Changes in brain muscarinic acetylcholine receptors and behavioral responses to atropine and apomorphine in chronic atropine-treated rats

Chronic blockade of cholinergic transmission with atropine resulted in a decrease in atropine-induced activity in the rats, whereas apomorphine - induced locomotion was enhanced. Maximal binding of ³H-quinuclidinyl benzilate (QNB), a muscarinic antagonist, to homogenate of cerebral cortex, striatum and hippocampus was significantly higher in chronic atropine-treated rats than in control animals. No difference was observed in K_D value of the specific ³H-QNB binding or in ID₅₀ value of oxotremorine in inhibiting ³H-QNB binding. No change in the specific binding of ³H-spiroperidol, a dopaminergic antagonist, was observed in those three regions of brains of chronic atropine-treated rats when it was compared with that of control animals. The role of brain muscarinic acetylcholine receptors in behavioral responses is discussed relating an effect of dopaminergic neurons on cholinergic activities.     

Regulation of putative muscarinic cholinergic receptor subtypes in rat brain

Transection of the fimbria/fornix, producing a 75% reduction in the activity of the cholinergic marker choline-o-acetyltransferase (CAT EC. 2.3.1.6) in rat hippocampus, did not change the binding characteristics of the non-subtype selective, muscarinic cholinergic receptor antagonist ligand [³H](−)quinuclidinyl benzilate {[³H](−)QNB}. Pirenzepine competition for [³H](−)QNB binding in the hippocampus was best described by a computer derived model assuming two binding sites of high affinity (putative M₁ receptors) and low affinity (putative M₂ receptors). There was no change in the proportion of high and low affinity pirenzepine binding sites in the hippocampus following cholinergic deafferentation. Thus, these data provide no evidence for a discrete localization of either putative subtype of muscarinic receptor discriminated by pirenzepine restricted to the terminals of CAT containing neurons innervating the rat hippocampus.Chronic scopolamine treatment produced a 48% increase in the B_max of [³H](−)QNB binding in the hippocampus, but again there was no change in the proportions of the sites discriminated by pirenzepine demonstrating that both putative subtypes were regulated identically. Similarly, carbachol competition for [³H](−)QNB was unaltered following cholinergic deafferentation or chronic scopolamine treatment. Furthermore, similar guanylyl-5′-imidodiphosphate [Gpp(NH)p] modulation of the proportions of high and low affinity carbachol binding sites was found in the hippocampus following transection of the fimbria/fornix or chronic scopolamine treatment. Thus there is no adaptation of receptor-effector coupling following these treatments that is reflected by changes in receptor recognition site characteristics.Carbachol competition for [³H]pirenzepine binding to putative M₁ receptors in the hippocampus was biphasic and was also modulated by Gpp(NH)p. In the brainstem, there was a homogeneous population of putative M₂ [³H](−)QNB binding sites having low affinity for pirenzepine. Carbachol competition for these binding sites was also biphasic and modulated by guanine nucleotides. Thus, both putative M₁ and M₂ muscarinic receptors, as defined by high or low affinity for pirenzepine respectively, may mediate their effects in rat brain via a guanine nucleotide regulatory subunit.     

STEREOSPECIFIC IN VlTRO BINDING OF [3H]DEXETIMIDE TO BRAIN MUSCARINIC RECEPTORS

A binding assay for muscarinic cholinergic receptors has been developed using labelled dexetimide as ligand and a filtration technique. The main features of this assay are its stereospecific nature, the very high affinity of the ligand for the specific receptors sitcs and its very low affinity for non-specific binding sites. The latter point was further investigated using labelled levitimide, the inactive enantiomer. The binding was found to be neither stereospecific nor saturable and displacement by both enantiomers revealed a particular curve with a very flattened course. Kinetic experiments with [³H]dexetimide suggest the occurrence of a heterogenous population of muscarinic receptors in the rat striatum. A study of the regional distribution of muscarinic receptors in rat brain showed a high concentration in the dopaminergic areas, the cortex and the hippocampus, but practically none in the cerebellum. The subcellular distribution pattern revealed a marked enrichment of [³H]dexetimide stereospecific binding sites in the microsomal fraction of rat striatum and hippocampus. Such a distribution was not found with [³H]levitimide. All the characteristics of this binding assay make dexetimide a very appropriate ligand for labelling muscarinic receptors in vitro as well as in vivo.     

Autoradiography of Serotonin 5-HT1A Receptor-Activated G Proteins in Guinea Pig Brain Sections by Agonist-Stimulated [35S]GTPγS Binding

Abstract: G protein activation mediated by serotonin 5-HT_1A and 5-HT_1B/D receptors in guinea pig brain was investigated by using quantitative autoradiography of agonist-stimulated [³⁵S]GTPγS binding to brain sections. [³⁵S]GTPγS binding was stimulated by the mixed 5-HT_1A/5-HT_1B/D agonist L694247 in brain structures enriched in 5-HT_1A binding sites, i.e., hippocampus (+140 ± 14%), dorsal raphe (+70 ± 8%), lateral septum (+52 ± 12%), cingulate (+36 ± 8%), and entorhinal cortex (+34 ± 5%). L694247 caused little or no stimulation of [³⁵S]GTPγS binding in brain regions with high densities of 5-HT_1B/D binding sites (e.g., substantia nigra, striatum, central gray, and dorsal subiculum). The [³⁵S]GTPγS binding response was antagonized by WAY100635 (10 µM) and methiothepin (10 µM). In contrast, the 5-HT_1B inverse agonist SB224289 (10 µM) did not affect the L694247-mediated [³⁵S]GTPγS binding response, and the mixed 5-HT_1B/D antagonist GR127935 (10 µM) yielded a partial blockade. The distribution pattern of the [³⁵S]GTPγS binding response and the antagonist profile suggest the L694247-mediated response in guinea pig brain to be mediated by 5-HT_1A receptors. In addition to L694247, 8-hydroxy-2-(di-n-propylamino)tetralin, and flesinoxan also stimulated [³⁵S]GTPγS binding; their maximal responses varied between 46 and 52% compared with L694247, irrespective of the brain structure being considered. Sumatriptan, rizatriptan, and zolmitriptan (10 µM) stimulated [³⁵S]GTPγS binding in the hippocampus by 20–50%. Naratriptan, CP122638, and dihydroergotamine stimulated [³⁵S]GTPγS binding to a similar level as L694247 in hippocampus, lateral septum, and dorsal raphe. It appears that under the present experimental conditions, G protein activation through 5-HT_1A but not 5-HT_1B/D receptors can be measured in guinea pig brain sections.     

Effects of 2α-DHET and its optical isomers on muscarinic and nicotinic receptors

The agent 2α-(2′, 2′-disubstituted-2′-hydroxy-ethoxy) tropane (2α-DHET), its optical isomers and atropine were compared in their ability to inhibit specific [³H]QNB binding to muscarinic receptors of guinea pig ileum and to antagonize oxotremorine- and nicotine—induced contractions of isolated guinea pig ileum. A good correlation was observed between the affinities to muscarinic receptors and the antimuscarinic potencies in isolated guinea pig ileum. The binding data for 2α-DHET and its isomers were also consistent with their central and peripheral pharmacological activity in vivo. Compounds with 2′R configuration are more suitable to the stereostruture of the binding sites of muscarinic receptors than that of 2′S configuration.     

Specific [3H]quinuclidinyl benzilate binding activity in Digitonin-solubilized preparations from bovine brain

Receptors for the specific muscarinic radioligand [³H]quinuclidinyl benzilate ([³H]QNB) were solubilized by digitonin from a particulate preparation of bovine brain without significant alteration in binding affinities for muscarinic antagonists. Electron microscopy and sucrose density gradient sedimentation analysis confirmed the solubility of these receptors in aqueous solutions of digitonin. Equilibrium and kinetic studies of [³H]QNB binding to solubilized receptors indicated that binding was stereoselective and was blocked by muscarinic compounds. These tests permit tentative identification of digitonin-solubilized [³H]QNB binding sites as muscarinic acetylcholine receptors. Digitonin-solubilized receptors were homogeneous with respect to sedimentation behavior and binding affinities for agonist and antagonist drugs, unlike membrane-bound receptors. Enzyme digestion studies and treatment with group-specific reagents indicated that muscarinic receptors are proteins whose binding activity could be disrupted by reduction with dithiothreitol or by modification of sulfhydryl residues.     

Use of ex vivo binding to measure the brain concentrations of putative radioligands

The development of radioligands capable of imaging brain receptors depends on, amongst other factors, the ability of such compounds to penetrate the blood-brain barrier. We describe an ex vivo binding technique for measuring the brain concentration of peripherally administered unlabeled compounds. This technique can be used early in the development of putative radioligands. The pharmacokinetics of brain penetration of three muscarinic antagonists are described: QNB, BrQNB and the 2-thienyl derivative of BrQNB and were found to compare favorably to previous studies using [³H]QNB. These studies demonstrate the effectiveness of ex vivo binding in assessing the brain concentration of peripherally administered unlabeled compounds.     

Muscarinic cholinergic binding in human erythrocyte membranes

Human erythrocyte ghosts contain a small population of muscarinic cholinergic receptors, as evidenced by their high affinity binding of radiolabeled quinuclinidinyl benzilate ([³H]QNB). The apparent K_D is 1.3 × 10^?9 M and the receptor sites are saturated at a QNB concentration of 5 nM. The number of sites is 23 fmoles/mg membrane protein. The pharmacological profile of the specific binding is similar to that of neural membranes. The binding is not stereoselective for the d and 1 isomers of QNB, a situation which prevails in the muscarinic receptors of another peripheral cholinergic system, the rat iris, but not in the central nervous system.     

Effect of long-term L-dopa administration on the dopaminergic and cholinergic (muscarinic) receptors of striatum in 6-hydroxydopamine lesioned rats

L-Dihydroxyphenylalanine (L-Dopa) (200 mg/kg/day) was administered for 30 days to the rats whose nigrostriatal dopamine pathway was lesioned unilaterally with 6-hydroxydopamine and the receptor binding of ³H-spiperone and ³H-quinuclidinyl benzilate (³HQNB) was measured in the dopaminergic and muscarinic cholinergic receptors of the striatum. ³H-spiperone binding increased by 73% and ³HQNB binding decreased by 14% in the lesioned side when compared to the control side of L-Dopa-non-treated rats. ³H-spiperone binding was measured in the lesioned sides of L-Dopa-treated and L-Dopa-non-treated rats and was found to have decreased by 21% in the former. In the control side of the L-Dopa-treated lesioned rats, however, ³H-spiperone binding increased by 27% when compared to the opposite striatum of the same rats. ³HQNB binding in the lesioned side of L-Dopa-treated rats was not significantly different from that of the control side statistically. These results suggest that changes in functional equilibrium between the dopaminergic and cholinergic mechanisms influence the muscarinic cholinergic receptors and that supersensitivity of dopamine receptors after lesion of the nigrostriatal pathway also remains after long-term L-Dopa treatment.     

Characterization of N-[3H]Methylcarbamylcholine Binding Sites and Effect of N-Methylcarbamylcholine on Acetylcholine Release in Rat Brain

The present experiments show that N-[3H]-methylcarbamylcholine ([3H]MCC) binds specifically and with high affinity to rat hippocampus, frontal cortex, and striatum. The highest maximal density of binding sites was apparent in frontal cortex and the lowest in hippocampus. [3H]MCC binding was potently inhibited by nicotinic, but not muscarinic, agonists and by the nicotinic antagonist dihydro-beta-erythroidine in all three brain regions studied. The effect of unlabeled MCC on acetylcholine (ACh) release from slices of rat brain was tested. The drug significantly enhanced spontaneous ACh release from slices of hippocampus and frontal cortex, but not from striatal slices. This effect of MCC to increase ACh release from rat hippocampus and frontal cortex was antagonized by the nicotinic antagonists dihydro-beta-erythroidine and d-tubocurarine, but not by alpha-bungarotoxin or by the muscarinic antagonist atropine. The MCC-induced increase in spontaneous ACh release from hippocampal and frontal cortical slices was not affected by tetrodotoxin. The results suggest that MCC might alter cholinergic transmission in rat brain by a direct activation of presynaptic nicotinic receptors on the cholinergic terminals. That this alteration of ACh release is apparent in hippocampus and frontal cortex, but not in striatum, suggests that there may be a regional specificity in the regulation of ACh by nicotinic receptors in rat brain.     

A comparison of the muscarinic cholinoceptors and benzodiazepine receptors of guinea-pig brain and rat brain

Some properties of muscarinic cholinoceptors and benzodiazepine receptors in selected brain regions of guinea-pigs and rats were compared under identical experimental conditions. The regions investigated were striatum, hippocampus and pons-medulla, and the properties examined were the concentrations of receptors; apparent dissociation constants of the ligands [³H]quinuclidinyl benzilate (for muscarinic receptors) and [³H]flunitrazepam (for benzodiazepine receptors); Hill coefficients for the interactions of the antagonist atropine and the agonist acetylcholine with the muscarinic receptors; the affinities of these compounds for the muscarinic receptors; and the effects of chronic administration of an organophosphate cholinesterase inhibitor (di-isopropylfluorophosphate) on the concentrations of receptors. Rat striatal and hippocampal muscarinic receptors were found to have a slightly higher affinity for acetylcholine than the corresponding guinea-pig receptors. Administration of di-isopropylfluorophosphate reduced the concentration of muscarinic receptors in rat brain by 30%, but had no significant effect on the concentration of receptors in guinea-pig brain. In all other aspects, the properties of the brain receptors of the two species were very similar. For both species, the affinities of the muscarinic receptors for acetylcholine were higher in the pons-medulla than in the striatum and hippocampus. This was found to be the result of differences in the values of the association constants of the high- and low-affinity states of the receptors, rather than because of varying proportions of two states which have the same association constant in all regions.The insensitivity of guinea-pig brain muscarinic receptors to chronic administration of an organophosphate confirms the results of a previous study on the guinea-pig alone, and makes this system unique. Many other studies on various species have all indicated that prolonged activation of a receptor by an agonist (caused in the present work by inactivation of acetyl-cholinesterase) leads to a decrease in the concentration of the receptor.     

Analysis of copper in brain by the mass-spectrometric integrated-ioncurrent procedure

Through use of the high-resolution double-focusing mass spectrometer, copper has been identified in various regions of the mouse, rat, guinea pig, rabbit, and human brain. The procedure depends on converting the copper (in ashed tissue) to its chloride salt, followed by derivatization with tetraphenylporphyrin (TPP) to yield a TPP chelate. After chromatographic separation, this chelate is assessed in the mass spectrometer by the integrated-ion-current procedure. Deuterated metal TPP chelates and the rare stable isotope⁶⁵Cu were used as internal standards. Whole brain values obtained were as follows: mouse, 6.67±0.16 (mean±SEM) g/g wet weight of tissue; rat, 1.06±0.05; guinea pig, 5.40±0.63; and rabbit, 7.52±0.76. In the rat, the cerebellum contained the highest concentration (1.25 g/g), and the striatum the lowest (0.70 g/g). In the human brain, the cortex (gray) and the striatum were relatively the highest copper-containing regions, with the cerebellum (white) being the lowest.     

Effect of mequitazine a non sedative antihistamine on brain H1 receptors

Marked species variations occured in the relative activity of mequitazine (10-[3-quinuclidinylmethyl]-phenothiazine) on the binding of [³H]mepyramine to brain cortical membranes. Mequitazine was as potent as promethazine in the mouse but about 6 times less effective than promethazine in the guinea pig and human. On the other hand in the guinea pig mequitazine was as potent as promethazine on [³H]mepyramine binding in a peripheral organ (lung). Although mequitazine did not displace [³H]mepyramine in vivo in the mouse and guinea pig, its brain concentration (measured by [³H]mequitazine) was largely sufficient and corresponds to 90% of inhibition in vitro. Moreover in the mouse the brain regional distribution of [³H]mequitazine was very different from that of [³H]mepyramine, highest level was obtained in the cerebellum and hypothalamus was the poorest region with mequitazine whereas the reverse was true with mepyramine. All these results could suggest that mequitazine possesses a greater affinity for peripheral H₁ receptors which could explain the absence of sedative side-effects of this potent H₁ antagonist.     

Changes in Nicotinic and Muscarinic Cholinergic Receptors in Alzheimer-Type Dementia

Nicotinic and muscarinic cholinergic receptors were studied in autopsied brains from four histologically normal controls and five histopathologically verified cases of Alzheimer-type dementia (ATD), using ligand binding techniques. Nicotinic and muscarinic cholinergic receptors were assessed by (-)-[3H]nicotine and [3H]quinuclidinyl benzilate [( 3H]QNB), respectively. Compared with the controls, (-)-[3H]nicotine binding sites in the ATD brain regions examined were significantly reduced in the putamen and the nucleus basalis of Meynert (NbM). [3H]QNB binding was significantly reduced in the hippocampus and NbM. These findings suggest that there are significant changes of nicotinic and muscarinic cholinergic receptors in selected regions of ATD brains.     

Reduced binding of (3H)-quinuclidinyl benzilate associated with chronically low acetylcholinesterase activity.

(³H)-Quinuclidinyl benzilate (QNB) binding was examined in the cortex, striatum and hippocampus of rats repeatedly exposed to the anticholinesterase, diisopropyl fluorophosphate (DFP). Compared to vehicle-treated controls, a reduction in maximal binding of 25–30% was observed in these brain regions. The reduction in binding was associated with regional acetylcholinesterase (AChE) inhibition of 80–90%. A tendency of lower muscarinic acetylcholine receptor (m-AChR) affinity for (³H)-QNB in control preparations, compared to those from DFP-treated rats, was observed. However, only in the case of the striatal control homogenate was there a significant increase in apparent K_D. A concomitant feature of chronically low AChE activity is therefore a reduction, mainly in number, of m-AChR's. These findings support the hypothesis that in the central nervous system (CNS) DFP tolerance and cholinomimetic subsensitivity may involve the m-AChR.     

Enhanced muscarinic M1 receptor gene expression in the corpus striatum of streptozotocin-induced diabetic rats

Acetylcholine (ACh), the first neurotransmitter to be identified, regulate the activities of central and peripheral functions through interactions with muscarinic receptors. Changes in muscarinic acetylcholine receptor (mAChR) have been implicated in the pathophysiology of many major diseases of the central nervous system (CNS). Previous reports from our laboratory on streptozotocin (STZ) induced diabetic rats showed down regulation of muscarinic M1 receptors in the brainstem, hypothalamus, cerebral cortex and pancreatic islets. In this study, we have investigated the changes of acetylcholine esterase (AChE) enzyme activity, total muscarinic and muscarinic M1 receptor binding and gene expression in the corpus striatum of STZ – diabetic rats and the insulin treated diabetic rats. The striatum, a neuronal nucleus intimately involved in motor behaviour, is one of the brain regions with the highest acetylcholine content. ACh has complex and clinically important actions in the striatum that are mediated predominantly by muscarinic receptors. We observed that insulin treatment brought back the decreased maximal velocity (V_max) of acetylcholine esterase in the corpus striatum during diabetes to near control state. In diabetic rats there was a decrease in maximal number (B_max) and affinity (K_d) of total muscarinic receptors whereas muscarinic M1 receptors were increased with decrease in affinity in diabetic rats. We observed that, in all cases, the binding parameters were reversed to near control by the treatment of diabetic rats with insulin. Real-time PCR experiment confirmed the increase in muscarinic M1 receptor gene expression and a similar reversal with insulin treatment. These results suggest the diabetes-induced changes of the cholinergic activity in the corpus striatum and the regulatory role of insulin on binding parameters and gene expression of total and muscarinic M1 receptors.     

[3H]Propyl β-Carboline-3-Carboxylate as a Selective Radioligand for the BZ1 Benzodiazepine Receptor Subclass

Abstract: Ethyl β-carboline-β-carboxylate (β-CCE) is a mixed-type inhibitor of [³H]flunitrazepam ([³H]FNM) binding to benzodiazepine receptors in noncerebellar regions of rat brain. These findings may represent the presence of either receptor multiplicity or negative cooperativity among benzodiazepine receptors. [³H]Propyl β-carboline-3-carboxylate ([³H]PrCC) has previously been shown to bind specifically to benzodiazepine receptors of rat cerebellum. In the present study we found no indication of the presence of true negative cooperativity among benzodiazepine receptors when [³H]PrCC was used as radioligand. However, we observed that [³H]PrCC labelled only 57% of [³H]FNM binding sites in rat hippocampus (B_max values) and 71% in rat cerebral cortex, whereas the number of receptors labelled by both ligands was equal in the cerebellum. Hofstee analyses of the shallow inhibition curves seen in hippocampus and cerebral cortex when [³H]FNM binding was inhibited by β-CCE indicate that β-CCE and some other β-carboline-3-carboxylate derivatives interact preferentially with a subclass of receptors, and that the percentage of this subclass is equivalent to the number of receptors labelled by [³H]PrCC. We conclude that [³H]PrCC at low concentration (0.3–0.4 × 10^-9 M) labels a subclass of benzodiazepine receptors, BZ₁, while another class, BZ₂ receptors, are not labelled by [³H]PrCC when filtration assays are used. By parallel determinations of the proportion between [³H]FNM and [³H]PrCC binding we calculated the percentage of BZ₁ receptors in several regions of rat, guinea pig and calf brain and in mouse forebrain. The values ranged from approximately 50% in hippocampus to 90% in the guinea pig pons.     

Alterations in central neurotransmitter receptor binding sites following estradiol implantation in female rats

The subcutaneous implantation of an estradiol pellet (10 mg) into female rats induced a hypophyseal hyperplasia with hyperprolactinaemia. Examination of neurotransmitter receptors in the hippocampus, striatum and cerebral cortex one month after the implantation revealed that estrogenization was associated with: an increased density of ³H-domperidone binding sites (D₂ receptors) in the striatum and reduced numbers of ³H-serotonin high affinity sites (5-HT₁ receptors) in the hippocampus and of ³H-muscimol binding sites (GABA receptors) in the hippocampus, striatum and cerebral cortex. In contrast, the characteristics of ³H-spiperone binding to 5-HT₂ receptors (in the cerebral cortex) and those of ³H-flunitrazepam binding to benzodiazepine sites (in the three brain regions examined) were not significantly different in estrogenized and in control female rats. However, the enhancing effect of GABA on ³H-flunitrazepam binding was markedly reduced in brain membranes from estrogenized animals. The respective roles of estradiol and prolactin in mediating these changes in neurotransmitter receptors are discussed notably with regard to the regional heterogeneity of estradiol binding capacity in the rat brain.     

Effect of aging on the interaction of quinuclidinyl benzilate,N-methylscopolamine,pirenzepine, and gallamine with brain muscarinic receptors

The objective of the present study was to investigate the effects of senescence on the binding characteristics of muscarinic receptors by using [³H]quinuclidinyl benzilate ([³H]QNB) and [³H]N-methylscopolamine ([³H]NMS) as ligands in young (3months), middle-age (10months) and old (24 months) male Fischer 344 rats. Muscarinic receptor density was found to decrease significantly with aging in certain brain regions, depending on the ligand employed. Moreover, the relative proportions of M₁ and M₂ muscarinic receptor subtypes was not significantly altered by aging, except in the aged striatum. Furthermore, the dissociation kinetics of [³H]NMS in the cerebral cortex and their allosteric modulation by gallamine were only slightly influenced by age.