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1.
Nature ; 581(7807): 204-208, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32405000

RESUMO

It has been speculated that brain activities might directly control adaptive immune responses in lymphoid organs, although there is little evidence for this. Here we show that splenic denervation in mice specifically compromises the formation of plasma cells during a T cell-dependent but not T cell-independent immune response. Splenic nerve activity enhances plasma cell production in a manner that requires B-cell responsiveness to acetylcholine mediated by the α9 nicotinic receptor, and T cells that express choline acetyl transferase1,2 probably act as a relay between the noradrenergic nerve and acetylcholine-responding B cells. We show that neurons in the central nucleus of the amygdala (CeA) and the paraventricular nucleus (PVN) that express corticotropin-releasing hormone (CRH) are connected to the splenic nerve; ablation or pharmacogenetic inhibition of these neurons reduces plasma cell formation, whereas pharmacogenetic activation of these neurons increases plasma cell abundance after immunization. In a newly developed behaviour regimen, mice are made to stand on an elevated platform, leading to activation of CeA and PVN CRH neurons and increased plasma cell formation. In immunized mice, the elevated platform regimen induces an increase in antigen-specific IgG antibodies in a manner that depends on CRH neurons in the CeA and PVN, an intact splenic nerve, and B cell expression of the α9 acetylcholine receptor. By identifying a specific brain-spleen neural connection that autonomically enhances humoral responses and demonstrating immune stimulation by a bodily behaviour, our study reveals brain control of adaptive immunity and suggests the possibility to enhance immunocompetency by behavioural intervention.


Assuntos
Comportamento Animal/fisiologia , Encéfalo/fisiologia , Imunidade Humoral/imunologia , Baço/imunologia , Baço/inervação , Acetilcolina/metabolismo , Acetilcolina/farmacologia , Neurônios Adrenérgicos/metabolismo , Tonsila do Cerebelo/citologia , Tonsila do Cerebelo/efeitos dos fármacos , Tonsila do Cerebelo/metabolismo , Animais , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Colina O-Acetiltransferase/metabolismo , Hormônio Liberador da Corticotropina/metabolismo , Hemocianinas/imunologia , Imunoglobulina G/imunologia , Ativação Linfocitária , Masculino , Camundongos , Núcleo Hipotalâmico Paraventricular/citologia , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos , Núcleo Hipotalâmico Paraventricular/metabolismo , Plasmócitos/citologia , Plasmócitos/efeitos dos fármacos , Plasmócitos/imunologia , Receptores Nicotínicos/deficiência , Receptores Nicotínicos/metabolismo , Baço/citologia , Baço/efeitos dos fármacos , Estresse Psicológico/imunologia , Estresse Psicológico/metabolismo , Linfócitos T/imunologia
2.
Med Sci Monit ; 26: e926016, 2020 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-32463026

RESUMO

The emergence of the novel ß-coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in a global pandemic of coronavirus disease 2019 (COVID-19). Clinical studies have documented that potentially severe neurological symptoms are associated with SARS-CoV-2 infection, thereby suggesting direct CNS penetration by the virus. Prior studies have demonstrated that the destructive neurological effects of rabies virus (RABV) infections are mediated by CNS transport of the virus tightly bound to the nicotinic acetylcholine receptor (nAChR). By comparison, it has been hypothesized that a similar mechanism exists to explain the multiple neurological effects of SARS-CoV-2 via binding to peripheral nAChRs followed by orthograde or retrograde transport into the CNS. Genetic engineering of the RABV has been employed to generate novel vaccines consisting of non-replicating RABV particles expressing chimeric capsid proteins containing human immunodeficiency virus 1 (HIV-1), Middle East respiratory syndrome (MERS-CoV), Ebolavirus, and hepatitis C virus (HCV) sequences. Accordingly, we present a critical discussion that integrates lessons learned from prior RABV research and vaccine development into a working model of a SARS-CoV-2 vaccine that selectively targets and neutralizes CNS penetration of a tightly bound viral nAChR complex.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Vírus da Raiva/fisiologia , Receptores Nicotínicos/metabolismo , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Replicação Viral , Betacoronavirus/química , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Humanos , Pandemias , Pneumonia Viral/virologia , Domínios Proteicos , Vírus da Raiva/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Vacinas Virais/química , Vacinas Virais/metabolismo
3.
ACS Chem Neurosci ; 11(9): 1206-1209, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32320211

RESUMO

The novel coronavirus SARS-CoV-2, which was identified after a recent outbreak in Wuhan, China, in December 2019, has kept the whole world in tenterhooks due to its severe life-threatening nature of the infection. The virus is unlike its previous counterparts, SARS-CoV and MERS-CoV, or anything the world has encountered before both in terms of virulence and severity of the infection. If scientific reports relevant to the SARS-CoV-2 virus are noted, it can be seen that the virus owes much of its killer properties to its unique structure that has a stronger binding affinity with the human angiotensin-converting enzyme 2 (hACE2) protein, which the viruses utilize as an entry point to gain accesses to its hosts. Recent reports suggest that it is not just the lung that the virus may be targeting; the human brain may soon emerge as the new abode of the virus. Already instances of patients with COVID-19 have been reported with mild (anosmia and ageusia) to severe (encephalopathy) neurological manifestations, and if that is so, then it gives us more reasons to be frightened of this killer virus. Keeping in mind that the situation does not worsen from here, immediate awareness and more thorough research regarding the neuroinvasive nature of the virus is the immediate need of the hour. Scientists globally also need to up their game to design more specific therapeutic strategies with the available information to counteract the pandemic. In this Viewpoint, we provide a brief outline of the currently known neurological manifestations of COVID-19 and discuss some probable ways to design therapeutic strategies to overcome the present global crisis.


Assuntos
Betacoronavirus/patogenicidade , Encéfalo/virologia , Infecções por Coronavirus/fisiopatologia , Pneumonia Viral/fisiopatologia , Idoso , Ageusia/virologia , Autopsia , Vacina BCG/administração & dosagem , Vacina BCG/imunologia , Betacoronavirus/química , Betacoronavirus/metabolismo , Encéfalo/patologia , Encéfalo/fisiopatologia , Encefalopatias/imunologia , Encefalopatias/patologia , Encefalopatias/virologia , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Citocinas/imunologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Inflamação/virologia , MicroRNAs/genética , Transtornos do Olfato/virologia , Mucosa Olfatória/patologia , Mucosa Olfatória/fisiopatologia , Mucosa Olfatória/virologia , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/diagnóstico , Pneumonia Viral/imunologia , Pneumonia Viral/patologia , Pneumonia Viral/transmissão , Pneumonia Viral/virologia , Interferência de RNA , Receptores Nicotínicos/metabolismo , Serina Endopeptidases/metabolismo , Fumar/metabolismo , Fumar/patologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
4.
Nat Commun ; 11(1): 1073, 2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-32103010

RESUMO

Denervation of skeletal muscles induces severe muscle atrophy, which is preceded by cellular alterations such as increased plasma membrane permeability, reduced resting membrane potential and accelerated protein catabolism. The factors that induce these changes remain unknown. Conversely, functional recovery following denervation depends on successful reinnervation. Here, we show that activation of nicotinic acetylcholine receptors (nAChRs) by quantal release of acetylcholine (ACh) from motoneurons is sufficient to prevent changes induced by denervation. Using in vitro assays, ACh and non-hydrolysable ACh analogs repressed the expression of connexin43 and connexin45 hemichannels, which promote muscle atrophy. In co-culture studies, connexin43/45 hemichannel knockout or knockdown increased innervation of muscle fibers by dorsal root ganglion neurons. Our results show that ACh released by motoneurons exerts a hitherto unknown function independent of myofiber contraction. nAChRs and connexin hemichannels are potential molecular targets for therapeutic intervention in a variety of pathological conditions with reduced synaptic neuromuscular transmission.


Assuntos
Acetilcolina/metabolismo , Gânglios Espinais/crescimento & desenvolvimento , Músculo Esquelético/inervação , Atrofia Muscular/patologia , Receptores Nicotínicos/metabolismo , Acetilcolina/análogos & derivados , Acetilcolina/farmacologia , Animais , Permeabilidade da Membrana Celular/fisiologia , Células Cultivadas , Conexina 43/metabolismo , Conexinas/metabolismo , Masculino , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Esquelético/metabolismo
5.
J Agric Food Chem ; 68(2): 523-529, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31908169

RESUMO

The present work sought to contribute to the development of new nematicides. Benzaldehydes were initially converted to nitrile oxides that underwent 1,3-dipolar cycloaddition reactions with methyl acrylate to generate 4,5-dihydroisoxazoles. In in vitro tests, methyl 3-phenyl-4,5-dihydroisoxazole-5-carboxylate (1) and methyl 3-(4-chlorophenyl)-4,5-dihydroisoxazole-5-carboxylate (4) increased the mortality of Meloidogyne exigua and Meloidogyne incognita second-stage juveniles (J2). Compounds 1 and 4 presented necessary concentrations of 398 and 501 µg mL-1, respectively, to kill 50% of M. incognita J2 (LC50 values), while the value for carbofuran (positive control) was 168 µg mL-1. In in vivo tests, compounds 1 and 4 reduced the number of M. incognita galls in tomato roots by 70 and 40%, respectively, and the number of eggs by 89 and 44%. Using an in silico approach, we showed that compounds 1 and 4 were toxic to the nematodes by binding to the allosteric binding sites of the agonist-binding domains of the nematode nicotinic acetylcholine receptors. These results opened up possibilities for further investigations aimed at developing novel commercial nematicides.


Assuntos
Antinematódeos/toxicidade , Isoxazóis/toxicidade , Doenças das Plantas/parasitologia , Tylenchoidea/efeitos dos fármacos , Animais , Antinematódeos/química , Simulação por Computador , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Isoxazóis/química , Lycopersicon esculentum/parasitologia , Raízes de Plantas/parasitologia , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Tylenchoidea/crescimento & desenvolvimento , Tylenchoidea/metabolismo
6.
Proc Natl Acad Sci U S A ; 117(3): 1788-1798, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31911476

RESUMO

The lipid dependence of the nicotinic acetylcholine receptor from the Torpedo electric organ has long been recognized, and one of the most consistent experimental observations is that, when reconstituted in membranes formed by zwitterionic phospholipids alone, exposure to agonist fails to elicit ion-flux activity. More recently, it has been suggested that the bacterial homolog ELIC (Erwinia chrysanthemi ligand-gated ion channel) has a similar lipid sensitivity. As a first step toward the elucidation of the structural basis of this phenomenon, we solved the structures of ELIC embedded in palmitoyl-oleoyl-phosphatidylcholine- (POPC-) only nanodiscs in both the unliganded (4.1-Å resolution) and agonist-bound (3.3 Å) states using single-particle cryoelectron microscopy. Comparison of the two structural models revealed that the largest differences occur at the level of loop C-at the agonist-binding sites-and the loops at the interface between the extracellular and transmembrane domains (ECD and TMD, respectively). On the other hand, the transmembrane pore is occluded in a remarkably similar manner in both structures. A straightforward interpretation of these findings is that POPC-only membranes frustrate the ECD-TMD coupling in such a way that the "conformational wave" of liganded-receptor gating takes place in the ECD and the interfacial M2-M3 linker but fails to penetrate the membrane and propagate into the TMD. Furthermore, analysis of the structural models and molecular simulations suggested that the higher affinity for agonists characteristic of the open- and desensitized-channel conformations results, at least in part, from the tighter confinement of the ligand to its binding site; this limits the ligand's fluctuations, and thus delays its escape into bulk solvent.


Assuntos
Microscopia Crioeletrônica , Canais Iônicos de Abertura Ativada por Ligante/química , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Animais , Sítios de Ligação , Ligantes , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Domínios Proteicos , Receptores Nicotínicos/metabolismo , Torpedo
7.
Phytochemistry ; 170: 112187, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31865001

RESUMO

Nicotinic acetylcholine receptor (nAChR) subtype-selective pharmacological profiles of tobacco alkaloids are essential for understanding the physiological effects of tobacco products. In this study, automated electrophysiology was used to functionally characterize the effects of distinct groups of tobacco alkaloids on human α4ß2 and α7 nAChRs. We found that, in tobacco alkaloids, pyridine as a hydrogen bond acceptor and a basic nitrogen atom at a distance of 4-7 Šare pharmacophoric elements necessary for molecular recognition by α4ß2 and α7 nAChRs with various degrees of selectivity, potency, and efficacy. While four alkaloids-nicotine, nornicotine, anabasine and R-anatabine-potently activated α4ß2, they were also weak agonists of α7 nAChRs. Nicotine was the most potent agonist of α4ß2, while anabasine elicited the highest activation of α7. None of the tobacco alkaloids enhanced nAChR activity elicited by the endogenous ligand acetylcholine; therefore, none was considered to be a positive allosteric modulator (PAM) of either α4ß2 or α7 nAChRs. In contrast, we identified tobacco alkaloids, such as the tryptophan metabolite 6-hydroxykynurenic acid, that decreased the activity of both α4ß2 and α7 nAChRs. Our study identified a class of alkaloids with positive and negative effects against human α4ß2 and α7 nAChRs. It also revealed human α4ß2 to be the principal receptor for sensing the most abundant alkaloids in tobacco leaves.


Assuntos
Alcaloides/farmacologia , Produtos Biológicos/farmacologia , Compostos Fitoquímicos/farmacologia , Receptores Nicotínicos/metabolismo , Tabaco/química , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Alcaloides/química , Alcaloides/isolamento & purificação , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Relação Dose-Resposta a Droga , Humanos , Ligantes , Estrutura Molecular , Compostos Fitoquímicos/química , Compostos Fitoquímicos/isolamento & purificação , Relação Estrutura-Atividade , Receptor Nicotínico de Acetilcolina alfa7/metabolismo
8.
Food Chem Toxicol ; 135: 111057, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31846720

RESUMO

Prenatal nicotine exposure (PNE) may lead to offspring's testicular dysplasia. Here, we confirmed the intergenerational effect of PNE on testosterone synthetic function and explored its epigenetic programming mechanism. Pregnant Wistar rats were injected subcutaneously with nicotine (2 mg/kg.d) from gestational day 9-20. Some dams were anesthetized to obtain fetal rats, the rest were allowed to spontaneous labor to generate F1 and F2 generation. In utero, PNE impaired testicular development and testosterone production. Meanwhile, the expression of steroidogenic acute regulatory protein (StAR) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) were decreased both in F1 and F2 generations. Furthermore, PNE enhanced the expression of fetal testicular nicotinic acetylcholine receptors (nAChRs) and histone deacetylase 4 (HDAC4), while obviously weakened histone 3 lysine 9 acetylation (H3K9ac) level of StAR/3ß-HSD promoter from GD20 to postnatal week 12 and even in F2 generation. In vitro, nicotine increased nAChRs and HDAC4 expression, and decreased the StAR/3ß-HSD H3K9ac level and expression, as well as the testosterone production in Leydig cells. Antagonism of nAChRs and inhibition of HDAC4 reversed the aforementioned changes. In conclusion, PNE programmed testicular low steroidogenesis and its heritability in male offspring rats. The underlying mechanism was associated to the low-level programming of StAR/3ß-HSD H3K9ac via nAChR/HDAC4.


Assuntos
Epigênese Genética/efeitos dos fármacos , Histona Desacetilases/metabolismo , Comportamento Materno , Nicotina/administração & dosagem , Receptores Nicotínicos/metabolismo , Testículo/efeitos dos fármacos , Testosterona/biossíntese , Idoso , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Camundongos , Nicotina/farmacologia , Gravidez , Ratos , Ratos Wistar , Testículo/embriologia , Testículo/metabolismo
9.
Yakugaku Zasshi ; 139(11): 1397-1402, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31685736

RESUMO

Patients with epilepsy are often affected by not only seizures but also a variety of cognitive and psychiatric comorbidities that further impair their quality of life. However, it is unclear whether epilepsy is associated with psychic function. The aim of the present study was to clarify the effects of kindling-induced epileptic seizures on psychic functioning, using behavioral pharmacological tests. Pentylenetetrazol (PTZ)-kindled mice displayed impaired motor coordination (in the rotarod test), and social approach impairment (in the three-chamber social test) compared with vehicle mice. Intraperitoneal ABT-418 treatment (0.05 mg/kg) alleviated these behavioral abnormalities in PTZ-kindled mice. Immunolabeling of tissue sections demonstrated that expression of the α4 subunit of the α4ß2 nicotinic acetylcholine receptor in the piriform cortex was significantly decreased in PTZ-kindled mice. In contrast, expression of the synaptic adhesion molecule neuroligin 3 (NLG3) was significantly higher in the piriform cortex of PTZ-kindled mice compared with vehicle mice. Collectively, our findings suggest that attention deficit/hyperactivity disorder (ADHD)-like or autistic-like behavioral abnormalities associated with epilepsy are closely linked to downregulation of the α4 subunit of the α4ß2 receptor and upregulation of NLG3 in the mouse piriform cortex. In summary, this study indicates that ABT-418 is a good candidate for the treatment of patients with epilepsy complicated by psychiatric symptoms such as autism and ADHD.


Assuntos
Epilepsia/psicologia , Excitação Neurológica , Transtornos Mentais/tratamento farmacológico , Transtornos Mentais/etiologia , Pentilenotetrazol , Receptores Nicotínicos/metabolismo , Convulsões/psicologia , Animais , Moléculas de Adesão Celular Neuronais/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Epilepsia/induzido quimicamente , Epilepsia/tratamento farmacológico , Epilepsia/etiologia , Isoxazóis/farmacologia , Isoxazóis/uso terapêutico , Proteínas de Membrana/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Córtex Piriforme/metabolismo , Desempenho Psicomotor/efeitos dos fármacos , Pirrolidinas/farmacologia , Pirrolidinas/uso terapêutico , Qualidade de Vida , Convulsões/induzido quimicamente , Convulsões/tratamento farmacológico , Convulsões/etiologia , Comportamento Social
10.
Proc Natl Acad Sci U S A ; 116(42): 21228-21235, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31570625

RESUMO

Charge selectivity forms the basis of cellular excitation or inhibition by Cys-loop ligand-gated ion channels (LGICs), and is essential for physiological receptor function. There are no reports of naturally occurring mutations in LGICs associated with the conversion of charge selectivity. Here, we report on a CHRNA1 mutation (α1Leu251Arg) in a patient with congenital myasthenic syndrome associated with transformation of the muscle acetylcholine receptor (AChR) into an inhibitory channel. Performing patch-clamp experiments, the AChR was found to be converted into chloride conductance at positive potentials, whereas whole-cell currents at negative potentials, although markedly reduced, were still carried by sodium. Umbrella sampling molecular dynamics simulations revealed constriction of the channel pore radius to 2.4 Å as a result of the mutation, which required partial desolvation of the ions in order to permeate the pore. Ion desolvation was associated with an energetic penalty that was compensated for by the favorable electrostatic interaction of the positively charged arginines with chloride. These findings reveal a mechanism for the transformation of the muscle AChR into an inhibitory channel in a clinical context.


Assuntos
Acetilcolina/metabolismo , Cloretos/metabolismo , Músculos/metabolismo , Mutação/genética , Receptores Colinérgicos/metabolismo , Linhagem Celular , Células HEK293 , Humanos , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/fisiologia , Síndromes Miastênicas Congênitas/metabolismo , Técnicas de Patch-Clamp/métodos , Receptores Nicotínicos/metabolismo , Sódio/metabolismo
11.
Nature ; 574(7778): 372-377, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31619789

RESUMO

Diabetes is far more prevalent in smokers than non-smokers, but the underlying mechanisms of vulnerability are unknown. Here we show that the diabetes-associated gene Tcf7l2 is densely expressed in the medial habenula (mHb) region of the rodent brain, where it regulates the function of nicotinic acetylcholine receptors. Inhibition of TCF7L2 signalling in the mHb increases nicotine intake in mice and rats. Nicotine increases levels of blood glucose by TCF7L2-dependent stimulation of the mHb. Virus-tracing experiments identify a polysynaptic connection from the mHb to the pancreas, and wild-type rats with a history of nicotine consumption show increased circulating levels of glucagon and insulin, and diabetes-like dysregulation of blood glucose homeostasis. By contrast, mutant Tcf7l2 rats are resistant to these actions of nicotine. Our findings suggest that TCF7L2 regulates the stimulatory actions of nicotine on a habenula-pancreas axis that links the addictive properties of nicotine to its diabetes-promoting actions.


Assuntos
Transtornos do Metabolismo de Glucose/genética , Habenula/metabolismo , Transdução de Sinais , Tabagismo/complicações , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Animais , AMP Cíclico/metabolismo , Glucose/metabolismo , Transtornos do Metabolismo de Glucose/metabolismo , Humanos , Camundongos , Mutagênese , Nicotina/metabolismo , Células PC12 , Pâncreas/metabolismo , Ratos , Receptores Nicotínicos/metabolismo , Tabagismo/genética , Tabagismo/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética
12.
Eur J Pharmacol ; 865: 172674, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31634461

RESUMO

Lung cancer is an aggressive tumor with high incidence and mortality rate. There was growing evidence supporting that nicotinic acetylcholine receptors (nAChRs) play vital role inlung cancer development. In this study, the expression of α3, α4, α5, α6, α7, α9, α10, ß2, ß3, ß4 nAChR subunits on protein and mRNA level were studied in A549, NCI-H1299, NCI-H1688, DMS114 and normal human embryonic lung fibroblast (HEL) cell lines by real-time quantitative PCR (qPCR) and Western blot assay respectively. The results indicated that most of these nAChR subunits were expressed in these five cell lines. Compared with normal cells, the expression of α3 and ß4 nAChR subunits were upregulated in A549 and NCI-H1299. Thus, we treated A549 and NCI-H1299 with an antagonist α-conotoxin TxID which potently and selectively blocks α3ß4 nAChRs. TxID treatment could inhibit A549 and NCI-H1299 cell growth and enhance the inhibitory effect of adriamycin when treated simultaneously. To sum up, our study identified the expression of nAChR subunits in different lung cells and the anti-tumor effect of α-conotoxin TxID, which may provide novel strategies for lung cancer therapy.


Assuntos
Antineoplásicos/farmacologia , Conotoxinas/farmacologia , Neoplasias Pulmonares/patologia , Receptores Nicotínicos/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Interações Medicamentosas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , RNA Mensageiro/genética , Receptores Nicotínicos/genética
13.
PLoS One ; 14(10): e0223272, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31584962

RESUMO

The nicotinic acetylcholine receptor (nAChR) is an excitatory pentameric ligand-gated ion channel (pLGIC), homologous to the inhibitory γ-aminobutyric acid (GABA) type A receptor targeted by pharmaceuticals and endogenous sedatives. Activation of the GABAA receptor by the neurosteroid allopregnanolone can be inhibited competitively by thyroid hormone (L-3,3',5-triiodothyronine, or T3), but modulation of nAChR by T3 or neurosteroids has not been investigated. Here we show that allopregnanolone inhibits the nAChR from Torpedo californica at micromolar concentrations, as do T3 and the anionic neurosteroid pregnenolone sulfate (PS). We test for the role of protein and ligand charge in mediated receptor inhibition by varying pH in a narrow range around physiological pH. We find that both T3 and PS become less potent with increasing pH, with remarkably similar trends in IC50 when T3 is neutral at pH < 7.3. After deprotonation of T3 (but no additional deprotonation of PS) at pH 7.3, T3 loses potency more slowly with increasing pH than PS. We interpret this result as indicating the negative charge is not required for inhibition but does increase activity. Finally, we show that both T3 and PS affect nAChR channel desensitization, which may implicate a binding site homologous to one that was recently indicated for accelerated desensitization of the GABAA receptor by PS.


Assuntos
Antagonistas Nicotínicos/farmacologia , Pregnenolona/farmacologia , Receptores Nicotínicos/metabolismo , Torpedo/metabolismo , Tri-Iodotironina/farmacologia , Animais , Relação Dose-Resposta a Droga , Antagonistas de Receptores de GABA-A/química , Antagonistas de Receptores de GABA-A/farmacologia , Concentração Inibidora 50 , Cinética , Estrutura Molecular , Antagonistas Nicotínicos/química , Oócitos/metabolismo , Pregnenolona/química , Receptores de GABA-A/metabolismo , Tri-Iodotironina/química
14.
Dokl Biochem Biophys ; 487(1): 251-255, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31559591

RESUMO

Three-finger snake neurotoxins are selective antagonists of some nicotinic acetylcholine receptor subtypes and are widely used to study these receptors. The peptide neurotoxin azemiopsin, recently isolated from the venom of Azemipos feae, is a selective blocker of muscle-type nicotinic acetylcholine receptor. In order to reduce their toxicity and increase resistance under physiological conditions, we have encapsulated these toxins into nanomaterials. The study of nanomaterials after interaction with neurotoxins by the methods of transmission electron microscopy and dynamic light scattering revealed an increase in the size of nanoparticles, which indicates the inclusion of neurotoxins in nanomaterials.


Assuntos
Portadores de Fármacos/química , Nanopartículas/química , Neurotoxinas/química , Antagonistas Nicotínicos/química , Polissacarídeos/química , Receptores Nicotínicos/metabolismo , Sulfatos/química , Cápsulas , Neurotoxinas/toxicidade , Antagonistas Nicotínicos/toxicidade , Tamanho da Partícula , Venenos de Serpentes/química
15.
Mar Drugs ; 17(9)2019 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-31527432

RESUMO

The a-Conotoxins are peptide toxins that are found in the venom of marine cone snails and they are potent antagonists of various subtypes of nicotinic acetylcholine receptors (nAChRs). Because nAChRs have an important role in regulating transmitter release, cell excitability, and neuronal integration, nAChR dysfunctions have been implicated in a variety of severe pathologies. We describe the isolation and characterization of α-conotoxin MilIA, the first conopeptide from the venom of Conus milneedwardsi. The peptide was characterized by electrophysiological screening against several types of cloned nAChRs that were expressed in Xenopus laevis oocytes. MilIA, which is a member of the α3/5 family, is an antagonist of muscle type nAChRs with a high selectivity for muscle versus neuronal subtype nAChRs. Several analogues were designed and investigated for their activity in order to determine the key epitopes of MilIA. Native MilIA and analogues both showed activity at the fetal muscle type nAChR. Two single mutations (Met9 and Asn10) allowed for MilIA to strongly discriminate between the two types of muscle nAChRs. Moreover, one analogue, MilIA [∆1,M2R, M9G, N10K, H11K], displayed a remarkable enhanced potency when compared to native peptide. The key residues that are responsible for switching between muscle and neuronal nAChRs preference were elucidated. Interestingly, the same analogue showed a preference for α9α10 nAChRs among the neuronal types.


Assuntos
Conotoxinas/farmacologia , Caramujo Conus/química , Neurotoxinas/farmacologia , Antagonistas Nicotínicos/farmacologia , Peptídeos/farmacologia , Sequência de Aminoácidos/genética , Animais , Conotoxinas/genética , Conotoxinas/isolamento & purificação , Mutação , Neurotoxinas/genética , Neurotoxinas/isolamento & purificação , Antagonistas Nicotínicos/isolamento & purificação , Oócitos , Técnicas de Patch-Clamp , Peptídeos/genética , Peptídeos/isolamento & purificação , Receptores Nicotínicos/metabolismo , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Xenopus laevis
16.
Neuron ; 104(3): 501-511.e6, 2019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31488329

RESUMO

Nicotinic acetylcholine receptors are pentameric ion channels that mediate fast chemical neurotransmission. The α3ß4 nicotinic receptor subtype forms the principal relay between the central and peripheral nervous systems in the autonomic ganglia. This receptor is also expressed focally in brain areas that affect reward circuits and addiction. Here, we present structures of the α3ß4 nicotinic receptor in lipidic and detergent environments, using functional reconstitution to define lipids appropriate for structural analysis. The structures of the receptor in complex with nicotine, as well as the α3ß4-selective ligand AT-1001, complemented by molecular dynamics, suggest principles of agonist selectivity. The structures further reveal much of the architecture of the intracellular domain, where mutagenesis experiments and simulations define residues governing ion conductance.


Assuntos
Nicotina/metabolismo , Agonistas Nicotínicos/metabolismo , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/ultraestrutura , Sódio/metabolismo , Microscopia Crioeletrônica , Gânglios Autônomos , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Oligopeptídeos/metabolismo , Técnicas de Patch-Clamp , Estrutura Terciária de Proteína
17.
Mol Pharmacol ; 96(5): 664-673, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31492697

RESUMO

Many peptide ligands of nicotinic acetylcholine receptors (nAChRs) contain a large number of positively charged amino acid residues, a striking example being conotoxins RgIA and GeXIVA from marine mollusk venom, with an arginine content of >30%. To determine whether peptides built exclusively from arginine residues will interact with different nAChR subtypes or with their structural homologs such as the acetylcholine-binding protein and ligand-binding domain of the nAChR α9 subunit, we synthesized a series of R3, R6, R8, and R16 oligoarginines and investigated their activity by competition with radioiodinated α-bungarotoxin, two-electrode voltage-clamp electrophysiology, and calcium imaging. R6 and longer peptides inhibited muscle-type nAChRs, α7 nAChRs, and α3ß2 nAChRs in the micromolar range. The most efficient inhibition of ion currents was detected for muscle nAChR by R16 (IC50 = 157 nM) and for the α9α10 subtype by R8 and R16 (IC50 = 44 and 120 nM, respectively). Since the R8 affinity for other tested nAChRs was 100-fold lower, R8 appears to be a selective antagonist of α9α10 nAChR. For R8, the electrophysiological and competition experiments indicated the existence of two distinct binding sites on α9α10 nAChR. Since modified oligoarginines and other cationic molecules are widely used as cell-penetrating peptides, we studied several cationic polymers and demonstrated their nAChR inhibitory activity. SIGNIFICANT STATEMENT: By using radioligand analysis, electrophysiology, and calcium imaging, we found that oligoarginine peptides are a new group of inhibitors for muscle nicotinic acetylcholine receptors (nAChRs) and some neuronal nAChRs, the most active being those with 16 and 8 Arg residues. Such compounds and other cationic polymers are cell-penetrating tools for drug delivery, and we also demonstrated the inhibition of nAChRs for several of the latter. Possible positive and negative consequences of such an action should be taken into account.


Assuntos
Arginina/metabolismo , Arginina/farmacologia , Antagonistas Nicotínicos/metabolismo , Antagonistas Nicotínicos/farmacologia , Peptídeos/metabolismo , Peptídeos/farmacologia , Animais , Arginina/química , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Feminino , Humanos , Camundongos , Antagonistas Nicotínicos/química , Peptídeos/química , Receptores Nicotínicos/metabolismo , Xenopus laevis
18.
Invert Neurosci ; 19(4): 11, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31486912

RESUMO

Parasitic nematode infections are treated using anthelmintic drugs, some of which target nicotinic acetylcholine receptors (nAChRs) located in different parasite tissues. The limited arsenal of anthelmintic agents and the prevalence of drug resistance imply that future defense against parasitic infections will depend on the discovery of novel targets and therapeutics. Previous studies have suggested that Ascaris suum ACR-16 nAChRs are a suitable target for the development of antinematodal drugs. In this study, we characterized the pharmacology of the Ancylostoma caninum ACR-16 receptor using two-electrode voltage-clamp electrophysiology. This technique allowed us to study the effects of cholinergic agonists and antagonists on the nematode nAChRs expressed in Xenopus laevis oocytes. Aca-ACR-16 was not sensitive to many of the existing cholinomimetic anthelmintics (levamisole, oxantel, pyrantel, and tribendimidine). 3-Bromocytisine was the most potent agonist (> 130% of the control acetylcholine current) on the Aca-ACR-16 nAChR but, unlike Asu-ACR-16, oxantel did not activate the receptor. The mean time constants of desensitization for agonists on Aca-ACR-16 were longer than the rates observed in Asu-ACR-16. In contrast to Asu-ACR-16, the A. caninum receptor was completely inhibited by DHßE and moderately inhibited by α-BTX. In conclusion, we have successfully reconstituted a fully functional homomeric nAChR, ACR-16, from A. caninum, a model for human hookworm infections. The pharmacology of the receptor is distinct from levamisole-sensitive nematode receptors. The ACR-16 homologue also displayed some pharmacological differences from Asu-ACR-16. Hence, A. caninum ACR-16 may be a valid target site for the development of anthelmintics against hookworm infections.


Assuntos
Ancylostoma/metabolismo , Anti-Helmínticos/farmacologia , Proteínas de Helminto/efeitos dos fármacos , Receptores Nicotínicos/efeitos dos fármacos , Ancilostomíase , Animais , Colinérgicos/farmacologia , Proteínas de Helminto/análise , Proteínas de Helminto/metabolismo , Receptores Nicotínicos/análise , Receptores Nicotínicos/metabolismo
19.
Cells ; 8(8)2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31434353

RESUMO

The protein kinase Csnk2/CK2 is important for cell proliferation, differentiation, and survival. Previously, we showed that CK2 binds distinctive proteins at neuromuscular junctions (NMJs) of mice and phosphorylates some of them. CK2 likely stabilizes clustered nicotinic acetylcholine receptors (AChRs). In the absence of the ß-subunit of CK2 in skeletal muscle fibers, mice develop an age-dependent decrease of grip strength accompanied by NMJ fragmentation and impairments of neuromuscular transmission. However, the precise role of CK2ß regarding the clustering of AChRs and downstream signaling at NMJs is unknown. Here, we compared conditional CK2ß-deficient mice with controls and found in the mutants (1) a lower decrement of endplate potentials after repetitive stimulation and decrements of nerve-evoked compound muscle action potentials decayed more rapidly after synaptic transmission was partially blocked, (2) that their muscle weakness was partially rescued by administration of an acetylcholine esterase inhibitor, (3) fragmented NMJs and impaired AChR clustering was detected in muscles and cultured muscle cells, (4) enlarged myonuclei, (5) impaired synaptic gene expression, and (6) a high turnover rate of their AChR clusters in vivo. Altogether, our data demonstrate a role for CK2 at the NMJ by maintaining a high density of AChRs and ensuring physiological synaptic gene expression.


Assuntos
Caseína Quinase II/metabolismo , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/fisiologia , Junção Neuromuscular/fisiologia , Receptores Nicotínicos/metabolismo , Animais , Expressão Gênica , Camundongos , Transmissão Sináptica
20.
Pharmacol Rep ; 71(5): 782-793, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31377559

RESUMO

BACKGROUND: Nicotine alleviates renal inflammation and injury induced by endotoxemia. This study investigated (i) the nicotine modulation of hemodynamic and renal vasodilatory responses to endotoxemia in rats, and (ii) roles of α7 or α4ß2-nAChRs and related HSP70/TNFα/iNOS signaling in the interaction. METHODS: Endotoxemia was induced by ip lipopolysaccharide (5 mg/kg/day, for 2 days) and changes in systolic blood pressure and vasodilator responsiveness of isolated perfused kidney to acetylcholine or 5'-N-ethylcarboxamidoadenosine (NECA, adenosine receptor agonist) were evaluated. RESULTS: Lipopolysaccharide had no effect on serum creatinine, reduced blood pressure, and increased renal vasodilations induced by acetylcholine or NECA in male and female preparations. Immunohistochemical analyses showed that lipopolysaccharide reduced renal HSP70 expression, but increased α7-nAChRs, α4ß2-nAChRs and iNOS expressions. The co-administration of aminoguanidine (iNOS inhibitor), pentoxifylline (TNFα inhibitor), or nicotine attenuated lipopolysaccharide mediation of renal vasodilations and elevations in α7/α4ß2-nAChR and iNOS expressions. Nicotine also reversed the downregulating effect of lipopolysaccharide on HSP70 expression. α7-nAChRs (methyllycaconitine citrate, MLA) or α4ß2-nAChRs (dihydro-ß-erythroidine, DHßE) blockade potentiated the lipopolysaccharide enhancement of renal vasodilations, and abolished the depressant effect of nicotine on lipopolysaccharide responses. A similar abolition of nicotine effects was seen after HSP70 inhibition by quercetin. Alternatively, lipopolysaccharide hypotension was eliminated in rats treated with DHßE/nicotine or quercetin/nicotine regimen in contrast to no effect for nicotine alone or combined with MLA. CONCLUSIONS: These findings establish that nicotine offsets lipopolysaccharide facilitation of renal vasodilations possibly through a crosstalk between HSP70 and nAChRs of the α7 and α4ß2 types.


Assuntos
Endotoxemia/fisiopatologia , Endotoxinas , Proteínas de Choque Térmico HSP70/metabolismo , Rim/irrigação sanguínea , Nicotina/farmacologia , Receptores Nicotínicos/metabolismo , Vasodilatação/efeitos dos fármacos , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Animais , Creatinina/sangue , Endotoxemia/induzido quimicamente , Endotoxemia/metabolismo , Feminino , Rim/efeitos dos fármacos , Masculino , Óxido Nítrico Sintase Tipo II/metabolismo , Ratos Wistar , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo
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