Inverse agonist activity of pirenzepine at M2 muscarinic acetylcholine receptors

1. The intrinsic properties of muscarinic ligands were studied through their binding properties and their abilities to modulate the GTPase activity of G proteins coupled to muscarinic M2 receptors in pig atrial sarcolemma. 2. Competition binding experiments were performed with [3H]-oxotremorine-M to assess the affinity of receptors coupled to G proteins (R*), with [3H]-N-methylscopolamine ([3H]-NMS) to estimate the affinities of coupled and uncoupled receptors (R*+R) and with [3H]-NMS in the presence of GppNHp to assess the affinity of uncoupled receptors (R). 3. The ranking of Ki values for the agonist carbachol was R*R*+R>R (174, 155, 115 nM), suggesting inverse agonism. 4. The Vmax of the basal high affinity GTPase activity of pig atrial sarcolemma was increased by mastoparan and decreased by GPAnt-2 indicating the relevance of this activity to G proteins coupled to receptors (R*). The K(M) value (0.26-0.33 microM) was not modified by mastoparan or GPAnt-2. 5. Carbachol increased the Vmax of GTP hydrolysis (EC50 8.1+/-0.3 microM), whereas atropine and AF-DX 116, up to 1 mM, did not modify it. Pirenzepine decreased the Vmax of GTP hydrolysis (EC50 77.5+/-10.3 microM). This effect was enhanced when KCI was substituted for NaCl (EC50 11.0+/-0.8 microM) and was antagonized by atropine and AF-DX 116 (IC50 0.91+/-0.71 and 197+/-85 nM). 6. Pirenzepine is proposed as an inverse agonist and atropine and AF-DX 116 as neutral antagonists at the muscarinic M2 receptor.     

Structure-function studies of allosteric agonism at M2 muscarinic acetylcholine receptors

The M2 muscarinic acetylcholine receptor (mAChR) possesses at least one binding site for allosteric modulators that is dependent on the residues (172)EDGE(175), Tyr(177), and Thr(423). However, the contribution of these residues to actions of allosteric agonists, as opposed to modulators, is unknown. We created mutant M2 mAChRs in which the charge of the (172)EDGE(175) sequence had been neutralized and each Tyr(177) and Thr(423) was substituted with alanine. Radioligand binding experiments revealed that these mutations had a profound inhibitory effect on the prototypical modulators gallamine, alcuronium, and heptane-1,7-bis-[dimethyl-3'-phthalimidopropyl]-ammonium bromide (C7/3-phth) but minimal effects on the orthosteric antagonist [3H]N-methyl scopolamine. In contrast, the allosteric agonists 4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride (McN-A-343), 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine hydrogen chloride (AC-42), and the novel AC-42 derivative 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1) demonstrated an increased affinity or proportion of high-affinity sites at the combined EDGE-YT mutation, indicating a different mode of binding to the prototypical modulators. Subsequent functional assays of extracellular signal-regulated kinase (ERK)1/2 phosphorylation and guanosine 5'-(gamma-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding revealed minimal effects of the mutations on the orthosteric agonists acetylcholine (ACh) and pilocarpine but a significant increase in the efficacy of McN-A-343 and potency of 77-LH-28-1. Additional mutagenesis experiments found that these effects were predominantly mediated by Tyr(177) and Thr(423), rather than the (172)EDGE(175) sequence. The functional interaction between each of the allosteric agonists and ACh was characterized by high negative cooperativity but was consistent with an increased allosteric agonist affinity at the combined EDGE-YT mutant M2 mAChR. This study has thus revealed a differential role of critical allosteric site residues on the binding and function of allosteric agonists versus allosteric modulators of M2 mAChRs.     

Thiochrome enhances acetylcholine affinity at muscarinic M4 receptors: receptor subtype selectivity via cooperativity rather than affinity

Thiochrome (2,7-dimethyl-5H-thiachromine-8-ethanol), an oxidation product and metabolite of thiamine, has little effect on the equilibrium binding of l-[3H]N-methyl scopolamine ([3H]NMS) to the five human muscarinic receptor subtypes (M1-M5) at concentrations up to 0.3 mM. In contrast, it inhibits [3H]NMS dissociation from M1 to M4 receptors at submillimolar concentrations and from M5 receptors at 1 mM. These results suggest that thiochrome binds allosterically to muscarinic receptors and has approximately neutral cooperativity with [3H]NMS at M1 to M4 and possibly M5 receptors. Thiochrome increases the affinity of acetylcholine (ACh) 3- to 5-fold for inhibiting [3H]NMS binding to M4 receptors but has no effect on ACh affinity at M1 to M3 or M5 receptors. Thiochrome (0.1 mM) also increases the direct binding of [3H]ACh to M4 receptors but decreases it slightly at M2 receptors. In agreement with the binding data, thiochrome does not affect the potency of ACh for stimulating the binding of guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTPgammaS) to membranes containing M1 to M3 receptors, but it increases ACh potency 3.5-fold at M4 receptors. It also selectively reduces the release of [3H]ACh from potassium-stimulated slices of rat striatum, which contain autoinhibitory presynaptic M4 receptors, but not from hippocampal slices, which contain presynaptic M2 receptors. We conclude that thiochrome is a selective M4 muscarinic receptor enhancer of ACh affinity and has neutral cooperativity with ACh at M1 to M3 receptors; it therefore demonstrates a powerful new form of selectivity, "absolute subtype selectivity", which is derived from cooperativity rather than from affinity.     

Determination of muscarinic agonist potencies at M1 and M2 muscarinic receptors in a modified pithed rat preparation

Summary The agonistic potencies of (±)muscarine, (±)cis - 2 - methyl - 5 - [(dimethylamino)methyl] - 1,3 -oxathiolane methiodide (cis-oxathiolane) and its two enantiomers were determined at muscarinic M₁ and M₂ receptors in the pithed rat. In non-pretreated animals, i.v. administration of these agents produced bradycardic effects mediated by cardiac M₂ receptors followed by increases in heart rate mediated by M1 receptors in sympathetic ganglia. As these responses have been shown to partly overlap, “true” M₁ and M₂ potencies were determined after selective blockade of M₁ and M₂ receptors by pirenzepine and methoctramine, respectively. A similar rank order of agonist potencies was obtained at M₁ and M₂ receptors: (+)cis-oxathiolane > (±)cis-oxathiolane > (±)muscarine > (-)cis-oxathiolane. At both receptor subtypes, (+)cis-oxathiolane was considerably more potent (ca. 30-fold) than its corresponding (−) enantiomer indicating that the agonist binding sites of the two receptor subtypes may have similar stereochemical properties. While (±)muscarine showed similar potencies at M₁ and M₂ receptors, racemic cis-oxathiolane and its two enantiomers showed a slight selectivity (3–7 fold) for M₁ receptors indicating the potential usefulness of these compounds in the development of selective M₁ receptor agonists. Send offprint requests to F. Cantalamessa at the above address     

Inhibition of m3 muscarinic acetylcholine receptors by local anaesthetics

1. Muscarinic m1 receptors are inhibited by local anaesthetics (LA) at nM concentrations. To elucidate in more detail the site(s) of LA interaction, we compared these findings with LA effects on m3 muscarinic receptors. 2. We expressed receptors in Xenopus oocytes. Using two-electrode voltage clamp, we measured the effects of lidocaine, QX314 (permanently charged) and benzocaine (permanently uncharged) on Ca(2+)-activated Cl(-)-currents (I(Cl(Ca))), elicited by acetyl-beta-methylcholine bromide (MCh). We also characterized the interaction of lidocaine with [(3)H]-quinuclydinyl benzylate ([(3)H]-QNB) binding to m3 receptors. Antisense-injection was used to determine the role of specific G-protein alpha subunits in mediating the inhibitory effects of LA. Using chimeric receptor constructs we investigated which domains of the muscarinic receptors contribute to the binding site for LA. 3. Lidocaine inhibited m3-signalling in a concentration-dependent, reversible, non-competitive manner with an IC(50) of 370 nM, approximately 21 fold higher than the IC(50) (18 nM) reported for m1 receptors. Intracellular inhibition of both signalling pathways by LA was similar, and dependent on the G(q)- protein alpha subunit. In contrast to results reported for the m1 receptor, the m3 receptor lacks the major extracellular binding site for charged LA. The N-terminus and third extracellular loop of the m1 muscarinic receptor molecule were identified as requirements to obtain extracellular inhibition by charged LA.     

Evaluation of an agonist index: affinity ratio for compounds active on muscarinic cholinergic M2 receptors

1 A protocol for predicting full agonist, partial agonist, and antagonist profiles of compounds with M₂ muscarinic cholinergic receptor activity was developed using radioligand binding assay techniques with [³H]-N-methyl scopolamine (NMS) and [³H]-Oxotremorine-M (Oxo-M) as radioligands. 2 Full muscarinic cholinergic receptor agonists such as muscarine and oxotremorine-M expressed a high agonist index (> 3000 for M₁ muscarinic cholinergic receptors and > 900 for M₂ muscarinic cholinergic receptor), whereas muscarinic receptor antagonists (selective or non-selective) for different receptor subtypes gave a low (0.5–10) agonist index. 3 Functional studies performed on preparations of guinea-pig ileum and heart were consistent with radioligand binding assay experiments. 4 The above results suggest that similarly as already established for the M₁ muscarinic cholinergic receptor subtype, evaluation of the [³H]-NMS/[³H]-Oxo-M ratio may provide useful information on the profile of compounds acting at the M₂ muscarinic cholinergic receptor subtype. 5 The availability of simple and predictive techniques for the characterization of muscarinic M₂ cholinergic receptor agonists, may help the identification of new compounds in therapeutic areas in which stimulation or inhibition of this receptor is desirable.     

Differences in kinetics of xanomeline binding and selectivity of activation of G proteins at M(1) and M(2) muscarinic acetylcholine receptors

Xanomeline is a functionally selective M(1)/M(4) muscarinic acetylcholine receptor agonist that nevertheless binds with high affinity to all five subtypes of muscarinic receptors. A novel mode of interaction of this ligand with the muscarinic M(1) receptors characterized by persistent binding and receptor activation after extensive washout has been shown previously. In the present study, using human M(1) and M(2) receptors expressed in Chinese hamster ovary cells and [(3)H]N-methylscopolamine as a tracer, we show that persistent binding of xanomeline also occurs at the M(2) receptor with similar affinity as at the M(1) receptor (K(I) = 294 and 296 nM, respectively). However, kinetics of formation of xanomeline wash-resistant binding to M(2) receptors was markedly slower than to M(1) receptors. Xanomeline was a potent fast-acting full agonist in stimulating guanosine 5'-O-(3-[(35)S]thio)triphosphate binding at M(1) receptors, whereas at M(2) receptors it behaved as a potent partial agonist (40% of carbachol maximal response) only upon preincubation for 1 h. Development of xanomeline agonistic effects at the M(2) receptor was slower than its ability to attenuate carbachol responses. We also demonstrate that xanomeline discriminates better between G protein subtypes at M(1) than at M(2) receptors. Our data support the notion that xanomeline interacts with multiple sites on the muscarinic receptor, resulting in divergent conformations that exhibit differential effects on ligand binding and receptor activation. These conformations are both time- and concentration-dependent and vary between the M(1) and the M(2) receptor.     

Inhibition of transmitter release from rat sympathetic neurons via presynaptic M1 muscarinic acetylcholine receptors

Background and purpose:

M₂, M₃ and/or M₄ muscarinic acetylcholine receptors have been reported to mediate presynaptic inhibition in sympathetic neurons. M₁ receptors mediate an inhibition of K_v7, Ca_V1 and Ca_V2.2 channels. These effects cause increases and decreases in transmitter release, respectively, but presynaptic M₁ receptors are generally considered facilitatory. Here, we searched for inhibitory presynaptic M₁ receptors.

Experimental approach:

In primary cultures of rat superior cervical ganglion neurons, Ca²⁺ currents were recorded via the perforated patch-clamp technique, and the release of [³H]-noradrenaline was determined.

Key results:

The muscarinic agonist oxotremorine M (OxoM) transiently enhanced ³H outflow and reduced electrically evoked release, once the stimulant effect had faded. The stimulant effect was enhanced by pertussis toxin (PTX) and was abolished by blocking M₁ receptors, by opening K_v7 channels and by preventing action potential propagation. The inhibitory effect was not altered by preventing action potentials or by opening K_v7 channels, but was reduced by PTX and ω-conotoxin GVIA. The inhibition remaining after PTX treatment was abolished by blockage of M₁ receptors or inhibition of phospholipase C. When [³H]-noradrenaline release was triggered independently of voltage-activated Ca²⁺ channels (VACCs), OxoM failed to cause any inhibition. The inhibition of Ca²⁺ currents by OxoM was also reduced by ω-conotoxin and PTX and was abolished by M₁ antagonism in PTX-treated neurons.

Conclusions and implications:

These results demonstrate that M₁, in addition to M₂, M₃ and M₄, receptors mediate presynaptic inhibition in sympathetic neurons using phospholipase C to close VACCs.     

A search for selective antagonists at M2 muscarinic receptors.

Isolated preparations of guinea-pig ileum and atria have been used to estimate the dose-ratios produced by antagonists at muscarinic receptors. Experiments with 4-diphenyl-acetoxy-N-methylpiperidine (4DAMP) metho-salts and with its isomer, 3DAMP methiodide, indicate that these are only slightly affected by the choice of physiological salt solution, the choice of agonist and the presence or absence of hexamethonium. Methyl or chloro groups in the p-position of the two benzene rings in 4DAMP metho-salts markedly reduce affinity and selectivity. When the two benzene rings are linked together, as in the fluorene-9-carboxylic ester, the affinity for the receptors in the atria is comparable with that of 4DAMP methobromide but that for the ileum is about half, so the selectivity is reduced. When the rings are linked as in the xanthene-9-carboxylic ester, the affinity for receptors in both tissues is greater than that of 4DAMP methobromide but there is less selectivity. When two molecules of 4DAMP are linked together by a polymethylene chain of from 4 to 12 carbon atoms the effects on affinity for muscarinic receptors in the guinea-pig ileum are different from those on affinity for muscarinic receptors in guinea-pig atria. The pentamethylene compound is the most selective: compared with 4DAMP methobromide it has slightly less affinity for receptors in the ileum but much less affinity for receptors in the atria. The effects of the compounds in antagonizing the actions of carbachol on atrial rate are not markedly different from their effects in antagonizing its actions on the force of the atrial contractions.     

Effects of halothane on high affinity agonist binding and guanine nucleotide sensitivity of muscarinic acetylcholine receptors from brainstem of rat

The influence of halothane on muscarinic receptors with a high affinity for agonists was studied using [3H]oxotremorine-M. [3H]Oxotremorine-M bound with high affinity (KD = 2.8 nM) to a subpopulation of muscarinic receptors in the brainstem of rat, representing 32% of the total receptor pool. Agonist affinity for binding sites for [3H]oxotremorine-M was not affected by a guanine nucleotide (5'-guanylylimidodidiphosphate; Gpp(NH)p), although the level of binding was decreased, presumably due to the conversion of receptors to lower affinity conformations. However, only 58% of 3 nM binding of [3H]oxotremorine-M was sensitive to Gpp(NH)p. Halothane had two effects on the binding of [3H]oxotremorine-M: halothane (1) decreased the level of binding of [3H]oxotremorine-M without affecting agonist affinity for the surviving sites, and (2) lowered the sensitivity of the binding of [3H]oxotremorine-M to Gpp(NH)p by a factor of 120. The decrease in binding of [3H]oxotremorine-M binding was nonselective with regard to the sensitivity of the receptors to the guanine nucleotide, insofar as Gpp(NH)p inhibited the binding of [3H]oxotremorine-M to the same extent in the presence and absence of halothane. These results suggest that halothane (1) converts both G protein-coupled and -uncoupled muscarinic receptors to states of lower agonist affinity and (2) lowers the affinity of receptor-G protein complexes for guanine nucleotides.     

An investigation of whether agonist-selective receptor conformations occur with respect to M2 and M4 muscarinic acetylcholine receptor signalling via Gi/o and Gs proteins

1. A range of muscarinic acetylcholine (mACh) receptor agonists (methacholine (MCh), oxotremorine-M (OXO-M), oxotremorine (OXO), arecoline (AREC), bethanechol (BETH), pilocarpine (PILO)) have been investigated with respect to their binding to, and activation of, M(2) and M(4) mACh receptors, recombinantly expressed in Chinese hamster ovary cells, to explore the possibility that these agonists may differentially affect mACh receptor-G(i/o) and -G(s) coupling. 2. M(2)/M(4) mACh receptor coupling to the adenylyl cyclase/cyclic AMP signalling pathway has been explored in intact cells. G(i/o)-mediated negative coupling to adenylyl cyclase was explored functionally by assessing the ability of the mACh receptor agonists to inhibit forskolin-stimulated enzymic activity. Following pertussis toxin treatment (100 ng ml(-1), 18-20 h) to inactivate G(i/o) proteins, each agonist caused a G(s)-mediated enhancement of forskolin-stimulated adenylyl cyclase activity. 3. At both M(2) and M(4) mACh receptors, all agonists tested were more potent in mediating G(i/o)- versus G(s)-coupled responses. This difference (determined as the pIC(50) (G(i/o) coupling) minus pEC(50) (G(s) coupling) value) was greatest for AREC (65-75-fold) and least for BETH and PILO (相似文献   

Tissue-dependent association of muscarinic acetylcholine receptors with guanine nucleotide-binding regulatory proteins.

The muscarinic acetylcholine receptors in heart and cerebellum form a stable association with guanine nucleotide-binding regulatory proteins (G proteins) in the presence of receptor agonists. This has been confirmed by purification of the muscarinic receptor-G protein complexes using an immunoprecipitation protocol. The isolated complexes were subjected to Western blotting to identify the G protein subunits present in the complexes. At saturating concentrations of carbachol, the muscarinic receptors in atrial membranes co-purified exclusively with Go, whereas in cerebellar and ventricular membranes an association with both Gi and Go was demonstrated. Further characterization of the G protein subunits allowed identification of the species of Gi alpha subunits present in the complexes of muscarinic receptor and G protein; in ventricle Gi alpha 2 was the only subtype present, whereas in cerebellum both Gi alpha 1 and Gi alpha 2 were present. These results demonstrate that a single muscarinic receptor subtype, depending on the tissue studied, is capable of interacting with more than one G protein subtype. The concentrations of agonist required to promote receptor-G protein association in atrial and ventricular membranes correlated with the high affinity component of receptor occupancy by agonist, as measured in equilibrium binding assays. Furthermore, incubation of cardiac membranes with saturating concentrations of pilocarpine or McN A343 resulted in reduced amounts of receptor-G protein complexes, compared with carbachol. Overall, our results suggest that the specificity of cellular effects of muscarinic agonists may relate, in part, to the selective interaction of receptor with G proteins.     

The interaction of methoctramine and himbacine at atrial, smooth muscle and endothelial muscarinic receptors in vitro.

1. The action of methoctramine and himbacine at muscarinic receptors has been studied using guinea-pig isolated trachea, oesophageal muscularis mucosae, paced left atria, and rat aortic preparations. 2. Methoctramine (1 x 10(-6)-3.2 x 10(-4) M), but not himbacine, elicited positive inotropic responses. These responses were enhanced by pretreating the animals with reserpine. The responses in reserpine-treated animals were not antagonized by phentolamine (1 x 10(-6) M) but were antagonized by propranolol (1 x 10(-6) M). 3. Methoctramine, but not himbacine, exhibited allosteric inhibitory effects at cardiac muscarinic receptors, resulting in a curvilinear Schild plot. Deviations from competitive antagonism were also observed in combination dose-ratio experiments using atropine and methoctramine. At 1 x 10(-6) M, the pKB value for methoctramine was 7.88 +/- 0.15 (mean +/- s.e.mean, n = 5). The pA2 value for himbacine at cardiac muscarinic receptors was 8.52 +/- 0.06 (n = 3). 4. At tracheal and oesophageal muscularis mucosal smooth muscle receptors, the Schild plots for both antagonists were linear. The pA2 values for methoctramine at receptors in these two preparations were similar (6.08 +/- 0.05 and 6.03 +/- 0.09 respectively, n = 4) and were approximately 60 fold less than those values observed at atrial receptors. Himbacine, also exhibited similar values at muscarinic receptors in the trachea and oesophageal muscularis mucosae (7.61 +/- 0.05 and 7.57 +/- 0.04 respectively, n = 4). 5. Muscarinic receptors mediating relaxation of the rat aortic endothelium exhibited pA2 values for methoctramine (5.87 +/- 0.12, n = 6) which were similar to those observed in the smooth muscle, but not the atria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Different agonist binding properties of M1 and M2 muscarinic receptors in calf brain cortex membranes

The muscarinic antagonist 1-[benzilic 4,4'-3H]-quinuclidinyl benzilate [3H]-QNB) bound to a single class of non-cooperative sites in calf cerebral cortex membranes (KD = 0.29 nM and Bmax = 1.06 pM/mg protein). Computer-assisted analysis of the shallow pirenzepine/[3H]-QNB competition binding curves indicated that 68% of these sites were of the M1-subtype and the remaining 32% of the M2 subtype. Respective Ki-values for pirenzepine were 27 nM and 1.14 microM. Binding characteristics of the antagonist atropine and of the agonist carbachol for M2 were evaluated by performing competition binding with 0.5 nM [3H]-QNB in the presence of 2 microM pirenzepine. The binding characteristics for the M1 receptors were obtained indirectly by subtracting the curve for M2 from the total curve, or directly by competition binding with 0.3 nM [3H]-pirenzepine. Atropine competition curves were steep for M1 and M2 and were not affected by 1 mM GTP nor by 1 mM N-ethylmaleimide. The carbachol competition curve was shallow for M2. The steep curves for M1 indicate that this receptor subclass was only composed of low agonist affinity sites. GTP, which caused a rightward shift and a steepening of the carbachol competition curve for M2, did not affect the curves for M1. N-ethylmaleimide provoked a leftward shift and a steepening of the carbachol competition curve for M2 and abolished GTP modulation. A leftward shift was also observed for M1, but of a smaller magnitude (i.e. 3-4-fold for M1 compared to 17-fold for M2). These data suggest that, in calf brain cortex, M1 and M2 receptors show different susceptibility towards GTP and N-ethylmaleimide modulation.     

Inhibition of nicotinic acetylcholine receptors by bicuculline.

A study was made on the effects of bicuculline, the classical gamma-aminobutyric acid-A receptor antagonist, on heteromeric mouse muscle alphabetagammadelta, heteromeric neuronal rat alpha2beta4 and alpha4beta2 and homomeric human alpha7 nicotinic acetylcholine receptors (nAChRs), expressed in Xenopus oocytes. Bicuculline reduced the ACh-induced currents in a rapid and reversible way, with IC50 values of 34+/-1.5 microM for mouse muscle alphabetagammadelta and 12.4+/-0.7 and 18+/-1 microM for rat neuronal alpha2beta4 and alpha4beta2 nAChRs, respectively. Therefore, the three types of heteromeric receptors are inhibited by bicuculline but the neuronal alpha2beta4 and alpha4beta2 receptors were more sensitive than the muscle alphabetagammadelta receptor. The Hill coefficients for ACh-current inhibition were close to one for all types of receptors, suggesting a single site of action for bicuculline inhibition of nAChRs.Bicuculline shifted the ACh-dose-current response curve to the right and the maximal current was reduced, a reduction that for the heteromeric receptors was not overcome by high concentrations of ACh. The effect of bicuculline was examined at different membrane potentials, and the ACh-current-membrane potential relationships obtained indicate that the inhibition by bicuculline is voltage-dependent for muscle alphabetagammadelta and neuronal alpha2beta4 and alpha4beta2 nAChRs. All these results are consistent with the notion that bicuculline blocks the heteromeric muscle and neuronal nAChRs in a non-competitive way. Studies were also made on the wild type (wt alpha7) and mutant leu-to-threo (L248T) homomeric human neuronal alpha7-nAChRs. In sharp contrast to the heteromeric ACh receptors examined, bicuculline blocked in a competitive way the homomeric wt alpha7-nAChRs, as evidenced by a parallel shift of the bicuculline dose-ACh-current inhibition on raising the ACh concentration. Moreover, similar to the effects of serotonin on wt and mutant alpha7 ACh receptors, the mutation converted bicuculline from an antagonist into a competitive agonist. All this suggests that bicuculline may serve as a lead molecule to design new anticholinergic substances.     

Putative M2 muscarinic receptors of rat heart have high affinity for organophosphorus anticholinesterases

The M2 subtype of muscarinic receptor is predominant in heart, and such receptors were reported to be located in muscles as well as in presynaptic cholinergic and adrenergic nerve terminals. Muscarinic receptors of rat heart were identified by the high affinity binding of the agonist (+)-[3H]cis-methyldioxolane ([3H]CD), which has been used to label a high affinity population of M2 receptors. A single population of sites (KD 2.74 nM; Bmax of 82 fmol/mg protein) was detected and [3H]CD binding was sensitive to the M2 antagonist himbacine but much less so to pirenzepine, the M1 antagonist. These cardiac receptors had different sensitivities to NiCl2 and N-ethylmaleimide from brain muscarinic receptors, that were also labeled with [3H]CD and considered to be of the M2 subtype. Up to 70% of the [3H]CD-labeled cardiac receptors had high affinities for several organophosphate (OP) anticholinesterases. [3H]CD binding was inhibited by the nerve agents soman, VX, sarin, and tabun, with K0.5 values of 0.8, 2, 20, and 50 nM, respectively. It was also inhibited by echothiophate and paraoxon with K0.5 values of 100 and 300 nM, respectively. The apparent competitive nature of inhibition of [3H]CD binding by both sarin and paraoxon suggests that the OPs bind to the acetylcholine binding site of the muscarinic receptor. Other OP insecticides had lower potencies, inhibiting less than 50% of 5 nM [3H]CD binding by 1 microM of EPN, coumaphos, dioxathion, dichlorvos, or chlorpyriphos. There was poor correlation between the potencies of the OPs in reversibly inhibiting [3H]CD binding, and their anticholinesterase activities and toxicities. Acetylcholinesterases are the primary targets for these OP compounds because of the irreversible nature of their inhibition, which results in building of acetylcholine concentrations that activate muscarinic and nicotinic receptors and desensitize them, thereby inhibiting respiration. Nevertheless, the high affinities that cardiac muscarinic receptors have for these toxicants point to their extra vulnerability. It is suggested that the success of iv administration of the muscarinic receptor inhibitor atropine in initial therapy of poisoning by OP anticholinesterases may be related in part to the extra sensitivity of M2 receptors to certain OPs.     

Effects of chain-length and unsaturation on affinity and selectivity at muscarinic receptors.

1. Lengthening the chain in diphenylacetylcholine decreases affinity for muscarinic cholinoceptors in guinea-pig ileum. Diphenylacetoxypropyldimethylamine and its quaternary trimethylammonium salt are roughly equiactive: the dimethylamine and the piperidine have some selectivity for ileum compared with atria, but are not as active nor as selective as 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) methobromide (MeBr). With the weaker diphenylacetoxybutyl compounds the base is more active than the quaternary salt. 2. The diphenylacetoxybutyl-, cis-butenyl and trans-butenyl compounds have similar affinities. The quaternary salts are less active than the tertiary bases, but they are less selective than the butynyl analogues studied in earlier work. 3. 1,1-Diphenyl-1-hydroxy-2,4-hexadiynyl dimethylamine and its trimethylammonium salt are inactive in concentrations below 100 microM, as are the (+)-camphor-sulphonyl ester of 4-hydroxy-N-methyl piperidine and its methiodide. The (+/-)-phenylcyclopentylacetyl ester of 4-hydroxy-N-methylpiperidine methobromide is more active than its cyclohexyl analogue and than 4-DAMP MeBr but it is less selective than 4-DAMP MeBr. 4. The high selectivity of p-fluoro-hexahydrosila-diphenidol is confirmed but this compound has relatively low affinity (for ileum log K = 7.8). 5. The results indicate steric constraints to binding at muscarinic receptors which could be used to check molecular modelling of the receptor based on its known amino acid sequence. The group binding the charged nitrogen is probably at the mouth of a cavity which can accommodate two large rings (as in 4-DAMP MeBr) but with a depth less than about 7 A so that the rod-like hexadiynes cannot fit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apparent down-regulation of muscarinic acetylcholine receptors of neuroblastoma cells by pilocarpine is due to occluded agonist

Down-regulation of muscarinic acetylcholine receptors on N1E 115 neuroblastoma cells caused by muscarinic agonists has been studied. Whereas the potent agonist carbachol provokes a true down-regulation of receptors, the down-regulation caused by the partial agonist pilocarpine is only apparent and is actually due to the presence of pilocarpine taken up by the cells and released into the binding assays upon cell breakage.