptake or GABA concentrations were observed 12 and 24 h after gamma-vinyl-GABA treatment. Increases in binding appeared to be due to increased apparent affinity at the receptor rather than a change in receptor number. These data indicate that binding of a benzodiazepine antagonist undergoes a GABA shift in vivo analogous to that observed with agonists in vitro.     

Effect of isopropanol on rat brain monoamine levels

Changes in monoamines were studied in discrete areas of brain with varying dose of isopropanol. Acute administration of isopropanol (0.463 g/kg, 0.925 g/kg and 1.85 g/kg) decreased dopamine level in hypothalamus, pons medulla and cerebral cortex and whereas it was increased in striatum and cerebellum. Noradrenaline level was reduced in all the brain areas studied. Adrenaline level was increased in hypothalamus, striatum, midbrain and pons medulla, and decreased in hippocampus and cerebral cortex. Serotonin level was increased in hypothalamus, midbrain, pons-medulla and cerebral cortex, and decreased in striatum and hippocampus. These changes were dose-dependent. It is concluded that isopropanol causes changes in brain monoamine content that this effect is not the same in all the regions of the brain.     

Chemotherapeutic targeting of etoposide to regions of the brain on the basis of polyamine level

The effects of etoposide on body weight, regional weights, and the concentrations of putrescine, spermidine and spermine in the cerebellum, hippocampus, corpus striatum, cortex, the combined regions of the thalamus and hypothalamus and the diencephalon of the brain were examined in rats. Etoposide seems to be a better choice for management of cortical and hippocampal tumors because it reduces polyamines, which are associated with tumor cell growth, but not for the management of tumors of the diencephalon and corpus striatum because it increases polyamines.     

Chemotherapeutic Targeting of Etoposide to Regions of the Brain on the Basis of Polyamine Level

The effects of etoposide on body weight, regional weights, and the concentrations of putrescine, spermidine and spermine in the cerebellum, hippocampus, corpus striatum, cortex, the combined regions of the thalamus and hypothalamus and the diencephalon of the brain were examined in rats. Etoposide seems to be a better choice for management of cortical and hippocampal tumors because it reduces polyamines, which are associated with tumor cell growth, but not for the management of tumors of the diencephalon and corpus striatum because it increases polyamines.     

Light microscopic autoradiographic localisation of [3H]glycine and [3H]strychnine binding sites in rat brain

Receptor autoradiography has been employed to determine the distribution of strychnine-insensitive glycine binding sites in rat brain using [3H]glycine as a ligand. The location was significantly different from and more widespread than glycine sensitive [3H]strychnine binding sites. Highest binding densities were observed in hippocampus, cortex, subiculum and amygdala followed by striatum, cerebellum and olfactory areas. Characterisation of the binding indicated that it was saturable, of high affinity, stereoselective and displaced by structurally related amino acids. The results support the existence of two glycine receptor subtypes: strychnine-sensitive and strychnine-insensitive.     

Effect of the convulsive agent 3-mercaptopropionic acid on the levels of GABA, other amino acids and glutamate decarboxylase in different regions of the rat brain

Intraperitoneal injection of 3-mercaptopropionic acid into rats caused severe convulsions which started after about 7 min. Of the amino acids examined only the level of GABA changed after 4 min and immediately before (6.5–7 min) the convulsions started. The decrease in GABA concentration detected immediately before the onset of convulsions was about 35 per cent in the cerebral cortex, corpus striatum and cerebellum, 30 per cent in pons-medulla and 20% in hippocampus. Concomitant with the fall in GABA there was a large, reversible inhibition of glutamate decarboxylase activity in the brain. The uptake of GABA into synaptosomes isolated after injection of the convulsive agent was not reduced, and the uptake of GABA into synaptosomes was not inhibited by high concentrations of 3-mercaptopropionic acid added in vitro. During convulsions levels of aspartate and taurine decreased significantly in all the brain regions investigated. A small increase in glutamine was detected in pons-medulla and in cerebellum. Major changes in the concentrations of other amino acids such as glutamate, alanine, serine and glycine were found only in corpus striatum.     

Binding of selective antagonists to four muscarinic receptors (M1 to M4) in rat forebrain

To compare the proportions of four muscarinic receptors in different rat brain regions, we used competition curves with four selective antagonists, at 1-[N-methyl-3H]scopolamine methyl chloride [( 3H]NMS) binding equilibrium and after allowing [3H]NMS dissociation for 35 min. Himbacine and methoctramine were shown to discriminate two muscarinic receptor subtypes having a high affinity for 4-diphenylacetoxy-N-methylpiperidine methiodide and hexahydrosiladifenidol, intermediate affinity for pirenzepine, and low affinity for AF-DX 116. One M4 subtype had a high affinity for himbacine and methoctramine; it was found predominantly in homogenates from rat striatum (46% of total [3H]NMS receptors) and in lower proportion in cortex (33% of [3H]NMS receptors) and hippocampus (16% of [3H]NMS receptors). Its binding properties were identical to those of muscarinic receptors in the neuroblastoma x glioma NG 108-15 hybrid, suggesting that it was encoded by m4 mRNA. The M3 subtype (typically found in rat pancreas, a tissue expressing the m3 mRNA) had a low affinity for himbacine and methoctramine and represented about 10% of all [3H]NMS receptors in rat brain cortex, hippocampus, striatum, and cerebellum. M1 and M2 receptors were identified in rat brain by their high affinity for pirenzepine and AF-DX 116, respectively.     

Effect of the combination of the benzodiazepine tranquilizer medazepam and the nootropic agent meclofenoxate on the activity of rat brain muscarinic receptors

1. The effect of 7-day treatment with the benzodiazepine tranquilizer medazepam (5 mg/kg), the nootropic agent meclofenoxate (100 mg/kg) and their combination in the same doses on the binding activity of muscarinic receptors in four rat brain structures (cerebral cortex, striatum, hippocampus and hypothalamus) were studied using the antagonist [3H]-1-quinuclidinyl benzylate [( 3H]-QNB) as radio-ligand. 2. Medazepam treatment caused significant decrease of muscarinic receptor binding affinity (Kd) and of the receptor binding capacity (Bmax) in the brain structures studied. The number of muscarinic binding sites was unsignificantly decreased only in the hippocampus. 3. Meclofenoxate treatment caused an increase of muscarinic receptor affinity and a decrease of the binding capacity in the cerebral cortex and hypothalamus and an increase of the binding affinity in the striatum and hippocampus. 4. The combination of medazepam and meclofenoxate caused no significant changes of both muscarinic receptor characteristics in the hippocampus and of the receptor affinity in the striatum and hypothalamus in comparison with control rats. The Bmax values were decreased in the cerebral cortex, striatum and hypothalamus when compared with control animals. The differences observed were slighter than those determined after the comparison of medazepam treated rats with control rats. 5. The results obtained afford an opportunity to suggest that the nootropic agent meclofenoxate acts to moderate the effect of the benzodiazepine tranquilizer medazepam on the activity of rat brain muscarinic receptors.     

Diversity of thyrotropin-releasing hormone receptors in the pituitary and discrete brain regions of rats

In order to analyze the receptor properties of central nervous system (CNS)-stimulant thyrotropin-releasing hormone (L-pyroglutamyl-L-histidyl-L-prolinamide, TRH), we evaluated the binding of TRH and its analog taltirelin hydrate ((-)-N-[(S)-hexahydro-1-methyl-2,6-dioxo-4-pyrimidinylcarbonyl]-L- histidyl-L-prolinamide tetrahydrate; taltirelin, TA-0910) in rat anterior pituitary and several brain regions. There was a specific binding of [3H]methyl TRH (MeTRH) in the anterior pituitary, hypothalamus, brain stem, cerebral cortex and cerebellum with Kd values of 1.0-1.6 nM. The inhibition of [3H]MeTRH binding by TRH and taltirelin was monophasic in the anterior pituitary, hypothalamus and brain stem with Ki values of 6.3-8.0 nM and 145.5-170.4 nM for TRH and taltirelin, respectively. In contrast, the biphasic inhibition was revealed in the cerebral cortex and cerebellum. The Ki values for TRH and taltirelin were 4.1-4.3 nM and 67.8-73.4 nM for the high affinity binding site and 3.6-4.2 microM and 82.3-197.5 microM for the low affinity binding site, respectively. Addition of 100 microM GTP or its analog 5'-guanylylimidodiphosphate (Gpp[NH]p) affected neither the biphasic inhibition by TRH nor that by taltirelin. Thus the results suggest the presence of distinct high and low affinity TRH receptors in the CNS in contrast to the pituitary.     

Down regulation of dihydroalprenolol and imipramine binding sites in brain of rats repeatedly treated with imipramine

In rats receiving repeated injections of imipramine, there is a reduction in the number of high affinity binding sites for [3H]imipramine and [3H]dihydroalprenolol present in crude synaptic membrane preparations from various brain structures. The location of the sites that become subsensitive to the two ligands did not coincide; the binding sites to [3H]imipramine became subsensitive in the hippocampus but not in cortex or cerebellum. In contrast the binding sites to [3H]dihydroalprenolol became subsensitive in cortex and cerebellum but not in hippocampus. It can be suggested that in rats repeatedly treated with imipramine the down regulation of beta-adrenergic receptors may not coincide with the down regulation of the high affinity binding sites for imipramine. Such a dissociation is supported further by experiments with rats treated with iprinidol.     

Time and brain region specific up-regulation of low affinity neuronal nicotinic receptors during chronic nicotine administration in mice

We studied the effects of chronic oral nicotine on brain low affinity nicotine binding sites. Mice received nicotine in the drinking water for 4 or 7 weeks. Receptor binding was measured at 24 or 48 h after cessation of nicotine administration with [3H]methyllycaconitine, an antagonist in alpha7 and alpha3/alpha6beta2beta3* nicotinic receptors in striatum, midbrain, hippocampus and cortex. Chronic nicotine for 4 weeks resulted in a significant increase in the [3H]methyllycaconitine binding in the striatum and cortex, whereas after 7 weeks the increase in binding could be found in the hippocampus but not in the other brain areas studied. For comparison, high affinity nicotine binding sites (mostly alpha4beta2) were measured with [3H]epibatidine after 7-week chronic nicotine treatment. [3H]Epibatidine binding sites were increased in the hippocampus, midbrain and cortex, but not in the striatum. The up-regulation of [3H]methyllycaconitine binding was significant at 24 h but that of [3H]epibatidine binding sites was not observed until at 48 h after cessation of chronic nicotine. These results suggest that up-regulation of low affinity nicotine binding sites does occur during chronic nicotine administration. Furthermore, the low affinity and high affinity binding differ clearly as regards regions and duration suggesting that different nicotinic receptors respond differently to nicotine administration.     

Characteristics of type 1 and type 2 benzodiazepine receptors in the ovine brain

Analysis of the displacement of 3H-diazepam binding to membranes prepared from the ovine frontal cortex by the triazolopyradiazine CL218,872 yielded a Hill coefficient significantly below unity. By analogy with similar studies of this drug in rat brain this suggested the existence of Types 1 and 2 benzodiazepine receptors. The degree of displacement of 3H-diazepam by CL218,872 (200 nM, Type 1; 800 nM, Type 2) in homogenates of brain regions differed, the rank order being cerebellum greater than parietal cortex greater than frontal cortex congruent to temporal cortex congruent to hippocampus greater than striatum. Displacement of 3H-diazepam by CL218,872 was enhanced by 10(-5) M GABA in the striatum (at 200 nM and 800 nM CL218,872) and cerebellum (at 200 nM CL218,872). Benzodiazepine receptors in the ovine frontal cortex were least sensitive to CL218,872 (200 nM) in young fetuses (54-68 days gestation) and achieved adult levels of sensitivity by late gestation. Finally, the potency of CL218,872 to displace 3H-diazepam was not effected by the 3H-ligand concentration (0.5 nM or 5.0 nM), suggesting that Types 1 and 2 benzodiazepine receptors are not identical to the high and low affinity 3H-diazepam binding sites we have previously identified in the ovine brain.     

Autoradiographic localization of high-affinity [3H]kainic acid binding sites in the rat forebrain

Utilizing in vitro autoradiographic techniques, we have studied the distribution of high affinity [3H]kainic acid ([3H]KA) binding sites in intact sections of the rat forebrain. These sites have the same kinetic and pharmacological characteristics as the [3H]KA site described in tissue homogenates. Moderate to high levels of specific binding were observed in several discrete brain regions. These include lamina I, V and VI of the neo- and cingulate cortex, superficial layers of the pyriform cortex, striatum, external plexiform and granule cell layers of the olfactory bulb, olfactory tubercle, the stratum lucidum of CA3 of the hippocampus, molecular layer of the dentate gyrus, reticular nucleus of the thalamus, the hypothalamic median eminence, and the granule cell layer of the cerebellum. Low levels of specific binding were associated with other discrete regions such as the lateral septum, bed nucleus of the stria terminalis, medial geniculate, superficial layers of the superior colliculus, nuclei of the central grey, interpeduncular nucleus and the molecular layer of the cerebellum. Moderate uniform levels of specific binding were observed over the hypothalamus, zona incerta and the amygdala. One of the important factors in KA neurotoxicity seems to be the presence of KA receptors, and regions that are susceptible to the toxic effects of KA after local administration, such as the striatum, hippocampus, amygdala and pyriform cortex, have moderate to high levels of binding. Thus, these data provide a useful map for studying the relationship between receptor-mediated and seizure-induced neuronal damage following KA administration.     

Chemotherapeutic choice of ranimustine or nimustine on the basis of regional polyamine levels in rat brain

The aim of the present study was to improve the chemotherapeutic efficacy of anticancer drugs by choosing on the basis of the polyamine level induced by the drug in each host cancer-bearing tissue. We propose an "organ-specific therapy" in the article. The polyamines, putrescine, spermidine and spermine are strongly associated with tumor cell growth. The effects of ranimustine (MCNU) and nimustine (ACNU) on body weight, regional brain weights and concentrations of putrescine, spermidine and spermine in the cerebellum, hippocampus, corpus striatum, cortex, combined thalamus and hypothalamus and diencephalon of the brain were examined in rats. MCNU and ACNU reduced spermidine and spermine in the corpus striatum, and spermine in the diencephalon, but increased putrescine in the corpus striatum and combined thalamus and hypothalamus. These results indicate that both MCNU and ACNU are suitable for the treatment of cancers of the corpus striatum, but ACNU is not suitable for cancers of the corpus striatum, thalamus and hypothalamus.