Muscarinic acetylcholine receptor M5 is involved in spermatogenesis through the modification of cell-cell junctions.

Muscarinic acetylcholine receptor (mAChR) antagonists have been reported to decrease male fertility; however, the roles of mAChRs in spermatogenesis and the underlying mechanisms are not understood yet. During spermatogenesis, extensive remodeling between Sertoli cells and/or germ cells interfaces takes place to accommodate the transport of developing germ cells across the blood-testis barrier (BTB) and adluminal compartment. The cell-cell junctions play a vital role in the spermatogenesis process. This study used ICR male mice and spermatogonial cells (C18-4) and Sertoli cells (TM-4). shRNA of control or M5 gene was injected into 5-week-old ICR mice testes. Ten days post-viral grafting, mice were deeply anesthetized with pentobarbital and the testes were collected. One testicle was fresh frozen for RNA-seq analysis or Western blotting (WB). The second testicle was fixed for immunofluorescence staining (IHF). C18-4 or TM-4 cells were treated with shRNA of control or M5 gene. Then, the cells were collected for RNA-seq analysis, WB, or IHF. Knockdown of mAChR M5 disrupted mouse spermatogenesis and damaged the actin-based cytoskeleton and many types of junction proteins in both Sertoli cells and germ cells. M5 knockdown decreased Phldb2 expression in both germ cells and Sertoli cells which suggested that Phldb2 may be involved in cytoskeleton and cell-cell junction formation to regulate spermatogenesis. Our investigation has elucidated a novel role for mAChR M5 in the regulation of spermatogenesis through the interactions of Phldb2 and cell-cell junctions. M5 may be an attractive future therapeutic target in the treatment of male reproductive disorders.

Simultaneous activation of mGlu2 and muscarinic receptors reverses MK-801-induced cognitive decline in rodents.

The activity of an allosteric agonist of muscarinic M1 receptor, VU0357017, and a positive allosteric modulator (PAM) of M5 receptor, VU0238429, were investigated alone or in combination with the mGlu2 receptor PAM, LY487379 using the following behavioural tests: prepulse inhibition (PPI), novel object recognition (NOR), and spatial delayed alternation (SDA). VU0357017 (10 and 20 mg/kg) and VU0238429 (5 and 10 mg/kg) reversed deficits in PPI while VU0238429 (2.5 and 5 mg/kg) was effective in SDA. The simultaneous administration of subeffective doses of M1 or M5 activators (5, 1, or 0.25 mg/kg) with LY487379 (0.5 mg/kg) induced the same effect as that observed for the active dose of each compound. Selective M1 or M5 receptor blockers antagonized the effect exerted by these combinations, and pharmacokinetic studies confirmed independent transport through the blood-brain barrier. The expression of both receptors (M1 and M5) was established in brain structures involved in cognition (neocortex, hippocampus, and entorhinal cortex) in both the rat and the mouse brains by immunofluorescence staining. Specifically, double neuronal staining of mGlu2-M1 and mGlu2-M5 receptors was observed in many areas of the rat brain, while the number of double-stained mGlu2-M1 receptors was moderate in the mouse brain with no mGlu2-M5 colocalization. Finally, the combined administration of subeffective doses of the compounds did not alter prolactin levels or motor coordination, in contrast to the compounds given alone at the highest dose or in combination with standard neuroleptics.

Crystal structure of the M5 muscarinic acetylcholine receptor.

The human M5 muscarinic acetylcholine receptor (mAChR) has recently emerged as an exciting therapeutic target for treating a range of disorders, including drug addiction. However, a lack of structural information for this receptor subtype has limited further drug development and validation. Here we report a high-resolution crystal structure of the human M5 mAChR bound to the clinically used inverse agonist, tiotropium. This structure allowed for a comparison across all 5 mAChR family members that revealed important differences in both orthosteric and allosteric sites that could inform the rational design of selective ligands. These structural studies, together with chimeric swaps between the extracellular regions of the M2 and M5 mAChRs, provided structural insight into kinetic selectivity, where ligands show differential residency times between related family members. Collectively, our study provides important insights into the nature of orthosteric and allosteric ligand interaction across the mAChR family that could be exploited for the design of selective drugs.

Acute Negative Allosteric Modulation of M5 Muscarinic Acetylcholine Receptors Inhibits Oxycodone Self-Administration and Cue-Induced Reactivity with No Effect on Antinociception.

Opioid use disorder (OUD) is a debilitating neuropsychiatric condition characterized by compulsive opioid use, dependence, and repeated relapse after periods of abstinence. Given the high risk of developing OUD following prescription opioid use, the continued need for opioid-induced analgesia, and the limitations of current OUD treatments, it is necessary to develop novel, non-opioid-based treatments for OUD and decrease abuse potential of prescription opioids. Recent evidence suggests that negative allosteric modulation (NAM) of the M5 muscarinic acetylcholine receptor (M5 mAChR) may provide an alternative therapeutic approach for the treatment of OUD. Previous studies demonstrated localization of M5 mAChR expression within the mesocorticolimbic reward circuitry and that the selective M5 NAM ML375 attenuates both cocaine and alcohol self-administration in rats. In the present study, the effects of ML375 were evaluated in rats self-administering the µ-opioid agonists oxycodone or remifentanil on a progressive ratio (PR) schedule or on cue reactivity (a rodent model of relapse) in the absence of oxycodone following 72 h of abstinence. ML375 reduced the PR break point for oxycodone and remifentanil self-administration and attenuated cue-elicited responding. Importantly, ML375 did not affect sucrose pellet-maintained responding on a PR schedule or opioid-induced antinociception using the hot-plate and tail-flick assays. We also confirm expression of M5 mAChR mRNA in the ventral tegmental area and show that this is primarily on dopamine (tyrosine hydroxylase mRNA-positive) neurons. Taken together, these findings suggest that selective functional antagonism of the M5 mAChR may represent a novel, non-opioid-based treatment for OUD.

Muscarinic M5 receptors trigger acetylcholine-induced Ca2+ signals and nitric oxide release in human brain microvascular endothelial cells.

Basal forebrain neurons control cerebral blood flow (CBF) by releasing acetylcholine (Ach), which binds to endothelial muscarinic receptors to induce nitric (NO) release and vasodilation in intraparenchymal arterioles. Nevertheless, the mechanism whereby Ach stimulates human brain microvascular endothelial cells to produce NO is still unknown. Herein, we sought to assess whether Ach stimulates NO production in a Ca2+ -dependent manner in hCMEC/D3 cells, a widespread model of human brain microvascular endothelial cells. Ach induced a dose-dependent increase in intracellular Ca2+ concentration ([Ca2+ ]i ) that was prevented by the genetic blockade of M5 muscarinic receptors (M5-mAchRs), which was the only mAchR isoform coupled to phospholipase Cß (PLCß) present in hCMEC/D3 cells. A comprehensive real-time polymerase chain reaction analysis revealed the expression of the transcripts encoding for type 3 inositol-1,4,5-trisphosphate receptors (InsP3 R3), two-pore channels 1 and 2 (TPC1-2), Stim2, Orai1-3. Pharmacological manipulation showed that the Ca2+ response to Ach was mediated by InsP3 R3, TPC1-2, and store-operated Ca2+ entry (SOCE). Ach-induced NO release, in turn, was inhibited in cells deficient of M5-mAchRs. Likewise, Ach failed to increase NO levels in the presence of l-NAME, a selective NOS inhibitor, or BAPTA, a membrane-permeant intracellular Ca2+ buffer. Moreover, the pharmacological blockade of the Ca2+ response to Ach also inhibited the accompanying NO production. These data demonstrate for the first time that synaptically released Ach may trigger NO release in human brain microvascular endothelial cells by stimulating a Ca2+ signal via M5-mAchRs.

Gßγ subunit activation promotes dopamine efflux through the dopamine transporter.

The dopamine transporter (DAT) is an important regulator of brain dopamine (DA) homeostasis, controlling the intensity and duration of DA signaling. DAT is the target for psychostimulants-like cocaine and amphetamine-and plays an important role in neuropsychiatric disorders, including attention-deficit hyperactivity disorder and drug addiction. Thus, a thorough understanding of the mechanisms that regulate DAT function is necessary for the development of clinical interventions to treat DA-related brain disorders. Previous studies have revealed a plethora of protein-protein interactions influencing DAT cellular localization and activity, suggesting that the fine-tuning of DA homeostasis involves multiple mechanisms. We recently reported that G-protein beta-gamma (Gßγ) subunits bind directly to DAT and decrease DA clearance. Here we show that Gßγ induces the release of DA through DAT. Specifically, a Gßγ-binding/activating peptide, mSIRK, increases DA efflux through DAT in heterologous cells and primary dopaminergic neurons in culture. Addition of the Gßγ inhibitor gallein or DAT inhibitors prevents this effect. Residues 582 to 596 in the DAT carboxy terminus were identified as the primary binding site of Gßγ. A TAT peptide containing the Gßγ-interacting domain of DAT blocked the ability of mSIRK to induce DA efflux, consistent with a direct interaction of Gßγ with the transporter. Finally, activation of a G-protein-coupled receptor, the muscarinic M5R, results in DAT-mediated DA efflux through a Gßγ-dependent mechanism. Collectively, our data show that Gßγ interacts with DAT to promote DA efflux. This novel mechanism may have important implications in the regulation of brain DA homeostasis.

Molecular Mechanisms of Action of M5 Muscarinic Acetylcholine Receptor Allosteric Modulators.

Recently, the first subtype-selective allosteric modulators of the M5 muscarinic acetylcholine receptor (mAChR) have been described, but their molecular mechanisms of action remain unknown. Using radioligand-binding and functional assays of inositol phosphate (IP) accumulation and Ca(2+) mobilization in a recombinant cell line stably expressing the human M5 mAChR, we investigated the effects of the positive allosteric modulator (PAM), ML380, and negative allosteric modulator, ML375. In functional assays, ML380 caused robust enhancements in the potency of the full agonists, acetylcholine (ACh), carbachol, and oxotremorine-M, while significantly increasing the maximal response to the partial agonist, pilocarpine. ML380 also demonstrated direct allosteric agonist activity. In contrast, ML375 displayed negative cooperativity with each of the agonists in a manner that varied with the pathway investigated and progressively reduced the maximal pilocarpine response. Radioligand-binding affinity cooperativity estimates were consistent with values derived from functional assays in some instances but not others, suggesting additional allosteric effects on orthosteric ligand efficacy. For ML375 this was confirmed in IP assays performed after reduction of receptor reserve by the alkylating agent, phenoxybenzamine, as it reduced the maximal ACh response. In contrast, ML380 enhanced only ACh potency after receptor alkylation, with no effect on maximal response, consistent with studies of the M1 mAChR with the prototypical PAM, BQZ12. Interaction studies between ML380 and ML375 also indicated that they most likely used an overlapping allosteric site. Our findings indicate that novel small-molecule modulators of the M5 mAChR display mixed mechanisms of action compared with previously characterized modulators of other mAChRs.

Muscarinic acetylcholine receptor binding affinities of pethidine analogs.

A series of pethidine analogs were synthesized and their affinities for the [(3)H]N-methyl-scopolamine (NMS) binding site on muscarinic acetylcholine receptors (mAChRs) were determined using M1, M3 or M5 human mAChRs expressed by Chinese hamster ovary (CHO) cell membranes. Compound 6b showed the highest binding affinities at M1, M3 and M5 mAChRs (Ki=0.67, 0.37, and 0.38 µM, respectively).

Further optimization of the M5 NAM MLPCN probe ML375: tactics and challenges.

This Letter describes the continued optimization of the MLPCN probe ML375, a highly selective M5 negative allosteric modulator (NAM), through a combination of matrix libraries and iterative parallel synthesis. True to certain allosteric ligands, SAR was shallow, and the matrix library approach highlighted the challenges with M5 NAM SAR within in this chemotype. Once again, enantiospecific activity was noted, and potency at rat and human M5 were improved over ML375, along with slight enhancement in physiochemical properties, certain in vitro DMPK parameters and CNS distribution. Attempts to further enhance pharmacokinetics with deuterium incorporation afforded mixed results, but pretreatment with a pan-P450 inhibitor (1-aminobenzotriazole; ABT) provided increased plasma exposure.

Development of a highly potent, novel M5 positive allosteric modulator (PAM) demonstrating CNS exposure: 1-((1H-indazol-5-yl)sulfoneyl)-N-ethyl-N-(2-(trifluoromethyl)benzyl)piperidine-4-carboxamide (ML380).

A functional high throughput screen identified a novel chemotype for the positive allosteric modulation (PAM) of the muscarinic acetylcholine receptor (mAChR) subtype 5 (M5). Application of rapid analog, iterative parallel synthesis efficiently optimized M5 potency to arrive at the most potent M5 PAMs prepared to date and provided tool compound 8n (ML380) demonstrating modest CNS penetration (human M5 EC50 = 190 nM, rat M5 EC50 = 610 nM, brain to plasma ratio (Kp) of 0.36).

Treatment with LPS plus INF-γ induces the expression and function of muscarinic acetylcholine receptors, modulating NIH3T3 cell proliferation: participation of NOS and COX.

BACKGROUND AND PURPOSE: LPS and IFN-γ are potent stimuli of inflammation, a process in which fibroblasts are frequently involved. We analysed the effect of treatment with LPS plus IFN-γ on the expression and function of muscarinic acetylcholine receptors in NIH3T3 fibroblasts with regards to proliferation of these cells. We also investigated the participation of NOS and COX, and the role of NF-κB in this process. EXPERIMENTAL APPROACH: NIH3T3 cells were treated with LPS (10 ng·mL(-1)) plus IFN-γ (0.5 ng·mL(-1)) for 72 h (iNIH3T3 cells). Cell proliferation was evaluated with MTT and protein expression by Western blot analysis. NOS and COX activities were measured by the Griess method and radioimmunoassay respectively. KEY RESULTS: The cholinoceptor agonist carbachol was more effective at stimulating proliferation in iNIH3T3 than in NIH3T3 cells, probably due to the de novo induction of M3 and M5 muscarinic receptors independently of NF-κB activation. iNIH3T3 cells produced higher amounts of NO and PGE2 than NIH3T3 cells, concomitantly with an up-regulation of NOS1 and COX-2, and with the de novo induction of NOS2/3 in inflamed cells. We also found a positive feedback between NOS and COX that could potentiate inflammation. CONCLUSIONS AND IMPLICATIONS: Inflammation induced the expression of muscarinic receptors and, therefore,stimulated carbachol-induced proliferation of fibroblasts. Inflammation also up-regulated the expression of NOS and COX-2, thus potentiating the effect of carbachol on NO and PGE2 production. A positive crosstalk between NOS and COX triggered by carbachol in inflamed cells points to muscarinic receptors as potential therapeutic targets in inflammation.

Discovery, synthesis and characterization of a highly muscarinic acetylcholine receptor (mAChR)-selective M5-orthosteric antagonist, VU0488130 (ML381): a novel molecular probe.

Of the five G-protein-coupled muscarinic acetylcholine receptors (mAChRs; M1-M5), M5 is the least explored and understood due to a lack of mAChR subtype-selective ligands. We recently performed a high-throughput functional screen and identified a number of weak antagonist hits that are selective for the M5 receptor. Here, we report an iterative parallel synthesis and detailed molecular pharmacologic profiling effort that led to the discovery of the first highly selective, central nervous system (CNS)-penetrant M5-orthosteric antagonist, with sub-micromolar potency (hM5 IC50=450 nM, hM5 Ki=340 nM, M1-M4 IC50>30 µM), enantiospecific inhibition, and an acceptable drug metabolism and pharmacokinetics (DMPK) profile for in vitro and electrophysiology studies. This compound will be a powerful tool and molecular probe for the further investigation into the role of M5 in addiction and other diseases.

M5 receptor activation produces opposing physiological outcomes in dopamine neurons depending on the receptor's location.

Of the five muscarinic receptor subtypes, the M5 receptor is the only one detectable in midbrain dopaminergic neurons, making it an attractive potential therapeutic target for treating disorders in which dopaminergic signaling is disrupted. However, developing an understanding of the role of M5 in regulating midbrain dopamine neuron function has been hampered by a lack of subtype-selective compounds. Here, we extensively characterize the novel compound VU0238429 and demonstrate that it acts as a positive allosteric modulator with unprecedented selectivity for the M5 receptor. We then used VU0238429, along with M5 knock-out mice, to elucidate the role of this receptor in regulating substantia nigra pars compacta (SNc) neuron physiology in both mice and rats. In sagittal brain slices that isolate the SNc soma from their striatal terminals, activation of muscarinic receptors induced Ca2+ mobilization and inward currents in SNc dopamine neurons, both of which were potentiated by VU0238429 and absent in M5 knock-out mice. Activation of M5 also increased the spontaneous firing rate of SNc neurons, suggesting that activation of somatodendritic M5 increases the intrinsic excitability of SNc neurons. However, in coronal slices of the striatum, potentiation of M5 with VU0238429 resulted in an inhibition in dopamine release as monitored with fast scan cyclic voltammetry. Accordingly, activation of M5 can lead to opposing physiological outcomes depending on the location of the receptor. Although activation of somatodendritic M5 receptors on SNc neurons leads to increased neuronal firing, activation of M5 receptors in the striatum induces an inhibition in dopamine release.

Discovery of the first M5-selective and CNS penetrant negative allosteric modulator (NAM) of a muscarinic acetylcholine receptor: (S)-9b-(4-chlorophenyl)-1-(3,4-difluorobenzoyl)-2,3-dihydro-1H-imidazo[2,1-a]isoindol-5(9bH)-one (ML375).

A functional high throughput screen and subsequent multidimensional, iterative parallel synthesis effort identified the first muscarinic acetylcholine receptor (mAChR) negative allosteric modulator (NAM) selective for the M5 subtype. ML375 is a highly selective M5 NAM with submicromolar potency (human M5 IC50 = 300 nM, rat M5 IC50 = 790 nM, M1-M4 IC50 > 30 µM), excellent multispecies PK, high CNS penetration, and enantiospecific inhibition.

Cholinergic control of morphine-induced locomotion in rostromedial tegmental nucleus versus ventral tegmental area sites.

M5 muscarinic acetylcholine receptors expressed on ventral tegmental dopamine (DA) neurons are needed for opioid activation of DA outputs. Here, the M5 receptor gene was bilaterally transfected into neurons in the ventral tegmental area (VTA) or the adjacent rostromedial tegmental nucleus (RMTg) in mice by means of a Herpes simplex viral vector (HSV) to increase the effect of endogenous acetylcholine. Three days after HSV-M5 gene infusion in VTA sites, morphine-induced locomotion more than doubled at two doses, while saline-induced locomotion was unaffected. When the HSV-M5 gene was infused into the adjacent RMTg, morphine-induced locomotion was strongly inhibited. The sharp boundary between these opposing effects was found where tyrosine hydroxylase (TH) and cholinesterase labelling decreases (-4.00 mm posterior to bregma). The same HSV-M5 gene transfections in M5 knockout mice induced even stronger inhibitory behavioural effects in RMTg but more variability in VTA sites due to stereotypy. The VTA sites where HSV-M5 increased morphine-induced locomotion receive direct inputs from many RMTg GAD-positive neurons, and from pontine ChAT-positive neurons, as shown by cholera-toxin B retrograde tracing. Therefore, morphine-induced locomotion was decreased by M5 receptor gene expression in RMTg GABA neurons that directly inhibit VTA DA neurons. Conversely, enhancing M5 receptor gene expression on VTA DA neurons increased morphine-induced locomotion via cholinergic inputs.

Somatodendritic targeting of M5 muscarinic receptor in the rat ventral tegmental area: implications for mesolimbic dopamine transmission.

Muscarinic modulation of mesolimbic dopaminergic neurons in the ventral tegmental area (VTA) plays an important role in reward, potentially mediated through the M5 muscarinic acetylcholine receptor (M5R). However, the key sites for M5R-mediated control of dopamine neurons within this region are still unknown. To address this question we examined the electron microscopic immunocytochemical localization of antipeptide antisera against M5R and the plasmalemmal dopamine transporter (DAT) in single sections through the rat VTA. M5R was located mainly to VTA somatodendritic profiles (71%; n = 627), at least one-third (33.2%; n = 208) of which also contained DAT. The M5R immunoreactivity was distributed along cytoplasmic tubulovesicular endomembrane systems in somata and large dendrites, but was more often located at plasmalemmal sites in small dendrites, the majority of which did not express DAT. The M5R-immunoreactive dendrites received a balanced input from unlabeled terminals forming either asymmetric or symmetric synapses. Compared with dendrites, M5R was less often seen in axon terminals, comprising only 10.8% (n = 102) of the total M5R-labeled profiles. These terminals were usually presynaptic to unlabeled dendrites, suggesting that M5R activation can indirectly modulate non-DAT-containing dendrites through presynaptic mechanisms. Our results provide the first ultrastructural evidence that in the VTA, M5R has a subcellular location conducive to major involvement in postsynaptic signaling in many dendrites, only some of which express DAT. These findings suggest that cognitive and rewarding effects ascribed to muscarinic activation in the VTA can primarily be credited to M5R activation at postsynaptic plasma membranes distinct from dopamine transport.

Muscarinic receptor M3 mediates human gallbladder contraction through voltage-gated Ca2+ channels and Rho kinase.

OBJECTIVE: Muscarinic receptors mediate contraction of the human gallbladder through unclear receptor subtypes. The aim of the present study was to characterize muscarinic acetylcholine receptors mediating contraction of the human gallbladder. MATERIALS AND METHODS: Contraction of human gallbladder muscle strips caused by agonists carbachol and muscarine was measured and the inhibition of carbachol-induced contraction by muscarinic receptor antagonists was evaluated. Reverse transcription polymerase chain reaction was performed to determine the existence of muscarinic receptor subtypes. RESULTS: Carbachol and muscarine caused concentration-dependent contraction of gallbladder strips. Four receptor antagonists, including atropine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), methoctramine, and pirenzepine, inhibited the carbachol-induced contraction. The relative inhibitory potency of these receptor antagonists was atropine > 4-DAMP > methoctramine > pirenzepine. The antagonist affinity estimates (pA(2) values) correlated with the known affinities at M(3), M(4), and M(5) muscarinic receptors. In addition, the M(4)-selective antagonist muscarinic toxin 3 did not inhibit and the M(5)-selective positive allosteric modulator VU0238429 did not potentiate carbachol-induced gallbladder contraction. This suggests that M(3) muscarinic receptors mediate the muscarinic response predominantly. The contractile response of carbachol was attenuated by the voltage-gated Ca(2+) channel inhibitor nifedipine and Rho-kinase inhibitor H-1152, but not affected by protein kinase C inhibitor chelerythrine. This implies the involvement of voltage-gated Ca(2+) channel and Rho kinase but not protein kinase C. CONCLUSIONS: These results suggest a major role of M(3) muscarinic receptors mediating the human gallbladder contraction through voltage-gated Ca(2+) channels and Rho kinase. M(3)-selective muscarinic receptor antagonists could be of therapeutic importance in the treatment of biliary motility disorders.

Adenosine receptor antagonism suppresses functional and histological inflammatory changes in the rat urinary bladder.

Cyclophosphamide (CYP) induces an interstitial cystitis-like inflammation. The resulting bladder dysfunction has been associated with increased release of adenosine-5'-triphosphate (ATP), structural bladder wall changes and contractile impairment. Due to the inflammatory modulatory effects of purines it was presently wondered if pre-treatment with P1 and P2 purinoceptor antagonists affect the CYP-induced alterations. Rats were pre-treated with saline or antagonists for five days, and 60 h before the in vitro functional examination the rats were administered either saline or CYP. Histological examination revealed CYP-induced bladder wall thickening largely depending on submucosal enlargement, mast cell invasion of the detrusor muscle, increase in muscarinic M5 receptor expression and macrophage migration inhibitory factor (MIF) occurrence in large parts of the urothelium. Functionally, methacholine- and ATP-evoked contractions were smaller in urinary bladders from CYP-treated rats. Pre-treatment with the P2 purinoceptor antagonist suramin and the P1A2B antagonist PSB1115 did not to any great extent affect the CYP-induced changes. The P1A1 antagonist DPCPX, however, abolished the difference of methacholine-evoked contractions between saline- and CYP-treated rats. ATP-evoked contractions were reduced in control after the DPCPX pre-treatment, but not in cystitis. The functional observations for DPCPX were supported by its suppression of CYP-induced submucosal thickening, muscarinic M5 receptor expression and, possibly, detrusor mast cell infiltration and the spread of urothelial MIF occurrence. Thus, P1A1 is an important pro-inflammatory receptor in the acute CYP-induced cystitis and a P1A1 blockade during the initial phase may suppress CYP-induced cystitis. P1A1 purinoceptors seem to regulate contractility in healthy and in inflamed rat urinary bladders.

Reconciling neuronally and nonneuronally derived acetylcholine in the regulation of immune function.

Immune cells, including lymphocytes, express muscarinic and nicotinic acetylcholine (ACh) receptors (mAChRs and nAChRs, respectively), and agonist stimulation of these AChRs causes functional and biochemical changes in the cells. The origin of the ACh that acts on immune cell AChRs has remained unclear until recently, however. In 1995, we identified choline acetyltransferase mRNA and protein in human T cells, and found that immunological T cell activation potentiated lymphocytic cholinergic transmission by increasing ACh synthesis and AChR expression. We also found that M(1) /M(5) mAChR signaling upregulates IgG(1) and proinflammatory cytokine production, whereas α7 nAChR signaling has the opposite effect. These findings suggest that ACh synthesized by T cells acts as an autocrine and/or paracrine factor via AChRs on immune cells to modulate immune function. In addition, a recently discovered endogenous allosteric α7 nAChR ligand, SLURP-1, also appears to be involved in modulating normal T cell function.

Competition radioligand binding assays for the investigation of bispyridinium compound affinities to the human muscarinic acetylcholine receptor subtype 5 (hM(5) ).

Standard treatment of poisoning by organophosphorus (OP) nerve agents with atropine and oximes lacks efficacy with some nerve agents. Promising in vitro and in vivo results were obtained with the bispyridinium compound SAD-128 which was partly attributed to its interaction with nicotinic acetylcholine receptors. Previous studies indicate that bispyridinium compounds interact with muscarinic acetylcholine receptors as well. The muscarinic M(5) receptor is not well investigated compared to other subtypes, but could be important in the search for new drugs for treating nerve agent poisoning. A set of bispyridinium compounds structurally related to SAD-128 were tested in competition binding experiments with recombinant human M(5) muscarinic acetylcholine receptors. Five of the six investigated bispyridinium compounds interacted with the orthosteric binding site, with affinities in the low micromolar range. These data indicate that interaction of bispyridinium compounds with muscarinic receptors may contribute to their therapeutic efficacy.