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1.
Int J Mol Sci ; 22(16)2021 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-34445737

RESUMO

Cholinergic neurotransmission is a key signal pathway in the peripheral nervous system and in several branches of the central nervous system. Despite the fact that it has been studied extensively for a long period of time, some aspects of its regulation still have not yet been established. One is the relationship between the nicotine-induced autoregulation of acetylcholine (ACh) release with changes in the concentration of presynaptic calcium levels. The mouse neuromuscular junction of m. Levator Auris Longus was chosen as the model of the cholinergic synapse. ACh release was assessed by electrophysiological methods. Changes in calcium transients were recorded using a calcium-sensitive dye. Nicotine hydrogen tartrate salt application (10 µM) decreased the amount of evoked ACh release, while the calcium transient increased in the motor nerve terminal. Both of these effects of nicotine were abolished by the neuronal ACh receptor antagonist dihydro-beta-erythroidine and Cav1 blockers, verapamil, and nitrendipine. These data allow us to suggest that neuronal nicotinic ACh receptor activation decreases the number of ACh quanta released by boosting calcium influx through Cav1 channels.


Assuntos
Acetilcolina/metabolismo , Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Neurônios Motores/metabolismo , Junção Neuromuscular/metabolismo , Animais , Eletrodiagnóstico , Feminino , Técnicas In Vitro , Masculino , Camundongos Endogâmicos BALB C , Nicotina , Canais de Potássio Cálcio-Ativados/metabolismo
2.
Int J Mol Sci ; 22(15)2021 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-34360687

RESUMO

In the intestine, the innate immune system excludes harmful substances and invading microorganisms. Tuft cells are taste-like chemosensory cells found in the intestinal epithelium involved in the activation of group 2 innate lymphoid cells (ILC2). Although tuft cells in other tissues secrete the neurotransmitter acetylcholine (ACh), their function in the gut remains poorly understood. In this study, we investigated changes in the expression of genes and cell differentiation of the intestinal epithelium by stimulation with interleukin-4 (IL-4) or IL-13 in macaque intestinal organoids. Transcriptome analysis showed that tuft cell marker genes were highly expressed in the IL-4- and IL-13-treated groups compared with the control, and the gene expression of choline acetyltransferase (ChAT), a synthesis enzyme of ACh, was upregulated in IL-4- and IL-13-treated groups. ACh accumulation was observed in IL-4-induced organoids using high-performance liquid chromatography-mass spectrometry (HPLC/MS), and ACh strongly released granules from Paneth cells. This study is the first to demonstrate ACh upregulation by IL-4 induction in primates, suggesting that IL-4 plays a role in Paneth cell granule secretion via paracrine stimulation.


Assuntos
Acetilcolina/metabolismo , Diferenciação Celular , Interleucina-4/farmacologia , Intestinos/fisiologia , Organoides/metabolismo , Animais , Perfilação da Expressão Gênica , Interleucina-4/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Macaca fuscata/fisiologia , Macaca mulatta/metabolismo , Macaca mulatta/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Organoides/efeitos dos fármacos , Organoides/fisiologia
3.
Eur J Med Chem ; 223: 113663, 2021 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-34198150

RESUMO

Acetylcholinesterase (AChE) inhibitors are currently the first-line drugs approved by the FDA for the treatment of Alzheimer's disease (AD). However, a short effective-window limits their therapeutic benefits. Clinical studies have confirmed that the combination of AChE inhibitors and neuroprotective agents exhibits better anti-AD effects. We have previously reported that the dual AChE/GSK3ß (Glycogen synthase kinase 3ß) modulators have both neuroprotective effects and cognitive impairment-improvement effects. In this study, we characterized a new backbone of the AChE/GSK3ß inhibitor 11c. It was identified as a highly potent AChE inhibitor and was found superior to donepezil, the first-line drug for the treatment of AD. In vivo studies confirmed that 11c significantly inhibited the activity of AChE in the brain but had little effect on the activity of AChE in the intestine. This advantage of 11c was expected to reduce the peripheral side effects caused by donepezil. Furthermore, biomarker studies have shown that 11c also improved the levels of acetylcholine and synaptophysin in the brain and exhibited neuroprotective effects. Preliminary in vivo and in vitro research results underline the exciting potential of compound 11c in the treatment of AD.


Assuntos
Acetilcolina/metabolismo , Acetilcolinesterase/química , Encéfalo/metabolismo , Inibidores da Colinesterase/química , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Niacinamida/química , Acetilcolinesterase/metabolismo , Doença de Alzheimer/tratamento farmacológico , Animais , Sítios de Ligação , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Encéfalo/efeitos dos fármacos , Inibidores da Colinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/uso terapêutico , Desenho de Fármacos , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Intestinos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Simulação de Dinâmica Molecular , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Niacinamida/metabolismo , Niacinamida/farmacologia , Niacinamida/uso terapêutico , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Relação Estrutura-Atividade , Regulação para Cima/efeitos dos fármacos
4.
Int J Mol Sci ; 22(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202925

RESUMO

Acetylcholine (ACh) is the classical neurotransmitter in the cholinergic nervous system. However, ACh is now known to regulate various immune cell functions. In fact, T cells, B cells, and macrophages all express components of the cholinergic system, including ACh, muscarinic, and nicotinic ACh receptors (mAChRs and nAChRs), choline acetyltransferase, acetylcholinesterase, and choline transporters. In this review, we will discuss the actions of ACh in the immune system. We will first briefly describe the mechanisms by which ACh is stored in and released from immune cells. We will then address Ca2+ signaling pathways activated via mAChRs and nAChRs on T cells and B cells, highlighting the importance of ACh for the function of T cells, B cells, and macrophages, as well as its impact on innate and acquired (cellular and humoral) immunity. Lastly, we will discuss the effects of two peptide ligands, secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1) and hippocampal cholinergic neurostimulating peptide (HCNP), on cholinergic activity in T cells. Overall, we stress the fact that ACh does not function only as a neurotransmitter; it impacts immunity by exerting diverse effects on immune cells via mAChRs and nAChRs.


Assuntos
Imunomodulação , Receptores Muscarínicos/metabolismo , Receptores Nicotínicos/metabolismo , Acetilcolina/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Regulação da Expressão Gênica , Humanos , Sistema Imunitário/imunologia , Sistema Imunitário/metabolismo , Imunidade , Linfócitos/imunologia , Linfócitos/metabolismo , Especificidade de Órgãos , Peptídeos/metabolismo , Peptídeos/farmacologia , Receptores Muscarínicos/genética , Receptores Nicotínicos/genética , Transdução de Sinais
5.
Molecules ; 26(9)2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-34062829

RESUMO

Betulinic acid (BA) is a major constituent of Zizyphus seeds that have been long used as therapeutic agents for sleep-related issues in Asia. BA is a pentacyclic triterpenoid. It also possesses various anti-cancer and anti-inflammatory effects. Current commercially available sleep aids typically use GABAergic regulation, for which many studies are being actively conducted. However, few studies have focused on acetylcholine receptors that regulate wakefulness. In this study, we utilized BA as an antagonist of α3ß4 nicotinic acetylcholine receptors (α3ß4 nAChRs) known to regulate rapid-eye-movement (REM) sleep and wakefulness. Effects of BA on α3ß4 nAChRs were concentration-dependent, reversible, voltage-independent, and non-competitive. Site-directed mutagenesis and molecular-docking studies confirmed the binding of BA at the molecular level and showed that the α3 subunit L257 and the ß4 subunit I263 residues affected BA binding. These data demonstrate that BA can bind to a binding site different from the site for the receptor's ligand, acetylcholine (ACh). This suggests that BA may be an effective antagonist that is unaffected by large amounts of ACh released during wakefulness and REM sleep. Based on the above experimental results, BA is likely to be a therapeutically useful sleep aid and sedative.


Assuntos
Acetilcolina/metabolismo , Triterpenos Pentacíclicos/farmacologia , Receptores Nicotínicos/metabolismo , Animais , Sítios de Ligação , Bovinos , Eletrofisiologia , Ligantes , Simulação de Acoplamento Molecular , Mutagênese , Mutação , Oócitos/citologia , Oócitos/metabolismo , Ligação Proteica , Conformação Proteica , Subunidades Proteicas/química , Sementes , Sono , Distúrbios do Início e da Manutenção do Sono/metabolismo , Transcrição Genética , Triterpenos/farmacologia , Xenopus laevis , Ziziphus , Ácido gama-Aminobutírico/metabolismo
6.
Int J Mol Sci ; 22(11)2021 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-34071104

RESUMO

Dorsal root ganglia (DRG) neurons synthesize acetylcholine (ACh), in addition to their peptidergic nature. They also release ACh and are cholinoceptive, as they express cholinergic receptors. During gangliogenesis, ACh plays an important role in neuronal differentiation, modulating neuritic outgrowth and neurospecific gene expression. Starting from these data, we studied the expression of choline acetyltransferase (ChAT) and vesicular ACh transporter (VAChT) expression in rat DRG neurons. ChAT and VAChT genes are arranged in a "cholinergic locus", and several splice variants have been described. Using selective primers, we characterized splice variants of these cholinergic markers, demonstrating that rat DRGs express R1, R2, M, and N variants for ChAT and V1, V2, R1, and R2 splice variants for VAChT. Moreover, by RT-PCR analysis, we observed a progressive decrease in ChAT and VAChT transcripts from the late embryonic developmental stage (E18) to postnatal P2 and P15 and in the adult DRG. Interestingly, Western blot analyses and activity assays demonstrated that ChAT levels significantly increased during DRG ontogenesis. The modulated expression of different ChAT and VAChT splice variants during development suggests a possible differential regulation of cholinergic marker expression in sensory neurons and confirms multiple roles for ACh in DRG neurons, both in the embryo stage and postnatally.


Assuntos
Colina O-Acetiltransferase/biossíntese , Neurônios Colinérgicos/metabolismo , Gânglios Espinais/citologia , Proteínas do Tecido Nervoso/biossíntese , Células Receptoras Sensoriais/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/biossíntese , Acetilcolina/metabolismo , Processamento Alternativo , Animais , Colina O-Acetiltransferase/genética , Neurônios Colinérgicos/citologia , Gânglios Espinais/embriologia , Gânglios Espinais/crescimento & desenvolvimento , Proteínas do Tecido Nervoso/genética , Neurogênese , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Receptoras Sensoriais/citologia , Vesículas Sinápticas/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/genética
7.
Molecules ; 26(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34070099

RESUMO

Wild ginseng has better pharmacological effects than cultivated ginseng. However, its industrialization is limited by the inability to grow wild ginseng on a large scale. Herein, we demonstrate how to optimize ginseng production through cultivation, and how to enhance the concentrations of specific ginsenosides through fermentation. In the study, we also evaluated the ability of fermented cultured wild ginseng root extract (HLJG0701-ß) to inhibit acetylcholinesterase (AChE), as well as its neuroprotective effects and antioxidant activity. In invitro tests, HLJG0701-ß inhibited AChE activity and exerted neuroprotective and antioxidant effects (showing increased catalyst activity but decreased reactive oxygen species concentration). In invivo tests, after HLJG0701-ß was orally administered at doses of 0, 125, 250, and 500 mg/kg in an animal model of memory impairment, behavioral evaluation (Morris water maze test and Y-maze task test) was performed. The levels of AChE, acetylcholine (ACh), blood catalase (CAT), and malondialdehyde (MDA) in brain tissues were measured. The results showed that HLJG0701-ß produced the best results at a dose of 250 mg/kg or more. The neuroprotective mechanism of HLJG0701-ß was determined to involve the inhibition of AChE activity and a decrease in oxidative stress. In summary, both invitro and invivo tests confirmed that HJG0701-ß administration can lead to memory improvement.


Assuntos
Antioxidantes/farmacologia , Fermentação , Fármacos Neuroprotetores/farmacologia , Panax/química , Extratos Vegetais/farmacologia , Raízes de Plantas/química , Acetilcolina/metabolismo , Acetilcolinesterase/metabolismo , Animais , Encéfalo/metabolismo , Catalase/sangue , Catalase/metabolismo , Inibidores da Colinesterase/farmacologia , Modelos Animais de Doenças , Feminino , Galactose , Ginsenosídeos/farmacologia , Masculino , Malondialdeído/sangue , Camundongos , Teste do Labirinto Aquático de Morris , Ovariectomia , Espécies Reativas de Oxigênio/metabolismo , Escopolamina
8.
FASEB J ; 35(7): e21724, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34133802

RESUMO

Neuromuscular junctions (NMJ) regulate cholinergic exocytosis through the M1 and M2 muscarinic acetylcholine autoreceptors (mAChR), involving the crosstalk between receptors and downstream pathways. Protein kinase C (PKC) regulates neurotransmission but how it associates with the mAChRs remains unknown. Here, we investigate whether mAChRs recruit the classical PKCßI and the novel PKCε isoforms and modulate their priming by PDK1, translocation and activity on neurosecretion targets. We show that each M1 and M2 mAChR activates the master kinase PDK1 and promotes a particular priming of the presynaptic PKCßI and ε isoforms. M1 recruits both primed-PKCs to the membrane and promotes Munc18-1, SNAP-25, and MARCKS phosphorylation. In contrast, M2 downregulates PKCε through a PKA-dependent pathway, which inhibits Munc18-1 synthesis and PKC phosphorylation. In summary, our results discover a co-dependent balance between muscarinic autoreceptors which orchestrates the presynaptic PKC and their action on ACh release SNARE-SM mechanism. Altogether, this molecular signaling explains previous functional studies at the NMJ and guide toward potential therapeutic targets.


Assuntos
Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Junção Neuromuscular/metabolismo , Proteína Quinase C/metabolismo , Receptores Muscarínicos/metabolismo , Acetilcolina/metabolismo , Animais , Regulação para Baixo/fisiologia , Exocitose/fisiologia , Fosforilação/fisiologia , Terminações Pré-Sinápticas/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Transmissão Sináptica/fisiologia , Proteína 25 Associada a Sinaptossoma/metabolismo
9.
Commun Biol ; 4(1): 603, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-34021245

RESUMO

Social discrimination in rats requires activation of the intrinsic bulbar vasopressin system, but it is unclear how this system comes into operation, as olfactory nerve stimulation primarily inhibits bulbar vasopressin cells (VPCs). Here we show that stimulation with a conspecific can activate bulbar VPCs, indicating that VPC activation depends on more than olfactory cues during social interaction. A series of in vitro electrophysiology, pharmacology and immunohistochemistry experiments implies that acetylcholine, probably originating from centrifugal projections, can enable olfactory nerve-evoked action potentials in VPCs. Finally, cholinergic activation of the vasopressin system contributes to vasopressin-dependent social discrimination, since recognition of a known rat was blocked by bulbar infusion of the muscarinic acetylcholine receptor antagonist atropine and rescued by additional bulbar application of vasopressin. Thus, our results implicate that top-down cholinergic modulation of bulbar VPC activity is involved in social discrimination in rats.


Assuntos
Acetilcolina/metabolismo , Potenciais de Ação , Neurônios/fisiologia , Bulbo Olfatório/fisiologia , Discriminação Social , Vasopressinas/metabolismo , Animais , Feminino , Masculino , Neurônios/citologia , Bulbo Olfatório/citologia , Ratos , Ratos Wistar
10.
Expert Opin Drug Metab Toxicol ; 17(7): 841-856, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33999717

RESUMO

Introduction: Alzheimer's disease and depression are health conditions affecting millions of people around the world. Both are strongly related to the level of the neurotransmitter acetylcholine. Since cholinergic deficit is characteristic of Alzheimer's disease, acetylcholinesterase inhibitors are applied as relevant drugs for the treatment of this disease, elevating the level of acetylcholine. On the other hand, a high level of acetylcholine is found to be associated with the symptoms of clinical depression.Areas covered: This article aims to discuss if acetylcholinesterase inhibitors used as anti-Alzheimer's drugs could be the cause of the symptoms of clinical depression often linked to this neurological disorder. Emphasis will be put on drugs currently in use and on newly investigated natural products, which can inhibit AChE activity.Expert opinion: Currently, it is not proven that the patient treated for Alzheimer's disease is prone to increased risk for depression due to the acetylcholinesterase inhibition, but there are strong indications. The level of acetylcholine is not the only factor in highly complicated diseases like AD and depression. Still, it needs to be considered isolated, keeping in mind the nature of presently available therapy, especially during a rational drug design process.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Inibidores da Colinesterase/efeitos adversos , Depressão/etiologia , Acetilcolina/metabolismo , Acetilcolinesterase/efeitos dos fármacos , Acetilcolinesterase/metabolismo , Doença de Alzheimer/psicologia , Animais , Produtos Biológicos/administração & dosagem , Produtos Biológicos/efeitos adversos , Produtos Biológicos/farmacologia , Inibidores da Colinesterase/administração & dosagem , Depressão/epidemiologia , Desenho de Fármacos , Humanos , Risco
11.
Cell Mol Life Sci ; 78(13): 5381-5395, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34028590

RESUMO

The α7 nicotinic acetylcholine receptor is involved in neurological, neurodegenerative, and inflammatory disorders. It operates both as a ligand-gated cationic channel and as a metabotropic receptor in neuronal and non-neuronal cells. As protein phosphorylation is an important cell function regulatory mechanism, deciphering how tyrosine phosphorylation modulates α7 dual ionotropic/metabotropic molecular function is required for understanding its integral role in physiological and pathological processes. α7 single-channel activity elicited by ACh appears as brief isolated openings and less often as episodes of few openings in quick succession. The reduction of phosphorylation by tyrosine kinase inhibition increases the duration and frequency of activation episodes, whereas the inhibition of phosphatases has the opposite effect. Removal of two tyrosine residues at the α7 intracellular domain recapitulates the effects mediated by tyrosine kinase inhibition. The tyrosine-free mutant receptor shows longer duration-activation episodes, reduced desensitization rate and significantly faster recovery from desensitization, indicating that phosphorylation decreases α7 channel activity by favoring the desensitized state. However, the mutant receptor is incapable of triggering ERK1/2 phosphorylation in response to the α7-agonist. Thus, while tyrosine phosphorylation is absolutely required for α7-triggered ERK pathway, it negatively modulates α7 ionotropic activity. Overall, phosphorylation/dephosphorylation events fine-tune the integrated cell response mediated by α7 activation, thus having a broad impact on α7 cholinergic signaling.


Assuntos
Acetilcolina/metabolismo , Neurônios/metabolismo , Tirosina/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Quinases da Família src/metabolismo , Células HEK293 , Humanos , Neurônios/citologia , Fosforilação , Transdução de Sinais , Receptor Nicotínico de Acetilcolina alfa7/genética , Quinases da Família src/genética
12.
Int J Mol Sci ; 22(9)2021 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-33924758

RESUMO

Adrenoceptor activators and blockers are widely used clinically for the treatment of cardiovascular and pulmonary disorders. More recently, adrenergic agents have also been used to treat neurodegenerative diseases. Recent studies indicate a location of sympathetic varicosities in close proximity to neuromuscular junctions. The pressing question is whether there could be any effects of endo- or exogenous catecholamines on cholinergic neuromuscular transmission. It was shown that the pharmacological stimulation of adrenoceptors, as well as sympathectomy, can affect both acetylcholine release from motor nerve terminals and the functioning of postsynaptic acetylcholine receptors. In this review, we discuss the recent data regarding the effects of adrenergic drugs on neurotransmission at the neuromuscular junction. The elucidation of the molecular mechanisms by which the clinically relevant adrenomimetics and adrenoblockers regulate quantal acetylcholine release from the presynaptic nerve terminals and postsynaptic sensitivity may help in the design of highly effective and well-tolerated sympathomimetics for treating a number of neurodegenerative diseases accompanied by synaptic defects.


Assuntos
Acetilcolina/metabolismo , Neurônios Colinérgicos/metabolismo , Junção Neuromuscular/metabolismo , Receptores Adrenérgicos/metabolismo , Transmissão Sináptica , Animais , Humanos , Receptores Nicotínicos/metabolismo , Simpatomiméticos
13.
FEBS J ; 288(18): 5331-5349, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33730374

RESUMO

Motoneurons (MNs) control muscle activity by releasing the neurotransmitter acetylcholine (ACh) at the level of neuromuscular junctions. ACh is packaged into synaptic vesicles by the vesicular ACh transporter (VAChT), and disruptions in its release can impair muscle contraction, as seen in congenital myasthenic syndromes (CMS). Recently, VAChT gene mutations were identified in humans displaying varying degrees of myasthenia. Moreover, mice with a global deficiency in VAChT expression display several characteristics of CMS. Despite these findings, little is known about how a long-term decrease in VAChT expression in vivo affects MNs structure and function. Using Cre-loxP technology, we generated a mouse model where VAChT is deleted in select groups of MNs (mnVAChT-KD). Molecular analysis revealed that the VAChT deletion was specific to MNs and affected approximately 50% of its population in the brainstem and spinal cord, with alpha-MNs primarily targeted (70% in spinal cord). Within each animal, the cell body area of VAChT-deleted MNs was significantly smaller compared to MNs with VAChT preserved. Likewise, muscles innervated by VAChT-deleted MNs showed atrophy while muscles innervated by VAChT-containing neurons appeared normal. In addition, mnVAChT KD mice had decreased muscle strength, were hypoactive, leaner and exhibited kyphosis. This neuromuscular dysfunction was evident at 2 months of age and became progressively worse by 6 months. Treatment of mutants with a cholinesterase inhibitor was able to improve some of the motor deficits. As these observations mimic what is seen in CMS, this new line could be valuable for assessing the efficacy of potential CMS drugs.


Assuntos
Acetilcolina/genética , Neurônios Motores/metabolismo , Síndromes Miastênicas Congênitas/genética , Proteínas Vesiculares de Transporte de Acetilcolina/genética , Acetilcolina/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Neurônios Motores/patologia , Contração Muscular/genética , Contração Muscular/fisiologia , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Síndromes Miastênicas Congênitas/metabolismo , Síndromes Miastênicas Congênitas/patologia , Junção Neuromuscular/genética , Junção Neuromuscular/metabolismo , Neurotransmissores/genética , Medula Espinal/metabolismo , Medula Espinal/fisiologia , Transmissão Sináptica/genética , Vesículas Sinápticas/metabolismo
14.
Nat Commun ; 12(1): 1374, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33654091

RESUMO

In many parts of the central nervous system, including the retina, it is unclear whether cholinergic transmission is mediated by rapid, point-to-point synaptic mechanisms, or slower, broad-scale 'non-synaptic' mechanisms. Here, we characterized the ultrastructural features of cholinergic connections between direction-selective starburst amacrine cells and downstream ganglion cells in an existing serial electron microscopy data set, as well as their functional properties using electrophysiology and two-photon acetylcholine (ACh) imaging. Correlative results demonstrate that a 'tripartite' structure facilitates a 'multi-directed' form of transmission, in which ACh released from a single vesicle rapidly (~1 ms) co-activates receptors expressed in multiple neurons located within ~1 µm of the release site. Cholinergic signals are direction-selective at a local, but not global scale, and facilitate the transfer of information from starburst to ganglion cell dendrites. These results suggest a distinct operational framework for cholinergic signaling that bears the hallmarks of synaptic and non-synaptic forms of transmission.


Assuntos
Acetilcolina/metabolismo , Sistema Nervoso Central/fisiologia , Transmissão Sináptica/fisiologia , Células Amácrinas/fisiologia , Células Amácrinas/ultraestrutura , Animais , Dendritos/fisiologia , Dendritos/ultraestrutura , Cinética , Camundongos Endogâmicos C57BL , Fótons , Células Ganglionares da Retina/ultraestrutura
15.
Toxicol Appl Pharmacol ; 419: 115512, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33785355

RESUMO

Nerve agents are highly toxic organophosphorus compounds that inhibit acetylcholinesterase resulting in rapid accumulation of the neurotransmitter acetylcholine (ACh) causing a cholinergic syndrome including respiratory failure. In the present study, respiratory responses and antimuscarinic treatment efficacy was evaluated ex vivo using rat precision-cut lung slices (PCLS) exposed to the nerve agent VX. The respiratory effects were evaluated either by adding exogenous ACh directly to the culture medium or by applying electric-field stimulation (EFS) to the PCLS to achieve a release of endogenous ACh from neurons in the lung tissue. The airway contraction induced by both methods was enhanced by VX and resulted in lingering airway recovery, in particular when airways were exposed to a high VX-dose. Both contractions induced by EFS and exogenously added ACh were significantly reduced by administration of the antimuscarinic drugs atropine or scopolamine. Two additions of atropine or scopolamine after maximal ACh-induced airway response was demonstrated effective to reverse the contraction. By adding consecutive doubled doses of antimuscarinics, high efficiency to reduce the cholinergic airway response was observed. However, the airways were not completely recovered by atropine or scopolamine, indicating that non-muscarinic mechanisms were involved in the smooth muscle contractions. In conclusion, it was demonstrated that antimuscarinic treatment reversed airway contraction induced by VX but supplemental pharmacological interventions are needed to fully recover the airways. Further studies should therefore clarify the mechanisms of physiological responses in lung tissue following nerve agent exposures to improve the medical management of poisoned individuals.


Assuntos
Atropina/farmacologia , Fibras Colinérgicas/efeitos dos fármacos , Inibidores da Colinesterase/toxicidade , Pulmão/inervação , Antagonistas Muscarínicos/farmacologia , Contração Muscular/efeitos dos fármacos , Músculo Liso/inervação , Compostos Organotiofosforados/toxicidade , Escopolamina/farmacologia , Acetilcolina/metabolismo , Acetilcolina/farmacologia , Acetilcolinesterase/metabolismo , Animais , Fibras Colinérgicas/enzimologia , Relação Dose-Resposta a Droga , Estimulação Elétrica , Feminino , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/metabolismo , Ratos Sprague-Dawley
16.
Molecules ; 26(5)2021 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-33652901

RESUMO

Slow-channel congenital myasthenic syndromes (SCCMSs) are rare genetic diseases caused by mutations in muscle nicotinic acetylcholine receptor (nAChR) subunits. Most of the known SCCMS-associated mutations localize at the transmembrane region near the ion pore. Only two SCCMS point mutations are at the extracellular domains near the acetylcholine binding site, α1(G153S) being one of them. In this work, a combination of molecular dynamics, targeted mutagenesis, fluorescent Ca2+ imaging and patch-clamp electrophysiology has been applied to G153S mutant muscle nAChR to investigate the role of hydrogen bonds formed by Ser 153 with C-loop residues near the acetylcholine-binding site. Introduction of L199T mutation to the C-loop in the vicinity of Ser 153 changed hydrogen bonds distribution, decreased acetylcholine potency (EC50 2607 vs. 146 nM) of the double mutant and decay kinetics of acetylcholine-evoked cytoplasmic Ca2+ rise (τ 14.2 ± 0.3 vs. 34.0 ± 0.4 s). These results shed light on molecular mechanisms of nAChR activation-desensitization and on the involvement of such mechanisms in channelopathy genesis.


Assuntos
Acetilcolina/genética , Sequência de Aminoácidos/genética , Síndromes Miastênicas Congênitas/genética , Receptores Nicotínicos/genética , Acetilcolina/metabolismo , Sítios de Ligação/genética , Cálcio/metabolismo , Humanos , Cinética , Síndromes Miastênicas Congênitas/patologia , Técnicas de Patch-Clamp , Mutação Puntual/genética , Ligação Proteica/genética
17.
Life Sci ; 273: 119300, 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-33662433

RESUMO

AIMS: Plasma hyperlipidemia is a protective factor in amyotrophic lateral sclerosis (ALS) while cholesterol-lowering drugs aggravate the pathology. We hypothesize that this phenomenon can be linked with membrane lipid alterations in the neuromuscular junctions (NMJs) occurring before motor neuron loss. METHODS: Neurotransmitter release in parallel with lipid membrane properties in diaphragm NMJs of SOD1G93A (mSOD) mice at nine weeks of age (pre-onset stage) were assessed. KEY FINDINGS: Despite on slight changes in spontaneous and evoked quantum release of acetylcholine, extracellular levels of choline at resting conditions, an indicator of non-quantum release, were significantly increased in mSOD mice. The use of lipid-sensitive fluorescent probes points to lipid raft disruption in the NMJs of mSOD mice. However, content of cholesterol, a key raft component was unchanged implying another pathway responsible for the loss of raft integrity. In the mSOD mice we found marked increase in levels of raft-destabilizing lipid ceramide. This was accompanied by enhanced ability to uptake of exogenous ceramide in NMJs. Acute and chronic administration of 25-hydroxycholesterol, whose levels increase due to hypercholesterolemia, recovered early alterations in membrane properties. Furthermore, chronic treatment with 25-hydroxycholesterol prevented increase in ceramide and extracellular choline levels as well as suppressed lipid peroxidation of NMJ membranes and fragmentation of end plates. SIGNIFICANCE: Thus, lipid raft disruption likely due to ceramide accumulation could be early event in ALS which may trigger neuromuscular abnormalities. Cholesterol derivative 25-hydroxycholesterol may serve as a molecule restoring the membrane and functional properties of NMJs at the early stage.


Assuntos
Esclerose Amiotrófica Lateral/tratamento farmacológico , Modelos Animais de Doenças , Hidroxicolesteróis/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Superóxido Dismutase-1/fisiologia , Acetilcolina/metabolismo , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Ceramidas/metabolismo , Colesterol/metabolismo , Feminino , Masculino , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Junção Neuromuscular , Transmissão Sináptica
18.
Cardiovasc Diabetol ; 20(1): 50, 2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33618724

RESUMO

BACKGROUND: Acetylcholine (ACh) plays a crucial role in the function of the heart. Recent evidence suggests that cardiomyocytes possess a non-neuronal cholinergic system (NNCS) that comprises of choline acetyltransferase (ChAT), choline transporter 1 (CHT1), vesicular acetylcholine transporter (VAChT), acetylcholinesterase (AChE) and type-2 muscarinic ACh receptors (M2AChR) to synthesize, release, degrade ACh as well as for ACh to transduce a signal. NNCS is linked to cardiac cell survival, angiogenesis and glucose metabolism. Impairment of these functions are hallmarks of diabetic heart disease (DHD). The role of the NNCS in DHD is unknown. The aim of this study was to examine the effect of diabetes on cardiac NNCS and determine if activation of cardiac NNCS is beneficial to the diabetic heart. METHODS: Ventricular samples from type-2 diabetic humans and db/db mice were used to measure the expression pattern of NNCS components (ChAT, CHT1, VAChT, AChE and M2AChR) and glucose transporter-4 (GLUT-4) by western blot analysis. To determine the function of the cardiac NNCS in the diabetic heart, a db/db mouse model with cardiac-specific overexpression of ChAT gene was generated (db/db-ChAT-tg). Animals were followed up serially and samples collected at different time points for molecular and histological analysis of cardiac NNCS components and prosurvival and proangiogenic signaling pathways. RESULTS: Immunoblot analysis revealed alterations in the components of cardiac NNCS and GLUT-4 in the type-2 diabetic human and db/db mouse hearts. Interestingly, the dysregulation of cardiac NNCS was followed by the downregulation of GLUT-4 in the db/db mouse heart. Db/db-ChAT-tg mice exhibited preserved cardiac and vascular function in comparison to db/db mice. The improved function was associated with increased cardiac ACh and glucose content, sustained angiogenesis and reduced fibrosis. These beneficial effects were associated with upregulation of the PI3K/Akt/HIF1α signaling pathway, and increased expression of its downstream targets-GLUT-4 and VEGF-A. CONCLUSION: We provide the first evidence for dysregulation of the cardiac NNCS in DHD. Increased cardiac ACh is beneficial and a potential new therapeutic strategy to prevent or delay the development of DHD.


Assuntos
Acetilcolina/metabolismo , Diabetes Mellitus Tipo 2/complicações , Cardiomiopatias Diabéticas/prevenção & controle , Glucose/metabolismo , Ventrículos do Coração/metabolismo , Acetilcolinesterase/metabolismo , Idoso , Animais , Estudos de Casos e Controles , Colina O-Acetiltransferase/genética , Colina O-Acetiltransferase/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Cardiomiopatias Diabéticas/etiologia , Cardiomiopatias Diabéticas/metabolismo , Cardiomiopatias Diabéticas/fisiopatologia , Modelos Animais de Doenças , Feminino , Proteínas Ligadas por GPI/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Humanos , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptor Muscarínico M2/metabolismo , Simportadores/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismo
19.
Int J Mol Sci ; 22(3)2021 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-33525357

RESUMO

Neurodegenerative diseases are a major public health problem worldwide with a wide spectrum of symptoms and physiological effects. It has been long reported that the dysregulation of the cholinergic system and the adrenergic system are linked to the etiology of Alzheimer's disease. Cholinergic neurons are widely distributed in brain regions that play a role in cognitive functions and normal cholinergic signaling related to learning and memory is dependent on acetylcholine. The Locus Coeruleus norepinephrine (LC-NE) is the main noradrenergic nucleus that projects and supplies norepinephrine to different brain regions. Norepinephrine has been shown to be neuroprotective against neurodegeneration and plays a role in behavior and cognition. Cholinergic and adrenergic signaling are dysregulated in Alzheimer's disease. The degeneration of cholinergic neurons in nucleus basalis of Meynert in the basal forebrain and the degeneration of LC-NE neurons were reported in Alzheimer's disease. The aim of this review is to describe current literature on the role of the cholinergic system and the adrenergic system (LC-NE) in the pathology of Alzheimer's disease and potential therapeutic implications.


Assuntos
Neurônios Adrenérgicos/metabolismo , Doença de Alzheimer/metabolismo , Núcleo Basal de Meynert/metabolismo , Neurônios Colinérgicos/metabolismo , Hipocampo/metabolismo , Locus Cerúleo/metabolismo , Acetilcolina/metabolismo , Neurônios Adrenérgicos/patologia , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Animais , Núcleo Basal de Meynert/patologia , Neurônios Colinérgicos/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Hipocampo/patologia , Humanos , Locus Cerúleo/patologia , Memória/fisiologia , Norepinefrina/metabolismo , Receptores Muscarínicos/genética , Receptores Muscarínicos/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Transdução de Sinais
20.
Yakugaku Zasshi ; 141(2): 245-254, 2021.
Artigo em Japonês | MEDLINE | ID: mdl-33518645

RESUMO

Distigmine bromide (distigmine) is a carbamate cholinesterase (ChE) inhibitor, which is mainly used for the treatment of myasthenia gravis. Distigmine is also used in Japan for the treatment for underactive bladder and glaucoma. The effectiveness of distigmine for underactive bladder treatment has been confirmed by many clinical reports, and this effect is thought to be caused by potentiating urinary bladder smooth muscle contraction due to inhibition of acetylcholine degradation during micturition. However, the pharmacological effects of distigmine on urinary bladder smooth muscle have not been well studied. The most distinctive pharmacological feature of distigmine is that it shows long-lasting effects than other ChE inhibitors; however, few studies have investigated the persistence of the enhancing effect of distigmine on the contractile function of urinary bladder smooth muscle. Moreover, this mechanism remains unclear. In this review, we present our findings on the mechanism of the potentiating effect of distigmine on isolated guinea pig urinary bladder smooth muscle contraction. We also discuss the long-lasting potentiating effect of distigmine on urinary bladder motility and the mechanism of these effects using guinea pig urinary bladder smooth muscle in vivo and in vitro. In addition, we present our investigations on the long-lasting mechanism of distigmine using recombinant human acetylcholinesterase.


Assuntos
Inibidores da Colinesterase/farmacologia , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Compostos de Piridínio/farmacologia , Bexiga Urinária/efeitos dos fármacos , Acetilcolina/metabolismo , Animais , Cobaias , Humanos , Técnicas In Vitro , Camundongos , Ratos , Estimulação Química , Micção/efeitos dos fármacos
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