Brain Muscarinic Acetylcholine Receptors Research Articles

Rivastigmine improves isolation rearing-induced prepulse inhibition deficits via muscarinic acetylcholine receptors in mice.

The acetylcholinesterase inhibitors donepezil, galantamine, and rivastigmine are used for the treatment of Alzheimer's disease. We previously demonstrated that donepezil and galantamine differentially affect isolation rearing-induced prepulse inhibition (PPI) deficits and that this might be due to differential effects on brain muscarinic acetylcholine (mACh) receptor function in mice. We examined the effects of rivastigmine on isolation rearing-induced PPI deficits, brain ACh levels, and mACh receptor function in mice. Acoustic startle responses were measured in a startle chamber. Microdialysis was performed, and the levels of dopamine and ACh in the prefrontal cortex were measured. Rivastigmine (0.3 mg/kg) improved PPI deficits, and this improvement was antagonized by the mACh receptor antagonist telenzepine but not by the nicotinic ACh receptor antagonist mecamylamine. Rivastigmine increased extracellular ACh levels by approximately 2-3-fold, less than the increase produced by galantamine. Rivastigmine enhanced the effect of the mACh receptor agonist N-desmethylclozapine on prefrontal dopamine release, a marker of mACh receptor function, and this increase was blocked by telenzepine. In contrast, galantamine did not affect N-desmethylclozapine-induced dopamine release. Furthermore, rivastigmine did not affect cortical dopamine release induced by the serotonin1A receptor agonist osemozotan, suggesting that the effect of rivastigmine has specificity for mACh receptors. Taken together with our previous finding that marked increases in ACh levels are required for the PPI deficit improvement induced by galantamine, our present results suggest that rivastigmine improves isolation rearing-induced PPI deficits by increasing ACh levels and by concomitantly enhancing mACh receptor function.

Xanomeline suppresses excessive pro-inflammatory cytokine responses through neural signal-mediated pathways and improves survival in lethal inflammation.

Inflammatory conditions characterized by excessive immune cell activation and cytokine release, are associated with bidirectional immune system-brain communication, underlying sickness behavior and other physiological responses. The vagus nerve has an important role in this communication by conveying sensory information to the brain, and brain-derived immunoregulatory signals that suppress peripheral cytokine levels and inflammation. Brain muscarinic acetylcholine receptor (mAChR)-mediated cholinergic signaling has been implicated in this regulation. However, the possibility of controlling inflammation by peripheral administration of centrally-acting mAChR agonists is unexplored. To provide insight we used the centrally-acting M1 mAChR agonist xanomeline, previously developed in the context of Alzheimer's disease and schizophrenia. Intraperitoneal administration of xanomeline significantly suppressed serum and splenic TNF levels, alleviated sickness behavior, and increased survival during lethal murine endotoxemia. The anti-inflammatory effects of xanomeline were brain mAChR-mediated and required intact vagus nerve and splenic nerve signaling. The anti-inflammatory efficacy of xanomeline was retained for at least 20h, associated with alterations in splenic lymphocyte, and dendritic cell proportions, and decreased splenocyte responsiveness to endotoxin. These results highlight an important role of the M1 mAChR in a neural circuitry to spleen in which brain cholinergic activation lowers peripheral pro-inflammatory cytokines to levels favoring survival. The therapeutic efficacy of xanomeline was also manifested by significantly improved survival in preclinical settings of severe sepsis. These findings are of interest for strategizing novel therapeutic approaches in inflammatory diseases.

Positive allosteric modulation of M1 muscarinic acetylcholine receptors suppresses lethal peripheral inflammation (P5082)

Abstract Peripheral inflammation can be regulated by activation of brain muscarinic acetylcholine receptor (mAChR)-dependent signaling functionally associated with a vagus-nerve mediated anti-inflammatory circuit (Proc Natl Acad Sci USA, 2006, 5219; Brain Behav Immun, 2009, 23:41). Here, we studied the specific role of the M1 mAChR subtype in this regulation by utilizing BQCA, a highly specific allosteric M1 mAChR activator that crosses the blood-brain barrier. Single drug (5,10 or 20 mg/kg, i.p) administration in mice 1h prior to endotoxin (8 mg/kg, i.p.) dose-dependently reduced serum and splenic TNF levels and significantly improved survival in mice as compared to vehicle-treated controls. Pharmacological blockade of brain mAChRs significantly abolished BQCA anti-inflammatory effects. Furthermore, BQCA (20 mg/kg, i.p.) significantly reduced serum and splenic TNF levels in wild type mice, but failed to alter TNF levels in M1 KO mice. Together these results indicate the anti-inflammatory role of increased functional activity of endogenous acetylcholine on the M1 mAChR by selective allosteric receptor activation. Our findings are of interest for further development of BQCA and other centrally-acting allosteric activators of the M1 mAChR as a novel class of experimental anti-inflammatory therapeutics.

Memory defect induced by beta-amyloid plus glutamate receptor agonist is alleviated by catalpol and donepezil through different mechanisms

Our previous studies demonstrate that a non-cholinesterase inhibitor (AChEI) compound catalpol, purified from a traditional Chinese medicinal herb Rehmannia glutinosa, could improve the symptoms and pathological changes in animal and cellular models of memory related neurodegenerative diseases. In this study, we compared catalpol with the most commonly used AChEI donepezil in respect to their mechanism of action on the neurodegenerative changes in an animal model induced by beta-amyloid (Aβ) plus glutamate receptor agonist. It was found that the model mice showed significant deficit in the learning ability and memory in Y maze avoidance test, and meanwhile both donepezil and catalpol greatly improve the learning ability and memory after 2 to 3months' administration. At the selected doses, donepezil only partially raised the declined brain muscarinic acetylcholine receptor (M receptor) density and choline acetyltransferase (ChAT) activity resulting in these levels still lower than normal control, while catalpol completely retrieved these two parameters. ELISA revealed that catalpol, instead of donepezil, possessed the capability of elevating the declined brain BDNF level of the animal model. The ELISA results on the BDNF protein level was confirmed by quantitative RT-PCR measurement of BDNF mRNA in Aβ25–35-treated primary culture of forebrain neurons. In combination with our previous work, we think the neuroprotective effects of donepezil and catalpol are mediated through different mechanisms. Since BDNF has been proved to be an important intrinsic factor in protecting neurodegenerative diseases, catalpol may be a hopefully effective compound against neurodegenerative changes induced by Aβ and glutamate receptor agonist.

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

Brain regional acetylcholinesterase activity and muscarinic acetylcholine receptors in rats after repeated administration of cholinesterase inhibitors and its withdrawal

Activity of acetylcholinesterase (AChE) and specific binding of [(3)H]quinuclidinyl benzilate (QNB), [(3)H]pirenzepine (PZP) and [(3)H]AF-DX 384 to muscarinic acetylcholine receptor (mAChR) preparations in the striatum, hippocampus and cortex of rats were determined 1, 6 and 11 days after the last treatment with an organophosphate DDVP, a carbamate propoxur or a muscarinic agonist oxotremorine as a reference for 7 and 14 days. AChE activity was markedly decreased in the three regions 1 day after the treatment with DDVP for 7 and 14 days with a gradual recovery 6 to 11 days, and much less decreased 1, 6 and 11 days after the treatment with propoxur for 7 days but not for 14 days in the hippocampus and cortex. The binding of [(3)H]-QNB, PZP and AF-DX 384 in the three regions was generally decreased by the treatment with DDVP for 7 and 14 days. Such down-regulations were generally restored 6 or 11 days after the treatment for 7 but not for 14 days. The down-regulation or up-regulation as measured by [(3)H]-QNB, PZP and AF-DX 384 was observed 1, 6 or 11 days after treatment with propoxur for 7 days and/or 14 days. Repeated treatment with oxotremorine produced similar effects except AChE activity to DDVP. These results suggest that repeated inhibition of AChE activity may usually cause down-regulation of mAChRs with some exception in the hippocampus when a reversible antiChE propoxur is injected.

Inactivation of the human brain muscarinic acetylcholine receptor by oxidative damage catalyzed by a low molecular weight endogenous inhibitor from Alzheimer’s brain is prevented by pyrophosphate analogs, bioflavonoids and other antioxidants

Oxidative stress has been implicated as a contributing factor to neurodegeneration in Alzheimer’s disease. An endogenous, low molecular weight (LMW) inhibitor from Alzheimer’s brain inactivates the human brain muscarinic acetylcholine receptor (mAChR). The inhibitor prevents agonist and antagonist binding to the mAChR as assesssed by radioligand binding studies. The LMW endogenous inhibitor, which has components with molecular weights between 100 and 1000 Da, requires dissolved oxygen and glutathione. Prevention of inactivation of the mAChR with peroxidase suggests that the LMW endogenous inhibitor generates peroxide. Heme, previously shown to be present in the LMW endogenous inhibitor, also inactivates the mAChR in the presence of peroxide. Free radical damage to the muscarinic receptor by the endogenous inhibitor can be prevented through the use of naturally occurring antioxidants including bilirubin, biliverdin, carnosol, myricetin and quericetin. In addition, pyrophosphate, imidodiphosphate, bisphosphonates and related compounds also protect the muscarinic receptor from free radical damage. Inactivation of the mAChR by the LMW endogenous inhibitor is likely to be a factor in the continual decline of Alzheimer’s patients, even those taking acetylcholinesterase inhibitors. Natural antioxidants and pyrophosphate analogs may improve the effectiveness of acetylcholinesterase inhibitors and prove useful in the treatment and prevention of Alzheimer’s disease since the muscarinic acetylcholine receptor is required for memory, and decreased cholinergic function is a critical deficit in Alzheimer’s disease.

Anandamides Inhibit Binding to the Muscarinic Acetylcholine Receptor

Loss of memory and cholinergic transmission are associated with both Alzheimer's disease (AD) and marijuana use. The human brain muscarinic acetylcholine receptor (mAChR), which is involved in memory function and is inhibited by arachidonic acid, is also inhibited by anandamides. Two agonists of the cannabinoid receptor derived from arachidonic acid, anandamide (AEA) and R-methanandamide, inhibit ligand binding to the mAChR. Binding of the mAChR antagonist [3H]quinuclidinyl benzilate ([3H]QNB) is inhibited up to 89% by AEA (half-maximal inhibition at 50 microM). Binding of the more polar antagonist [N-methyl-3H]scopolamine ([3H]NMS) is inhibited by AEA up to 76% (half-maximal inhibition at 44 microM). R-methanandamide inhibits more than 90% of both [3H]QNB binding (I50 = 34 microM) and [3H]NMS binding (I50 = 15 microM) to the mAChR. Both AEA and R-methanandamide stimulate mAChR binding of the agonist [3H]oxotremorine-M at low concentrations (25-75 microM), but significantly inhibit agonist binding at higher concentrations (I50 = 150 microM). The cannabinoid antagonist SR141716A did not alter AEA or R-methanandamide inhibition of [3H]NMS binding to the mAChR, even at concentrations as high as 1 microM. Further, the cannabinoid agonist WIN 55212-2 does not alter antagonist binding to the mAChR. This demonstrates that mAChR inhibition by the anandamides is not mediated by the cannabinoid receptor. Since AEA and R-methanandamide are structurally similar to arachidonic acid, they may interact with the mAChR in a similar manner to inhibit receptor function.

Involvement of endogenous nitric oxide signalling system in brain muscarinic acetylcholine receptor activation.

Biochemical signalling events coupled to muscarinic cholinergic receptors (mAChR), specifically those related to nitric oxide (NO) production, were studied on rat cerebral frontal cortex. The mAChR agonist carbachol was found to exert a specific biphasic action on NO synthase (NOS) activity: low doses ranging between 10(-9) M to 10(-7) M lead to NOS activation while higher doses (>10(-6) M) inhibited enzymatic activity. Carbachol stimulatory action was blunted by agents that interfere with calcium-calmodulin while a protein kinase (PKC) inhibitor, staurosporine was able to abrogate the inhibitory effect. Moreover, PKC activity showed maximum translocation to cerebral frontal cortex membranes with carbachol concentrations that inhibited NO production. Products from phosphoinosite (PI) hydrolysis are involved in these actions as carbachol was found to increase PI turnover in a dose dependent manner. These results would serve as an example of cross-talk between both enzymatic pathways.

Regulatory effects of acutely repeated nicotine treatment towards central muscarinic receptors.

Sensitivity of brain muscarinic acetylcholine receptors to the agonists was examined in nicotine tolerant animals which were developed by acutely repeated injections of nicotine. In conscious rats, the dose-response curves of muscarinic agonists arecoline and pilocarpine, cholinesterase inhibitors soman and physostigmine rather than GABA receptor antagonist pentylenetetrazol or glycine receptor antagonist strychnine for producing EEG seizures were shifted leftwards by acutely repeated injections of nicotine. This phenomenon could be prevented by nicotinic antagonist mecamylamine. Similar results were obtained in acute nicotine tolerant mice and rabbits. In other experiments, the dose-response curve of arecoline-induced convulsions or oxotremorine-induced tremors was also shifted leftwards, and the durations of arecoline- and oxotremorine-induced tremors were prolonged in acute nicotine tolerant mice. In addition, the effects of arecoline for producing down-regulation of muscarinic receptors of rat cerebrum and hippocampus rather than brain stem were potentiated in acute nicotine tolerant rats. It is concluded that the sensitivity of brain muscarinic receptor to its agonists is increased in acute nicotine tolerant rats, mice and rabbits.

Down-regulation of brain muscarinic cholinergic receptor promoted by diacylglycerols and phorbol ester.

Sustained agonist stimulation induces an asymmetric down-regulation of brain muscarinic acetylcholine receptor (mAChR): 43 +/- 2% in the right and 26 +/- 2% in the left cerebral hemisphere, respectively (Ref. 1). In order to determine the possible involvement of endogenous diacylglycerols produced under muscarinic stimulation in the down-regulation phenomenon, here we have studied the effects of synthetic diacylglycerols and a phorbol ester on cells dissociated from rat cerebral cortex. Oleylacetylglycerol decreased the amount of cell-surface mAChR by 37 +/- 2% and 25 +/- 2% in right and left cerebral cortex, respectively. Long-term treatment with phorbol dibutyrate also produced internalization of the mAChR (25 +/- 1.5% and 33 +/- 2% in right and left cortical cells, respectively). These changes occurred without modification of the Kdapp for the selective antagonist pirenzepine. The action of calcium ions was also studied using incubation of cells with the ionophore A23187. No changes were observed in the amount of mAChR detected at the plasma membrane with the ionophore alone, but when used in combination with phorbol dibutyrate and the agonist carbamylcholine a sinergistic decrease in mAChR was apparent. It is concluded that long-term exposure to exogenously added diacyglycerols and phorbol ester significantly reduces the amount of mAChR detected at the plasma membrane and abolishes the asymmetry of the down-regulation phenomenon observed under specific muscarinic stimulation, suggesting that diacylglycerols may be one of the factors responsible for such asymmetry.

Characterization of in vivo brain muscarinic acetylcholine receptor subtype selectivity by competition studies against ( [formula omitted

We have studied the in vivo rat brain muscarinic acetylcholine receptor (mAChR) m2 subtype selectivities of three quinuclidine derivatives: ( R)-3-quinuclidinyl benzilate (QNB), E-(+,+)-1-azabicyclo[2.2.2]oct-3-yl α-hydroxy-α-(1-iodo-1-propen-3-yl)-α-phenyl-acetate (E-(+,+)-IQNP), and E-(+,−)-1-azabicyclo[2.2.2]oct-3-yl α-hydroxy-α-(1-iodo-1-propen-3-yl)-α-phenylacetate (E-(+,−)-IQNP), and two tricyclic ring compounds: 5-[[4-[4-(diisobutylamino)butyl]-1-phenyl]-10,11-dihydro-5H-dibenzo[b,e][1,4]diazepin-11-one (DIBD) and 11-[[4-4-(diisobutylamino)butyl-1-phenyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one (PBID), by correlating the regional inhibition of ( R,S)-[ 125 I]IQNB with the regional composition of the m1–m4 subtypes. Subtle effects are demonstrated after reduction of the between-animal variability by normalization to corpus striatum. Substantial in vivo m2-selectivity is exhibited by QNB and DIBD, modest in vivo m2-selectivity is exhibited by E-(+,+)-IQNP, and little or no in vivo m2-selectivity is exhibited by PBID and E-(+,−)-IQNP. Surrisingly, the in vivo m2-selectivity is not correlated with the in vitro m2-selectivity. For example, QNB, which appears to be the most strongly in vivo m2-selective compound, exhibits negligible in vitro m2-selectivity. These examples indicate that a strategy which includes only preliminary in vitro screening may very well preclude the discovery of a novel compound which would prove useful in vivo.

Modification of muscarinic acetylcholine receptors in the rat brain following chronic immobilization stress: an autoradiographic study

The modifications of rat brain muscarinic acetylcholine receptors induced by chronic immobilization stress lasting 10 min/daily or 2 h/daily for 3, 7 or 21 days were analyzed by quantitative in vitro autoradiography. [ 3H]N-Methylscopolamine ([ 3H]NMS) was used as ligand. Chronic immobilization stress for 10 min/day did not produce any significant change in the properties of [ 3H]NMS binding sites throughout the rat brain. In contrast, 2 h/day immobilization caused a significant increase in the maximal number of muscarinic receptors (B max) in several brain areas such as the cortical layers, the CA 1 field of the hippocampus and caudate-putamen, among others. Affinity values (k d) were not modified. These results suggest that chronic immobilization stress induces supersensitivity of muscarinic receptors in certain cholinergic pathways in rat brain, the pattern of response being different to that previously found for acute stress.

Release of Alzheimer amyloid precursor derivatives stimulated by activation of muscarinic acetylcholine receptors.

Altered processing of the amyloid precursor protein (APP) is a central event in the formation of amyloid deposits in the brains of individuals with Alzheimer's disease. To investigate whether cellular APP processing is controlled by cell-surface neurotransmitter receptors, human embryonic kidney (293) cell lines were transfected with the genes for human brain muscarinic acetylcholine receptors. Stimulation of m1 and m3 receptor subtypes with carbachol increased the basal release of APP derivatives within minutes of treatment, indicating that preexisting APP is released in response to receptor activation. Receptor-activated APP release was blocked by staurosporine, suggesting that protein kinases mediate neurotransmitter receptor-controlled APP processing.

Affinity changes in muscarinic acetylcholine receptors in the rat brain following acute immobilization stress: an autoradiographic study

The modifications in rat brain muscarinic acetylcholine receptors induced by acute immobilization stress lasting 10 min or 2 h were analyzed by quantitative in vitro autoradiography. [ 3H]N-Methylscopolamine ([ 3H]NMS) was used as a ligand. Immobilization stress for 10 min did not produce any significant change in the properties of [ 3H]NMS binding sites throughout the brain. In contrast, 2 h immobilization caused a significant increase in receptor affinity (K d) without modification in the maximal number of receptors (B max) in several brain areas such as the caudate-putamen, cortical layers and CA 1 field of the hippocampus, among others. These results, found even in animals killed immediately after the end of the immobilization sessions, suggest that immobilization stress induces supersensitivity of muscarinic receptors in certain cholinergic pathways in rat brain.

N-Substituted derivatives of 4-piperidinyl benzilate: Affinities for brain muscarinic acetylcholine receptors

N-Substituted derivatives of 4- piperidinyl benzilate were synthesized and their affinities for central muscarinic cholinergic receptors determined using an in vitro radioligand binding assay. 4- Piperidinyl benzilate exhibited a K i value of 2.0nM. N-Substitution with a methyl or an ethyl group increased the affinity to 0.2nM, whereas substitution with a n-propyl or isopropyl group decreased the binding affinity over 100 fold. Compounds with aralkyl substitutions at the nitrogen atom of piperidinyl benzilate were also synthesized and evaluated. The K i values (nM) obtained for these compounds were: benzyl, 0.2; p-nitrobenzyl, 13.0; p- flourobenzyl, 3.0; phenethyl, 8.0; p- nitrophenethyl, 15.0. These data suggest that a binding region near the piperidinyl nitrogen may tolerate bulky aromatic substitutions (e.g., benzyl or phenethyl) as well or better than straight chain or branched alkyl substitutions (e.g., n-propyl or isopropyl).

Actions of the insecticide 2 (nitromethylene) tetrahydro-1, 3-thiazine on insect and vertebrate nicotinic acetylcholine receptors

The nitromethylene heterocyclic compound 2(nitromethylene)tetrahydro)1,3-thiazine (NMTHT) inhibits the binding of [125I]alpha-bungarotoxin to membranes prepared from cockroach (Periplaneta americana) nerve cord and fish (Torpedo californica) electric organ. Electrophysiological studies on the cockroach fast coxal depressor motorneuron (Df) reveal a dose-dependent depolarization in response to bath-applied NMTHT. Responses to ionophoretic application of NMTHT onto the cell-body membrane of motorneuron Df are suppressed by bath-applied mecamylamine (1.0 x 10(-4) M) and alpha-bungarotoxin (1.0 x 10(-7) M). These findings, together with the detection of a reversal potential close to that estimated for acetylcholine, provide evidence for an agonist action of this nitromethylene on an insect neuronal nicotinic acetylcholine receptor. The binding of [3H]H12-histrionicotoxin to Torpedo membranes was enhanced in the presence of NMTHT indicating an agonist action at this vertebrate peripheral nicotinic acetylcholine receptor. NMTHT is ineffective in radioligand binding assays for rat brain GABAA receptors, rat brain L-glutamate receptors and insect (Musca domestica) L-glutamate receptors. Partial block of rat brain muscarinic acetylcholine receptors is detected at millimolar concentrations of NMTHT. Thus nitromethylenes appear to exhibit selectivity for acetylcholine receptors and exhibit an agonist action at nicotinic acetylcholine receptors.

Direct and energy-transfer photolabelling of brain muscarinic acetylcholine receptors.

Efficient photolabelling of muscarinic acetylcholine receptors was obtained using either two aryldiazonium salts or an azido derivative. These probes did not discriminate between muscarinic binding subtypes or affinity states and became irreversibly bound to the receptor sites, in an entirely atropine-protectable manner, upon ultraviolet irradiation. The extent of labelling was dependent both on probe concentration and on time of irradiation and reached up to 80% of the receptor population, under optimal alkylating conditions. In contrast to the azido derivative, both diazonium salts behave as potent irreversible labels of muscarinic receptors, provided energy-transfer photolabelling conditions were followed. Such an indirect activation of diazonium ligands, through an energy transfer from photoexcited tryptophan residues, has been previously found to increase the site-specificity and the rate of labelling of other acetylcholine binding proteins. Analogies in the photolabelling process of acetylcholinesterase or of nicotinic and muscarinic receptors by the two diazonium salts are discussed. Altogether, these findings suggest that these new probes may be promising tools to investigate the location and the topography of the agonist-antagonist binding domain on purified muscarinic receptors, through amino acid and/or sequence analyses of radioactive, photolabelled residues.

P-006 - Possible allosteric interaction of calcium antagonists with rat brain muscarinic acetylcholine receptor subtypes

P-006 - Possible allosteric interaction of calcium antagonists with rat brain muscarinic acetylcholine receptor subtypes

Lack of alterations in muscarinic receptor subtypes and phosphoinositide hydrolysis upon acute DFP treatment

There was a 25 and 27% reduction in the density of mouse brain muscarinic acetylcholine receptors 18 and 24 h following a single injection of the organophosphate diisopropylfluorophosphate (DFP) when the muscarinic antagonist [ 3H]N-methylscopolamine ([ 3H]NMS) was used as the ligand. Down-regulation of specific [ 3H]NMS binding was rapidly reversible reaching control levels 36 h after DFP administration. Carbamylcholine and pirenzepine competition for the specific binding of either [ 3H]NMS or [ 3H]quinuclidinyl benzilate ([ 3H]QNB) in brain homogenates from untreated and DFP-treated mice demonstrated that the alteration in muscarinic receptor density following acute DFP treatment was not accompanied by a change in a particular muscarinic receptor binding conformation. Furthermore, the magnitude of muscarinic receptor-mediated phosphoinositide hydrolysis was unchanged following short-term DFP treatment suggesting that a physiological desensitization in this response does not accompany acute down-regulation of [ 3H]NMS binding sites.