Rat Brain Muscarinic Receptors Research Articles

Agonist-mediated conformational changes of muscarinic receptors in rat brain.

Muscarinic acetylcholine receptors were identified in the microsomal P fraction of rat forebrain by the specific binding of the radiolabeled antagonist [3H]dexetimide. Binding occurred to a single class of noncooperative sites (3.25 mumol/mg protein) with an equilibrium dissociation constant of 1.1 nM. Agonist/[3H]-dexetimide competition binding experiments allowed the distinction between two major muscarinic receptor subpopulations, having respectively high affinity (20% of the total receptor population) and low affinity for agonists, but with the same affinity for antagonists. A 610-fold difference in affinity was calculated for carbamoyl-choline, the agonist extensively investigated in this study. The alkylating reagent N-ethylmaleimide did not affect the total receptor number, antagonist binding to the high-affinity and low-affinity sites, nor agonist binding to the high-affinity sites. The reagent, however, caused a net increase in agonist affinity for the low-affinity sites. This process was dependent on time and dose of N-ethylmaleimide, until a maximal increase in affinity (fourfold increase for carbamoylcholine) was attained. This suggests a quantal conversion of the low-affinity sites by the reagent into an alkylated form, which possesses a higher affinity for agonists but an unchanged affinity for antagonists. The rate of alkylation was enhanced by the presence of agonists but not of antagonists, which is indicative for the ability of agonists to mediate a conformational change of these sites. The close correlation between the N-ethylmaleimide-mediated increase in drug affinity for the low-affinity sites and the ability of the drugs to enhance alkylation of these sites by N-ethylmaleimide can be explained by the ability of (a) muscarinic drugs to interact with the low-affinity sites according to the Monod-Wyman-Changeux 'Plausible Model' and (b) N-ethylmaleimide to freeze these sites in the 'active' conformation by alkylation.

(+/-)-4-Aryl-4,5-dihydro-3H-1,3-benzodiazepines. 1. Synthesis and evaluation of (+/-)-4,5-dihydro-2,3-dimethyl-4-phenyl-3H-1,3-benzodiazepine and analogues as potential antidepressant agents.

A series of (+/-)-4,5-dihydro-4-phenyl-3H-1,3-benzodiazepines and (+/-)-4,5-dihydro-4-phenyl-1H-1,3-benzodiazepines was synthesized as part of a program to develop novel psychotropics. Of these compounds, (+/-)-4,5-dihydro-2,3-dimethyl-4-phenyl-3H-1,3-benzodiazepine (10a, HRP 543) emerged as a potential antidepressant. In in vivo mouse tests (inhibition of tetrabenazine-induced ptosis and potentiation of yohimbine toxicity) which are predictive of antidepressant-like activity, 10a is comparable to amitriptyline. The similarity is also maintained in vitro, as both 10a and amitriptyline inhibit norepinephrine and serotonin uptake into rat brain synaptosomes. No significant inhibition of rat brain monoamine oxidase A or B was found with 10a, nor did the compound potentiate tryptamine-induced seizures. On chronic administration, the number of cortical beta-adrenergic receptor sites was similarly reduced by 10a and desipramine. The anticholinergic activity of clinically useful antidepressants, such as amitriptyline, is a proposed cause of side effects which reduce patient compliance. In contrast to the tricyclics, 10a apparently lacks anticholinergic activity, as evidenced in vitro by negligible displacement of [3H]quinuclidinyl benzylate from rat brain muscarinic receptors and in vivo by insignificant antagonism of the cholinergic stimulation produced by physostigmine or oxotremorine. These data suggest that 10a may be clinically useful as a novel nontricyclic antidepressant which is devoid of anticholinergic side-effect liability. Further evaluation of 10a in nonrodent species is in progress.

Antibodies to muscarinic acetylcholine receptors in myasthenia gravis

IgG obtained from patients with myasthenia gravis block the specific binding of the muscarinic antagonists ( 3H)-N-methyl-4-piperidyl benzilate (4NMPB) and ( 3H)-Quinuclidinyl benzilate to rat brain muscarinic acetylcholine receptors. IgG obtained from healthy controls have a much smaller effect. The inhibitory effect of the myasthenic IgG increases linearly with immunoglobulin concentration and has no effect on the affinity of the muscarinic receptors towards ( 3H)-4NMPB (K D = 0.7 ± 0.1 nM). This implies that the inhibition is probably due to the blocking of some of the muscarinic receptors by the myasthenic IgG, and not due to alteration in affinity of all the receptors. it remains to be established whether the presence of antimuscarinic receptor activity in the serum of myasthenic patients is of importance in the pathophysiology and diagnosis of myasthenia gravis.

Acute extrapyramidal side effects: serum levels of neuroleptics and anticholinergics.

An assay technique for measuring anticholinergic drugs in human serum based upon their inhibition of the specific binding of [3H]-quinuclidinyl benzilate to rat brain muscarinic receptors is described. The assay was validated by demonstrating a close correlation (r = 0.99) between serum levels of nortriptyline measured by the radioenzymatic assay and a GLC technique. The assay measures free anticholinergics, and under standard assay conditions, approximately 95% of benztropine is bound to serum protein. Marked variation in serum anticholinergic levels in patients receiving the same oral dose was observed, and in individual patients there was a non-linear relationship between increasing oral dose and serum anticholinergic levels. In a cross-sectional study of 109 patients receiving concurrently neuroleptics and anticholinergics, there was no correlation (r = 0.029) between serum neuroleptic levels measured by radioreceptor assay and extrapyramidal side effects (EPS). In the patients whose serum anticholinergic levels were also determined, there was a significant inverse correlation (r = 0.44) between anticholinergic levels of EPS. In this cohort of patients, there was no significant correlation between serum anticholinergic and serum neuroleptic levels (r = 0.16) and the ratio of serum anticholinergic to serum neuroleptic was a poor predictor of EPS (r = 0.26). The results suggest a marked variation in sensitivity of patients to the EPS-inducing of neuroleptics; nevertheless, the incidence of EPS decreases with increasing serum levels of anticholinergics. An optimal serum anticholinergic level of 10 pmole atropine equivalent per ml was associated with a low incidence of EPS and is relevant to drug action at the striatal muscarinic receptor.

In vivo modulation of the number of muscarinic receptors in rat brain by cholinergic ligands

Administration of the muscarinic agonist oxotremorine led to a decrease in the number of muscarinic receptors, as determined by specific binding of [ 3H]quinuclidinyl benzilate ([ 3H]QNB), in several rat brain regions both during development and at maturity. In contrast, administration of the muscarinic antagonist scopolamine led to an increase in the number of [ 3H]QNB-binding sites in various brain regions. Scopolamine also prevented the decrease in the number of [ 3H]QNB-binding sites induced by administration of an organophosphorus drug. The results are compatible with the hypothesis that the number of brain muscarinic receptors, or at least of a sub-class of them, is regulated by their transmitter.

Quinuclidinyl benzilate binding in house fly heads and rat brain.

House fly heads contain a binding site for 3-quinuclidinyl benzilate (QNB) that is quite similar in pharmacology to the muscarinic acetylcholine receptor of vertebrate tissues. The house fly site binds [3H]QNB reversibly with a Kd of 260 pM and Bmax of 1 pmol/g of heads from direct binding measurements. The Kd calculated from the ratio of the dissociation rate constant (2 x 10(-4)sec-1) to the association rate constant (2.5 x 10(6) M-1sec-1) was 80 pM. The house fly site binds (-)quinuclidinyl benzilate preferentially, as do classic muscarinic receptors. The binding is also sensitive to other muscarinic antagonists and agonists. Nicotinic and other drugs are no more effective on the house fly site than they are on the rat brain muscarinic receptor itself. These binding studies suggest that the house fly QNB binding site is a muscarinic receptor.

Methylmercury inhibition of rat brain muscarinic receptors

The distribution of methylmercury (MeHg) in subcellular organelles of the rat brain varied slightly in relation to the in vivo or the in vitro mode of labeling with Me 203HgCl. The differences may be explained by disruption of the normal diffusion path into the brain cells and the amount of MeHg that is bound along this diffusion path. When Me 203Hg and [ 3H]QNB (quinuclidinyl benzilate) were added directly to brain homogenates, the subcellular fractions demonstrated a molar rmQNB MeHg binding ratio greater than 1:100. This suggests that MeHg binding sites are in considerable excess over QNB binding sites and represent a relatively large sink for decreasing effective MeHg concentration. HgCl 2 was 100 times as potent as MeHg for blocking specific QNB binding sites. (MeHg, ID50 = 10 −5 m; HgCl 2, ID50 = 10 −7 m) On the basis of the respective ID50 points, a 1% conversion of MeHg to the inorganic form would produce a separate but equivalent block of muscarinic binding sites. Therefore, it is suggested that the early theory involving organic to inorganic Hg conversion be reexamined as a possibility for producing the selective toxicity to the central nervous system observed in cases of MeHg poisoning.

Aging and rat brain muscarinic receptors as measured by quinuclidinyl benzilate binding.

Measurement of cholinergic muscarinic receptor binding in various rat brain areas using the ligand [3H]quinuclidinyl benzilate indicates that receptor binding is decreased in striatum and cerebellum of aged female rats (22 months old) as compared to younger rats (4 months old). Decreases were not observed in cortex, hippocampus, hypothalamus, or amygdala areas. Further examination o [3H]quinuclidinyl benzilate binding in subcellular fractions of aged and young rat cerebellum and striatum indicated a decrease in binding in the crude nuclear and crude synaptosomal fractions. Binding data indicate the observed decrease in specific ligand binding is due to a decrease in number of binding sites while receptor affinity does not appear to change.

Ontogeny of muscarinic receptors in rat brain

The ontogeny of muscarinic acetylcholine receptors in the rat brain has been examined using the radioligards, [ 3H]N-methylscopolamine, [ 3H]propylbenzilylcholine and [ 3H]oxotremorine-M. In the 3 regions of the brain selected for study, the cerebral cortex, the diencephalon and the medulla-pons, the receptors develop at different rates. The most rapid development takes place in the medulla with considerably slower maturation in the diencephalon and cerebral cortex. In the cortex, the agonist binding properties of the muscarinic receptors vary during development. There appears to be a 6–7 day lag in the appearance of high affinity sites following formation of low affinity sites.

The effects of ions on the binding of agonists and antagonists to muscarinic receptors.

1 There are no selective effects of Na+, K+, Ca2+, Mg2+ or Cl- on the binding of antagonists or agonists to muscarinic receptors in rat brain. A decrease in affinity related to ionic strength is found for all these ions. 2 Larger effects were produced by T1+, La3+, and some transition metal ions.

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.

On the ability of choline and its analogues to interact with muscarinic cholinergic receptors in the rat brain

Choline displaced [ 3H]QNB binding from rat brain muscarinic receptors competitively, (K i = 460 μM) but it was only 1/1000th as potent as ACh. Deanol was an extremely weak displacer of [ 3H]QNB binding while hemicholinium-3 was 50 times more potent than choline. Although brain levels of choline are well below its K i value for muscarinic receptors, choline may directly interact with rat brain muscarinic receptors in some circumstances.

Effects of alkanols and halothane on rat brain muscarinic and α-adrenergic receptors

Effects of the primary alcohols ethanol, butanol, pentanol and of halothane were measured on the binding functions of muscarinic and α-adrenergic receptor preparatoins in rat brain homogenates, with the use of the antagonists 3H-quinuclidinyl benzilate and 3H-WB-4101. IC 50 concentrations of the alkanols for the muscarinic and α-receptors respectively were: ethanol, 2.0 M and 1.4 M; butanol, 0.24 M and 0.16 M; heptanol, 3.7 × 10 −3 M and 2.6 × 10 −3. The plot of IC 50 values versus number of carbon atoms in the alkanol was linear and of the same slope as the plot of membrane fluidity changes, thus indicating the importance of the membrane/water partition coefficient of the alkanol. Halothane at clinical concenrations had no effect on the receptors, although significant inhibitino of radioligand binding was produced by 2.5 M halothane, and inhibition was complete in presence of 17.5 mM anesthetic. From the correlation of receptor binding inhibitions with membrane fluidity changes reported by other workers, it is suggested that the activity of membrane receptors may be modulated by the fluidity of their membranes.

Effect of acute and chronic treatment of tandamine, a new heterocyclic antidepressant, on biogenic amine metabolism and related activities.

The effects of tandamine, a clinically effective heterocyclic antidepressant, administered either acutely (10 mg/kg i.p.) or chronically (10 mg/kg i.p. daily for 21 days) on biogenic amine uptake and metabolism in the rat were determined and a comparison with desipramine was made. Tandamine, similarly to desipramine, blocked norepinephrine (NE) uptake in rat brain and heart following both acute and chronic administration. No effect of tandamine on dopamine (DA) or serotonin (5-HT) uptake was observed. Both drugs lowered endogenous brain NE when given chronically but not acutely. In contrast, no such effect on brain DA and 5-HT or heart NE was observed. Tandamine, like desipramine, administered chronically prior to an intraventricular injection of 3H-NE, produced increases in the decline of 3H-NE as indicated by decreased 3H-NE with increased levels of 3H-normetanephrine in brain stem of rats, suggesting an increased turnover of NE. No such effect was observed following acute treatment. Both drugs increased the behavioural effects of L-Dopa following and acute oral administration, with tandamine appearing superior to desipramine at the lower dose examined (10 mg/kg). Tandamine was 57--833 times less effective in binding to rat brain muscarinic receptors than desipramine, imipramine, butriptyline and amitriptyline, respectively. Thus, tandamine affects biogenic amine mechanism following either acute or chronic administration in a fashion similar to desipramine, but unlike desipramine, it exhibits relatively little anticholinergic properties, a further indication of the potential use of tandamine in the treatment of human depression, particularly where an increase in drive is desired.

Stereospecific in vitro binding of [3H]dexetimide to brain muscarinic receptors.

Abstract—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.

Muscarinic cholinergic receptor binding in bovine retina

Using [ 3H] quinuclidinyl benzylate ([ 3H] QNB) muscarinic cholinergic receptors have been demonstrated in crude membrane fractions of bovine retina. Specific [ 3H] QNB binding is saturable with a K D of 0.5 nM and a maximal number of muscarinic agonists and antagonists for displacing specific [ 3H] QNB binding closely parallel the affinities for muscarinic receptors in rat brain and guinea pig ileum. The findings may explain atropine sensitive effects of muscarinic agonists on the electroretinogram and on retinal cells in vitro.

Localization of cholinergic muscarinic receptors in rat brain by light microscopic radioautography

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.

Light autoradiographic localisation of cholinergic muscarinic receptors in rat brain by specific binding of a potent antagonist

THE presence of cholinergic muscarinic receptors on neurones in many regions of the mammalian brain is known from classical electrophysiological studies, which showed that neuronal firing rates are altered by the microiontophoretic application of cholinergic muscarinic agonists and antagonists1. It is now possible to measure these receptors directly by biochemical procedures, some of which involve studying the responses of the cyclic guanosine monophosphate system in neuronal tissue to agonists and antagonists2–5. Other procedures utilised radiolabelled muscarinic agonists and antagonists that bind specifically and directly to presumed receptor sites6–12. One of these agonists13,14, 3-quinuclidinyl-benzilate (QNB) binds in a radiolabelled form to apparent muscarinic receptors in vitro6,7. Recent experiments indicate that it is also possible to demonstrate specific 3H-QNB binding to cholinergic muscarinic receptors in rat brain in vivo soon after intravenous administration15. Because of this latter factor, we have been able to examine the in vivo localisation of 3H-QNB in regions of rat brains by light microscopic autoradiography. We have found receptor sites associated with various groups of cells, some of which are known to be cholinoceptive. In general, the greatest densities of 3H-QNB sites were in telencephalic regions.