Acetylcholine In Tissue Research Articles

Cholinergic Mechanisms in Gastrointestinal Neoplasia.

Acetylcholine-activated receptors are divided broadly into two major structurally distinct classes: ligand-gated ion channel nicotinic and G-protein-coupled muscarinic receptors. Each class encompasses several structurally related receptor subtypes with distinct patterns of tissue expression and post-receptor signal transduction mechanisms. The activation of both nicotinic and muscarinic cholinergic receptors has been associated with the induction and progression of gastrointestinal neoplasia. Herein, after briefly reviewing the classification of acetylcholine-activated receptors and the role that nicotinic and muscarinic cholinergic signaling plays in normal digestive function, we consider the mechanics of acetylcholine synthesis and release by neuronal and non-neuronal cells in the gastrointestinal microenvironment, and current methodology and challenges in measuring serum and tissue acetylcholine levels accurately. Then, we critically evaluate the evidence that constitutive and ligand-induced activation of acetylcholine-activated receptors plays a role in promoting gastrointestinal neoplasia. We focus primarily on adenocarcinomas of the stomach, pancreas, and colon, because these cancers are particularly common worldwide and, when diagnosed at an advanced stage, are associated with very high rates of morbidity and mortality. Throughout this comprehensive review, we concentrate on identifying novel ways to leverage these observations for prognostic and therapeutic purposes.

Vagus nerve stimulation-induced stromal cell-derived factor-l alpha participates in angiogenesis and repair of infarcted hearts.

We aim to explore the role and mechanism of vagus nerve stimulation (VNS) in coronary endothelial cells and angiogenesis in infarcted hearts. Seven days after rat myocardial infarction (MI) was prepared by ligation of the left anterior descending coronary artery, the left cervical vagus nerve was treated with electrical stimulation 1h after intraperitoneal administration of the α7-nicotinic acetylcholine inhibitor mecamylamine or the mAChR inhibitor atropine or 3days after local injection of Ad-shSDF-1α into the infarcted heart. Cardiac tissue acetylcholine (ACh) and serum ACh, tumour necrosis factor α (TNF-α), interleukin 1β (IL-1β) and interleukin 6 (IL-6) levels were detected by ELISA to determine whether VNS was successful. An inflammatory injury model in human coronary artery endothelial cells (HCAECs) was established by lipopolysaccharide and identified by evaluating TNF-α, IL-1β and IL-6 levels and tube formation. Immunohistochemistry staining was performed to evaluate CD31-positive vessel density and stromal cell-derived factor-l alpha (SDF-1α) expression in the MI heart in vivo and the expression and distribution of SDF-1α, C-X-C motif chemokine receptor 4 and CXCR7 in HCAECs in vitro. Western blotting was used to detect the levels of SDF-1α, V-akt murine thymoma viral oncogene homolog (AKT), phosphorylated AKT (pAKT), specificity protein 1 (Sp1) and phosphorylation of Sp1 in HCAECs. Left ventricular performance, including left ventricular systolic pressure, left ventricular end-diastolic pressure and rate of the rise and fall of ventricular pressure, should be evaluated 28days after VNS treatment. VNS was successfully established for MI therapy with decreases in serum TNF-α, IL-1β and IL-6 levels and increases in cardiac tissue and serum ACh levels, leading to increased SDF-1α expression in coronary endothelial cells of MI hearts, triggering angiogenesis of MI hearts with increased CD31-positive vessel density, which was abolished by the m/nAChR inhibitors mecamylamine and atropine or knockdown of SDF-1α by shRNA. ACh promoted SDF-1α expression and its distribution along with the branch of the formed tube in HCAECs, resulting in an increase in the number of tubes formed in HCAECs. ACh increased the levels of pAKT and phosphorylation of Sp1 in HCAECs, resulting in inducing SDF-1α expression, and the specific effects could be abolished by mecamylamine, atropine, the PI3K/AKT blocker wortmannin or the Sp1 blocker mithramycin. Functionally, VNS improved left ventricular performance, which could be abolished by Ad-shSDF-1α. VNS promoted angiogenesis to repair the infarcted heart by inducing SDF-1α expression and redistribution along new branches during angiogenesis, which was associated with the m/nAChR-AKT-Sp1 signalling pathway.

Behavioural Fever Promotes an Inflammatory Reflex Circuit in Ectotherms.

Background: The communication between the brain and the immune system is a cornerstone in animal physiology. This interaction is mediated by immune factors acting in both health and pathogenesis, but it is unclear how these systems molecularly and mechanistically communicate under changing environmental conditions. Behavioural fever is a well-conserved immune response that promotes dramatic changes in gene expression patterns during ectotherms’ thermoregulatory adaptation, including those orchestrating inflammation. However, the molecular regulators activating the inflammatory reflex in ectotherms remain unidentified. Methods: We revisited behavioural fever by providing groups of fish a thermal gradient environment during infection. Our novel experimental setup created temperature ranges in which fish freely moved between different thermal gradients: (1) wide thermoregulatory range; T° = 6.4 °C; and (2) restricted thermoregulatory range; T° = 1.4 °C. The fish behaviour was investigated during 5-days post-viral infection. Blood, spleen, and brain samples were collected to determine plasmatic pro- and anti-inflammatory cytokine levels. To characterize genes’ functioning during behavioural fever, we performed a transcriptomic profiling of the fish spleen. We also measured the activity of neurotransmitters such as norepinephrine and acetylcholine in brain and peripheral tissues. Results: We describe the first set of the neural components that control inflammatory modulation during behavioural fever. We identified a neuro-immune crosstalk as a potential mechanism promoting the fine regulation of inflammation. The development of behavioural fever upon viral infection triggers a robust inflammatory response in vivo, establishing an activation threshold after infection in several organs, including the brain. Thus, temperature shifts strongly impact on neural tissue, specifically on the inflammatory reflex network activation. At the molecular level, behavioural fever causes a significant increase in cholinergic neurotransmitters and their receptors’ activity and key anti-inflammatory factors such as cytokine Il10 and Tgfβ in target tissues. Conclusion: These results reveal a cholinergic neuronal-based mechanism underlying anti-inflammatory responses under induced fever. We performed the first molecular characterization of the behavioural fever response and inflammatory reflex activation in mobile ectotherms, identifying the role of key regulators of these processes. These findings provide genetic entry points for functional studies of the neural–immune adaptation to infection and its protective relevance in ectotherm organisms.

27414 A novel blend of neuromodulating peptides and gamma aminobutyric acid to regulate the release of acetylcholine

Background: The mechanism of action of topically applied neuromodulating peptides (NPs) has been demonstrated in vitro using neuronal and muscular cells. Nonneuronal tissues have been shown to synthesize, secrete, and degrade acetylcholine (ACh). The mechanism of NPs in regulating ACh in nonneuronal tissue is studied using a synergistic patent pending blend (PPB) of five NPs and gamma aminobutyric acid (GABA).

Neuro-Immune Modulation Effects of Sacral Nerve Stimulation for Visceral Hypersensitivity in Rats.

Background: Visceral hypersensitivity (VH) is one of the underlying pathophysiologies of irritable bowel syndrome. Mast cell overactivation has been found to be one of the main causes of VH. We investigated the effects and mechanisms of actions of sacral nerve stimulation (SNS) on visceral pain in a rodent model of VH.Methods: The VH was established by an intrarectal infusion of AA in 10-day-old pups. Rats were chronically implanted with electrodes for SNS and recording electromyogram (EMG) and electrocardiogram. The acute study was performed in 2-randomized sessions with SNS (14 Hz, 330 μs, 40% motor threshold or MT, 30 min) or sham-SNS. Later on, rats were randomized into SNS/sham-SNS groups and a chronic study was performed with 2 h-daily SNS or sham-SNS for 21 days. Visceromotor reflexes were assessed by abdominal EMG and withdrawal reflex (AWR). Colon tissues were collected to study colonic acetylcholine (ACh), the enteric neurons (ChAT, nNOS, and PGP9.5), mast cells activity [Tryptase, prostaglandins E2 (PGE2), and cyclooxygenases-2 (COX2)] and pain markers [nerve growth factor (NGF) and Sub-P].Key Results: Sacral nerve stimulation significantly improved visceromotor reflexes assessed by the EMG and AWR, compared with sham-SNS. SNS normalized the protein expressions of ChAT and nNOS and regulated mast cells activity by downregulating Tryptase, COX2, and PGE2. Neonatal AA administration upregulated NGF and Sub-P; chronic SNS significantly decreased these pain biomarkers. Concurrently, chronic SNS increased ACh in colon tissues and vagal efferent activity.Conclusions: Sacral nerve stimulation reduces VH in rats and this ameliorating effect might be attributed to the suppression of mast cell overactivation in the colon tissue via the modulation of autonomic nervous system functions.

Neuroprotective Effect and Antioxidant Potency of Fermented Cultured Wild Ginseng Root Extracts of Panax ginseng C.A. Meyer in Mice.

Wild ginseng has better pharmacological effects than cultivated ginseng. However, its industrialization is limited by the inability to grow wild ginseng on a large scale. Herein, we demonstrate how to optimize ginseng production through cultivation, and how to enhance the concentrations of specific ginsenosides through fermentation. In the study, we also evaluated the ability of fermented cultured wild ginseng root extract (HLJG0701-β) to inhibit acetylcholinesterase (AChE), as well as its neuroprotective effects and antioxidant activity. In in vitro tests, HLJG0701-β inhibited AChE activity and exerted neuroprotective and antioxidant effects (showing increased catalyst activity but decreased reactive oxygen species concentration). In in vivo tests, after HLJG0701-β was orally administered at doses of 0, 125, 250, and 500 mg/kg in an animal model of memory impairment, behavioral evaluation (Morris water maze test and Y-maze task test) was performed. The levels of AChE, acetylcholine (ACh), blood catalase (CAT), and malondialdehyde (MDA) in brain tissues were measured. The results showed that HLJG0701-β produced the best results at a dose of 250 mg/kg or more. The neuroprotective mechanism of HLJG0701-β was determined to involve the inhibition of AChE activity and a decrease in oxidative stress. In summary, both in vitro and in vivo tests confirmed that HJG0701-β administration can lead to memory improvement.

Ultrasound Combined With Microbubbles Loading BDNF Retrovirus to Open BloodBrain Barrier for Treatment of Alzheimer's Disease.

Background: Brain-derived nerve growth factor (BDNF) is a promising effective target for the treatment of Alzheimer’s disease (AD). BDNF, which has a high molecular weight, has difficulty in crossing the blood–brain barrier (BBB). The study aimed to prepare microbubbles loading brain-derived nerve growth factor (BDNF) retrovirus (MpLXSN-BDNF), to verify the characteristics of the microbubbles, and to study the therapeutic effect of the microbubbles combined with ultrasound on the opening of the blood–brain barrier in an AD rat model. Methods: 32 adult male SD rats were randomly divided into four groups: control group, ultrasound + pLXSN-EGFP microbubble group (U + MpLXSN-BDNF), ultrasound + pLXSN-BDNF microbubble group, and ultrasound + microbubble + pLXSN-BDNF virus group (U + MpLXSN-BDNF), with eight rats in each group. At the same time, the left hippocampus of rats was irradiated with low-frequency focused ultrasound guided by MRI to open the blood–brain barrier (BBB). The effects of BDNF overexpression on AD rats were evaluated behaviorally before and 1 month after the treatment. The number of acetylcholinesterase (ChAT)-positive cells and the content of acetylcholine (ACh) in brain tissues were determined by immunohistochemistry and high-performance liquid chromatography (HPLC), respectively. IF staining of synaptic spines and Western blot of synaptophysin presented herein detected synaptic density recovery. Results: Signal intensity enhancement at the BBB disruption sites could be observed on the MR images. The behavioral evaluation showed that the times of crossing the original platform in the U + MpLXSN-BDNF group increased significantly after treatment. Immunohistochemistry and HPLC revealed that the number of ChAT-positive neurons and the contents of ACh in the brain were significantly decreased in the treated groups compared with the controls. IF staining of synaptic spines and Western blot data of synaptophysin showed that the U + MpLXSN-BDNF group can recover the synaptic loss better by BDNF supplementation than the other treatment groups. Conclusion: Ultrasound combined with viral microbubbles carrying BDNF can increase the transfection efficiency of brain neurons, promote the high expression of exogenous gene BDNF, and play a therapeutic role in the AD model rats.

A study of neurotoxicity of crotonaldehyde in male rats

Objective: To investigate the neurotoxicity of crotonaldehyde exposure in male rats and its possible mechanism of action. Methods: From July to October 2019, 24 specific pathogen-free male Wistar rats were randomly divided into control group and 2.5, 4.5, and 8.5 mg/kg exposure groups, with 6 rats in each group, and the rats in these groups were given oral administration of crotonaldehyde solution at doses of 0.0, 2.5, 4.5, and 8.5 mg/kg, respectively, 5 times a week for 90 consecutive days. Body weight was measured after exposure, and brain tissue and liver tissue were collected. The activity of acetylcholinesterase (AChE) in brain tissue and the level of acetylcholine (ACh) in liver tissue were measured; The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and the levels of malondialdehyde (MDA) and reduced glutathione (GSH) in brain tissue were measured; ELISA was used to measure the levels of interleukin-6 (IL-6) , interleukin-1β (IL-1β) , and tumor necrosis factor-α (TNF-α) in brain tissue. Results: Compared with the control group, the 2.5, 4.5, and 8.5 mg/kg exposure groups had a significant reduction in the activity of AChE in brain tissue, and the 8.5 mg/kg exposure group had a significant increase in the level of ACh in liver tissue (P<0.05) . Compared with the control group, the 4.5 and 8.5 mg/kg exposure groups had a significant increase in the level of MDA and significant reductions in the level of GSH and the activities of SOD and GSH-Px in brain tissue (P<0.05) . Compared with the control group, the 2.5, 4.5, and 8.5 mg/kg exposure groups had significant increases in the levels of TNF-α and IL-6 in brain tissue, and the 4.5 and 8.5 mg/kg exposure groups had a significant increase in the level of IL-1β (P<0.05) . Conclusion: Crotonaldehyde exposure can induce nervous system injury in rats, possibly by altering oxidative balance and upregulating the expression of inflammatory factors in brain tissue.

Sacral nerve stimulation ameliorates colonic barrier functions in a rodent model of colitis.

The mucosal barrier damage is recognized as one of the key factors in the pathogenesis of colitis. While sacral nerve stimulation (SNS) was reported to have therapeutic potential for colitis, its mechanisms of actions on colonic permeability remained largely unknown. In this study, colitis was induced by intrarectal administration of TNBS in rats. Five days later, they were treated with SNS or sham-SNS for 10days. The effects of SNS on colonic permeability were assessed by measuring the expression of tight-junction proteins involved in regulating permeability and the FITC-dextran test. The mechanism of actions of SNS was investigated by studying the function of the enteric nervous system (ENS) cells and analyzing the autonomic nervous system. SNS decreased the disease activity index, microscopic and macroscopic scores, myeloperoxidase activity, and pro-inflammatory cytokines (TNF-α, IL-6). SNS increased the expression of Zonula Occludens-1, Occludin, Claudin-1, and Junctional adhesion molecule-A in the colon tissue. The FITC-dextran test showed that the colonic permeability was lower with SCS than sham-SNS. SNS increased ChAT, pancreatic polypeptide, and GDNF and reduced norepinephrine NGF, sub-P, and mast cell overactivation in the colon tissue. Concurrently, SNS increased acetylcholine in colon tissues and elevated vagal efferent activity. SNS ameliorates colonic inflammation and enhances colonic barrier function with the proposed mechanisms involving the increase in parasympathetic activity and modulation of the activity of the ENS and immune system, including mast cells.

Association of Salivary Cholinesterase With Arthropod Vectors of Disease

Acetylcholinesterase (AChE) was previously reported to be present in saliva of the southern cattle tick, Rhipicephalus (Boophilus) microplus (Canestrini), with proposed potential functions to 1) reduce acetylcholine toxicity during rapid engorgement, 2) modulate host immune responses, and 3) to influence pathogen transmission and establishment in the host. Potential modulation of host immune responses might include participation in salivary-assisted transmission and establishment of pathogens in the host as has been reported for a number of arthropod vector-borne diseases. If the hypothesis that tick salivary AChE may alter host immune responses is correct, we reasoned that similar cholinesterase activities might be present in saliva of additional arthropod vectors. Here, we report the presence of AChE-like activity in the saliva of southern cattle ticks, Rhipicephalus (Boophilus) microplus; the lone star tick, Amblyomma americanum (Linnaeus); Asian tiger mosquitoes, Aedes albopictus (Skuse); sand flies, Phlebotomus papatasi (Scopoli); and biting midges, Culicoides sonorensis Wirth and Jones. Salivary AChE-like activity was not detected for horn flies Haematobia irritans (L.), stable flies Stomoxys calcitrans (L.), and house flies Musca domestica L. Salivary cholinesterase (ChE) activities of arthropod vectors of disease-causing agents exhibited various Michaelis-Menten KM values that were each lower than the KM value of bovine serum AChE. A lower KM value is indicative of higher affinity for substrate and is consistent with a hypothesized role in localized depletion of host tissue acetylcholine potentially modulating host immune responses at the arthropod bite site that may favor ectoparasite blood-feeding and alter host defensive responses against pathogen transmission and establishment.

Sacral nerve stimulation prompts vagally-mediated amelioration of rodent colitis.

Neuromodulation based on the vagal anti‐inflammatory reflex has emerged as an exciting therapeutic approach for chronic inflammatory diseases. However, it is unclear whether direct stimulation of the vagus or of pelvic nerves coming from sacral roots, providing the bulk of colonic parasympathetic innervation, is the best approach. We hypothesized that sacral nerve stimulation (SNS) would be an effective treatment for colitis. Age and sex‐matched Sprague‐Dawley rats were administered 5% dextran sulphate sodium (DSS) in drinking water ad libitum for 7 days. A group of rats was sacrificed after DSS treatment, and the remaining rats were randomized to either sham‐SNS or SNS groups, which were performed for 1 hr daily for 10 days. Stimulations were delivered via chronically implanted electrodes using an 8‐channel universal pulse generator. Sacral nerve stimulation promoted recovery of colitis demonstrated by decreased disease activity index, myeloperoxidase activity, tissue TNF‐alpha, and histological scores as well as an increased colonic M2 macrophage population. Heart rate variability analysis demonstrated a decrease in low frequency and increase in high frequency with SNS, corresponding to increased vagal tone. Additionally, plasma pancreatic peptide was increased and norepinephrine was decreased after SNS in colitis while colon tissue acetylcholine was increased with SNS. This is the first study to the best of our knowledge that demonstrates the benefit of SNS with autonomic mediation. SNS alters the expression of inflammatory cytokines and macrophages as well as modulates neurotransmitters involved in systemic inflammation.

Lactobacillus casei Strain Shirota Alleviates Constipation in Adults by Increasing the Pipecolinic Acid Level in the Gut.

The benefits of probiotics for constipation are widely accepted, but the mechanisms involving gut metabolites are unclear. In this study, we investigated the effects of Lactobacillus casei strain Shirota (LcS) on constipated patients and revealed that a metabolite mediator is involved in the LcS-induced constipation alleviation. Sixteen constipated patients and 22 non-constipated participants were recruited. The subjects consumed 100 mL of an LcS beverage (108 CFU/mL) per day for 28 days. The fecal non-volatile metabolites were determined by GC/MS, and the targeted metabolites were further verified in a constipated mouse model. In constipated patients, LcS intervention significantly improved defecation frequency (from 4.81 to 7.81 times per week, p < 0.05), stool consistency (from 2.52 to 3.68, p < 0.05) and constipation-related symptoms. A total of 14 non-volatile fecal metabolites were obtained as potential constipation-related metabolites that were regulated by LcS. Among these metabolites, pipecolinic acid (PIPA) had a significant positive correlation with defecation frequency in constipated patients. PIPA significantly promoted the small intestinal propulsive rate (from 25.45 to 39.68%) and increased the number of fecal pellets (from 30.38 to 57.38 pellets) in constipated mice (p < 0.05). The 5-hydroxytryptamine (5-HT) and acetylcholine (ACh) in colonic tissue may be partly involved in PIPA-mediated constipation alleviation. In conclusion, PIPA was a metabolic mediator in the gut that participated in LcS-induced constipation alleviation.

The analysis of hippocampus neuronal density (CA1 and CA3) after Ocimum sanctum ethanolic extract treatment on the young adulthood and middle age rat model

Aim:This study aimed to assess the changes in neuronal density in CA1 and CA3 regions in the hippocampus of young adulthood and middle age rat model after feeding by Ocimum sanctum ethanolic extract.Materials and Methods:In this research, 30 male Wistar rats consist of young to middle-aged rats were divided into three groups (3, 6, and 9 months old) and treated with a different dosage of O. sanctum ethanolic extract (0, 50, and 100 mg/kg b.w.) for 45 days. Furthermore, cresyl violet staining was performed to analyze hippocampus formation mainly in CA1 and CA3 area. The concentrations of acetylcholine (Ach) in brain tissues were analyzed by enzyme-linked immunosorbent assay.Results:In our in vivo models using rat model, we found that the administration of O. sanctum ethanolic extract with a dosage of 100 mg/kg b.w. for 45 days induced the density of pyramidal cells significantly in CA1 and CA3 of the hippocampus. These results were supported by an increase of Ach concentrations on the brain tissue.Conclusions:The administration of O. sanctum ethanolic extract may promote the density of the pyramidal cells in the CA1 and CA3 mediated by the up-regulated concentration of Ach.

A novel liquid chromatography/mass spectrometry method for determination of neurotransmitters in brain tissue: Application to human tauopathies

Neurotransmitters, small molecules widely distributed in the central nervous system are essential in transmitting electrical signals across neurons via chemical communication. Dysregulation of these chemical signaling molecules is linked to numerous neurological diseases including tauopathies. In this study, a precise and reliable liquid chromatography method was established with tandem mass spectrometry detection for the simultaneous determination of aspartic acid, asparagine, glutamic acid, glutamine, γ-aminobutyric acid, N-acetyl-l-aspartic acid, pyroglutamic acid, acetylcholine and choline in human brain tissue. The method was successfully applied to the analysis of human brain tissues from three different tauopathies; corticobasal degeneration, progressive supranuclear palsy and parkinsonism-dementia complex of Guam. Neurotransmitters were analyzed on ultra-high performance chromatography (UHPLC) using an ethylene bridged hybrid amide column coupled with tandem mass spectrometry (MS/MS). Identification and quantification of neurotransmitters was carried out by ESI+ mass spectrometry detection. We optimized sample preparation to achieve simple and fast extraction of all nine analytes. Our method exhibited an excellent linearity for all analytes (all coefficients of determination >0.99), with inter-day and intra-day precision yielding relative standard deviations 3.2%–11.2% and an accuracy was in range of 92.6%–104.3%. The present study, using the above method, is the first to demonstrate significant alterations of brain neurotransmitters caused by pathological processes in the brain tissues of patient with three different tauopathies.

Elastase alters contractility and promotes an inflammatory synthetic phenotype in airway smooth muscle tissues.

Neutrophil elastase is secreted by inflammatory cells during airway inflammation and can elicit airway hyperreactivity in vivo. Elastase can degrade multiple components of the extracellular matrix. We hypothesized that elastase might disrupt the connections between airway smooth muscle (ASM) cells and the extracellular matrix and that this might have direct effects on ASM tissue responsiveness and inflammation. The effect of elastase treatment on ASM contractility was assessed in vitro in isolated strips of canine tracheal smooth muscle by stimulation of tissues with cumulatively increasing concentrations of acetylcholine (ACh) and measurement of contractile force. Elastase treatment potentiated contractile responses to ACh at low concentrations but suppressed the maximal contractile force generated by the tissues without affecting the phosphorylation of myosin regulatory light chain (RLC). Elastase also promoted the secretion of eotaxin and the activation of Akt in ASM tissues and decreased expression of smooth muscle myosin heavy chain, consistent with promotion of a synthetic inflammatory phenotype. As the degradation of matrix proteins can alter integrin engagement, we evaluated the effect of elastase on the assembly and activation of integrin-associated adhesion junction complexes in ASM tissues. Elastase led to talin cleavage, reduced talin binding to vinculin, and suppressed activation of the adhesome proteins paxillin, focal adhesion kinase, and vinculin, indicating that elastase causes the disassembly of adhesion junction complexes and the inactivation of adhesome signaling proteins. We conclude that elastase promotes an inflammatory phenotype and increased sensitivity to ACh in ASM tissues by disrupting signaling pathways mediated by integrin-associated adhesion complexes.

The interaction of l-cysteine/H2S pathway and muscarinic acetylcholine receptors (mAChRs) in mouse corpus cavernosum

The aim of this study was to investigate the possible interaction of l-cysteine/H2S pathway and muscarinic acetylcholine receptors (mAChRs) in the mouse corpus cavernosum (CC). l-cysteine (endogenous H2S substrate; 10−6-10−3 M), sodium hydrogen sulfide (NaHS; exogenous H2S; 10−6–10−3 M) and acetylcholine (10−9-10−4 M) produced concentration-dependent relaxation in isolated mouse CC tissues. Relaxations to endogenous and exogenous H2S were reduced by non-selective mAChR antagonist atropine (5 × 10−5 M), selective M1 mAChR antagonist pirenzepine (5 × 10−5 M) and selective M3 mAChR antagonist 4-DAMP (10−7 M) but not by selective M2 mAChR antagonist AF-DX 116 (10−6 M). Also, acetylcholine-induced relaxations were reduced by atropine, pirenzepine, 4-DAMP and AF-DX 116, confirming the selective effects of mAChR antagonists. Furthermore, acetylcholine-induced relaxations were attenuated by cystathionine-gamma-lyase (CSE) inhibitor d,l-propargylglycine (PAG, 10−2 M) and cystathionine-β-synthase inhibitor (CBS) aminooxyacetic acid (AOAA, 10−3 M). l-nitroarginine, nitric oxide synthase inhibitor, augmented the inhibitory effects of mAChR antagonists and H2S enzyme inhibitors on acetylcholine-induced relaxations. In addition, the existence and localization of CSE, CBS and 3-MST were demonstrated in mouse CC. Furthermore, tissue acetylcholine release was significantly increased by l-cysteine but not by exogenous H2S. The increase in acetylcholine level was completely inhibited by AOAA and PAG. These results suggest that M1 and M3 mAChRs contributes to relaxant effect mediated by endogenous H2S but at same time l-cysteine triggers acetylcholine release from cavernosal tissue. Also, the role of NO in the interaction of l-cysteine/H2S pathway and muscarinic acetylcholine receptors (mAChRs) could not be excluded.

Effect of Ginkgo biloba extract on cognitive function and neurotransmitter levels in rats with vascular dementia

This study aimed to investigate the effect of Ginkgo biloba extract on the cognitive function and neurotransmitter levels in rats with vascular dementia (VD) and its mechanism of action. 50 male Sprague Dawley rats were selected for this study, and the vascular dementia model was established by permanent occlusion of bilateral common carotid arteries of rats. Of which 40 rats were then divided into four groups (n=10): vascular dementia group, low-dose, middle-dose and high-dose ginkgo biloba extract groups respectively; another 10 rats were allocated to sham operation group. Rats in low, middle and high dose groups were received 5, 10 and 20 mg/kg/d Ginkgo biloba extract via tail vein, respectively, while model group and sham group received an equal volume of normal saline. Morris water maze was used to evaluate the cognitive function of rats. After behavioral observation, these rats were sacrificed for detecting the level of acetylcholine (ACh), dopamine (DA) and 5-hydroxytryptamine (5-HT) in brain tissue. Ginkgo biloba extract can significantly improve the cognitive function of rats with vascular dementia, the mechanism may be correlated with the fact that the extract can obviously increase the levels of ACh, 5-HT and DA, as well as inhibit the activity of AChE.

Botulinum neurotoxin for sialorrhea: a first choice therapy

This article emphasizes the therapeutic potential of botulinum neurotoxin (BoNT) in the autonomous nerve system disorder of sialorrhea, a pathologic situation found mainly in patients with neurologic deficits. Major applications that are well known to be treated with botulinum toxin in the head and neck area, include undesirable muscular dysfunctions and spasms, based on the toxins blocking effect on the neuromuscular junction transmitter acetylcholine. In the 90’s it became clear that BoNT could block the release of acetylcholine in gland tissues, too. A new era in the management of autonomic dysfunctions began. There have been numerous of treatments such as Frey’s syndrome, sialorrhea, facial and cranial hyperhidrosis reviewed. Ultrasound guided intraglandular injections of botulinum toxin type A into both the submandibular and parotid glands, have been noted to be effective at controlling sialorrhea in practice. The doses and injection techniques are described and further discussed in this article. The option of the minimal invasive, well tolerated BoNT injections into the major salivary glands is crucial for the best management of sialorrhea patients, avoiding the side-effects of anticholinergic medications. The indications of BoNT in the head and neck region are still expanding as physicians are finding out the therapeutic valuability of the toxin in otorhinolaryngology.

Effect of cholinotropic agents to change the content of oxygen triplet forms of liver tissue and the ability of the liver homogenate to produce active oxygen in rat cooling

The method of polarographic analysis of elements in the tissue solutions of living organisms, and the method of checking the ability of the liver homogenate to produce active forms of oxygen are among the methods for assessing the content of triplet forms of oxygen in the tissue and the ability of the tissue homogenate to initiate lipid peroxidation (POL) after a cold load and administration of cholinotropic agents to rats. In the study, the content of triplet form of oxygen of liver homogenate and the ability of the liver homogenate to produce active oxygen in the period of 3 hour and 5 days cooling of experimental animals were determined. The effect of cholinotropic agent accumulating the endogenous acetylcholine in liver tissue, pharmacological agents that stimulate and block the work of muscarine-sensitive cholinoreactive structures of hepatocyte plasmatic membranes with the assessment of their impact on the content of triplet forms of oxygen, the ability of the liver homogenate to produce active oxygen in supercooling of animals were investigated. Neostigmine on the background of 3 hours cold exposure led to a decrease in the content of triplet forms of oxygen but increased the ability of the liver homogenate to produce active forms of oxygen. Pilocarpine and atropine on the background of cooling animals in 5 days period caused the manifestation of reciprocity as in 3 minutes, so as in 30 minutes of experiment for the determination of triplet forms of oxygen of the liver tissue. Besides pilocarpine and atropine reduced the ability of liver tissue to produce active forms of oxygen. The obtained results indicate the change in the content of triplet forms of oxygen in the liver tissue when pilocarpine and atropine administrated to animals on the backdrop of 5 days cooling. Additionally the results show that the injection of neostigmine to animals on the background of 3 hours cooling promotes the increase of active forms of oxygen.

Age-dependent modulation of fasting and long-term dietary restriction on acetylcholinesterase in non-neuronal tissues of mice.

Dietary restriction (DR) without malnutrition is a robust intervention that extends lifespan and slows the onset of nervous system deficit and age-related diseases in diverse organisms. Acetylcholinesterase (AChE), a thoroughly studied enzyme better known for hydrolyzing acetylcholine (ACh) in neuronal tissues, has recently been linked with multiple unrelated biological functions in different non-neuronal tissues. In the present study, the activity and protein expression level of AChE in liver, heart, and kidney of young (1 month), adult (6 month), and aged (18 month) mice were investigated. We also studied age- and tissue-specific changes in AChE activity and protein expression level after the mice were subjected to 24-h fasting and long-term DR. Our results showed that AChE activity and protein expression in kidney and heart of aged mice decreased significantly in comparison with young mice. On the contrary, long-term DR decreases the AChE activity and the protein expression level in all tissues irrespective of ages studied. We summarized that changes in AChE with age in different tissues studied reflects its different roles at different phases of an organism's life. Conversely, the cumulative modulation manifested in the form of lowering AChE by long-term DR may prevent the futile synthesis and accumulation of unwanted AChE besides the added compensatory benefit of enhanced ACh availability needed during the period of starvation. This, in turn, may help in preventing the declining homeostatic roles of this important neurotransmitter in different tissues.