RESUMEN

There is growing concern that some managed and wild insect pollinator populations are in decline, potentially threatening biodiversity and sustainable food production on a global scale. In recent years, there has been increasing evidence that sub-lethal exposure to neurotoxic, neonicotinoid pesticides can negatively affect pollinator immunocompetence and could amplify the effects of diseases, likely contributing to pollinator declines. However, a direct pathway connecting neonicotinoids and immune functions remains elusive. In this study we show that haemocytes and non-neural tissues of the honeybee Apis mellifera express the building blocks of the nicotinic acetylcholine receptors that are the target of neonicotinoids. In addition, we demonstrate that the haemocytes, which form the cellular arm of the innate immune system, actively express choline acetyltransferase, a key enzyme necessary to synthesize acetylcholine. In a last step, we show that the expression of this key enzyme is affected by field-realistic doses of clothianidin, a widely used neonicotinoid. These results support a potential mechanistic framework to explain the effects of sub-lethal doses of neonicotinoids on the immune function of pollinators.

Asunto(s)

Acetilcolina , Guanidinas , Hemocitos , Insecticidas , Neonicotinoides , Animales , Abejas/efectos de los fármacos , Abejas/inmunología , Insecticidas/toxicidad , Neonicotinoides/toxicidad , Acetilcolina/metabolismo , Hemocitos/efectos de los fármacos , Hemocitos/inmunología , Hemocitos/metabolismo , Guanidinas/toxicidad , Tiazoles , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/efectos de los fármacos , Colina O-Acetiltransferasa/metabolismo

RESUMEN

BACKGROUND: Intracellular tracking is commonly used in trafficking research. Until today, the respective techniques have remained complex, and complicated, mostly transgenic target protein changes are necessary, often requiring expensive equipment and expert knowledge. METHODS: We present a novel method, which we term "cell-sonar", that enables the user to track expression changes of specific protein markers that serve as points of interaction. Our study provides comparable analyses of expression changes of these marker proteins by in-cell Western analyses in two otherwise isogenic cell lines that only differ in the overexpression of the tracked target protein. Using the overexpressed human adult muscle-type nicotinic acetylcholine receptor as an example, we demonstrate that cell-sonar can cover multiple intracellular compartments such as the endoplasmic reticulum, the pathway between it and the Golgi apparatus, and the endocytic pathway. RESULTS: We provide evidence for receptor maturation in the Golgi and storage in recycling endosomes, rather than the fate of increased insertion into the plasma membrane. Additionally, we demonstrate with the implementation of nicotine that the receptor's destiny is exasperated up to secondary degradation. CONCLUSIONS: Cell-sonar is an affordable, easy-to-implement, and cheap method that can be adapted to a broad variety of proteins and cellular pathways of interest to researchers.

Asunto(s)

Aparato de Golgi , Vías Secretoras , Humanos , Aparato de Golgi/metabolismo , Retículo Endoplásmico/metabolismo , Receptores Nicotínicos/metabolismo , Transporte de Proteínas , Endosomas/metabolismo , Línea Celular

RESUMEN

AIMS: In contrast to G protein-coupled receptors or receptor tyrosine kinases, the mechanism underlying ERK activation through nicotine acetylcholine receptors (nAChRs), members of the ligand-gated ion channel family, remains poorly elucidated. This study aimed to delineate the signaling pathway responsible for ERK activation by the α4ß2 nAChR subtype, which is implicated in nicotine addiction and various mental disorders. MATERIALS AND METHODS: Loss-of-function strategies and mutants of arrestin2/PKCßII with distinct functional characteristics were employed to identify the cellular components and processes involved in ERK activation. KEY FINDINGS: ERK activation via α4ß2 nAChR was observed within the nucleus and necessitated the nuclear translocation of arrestin2 and PKCßII, which exhibited mutual augmentation. Activation of PKCßII by α4ß2 nAChR stimulation facilitated the nuclear translocation of arrestin2 by enhancing its interaction with importin ß1. Apart from scaffolding ERK activation in the nucleus, arrestin2, in cooperation with GRK2, facilitated the activation of the Src/Syk/PKCßII signaling cascade, leading to the nuclear entry of PKCßII in a Gßγ-dependent manner. Upon nuclear localization, PKCßII underwent ubiquitination by Mdm2 and interacted with MEK1, resulting in ERK activation. In summary, α4ß2 nAChR-mediated ERK activation in the nucleus involves the nuclear translocation of arrestin2 and PKCßII, which is reciprocally facilitated via positive feedback augmentation. SIGNIFICANCE: As α4ß2 nAChRs play a pivotal role in various cellular processes including drug addiction and mental disorders, our findings will offer insights into understanding the pathogenesis of α4ß2 nAChR-related disorders and may facilitate the development of targeted therapeutic interventions.

Asunto(s)

Subunidades beta de la Proteína de Unión al GTP , Proteína Quinasa C beta , Receptores Nicotínicos , Receptores Nicotínicos/metabolismo , Proteína Quinasa C beta/metabolismo , Humanos , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Células HEK293 , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Animales , Transducción de Señal , Sistema de Señalización de MAP Quinasas/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Núcleo Celular/metabolismo

RESUMEN

Age-related impairment of the diaphragm causes respiratory complications. Neuromuscular junction (NMJ) dysfunction can be one of the triggering events in diaphragm weaknesses in old age. Prominent structural and functional alterations in diaphragm NMJs were described in elderly rodents, but NMJ changes in middle age remain unclear. Here, we compared diaphragm muscles from young adult (3 months) and middle-aged (12 months) BALB/c mice. Microelectrode recordings, immunofluorescent staining, electron microscopy, myography, and whole-body plethysmography were used. We revealed presynaptic (i) and postsynaptic (ii) changes. The former (i) included an increase in both action potential propagation velocity and neurotransmitter release evoked by low-, moderate-, and high-frequency activity but a decrease in immunoexpression of synapsin 1 and synaptic vesicle clustering. The latter (ii) consisted of a decrease in currents via nicotinic acetylcholine receptors and the area of their distribution. These NMJ changes correlated with increased contractile responses to moderate- to high-frequency nerve activation. Additionally, we found alterations in the pattern of respiration (an increase in peak inspiratory flow and a tendency of elevation of the tidal volume), which imply increased diaphragm activity in middle-aged mice. We conclude that enhancement of neuromuscular communication (due to presynaptic mechanism) accompanied by improved contractile responses occurs in the diaphragm in early aging.

Asunto(s)

Envejecimiento , Diafragma , Ratones Endogámicos BALB C , Unión Neuromuscular , Animales , Unión Neuromuscular/metabolismo , Diafragma/metabolismo , Envejecimiento/metabolismo , Envejecimiento/fisiología , Ratones , Receptores Nicotínicos/metabolismo , Masculino , Transmisión Sináptica/fisiología , Potenciales de Acción , Vesículas Sinápticas/metabolismo , Contracción Muscular/fisiología , Sinapsinas/metabolismo

RESUMEN

A new series of substituted benzo[h]chromene, benzochromenopyrimidine, and benzochromenotriazolopyrimidine derivatives were synthesized via chemical transformations of iminonitrile, ethoxymethylene amino, and cyanomethylene functionalities. The chemical structures of the synthesized compounds were assured by spectroscopic data and elemental analysis. The larvicidal efficacy of these compounds against Culex pipiens L. larvae was investigated, revealing potent insecticidal activity, particularly for compounds 6, 10, and 16, exceeding that of the standard insecticide chlorpyrifos. The mode of action of these compounds was explored through molecular docking studies, indicating their potential as acetylcholine esterase (AChE) inhibitors and nicotinic acetylcholine receptors (nAChR) blockers. The structure-activity relationship analysis highlighted the influence of substituents and fused heterocyclic rings on larvicidal potency. These findings suggest that the synthesized compounds hold promise as potential candidates for developing novel and effective mosquito control agents.

Asunto(s)

Benzopiranos , Culex , Insecticidas , Larva , Simulación del Acoplamiento Molecular , Animales , Culex/efectos de los fármacos , Larva/efectos de los fármacos , Insecticidas/farmacología , Insecticidas/química , Insecticidas/síntesis química , Relación Estructura-Actividad , Benzopiranos/farmacología , Benzopiranos/química , Benzopiranos/síntesis química , Modelos Moleculares , Inhibidores de la Colinesterasa/farmacología , Inhibidores de la Colinesterasa/síntesis química , Inhibidores de la Colinesterasa/química , Receptores Nicotínicos/metabolismo , Estructura Molecular

RESUMEN

The α9α10 nicotinic cholinergic receptor (nAChR) is a ligand-gated pentameric cation-permeable ion channel that mediates synaptic transmission between descending efferent neurons and mechanosensory inner ear hair cells. When expressed in heterologous systems, α9 and α10 subunits can assemble into functional homomeric α9 and heteromeric α9α10 receptors. One of the differential properties between these nAChRs is the modulation of their ACh-evoked responses by extracellular calcium (Ca2+). While α9 nAChRs responses are blocked by Ca2+, ACh-evoked currents through α9α10 nAChRs are potentiated by Ca2+ in the micromolar range and blocked at millimolar concentrations. Using chimeric and mutant subunits, together with electrophysiological recordings under two-electrode voltage-clamp, we show that the TM2-TM3 loop of the rat α10 subunit contains key structural determinants responsible for the potentiation of the α9α10 nAChR by extracellular Ca2+. Moreover, molecular dynamics simulations reveal that the TM2-TM3 loop of α10 does not contribute to the Ca2+ potentiation phenotype through the formation of novel Ca2+ binding sites not present in the α9 receptor. These results suggest that the TM2-TM3 loop of α10 might act as a control element that facilitates the intramolecular rearrangements that follow ACh-evoked α9α10 nAChRs gating in response to local and transient changes of extracellular Ca2+ concentration. This finding might pave the way for the future rational design of drugs that target α9α10 nAChRs as otoprotectants.

Asunto(s)

Calcio , Receptores Nicotínicos , Animales , Ratas , Acetilcolina/metabolismo , Acetilcolina/farmacología , Secuencia de Aminoácidos , Sitios de Unión , Calcio/metabolismo , Simulación de Dinámica Molecular , Técnicas de Placa-Clamp , Subunidades de Proteína/metabolismo , Subunidades de Proteína/genética , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/química , Xenopus laevis

RESUMEN

The neurotoxic effects of neonicotinoids (NEOs) have been widely reported in relation to the poisoning of wild birds, yet the underlying molecular mechanism has remained elusive. This study employed Japanese quails (Coturnix japonica) and primary quail embryonic neurons as in vivo and ex vivo models, respectively, to investigate the neurotoxic effects and mechanism of thiamethoxam (TMX), a representative neonicotinoid insecticide, at environmentally relevant concentrations. Following a 28-day exposure to TMX, metabolomic analysis of quail brain revealed TMX-induced changes in glutamatergic, GABA-ergic, and dopaminergic function. Subsequent ex vivo and in silico experimentation revealed that the activation of nicotinic acetylcholine receptors and calcium signaling, induced by clothianidin (CLO), the primary metabolite of TMX, served as upstream events for the alterations in neurotransmitter synthesis, metabolism, release, and uptake. Our findings propose that the disruption of the central nervous system, caused by environmentally significant concentrations of NEOs, may account for the avian poisoning events induced by NEOs.

Asunto(s)

Coturnix , Insecticidas , Tiametoxam , Animales , Tiametoxam/toxicidad , Coturnix/metabolismo , Insecticidas/toxicidad , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/metabolismo , Neonicotinoides/toxicidad , Tiazoles/toxicidad , Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Simulación por Computador , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Guanidinas/toxicidad , Oxazinas/toxicidad , Receptores Nicotínicos/metabolismo

RESUMEN

Nicotinic acetylcholine receptors (nAChRs) are expressed in excitable and non-excitable cells of the organism. Extensive studies suggest that nAChR ligands have therapeutic potential, notably for neurological and psychiatric disorders. Organometallic ruthenium complexes are known to inhibit several medically important enzymes such as cholinesterases. In addition, they can also interact with muscle- and neuronal-subtype nAChRs. The present study aimed to investigate the direct effects of three organometallic ruthenium complexes, [(η6-p-cymene)Ru(II)(5-nitro-1,10-phenanthroline)Cl]Cl (C1-Cl), [(η6-p-cymene)Ru(II)(1-hydroxypyridine-2(1H)-thionato)Cl] (C1a) and [(η6-p-cymene)Ru(II)(1-hydroxy-3-methoxypyridine-2(1H)-thionato)pta]PF6 (C1), on muscle-subtype (Torpedo) nAChRs and on the two most abundant human neuronal-subtype nAChRs in the CNS (α4ß2 and α7) expressed in Xenopus laevis oocytes, using the two-electrode voltage-clamp. The results show that none of the three compounds had agonistic activity on any of the nAChR subtypes studied. In contrast, C1-Cl reversibly blocked Torpedo nAChR (half-reduction of ACh-evoked peak current amplitude by 332 nM of compound). When tested at 10 µM, C1-Cl was statistically more potent to inhibit TorpedonAChR than α4ß2 and α7 nAChRs. Similar results of C1 effects were obtained on Torpedo and α4ß2 nAChRs, while no action of the compound was detected on α7 nAChRs. Finally, the effects of C1a were statistically similar on the three nAChR subtypes but, in contrast to C1-Cl and C1, the inhibition was hardly reversible. These results, together with our previous studies on isolated mouse neuromuscular preparations, strongly suggest that C1-Cl is, among the three compounds studied, the only molecule that could be used as a potential myorelaxant drug.

Asunto(s)

Oocitos , Receptores Nicotínicos , Xenopus laevis , Animales , Receptores Nicotínicos/metabolismo , Humanos , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Rutenio/química , Rutenio/farmacología , Torpedo , Compuestos Organometálicos/farmacología , Compuestos Organometálicos/química , Técnicas de Placa-Clamp , Complejos de Coordinación/farmacología , Complejos de Coordinación/química , Piridinas/farmacología , Piridinas/química

RESUMEN

Despite its efficacy in human epidermal growth factor receptor 2 positive cancer treatment, trastuzumab-induced cardiotoxicity (TIC) has become a growing concern. Due to the lack of cardiomyocyte regeneration and proliferation in adult heart, cell death significantly contributes to cardiovascular diseases. Cardiac autonomic modulation by vagus nerve stimulation (VNS) has shown cardioprotective effects in several heart disease models, while the effects of VNS and its underlying mechanisms against TIC have not been found. Forty adult male Wistar rats were divided into 5 groups: (i) control without VNS (CSham) group, (ii) trastuzumab (4 mg/kg/day, i.p.) without VNS (TSham) group, (iii) trastuzumab + VNS (TVNS) group, (iv) trastuzumab + VNS + mAChR blocker (atropine; 1 mg/kg/day, ip, TVNS + Atro) group, and (v) trastuzumab + VNS + nAChR blocker (mecamylamine; 7.5 mg/kg/day, ip, TVNS + Mec) group. Our results showed that trastuzumab induced cardiac dysfunction by increasing autonomic dysfunction, mitochondrial dysfunction/dynamics imbalance, and cardiomyocyte death including apoptosis, autophagic deficiency, pyroptosis, and ferroptosis, which were notably alleviated by VNS. However, mAChR and nAChR blockers significantly inhibited the beneficial effects of VNS on cardiac autonomic dysfunction, mitochondrial dysfunction, cardiomyocyte apoptosis, pyroptosis, and ferroptosis. Only nAChR could counteract the protective effects of VNS on cardiac mitochondrial dynamics imbalance and autophagy insufficiency. Therefore, VNS prevented TIC by rebalancing autonomic activity, ameliorating mitochondrial dysfunction and cardiomyocyte death through mAChR and nAChR activation. The current study provides a novel perspective elucidating the potential treatment of VNS, thus also offering other pharmacological therapeutic promises in TIC patients.

Asunto(s)

Apoptosis , Cardiotoxicidad , Miocitos Cardíacos , Ratas Wistar , Receptores Muscarínicos , Receptores Nicotínicos , Trastuzumab , Estimulación del Nervio Vago , Animales , Estimulación del Nervio Vago/métodos , Masculino , Ratas , Trastuzumab/toxicidad , Trastuzumab/farmacología , Apoptosis/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Receptores Muscarínicos/metabolismo , Receptores Muscarínicos/efectos de los fármacos , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/efectos de los fármacos , Antagonistas Nicotínicos/farmacología , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/toxicidad , Nervio Vago/efectos de los fármacos

RESUMEN

During development, motor neurons originating in the brainstem and spinal cord form elaborate synapses with skeletal muscle fibres1. These neurons release acetylcholine (ACh), which binds to nicotinic ACh receptors (AChRs) on the muscle, initiating contraction. Two types of AChR are present in developing muscle cells, and their differential expression serves as a hallmark of neuromuscular synapse maturation2-4. The structural principles underlying the switch from fetal to adult muscle receptors are unknown. Here, we present high-resolution structures of both fetal and adult muscle nicotinic AChRs, isolated from bovine skeletal muscle in developmental transition. These structures, obtained in the absence and presence of ACh, provide a structural context for understanding how fetal versus adult receptor isoforms are tuned for synapse development versus the all-or-none signalling required for high-fidelity skeletal muscle contraction. We find that ACh affinity differences are driven by binding site access, channel conductance is tuned by widespread surface electrostatics and open duration changes result from intrasubunit interactions and structural flexibility. The structures further reveal pathogenic mechanisms underlying congenital myasthenic syndromes.

Asunto(s)

Acetilcolina , Modelos Moleculares , Músculo Esquelético , Receptores Nicotínicos , Animales , Acetilcolina/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/citología , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/química , Bovinos , Sitios de Unión , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/química , Feto/metabolismo , Electricidad Estática , Síndromes Miasténicos Congénitos/metabolismo , Microscopía por Crioelectrón , Humanos , Receptores Colinérgicos/metabolismo , Receptores Colinérgicos/química , Desarrollo de Músculos , Contracción Muscular

RESUMEN

Lung adenocarcinoma (LUAD) is the leading cause of cancer death worldwide, with high incidence and low survival rates. Nicotinic acetylcholine receptors play an important role in the progression of LUAD. In this study, a screening of 17 nicotinic acetylcholine receptor allosteric agents revealed that spinosad effectively suppressed the proliferation of LUAD cells. The experiments demonstrated that spinosad induced cell cycle arrest in the G1 phase and stimulated apoptosis, thereby impeding the growth of LUAD and enhancing the responsiveness to gefitinib in vitro and vivo. Mechanistic insights obtained through transcriptome sequencing, Co-IP, and protein immunoblots indicated that spinosad disrupted the interaction between CHRNA5 and EGFR, thereby inhibiting the formation of downstream complexes and activation of the EGFR signaling pathway. The supplementation of exogenous acetylcholine showed to mitigate the inhibition of LUAD cell proliferation induced by spinosad. This study elucidates the therapeutic effects and mechanisms of spinosad in LUAD, and offers a theoretical and experimental foundation for novel LUAD treatments.

Asunto(s)

Adenocarcinoma del Pulmón , Apoptosis , Proliferación Celular , Combinación de Medicamentos , Receptores ErbB , Neoplasias Pulmonares , Macrólidos , Receptores Nicotínicos , Transducción de Señal , Humanos , Transducción de Señal/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Receptores Nicotínicos/metabolismo , Receptores ErbB/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Macrólidos/farmacología , Adenocarcinoma del Pulmón/tratamiento farmacológico , Adenocarcinoma del Pulmón/patología , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/genética , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Ratones Desnudos , Ratones , Ratones Endogámicos BALB C , Células A549

RESUMEN

Understanding the agonist concentration-response curve (CRC) is the cornerstone in pharmacology. While CRC parameters, agonist potency (EC50) and efficacy (maximum response, Imax) are well-studied, the role of unliganded gating (minimum response, Imin) on CRC is often overlooked. This study explores the effect of unliganded gating on agonist response in muscle-type acetylcholine (ACh) receptors, focusing on the underexplored role of Imin in modulating EC50 and Imax. Three Gain-of-Function (GOF) mutations that increase, and two Loss-of-Function (LOF) mutations that decrease the unliganded gating equilibrium constant (L0) were studied using automated patch-clamp electrophysiology. GOF mutations enhanced agonist potency, whereas LOF mutations reduced it. The calculated CRC aligned well with empirical results, indicating that agonist CRC can be estimated from knowledge of L0. Reduction in agonist efficacy due to LOF mutations was calculated and subsequently validated using single-channel patch-clamp electrophysiology, a factor often obscured in normalized CRC. The study also evaluated the combined impact of mutations (L0) on CRC, confirming the predictive model. Further, no significant energetic coupling between distant residues (>15 Å) was found, indicating that the mutations' effects are localized and do not alter overall agonist affinity. These findings substantiate the role of unliganded gating in modulating agonist responses and establishes a predictive model for estimating CRC parameters from known changes in L0. The study highlights the importance of intrinsic activity in receptor theory.

Asunto(s)

Activación del Canal Iónico , Mutación , Agonistas Nicotínicos , Receptores Nicotínicos , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/genética , Activación del Canal Iónico/efectos de los fármacos , Humanos , Agonistas Nicotínicos/farmacología , Ligandos , Relación Dosis-Respuesta a Droga , Animales , Células HEK293

RESUMEN

Adolescence is characterized by a critical period of maturation and growth, during which regions of the brain are vulnerable to long-lasting cognitive disturbances. Adolescent exposure to nicotine can lead to deleterious neurological and psychological outcomes. Moreover, the nicotinic acetylcholine receptor (nAChR) has been shown to play a functionally distinct role in the development of the adolescent brain. CHRNA2 encodes for the α2 subunit of nicotinic acetylcholine receptors associated with CA1 oriens lacunosum moleculare GABAergic interneurons and is associated with learning and memory. Previously, we found that adolescent male hypersensitive CHRNA2L9'S/L9' mice had impairments in learning and memory during a pre-exposure-dependent contextual fear conditioning task that could be rescued by low-dose nicotine exposure. In this study, we assessed learning and memory in female adolescent hypersensitive CHRNA2L9'S/L9' mice exposed to saline or a subthreshold dose of nicotine using a hippocampus-dependent task of pre-exposure-dependent contextual fear conditioning. We found that nicotine-treated wild-type female mice had significantly greater improvements in learning and memory than both saline-treated wild-type mice and nicotine-treated CHRNA2L9'S/L9' female mice. Thus, hyperexcitability of CHRNA2 in female adolescent mice ablated the nicotine-mediated potentiation of learning and memory seen in wild-types. Our results indicate that nicotine exposure during adolescence mediates sexually dimorphic patterns of learning and memory, with wild-type female adolescents being more susceptible to the effects of sub-threshold nicotine exposure. To understand the mechanism underlying sexually dimorphic behavior between hyperexcitable CHRNA2 mice, it is critical that further research be conducted.

Asunto(s)

Miedo , Hipocampo , Memoria , Nicotina , Receptores Nicotínicos , Animales , Receptores Nicotínicos/metabolismo , Nicotina/farmacología , Femenino , Ratones , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Miedo/efectos de los fármacos , Miedo/fisiología , Memoria/efectos de los fármacos , Memoria/fisiología , Agonistas Nicotínicos/farmacología , Aprendizaje/efectos de los fármacos , Aprendizaje/fisiología , Condicionamiento Clásico/efectos de los fármacos , Condicionamiento Clásico/fisiología , Ratones Endogámicos C57BL

RESUMEN

Neuromuscular signal transmission is affected in various diseases including myasthenia gravis, congenital myasthenic syndromes, and sarcopenia. We used an ATF2-luciferase system to monitor the phosphorylation of MuSK in HEK293 cells introduced with MUSK and LRP4 cDNAs to find novel chemical compounds that enhanced agrin-mediated acetylcholine receptor (AChR) clustering. Four compounds with similar chemical structures carrying benzene rings and heterocyclic rings increased the luciferase activities 8- to 30-folds, and two of them showed continuously graded dose dependence. The effects were higher than that of disulfiram, a clinically available aldehyde dehydrogenase inhibitor, which we identified to be the most competent preapproved drug to enhance ATF2-luciferase activity in the same assay system. In C2C12 myotubes, all the compounds increased the area, intensity, length, and number of AChR clusters. Three of the four compounds increased the phosphorylation of MuSK, but not of Dok7, JNK. ERK, or p38. Monitoring cell toxicity using the neurite elongation of NSC34 neuronal cells as a surrogate marker showed that all the compounds had no effects on the neurite elongation up to 1 µM. Extensive docking simulation and binding structure prediction of the four compounds with all available human proteins using AutoDock Vina and DiffDock showed that the four compounds were unlikely to directly bind to MuSK or Dok7, and the exact target remained unknown. The identified compounds are expected to serve as a seed to develop a novel therapeutic agent to treat defective NMJ signal transmission.

Asunto(s)

Fibras Musculares Esqueléticas , Receptores Nicotínicos , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Animales , Ratones , Línea Celular , Humanos , Factor de Transcripción Activador 2/genética , Factor de Transcripción Activador 2/metabolismo , Genes Reporteros , Proteínas Relacionadas con Receptor de LDL/genética , Proteínas Relacionadas con Receptor de LDL/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Familia de Multigenes , Transducción de Señal/efectos de los fármacos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Neuritas , Bungarotoxinas/farmacología , Benceno/farmacología , Compuestos Heterocíclicos/farmacología , Simulación del Acoplamiento Molecular

RESUMEN

With the spread of resistance to long-established insecticides targeting Anopheles malaria vectors, understanding the actions of compounds newly identified for vector control is essential. With new commercial vector-control products containing neonicotinoids under development, we investigate the actions of 6 neonicotinoids (imidacloprid, thiacloprid, clothianidin, dinotefuran, nitenpyram and acetamiprid) on 13 Anopheles gambiae nicotinic acetylcholine receptor (nAChR) subtypes produced by expression of combinations of the Agα1, Agα2, Agα3, Agα8 and Agß1 subunits in Xenopus laevis oocytes, the Drosophila melanogaster orthologues of which we have previously shown to be important in neonicotinoid actions. The presence of the Agα2 subunit reduces neonicotinoid affinity for the mosquito nAChRs, whereas the Agα3 subunit increases it. Crystal structures of the acetylcholine binding protein (AChBP), an established surrogate for the ligand-binding domain, with dinotefuran bound, shows a unique target site interaction through hydrogen bond formation and CH-N interaction at the tetrahydrofuran ring. This is of interest as dinotefuran is also under trial as the toxic element in baited traps. Multiple regression analyses show a correlation between the efficacy of neonicotinoids for the Agα1/Agα2/Agα8/Agß1 nAChR, their hydrophobicity and their rate of knockdown of adult female An. gambiae, providing new insights into neonicotinoid features important for malaria vector control.

Asunto(s)

Anopheles , Guanidinas , Insecticidas , Mosquitos Vectores , Neonicotinoides , Nitrocompuestos , Receptores Nicotínicos , Animales , Anopheles/metabolismo , Anopheles/genética , Anopheles/efectos de los fármacos , Neonicotinoides/farmacología , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/química , Insecticidas/farmacología , Insecticidas/química , Nitrocompuestos/farmacología , Nitrocompuestos/química , Guanidinas/farmacología , Mosquitos Vectores/efectos de los fármacos , Mosquitos Vectores/genética , Xenopus laevis , Ligandos , Piridinas/farmacología , Malaria/transmisión , Malaria/parasitología , Tiazoles/farmacología , Tiazoles/química , Tiazoles/metabolismo , Tiazinas/farmacología , Tiazinas/química , Oocitos/metabolismo , Oocitos/efectos de los fármacos , Femenino , Proteínas de Insectos/metabolismo , Proteínas de Insectos/genética , Proteínas de Insectos/química , Imidazoles/farmacología , Imidazoles/química

RESUMEN

Thiacloprid, a neonicotinoid insecticide, has become one of the major control agents for the pine sawyer beetle, Monochamus alternatus Hope, however, the mechanism of detoxification is unknown. We demonstrate that glutathione S-transferases (GSTs) and nicotinic acetylcholine receptors (nAChRs) are involved in the rapid detoxification of thiacloprid in M. alternatus larvae. The activity of detoxification enzyme GSTs was significantly higher, while the activity of acetylcholinesterase (AChE) was inhibited under thiacloprid exposure. The inhibition of AChE activity led to lethal over-stimulation of the cholinergic synapse, which was then released by the rapid downregulation of nAChRs. Meanwhile, GSTs were overexpressed to detoxify thiacloprid accordingly. A total of 3 nAChR and 12 GST genes were identified from M. alternatus, among which ManAChRα2 and MaGSTs1 were predicted to confer thiacloprid tolerance. RNA interference (RNAi) was subsequently conducted to confirm the function of ManAChRα2 and MaGSTs1 genes in thiacloprid detoxification. The successful knock-down of the ManAChRα2 gene led to lower mortality of M. alternatus under LC30 thiacloprid treatment, and the suppression of the MaGSTs1 gene increased the mortality rate of M. alternatus. However, the mortality rate has no significant difference with controls when thiacloprid was fed together with both dsMaGSTs1 and dsManAChRα2. Molecular docking modeled the molecular basis for interaction between MaGSTs1/ManAChR and thiacloprid. This study highlights the important roles that ManAChRα2 and MaGSTs1 genes play in thiacloprid detoxification through transcriptional regulation and enzymatic metabolization, and proposes a new avenue for integrated pest management that combines pesticides and RNAi technology as an efficient strategy for M. alternatus control.

Asunto(s)

Escarabajos , Glutatión Transferasa , Insecticidas , Neonicotinoides , Receptores Nicotínicos , Tiazinas , Animales , Neonicotinoides/farmacología , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/genética , Escarabajos/efectos de los fármacos , Escarabajos/genética , Escarabajos/metabolismo , Tiazinas/farmacología , Tiazinas/metabolismo , Tiazinas/toxicidad , Glutatión Transferasa/metabolismo , Glutatión Transferasa/genética , Insecticidas/toxicidad , Insecticidas/farmacología , Insecticidas/metabolismo , Larva/efectos de los fármacos , Larva/metabolismo , Proteínas de Insectos/metabolismo , Proteínas de Insectos/genética , Inactivación Metabólica , Acetilcolinesterasa/metabolismo , Acetilcolinesterasa/genética , Piridinas/farmacología

RESUMEN

Metabolic dysfunction-associated steatohepatitis (MASH) is the progressive form of liver steatosis, the most common liver disease, and substantially increases the mortality rate. However, limited therapies are currently available to prevent MASH development. Identifying potential pharmacological treatments for the condition has been hampered by its heterogeneous and complex nature. Here, we identified a hepatic nonneuronal cholinergic signaling pathway required for metabolic adaptation to caloric overload. We found that cholinergic receptor nicotinic alpha 2 subunit (CHRNA2) is highly expressed in hepatocytes of mice and humans. Further, CHRNA2 is activated by a subpopulation of local acetylcholine-producing macrophages during MASH development. The activation of CHRNA2 coordinates defensive programs against a broad spectrum of MASH-related pathogenesis, including steatosis, inflammation, and fibrosis. Hepatocyte-specific loss of CHRNA2 signaling accelerates the disease onset in different MASH mouse models. Activation of this pathway via pharmacological inhibition of acetylcholine degradation protects against MASH development. Our study uncovers a hepatic nicotinic cholinergic receptor pathway that constitutes a cell-autonomous self-defense route against prolonged metabolic stress and holds therapeutic potential for combatting human MASH.

Asunto(s)

Hígado Graso , Hepatocitos , Hígado , Receptores Nicotínicos , Transducción de Señal , Animales , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/genética , Humanos , Hígado/metabolismo , Hígado/patología , Ratones , Hígado Graso/metabolismo , Hepatocitos/metabolismo , Ratones Endogámicos C57BL , Masculino , Macrófagos/metabolismo , Acetilcolina/metabolismo , Ratones Noqueados , Modelos Animales de Enfermedad

RESUMEN

α-Conotoxins, as selective nAChR antagonists, can be valuable tools for targeted drug delivery and fluorescent labeling, while conotoxin-drug or conotoxin-fluorescent conjugates through the disulfide bond are rarely reported. Herein, we demonstrate the [2,4] disulfide bond of α-conotoxin as a feasible new chemical modification site. In this study, analogs of the α-conotoxin LsIA cysteine[2,4] were synthesized by stapling with five linkers, and their inhibitory activities against human α7 and rat α3ß2 nAChRs were maintained. To further apply this method in targeted delivery, the alkynylbenzyl bromide linker was synthesized and conjugated with Coumarin 120 (AMC) and Camptothecin (CPT) by copper-catalyzed click chemistry, and then stapled between cysteine[2,4] of the LsIA to construct a fluorescent probe and two peptide-drug conjugates. The maximum emission wavelength of the LsIA fluorescent probe was 402.2 nm, which was essentially unchanged compared with AMC. The cytotoxic activity of the LsIA peptide-drug conjugates on human A549 was maintained in vitro. The results demonstrate that the stapling of cysteine[2,4] with alkynylbenzyl bromide is a simple and feasible strategy for the exploitation and utilization of the α-conotoxin LsIA.

Asunto(s)

Conotoxinas , Cisteína , Humanos , Conotoxinas/química , Conotoxinas/farmacología , Cisteína/química , Animales , Disulfuros/química , Células A549 , Sistemas de Liberación de Medicamentos , Ratas , Antagonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/química , Colorantes Fluorescentes/química , Receptores Nicotínicos/metabolismo , Cumarinas/química , Cumarinas/farmacología , Química Clic

RESUMEN

Wrinkles, one of the most common signs of aging, are primarily caused by the continuous contraction of muscles. Muscle contraction is induced by the binding of acetylcholine (ACh), released at the neuromuscular junction, to nicotinic acetylcholine receptor (nAChR) present on the muscle cell surface. In this study, we aimed to develop a wrinkle-improving peptide that inhibits the binding of ACh to nAChR using peptide phage display technology. Our peptide showed a remarkably high binding affinity to nAChR subunit α1, with a value below 1 µM, and was found to inhibit the action of ACh through its interaction with these receptors. Furthermore, it increased collagen synthesis in skin cells and upregulated the expression of the aquaporin-3 (AQP3) and hyaluronan synthase-2 (HAS2) genes. These results confirm that the peptide effectively inhibits muscle contraction and enhances skin elasticity and hydration, contributing to its wrinkle-reducing effects. Clinical studies on humans observed significant improvement in wrinkles after three weeks of use, with substantial reduction observed after six weeks. In conclusion, these findings demonstrate the efficacy of the peptide (named Medipep) in reducing wrinkles.

Asunto(s)

Péptidos , Receptores Nicotínicos , Envejecimiento de la Piel , Receptores Nicotínicos/metabolismo , Envejecimiento de la Piel/efectos de los fármacos , Humanos , Péptidos/farmacología , Péptidos/metabolismo , Acetilcolina/metabolismo , Acetilcolina/farmacología , Femenino , Colágeno/metabolismo , Unión Proteica , Piel/metabolismo , Piel/efectos de los fármacos , Animales , Persona de Mediana Edad , Adulto

RESUMEN

Adolescence is characterized by changes in reward-related behaviors, social behaviors, and decision-making. These behavioral changes are necessary for the transition into adulthood, but they also increase vulnerability to the development of a range of psychiatric disorders. Major reorganization of the dopamine system during adolescence is thought to underlie, in part, the associated behavioral changes and increased vulnerability. Here, we utilized fast scan cyclic voltammetry and microdialysis to examine differences in dopamine release as well as mechanisms that underlie differential dopamine signaling in the nucleus accumbens (NAc) core of adolescent (P28-35) and adult (P70-90) male rats. We show baseline differences between adult and adolescent-stimulated dopamine release in male rats, as well as opposite effects of the α6 nicotinic acetylcholine receptor (nAChR) on modulating dopamine release. The α6-selective blocker, α-conotoxin, increased dopamine release in early adolescent rats, but decreased dopamine release in rats beginning in middle adolescence and extending through adulthood. Strikingly, blockade of GABAA and GABAB receptors revealed that this α6-mediated increase in adolescent dopamine release requires NAc GABA signaling to occur. We confirm the role of α6 nAChRs and GABA in mediating this effect in vivo using microdialysis. Results herein suggest a multisynaptic mechanism potentially unique to the period of development that includes early adolescence, involving acetylcholine acting at α6-containing nAChRs to drive inhibitory GABA tone on dopamine release.

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During adolescence, chemicals and cells in the brain undergo significant reorganization. These changes are thought to be why teenagers are often more vulnerable to developing drug addictions and psychiatric disorders. However, it is not fully understood how the brain transforms during this transitional period. Most of this reorganization takes place in the dopamine system which is responsible for triggering pleasurable sensations, including the feeling of reward after taking drugs. In 2020, a group of researchers found that adolescent male rats released less of the chemical dopamine in a part of the brain involved in the reward pathway than adult rats. But it was unclear what was causing this age-related effect. To investigate, Iacino et al. ­ including some of the researchers involved in the 2020 study ­blocked a family of receptors called nAChRs (short for nicotinic acetylcholine receptors) in the brain cells of male rats. These receptors bind to a neurotransmitter called acetylcholine which stimulates cells to release dopamine. Iacino et al. found that inhibiting a specific type of nAChR led to a decrease in dopamine in adult rats, but an increase in early adolescent rats. However, this effect was not observed when other types of nAChRs were inhibited. Iacino et al. found that the adolescent male rats also had higher levels of another neurotransmitter called GABA which blocks the release of dopamine. This led them to hypothesize that the reduced levels of dopamine in early adolescence may be due to increased levels of GABA, which is secreted by specialized cells which also have nAChRs on their surface. To investigate, Iacino et al. blocked two receptors for GABA that are found on dopamine-releasing neurons before exposing the rats to the nAChR inhibitor. This caused the adolescent rats to release less dopamine following nAChR inhibition, similar to the levels observed in adult rats. These findings suggest that the nAChR inhibitor leads to a rise in dopamine by stopping cells from releasing GABA ­ but only in adolescent rats. The work of Iacino et al. demonstrates how the dopamine system differs in adolescence, which may provide new insights in to why teenagers are often more susceptible to addiction. For instance, nicotine, the addictive substance in cigarettes, can also bind to nAChRs and make them less sensitive to acetylcholine. This may reduce the release of GABA, resulting in more dopamine being released which is then sensed as a reward by the teenage brain. However, more research is needed to fully understand how this brain circuit is modulated by nicotine intake.

Asunto(s)

Acetilcolina , Dopamina , Núcleo Accumbens , Ácido gamma-Aminobutírico , Masculino , Ratas , Acetilcolina/metabolismo , Dopamina/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Núcleo Accumbens/metabolismo , Ratas Sprague-Dawley , Receptores Nicotínicos/metabolismo , Animales