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Introduction: Sedatives and analgesics are commonly utilized as continuous infusions in the ICU but have complications, including an increase in mechanical ventilation days, ICU length of stay, and delirium. Atypical antipsychotics (AAPs) affect several receptors including muscarinic, histamine, and α-1 adrenergic receptors, which may allow them to act as adjunctive agents to facilitate weaning of continuous infusions. Objective: To determine if there is a decrease in sedatives/analgesics requirements with the use of quetiapine and olanzapine in mechanically ventilated critically ill patients. Methods: A single-center, retrospective study conducted at Brigham and Women's Hospital from 1/1/2018 to 12/31/2019. Patients were included if they were mechanically ventilated for at least 48 hours before and following AAP initiation, were receiving at least one sedative/analgesic by continuous infusion and received AAP for at least 48 hours. The major endpoint was the percentage of patients with ≥20% reduction in the cumulative dose (CD) of midazolam, propofol, or opioids using morphine mg equivalent (MME), 48 hours from AAP initiation. Minor endpoints included median changes in CD at 24 and 48 hours, and Richmond Agitation–Sedation Scale (RASS), and Critical Care Pain Observation Tool (CPOT) changes at 48 hours. Results: A total of 1177 encounters were screened and 107 were included. Within the 48 hours after AAP initiation, 77.6% had ≥20% reduction in the CD of a sedative/analgesic. There was a significant reduction in propofol, no change in MME, and significant increase in dexmedetomidine median CD at 48 hours from AAP start. No difference was found in pain scores, but patients had significantly lighter sedation scores in the 48 hours after AAP initiation. A multivariate analysis showed that earlier initiation of antipsychotics was associated with a higher likelihood of achieving a 20% reduction in a sedative/analgesic. Conclusion: AAP use was associated with a significant reduction in sedatives/analgesics doses. Future studies are warranted to confirm the results. [ABSTRACT FROM AUTHOR]

Schizophrenia is a severe mental illness that has its onset in late adolescence or early adulthood and is associated with significant dysfunction across multiple domains. The pathogenesis of schizophrenia remains unknown, but physiologic understanding of the illness has been driven by the dopamine hypothesis. However, acetylcholine (ACh) clearly plays a role with mixed results regarding effect on psychosis. Selective muscarinic M1 and M4 agonists, such as xanomeline, originally developed to aid in cognitive loss with Alzheimer's, showed promise in proof-of-concept study in 20 patients with schizophrenia. Unfortunately, tolerability problems made muscarinic agonists impractical in either condition. However, coadministration of trospium, a lipophobic, non-selective muscarinic antagonist previously used for the treatment of overactive bladder, with xanomeline resulted in a significant reduction of cholinergic adverse effects. A recent randomized, placebo-controlled study of the antipsychotic effects of this combination in 182 patients with acute psychosis revealed improved tolerability with 80% of subjects staying to the end of the 5 weeks study. At the end of the trial, the treatment group saw a − 17.4 change in the positive and negative symptom scale (PANSS) score from baseline compared to a − 5.9 change in the placebo arm (P < 0.001). Furthermore, the negative symptom subscore, was also superior in the active arm (P < 0.001). These early studies are exciting because they suggest that the cholinergic system may be recruited to treat a severe and disabling disorder with suboptimal treatment options. Xanomeline-trospium combination is currently in phase III studies. [ABSTRACT FROM AUTHOR]

Context affects the salience and visibility of image elements in visual scenes. Collinear flankers can enhance or decrease the perceptual and neuronal sensitivity to flanked stimuli. These effects are mediated through lateral interactions between neurons in the primary visual cortex (area V1), in conjunction with feedback from higher visual areas. The strength of lateral interactions is affected by cholinergic neuromodulation. Blockade of muscarinic receptors should increase the strength of lateral intracortical interactions, while nicotinic blockade should reduce thalamocortical feed-forward drive. Here we test this proposal through local iontophoretic application of the muscarinic receptor antagonist scopolamine and the nicotinic receptor antagonist mecamylamine, while recording single cells in parafoveal representations in awake fixating macaque V1. Collinear flankers generally reduced neuronal contrast sensitivity. Muscarinic and nicotinic receptor blockade equally reduced neuronal contrast sensitivity. Contrary to our hypothesis, flanker interactions were not systematically affected by either receptor blockade. [ABSTRACT FROM AUTHOR]

Aim: The aim of this study was to evaluate the clinical efficacy and safety of intravesical botulinum neurotoxin-A injection in patients with refractory overactive bladder to conservative treatment and anti-muscarinic drugs. Material and Methods: The data of 62 patients, who received botulinum neurotoxin-A (BonTA) injection (100 U) for overactive bladder (OAB) between 2015 and 2019, were reviewed retrospectively. For 55 patients included into the study, the urinary frequency and urgency, the number of incontinence and nocturia episodes, the maximum flow rate (Qmax), the post-void residual volume (PVR), and the Urinary Incontinence Quality of Life (I-QoL) scores were evaluated before the treatment and at the third month after the treatment. Results: The comparison of the pre-treatment and posttreatment 3rd-month follow-up data revealed a statistically significant decrease in the urinary frequency and urgency, and the number of incontinence and nocturia episodes (p <0.05). There were no statistically significant differences in Qmax and PVR in the posttreatment 3rd month (p> 0.05). The mean I-QoL score increased significantly in the posttreatment 3rd month compared to the pre-treatment scores (43.62 ± 11.2 and 75.2 ± 12.6, p = 0.001, respectively). After the treatment, hematuria developed in 3 female and 2 male patients and urinary tract infections developed in 4 females and 2 male patients. Conclusion: BonTA injection significantly improves the daily urinary frequency, incontinence, and quality of life scores in patients with OAB. [ABSTRACT FROM AUTHOR]

The impact of rare and damaging variants in genes associated with platelet function in large-vessel ischemic stroke (LVIS) remains unknown. The aim of this study was to investigate the contribution of some of these variants to the genetic susceptibility to LVIS in Polish patients using a deep re-sequencing of 54 selected genes, coding for proteins associated with altered platelet function. Targeted pooled re-sequencing (Illumina HiSeq 2500) was performed on genomic DNA of 500 cases (patients with history of clinically proven diagnosis of LVIS) and 500 age-, smoking status-, and sex-matched controls (no history of any type of stroke), and from the same population as patients with LVIS. After quality control and prioritization based on allele frequency and damaging probability, individual genotyping of all deleterious rare variants was performed in patients from the original cohort, and stratified to concomitant cardiac conditions differing between the study and stroke groups. We demonstrated a statistically significant increase in the number of rare and potentially damaging variants in some of the investigated genes in the LVIS pool (an increase in the genomic variants burden). Furthermore, we identified an association between LVIS and 6 rare functional and damaging variants in the Kv7.1 potassium channel gene (KCNQ1). The predicted functional properties (partial loss-of function) for the three most damaging variants in KCNQ1 coding locus were further confirmed in vitro by analyzing the membrane potential changes in cell lines co-transfected heterogeneously with human muscarinic type 1 receptor and wild-type or mutated KCNQ1 cDNA constructs using fluorescence imaging plate reader. The study demonstrated an increased rare variants burden for 54 genes associated with platelet function, and identified a putative role for rare damaging variants in the KCNQ1 gene on LVIS susceptibility in the Polish population. [ABSTRACT FROM AUTHOR]

Summary: Azithromycin (AZM) has been used for the treatment of asthma and chronic obstructive pulmonary disease (COPD); however, the effects and underlying mechanisms of AZM remain largely unknown. The effects of AZM on airway smooth muscles (ASMs) and the underlying mechanisms were studied using isometric muscle force measurements, the examination of lung slices, imaging, and patch‐clamp techniques. AZM completely inhibited acetylcholine (ACH)‐induced precontraction of ASMs in animals (mice, guinea pigs, and rabbits) and humans. Two other macrolide antibiotics, roxithromycin and Klaricid, displayed a decreased inhibitory activity, and the aminoglycoside antibiotics penicillin and streptomycin did not have an inhibitory effect. Precontractions were partially inhibited by nifedipine (selective inhibitor of L‐type voltage‐dependent Ca2+ channels (LVDCCs)), Pyr3 (selective inhibitor of TRPC3 and/or STIM/Orai channels, which are nonselective cation channels (NSCCs)), and Y‐27632 (selective inhibitor of Rho‐associated kinase (ROCK)). Moreover, LVDCC‐ and NSCC‐mediated currents were inhibited by AZM, and the latter were suppressed by the muscarinic (M) 2 receptor inhibitor methoctramine. AZM inhibited LVDCC Ca2+ permeant ion channels, M2 receptors, and TRPC3 and/or STIM/Orai, which decreased cytosolic Ca2+ concentrations and led to muscle relaxation. This relaxation was also enhanced by the inhibition of Ca2+ sensitization. Therefore, AZM has potential as a novel and potent bronchodilator. The findings of this study improve the understanding of the effects of AZM on asthma and COPD. [ABSTRACT FROM AUTHOR]

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Group IVa cytosolic phospholipase A2 (cPLA2α) mediates GPCR-stimulated arachidonic acid (AA) release from phosphatidylinositol 4,5-bisphosphate (PIP2) located in plasma membranes. We previously found in superior cervical ganglion (SCG) neurons that PLA2 activity is required for voltage-independent N-type Ca2+ (N-) current inhibition by M1 muscarinic receptors (M1Rs). These findings are at odds with an alternative model, previously observed for M-current inhibition, where PIP2 dissociation from channels and subsequent metabolism by phospholipase C suffices for current inhibition. To resolve cPLA2α’s importance, we have investigated its role in mediating voltage-independent N-current inhibition (~40%) that follows application of the muscarinic agonist oxotremorine-M (Oxo-M). Preincubation with different cPLA2α antagonists or dialyzing cPLA2α antibodies into cells minimized N-current inhibition by Oxo-M, whereas antibodies to Ca2+-independent PLA2 had no effect. Taking a genetic approach, we found that SCG neurons from cPLA2α-/- mice exhibited little N-current inhibition by Oxo-M, confirming a role for cPLA2α. In contrast, cPLA2α antibodies or the absence of cPLA2α had no effect on voltage-dependent N-current inhibition by M2/M4Rs or on M-current inhibition by M1Rs. These findings document divergent M1R signaling mediating M-current and voltage-independent N-current inhibition. Moreover, these differences suggest that cPLA2α acts locally to metabolize PIP2 intimately associated with N- but not M-channels. To determine cPLA2α’s functional importance more globally, we examined action potential firing of cPLA2α+/+ and cPLA2α-/- SCG neurons, and found decreased latency to first firing and interspike interval resulting in a doubling of firing frequency in cPLA2α-/- neurons. These unanticipated findings identify cPLA2α as a tonic regulator of neuronal membrane excitability. [ABSTRACT FROM AUTHOR]

Neuropathic pain is associated with persistent changes in gene expression in primary sensory neurons, but the underlying epigenetic mechanisms that cause these changes remain unclear. The muscarinic cholinergic receptors (mAChRs), particularly the M2 subtype (encoded by the cholinergic receptor muscarinic 2 (Chrm2) gene), are critically involved in the regulation of spinal nociceptive transmission. However, little is known about how Chrm2 expression is transcriptionally regulated. Here we show that nerve injury persistently increased the expression of RE1-silencing transcription factor (REST, also known as neuron-restrictive silencing factor [NRSF]), a genesilencing transcription factor, in the dorsal root ganglion (DRG). Remarkably, nerve injury-induced chronic but not acute pain hypersensitivity was attenuated in mice with Rest knockout in DRG neurons. Also, siRNA-mediated Rest knockdown reversed nerve injury-induced chronic pain hypersensitivity in rats. Nerve injury persistently reduced Chrm2 expression in theDRG and diminished the analgesic effect of muscarine. The RE1 binding site on the Chrm2 promoter is required for REST-mediated Chrm2 repression, and nerve injury increased the enrichment of REST in the Chrm2 promoter in the DRG. Furthermore, Rest knockdown or genetic ablation in DRG neurons normalized Chrm2 expression and augmented muscarine's analgesic effect on neuropathic pain and fully reversed the nerve injury-induced reduction in the inhibitory effect of muscarine on glutamatergic input to spinal dorsal horn neurons. Our findings indicate that nerve injury-induced REST up-regulation in DRG neurons plays an important role in the acute-to-chronic pain transition and is essential for the transcriptional repression of Chrm2 in neuropathic pain. [ABSTRACT FROM AUTHOR]

Background: Pesticide poisoning is very common in Tunisia. Various factors are involved in the analysis of the clinical presentations and the severity of this condition. Major factors are the chemical nature of the pesticides and the quantity entered the body. Methods: This is a retrospective study, reporting the pediatric cases that presented to us with signs and symptoms of pesticide poisoning. Fifty cases pesticide poisoning were admitted to the hospital between January 2013 and October 2016. Results: A total of 50 pediatric cases were included in this study with the mean age of 3 years and 4months. The poisoning was accidental in 49 cases and self-inflicted in one, with the mode being oral (N=45), respiratory (N=2) and cutaneous (N=3). The average duration of hospital care for these patients was 2 hours and 30 minutes (range: 30 min-24 hr). The clinical manifestations of poisoning noted were due to muscarinic and nicotinic receptors inhibition. Upon clinical examination, 29 patients had no pesticide in the gastric lavage fluid and urine and demonstrated no abnormal cholinesterase activity. The therapeutic management was mainly symptomatic with antidote medications prescribed (atropine and oxime). All patients had favorable outcomes and no death occurred. Conclusion: This study demonstrated the frequency of pesticide poisoning in a pediatric setting and the importance of early management. Optimal therapeutic approaches were evaluated, demonstrating that prevention still remains the best solution in such cases. [ABSTRACT FROM AUTHOR]

Abstract G-Protein-Coupled Receptors (GPCRs) are an underdeveloped target in the search for agrochemicals with octopamine receptors, a GPCR, being the target of a single insecticide/acaricide class (formamidines). The evolution of insecticide resistance has resulted in the need to identify new or underutilized targets for the development of agrochemicals, with the goal of controlling arthropod pests that affect agriculture or human and animal health. The insect cholinergic system has been a fruitful target for the development of insecticides/acaricides viz. acetylcholinesterase inhibitors and agonists/modulators of the nicotinic acetylcholine receptor. However, the muscarinic acetylcholine receptors (mAChRs), which are GPCRs, have not been successfully developed as a target for agrochemicals. Others have recently identified three subtypes of insect mAChRs in Drosophila melanogaster , and extracellular recordings from transected D. melanogaster larval central nervous system (CNS) were performed to investigate the electrogenesis of the octopaminergic and muscarinic systems. Octopamine (10 μM) resulted in a sustained neuroexcitation during a 30 min exposure, and neuroexcitation after 21 min was blocked by octopamine receptor antagonist, phentolamine (100 μM). Exposure of this preparation to the non-selective mAChR agonist, pilocarpine (10 μM), resulted in a biphasic response, characterized by neuroexcitation followed by a decrease in the CNS firing rate below initial control levels. This biphasic effect was antagonized by the classical mAChR antagonist atropine (10 μM). It was also found that atropine (10 μM) blocked octopamine's sustained neuroexcitation, indicating the possibility of cross-talk between these two GPCR pathways. Graphical abstract Unlabelled Image Highlights • GPCRs remain an underutilized target for agrochemical development. • In situ extracellular recordings of Drosophila transected CNS provides a way to investigate electrogenesis of GPCR targets in intact tissue • Octopamine results in neuroexcitation and is partially blocked by phentolamine. • Pilocarpine has a biphasic effect on the CNS firing rate, which is antagonized by atropine (10 μM). [ABSTRACT FROM AUTHOR]

3-(4-(3-[18F]Fluoropropylthio)-1,2,5-thiadiazol-3-yl)-1-methyl-1,2,5,6-tetrahydropyridine ([18F]FP-TZTP) is a selective 18F-radiotracer for the muscarinic acetylcholine receptor subtype M2, which can be used to perform positron emission tomography (PET) scans on patients with neurological disorders such as Alzheimer's disease. [18F]FP-TZTP was produced using continuous-flow microfluidics, a technique that uses reduced amounts of chemical reagents, shorter reaction times and in general, results in higher radiochemical yields compared to currently used techniques. The optimal 18F-radiolabelling conditions consisted of a total flow rate of 40 µL min−1 and 190°C, which produced [18F]FP-TZTP in 26 ± 10 % radiochemical yield with a molar activity of 182 ± 65 GBq µmol−1 and >99 % radiochemical purity. The fluorine-18 radiolabelling optimisation of the selective muscarinic M2 imaging agent [18F]FP-TZTP was performed using continuous-flow microfluidics. When scaled up to production-level quantities, [18F]FP-TZTP could be produced using decreased amounts of reagents in good radiochemical yield and molar activity. [ABSTRACT FROM AUTHOR]

Introduction: Many studies are studying the pharmacology of important ligands on different receptors. Cholinergic receptors are one of the interested fields to study; as it is connected to many diseases and particularly Parkinson and Alzheimer. Objective: The aim of the research is to study the molecular pharmacology of atropine and methacholine on muscarinic acetylcholine receptors using confocal microscope to measure Ca level. Moreover, atropine and methacholine are used to study their molecular pharmacology on cholinergic receptors and especially muscarinic one on neuroblastoma cells (SHSY5Y). Materials and Methods: The method was designed to treat SH-SY5Y cells with two different ligands (methacholine and atropine) separately, and then, atropine was added before loaded the cells with methacholine. Results: The results are as follows: Methacholine shows agonist effect with high potency while atropine is very controversial because it can be explained as either allosteric modulator or non-competitive antagonist in addition to its already approved competitive mechanism. Conclusion: In conclusion, atropine can be investigated for more pharmacological mechanisms which can help in different pathological conditions and specifically in neurological disorder. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the cost-effectiveness of solifenacin 5 mg/day versus other oral antimuscarinic agents used for overactive bladder (OAB) from a UK National Health Service (NHS) perspective. Study design: In a Markov model, hypothetical patients received solifenacin 5 mg/day or a comparator antimuscarinic, after which they could switch to an alternative antimuscarinic. The model estimated incremental cost-effectiveness ratios (ICER), expressed as cost per quality-adjusted life year (QALY) over a 5-year period. Results: Solifenacin 5 mg/day was the dominant treatment strategy (i.e., less costly and more effective) versus tolterodine extended-release (ER) 4 mg/day, fesoterodine 4 and 8 mg/day, oxybutynin ER 10 mg/day and solifenacin 10 mg/day, and was cost-effective (i.e., ICERs below the £30,000 per QALY threshold generally applied in the NHS) versus oxybutynin immediate release (IR) 10 mg/day, tolterodine IR 4 mg/day and trospium chloride 60 mg/day. The probability of solifenacin 5 mg/day being dominant/cost-effective at a willingness-to-pay threshold of £30,000 per QALY was 57–98%. Conclusions: Solifenacin 5 mg/day appears to be a cost-effective strategy for the treatment of OAB over a 5-year timeframe compared with other oral antimuscarinic agents in the UK. These findings are important for decision-makers considering the economic implications of selecting treatments for OAB.

This study used an anaesthetized rat model to directly observe changes in diameter of the vessels supplying the sciatic nerve in response to acetylcholine (10−4 M), a muscarinic receptor agonist, and atropine (10−5 M), a muscarinic receptor antagonist. Topical application of acetylcholine resulted in increases in vessel diameter (baseline: 22.0 ± 2.5 μm, acetylcholine: 28.8 ± 3.3 μm), while topical application of atropine resulted in a decrease in diameter (baseline: 26.6 ± 3.2 μm, atropine: 15.5 ± 3.6 μm) of the epineurial vessels. Mean arterial pressure was not affected by either acetylcholine (baseline: 103.8 ± 1.8 mm Hg, acetylcholine: 102.8 ± 3.2 mm Hg) or atropine (baseline: 104.0 ± 1.9 mm Hg, atropine: 105.2 ± 2.2 mm Hg). These data suggest that muscarinic-receptor-mediated responses can affect the diameter of the epineurial vessels at the sciatic nerve. In addition, muscarinic-receptor-mediated responses appear to contribute to baseline diameter of epineurial vessels at the sciatic nerve. [ABSTRACT FROM AUTHOR]

Abstract: Long‐acting bronchodilators and inhaled corticosteroids (ICS) are the cornerstones in treatment of chronic obstructive and inflammatory pulmonary diseases. However, non‐adherence to guidelines is widespread. Detailed information on real‐life treatment patterns is needed to promote rational use. We aimed to investigate nationwide time trends in individual‐level treatment patterns of long‐acting bronchodilators and ICS. Using nationwide Danish health registries, we identified all Danish adults with a prescription for long‐acting bronchodilators and/or ICS from 2000 to 2016. We investigated the total use of long‐acting bronchodilators and ICS, the proportion of current users and the rate of new users over time. Finally, we assessed treatment persistence. We included 23,061,681 prescriptions for long‐acting bronchodilators and ICS issued to 805,860 individuals from 2000 to 2016. Over this period, the total annual amount of prescribed long‐acting bronchodilators and ICS increased by 39%. Similarly, the proportion of adult users increased from 2.6% to 4.5%, mainly driven by the introduction of combination therapy and long‐acting muscarinic antagonist (LAMA). Although the rate of new users of fixed‐dose combination drugs increased substantially over time, the overall rate of new users was stable. In general, the proportion of patients on therapy after 1 year was low (25–53%), especially among young individuals and users of ICS. We document a pronounced increase in the total use of long‐acting bronchodilators and ICS over time, mainly driven by the introduction of combination drugs and LAMA. Special attention should be paid to the low level of persistence, especially among young individuals and users of ICS. [ABSTRACT FROM AUTHOR]

The endocannabinoid system, which modulates emotional learning and memory through CB1 receptors, has been found to be deregulated in Alzheimer's disease (AD). AD is characterized by a progressive decline in memory associated with selective impairment of cholinergic neurotransmission. The functional interplay of endocannabinoid and muscarinic signaling was analyzed in seven-month-old 3xTg-AD mice following the evaluation of learning and memory of an aversive stimulus. Neurochemical correlates were simultaneously studied with both receptor and functional autoradiography for CB1 and muscarinic receptors, and regulations at the cellular level were depicted by immunofluorescence. 3xTg-AD mice exhibited increased acquisition latencies and impaired memory retention compared to age-matched non-transgenic mice. Neurochemical analyses showed changes in CB1 receptor density and functional coupling of CB1 and muscarinic receptors to Gi/o proteins in several brain areas, highlighting that observed in the basolateral amygdala. The subchronic (seven days) stimulation of the endocannabinoid system following repeated WIN55,212-2 (1 mg/kg) or JZL184 (8 mg/kg) administration induced a CB1 receptor downregulation and CB1-mediated signaling desensitization, normalizing acquisition latencies to control levels. However, the observed modulation of cholinergic neurotransmission in limbic areas did not modify learning and memory outcomes. A CB1 receptor-mediated decrease of GABAergic tone in the basolateral amygdala may be controlling the limbic component of learning and memory in 3xTg-AD mice. CB1 receptor desensitization may be a plausible strategy to improve behavior alterations associated with genetic risk factors for developing AD. [ABSTRACT FROM AUTHOR]

Gastrointestinal tract motility may be demoted significantly after surgery operations at least in part due to anaesthetic agents, but there is no comprehensive explanation of the molecular mechanism(s) of such adverse effects. Anesthetics are known to interact with various receptors and ion channels including several subtypes of transient receptor potential (TRP) channels. Two members of the canonical subfamily of TRP channels (TRPC), TRPC4 and TRPC6 are Ca 2+ -permeable cation channels involved in visceral smooth muscle contractility induced by acetylcholine, the primary excitatory neurotransmitter in the gut. In the present study, we aimed to study the effect of anesthetics on muscarinic receptor-mediated excitation and contraction of intestinal smooth muscle. Here we show that muscarinic cation current (mI CAT ) mediated by TRPC4 and TRPC6 channels in mouse ileal myocytes was strongly inhibited by isoflurane (0.5 mM), one of the most commonly used inhalation anesthetics. Carbachol-activated mI CAT was reduced by 63 ± 11% (n = 5), while GTPγS-induced (to bypass muscarinic receptors) current was inhibited by 44 ± 9% (n = 6). Furthermore, carbachol-induced ileum and colon contractions were inhibited by isoflurane by about 30%. We discuss the main sites of isoflurane action, which appear to be G-proteins and muscarinic receptors, rather than TRPC4/6 channels. These results contribute to our better understanding of the signalling pathways affected by inhalation anesthetics, which may cause ileus, and thus may be important for the development of novel treatment strategies during postoperative recovery. [ABSTRACT FROM AUTHOR]

New Findings: What is the central question of this study? It remains to be determined whether type 2 diabetes attenuates muscarinic and nicotinic cutaneous vasodilatation and sweating as well as purinergic cutaneous vasodilatation. What is the main finding and its importance? We show that type 2 diabetes specifically attenuates purinergic cutaneous vasodilatation without influencing muscarinic and nicotinic cutaneous vasodilatation and sweating. Our results provide valuable new information regarding the receptor‐specific influence of type 2 diabetes on microvascular and sudomotor function. Abstract: The present study evaluated whether type 2 diabetes (T2D) attenuates muscarinic and/or nicotinic cutaneous vasodilatation and sweating as well as purinergic cutaneous vasodilatation. Cutaneous vascular conductance and sweat rate were evaluated in 12 healthy non‐diabetic older adults (Control, 60 ± 8 years) and 13 older adults with T2D (62 ± 10 years) at three intradermal forearm skin sites perfused with the following: (i) methacholine (muscarinic receptor agonist, five doses: 0.0125, 0.25, 5, 100 and 2000 m m); (ii) nicotine (nicotinic receptor agonist, five doses: 1.2, 3.6, 11, 33 and 100 m m); or (iii) ATP (purinergic receptor agonist, five doses: 0.03, 0.3, 3, 30 and 300 m m). Each agonist was administered for 25 min per dose. At the end of the protocol, 50 m m sodium nitroprusside was administered to all skin sites to elicit maximal cutaneous vasodilatation. Cutaneous vascular conductance during methacholine and nicotine administration did not differ between groups (all P > 0.05). In contrast, cutaneous vascular conductance during administration of 30 m m (42 ± 28 versus 63 ± 26% maximum, P ≤ 0.05) and 300 m m ATP (56 ± 24 versus 71 ± 20% maximum, P ≤ 0.05) was attenuated in individuals with T2D in comparison to the Control participants. Furthermore, cutaneous vascular conductance during administration of 50 m m sodium nitroprusside was lower in individuals with T2D relative to Control subjects (P = 0.04). Methacholine‐ and nicotine‐induced sweating was similar between groups (all P > 0.05). Thus, T2D attenuates purinergic cutaneous vasodilatation without affecting muscarinic and nicotinic cutaneous vascular and sweating responses. [ABSTRACT FROM AUTHOR]

Background Patients with myocardial infarction and concomitant COPD are at increased risk of poor clinical outcomes, including death, as compared to patients without COPD. Aim To investigate and compare the severity of the clinical presentation of ST-segment elevation myocardial infarction (STEMI) and of the short-(7 days) and long-term-(end of follow up) mortality in COPD patients treated with inhaled corticosteroids (ICS)/long-acting bronchodilator (LABD) - either long-acting beta2 agonist (LABA) or long-acting muscarinic antagonist (LAMA) - vs. any other inhaled treatments. Methods Data from the REAL (Registro Angioplastiche dell'Emilia-Romagna) Registry were obtained from a large prospective study population of 11,118 patients admitted to hospital for STEMI. Results From January 2003 to June 2009 we identified 2032 COPD patients admitted to hospital for STEMI. Eight hundred and twenty (40%) COPD patients were on ICS/LABD treatment (of which 55% on ICS/LABA) prior to admission. After adjustment for potential confounding factors, ICS/LABD treatment before STEMI was an independent predictor of reduced risk of pulmonary oedema and cardiogenic shock (OR 0.5, 95%CI 0.3–0.72, p < 0.01; OR 0.7, 95%CI 0.4–0.9, p = 0.03, respectively). ICS/LABD treatment was associated to reduced 7-days mortality (OR 0.54, 95%CI 0.29–0.98, p = 0.045) compared to other inhaled regimens. ICS/LABD-treated did not affect long-term (median 4 years) mortality. After hospital discharge, the proportion of ICS/LABD treated patients decreased significantly at 6 months and afterwards after the STEMI episode. Conclusion Our data provide preliminary evidence that in COPD patients ICS/LABD treatment reduces the severity of STEMI acute-phase clinical manifestations compared to other inhaled treatments. [ABSTRACT FROM AUTHOR]

This article provides a review of the effects of activation of muscarinic and nicotinic receptors on the physiological properties of circuits in the hippocampal formation. Previous articles have described detailed computational hypotheses about the role of cholinergic neuromodulation in enhancing the dynamics for encoding in cortical structures and the role of reduced cholinergic modulation in allowing consolidation of previously encoded information. This article will focus on addressing the broad scope of different modulatory effects observed within hippocampal circuits, highlighting the heterogeneity of cholinergic modulation in terms of the physiological effects of activation of muscarinic and nicotinic receptors and the heterogeneity of effects on different subclasses of neurons. [ABSTRACT FROM AUTHOR]

Background: This study determined whether changes in the expression of neuregulin (NRG) 1, erbB2 tyrosine kinase (ErbB2) and the M2 muscarinic receptor in the urothelium and detrusor muscle of the rat bladder were associated with partial bladder outlet obstruction (PBOO).Methods: Male Sprague-Dawley rats (body weight 250-300 g) were used and subdivided into control (n = 10) and PBOO groups (n = 20). PBOO was induced for 21 days, and the expression of NRG1, ErbB2 and M2 muscarinic receptor mRNA and protein was evaluated using reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting, respectively.Results: In the urothelium of rat bladder samples, protein expression and mRNA expression of NRG1, ErbB2 and M2 muscarinic receptor were significantly increased in the PBOO group compared to the control group (p < 0.05). Only mRNA expression levels of NRG1/ ErbB2 were higher in the detrusor muscle of the PBOO group compared to the control group (p < 0.05).Conclusions: Our study demonstrated remarkable changes in the expression of NRG1/ErbB2 receptor mRNA and protein in the urothelium and muscle layer. These results suggest that NRG1 overexpression plays some kind of role against the PBOO-induced upregulated muscarinic receptors in detrusor overactivity. [ABSTRACT FROM AUTHOR]

Key points The muscarinic acetylcholine receptor (mAChR)-mediated increase in excitability in rat adrenal medullary cells is at least in part due to inhibition of TWIK (tandem of P domains in a weak inwardly rectifying K+ channel)-related acid-sensitive K+ (TASK)1 channels., In this study we focused on the molecular mechanism of mAChR-mediated inhibition of TASK1 channels., Exposure to muscarine resulted in a clathrin-dependent endocytosis of TASK1 channels following activation of the muscarinic M1 receptor (M1R)., This muscarinic signal for the endocytosis was mediated in sequence by phospholipase C (PLC), protein kinase C (PKC), and then the non-receptor tyrosine kinase Src with the consequent tyrosine phosphorylation of TASK1., The present results establish that TASK1 channels are tyrosine phosphorylated and internalized in a clathrin-dependent manner in response to M1R stimulation and this translocation is at least in part responsible for muscarinic inhibition of TASK1 channels in rat AM cells., Abstract Activation of muscarinic receptor (mAChR) in rat adrenal medullary (AM) cells induces depolarization through the inhibition of TWIK-related acid-sensitive K+ (TASK)1 channels. Here, pharmacological and immunological approaches were used to elucidate the molecular mechanism for this mAChR-mediated inhibition. TASK1-like immunoreactive (IR) material was mainly located at the cell periphery in dissociated rat AM cells, and its majority was internalized in response to muscarine. The muscarine-induced inward current and translocation of TASK1 were suppressed by dynasore, a dynamin inhibitor. The muscarinic translocation was suppressed by MT7, a specific M1 antagonist, and the dose-response curves for muscarinic agonist-induced translocation were similar to those for the muscarinic inhibition of TASK1 currents. The muscarine-induced inward current and/or translocation of TASK1 were suppressed by inhibitors for phospholipase C (PLC), protein kinase C (PKC), and/or Src. TASK1 channels in AM cells and PC12 cells were transiently associated with Src and were tyrosine phosphorylated in response to muscarinic stimulation. After internalization, TASK1 channels were quickly dephosphorylated even while they remained in the cytoplasm. The cytoplasmic TASK1-like IR material quickly recycled back to the cell periphery after muscarine stimulation for 0.5 min, but not 10 min. We conclude that M1R stimulation results in internalization of TASK1 channels through the PLC-PKC-Src pathway with the consequent phosphorylation of tyrosine and that this M1R-mediated internalization is at least in part responsible for muscarinic inhibition of TASK1 channels in rat AM cells. [ABSTRACT FROM AUTHOR]

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease characterized by pathophysiological factors including airflow limitation, hyperinflation and reduced gas exchange. Treatment consists of lifestyle changes, lung rehabilitation and pharmacological therapies such as long acting beta-2-agonists (LABA) and long acting muscarinic antagonists (LAMA). More recently bronchoscopic treatments are emerging for COPD. Among them endobronchial valves (EBV) and endobronchial coils (EBC), next to endobronchial stents, sclerosing agents, targeted lung denervation and liquid nitrogen metered cryospray. In this review we aim to summarize the new emerging bronchoscopic treatments and their effect sizes compared with lung rehabilitation and pharmacological therapies. [ABSTRACT FROM AUTHOR]

The study was conducted to provide the ethnopharmacological bases of the crude extract of seed pods of Ceratonia siliqua in the gastrointestinal spasm, diarrhea and emesis. In segregated rabbit jejunum, it showed dose-dependent (0.01-10 mg/mL) relaxation of spontaneous as well as carbachol (1 µM)- induced contraction. Pre-treatment of segregated rat ileum with C. siliqua, significantly (p<0.0001) suppressed the carbachol (1 µM)-induced contraction similar to atropine (1 µM). These results indicated that C. siliqua possesses spasmolytic activity through possible blockage of muscarinic receptor in jejunum preparations. Furthermore, the crude extract inhibited the castor oilinduced diarrhea, charcoal meal propulsion in mice and copper sulfateinduced retches in chicks in a dose-dependent manner (100, 200, 300 mg/kg). These in vitro and in vivo results indicate that C. siliqua possesses the spasmolytic and antidiarrheal activities mediated possibly through blockage of muscarinic receptors. Thus, this study provides a rationale for its folkloric use. [ABSTRACT FROM AUTHOR]

Standard therapeutic options in organophosphate (OP) poisoning are limited to the administration of atropine and oximes, a regimen often lacking in efficacy and applicability. Treatment alternatives are needed, preferably covering a broad spectrum of OP intoxications. Although recent research yielded several promising compounds, e.g. bioscavengers, modulators of the muscarinic acetylcholine (ACh) receptor or bispyridinium non-oximes, these substances still need further evaluation, especially regarding effects on the potentially lethal respiratory symptoms of OP poisoning. Aim of this study was the development of an applicable and easy method to test the therapeutic efficiency of such substances. For this purpose, airway responsiveness in viable precision cut lung slices (PCLS) from rats was analysed. We showed that ACh-induced airway contractions were spontaneously reversible in non-poisoned PCLS, whereas in OP poisoned PCLS, contractions were irreversible. This effect could be antagonized by addition of the standard therapeutic atropine, thereby presenting a clear indication for treatment efficiency. Now, candidate therapeutic compounds can be evaluated, based on their ability to counteract the irreversible airway contraction in OP poisoned PCLS. [ABSTRACT FROM AUTHOR]

Anhydroecgonine methyl ester (AEME), also called methylecgonidine, is a pyrolysis product of crack cocaine that is neurotoxic and potentiates cocaine-induced sensitization. The sensitization induced by drugs of abuse can be influenced by melatonin, a neuroprotective pineal hormone. In the same way, drugs of abuse like alcohol and methamphetamine can modify melatonin synthesis. The aim of the present work was to investigate the AEME effects on melatonin synthesis in the rat pineal gland. Neurotransmitter systems involved in its effects, antioxidant enzyme activities and the melatonin protective role in AEME-induced toxicity were also evaluated. The animals were injected with AEME i.p. (1.12 mg per kg of body weight per day) or vehicle for 10 consecutive days and the nocturnal pineal melatonin synthesis profile and SOD, GPx and GR activities in the cerebral cortex and hippocampus were assessed. Cultured pineal glands were incubated with AEME for 30 min or 48 h before norepinephrine stimulation and melatonin synthesis, arylalkylamine N-acetyltransferase activity, cAMP and [Ca2+]i were determined. The involvement of cholinergic and glutamatergic systems was analyzed using different antagonists. The protective role of melatonin in AEME toxicity on hippocampal neurons was evaluated by a viability assay. AEME impaired melatonin synthesis both in vivo and in vitro and this effect seems to be mediated by muscarinic receptors and [Ca2+]i elevation. AEME reduced neuronal viability and melatonin was able to protected hippocampal neurons against AEME toxicity. The melatonin synthesis impairment observed could lead to the worsening of the direct AEME neurotoxicity and to the exacerbation of the crack cocaine addiction and sensitization. [ABSTRACT FROM AUTHOR]

The ventral tegmental area (VTA) and the rostromedial tegmental nucleus (RMTg) each contribute to opiate reward and each receive inputs from the laterodorsal tegmental and pedunculopontine tegmental nuclei, the two principle brainstem cholinergic cell groups. We compared the contributions of VTA or RMTg muscarinic cholinergic receptors to locomotion induced by morphine infusions into the same sites. VTA co-infusion of atropine completely blocked VTA morphine-induced locomotion providing additional support for the important role of VTA muscarinic cholinergic receptors in the stimulant effects of opiates. By contrast, RMTg co-infusion of atropine increased RMTg morphine-induced locomotion. Furthermore, RMTg co-infusion of the M3-selective antagonist 4-DAMP, but not the M4-selective antagonist Tropicamide, strongly increased RMTg morphine-induced locomotion. RMTg infusions of 4-DAMP, but not of Tropicamide, by themselves strongly increased drug-free locomotion. Muscarinic cholinergic receptors in the RMTg thus also contribute to the stimulant effects of morphine, but in a way opposite to those in VTA. We suggest that the net effect of endogenous cholinergic input to the RMTg on drug-free and on RMTg morphine-induced locomotion is inhibitory. [ABSTRACT FROM AUTHOR]

The aim of this study was to develop two indirect ELISAs (i-ELISA) using heat-extracted antigens prepared with the B. canis M- and B. abortus RB51 strains for the serologic diagnosis of B. canis infection in dogs. Reference sera consisted of 16 agar gel immunodiffusion (AGID)-positive sera from animals with clinical signs and positive bacterial isolation and 24 AGID-negative sera from healthy animals with no history or evidence of canine brucellosis were used to assess diagnostic sensitivity and specificity of the tests. Using validation panel, the cut-off values of OD450 0.243 and 0.354 were selected to give 100 % diagnostic specificity and sensitivity for B. abortus RB51 i-ELISA and B. canis M- i-ELISA, respectively. Based on these cut-off values, 113 serum samples taken from dog shelters in Şanlıurfa and Bursa metropolitan municipalities tested by rapid slide agglutination test (RSAT) and both i-ELISAs. Of the 47 dog sera tested from Şanlıurfa, 6 (12.8%) were positive by RSAT, 3 (6.4%) were positive by B. abortus RB51-i-ELISA and two of the samples (4.2%) were found positive by B. canis M- i-ELISA. Of the 66 serum samples from Bursa, 11 (16.6%) were found positive by RSAT, 5 (7.6%) and 4 (6.1%) of which were confirmed by B. abortus RB51 i-ELISA and B. canis M- i-ELISA, respectively. In conclusion, both B. abortus RB51 i-ELISA and B. canis M- i-ELISA yielded the same diagnostic specificity and sensitivity with minor differences and it was thought that they could be used successfully as a confirmatory test together with RSAT as a screening test. [ABSTRACT FROM AUTHOR]

Rats display conditioned flavor preferences (CFP) for fats. Previous studies demonstrated that whereas expression of an already-acquired corn oil (CO)-CFP was mildly reduced by dopamine (DA) D1, DA D2, NMDA or opioid receptor antagonists, the acquisition or learning of CO-CFP was eliminated by NMDA antagonists, and significantly reduced by DA D1 and D2, but not opioid antagonists. Previous studies of fructose-CFP demonstrated that muscarinic (scopolamine) and nicotinic (mecamylamine) cholinergic receptor antagonists and GABA B (baclofen) receptor agonism reduced the expression of this acquired response, and that scopolamine, but not mecamylamine or baclofen eliminated the acquisition or learning of this response. The present study examined scopolamine, mecamylamine or baclofen effects upon expression or acquisition of CO-CFP. For expression, rats were trained over 10 sessions with CS + (3.5% CO) and CS − (0.9% CO) flavored solutions without drugs. Two-bottle choice tests with CS + and CS − flavors in 0.9% CO examined preferences following vehicle, scopolamine (1–10 mg/kg), mecamylamine (1–8 mg/kg) and baclofen (1.5–5 mg/kg). In acquisition, eight groups of rats received vehicle, scopolamine (1, 2.5 mg/kg), mecamylamine (4, 6 mg/kg), baclofen (3, 5 mg/kg) or a limited intake vehicle control 0.5 h prior to all 10 CS + and CS − training sessions followed by six 2-bottle CS + and CS − choice tests in 0.9% CO. CO-CFP expression (percent CS + intake) was significantly but marginally reduced by scopolamine (70%), mecamylamine (85%) and baclofen (74%) relative to vehicle (98%). CO-CFP acquisition was eliminated (41%) by scopolamine relative to vehicle (88%) and limited control (98%) conditions. Neither mecamylamine nor baclofen altered CO-CFP acquisition. Thus, the muscarinic cholinergic receptor system is essential for acquisition (learning) of both fat-induced and sugar (fructose)-induced preferences. In contrast, muscarinic, nicotinic and GABA B receptors were minimally involved in the expression (maintenance) of fat- and fructose-CFP. [ABSTRACT FROM AUTHOR]

Aim Subjects with increasing age are more sensitive to the effects of the anti-muscarinic agent scopolamine, which is used (among other indications) to induce temporary cognitive dysfunction in early phase drug studies with cognition enhancing compounds. The enhanced sensitivity has always been attributed to incipient cholinergic neuronal dysfunction, as a part of the normal aging process. The aim of the study was to correlate age-dependent pharmacodynamic neuro-physiologic effects of scopolamine after correcting for differences in individual exposure. Methods We applied a pharmacokinetic and pharmacodynamic modelling approach to describe individual exposure and neurocognitive effects of intravenous scopolamine administration in healthy subjects. Results A two-compartment linear kinetics model best described the plasma concentrations of scopolamine. The estimated scopolamine population mean apparent central and peripheral volume of distribution was 2.66 ± 1.050 l and 62.10 ± 10.100 l, respectively and the clearance was 1.09 ± 0.096 l min−1. Age was not related to a decrease of performance in the tests following scopolamine administration in older subjects. Only the saccadic peak velocity showed a positive correlation between age and sensitivity to scopolamine. Age was, however, correlated at baseline with an estimated slower reaction time while performing the cognitive tests and to higher global δ and frontal θ frequency bands measured with the surface EEG. Conclusions Most of the differences in response to scopolamine administration between young and older subjects could be explained by pharmacokinetic differences (lower clearance) and not to an enhanced sensitivity when corrected for exposure levels. [ABSTRACT FROM AUTHOR]

The current study was designed to examine the involvement of cannabinoid CB1 receptors in the basolateral amygdala (BLA) in scopolamine-induced memory impairment in adult male Wistar rats. The animals were bilaterally implanted with the cannulas in the BLA and submitted to a step-through type passive avoidance task to measure the memory formation. The results showed that intraperitoneal (i.p.) administration of different doses of scopolamine (0.5–1.5 mg/kg) immediately after the training phase (post-training) impaired memory consolidation. Bilateral microinjection of the cannabinoid CB1 receptor agonist, arachydonilcyclopropylamide (ACPA; 1–4 ng/rat), into the BLA significantly improved scopolamine-induced memory consolidation impairment. On the other hand, co-administration of AM251, a cannabinoid CB1 receptor antagonist (0.25–1 ng/rat, intra-BLA), with an ineffective dose of scopolamine (0.5 mg/kg, i.p.), significantly impaired memory consolidation and mimicked the response of a higher dose of scopolamine. It is important to note that post-training intra-BLA microinjections of the same doses of ACPA or AM251 alone had no effect on memory consolidation. Moreover, the blockade of the BLA CB1 receptors by 0.3 ng/rat of AM251 prevented ACPA-induced improvement of the scopolamine response. In view of the known actions of the drugs used, the present data pointed to the involvement of the BLA CB1 receptors in scopolamine-induced memory consolidation impairment. Furthermore, it seems that a functional interaction between the BLA endocannabinoid and cholinergic muscarinic systems may be critical for memory formation. [ABSTRACT FROM AUTHOR]

Long-acting muscarinic antagonists (LAMAs) and short-acting 2-adrenoceptor agonists (SABAs) play important roles in remedy for COPD. To propel a translational research for development of bronchodilator therapy, synergistic effects between SABAs with LAMAs were examined focused on Ca2+ signaling using simultaneous records of isometric tension and F340/F380 in fura-2-loaded tracheal smooth muscle. Glycopyrronium (3 nM), a LAMA, modestly reduced methacholine (1 μM)-induced contraction. When procaterol, salbutamol and SABAs were applied in the presence of glycopyrronium, relaxant effects of these SABAs are markedly enhanced, and percent inhibition of tension was much greater than the sum of those for each agent and those expected from the BI theory. In contrast, percent inhibition of F340/F380 was not greater than those values. Bisindolylmaleimide, an inhibitor of protein kinase C (PKC), significantly increased the relaxant effect of LAMA without reducing F340/F380. Iberiotoxin, an inhibitor of large-conductance Ca2+-activated K+ (KCa) channels, significantly suppressed the effects of these combined agents with reducing F340/F380. In conclusion, combination of SABAs with LAMAs synergistically enhances inhibition of muscarinic contraction via decreasing both Ca2+ sensitization mediated by PKC and Ca2+ dynamics mediated by KCa channels. PKC and KCa channels may be molecular targets for cross talk between β2-adrenoceptors and muscarinic receptors. [ABSTRACT FROM AUTHOR]

Opioids induce rewarding and locomotor effects by inhibiting rostromedial tegmental GABA neurons that express l-opioid and nociceptin receptors. These GABA neurons then strongly inhibit dopamine neurons. Opioid-induced reward, locomotion and dopamine release also depend on pedunculopontine and laterodorsal tegmental cholinergic and glutamate neurons, many of which project to and activate ventral tegmental area dopamine neurons. Here we show that laterodorsal tegmental and pedunculopontine cholinergic neurons project to both rostromedial tegmental nucleus and ventral tegmental area, and that M4 muscarinic receptors are co-localized with l-opioid receptors associated with rostromedial tegmental GABA neurons. To inhibit or excite rostromedial tegmental GABA neurons, we utilized adeno-associated viral vectors and DREADDs to express designed muscarinic receptors (M4D or M3D respectively) in GAD2::Cre mice. In M4D-expressing mice, clozapine-N-oxide increased morphineinduced, but not vehicle-induced, locomotion. In M3D-expressing mice, clozapine-N-oxide blocked morphine-induced, but not vehicle-induced, locomotion. We propose that cholinergic inhibition of rostromedial tegmental GABA neurons via M4 muscarinic receptors facilitates opioid inhibition of the same neurons. This model explains how mesopontine cholinergic systems and muscarinic receptors in the rostromedial tegmental nucleus and ventral tegmental area are important for dopamine-dependent and dopamine-independent opioid-induced rewards and locomotion. [ABSTRACT FROM AUTHOR]

The striatum is the most prominent nucleus in the basal ganglia and plays an important role in motor movement regulation. The cholinergic interneurons (ChIs) in striatum are involved in the motion regulation by releasing acetylcholine (ACh) and modulating the output of striatal projection neurons. Here, we report that muscarinic ACh receptor (M receptor) agonists, ACh and Oxotremorine (OXO-M), decreased the firing frequency of ChIs by blocking the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Scopolamine (SCO), a nonselective antagonist of M receptors, abolished the inhibition. OXO-M exerted its function by activating the Gi/o cAMP signaling cascade. The single-cell reverse transcription polymerase chain reaction (scRT-PCR) revealed that all the five subtypes of M receptors and four subtypes of HCN channels were expressed on ChIs. Among them, M2 receptors and HCN2 channels were the most dominant ones and expressed in every single studied cholinergic interneuron (ChI).Our results suggest that ACh regulates not only the output of striatal projection neurons, but also the firing activity of ChIs themselves by activating presynaptic M receptors in the dorsal striatum. The activation of M2 receptors and blockage of HCN2 channels may play an important role in ACh inhibition on the excitability of ChIs. This finding adds a new G-protein coupled receptor mediated regulation on ChIs and provides a cellular mechanism for control of cholinergic activity and ACh release in the dorsal striatum. [ABSTRACT FROM AUTHOR]

Accumulating evidence indicates direct relationships between sleep abnormalities and the severity and prevalence of other symptom clusters in schizophrenia. Assessment of potential state-dependent alterations in sleep architecture and arousal relative to antipsychotic-like activity is critical for the development of novel antipsychotic drugs (APDs). Recently, we reported that VU0467154, a selective positive allosteric modulator (PAM) of the M 4 muscarinic acetylcholine receptor (mAChR), exhibits robust APD-like and cognitive enhancing activity in rodents. However, the state-dependent effects of VU0467154 on sleep architecture and arousal have not been examined. Using polysomnography and quantitative electroencephalographic recordings from subcranial electrodes in rats, we evaluated the effects of VU0467154, in comparison with the atypical APD clozapine and the M 1 /M 4 -preferring mAChR agonist xanomeline. VU0467154 induced state-dependent alterations in sleep architecture and arousal including delayed Rapid Eye Movement (REM) sleep onset, increased cumulative duration of total and Non-Rapid Eye Movement (NREM) sleep, and increased arousal during waking periods. Clozapine decreased arousal during wake, increased cumulative NREM, and decreased REM sleep. In contrast, xanomeline increased time awake and arousal during wake, but reduced slow wave activity during NREM sleep. Additionally, in combination with the N -methyl- d -aspartate subtype of glutamate receptor (NMDAR) antagonist MK-801, modeling NMDAR hypofunction thought to underlie many symptoms in schizophrenia, both VU0467154 and clozapine attenuated MK-801-induced elevations in high frequency gamma power consistent with an APD-like mechanism of action. These findings suggest that selective M 4 PAMs may represent a novel mechanism for treating multiple symptoms of schizophrenia, including disruptions in sleep architecture without a sedative profile. [ABSTRACT FROM AUTHOR]

A stereoselective synthesis of (1RS,2SR,6SR)-7-benzyl-6-cyclobutyl-2-methoxymethyl-4,7-diaza-9-oxobicyclo[4.3.0]nonan-8-one, which is representative of a novel series of selective muscarinic (M1) receptor agonists, is described. [ABSTRACT FROM AUTHOR]

Acetylcholine can modulate hippocampal network function through activation of both nicotinic and muscarinic acetylcholine receptors (mAChRs). All five mAChR subtypes have been identified in the hippocampus. Besides by their involvement in excitability of hippocampal cells, synaptic plasticity and memory, a large body of research has demonstrated the involvement of presynaptic mAChRs in the inhibition of glutamatergic transmission in the hippocampus. Over the years, however, pharmacological and molecular genetic studies have yielded quite contradictory results regarding the mAChR subtype(s) involved. In this study, multi-electrode array technology was used for the pharmacological elucidation of the subtype of mAChR mediating the depression of excitatory synaptic transmission at the SC-CA1 synapse. Using selective antagonists (VU0255035, MT7, tripinamide, MT3) and allosteric potentiators (VU 10010, VU 0238429) the involvement of M 1 , M 2 , M 4 , and M 5 subtypes was ruled out thereby implying a major modulatory role for M 3 receptors in the inhibition of excitatory synaptic transmission in area CA1 of rat hippocampus. [ABSTRACT FROM AUTHOR]

Background: Sugammadex is a novel neuromuscular reversal agent, but its associated hypersensitivity reaction and high cost have been obstacles to its widespread use. In the interest of reducing the necessary dosage of sugammadex, the reversal time of the combined use of sugammadex and neostigmine from moderate neuromuscular blockade were investigated. Methods: The patients enrolled ranged in age from 18 to 65 years old with American Society of Anesthesiologists class 1 or 2. The subjects were randomly assigned into one of the four groups (Group S2, S1, SN, and N; n = 30 per group). The reversal agents of each groups were as follows: S2 - sugammadex 2 mg/kg, S1 - sugammadex 1 mg/kg, SN - sugammadex 1 mg/kg + neostigmine 50 μg/kg + glycopyrrolate 10 μg/kg, N - neostigmine 50 μg/kg + glycopyrrolate 10 μg/kg. The time to recovery of the train-of-four (TOF) ratio was checked in each group. Results: The time to 90% recovery of TOF ratio was 182.6 ± 88.9, 371.1 ± 210.4, 204.3 ± 103.2, 953.2 ± 379.7 sec in group S2, S1, SN and N, respectively. Group SN showed a significantly shorter recovery time than did group S1 and N (P < 0.001). However, statistically significant differences between the S2 and SN groups were not be observed (P = 0.291). No hypersensitivity reactions occurred in all groups. Conclusions: For the reversal from rocuronium-induced moderate neuromuscular blockade, the combined use of sugammadex and neostigmine may be helpful to decrease the recovery time and can also reduce the required dosage of sugammadex. However, the increased incidence of systemic muscarinic side effects must be considered. [ABSTRACT FROM AUTHOR]

Impaired intestinal barrier function is one of the critical issues in inflammatory bowel diseases. The aim of this study is to investigate muscarinic cholinoceptor (mAChR)-mediated signaling for the amelioration of cytokine-induced barrier dysfunction in intestinal epithelium. Rat colon challenged with TNF-α and interferon γ reduced transepithelial electrical resistance (TER). This barrier injury was attenuated by muscarinic stimulation. In HT-29/B6 intestinal epithelial cells, muscarinic stimulation suppressed TNF-α-induced activation of NF-κB signaling and barrier disruption. Finally, muscarinic stimulation promoted the shedding of TNFR1, which would be a mechanism for the attenuation of TNF-α/NF-κB signaling and barrier disruption via mAChR. [ABSTRACT FROM AUTHOR]

The existence of interactions between the immune and the sympathetic nervous systems is well established. Noradrenaline can promote or inhibit the immune response, and conversely, the immune response itself can affect noradrenaline concentration in lymphoid organs, such as the spleen. It is also well known that acetylcholine released by pre-ganglionic neurons can modulate noradrenaline release by the postsynaptic neuron. The spleen does not receive cholinergic innervation, but it has been reported that lymphocytes themselves can produce acetylcholine, and express acetylcholine receptors and acetylcholinesterase. We found that the spleen of not overtly immunized mice in which muscarinic type 1 acetylcholine receptors have been knocked out (M1KO) has higher noradrenaline concentrations than that of the wildtype mice, without comparable alterations in the heart, in parallel to a decreased number of IgG-producing B cells. Splenic lymphocytes from M1KO mice displayed increased in vitro -induced cytotoxicity, and this was observed only when CD4 + T cells were present. In contrast, heterozygous acetylcholinesterase (AChE +/−) mice, had no alterations in splenic noradrenaline concentration, but the in vitro proliferation of AChE +/− CD4 + T cells was increased. It is theoretically conceivable that reciprocal effects between neuronally and non-neuronally derived acetylcholine and noradrenaline might contribute to the results reported. Our results emphasize the need to consider the balance between the effects of these mediators for the final immunoregulatory outcome. [ABSTRACT FROM AUTHOR]

Corneal epithelial erosion is one of the most common problems in clinical ophthalmology. Despite significant progress in understanding how the cornea heals, clinically available pharmacological therapies that can promote repair and prevent visual complications remain limited. We have recently demonstrated that the acetylcholine (ACh) axis of corneal epithelium plays an important role in regulation and coordination of distinct activities of corneal epithelial cells (CEC) mediating re-epithelialization, but mechanisms remained unclear. We hypothesized that the grounds for synergistic effects of corneal ACh receptors lie within the signaling pathways linking different receptors to specific elements of the CEC pro-epithelialization activities. In this study, we sought to elucidate the molecular mechanisms of cooperation of corneal muscarinic and nicotinic ACh receptors (mAChRs and nAChRs) in upregulation of E-cadherin expression. The roles of individual corneal mAChRs and nAChRs subtypes were investigated by in-cell western assay of the ACh-treated CEC, in which different ACh receptor genes were silenced by receptor-specific shRNAs. Functional inactivation of M 3 , but not M 4 , mAChR subtype, or α3 or α7, but not α9, nAChR subunit significantly inhibited E-cadherin expression. To gain a mechanistic insight, we blocked the key steps of the downstream signaling pathways. Results demonstrated that cholinergic agonists can upregulate E-cadherin expression by activating M 3 mAChR, and α3β2 and α7 nAChRs via the common signaling cascade Ca 2 + –CaMKII–PKC–Ras–Raf–MEK–ERK. Activation of α7 nAChR can launch the Ras–Raf–MEK–ERK cascade both indirectly, through the Ca 2 + –CaMKII–PKC step, and directly, perhaps, due to its direct interaction with Ras. Although the biological significance of such redundancy remained to be elucidated, results of the present study point to a new direction to pharmacologically accelerate corneal re-epithelialization, and should have salient clinical implication. [ABSTRACT FROM AUTHOR]

Previous reports have demonstrated that a non-neuronal cholinergic system is expressed aberrantly in airways. A proliferative effect is exerted directly by cholinergic agonists through the activation of nicotinic and muscarinic receptors. In cancer, particularly those related with smoking, the mechanism through which tumour cells respond to aberrantly activated cholinergic signalling is a key question. Fifty paired pieces of larynx squamous cell carcinoma and adjacent non-cancerous tissue were compared in terms of their acetylcholinesterase activity (AChE). The AChE activity in non-cancerous tissues (0.248 ± 0.030 milliunits per milligram of wet tissue; mU/mg) demonstrates that upper respiratory tissues express sufficient AChE activity for controlling the level of acetylcholine (ACh). In larynx carcinomas, the AChE activity decreased to 0.157 ± 0.024 mU/mg (p = 0.009). Larynx cancer patients exhibiting low ACh-degrading enzymatic activity had a significantly shorter overall survival (p = 0.031). Differences in the mRNA levels of alternatively spliced AChE isoforms and molecular compositions were noted between glottic and supraglottic cancers. Our results suggest that the low AChE activity observed in larynx squamous cell carcinoma may be useful for predicting the outcome of patients. [ABSTRACT FROM AUTHOR]

Elucidating the detailed process of ligand binding to a receptor is pharmaceutically important for identifying druggable binding sites. With the ability to provide atomistic detail, computational methods are well poised to study these processes. Here, accelerated molecular dynamics (aMD) is proposed to simulate processes of ligand binding to a G-protein-coupled receptor (GPCR), in this case the M3 muscarinic receptor, which is a target for treating many human diseases, including cancer, diabetes and obesity. Long-timescale aMD simulations were performed to observe the binding of three chemically diverse ligand molecules: antagonist tiotropium (TTP), partial agonist arecoline (ARc) and full agonist acetylcholine (ACh). In comparison with earlier microsecond-timescale conventional MD simulations, aMD greatly accelerated the binding of ACh to the receptor orthosteric ligand-binding site and the binding of TTP to an extracellular vestibule. Further aMD simulations also captured binding of ARc to the receptor orthosteric site. Additionally, all three ligands were observed to bind in the extracellular vestibule during their binding pathways, suggesting that it is a metastable binding site. This study demonstrates the applicability of aMD to protein–ligand binding, especially the drug recognition of GPCRs. [ABSTRACT FROM PUBLISHER]

The observation that cholinergic deafferentation of circuits projecting from forebrain basal nuclei to frontal and hippocampal circuits occurs in Alzheimer’s disease has led to drug-targeting of muscarinic M 1 receptors to alleviate cognitive symptoms. The high homology within the acetylcholine binding domain of this family however has made receptor-selective ligand development challenging. This work presents the synthesis scheme, pharmacokinetic and structure–activity-relationship study findings for M 1 -selective ligand, LY593093. Pharmacologically the compound acts as an orthosteric ligand. The homology modeling work presented however will illustrate that compound binding spans from the acetylcholine pocket to the extracellular loops of the receptor, a common allosteric vestibule for the muscarinic protein family. Altogether LY593093 represents a growing class of multi-topic ligands which interact with the receptors in both the ortho- and allosteric binding sites, but which exert their activation mechanism as an orthosteric ligand. [ABSTRACT FROM AUTHOR]

The mesolimbic dopamine (DA) system is known to play a role in cue-mediated reward-seeking for natural rewards and drugs of abuse. Specifically, cholinergic and glutamatergic receptors in the ventral tegmental area (VTA) have been shown to regulate cue-induced drug-seeking. However, the potential role of these VTA receptors in regulating cue-induced reward seeking for natural rewards is unknown. Here, we examined whether blockade of VTA acetylcholine receptors (AChRs) and N-methyl- d -aspartate receptors (NMDARs) would alter cue-induced sucrose seeking in male Sprague-Dawley rats. Subjects underwent 10 days of sucrose self-administration training (fixed ratio 1 schedule) followed by 7 days of forced abstinence. On withdrawal day 7, rats received bilateral VTA infusion of vehicle, the muscarinic AChR antagonist scopolamine (2.4 or 24 μg/side), the nicotinic AChR antagonist mecamylamine (3 or 30 μg/side), or the NMDAR antagonist AP-5 (0.1 or 1 μg/side) immediately prior to examination of cue-induced sucrose-seeking. Scopolamine infusion led to robust attenuation, but did not completely block, sucrose-seeking behavior. In contrast, VTA administration of mecamylamine or AP-5 did not alter cue-induced sucrose-seeking. Together, the data suggest that VTA muscarinic AChRs, but not nicotinic AChRs nor NMDARs, facilitate the ability of food-associated cues to drive seeking behavior for a food reward. [ABSTRACT FROM AUTHOR]

The importance of vagal efferent signaling for the insulinotropic and glucagonostatic effects of glucagon-like peptide-1 (GLP-1) was investigated in a randomized single-blinded study. Healthy male participants (n = 10) received atropine to block vagal cholinergic transmission or saline infusions on separate occasions. At t = 15 min, plasma glucose was clamped at 6 mmol/l. GLP-1 was infused at a low dose (0.3 pmol-kg-1-min-1) from t = 45-95 min and at a higher dose (1 pmol-kg-1-min-1) from t = 95-145 min. Atropine blocked muscarinic, cholinergic transmission, as evidenced by an increase in heart rate [peak: 70 ± 2 (saline) vs. 90 ± 2 (atropine) beats/min, P < 0.002] and suppression of pancreatic polypeptide levels [area under the curve during the GLP-1 infusions (AUC45-145): 492 ± 85 (saline) vs. 247 ± 59 (atropine) pmol/l × min, P < 0.0001]. More glucose was needed to maintain the clamp during the high-dose GLP-1 infusion steadystate period on the atropine day [6.4 ± 0.9 (saline) vs. 8.7 ± 0.8 (atropine) mg-kg--min-1, P < 0.0023]. GLP-1 dose-dependently increased insulin secretion on both days. The insulinotropic effect of GLP-1 was not impaired by atropine [C-peptide A U C S 4 5 - I 4 5 : 99 ± 8 (saline) vs. 113 ± 13 (atropine) nmol/l × min, P = 0.19]. Atropine suppressed glucagon levels additively with GLP-1 [AUC45-145 : 469 ± 70 (saline) vs. 265 ± 50 (atropine) pmol/l × min, P = 0.018], resulting in hypoglycemia when infusions were suspended [3.6 ± 0.2 (saline) vs. 2.7 ± 0.2 (atropine) mmol/l, P < 0.0001]. To ascertain whether atropine could independently suppress glucagon levels, control experiments (n = 5) were carried out without GLP-1 infusions [AUC45-145 : 558 ± 103 (saline) vs. 382 ± 76 (atropine) pmol/l × min, P = 0.06]. Our results suggest that efferent muscarinic activity is not required for the insulinotropic effect of exogenous GLP-1 but that blocking efferent muscarinic activity independently suppresses glucagon secretion. In combination, GLP-1 and muscarinic blockade strongly affect glucose turnover. [ABSTRACT FROM AUTHOR]