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acetylcholine chloride [keywords]
- Mechanistic Insights into Allosteric Structure-Function Relationships at the M1 Muscarinic Acetylcholine Receptor. [JOURNAL ARTICLE]
- J Biol Chem 2014 Oct 17.
Benzylquinolone carboxylic acid (BQCA) is the first highly selective positive allosteric modulator (PAM) for the M1 muscarinic acetylcholine receptor (mAChR), but possesses low affinity for the allosteric site on the receptor. More recent drug discovery efforts identified 3-((1S,2S)-2-hydroxycyclohexyl)-6-((6-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methyl)benzo[h]quinazolin-4(3H)-one (referred to herein as benzoquinazolinone 12) as a more potent M1 mAChR PAM with a structural ancestry originating from BQCA and related compounds. In the current study, we optimized the synthesis of and fully characterized the pharmacology of benzoquinazolinone 12, finding that its improved potency derived from a 50-fold increase in allosteric site affinity as compared to BQCA, while retaining a similar level of positive cooperativity with acetylcholine (ACh). We then utilized site-directed mutagenesis and molecular modeling to validate the allosteric binding pocket we previously described for BQCA as a shared site for benzoquinazolinone 12, and provide a molecular basis for its improved activity at the M1 mAChR. This includes a key role for hydrophobic and polar interactions with residues Tyr179, in the second extracellular loop (ECL2), and Trp400(7.35) in transmembrane (TM) domain 7. Collectively, this study highlights how the properties of affinity and cooperativity can be differentially modified on a common structural scaffold, and identifies molecular features that can be exploited to tailor the development of M1 mAChR-targeting PAMs.
- α7 Nicotinic Acetylcholine Receptor Is Expressed in Human Atherosclerosis and Inhibits Disease in Mice. [JOURNAL ARTICLE]
- Arterioscler Thromb Vasc Biol 2014 Oct 16.
Cholinergic pathways of the autonomic nervous system are known to modulate inflammation. Because atherosclerosis is a chronic inflammatory condition, we tested whether cholinergic signaling operates in this disease. We have analyzed the expression of the α7 nicotinic acetylcholine receptor (α7nAChR) in human atherosclerotic plaques and studied its effects on the development of atherosclerosis in the hypercholesterolemic Ldlr(-/-) mouse model.α7nAChR protein was detected on T cells and macrophages in surgical specimens of human atherosclerotic plaques and in aortic lesions of hypercholesterolemic mice. To study the role of α7nAChR signaling in atherosclerosis, male Ldlr(-/-) mice were lethally irradiated and reconstituted with bone marrow from wild-type or α7nAChR-deficient animals. Ablation of hematopoietic cell α7nAChR increased aortic atherosclerosis by 72%. This was accompanied by increased aortic interferon-γ mRNA, implying increased Th1 activity in the absence of α7nAChR signaling.The present study shows that signaling through hematopoietic α7nAChR inhibits atherosclerosis and suggests that it operates by modulating immune inflammation. Given the observation that α7nAChR is expressed by T cells and macrophages in human plaques, our findings support the notion that cholinergic regulation may act to inhibit disease development also in man.
- Bronchoprotective Effect of Simulated Deep Inspirations in Tracheal Smooth Muscle. [JOURNAL ARTICLE]
- J Appl Physiol (1985) 2014 Oct 16.
Deep inspirations (DIs) taken prior to an inhaled challenge with a spasmogen limit airway responsiveness in non-asthmatics. This phenomenon is called bronchoprotection and is severely impaired in asthmatics. The ability of DIs to prevent a decrease in FEV1 was initially attributed to inhibition of airway narrowing. However, DIs taken prior to methacholine challenge limit airway responsiveness only when a test of lung function requiring a DI is used (FEV1). Therefore it has been suggested that prior DIs enhance the compliance of the airways or airway smooth muscle (ASM). This would increase the strain the airway wall undergoes during the subsequent DI which is part of the FEV1 maneuver. To investigate this phenomenon, we used ovine tracheal smooth muscle strips that were subjected to shortening elicited by acetylcholine with or without prior strain mimicking two DIs. The compliance of the shortened strip was then measured in response to a stress mimicking one DI. Our results show that the presence of "DIs" prior to acetylcholine-induced shortening resulted in 11% greater re-lengthening in response to the third DI; compared to without the prior DIs. This effect, although small, is shown to be potentially important for the reopening of closed airways. The effect of prior DIs was abolished by the adaptation of ASM to either shorter or longer lengths or to a low baseline tone. These results suggest that DIs confer bronchoprotection because they increase the compliance of ASM, which consequently promotes greater strain from subsequent DI and fosters the reopening of closed airways.
- Cholinergic Receptors as Target for Cancer Therapy in a Systems Medicine Perspective. [JOURNAL ARTICLE]
- Curr Mol Med 2014 Oct 15.
Epithelial cells not innervated by cholinergic neurons express nicotinic and muscarinic acetylcholine (ACh) receptors (nAChR, mAChR). nAChR and mAChR are components of the auto-/paracrine-regulatory loop of non-neuronal ACh release. The cholinergic control of non-neuronal cells may be mediated by different effects (synergistic, additive, or reciprocal) triggered by these receptors. The ionic events (Ca+2 influx) are generated by the ACh-opening of nAChR channels, while the metabolic events by ACh-binding to G-protein-coupled mAChR. Effective inter- and intracellular signaling is crucial for valuable cancer cells proliferation and survival. Depending on cancer cell type, different AChR have been identified. The proliferation of airways epithelial cancer cells and pancreatic cancer cells may be under the control of α7-nAChR and M3-mAChR, while breast cancer cells and colon cancer cells are regulated by α9-nAChR, and M3-mAChR, respectively. In turn, these receptors may activate different pathways (Ras-Raf-1-Erk-AKT) as well as other receptors (β-adrenergicR). nAChR or mAChR antagonists may inhibit cancer growth. Inhibition of M3 by antisense or antagonists (Darifenacin, Tiotropium) reduces lung or colon cancer proliferation, as well as inhibition of α9-nAChR [polyphenol (-)-epigallocatechin-3-gallate] diminishes breast cancer cells growth. α7-nAChR silencing inhibits lung cancer proliferation. Moreover, inhibition of the nAChR-β-adrenergicR pathway (β-blockers) could be also useful. This review will describe the future translational perspectives of cholinergic receptors drug-inhibition in a complex disease such as cancer that poses compelling treatment challenges. Cancer happens as consequence of disease-perturbed molecular networks in relevant organ cells that change during progression. The framework for approaching these challenges is a systems approach.
- Beer elicits vasculoprotective effects through Akt/eNOS activation. [JOURNAL ARTICLE]
- Eur J Clin Invest 2014 Oct 16.
There is controversy regarding the effect of alcohol beverage intake in vascular vasodilatory function in peripheral arteries. The effects of beer intake in coronary vasodilation remain unknown. We investigated whether regular beer intake (alcohol and alcohol-free) protects against hypercholesterolemia-induced coronary endothelial dysfunction and the mechanisms behind this effect.Pigs were fed 10days: I) a Western-type hypercholesterolemic diet (WD; II) WD+low-dose beer (12.5g alcohol/day); III) WD+moderate-dose beer (25g alcohol/day); or IV) WD+moderate-dose alcohol-free-beer (0.0g alcohol/day). Coronary responses to endothelium-dependent vasoactive drugs (acetylcholine: receptor-mediated; calcium ionophore-A23189: non-receptor mediated), endothelium-independent vasoactive drug (SNP); and to L-NMMA (NOS-antagonist) were evaluated in the LAD coronary artery by flow Doppler. Coronary Akt/eNOS activation, MCP-1 expression, oxidative DNA-damage and superoxide production were assessed. Lipid profile, lipoproteins resistance to oxidation and urinary isoxanthohumol concentration were evaluated.Alcoholic and non-alcoholic beer intake prevented WD-induced impairment of receptor- and non-receptor operated endothelial-dependent coronary vasodilation. All animals displayed a similar vasodilatory response to SNP and L-NMMA blunted all endothelial-dependent vasorelaxation responses. Haemodynamic parameters remained unchanged. Coronary arteries showed lower DNA damage and increased Akt/eNOS-axis activation in beer-fed animals. Animals taking beer showed HDL with higher antioxidant capacity, higher LDL resistance to oxidation and increased isoxanthohumol levels. Weight, lipids levels, liver enzymes and MCP-1 expression were not affected by beer intake.Non-alcoholic related beer components protect against hyperlipemia-induced coronary endothelial dysfunction by counteracting vascular oxidative damage and preserving the Akt/eNOS pathway. Light-to-moderate beer consumption prevents and/or reduces the endothelial dysfunction associated with cardiovascular risk factors. This article is protected by copyright. All rights reserved.
- Abrogation of Airway Hyperresponsiveness but not Inflammation by Rho kinase Insufficiency. [JOURNAL ARTICLE]
- Clin Exp Allergy 2014 Oct 16.
Major features of allergic asthma include airway hyperresponsiveness (AHR), eosinophilic inflammation, and goblet cell metaplasia. Rho kinase (ROCK) is a serine/threonine protein kinase that regulates the actin cytoskeleton. By doing so, it can modulate airway smooth muscle cell contraction and leukocyte migration and proliferation. This study was designed to determine the contributions of the two ROCK isoforms, ROCK1 and ROCK2, to AHR, inflammation and goblet cell metaplasia in a mast-cell dependent model of allergic airways disease.Repeated intranasal challenges with OVA caused AHR, eosinophilic inflammation, and goblet cell hyperplasia in wildtype (WT) mice. OVA- induced AHR was partially or completely abrogated in mice haploinsufficient for ROCK2 (ROCK2(+/-) ) or ROCK1 (ROCK1(+/-) ), respectively. In contrast, there was no effect of ROCK insufficiency on allergic airways inflammation, although both ROCK1 and ROCK2 insufficiency attenuated mast cell degranulation. Goblet cell hyperplasia, as indicated by PAS staining, was not different in ROCK1(+/-) versus WT mice. However, in ROCK2(+/-) mice, goblet cell hyperplasia was reduced in medium but not large airways. Maximal acetylcholine-induced force generation was reduced in tracheal rings from ROCK1(+/-) and ROCK2(+/-) versus WT mice. The ROCK inhibitor, fasudil, also reduced airway responsiveness in OVA-challenged mice, without affecting inflammatory responses.In a mast cell model of allergic airways disease, ROCK1 and ROCK2 both contribute to AHR, likely through direct effects on smooth muscle cell and effects on mast-cell degranulation. In addition, ROCK2 but not ROCK1 plays a role in allergen-induced goblet cell hyperplasia. This article is protected by copyright. All rights reserved.
- Target-site resistance to neonicotinoids. [Journal Article]
- J Chem Biol 2014 Oct; 7(4):125-8.
Neonicotinoid insecticides selectively target the invertebrate nicotinic acetylcholine receptor and disrupt excitatory cholinergic neurotransmission. First launched over 20 years ago, their broad pest spectrum, variety of application methods and relatively low risk to nontarget organisms have resulted in this class dominating the insecticide market with global annual sales in excess of $3.5 bn. This remarkable commercial success brings with it conditions in the field that favour selection of resistant phenotypes. A number of important pest species have been identified with mutations at the nicotinic acetylcholine receptor associated with insensitivity to neonicotinoids. The detailed characterization of these mutations has facilitated a greater understanding of the invertebrate nicotinic acetylcholine receptor.
- The Matrix Protein Hikaru genki Localizes to Cholinergic Synaptic Clefts and Regulates Postsynaptic Organization in the Drosophila Brain. [Journal Article]
- J Neurosci 2014 Oct 15; 34(42):13872-7.
The synaptic cleft, a crucial space involved in neurotransmission, is filled with extracellular matrix that serves as a scaffold for synaptic differentiation. However, little is known about the proteins present in the matrix and their functions in synaptogenesis, especially in the CNS. Here, we report that Hikaru genki (Hig), a secreted protein with an Ig motif and complement control protein domains, localizes specifically to the synaptic clefts of cholinergic synapses in the Drosophila CNS. The data indicate that this specific localization is achieved by capture of secreted Hig in synaptic clefts, even when it is ectopically expressed in glia. In the absence of Hig, the cytoskeletal scaffold protein DLG accumulated abnormally in cholinergic postsynapses, and the synaptic distribution of acetylcholine receptor (AchR) subunits Dα6 and Dα7 significantly decreased. hig mutant flies consistently exhibited resistance to the AchR agonist spinosad, which causes lethality by specifically activating the Dα6 subunit, suggesting that loss of Hig compromises the cholinergic synaptic activity mediated by Dα6. These results indicate that Hig is a specific component of the synaptic cleft matrix of cholinergic synapses and regulates their postsynaptic organization in the CNS.
- Neuromuscular synapse integrity requires linkage of acetylcholine receptors to postsynaptic intermediate filament networks via rapsyn-plectin 1f complexes. [JOURNAL ARTICLE]
- Mol Biol Cell 2014 Oct 15.
Mutations in the cytolinker protein plectin lead to grossly distorted morphology of neuro-muscular junctions (NMJs) in patients suffering from EBS-MD with myasthenic syn-drome (MyS). Here we investigated whether plectin contributes to the structural integrity of NMJs by linking them to the postsynaptic intermediate filament (IF) network. Live imag-ing of acetylcholine receptors (AChRs) in cultured myotubes differentiated ex vivo from immortalized plectin-deficient myoblasts revealed them to be highly mobile and unable to coalesce into stable clusters, contrary to wild-type cells. We found plectin iso-form P1f to bridge AChRs and IFs via direct interaction with the AChR-scaffolding pro-tein rapsyn, in an isoform-specific fashion; forced expression of P1f in plectin-deficient cells rescued both, compromised AChR-clustering and IF network-anchoring. In condi-tional plectin knockout mice with gene disruption in muscle precursor/satellite cells (Pax7-Cre/cKO), uncoupling of AChRs from IFs was shown to lead to loss of postsyn-aptic membrane infoldings and disorganization of the NMJ microenvironment including its invasion by microtubules. In their phenotypic behavior, mutant mice closely mimicked EBS-MD-MyS patients, including impaired body balance, severe muscle weakness, and reduced life span. Our study demonstrates that linkage to desmin IF networks via plectin is crucial for formation and maintenance of AChR clusters, postsynaptic NMJ organization, and body-locomotion.
- [Factors influencing intraocular pressure elevation on the first postoperative day following small-incision cataract surgery--the effect of apraclonidine]. [English Abstract, Journal Article]
- Nihon Ganka Gakkai Zasshi 2014 Sep; 118(9):768-72.
To investigate the effects of apraclonidine on intraocular pressure elevation after cataract surgery and the factors associated with elevated intraocular pressure.A group of patients (apraclonidine group) was administered a drop of apraclonidine before and one drop after surgery, and the difference between the intraocular pressure in the apraclonidine group and the non-use group was investigated postoperatively. On the first postoperative day, multivariate analysis was performed using intraocular pressure as the objective value and other variable factors involved in the surgery as the explanatory variables.On the first postoperative day, the intraocular pressure in the apraclonidine group (520 eyes: 15.5 +/- 4.9 mmHg) was significantly lower than that in the non-use group (577 eyes: 18.7 +/- 7.2 mmHg) (p < 0.001). The significant variables included preoperative intraocular pressure, apraclonidine use, sex (men > women), poor mydriasis, acetylcholine use, pseudoexfoliation, and diabetes mellitus.Apraclonidine is useful in suppressing postoperative elevation of intraocular pressure.