Concentrations Of ACh Research Articles

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

BackgroundNon-neuronal cholinergic system (NNCS) contributes to various inflammatory airway diseases. However, the role of NNCS in severe asthma (SA) remains largely unexplored. ObjectiveTo explore airway NNCS in SA. MethodsIn 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. ResultsPatients 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. ConclusionThis 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 RegistrationChiCTR-OOC-16009529 (http://www.chictr.org.cn).

Effects of Cycloastragenol on Alzheimer's Disease in Rats by Reducing Oxidative Stress, Inflammation, and Apoptosis.

As individuals age, they may develop Alzheimer's disease (AD), which is characterized by difficulties in speech, memory loss, and other issues related to neural function. Cycloastragenol is an active ingredient of Astragalus trojanus and has been used to treat inflammation, aging, heart disease, and cancer. This study aimed to explore the potential therapeutic benefits of cycloastragenol in rats with experimentally induced AD. Moreover, the underlying molecular mechanisms were also evaluated by measuring Nrf2 and HO-1, which are involved in oxidative stress, NFκB and TNF-α, which are involved in inflammation, and BCL2, BAX, and caspase-3, which are involved in apoptosis. Sprague-Dawley rats were given 70 mg/kg of aluminum chloride intraperitoneally daily for six weeks to induce AD. Following AD induction, the rats were given 25 mg/kg of cycloastragenol daily by oral gavage for three weeks. Hippocampal sections were stained with hematoxylin/ eosin and with anti-caspase-3 antibodies. The Nrf2, HO-1, NFκB, TNF-α, BCL2, BAX, and caspase-3 gene expressions and protein levels in the samples were analyzed. Cycloastragenol significantly improved rats' behavioral test performance. It also strengthened the organization of the hippocampus. Cycloastragenol significantly improved behavioral performance and improved hippocampal structure in rats. It caused a marked decrease in the expression of NFκB, TNF-α, BAX, and caspase-3, which was associated with an increase in the expression of BCL2, Nrf2, and HO-1. Cycloastragenol improved the structure of the hippocampus in rats with AD. It enhanced the outcomes of behavioral tests, decreased the concentration of AChE in the brain, and exerted antioxidant and anti-inflammatory effects. Antiapoptotic effects were also noted, leading to significant improvements in cognitive function, memory, and behavior in treated rats.

Mercury chloride causes cognitive impairment, oxidative stress and neuroinflammation in male Wistar rats: The potential protective effect of 6-gingerol-rich fraction of Zingiber officinale via regulation of antioxidant defence system and reversal of pro-inflammatory markers increase

This study investigated the effects of 6-gingerol-rich fraction of Zingiber officinale (6-GIRIFZO) on mercury chloride (HgCl2)-induced neurotoxicity in Wistar rats. Thirty -five male Wistar rats weighing between (150–200 g) were divided randomly into five groups (n = 7): group 1: control, received 0.5 mL of normal saline, group 2: received HgCl2 (5 mg/kg), group 3: received N-acetylcysteine (NAC) (50 mg/kg) as well as HgCl2 (5 mg/kg), group 4: received 6-GIRIFZO (100 mg/kg) and HgCl2 (5 mg/kg), group 5: had 6-GIRIFZO (200 mg/kg) and HgCl2 (5 mg/kg), consecutively for 14 days. On the day14, the rats were subjected to behavioural tests using a Morris water maze and novel object recognition tests. The rats were then euthanized to obtain brain samples for the determination of biochemical parameters (acetylcholinesterase (AchE), nitric oxide (NO), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), glutathione (GSH), tumor necrosis factor- alpha (TNF-α), nuclear factor kappa-B (NF-κB), interleukin-1β (IL-1β) and interleukin-6 (IL-6)) using standard methods. The result revealed a significant increase in escape latency and a significant decrease in recognition ratio in the rats that were exposed to HgCl2 only. However, 6-GIRIFZO produced a significant reduction in the escape latency and (p < 0.05) increase in the recognition ratio. Similarly, HgCl2 exposure caused a significant (p < 0.05) decrease in the brain SOD, GPx, CAT, GSH with increased brain levels of MDA, NO, AchE, TNF-α, NF-κB, IL-1β and IL-6. Similarly to the standard drug, NAC, 6-GIRIFZO (100 and 200 mg/kg) significantly (p < 0.05) increased brain SOD, GPx, CAT, and GSH levels with decreased concentrations of MDA, NO, AchE, TNF-α, NF-κB, IL-1β and IL-6. Also, pre-treatment with 6-GIRIFZO prevented the HgCl2-induced morphological aberrations in the rats. This study concludes that 6-GIRIFZO prevents HgCl2-induced cognitive deficit via reduction of brain inflammation as well as oxidative stress in rats.

Highly Sensitive Acetylcholine Biosensing Via Chemical Amplification of Enzymatic Processes in Single Track-Etched Nanochannels

Neurotransmitters are endogenous chemical messengers that play crucial roles in the transmission, enhancement and conversion of specific signals between neurons and other cells and are considered biomarkers of neurodegenerative diseases.[1] Neurodegenerative diseases cause progressive loss of cognitive and/or motor function and, as life expectancy rises, they imply major challenges for societies with rapidly aging populations.[2] As a result, monitoring neurotransmitters is becoming increasingly relevant in clinical environments. Acetylcholine (Ach) is a neurotransmitter that plays a key role in the communication between neurons in the spinal cord and nerve skeletal junctions in vertebrates. [3] Since an abnormal concentration of Ach has proven to be related to neurodegenerative diseases, novel strategies for highly sensitive and specific Ach (bio)sensing are in great demand.In this work we present the construction of Ach enzymatic biosensors with remarkable sensitivity by acetylcholinesterase immobilization on polyamine-modified single nanochannels via electrostatic self-assembly. [4] To fabricate bullet-shaped single nanochannels in polyethylene terephthalate (PET), 12 µm thick PET foils are irradiated with a single swift heavy ion. The resulting ion track consists of highly localized damaged material along the ion trajectory and is selectively dissolved in a highly basic aqueous solution under asymmetric conditions to obtain a bullet-shaped channel. The controlled PET hydrolysis exposes carboxylic groups at the channel surface (negative charge at pH < 4), which allows the immobilization of further functional groups to design and construct the biosensor.Here, we will discuss in detail the sensor design, the applied functionalization strategy, and the performance of the sensor. In short, changes in the surface charge of the nanochannel lead to measurable changes in the transmembrane current in a steady-state configuration. We will show experimental results that evidence that the designed sensor successfully transduces the presence of acetylcholine into measurable ionic signals with a 16 nM low limit of detection.[1] World Health Organization, Neurological Disorders: Public Health Challenges, World Health Organization Press, Geneva, Switzerland,2007.[2] L. Gan, M. R. Cookson, L. Petrucelli and A. R. La Spada,Nat. Neurosci., 2018, 21, 1300–1309.[3] M. Hasanzadeh, N. Shadjou and M. de la Guardia, TrAC, Trends Anal. Chem., 2017, 86, 107–121.[4] Yamili Toum Terrones, Gregorio Laucirica, Vanina M. Cayón, Gonzalo E. Fenoy, M. Lorena Cortez, María Eugenia Toimil-Molares, Christina Trautmann, Waldemar A. Mamisollé and Omar Azzaroni, Chem. Commun., 2022, 58, 10166

Environmental levels of azoxystrobin disturb male zebrafish behavior: Possible roles of oxidative stress, cholinergic system, and dopaminergic system

A widely applied pesticide of azoxystrobin, is increasingly detected in the water environment. Concern has been raised against its potential detriment to aquatic ecosystems. It has been shown that exposure to azoxystrobin interfere with the locomotor behavior of zebrafish larvae. This study aims to investigate whether exposure to environmental levels of azoxystrobin (2 μg/L, 20 μg/L, and 200 μg/L) changes the behavior of male adult zebrafish. Herein, we evaluated behavioral response (locomotor, anxiety-like, and exploratory behaviors), histopathology, biochemical indicators, and gene expression in male adult zebrafish upon azoxystrobin exposure. The study showed that exposure to azoxystrobin for 42 days remarkably increased the locomotor ability of male zebrafish, resulted in anxiety-like behavior, and inhibited exploratory behavior. After treatment with 200 μg/L azoxystrobin, vasodilatation, and congestion were observed in male zebrafish brains. Exposure to 200 μg/L azoxystrobin notably elevated ROS level, MDA concentration, CAT activity, and AChE activity, while inhibiting SOD activity, GPx activity, ACh concentration, and DA concentration in male zebrafish brains. Moreover, the expression levels of genes related to the antioxidant, cholinergic, and dopaminergic systems were significantly changed. This suggests that azoxystrobin may interfere with the homeostasis of neurotransmitters by causing oxidative stress in male zebrafish brains, thus affecting the behavioral response of male zebrafish.

Effects of Hydrolysates from Silkworms Fed Cudrania tricuspidata Leaves on Improvement of Memory in Rats with Impaired Memory Induced by Scopolamine

This study investigated the memory-improving effects and mechanisms of action of hydrolysate from silkworm fed Cudrania tricuspidata leaves (HSCT) in rats with scopolamine-induced memory impairment. Thirty-two rats were categorized into 4 groups and the experiment was conducted for 6 weeks. The experimental diet groups are as follows: control, control diet; scopolamine, control diet + scopolamine (0.7 mg/kg); HSCT, HSCT diet + scopolamine (0.7 mg/kg); and donepezil, control diet + scopolamine (0.7 mg/kg) + donepezil (1.0 mg/kg). Except for the control group, the experimental groups received scopolamine 0.7 mg/kg intraperitoneally to induce decline in memory. Animal behavioral tests such as the Barnes maze, novel object recognition test, and Y-maze were performed to investigate the effects of HSCT on memory improvement. In addition, brain acetylcholine (ACh) concentration and acetylcholinesterase (AChE) activity were assessed to investigate the mechanism of memory improvement. Results of the animal behavior test suggested that the rats administered HSCT displayed improved memory behavior compared to those administered scopolamine (p &lt; 0.05). The concentration of Ach was increased in the HSCT group compared to the scopolamine group (p &lt; 0.05), and the activity of AChE was significantly reduced (p &lt; 0.05). These results suggest that HSCT effectively improves memory by reducing AChE activity and increasing ACh content in the brains of rats with scopolamine-induced memory impairment. As a natural compound, HSCT has the potential to be used as a material to improve memory.

Region-specific remodeling of the enteric nervous system and enteroendocrine cells in the colon of spinal cord injury patients

Patients with spinal cord injury (SCI) suffer from major bowel dysfunction, whose exact pathophysiology, particularly the involvement of the enteric nervous system or epithelial dysfunction is poorly understood. Herein, we aimed to characterize the mucosal biopsies of the right and left colon in SCI patients vs controls (CT): (1) remodeling of key enteric neurotransmitters, (2) remodeling of enteroendocrine cells, and (3) mucosal inflammation compared to those in controls. In SCI, mucosal ACh concentration was lower in the right colon as compared to CT, but no change was observed in the left colon, and AChE expression was lower in both the right and left colons than in CT. While the VIP concentration was similar in the right and left colons, VIP mRNA expression was increased in the right colon and decreased in the left colon, in SCI patients as compared to CT. Interestingly, 5-HT concentration was reduced in the left colon but not in the right colon in SCI patients. Moreover, in SCI patients, as compared to CT, SERT mRNA expression was selectively increased in the left colon while TPH1 mRNA expression was increased in the right and left colons. Although mucosal TNFα and IL-1β mRNA expression did not significantly differ between SCI and CT groups, we identified a significant positive correlation between TNFα and IL-1β mRNA expression and left colon transit time in the SCI group. In conclusion, region-specific changes occur in the enteric neurotransmitter, serotonergic, and inflammatory pathways in the colon of SCI patients. The significant correlations between these pathways and clinical parameters in the left colon further set a scientific basis for designing therapeutic targets to improve colonic motor dysfunction in patients.Biobank information: Spinal cord injury patients: PHRC ConstiCAPE—clinical trial NCT02566746. Controls: Anosain—clinical trial NCT03054415 and biobank of the “Institut des Maladies de l’Appareil Digestif (IMAD)” registered under number DC-2008-402.

Label free impedance based acetylcholinesterase enzymatic biosensors for the detection of acetylcholine

Realtime monitoring of neurotransmitters is of great interest for understanding their fundamental role in a wide range of biological processes in the central and peripheral nervous system, as well as their role, in several degenerative brain diseases. The measurement of acetylcholine in the brain is particularly challenging due to the complex environment of the brain and the low concentration and short lifetime of acetylcholine. In this paper, we demonstrated a novel, label-free biosensor for the detection of Ach using a single enzyme, acetylcholinesterase (ACHE), and electrochemical impedance spectroscopy (EIS). Acetylcholinesterase was covalently immobilized onto the surface of gold microelectrodes through an amine-reactive crosslinker dithiobis(succinimidyl propionate) (DSP). Passivation of the gold electrode with SuperBlock eliminated or reduced any non-specific response to other major interfering neurotransmitter molecules such as dopamine (DA), norepinephrine (NE) and epinephrine (EH). The sensors were able to detect acetylcholine over a wide concentration range (5.5–550 μM) in sample volumes as small as 300 μL by applying a 10 mV AC voltage at a frequency of 500 Hz. The sensors showed a linear relationship between Ach concentration and ΔZmod(R2 = 0.99) in PBS. The sensor responded to acetylcholine not only when evaluated in a simple buffer (PBS buffer) but in several more complex environments such as rat brain slurry and rat whole blood. The sensor remained responsive to acetylcholine after being implanted ex vivo in rat brain tissue. These results bode well for the future application of these novel sensors for real time in vivo monitoring of acetylcholine.

Effects of Danggui-Shaoyao-San on central neuroendocrine and pharmacokinetics in female ovariectomized rats

Ethnopharmacological relevanceThe traditional Chinese medicine formula Danggui-Shaoyao-San (DSS) has been reported to have estrogen-like effects and therapeutic effects on the symptoms of Alzheimer's disease (AD). Aim of the studyTo explore whether the central oxytocin and neuroendocrine system is involved in the modulating effects of DSS on the cognition and neuropsychiatric hebaviors in female AD rats, and to investigate the pharmacokinetics of paeoniflorin and ferulic acid in female AD rats with DSS treatment. Material and methodsDSS (1.2, 3.2, 8.6 g/kg/day) was orally administered to ovariectomized (OVX) rats, and saline was orally administered to sham operation rats as control group. The Morris water maze test, novel object recognition test, and passive avoidance test were conducted for evaluation of learning and memory abilities, while elevated plus maze test and forced swim test were performed to assess anxiety- and depressive-like behaviors. ELISA kits were used to detect the levels of estrogen (E), estrogen receptor α (ERα), oxytocin (OT), oxytocin receptor (OTR), acetylcholine (Ach), acetylcholin esterase (AchE), and choline acetyl transferase (ChAT) in the cortex. The concentrations of Ach, glutamate (Glu), γ-aminobutyric acid (GABA), 5-hydroxytryptamine (5-HT), norepinephrine (NE) and dopamine (DA) in the hippocampus were assessed by HPLC-MS. The changes of neuronal morphology in the hippocampus were observed by Nissl staining. The pharmacokinetics of paeoniflorin and ferulic acid in OVX rats with DSS treatment were studied by HPLC. ResultsIn the Morris water maze test, novel object recognition test, and passive avoidance test, OVX rats showed cognitive impairment. In the elevated plus maze test and forced swim test, the anxiety- and depressive-like behaviors of OVX rats were significant as compared to the control group. Treatment of DSS significantly imporved the cognitive deficits, and ameliorated anxiety- and depressive-like behaviors of OVX rats. The expression of E, ERα, OT, OTR, AchE and ChAT in the cortex of model group were significantly decreased, and DSS significantly reversed these changes. The concentrations of Ach, Glu, GABA, 5-HT and NE in the hippocampus of OVX rats were significantly decreased, whereas DSS significantly increased the levels of Ach, Glu, GABA, 5-HT and NE. There was no significant difference in the concentration of DA in the hippocampus among groups. Degenerating neurons in the hippocampal CA3 region were observed in OVX rats, and the number of neurons was decreased. DSS treatment reduced the degenerating neurons, and incresed the number of neurons. The MRT (0 - ∞), AUC (0 - ∞), Cmax and t1/2z values of paeoniflorin, and the AUC 0-∞ and Cmax value of ferulic acid were higher in DSS-treated OVX rats than those in the DSS-treated control rats. ConclusionsDSS improves the learning and memory ability, and attenuates anxiety- and depressive-like behaviors of OVX rats. The mechanism may be through increasing estrogen, reducing cholinergic damage, and modulating neurotransmitters. The increase in absorption and elimination time of paeoniflorin and ferulic acid in OVX rats may enhance the efficacy of DSS.

High antioxidant load impairs cutaneous microvascular function in healthy women

PURPOSE: Human cutaneous circulation is used as an in vivo bioassay to examine mechanisms of systemic vascular dysfunction in cohorts of patients with accelerated cardiovascular disease risk. Local drug infusions, including atorvastatin (statin; LOX inhibitor) and systemic interventions to investigate anti-inflammatory mechanisms (e.g., oral salsalate) have been used to elucidate mechanisms underlying microvascular dysfunction. However, in healthy cohorts, high antioxidant load can attenuate total vasodilation and nitric oxide (NO)-dependent vasodilation. Thus, we aimed to examine endothelial function during high antioxidant load from these localized and systemic treatments in healthy control groups. We hypothesized that combined localized and systemic treatments will attenuate endothelial function in healthy women. METHODS: In a randomized placebo control design, oral salsalate (1500mg/twice daily/5 days) was used to assess the impact of systemic inflammation on cutaneous vascular function. Four intradermal microdialysis fibers were placed into the intradermal space of the ventral forearm of six healthy women (32±7 years). Laser-Doppler flowmeter probes, used to measure cutaneous blood flow, were placed in local heating units set to 33°C. Increasing concentrations of acetylcholine (ACh; endothelium-dependent vasodilator) from 10 -10 to 10 -1 M were co-perfused with one of the following: Lactated Ringer’s (control), 0.02 mM statin, 15 mM N G -nitro-L-arginine methyl ester (L-NAME; nitric oxide synthase inhibitor), and statin + L-NAME (COMBO) sequentially for 5 min each. Following the dose-response protocol, maximal cutaneous vasodilation was induced by increasing local skin temperature to 43°C and perfusing 28 mM sodium nitroprusside. Cutaneous vascular conductance (CVC) was calculated as the quotient of red blood cell flux and mean arterial pressure and normalized as a percentage of the site-specific maximum (%CVC max ). Data at each intervention were compared with control using a two-way (dose x site) analysis of variance. Responses at each dosage were compared using paired t-tests and corrected for multiple comparisons. RESULTS: Following oral salsalate, there was no difference in the %CVC max response to ACh with local L-NAME (p=0.896) or COMBO interventions (p=0.617) as compared to the placebo. However, following oral salsalate, there was a significant reduction in %CVC max in the statin treated site at higher ACh concentrations (10 -5 M, 10 -4 M, 10 -3 M; all p&lt;0.01). CONCLUSION: Endothelial function was attenuated when subjects were introduced to a combination of oral salsalate and statin localized treatments. Five-day oral salsalate intake and local statin may result in high antioxidant load in healthy women creating a shift in redox balance, thus leading to impaired endothelial function. Supported by NIH Grant R01 HL161000 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Examining the effect of a substituted carbamate positive allosteric modulator on the (α4)3(β2)2 and (α4)2(β2)3 nicotinic acetylcholine receptors.

The α4β2 nicotinic acetylcholine receptors (nAChR) are the most abundant nAChR subtypes in the brain and exist in two distinct isoforms: (α4)2(β2)3 nAChR, the high sensitivity (HS) isoform, and (α4)3(β2)2 nAChR, the low sensitivity (LS) isoform. Given the pathophysiological and pharmacological importance of these brain receptors, there are ongoing efforts to develop pharmacophores that selectively potentiate α4β2 nAChRs (positive allosteric modulators, PAMs). PAMs selective to the α4β2 nAChRs would be beneficial for neuropsychiatric conditions such as nicotine addiction and Alzheimer's disease. A chemical synthesis and high throughput screens at Amgen laboratories have identified series of compounds that preferentially potentiate the α4β2 nAChRs (Albrecht, Berry et al. 2008, Springer, Woodin et al. 2008). Notable PAMs identified in these screens include CMPI, which selectively potentiates the (α4)3(β2)2 nAChR isoform (Wang, Deba et al. 2017). In this study, we started to pharmacologically characterize additional derivatives (C9M) from these screens, focusing on its effects on the LS and HS nAChR isoforms. Unlike CMPI, C9M potentiated current response of Xenopus oocytes expressing LS and HS nAChR isoforms elicited by submaximal and saturating concentrations of ACh. This potentiation indicates that C9M enhances the agonist's efficacy at both α4β2 nAChR isoforms. Furthermore, mutations known to alter CMPI potentiation of LS nAChR (e.g. α4G41M) did not abolish the potentiating effects of C9M. Additionally, C9M enhancement of agonist's efficacy at HS (α4)2(β2)3 nAChR was notably greater than that for a panel of known nAChR PAMs including dFBr and LY2087101 (all co-applied at 1 µM with 10 µM ACh). Experiments to determine the concentration dependent effect of C9M at both LS and HS nAChR isoforms and to determine the extent of C9M effect on the agonist's potency are ongoing.

Acetylcholine suppresses LPS-induced endothelial cell activation by inhibiting the MAPK and NF-κB pathways.

Endothelial cell activation plays a critical role in leukocyte recruitment during inflammation and infection. We previously found that cholinergic stimulation (via vagus nerve stimulation) attenuates vascular endothelial impairment and reduces the inflammatory profile in ovariectomized rats. However, the specific molecular mechanism is unclear. This study was designed to explore the effects and molecular mechanisms of cholinergic agonists (acetylcholine [ACh]) on lipopolysaccharide (LPS)-induced endothelial cell activation in vitro. Human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of LPS (10/100/1000ng/mL) to activate endothelial cells. HUVECs were untreated, treated with ACh (10-5 M) alone, treated with 100ng/mL LPS alone, or treated with different concentrations of ACh (10-9/10-8/10-7/10-6/10-5 M) before LPS stimulation. HUVECs were also pre-treated with 10-6 M ACh with or without mecamylamine (an nAChR blocker) (10μΜ) and methyllycaconitine (a specific α7 nAChR blocker) (10μΜ) and incubated with or without LPS. ELISA, western blotting, cell immunofluorescence, and cell adhesion assays were used to examine inflammatory cytokine production, adhesion molecule expression, monocyte-endothelial cell adhesion and activation of the MAPK/NF-κB pathways. LPS (at 10ng/mL, 100ng/mL and 1,000ng/mL) increased VCAM-1 expression in HUVECs in a dose-dependent manner (with no significant difference between LPS at 100ng/mL and 1,000ng/mL). ACh (10-9 M-10-5 M) blocked adhesion molecule expression (VCAM-1, ICAM-1, and E-selectin) and inflammatory cytokine production (TNF-α, IL-6, MCP-1, IL-8) in response to LPS in a dose-dependent manner (with no significant difference between 10-5 and 10-6 M Ach). LPS was also shown to significantly enhance monocyte-endothelial cell adhesion, which was largely abrogated by treatment with ACh (10-6M). VCAM-1 expression was blocked by mecamylamine rather than methyllycaconitine. Lastly, ACh (10-6 M) significantly reduced LPS-induced phosphorylation of NF-κB/p65, IκBα, ERK, JNK and p38MAPK in HUVECs, which was blocked by mecamylamine. ACh protects against LPS-induced endothelial cell activation by inhibiting the MAPK and NF-κB pathways, which are mediated by nAChR, rather than α7 nAChR. Our results may provide novel insight into the anti-inflammatory effects and mechanisms of ACh.

Evaluation of effectiveness of ginkgo biloba aqueous extract on some physiological parameters and cerebrum tissue in male albino rats treated with aluminum chloride

The current study aims to know the protective role of the aqueous extract of the Ginkgo biloba against cerebrum damage induced by Aluminum chloride (AlCl3) in male rats. 40 adult male rats were used, which were randomly divided into four equal groups (10 animals/group), the first group (G1) administered 1 ml/kg of tap water, the second group (G2) administered 10 mg / kg of AlCl3, the third group (G3) was administered 10 mg/kg of aqueous extract of the Ginkgo biloba ,while the fourth group animals (G4) were administered 10 mg/kg of AlCl3 and 10 mg/kg of Ginkgo biloba aqueous extract orally and daily for 30 days. Fasting blood samples were collected after the end of the experiment to study the following parameters: concentration of soluble acetylcholinesterase AChE(G1), membrane bound acetylcholinesterase AChE (G4), Neuronal Mitochondrial ATPase (Nmit ATPase), Cytochrome C oxidase (COX) , Tumor Necrosis Factor-α (TNF-α). The results showed that oral administration of AlCl3 caused significant increase (P&lt;0.05) in the concentration of AChE (G1), TNF-α, and significant decrease (P&lt;0.05) in the concentration of AChE (G4), Nmit ATPase and COX compared with the control group.

Spasmolytic, Gastroprotective and Antioxidant Activities of Dry Methanol Extract of Ferula heuffelii Underground Parts

AbstractDry MeOH extract of Ferula heuffelii (Apiaceae) underground parts was tested for spasmolytic, gastroprotective and antioxidant activities. HPLC analysis revealed that chlorogenic acid (CGA; 34.6 mg/g) was its main constituent. Extract in vitro exhibited notable total antioxidant activity (FRAP value=1.0 μmol Fe2+/mg), and scavenging of DPPH (SC50=62.5 μg/ml) and •OH radicals (49.5 % at 20 μg/ml in 2‐deoxyribose assay). In vitro on isolated rat ileum, extract exhibited significant spasmolytic activity, i. e., it showed 124.6 % of maximal atropine effect on spontaneous contractions (at 100 μg/ml), and reduced spasmogenic effect of KCl (80 mm) to 44.4 % (at 60 μg/ml) and of highest applied concentration of ACh to 26.3 % (at 120 μg/ml). In parallel experiments, spasmolytic effect of CGA was also demonstrated. In acute EtOH‐induced gastric ulceration model in rats, extract (100 mg/kg p.o.) showed significant gastroprotective effect (gastric damage score 0.50), similar to ranitidine (20 mg/kg p.o.). Obtained results showed that tested F. heuffelii polar extract represents new herbal preparation with potential use against some gastrointestinal complaints.

Grape seed alleviates lindane-induced oxidative stress and improves growth performance, caecal fermentation and antioxidant capacity in growing rabbits.

This study evaluated the protective effect of grape seed on performance, caecal characteristics, blood metabolites and liver antioxidant status in lindane-treated rabbits. Four-week-old New Zealand White rabbits (n=96) with an initial body weight of 0.750 ± 0.23g were randomly divided into four groups (n=24). One group was the control received only corn oil orally, while group L were received lindane daily via gavage in corn oil (4mg/kg BW; 1/50 LD50 for oral dose), group GS was treated with 50g grape seed /kg diet, and group LGS treated with a combination of both L and GS for 98days. Results revealed that final body weight (FBW), average daily gain (ADG), dry matter intake and feed efficiency (FE) were similar between GS and control groups, and achieved the highest FBW and ADG, and the best FE. Caecum pH of the L group increased, while the caecum pH of the GS group decreased sharply. There was a significant increase in the concentration of total VFA, acetic acid, propionic acid and NH3 -N in the GS group, but butyric acid level decreased. GS treatment resulted in a significant increase in the concentrations of total protein, albumin and AChE. GPx, GST, CAT and SOD activity decreased, but TBARS activity significantly increased in the group L, while GS caused a significant elevation of antioxidant activity in the liver. These results confirm that the antioxidant compounds present in grape seed can alleviate the oxidative stress caused by lindane-induced hepatotoxicity and could be a useful supplement to maintain health and improve performance in rabbits.

Stoichiometry-selective modulation of α4β2 nicotinic ACh receptors by divalent cations.

α4β2 nicotinic ACh receptors (nAChRs) comprise the most abundant class of nAChRs in the nervous system. They assemble in two stoichiometric forms, each exhibiting distinct functional and pharmacological signatures. However, whether one or both forms are modulated by calcium or magnesium has not been established. To assess the functional consequences of calcium and magnesium, each stoichiometric form was expressed in clonal mammalian fibroblasts and single-channel currents were recorded in the presence of a range of ACh concentrations. In the absence of divalent cations, each stoichiometric form exhibits high unitary conductance and simple gating kinetics composed of solitary channel openings or short bursts of openings. However, in the presence of calcium and magnesium, the conductance and gating kinetics change in a stoichiometry-dependent manner. Calcium and magnesium reduce the conductance of both stoichiometric forms, with each cation producing an equivalent reduction, but the reduction is greater for the (α4)2 (β2)3 form. Moreover, divalent cations promote efficient channel opening of the (α4)3 (β2)2 stoichiometry, while minimally affecting the (α4)2 (β2)3 stoichiometry. For the (α4)3 (β2)2 stoichiometry, at high but not low ACh concentrations, calcium in synergy with magnesium promote clustering of channel openings into episodes of many openings in quick succession. Modulation of the α4β2 nAChR by divalent cations depends on the ACh concentration, the type of cation and the subunit stoichiometry. The functional consequences of modulation are expected to depend on the regional distributions of the stoichiometric forms and synaptic versus extrasynaptic locations of the receptors.

Role of Bmal1 in mediating the cholinergic system to regulate the behavioral rhythm of nocturnal marine molluscs

The circadian rhythm is one of the most general and important rhythms in biological organisms. In this study, continuous 24-h video recordings showed that the cumulative movement distance and duration of the abalone, Haliotis discus hannai, reached their maximum values between 20:00–00:00, but both were significantly lower between 08:00–12:00 than at any other time of day or night (P < 0.05). To investigate the causes of these diel differences in abalone movement behavior, their cerebral ganglia were harvested at 00:00 (group D) and 12:00 (group L) to screen for differentially expressed proteins using tandem mass tagging (TMT) quantitative proteomics. Seventy-five significantly different proteins were identified in group D vs. group L. The differences in acetylcholinesterase (AchE) expression levels between day- and nighttime and the key role in the cholinergic nervous system received particular attention during the investigation. A cosine rhythm analysis found that the concentration of acetylcholine (Ach) and the expression levels of AchE tended to be low during the day and high at night, and high during the day and low at night, respectively. However, the rhythmicity of the diel expression levels of acetylcholine receptor (nAchR) appeared to be insignificant (P > 0.05). Following the injection of three different concentrations of neostigmine methylsulfate, as an AchE inhibitor, the concentration of Ach in the hemolymph, and the expression levels of nAchR in the cerebral ganglia increased significantly (P < 0.05). Four hours after drug injection, the cumulative movement distance and duration of abalones were significantly higher than those in the uninjected control group, and the group injected with saline (P < 0.05). The expression levels of the core diurnal clock Bmal1 over a 24-h period also tended to be high during the day and low at night. First, a co-immunoprecipitation assay demonstrated the binding between Bmal1 and AchE or nAchR. A dual-luciferase gene test and electrophoretic mobility shift assay showed that Bmal1 bound to the promoter regions of AchE and nAchR. Twenty-four hours after silencing the Bmal1 gene, the expression levels of AchE and nAchR decreased significantly compared to those of the dsEGFP and PBS control groups, further showing that Bmal1 mediates the cholinergic system to regulate the behavioral rhythm of abalone. These findings shed light on the endocrine mechanism regulating the rhythmic behavior of abalone, and provide a reference for understanding the life history adaptation strategies of nocturnal organisms and the proliferation and protection of bottom dwelling economically important organisms.

Abstract 14465: Sex Has an Effect on the Relationship Between Intracoronary Acetylcholine and Coronary Blood Flow

Introduction: The endothelium controls coronary blood flow (CBF) by regulating microvascular tone, which is an important index of microcirculatory health. The difference in vasomotor response of the microvasculature between males and females is not well described. Hypothesis: Males and females react differently to different doses of intracoronary (IC) acetylcholine (Ach) injections. Methods: Patients with angina and non-obstructive coronary artery disease (&lt;50% stenosis) that underwent coronary reactivity study were enrolled. A doppler wire was positioned and incremental IC Ach doses were injected (10 -6 , 10 -5 , and 10 -4 mol/L). CBF (mL/min) was then calculated from CAD and doppler-derived flow velocity. Comparisons between the 2 sexes were performed. Results: A total of 1927 patients (mean age 51±13 years, 67% females) were included. Males were significantly younger (49±13 vs 53±12, P&lt;0.0001) than females and had a higher smoking exposure rate (56% vs 41%, P&lt;0.0001). Baseline (B) CBF was significantly lower in males (27.7 [19.2; 40.8] vs 30.2 [20.6; 43.4], P=0.04). The gap between females and males widened the first 2 doses, but then diminished after the highest dose (41.2 [24.5; 67.8] vs 44.5 [25.9; 72.0], P=0.09) (Fig 1A) . On multivariable analyses, female sex was an independent predictor of higher CBF at all doses (Estimate±StdError) (B= 1.14±0.53; 10 -6 : 1.59±0.61; 10 -5 : 2.54±0.82; 10 -4 : 2.70±1.2; P&lt;0.05 for all). When calculating the %ΔCBF, females had a significantly larger augmentation of flow after the first 2 doses of IC Ach (10 -6 : 3 [-14; 24] vs. -2 [-16; 19], P=0.02; 10 -5 : 21 [-7;68] vs. 12 [-10; 50], P=0.003). However, with the highest Ach dose, there was an equalization of overall augmentation (10 -4 : 49 [-1; 109] vs. 46 [-5; 106], P=0.46) (Fig 1B). Conclusions: Females had higher CBF regardless of given Ach concentration. Females have a higher sensitivity to Ach at smaller doses, while males exhibit a greater vasodilatory reaction at higher doses.

Neuroprotective Activity of the Methanolic Extract of Indigofera aspalathoides against Scopalamine induced Alzheimer's Disease in Experimental Rats

The plant Indigofera aspalathoides is a traditional medicine with tremendous therapeutic potential which finds it use in treatment of various ailments such as antibacterial, antioxidant, anti-inflammatory, antidiabetic, and anticancer activities. There are no reports that related to the use of this plant in treating patients with Alzheimer’s disease (AD). Hence present study was aimed to scientifically evaluate the neuroprotective effect of the methanolic extract of Indigofera aspalathoides against scopalamine induced Alzheimer’s disease in experimental rats using behavioral tests like elevated plus maze, Y-maze, and rota-rod tests. In addition to this, biochemical evaluation for acetylcholinesterase activity and histopathological evaluation of brain were done. The results suggests that methanolic extract Indigofera aspalathoides (200mg/kg B.wt and 400mg/kg B.wt) used in this study shows significant improvement of various behavioral parameters like locomotion, anxiety, memory, motor integrity and coordination etc when compared to control group. MEIA inhibited brain AChE enzyme, thereby elevating Ach concentration in brain homogenate and ultimately improved memory of rats. Further, more or less normal histological structure of the hippocampus and all amyloid plaques and neurofibrillary tangles that are formed under the influence of scopolamine disappeared in the rats pretreated with MEIA (200mg/kg B.wt and 400mg/kg B.wt). It can be concluded that our results strongly support the anti-Alzheimer’s potential of the methanolic extract of the plant I.aspalathoides and its use in traditional medicine.

Dual Mode of Action of Acetylcholine on Cytosolic Calcium Oscillations in Pancreatic Beta and Acinar Cells In Situ.

Cholinergic innervation in the pancreas controls both the release of digestive enzymes to support the intestinal digestion and absorption, as well as insulin release to promote nutrient use in the cells of the body. The effects of muscarinic receptor stimulation are described in detail for endocrine beta cells and exocrine acinar cells separately. Here we describe morphological and functional criteria to separate these two cell types in situ in tissue slices and simultaneously measure their response to ACh stimulation on cytosolic Ca2+ oscillations [Ca2+]c in stimulatory glucose conditions. Our results show that both cell types respond to glucose directly in the concentration range compatible with the glucose transporters they express. The physiological ACh concentration increases the frequency of glucose stimulated [Ca2+]c oscillations in both cell types and synchronizes [Ca2+]c oscillations in acinar cells. The supraphysiological ACh concentration further increases the oscillation frequency on the level of individual beta cells, inhibits the synchronization between these cells, and abolishes oscillatory activity in acinar cells. We discuss possible mechanisms leading to the observed phenomena.