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.

The Cholinergic System Contributes to the Immunopathological Progression of Experimental Pulmonary Tuberculosis.

The cholinergic system is present in both bacteria and mammals and regulates inflammation during bacterial respiratory infections through neuronal and non-neuronal production of acetylcholine (ACh) and its receptors. However, the presence of this system during the immunopathogenesis of pulmonary tuberculosis (TB) in vivo and in its causative agent Mycobacterium tuberculosis (Mtb) has not been studied. Therefore, we used an experimental model of progressive pulmonary TB in BALB/c mice to quantify pulmonary ACh using high-performance liquid chromatography during the course of the disease. In addition, we performed immunohistochemistry in lung tissue to determine the cellular expression of cholinergic system components, and then administered nicotinic receptor (nAChR) antagonists to validate their effect on lung bacterial burden, inflammation, and pro-inflammatory cytokines. Finally, we subjected Mtb cultures to colorimetric analysis to reveal the production of ACh and the effect of ACh and nAChR antagonists on Mtb growth. Our results show high concentrations of ACh and expression of its synthesizing enzyme choline acetyltransferase (ChAT) during early infection in lung epithelial cells and macrophages. During late progressive TB, lung ACh upregulation was even higher and coincided with ChAT and α7 nAChR subunit expression in immune cells. Moreover, the administration of nAChR antagonists increased pro-inflammatory cytokines, reduced bacillary loads and synergized with antibiotic therapy in multidrug resistant TB. Finally, in vitro studies revealed that the bacteria is capable of producing nanomolar concentrations of ACh in liquid culture. In addition, the administration of ACh and nicotinic antagonists to Mtb cultures induced or inhibited bacterial proliferation, respectively. These results suggest that Mtb possesses a cholinergic system and upregulates the lung non-neuronal cholinergic system, particularly during late progressive TB. The upregulation of the cholinergic system during infection could aid both bacterial growth and immunomodulation within the lung to favor disease progression. Furthermore, the therapeutic efficacy of modulating this system suggests that it could be a target for treating the disease.

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.

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.

The Cholinergic System as a Treatment Target for Opioid Use Disorder.

Opioid overdoses recently became the leading cause of accidental death in the US, marking an increase in the severity of the opioid use disorder (OUD) epidemic that is impacting global health. Current treatment protocols for OUD are limited to opioid medications, including methadone, buprenorphine, and naltrexone. While these medications are effective in many cases, new treatments are required to more effectively address the rising societal and interpersonal costs associated with OUD. In this article, we review the opioid and cholinergic systems, and examine the potential of acetylcholine (ACh) as a treatment target for OUD. The cholinergic system includes enzymes that synthesize and degrade ACh and receptors that mediate the effects of ACh. ACh is involved in many central nervous system functions that are critical to the development and maintenance of OUD, such as reward and cognition. Medications that target the cholinergic system have been approved for the treatment of Alzheimer's disease, tobacco use disorder, and nausea. Clinical and preclinical studies suggest that medications such as cholinesterase inhibitors and scopolamine, which target components of the cholinergic system, show promise for the treatment of OUD and further investigations are warranted.

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.

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.

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.

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.

Sodium Tanshinone IIA Sulfonate Attenuates Scopolamine-Induced Cognitive Dysfunctions via Improving Cholinergic System.

Sodium Tanshinone IIA sulfonate (STS) is a derivative of Tanshinone IIA (Tan IIA). Tan IIA has been reported to possess neuroprotective effects against Alzheimer's disease (AD). However, whether STS possesses effect on AD remains unclear. This study aims to estimate whether STS could protect against scopolamine- (SCOP-) induced learning and memory deficit in Kunming mice. Morris water maze results showed that oral administration of STS (10 mg/kg and 20 mg/kg) and Donepezil shortened escape latency, increased crossing times of the original position of the platform, and increased the time spent in the target quadrant. STS decreased the activity of acetylcholinesterase (AChE) and increased the activity of choline acetyltransferase (ChAT) in the hippocampus and cortex of SCOP-treated mice. Oxidative stress results showed that STS increased the activity of superoxide dismutase (SOD) and decreased the levels of malondialdehyde (MDA) and reactive oxygen species (ROS) in hippocampus and cortex. In addition, western blot was carried out to detect the expression of apoptosis related proteins (Bcl-2, Bax, and Caspase-3). STS upregulated the protein expression of Bcl-2 and downregulated the proteins expression of Bax and Caspase-3. These results indicated that STS might become a promising therapeutic candidate for attenuating AD-like pathological dysfunction.