Overcoming Obstacles to Targeting Muscarinic Receptor Signaling in Colorectal Cancer.

Despite great advances in our understanding of the pathobiology of colorectal cancer and the genetic and environmental factors that mitigate its onset and progression, a paucity of effective treatments persists. The five-year survival for advanced, stage IV disease remains substantially less than 20%. This review examines a relatively untapped reservoir of potential therapies to target muscarinic receptor expression, activation, and signaling in colorectal cancer. Most colorectal cancers overexpress M3 muscarinic receptors (M3R), and both in vitro and in vivo studies have shown that activating these receptors stimulates cellular programs that result in colon cancer growth, survival, and spread. In vivo studies using mouse models of intestinal neoplasia have shown that using either genetic or pharmacological approaches to block M3R expression and activation, respectively, attenuates the development and progression of colon cancer. Moreover, both in vitro and in vivo studies have shown that blocking the activity of matrix metalloproteinases (MMPs) that are induced selectively by M3R activation, i.e., MMP1 and MMP7, also impedes colon cancer growth and progression. Nonetheless, the widespread expression of muscarinic receptors and MMPs and their importance for many cellular functions raises important concerns about off-target effects and the safety of employing similar strategies in humans. As we highlight in this review, highly selective approaches can overcome these obstacles and permit clinicians to exploit the reliance of colon cancer cells on muscarinic receptors and their downstream signal transduction pathways for therapeutic purposes.

[Subtypes of muscarinic acetylcholine receptors involved in the persistent activity of layer V pyramidal neurons in the primary auditory cortex of young mice].

Cholinergic receptor activation and intracellular current injection lead to the persistent activity (PA), which may be involved in inducing neural plasticity. Our previous study showed that PA is closely related to the activation of muscarinic acetylcholine receptors (mAChRs) in pyramidal neurons of mouse primary auditory cortex (AI). However, the subtypes of mAChRs involved in PA remain unclear. Thus, using whole-cell patch-clamp recording and pharmacological methods, we investigated the role of different mAChR subtypes in inducing PA in AI layer V pyramidal neurons of young mice. The results showed that activation of mAChRs with intracellular depolarizing current induced PA in layer V pyramidal neurons. Blockade of M1, M2 or M3 subtypes prevented the PA, whereas M4 receptor antagonists did not affect the production of PA. The results suggest that the PA may be induced through a mechanism involving M1, M2 and M3 muscarinic receptors, but not M4 subtype.

Muscarinic acetylcholine receptors: novel opportunities for drug development.

The muscarinic acetylcholine receptors are a subfamily of G protein-coupled receptors that regulate numerous fundamental functions of the central and peripheral nervous system. The past few years have witnessed unprecedented new insights into muscarinic receptor physiology, pharmacology and structure. These advances include the first structural views of muscarinic receptors in both inactive and active conformations, as well as a better understanding of the molecular underpinnings of muscarinic receptor regulation by allosteric modulators. These recent findings should facilitate the development of new muscarinic receptor subtype-selective ligands that could prove to be useful for the treatment of many severe pathophysiological conditions.

Effects of ovarian hormone treatment on the gene expression of muscarinic acetylcholine receptors in the ovariectomized rat myometrium.

We investigate the effects of ovarian hormone on the gene expression of muscarinic acetylcholine receptors (M1-M5) in the myometrium using real-time PCR and evaluate the relationships between their expression and that of ovarian hormone receptors (ERα, ERß, and PgR). Wistar rats were sham operated (SO) or ovariectomized (OVX) and treated with vehicle, estradiol (E2), progesterone (P4), or both E2 and P4 for 2 days beginning on postoperative day 33. M1 and M4 mRNA expressions were not detected in the myometrium. M2 mRNA expression did not change significantly in the OVX and OVX+P4 groups compared to the SO group, but increased significantly in the OVX+E2 group and was normalized in the OVX+E2P4 group. M3 mRNA expression increased significantly in the OVX and OVX+P4 groups compared to the SO group, but was normalized in the OVX+E2 and OVX+E2P4 groups. M5 mRNA expression did not change significantly in all experimental groups. ERα mRNA expression increased significantly in the OVX, OVX+E2, and OVX+P4 groups compared to the SO group, but was normalized in the OVX+E2P4 group. The changes in ERß mRNA expression were similar to those of M3 mRNA expression in all experimental groups. In contrast, the changes in PgR mRNA expression did not correspond with that of M2, M3, or M5 mRNA expression in any of the experimental groups. Additionally, we evaluated the relationship between the expression of muscarinic acetylcholine receptors and ovarian hormone receptors in estrus cycle. M2 mRNA expression increased significantly in diestus and metaestrus compared in proestrus and estrus. M3 mRNA expression increased significantly in only diestrus compared in the other stages. In contrast, M5 mRNA expression did not change in estrus cycle. The changes in ERα mRNA expression appeared to be similar to those of M2 in estrus cycle, but no significant difference was found. The changes in ERß mRNA expression were similar to those of M3 mRNA expression. The change in PgR mRNA expression increased significantly in diestrus compared in metaestrus, but did not correspond with that of M2, M3, or M5 mRNA expression in estrus cycle. When acetylcholine sensitivity in the myometrium was compared between diestrus and estrus, the sensitivity is significantly lower in estrus than in diestrus. These results suggest that ovarian hormones influence the expression of M2 and M3 in the myometrium by regulating the expression of hormone receptors. E2 may upregulate M2 via ERα, but P4 may downregulate M2 by inhibiting ERα via PgR. E2 may downregulate M3 by inhibiting ERß, but P4 may not regulate the expression of M3 and ERß. M5 may be a constitutive muscarinic receptor in the myometrium because neither E2 nor P4 influence the expression of M5. The combination of E2 and P4 may contribute the reproduction by quieting down the acetylcholine-induced myometrial contraction.

Impaired ß-cell function in the adult offspring of rats fed a protein-restricted diet during lactation is associated with changes in muscarinic acetylcholine receptor subtypes.

Impaired pancreatic ß-cell function, as observed in the cases of early nutrition disturbance, is a major hallmark of metabolic diseases arising in adulthood. In the present study, we aimed to investigate the function/composition of the muscarinic acetylcholine receptor (mAChR) subtypes, M2 and M3, in the pancreatic islets of adult offspring of rats that were protein malnourished during lactation. Neonates were nursed by mothers that were fed either a low-protein (4 %, LP) or a normal-protein (23 %, NP) diet. Adult rats were pre-treated with anti-muscarinic drugs and subjected to the glucose tolerance test; the function and protein expression levels of M2mAChR and M3mAChR were determined. The LP rats were lean and hypoinsulinaemic. The selective M2mAChR antagonist methoctramine increased insulinaemia by 31 % in the NP rats and 155 % in the LP rats, and insulin secretion was increased by 32 % in the islets of the NP rats and 88 % in those of the LP rats. The selective M3mAChR antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide decreased insulinaemia by 63 % in the NP rats and 40 % in the LP rats and reduced insulin release by 41 % in the islets of the NP rats and 28 % in those of the LP rats. The protein expression levels of M2mAChR and M3mAChR were 57 % higher and 53 % lower, respectively, in the islets of the LP rats than in those of the NP rats. The expression and functional compositions of M2mAChR and M3mAChR were altered in the islets of the LP rats, as a result of metabolic programming caused by the protein-restricted diet, which might be another possible effect involved in the weak insulin secretion ability of the islets of the programmed adult rats.

Distribution of muscarinic acetylcholine receptor mRNA in the brain of the weakly electric fish Apteronotus leptorhynchus.

Various neuromodulators have been shown to be involved in shaping the sensory information available to the brain. Acetylcholine (ACh) modulation, through muscarinic receptors, is a particularly widespread mechanism of controlling sensory information transmission. The precise effects of ACh modulation depend on the subtype of muscarinic ACh receptors that are activated. In weakly electric fish, previous work suggested a role of ACh, via muscarinic receptors, in the modulation of information transmission in the electrosensory lateral line lobe (ELL) of the hindbrain. In this study, we determined which muscarinic receptor (mAChR) subtypes are present in the brain of Apteronotus leptorhynchus as well as their spatial distribution. We partially cloned three subtypes of muscarinic receptors (mAChR2, -3, and -4) from brain tissue of A. leptorhynchus and used in situ hybridization in transverse sections of the brain to determine their distributions. Sites labeled for the three muscarinic receptor mRNAs were found in various brain regions devoted to the processing of different sensory modalities. The mRNA probes for the three receptor types showed differential distribution but also overlapping presence of two or more receptors in particular nuclei. In addition to the presence of mAChR3 in the ELL region, electrosensory nuclei including the nucleus praeeminentialis, dorsal torus semicircularis and optic tectum showed expression of one or more mAChRs. Thus, the overall pattern of mAChR expression found is in agreement with mAChR expression in other species, with additional presence evident in specialized regions of the electrosensory system, which suggests an important modulating role of ACh in this sensory modality.

Muscarinic receptors and their antagonists in COPD: anti-inflammatory and antiremodeling effects.

Muscarinic receptors are expressed by most cell types and mediate cellular signaling of their natural ligand acetylcholine. Thereby, they control numerous central and peripheral physiological organ responses to neuronal activity. In the human lung, muscarinic receptors are predominantly expressed by smooth muscle cells, epithelial cells, and fibroblasts. Antimuscarinic agents are used for the treatment of chronic obstructive pulmonary disease and to a lesser extent for asthma. They are primarily used as bronchodilators, but it is now accepted that they are also associated with anti-inflammatory, antiproliferative, and antiremodeling effects. Remodeling of the small airways is a major pathology in COPD and impairs lung function through changes of the extracellular matrix. Glycosaminoglycans, particularly hyaluronic acid, and matrix metalloproteases are among extracellular matrix molecules that have been associated with tissue inflammation and remodeling in lung diseases, including chronic obstructive pulmonary disease and asthma. Since muscarinic receptors have been shown to influence the homeostasis of glycosaminoglycans and matrix metalloproteases, these molecules may be proved valuable endpoint targets in clinical studies for the pharmacological exploitation of the anti-inflammatory and antiremodeling effects of muscarinic inhibitors in the treatment of chronic obstructive pulmonary disease and asthma.

M-type potassium channels modulate Schaffer collateral-CA1 glutamatergic synaptic transmission.

Previous studies have suggested that muscarinic receptor activation modulates glutamatergic transmission. M-type potassium channels mediate the effects of muscarinic activation in the hippocampus, and it has been proposed that they modulate glutamatergic synaptic transmission. We tested whether M1 muscarinic receptor activation enhances glutamatergic synaptic transmission via the inhibition of the M-type potassium channels that are present in Schaffer collateral axons and terminals. Miniature excitatory postsynaptic currents (mEPSCs) were recorded from CA1 pyramidal neurons. The M1 receptor agonist, NcN-A-343, increased the frequency of mEPSCs, but did not alter their amplitude. The M-channel blocker XE991 and its analogue linopirdine also increased the frequency of mEPSCs. Flupirtine, which opens M-channels, had the opposite effect. XE991 did not enhance mEPSCs frequency in a calcium-free external medium. Blocking P/Q- and N-type calcium channels abolished the effect of XE991 on mEPSCs. These data suggested that the inhibition of M-channels increases presynaptic calcium-dependent glutamate release in CA1 pyramidal neurons. The effects of these agents on the membrane potentials of presynaptic CA3 pyramidal neurons were studied using current clamp recordings; activation of M1 receptors and blocking M-channels depolarized neurons and increased burst firing. The input resistance of CA3 neurons was increased by the application of McN-A-343 and XE991; these effects were consistent with the closure of M-channels. Muscarinic activation inhibits M-channels in CA3 pyramidal neurons and its efferents ­ Schaffer collateral, which causes the depolarization, activates voltage-gated calcium channels, and ultimately elevates the intracellular calcium concentration to increase the release of glutamate on CA1 pyramidal neurons.

Distinct muscarinic acetylcholine receptor subtypes mediate pre- and postsynaptic effects in rat neocortex.

BACKGROUND: Cholinergic transmission has been implicated in learning, memory and cognition. However, the cellular effects induced by muscarinic acetylcholine receptors (mAChRs) activation are poorly understood in the neocortex. We investigated the effects of the cholinergic agonist carbachol (CCh) and various agonists and antagonists on neuronal activity in rat neocortical slices using intracellular (sharp microelectrode) and field potential recordings. RESULTS: CCh increased neuronal firing but reduced synaptic transmission. The increase of neuronal firing was antagonized by pirenzepine (M1/M4 mAChRs antagonist) but not by AF-DX 116 (M2/M4 mAChRs antagonist). Pirenzepine reversed the depressant effect of CCh on excitatory postsynaptic potential (EPSP) but had marginal effects when applied before CCh. AF-DX 116 antagonized the depression of EPSP when applied before or during CCh. CCh also decreased the paired-pulse inhibition of field potentials and the inhibitory conductances mediated by GABA(A) and GABA(B) receptors. The depression of paired-pulse inhibition was antagonized or prevented by AF-DX 116 or atropine but only marginally by pirenzepine. The inhibitory conductances were unaltered by xanomeline (M1/M4 mAChRs agonist), yet the CCh-induced depression was antagonized by AF-DX 116. Linopirdine, a selective M-current blocker, mimicked the effect of CCh on neuronal firing. However, linopirdine had no effect on the amplitude of EPSP or on the paired-pulse inhibition, indicating that M-current is involved in the increase of neuronal excitability but neither in the depression of EPSP nor paired-pulse inhibition. CONCLUSIONS: These data indicate that the three effects are mediated by different mAChRs, the increase in firing being mediated by M1 mAChR, decrease of inhibition by M2 mAChR and depression of excitatory transmission by M4 mAChR. The depression of EPSP and increase of neuronal firing might enhance the signal-to-noise ratio, whereas the concomitant depression of inhibition would facilitate long-term potentiation. Thus, this triade of effects may represent a "neuronal correlate" of attention and learning.

Overview of muscarinic receptor subtypes.

The physiological role of muscarinic receptors is highly complex and, although not completely understood, has become clearer over the last decade. Recent pharmacological evidence with novel compounds, together with data from transgenic mice, suggests that all five subtypes have defined functions in the nervous system as well as mediating the non neuronal, hormonal actions of acetylcholine. Numerous novel agonists, allosteric regulators, and antagonists have now been identified with authentic subtype specificity in vitro and in vivo. These compounds provide additional pharmacological opportunities for selective subtype modulation as well as a new generation of muscarinic receptor-based therapeutics.

Structure-function studies of muscarinic acetylcholine receptors.

There has been great interest in the structure-function relationships of the muscarinic acetylcholine receptors (mAChRs) because these prototypical Family A/class 1 G protein-coupled receptors (GPCRs) are attractive therapeutic targets for both peripheral and central nervous system disorders. A multitude of drugs that act at the mAChRs have been identified over the years, but many of these show minimal selectivity for any one of the five mAChR subtypes over the others, which has hampered their development into therapeutics due to adverse side effects. The lack of drug specificity is primarily due to high sequence similarity in this family of receptor, especially in the orthosteric binding pocket. Thus, there remains an ongoing need for a molecular understanding of how mAChRs bind their ligands, and how selectivity in binding and activation can be achieved. Unfortunately, there remains a paucity of solved high-resolution structures of GPCRs, including the mAChRs, and thus most of our knowledge of structure-function mechanisms related to this receptor family to date has been obtained indirectly through approaches such as mutagenesis. Nonetheless, such studies have revealed a wealth of information that has led to novel insights and may be used to guide future rational drug design campaigns.

Muscarinic acetylcholine receptor subtype expression in type vestibular hair cells of guinea pigs.

Recent studies have demonstrated that five subtypes (M1-M5) of muscarinic acetylcholine receptor (mAChR) are expressed in the vestibular periphery. However, the exact cellular location of the mAChRs is not clear. In this study, we investigated whether there is the expression of M1-M5 muscarinic receptor mRNA in isolated type II vestibular hair cells of guinea pig by using single-cell RT-PCR. In vestibular end-organ, cDNA of the expected size was obtained by RT-PCR. Moreover, mRNA was identified by RT-PCR from individually isolated type II vestibular hair cells (single-cell RT-PCR). Sequence analysis confirmed that the products were M1-M5 mAChR. These results demonstrated that M1-M5 mAChR was expressed in the type II vestibular hair cells of the guinea pig, which lends further support for the role of M1-M5 mAChR as a mediator of efferent cholinergic signalling pathway in vestibular hair cells.

Muscarinic receptor immunoreactivity in the superior salivatory nucleus neurons innervating the salivary glands of the rat.

The superior salivatory nucleus (SSN) contains preganglionic parasympathetic neurons to the submandibular and sublingual salivary glands. Cevimeline, a muscarinic acetylcholine receptor agonist, stimulates the salivary glands and is presently used as sialogogue in the treatment of dry mouth. Since cevimeline passes through the blood-brain barrier, it is also able to act on muscarinic acetylcholine receptors in the central nervous system. Our preliminary experiment using the whole-cell patch-clamp technique has shown that cevimeline excites SSN neurons in rat brain slices, suggesting that SSN neurons have muscarinic acetylcholine receptors; however, it is unclear which subtypes of muscarinic acetylcholine receptors exist in SSN neurons. In the present study, we investigated immunohistochemically muscarinic acetylcholine receptor subtypes, M1 receptor (M1R), M2R, M3R, M4R, and M5R in SSN neurons. SSN neurons innervating the salivary glands, retrogradely labeled with a fluorescent tracer from the chorda-lingual nerve, mostly expressed M3R immunoreactivity (-ir) (92.3%) but not M1R-ir. About half of such SSN neurons also showed M2R- (40.1%), M4R- (54.0%) and M5R-ir (46.0%); therefore, it is probable that SSN neurons co-express M3R-ir with at least two of the other muscarinic receptor subtypes. This is the first report to show that SSN neurons contain muscarinic acetylcholine receptors.

Antimuscarinics for the treatment of overactive bladder: understanding the role of muscarinic subtype selectivity.

INTRODUCTION AND HYPOTHESIS: Antimuscarinic agents appear to exert their therapeutic activity in overactive bladder (OAB) via blockade of the M(3) muscarinic receptor subtype. Antimuscarinics are broadly similar in efficacy, but their safety and tolerability profiles vary, which may reflect differences in muscarinic receptor selectivity profiles. METHODS: This review of available literature aims to determine whether antimuscarinic agents with greater M(3) selectivity have clinical advantages over less selective drugs. RESULTS: Antimuscarinic agents differ widely in their propensity to cause cognitive and cardiovascular (CV) effects, which appear mainly to be related to differences in their relative selectivity for binding to non-M(3) receptors, including M(1) receptors in the brain and cardiac M(2) receptors. CONCLUSIONS: Cognitive and CV effects are especially pertinent for the OAB patient who tends to be older with various comorbidities and is often taking multiple medications. Hence, it is important to consider the risk/benefit balance of antimuscarinic agents when selecting OAB treatment.

An update on the efficacy and safety of aclidinium bromide in patients with COPD.

Aclidinium is a potent and selective muscarinic antagonist, which interacts rapidly with muscarinic receptors and shows subnanomolar affinity for the five human muscarinic receptors (M(1)-M(5)); its association rate for the M(3) receptor is similar to that of ipratropium and 2.6 times faster than that of tiotropium. Aclidinium dissociates slightly faster from M(2) and M(3) receptors than tiotropium but much more slowly than ipratropium. A potent bronchodilatory activity has been observed after inhaled administration of aclidinium. Aclidinium undergoes rapid hydrolysis in the plasma into two major compounds, the alcohol (LAS34823) and the carboxylic acid (LAS34850) metabolites, resulting in low and transient systemic exposure to the active drug. The two major metabolites show no significant affinity for human muscarinic receptors. A potent bronchodilatory activity has been observed after inhaled administration of aclidinium. Clinical trials have provided evidence of sustained bronchodilation similar to that observed with tiotropium. Trial results have confirmed the positive safety profile of aclidinium, particularly in terms of a very low propensity to cause anticholinergic adverse events. Aclidinium is now moving to phase III clinical development for chronic obstructive pulmonary disease (COPD).

Effects of 17ß-estradiol on expression of muscarinic acetylcholine receptor subtypes and estrogen receptor alpha in rat hippocampus.

The aim of the present study was to investigate the effects of 17beta-estradiol on expression of muscarinic acetylcholine receptor subtypes (M1 to M5) and estrogen receptor alpha, in the rat hippocampus. Hippocampi were obtained from rats in proestrus, rats ovariectomized for 15 days, rats ovariectomized for 15 days and then treated with 17beta-estradiol for 7 days, and rats ovariectomized and immediately treated with 17beta-estradiol for 21 days. Expression of M1 to M5 was increased in hippocampi of rats ovariectomized for 15 days compared to rats in proestrus. Although this effect was abolished when replacement with 17beta-estradiol started immediately after ovariectomy, the increased expression of M1, M3 and M5 receptor subtypes was unchanged when replacement with 17beta-estradiol started only 15 days after ovariectomy. The expression of estrogen receptor alpha in the hippocampus was also upregulated after ovariectomy when compared to rats in proestrus. This effect was abolished when 17beta-estradiol was replaced immediately after ovariectomy, and slightly reduced when the replacement started 15 days after ovariectomy. The replacement with estrogen also had beneficial effects on cognitive function, as suggested by data obtained in the plus-maze discriminative avoidance task. In conclusion, the present results provide evidence that 17beta-estradiol regulates the expression of muscarinic acetylcholine receptor subtypes and estrogen receptor alpha. The immediate replacement with estrogen seems critical to restore the expression of these receptors after hormonal deprivation. The understanding of the regulation of expression and intracellular signaling of the muscarinic acetylcholine receptor subtype M1 and the estrogen receptor alpha may be helpful to elucidate the mechanisms involved in changes of cognitive function in postmenopausal women and in neurodegenerative diseases.

Gain of new exons and promoters by lineage-specific transposable elements-integration and conservation event on CHRM3 gene.

The CHRM3 gene is a member of the muscarinic acetylcholine receptor family that plays important roles in the regulation of fundamental physiological functions. The evolutionary mechanism of exon-acquisition and alternative splicing of the CHRM3 gene in relation to transposable elements (TEs) were analyzed using experimental approaches and in silico analysis. Five different transcript variants (T1, T2, T3, T3-1, and T4) derived from three distinct promoter regions (T1: L1HS, T2, T4: original, T3, T3-1: THE1C) were identified. A placenta (T1) and testis (T3 and T3-1)-dominated expression pattern appeared to be controlled by different TEs (L1HS and THE1C) that were integrated into the common ancestor genome during primate evolution. Remarkably, the T1 transcript was formed by the integration event of the human specific L1HS element. Among the 12 different brain regions, the brain stem, olfactory region, and cerebellum showed decreased expression patterns. Evolutionary analysis of splicing sites and alternative splicing suggested that the exon-acquisition event was determined by a selection and conservation mechanism. Furthermore, continuous integration events of transposable elements could produce lineage specific alternative transcripts by providing novel promoters and splicing sites. Taken together, exon-acquisition and alternative splicing events of CHRM3 genes were shown to have occurred through the continuous integration of transposable elements following conservation.

Functional characterization of muscarinic autoreceptors in rat and human neocortex.

Electrically evoked overflow of [(3)H]acetylcholine in slices of rat neocortex and of human neocortex (freshly obtained during neurosurgical treatment of epilepsy or deep-seated tumors) was used to functionally characterize the muscarinic receptor subtype, which mediates autoinhibition of acetylcholine release in these tissues. In the rat neocortex, the following pK(B) values [CI(95)] were calculated from the shifts to the right of the concentration-response curves of the full agonist oxotremorine in presence of subtype preferring muscarinic receptor antagonists: tripitramine: 9.1 [8.8, 9.4], tripinamide: 8.6 [8.5, 8.7], AQ-RA 741 (11-[[4-[4-(diethylamino)butyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido(2,3-b)[1,4]benzodiazepine-6-one): 8.2 [8.0, 8.4], himbacine: 8.0 [7.9, 8.1], 4-diphenylacetoxy-N-methylpiperidine methobromide: 8.0 [7.8, 8.1], methoctramine: 7.5 [7.4, 7.6], AF-DX 116 (11[[2-[(diethyl-amino)methyl]-1-piperidinyl] acetyl] 5,11-dihydro-6H-pyrido(2,3-b)[1,4]benzodiazepine-6-one): 7.1 [7.0, 7.3], hexahydro-sila-difenidol: 6.8 [6.7, 6.9], pirenzepine: 6.6 [6.4, 6.7], and 3,6a,11,14-tetrahydro-9-methoxy-2-methyl-12H-isoquino[1,2-b]pyrrolo[3,2-f] [1,3]benzoxazine-1-carboxylic acid ethyl ester (PD 102807): 6.0 [5.8, 6.2]. In the human neocortex the following values were found: tripitramine: 9.4 [9.3, 9.6], tripinamide: 9.0 [8.9, 9.2], AF-DX 116: 6.7 [6.4, 6.9], hexahydro-sila-difenidol: 6.6 [6.2, 6.9], and PD 102807: 6.5 [6.3, 6.6]. In correlation plots, these pK(B) values correspond best to published binding data on native or recombinant M(2) receptors but not to those on M(1), M(3), M(4), and M(5) receptors, suggesting that muscarinic autoreceptors of both the rat and human neocortex belong to the M(2) subtype. This observation lends further support to the development of M(2) receptor selective brain penetrating antagonists for application in Alzheimer's disease.

Muscarinic toxins: tools for the study of the pharmacological and functional properties of muscarinic receptors.

Muscarinic receptors mediate metabotropic actions of acetylcholine in the CNS and PNS and autocrine functions of acetylcholine in non-neuronal systems. Because of the lack of highly selective muscarinic ligands, the precise location, functional role, and roles in various diseases of the five muscarinic receptor subtypes remain unclear. Muscarinic toxins isolated from the venom of Dendroaspis snakes have a natural high affinity and selectivity, associated with roles as competitive antagonists, allosteric modulators, and potential agonists. These toxins may therefore be invaluable tools for studying muscarinic receptors. We review data on the structural and pharmacological characterization of the muscarinic toxins, focusing on recent structure-function studies on toxin-receptor interactions. We discuss the potential benefits of using these toxins for investigating muscarinic function in vivo.