Rat Brain Muscarinic Receptors Research Articles

P.5.a.001 Novel cholinesterase inhibitors 1-PC-37 and 1-PC-48 in the treatment of 3-quinuclidinyl benzilate-induced behavioural deficit in rats

Injection of a potent, cholinergic muscarinic antagonist, [3H]3-quinuclidinyl benzilate ([3H]QNB), results in a localization of the drug to muscarinic receptors in rat brain. The distribution of these drug receptors was examined in various brain regions previously thought to contain cholinergic neurons. They were localized to dendritic regions in the hippocampus, corpus striatum, nucleus accumbens and cerebral cortex. Particularly in the hippocampys, the receptor distribution may correspond to that for cholinergic nerve terminals.

Effects of chlorine substituents on the benzene rings of an analogue of the antipsychotic drug butaclamol on the interaction with dopamine and muscarinic receptors in rat brain.

Effects of chlorine substituents on the benzene rings of an analogue of the antipsychotic drug butaclamol on the interaction with dopamine and muscarinic receptors in rat brain.

Noninvasive evaluation of brain muscarinic receptor occupancy of oxybutynin, darifenacin and imidafenacin in rats by positron emission tomography

Aims The current study was conducted to evaluate, by the noninvasive positron emission tomography (PET), the binding of antimuscarinic agents used to treat overactive bladder (OAB) to muscarinic receptors in rat brain. Main methods Muscarinic receptor occupancy in the rat brain after the intravenous (i.v.) injection of oxybutynin, darifenacin and imidafenacin was evaluated by using a small animal PET system, and compared with the results by ex vivo autoradiographic and ex vivo radioligand binding experiments. Key findings In PET study, the i.v. injection of oxybutynin but not darifenacin or imidafenacin at pharmacological doses decreased significantly binding potential (BP) of (+) N-[ 11C]methyl-3-piperidyl benzilate ([ 11C](+)3-MPB) in the rat cerebral cortex and corpus striatum in a dose-dependent manner. Similarly, in the in vivo autoradiographic experiment, oxybutynin dose-dependently reduced binding of [ 11C](+)3-MPB in the brain, whereas darifenacin and imidafenacin did not. Following the i.v. injection of oxybutynin, darifenacin and imidafenacin, there was a similar degree of binding to muscarinic receptors in the bladder as demonstrated by a significant increase in apparent dissociation constant ( K d ) values for specific [N-methyl- 3H]scopolamine methyl chloride ([ 3H]NMS) binding. Significant binding of muscarinic receptors in the brain was observed after the injection of oxybutynin but not darifenacin or imidafenacin. Significance Oxybutynin but not darifenacin or imidafenacin has potential side effects on the central nervous system (CNS) in patients with OAB. The results reveal the noninvasive characterization of brain receptor occupancy by PET to be a powerful tool for precise evaluation of adverse CNS effects of antimuscarinic agents in pre-clinical and clinical evaluations.

Flaws in the Serum Anticholinergic Activity Assay: Implications for the Study of Delirium

Flaws in the Serum Anticholinergic Activity Assay: Implications for the Study of Delirium

Effects of Typical and Atypical Antipsychotic Drugs on Rat Brain Muscarinic Receptors

Quantitative in vitro autoradiography was used to examine changes in muscarinic M1/M4 and M2/M4 receptors (targeted with [3H]pirenzepine and [3H]AF-DX384 respectively), in rats treated with the typical (haloperidol) and atypical (clozapine and olanzapine) antipsychotic medications for a period of 36 days. Rats were sacrificed at either 2 h or 48 h after the last drug administration to examine immediate effects as well as the effects at 48 h after drug withdrawal. Haloperidol significantly increased [3H]pirenzepine binding in the dentate gyrus (37%) and in the CA1 region of the hippocampus (34%) in animals sacrificed 2 h after the last drug administration compared to controls. Similarly, clozapine significantly increased [3H]pirenzepine binding in dentate gyrus (29%) in rats sacrificed 2 h after the last drug administration compared to controls. Haloperidol decreased [3H]AF-DX384 binding in the basolateral nucleus of the amygdala (20%) in the rats sacrificed 48 h after the last drug administration compared to controls. These findings suggest that muscarinic receptors and limbic brain regions such as hippocampus and amygdala might represent common targets that mediate beneficial clinical effects of antipsychotic drugs.

Methylmercury interaction with lymphocyte cholinergic muscarinic receptors in developing rats

Cerebral cholinergic muscarinic receptors (MR) have been suggested as one of the sensitive biochemical endpoints of the central nervous system altered by developmental exposure to the widespread seafood contaminant methylmercury (MeHg). In adult rats, MeHg has been shown to alter MR binding both in the brain and lymphocytes, supporting the use of MR in blood cells as a surrogate marker of CNS changes. The effects of MeHg have been evaluated on rat lymphocyte MR binding (using [ 3H]QNB as specific muscarinic ligand) in vivo (after perinatal exposure) and in vitro. For comparison, in vitro studies were also performed on human lymphocytes. Exposure to 1 mg MeHg/kg/day during pregnancy and lactation (from GD7 to PND7) significantly enhanced lymphocyte MR density in both adult and young rats 21 days after delivery, with a more pronounced effect in the mothers ( B max increase of 139%) than in the male offspring (+49%) and female offspring (+73%) as compared with their respective controls (33±4, 41±8, and 37±4 fmol/million cells), in accordance with the higher Hg levels detected in the adult blood (11.3±2.2 μg/mL) than in pups (1.3±0.4 μg/L in both genders). A lower MeHg dose (0.5 mg/kg/day) was without any effect on lymphocyte MRs. In in vitro studies, MeHg was an almost equipotent inhibitor of 3H-QNB binding to rat and human lymphocyte MRs (IC 50 values were 4.1±0.29, 5.2±0.51, and 5.0±0.9 μM for total rat lymphocytes, rat T lymphocytes, and total human lymphocytes, respectively). Notably, the IC 50 values for MeHg to lymphocyte MRs were comparable to the Hg levels reached in blood (5–50 μM) of the PND21 rats exposed to MeHg. The finding that the MR binding is a target for the effects of MeHg in peripheral blood cells is in accordance with our previous data in brain [Coccini et al., 2006. Effects of developmental co-exposure to methylmercury and 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB153) on cholinergic muscarinic receptors in rat brain. Neurotoxicology, in press], and supports the use of this peripheral endpoint as a biomarker of MeHg-induced cerebral muscarinic alterations. The similarity of MeHg IC 50 binding data between human and rat in peripheral tissues suggests the possible application of such biomarker to humans exposed to environmental chemicals.

Effects of developmental co-exposure to methylmercury and 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB153) on cholinergic muscarinic receptors in rat brain

The developing nervous system is thought to be particularly sensitive to polychlorinated biphenyls (PCBs) present as food contaminants together with methylmercury (MeHg). Effects of perinatal co-exposure to PCB153 and MeHg on brain cholinergic muscarinic receptors (MRs) were investigated by saturation binding studies in mature and immature rats. MeHg alone (1 mg/kg/day, GD7–PND7) enhanced cerebral MRs more in dams (87% and 60% in cerebellum and cerebral cortex, respectively) than in PND21 pups (0–50%) in accordance with the higher Hg levels detected in the adult brain (7–9 μg/g) than in the male and female offspring's brain (1.5–2.8 μg/g). Prenatal administration of PCB153 (20 mg/kg/day, GD10–GD16), leading to higher contaminant levels in the offspring brain than in that of adults (25–66 μg/g versus 3 μg/g), induced cerebral MR changes of similar extent at both ages, namely decreased cerebellar (20–30%) and increased cortical MR density (40–50%). Co-exposure to PCB and MeHg had no more effect than exposure to either compound alone on cerebral cortex MRs, whereas, in the cerebellum, the combined treatment induced a PCB-like lowering of the MR density that masked the MeHg-induced receptor increase. None of the treatments affected the striatal and hippocampal MRs. A lower MeHg dose (0.5 mg/kg/day) was without any effect on cerebral MRs. These results show that MRs are one of the sensitive biochemical endpoints of the central nervous system altered by developmental exposure to MeHg and PCB153. Cerebral cortex and cerebellum were the most susceptible targets in the response to these neurotoxicants. MR changes were detected in both immature and adult animals and the interaction of MeHg and PCB153 at the level of these receptors occurred in a non-additive manner.

Central Muscarinic Receptor Subtypes Regulating Voiding in Rats

Muscarinic receptors are distributed widely in the brain. A recent study revealed that central muscarinic receptors are involved in voiding regulation. However, to our knowledge the role of each muscarinic receptor subtype has not been resolved. Therefore, we evaluated the effect of intracerebroventricular administration of selective muscarinic M1 to M4 receptor antagonists on voiding function in rats. Female Sprague-Dawley rats were cannulated for intracerebroventricular infusion under halothane anesthesia. In experiment 1 cystometry was performed in conscious rats, and BC and maximal voiding pressure were measured. In experiment 2 a catheter was inserted via the bladder dome to the bladder neck and UPP was measured by saline infusion. Repeat cystostomy was performed, and saline infusion and discharge saline, BC, maximal IVP and minimal UPP were measured in conscious rats. Pirenzepine, methoctramine, pFHHSiD and MT-3 were used as selective M1, M2, M3 and M4 muscarinic receptor antagonists, respectively, which were injected intracerebroventricularly. In experiment 1 pirenzepine and pFHHSiD increased BC and decreased maximal voiding pressure. Methoctramine and MT-3 decreased BC. In experiment 2 pirenzepine and pFHHSiD increased BC and minimal UPP, and decreased maximal IVP. Methoctramine and MT-3 decreased BC and maximal IVP. Minimal UPP remained unchanged. Intracerebroventricular administration of muscarinic M1 and M3 receptor antagonists inhibited urination in conscious rats, while M2 and M4 receptor antagonists induced excitatory changes.

The effects of repeated oral exposures to methyl parathion on rat brain cholinesterase and muscarinic receptors during postnatal development.

Dimethyl phosphorylated cholinesterase (ChE) is known to be more rapidly reactivated, spontaneously, and have a higher aging rate than diethyl phosphorylated ChE. This may result in differences in toxic signs and tolerance development after treatment of juvenile rats with methyl parathion (MPS), a dimethyl phosphorothionate, than after treatment with chlorpyrifos (CPS), a diethyl phosphorothionate. The effects of repeated MPS exposures on brain ChE activity and surface and total muscarinic acetylcholine receptor (mAChR) density were studied in postnatal rats gavaged daily from postnatal day 1 (PND1) through PND 21. Results of this study were compared to our recent report with CPS (Tang et al., 1999, Toxicol. Sci. 51, 265-272). Rats received MPS daily starting at 0.3 mg/kg and increasing gradually to 0.6 mg/kg (for the medium-dosage groups) and then to 0.9 mg/kg (for the high-dosage group). ChE activity was assayed in brain homogenates. Synaptosomal mAChR densities, surface, and total were assayed using 3H-N-methylscopolamine (NMS) and 3H-quinuclidinyl benzilate (QNB), respectively, as ligands. Developmental increases in brain ChE activities and mAChR densities were observed from PND 6 through PND 22. On PND 22, inhibition of ChE activity was observed in the low (26%)-, medium (42%)-, and high (55%)-dosage groups. Significant inhibition was still present on PND 30 (16-24%) and PND 40 (12-14%), which were 9 and 19 days after the last treatment, respectively. Densities of 3H-NMS and 3H-QNB binding sites in treated groups were significantly reduced by PND 22, 1 day following cessation of treatment, and were significantly increased during the recovery period. After MPS exposure, the initial recovery of phosphorylated ChE was more rapid and the density of 3H-NMS binding sites was less readily reduced than following CPS exposure. The lesser effects on surface mAChR may explain why more severe signs appeared after each treatment with the high dosage of MPS than were observed previously with CPS, indicating little or no tolerance had developed to MPS.

Deficits in avoidance responding after paradoxical sleep deprivation are not associated with altered [ 3H]pirenzepine binding to M1 muscarinic receptors in rat brain

Previous work had indicated that animals that were sleep-deprived and then trained on a passive avoidance task show poor retention of the task 24 h later after being allowed to sleep freely again. Cholinergic involvement is suggested by the fact that this effect is prevented by treatment with the muscarinic agonist pilocarpine during sleep deprivation. The observation that similar deficits are observed in non-deprived rats after treatment with M1-selective antagonist compounds such as dicyclomine or pirenzepine cause similar impairments, and gave rise to the hypothesis that sleep deprivation might induce significant reductions in M1 binding in brain areas involved in learning and memory processes. Rats were deprived of sleep for 96 h and then either immediately killed, or allowed to recover sleep for 24 h before being killed. [ 3H]pirenzepine binding to M1 sites was examined by quantitative autoradiography in 39 different brain areas in cage controls, sleep-deprived and sleep-recovered animals ( N=8 per group). No significant differences among groups were found in any brain region. A separate group of animals was subjected to the sleep deprivation procedure and then trained in a simple avoidance task. Animals were then allowed to sleep and retested 24 h later. This group showed a significant impairment in the avoidance task compared to cage controls, in agreement with previous observations. These data suggest that proactive learning/memory deficits induced by sleep deprivation cannot be attributed to altered M1 binding either immediately after deprivation (when avoidance training occurs) or after sleep has recovered (when acquisition/retention are tested). The possibility remains that alterations in M1 function occur at post-membrane second messenger systems.

O-Substituted derivatives of pralidoxime: muscarinic properties and protection against soman effects in rats

O-Substituted aldoximes of the cholinesterase reactivator pralidoxime ( O-methyl 1, O-benzyl 2, O-propynyl 3 and O-butynyl 4 derivatives) were synthesized and found to exhibit strong binding affinities for muscarinic receptors in rat brain, heart and submandibulary glands. The aldoximes were noncompetitive antagonists of acetylcholine-induced contraction of the guinea pig ileum. A good correlation was observed between binding affinity and p K B. Weak anticholinesterase activities were observed for these compounds. When given intracerebroventricularly into conscious rats before soman administration (0.9 LD 50, subcutaneously), the aldoximes, like atropine but not pralidoxime, protected against respiratory depression ( 3, 4) and bradycardia ( 2). No protection against soman-induced pressor effects was noted. The protective effects of these aldoximes may be the outcome of compensatory mechanisms, of which the cholinergic receptor agonist and antagonist properties of these compounds may be important.

Interaction of a new potent anticholinesterasic compound (±)huprine X with muscarinic receptors in rat brain

The interaction of rac-12-amine-3-clor-6,7,10,11-tetrahydro-9-ethyl-7-11-methanecyclo-octane[ b]quinoline ((±)huprine X) with M 1 and M 2 receptors has been studied in rat brain. Specific binding of [ 3H]pirenzepine or [ 3H]quinuclinidylbenzylate to hippocampus preparations was inhibited by (±)huprine X. This drug displayed a greater affinity for M 1 ( K i =0.338±0.41 μM) than M 2 ( K i =4.66±0.32 μM) receptors. In functional studies, (±)huprine X (1 μM) increased the release of [ 3H]dopamine in cortical synaptosomes, and this effect was partially reverted by atropine and mecamylamine, suggesting an agonistic effect on both M 1 and nicotinic receptors. The inhibitory effect of (±)huprine X (10 μM) on [ 3H]acetylcholine release and the subsequent reversion by atropine suggests that the drug also has an agonist effect on M 2 receptors. The present results demonstrate that this acetylcholinesterase inhibitor has an ample cholinergic profile, which suggests a potential source of interest of (±)huprine X in Alzheimer's disease therapy.

Differential inhibition of [3H]-oxotremorine-M and [3H]-quinuclinidyl benzilate binding to muscarinic receptors in rat brain membranes with acetylcholinesterase inhibitors.

The potential interaction of acetylcholinesterase inhibitors with cholinergic receptors may play a significant role in the therapeutic and/or side-effects associated with this class of compound. In the present study, the capacity of acetylcholinesterase inhibitors to interact with muscarinic receptors was assessed by their ability to displace both [3H]-oxotremorine-M and [3H]-quinuclinidyl benzilate binding in rat brain membranes. The [3H]-quinuclinidyl benzilate/[3H]-oxotremorine-M affinity ratios permitted predictions to be made of either the antagonist or agonist properties of the different compounds. A series of compounds, representative of the principal classes of acetylcholinesterase inhibitors, displaced [3H]-oxotremorine-M binding with high-to-moderate potency (ambenonium>neostigmine=pyridostigmine=tacrine>physostigmine> edrophonium=galanthamine>desoxypeganine) whereas only ambenonium and tacrine displaced [3H]-quinuclinidyl benzilate binding. Inhibitors such as desoxypeganine, parathion and gramine demonstrated negligible inhibition of the binding of both radioligands. Scatchard plots constructed from the inhibition of [3H]-oxotremorine-M binding in the absence and presence of different inhibitors showed an unaltered Bmax and a reduced affinity constant, indicative of potential competitive or allosteric mechanisms. The capacity of acetylcholinesterase inhibitors, with the exception of tacrine and ambenonium, to displace bound [3H]-oxotremorine-M in preference to [3H]quinuclinidyl benzilate predicts that the former compounds could act as potential agonists at muscarinic receptors. Moreover, the rank order for potency in inhibiting acetylcholinesterase (ambenonium>neostigmine=physostigmine =tacrine>pyridostigmine=edrophonium=galanthamine >desoxypeganine>parathion>gramine) indicated that the most effective inhibitors of acetylcholinesterase also displaced [3H]-oxotremorine-M to the greatest extent. The capacity of these inhibitors to displace [3H]-oxotremorine-M binding preclude their utilisation for the prevention of acetylcholine catabolism in rat brain membranes, the latter being required to estimate the binding of acetylcholine to [3H]-oxotremorine-M-labelled muscarinic receptors. However, fasciculin-2, a potent peptide inhibitor of acetylcholinesterase (IC50 24 nM), did prevent catabolism of acetylcholine in rat brain membranes with an atypical inhibition isotherm of [3H]-oxotremorine-M binding, thus permitting an estimation of the "global affinity" of acetylcholine (Ki 85 nM) for [3H]-oxotremorine-M-labelled muscarinic receptors in rat brain.

Design and development of selective muscarinic agonists for the treatment of Alzheimer's disease: characterization of tetrahydropyrimidine derivatives and development of new approaches for improved affinity and selectivity for M1 receptors

Cholinergic neurons degenerate in Alzheimer's disease, resulting in cognitive impairments and memory deficits, and drug development efforts have focused on selective M1 muscarinic agonists. 5-(3-Ethyl-1,2,4-oxadiazol-5-yl)-1,4,5,6-tetrahydropyrimidine trifluoroacetic acid (CDD-0102) stimulates M1 muscarinic receptors in rat brain [Messer, W.S., Jr., Abuh, Y.F., Liu, Y., Periyasamy, S., Ngur, D.O., Edgar, M.A., El-Assadi, A.A., Sbeih, S., Dunbar, P.G., Roknich, S., Rho, T., Fang, Z., Ojo, B., Zhang, H., Huzl, J.J., III, Nagy, P.I., 1997a. J. Med. Chem. 40, 1230–1246.] and improves memory function in rats with lesions of the basal forebrain cholinergic system. Moreover, CDD-0102 exhibits oral bioavailability, few side effects and low toxicity, and thus represents a viable candidate for clinical studies. Despite the development of functionally selective agonists such as xanomeline and CDD-0102, there is room for improvements in ligand affinity and selectivity. The high degree of amino acid homology within transmembrane domains has hindered the development of truly selective agonists. Site-directed mutagenesis, biochemical and molecular modeling studies have identified key amino acid residues such as Thr192 and Asn382 in the binding of agonists to M1 receptors [Huang, X.P., Nagy, P.I., Williams, F.E., Peseckis, S.M., Messer, W.S., Jr., 1999. Br. J. Pharmacol. 126, 735–745.]. Recent work has implicated residues at the top of transmembrane domain VI in the binding of muscarinic agonists and activation of M1 receptors [Huang, X.P., Williams, F.E., Peseckis, S.M., Messer, W.S., Jr., 1998. J. Pharmacol. Exp. Ther. 286, 1129–1139.]. Thus, residues such as Ser388 represent molecular targets for the further development of agonists with improved M1 receptor affinity, selectivity and activity.

The in vivo effects of olanzapine and other antipsychotic agents on receptor occupancy and antagonism of dopamine D1, D2, D3, 5HT2A and muscarinic receptors.

The atypical antipsychotic olanzapine was compared to other atypical as well as typical antipsychotic agents for in vivo occupancy of D1, D2, D3, 5HT2, and muscarinic receptors in rat brain. Blockade of D2 receptors was determined by measuring the levels of the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). To assess the interaction with phosphoinositide (PI)-coupled 5HT2A and muscarinic receptors in vivo, we used a novel radiometric technique to measure in vivo PI hydrolysis. The antagonism of olanzapine and other antipsychotic agents on 5HT2A and muscarinic receptors was determined by in vivo blockade of PI hydrolysis, stimulated by the 5HT2 agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) or the muscarinic agonist pilocarpine. Olanzapine inhibited 5HT2, D2, and D3 in vivo binding with high potency (ID50=0.15, 0.6 and 1.2 mg/kg, IP, respectively), while inhibiting D1 and muscarinic in vivo binding with much less potency (ID50 > 10 mg/kg, IP). The binding of olanzapine to D2 receptors in neostriatum was well correlated with the increase of DOPAC (ED200 = 0.8 mg/kg, IP) in vivo, indicating dopamine D2 antagonism. In vivo PI hydrolysis was increased by DOI in frontal cortex and by pilocarpine in hippocampus up to 2- and 7-fold above the basal level, respectively. The agonist-induced increases in PI hydrolysis were fully blocked by the 5HT2A antagonist MDL100907 and the muscarinic antagonist scopolamine, indicating the mediation by 5HT2A receptors in frontal cortex and PI-coupled muscarinic receptors (ml, m3, and m5) in hippocampus, respectively. Olanzapine was about 8-fold more potent in vivo in blocking DOI-induced stimulation of PI hydrolysis (ID50 = 0.1 mg/kg, IP) than pilocarpine-induced stimulation of PI hydrolysis (ID50 = 0.8 mg/kg, IP). In conclusion, olanzapine is more potent in blocking the 5HT2A receptor than D1, D2, D3 and muscarinic receptors in vivo, consistent with its favorable clinical profiles. In addition, the novel in vivo PI hydrolysis assay proved to be a useful and reliable in vivo method to assess the functional efficacy of compounds that interact with the 5HT2 and muscarinic receptors.

Localization of M 1 Muscarinic Receptors in Rat Brain Using Selective Muscarinic Toxin-1

Mambas, African snakes of the genus Dendroaspis, produce several types of toxins that are of pharmacological interest. The novel muscarinic toxin-1 (MT-1), from the green mamba Dendroaspis angusticeps, binds specifically to muscarinic M 1 receptors in homogenates of rat cerebral cortex. Iodination of the toxin, 125I-muscarinic toxin-1 ( 125I-MT-1), renders the toxin selective for M 1 muscarinic receptors. Quantitative measurement of 125I-MT-1 autoradiography in rat brain sections indicated highest labeling in the nucleus accumbens, striatum, and dentate gyrus. High densities of 125I-MT-1 binding sites were located in the CA1 region of the hippocampus, frontal, and parietal cortices. Moderate densities of binding sites were seen in temporal cortex, and hippocampal subregions CA2, CA3, and CA4, whereas low labeling was observed in the cerebellum and spinal cord.

Synthesis and biochemical activity of novel amidine derivatives as m1 muscarinic receptor agonists

As part of a continuing effort aimed at the development of selective, efficacious, and centrally active m1 muscarinic agonists for the treatment of Alzheimer's disease, a series of amide and hydrazide amidine derivatives ( 2a–e and 3b–d) was synthesized and examined for muscarinic agonist activity. Preliminary biochemical studies indicated that 2b, 2d, and 3d bound to muscarinic receptors in rat brain and stimulated phosphoinositide (PI) metabolism in rat cerebral cortex. Compounds 2b and 2d were also highly efficacious at m1 muscarinic receptors expressed in cultured A9 L cells. Molecular modeling studies suggest slightly different modes of interaction with m1 receptors for the ester and amide derivatives. Also, hydrogen-bond formation with a Thr residue may be important for m1 muscarinic agonist potency. The data suggest that the amide moiety can replace the ester group found in muscarinic agonists and provide further support for the utility of amidine derivatives in the development of efficacious m1 agonists.

Specific binding component of the “inactive” stereoisomer (S,S)-[ 125I]IQNB to rat brain muscarinic receptors in vivo

In vivo nonspecific binding can be estimated using the inactive stereoisomer of a receptor radioligand. However, the binding of the inactive stereoisomer may be partially specific. Specific binding of the inactive (S,S)-[ 125I]IQNB was estimated from the inhibition induced by a competing nonradioactive ligand. This technique differed from the usual approach, since it was used to study the inactive rather than the active stereoisomer. The results indicate that there is substantial specific binding for (S,S)-[ 125I]IQNB.

Changes in rat brain muscarinic receptors after inhibitory avoidance learning

It is widely accepted that cerebral acetylcholine is necessary for learning and memory, but little is known about the type of muscarinic receptors involved in these functions. To investigate this problem, [ 3H]-N-methyl-scopolamine which binds to different types of muscarinic receptors, [ 3H]-Pirenzepine an M 1 receptor antagonist, and [ 3H]-Oxotremorine-M which binds mainly to M 2 receptors, were used as ligands to look for possible changes in muscarinic receptor density in neostriatum (NEO), hippocampus (HIP), amygdala (AMY), and temporo-parietal neocortex (CTX), after testing for retention of inhibitory avoidance, trained with high or low footshock intensities. After low reinforcement there was an M 1 postsynaptic receptor up-regulation in NEO, HIP, and CTX, and an M 2 presynaptic receptor down-regulation in HIP, which suggests a concerted pre-and postsynaptic cholinergic activation in this area. An up-regulation of both M 1 and M 2 receptors was detected in CTX of low and high footshocked animals, which indicates the presence of a cortical postsynaptic M 2 receptor.

Purification and sequence determination of a new muscarinic toxin (MT4) from the venom of the green mamba ( Dendroaspis angusticeps)

A toxin which partially inhibited [ 3H]N- methylscopolamine binding to rat brain muscarinic receptors was purified from the venom of green mamba, Dendroaspis angusticeps. The N-terminal sequence (up to 45 amino acids) was determined by automated Edman degradation of the whole molecule. The complete sequence was elucidated after enzymatic cleavage with endoproteinase Arg-C or endoproteinase Lys-C and peptide fragments purification. The identity of the C-terminal amino acid was confirmed by hydrazinolysis. The new toxin (MT4) had eight half-cystines and 66 amino acids. It differed from muscarinic toxin MT1 by a single substitution in position 57 (arginine in MT1, histidine in MT4), proximal to the sixth half-cystine.