RESUMO
Mucociliary clearance through coordinated ciliary beating is a major innate defense removing pathogens from the lower airways, but the pathogen sensing and downstream signaling mechanisms remain unclear. We identified virulence-associated formylated bacterial peptides that potently stimulated ciliary-driven transport in the mouse trachea. This innate response was independent of formyl peptide and taste receptors but depended on key taste transduction genes. Tracheal cholinergic chemosensory cells expressed these genes, and genetic ablation of these cells abrogated peptide-driven stimulation of mucociliary clearance. Trpm5-deficient mice were more susceptible to infection with a natural pathogen, and formylated bacterial peptides were detected in patients with chronic obstructive pulmonary disease. Optogenetics and peptide stimulation revealed that ciliary beating was driven by paracrine cholinergic signaling from chemosensory to ciliated cells operating through muscarinic M3 receptors independently of nerves. We provide a cellular and molecular framework that defines how tracheal chemosensory cells integrate chemosensation with innate defense.
Assuntos
Acetilcolina/imunologia , Proteínas de Bactérias/farmacologia , Cílios/imunologia , Depuração Mucociliar/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Canais de Cátion TRPM/imunologia , Traqueia/imunologia , Acetilcolina/metabolismo , Animais , Proteínas de Bactérias/imunologia , Transporte Biológico , Cílios/efeitos dos fármacos , Cílios/metabolismo , Feminino , Formiatos/metabolismo , Expressão Gênica , Humanos , Imunidade Inata , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Optogenética/métodos , Comunicação Parácrina/imunologia , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/patologia , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/imunologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/imunologia , Canais de Cátion TRPM/deficiência , Canais de Cátion TRPM/genética , Papilas Gustativas/imunologia , Papilas Gustativas/metabolismo , Traqueia/efeitos dos fármacos , Traqueia/patologia , VirulênciaRESUMO
Stroke is a leading cause of death and disability. Recovery depends on a delicate balance between inflammatory responses and immune suppression, tipping the scale between brain protection and susceptibility to infection. Peripheral cholinergic blockade of immune reactions fine-tunes this immune response, but its molecular regulators are unknown. Here, we report a regulatory shift in small RNA types in patient blood sequenced 2 d after ischemic stroke, comprising massive decreases of microRNA levels and concomitant increases of transfer RNA fragments (tRFs) targeting cholinergic transcripts. Electrophoresis-based size-selection followed by qRT-PCR validated the top six up-regulated tRFs in a separate cohort of stroke patients, and independent datasets of small and long RNA sequencing pinpointed immune cell subsets pivotal to these responses, implicating CD14+ monocytes in the cholinergic inflammatory reflex. In-depth small RNA targeting analyses revealed the most-perturbed pathways following stroke and implied a structural dichotomy between microRNA and tRF target sets. Furthermore, lipopolysaccharide stimulation of murine RAW 264.7 cells and human CD14+ monocytes up-regulated the top six stroke-perturbed tRFs, and overexpression of stroke-inducible tRF-22-WE8SPOX52 using a single-stranded RNA mimic induced down-regulation of immune regulator Z-DNA binding protein 1. In summary, we identified a "changing of the guards" between small RNA types that may systemically affect homeostasis in poststroke immune responses, and pinpointed multiple affected pathways, which opens new venues for establishing therapeutics and biomarkers at the protein and RNA level.
Assuntos
AVC Isquêmico/genética , AVC Isquêmico/imunologia , MicroRNAs/imunologia , Sistema Colinérgico não Neuronal/imunologia , RNA de Transferência/imunologia , Idoso , Animais , Estudos de Casos e Controles , Feminino , Humanos , Inflamação/etiologia , Inflamação/genética , Inflamação/imunologia , AVC Isquêmico/fisiopatologia , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , MicroRNAs/sangue , MicroRNAs/genética , Pessoa de Meia-Idade , Monócitos/fisiologia , Sistema Colinérgico não Neuronal/genética , Estudos Prospectivos , Células RAW 264.7 , RNA de Transferência/sangue , RNA de Transferência/genéticaRESUMO
Tau protein aggregations are important contributors to the etiology of Alzheimer's disease (AD). Hydromethylthionine (HMT) is a potent inhibitor of tau aggregation in vitro and in vivo and is being developed as a possible anti-dementia medication. HMT was also shown to affect the cholinergic system and to interact with mitochondria. Here, we used tau-transgenic (L1 and L66) and wild-type NMRI mice that were treated with HMT, rivastigmine and memantine and with combinations thereof, for 2-4 weeks. We measured HMT concentrations in both brain homogenates and isolated mitochondria and concentrations of glucose, lactate and pyruvate in brain by microdialysis. In isolated brain mitochondria, we recorded oxygen consumption of mitochondrial complexes by respirometry. While rivastigmine and memantine lowered mitochondrial respiration, HMT did not affect respiration in wild-type animals and increased respiration in tau-transgenic L1 mice. Glucose and lactate levels were not affected by HMT administration. The presence of HMT in isolated mitochondria was established. In summary, traditional anti-dementia drugs impair mitochondrial function while HMT has no adverse effects on mitochondrial respiration in tau-transgenic mice. These results support the further development of HMT as an anti-dementia drug.
Assuntos
Doença de Alzheimer , Memantina , Camundongos , Animais , Rivastigmina/farmacologia , Memantina/farmacologia , Memantina/uso terapêutico , Proteínas tau/genética , Proteínas tau/metabolismo , Camundongos Transgênicos , Inibidores da Colinesterase/farmacologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Doença de Alzheimer/induzido quimicamente , Mitocôndrias/metabolismoRESUMO
The prevention of tau protein aggregations is a therapeutic goal for the treatment of Alzheimer's disease (AD), and hydromethylthionine (HMT) (also known as leucomethylthioninium-mesylate [LMTM]), is a potent inhibitor of tau aggregation in vitro and in vivo. In two Phase 3 clinical trials in AD, HMT had greater pharmacological activity on clinical endpoints in patients not receiving approved symptomatic treatments for AD (acetylcholinesterase (AChE) inhibitors and/or memantine) despite different mechanisms of action. To investigate this drug interaction in an animal model, we used tau-transgenic L1 and wild-type NMRI mice treated with rivastigmine or memantine prior to adding HMT, and measured changes in hippocampal acetylcholine (ACh) by microdialysis. HMT given alone doubled hippocampal ACh levels in both mouse lines and increased stimulated ACh release induced by exploration of the open field or by infusion of scopolamine. Rivastigmine increased ACh release in both mouse lines, whereas memantine was more active in tau-transgenic L1 mice. Importantly, our study revealed a negative interaction between HMT and symptomatic AD drugs: the HMT effect was completely eliminated in mice that had been pre-treated with either rivastigmine or memantine. Rivastigmine was found to inhibit AChE, whereas HMT and memantine had no effects on AChE or on choline acetyltransferase (ChAT). The interactions observed in this study demonstrate that HMT enhances cholinergic activity in mouse brain by a mechanism of action unrelated to AChE inhibition. Our findings establish that the drug interaction that was first observed clinically has a neuropharmacological basis and is not restricted to animals with tau aggregation pathology. Given the importance of the cholinergic system for memory function, the potential for commonly used AD drugs to interfere with the treatment effects of disease-modifying drugs needs to be taken into account in the design of clinical trials.
Assuntos
Hipocampo/efeitos dos fármacos , Memantina/farmacologia , Azul de Metileno/análogos & derivados , Rivastigmina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Acetilcolina/metabolismo , Acetilcolinesterase/metabolismo , Animais , Inibidores da Colinesterase/farmacologia , Dopaminérgicos/farmacologia , Interações Medicamentosas , Feminino , Hipocampo/metabolismo , Azul de Metileno/farmacologia , Camundongos , Camundongos TransgênicosRESUMO
ß-Hydroxybutyrate (BHB) is a ketone body formed in high amounts during lipolysis and fasting. Ketone bodies and the ketogenic diet were suggested as neuroprotective agents in neurodegenerative disease. In the present work, we induced transient ischemia in mouse brain by unilaterally occluding the middle cerebral artery for 90 min. BHB (30 mg/kg), given immediately after reperfusion, significantly improved the neurological score determined after 24 h. In isolated mitochondria from mouse brain, oxygen consumption by the complexes I, II and IV was reduced immediately after ischemia but recovered slowly over 1 week. The single acute BHB administration after reperfusion improved complex I and II activity after 24 h while no significant effects were seen at later time points. After 24 h, plasma and brain BHB concentrations were strongly increased while mitochondrial intermediates (citrate, succinate) were unchanged in brain tissue. Our data suggest that a single administration of BHB may improve mitochondrial respiration for 1-2 days but not for later time points. Endogenous BHB formation seems to complement the effects of exogenous BHB administration.
Assuntos
Doenças Neurodegenerativas , Fármacos Neuroprotetores , Ácido 3-Hidroxibutírico/farmacologia , Ácido 3-Hidroxibutírico/uso terapêutico , Animais , Citratos , Hidroxibutiratos , Isquemia , Corpos Cetônicos , Camundongos , Mitocôndrias , Fármacos Neuroprotetores/farmacologia , SuccinatosRESUMO
As glucose hypometabolism in the brain is an early sign of Alzheimer´s dementia (AD), the diabetogenic drug streptozotocin (STZ) has been used to induce Alzheimer-like pathology in rat brain by intracereboventricular injection (icv-STZ). However, many details of the pathological mechanism of STZ in this AD model remain unclear. Here, we report metabolic and cholinergic effects of icv-STZ using microdialysis in freely moving animals. We found that icv-STZ at a dose of 3 mg/kg (2 × 1.5 mg/kg) causes overt toxicity reflected in body weight loss. Three weeks after STZ administration, histological examination revealed a high number of glial fibrillary acidic protein reactive cells in the hippocampus, accompanied by Fluoro-Jade C-positive cells in the CA1 region. Glucose and lactate levels in microdialysates were unchanged, but mitochondrial respiration measured ex vivo was reduced by 9%-15%. High-affinity choline uptake, choline acetyltransferase, and acetylcholine esterase (AChE) activities in the hippocampus were reduced by 16%, 28%, and 30%, respectively. Importantly, extracellular acetylcholine (ACh) levels in the hippocampus were unchanged and responded to behavioral and pharmacological challenges. In comparison, extracellular ACh levels and cholinergic parameters in the striatum were unchanged or slightly increased. We conclude that the icv-STZ model poorly reflects central cholinergic dysfunction, an important characteristic of dementia. The icv-STZ model may be more aptly described as an animal model of hippocampal gliosis.
Assuntos
Acetilcolina/metabolismo , Lesões Encefálicas/induzido quimicamente , Lesões Encefálicas/metabolismo , Neurônios Colinérgicos/metabolismo , Modelos Animais de Doenças , Estreptozocina/toxicidade , Animais , Colina O-Acetiltransferase/metabolismo , Colinérgicos/administração & dosagem , Neurônios Colinérgicos/efeitos dos fármacos , Injeções Intraventriculares , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Microdiálise/métodos , Ratos , Ratos Wistar , Estreptozocina/administração & dosagemRESUMO
PURPOSE: Status epilepticus (SE) is characterized by recurrent seizure activity and can be drug- resistant. Knowledge of neuronal and metabolic activity of the brain during SE may be helpful to improve medical care. We here report the effects of three anti-seizure drugs on changes of acetylcholine energy metabolites and oxidative stress during SE. METHODS: We used the lithium-pilocarpine model in rats to induce SE and in vivo- microdialysis to monitor cholinergic and metabolic activity in the hippocampus. We measured extracellular concentrations of acetylcholine, glucose, lactate, pyruvate, glycerol and isoprostanes before and during SE, and after acute treatment with pregabalin, valproic acid, and levetiracteam. RESULTS: Upon onset of SE, acetylcholine (ACh) release increased six- to eightfold. Glucose was increased only transiently by 30% but lactate levels rose four-fold, and extracellular concentrations of glycerol ten-fold. Isoprostanes are markers of oxidative stress and increased more than 20-fold. Two hours after pilocarpine adminstration, rats were treated with pregabalin (100 mg/kg), levetiracetam (200 mg/kg) or valproic acid (400 mg/kg) by i.p. injection. All three drugs stopped seizure activity in a delayed fashion, but at the doses indicated, only animals that received levetiracetam reached consciousness. All drugs reduced ACh release within 60-120 minutes. Lactate/pyruvate ratios, glycerol and isoprostanne levels were also reduced significantly after drug administration. CONCLUSIONS: Hippocampal ACh release closely follows seizure activity in SE and is attenuated when SE subsides. Pregabalin, valproic acid and levetiracetam all terminate seizures in the rat SE model and attenuate cholinergic and metabolic changes within two hours.
Assuntos
Anticonvulsivantes/farmacologia , Colinérgicos/farmacologia , Convulsões/tratamento farmacológico , Estado Epiléptico/tratamento farmacológico , Acetilcolina/análise , Animais , Anticonvulsivantes/química , Anticonvulsivantes/metabolismo , Comportamento Animal , Colinérgicos/química , Colinérgicos/metabolismo , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Levetiracetam/química , Levetiracetam/metabolismo , Levetiracetam/farmacologia , Masculino , Estresse Oxidativo/efeitos dos fármacos , Pregabalina/química , Pregabalina/metabolismo , Pregabalina/farmacologia , Ratos , Ratos Sprague-Dawley , Ácido Valproico/química , Ácido Valproico/metabolismo , Ácido Valproico/farmacologiaRESUMO
IL-1ß is a potent proinflammatory cytokine of the innate immune system that is involved in host defense against infection. However, increased production of IL-1ß plays a pathogenic role in various inflammatory diseases, such as rheumatoid arthritis, gout, sepsis, stroke, and transplant rejection. To prevent detrimental collateral damage, IL-1ß release is tightly controlled and typically requires two consecutive danger signals. LPS from Gram-negative bacteria is a prototypical first signal inducing pro-IL-1ß synthesis, whereas extracellular ATP is a typical second signal sensed by the ATP receptor P2X7 that triggers activation of the NLRP3-containing inflammasome, proteolytic cleavage of pro-IL-1ß by caspase-1, and release of mature IL-1ß. Mechanisms controlling IL-1ß release, even in the presence of both danger signals, are needed to protect from collateral damage and are of therapeutic interest. In this article, we show that acetylcholine, choline, phosphocholine, phosphocholine-modified LPS from Haemophilus influenzae, and phosphocholine-modified protein efficiently inhibit ATP-mediated IL-1ß release in human and rat monocytes via nicotinic acetylcholine receptors containing subunits α7, α9, and/or α10. Of note, we identify receptors for phosphocholine-modified macromolecules that are synthesized by microbes and eukaryotic parasites and are well-known modulators of the immune system. Our data suggest that an endogenous anti-inflammatory cholinergic control mechanism effectively controls ATP-mediated release of IL-1ß and that the same mechanism is used by symbionts and misused by parasites to evade innate immune responses of the host.
Assuntos
Trifosfato de Adenosina/farmacologia , Interleucina-1beta/metabolismo , Lipopolissacarídeos/farmacologia , Monócitos/efeitos dos fármacos , Agonistas Nicotínicos/farmacologia , Acetilcolina/farmacologia , Trifosfato de Adenosina/análogos & derivados , Animais , Western Blotting , Células Cultivadas , Colina/farmacologia , Relação Dose-Resposta a Droga , Humanos , Lipopolissacarídeos/química , Potenciais da Membrana/efeitos dos fármacos , Monócitos/metabolismo , Nicotina/farmacologia , Fosforilcolina/química , Interferência de RNA , Ratos , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células U937 , Receptor Nicotínico de Acetilcolina alfa7/genética , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
Chemosensory cells in the mucosal surface of the respiratory tract ("brush cells") use the canonical taste transduction cascade to detect potentially hazardous content and trigger local protective and aversive respiratory reflexes on stimulation. So far, the urogenital tract has been considered to lack this cell type. Here we report the presence of a previously unidentified cholinergic, polymodal chemosensory cell in the mammalian urethra, the potential portal of entry for bacteria and harmful substances into the urogenital system, but not in further centrally located parts of the urinary tract, such as the bladder, ureter, and renal pelvis. Urethral brush cells express bitter and umami taste receptors and downstream components of the taste transduction cascade; respond to stimulation with bitter (denatonium), umami (monosodium glutamate), and uropathogenic Escherichia coli; and release acetylcholine to communicate with other cells. They are approached by sensory nerve fibers expressing nicotinic acetylcholine receptors, and intraurethral application of denatonium reflexively increases activity of the bladder detrusor muscle in anesthetized rats. We propose a concept of urinary bladder control involving a previously unidentified cholinergic chemosensory cell monitoring the chemical composition of the urethral luminal microenvironment for potential hazardous content.
Assuntos
Acetilcolina/metabolismo , Células Quimiorreceptoras/metabolismo , Uretra/citologia , Uretra/metabolismo , Bexiga Urinária/fisiologia , Animais , Células Quimiorreceptoras/citologia , Feminino , Proteínas de Fluorescência Verde/genética , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Microvilosidades/fisiologia , Comunicação Parácrina/fisiologia , Técnicas de Patch-Clamp , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Células Receptoras Sensoriais/citologia , Células Receptoras Sensoriais/fisiologia , Paladar/fisiologia , Língua/citologia , Língua/inervação , Língua/fisiologia , Uretra/inervação , Bexiga Urinária/inervação , Urodinâmica/fisiologia , Urotélio/citologia , Urotélio/metabolismoRESUMO
Proteins and glycolipids have been found to be decorated with phosphorylcholine (PC) both in protozoa and nematodes that parasitize humans and animals. PC epitopes can provoke various effects on immune cells leading to an immunomodulation of the host's immune system that allows long-term persistence of the parasites. So far, only a limited number of PC-modified proteins, mainly from nematodes, have been identified. Infections caused by Leishmania spp. (e.g., L. infantum in southern Europe) affect about 12 million people worldwide and are characterized by a wide spectrum of clinical forms in humans, ranging from cutaneous to fatal visceral leishmaniasis. To establish and maintain the infection, these protozoa are dependent on the secretion of effector molecules into the host for modulating their immune system. In this project, we analyzed the PC modification of L. infantum promastigotes by 2D-gel based proteomics. Western blot analysis with the PC-specific antibody TEPC-15 revealed one PC-substituted protein in this organism, identified as eEF1α. We could demonstrate that the binding of eEF1α to one of its downstream effectors is dependent on its PC-modification. In this study we provide evidence that in this parasite the modification of eEF1α with PC may be essential for its function as an important virulence factor.
Assuntos
Leishmania infantum/metabolismo , Fator 1 de Elongação de Peptídeos/química , Fator 1 de Elongação de Peptídeos/metabolismo , Fosforilcolina/química , Epitopos/química , Epitopos/imunologia , Imunomodulação/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/imunologia , Estrutura Molecular , Fator 1 de Elongação de Peptídeos/imunologia , Fosforilcolina/farmacologiaRESUMO
The lithium-pilocarpine model of status epilepticus is a well-known animal model of temporal lobe epilepsy. We combined this model with in vivo microdialysis to investigate energy metabolites and acute cellular membrane damage during seizure development. Rats were implanted with dialysis probes and pretreated with lithium chloride (127 mg/kg i.p.). Twenty-four hours later, they received pilocarpine (30 mg/kg s.c.) which initiated seizures within 30 min. In the dialysate from rat hippocampus, we observed a transient increase in glucose and a prominent, five-fold increase in lactate during seizures. Lactate release was because of neuronal activation as it was strongly reduced by infusion of tetrodotoxin, administration of atropine or when seizures were terminated by diazepam or ketamine. In ex vivo assays, mitochondrial function as measured by respirometry was not affected by 90 min of seizures. Extracellular levels of choline, however, increased two-fold and glycerol levels 10-fold, which indicate cellular phospholipid breakdown during seizures. Within 60 min of pilocarpine administration, hydroxylation of salicylate increased two-fold and formation of isoprostanes 20-fold, revealing significant oxidative stress in hippocampal tissue. Increases in lactate, glycerol and isoprostanes were abrogated, and increases in choline were completely prevented, when hippocampal probes were perfused with calcium-free solution. Similarly, administration of pregabalin (100 mg/kg i.p.), a calcium channel ligand, 15 min prior to pilocarpine strongly attenuated parameters of membrane damage and oxidative stress. We conclude that seizure development in a rat model of status epilepticus is accompanied by increases in extracellular lactate, choline and glycerol, and by oxidative stress, while mitochondrial function remains intact for at least 90 min. Membrane damage depends on calcium influx and can be prevented by treatment with pregabalin. Status epilepticus (SE) was induced in rats by lithium-pilocarpine ('Pilo') administration, and extracellular metabolites were measured by microdialysis. Seizures caused several-fold increases in lactate levels which were attenuated by diazepam ('Diaz'), ketamine, atropine and tetrodotoxin (TTX). Indicators of oxidative stress and membrane damage were also increased during seizures. Omission of calcium and pregabalin, a calcium channel blocker, reduced cellular damage induced by SE.
Assuntos
Encéfalo/metabolismo , Convulsivantes/toxicidade , Lítio/toxicidade , Pilocarpina/toxicidade , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/patologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Colina , Modelos Animais de Doenças , Eletroquímica , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Glucose/metabolismo , Ácido Láctico/metabolismo , Masculino , Microdiálise , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
PURPOSE: Ginkgo extract EGb 761 and cholinesterase inhibitors have been shown to be effective in the treatment of dementia patients. In addition to neuroprotective effects, Ginkgo extract EGb 761 has been reported to elevate brain levels of certain neurotransmitters such as dopamine, noradrenaline, and acetylcholine. In the present study, we investigated the impact of EGb 761, donepezil and the combination of both drugs on the central cholinergic system in aged rats. METHODS: 24 month old rats received EGb 761 (100 mg/kg/day), donepezil (1.5 mg/kg/day), the combination of both drugs or vehicle control by oral gavage for 14 days. We used microdialysis in rat hippocampus to monitor extracellular concentrations of acetylcholine (ACh), choline, glucose and lactate. Brain homogenates were prepared to measure activities of acetylcholinesterase (AChE), choline acetyltransferase (ChAT) and high affinity choline uptake (HACU). RESULTS: While EGb 761 alone had no effect, donepezil and the combination of donepezil and EGb 761 increased basal ACh levels by 2- to 3-fold. Concomitantly, significant reductions of AChE and HACU were measured in both groups. No differences were seen between donepezil and the combination in these parameters. Treatment with EGb 761 decreased extracellular choline release and showed a tendency to moderately elevate ChAT activity. CONCLUSIONS: We found that donepezil and EGb 761 do not display a pharmacological interaction when given together. Adding EGb 761 did not modify the effects of donepezil on the hippocampal cholinergic system. Reduced choline levels indicate neuroprotective properties of EGb 761. Therefore, the combination of EGb 761 and donepezil may be beneficial in the treatment of Alzheimer's disease (AD). This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.
Assuntos
Inibidores da Colinesterase/farmacologia , Indanos/farmacologia , Fármacos Neuroprotetores/farmacologia , Piperidinas/farmacologia , Extratos Vegetais/farmacologia , Acetilcolina/metabolismo , Envelhecimento , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Colina/metabolismo , Colina O-Acetiltransferase/metabolismo , Inibidores da Colinesterase/administração & dosagem , Donepezila , Interações Medicamentosas , Ginkgo biloba , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Indanos/administração & dosagem , Masculino , Microdiálise , Fármacos Neuroprotetores/administração & dosagem , Piperidinas/administração & dosagem , Extratos Vegetais/administração & dosagem , Ratos , Ratos Sprague-DawleyRESUMO
The lithium-pilocarpine model is a rat model of epilepsy that mimics status epilepticus in humans. Here, we report changes of acetylcholine (ACh) release in the hippocampus before, during and after status epilepticus as monitored by microdialysis in unanesthetized rats. Administration of pilocarpine (30 mg/kg s.c.) to rats pretreated with lithium chloride (127 mg/kg i.p.) caused a massive, six-fold increase of hippocampal ACh release, paralleling the development of tonic seizures. When seizures were stopped by administration of diazepam (10 mg/kg i.p.) or ketamine (75 mg/kg i.p.), ACh levels returned to normal. Extracellular concentrations of glutamate remained unchanged during this procedure. Administration of atropine (1 mg/kg i.p.) 2 h after pilocarpine caused a further increase of ACh but did not affect seizures, whereas injection of mecamylamine (5 mg/kg i.p.) reduced ACh levels and seizures in a delayed fashion. Local infusion of tetrodotoxin, 1 µM locally) or hemicholinium (10 µM locally) strongly reduced ACh release and had delayed effects on seizures. Administration of glucose or inositol (250 mg/kg each i.p.) had no visible consequences. In parallel experiments, lithium-pilocarpine-induced status epilepticus also enhanced striatal ACh release, and hippocampal ACh levels equally increased when status epilepticus was induced by kainate (30 mg/kg i.p.). Taken together, our results demonstrate that seizure development in status epilepticus models is accompanied by massive increases of extracellular ACh, but not glutamate, levels. Treatments that reduce seizure activity also reliably reduce extracellular ACh levels.
Assuntos
Acetilcolina/metabolismo , Hipocampo/metabolismo , Lítio/toxicidade , Pilocarpina/toxicidade , Estado Epiléptico/metabolismo , Animais , Líquido Extracelular/efeitos dos fármacos , Líquido Extracelular/metabolismo , Hipocampo/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-Dawley , Estado Epiléptico/induzido quimicamenteRESUMO
Nursing honeybees produce brood food with millimolar concentrations of acetylcholine (ACh), which is synthesized through head gland secretions mixed with honey stomach contents. While we previously demonstrated the necessity of ACh for proper larval development, the dynamics of ACh levels throughout ontogenesis and their seasonal variations have remained unclear until now. Our HPLC analysis reveals dependencies of choline and ACh levels on larval development days (LDDs), influenced by seasonal (April-September) variations. Median ACh concentrations peak on LDD 2, declining significantly toward cell capping, while choline levels are lowest during the initial LDDs, rising markedly toward cell capping. Seasonal patterns show peak ACh levels from April to June and a low in August, paralleling choline's peak in July and low in August. This seasonality holds consistently across multiple years (2020-2022) and colonies, despite potential variations in colony performance and environmental conditions. Our analysis found no correlation between temperature, sunshine, precipitation, or favourable foraging days and ACh/choline levels, suggesting the involvement of additional factors. These findings underscore the seasonal fluctuation of ACh levels and its potential implications for the genetic programs governing winter bee development.
Assuntos
Acetilcolina , Colina , Estações do Ano , Animais , Abelhas/metabolismo , Abelhas/fisiologia , Colina/metabolismo , Colina/análise , Acetilcolina/metabolismo , Acetilcolina/análise , Larva/metabolismo , Mel/análise , Fatores EtáriosRESUMO
General anesthetic drugs have been associated with various unwanted effects including an interference with mitochondrial function. We had previously observed increases of lactate formation in the mouse brain during anesthesia with volatile anesthetic agents. In the present work, we used mitochondria that were freshly isolated from mouse brain to test mitochondrial respiration and ATP synthesis in the presence of six common anesthetic drugs. The volatile anesthetics isoflurane, halothane, and (to a lesser extent) sevoflurane caused an inhibition of complex I of the electron transport chain in a dose-dependent manner. Significant effects were seen at concentrations that are reached under clinical conditions (< 0.5 mM). Pentobarbital and propofol also inhibited complex I but at concentrations that were two-fold higher than clinical EC50 values. Only propofol caused an inhibition of complex II. Complex IV respiration was not affected by either agent. Ketamine did not affect mitochondrial respiration. Similarly, all anesthetic agents except ketamine suppressed ATP production at high concentrations. Only halothane increased cytochrome c release indicating damage of the mitochondrial membrane. In summary, volatile general anesthetic agents as well as pentobarbital and propofol dose-dependently inhibit mitochondrial respiration. This action may contribute to depressive actions of the drugs in the brain.
Assuntos
Anestésicos Gerais , Isoflurano , Ketamina , Propofol , Camundongos , Animais , Halotano/farmacologia , Ketamina/farmacologia , Propofol/farmacologia , Pentobarbital , Anestésicos Gerais/farmacologia , Isoflurano/farmacologia , Mitocôndrias , Complexo I de Transporte de Elétrons , Trifosfato de AdenosinaRESUMO
Antioxidants have been proposed as a treatment for diseases of the central nervous system. However, few studies actually studied their effects in the brain. To test central actions of antioxidants, we used the lithium-pilocarpine (Li-Pilo) model of status epilepticus (SE) in the rat in which seizures are accompanied by significant oxidative stress. We used in vivo microdialysis to determine isoprostane levels during SE in real time and brain homogenates for other measures of oxidative stress. Six different antioxidants were tested in acute and preventive experiments (vitamin C, vitamin E, ebselen, resveratrol, n-tert-butyl-α-phenylnitrone and coenzyme Q10). None of the antioxidants had an effect when given acutely during SE. In contrast, when antioxidants were given for 3 days prior to seizure induction, vitamins C and E reduced isoprostane formation by 58% and 65%, respectively. Pretreatment with the other antioxidants was ineffective. In brain homogenates prepared after 90 min of seizures, SE decreased the ratio of reduced vs. oxidized glutathione (GSH/GSSG ratio) from 60.8 to 7.50 and caused a twofold increase of 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels and protein carbonyls. Pretreatment with vitamin C or vitamin E mitigated these effects and increased the GSH/GSSG ratio to 23.9 and 28.3, respectively. Again, the other antioxidants were not effective. We conclude that preventive treatment with vitamin C or vitamin E ameliorates seizure-induced oxidative damage in the brain. Several well-studied antioxidants were inactive, possibly due to limited brain permeability or a lack of chain-breaking antioxidant activity in hydrophilic compounds.
RESUMO
Acetylcholinesterase (AChE) rapidly hydrolyzes acetylcholine. At the neuromuscular junction, AChE is mainly anchored in the extracellular matrix by the collagen Q, whereas in the brain, AChE is tethered by the proline-rich membrane anchor (PRiMA). The AChE-deficient mice, in which AChE has been deleted from all tissues, have severe handicaps. Surprisingly, PRiMA KO mice in which AChE is mostly eliminated from the brain show very few deficits. We now report that most of the changes observed in the brain of AChE-deficient mice, and in particular the high levels of ambient extracellular acetylcholine and the massive decrease of muscarinic receptors, are also observed in the brain of PRiMA KO. However, the two groups of mutants differ in their responses to AChE inhibitors. Since PRiMA-KO mice and AChE-deficient mice have similar low AChE concentrations in the brain but differ in the AChE content of the peripheral nervous system, these results suggest that peripheral nervous system AChE is a major target of AChE inhibitors, and that its absence in AChE- deficient mice is the main cause of the slow development and vulnerability of these mice. At the level of the brain, the adaptation to the absence of AChE is nearly complete.
Assuntos
Acetilcolinesterase/deficiência , Adaptação Fisiológica/genética , Encéfalo/enzimologia , Regulação da Expressão Gênica/genética , Proteínas de Membrana/deficiência , Proteínas do Tecido Nervoso/deficiência , Acetilcolina/metabolismo , Acetilcolinesterase/metabolismo , Adaptação Fisiológica/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Temperatura Corporal/efeitos dos fármacos , Temperatura Corporal/genética , Encéfalo/anatomia & histologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacocinética , Bungarotoxinas/farmacocinética , Colina/metabolismo , Colinérgicos/farmacologia , Inibidores da Colinesterase/farmacologia , Colágeno/deficiência , Di-Hidro-beta-Eritroidina/farmacologia , Relação Dose-Resposta a Droga , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Marcha/efeitos dos fármacos , Marcha/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas In Vitro , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Knockout , Microdiálise , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Antagonistas Muscarínicos/farmacocinética , Proteínas Musculares/deficiência , Unhas Encravadas , Neostigmina/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Pirenzepina/análogos & derivados , Pirenzepina/farmacocinética , Ligação Proteica/efeitos dos fármacos , Piridinas/farmacocinética , Radioisótopos/farmacocinética , Receptores Muscarínicos/metabolismo , Teste de Desempenho do Rota-Rod , Escopolamina/farmacologia , Medula Espinal/citologia , Estatísticas não Paramétricas , Trítio/farmacocinéticaRESUMO
PURPOSE: Ginkgo extract EGb761 has shown anti-edema and anti-ischemic effects in various experimental models. In the present study, we demonstrate neuroprotective effects of EGb761 in experimental stroke while monitoring brain metabolism by microdialysis. METHODS: We have used oxygen-glucose deprivation in brain slices in vitro and middle cerebral artery occlusion (MCAO) in vivo to induce ischemia in mouse brain. We used microdialysis in mouse striatum to monitor extracellular concentrations of glucose and glutamate. RESULTS: In vitro, EGb761 reduced ischemia-induced cell swelling in hippocampal slices by 60%. In vivo, administration of EGb761 (300 mg/kg) reduced cell degeneration and edema formation after MCAO by 35-50%. Immediately following MCAO, striatal glucose levels dropped to 25% of controls, and this reduction was not significantly affected by EGb761. Striatal glutamate levels, in contrast, increased 15-fold after MCAO; after pretreatment with EGb761, glutamate levels only increased by 4-5fold. CONCLUSIONS: We show that pretreatment with EGb761 strongly reduces cellular edema formation and neurodegeneration under conditions of ischemia. The mechanism of action seems to be related to a reduction of excitotoxicity, because ischemia-induced release of glutamate was strongly suppressed. Ginkgo extracts such as EGb761 may be valuable to prevent ischemia-induced damage in stroke-prone patients.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Ácido Glutâmico/efeitos dos fármacos , Extratos Vegetais/farmacologia , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Edema Encefálico/tratamento farmacológico , Edema Encefálico/etiologia , Edema Encefálico/patologia , Isquemia Encefálica/fisiopatologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/patologia , Modelos Animais de Doenças , Feminino , Ginkgo biloba , Glucose/metabolismo , Ácido Glutâmico/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/fisiopatologia , Masculino , Camundongos , Microdiálise , Fármacos Neuroprotetores/farmacologia , Ratos , Ratos Wistar , Acidente Vascular Cerebral/fisiopatologiaRESUMO
The gallbladder stores bile between meals and empties into the duodenum upon demand and is thereby exposed to the intestinal microbiome. This exposure raises the need for antimicrobial factors, among them, mucins produced by cholangiocytes, the dominant epithelial cell type in the gallbladder. The role of the much less frequent biliary tuft cells is still unknown. We here show that propionate, a major metabolite of intestinal bacteria, activates tuft cells via the short-chain free fatty acid receptor 2 and downstream signaling involving the cation channel transient receptor potential cation channel subfamily M member 5. This results in corelease of acetylcholine and cysteinyl leukotrienes from tuft cells and evokes synergistic paracrine effects upon the epithelium and the gallbladder smooth muscle, respectively. Acetylcholine triggers mucin release from cholangiocytes, an epithelial defense mechanism, through the muscarinic acetylcholine receptor M3. Cysteinyl leukotrienes cause gallbladder contraction through their cognate receptor CysLTR1, prompting emptying and closing. Our results establish gallbladder tuft cells as sensors of the microbial metabolite propionate, initiating dichotomous innate defense mechanisms through simultaneous release of acetylcholine and cysteinyl leukotrienes.
Assuntos
Acetilcolina , Propionatos , Acetilcolina/metabolismo , Células Epiteliais/metabolismo , LeucotrienosRESUMO
The current study used measurements of metabolites and markers of membrane integrity to determine the most suitable time point for microdialysis experiments following probe implantation. Leakage of Evans blue and sodium fluorescein indicated increased BBB permeability only immediately (15 min), but not 1.5 and 24 h following probe implantation. Acute implantation decreased glucose and lactate levels relative to the levels after 24 h (to 13-37% and 25-60%, respectively). No change in extracellular levels of glutamate or glycerol was seen. In comparison to acute probe implantation, the pattern of damage under brain ischemia (middle cerebral artery occlusion) differed: While glucose levels dropped, lactate levels rose after ischemia, and glutamate (tenfold) and glycerol (eightfold) increased sharply. In conclusion, acute implantation of a microdialysis probe causes transient depression of the energy metabolites, glucose and lactate, likely due to injury-induced hypermetabolism. However, no massive tissue damage or severe ischemic conditions around the probe occur.