Muscarinic Receptor Subtypes Research Articles

The structural study of mutation-induced inactivation of human muscarinic receptor M4.

Human muscarinic receptor M4 belongs to the class A subfamily of the G-protein-coupled receptors (GPCRs). M4 has emerged as an attractive drug target for the treatment of Alzheimer's disease and schizophrenia. Recent results showed that M4-mediated cholinergic transmission is related to motor symptoms in Parkinson's disease. Selective ligand design for the five muscarinic acetylcholine receptor (mAchR) subtypes currently remains challenging owing to the high sequence and structural similarity of their orthosteric binding pockets. In order to obtain M4-selective antagonists, a new approach was tried to lock M4 into an inactive form by rationally designing an N4497.49R mutation, which mimics the allosteric sodium binding in the conserved sodium site usually found in class A GPCRs. In addition, the crystal structure of the mutation-induced inactive M4 was determined. By comparative analysis with other mAchR structures, followed by functional assays, the N4497.49R mutation was shown to stabilize M4 into an inactive state. Virtual screening of a focused ligand library using the crystal structure showed that the inactive M4 prefers antagonists much more than agonists. This study provides a powerful mutation strategy to stabilize GPCRs in inactive states and facilitate their structure determination.

Structure-Activity Relationship Study of Psychostimulant Synthetic Cathinones Reveals Nanomolar Antagonist Potency of α-Pyrrolidinohexiophenone at Human Muscarinic M2 Receptors.

Synthetic cathinones (SCs) are designer, psychostimulant drugs of abuse that primarily act on monoamine transporters; little is known about their off-target liability. Abuse of pyrrolidine-containing SCs, such as α-PHP, has been linked to clinical features, including tachycardia and hypertension, and psychiatric events, including delusions and memory impairments-effects mimicking deliriant hallucinogens that are acetylcholine muscarinic receptor (MR) antagonists. α-PHP and nine analogs with modifications in the α-carbon side chain length and/or containing a methylenedioxy moiety were screened for activity at each of the five human MRs. Increasing the length of the α-carbon side chain of 1-phenyl-2-(pyrrolidin-1-yl)ethan-1-one analogs from a methyl (α-PPP) to a propyl (α-PVP) group caused a steep increase in affinity at all MR subtypes, and one extra carbon (α-PHP) further enhanced MR affinity; the presence of a methylenedioxy moiety generally hindered this effect. Highest MR affinity was observed with α-PHP at M2Rs-its M2R affinity (Ki = 251 nM) was 302-fold higher than α-PPP's. M2R-cAMP inhibition and β-arrestin recruitment assays showed that α-PHP is an M2R antagonist (Kb = 120 and 502 nM, respectively). Additional experiments showed α-PHP is also an antagonist of M1R-inositol phosphate production (Kb = 1.4 μM). Human toxicology studies report blood concentrations of pyrrolidine-containing SCs, including α-PHP, that reach micromolar levels during intoxication, indicating α-PHP's MR activity might have physiological relevance. As M2Rs and M1Rs are widely expressed in the autonomic and central nervous systems, α-PHP's anticholinergic activity might be relevant to adverse events associated with α-PHP intoxication.

Novel M2 -selective, Gi -biased agonists of muscarinic acetylcholine receptors.

More than 30% of currently marketed medications act via GPCRs. Thus, GPCRs represent one of the most important pharmacotherapeutic targets. In contrast to traditional agonists activating multiple signalling pathways, agonists activating a single signalling pathway represent a new generation of drugs with increased specificity and fewer adverse effects. We have synthesized novel agonists of muscarinic ACh receptors and tested their binding and function (on levels of cAMP and inositol phosphates) in CHO cells expressing individual subtypes of muscarinic receptors, primary cultures of rat aortic smooth muscle cells and suspensions of digested native tissues from rats. Binding of the novel compounds to M2 receptors was modelled in silico. Two of the tested new compounds (1-(thiophen-2-ylmethyl)-3,6-dihydro-2H-pyridinium and 1-methyl-1-(thiophen-2-ylmethyl)-3,6-dihydro-2H-pyridinium) only inhibited cAMP synthesis in CHO cells, primary cultures, and native tissues, with selectivity for M2 muscarinic receptors and displaying bias towards the Gi signalling pathway at all subtypes of muscarinic receptors. Molecular modelling revealed interactions with the orthosteric binding site in a way specific for a given agonist followed by agonist-specific changes in the conformation of the receptor. The identified compounds may serve as lead structures in the search for novel non-steroidal and non-opioid analgesics acting via M2 and M4 muscarinic receptors with reduced side effects associated with activation of the phospholipase C signalling pathway.

The M2 muscarinic receptor, in association to M1 , regulates the neuromuscular PKA molecular dynamics.

Muscarinic acetylcholine receptor 1 subtype (M1 ) and muscarinic acetylcholine receptor 2 subtype (M2 ) presynaptic muscarinic receptor subtypes increase and decrease, respectively, neurotransmitter release at neuromuscular junctions. M2 involves protein kinase A (PKA), although the muscarinic regulation to form and inactivate the PKA holoenzyme is unknown. Here, we show that M2 signaling inhibits PKA by downregulating Cβ subunit, upregulating RIIα/β and liberating RIβ and RIIα to the cytosol. This promotes PKA holoenzyme formation and reduces the phosphorylation of the transmitter release target synaptosome-associated protein 25 and the gene regulator cAMP response element binding. Instead, M1 signaling, which is downregulated by M2 , opposes to M2 by recruiting R subunits to the membrane. The M1 and M2 reciprocal actions are performed through the anchoring protein A kinase anchor protein 150 as a common node. Interestingly, M2 modulation on protein expression needs M1 signaling. Altogether, these results describe the dynamics of PKA subunits upon M2 muscarinic signaling in basal and under presynaptic nerve activity, uncover a specific involvement of the M1 receptor and reveal the M1 /M2 balance to activate PKA to regulate neurotransmission. This provides a molecular mechanism to the PKA holoenzyme formation and inactivation which could be general to other synapses and cellular models.

Mechanisms of synergy of autoantibodies to M3 muscarinic acetylcholine receptor and secretory pathway Ca2+/Mn2+-ATPase isoform 1 in patients with non-desmoglein pemphigus vulgaris

Pemphigus vulgaris (PV) is a potentially lethal mucocutaneous blistering disease characterized by IgG autoantibodies (AuAbs) binding to epidermal keratinocytes and inducing a devastating blistering disease affecting oral and/or esophageal surfaces and, sometimes, also the skin. Anti-keratinocyte AuAbs developed by the desmoglein (Dsg) 1/3 AuAb-negative acute PV patients are pathogenic, as they induced acantholysis and epidermal split in the experimental models of PV in vitro and in vivo. These PV patients have various combinations of AuAbs to keratinocyte muscarinic acetylcholine receptor subtype M3 (M3AR), the secretory pathway Ca2+/Mn2+-ATPase isoform 1 (SPCA1), and desmocollin 3 whose relative concentrations correlate with the disease activity. In this study, we identified new molecular mechanisms of the synergistic cooperation of AuAbs to M3AR and SPCA1 in inducing acantholysis in the anti-Dsg 1/3 AuAb-negative PV patients. Anti-M3AR AuAb was found to play an important role in determining the level of intraepidermal split just above the basal cells, caspase to mediate early pro-apoptotic events triggered by anti-SPCA1 AuAb, and the neonatal Fc receptor (FcRn) to contribute to the pathobiological actions of both anti-M3AR and anti-SPCA1 AuAbs. Altogether, these novel results support our original hypothesis that pemphigus acantholysis is a complex disease process (also known as apoptolysis) initiated by AuAbs directed against different keratinocyte proteins that play important roles in supporting cell viability and regulating vital cell functions.

Muscarinic modulation of M and h currents in gerbil spherical bushy cells.

Descending cholinergic fibers innervate the cochlear nucleus. Spherical bushy cells, principal neurons of the anterior part of the ventral cochlear nucleus, are depolarized by cholinergic agonists on two different time scales. A fast and transient response is mediated by alpha-7 homomeric nicotinic receptors while a slow and long-lasting response is mediated by muscarinic receptors. Spherical bushy cells were shown to express M3 receptors, but the receptor subtypes involved in the slow muscarinic response were not physiologically identified yet. Whole-cell patch clamp recordings combined with pharmacology and immunohistochemistry were performed to identify the muscarinic receptor subtypes and the effector currents involved. Spherical bushy cells also expressed both M1 and M2 receptors. The M1 signal was stronger and mainly somatic while the M2 signal was localized in the neuropil and on the soma of bushy cells. Physiologically, the M-current was observed for the gerbil spherical bushy cells and was inhibited by oxotremorine-M application. Surprisingly, long application of carbachol showed only a transient depolarization. Even though no muscarinic depolarization could be detected, the input resistance increased suggesting a decrease in the cell conductance that matched with the closure of M-channels. The hyperpolarization-activated currents were also affected by muscarinic activation and counteracted the effect of the inactivation of M-current on the membrane potential. We hypothesize that this double muscarinic action might allow adaptation of effects during long durations of cholinergic activation.

Biological behaviors of muscarinic receptors in mesenchymal stem cells derived from human placenta and bone marrow.

Objective(s):Cells perform their functional activities by communicating with each other through endogenous substances and receptors. Post-translation, stem cells function properly in new host tissue by carrying specific cell surface receptors. We aimed to characterize muscarinic receptor subtypes in mesenchymal stem cells (MSCs) together with osteogenic and adipogenic differentiation markers.Materials and Methods:mRNA levels of 5 muscarinic receptor subtypes (CHRM1 to 5), BMP-6, and PPARγ during osteogenic and adipogenic differentiation, under the effect of atropine blockade, were measured in MSCs obtained from human fetal membrane (FM) and bone marrow (BM). Additionally, the effect of atropine on differentiation in the 1st, 2nd, and 3rd passages of MSCs, obtained from human FM and BM, were analyzed by RT-qPCR.Results: CHRM1 mRNA levels increased in the FM group, while decreasing in the BM group. We found significant decreases in CHRM3 and CHRM5 mRNA levels in FM and BM groups, respectively. Atropine had variable effects based on cell source and receptor type. BMP-6 mRNA levels in differentiated osteogenic cells increased significantly compared to undifferentiated cells in both FM and BM groups. In MSCs derived from both sources, PPARγ mRNA levels in differentiated adipogenic cells increased significantly. Atropine showed no effect on MSCs differentiation.Conclusion:These results indicate that expressions of muscarinic receptors in MSCs derived from BM and FM can vary and these cells keep the potential of osteogenic and adipogenic differentiation in vitro. Besides, atropine had no effect on adipogenic and osteogenic differentiation of MSCs.

The antidepressant efficacy of the muscarinic antagonist scopolamine: Past findings and future directions.

Scopolamine is a nonselective muscarinic antagonist that has shown relatively rapid antidepressant effects, although to date the results are from limited clinical studies. Scopolamine reportedly has downstream signaling effects thought to be linked to neuroplasticity within glutamatergic synapses and consequent antidepressant action. In psychiatry, clinically validated pathways are unusual and thus merit further research in an effort develop more effect medicines for patients with mood disorders. Thus, we are faced with a unique opportunity to build on the clinical observation associated with scopolamine through reverse translation to identify of targets that retain the clinical efficacy while reducing the side effect profile. This chapter reviews the clinical antidepressant findings with scopolamine, including discussion of differential response across patient subgroups, as well as a review of biomarkers that predict clinical outcome. The preclinical data associated with scopolamine also are reviewed and convey a vision for narrowing in on the therapeutic muscarinic receptor subtype(s) that support the antidepressant effects to guide the development of next generation antimuscarinic drug targets for depression.

Biased M1 receptor-positive allosteric modulators reveal role of phospholipase D in M1-dependent rodent cortical plasticity.

Highly selective, positive allosteric modulators (PAMs) of the M1 subtype of muscarinic acetylcholine receptor have emerged as an exciting new approach to potentially improve cognitive function in patients suffering from Alzheimer's disease and schizophrenia. Discovery programs have produced a structurally diverse range of M1 receptor PAMs with distinct pharmacological properties, including different extents of agonist activity and differences in signal bias. This includes biased M1 receptor PAMs that can potentiate coupling of the receptor to activation of phospholipase C (PLC) but not phospholipase D (PLD). However, little is known about the role of PLD in M1 receptor signaling in native systems, and it is not clear whether biased M1 PAMs display differences in modulating M1-mediated responses in native tissue. Using PLD inhibitors and PLD knockout mice, we showed that PLD was necessary for the induction of M1-dependent long-term depression (LTD) in the prefrontal cortex (PFC). Furthermore, biased M1 PAMs that did not couple to PLD not only failed to potentiate orthosteric agonist-induced LTD but also blocked M1-dependent LTD in the PFC. In contrast, biased and nonbiased M1 PAMs acted similarly in potentiating M1-dependent electrophysiological responses that were PLD independent. These findings demonstrate that PLD plays a critical role in the ability of M1 PAMs to modulate certain central nervous system (CNS) functions and that biased M1 PAMs function differently in brain regions implicated in cognition.

Transcriptomic analysis of \u03b1-synuclein knockdown after T3 spinal cord injury in rats

BackgroundEndogenous α-synuclein (α-Syn) is involved in many pathophysiological processes in the secondary injury stage after acute spinal cord injury (SCI), and the mechanism governing these functions has not been thoroughly elucidated to date. This research aims to characterize the effect of α-Syn knockdown on transcriptional levels after SCI and to determine the mechanisms underlying α-Syn activity based on RNA-seq.ResultThe establishment of a rat model of lentiviral vector-mediated knockdown of α-Syn in Sprague-Dawley rats with T3 spinal cord contusion (LV_SCI group). The results of the RNA-seq analysis showed that there were 337 differentially expressed genes (DEGs) between the SCI group and the LV_SCI group, and 153 DEGs specific to LV_SCI between the (SCI vs LV_SCI) and (SCI vs CON) comparisons. The top 20 biological transition terms were identified by Gene ontology (GO) analysis. The Kyoto Gene and Genomic Encyclopedia (KEGG) analysis showed that the LV_SCI group significantly upregulated the cholinergic synaptic & nicotine addiction and the neuroactive ligand receptor interaction signaling pathway. Enriched chord analysis analyzes key genes. Further cluster analysis, gene and protein interaction network analysis and RT-qPCR results showed that Chrm2 and Chrnb2 together significantly in both pathways. The proliferation of muscarinic cholinergic receptor subtype 2 (Chrm2) and nicotinic cholinergic receptor subtype β2 (Chrnb2), and the neurogenesis were elevated in the injury site of LV_SCI group by immunofluorescence. Further by subcellular localization, the LV_SCI group enhanced the expression of Chrnb2 at the cell membrane.ConclusionKnockdown of α-Syn after SCI enhance motor function and promote neurogenesis probably through enhancing cholinergic signaling pathways and neuroreceptor interactions. This study not only further clarifies the understanding of the mechanism of knockdown of α-Syn on SCI but also helps to guide the treatment strategy for SCI.

Blockade of muscarinic acetylcholine receptor by scopolamine impairs the memory formation of filial imprinting in domestic chicks (Gallus Gallus domesticus)

Filial imprinting in precocial birds is a useful model for studying memory formation in early learning. The intermediate medial mesopallium (IMM) in the dorsal telencephalon is one of the critical brain regions where the releases of several neurotransmitters increase after the start of imprinting training. Among the increased neurotransmitters, the role of acetylcholine in imprinting has remained unclear. Acetylcholine in the mammalian brain plays an important role in encoding new memories. The muscarinic acetylcholine receptor subtype 1 (M1 receptor) and subtype 3 (M3 receptor) in the hippocampus and cortex of mammalian brain have been shown to be necessary for memory encoding. In this study, we examined whether the imprinting acquisition in chick can be impaired by injecting muscarinic acetylcholine receptor (mAChR) antagonist scopolamine into the bilateral IMM. We show that the injection of scopolamine decreased the preference for the imprinting object in the test, but did not affect the number of approaches to the imprinting object during training. Immunoblotting and immunohistochemistry revealed that M3 receptors were expressed in the IMM. Our data suggest that acetylcholine is involved in the memory formation of imprinting through M3 receptors in the IMM. The scopolamine-injected chicks may be useful as an animal model for dementia such as Alzheimer’s disease.

Central muscarinic receptor subtypes (M1 and M3) involved in carbacol-induced hypophagia in neonatal broiler chicken

Aim: Food intake regulated by a complex of physiologic mechanisms in the nervous system. Muscarinergic system has an important role in the central regulation of appetite in mammals, but there is no information for Muscarinic receptors in avian. The purpose of this study was to examine the effects of intracerebroventricular injection of carbachol (cholinergic agonist), Telenzepine (M1 receptor antagonist), AF-DX116 (M2 receptor antagonist), 4-DAMP (M3 receptor antagonist), and PD102807 (M4 receptor antagonist) on feeding behavior in 3-h food-deprived (FD3) neonatal broiler chicken.Materials and Methods: In experiment 1, chicken intracerebroventricular injected with carbachol (125, 250, and 500 nmol). In experiment 2, birds intracerebroventricular injected with telenzepine (125, 250, and 500 nmol). In experiments 3–5, birds intracerebroventricular injected with AF-DX 116 (125, 250, and 500 nmol), 4-DAMP (125, 250, and 500 nmol), and PD102807 (125, 250, and 500 nmol), respectively. In experiment 6, broilers intracerebroventricular injected with carbacol (500 nmol), co-injection of telenzepine (125 nmol)+carbacol (500 nmol), and 4-DAMP (125 nmol)+carbacol (500 nmol). In experiment 7, injection procedure was carbacol (500 nmol), co-injection of AF-DX116 (125 nmol)+carbacol (500 nmol), and PD102807 (125 nmol)+carbacol (500 nmol). Then, food intake measured until 120 min after injection.Results: According to the data, carbachol (250 and 500 nmol) significantly decreased food intake in comparison with control group (P < 0.05). Intracerebroventricular injection of telenzepine (250 and 500 nmol) and 4-DAMP (250 and 500 nmol) significantly increased food intake (P < 0.05). In addition, carbacol-induced hypophagia was significantly attenuated by co-injection of telenzepine + carbacol (P < 0.05). Also, co-injection of 4-DAMP + carbacol decreased the effect of carbacol on food intake (P < 0.05). However, AF-DX116 and PD102807 had no effect on hypophagia induced by carbacol (P > 0.05).Conclusion: These results suggest, hypophagic effect of muscarinergic system is mediated via M1 and M3 receptors in neonatal chicken.

Abstract 501: Selective i n vivo Modulation of Vagal-Muscarinic Signaling Prevents Sudden Cardiac Death in a Pressure Overload Model of Heart Failure

Introduction: Sudden cardiac death (SCD) from ventricular tachyarrhythmias (VT/VF) claims a quarter millions lives/year in USA. Patients with heart failure (HF) have high SCD risk. We previously demonstrated that differential remodeling of cardiac muscarinic receptors plays a critical role in pathogenesis and for effective therapy of HF and SCD risk [PMID: 25733594]. Ongoing clinical trials of chronic vagus nerve stimulation (cVNS) aimed at improving left ventricular (LV) function in HF patients have had mixed preliminary results. However, the effect of cVNS on SCD risk is unexplored. While acute VNS terminates VT/VF, cVNS likely alters the arrhythmogenic substrate. A better understanding of the underlying mechanisms is necessary for the design of new, more effective therapies. Hypothesis: cVNS prevents SCD by reducing calcium, autonomic and redox derangements in LV myocytes and improving repolarization reserve in pressure overloaded hearts. Methods: Guinea pigs are randomized to Sham or aortic constriction (AC) with daily brief (30-min) low-dose β-adrenergic stress (Shami; ACi), with and without in vivo cVNS therapy via an implanted device, with and without in vivo end-organ muscarinic receptor blockade (MRB) using glycopyrrolate. Echocardiography and continuous 24-hour ECG analyses are performed. Animals are sacrificed at 4 weeks for biochemical, excised perfused heart (pressure-volume, electrophysiology, optical mapping) and isolated LV myocyte studies at 37°C. PRELIMINARY RESULTS: Compared to Sham, the M2 subtype of muscarinic receptors are upregulated in AC and ACi, along with reduced calcium cycling, sarcomere shortening and β-adrenergic responsiveness. Whereas ACi+MRB markedly increased SCD incidence, ACi+cVNS prevented SCD. Interestingly, cVNS prolonged but also stabilized the ECG QT interval by reducing dispersion of repolarization in ACi model. Echocardiographic fractional shortening was also higher in ACi+cVNS (52±1%; 4 weeks) compared to ACi (27±2%; 4 weeks) and ACi+MRB (23±5%; 2 weeks). Conclusions: Chronic in vivo perturbation of cholinergic signaling may be an effective strategy for SCD therapy. Ongoing experiments are aimed at dissecting underlying mechanisms and identifying potential targets for SCD therapy.

Alpha2‐adrenoceptor agonists inhibit form‐deprivation myopia in the chick

BackgroundThe putative myopia‐controlling receptor is thought to be muscarinic acetylcholine receptor subtype M4, because mamba toxin‐3 can inhibit form‐deprivation myopia in chicks at a far lower concentration than atropine. However, mamba toxin‐3 is equally potent at the human α1A‐, α1D‐, and α2A‐adrenoceptors. To test the hypothesis that α‐adrenoceptors might be involved in regulation of eye growth, the treatment effects of α2‐adrenoceptor agonists brimonidine, clonidine, and guanfacine, and antagonist yohimbine, on form‐deprivation myopia in the chick were measured.MethodsRight eyes of White Leghorn chicks were goggled with diffusers to induce form‐deprivation myopia; left eyes were left open as controls. Goggled eyes were injected intravitreally with 20 μL of vehicle, or 2, 20, or 200-nmol of brimonidine, clonidine, guanfacine, or yohimbine, 24, 72, and 120-hours after goggle application. Alternatively, myopia was inhibited physiologically by goggle removal for two-hours, and the α2‐adrenoceptor antagonist, yohimbine, was injected to test whether it could block this type of myopia inhibition. One day after the last injection, refractive error and axial length were measured.ResultsBrimonidine (20 and 200-nmol) and clonidine (200-nmol) effectively inhibited experimentally induced increases in negative refractive error and axial elongation. All doses of guanfacine significantly inhibited induced negative refractive error, but only 20 and 200-nmol significantly inhibited axial elongation. Yohimbine had no effect on form‐deprivation myopia, but 200-nmol reduced the myopia‐inhibiting effect of goggle removal.ConclusionHigh concentrations of α2‐adrenoceptor agonists, similar to those required by atropine, inhibited chick form‐deprivation myopia; antagonism by yohimbine had no effect. High‐concentration yohimbine partially interfered with emmetropisation in form‐deprived chicks experiencing normal vision for two-hours per day. These data support the hypothesis that treatment with high concentrations of adrenergic drugs can affect experimentally induced myopia and normal visual processes.

Effects mediated by M2 muscarinic orthosteric agonist on cell growth in human neuroblastoma cell lines

Abstract The role of muscarinic receptors has been largely documented over the past few decades. Recently we demonstrated that the activation of M2 muscarinic receptors arrested cell proliferation and induced apoptosis in glioblastoma and in other tumour types. This paper aims to evaluate the expression of the M2 muscarinic receptor subtypes in different neuroblastoma cell lines and its role in the control of cell proliferation and survival. Neuroblastoma is the most common solid extracranial tumour, appearing during childhood and displaying a differentiated clinical behaviour. Considering the high homology between muscarinic receptor subtypes, we have identified Arecaidine Propargyl Ester (APE) as a selective orthosteric agonist for M2 muscarinic receptors. Using this agonist, we demonstrate how a selective activation of the M2 receptor subtype negatively modulates cell growth without affecting cell survival in different human neuroblastoma cell lines. As similarly demonstrated in other cell types, following the M2 receptor silencing by short-interference RNA, the effects of APE are completely abolished. We conclude by confirming the ability of APE to bind selectively M2 muscarinic receptor subtypes. Moreover, for the first time we demonstrate that M2 receptor activation inhibits cell growth also in human neuroblastoma cells, indicating that M2 receptors may be an interesting therapeutic target in several solid tumours.

Muscarinic M1 and M2 receptor subtypes play opposite roles in LPS-induced septic shock

BackgroundTo compare pharmacologic effects of pirenzepine and AF-DX116, a selective competitive antagonist for M1 and M2 subtype muscarinic cholinergic receptors (mAChRs), respectively, with atropine, a non-selective competitive antagonist for mAChRs, on Lipopolysaccharide (LPS).MethodsMale C57BL/6 mice were used to establish models of LPS-induced experimental endotoxemia. Mice were intraperitoneally injected 10 min prior to LPS injection with control (saline), atropine, pirenzepine and AF-DX116, respectively. Overall survival time was estimated using Kaplan-Meier plots. Inflammatory cytokine tumor necrosis factor-α (TNF-α) was monitored at various intervals after LPS injection and individual reagent administration. Pathological alternations in lungs and liver were analyzed.ResultsPirenzepine and atropine pretreatment improved survival rate of LPS-induced septic shock; in contrast, AF-DX116 accelerated death from sepsis. Moreover, TNF-α plasma level was decreased in response to pirenzepine or atropine, whereas increased in response to AF-DX116. Pirenzepine and atropine relieved whereas AF-DX116 accelerated LPS-induced pulmonary and hepatic injury. Pirenzepine reduced proportion of M1 subtype of macrophages, while AF-DX116 promoted polarization of macrophages to M1 subtype. Pirenzepine pretreatment reduced while AF-DX116 enhanced expression of SOCS3 at mRNA level.ConclusionsThe administration of pirenzepine and atropine may have beneficial effects on septic shock.

Choosing a partner

Structural Biology G protein–coupled receptors (GPCRs) bind ligands outside the cell and trigger events inside the cell by selectively binding and activating specific G proteins. The selectivity occurs even among highly related GPCRs. For example, five subtypes of muscarinic acetylcholine receptors (M1R to M5R) play different roles in the nervous system by binding to different G proteins. Maeda et al. determined cryo–electron microscopy structures of M1R and M2R bound to their respective G proteins. A side-by-side comparison provided a molecular understanding of G protein–coupling selectivity. Science , this issue p. [552][1] [1]: /lookup/doi/10.1126/science.aaw5188

Structures of the M1 and M2 muscarinic acetylcholine receptor/G-protein complexes.

Muscarinic acetylcholine receptors are G protein-coupled receptors that respond to acetylcholine and play important signaling roles in the nervous system. There are five muscarinic receptor subtypes (M1R to M5R), which, despite sharing a high degree of sequence identity in the transmembrane region, couple to different heterotrimeric GTP-binding proteins (G proteins) to transmit signals. M1R, M3R, and M5R couple to the Gq/ 11 family, whereas M2R and M4R couple to the Gi/ o family. Here, we present and compare the cryo-electron microscopy structures of M1R in complex with G11 and M2R in complex with GoA The M1R-G11 complex exhibits distinct features, including an extended transmembrane helix 5 and carboxyl-terminal receptor tail that interacts with G protein. Detailed analysis of these structures provides a framework for understanding the molecular determinants of G-protein coupling selectivity.

Mechanisms of G Protein‐Selectivity in Muscarinic Acetylcholine Receptor Family

Muscarinic acetylcholine receptors are integral membrane proteins that regulate physiological activity. Acetylcholine is a neurotransmitter that binds to the extracellular side of these G protein coupled receptors (GPCRs) activating the Gα subunit of the trimeric G protein inside the cell. There are 5 different muscarinic receptor subtypes (M1–M5), where the same ligand acetylcholine causes M2 and M4 receptors to preferentially couple to the Gαi subunit and the receptors M1, M3, and M5 to preferentially couple to the Gαq subunit. This selectivity of Gα subunits mediates different secondary messenger pathways in the cell: Gαi being responsible for deactivating adenyl cyclase activity and Gαq being responsible for activating phospholipase C. We are using computational biophysical methods to probe the mechanisms of G protein selectivity of this muscarinic receptor family. We have built structural models for the M1 and M2 receptors coupled to Gαi or Gαq proteins and used molecular dynamics simulations to obtain relaxed structures of these complexes, so that the thermodynamic analysis of the receptor G protein interactions can be conducted. The analyses are beginning to show that thermodynamically, some complexes are more stable than others in agreement with corresponding biochemical experiments. In addition, some receptor ‐ G protein interactions may be kinetically controlled. Overall, these studies are beginning to provide us with the missing mechanistic insight into the G protein selectivity of the muscarinic receptors.Support or Funding InformationWe acknowledge the CSUPERB New Investigator Grant. We thank the RISE (GM063787) program for their student support.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The M1 muscarinic acetylcholine receptor subtype is important for retinal neuron survival in aging mice

Muscarinic acetylcholine receptors have been implicated as potential neuroprotective targets for glaucoma. We tested the hypothesis that the lack of a single muscarinic receptor subtype leads to age-dependent neuron reduction in the retinal ganglion cell layer. Mice with targeted disruption of single muscarinic acetylcholine receptor subtype genes (M1 to M5) and wild-type controls were examined at two age categories, 5 and 15 months, respectively. We found no differences in intraocular pressure between individual mouse groups. Remarkably, in 15-month-old mice devoid of the M1 receptor, neuron number in the retinal ganglion cell layer and axon number in the optic nerve were markedly reduced. Moreover, mRNA expression for the prooxidative enzyme, NOX2, was increased, while mRNA expression for the antioxidative enzymes, SOD1, GPx1 and HO-1, was reduced in aged M1 receptor-deficient mice compared to age-matched wild-type mice. In line with these findings, the reactive oxygen species level was also elevated in the retinal ganglion cell layer of aged M1 receptor-deficient mice. In conclusion, M1 receptor deficiency results in retinal ganglion cell loss in aged mice via involvement of oxidative stress. Based on these findings, activation of M1 receptor signaling may become therapeutically useful to promote retinal ganglion cell survival.