Antagonist N-methylscopolamine Research Articles

Characterization of methanthelinium binding and function at human M1-M5 muscarinic acetylcholine receptors.

Firstly, it was determined whether methanthelinium bromide (MB) binds to human M1-M5 (hM1-hM5) muscarinic acetylcholine receptors in comparison to the classical muscarinic antagonist N-methylscopolamine (NMS). [3H]NMS dissociation binding experiments revealed an allosteric retardation of dissociation at 100μM of MB ranging from none in hM3 to 4.6-fold in hM2 receptors. Accordingly, global non-linear regression analysis of equilibrium inhibition binding curves between [3H]NMS (0.2 and 2.0nM) and MB was applied and compared using either an allosteric or a competitive model. The allosteric cooperativity of MB binding within MB/NMS/hM receptor complexes was strongly negative and undistinguishable from a competitive interaction throughout all subtypes. Applying the competitive model to the equilibrium binding data of MB and NMS, suggested competition at all hM subtypes: logKI (± S.E.) hM3 = 8.71 ± 0.15, hM1 = 8.68 ± 0.14, hM5 = 8.58 ± 0.07, hM2 = 8.27 ± 0.07 to hM4 = 8.25 ± 0.11. Secondly, the effects of MB on acetylcholine (ACh) induced hM receptor function showed very strong negative allosteric cooperativity at all subtypes pointing against an allosteric antagonism of MB with ACh. Competition with ACh was characterized by logKB: hM1 = 9.53 ± 0.05, hM4 = 9.33 ± 0.05, hM5 = 8.80 ± 0.05, hM2 = 8,79± 0.06, to hM3 = 8.43 ± 0.04. In conclusion, MB, below 1μM, binds competitively and non-selectively (except for the difference between hM3 vs. hM4) to all five hM receptor subtypes with nanomolar affinity and is able to functionally inhibit ACh responses in a competitive fashion, with a slight subtype preference for hM1 and hM4.

Structure-based discovery of selective positive allosteric modulators of antagonists for the M2 muscarinic acetylcholine receptor

Subtype-selective antagonists for muscarinic acetylcholine receptors (mAChRs) have long been elusive, owing to the highly conserved orthosteric binding site. However, allosteric sites of these receptors are less conserved, motivating the search for allosteric ligands that modulate agonists or antagonists to confer subtype selectivity. Accordingly, a 4.6 million-molecule library was docked against the structure of the prototypical M2 mAChR, seeking molecules that specifically stabilized antagonist binding. This led us to identify a positive allosteric modulator (PAM) that potentiated the antagonist N-methyl scopolamine (NMS). Structure-based optimization led to compound '628, which enhanced binding of NMS, and the drug scopolamine itself, with a cooperativity factor (α) of 5.5 and a KB of 1.1 μM, while sparing the endogenous agonist acetylcholine. NMR spectral changes determined for methionine residues reflected changes in the allosteric network. Moreover, '628 slowed the dissociation rate of NMS from the M2 mAChR by 50-fold, an effect not observed at the other four mAChR subtypes. The specific PAM effect of '628 on NMS antagonism was conserved in functional assays, including agonist stimulation of [35S]GTPγS binding and ERK 1/2 phosphorylation. Importantly, the selective allostery between '628 and NMS was retained in membranes from adult rat hypothalamus and in neonatal rat cardiomyocytes, supporting the physiological relevance of this PAM/antagonist approach. This study supports the feasibility of discovering PAMs that confer subtype selectivity to antagonists; molecules like '628 can convert an armamentarium of potent but nonselective GPCR antagonist drugs into subtype-selective reagents, thus reducing their off-target effects.

Structural Insight into Specificity of Interactions between Nonconventional Three-finger Weak Toxin from Naja kaouthia (WTX) and Muscarinic Acetylcholine Receptors

Weak toxin from Naja kaouthia (WTX) belongs to the group of nonconventional "three-finger" snake neurotoxins. It irreversibly inhibits nicotinic acetylcholine receptors and allosterically interacts with muscarinic acetylcholine receptors (mAChRs). Using site-directed mutagenesis, NMR spectroscopy, and computer modeling, we investigated the recombinant mutant WTX analogue (rWTX) which, compared with the native toxin, has an additional N-terminal methionine residue. In comparison with the wild-type toxin, rWTX demonstrated an altered pharmacological profile, decreased binding of orthosteric antagonist N-methylscopolamine to human M1- and M2-mAChRs, and increased antagonist binding to M3-mAChR. Positively charged arginine residues located in the flexible loop II were found to be crucial for rWTX interactions with all types of mAChR. Computer modeling suggested that the rWTX loop II protrudes to the M1-mAChR allosteric ligand-binding site blocking the entrance to the orthosteric site. In contrast, toxin interacts with M3-mAChR by loop II without penetration into the allosteric site. Data obtained provide new structural insight into the target-specific allosteric regulation of mAChRs by "three-finger" snake neurotoxins.

Molecular mechanisms of methoctramine binding and selectivity at muscarinic acetylcholine receptors.

Methoctramine (N,N'-bis[6-[[(2-methoxyphenyl)-methyl]hexyl]-1,8-octane] diamine) is an M(2)-selective competitive antagonist of muscarinic acetylcholine receptors and exhibits allosteric properties at high concentrations. To reveal the molecular mechanisms of methoctramine binding and selectivity we took advantage of reciprocal mutations of the M(2) and M(3) receptors in the second and third extracellular loops that are involved in the binding of allosteric ligands. To this end we performed measurements of kinetics of the radiolabeled antagonists N-methylscopolamine (NMS) in the presence of methoctramine and its precursors, fluorescence energy transfer between green fluorescent protein-fused receptors and an Alexa-555-conjugated precursor of methoctramine, and simulation of molecular dynamics of methoctramine association with the receptor. We confirm the hypothesis that methoctramine high-affinity binding to the M(2) receptors involves simultaneous interaction with both the orthosteric binding site and the allosteric binding site located between the second and third extracellular loops. Methoctramine can bind solely with low affinity to the allosteric binding site on the extracellular domain of NMS-occupied M(2) receptors by interacting primarily with glutamate 175 in the second extracellular loop. In this mode, methoctramine physically prevents dissociation of NMS from the orthosteric binding site. Our results also demonstrate that lysine 523 in the third extracellular loop of the M(3) receptors forms a hydrogen bond with glutamate 219 of the second extracellular loop that hinders methoctramine binding to the allosteric site at this receptor subtype. Impaired interaction with the allosteric binding site manifests as low-affinity binding of methoctramine at the M(3) receptor.

Long-Term Activation upon Brief Exposure to Xanomleline Is Unique to M1 and M4 Subtypes of Muscarinic Acetylcholine Receptors

Xanomeline is an agonist endowed with functional preference for M1/M4 muscarinic acetylcholine receptors. It also exhibits both reversible and wash-resistant binding to and activation of these receptors. So far the mechanisms of xanomeline selectivity remain unknown. To address this question we employed microfluorometric measurements of intracellular calcium levels and radioligand binding to investigate differences in the short- and long-term effects of xanomeline among muscarinic receptors expressed individually in Chinese hamster ovary cells. 1/One-min exposure of cells to xanomeline markedly increased intracellular calcium at hM1 and hM4, and to a lesser extent at hM2 and hM3 muscarinic receptors for more than 1 hour. 2/Unlike the classic agonists carbachol, oxotremorine, and pilocarpine 10-min exposure to xanomeline did not cause internalization of any receptor subtype. 3/Wash-resistant xanomeline selectively prevented further increase in intracellular calcium by carbachol at hM1 and hM4 receptors. 4/After transient activation xanomeline behaved as a long-term antagonist at hM5 receptors. 5/The antagonist N-methylscopolamine (NMS) reversibly blocked activation of hM1 through hM4 receptors by xanomeline. 6/NMS prevented formation of xanomeline wash-resistant binding and activation at hM2 and hM4 receptors and slowed them at hM1, hM3 and hM5 receptors. Our results show commonalities of xanomeline reversible and wash-resistant binding and short-time activation among the five muscarinic receptor subtypes. However long-term receptor activation takes place in full only at hM1 and hM4 receptors. Moreover xanomeline displays higher efficacy at hM1 and hM4 receptors in primary phasic intracellular calcium release. These findings suggest the existence of particular activation mechanisms specific to these two receptors.

Synthesis ofN-Substituted Piperidine Salts as Potential Muscarinic Ligands for Alzheimer's Applications

Several heterocyclic N-piperidine substituted salts were synthesized that were found to inhibit the specific binding of the antagonist [3H] quinuclidinyl benzilate in radioligand muscarinic binding assays (3H-QNB) in bioassays. One of the heterocyclic salts, compound 7, met the significance criteria in these assays (>50% inhibition) at 10 μM of the nonselective muscarinic antagonist (3H-QNB) in cells of the Wistar rat cerebral cortex. Furthermore, this compound displayed 61% inhibition at 10 μM of the antagonist (3H-QNB) for the M5 receptor (IC50 6.34 μM, Ki 3.93 μM, nH = 0.996) in human recombinant CHO cell lines. These data obtained from Ricerca Biosciences suggested that compound 7 was selective for the M5 receptor. Another study from the Czech Academy of Sciences demonstrated that compound 7 was 3 to 8 times more potent at M2 than other subtypes of muscarinic receptors in competition with antagonist N-methylscopolamine and selective for the M1 receptors over M3 and M5 in antagonizing accumulation of inositol phosphates induced by muscarinic agonist carbachol.

Uncoupling of M1 muscarinic receptor/G-protein interaction by amyloid β1–42

The overproduction of β-amyloid (Aβ) fragments in transgenic APPswe/PS1dE9 mice results in formation of amyloid deposits in the cerebral cortex and hippocampus starting around four months of age and leading to cognitive impairment much later. We have previously found an age and transgene-dependent weakening of muscarinic receptor-mediated transmission that was not present in young (6–10-week-old) animals but preceded both amyloid deposits and cognitive deficits. Now we investigated immediate and prolonged in vitro effects of non-aggregated Aβ1–42 on coupling of individual muscarinic receptor subtypes expressed in CHO (Chinese hamster ovary) cells and their underlying mechanisms. Immediate application of 1 μM Aβ1–42 had no effect on the binding of the muscarinic antagonist N-methylscopolamine or the agonist carbachol. In contrast, 4-day treatment of CHO cells expressing the M1 muscarinic receptor with 100 nM Aβ1–42 significantly changed the binding characteristics of the muscarinic agonist carbachol and reduced the extent of the M1 receptor-stimulated breakdown of phosphatidylinositol while it did not demonstrate overt toxic effects. The treatment had no influence on the expression of either G-proteins or muscarinic receptors. In concert, we found no change in the gene expression of muscarinic receptor subtypes and gene or protein expression of the Gs, Gq/11, and Gi/o G-proteins in the cerebral cortex of young adult APPswe/PS1dE9 mice that demonstrate high concentrations of soluble Aβ1–42 and impaired muscarinic receptor-mediated G-protein activation. Our results provide strong evidence that the initial injurious effects of Aβ1–42 on M1 muscarinic receptor-mediated transmissionis is due to compromised coupling of the receptor with Gq/11 G-protein.

Molecular Modeling of the M3 Acetylcholine Muscarinic Receptor and Its Binding Site

The present study reports the results of a combined computational and site mutagenesis study designed to provide new insights into the orthosteric binding site of the human M3 muscarinic acetylcholine receptor. For this purpose a three-dimensional structure of the receptor at atomic resolution was built by homology modeling, using the crystallographic structure of bovine rhodopsin as a template. Then, the antagonist N-methylscopolamine was docked in the model and subsequently embedded in a lipid bilayer for its refinement using molecular dynamics simulations. Two different lipid bilayer compositions were studied: one component palmitoyl-oleyl phosphatidylcholine (POPC) and two-component palmitoyl-oleyl phosphatidylcholine/palmitoyl-oleyl phosphatidylserine (POPC-POPS). Analysis of the results suggested that residues F222 and T235 may contribute to the ligand-receptor recognition. Accordingly, alanine mutants at positions 222 and 235 were constructed, expressed, and their binding properties determined. The results confirmed the role of these residues in modulating the binding affinity of the ligand.

Amiodarone and N‐ethyl‐amiodarone exert opposing allosteric effects via a novel site on muscarinic receptors

Allosteric modulation provides several advantages over the classical (orthosteric) approach to receptor activation; further, the characterization of new allosteric sites provides additional opportunities for the development of subtype‐selective and functionally selective compounds. We have been investigating the allosteric actions of amiodarone and N‐ethyl‐amiodarone (NEA) at muscarinic receptors. Previous studies have demonstrated that amiodarone interacts with a site that is distinct from the one that has been defined by gallamine.We have shown that amiodarone enhances stimulation of [3H]arachidonic acid release induced by agonists acting at Gq‐coupled muscarinic receptors. Evaluation of this action in light of Hall's allosteric two‐state model indicates that amiodarone's enhancement of agonist efficacy is tempered by a concomitant reduction in agonist binding affinity. The enhancement of efficacy is attenuated by NEA but not by the orthosteric antagonist N‐methylscopolamine; further evaluation of this finding using an elaboration of Hall's model suggests that amiodarone and NEA exert their functional effects through a common allosteric site.In conclusion, we have demonstrated that amiodarone and NEA interact allosterically with a novel, extracellular, site on muscarinic receptors, through which they modulate agonist potency and efficacy in a complex manner.

Efficacy As An Intrinsic Property of the M2 Muscarinic Receptor in Its Oligomeric State

G protein-coupled receptors exhibit a characteristic dispersion of affinities for agonists, and the breadth of that dispersion is correlated with efficacy. The implied heterogeneity is commonly attributed to a ligand-regulated transient complex between the receptor and the G protein; that is, agonists bind with higher affinity to the complex than to the receptor alone. Such an arrangement is at odds with observations that the receptor-G protein complex is less transient than required by such models, and that GPCRs exist as oligomers. The present investigation has been directed toward the alternative notion that heterogeneity emerges as a property of the oligomer and is independent of G proteins. M2 muscarinic receptors were purified as monomers from Sf9 cells and reconstituted as tetramers devoid of G proteins in phospholipid vesicles (POPC/POPS/cholesterol). The antagonists N-methylscopolamine and quinuclidinylbenzilate recognized a single class of sites in assays with N-[3H]methylscopolamine; in contrast, seven agonists recognized at least two classes (log KH and log KL). The magnitude of that dispersion was quantified empirically as the product of the difference in affinity for the two classes (Δlog K) and the fraction exhibiting higher affinity (FH). The value of FHΔlog K equals the area between the observed curve and a curve of equal amplitude and a single affinity corresponding to log KL. It therefore prefigures the shift that could be effected by GTP acting via a G protein, were it present. The values of FHΔlog K generally were higher for full agonists than partial agonists. This distinction among agonists and antagonists recalls the propensity of those ligands to elicit a response. It follows that the heterogeneity revealed by agonists, which is predictive of efficacy, is a property intrinsic to the receptor in its oligomeric state.

Binding of Orthosteric Ligands to the Allosteric Site of the M2Muscarinic Cholinergic Receptor

The M(2) muscarinic receptor has two topographically distinct sites: the orthosteric site and an allosteric site recognized by compounds such as gallamine. It also can exhibit cooperative effects in the binding of orthosteric ligands, presumably to the orthosteric sites within an oligomer. Such effects would be difficult to interpret, however, if those ligands also bound to the allosteric site. Monomers of the hemagglutinin (HA)- and FLAG-tagged human M(2) receptor therefore have been purified from coinfected Sf9 cells and examined for any effect of the antagonist N-methyl scopolamine or the agonist oxotremorine-M on the rate at which N-[(3)H]methyl scopolamine dissociates from the orthosteric site (k(obsd)). The predominantly monomeric status was confirmed by coimmunoprecipitation and by cross-linking with bis(sulfosuccinimidyl)suberate. Both N-methyl scopolamine and oxotremorine-M acted in a cooperative manner to decrease k(obsd) by 4.5- and 9.1-fold, respectively; the corresponding estimates of affinity (log K(L)) are -2.55 +/- 0.13 and -2.29 +/- 0.14. Gallamine and the allosteric ligand obidoxime decreased k(obsd) by more than 100-fold (log K(L) = -4.12 +/- 0.04) and by only 1.1-fold (log K(L) = -1.73 +/- 0.91), respectively. Obidoxime reversed the effect of N-methyl scopolamine, oxotremorine-M, and gallamine in a manner that could be described by a model in which all four ligands compete for a common allosteric site. Ligands generally assumed to be exclusively orthosteric therefore can act at the allosteric site of the M(2) receptor, albeit at comparatively high concentrations.

Determinants of Positive Cooperativity Between Strychnine-Like Allosteric Modulators and N-Methylscopolamine at Muscarinic Receptors

It has been shown previously that the third extracellular loop (o3) and its vicinity play a critical role in allosteric modulation at muscarinic acetylcholine receptors (mAChRs) (Ellis et al., 1993; Krejçí and Tuçek, 2001; Buller et al., 2002). In this study interaction of four chemically related substances (strychnine, its dimethoxy derivate brucine, precursor for synthesis of strychnine Wieland-Gumlich aldehyde (WGA), and precursor for synthesis of alcuronium propargyl-WGA) with orthosteric antagonist N-methylscopolamine (NMS) was investigated on the M3 subtype of mAChRs mutated at the o3 loop.

Allosteric Modulators and Selective Agonists of Muscarinic Receptors

Allosteric modulators of ligand-receptor interactions are found for a variety of receptors (Christopoulos, 2002). Allosteric agents attach to a binding site being topographically distinct from the site for conventional (orthosteric) agonists or antagonists. In the case of the muscarinic receptor, a huge selection of structurally divergent modulators has been described for different receptor subtypes (Mohr et al., 2003). Alkane-bisammonio-type compounds carrying lateral phthalimido substituents are known to have a high affinity for the common allosteric binding site of the muscarinic acetylcholine M2 receptor (mAChR M2), which is already occupied by the orthosteric antagonist N-methylscopolamine (NMS). The resulting allosteric inhibition of the dissociation of [3H]NMS from the M2 receptors in porcine cardiac homogenates served to indicate binding of the test compounds to the allosteric site. Additionally, allosteric modulators can strongly influence equilibrium binding of the orthosteric ligand: Its binding can be reduced, left unaltered or elevated, and encoded as negative, neutral, and positive cooperativity, respectively (Christopoulos and Kenakin, 2002). The cooperativity is strongly dependent on the pair of allosteric/orthosteric ligands and on the receptor subtype.

Allosteric modulation by persistent binding of xanomeline of the interaction of competitive ligands with the M1 muscarinic acetylcholine receptor.

Xanomeline is a potent agonist that is functionally selective for muscarinic M(1) receptors. We have shown previously that a significant fraction of xanomeline binding to membranes of Chinese hamster ovary (CHO) cells expressing the M(1) receptors occurs in a wash-resistant manner and speculated that this persistent binding likely does not take place at the primary binding site on the receptor. In the present work we investigated in depth the pharmacological characteristics of this unique mode of xanomeline binding and the effects of this binding on the interaction of classical competitive ligands with the receptor in CHO cells that express the M(1) muscarinic receptor. Onset of persistent binding of xanomeline to the M(1) muscarinic receptor was fast and was only slightly hindered by atropine. Its dissociation was extremely slow, with a half-life of over 30 h. Although persistently bound xanomeline strongly inhibited binding of the classical antagonist N-methylscopolamine (NMS) to the receptor, there are multiple indications that this is not the result of competition at the same binding domain. Namely, wash-resistant binding of xanomeline only slightly slowed the rate of NMS association, but enhanced the rate of NMS dissociation. Moreover, preincubation with xanomeline followed by extensive washing brought about an apparent decrease in the number of NMS binding sites. Our findings are best interpreted in terms of allosteric interactions between xanomeline-persistent binding to the M(1) muscarinic receptor and competitive ligands bound to the classical receptor binding site.

Substituted pentacyclic carbazolones as novel muscarinic allosteric agents: synthesis and structure-affinity and cooperativity relationships.

Two series of pentacyclic carbazolones, 22 and 23, have been synthesized utilizing a facile intramolecular Dielsminus signAlder reaction and are allosteric modulators at muscarinic acetylcholine receptors. Their affinities and cooperativities with acetylcholine and the antagonist N-methylscopolamine (NMS) at M(1)minus signM(4) receptors have been analyzed and compared. All of the synthesized compounds are negatively cooperative with acetylcholine. In contrast, the majority of the compounds exhibit positive cooperativity with NMS, particularly at M(2) and M(4) receptors. The subtype selectivity, in terms of affinity, was in general M(2) > M(1) > M(4) > M(3). The largest increases in affinity produced by a single substitution of the core structure were given by the 1-OMe (22b) and 1-Cl (22d) derivatives. The position of the N in the ring did not appear to be important for binding affinity or cooperativity. Two compounds 22y and 23i, both trisubstituted analogues, were the most potent compounds synthesized, with dissociation constants of 30minus sign100 nM for the M(2) NMS-liganded and unliganded receptor, respectively. The results indicate that the allosteric site, like the primary binding site, is capable of high-affinity interactions with molecules of relatively low molecular weight.

Trafficking of green fluorescent protein-tagged muscarinic M 4 receptors in NG108-15 cells

The muscarinic M 4 receptor for acetylcholine was tagged at its C terminus with green fluorescent protein (GFP) and expressed in NG108-15 cells, which normally express this receptor subtype. The binding affinity of the antagonist N-methylscopolamine was not significantly affected by the presence of the GFP tag, whereas the affinity of the receptor for the agonist carbachol was reduced by four-fold. Stimulation of the tagged receptor resulted in inhibition of adenylyl cyclase. Following agonist stimulation, the tagged receptor was slowly internalized, and became partially co-localized with the endosomal marker Texas Red–transferrin after 30 min. There was little co-localization with the lysosomal marker lgp120 even after 60 min of internalization. Finally, the tagged receptor, unlike the endogenous receptor, failed to recycle to the plasma membrane on removal of the agonist. We conclude that the GFP-tagged muscarinic M 4 receptor does not traffic normally in NG108-15 cells, most likely because of its gross overexpression.

The allosteric interaction of otenzepad (AF-DX 116) at muscarinic M 2 receptors in guinea pig atria

The effects of the muscarinic receptor antagonist, otenzepad, in combination with the competitive antagonists N-methylscopolamine, dexetimide and atropine, or the allosteric modulators, C 7/3′-phth, gallamine and alcuronium, were measured in the guinea pig electrically driven left atrium using the agonists, carbachol or acetylcholine. Otenzepad, in combination with C 7/3′-phth or gallamine, gave concentration-ratios close to additive and in agreement with theoretical model predictions for combination of two allosteric modulators acting at a common site. However, when otenzepad was combined with alcuronium, dexetimide or N-methylscopolamine, supra-additive effects were observed. For either competitive antagonist in combination with otenzepad, the degree of supra-additivity was more evident after 2-h equilibration than after 40 min. When otenzepad was combined with atropine, no supra-additivity was observed with carbachol as the agonist, but was evident with acetylcholine. Otenzepad was also unable to fully inhibit [ 3H] N-methylscopolamine binding when the radioligand was employed at a concentration of ∼100× K D. It is concluded that the action of otenzepad involves an allosteric site and a number of possibilities are discussed for its location.

Interactions of alcuronium, TMB-8, and other allosteric ligands with muscarinic acetylcholine receptors: studies with chimeric receptors.

A series of ligands that allosterically modulate the binding of classical ligands to muscarinic receptors was evaluated at wild-type and chimeric receptors. All of the ligands studied had highest affinity toward the M(2) subtype and lowest affinity toward the M(5) subtype. The chimeric receptors were mostly M(5) sequence; the amount of M(2) sequence ranged from about 6 to just under 30%. Alcuronium and TMB-8 had much higher affinity for the chimeric receptor that included the M(2) second outer loop of the receptor plus flanking regions of TM4 and TM5 than for any of the other chimeric receptors (the affinities of which remained similar to that of the M(5) subtype). However, this chimera retained the negative cooperativity between alcuronium and the classical antagonist N-methylscopolamine that is characteristic of M(5) (these ligands are positively cooperative at M(2)). Verapamil, tetrahydroaminoacridine, and d-tubocurarine were also sensitive to that chimeric substitution, although verapamil and tetrahydroaminoacridine had even higher affinity for a chimera with M(2) sequence in TM7. None of these ligands shared gallamine's sensitivity to a region of the third outer loop, but studies in which obidoxime reversed the allosteric effects of gallamine and other ligands suggested that they nevertheless compete for a common site. In summary, although the present data are consistent with previous studies that have suggested that allosteric ligands bind to the outermost regions of muscarinic receptors, it appears that different allosteric ligands may derive subtype selectivity from different regions of the receptor.

Cholinergic receptor up-regulates COX-2 expression and prostaglandin E(2) production in colon cancer cells.

The M(3) muscarinic cholinergic receptor has important physiological functions on normal colonic cells. It is frequently expressed on human colon cancer cells and is biologically active. Although it is mitogenic in certain cell models, the importance of this receptor on colon carcinogenesis is unknown. In the present study we have determined expression of the M(3) receptor on human colon cancer tissue compared with matched normal tissue and examined the downstream effect of receptor activation in the HT-29 human colon carcinoma cell line. Using reverse transcription-PCR, M(3) receptor RNA expression was detected in all matched colon carcinoma and normal specimens from eight patients. Five of the eight (62%) patients showed an up to 8-fold greater level of M(3) receptor expression in cancer compared with the matched normal tissue. Exposure of HT-29 cells to carbachol, a stable receptor agonist, results in a 10-fold increase in cyclooxygenase-2 (COX-2) protein. This induction of COX-2 protein was dose dependent and was inhibited by the cholinergic receptor antagonist N-methylscopolamine (NMS). Carbachol caused a dose-dependent increase in prostaglandin E(2) (PGE(2)), the main product of cyclooxygenase activity. The maximum stimulatory effect (40-fold increase) was noted with 1mM carbachol. The increase in PGE(2) was completely abolished by NMS and by the COX-2 selective inhibitor NS398. This suggests that the M(3) receptor mediates PGE(2) production by a mechanism involving COX-2. As COX-2 and PGE(2) are known promoters of gastrointestinal cancer, these data suggest that M(3) receptor activation may facilitate progression of colon carcinoma, in part by a COX-2-mediated cellular mechanism.

Functional role of muscarinic M(2) receptors in alpha,beta-methylene ATP induced, neurogenic contractions in guinea-pig ileum.

1. The muscarinic acetylcholine receptors mediating the contractile response elicited to endogenous acetylcholine released by the selective P2X receptor agonist alpha,beta-methylene ATP (mATP) were investigated in guinea-pig ileum. 2. mATP (0.1 - 30 microM) elicited a concentration-dependent neurogenic contractile response inhibited by tetrodotoxin (TTX) and antagonized by the non-selective muscarinic receptor antagonist N-methylscopolamine (NMS). 3. The contractile response to mATP was pertussis toxin-insensitive, irreversibly antagonized by N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard), and unaffected by the muscarinic M(2)/M(4) receptor selective antagonist AF-DX 116 (1 microM). 4. When measured in the presence of histamine and isoproterenol after treatment with 4-DAMP mustard, mATP elicited a pertussis toxin-sensitive contractile response potently antagonized by AF-DX 116. 5. Collectively, our data suggest that endogenous acetylcholine released by mATP can elicit a direct contractile response through the muscarinic M(3) receptor and an indirect contractile response through the muscarinic M(2) receptor by antagonizing the relaxant effects of isoproterenol on histamine induced contraction.