Development of a Selective and High Affinity Radioligand, [3H]VU6013720, for the M4 Muscarinic Receptor.

M4 muscarinic receptors are highly expressed in the striatum and cortex, brain regions that are involved in diseases such as Parkinson's disease, schizophrenia, and dystonia. Despite potential therapeutic advantages of specifically targeting the M4 receptor, it has been historically challenging to develop highly selective ligands, resulting in undesired off-target activity at other members of the muscarinic receptor family. Recently, we have reported first-in-class, potent, and selective M4 receptor antagonists. As an extension of that work, we now report the development and characterization of a radiolabeled M4 receptor antagonist, [3H]VU6013720, with high affinity (pKd of 9.5 ± 0.2 at rat M4, 9.7 at mouse M4, and 10 ± 0.1 at human M4 with atropine to define nonspecific binding) and no significant binding at the other muscarinic subtypes. Binding assays using this radioligand in rodent brain tissues demonstrate loss of specific binding in Chrm4 knockout animals. Dissociation kinetics experiments with various muscarinic ligands show differential effects on the dissociation of [3H]VU6013720 from M4 receptors, suggesting a binding site that is overlapping but may be distinct from the orthosteric site. Overall, these results demonstrate that [3H]VU6013720 is the first highly selective antagonist radioligand for the M4 receptor, representing a useful tool for studying the basic biology of M4 as well for the support of M4 receptor-based drug discovery. SIGNIFICANCE STATEMENT: This manuscript describes the development and characterization of a novel muscarinic (M) acetylcholine subtype 4 receptor antagonist radioligand, [3H]VU6013720. This ligand binds to or overlaps with the acetylcholine binding site, providing a highly selective radioligand for the M4 receptor that can be used to quantify M4 protein expression in vivo and probe the selective interactions of acetylcholine with M4 versus the other members of the muscarinic receptor family.

Investigating the Unbinding of Muscarinic Antagonists from the Muscarinic 3 Receptor.

Patient symptom relief is often heavily influenced by the residence time of the inhibitor-target complex. For the human muscarinic receptor 3 (hMR3), tiotropium is a long-acting bronchodilator used in conditions such as asthma or chronic obstructive pulmonary disease (COPD). The mechanistic insights into this inhibitor remain unclear; specifically, the elucidation of the main factors determining the unbinding rates could help develop the next generation of antimuscarinic agents. Using our novel unbinding algorithm, we were able to investigate ligand dissociation from hMR3. The unbinding paths of tiotropium and two of its analogues, N-methylscopolamin and homatropine methylbromide, show a consistent qualitative mechanism and allow us to identify the structural bottleneck of the process. Furthermore, our machine learning-based analysis identified key roles of the ECL2/TM5 junction involved in the transition state. Additionally, our results point to relevant changes at the intracellular end of the TM6 helix leading to the ICL3 kinase domain, highlighting the closest residue L482. This residue is located right between two main protein binding sites involved in signal transduction for hMR3's activation and regulation. We also highlight key pharmacophores of tiotropium that play determining roles in the unbinding kinetics and could aid toward drug design and lead optimization.

Muscarinic acetylcholine receptor activation synergizes the knockdown and toxicity of GABA-gated chloride channel insecticides.

BACKGROUND: Pest management requires continual identification of new physiological targets and strategies to control pests affecting agriculture and public/animal health. We propose the muscarinic system as a target for agrochemicals because of its physiological importance. Unlike the muscarinic system, gamma-amino butyric acid (GABA) receptors are an established insecticide target. Here, we investigated target-site synergism using small molecule probes (agonist and antagonist) against the muscarinic system and their ability to enhance the toxicity of GABAergic insecticides in Drosophila melanogaster (Meigen). RESULTS: Oral delivery of pilocarpine (muscarinic agonist) enhanced the toxicity of dieldrin, fipronil, and lindane, resulting in synergist ratios (SRs) between 4-32-fold (orally delivered) or between 2-67-fold when insecticides were topically applied. The synergism between pilocarpine and the GABA-insecticides was greater than the synergism observed with atropine (muscarinic antagonist), and was greater, or comparable, to the synergism observed with the metabolic inhibitor piperonyl butoxide. In addition to lethality, pilocarpine increased the knockdown of lindane. The mechanism of synergism was also investigated in the central nervous system using extracellular electrophysiology, where pilocarpine (3 µmo/L) lowered the half-maximal inhibitory concentration (IC50 ) of lindane from 1.3 (0.86-1.98) µmol/L to 0.17 (0.14-0.21) µmol/L and fipronil's IC50 from 2.2 (1.54-3.29) µmol/L to 0.56 (0.40-0.77) µmol/L. CONCLUSION: Convergence of the cellular function between the muscarinic and GABAergic systems enhanced the insecticidal activity of GABA receptor blocking insecticides through the modulation of the central nervous system (CNS). The future impact of the findings could be the reduction of the active ingredient needed in a formulation with the development of muscarinic synergists. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

3-Quinuclidinyl benzilate (agent BZ) toxicokinetics in rats.

3-Quinuclidinyl benzilate (BZ) ranks among incapacitating military warfare agents. It acts as a competitive inhibitor on muscarinic receptors leading to non-lethal mental impairment. The present study aimed to investigate toxicokinetics of BZ in rats. Moreover, BZ can be exploited to produce a pharmacological model of Alzheimer's disease; thus, this paper focuses mainly on the BZ distribution to the brain. Wistar rats were administered i.p. with BZ (2 and 10 mg/kg). The BZ concentration was determined using LC-MS/MS in plasma, urine, bile, brain, kidney and liver. The sample preparation was based on a solid phase extraction (liquids) or protein precipitation (organ homogenates). The plasma concentration peaked at 3 min (204.5 ± 55.4 and 2185.5 ± 465.4 ng/ml). The maximal concentration in the brain was reached several minutes later. Plasma elimination half-life was 67.9 ± 3.4 in the 2 mg/kg group and 96.6 ± 27.9 in the 10 mg/kg group. BZ concentrations remained steady in the brain, with slow elimination (t1/2 506.9 ± 359.5 min). Agent BZ is excreted mainly via the urine. Steady BZ concentration in the brain could explain the previously published duration of the significant impairment in passive avoidance tasks in rats after an injection of BZ.

Trospium Chloride Transport by Mouse Drug Carriers of the Slc22 and Slc47 Families.

BACKGROUND: The muscarinic receptor antagonist trospium chloride (TCl) is used for pharmacotherapy of the overactive bladder syndrome. TCl is a hydrophilic positively charged drug. Therefore, it has low permeability through biomembranes and requires drug transporters for distribution and excretion. In humans, the organic cation transporters OCT1 and OCT2 and the multidrug and toxin extrusion MATE1 and MATE2-K carriers showed TCl transport. However, their individual role for distribution and excretion of TCl is unclear. Knockout mouse models lacking mOct1/mOct2 or mMate1 might help to clarify their role for the overall pharmacokinetics of TCl. METHOD: In preparation of such experiments, TCl transport was analyzed in HEK293 cells stably transfected with the mouse carriers mOct1, mOct2, mMate1, and mMate2, respectively. RESULTS: Mouse mOct1, mOct2, and mMate1 showed significant TCl transport with Km values of 58.7, 78.5, and 29.3 µM, respectively. In contrast, mMate2 did not transport TCl but showed MPP+ transport with Km of 60.0 µM that was inhibited by the drugs topotecan, acyclovir, and levofloxacin. CONCLUSION: TCl transport behavior as well as expression pattern were quite similar for the mouse carriers mOct1, mOct2, and mMate1 compared to their human counterparts.

Carbachol dimers with primary carbamate groups as homobivalent modulators of muscarinic receptors.

Although agonists and antagonists of muscarinic receptors have been known for long time, there is renewed interest in compounds (such as allosteric or bitopic ligands, or biased agonists) able to differently and selectively modulate these receptors. As a continuation of our previous research, we designed a new series of dimers of the well-known cholinergic agonist carbachol. The new compounds were tested on the five cloned human muscarinic receptors (hM1-5) expressed in CHO cells by means of equilibrium binding experiments, showing a dependence of the binding affinity on the length and position of the linker connecting the two monomers. Kinetic binding studies revealed that some of the tested compounds were able to slow the rate of NMS dissociation, suggesting allosteric behavior, also supported by docking simulations. Assessment of ERK1/2 phosphorylation on hM1, hM2 and hM3 activation showed that the new compounds are endowed with muscarinic antagonist properties. At hM2 receptors, some compounds were able to stimulate GTPγS binding but not cAMP accumulation, suggesting a biased behavior. Classification, Molecular and cellular pharmacology.

Novel Potent Muscarinic Receptor Antagonists: Investigation on the Nature of Lipophilic Substituents in the 5- and/or 6-Positions of the 1,4-Dioxane Nucleus.

A series of novel 1,4-dioxane analogues of the muscarinic acetylcholine receptor (mAChR) antagonist 2 was synthesized and studied for their affinity at M1-M5 mAChRs. The 6-cyclohexyl-6-phenyl derivative 3b, with a cis configuration between the CH2N+(CH3)3 chain in the 2-position and the cyclohexyl moiety in the 6-position, showed pKi values for mAChRs higher than those of 2 and a selectivity profile analogous to that of the clinically approved drug oxybutynin. The study of the enantiomers of 3b and the corresponding tertiary amine 33b revealed that the eutomers are (2S,6S)-(-)-3b and (2S,6S)-(-)-33b, respectively. Docking simulations on the M3 mAChR-resolved structure rationalized the experimental observations. The quaternary ammonium function, which should prevent the crossing of the blood-brain barrier, and the high M3/M2 selectivity, which might limit cardiovascular side effects, make 3b a valuable starting point for the design of novel antagonists potentially useful in peripheral diseases in which M3 receptors are involved.

Red-Emitting Dibenzodiazepinone Derivatives as Fluorescent Dualsteric Probes for the Muscarinic Acetylcholine M2 Receptor.

Fluorescently labeled dibenzodiazepinone-type muscarinic acetylcholine receptor (MR) antagonists, including dimeric ligands, were prepared using red-emitting cyanine dyes. Probes containing a fluorophore with negative charge showed high M2R affinities (pKi (radioligand competition binding): 9.10-9.59). Binding studies at M1 and M3-M5 receptors indicated a M2R preference. Flow cytometric and high-content imaging saturation and competition binding (M1R, M2R, and M4R) confirmed occupation of the orthosteric site. Confocal microscopy revealed that fluorescence was located mainly at the cell membrane (CHO-hM2R cells). Results from dissociation and saturation binding experiments (M2R) in the presence of allosteric M2R modulators (dissociation: W84, LY2119620, and alcuronium; saturation binding: W84) were consistent with a competitive mode of action between the fluorescent probes and the allosteric ligands. Taken together, these lines of evidence indicate that these ligands are useful fluorescent molecular tools to label the M2R in imaging and binding studies and suggest that they have a dualsteric mode of action.

New Nocobactin Derivatives with Antimuscarinic Activity, Terpenibactins A-C, Revealed by Genome Mining of Nocardia terpenica IFM 0406.

We report a genomics-guided exploration of the metabolic potential of the brasilicardin producer strain Nocardia terpenica IFM 0406. Bioinformatics analysis of the whole genome sequence revealed the presence of a biosynthetic gene cluster presumably responsible for the generation of formerly unknown nocobactin derivatives. Mass spectrometry-assisted isolation led to the identification of three new siderophores, terpenibactins A (1), B (2) and C (3), which belong to the class of nocobactins. Their structures were elucidated by employing spectroscopic techniques. Compounds 1-3 demonstrated inhibitory activity towards the muscarinic M3 receptor, while exhibiting only a low cytotoxicity.

Current Advances in Allosteric Modulation of Muscarinic Receptors.

Allosteric modulators are ligands that bind to a site on the receptor that is spatially separated from the orthosteric binding site for the endogenous neurotransmitter. Allosteric modulators modulate the binding affinity, potency, and efficacy of orthosteric ligands. Muscarinic acetylcholine receptors are prototypical allosterically-modulated G-protein-coupled receptors. They are a potential therapeutic target for the treatment of psychiatric, neurologic, and internal diseases like schizophrenia, Alzheimer's disease, Huntington disease, type 2 diabetes, or chronic pulmonary obstruction. Here, we reviewed the progress made during the last decade in our understanding of their mechanisms of binding, allosteric modulation, and in vivo actions in order to understand the translational impact of studying this important class of pharmacological agents. We overviewed newly developed allosteric modulators of muscarinic receptors as well as new spin-off ideas like bitopic ligands combining allosteric and orthosteric moieties and photo-switchable ligands based on bitopic agents.

The muscarinic receptor antagonist tolterodine inhibits voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells.

We explored the effect of the muscarinic receptor antagonist tolterodine on voltage-dependent K+ (Kv) channels using the patch-clamp technique in coronary arterial smooth muscle cells freshly isolated from rabbits. Tolterodine inhibited Kv channels in a concentration-dependent manner, with an IC50 of 1.71 ± 0.33 µM and Hill coefficient of 0.69 ± 0.03. Tolterodine accelerated the decay rate of Kv channel inactivation. The apparent rate constants of association and dissociation for tolterodine were 1.79 ± 0.13 µM-1s-1, and 3.13 ± 0.96 s-1, respectively. Although 3 µM tolterodine had no effect on the steady-state activation of the Kv current, it shifted the steady-state inactivation curve towards a negative potential. Application of consecutive train steps (1 or 2 Hz) progressively decreased the Kv current and promoted its inhibition. Furthermore, the recovery time constant was augmented in the presence of tolterodine, indicating that tolterodine-induced Kv channel blockade is use (state) dependent. Pretreatment with inhibitors of the Kv1.5, Kv2.1, and Kv7 subtypes (DPO-1, guangxitoxin, and linopirdine) partially reduced the inhibitory effect of tolterodine on Kv channels. The alternative muscarinic receptor antagonist atropine did not inhibit the Kv current nor influence tolterodine-induced inhibition of the Kv current. Tolterodine induced vasoconstriction and membrane depolarization. Based on these results, we conclude that tolterodine inhibits Kv channels in concentration-, time-, and use (state)-dependent manners, irrespective of its antagonism of muscarinic receptors.

Muscarinic M1 receptors stimulated by intracerebroventricular administration of McN-A-343 reduces the nerve injury-induced mechanical hypersensitivity via GABAB receptors rather than GABAA receptors in mice.

Cholinergic neurons play an important role in the higher functions of the brain, such as the memory, cognition, and nociception. However, the exact mechanism behind how the stimulation of all the muscarinic M1 receptors in the entire brain results in the alleviation of partial sciatic nerve ligation (PSNL)-induced mechanical hypersensitivity has not been investigated. Thus, we examined which subtype of GABA receptor was involved in the alleviation of PSNL-induce mechanical hypersensitivity produced by an intracerebroventricular administration of a muscarinic M1 receptor agonist, McN-A-343. Administering a GABAA receptor antagonist, bicuculline, resulted in no changes to the McN-A-343-induced anti-hypersensitivity in PSNL mice whereas a GABAB receptor antagonist, CGP35348, dose-dependently inhibited the anti-hypersensitivity. Furthermore, CGP35348 increased mechanical hypersensitivity in naïve mice, and the hypersensitivity was blocked by NMDA receptor antagonists, MK-801 and D-AP5. Additionally, muscarinic M1 receptors colocalized with GABAB1 receptors and an NMDA receptor subunit, GluN2A, in a large region of the brain. Consequently, these results suggest that the activation of muscarinic M1 receptors in the entire brain reduces nerve injury-induced mechanical hypersensitivity via the GABAB receptors, and the activation of the GABAB receptors regulates glutamatergic transmission via NMDA receptors.

Corticolimbic analysis of microRNAs and protein expressions in scopolamine-induced memory loss under stress.

The aim of the present study was to assess thealterations of corticolimbic microRNAs and protein expressions in the effect of scopolamine with or without stress on passive-avoidance memory in male Wistar rats. The expressions of miR-1, miR-10 and miR-26 and also the levels of p-CREB, CREB, C-FOS and BDNF in the prefrontal cortex (PFC), the hippocampus and the amygdala were evaluated using RT-qPCR and Western blotting techniques. The data showed that the administration of a muscarinic receptor antagonist, scopolamine or the exposure to 30 min stress significantly induced memory loss. Interestingly, the injection of an ineffective dose of scopolamine (0.5 mg/kg) alongside with exposure to an ineffective time of stress (10 min) impaired memory formation, suggesting a potentiative effect of stress on scopolamine response. Our results showed that memory formation was associated with the down-regulated expression of miR-1, miR-10 and miR-26 in the PFC and the hippocampus, but not the amygdala. The relative expression increase of miR-1 and miR-10 in the PFC and the hippocampus was shown in memory loss induced by scopolamine administration or 30-min stress. The PFC level of miR-10 and also hippocampal level of miR-1 and miR-10 were significantly up-regulated, while amygdala miR-1 and miR-26 were down-regulated in scopolamine-induced memory loss under stress. Memory formation increased BDNF, C-FOS and p-CREB/CREB in the PFC, the hippocampus and the amygdala. In contrast, the PFC, hippocampal and amygdala protein expressions were significantly decreased in memory loss induced by scopolamine administration (2 mg/kg), stress exposure (for 30 min) or scopolamine (0.5 mg/kg) plus stress (10 min). One of the most significant findings to emerge from this study is that the stress exposure potentiated the amnesic effect of scopolamine may via affecting the expressions of miRs and proteins in the PFC, the hippocampus and the amygdala. It is possible to hypothesis that corticolimbic signaling pathways play a critical role in relationship between stress and Alzheimer's disease.

Interplay of the Organic Cation Transporters OCT1 and OCT2 with the Apically Localized Export Protein MATE1 for the Polarized Transport of Trospium.

The anticholinergic drug trospium is secreted into urine and, to a smaller extent, into bile. Chemically, it is an organic cation, and it is a substrate of the uptake transporters OCT1 and OCT2 as well as for the export proteins MATE1 and MATE2-K as determined in uptake studies using HEK293 cells. So far, neither MATE-mediated export nor the interplay of OCT-mediated uptake and MATE-mediated export have been investigated. Therefore, we used polarized monolayers of single- and double-transfected MDCKII cells (MDCK-OCT1, MDCK-OCT2, MDCK-MATE1, MDCK-OCT1-MATE1, and MDCK-OCT2-MATE1) and the respective control cells (MDCK-Co) for transcellular transport assays. We demonstrate that the transcellular, basal-to-apical transport of trospium is significantly higher in all cell lines compared to control cells over nearly the complete concentration range tested. The transcellular transport mediated by double-transfected MDCK-OCT1-MATE1 and MDCK-OCT2-MATE1 exceeded that in the single-transfected cells (MDCK-OCT1-MATE1 vs MDCK-OCT1: 2.2-fold; MDCK-OCT1-MATE1 vs MDCK-MATE1: 1.7-fold; MDCK-OCT2-MATE1 vs MDCK-OCT2: 6.1-fold; MDCK-OCT2-MATE1 vs MDCK-MATE1: 1.8-fold at a trospium concentration of 1.0 µM; p < 0.001 each). Thus, we show that MATE1 does not only mediate the uptake of trospium into HEK293 cells but also the efflux of trospium out of polarized MDCKII-cells. Furthermore, our results indicate that OCT1 or OCT2 as uptake transporters and MATE1 as an export protein contribute to the transcellular transport of trospium at concentrations normally reached during trospium therapy. These data suggest that both, OCT-mediated uptake as well as MATE1-mediated efflux may contribute to trospium renal and biliary elimination.

Structure-guided development of selective M3 muscarinic acetylcholine receptor antagonists.

Drugs that treat chronic obstructive pulmonary disease by antagonizing the M3 muscarinic acetylcholine receptor (M3R) have had a significant effect on health, but can suffer from their lack of selectivity against the M2R subtype, which modulates heart rate. Beginning with the crystal structures of M2R and M3R, we exploited a single amino acid difference in their orthosteric binding pockets using molecular docking and structure-based design. The resulting M3R antagonists had up to 100-fold selectivity over M2R in affinity and over 1,000-fold selectivity in vivo. The crystal structure of the M3R-selective antagonist in complex with M3R corresponded closely to the docking-predicted geometry, providing a template for further optimization.

Structural insights into the subtype-selective antagonist binding to the M2 muscarinic receptor.

Human muscarinic receptor M2 is one of the five subtypes of muscarinic receptors belonging to the family of G-protein-coupled receptors. Muscarinic receptors are targets for multiple neurodegenerative diseases. The challenge has been designing subtype-selective ligands against one of the five muscarinic receptors. We report high-resolution structures of a thermostabilized mutant M2 receptor bound to a subtype-selective antagonist AF-DX 384 and a nonselective antagonist NMS. The thermostabilizing mutation S110R in M2 was predicted using a theoretical strategy previously developed in our group. Comparison of the crystal structures and pharmacological properties of the M2 receptor shows that the Arg in the S110R mutant mimics the stabilizing role of the sodium cation, which is known to allosterically stabilize inactive state(s) of class A GPCRs. Molecular dynamics simulations reveal that tightening of the ligand-residue contacts in M2 receptors compared to M3 receptors leads to subtype selectivity of AF-DX 384.

Interaction of pirenzepine with bovine serum albumin and effect of ß-cyclodextrin on binding: A biophysical and molecular docking approach.

Studying the interaction of therapeutic molecules with serum albumin is important to understand their biopharmaceutics, pharmacokinetics and toxicity as well as their relation with the structure and function of protein. The biomolecular interaction of an anti-spasmodic drug, pirenzepine with bovine serum albumin (BSA) was investigated using multi-spectroscopic, calorimetric and docking studies. Fluorescence quenching of BSA on interaction with pirenzepine revealed the static mode of quenching. Pirenzepine exhibited a moderate binding to serum albumin with the binding constant value in the order of 104 M-1. Based on the Forster's non-radiative energy transfer theory, the average binding distance between BSA and pirenzepine was calculated. Competitive site marker experiments demonstrated that pirenzepine binds to the sudlow site III located in subdomain IB of BSA. Circular dichroic spectroscopy indicated secondary structural changes in BSA while three-dimensional fluorescence spectroscopy showed the microenvironmental perturbations in the structure of BSA on interaction with pirenzepine. Moreover, thermodynamic parameters obtained from isothermal titration calorimetry suggested that the interaction between pirenzepine and BSA was spontaneous and hydrophobic interactions played the major role in stabilizing the complex. Additionally, the effect of inclusion compound, ß-cyclodextrin on pirenzepine-BSA interaction was studied. As pirenzepine is involved in drug-drug interactions, ß-cyclodextrin forms an inclusion complex with pirenzepine and prevents drug-drug interactions, thereby, enhancing the therapeutic effect of pirenzepine. Some common metal ions have also been found to interfere with pirenzepine-BSA interaction. The above experimental results further corroborated the molecular modelling studies.

Synthesis of novel and functionally selective non-competitive muscarinic antagonists as chemical probes.

Muscarinic receptors are known to play important biological roles and are drug targets for several human diseases. In a pilot study, novel muscarinic antagonists were synthesized and used as chemical probes to obtain additional information of the muscarinic pharmacophore. The design of these ligands made use of current orthosteric and allosteric models of drug-receptor interactions together with chemical motifs known to achieve muscarinic receptor selectivity. This approach has led to the discovery of several non-competitive muscarinic ligands that strongly bind at a secondary receptor site. These compounds were found to be non-competitive antagonists that completely abolished carbachol activation in functional assays. Several of these compounds antagonized functional response to carbachol with great potency at M1 and M4 than at the rest of receptor subtypes.

Teaching an Old Drug New Tricks: Agonism, Antagonism, and Biased Signaling of Pilocarpine through M3 Muscarinic Acetylcholine Receptor.

Pilocarpine is a prototypical drug used to treat glaucoma and dry mouth and is classified as either a full or partial muscarinic agonist. Here, we report several unexpected results pertaining to its interaction with muscarinic M3 receptor (M3R). We found that pilocarpine was 1000 times less potent in stimulating mouse-eye pupil constriction than muscarinic agonists oxotremorin-M (Oxo-M) or carbachol (CCh), although all three ligands have similar Kd values for M3R. In contrast to CCh or Oxo-M, pilocarpine does not induce Ca2+ mobilization via endogenous M3R in human embryonic kidney cell line 293T (HEK293T) or mouse insulinoma (MIN6) cells. Pilocarpine also fails to stimulate insulin secretion and, instead, antagonizes the insulinotropic effect of Oxo-M and CCh-induced Ca2+ upregulation; however, in HEK293T or Chinese hamster ovary-K1 cells overexpressing M3R, pilocarpine induces Ca2+ transients like those recorded with another cognate G protein-coupled muscarinic receptor, M1R. Stimulation of cells overexpressing M1R or M3R with CCh resulted in a similar reduction in phosphatidylinositol 4,5-bisphosphate (PIP2). In contrast to CCh, pilocarpine stimulated PIP2 hydrolysis only in cells overexpressing M1R but not M3R. Moreover, pilocarpine blocked CCh-stimulated PIP2 hydrolysis in M3R-overexpressing cells, thus, it acted as an antagonist. Pilocarpine activates extracellular regulated kinase 1/2 in MIN6 cells. The stimulatory effect on extracellular regulated kinase (ERK1/2) was blocked by the Src family kinase inhibitor PP2, indicating that the action of pilocarpine on endogenous M3R is biased toward ß-arrestin. Taken together, our findings show that pilocarpine can act as either an agonist or antagonist of M3R, depending on the cell type, expression level, and signaling pathway downstream of this receptor.

Acetylcholine-Atropine Interactions: Paradoxical Effects on Atrial Fibrillation Inducibility.

Atropine (ATr) is well known as a cholinergic antagonist, however, at low concentrations ATr could paradoxically accentuate the parasympathetic actions of acetylcholine (ACh). In 22 pentobarbital anesthetized dogs, via a left and right thoracotomy, a leak-proof barrier was attached to isolate the atrial appendages (AAs) from the rest of the atria. In group 1 (Ach+ATr+Ach), ACh, 100 mM, was placed on the AA followed by the application of ATr, 2 mg/mL. The average atrial fibrillation (AF) duration was 17 ± 7 minutes. After ATr was applied to the AA and ACh again tested, the AF duration was markedly attenuated (2 ± 2 minutes, P < 0.05). In group 2 (ATr+Ach), ATr was initially applied to the AA followed by the application of ACh, 100 mM. There was no significant difference in AF duration (16 ± 4 minutes vs. 18 ± 2 minutes, P = NS). The inhibitory effect of ATr on induced HR reduction (electrical stimulation of the anterior right ganglionated plexi and vagal nerves) was similar between groups 1 and 2. These observations suggest that when ATr is initially administered it attaches to the allosteric site of the muscarinic ACh receptor (M2) leaving the orthosteric site free to be occupied by ACh. The M3 receptor that controls HR slowing does not show the same allosteric properties.