The activation of M3 mAChR in airway epithelial cells promotes IL-8 and TGF-ß1 secretion and airway smooth muscle cell migration.

BACKGROUND: Muscarinic acetylcholine receptors (mAChRs) have been identified in airway epithelium, and epithelium-derived chemokines can initiate the migration of airway smooth muscle (ASM) cells. However, the mAChRs that are expressed in airway epithelium and the mechanism underlying the regulation of ASM cell migration are not clear. The aim of this study was to test whether the effects of the epithelium-derived chemokines on ASM cell migration could be modulated by mAChRs. METHOD: Human epithelial cells (A549 cells) were stimulated with cigarette smoke extract (CSE) or the mAChRs agonist carbachol. IL-8 and TGF-ß1 production were measured by ELISA, and human ASM cell migration was measured using the transwell migration assay and scratch assay. The mRNA levels of the mAChRs subtypes and the acetylcholine concentrations were measured using RT-PCR and LC-MS/MS, respectively. RESULTS: ASM cell migration toward CSE-stimulated A549 cells was markedly reduced by Ac-RRWWCR-NH2 (IL-8 inhibitor) and SB431542 (TGF-ß1 inhibitor). CSE-induced ASM cell migration was also suppressed by the mAChRs antagonist tiotropium. Interestingly, carbachol-stimulated A549 cells also induced ASM cell migration; this migration event was suppressed by tiotropium, Ac-RRWWCR-NH2 and SB431542. In addition, the effects of CSE on ASM cell migration were significantly and cooperatively enhanced by carbachol compared to CSE alone. Carbachol-induced ASM cell migration was reduced by selective inhibitors of PI3K/Akt (LY294002) and p38 (SB203580), suggesting that it occurred through p38 and Akt phosphorylation, which was inhibited by the M3 mAChR antagonist 4-DAMP. CONCLUSIONS: These findings indicate that M3 mAChR may be important therapeutic target for obstructive airway diseases, as it regulates the effects of the epithelial-derived chemokines on ASM cell migration, which results in lung remodeling.

A mucoactive drug carbocisteine ameliorates steroid resistance in rat COPD model.

Steroid insensitivity has been commonly found in chronic obstructive pulmonary disease (COPD) patients, which is mediated by the reduction of histone deacetylase (HDAC) 2. Here we aimed to establish a steroid resistant model on experimental COPD rats and evaluate the effect of carbocisteine (S-CMC), a mucoactive drug. Exposure to cigarette smoke (CS) caused marked pathological features of COPD which are insensitive to DEX associated with the down-regulation of HDAC2 expression/activity. The DEX insensitivity observed in COPD featured rats was improved by S-CMC in the aspects of inhibiting chronic lung inflammation (total and differential inflammatory cell counts, inflammatory cytokines release and inflammatory cells infiltration); ameliorating airway remodeling (thickness of airway epithelium and smooth muscle, airway fibrosis, and the level of α-SMA and TGF-ß1); improving emphysema (emphysema index D2, level of MMP-9 in BALF and the expression of alpha-1 antitrypsin) and preventing impairments of lung function (PEF, IP and IP-slope). Simultaneously, down-regulation of HDAC2 expression/activity was ameliorated by S-CMC treatment. These results indicate that the rat COPD model with steroid resistance was established by active smoking in a short time frame and demonstrate that the failure of steroid therapy can be restored by S-CMC accompanied by increasing HDAC2 expression/activity, providing additional evidence that S-CMC might be used for GC resistance in COPD.

Carbocysteine restores steroid sensitivity by targeting histone deacetylase 2 in a thiol/GSH-dependent manner.

Steroid insensitivity is commonly observed in patients with chronic obstructive pulmonary disease. Here, we report the effects and mechanisms of carbocysteine (S-CMC), a mucolytic agent, in cellular and animal models of oxidative stress-mediated steroid insensitivity. The following results were obtained: oxidative stress induced higher levels of interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-α), which are insensitive to dexamethasone (DEX). The failure of DEX was improved by the addition of S-CMC by increasing histone deacetylase 2 (HDAC2) expression/activity. S-CMC also counteracted the oxidative stress-induced increase in reactive oxygen species (ROS) levels and decreases in glutathione (GSH) levels and superoxide dismutase (SOD) activity. Moreover, oxidative stress-induced events were decreased by the thiol-reducing agent dithiothreitol (DTT), enhanced by the thiol-oxidizing agent diamide, and the ability of DEX was strengthened by DTT. In addition, the oxidative stress-induced decrease in HDAC2 activity was counteracted by S-CMC by increasing thiol/GSH levels, which exhibited a direct interaction with HDAC2. S-CMC treatment increased HDAC2 recruitment and suppressed H4 acetylation of the IL-8 promoter, and this effect was further ablated by addition of buthionine sulfoximine, a specific inhibitor of GSH synthesis. Our results indicate that S-CMC restored steroid sensitivity by increasing HDAC2 expression/activity in a thiol/GSH-dependent manner and suggest that S-CMC may be useful in a combination therapy with glucocorticoids for treatment of steroid-insensitive pulmonary diseases.

M3 mAChR-mediated IL-8 expression through PKC/NF-κB signaling pathways.

OBJECTIVE: M3 muscarinic acetylcholine receptor (mAChR) plays an important role in the regulation of cytokine production in inflammatory diseases. In this study, we explored the precise role of M3 mAChR under stimulation with agonist in IL-8 expression and of the signaling pathway involved in this process. MATERIALS AND METHODS: Recombinant U2OS cells stably expressing M3 mAChR as a model system were stimulated by carbachol to evaluate the role of M3 mAChR in the expression of IL-8. RESULTS: Activation of M3 mAChR with carbachol increased both IL-8 mRNA and protein expression in a concentration-dependent manner. Elevated IL-8 expression was completely antagonized by atropine, 4-DAMP and tiotropium. M3 mAChR-mediated IL-8 expression was almost completely inhibited by the NF-κB inhibitor BAY11-7082 and, to a lesser extent, by U0126, SB203580, and SP600125, which are inhibitors for ERK1/2, p38, and JNK, respectively. Furthermore, M3 mAChR-mediated NF-κB activation and IL-8 expression were simultaneously attenuated by the PKC inhibitor calphostin C, whereas PMA, a PKC activator, mimicked the effects of carbachol, inducing IL-8 expression. CONCLUSIONS: Our findings offer insights into the specific and critical role of M3 mAChR in regulating inflammatory response and indicate M3 mAChR/PKC/NF-κB signaling axis driven by endogenous acetylcholine as a potential therapeutic targets for inflammatory diseases.

mAChRs activation induces epithelial-mesenchymal transition on lung epithelial cells.

BACKGROUND: Epithelial-mesenchymal transition (EMT) has been proposed as a mechanism in the progression of airway diseases and cancer. Here, we explored the role of acetylcholine (ACh) and the pathway involved in the process of EMT, as well as the effects of mAChRs antagonist. METHODS: Human lung epithelial cells were stimulated with carbachol, an analogue of ACh, and epithelial and mesenchymal marker proteins were evaluated using western blot and immunofluorescence analyses. RESULTS: Decreased E-cadherin expression and increased vimentin and α-SMA expression induced by TGF-ß1 in alveolar epithelial cell (A549) were significantly abrogated by the non-selective mAChR antagonist atropine and enhanced by the acetylcholinesterase inhibitor physostigmine. An EMT event also occurred in response to physostigmine alone. Furthermore, ChAT express and ACh release by A549 cells were enhanced by TGF-ß1. Interestingly, ACh analogue carbachol also induced EMT in A549 cells as well as in bronchial epithelial cells (16HBE) in a time- and concentration-dependent manner, the induction of carbachol was abrogated by selective antagonist of M1 (pirenzepine) and M3 (4-DAMP) mAChRs, but not by M2 (methoctramine) antagonist. Moreover, carbachol induced TGF-ß1 production from A549 cells concomitantly with the EMT process. Carbachol-induced EMT occurred through phosphorylation of Smad2/3 and ERK, which was inhibited by pirenzepine and 4-DAMP. CONCLUSIONS: Our findings for the first time indicated that mAChR activation, perhaps via M1 and M3 mAChR, induced lung epithelial cells to undergo EMT and provided insights into novel therapeutic strategies for airway diseases in which lung remodeling occurs.

Targeted inhibition of KCa3.1 channel attenuates airway inflammation and remodeling in allergic asthma.

KCa3.1 has been suggested to be involved in regulating cell activation, proliferation, and migration in multiple cell types, including airway inflammatory and structural cells. However, the contributions of KCa3.1 to airway inflammation and remodeling and subsequent airway hyperresponsiveness (AHR) in allergic asthma remain to be explored. The main purpose of this study was to elucidate the roles of KCa3.1 and the potential therapeutic value of KCa3.1 blockers in chronic allergic asthma. Using real-time PCR, Western blotting, or immunohistochemical analyses, we explored the precise role of KCa3.1 in the bronchi of allergic mice and asthmatic human bronchial smooth muscle cells (BSMCs). We found that KCa3.1 mRNA and protein expression were elevated in the bronchi of allergic mice, and double labeling revealed that up-regulation occurred primarily in airway smooth muscle cells. Triarylmethane (TRAM)-34, a KCa3.1 blocker, dose-dependently inhibited the generation and maintenance of the ovalbumin-induced airway inflammation associated with increased Th2-type cytokines and decreased Th1-type cytokine, as well as subepithelial extracellular matrix deposition, goblet-cell hyperplasia, and AHR in a murine model of asthma. Moreover, the pharmacological blockade and gene silencing of KCa3.1, which was evidently elevated after mitogen stimulation, suppressed asthmatic human BSMC proliferation and migration, and arrested the cell cycle at the G0/G1 phase. In addition, the KCa3.1 activator 1-ethylbenzimidazolinone-induced membrane hyperpolarization and intracellular calcium increase in asthmatic human BSMCs were attenuated by TRAM-34. We demonstrate for the first time an important role for KCa3.1 in the pathogenesis of airway inflammation and remodeling in allergic asthma, and we suggest that KCa3.1 blockers may represent a promising therapeutic strategy for asthma.

The effect of absolute configuration on activity, subtype selectivity (M3/M2) of 3α-acyloxy-6ß-acetoxyltropane derivatives as muscarinic M3 receptor antagonists.

Both enantiomers of 3α-acyloxy-6ß-acetoxyltropane derivatives 1-4 were prepared respectively and underwent functional studies and radioreceptor binding assays. 6S Enantiomers showed obvious muscarinic M3, M2 antagonistic activity, while the 6R ones elicited little muscarinic activity by functional studies. Besides, the affinity of 6S enantiomers to muscarinic M3 receptors of rat submandibulary gland, M2 receptors of rat left atria was much larger than that of corresponding 6R enantiomers. All these pharmalogical results indicated 6S configuration was favorable for 3α-acyloxy-6ß-acetoxyltropane derivatives to bind with muscarinic M3 or M2 receptors and elicited antagonistic activity. Furthermore, the muscarinic M3 activity and subtype selectivity (M3/M2) of 6S enantiomers could be improved by increasing the electron density of carbonyl oxygen or introducing methylene group between the carbonyl and phenyl ring in C-3α position. Understanding the effect of absolute configuration on activity, subtype selectivity (M3/M2) of 3α-acyloxy-6ß-acetoxyltropane derivatives will provide the clues for designing muscarinic M3 antagonists with high activity and low side effects or toxicity.

Protective effects of anisodamine on cigarette smoke extract-induced airway smooth muscle cell proliferation and tracheal contractility.

Anisodamine, an antagonist of muscarinic acetylcholine receptors (mAChRs), has been used therapeutically to improve smooth muscle function, including microvascular, intestinal and airway spasms. Our previous studies have revealed that airway hyper-reactivity could be prevented by anisodamine. However, whether anisodamine prevents smoking-induced airway smooth muscle (ASM) cell proliferation remained unclear. In this study, a primary culture of rat ASM cells was used to evaluate an ASM phenotype through the ability of the cells to proliferate and express contractile proteins in response to cigarette smoke extract (CSE) and intervention of anisodamine. Our results showed that CSE resulted in an increase in cyclin D1 expression concomitant with the G0/G1-to-S phase transition, and high expression of M2 and M3. Functional studies showed that tracheal hyper-contractility accompanied contractile marker α-SMA high-expression. These changes, which occur only after CSE stimulation, were prevented and reversed by anisodamine, and CSE-induced cyclin D1 expression was significantly inhibited by anisodamine and the specific inhibitor U0126, BAY11-7082 and LY294002. Thus, we concluded that the protective and reversal effects and mechanism of anisodamine on CSE-induced events might involve, at least partially, the ERK, Akt and NF-κB signaling pathways associated with cyclin D1 via mAChRs. Our study validated that anisodamine intervention on ASM cells may contribute to anti-remodeling properties other than bronchodilation.

Huperzine A lowers intraocular pressure via the M3 mAChR and provides retinal neuroprotection via the M1 mAChR: a promising agent for the treatment of glaucoma.

BACKGROUND: Glaucoma is a neurodegenerative disease that shares similar pathological mechanisms with Alzheimer's disease (AD). Drug treatments for glaucoma increasingly rely upon both lowering of intraocular pressure (IOP) and optic nerve protection, as lowering of IOP alone has been unsatisfactory. Huperzine A (HupA) is an acetylcholinesterase inhibitor (AChEI) used for AD. This study investigated the potential of HupA as a treatment for glaucoma. METHODS: The ability of HupA to lower IOP via causing pupil constriction was assessed using New Zealand rabbits. The retinal neuroprotective effects of HupA were assessed in vivo using rat retinas subjected to ischemia-reperfusion (I/R) and in vitro using primary retinal neurons (PRNs) suffering from oxygen-glucose deprivation (OGD). RESULTS: HupA caused pupil constriction in a dose-time dependent manner which was reversed by the nonselective muscarinic acetylcholine receptor (mAChR) antagonist atropine and the selective M3 mAChR antagonist 4-DAMP. However, HupA had no effect on isolated iris muscle tension and calcium flow indicating an indirect M3 mAChR mediated effect. HupA exerted a neuroprotective effect against I/R and OGD to attenuate the retinal pathological lesion, improve retinal neuronal cell viability, reverse oxidative stress injury by increasing GSH levels and SOD activity, and decreasing MDA content and reduce the retinal neuronal apoptosis by decreasing Bax/Bcl-2 ratio and caspase-3 expression with no effect on the calcium flow tests. The effects were abolished by atropine and the selective M1 mAChR antagonist pirenzepine in OGD-induced PRNs suggesting an indirect M1 mAChR-mediated effect via inhibiting AChE activity to increase endogenous ACh level. Furthermore, HupA increased phosphorylated AKT level and decreased the levels of phosphorylated JNK, P38 MAPK and ERK via M1 mAChR antagonists indicating an involvement of activating the M1 mAChR and the downstream AKT/MAPK signaling pathway in the protective effects of HupA. CONCLUSIONS: HupA could significantly decrease IOP via activating M3 mAChR indirectly and produce retinal neuroprotective effect through M1 mAChR/AKT/MAPK by increasing endogenous ACh level. These investigations demonstrated that HupA was an effective drug in glaucoma treatment and the clinical application of HupA and other AChEIs for glaucoma patients should be further investigated.

Novel piperazine derivative PMS1339 exhibits tri-functional properties and cognitive improvement in mice.

Amyloid-beta-induced neuroinflammation plays a central role in the extensive loss of cholinergic neurons and cognitive decline in Alzheimer's disease. The acetylcholinesterase (AChE) inhibitors are the first class of drugs used to enhance surviving cholinergic activities. However, their limited effectiveness following long-term treatment raises a need for new multi-target therapies. We report herein a novel piperazine derivative compound PMS1339 possesses multifunctional properties including anti-platelet-activating factor, AChE inhibition, Abeta aggregation inhibition and cognitive improvement. PMS1339 could significantly inhibit both mice brain AChE (IC50=4.41+/-0.63 microM) and sera butyrylcholinesterase (BuChE, IC50=1.09+/-0.20 microM). PMS1339 was also found to inhibit neuronal AChE secreted by SH-SY5Y cell line (IC50=17.95+/-2.31 microM). Enzyme kinetics experiments performed on electric eel AChE indicated that PMS1339 acts as a mixed type competitive AChE inhibitor. Molecular docking studies using the X-ray crystal structure of AChE from Torpedo californica elucidated the interactions between PMS1339 and AChE: PMS1339 is well buried inside the active-site gorge of AChE interacting with Trp84 at the bottom, Tyr121 halfway down and Trp279 at the peripheral anionic site (PAS). Thioflavin T-based fluorimetric assay revealed the ability of PMS1339 to inhibit AChE-induced Abeta aggregation. In-vivo study indicated PMS1339 (1 mg/kg i.p.) reversed scopolamine-induced memory impairment in mice. Overall, these findings indicated that PMS1339 exhibits tri-functional properties in vitro and cognitive improvement in vivo, and revealed the emergence of a multi-target-directed ligand to tackle the determinants of Alzheimer's disease.

Peripheral cholinoceptor antagonist anisodamine counteracts cholinergic adverse effects and facilitates cognitive amelioration of rivastigmine.

Rivastigmine is a potent acetyl- and butyrylcholinesterase inhibitor widely used for cognitive improvement in Alzheimer's disease (AD) therapy. However, dose-limiting adverse effects restrict its tolerability and clinical outcomes. This study explored new combined therapy, in which peripheral cholinergic adverse effects and central cognitive amelioration of rivastigmine were differentiated by a peripheral cholinoceptor antagonist anisodamine. The results demonstrated that rivastigmine (0.75 and 2.0 mg/kg) could significantly reverse the scopolamine-induced cognitive deficit in mice through passive avoidance test. Nevertheless, a high dose of rivastigmine (3.25 mg/kg) would compromise cognitive amelioration and produce obvious adverse effects, including hypersalivation, intestinal hyperperistalsis and muscle cramp. Interestingly, concomitant administration of anisodamine (10 mg/kg) effectively counteracted both the muscarinergic and nicotinergic adverse effects, while facilitating cognitive amelioration of rivastigmine (3.25 mg/kg). These findings provide an insight into the feasibility of combined therapy with cholinesterase inhibitors and peripheral cholinoceptor antagonists for the treatment of AD.

Quantitative determination of a novel enantiomeric tropane analog, (-)-satropane, in biological fluids using liquid chromatography/tandem mass spectrometry.

A sensitive, accurate and precise liquid chromatography-tandem mass spectrometry method was developed for the determination of (-)-satropane (3alpha-paramethyl-benzenesulfonyloxy-6beta-acetoxy-tropane) in rabbit aqueous humor. Since (-)-satropane may be absorbed from the aqueous humour with resultant systemic side effects, the LC-MS/MS method was also evaluated for its applicability in analyzing plasma samples containing this compound. (-)-Satropane and phentolamine (the internal standard, represented as IS) were detected by multiple reaction monitoring using the transitions m/z 354-182 and 282-212, respectively. The calibration curve was linear over the ranges 2-500 and 5-1000 ng/mL, and the values of the lower limit of quantification were 2 and 5 ng/mL for the microdialysis dialysate and rat plasma samples, respectively. The intra-day and inter-day precision and accuracy were better than 8.6 and 6.00%, respectively, in both matrices investigated. The absolute recovery of the plasma samples was more than 76.30%. The average matrix effects of (-)-satropane were 91.72 and 83.05% in the microdialysis dialysate and plasma samples, respectively. The validated method was successfully applied to analyze (-)-satropane in microdialysis dialysate and rat plasma samples, and this assay has been used to quantify (-)-satropane in the pharmacokinetic and toxicokinetic studies in our laboratory.

Carbocisteine Improves Histone Deacetylase 2 Deacetylation Activity via Regulating Sumoylation of Histone Deacetylase 2 in Human Tracheobronchial Epithelial Cells.

Histone deacetylase (HDAC) 2 plays a vital role in modifying histones to mediate inflammatory responses, while HDAC2 itself is commonly regulated by post-translational modifications. Small ubiquitin-related modifier (SUMO), as an important PTM factor, is involved in the regulation of multiple protein functions. Our previous studies have shown that carbocisteine (S-CMC) reversed cigarette smoke extract (CSE)-induced down-regulation of HDAC2 expression/activity in a thiol/GSH-dependent manner and enhanced sensitivity of steroid therapy. However, the mechanism by which S-CMC regulates HDAC2 is worth further exploring. Our study aimed to investigate the relationships between HDAC2 sumoylation and its deacetylase activity under oxidative stress and the molecular mechanism of S-CMC to regulate HDAC2 activity that mediates inflammatory responses in human bronchial epithelial cells. We found that modification of HDAC2 by SUMO1 and SUMO2/3 occurred in 16HBE cells under physiological conditions, and CSE induced SUMO1 modification of HDAC2 in a dose and time-dependent manner. K462 and K51 of HDAC2 were the two major modification sites of SUMO1, and the K51 site mediated deacetylation activity and function of HDAC2 on histone H4 that regulates IL-8 secretion. S-CMC inhibited CSE-induced SUMO1 modification of HDAC2 in the presence of thiol/GSH, increased HDAC activity, and decreased IL-8 expression. Our study may provide novel mechanistic explanation of S-CMC to ameliorate steroid sensitivity treatment in chronic obstructive pulmonary disease.

Differential neuropsychopharmacological influences of naturally occurring tropane alkaloids anisodamine versus scopolamine.

Two naturally occurring tropane alkaloids, anisodamine and scopolamine, structurally dissimilar in one OH group, are well established as muscarinic acetylcholine receptor (mAChR) antagonists in clinic and basic research. However, experimental evidence for central effects of anisodamine is limited and conflicting compared with that of scopolamine. In the present study, Morris water maze test, long-term potentiation (LTP) recording and receptor radioligand binding assays were used to explore the disparity in neuropsychopharmacological influences of anisodamine versus scopolamine and possible mechanisms. Anisodamine, at 10-40-fold higher doses than those of scopolamine, did not produce any spatial cognitive deficits as scopolamine, but tended to improve cognition at the repeated high doses. LTP in vivo was then adopted to predict BBB permeability of the muscarinic antagonists following systemic drug administration. Contrary to scopolamine, anisodamine did not influence the formation of LTP in the CA(1) region of rat hippocampus at 40-fold higher dose than that of scopolamine. Additionally, receptor radioligand binding assays (RRLBA) revealed that the binding affinity of anisodamine to mice brain mAChR was much lower than that of scopolamine. The findings suggested that anisodamine did not impair cognition nor depress LTP primarily due to its poor BBB permeability. This work enlarged knowledge of structure-activity relationship among tropane alkaloids, meanwhile providing evidence for more reasonable drug prescription in clinic.

Is acupuncture beneficial in depression: a meta-analysis of 8 randomized controlled trials?

BACKGROUND: Depression is one of the most common mental health disorders. Acupuncture is a popular complementary and alternative medicine intervention suggested in the treatment of depression, but its effectiveness is uncertain. This updated meta-analysis was conducted to more precisely assess the beneficial effect of acupuncture in depression therapy. METHODS: The following databases were searched: MEDLINE, EMBASE, BIOSIS, Cochrane Central Register of Controlled Trials, and Chinese Scientific Journal Database. The following terms were used: acupuncture, acupressure, depression, depressive disorder, clinical trial, and randomized controlled trial. RESULTS: Eight small-randomized controlled trials comparing 477 subjects were included in the meta-analysis. Our results confirmed that acupuncture could significantly reduce the severity of depression, which was indicated by decreased scores of Hamilton rating scale for depression (HAMD) or Beck Depression Inventory (BDI). The pooled standardized mean difference of the 'Improvement of depression' was -0.65 (95% CI -1.18, -0.11; P=0.02) by random effect model. However, no significant effect of active acupuncture was found on the response rate (RR 1.32, 95% CI 0.83 to 2.10; P=0.25) and remission rate (RR 1.30, 95% CI 0.57 to 2.95; P=0.53). CONCLUSION: Although this meta-analysis might be discounted due to the low quality of individual trials, it supported that acupuncture was an effective treatment that could significantly reduce the severity of disease in the patients with depression. More full-scale randomized clinical trials with reliable designs are recommended to further warrant the effectiveness of acupuncture.

Muscarinic receptors involved in airway vascular leakage induced by experimental gastro-oesophageal reflux.

Gastro-oesophageal acid reflux may cause airway responses such as cough, bronchoconstriction and inflammation in asthmatic patients. Studies in humans or in animals have suggested that these responses involve cholinergic nerves. The purpose of this study was to investigate the role of the efferent vagal component on airway microvascular leakage induced by instillation of hydrochloric acid (HCl) into the oesophagus of guinea-pigs and the subtype of muscarinic receptors involved. Airway microvascular leakage induced by intra-oesophageal HCl instillation was abolished by bilateral vagotomy or by the nicotinic receptor antagonist, hexamethonium. HCl-induced leakage was inhibited by pretreatment with atropine, a non-specific muscarinic receptor antagonist, and also by pretreatment with either pirenzepine, a muscarinic M(1) receptor antagonist, or 4-DAMP, a muscarinic M(3) receptor antagonist. Pirenzepine was more potent than atropine and 4-DAMP. These antagonists were also studied on airway microvascular leakage or bronchoconstriction induced by intravenous administration of acetylcholine (ACh). Atropine, pirenzepine and 4-DAMP inhibited ACh-induced airway microvascular leakage with similar potencies. In sharp contrast, 4-DAMP and atropine were more potent inhibitors of ACh-induced bronchoconstriction than pirenzepine. Methoctramine, a muscarinic M(2) receptor antagonist, was ineffective in all experimental conditions. These results suggest that airway microvascular leakage caused by HCl intra-oesophageal instillation involves ACh release from vagus nerve terminals and that M(1) and M(3) receptors play a major role in cholinergic-mediated microvascular leakage, whereas M(3) receptors are mainly involved in ACh-induced bronchoconstriction.

The absolute configuration plays an important role in muscarinic activity of BGT-A and its analogs.

Both enantiomers of 2, 3, and 4, three bioactive analogs of muscarinic agonist BGT-A were prepared respectively and underwent functional studies and radioreceptor binding assays. 6S enantiomers of 2, 3, and 4 showed obvious muscarinic activity, while 6R ones elicited little muscarinic activity by functional studies. Besides, the affinity of 6S enantiomers of 2, 3, and 4 was greatly larger than that of their 6R enantiomers respectively. All these pharmacological results indicated the 6S configuration was beneficial for the active BGT-A analogs to bind with the muscarinic receptors. The finding was in good agreement with our previous SAR study to BGT-A and its active analogs by computational approach. The understanding to the relationship between muscarinic activity and absolute configuration will provide the basis for successive screening of BGT-A analogs as effective muscarinic agonists or antagonists in clinical use.

Cannabinoid CB(2) receptor activation prevents bronchoconstriction and airway oedema in a model of gastro-oesophageal reflux.

Cannabinoids have been shown to inhibit sensory nerve activation in guinea-pigs and humans. Their effects are mediated by specific activation of two types of receptors, named CB(1) and CB(2). The purpose of this study was to investigate the effects of WIN 55,212-2, (R)-(+)-[2,3-dihydro-5methyl-3-[(4-morpholino)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthyl)methanone, a non selective agonist of cannabinoid receptors, and JWH 133, (6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran a selective cannabinoid CB(2) receptor agonist, on the sensory nerve component of intraoesophageal (i.oe.) HCl-induced airway microvascular leakage and bronchoconstriction in guinea-pigs. We also tested the effect of WIN 55,212-2 on substance P-induced plasma extravasation and bronchoconstriction. Airway microvascular leakage and bronchoconstriction induced by i.oe. HCl was inhibited by the cannabinoid CB(1)/CB(2) agonist WIN 55,212-2 (0.3-3 mg/kg i.p.) in a dose-dependent manner (maximal inhibition at the dose of 3 mg kg(-1), P<0.01). The effect of WIN 55,212-2 was inhibited by a cannabinoid CB(2) receptor antagonist SR 144528, [N-[(1S)-endo-1,3,3-trimethylbicyclo[2,2,1] heptan-2yl]-5-(-4-chloro-3-methylphenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide], but not by a CB(1) receptor antagonist, SR 141716, [N-(piperidin-1yl)-5-(-4-chlorophenyl)-1-(2,4dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride]. The cannabinoid CB(2) agonist JWH 133 (0.3-3 mg/kg i.p.) mimicked the inhibitory effect of WIN 55,212-2 on HCl-induced microvascular leakage. Under similar conditions, WIN 55,212-2 (1 mg kg (-1) i.p.) was unable to counteract the airway microvascular leakage and bronchoconstriction induced by substance P. These results suggest that inhibition by WIN 55,212-2 of airway plasma extravasation and bronchoconstriction induced by i.oe. HCl instillation in guinea-pigs is mediated through cannabinoid CB(2) receptor activation.

L-Satropane Prevents Retinal Neuron Damage by Attenuating Cell Apoptosis and Aß Production via Activation of M1 Muscarinic Acetylcholine Receptor.

Muscarinic acetylcholine receptor (mAChR) agonists have been used to treat glaucoma due to their intraocular pressure-lowering effects. Recently, it has been reported that retinal mAChRs activation can also stimulate neuroprotective pathways. PURPOSE: In our study, we evaluated the potential neuroprotective effect of L-satropane, a novel mAChR agonist, on retinal neuronal injury induced by cobalt chloride (CoCl2) and ischemia/reperfusion (I/R). METHODS: CoCl2-induced hypoxia injury in cultured cell models and I/R-induced retinal neuronal damage in rats in vivo were used to evaluate the abilities of L-satropane. In detail, we measured the occurrence of retinal pathological changes including molecular markers of neuronal apoptosis and Aß expression. RESULTS: Pretreatment with L-satropane protects against CoCl2-induced neurotoxicity in PC12 and primary retinal neuron (PRN) cells in a dose-dependent manner by increasing retinal neuron survival. CoCl2 or I/R-induced cell apoptosis by upregulating Bax expression and downregulating Bcl-2 expression, which resulted in an increased Bax/Bcl-2 ratio, and upregulating caspase-3 expression/activity was significantly reversed by L-satropane treatment. In addition, L-satropane significantly inhibited the upregulation of Aß production in both retinal neurons and tissue. We also found that I/R-induced histopathological retinal changes including cell loss in the retinal ganglion cell layer (GCL) and increased TUNEL positive retinal ganglion cells in GCL and thinning of the inner plexiform layer (IPL) and inner nuclear layer (INL) were markedly improved by L-satropane. The effects of L-satropane were largely abolished by the nonselective mAChRs antagonist atropine and M1-selective mAChR antagonist pirenzepine. CONCLUSION: These results demonstrated that L-satropane might be effective in preventing retinal neuron damage caused by CoCl2 or I/R. The neuroprotective effects of L-satropane may be attributed to decreasing cell apoptosis and Aß production through activation of M1 mAChR.

Aceclidine and pilocarpine interact differently with muscarinic receptor in isolated rabbit iris muscle.

The relationship between muscarinic receptor affinity states and the contractile response to the muscarinic agonists carbachol, aceclidine, and pilocarpine, has been examined in the isolated rabbit iris muscle. Contraction of the iris muscle by carbachol and aceclidine was more potent and/or more efficacious than the response to pilocarpine. Analysis of [3H]-Quinuclidinyl benzilate (QNB) binding showed that while both carbachol and aceclidine bound to high- and low-affinity forms of the muscarinic receptor, pilocarpine bound to one affinity state. The efficacy of carbachol and aceclidine to stimulate contraction of the iris muscle was consistent with receptor occupancy theory only when considering the low-affinity state of the muscarinic receptor, and activation of the low-affinity rather than high-affinity binding state of the receptor is likely to mediate the contraction of iris muscle. Therefore, the typical anti-glaucoma muscarinic agonists aceclidine and pilocarpine may interact differently with their target receptors in isolated rabbit iris muscle.