Activation of the nonneuronal cholinergic cardiac system by hypoxic preconditioning protects isolated adult cardiomyocytes from hypoxia/reoxygenation injury.

Activation of the vagus nerve mediates cardioprotection and attenuates myocardial ischemia/reperfusion (I/R) injury. In response to vagal activation, acetylcholine (ACh) is released from the intracardiac nervous system (ICNS) and activates intracellular cardioprotective signaling cascades. Recently, however, a nonneuronal cholinergic cardiac system (NNCCS) in cardiomyocytes has been described as an additional source of ACh. To investigate whether the NNCCS mediates cardioprotection in the absence of vagal and ICNS activation, we used a reductionist approach of isolated adult rat ventricular cardiomyocytes without neuronal cells, using hypoxic preconditioning (HPC) as a protective stimulus. Adult rat ventricular cardiomyocytes were isolated, the absence of neuronal cells was confirmed, and HPC was induced by 10/20 min hypoxia/reoxygenation (H/R) before subjection to 30/5 min H/R to simulate I/R injury. Cardiomyocyte viability was assessed by trypan blue staining at baseline and after HPC+H/R or H/R. Intra- and extracellular ACh was quantified using liquid chromatography-coupled mass spectrometry at baseline, after HPC, after hypoxia, and after reoxygenation, respectively. In a subset of experiments, muscarinic and nicotinic ACh receptor (m- and nAChR) antagonists were added during HPC or during H/R. Cardiomyocyte viability at baseline (69 ± 4%) was reduced by H/R (10 ± 3%). With HPC, cardiomyocyte viability was preserved after H/R (25 ± 6%). Intra- and extracellular ACh increased during hypoxia; HPC further increased both intra- and extracellular ACh (from 0.9 ± 0.7 to 1.5 ± 1.0 nmol/mg; from 0.7 ± 0.6 to 1.1 ± 0.7 nmol/mg, respectively). The addition of mAChR and nAChR antagonists during HPC had no impact on HPC's protection; however, protection was abrogated when antagonists were added during H/R (cardiomyocyte viability after H/R: 23 ± 5%; 13 ± 4%). In conclusion, activation of the NNCCS is involved in cardiomyocyte protection; HPC increases intra- and extracellular ACh during H/R, and m- and nAChRs are causally involved in HPC's cardiomyocyte protection during H/R. The interplay between upstream ICNS activation and NNCCS activation in myocardial cholinergic metabolism and cardioprotection needs to be investigated in future studies.NEW & NOTEWORTHY The intracardiac nervous system is considered to be involved in ischemic conditioning's cardioprotection through the release of acetylcholine (ACh). However, we demonstrate that hypoxic preconditioning (HPC) protects from hypoxia/reoxygenation injury and increases intra- and extracellular ACh during hypoxia in isolated adult ventricular rat cardiomyocytes. HPC's protection involves cardiomyocyte muscarinic and nicotinic ACh receptor activation. Thus, besides the intracardiac nervous system, a nonneuronal cholinergic cardiac system may also be causally involved in cardiomyocyte protection by ischemic conditioning.

Activated non-neuronal cholinergic system correlates with non-type 2 inflammation and exacerbations in severe asthma.

BACKGROUND: Non-neuronal cholinergic system (NNCS) contributes to various inflammatory airway diseases. However, the role of NNCS in severe asthma (SA) remains largely unexplored. OBJECTIVE: To explore airway NNCS in SA. METHODS: In this prospective cohort study based on the Australasian Severe Asthma Network in a real-world setting, patients with SA (n = 52) and non-SA (n = 104) underwent clinical assessment and sputum induction. The messenger RNA (mRNA) levels of NNCS components and proinflammatory cytokines in the sputum were detected using real-time quantitative polymerase chain reaction, and the concentrations of acetylcholine (Ach)-related metabolites were evaluated using liquid chromatography coupled with tandem mass spectrometry. Asthma exacerbations were prospectively investigated during the next 12 months. The association between NNCS and future asthma exacerbations was also analyzed. RESULTS: Patients with SA were less controlled and had worse airway obstruction, a lower bronchodilator response, higher doses of inhaled corticosteroids, and more add-on treatments. The sputum mRNA levels of NNCS components, such as muscarinic receptors M1R-M5R, OCT3, VACHT, and ACHE; proinflammatory cytokines; and Ach concentration in the SA group were significantly higher than those in the non-SA group. Furthermore, most NNCS components positively correlated with non-type (T) 2 inflammatory profiles, such as sputum neutrophils, IL8, and IL1B. In addition, the mRNA levels of sputum M2R, M3R, M4R, M5R, and VACHT were independently associated with an increased risk of moderate-to-severe asthma exacerbations. CONCLUSION: This study indicated that the NNCS was significantly activated in SA, leading to elevated Ach and was associated with clinical features, non-T2 inflammation, and future exacerbations of asthma, highlighting the potential role of the NNCS in the pathogenesis of SA. CLINICAL TRIAL REGISTRATION: ChiCTR-OOC-16009529 (http://www.chictr.org.cn).

Contributions of Non-Neuronal Cholinergic Systems to the Regulation of Immune Cell Function, Highlighting the Role of α7 Nicotinic Acetylcholine Receptors.

Loewi's discovery of acetylcholine (ACh) release from the frog vagus nerve and the discovery by Dale and Dudley of ACh in ox spleen led to the demonstration of chemical transmission of nerve impulses. ACh is now well-known to function as a neurotransmitter. However, advances in the techniques for ACh detection have led to its discovery in many lifeforms lacking a nervous system, including eubacteria, archaea, fungi, and plants. Notably, mRNAs encoding choline acetyltransferase and muscarinic and nicotinic ACh receptors (nAChRs) have been found in uninnervated mammalian cells, including immune cells, keratinocytes, vascular endothelial cells, cardiac myocytes, respiratory, and digestive epithelial cells. It thus appears that non-neuronal cholinergic systems are expressed in a variety of mammalian cells, and that ACh should now be recognized not only as a neurotransmitter, but also as a local regulator of non-neuronal cholinergic systems. Here, we discuss the role of non-neuronal cholinergic systems, with a focus on immune cells. A current focus of much research on non-neuronal cholinergic systems in immune cells is α7 nAChRs, as these receptors expressed on macrophages and T cells are involved in regulating inflammatory and immune responses. This makes α7 nAChRs an attractive potential therapeutic target.

Transfer RNA fragments replace microRNA regulators of the cholinergic poststroke immune blockade.

Stroke is a leading cause of death and disability. Recovery depends on a delicate balance between inflammatory responses and immune suppression, tipping the scale between brain protection and susceptibility to infection. Peripheral cholinergic blockade of immune reactions fine-tunes this immune response, but its molecular regulators are unknown. Here, we report a regulatory shift in small RNA types in patient blood sequenced 2 d after ischemic stroke, comprising massive decreases of microRNA levels and concomitant increases of transfer RNA fragments (tRFs) targeting cholinergic transcripts. Electrophoresis-based size-selection followed by qRT-PCR validated the top six up-regulated tRFs in a separate cohort of stroke patients, and independent datasets of small and long RNA sequencing pinpointed immune cell subsets pivotal to these responses, implicating CD14+ monocytes in the cholinergic inflammatory reflex. In-depth small RNA targeting analyses revealed the most-perturbed pathways following stroke and implied a structural dichotomy between microRNA and tRF target sets. Furthermore, lipopolysaccharide stimulation of murine RAW 264.7 cells and human CD14+ monocytes up-regulated the top six stroke-perturbed tRFs, and overexpression of stroke-inducible tRF-22-WE8SPOX52 using a single-stranded RNA mimic induced down-regulation of immune regulator Z-DNA binding protein 1. In summary, we identified a "changing of the guards" between small RNA types that may systemically affect homeostasis in poststroke immune responses, and pinpointed multiple affected pathways, which opens new venues for establishing therapeutics and biomarkers at the protein and RNA level.

Alterations in the non-neuronal cholinergic system induced by in-vitro exposure to diazoxon in spleen mononuclear cells of Nile tilapia (O. niloticus).

Organophosphate pesticides as diazinon disrupt the neuroimmune communication, affecting the innate and adaptive immune response of the exposed organisms. Since the target molecule of diazinon is typically the acetylcholinesterase enzyme (AChE), the existence of a non-neuronal cholinergic system in leukocytes makes them susceptible to alterations by diazinon. Therefore, the aim of this work was to evaluate the activity of AChE, acetylcholine (ACh) concentration, and the expression of nicotinic ACh receptors (nAChR) and muscarinic ACh receptors (mAChR) in spleen mononuclear cells (SMNC) of Nile tilapia (O. niloticus) exposed in vitro to diazoxon, a diazinon metabolite. SMNC were exposed in-vitro to 1 nM, 1 µM, and 10 µM diazoxon for 24 h. The enzyme activity of AChE was then evaluated by spectrophotometry, followed by ACh quantification by ultra-performance liquid chromatography. Finally, mAChR and nAChR expression was evaluated by RT-qPCR. The results indicate that AChE levels are significantly inhibited at 1 and 10 µM diazoxon, while the relative expression of (M3, M4, and M5) mAChR and (ß2) nAChR is reduced significantly as compared against SMNC not exposed to diazoxon. However, ACh levels show no significant difference with respect to the control group. The data indicate that diazoxon directly alters elements in the cholinergic system of SMNC by AChE inhibition or indirectly through the interaction with AChR, which is likely related to the immunotoxic properties of diazinon and its metabolites.

Cholinergic-induced anion secretion in murine jejunal enteroids involves synergy between muscarinic and nicotinic pathways.

Acetylcholine induces robust electrogenic anion secretion in mammalian intestine and it has long been hypothesized that it mediates the epithelial response through the M3 and, to a lesser extent, the M1 muscarinic receptors in the mouse. However, nicotinic receptors have recently been identified in intestinal enterocytes by quantitative real-time (qRT)-PCR/RNAseq, although any direct influence on intestinal transport has not been identified. We tested the hypothesis that cholinergic-induced anion secretion in the intestine is a result of both muscarinic and nicotinic pathways that are intrinsic to the intestinal epithelia. We developed a method to generate mouse jejunal enteroid monolayers which were used to measure active electrogenic anion secretion by the Ussing chamber/voltage-clamp technique. Here, we show that the cholinergic agonist carbachol (CCh) and the muscarinic agonist bethanechol (BCh) stimulate short-lived, concentration-dependent anion secretion in the epithelial cell-only enteroid monolayers. The muscarinic antagonist atropine completely inhibited CCh- and BCh-induced secretion, while the nicotinic antagonist hexamethonium reduced the CCh response by ~45%. While nicotine alone did not alter anion secretion, it increased the BCh-induced increase in short-circuit current in a concentration-dependent manner; this synergy was prevented by pretreatment with hexamethonium. In addition to being sensitive to hexamethonium, monolayers express both classes of cholinergic receptor by qRT-PCR, including 13 of 16 nicotinic receptor subunits. Our findings indicate that an interaction between muscarinic and nicotinic agonists synergistically stimulates anion secretion in mouse jejunal epithelial cells and identify a role for epithelial nicotinic receptors in anion secretion.

Acetylcholine-producing NK cells attenuate CNS inflammation via modulation of infiltrating monocytes/macrophages.

The nonneural cholinergic system of immune cells is pivotal for the maintenance of immunological homeostasis. Here we demonstrate the expression of choline acetyltransferase (ChAT) and cholinergic enzymes in murine natural killer (NK) cells. The capacity for acetylcholine synthesis by NK cells increased markedly under inflammatory conditions such as experimental autoimmune encephalomyelitis (EAE), in which ChAT expression escalated along with the maturation of NK cells. ChAT+ and ChAT- NK cells displayed distinctive features in terms of cytotoxicity and chemokine/cytokine production. Transfer of ChAT+ NK cells into the cerebral ventricles of CX3CR1-/- mice reduced brain and spinal cord damage after EAE induction, and decreased the numbers of CNS-infiltrating CCR2+Ly6Chi monocytes. ChAT+ NK cells killed CCR2+Ly6Chi monocytes directly via the disruption of tolerance and inhibited the production of proinflammatory cytokines. Interestingly, ChAT+ NK cells and CCR2+Ly6Chi monocytes formed immune synapses; moreover, the impact of ChAT+ NK cells was mediated by α7-nicotinic acetylcholine receptors. Finally, the NK cell cholinergic system up-regulated in response to autoimmune activation in multiple sclerosis, perhaps reflecting the severity of disease. Therefore, this study extends our understanding of the nonneural cholinergic system and the protective immune effect of acetylcholine-producing NK cells in autoimmune diseases.

Anxiety and depression in adolescents with asthma and in their parents. Is an increased basal cholinergic tone a possible further reason to explain the negative impact on asthma control?

To the Editor We read with interest the excellent article of Licari et al.  reporting that anxiety and depression are common in adolescents suffering from asthma as well as in their parents, mainly in mothers. The consequence of this relationship is that emotional disorders might negatively affect also the control of asthma.

Comparison of the efficacy between bilateral proximal tubal occlusion and total salpingectomy on ovarian reserve and the cholinergic system: an experimental study

Background and aim: To compare the effects of bilateral proximal tubal occlusion and bilateral total salpingectomy on ovarian reserve and the cholinergic system via rat experiment. Materials and methods: Twenty-one adult female rats were randomly divided into the following three groups:G1 (n = 7), sham group;G2 (n = 7), bilateral total salpingectomy group; and G3 (n = 7), bilateral proximal tubal occlusion group. Four weeks later, the abdomen of the rats was opened. The right ovarian tissues were stored in 10% formaldehyde, whereas the left ovarian tissues were stored at ­80 °C in aluminum foil. Serum samples were evaluated for antimullerian hormone. The right ovary was used for histological and immunoreactive examination, and the left ovary was used for tissue MDA analysis. Tissue samples were analyzed for MDA levels with spectrophotometric measurement, apoptosis with TUNEL staining, fibrosis score with Mason trichrome staining, ovarian reserve with HE staining, and cholinergic receptor muscarinic 1 (CHRM1) level with immunoreactivity method. Results: Compared to G1 and G3, the number of corpus luteum with secondary follicles was significantly lower in G2, whereas the number of ovarian cysts and fibrosis and apoptosis scores increased significantly. The CHRM1 immunoreactivity was significantly lower in G2 than in G1 and G3. Conclusions: Compared to the bilateral proximal tubal occlusion performed by using bipolar cautery, bilateral total salpingectomy in rats leads to a significant damage in ovarian histopathology and the cholinergic system.

Robustness of the non-neuronal cholinergic system in rat large intestine against luminal challenges.

Acetylcholine and atypical esters of choline such as propionyl- and butyrylcholine are produced by the colonic epithelium and are released when epithelial receptors for short-chain fatty acids (SCFA) are stimulated by propionate. It is assumed that the SCFA used by the choline acetyltransferase (ChAT), the central enzyme for the production of these choline esters, originate from the colonic lumen, where they are synthesized during the bacterial fermentation of carbohydrates. Therefore, it seemed to be of interest to study whether the non-neuronal cholinergic system in the colonic epithelium is affected by maneuvers intended to stimulate or to inhibit colonic fermentation by changing the intestinal microbiota. In two series of experiments, rats were either fed with a high fiber diet (15.5% (w/v) crude fibers in comparison to 4.6% (w/w) in the control diet) or treated orally with the antibiotic vancomycin. High fiber diet induced an unexpected decrease in the luminal concentration of SCFA in the colon, but an increase in the caecum, suggesting an upregulation of colonic SCFA absorption, whereas vancomycin treatment resulted in the expected strong reduction of SCFA concentration in colon and caecum. MALDI MS analysis revealed a decrease in the colonic content of propionylcholine by high fiber diet and by vancomycin. High fiber diet caused a significant downregulation of ChAT expression on protein and mRNA level. Despite a modest increase in tissue conductance during the high fiber diet, main barrier and transport properties of the epithelium such as basal short-circuit current (Isc), the flux of the paracellularly transported marker, fluorescein, or the Isc induced by epithelial acetylcholine release evoked by propionate remained unaltered. These results suggest a remarkable stability of the non-neuronal cholinergic system in colonic epithelium against changes in the luminal environment underlying its biological importance for intestinal homeostasis.

A Novel Nitric Oxide Donor, S-Nitroso-NPivaloyl-D-Penicillamine, Activates a Non-Neuronal Cardiac Cholinergic System to Synthesize Acetylcholine and Augments Cardiac Function.

BACKGROUND/AIMS: In a previous study, we reported that cardiomyocytes were equipped with non-neuronal cardiac cholinergic system (NNCCS) to synthesize acetylcholine (ACh), which is indispensable for maintaining the basic physiological cardiac functions. The aim of this study was to identify and characterize a pharmacological inducer of NNCCS. METHODS: To identify a pharmacological inducer of NNCCS, we screened several chemical compounds with chemical structures similar to the structure of S-nitroso-N-acetyl-DL-penicillamine (SNAP). Preliminary investigation revealed that SNAP is an inducer of non-neuronal ACh synthesis. We screened potential pharmacological inducers in H9c2 and HEK293 cells using western blot analysis, luciferase assay, and measurements of intracellular cGMP, NO2 and ACh levels. The effects of the screened compound on cardiac function of male C57BL6 mice were also evaluated using cardiac catheter system. RESULTS: Among the tested compounds, we selected S-nitroso-Npivaloyl-D-penicillamine (SNPiP), which gradually elevated the intracellular cGMP levels and nitric oxide (NO) levels in H9c2 and HEK293 cells. These elevated levels resulted in the gradual transactivation and translation of the choline acetyltransferase gene. Additionally, in vitro and in vivo SNPiP treatment elevated ACh levels for 72 h. SNPiP-treated mice upregulated their cardiac function without tachycardia but with enhanced diastolic function resulting in improved cardiac output. The effect of SNPiP was dependent on SNPiP nitroso group as verified by the ineffectiveness of N-pivaloyl-D-penicillamine (PiP), which lacks the nitroso group. CONCLUSION: SNPiP is identified to be one of the important pharmacological candidates for induction of NNCCS.

The non-neuronal cholinergic system in the heart: A comprehensive review.

The autonomic influences on the heart have a ying-yang nature, albeit oversimplified, the interplay between the sympathetic and parasympathetic system (known as the cholinergic system) is often complex and remain poorly understood. Recently, the heart has been recognized to consist of neuronal and non-neuronal cholinergic system (NNCS). The existence of cardiac NNCS has been confirmed by the presence of cholinergic markers in the cardiomyocytes, which are crucial for synthesis (choline acetyltransferase, ChAT), storage (vesicular acetylcholine transporter, VAChT), reuptake of choline for synthesis (high-affinity choline transporter, CHT1) and degradation (acetylcholinesterase, AChE) of acetylcholine (ACh). The non-neuronal ACh released from cardiomyocytes is believed to locally regulate some of the key physiological functions of the heart, such as regulation of heart rate, offsetting hypertrophic signals, maintenance of action potential propagation as well as modulation of cardiac energy metabolism via the muscarinic ACh receptor in an auto/paracrine manner. Apart from this, several studies have also provided evidence for the beneficial role of ACh released from cardiomyocytes against cardiovascular diseases such as sympathetic hyperactivity-induced cardiac remodeling and dysfunction as well as myocardial infarction, confirming the important role of NNCS in disease prevention. In this review, we aim to provide a fundamental overview of cardiac NNCS, and information about its physiological role, regulatory factors as well as its cardioprotective effects. Finally, we propose the different approaches to target cardiac NNCS as an adjunctive treatment to specifically address the withdrawal of neuronal cholinergic system in cardiovascular disease such as heart failure.

Cigarette smoke and non-neuronal cholinergic system in the airway epithelium of COPD patients.

Acetylcholine (ACh), synthesized by Choline Acetyl-Transferase (ChAT), exerts its physiological effects via mAChRM3 in epithelial cells. We hypothesized that cigarette smoke affects ChAT, ACh, and mAChRM3 expression in the airways from COPD patients promoting airway disease. ChAT, ACh, and mAChRM3 were assessed: "ex vivo" in the epithelium from central and distal airways of COPD patients, Healthy Smoker (S) and Healthy Subjects (C), and "in vitro" in bronchial epithelial cells stimulated with cigarette smoke extract (CSE). In central airways, mAChRM3, ChAT, and ACh immunoreactivity was significantly higher in the epithelium from S and COPD than in C subjects. mAChRM3, ChAT, and ACh score of immunoreactivity was high in the metaplastia area of COPD patients. mAChRM3/ChAT and ACh/ChAT co-localization of immunoreactivity was observed in the bronchial epithelium from COPD. In vitro, CSE stimulation significantly increased mAChRM3, ChAT, and ACh expression and mAChRM3/ChAT and ACh/ChAT co-localization in 16HBE and NHBE, and increased 16HBE proliferation. Cigarette smoke modifies the levels of mAChMR3, ChAT expression, and ACh production in bronchial epithelial cells from COPD patients. Non-neuronal components of cholinergic system may have a role in the mechanism of bronchial epithelial cell proliferation, promoting alteration of normal tissue, and of related pulmonary functions.

Segmental differences in the non-neuronal cholinergic system in rat caecum.

Acetylcholine is not only a neurotransmitter but is also produced by several non-neuronal cell types with barrier or defence function. One of the non-neuronal tissues with expression of the key enzyme for production of acetylcholine, the choline acetyltransferase (ChAT), is the colonic surface epithelium, which releases acetylcholine after contact with the short-chain fatty acid propionate produced physiologically in the colonic lumen during the microbial fermentation of carbohydrates. Despite the fact that the caecum is the largest fermentation chamber in non-ruminant mammals, nothing is known about the expression and function of a non-neuronal cholinergic system in this part of the large intestine, which was addressed in the present study. In Ussing chamber experiments, propionate induced a concentration-dependent Cl- secretion leading to an increase in short-circuit current (Isc), which was stronger in the aboral part (near the blind ending sac of the caecum) compared to the oral part of caecum. The propionate-induced Isc was blocked by atropine, but was resistant against tetrodotoxin, conotoxins (MVIIC and SVIB) or hexamethonium indicating that propionate acts via non-neuronal acetylcholine. Immunohistochemical staining revealed the expression of ChAT in the caecal surface epithelium with a significant gradient between aboral (high) and oral (low) expression. This difference combined with a higher efficiency of cholinergically induced anion secretion (as revealed by the Isc evoked by the cholinergic agonist carbachol) is probably responsible for the segment dependency of the response to propionate. In summary, propionate stimulates anion secretion in rat caecum via non-neuronal acetylcholine emphasizing the physiological importance of the non-neuronal cholinergic system in the communication between the gastrointestinal microbiome and the mammalian host.

Detection of intrinsic cholinergic system in the human lacrimal drainage system: evidence and potential implications.

PURPOSE: To investigate the presence and distribution of epithelial and non-epithelial cholinergic system and cholinergic brush cells in the human lacrimal drainage system. METHODS: The study was performed on fresh frozen human cadaveric samples of the lacrimal drainage system. Immunohistochemistry was performed for assessing the presence and distribution of cholinergic brush cell proteins-villin, acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT); vesicular acetylcholine transporter (VAChT); components of canonical taste transduction signaling cascade, phospholipase C ß2 (PLCß2), and transient receptor potential cation channel, subfamily M, and member 5 (TRPM5). In addition, immunoreactivity to carbonic anhydrase 4 (CA4) was assessed. The immunoreactivity was scored as positive or negative and the distribution patterns in the canaliculi, lacrimal sac, and nasolacrimal duct were investigated. In addition, ultrastructural analysis was performed to ascertain the presence of brush cells by means of scanning electron microscopy (SEM). RESULTS: Villin revealed immunoreactivity in the superficial epithelial cells of lacrimal sac and nasolacrimal ducts. Positive immunoreactivity was also found for ChAT, VAChT, TRPM5, and PLCß2. ChAT expression was limited to the superficial epithelial layers of the lacrimal sac epithelium. TRPM5 and PLCß2 were expressed on the cell membranes, cytoplasm, and basolateral surfaces of the lacrimal sac epithelium and also showed strong expression in the submucosal glandular acinar cells. VAChT showed strong expression in the canaliculus and lacrimal sac and was expressed on the surface of the superficial epithelial cells and the submucosal glandular acinar cells and lining of the blood vessels. There was a uniformly negative immunoreactivity for CA4. SEM revealed single epithelial cells with dense tuft of rigid apical microvilli in the lacrimal sac and nasolacrimal ducts. CONCLUSIONS: This study provides a proof of principle for the presence of an intrinsic epithelial cholinergic mechanism in the lacrimal drainage system.

Stimulation of the Nonneuronal Cholinergic System by Highly Diluted Acetylcholine in Keratinocytes.

The physiological effects of acetylcholine on keratinocytes depend on the presence of nicotinic and muscarinic receptors. The role of nonneuronal acetylcholine in keratinocytes could have important clinical implications for patients with various skin disorders such as nonhealing wounds. In order to evaluate the efficacy of highly diluted acetylcholine solutions obtained by sequential kinetic activation, we aimed to investigate the effects of these solutions on normal human keratinocytes. Two different concentrations (10 fg/mL and 1 pg/mL) and formulations (kinetically activated and nonkinetically activated) of acetylcholine were used to verify keratinocyte viability, proliferation, and migration and the intracellular pathways involved using MTT, crystal violet, wound healing, and Western blot compared to 147 ng/mL acetylcholine. The activated formulations (1 pg/mL and 10 fg/mL) revealed a significant capacity to increase migration, cell viability, and cell proliferation compared to 147 ng/mL acetylcholine, and these effects were more evident after a single administration. Sequential kinetic activation resulted in a statistically significant decrease in reactive oxygen species production accompanied by an increase in mitochondrial membrane potential and a decrease in oxygen consumption compared to 147 ng/mL acetylcholine. The M1 muscarinic receptor was involved in these effects. Finally, the involvement of ERK/mitogen-activated protein kinases (MAPK) and KI67 confirmed the effectiveness of the single treatment on cell proliferation. The intracellular pathways of calcium were investigated as well. Our results indicate for the first time that highly diluted and kinetically activated acetylcholine seems to play an active role in an in vitro model of wound healing. Moreover, the administration of acetylcholine within the physiological range may not only be effective but is also likely to be safe.

[The role of central cholinergic system in epilepsy].

Epilepsy is a chronic neurological disorder, which is not only related to the imbalance between excitatory glutamic neurons and inhibitory GABAergic neurons, but also related to abnormal central cholinergic regulation. This article summarizes the scientific background and experimental data about cholinergic dysfunction in epilepsy from both cellular and network levels, further discusses the exact role of cholinergic system in epilepsy. In the cellular level, several types of epilepsy are believed to be associated with aberrant metabotropic muscarinic receptors in several different brain areas, while the mutations of ionotropic nicotinic receptors have been reported to result in a specific type of epilepsy-autosomal dominant nocturnal frontal lobe epilepsy. In the network level, cholinergic projection neurons as well as their interaction with other neurons may regulate the development of epilepsy, especially the cholinergic circuit from basal forebrain to hippocampus, while cholinergic local interneurons have not been reported to be associated with epilepsy. With the development of optogenetics and other techniques, dissect and regulate cholinergic related epilepsy circuit has become a hotspot of epilepsy research.

Non-neuronal cholinergic system contributes to corticosteroid resistance in chronic obstructive pulmonary disease patients.

BACKGROUND: Inhaled corticosteroid (ICS) with long-acting beta-2 agonists is a well-documented combination therapy for chronic obstructive pulmonary disease (COPD) based on its additive anti-inflammatory properties. By contrast, the recommendation of ICS in combination with long-acting muscarinic antagonist (LAMA) is not evidence-based. In this study, neutrophils obtained from COPD patients were used to compare the anti-inflammatory effects of aclidinium bromide (a long-acting muscarinic antagonist) with corticosteroids and their potential additive effect. METHODS: Human sputum and blood neutrophils were isolated from healthy individuals (n = 37), patients with stable COPD (n = 52) and those with exacerbated COPD (n = 16). The cells were incubated with corticosteroid fluticasone propionate (0.1 nM-1 µM), aclidinium bromide (0.1 nM-1 µM) or a combination thereof and stimulated with 1 µg of lipopolysaccharide/ml or 5 % cigarette smoke extract. Levels of the pro-inflammatory mediators interleukin-8, matrix metalloproteinase-9, CCL-5, granulocyte-macrophage colony-stimulating factor and interleukin-1ß were measured and the mechanisms of corticosteroid resistance evaluated at the end of the incubation. RESULTS: The non-neuronal cholinergic system was over-expressed in neutrophils from COPD patients, as evidenced by increases in the expression of muscarinic receptors (M2, M4 and M5), choline acetyltransferase and vesicular acetylcholine transporter. Aclidinium bromide demonstrated anti-inflammatory effects on neutrophils from COPD patients, reversing their resistance to corticosteroids. Additive effects of combined aclidinium bromide and fluticasone propionate in blocking M2 receptor levels, inhibiting phosphoinositide 3-kinase-δ and enhancing the glucocorticoid response element transcription factor were demonstrated and were accompanied by an increase in the corticosteroid-induced expression of anti-inflammatory-related genes. CONCLUSIONS: LAMAs potentiate the anti-inflammatory effects of corticosteroids in neutrophils from COPD patients in vitro, thus providing a scientific rationale for their use in combination with corticosteroids in the treatment of COPD.