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The chemogenetic technology designer receptors exclusively activated by designer drugs (DREADDs) afford remotely reversible control of cellular signaling, neuronal activity and behavior. Although the combination of muscarinic-based DREADDs with clozapine-N-oxide (CNO) has been widely used, sluggish kinetics, metabolic liabilities and potential off-target effects of CNO represent areas for improvement. Here, we provide a new high-affinity and selective agonist deschloroclozapine (DCZ) for muscarinic-based DREADDs. Positron emission tomography revealed that DCZ selectively bound to and occupied DREADDs in both mice and monkeys. Systemic delivery of low doses of DCZ (1 or 3 μg per kg) enhanced neuronal activity via hM3Dq within minutes in mice and monkeys. Intramuscular injections of DCZ (100 μg per kg) reversibly induced spatial working memory deficits in monkeys expressing hM4Di in the prefrontal cortex. DCZ represents a potent, selective, metabolically stable and fast-acting DREADD agonist with utility in both mice and nonhuman primates for a variety of applications. Deschloroclozapine (DCZ) is a broadly useful chemogenetic agonist for studies using nonhuman primates and mice. DCZ rapidly and reversibly activates DREADDs, and its binding can be visualized noninvasively by positron emission tomography.

Dopamine (DA) signaling in the basal ganglia plays important roles in motor control. Motor deficiencies were previously reported in dopamine receptor D1 (D1R) and D2 (D2R) knockout mice. While these results indicate the involvement of DA receptors in motor execution, the null knockout (KO) mouse lacks the specificity necessary to determine when and where in the brain D1R and D2R function in motor execution. To address these questions, we restricted the loss of function temporally and spatially by using D1R conditional knockdown (cKD) mice and mice injected with antagonists against DA receptors directly into the dorsal striatum. In addition, we address the DA and acetylcholine (ACh) balance hypothesis by using antagonists against ACh receptors. We tested the motor ability of the mice with a newly devised task named the accelerating step-wheel. In this task, the maximum running speed was measured in a situation where the wheel rotation speed was gradually accelerated in one trial. We found significant decreases in the maximum running speed of D1R cKD mice and the mice injected with the antagonist against D1R or muscarinic ACh receptor. These results indicated that D1R and muscarinic ACh receptor in the dorsal striatum play pivotal roles in the execution of walking/running.

OBJECTIVE To investigate the mechanism of cAMP-PKA signaling pathway mediated by Chinese medicine formula Shaoyao Gancao Decoction (, SGD) on the regulation of aquaporin 5 (AQP5) and muscarinic receptor 3 (M3R) levels in Sjogren's syndrome (SS). METHODS Of the 30 mice, 5 were randomly selected as control, and others were used for creating SS model. After successful modeling, mice were randomly divided into 5 groups (n=5 per group) and intragastrically administered with saline (8 mL/kg), pilocarpine (1.4 mg/kg), or low, medium and high doses SGD (0.14, 0.21, 0.35 g/kg Radix paeoniae with 0.01 g/kg Radix glycyrrhizae, respectively) for 6 weeks. Human labial gland acinar cells were treated with pilocarpine or varying doses of SGD with saline as the placebo. Hematoxylin and eosin staining was used to observe the histopathological changes of the submandibular glands of mice. The serum levels of anti-SS antigen A (SS-A), anti-SS antigen B (SS-B), M3R, and α-fodrin in submandibular glands of mice were measured by enzyme-linked immunosorbent assay. Immunofluorescence staining was used to observe the spatial localization of AQP5 and M3R in acinar cells. Reverse transcriptase polymerase chain reaction and Western blot were used to detect the expressions of PKA, cAMP, Epac1, AQP5, M3R, nuclear factor kappa-B (NF-κB), and tumor necrosis factor (TNF)-α in submandibular gland tissues and cells of each group. RESULTS Compared to normal mice, body weight, 5-min salivary secretion, 30-min secretion of tears and breakup time of tear film of model mice decreased at 1-6 weeks after immunization (all P

Though immunomodulation via cholinergic neurotransmitter acetylcholine (ACh), an important part of neuroendocrine-immune (NEI) regulatory network, has been well established in vertebrate species, the mechanisms remain poorly understood in invertebrates. In the present study, the immunomodulatory effect of ACh on haemocyte phagocytosis was investigated in an invertebrate bivalve species, Tegillarca granosa. Data obtained showed that in vitro ACh incubation suppressed phagocytic activity of haemocytes along with a significant elevation in intracellular Ca2+. In addition, the expressions of genes from Ca2+ signaling pathway were significantly induced whereas those from NF-κB signaling pathway were significantly down-regulated by ACh incubation. Furthermore, these adverse impacts of ACh were significantly relieved by the blocking of muscarinic acetylcholine receptors (mAChRs) or nicotinic acetylcholine receptors (nAChRs) using corresponding antagonists. Our study suggests that ACh suppresses phagocytosis via binding to both mAChRs and nAChRs, which disrupts intracellular Ca2+ homeostasis and subsequently interferes with downstream Ca2+ and NF-κB signaling pathways.

Aims Pharmaco‐genetics emerges as a new promising approach for epileptic seizures. Whether it can modulate epileptogenesis is still unknown. Methods Here, parvalbumin neurons and pyramidal neurons of the seizure focus were transfected with engineered excitatory Gq‐coupled human muscarinic receptor hM3Dq and engineered inhibitory Gi‐coupled human muscarinic receptor hM4Di, respectively. And their therapeutic value in mouse hippocampal kindling‐induced epileptogenesis was tested. Results Pharmaco‐genetic activating parvalbumin neurons limitedly retarded the progression of behavioral seizure stage and afterdischarge duration (ADD) during epileptogenesis induced by kindling. Activating parvalbumin neurons delayed seizure development only in the early stage, but accelerated it in late stages. On the contrary, pharmaco‐genetic inhibiting pyramidal neurons robustly retarded the progression of seizure stages and ADDs, which greatly delayed seizure development in both early and late stages. Although both pharmaco‐genetic therapeutics efficiently alleviated the severity of acute kindling‐induced seizures, pharmaco‐genetic inhibiting pyramidal neurons were able to reverse the enhanced synaptic plasticity during epileptogenesis, compared with that of pharmaco‐genetic activating parvalbumin neurons. Conclusion Our results demonstrated that pharmaco‐genetic inhibiting pyramidal neurons retard hippocampal kindling‐induced epileptogenesis and reverse the enhanced synaptic plasticity during epileptogenesis, compared with that of pharmaco‐genetic activating parvalbumin neurons. It suggests that pharmaco‐genetics targeting pyramidal neurons may be a promising treatment for epileptogenesis., Pharmaco‐genetic activating parvalbumin neurons of the seizure focus limitedly retarded the progression of behavioral seizure stage and afterdischarge duration (ADD) during epileptogenesis in hippocampal kindling mice. On the contrary, Pharmaco‐genetic inhibiting pyramidal neurons of the seizure focus robustly retarded epileptogenesis.

To examine the effects of intravenous and intravesical application of vibegron, a new β3-adrenoceptor (β3-AR) agonist, on bladder function in rats with oxotremorine methiodide (oxo-M: a nonselective muscarinic receptor agonist)-induced bladder overactivity. Cystometry was performed in conscious female rats with intravesical instillation of oxo-M (200 μM). In oxo-M-treated rats, vehicle or vibegron (1 and 10 mg/kg) was cumulatively applied intravenously at 30-min intervals. In other groups of rats, oxo-M + vehicle or oxo-M + vibegron (10, 100 μM, and 1 mM) was cumulatively instilled intravesically at 60-min intervals followed by intravenous application of vibegron (10 mg/kg). Expression of β3-ARs in the bladder was also evaluated using immunohistochemical staining. Intravenous application of vibegron (10 mg/kg) significantly increased bladder capacity (1.3 times) and decreased baseline, threshold, and maximal voiding pressure compared with vehicle. Next, intravesical application of vibegron (1 mM) significantly increased threshold pressure and bladder capacity (1.2 times) compared with vehicle. Combined treatments of intravesical (1 mM) and intravenous (10 mg/kg) application of vibegron induced a significantly larger degree of increases in bladder capacity (1.4 times) compared with vehicle. In addition, β3-ARs were expressed throughout the rat bladder, mainly in the urothelium. These results suggest that vibegron excreted in urine as an unchanged compound can induce the additive inhibitory effects on bladder overactivity possibly through urothelial β3-AR activation, which inhibits the afferent limb of micturition reflex rather than the efferent function as evidenced by the increases in threshold pressure and bladder capacity without affecting bladder contractile function after intravesical vibegron application.

BACKGROUND AND PURPOSE: NALCN is a Na(+) leak, GPCR‐activated channel that regulates the resting membrane potential and neuronal excitability. Despite numerous possible roles for NALCN in both normal physiology and disease processes, lack of specific blockers hampers further investigation. EXPERIMENTAL APPROACH: The effect of N‐benzhydryl quinuclidine compounds on NALCN channels was demonstrated using whole‐cell patch‐clamp recordings in HEK293T cells overexpressing NALCN and acutely isolated nigral dopaminergic neurons that express NALCN endogenously. Src kinase activity was measured using a Src kinase assay kit, and voltage and current‐clamp recordings from nigral dopaminergic neurons were used to measure NALCN currents and membrane potentials. KEY RESULTS: N‐benzhydryl quinuclidine compounds inhibited NALCN channels without affecting TRPC channels, another important route for Na(+) leak. In HEK293T cells overexpressing NALCN, N‐benzhydryl quinuclidine compounds potently suppressed muscarinic M(3) receptor‐activated NALCN currents. Structure–function relationship studies suggest that the quinuclidine ring with a benzhydryl group imparts the ability to inhibit NALCN currents regardless of Src family kinases. Moreover, N‐benzhydryl quinuclidine compounds inhibited not only GPCR‐activated NALCN currents but also background Na(+) leak currents and hyperpolarized the membrane potential in native midbrain dopaminergic neurons that express NALCN endogenously. CONCLUSION AND IMPLICATIONS: These findings suggest that N‐benzhydryl quinuclidine compounds have a pharmacological potential to directly inhibit NALCN channels and could be a useful tool to investigate functions of NALCN channels.

The work is devoted to the problem of general anesthetics side effects during surgical interventions. In particular, some of the common complications of anesthetics usage are intestinal motility disorders (eg, ileus), nausea and vomiting. We analyzed a clinical data of 60 patients who underwent analgosedation (AS) during coronary artery stenting. They were divided into 2 groups (1 group - AS with Diazepam and Fentanyl, 2 group - AS with Ketamine, Fentanyl and Propofol). Significantly more frequent manifestations of nausea in the perioperative period were identified in the group where ketamine was used. This formed the basis of the study of the molecular and cellular mechanisms of the effects of ketamine on the contractile activity of the smooth muscles of the small intestine.Anesthetics are known to interact with receptors, G-proteins and ion channels, including transient receptor potential channels (TRP channels). The member of this superfamily, TRPC4 channel, which is coupled to muscarinic receptors (M2/M3 type) through G-protein activation and causes cholinergic excitation and contraction of small intestinal smooth muscles, may be a potential target for ketamine.Thus, we aimed to investigate the effects of ketamine (100 μM) on the muscarinic cation current (mICAT), which underlies cholinergic excitation-contraction coupling of visceral smooth muscles. All experiments were performed on single ileal myocytes freshly isolated from the longitudinal layer of the mouse ileum using patch-clamp techniques in the whole-cell configuration. mICAT was recorded using symmetrical Cs+ solutions (containing Cs+125 mM). [Ca2+]i was ‘clamped’ at 100 nM using 10 mM BAPTA/4.6 mM CaCl2 mixture. Measurements of isometric contractile force of the small intestinal smooth muscles were recorded using tensiometry techniques. It was showed that 100 μM ketamine inhibits mICAT . mICAT initiated by the application of CCh (50 μM) was suppressed on 64% (n=5) and mICAT, induced by intracellular GTP γ s (200 μM) that interacts directly with the G-proteins (when muscarinic receptors are bypassed) was inhibited on 42% (n=5). Ketamine inhibited intestinal smooth muscle contractions evoked by carbachol (50 μM) by about 40 % (n=5). Thus, we can conclude that both muscarinic receptors and G-proteins (or their coupling) are affected by ketamine, but the main sites of ketamine action appear to be the G-proteins. These data will provide basis for the molecular mechanisms of postoperative motility disorders and thus may be important for the development of novel approaches to the correction of such states.

Recent clinical and experimental evidence has evoked the concept of the gut–brain axis to explain mutual interactions between the central nervous system and gut microbiota that are closely associated with the bidirectional effects of inflammatory bowel disease and central nervous system disorders1–4. Despite recent advances in our understanding of neuroimmune interactions, it remains unclear how the gut and brain communicate to maintain gut immune homeostasis, including in the induction and maintenance of peripheral regulatory T cells (pTreg cells), and what environmental cues prompt the host to protect itself from development of inflammatory bowel diseases. Here we report a liver–brain–gut neural arc that ensures the proper differentiation and maintenance of pTreg cells in the gut. The hepatic vagal sensory afferent nerves are responsible for indirectly sensing the gut microenvironment and relaying the sensory inputs to the nucleus tractus solitarius of the brainstem, and ultimately to the vagal parasympathetic nerves and enteric neurons. Surgical and chemical perturbation of the vagal sensory afferents at the hepatic afferent level reduced the abundance of colonic pTreg cells; this was attributed to decreased aldehyde dehydrogenase (ALDH) expression and retinoic acid synthesis by intestinal antigen-presenting cells. Activation of muscarinic acetylcholine receptors directly induced ALDH gene expression in both human and mouse colonic antigen-presenting cells, whereas genetic ablation of these receptors abolished the stimulation of antigen-presenting cells in vitro. Disruption of left vagal sensory afferents from the liver to the brainstem in mouse models of colitis reduced the colonic pTreg cell pool, resulting in increased susceptibility to colitis. These results demonstrate that the novel vago-vagal liver–brain–gut reflex arc controls the number of pTreg cells and maintains gut homeostasis. Intervention in this autonomic feedback feedforward system could help in the development of therapeutic strategies to treat or prevent immunological disorders of the gut. A liver–brain–gut neural circuit responds to the gut microenvironment and regulates the activity of peripheral regulatory T cells in the colon by controlling intestinal antigen-presenting cells in a muscarinic signalling-dependent manner.

Racemic 3-quinuclidinyl-α-methoxydiphenylacetate (MB266) was synthesised. Its activity at muscarinic acetylcholine receptors (mAChRs), and muscle and neuronal nicotinic acetylcholine receptors (nAChRs), was compared to that of atropine and racemic 3-quinucidinyl benzilate (QNB) using a functional assay based on agonist-induced elevation of intracellular calcium ion concentration in CN21, Chinese Hamster Ovary (CHO) and SHSY5Y human cell lines. MB266 acted as an antagonist at acetylcholine receptors, displaying 18-fold selectivity for mAChR versus nAChR (compared to the 15,200-fold selectivity observed for QNB). Thus O-methylation of QNB reduced the affinity for mAChR antagonism and increased the relative potency at both muscle and neuronal nAChRs. Despite MB266 having a pharmacological profile potentially useful for the treatment of anticholinesterase poisoning, its administration did not improve the neuromuscular function in a soman-poisoned guinea-pig diaphragm preparation pretreated with the organophosphorus nerve agent soman. Consideration should be given to exploring the potential of MB266 for possible anticonvulsant action in vitro as part of a multi-targeted ligand approach.

Heterotrimeric G proteins are categorized into four main families based on their function and sequence, Gs, Gi/o, Gq/11, and G12/13. One receptor can couple to more than one G protein subtype, and the coupling efficiency varies depending on the GPCR-G protein pair. However, the precise mechanism underlying different coupling efficiencies is unknown. Here, we study the structural mechanism underlying primary and secondary Gi/o coupling, using the muscarinic acetylcholine receptor type 2 (M2R) as the primary Gi/o-coupling receptor and the β2-adrenergic receptor (β2AR, which primarily couples to Gs) as the secondary Gi/o-coupling receptor. Hydrogen/deuterium exchange mass spectrometry and mutagenesis studies reveal that the engagement of the distal C-terminus of Gαi/o with the receptor differentiates primary and secondary Gi/o couplings. This study suggests that the conserved hydrophobic residue within the intracellular loop 2 of the receptor (residue 34.51) is not critical for primary Gi/o-coupling; however, it might be important for secondary Gi/o-coupling., G protein-coupled receptors (GPCRs) can couple to more than one G protein subtype, and the coupling efficiency varies depending on the GPCR-G protein pair. Here authors use hydrogen/deuterium exchange mass spectrometry and mutagenesis to study the structural mechanism underlying primary and secondary Gi/o coupling.

Background The radioligand [11C]VC-002 was introduced in a small initial study long ago for imaging of muscarinic acetylcholine receptors (mAChRs) in human lungs using positron emission tomography (PET). The objectives of the present study in control subjects were to advance the methodology for quantification of [11C]VC-002 binding in lung and to examine the reliability using a test-retest paradigm. This work constituted a self-standing preparatory step in a larger clinical trial aiming at estimating mAChR occupancy in the human lungs following inhalation of mAChR antagonists. Methods PET measurements using [11C]VC-002 and the GE Discovery 710 PET/CT system were performed in seven control subjects at two separate occasions, 2–19 days apart. One subject discontinued the study after the first measurement. Radioligand binding to mAChRs in lung was quantified using an image-derived arterial input function. The total distribution volume (VT) values were obtained on a regional and voxel-by-voxel basis. Kinetic one-tissue and two-tissue compartment models (1TCM, 2TCM), analysis based on linearization of the compartment models (multilinear Logan) and image analysis by data-driven estimation of parametric images based on compartmental theory (DEPICT) were applied. The test-retest repeatability of VT estimates was evaluated by absolute variability (VAR) and intraclass correlation coefficients (ICCs). Results The 1TCM was the statistically preferred model for description of [11C]VC-002 binding in the lungs. Low VAR (< 10%) across analysis methods indicated good reliability of the PET measurements. The VT estimates were stable after 60 min. Conclusions The kinetic behaviour and good repeatability of [11C]VC-002 as well as the novel lung image analysis methodology support its application in applied studies on drug-induced mAChR receptor occupancy and the pathophysiology of pulmonary disorders. Trial registration ClinicalTrials.gov identifier: NCT03097380, registered: 31 March 2017.

The molecular processes underlying neurodegenerative diseases (such as Alzheimer's Disease - AD) remain poorly understood. There is also an imperative need for disease-modifying therapies in AD since the present treatments, acetylcholinesterase inhibitors and NMDA antagonists, do not halt its progression. AD and other dementias present unique pathological features such as that of microtubule associated protein tau metabolic regulation. Tau has numerous binding partners, including signaling molecules, cytoskeletal elements and lipids, which suggests that it is a multifunctional protein. AD has also been associated with severe loss of cholinergic markers in the brain and such loss may be due to the toxic interaction of tau with cholinergic muscarinic receptors. By using specific antagonists of muscarinic receptors it was found in vitro that extracellular tau binds to M1 and M3 receptors and which the increase of intracellular calcium found in neuronal cells upon tau-binding. However, so far, the significance of tau signaling through muscarinic receptor in vivo in tauopathic models remains uncertain. The data reviewed in the present paper highlight the significant effect of M1 receptor/tau interaction in exacerbating tauopathy related pathological features and suggest that selective M1 agonists may serve as a prototype for future therapeutic development toward modification of currently intractable neurodegenerative diseases, such as tauopathies.

The deletion of M4 muscarinic receptors (MRs) changes biological rhythm parameters in females. Here, we searched for the mechanisms responsible for these changes. We performed biological rhythm analysis in two experiments: in experiment 1, the mice [C57Bl/6NTac (WT) and M4 MR −/− mice (KO)] were first exposed to a standard LD regime (12/12-h light/dark cycle) for 8 days and then subsequently exposed to constant darkness (for 24 h/day, DD regime) for another 16 days. In experiment 2, the mice (after the standard LD regime) were exposed to the DD regime and to one light pulse (zeitgeber time 14) on day 9. We also detected M1 MRs in brain areas implicated in locomotor biological rhythm regulation. In experiment 1, the biological rhythm activity curves differed: the period (τ, duration of diurnal cycle) was shorter in the DD regime. Moreover, the day mean, mesor (midline value), night mean and their difference were higher in KO animals. The time in which the maximal slope occurred was lower in the DD regime than in the LD regime in both WT and KO but was lower in KO than in WT mice. In experiment 2, there were no differences in biological rhythm parameters between WT and KO mice. The densities of M1 MRs in the majority of areas implicated in locomotor biological rhythm were low. A significant amount of M1 MR was found in the striatum. These results suggest that although core clock output is changed by M4 MR deletion, the structures involved in biological rhythm regulation in WT and KO animals are likely the same, and the most important areas are the striatum, thalamus and intergeniculate leaflet.

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.

Motion sickness (MS) is caused due to sensory mismatch. Sensory conflict theory which proves acetylcholine, histamine elevations as the primary reason for MS provides a path for an effective pharmaco-therapy, by an attempt to inhibit the elevation of acetylcholine, histamine during travel. The present study aimed to screen herbal extracts and their bioactive compounds for anti-MS effect by using in vitro and in vivo models. Ten herbal extracts and their bioactive compounds were tested for its effect on acetylcholine esterase (AChE) activity, histamine and dopamine release in human leukaemia KU812 and rat pheochromocytoma PC12 cells respectively. Rutin hydrate, menthol, eugenol, linalool and hesperidin showed less inhibitory effect on AChE activity and promoting the degradation of acetylcholine and inhibited the release of histamine and dopamine from KU812 and PC12 cells. These bioactive compounds being present in Ocimum basilicum L. var. basilicum seed extract (OBSE) was chosen for further validation studies in vivo. Anti-MS effect was evaluated in BALB/c mice model using conditioned taste aversion. Pre-treatment with OBSE at 300 mg/kg bodyweight concentration prevented MS by reducing acetylcholine, histamine, dopamine, epinephrine levels, cholinergic receptor muscarinic 1 gene expression and increasing serotonin and norepinephrine levels and thus it could serve as a potent herbal antidote for MS.

Syzygium myrtifolium Walp. (Syzygium campanulatum Korth), Myrtaceae, is locally known as “Kelat paya” in Malaysia. Traditionally, it is used as a remedy for stomach pain. The goal of the present study was to investigate the toxicological potential as well as the antidiarrhoeal and antispasmodic activities of betulinic acid, dimethyl cardamonin and standardized non-formulated and nano-formulated ethanol and supercritical fluid extracts prepared from leaves of S. myrtifolium. The standardized extracts did not produce in vivo toxicity. Both the compounds and standardized extracts showed dose-dependent antidiarrhoeal activity by examining changes in the percentage of liquid stools and percentage of defecation frequency. Dimethyl cardamonin and standardized extracts showed antispasmodic activity on the isolated ileum of guinea pigs. Compared with hyoscine-N-butylbromide, dimethyl cardamonin and standardized extracts produced significant, potent antagonizing activity in ileum contractions induced with acetylcholine. Furthermore, the antagonistic potential of S. myrtifolium active markers against muscarinic type M2 and M3 receptors was investigated by molecular docking.

Urinary incontinence is defined as an involuntary leakage of urine and is categorized into three types: stress urinary incontinence (SUI), urge urinary incontinence (UUI), and mixed urinary incontinence, which includes symptoms of SUI and UUI. As the underlying mechanisms of SUI and UUI are different, no drug is approved to treat all three types of urinary incontinence. TAS-303 is a selective norepinephrine reuptake inhibitor and has therapeutic potential for patients with SUI. In this report, we describe newly discovered pharmacological properties of TAS-303 and its effects on bladder function. Radioligand binding studies showed that TAS-303 inhibits M3 muscarinic receptor binding, with a Ki value of 547 nM. TAS-303 at 1, 3, and 10 mg/kg dose-dependently prolonged the intercontraction interval of carbachol-induced detrusor overactivity in rats, exhibiting a maximal effect that was comparable to tolterodine. These effects may result from coordinated regulation of bladder afferent activity via M3 muscarinic inhibition and β3 adrenoreceptor activation by norepinephrine elevation due to norepinephrine transporter inhibition. Moreover, TAS-303 at the effective dose for bladder function did not induce dry mouth or constipation in rats, showing that this compound may have a lower risk of antimuscarinic side effects. Thus, TAS-303 is expected to be a new profile agent with therapeutic potential for all types of urinary incontinence. SIGNIFICANCE STATEMENT: Urinary incontinence is categorized into stress, urge, and mixed urinary incontinence, but because the underlying mechanisms of each differ, no drugs are available that treat all three. TAS-303 has therapeutic potential for stress urinary incontinence. This study describes newly discovered pharmacological properties of TAS-303, which ameliorated bladder afferent activity partly via M3 muscarinic inhibition, indicating improvement in urge urinary incontinence, and highlights the potential of TAS-303 as a new therapeutic agent for all types of urinary incontinence.

Myopia (long-sightedness) is one of the many problems of vision disorder occurring around the world and being 1 in 5 eye conditions that becoming a priority at the World Health Organization's Global Intitiative for the Elimination of Avoidable Blindness. Many methods of preventing myopia development have been sought. Atropine is an alkaloid derived herbal plant Atropa belladonna which acts as a muscarinic acetylcholine receptor antagonist that has a strong nonselective competitive affinity on the five types of muskarinic acetylcholine receptors (M1-M5) that weaken the muscles of the ciliary and iris and causes the reduction of accommodation and the occurrence of pupil dilation. Recent research found that with the use of low dose atropine (0.01%), in addition to the side effects inflicted more minimal, effectiveness in the long time period shows better results. Until now, the mechanism of atropine is still experiencing debate whether working on an accommodating or non-accommodating pathway. But recent research suggests the possibility of working mechanism of this drug is through a non-accommodating pathway i.e. on GABAergic, dopaminergic as well as involving nitric oxide (NO) role to help inhibit the progression of myopia. In research and advanced scientific studies, it can be researched regarding the pathways of atropine mechanism and its advantages and disadvantages at any dose of atropine given to myopia sufferers using the latest literature sources.

Purpose: To compare the effect of bethanechol versus selegiline in ameliorating spinal cord injury (SCI) in a rat model.Methods: Male adult Wistar rats (200 – 250 g) were equally divided into 3 groups: test (SCI rats treated with bethanechol), and control reference (SCI rats treated with selegiline) and control (SCI rats treated with vehicle). SCI was induced in the rats using the clipping method. Thereafter, motor function was assessed in the rats using a rotarod. Each rat was sacrificed by decapitation, and the cortex was excised for use in the study of the involvement of cholinergic and monoaminergic transmission in SCI rats using real-time quantitative polymerase chain reaction and western blot analysis.Results: Retention time was numerically greater in rats treated with acetyl choline agonist at all rotations (10, 15 and 25 rpm) when compared to MAO A inhibitor group, but the difference was not statistically significant (p > 0.05). Both bethanechol and selegiline improved motor function by increasing cholinergic and monoaminergic transmission. Both drugs (bethanechol and selegiline) were effective in ameliorating the motor function deficit caused by spinal cord injury. A significant upregulation in acetylcholine esterase (AChE) was observed in the cortex of the SCI rats, relative to non-SCI rats (p < 0.005). Results from cholinergic receptor binding studies revealed significantly decreased Bmax and kd values for muscarinic receptors in SCI rats, when compared to non-SCI rats. Moreover, the reduction in the intensity of cholinergic receptors was significantly higher in the cerebral cortex of SCI rats than in non-SCI rats.Conclusion: Bethanechol and selegiline are effective in ameliorating motor function deficit caused by spinal cord injury in rats. Both drugs also improve motor function in SCI rats. Therefore, the drugs have potentials for use in the therapeutical management of spinal cord injury. Keywords: Spinal cord injury, Bethanechol, Selegiline, Motor functions, Monoaminergic transmission, Cholinergic transmission

Currently, metabolic disorders are one of the major health problems worldwide, which have been shown to be related to perinatal nutritional insults, and the autonomic nervous system and endocrine pancreas are pivotal targets of the malprogramming of metabolic function. We aimed to assess glucose–insulin homeostasis and the involvement of cholinergic responsiveness (vagus nerve activity and insulinotropic muscarinic response) in pancreatic islet capacity to secrete insulin in weaned rat offspring whose mothers were undernourished in the first 2 weeks of the suckling phase. At delivery, dams were fed a low-protein (4% protein, LP group) or a normal-protein diet (20.5% protein, NP group) during the first 2 weeks of the suckling period. Litter size was adjusted to six pups per mother, and rats were weaned at 21 days old. Weaned LP rats presented a lean phenotype (P < 0.01); hypoglycaemia, hypoinsulinaemia and hypoleptinaemia (P < 0.05); and normal corticosteronaemia (P > 0.05). In addition, milk insulin levels in mothers of the LP rats were twofold higher than those of mothers of the NP rats (P < 0.001). Regarding glucose–insulin homeostasis, weaned LP rats were glucose-intolerant (P < 0.01) and displayed impaired pancreatic islet insulinotropic function (P < 0.05). The M3 subtype of the muscarinic acetylcholine receptor (M3mAChR) from weaned LP rats was less responsive, and the superior vagus nerve electrical activity was reduced by 30% (P < 0.01). A low-protein diet in the suckling period malprogrammes the vagus nerve to low tonus and impairs muscarinic response in the pancreatic β-cells of weaned rats, which are imprinted to secrete inadequate insulin amounts from an early age.

Compounds 3a [1-(3′,4′-methylenedioxyphenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline] and 3b [1-(6′-bromo-3′,4′-dimethoxyphenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline] demonstrated dose-dependent (5-75 μM) negative inotropic effects. Compound 5 [1,3-bis-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin- 1-yl)propane] demonstrated a two-phase inotropic effect. The experimental results and a literature analysis suggested that the negative inotropic effects of the tested compounds (3a, 3b, 5) could be due to their influences on the [Ca2+]i concentration through blocking of cardiomyocyte Na+- and Ca2+-channels. The positive inotropic effect of free base 5 could be related to modulation of cardiomyocyte muscarinic receptor activity.

—We have analyzed the influence of acetylcholine and GABAergic inhibitory transmission on the functioning of parallel topically organized cortico-basal ganglia–thalamo-cortical neural loops including prefrontal and sensory neocortical areas and the hippocampo-basal ganglia–thalamo–hippocampal loop. Abnormal changes in the acetylcholine concentration as well as in the efficiency of excitation and inhibition in these circuits may lead to various types of epilepsy. Epileptiform activity may develop with the participation of long-range GABAergic cells which promote the synchronization of discharges of excitatory neurons in different structures. We discuss the role of GABAergic interactions between the nuclei of the basal ganglia; GABAergic inputs from hippocampal CA1 field to the medial septum and retrosplenial cortex; from the neocortex to the striatum; and from the external segment of the globus pallidus to the striatum and neocortex. We hypothesized that the limited duration of epileptiform activity is a result of the functioning of several negative feedback loops. These loops are under the influence of GABAergic inputs from the pedunculopontine nucleus to the basal forebrain, thalamus, striatum, basal ganglia outputs, and external segment of the globus pallidus. The influence of acetylcholine is complex because different types of cholinoreceptors are present on excitatory and inhibitory neurons in all structures, and different types of receptors show different affinity for acetylcholine. Our analysis suggests that epileptiform activity may be suppressed by reinforcing the negative feedback loop that is formed by the inhibitory input from the external segment of the globus pallidus to the neocortex. This requires a reduction in the activity of striatal GABAergic spiny cells which project to the neurons of the external segment of the globus pallidus. Since this reduction may be achieved by M1 muscarinic receptor antagonists, they may be used to suppress epileptiform activity. The paper discusses possible side effects of targeting muscarinic receptors in the treatment of various diseases including neurodegenerative disorders. In order to reduce side effects caused by M1 receptor antagonists and other antiepileptic drugs, it is suggested to reduce their dose and use adenosine A2A receptor antagonists. Our conclusions are consistent with the known results of experimental and clinical studies, which means that they may be used in a targeted search for drugs that alleviate the symptoms of various types of epilepsy.

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.

Regenerative capability of the peripheral nervous system after injury is enhanced by Schwann cells (SCs) producing several growth factors. The clinical use of SCs in nerve regeneration strategies is hindered by the necessity of removing a healthy nerve to obtain the therapeutic cells. Adipose-derived stem cells (ASCs) can be chemically differentiated towards a SC-like phenotype (dASCs), and represent a promising alternative to SCs. Their physiology can be further modulated pharmacologically by targeting receptors for neurotransmitters such as acetylcholine (ACh). In this study, we compare the ability of rat dASCs and native SCs to produce NGF in vitro. We also evaluate the ability of muscarinic receptors, in particular the M2 subtype, to modulate NGF production and maturation from the precursor (proNGF) to the mature (mNGF) form. For the first time, we demonstrate that dASCs produce higher basal levels of proNGF and mature NGF compared to SCs. Moreover, muscarinic receptor activation, and in particular M2 subtype stimulation, modulates NGF production and maturation in both SCs and dASCs. Indeed, both cell types express both proNGF A and B isoforms, as well as mNGF. After M2 receptor stimulation, proNGF-B (25 kDa), which is involved in apoptotic processes, is strongly reduced at transcript and protein level. Thus, we demonstrate that dASCs possess a stronger neurotrophic potential compared to SCs. ACh, via M2 muscarinic receptors, contributes to the modulation and maturation of NGF, improving the regenerative properties of dASCs.

Current therapies for Alzheimer's disease seek to correct for defective cholinergic transmission by preventing the breakdown of acetylcholine through inhibition of acetylcholinesterase, these however have limited clinical efficacy. An alternative approach is to directly activate cholinergic receptors responsible for learning and memory. The M1-muscarinic acetylcholine (M1) receptor is the target of choice but has been hampered by adverse effects. Here we aimed to design the drug properties needed for a well-tolerated M1-agonist with the potential to alleviate cognitive loss by taking a stepwise translational approach from atomic structure, cell/tissue-based assays, evaluation in preclinical species, clinical safety testing, and finally establishing activity in memory centers in humans. Through this approach, we rationally designed the optimal properties, including selectivity and partial agonism, into HTL9936-a potential candidate for the treatment of memory loss in Alzheimer's disease. More broadly, this demonstrates a strategy for targeting difficult GPCR targets from structure to clinic.

Background: In obstructive sleep apnea (OSA), intermittent hypoxemia and intrathoracic pressure fluctuations may increase atrial fibrillation (AF) susceptibility by cholinergic activation.Objective: To investigate short-term atrial electrophysiological consequences of obstructive respiratory events, simulated by intermittent negative upper airway pressure (INAP), and the role of atrial acetylcholine-regulated potassium current (I K,ACh) activated by the M2 receptor.Methods: In sedated (2% isoflurane), spontaneously breathing rats, INAP was applied noninvasively by a negative pressure device for 1 minute, followed by a resting period of 4 minutes. INAP was applied repeatedly throughout 70 minutes, followed by a 2-hour recovery period. Atrial effective refractory period (AERP) and AF inducibility were determined throughout the protocol. To study INAP-induced I K,ACh activation, protein levels of protein kinase C (PKCƐ) were determined in membrane and cytosolic fractions of left atrial (LA) tissue by Western blotting. Moreover, an I K,ACh inhibitor (XAF-1407: 1 mg/kg) and a muscarinic receptor inhibitor (atropine: 1 μg/kg) were investigated.Results: In vehicle-treated rats, repetitive INAP shortened AERP (37 ± 3 ms vs baseline 44 ± 3 ms; P = .001) and increased LA membrane PKCƐ content relative to cytosolic levels. Upon INAP recovery, ratio of PKCƐ membrane to cytosol content normalized and INAP-induced AERP shortening reversed. Both XAF-1407 and atropine increased baseline AERP (control vs XAF-1407: 61 ± 4 ms; P > .001 and control vs atropine: 58 ± 3 ms; P = .011) and abolished INAP-associated AERP shortening.Conclusion: Short-term simulated OSA is associated with a progressive, but transient, AERP shortening and a PKCƐ translocation to LA membrane. Pharmacological I K,ACh and muscarinic receptor inhibition prevented transient INAP-induced AERP shortening, suggesting an involvement of I K,ACh in the transient arrhythmogenic AF substrate in OSA.

Atropine is an established antidote in organophosphorus poisoning. It is an anticholinergic that inhibits the muscarinic actions of acetylcholine at postganglionic parasympathetic neuroeffector sites including secretary glands, smooth muscle, and CNS sites. In the literature, the cases of atropine intoxication are not uncommon. We report a case series of five cases of atropine induced psychotic disorder in patients, who manifested with delirium, hallucinations, nervousness, drowsiness, weakness, irrelevant talk, flushing along with tachycardia, and non-reactive dilated pupils suggesting possible anti-cholinergic abuse. The patients reported at the Department of Medicine-Unit II, Sheikh Zayed Medical College/Hospital, Rahim Yar Khan, from January to June 2019. The patients were managed symptomatically by titrating down the atropine's dose, administration of benzodiazepine, and antipsychotics. "This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited".