Combined administration of anisodamine and neostigmine produces anti-shock effects: involvement of α7 nicotinic acetylcholine receptors.
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ABSTRACT:
SUBMITTER: Sun L
PROVIDER: S-EPMC4010367 | biostudies-other | 2012 Jun
REPOSITORIES: biostudies-other
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Project description:α7 Nicotinic acetylcholine receptors (nAChRs) reportedly reduce inflammation by blocking effects of the important pro-inflammatory transcription factor, nuclear factor kappa-light chain-enhancer of B cells (NFκB). The α7 nAChR partial agonist GTS-21 reduces secretion of pro-inflammatory cytokines including interleukin-6 (IL6) and tumor-necrosis factor (TNF) in models of endotoxemia and sepsis, and its anti-inflammatory effects are widely ascribed to α7 nAChR activation. However, mechanistic details of α7 nAChR involvement in GTS-21 effects on inflammatory pathways remain unclear. Here, we investigate how GTS-21 acts in two cell systems including the non-immune rat pituitary cell line GH4C1 expressing an NFκB-driven reporter gene and cytokine secretion by ex vivo cultures of primary mouse macrophages activated by lipopolysaccharide (LPS). GTS-21 does not change TNF-stimulated NFκB signaling in GH4C1 cells expressing rat α7 nAChRs, suggesting that GTS-21 requires additional unidentified factors besides α7 nAChR expression to allow anti-inflammatory effects in these cells. In contrast, GTS-21 dose-dependently suppresses LPS-induced IL6 and TNF secretion in primary mouse macrophages endogenously expressing α7 nAChRs. GTS-21 also blocks TNF-induced phosphorylation of NFκB inhibitor alpha (IκBα), an important intermediary in NFκB signaling. However, α7 antagonists methyllycaconitine and α-bungarotoxin only partially reverse GTS-21 blockade of IL6 and TNF secretion. Further, GTS-21 significantly inhibited LPS-induced IL6 and TNF secretion in macrophages isolated from knockout mice lacking α7 nAChRs. These data indicate that even though a discrete component of the anti-inflammatory effects of GTS-21 requires expression of α7 nAChRs in macrophages, GTS-21 also has anti-inflammatory effects independent of these receptors depending on the cellular context.
| S-EPMC6448884 | biostudies-literature
Project description:Virus entry into animal cells is initiated by attachment to target macromolecules located on host cells. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) trimeric spike glycoprotein targets host angiotensin converting enzyme 2 to gain cellular access. The SARS-CoV-2 glycoprotein contains a neurotoxin-like region that has sequence similarities to the rabies virus and the human immunodeficiency virus glycoproteins, as well as to snake neurotoxins, which interact with nicotinic acetylcholine receptor (nAChR) subtypes via this region. Using a peptide of the neurotoxin-like region of SARS-CoV-2 (SCoV2P), we identified that this area moderately inhibits α3β2, α3β4 and α4β2 subtypes, while potentiating and inhibiting α7 nAChRs. These nAChR subtypes are found in target tissues including the nose, lung, central nervous system and immune cells. Importantly, SCoV2P potentiates and inhibits ACh-induced α7 nAChR responses by an allosteric mechanism, with nicotine enhancing these effects. Live-cell confocal microscopy was used to confirm that SCoV2P interacts with α7 nAChRs in transfected neuronal-like N2a and human embryonic kidney 293 cells. The SARS-CoV-2 ectodomain functionally potentiates and inhibits the α7 subtype with nanomolar potency. Our functional findings identify that the α7 nAChR is a target for the SARS-CoV-2 glycoprotein, providing a new aspect to our understanding of SARS-CoV-2 and host cell interactions, in addition to disease pathogenesis.
| S-EPMC10101490 | biostudies-literature
Project description:A series of 9H-fluoren-9-one substituents were synthesized and evaluated for imaging cerebral α7-nAChRs. Meta-iodine substituted 9-fluorenone 5 with high binding affinity (K i = 9.3 nM) and selectivity was radiolabeled with 125I. Fully in vitro and in vivo studies of [125I]5 have been performed. [125I]5 exhibited well brain uptake with a peak concentration of 7.5 ± 0.9% ID/g in mice brains. Moreover, ex vivo autoradiography studies and micro single-photon emission computed tomography (micro-SPECT/CT) dynamic imaging in mice confirmed its in vivo imaging properties. Besides, molecular docking and MD studies were also performed to interpret the binding mechanisms of the two series of ligands towards α7-nAChRs. To conclude, the meta-iodine substituted 9-fluorenone [125I]5 could be a promising tracer for imaging α7-nAChRs.
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Project description:PurposeThe α7 nicotinic acetylcholine receptor (nAChR) is widely expressed in the nervous system, including in the inner retinal neurons in all species studied to date. Although reductions in the expression of α7 nAChRs are thought to contribute to the memory and visual deficits reported in Alzheimer's disease (AD) and schizophrenia , the α7 nAChR knockout (KO) mouse is viable and has only slight visual dysfunction. The absence of a major phenotypic abnormality may be attributable to developmental mechanisms that serve to compensate for α7 nAChR loss. We hypothesized that the upregulation of genes encoding other nAChR subunits or muscarinic acetylcholine receptor (mAChR) subtypes during development partially accounts for the absence of major deficiencies in the α7 nAChR KO mouse. The purpose of this study was to determine whether the deletion of the α7 nAChR subunit in a mouse model resulted in changes in the regulation of other cholinergic receptors or other ion channels in an α7 nAChR KO mouse when compared to a wild-type (WT) mouse.MethodsTo examine gene expression changes, we employed a quantitative real-time polymerase chain reaction (qPCR) using whole retina RNA extracts as well as RNA extracted from selected regions of the retina. These extracts were collected using laser capture microdissection (LCM). The presence of acetylcholine receptor (AChR) subunit and subtype proteins was determined via western blotting. To determine any differences in the number and distribution of choline acetyltransferase (ChAT) amacrine cells, we employed wholemount and vertical immunohistochemistry (IHC) and cell counting. Additionally, in both WT and α7 nAChR KO mouse retinas, the distribution of the nAChR subunit and mAChR subtype proteins were determined via IHC for those KO mice that experienced mRNA changes.ResultsIn the whole retina, there was a statistically significant upregulation of α2, α9, α10, β4, nAChR subunit, and m1 and m4 mAChR subtype transcripts in the α7 nAChR KO mice. However, the retinal layers showed complex patterns of transcript expression. In the ganglion cell layer (GCL), m2 and m4 mAChR subtype transcripts were significantly upregulated, while β3 and β4 nAChR subunit transcripts were significantly downregulated. In the inner portion of the inner nuclear layer (iINL), α2, α9, β4, nAChR subunit, and m3 and m4 mAChR subtype transcripts were significantly downregulated. In the outer portion of the inner nuclear layer (oINL), β2, β4, and m4 AChR subunit transcripts were significantly upregulated. Western blot experiments confirmed the protein expression of α3-α5 and α9-containing nAChR subunits and m1-m2 mAChR subtypes in mouse retinas. IHC results supported many of the mRNA changes observed. Finally, this is the first report of α9 and α10 nAChR subunit expressions in the retina of any species.ConclusionsRather than a simple upregulation of a single AChR subunit or subtype, the absence of the α7 nAChR in the KO mice was associated with complex layer-specific changes in the expression of AChR subunits and subtypes.
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Project description:The α7 nicotinic acetylcholine receptor (α7 nAChR) is involved in various intracellular signaling pathways that mediate addiction, chronic pain, and other diseases, but its intracellular domain structures remain undetermined. The presence of 17 native cysteines in α7 nAChR provides opportunities for extracting structural information through site-directed labeling of chemical probes in strategic locations, but it also creates uncertainties in channel function when those native cysteines must be mutated. Using site-directed mutagenesis and two-electrode voltage clamp electrophysiology measurements, we found that α7 nAChR's function was well tolerated for mutations of all 13 cysteines as long as two pairs of disulfide-bond cysteines remained in the extracellular domain. Furthermore, surface plasmon resonance measurements showed that the cysteine mutations did not affect α7 nAChR binding to the intracellular protein PICK1. The study suggests that a high native cysteine content does not necessarily preclude the use of single cysteine labeling for acquiring structural information on functional proteins.
| S-EPMC7057392 | biostudies-literature
Project description:We investigated assembly and function of nicotinic acetylcholine receptors (nAChRs) composed of α7 and β2 subunits. We measured optical and electrophysiological properties of wild-type and mutant subunits expressed in cell lines and Xenopus laevis oocytes. Laser scanning confocal microscopy indicated that fluorescently tagged α7 and β2 subunits colocalize. Förster resonance energy transfer between fluorescently tagged subunits strongly suggested that α7 and β2 subunits coassemble. Total internal reflection fluorescence microscopy revealed that assemblies localized to filopodia-like processes of SH-EP1 cells. Gain-of-function α7 and β2 subunits confirmed that these subunits coassemble within functional receptors. Moreover, α7β2 nAChRs composed of wild-type subunits or fluorescently tagged subunits had pharmacological properties similar to those of α7 nAChRs, although amplitudes of α7β2 nAChR-mediated, agonist-evoked currents were generally ~2-fold lower than those for α7 nAChRs. It is noteworthy that α7β2 nAChRs displayed sensitivity to low concentrations of the antagonist dihydro-β-erythroidine that was not observed for α7 nAChRs at comparable concentrations. In addition, cysteine mutants revealed that the α7-β2 subunit interface does not bind ligand in a functionally productive manner, partly explaining lower α7β2 nAChR current amplitudes and challenges in identifying the function of native α7β2 nAChRs. On the basis of our findings, we have constructed a model predicting receptor function that is based on stoichiometry and position of β2 subunits within the α7β2 nAChRs.
| S-EPMC3263954 | biostudies-literature
Project description:Nicotinic acetylcholine receptors (nAChRs) mediate and modulate synaptic transmission throughout the brain, and contribute to learning, memory, and behavior. Dysregulation of α7-type nAChRs in neuropsychiatric as well as immunological and oncological diseases makes them attractive targets for pharmaceutical development. Recently, we identified NACHO as an essential chaperone for α7 nAChRs. Leveraging the robust recombinant expression of α7 nAChRs with NACHO, we utilized genome-wide cDNA library screening and discovered that several anti-apoptotic Bcl-2 family proteins further upregulate receptor assembly and cell surface expression. These effects are mediated by an intracellular motif on α7 that resembles the BH3 binding domain of pro-apoptotic Bcl-2 proteins, and can be blocked by BH3 mimetic Bcl-2 inhibitors. Overexpression of Bcl-2 member Mcl-1 in neurons enhanced surface expression of endogenous α7 nAChRs, while a combination of chemotherapeutic Bcl2-inhibitors suppressed neuronal α7 receptor assembly. These results demonstrate that Bcl-2 proteins link α7 nAChR assembly to cell survival pathways.
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Project description:Background and purposeNeonicotinoid insecticides are described as poor agonists of mammalian nicotinic ACh receptors. In this paper, we show that their effects on mammalian nicotinic receptors differ between compounds.Experimental approachTwo-electrode voltage-clamp electrophysiology was used to characterize the pharmacology of three neonicotinoid insecticides on nicotinic α7 receptors expressed in Xenopus oocytes. Single and combined application of clothianidin, acetamiprid and thiamethoxam were tested.ResultsTwo neonicotinoid insecticides, clothianidin and acetamiprid, were partial agonists of mammalian neuronal α7 nicotinic receptors, whereas another neonicotinoid insecticide, thiamethoxam, which is converted to clothianidin in insect and plant tissues, had no effect. Pretreatment with clothianidin and acetamiprid (10 μM) ACh significantly enhanced the subsequent currents evoked by ACh (100 μM ) whereas pretreatment with thiamethoxam (10 μM) reduced ACh-induced current amplitudes.A combination of the three neonicotinoids decreased the ACh-evoked currents.Conclusions and implicationsThe present findings suggest that neonicotinoid insecticides differ markedly in their direct effects on mammalian α7 nicotinic ACh receptors and can also modulate ACh-induced currents. Furthermore, our data indicate a previously unknown modulation of mammalian α7 nicotinic receptors by a combination of clothianidin, acetamiprid and thiamethoxam.Linked articlesThis article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc.
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Project description:Prototoxins are a diverse family of membrane-tethered molecules expressed in the nervous system that modulate nicotinic cholinergic signaling, but their functions and specificity have yet to be completely explored. We tested the selectivity and efficacy of leukocyte antigen, PLAUR (plasminogen activator, urokinase receptor) domain-containing (LYPD)-6B on α3β4-, α3α5β4-, and α7-containing nicotinic acetylcholine receptors (nAChRs). To constrain stoichiometry, fusion proteins encoding concatemers of human α3, β4, and α5 (D and N variants) subunits were expressed in Xenopus laevis oocytes and tested with or without LYPD6B. We used the 2-electrode voltage-clamp method to quantify responses to acetylcholine (ACh): agonist sensitivity (EC50), maximal agonist-induced current (Imax), and time constant (τ) of desensitization. For β4-α3-α3-β4-α3 and β4-α3-β4-α3-α3, LYPD6B decreased EC50 from 631 to 79 μM, reduced Imax by at least 59%, and decreased τ. For β4-α3-α5D-β4-α3 and β4-α3-β4-α-α5D, LYPD6B decreased Imax by 63 and 32%, respectively. Thus, LYPD6B acted only on (α3)3(β4)2 and (α3)2(α5D)(β4)2 and did not affect the properties of (α3)2(β4)3, α7, or (α3)2(α5N)(β4)2 nAChRs. Therefore, LYPD6B acts as a mixed modulator that enhances the sensitivity of (α3)3(β4)2 nAChRs to ACh while reducing ACh-induced whole-cell currents. LYPD6B also negatively modulates α3β4 nAChRs that include the α5D common human variant, but not the N variant associated with nicotine dependence.
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Project description:Nicotinic acetylcholine receptors (nAChRs) are best known to function as ligand-gated ion channels in the nervous system. However, recent evidence suggests that nicotine modulates inflammation by desensitizing non-neuronal nAChRs, rather than by inducing channel opening. Silent agonists are molecules that selectively induce the desensitized state of nAChRs while producing little or no channel opening. A silent agonist of α7 nAChRs has recently been shown to reduce inflammation in an animal model of inflammatory pain. The objective of this study was to determine whether a silent agonist of α7 nAChRs can also effectively modulate inflammation and disease manifestation in an animal model of multiple sclerosis. We first evaluated the effects of various nAChR ligands and of an α7 nAChR-selective silent agonist, 1-ethyl-4-(3-(bromo)phenyl)piperazine (m-bromo PEP), on the modulation of mouse bone marrow-derived monocyte/macrophage (BMDM) numbers, phenotype and cytokine production. The non-competitive antagonist mecamylamine and the silent agonist m-bromo PEP reduced pro-inflammatory BMDM numbers by affecting their viability and proliferation. Both molecules also significantly reduced cytokine production by mouse BMDMs and significantly ameliorated disease in experimental autoimmune encephalomyelitis. Finally, m-bromo PEP also reduced chronic inflammatory pain in mice. Taken together, our results further support the hypothesis that nAChRs may modulate inflammation via receptor desensitization rather than channel opening. α7 nAChR-selective silent agonists may thus be a novel source of anti-inflammatory compounds that could be used for the treatment of inflammatory disorders.
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