RESUMO
Despite the identification of Aß plaques and NFTs as biomarkers for Alzheimer's disease (AD) pathology, therapeutic interventions remain elusive, with neither an absolute prophylactic nor a curative medication available to impede the progression of AD presently available. Current approaches focus on symptomatic treatments to maintain AD patients' mental stability and behavioral symptoms by decreasing neuronal degeneration; however, the complexity of AD pathology requires a wide range of therapeutic approaches for both preventive and curative treatments. In this regard, this review summarizes the role of receptors as a potential target for treating AD and focuses on the path of major receptors which are responsible for AD progression. This review gives an overall idea centering on major receptors, their agonist and antagonist and future prospects of viral mimicry in AD pathology. This article aims to provide researchers and developers a comprehensive idea about the different receptors involved in AD pathogenesis that may lead to finding a new therapeutic strategy to treat AD.
Assuntos
Doença de Alzheimer/terapia , Antagonistas GABAérgicos/farmacologia , Placa Amiloide/patologia , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores Nicotínicos/biossíntese , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Humanos , Emaranhados Neurofibrilares/patologia , Ácido gama-Aminobutírico/metabolismoRESUMO
The construction and maturation of the postsynaptic apparatus are crucial for synapse and dendrite development. The fundamental mechanisms underlying these processes are most often studied in glutamatergic central synapses in vertebrates. Whether the same principles apply to excitatory cholinergic synapses, such as those found in the insect central nervous system, is not known. To address this question, we investigated a group of projection neurons in the Drosophila larval visual system, the ventral lateral neurons (LNvs), and identified nAchRα1 (Dα1) and nAchRα6 (Dα6) as the main functional nicotinic acetylcholine receptor (nAchR) subunits in the larval LNvs. Using morphological analyses and calcium imaging studies, we demonstrated critical roles of these two subunits in supporting dendrite morphogenesis and synaptic transmission. Furthermore, our RNA sequencing analyses and endogenous tagging approach identified distinct transcriptional controls over the two subunits in the LNvs, which led to the up-regulation of Dα1 and down-regulation of Dα6 during larval development as well as to an activity-dependent suppression of Dα1 Additional functional analyses of synapse formation and dendrite dynamics further revealed a close association between the temporal regulation of individual nAchR subunits and their sequential requirements during the cholinergic synapse maturation. Together, our findings support transcriptional control of nAchR subunits as a core element of developmental and activity-dependent regulation of central cholinergic synapses.
Assuntos
Neurônios Colinérgicos/metabolismo , Dendritos/metabolismo , Proteínas de Drosophila/biossíntese , Morfogênese , Receptores Nicotínicos/biossíntese , Sinapses/metabolismo , Transmissão Sináptica , Animais , Drosophila melanogaster , Larva/metabolismoRESUMO
OBJECTIVE: To understand the characteristics of DNA methyltransferase 3a (DNMT3a) in thymoma associated Myasthenia Gravis reveal its transcriptional regulator network as while as analyze the effect of DNMT3a on Rel/ nuclear factor-kappaB family (RelA/RelB) and its downstream autoimmune regulatory factor (Aire). METHODS: Tissues of 30 patients with thymoma, with or without myasthenia gravis (MG), were collected and the DNMT3a protein expression were evaluated through immunohistochemistry. We performed mRNA expression profiling microarray detection and analysis, and integrated the analysis by constructing protein-protein interaction networks and the integration with other database. We identified molecular difference between low and high DNMT3a in the thymoma by heatmap. We also performed PCR validation in thymoma tissues. The DNMT3a-shRNA plasmid was transfected into TEC cells, and these cells were treated with 5-aza-2-deoxycytidine, a blocker of DNMT3a. After the down-regulation of DNMT3a in TEC cells, the transcript and protein levels of RelA, RelB, Aire, and CHRNA3 were evaluated by western blotting. In addition, changes in gene expression profiles were screened through microarray technology. We performed differential gene analysis in the thymoma cohort by heatmap with R (v.4.3.0) software. RESULTS: In 30 matched tissue specimens, the expression of DNMT3a protein in thymoma with MG was lower than that in thymoma. Through mRNA expression profiling analysis, we constructed a co-expression network of DNMT3a and found direct interaction between IKZF1 and DNMT3a, and this co-expression relationship was overlappted with Cistrome DB database. We found up-regulation of 149 mRNAs and repression of 177 mRNAs in thymoma with MG compared with thymoma. Gene ontology and pathway analysis show the involvement of a multitude of genes in the mis-regulation of MG-related pathways. RNA interference significantly reduced the level of mRNA of DNMT3a, which proved that plasmid DNMT3a was effective. In comparison to the control group, the levels of DNMT3a, Aire, and CHRNA3 mRNA and protein in TEC cells transfected with DNMT3a-shRNA interference plasmid were significantly decreased, while the expression level of RelA and RelA/RelB was significantly increased. CONCLUSIONS: Our study reveals the DNMT3a-NF-κB pathway has a major effect on MG, and can be used as a marker for diagnosis as well as a target for MG treatment.
Assuntos
DNA Metiltransferase 3A/biossíntese , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Miastenia Gravis/metabolismo , NF-kappa B/biossíntese , Proteínas de Neoplasias/biossíntese , Interferência de RNA , Timoma/metabolismo , Timo/metabolismo , Neoplasias do Timo/metabolismo , Adolescente , Adulto , DNA Metiltransferase 3A/antagonistas & inibidores , DNA Metiltransferase 3A/genética , Decitabina/farmacologia , Ontologia Genética , Humanos , Masculino , Pessoa de Meia-Idade , Miastenia Gravis/etiologia , Miastenia Gravis/genética , NF-kappa B/genética , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Mapas de Interação de Proteínas , RNA Neoplásico/genética , RNA Interferente Pequeno/genética , Receptores Nicotínicos/biossíntese , Receptores Nicotínicos/genética , Timoma/complicações , Timoma/genética , Neoplasias do Timo/complicações , Neoplasias do Timo/genética , Análise Serial de Tecidos , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Transcriptoma , Proteína AIRERESUMO
To better comprehend the relationship between left/right (L/R) differences and hippocampus functions is necessary knowledge of lateral asymmetry and regional distribution. This research was design to examine hippocampal L/R asymmetry and regional distribution profile of the alpha7 and alpha4 subtypes of nicotinic acetylcholine receptors (nAChRs) in the adult rat. 10-12-week-old twenty-four male wistar rats were randomly selected. After removing the brains, immunohistochemistry, real-time PCR, and western blot methods were applied to distinguish the presence of the receptors in the hippocampus. Outcomes stated that the mentioned receptors expression profile was spatial-dependent. As, the hippocampal dispersal of alpha7 and alpha4 subtypes in the left hippocampus (LH) was remarkably maximum compare with the right hippocampus (RH) (pâ¯=â¯0.001, pâ¯=â¯0.005 respectively). Furthermore, the alpha7 optical density (OD) was not significantly different in the diverse regions in hippocampus of adult rat (pâ¯=â¯0.057), while the maximum OD of the alpha4 was detected in the hippocampal dentate gyrus and CA3 regions of LH (pâ¯=â¯0.007, pâ¯=â¯0.009 respectively) and the minimum OD was in the CA1 of the RH (pâ¯=â¯0.019). In real time PCR evaluation, there is a significantly higher expression of alpha7 and alpha4 in LH compared to RH (pâ¯=â¯0.043, pâ¯=â¯0.049 respectively), also, for western blot (pâ¯=â¯0.042, pâ¯=â¯0.030 respectively). According to present data, the alpha7 and alpha4 nAChR subtypes expression profile demonstrated lateral asymmetry, the uniform regional dispersal for alpha7 and different regional dispersal for alpha4 in the adult rat hippocampus.
Assuntos
Lateralidade Funcional/fisiologia , Hipocampo/metabolismo , Receptores Nicotínicos/biossíntese , Receptor Nicotínico de Acetilcolina alfa7/biossíntese , Animais , Hipocampo/química , Hipocampo/citologia , Masculino , Ratos , Ratos Wistar , Receptores Nicotínicos/análise , Receptor Nicotínico de Acetilcolina alfa7/análiseRESUMO
Evidence in humans suggests a correlation between nicotine smoking and severe respiratory symptoms with COVID-19 infection. In lung tissue, angiotensin-converting enzyme 2 (ACE2) appears to mechanistically underlie viral entry. Here, we investigated whether e-cigarette vapor inhalation alters ACE2 and nicotinic acetylcholine receptor (nAChR) expression in male and female mice. In male lung, nicotine vapor inhalation induced a significant increase in ACE2 mRNA and protein, but surprisingly, these differences were not found in females. Further, both vehicle and nicotine vapor inhalation downregulated α5 nAChR subunits in both sexes, while differences were not found in α7 nAChR subunit expression. Finally, blood ACE2 levels did not differ with exposure, indicating that blood sampling is not a sufficient indicator of lung ACE2 changes. Together, these data indicate a direct link between e-cigarette vaping and increased ACE2 expression in male lung tissue, which thereby reveals an underlying mechanism of increased vulnerability to coronavirus infection in individuals vaping nicotine.
Assuntos
Enzima de Conversão de Angiotensina 2/biossíntese , COVID-19/epidemiologia , Sistemas Eletrônicos de Liberação de Nicotina , Pulmão/enzimologia , Vaping/efeitos adversos , Enzima de Conversão de Angiotensina 2/sangue , Enzima de Conversão de Angiotensina 2/genética , Animais , DNA Complementar/biossíntese , Feminino , Pulmão/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nicotina/administração & dosagem , Nicotina/farmacologia , Agonistas Nicotínicos/administração & dosagem , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/biossíntese , Caracteres Sexuais , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
Neurons of the medial olivary complex inhibit cochlear hair cells through the activation of α9α10-containing nicotinic acetylcholine receptors (nAChRs). Efforts to study the localization of these proteins have been hampered by the absence of reliable antibodies. To overcome this obstacle, CRISPR-Cas9 gene editing was used to generate mice in which a hemagglutinin tag (HA) was attached to the C-terminus of either α9 or α10 proteins. Immunodetection of the HA tag on either subunit in the organ of Corti of adult mice revealed immunopuncta clustered at the synaptic pole of outer hair cells. These puncta were juxtaposed to immunolabeled presynaptic efferent terminals. HA immunopuncta also occurred in inner hair cells of pre-hearing (P7) but not in adult mice. These immunolabeling patterns were similar for both homozygous and heterozygous mice. All HA-tagged genotypes had auditory brainstem responses not significantly different from those of wild type littermates. The activation of efferent neurons in heterozygous mice evoked biphasic postsynaptic currents not significantly different from those of wild type hair cells. However, efferent synaptic responses were significantly smaller and less frequent in the homozygous mice. We show that HA-tagged nAChRs introduced in the mouse by a CRISPR knock-in are regulated and expressed like the native protein, and in the heterozygous condition mediate normal synaptic function. The animals thus generated have clear advantages for localization studies.
Assuntos
Células Ciliadas Auditivas Externas/metabolismo , Receptores Nicotínicos/biossíntese , Animais , Sistemas CRISPR-Cas , Feminino , Edição de Genes , Células Ciliadas Auditivas Externas/citologia , Masculino , Camundongos , Camundongos Knockout , Receptores Nicotínicos/genéticaRESUMO
Adrenal chromaffin cells release neurotransmitters in response to stress and may be involved in conditions such as post-traumatic stress and anxiety disorders. Neurotransmitter release is triggered, in part, by activation of nicotinic acetylcholine receptors (nAChRs). However, despite decades of use as a model system for studying exocytosis, the nAChR subtypes involved have not been pharmacologically identified. Quantitative real-time PCR of rat adrenal medulla revealed an abundance of mRNAs for α3, α7, ß2, and ß4 subunits. Whole-cell patch-clamp electrophysiology of chromaffin cells and subtype-selective ligands were used to probe for nAChRs derived from the mRNAs found in adrenal medulla. A novel conopeptide antagonist, PeIA-5469, was created that is highly selective for α3ß2 over other nAChR subtypes heterologously expressed in Xenopus laevis oocytes. Experiments using PeIA-5469 and the α3ß4-selective α-conotoxin TxID revealed that rat adrenal medulla contain two populations of chromaffin cells that express either α3ß4 nAChRs alone or α3ß4 together with the α3ß2ß4 subtype. Conclusions were derived from observations that acetylcholine-gated currents in some cells were sensitive to inhibition by PeIA-5469 and TxID, while in other cells, currents were sensitive only to TxID. Expression of functional α7 nAChRs was determined using three α7-selective ligands: the agonist PNU282987, the positive allosteric modulator PNU120596, and the antagonist α-conotoxin [V11L,V16D]ArIB. The results of these studies identify for the first time the expression of α3ß2ß4 nAChRs as well as functional α7 nAChRs by rat adrenal chromaffin cells.
Assuntos
Medula Suprarrenal/metabolismo , Células Cromafins/metabolismo , Antagonistas Nicotínicos/farmacologia , Receptores Nicotínicos/biossíntese , Animais , Células Cultivadas , Conotoxinas/farmacologia , Masculino , Ratos , Ratos Sprague-Dawley , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa7/antagonistas & inibidores , Receptor Nicotínico de Acetilcolina alfa7/biossínteseRESUMO
Cannabis is the most frequently used illicit psychoactive substance worldwide; around one in ten users become dependent. The risk for cannabis use disorder (CUD) has a strong genetic component, with twin heritability estimates ranging from 51 to 70%. Here we performed a genome-wide association study of CUD in 2,387 cases and 48,985 controls, followed by replication in 5,501 cases and 301,041 controls. We report a genome-wide significant risk locus for CUD (P = 9.31 × 10-12) that replicates in an independent population (Preplication = 3.27 × 10-3, Pmeta-analysis = 9.09 × 10-12). The index variant (rs56372821) is a strong expression quantitative trait locus for cholinergic receptor nicotinic α2 subunit (CHRNA2); analyses of the genetically regulated gene expression identified a significant association of CHRNA2 expression with CUD in brain tissue. At the polygenic level, analyses revealed a significant decrease in the risk of CUD with increased load of variants associated with cognitive performance. The results provide biological insights and inform on the genetic architecture of CUD.
Assuntos
Abuso de Maconha/genética , Proteínas do Tecido Nervoso/fisiologia , Receptores Nicotínicos/fisiologia , Idade de Início , Alelos , Transtorno do Deficit de Atenção com Hiperatividade/genética , Encéfalo/metabolismo , Estudos de Casos e Controles , Cromossomos Humanos Par 8/genética , Cognição/fisiologia , Estudos de Coortes , Fatores de Confusão Epidemiológicos , Dinamarca , Escolaridade , Feminino , Perfilação da Expressão Gênica , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Islândia , Masculino , Herança Multifatorial , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Receptores Nicotínicos/biossíntese , Receptores Nicotínicos/genética , Esquizofrenia/genética , Fumar/genética , TranscriptomaRESUMO
Nicotine improves endotoxic manifestations of hypotension and cardiac autonomic dysfunction in rats. Here, we test the hypothesis that brainstem antiinflammatory pathways of α7/α4ß2 nicotinic acetylcholine receptors (nAChRs) modulate endotoxic cardiovascular derangements. Pharmacologic and molecular studies were performed to determine the influence of nicotine or selective α7/α4ß2-nAChR ligands on cardiovascular derangements and brainstem neuroinflammation caused by endotoxemia in conscious rats. The i.v. administration of nicotine (50⯵g/kg) abolished the lipopolysaccharide (LPS, 10â¯mg/kg i.v.)-evoked: (i) falls in blood pressure and spectral measure of cardiac sympathovagal balance (ratio of the low-frequency to high-frequency power, LF/HF), (ii) elevations in immunohistochemical protein expressions of NFκB and α4ß2-nAChR in medullary neurons of the nucleus tractus solitarius (NTS) and rostral ventrolateral medulla (RVLM), and (iii) decreases in medullary α7-nAChR protein expression. These favorable nicotine influences were replicated in rats treated intracisternally (i.c.) with PHA-543613 (selective α7-nAChR agonist) or 5-iodo-A-85380 (5IA, selective α4ß2-nAChRs agonist). Measurement of arterial baroreflex activity by the vasoactive method revealed that nicotine, PHA, or 5IA reversed the LPS depression of reflex bradycardic, but not tachycardic, activity. Moreover, the counteraction by nicotine of LPS hypotension was mostly inhibited after treatment with i.c. methyllycaconitine (MLA, α7-nAChR antagonist) in contrast to a smaller effect for dihydro-ß-erythroidine (DHßE, α4ß2-nAChR antagonist), whereas the associated increases in LF/HF ratio remained unaltered. The data signifies the importance of brainstem α7, and to a lesser extent α4ß2, receptors in the nicotine counteraction of detrimental cardiovascular and neuroinflammatory consequences of endotoxemia.
Assuntos
Fibras Colinérgicas/fisiologia , Endotoxemia/prevenção & controle , Hipotensão/prevenção & controle , NF-kappa B/biossíntese , Inflamação Neurogênica/prevenção & controle , Receptores Nicotínicos/biossíntese , Receptor Nicotínico de Acetilcolina alfa7/biossíntese , Aconitina/análogos & derivados , Aconitina/farmacologia , Animais , Azetidinas/farmacologia , Bradicardia/complicações , Bradicardia/prevenção & controle , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Di-Hidro-beta-Eritroidina/farmacologia , Endotoxemia/complicações , Hipotensão/induzido quimicamente , Hipotensão/complicações , Infusões Intraventriculares , Lipopolissacarídeos , Masculino , Bulbo/metabolismo , Vias Neurais/fisiologia , Nicotina/farmacologia , Piridinas/farmacologia , Quinuclidinas/administração & dosagem , Quinuclidinas/farmacologia , Ratos , Transdução de Sinais , Núcleo Solitário/metabolismo , Taquicardia/induzido quimicamente , Taquicardia/prevenção & controleRESUMO
Organophosphate pesticides are developmental neurotoxicants. We gave diazinon via osmotic minipumps implanted into dams prior to conception, with exposure continued into the second postnatal week, at doses (0.5 or 1â¯mg/kg/day) that did not produce detectable brain cholinesterase inhibition. We evaluated the impact on acetylcholine (ACh) and serotonin (5-hydroxytryptamine, 5HT) systems in brain regions from adolescence through full adulthood. Diazinon produced deficits in presynaptic ACh activity with regional and sex selectivity: cerebrocortical regions and the hippocampus were affected to a greater extent than were the striatum, midbrain or brainstem, and females were more sensitive than males. Diazinon also reduced nicotinic ACh receptors and 5HT1A receptors, with the same regional and sex preferences. These patterns were similar to those of diazinon given in a much more restricted period (postnatal day 1-4) but were of greater magnitude and consistency; this suggests that the brain is vulnerable to diazinon over a wide developmental window. Diazinon's effects differed from those of the related organophosphate, chlorpyrifos, with regard to regional and sex selectivity, and more importantly, to the effects on receptors: chlorpyrifos upregulates nicotinic ACh receptors and 5HT receptors, effects that compensate for the presynaptic ACh deficits. Diazinon can thus be expected to have worse neurodevelopmental outcomes than chlorpyrifos. Further, the disparities between diazinon and chlorpyrifos indicate the problems of predicting the developmental neurotoxicity of organophosphates based on a single compound, and emphasize the inadequacy of cholinesterase inhibition as an index of safety.
Assuntos
Acetilcolina/metabolismo , Inibidores da Colinesterase/toxicidade , Diazinon/toxicidade , Inseticidas/toxicidade , Serotonina/metabolismo , Animais , Animais Recém-Nascidos , Química Encefálica/efeitos dos fármacos , Clorpirifos/toxicidade , Feminino , Masculino , Gravidez , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Nicotínicos/biossíntese , Receptores Nicotínicos/efeitos dos fármacos , Receptores Nicotínicos/genética , Receptores de Serotonina/biossíntese , Receptores de Serotonina/efeitos dos fármacos , Receptores de Serotonina/genética , Caracteres Sexuais , Regulação para Cima/efeitos dos fármacosRESUMO
Sensitization of motor activity is a behavioural test to evaluate the effects of psychostimulants. Conditioned place preference (CPP) is an associative learning procedure to examine the rewarding properties of drugs. We aimed to assess whether motor sensitization to drugs of abuse can make zebrafish more vulnerable to establishing drug-induced CPP. We first evaluated sensitization of locomotor activity of zebrafish to repeated administrations of nicotine and cocaine during 5â¯days and after 5â¯days of withdrawal. After withdrawal, when zebrafish were re-exposed to the same dose of nicotine or cocaine locomotor activity was increased by 103% and 166%, respectively. Different groups of zebrafish were sensitized to nicotine or cocaine and trained on a nicotine-CPP task the day after withdrawal. The nicotine dose selected for sensitization was not effective for developing CPP in naïve zebrafish whereas it elicited CPP in zebrafish that were previously sensitized to nicotine or cocaine. Levels of nicotinic acetylcholine receptor ß2, α6 and α7 subunit, Pitx3, and tyrosine hydroxylase 1 (TH1) mRNAs were increased in the brain of nicotine- and cocaine-sensitized zebrafish. Nicotine-CPP performed with drug-sensitized zebrafish provoked further enhancements in the expression of α6 and α7 subunit, Pitx3, and TH1 mRNAs suggesting that the expression of these molecules in the reward pathway is involved in both processes. Our findings indicate that repeated exposures to low doses of drugs of abuse can increase subject's sensitivity to the rewarding properties of the same or different drugs. This further suggests that casual drug intake increases the probability of becoming addict.
Assuntos
Sensibilização do Sistema Nervoso Central/efeitos dos fármacos , Cocaína/farmacologia , Condicionamento Clássico/efeitos dos fármacos , Nicotina/farmacologia , Receptores Nicotínicos/biossíntese , Peixe-Zebra , Animais , Encéfalo/metabolismo , Proteínas de Homeodomínio/biossíntese , Locomoção/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/biossíntese , Proteínas de Peixe-Zebra/biossínteseRESUMO
SIDS occurs in early infancy and predominantly during a sleep period. Abnormalities in nicotine receptor binding and in the expression of the nicotinic acetylcholine receptor (nAChR) subunits α7 and ß2 have been reported in the brainstem of SIDS infants. This study focuses on the α3 and α4 nAChR subunits as α3 is important for early postnatal survival while α4 is crucial for nicotine-elicited antinociception and sleep-wake cycle regulation. Tissue from the rostral medulla of infants who died with a known cause of death (eSUDI, nâ¯=â¯7), and from SIDS classified as SIDS I (nâ¯=â¯8) and SIDS II (nâ¯=â¯27), was immunohistochemically stained for the α3 and α4 nAChR subunits and quantified in 9 nuclei comparing amongst these groups. The association with risk factors of sex, cigarette smoke exposure, upper respiratory tract infection (URTI), prone sleeping and bedsharing was also evaluated. Results showed that only α4 changes (increase) were evident in SIDS, occurring in the hypoglossal and cuneate nuclei of SIDS II infants and the nucleus of the spinal trigeminal tract of SIDS I infants. Amongst the SIDS infants, cigarette smoke exposure was only associated with decreased α4 in cribriform fibre tracts, while sex and bedsharing were associated with increases in α3 in the dorsal motor nucleus of the vagus and solitary nucleus, respectively. Combined, these findings suggest that abnormalities in endogenous acetylcholine synthesis and regulation may underlie the altered α3 and α4 nAChR subunit expressions in the SIDS brainstem medulla since the changes were not related to cigarette smoke exposure.
Assuntos
Bulbo/metabolismo , Receptores Nicotínicos/biossíntese , Morte Súbita do Lactente/etiologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Receptores Nicotínicos/análise , Fumaça/efeitos adversos , Nicotiana/efeitos adversosRESUMO
The present study was designed to explore the role of transient receptor potential canonical 3 (TRPC3) in nicotine-induced chronic obstructive pulmonary disease (COPD) and its underlying mechanism. In this study, the expression and localization of α5 nicotinic acetylcholine receptor (α5-nAchR) in lung tissues were determined by western blotting and immunohistochemistry. The quantitative real-time PCR (qRT-PCR) analysis was performed to examine the mRNA expression levels of α5-nAchR and TRPC3 in human airway smooth muscle cells (HASMCs). Cell viability was assessed by CCK-8 assay. Proliferation was detected by cell counting and EdU immunofluorescent staining. Fluorescence calcium imaging was carried out to measure cytosolic Ca2+ ([Ca2+]cyt) concentration. The results showed that the α5-nAchR and TRPC3 expressions were significantly up-regulated in lung tissues of COPD smokers. Nicotine promoted HASMC proliferation, which was accompanied by elevated α5-nAchR and TRPC3 expressions, basal [Ca2+]cyt, store-operated calcium entry (SOCE) and the rate of Mn2+ quenching in HASMCs. Further investigation indicated that nicotine-induced Ca2+ response and TRPC3 up-regulation was reversibly blocked by small interfering RNA (siRNA) suppression of α5-nAChR. The knockdown of TRPC3 blunted Ca2+ response and HASMC proliferation induced by nicotine. In conclusion, nicotine-induced HASMC proliferation was mediated by TRPC3-dependent calcium entry via α5-nAchR, which provided a potential target for treatment of COPD.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Nicotina/farmacologia , Receptores Nicotínicos/biossíntese , Sistema Respiratório/metabolismo , Canais de Cátion TRPC/biossíntese , Idoso , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Miócitos de Músculo Liso/patologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Doença Pulmonar Obstrutiva Crônica/terapia , Sistema Respiratório/patologiaRESUMO
Inhibitory interneurons participate in mnemonic processes. However, defined roles for identified interneuron populations are scarce. A subpopulation of oriens lacunosum-moleculare (OLM) interneurons genetically defined by the expression of the nicotinic receptor α2 subunit has been shown to gate information carried by either the temporoammonic pathway or Schaffer collaterals in vitro. Here we set out to determine whether selective modulation of OLMα2 cells in the intermediate CA1 affects learning and memory in vivo. Our data show that intermediate OLMα2 cells can either enhance (upon their inhibition) or impair (upon their activation) object memory encoding in freely moving mice, thus exerting bidirectional control. Moreover, we find that OLMα2 cell activation inhibits fear-related memories and that OLMα2 cells respond differently to nicotine in the dorsoventral axis. These results suggest that intermediate OLMα2 cells are an important component in the CA1 microcircuit regulating learning and memory processes. VIDEO ABSTRACT.
Assuntos
Aprendizagem da Esquiva/fisiologia , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/fisiologia , Receptores Nicotínicos/biossíntese , Animais , Região CA1 Hipocampal/química , Feminino , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores Nicotínicos/genéticaRESUMO
Communications across chemical synapses are primarily mediated by neurotransmitters and their postsynaptic receptors. There are diverse molecular systems to localize and regulate the receptors at the synapse. Here, we identify HPO-30, a member of the claudin superfamily of membrane proteins, as a positive regulator for synaptic localization of levamisole-dependent AChRs (LAChRs) at the Caenorhabditis elegans neuromuscular junction (NMJ). The HPO-30 protein localizes at the NMJ and shows genetic and physical association with the LAChR subunits LEV-8, UNC-29, and UNC-38. Using genetic and electrophysiological assays in the hermaphrodite C. elegans, we demonstrate that HPO-30 functions through Neuroligin at the NMJ to maintain postsynaptic LAChR levels at the synapse. Together, this work suggests a novel function for a tight junction protein in maintaining normal receptor levels at the NMJ.SIGNIFICANCE STATEMENT Claudins are a large superfamily of membrane proteins. Their role in maintaining the functional integrity of tight junctions has been widely explored. Our experiments suggest a critical role for the claudin-like protein, HPO-30, in maintaining synaptic levamisole-dependent AChR (LAChR) levels. LAChRs contribute to <20% of the acetylcholine-mediated conductance in adult Caenorhabditis elegans; however, they play a significant functional role in worm locomotion. This study provides a new perspective in the study of LAChR physiology.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/metabolismo , Proteínas de Transporte/biossíntese , Proteínas de Membrana/fisiologia , Junção Neuromuscular/metabolismo , Receptores Nicotínicos/biossíntese , Junções Íntimas/fisiologia , Aldicarb/toxicidade , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/biossíntese , Proteínas de Caenorhabditis elegans/genética , Proteínas de Transporte/genética , Moléculas de Adesão Celular Neuronais/deficiência , Moléculas de Adesão Celular Neuronais/fisiologia , Resistência a Medicamentos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Levamisol/farmacologia , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Atividade Motora/efeitos dos fármacos , Muscimol/farmacologia , Músculos/efeitos dos fármacos , Músculos/metabolismo , Domínios PDZ , Mapeamento de Interação de Proteínas , Receptores Nicotínicos/genéticaRESUMO
BACKGROUND/AIMS: Butyric acid plays an important role in maintaining intestinal health. Butyric acid has received special attention as a short-chain fatty acid, but its role in protecting the intestinal barrier is poorly characterized. Butyric acid not only provides energy for epithelial cells but also acts as a histone deacetylase inhibitor; it is also a natural ligand for G protein-coupled receptor 109A (GPR109A). A GPR109A analog was expressed in Sus scrofa and mediated the anti-inflammatory effects of beta-hydroxybutyric acid. This study investigated the effects of butyrate on growth performance, diarrhea symptoms, and tight junction protein levels in 21-day-old weaned piglets. We also studied the mechanism by which butyric acid regulates intestinal permeability. METHODS: Twenty-four piglets that had been weaned at an age of 21 days were divided randomly into 2 equal groups: basal diet group and sodium butyrate + basal diet group. Diarrhea rate, growth performance during 3 weeks of feeding on these diets were observed, the lactulose-mannitol ratio in urine were detected by High Performance Liquid Chromatography, the expression levels of tight junction proteins in the intestinal tract and related signaling molecules, such as GPR109A and Akt, in the colon were examined by quantitative real-time PCR or western blot analyses on day 21. Caco-2 cells were used as a colon cell model and cultured with or without sodium butyrate to assess the expression of tight junction proteins and the activation of related signaling molecules. GPR109A-short hairpin RNA (shRNA) and specific antagonists of Akt and ERK1/2 were used as signaling pathway inhibitors to elucidate the mechanism by which butyric acid regulates the expression of tight junction proteins and the colonic epithelial barrier. RESULTS: The sodium butyrate diet alleviated diarrhea symptoms and decreased intestinal permeability without affecting the growth of early weaned piglets. The expression levels of the tight junction proteins Claudin-3, Occludin, and zonula occludens 1 were up-regulated by sodium butyrate in the colon and Caco-2 cells. GPR109A knockdown using shRNA or blockade of the Akt signaling pathway in Caco-2 cells suppressed sodium butyrate-induced Claudin-3 expression. CONCLUSIONS: Sodium butyrate acts on the Akt signaling pathway to facilitate Claudin-3 expression in the colon in a GPR109A-dependent manner.
Assuntos
Ácido Butírico/farmacologia , Colo/metabolismo , Diarreia , Regulação da Expressão Gênica/efeitos dos fármacos , Receptores Nicotínicos/biossíntese , Junções Íntimas/metabolismo , Animais , Células CACO-2 , Colo/patologia , Diarreia/tratamento farmacológico , Diarreia/metabolismo , Diarreia/patologia , Humanos , Suínos , Junções Íntimas/patologiaRESUMO
BACKGROUND: Several studies demonstrate that cigarette smoking has a negative effect on the reproductive health of both genders. The mechanisms by which it alters male gonadic function are not entirely clear. The combustion of cigarette produces a lot of chemical compounds that may be responsible for the negative impact of cigarette smoke on sperm parameters. In particular, the effects on semen of nicotine, a substance present in the tobacco plant and the main constituent of cigarette smoke, have been studied, showing that this alkaloid alters sperm parameters. Recently we investigated the mechanism by which nicotine damages sperm through the evaluation of the expression of nicotinic receptors subunits in human spermatozoa. CONCLUSION: 8 nAChR subunits found to date in mammals are expressed in human spermatozoa but, in non-smokers subjects, only α7 subunit is translated. Cigarette smoking may stimulate the expression of some subunits, not translated in non-smokers. Therefore, the presence in sperm of other nAChR subunits than α7 could represent a marker for smoking-related sperm damage.
Assuntos
Regulação da Expressão Gênica , Infertilidade Masculina , Nicotina/metabolismo , Receptores Nicotínicos/biossíntese , Fumar , Espermatozoides/metabolismo , Humanos , Infertilidade Masculina/etiologia , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Masculino , Fumar/efeitos adversos , Fumar/metabolismo , Fumar/patologia , Espermatozoides/patologiaRESUMO
In rodents, CHRNs are involved in bitter taste transduction of nicotine and ethanol. Currently, it is not clear if CHRNs are expressed in human taste cells and if they play a role in transducing the bitter taste of nicotine and ethanol or in the synthesis and release of neurohumoral peptides. Accordingly, we investigated the expression and functional role of CHRNs in HBO cells. Using molecular techniques, we demonstrate that a subset of HBO cells express CHRNs that also co-express TRPM5, T1R3 or T2R38. Exposing HBO cells to nicotine or ethanol acutely or to nicotine chronically induced a differential increase in the expression of CHRN mRNA and protein in a dose- and time-dependent manner. Acutely exposing HBO cells to a mixture containing nicotine plus ethanol induced a smaller increase in CHRN mRNAs relative to nicotine or ethanol treatment alone. A subset of HBO cells responded to nicotine, acetylcholine and ATP with a transient increase in [Ca2+]i. Nicotine effects on [Ca2+]i were mecamylamine sensitive. Brain-derived neurotrophic factor (BDNF) protein was detected in HBO cells using ELISA. Acute nicotine exposure decreased BDNF in HBO cells and increased BDNF release in the medium. CHRNs were also detected in HEK293 cells by RT-PCR. Unlike HBO cells, CHRNs were localized in most of HEK293 cells and majority of HEK293 cells responded to nicotine and ethanol stimulation with a transient increase in [Ca2+]i. BDNF levels in HEK293 cells were significantly higher than in HBO cells but the nicotine induced release of BDNF in the media was a fraction of the BDNF cellular content. We conclude that CHRNs are expressed in TRPM5 positive HBO cells. CHRN mRNA expression is modulated by exposure to nicotine and ethanol in a dose- and time-dependent manner. Nicotine induces the synthesis and release of BDNF in HBO cells.
Assuntos
Receptores Nicotínicos/biossíntese , Papilas Gustativas/metabolismo , Adulto , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Fator Neurotrófico Derivado do Encéfalo/genética , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Etanol/farmacologia , Regulação da Expressão Gênica , Células HEK293 , Humanos , Nicotina/farmacologia , Subunidades Proteicas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores Nicotínicos/genéticaRESUMO
Principal neurons in the ventral cochlear nucleus (VCN) receive powerful ascending excitation and pass on the auditory information with exquisite temporal fidelity. Despite being dominated by ascending inputs, the VCN also receives descending cholinergic connections from olivocochlear neurons and from higher regions in the pontomesencephalic tegmentum. In Mongolian gerbils, acetylcholine acts as an excitatory and modulatory neurotransmitter on VCN neurons, but the anatomical structure of cholinergic innervation of gerbil VCN is not well described. We applied fluorescent immunohistochemical staining to elucidate the development and the cellular localization of presynaptic and postsynaptic components of the cholinergic system in the VCN of the Mongolian gerbil. We found that cholinergic fibers (stained with antibodies against the vesicular acetylcholine transporter) were present before hearing onset at P5, but innervation density increased in animals after P10. Early in development cholinergic fibers invaded the VCN from the medial side, spread along the perimeter and finally innervated all parts of the nucleus only after the onset of hearing. Cholinergic fibers ran in a rostro-caudal direction within the nucleus and formed en-passant swellings in the neuropil between principal neurons. Nicotinic and muscarinic receptors were expressed differentially in the VCN, with nicotinic receptors being mostly expressed in dendritic areas while muscarinic receptors were located predominantly in somatic membranes. These anatomical data support physiological indications that cholinergic innervation plays a role in modulating information processing in the cochlear nucleus.
Assuntos
Núcleo Coclear/citologia , Gerbillinae/fisiologia , Neurônios/fisiologia , Sistema Nervoso Parassimpático/citologia , Acetilcolina/metabolismo , Animais , Núcleo Coclear/crescimento & desenvolvimento , Dendritos/metabolismo , Dendritos/ultraestrutura , Fenômenos Eletrofisiológicos , Imuno-Histoquímica , Fibras Nervosas/ultraestrutura , Sistema Nervoso Parassimpático/crescimento & desenvolvimento , Receptor Muscarínico M3/biossíntese , Receptores Muscarínicos/biossíntese , Receptores Nicotínicos/biossíntese , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismoRESUMO
Mathematical models predicting in vivo pharmacodynamic effects from in vitro data can accelerate drug discovery, and reduce costs and animal use. However, data integration and modeling is non-trivial when more than one drug-target receptor is involved in the biological response. We modeled the inhibition of non-esterified fatty acid release by dual G-protein-coupled receptor 81/109A (GPR81/GPR109A) agonists in vivo in the rat, to estimate the in vivo EC50 values for 12 different compounds. We subsequently predicted those potency estimates using EC50 values obtained from concentration-response data in isolated primary adipocytes and cell systems overexpressing GPR81 or GPR109A in vitro. A simple linear regression model based on data from primary adipocytes predicted the in vivo EC50 better than simple linear regression models based on in vitro data from either of the cell systems. Three models combining the data from the overexpressing cell systems were also evaluated: two piecewise linear models defining logical OR- and AND-circuits, and a multivariate linear regression model. All three models performed better than the simple linear regression model based on data from primary adipocytes. The OR-model was favored since it is likely that activation of either GPR81 or GPR109A is sufficient to deactivate the cAMP pathway, and thereby inhibit non-esterified fatty acid release. The OR-model was also able to predict the in vivo selectivity between the two receptors. Finally, the OR-model was used to predict the in vivo potency of 1651 new compounds. This work suggests that data from the overexpressing cell systems are sufficient to predict in vivo potency of GPR81/GPR109A agonists, an approach contributing to faster and leaner drug discovery.