RESUMO
Nicotinic acetylcholine receptors are not only expressed by the nervous system and at the neuro-muscular junction but also by mononuclear phagocytes, which belong to the innate immune system. Mononuclear phagocyte is an umbrella term for monocytes, macrophages, and dendritic cells. These cells play pivotal roles in host defense against infection but also in numerous often debilitating diseases that are characterized by exuberant inflammation. Nicotinic acetylcholine receptors of the neuronal type dominate in these cells, and their stimulation is mainly associated with anti-inflammatory effects. Although the cholinergic modulation of mononuclear phagocytes is of eminent clinical relevance for the prevention and treatment of inflammatory diseases and neuropathic pain, we are only beginning to understand the underlying mechanisms on the molecular level. The purpose of this review is to report and critically discuss the current knowledge on signal transduction mechanisms elicited by nicotinic acetylcholine receptors in mononuclear phagocytes.
Assuntos
Receptores Nicotínicos , Humanos , Receptores Nicotínicos/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Transdução de Sinais , InflamaçãoRESUMO
Activation of the NLRP3 inflammasome induces maturation of IL-1ß and IL-18, both validated targets for treating acute and chronic inflammatory diseases. Here, we demonstrate that OLT1177, an orally active ß-sulfonyl nitrile molecule, inhibits activation of the NLRP3 inflammasome. In vitro, nanomolar concentrations of OLT1177 reduced IL-1ß and IL-18 release following canonical and noncanonical NLRP3 inflammasome activation. The molecule showed no effect on the NLRC4 and AIM2 inflammasomes, suggesting specificity for NLRP3. In LPS-stimulated human blood-derived macrophages, OLT1177 decreased IL-1ß levels by 60% and IL-18 by 70% at concentrations 100-fold lower in vitro than plasma concentrations safely reached in humans. OLT1177 also reduced IL-1ß release and caspase-1 activity in freshly obtained human blood neutrophils. In monocytes isolated from patients with cryopyrin-associated periodic syndrome (CAPS), OLT1177 inhibited LPS-induced IL-1ß release by 84% and 36%. Immunoprecipitation and FRET analysis demonstrated that OLT1177 prevented NLRP3-ASC, as well as NLRP3-caspase-1 interaction, thus inhibiting NLRP3 inflammasome oligomerization. In a cell-free assay, OLT1177 reduced ATPase activity of recombinant NLRP3, suggesting direct targeting of NLRP3. Mechanistically, OLT1177 did not affect potassium efflux, gene expression, or synthesis of the IL-1ß precursor. Steady-state levels of phosphorylated NF-κB and IkB kinase were significantly lowered in spleen cells from OLT1177-treated mice. We observed reduced IL-1ß content in tissue homogenates, limited oxidative stress, and increased muscle oxidative metabolism in OLT1177-treated mice challenged with LPS. Healthy humans receiving 1,000 mg of OLT1177 daily for 8 d exhibited neither adverse effects nor biochemical or hematological changes.
Assuntos
Anti-Inflamatórios/farmacologia , Inflamassomos/antagonistas & inibidores , Inflamação/prevenção & controle , Macrófagos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Nitrilas/farmacologia , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/uso terapêutico , Caspase 1/metabolismo , Células Cultivadas , Humanos , Inflamação/induzido quimicamente , Inflamação/imunologia , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Lipopolissacarídeos/toxicidade , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Nitrilas/química , Nitrilas/uso terapêuticoRESUMO
This study reveals that it is possible to secrete truncated versions of outer membrane cytochromes into the culture supernatant and that these proteins can provide a basis for the export of heterologously produced proteins. Different soluble and truncated versions of the outer membrane cytochrome MtrF were analyzed for their suitability to be secreted. A protein version with a very short truncation of the N-terminus to remove the recognition sequence for the addition of a lipid anchor is secreted efficiently to the culture supernatant, and moreover this protein could be further truncated by a deletion of 160 amino acid and still is detectable in the supernatant. By coupling a cellulase to this soluble outer membrane cytochrome, the export efficiency was measured by means of relative cellulase activity. We conclude that outer membrane cytochromes of S. oneidensis can be applied as transporters for the export of target proteins into the medium using the type II secretion pathway.
Assuntos
Proteínas da Membrana Bacteriana Externa/biossíntese , Membrana Externa Bacteriana/metabolismo , Citocromos/metabolismo , Shewanella/metabolismo , Shewanella/química , SolubilidadeRESUMO
While interleukin-1ß (IL-1ß) is a potent pro-inflammatory cytokine essential for host defense, high systemic levels cause life-threatening inflammatory syndromes. ATP, a stimulus of IL-1ß maturation, is released from damaged cells along with ß-nicotinamide adenine dinucleotide (ß-NAD). Here, we tested the hypothesis that ß-NAD controls ATP-signaling and, hence, IL-1ß release. Lipopolysaccharide-primed monocytic U937 cells and primary human mononuclear leukocytes were stimulated with 2'(3')-O-(4-benzoyl-benzoyl)ATP trieethylammonium salt (BzATP), a P2X7 receptor agonist, in the presence or absence of ß-NAD. IL-1ß was measured in cell culture supernatants. The roles of P2Y receptors, nicotinic acetylcholine receptors (nAChRs), and Ca2+-independent phospholipase A2 (iPLA2ß, PLA2G6) were investigated using specific inhibitors and gene-silencing. Exogenous ß-NAD signaled via P2Y receptors and dose-dependently (IC50 = 15 µM) suppressed the BzATP-induced IL-1ß release. Signaling involved iPLA2ß, release of a soluble mediator, and nAChR subunit α9. Patch-clamp experiments revealed that ß-NAD inhibited BzATP-induced ion currents. In conclusion, we describe a novel triple membrane-passing signaling cascade triggered by extracellular ß-NAD that suppresses ATP-induced release of IL-1ß by monocytic cells. This cascade links activation of P2Y receptors to non-canonical metabotropic functions of nAChRs that inhibit P2X7 receptor function. The biomedical relevance of this mechanism might be the control of trauma-associated systemic inflammation.
Assuntos
Interleucina-1beta/metabolismo , Monócitos/metabolismo , NAD/farmacologia , Trifosfato de Adenosina/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Lipopolissacarídeos/farmacologia , Antagonistas Nicotínicos/farmacologia , Inibidores de Fosfolipase A2/farmacologia , Fosfolipases A2/genética , Fosfolipases A2/metabolismo , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Receptores Purinérgicos P2Y/genética , Receptores Purinérgicos P2Y/metabolismoRESUMO
Phosphocholine-modified bacterial cell wall components are virulence factors enabling immune evasion and permanent colonization of the mammalian host, by mechanisms that are poorly understood. Recently, we demonstrated that free phosphocholine (PC) and PC-modified lipooligosaccharides (PC-LOS) from Haemophilus influenzae, an opportunistic pathogen of the upper and lower airways, function as unconventional nicotinic agonists and efficiently inhibit the ATP-induced release of monocytic IL-1ß. We hypothesize that H. influenzae PC-LOS exert similar effects on pulmonary epithelial cells and on the complex lung tissue. The human lung carcinoma-derived epithelial cell lines A549 and Calu-3 were primed with lipopolysaccharide from Escherichia coli followed by stimulation with ATP in the presence or absence of PC or PC-LOS or LOS devoid of PC. The involvement of nicotinic acetylcholine receptors was tested using specific antagonists. We demonstrate that PC and PC-LOS efficiently inhibit ATP-mediated IL-1ß release by A549 and Calu-3 cells via nicotinic acetylcholine receptors containing subunits α7, α9, and/or α10. Primed precision-cut lung slices behaved similarly. We conclude that H. influenzae hijacked an endogenous anti-inflammatory cholinergic control mechanism of the lung to evade innate immune responses of the host. These findings may pave the way towards a host-centered antibiotic treatment of chronic airway infections with H. influenzae.
Assuntos
Trifosfato de Adenosina/farmacologia , Células Epiteliais/metabolismo , Haemophilus influenzae/química , Interleucina-1beta/metabolismo , Lipopolissacarídeos/química , Lipopolissacarídeos/farmacologia , Pulmão/citologia , Fosforilcolina/química , Células A549 , Animais , Células Epiteliais/efeitos dos fármacos , Humanos , Camundongos , Nicotina/farmacologia , Receptores Nicotínicos/metabolismoRESUMO
Interleukin (IL)-1ß is a potent pro-inflammatory cytokine of innate immunity involved in host defense. High systemic IL-1ß levels, however, cause life-threatening inflammatory diseases, including systemic inflammatory response syndrome. In response to various danger signals, the pro-form of IL-1ß is synthesized and stays in the cytoplasm unless a second signal, such as extracellular ATP, activates the inflammasome, which enables processing and release of mature IL-1ß. As pulmonary surfactant is known for its anti-inflammatory properties, we hypothesize that surfactant inhibits ATP-induced release of IL-1ß. Lipopolysaccharide-primed monocytic U937 cells were stimulated with an ATP analog in the presence of natural or synthetic surfactant composed of recombinant surfactant protein (rSP)-C, palmitoylphosphatidylglycerol, and dipalmitoylphosphatidylcholine (DPPC). Both surfactant preparations dose-dependently inhibited IL-1ß release from U937 cells. DPPC was the active constituent of surfactant, whereas rSP-C and palmitoylphosphatidylglycerol were inactive. DPPC was also effective in primary mononuclear leukocytes isolated from human blood. Experiments with nicotinic antagonists, siRNA technology, and patch-clamp experiments suggested that stimulation of nicotinic acetylcholine receptors (nAChRs) containing subunit α9 results in a complete inhibition of the ion channel function of ATP receptor, P2X7. In conclusion, the surfactant constituent, DPPC, efficiently inhibits ATP-induced inflammasome activation and maturation of IL-1ß in human monocytes by a mechanism involving nAChRs.
Assuntos
Trifosfato de Adenosina/farmacologia , Interleucina-1beta/metabolismo , Surfactantes Pulmonares/farmacologia , Receptores Nicotínicos/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Trifosfato de Adenosina/química , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Subunidades Proteicas/metabolismo , Células U937RESUMO
Anode-associated multispecies exoelectrogenic biofilms are essential for the function of bioelectrochemical systems (BESs). The individual activities of anode-associated organisms and physiological responses resulting from coculturing are often hard to assess due to the high microbial diversity in these systems. Therefore, we developed a model multispecies biofilm comprising three exoelectrogenic proteobacteria, Shewanella oneidensis, Geobacter sulfurreducens, and Geobacter metallireducens, with the aim to study in detail the biofilm formation dynamics, the interactions between the organisms, and the overall activity of an exoelectrogenic biofilm as a consequence of the applied anode potential. The experiments revealed that the organisms build a stable biofilm on an electrode surface that is rather resilient to changes in the redox potential of the anode. The community operated at maximum electron transfer rates at electrode potentials that were higher than 0.04 V versus a normal hydrogen electrode. Current densities decreased gradually with lower potentials and reached half-maximal values at -0.08 V. Transcriptomic results point toward a positive interaction among the individual strains. S. oneidensis and G. sulfurreducens upregulated their central metabolisms as a response to cultivation under mixed-species conditions. G. sulfurreducens was detected in the planktonic phase of the bioelectrochemical reactors in mixed-culture experiments but not when it was grown in the absence of the other two organisms.IMPORTANCE In many cases, multispecies communities can convert organic substrates into electric power more efficiently than axenic cultures, a phenomenon that remains unresolved. In this study, we aimed to elucidate the potential mutual effects of multispecies communities in bioelectrochemical systems to understand how microbes interact in the coculture anodic network and to improve the community's conversion efficiency for organic substrates into electrical energy. The results reveal positive interactions that might lead to accelerated electron transfer in mixed-species anode communities. The observations made within this model biofilm might be applicable to a variety of nonaxenic systems in the field.
Assuntos
Fontes de Energia Bioelétrica/microbiologia , Biofilmes/crescimento & desenvolvimento , Técnicas Eletroquímicas/métodos , Geobacter/metabolismo , Shewanella/metabolismo , Técnicas de Cocultura , Eletricidade , Eletrodos/microbiologia , Transporte de Elétrons , Geobacter/crescimento & desenvolvimento , Oxirredução , Shewanella/crescimento & desenvolvimentoRESUMO
IL-1ß is a potent proinflammatory cytokine of the innate immune system that is involved in host defense against infection. However, increased production of IL-1ß plays a pathogenic role in various inflammatory diseases, such as rheumatoid arthritis, gout, sepsis, stroke, and transplant rejection. To prevent detrimental collateral damage, IL-1ß release is tightly controlled and typically requires two consecutive danger signals. LPS from Gram-negative bacteria is a prototypical first signal inducing pro-IL-1ß synthesis, whereas extracellular ATP is a typical second signal sensed by the ATP receptor P2X7 that triggers activation of the NLRP3-containing inflammasome, proteolytic cleavage of pro-IL-1ß by caspase-1, and release of mature IL-1ß. Mechanisms controlling IL-1ß release, even in the presence of both danger signals, are needed to protect from collateral damage and are of therapeutic interest. In this article, we show that acetylcholine, choline, phosphocholine, phosphocholine-modified LPS from Haemophilus influenzae, and phosphocholine-modified protein efficiently inhibit ATP-mediated IL-1ß release in human and rat monocytes via nicotinic acetylcholine receptors containing subunits α7, α9, and/or α10. Of note, we identify receptors for phosphocholine-modified macromolecules that are synthesized by microbes and eukaryotic parasites and are well-known modulators of the immune system. Our data suggest that an endogenous anti-inflammatory cholinergic control mechanism effectively controls ATP-mediated release of IL-1ß and that the same mechanism is used by symbionts and misused by parasites to evade innate immune responses of the host.
Assuntos
Trifosfato de Adenosina/farmacologia , Interleucina-1beta/metabolismo , Lipopolissacarídeos/farmacologia , Monócitos/efeitos dos fármacos , Agonistas Nicotínicos/farmacologia , Acetilcolina/farmacologia , Trifosfato de Adenosina/análogos & derivados , Animais , Western Blotting , Células Cultivadas , Colina/farmacologia , Relação Dose-Resposta a Droga , Humanos , Lipopolissacarídeos/química , Potenciais da Membrana/efeitos dos fármacos , Monócitos/metabolismo , Nicotina/farmacologia , Fosforilcolina/química , Interferência de RNA , Ratos , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células U937 , Receptor Nicotínico de Acetilcolina alfa7/genética , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
SCN5A encodes for the α-subunit of the cardiac voltage-gated sodium channel Nav1.5. Gain-of-function mutations in SCN5A are related to congenital long QT syndrome (LQTS3) characterized by delayed cardiac repolarization, leading to a prolonged QT interval in the ECG. Loss-of-function mutations in SCN5A are related to Brugada syndrome (BrS), characterized by an ST-segment elevation in the right precordial leads (V1-V3). The aim of this study was the characterization of a large set of novel SCN5A variants found in patients with different cardiac phenotypes, mainly LQTS and BrS. SCN5A variants of 13 families were functionally characterized in Xenopus laevis oocytes using the two-electrode voltage-clamp technique. We found in most of the cases, but not all, that the electrophysiology of the variants correlated with the clinically diagnosed phenotype. A susceptibility to develop LQTS can be suggested in patients carrying the variants S216L, K480N, A572D, F816Y, and G983D. However, taking the phenotype into account, the presence of the variants in genomic data bases, the mutational segregation, combined with our in vitro and in silico experiments, the variants S216L, S262G, K480N, A572D, F816Y, G983D, and T1526P remain as variants of unknown significance. However, the SCN5A variants R568H and A993T can be classified as pathogenic LQTS3 causing mutations, while R222stop and R2012H are novel BrS causing mutations.
Assuntos
Fenômenos Eletrofisiológicos/genética , Mutação/genética , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Adulto , Animais , Síndrome de Brugada/genética , Doença do Sistema de Condução Cardíaco , Sistema de Condução Cardíaco/metabolismo , Humanos , Masculino , Oócitos/metabolismo , Fenótipo , Xenopus laevis/metabolismoRESUMO
Lungs of air-breathing vertebrates are constantly exposed to mechanical forces and therefore are suitable for investigation of mechanotransduction processes in nonexcitable cells and tissues. Freshly dissected Xenopus laevis lungs were used for transepithelial short-circuit current (ISC) recordings and were exposed to increased hydrostatic pressure (HP; 5 cm fluid column, modified Ussing chamber). I(SC) values obtained under HP (I(5cm)) were normalized to values before HP (I(0cm)) application (I(5cm)/I(0cm)). Under control conditions, HP decreased I(SC) (I(5cm)/I(0cm)=0.84; n=68; P<0.0001). This effect was reversible and repeatable ≥30 times. Preincubation with ATP-sensitive K(+) channel (K(ATP)) inhibitors (HMR1098 and glibenclamide) prevented the decrease in I(SC) (I(5cm)/I(0cm): HMR1098=1.19, P<0.0001; glibenclamide=1.11, P<0.0001). Similar effects were observed with hemichannel inhibitors (I(5cm)/I(0cm): meclofenamic acid=1.09, P<0.0001; probenecid=1.0, P<0.0001). The HP effect was accompanied by release of ATP (P<0.05), determined by luciferin-luciferase luminescence in perfusion solution from the luminal side of an Ussing chamber. ATP release was abrogated by both meclofenamic acid and probenecid. RT-PCR experiments revealed the expression of pannexin and connexin hemichannels and KATP subunit transcripts in X. laevis lung. These data show an activation of KATP in pulmonary epithelial cells in response to HP that is induced by ATP release through mechanosensitive pannexin and connexin hemichannels. These findings represent a novel mechanism of mechanotransduction in nonexcitable cells.
Assuntos
Conexinas/metabolismo , Epitélio/metabolismo , Pressão Hidrostática , Canais KATP/metabolismo , Pulmão/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Feminino , Xenopus laevisRESUMO
Several lines of evidence have indicated that nicotinic acetylcholine receptors (nAChR) that contain α9 subunits, probably in combination with α10 subunits, may be valuable targets for the management of pain associated with inflammatory diseases through a cholinergic anti-inflammatory system (CAS), which has also been associated with α7 nAChR. Both α7- and α9-containing neuronal nAChR can be pharmacologically distinguished from the high-affinity nicotinic receptors of the brain by their sensitivity to α-bungarotoxin, but in other ways, they have quite distinct pharmacological profiles. The early association of α7 with CAS led to the development of numerous new ligands, variously characterized as α7 agonists, partial agonists, or silent agonists that desensitized α7 receptors without activation. Subsequent reinvestigation of one such family of α7 ligands based on an N,N-diethyl-N'-phenylpiperazine scaffold led to the identification of potent agonists and antagonists for α9. In this paper, we characterize the α9/α10 activity of a series of compounds based on a 5-(quinuclidin-3-ylmethyl)-1,2,4-oxadiazole (QMO) scaffold and identify two new potent ligands of α9, QMO-28, an agonist, and QMO-17, an antagonist. We separated the stereoisomers of these compounds to identify the most potent agonist and discovered that only the 3R isomer of QMO-17 was an α9 antagonist, permitting an in silico model of α9 antagonism to be developed. The α9 activity of these compounds was confirmed to be potentially useful for CAS management of inflammatory pain in cell-based assays of cytokine release.
Assuntos
Receptores Nicotínicos , Humanos , Oxidiazóis/farmacologia , Receptor Nicotínico de Acetilcolina alfa7 , Ligantes , DorRESUMO
There is an urgent need for nonopioid treatments for chronic and neuropathic pain to provide effective alternatives amid the escalating opioid crisis. This study introduces novel compounds targeting the α9 nicotinic acetylcholine receptor (nAChR) subunit, which is crucial for pain regulation, inflammation, and inner ear functions. Specifically, it identifies novel substituted carbamoyl/amido/heteroaryl dialkylpiperazinium iodides as potent agonists selective for human α9 and α9α10 over α7 nAChRs, particularly compounds 3f, 3h, and 3j. Compound 3h (GAT2711) demonstrated a 230 nM potency as a full agonist at α9 nAChRs, being 340-fold selective over α7. Compound 3c was 10-fold selective for α9α10 over α9 nAChR. Compounds 2, 3f, and 3h inhibited ATP-induced interleukin-1ß release in THP-1 cells. The analgesic activity of 3h was fully retained in α7 knockout mice, suggesting that analgesic effects were potentially mediated through α9* nAChRs. Our findings provide a blueprint for developing α9*-specific therapeutics for pain.
Assuntos
Analgésicos , Inflamação , Piperazinas , Receptores Nicotínicos , Animais , Humanos , Masculino , Camundongos , Analgésicos/farmacologia , Analgésicos/química , Analgésicos/síntese química , Analgésicos/uso terapêutico , Inflamação/tratamento farmacológico , Camundongos Knockout , Agonistas Nicotínicos/farmacologia , Agonistas Nicotínicos/química , Agonistas Nicotínicos/uso terapêutico , Agonistas Nicotínicos/síntese química , Dor/tratamento farmacológico , Piperazinas/farmacologia , Piperazinas/química , Piperazinas/síntese química , Piperazinas/uso terapêutico , Receptores Nicotínicos/metabolismo , Sais/química , Sais/farmacologia , Relação Estrutura-Atividade , Iodetos/químicaRESUMO
Mucociliary clearance is a pivotal physiological mechanism that protects the lung by cleaning the airways from pollution and colonization, thereby preventing infection. Ciliary function is influenced by various signal transduction cascades, and Ca2+ represents a key second messenger. A fixed 20:1 combination of cafedrine and theodrenaline has been widely used to treat perioperative hypotension and emergency hypotensive states since the 1960s; however, its effect on the intracellular Ca2+ concentration ([Ca2+]i) of respiratory epithelium remains unknown. Therefore, human tracheal epithelial cells were exposed to the clinically applied 20:1 mixture of cafedrine/theodrenaline and the individual substances separately. [Ca2+]i was assessed by FURA-2 340/380 fluorescence ratio. Pharmacological inhibitors were applied to elucidate relevant signal transduction cascades, and reverse transcription polymerase chain reaction (RT-PCR) was performed on murine tracheal epithelium to analyze ryanodine receptor (RyR) subtype expression. All three pharmacological preparations instantaneously induced a steep increase in [Ca2+]i that quickly returned to its baseline value despite the persistence of each substance. Peak [Ca2+]i following the administration of 20:1 cafedrine/theodrenaline, cafedrine alone, and theodrenaline alone increased in a dose-dependent manner, with median effective concentrations of 0.35 mM (7.32 mM cafedrine and 0.35 mM theodrenaline), 3.14 mM, and 3.45 mM, respectively. When extracellular Ca2+ influx was inhibited using a Ca2+-free buffer solution, the peak [Ca2+]i following the administration of cafedrine alone and theodrenaline alone were reduced but not abolished. No alteration in [Ca2+]i compared with baseline [Ca2+]i was observed during ß-adrenergic receptor inhibition. Depletion of caffeine-sensitive stores and inhibition of RyR, but not IP3 receptors, completely abolished any increase in [Ca2+]i. However, [Ca2+]i still increased following the depletion of mitochondrial Ca2+ stores using 2,4-dinitrophenol. RT-PCR revealed RyR-2 and RyR-3 expression on murine tracheal epithelium. Although our experiments showed that cafedrine/theodrenaline, cafedrine alone, or theodrenaline alone release Ca2+ from intracellular stores through mechanisms that are exclusively triggered by ß-adrenergic receptor stimulation, which most probably lead to RyR activation, clinical plasma concentrations are considerably lower than those used in our experiments to elicit an increase in [Ca2+]i; therefore, further studies are needed to evaluate the ability of cafedrine/theodrenaline to alter mucociliary clearance in clinical practice.
Assuntos
Canal de Liberação de Cálcio do Receptor de Rianodina , Traqueia , Humanos , Animais , Camundongos , Células EpiteliaisRESUMO
Pain due to inflammation can be reduced by targeting the noncanonical nicotinic receptors (NCNR) in cells of the immune system that regulate the synthesis and release of pro- and anti-inflammatory cytokines. Although NCNR do not generate ion channel currents, the pharmacology of ion-channel forms of the receptors can predict drugs which may be effective regulators of the cholinergic anti-inflammatory system (CAS). Agonists of α7 type receptors have been definitively associated with CAS. Receptors containing α9 and α10 subunits have also been implicated. We have recently characterized two small molecules, pCN-diEPP and mCN-diEPP, as selective α9α10 agonists and antagonists, respectively. We used these drugs, along with nicotine, an α7 agonist and α9α10 antagonist, to probe the mixed populations of receptors that are formed when α7, α9, and α10 are all expressed together in Xenopus oocytes. We also evaluated the effects of the CN-diEPP compounds on regulating the ATP-induced release of interleukin-1ß from monocytic THP-1 cells, which express NCNR. The compounds successfully identified separate populations of receptors when all three subunits were co-expressed, including a potential population of homomeric α10 receptors. The α9α10 agonist pCN-diEPP was the more effective regulator of interleukin-1ß release in THP-1 cells. pCN-diEPP was also fully effective in a mouse model of inflammatory pain, while mCN-diEPP had only partial effects, requiring a higher dosage. The analgetic effects of pCN-diEPP and mCN-diEPP were retained in α7 knockout mice. Taken together, our results suggest that drugs that selectively activate α9α10 receptors may useful to reduce inflammatory pain through the CAS.
RESUMO
Objective: The pro-inflammatory cytokine interleukin-1ß (IL-1ß) plays a central role in host defense against infections. High systemic IL-1ß levels, however, promote the pathogenesis of inflammatory disorders. Therefore, mechanisms controlling IL-1ß release are of substantial clinical interest. Recently, we identified a cholinergic mechanism inhibiting the ATP-mediated IL-1ß release by human monocytes via nicotinic acetylcholine receptor (nAChR) subunits α7, α9 and/or α10. We also discovered novel nAChR agonists that trigger this inhibitory function in monocytic cells without eliciting ionotropic functions at conventional nAChRs. Here, we investigate the ion flux-independent signaling pathway that links nAChR activation to the inhibition of the ATP-sensitive P2X7 receptor (P2X7R). Methods: Different human and murine mononuclear phagocytes were primed with lipopolysaccharide and stimulated with the P2X7R agonist BzATP in the presence or absence of nAChR agonists, endothelial NO synthase (eNOS) inhibitors, and NO donors. IL-1ß was measured in cell culture supernatants. Patch-clamp and intracellular Ca2+ imaging experiments were performed on HEK cells overexpressing human P2X7R or P2X7R with point mutations at cysteine residues in the cytoplasmic C-terminal domain. Results: The inhibitory effect of nAChR agonists on the BzATP-induced IL-1ß release was reversed in the presence of eNOS inhibitors (L-NIO, L-NAME) as well as in U937 cells after silencing of eNOS expression. In peripheral blood mononuclear leukocytes from eNOS gene-deficient mice, the inhibitory effect of nAChR agonists was absent, suggesting that nAChRs signal via eNOS to inhibit the BzATP-induced IL-1ß release. Moreover, NO donors (SNAP, S-nitroso-N-acetyl-DL-penicillamine; SIN-1) inhibited the BzATP-induced IL-1ß release by mononuclear phagocytes. The BzATP-induced ionotropic activity of the P2X7R was abolished in the presence of SIN-1 in both, Xenopus laevis oocytes and HEK cells over-expressing the human P2X7R. This inhibitory effect of SIN-1 was absent in HEK cells expressing P2X7R, in which C377 was mutated to alanine, indicating the importance of C377 for the regulation of the P2X7R function by protein modification. Conclusion: We provide first evidence that ion flux-independent, metabotropic signaling of monocytic nAChRs involves eNOS activation and P2X7R modification, resulting in an inhibition of ATP signaling and ATP-mediated IL-1ß release. This signaling pathway might be an interesting target for the treatment of inflammatory disorders.
Assuntos
Leucócitos Mononucleares , Receptores Purinérgicos P2X7 , Humanos , Camundongos , Animais , Interleucina-1beta/metabolismo , Leucócitos Mononucleares/metabolismo , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Monócitos/metabolismo , Trifosfato de Adenosina/metabolismo , Óxido Nítrico Sintase/metabolismoRESUMO
Host-derived succinate accumulates in the airways during bacterial infection. Here, we show that luminal succinate activates murine tracheal brush (tuft) cells through a signaling cascade involving the succinate receptor 1 (SUCNR1), phospholipase Cß2, and the cation channel transient receptor potential channel subfamily M member 5 (TRPM5). Stimulated brush cells then trigger a long-range Ca2+ wave spreading radially over the tracheal epithelium through a sequential signaling process. First, brush cells release acetylcholine, which excites nearby cells via muscarinic acetylcholine receptors. From there, the Ca2+ wave propagates through gap junction signaling, reaching also distant ciliated and secretory cells. These effector cells translate activation into enhanced ciliary activity and Cl- secretion, which are synergistic in boosting mucociliary clearance, the major innate defense mechanism of the airways. Our data establish tracheal brush cells as a central hub in triggering a global epithelial defense program in response to a danger-associated metabolite.
Assuntos
Acetilcolina , Traqueia , Camundongos , Animais , Traqueia/metabolismo , Transdução de Sinais , Succinatos/metabolismo , Epitélio/metabolismoRESUMO
Heterologous multiprotein expression is the tool to answer a number of questions in basic science as well as to convert strains into producers and/or consumers of certain compounds in applied sciences. Multiprotein expression can be driven by plasmids with the disadvantages that the gene dosage might, in some cases, lead to toxic effects and that the continuous addition of antibiotics is undesirable. Stable genomic expression of proteins can forgo these problems and is a helpful and promising tool in synthetic biology. In the present paper, we provide an extract of methods from the toolbox for chromosome-based heterologous expression in Escherichia coli.
Assuntos
Clonagem Molecular , Escherichia coli/genética , Elementos de DNA Transponíveis , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Engenharia Genética , Mutagênese Insercional , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Transformação BacterianaRESUMO
An extension of the respiratory chain to the cell surface is necessary to reduce extracellular electron acceptors like ferric iron or manganese oxides. In the past few years, more and more compounds were revealed to be reduced at the surface of the outer membrane of Gram-negative bacteria, and the list does not seem to have an end so far. Shewanella as well as Geobacter strains are model organisms to discover the biochemistry that enables the dissimilatory reduction of extracellular electron acceptors. In both cases, c-type cytochromes are essential electron-transferring proteins. They make the journey of respiratory electrons from the cytoplasmic membrane through periplasm and over the outer membrane possible. Outer membrane cytochromes have the ability to catalyze the last step of the respiratory chains. Still, recent discoveries provided evidence that they are accompanied by further factors that allow or at least facilitate extracellular reduction. This review gives a condensed overview of our current knowledge of extracellular respiration, highlights recent discoveries, and discusses critically the influence of different strategies for terminal electron transfer reactions.
Assuntos
Anaerobiose , Transporte de Elétrons , Compostos Férricos/metabolismo , Bactérias Gram-Negativas/metabolismo , Compostos de Manganês/metabolismo , Óxidos/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Membrana/metabolismo , Oxirredução , Proteínas Periplásmicas/metabolismoRESUMO
The expression of the acute-phase reactants C-reactive protein (CRP), α1-antitrypsin (AAT), and secretory leukocyte protease inhibitor (SLPI), is induced in response to inflammation by pro-inflammatory mediators, including interleukin-1ß. It is conceivable that acute-phase proteins exert protective functions, when the integrity of an organism is challenged by pathogens or trauma, which result in uncontrolled release of endogenous damage-associated molecular patterns like Toll-like receptor agonists and ATP. Acute-phase proteins can enhance or down-modulate immunity against infections or protect the host against damage caused by over-shooting effector functions of the immune system. CRP is mainly regarded as a pro-inflammatory opsonizing agent that binds to bacteria and damaged host cells thereby contributing to their inactivation and elimination. AAT and SLPI are well known for their anti-protease activity, which protects the lung extracellular matrix against degradation by proteases that are released by activated neutrophil granulocytes. In addition, there is growing evidence, that CRP, AAT, and SLPI can control the biosynthesis, maturation, and secretion of pro-inflammatory cytokines. The purpose of this narrative mini review is to summarize these anti-inflammatory functions with a focus on the negative control of the ATP-induced, inflammasome-dependent secretion of interleukin-1ß by monocytes. CRP-, AAT- and SLPI-mediated control of interleukin-1ß release involves the activation of unconventional nicotinic acetylcholine receptors that inhibits the ionotropic function of the ATP receptor P2X7. Apart from other functions, CRP, AAT, and SLPI seem to be central elements of systemic negative feedback loops that protect the host against systemic hyperinflammation, barrier dysfunction, and death by multiple organ damage.
RESUMO
BACKGROUND AND PURPOSE: ATP plays an important role as an extracellular messenger acting via different types of purinoceptors. Whereas most of the actions of ATP at intestinal epithelia are thought to be mediated by metabotropic P2Y receptors, the role of ionotropic P2X receptors remains unclear. Consequently, we investigated the role of P2X4 and P2X7 receptors on ion transport across rat colonic epithelia by using BzATP, a potent agonist at P2X7 (and weak agonist at P2X4). EXPERIMENTAL APPROACH: Ussing chamber and Ca2+ imaging experiments were performed on rat colonic epithelia, combined with P2X receptor expression studies. KEY RESULTS: Ussing chamber experiments revealed that serosal BzATP induced a neuronally mediated increase in short-circuit current caused by Cl- secretion. In contrast, the effect of mucosal BzATP was smaller, insensitive to tetrodotoxin and Cl- -independent. When epithelia were basolaterally depolarized to measure currents across the apical membrane, BzATP stimulated a cation current consistent with the activation of apical nonselective cation channels. Experiments with isolated colonic crypts revealed a BzATP-induced increase in the cytosolic Ca2+ concentration. Sensitivity to antagonists indicates stimulation of P2X4 and P2X7 receptors by serosal BzATP and of P2X7 receptors by mucosal BzATP. A similar pattern was observed with native ATP, which induced larger transepithelial currents in comparison to BzATP. RT-PCR and immunohistochemistry experiments confirmed the expression of P2X4 and P2X7 receptors in the colon localized in the epithelium and in submucosal ganglia. CONCLUSIONS AND IMPLICATIONS: Epithelial and neuronal ionotropic P2X receptors are involved in the regulation of intestinal ion transport.