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1.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 40(7): 972-980, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32701228

RESUMO

OBJECTIVE: To investigate the effects of acid-sensing ion channels (ASICs) on electrophysiological epileptic activities of mouse hippocampal pyramidal neurons in the extracellular acidotic condition. METHODS: We investigated effects of extracellular acidosis on epileptic activities induced by elevated extracellular K + concentration or the application of an antagonist of GABAA receptors in perfusate of mouse hippocampal slices under field potential recordings. We also tested the effects of extracellular acidosis on neuronal excitability under field potential recording and evaluated the changes in epileptic activities of the neurons in response to pharmacological inhibition of ASICs using a specific inhibitor of ASICs. RESULTS: Extracellular acidosis significantly suppressed epileptic activities of the hippocampal neurons by converting ictal-like epileptic activities to non-ictal-like epileptic activities in both high [K +]o and disinhibition models, and also suppressed the intrinsic excitability of the neurons. ASICs inhibitor did not antagonize the inhibitory effect of extracellular acidosis on ictal epileptic activities and intrinsic neuronal excitability, but exacerbated non-ictal epileptic activities of the neurons in extracellular acidotic condition in both high [K+]o and disinhibition models. CONCLUSIONS: ASICs can differentially modulate ictal-like and non-ictallike epileptic activities via its direct actions on excitatory neurons.


Assuntos
Canais Iônicos Sensíveis a Ácido , Acidose , Epilepsia , Células Piramidais , Canais Iônicos Sensíveis a Ácido/metabolismo , Animais , Epilepsia/fisiopatologia , Concentração de Íons de Hidrogênio , Camundongos , Células Piramidais/patologia , Células Piramidais/fisiologia
2.
Am J Physiol Heart Circ Physiol ; 318(5): H1316-H1324, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32302492

RESUMO

The exercise pressor reflex arises from contracting muscle and is manifested by increases in arterial pressure, heart rate, and cardiac contractility. In patients with peripheral artery disease, the exercise pressor reflex is exaggerated. This effect is believed to be caused by a metabolite whose concentration is increased when the working muscles are inadequately perfused. Previous work in rats with simulated peripheral artery disease has shown that pharmacological blockade of acid-sensing ion channel 3 (ASIC3), which is found on group III and IV afferents, prevented the exaggeration of the exercise pressor reflex. Blockade of ASIC3, however, may have off-target effects that preclude a conclusion that ASIC3 plays a role in evoking the reflex in rats with simulated peripheral artery disease. In the present experiments performed in decerebrated rats with simulated peripheral artery disease, we compared the exercise pressor reflex in rats with a functional knockout of the ASIC3 (KO) with the reflex in their wild-type counterparts (WT). We found that the exercise pressor reflex in ASIC3 KO rats was significantly lower than the exercise pressor reflex in their WT counterparts (P < 0.05). ASIC 3 KO rats demonstrated lower pressor responses to intra-arterial injection of diprotonated phosphate (86 mM; pH 6.0), lactic acid (12 mM; pH 2.85), and capsaicin (0.2 µg; pH 7.2) (P < 0.05). In contrast, both ligated WT and ASIC3 KO rats displayed similar pressor responses to tendon stretch (P > 0.05). We conclude that ASIC3 play an important role in evoking the exaggerated exercise pressor reflex in rats with peripheral artery disease.NEW & NOTEWORTHY We used a genetic approach to test the hypothesis that the magnitude of the exercise pressor reflex evoked in ligated ASIC3 KO rats was significantly lower than the magnitude of the exercise pressor reflex evoked in their ligated wild-type (WT) counterparts. The pressor response to contraction in ligated ASIC3 KO rats was significantly smaller than was the pressor response to contraction in ligated WT rats.


Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Artéria Femoral/fisiopatologia , Contração Muscular , Doença Arterial Periférica/metabolismo , Reflexo , Canais Iônicos Sensíveis a Ácido/genética , Animais , Pressão Sanguínea , Masculino , Doença Arterial Periférica/fisiopatologia , Ratos , Ratos Wistar
3.
Am J Physiol Lung Cell Mol Physiol ; 318(5): L873-L887, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32160007

RESUMO

Tenacious mucus produced by tracheal and bronchial submucosal glands is a defining feature of several airway diseases, including cystic fibrosis (CF). Airway acidification as a driving force of CF airway pathology has been controversial. Here we tested the hypothesis that transient airway acidification produces pathologic mucus and impairs mucociliary transport. We studied pigs challenged with intra-airway acid. Acid had a minimal effect on mucus properties under basal conditions. However, cholinergic stimulation in acid-challenged pigs revealed retention of mucin 5B (MUC5B) in the submucosal glands, decreased concentrations of MUC5B in the lung lavage fluid, and airway obstruction. To more closely mimic a CF-like environment, we also examined mucus secretion and transport following cholinergic stimulation under diminished bicarbonate and chloride transport conditions ex vivo. Under these conditions, airways from acid-challenged pigs displayed extensive mucus films and decreased mucociliary transport. Pretreatment with diminazene aceturate, a small molecule with ability to inhibit acid detection through blockade of the acid-sensing ion channel (ASIC) at the doses provided, did not prevent acid-induced pathologic mucus or transport defects but did mitigate airway obstruction. These findings suggest that transient airway acidification early in life has significant impacts on mucus secretion and transport properties. Furthermore, they highlight diminazene aceturate as an agent that might be beneficial in alleviating airway obstruction.


Assuntos
Ácido Acético/administração & dosagem , Bloqueadores do Canal Iônico Sensível a Ácido/farmacologia , Canais Iônicos Sensíveis a Ácido/genética , Obstrução das Vias Respiratórias/induzido quimicamente , Fibrose Cística/induzido quimicamente , Diminazena/análogos & derivados , Canais Iônicos Sensíveis a Ácido/metabolismo , Obstrução das Vias Respiratórias/tratamento farmacológico , Obstrução das Vias Respiratórias/metabolismo , Obstrução das Vias Respiratórias/patologia , Animais , Animais Recém-Nascidos , Bicarbonatos/metabolismo , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Brônquios/patologia , Líquido da Lavagem Broncoalveolar/química , Cloretos/metabolismo , Fibrose Cística/tratamento farmacológico , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Diminazena/farmacologia , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Masculino , Mucina-5AC/genética , Mucina-5AC/metabolismo , Mucina-5B/genética , Mucina-5B/metabolismo , Depuração Mucociliar/efeitos dos fármacos , Muco/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Suínos , Traqueia/efeitos dos fármacos , Traqueia/metabolismo , Traqueia/patologia
4.
Am J Physiol Renal Physiol ; 318(3): F531-F543, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31984789

RESUMO

Acid-sensing ion channels (ASICs) are cation-permeable channels that in the periphery are primarily expressed in sensory neurons that innervate tissues and organs. Soon after the cloning of the ASIC subunits, almost 20 yr ago, investigators began to use genetically modified mice to assess the role of these channels in physiological processes. These studies provide critical insights about the participation of ASICs in sensory processes, including mechanotransduction, chemoreception, and nociception. Here, we provide an extensive assessment of these findings and discuss the current gaps in knowledge with regard to the functions of ASICs in the peripheral nervous system.


Assuntos
Canais Iônicos Sensíveis a Ácido/fisiologia , Células Receptoras Sensoriais/fisiologia , Transdução de Sinais/fisiologia , Animais , Dor/fisiopatologia , Tato/fisiologia
5.
Nat Commun ; 11(1): 475, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980622

RESUMO

We reported previously that acid-sensing ion channel 1a (ASIC1a) mediates acidic neuronal necroptosis via recruiting receptor-interacting protein kinase 1 (RIPK1) to its C terminus (CT), independent of its ion-conducting function. Here we show that the N-terminus (NT) of ASIC1a interacts with its CT to form an auto-inhibition that prevents RIPK1 recruitment/activation under resting conditions. The interaction involves glutamate residues at distal NT and is disrupted by acidosis. Expression of mutant ASIC1a bearing truncation or glutamate-to-alanine substitutions at distal NT causes constitutive cell death. The NT-CT interaction is further disrupted by N-ethylmaleimide-sensitive fusion ATPase (NSF), which associates with ASIC1a-NT under acidosis, facilitating RIPK1 interaction with ASIC1a-CT. Importantly, a membrane-penetrating synthetic peptide representing the distal 20 ASIC1a NT residues, NT1-20, reduced neuronal damage in both in vitro model of acidotoxicity and in vivo mouse model of ischemic stroke, demonstrating the therapeutic potential of targeting the auto-inhibition of ASIC1a for neuroprotection against acidotoxicity.


Assuntos
Canais Iônicos Sensíveis a Ácido/química , Canais Iônicos Sensíveis a Ácido/fisiologia , Necroptose/fisiologia , Neurônios/fisiologia , Canais Iônicos Sensíveis a Ácido/genética , Acidose/patologia , Acidose/fisiopatologia , Substituição de Aminoácidos , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Modelos Neurológicos , Proteínas Sensíveis a N-Etilmaleimida/farmacologia , Proteínas Sensíveis a N-Etilmaleimida/fisiologia , Neurônios/citologia , Neuroproteção/efeitos dos fármacos , Neuroproteção/fisiologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/fisiologia , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia , Transdução de Sinais , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/fisiopatologia
6.
Am J Physiol Heart Circ Physiol ; 318(1): H78-H89, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31675256

RESUMO

The role of the acid-sensing ion channel 1a (ASIC1a) in evoking the exercise pressor reflex is unknown, despite the fact that ASIC1a is opened by decreases in pH in the physiological range. This fact prompted us to test the hypothesis that ASIC1a plays an important role in evoking the exercise pressor reflex in decerebrated rats with freely perfused hindlimb muscles. To test this hypothesis, we measured the effect of injecting two ASIC1a blockers into the arterial supply of the triceps surae muscles on the reflex pressor responses to four maneuvers, namely 1) static contraction of the triceps surae muscles (i.e., the exercise pressor reflex), 2) calcaneal tendon stretch, 3) intra-arterial injection of lactic acid, and 4) intra-arterial injection of diprotonated phosphate. We found that the 2 ASIC1a blockers, psalmotoxin-1 (200 ng/kg) and mambalgin-1 (6.5 µg/kg), decreased the pressor responses to static contraction as well as the peak pressor responses to injection of lactic acid and diprotonated phosphate. In contrast, neither ASIC1a blocker had any effect on the pressor responses to tendon stretch. Importantly, we found that ASIC1a blockade significantly decreased the pressor response to static contraction after a latency of at least 8 s. Our results support the hypothesis that ASIC1a plays a key role in evoking the metabolic component of the exercise pressor reflex.NEW & NOTEWORTHY The role played by acid-sensing ion channel 1a (ASIC1a) in evoking the exercise pressor reflex remains unknown. In decerebrated rats with freely perfused femoral arteries, blocking ASIC1a with psalmotoxin-1 or mambalgin-1 significantly attenuated the pressor response to static contraction, lactic acid, and diprotonated phosphate injection but had no effect on the pressor response to stretch. We conclude that ASIC1a plays a key role in evoking the exercise pressor reflex by responding to contraction-induced metabolites, such as protons.


Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Sistema Nervoso Autônomo/fisiologia , Células Quimiorreceptoras/metabolismo , Contração Muscular , Fusos Musculares/metabolismo , Músculo Esquelético/inervação , Músculo Esquelético/metabolismo , Reflexo , Canais Iônicos Sensíveis a Ácido/efeitos dos fármacos , Animais , Células Quimiorreceptoras/efeitos dos fármacos , Estado de Descerebração , Venenos Elapídicos/farmacologia , Membro Posterior , Concentração de Íons de Hidrogênio , Masculino , Moduladores de Transporte de Membrana/farmacologia , Fusos Musculares/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Peptídeos/farmacologia , Ratos Sprague-Dawley , Venenos de Aranha/farmacologia
7.
Handb Exp Pharmacol ; 260: 161-186, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31820179

RESUMO

A large series of different ion channels have been identified and investigated as potential targets for new medicines for the treatment of a variety of human diseases, including pain. Among these channels, the voltage gated calcium channels (VGCC) are inhibited by drugs for the treatment of migraine, neuropathic pain or intractable pain. Transient receptor potential (TRP) channels are emerging as important pain transducers as they sense low pH media or oxidative stress and other mediators and are abundantly found at sites of inflammation or tissue injury. Low pH may also activate acid sensing ion channels (ASIC) and mechanical forces stimulate the PIEZO channels. While potent agonists of TRP channels due to their desensitizing action on pain transmission are used as topical applications, the potential of TRP antagonists as pain therapeutics remains an exciting field of investigation. The study of ASIC or PIEZO channels in pain signaling is in an early stage, whereas antagonism of the purinergic P2X3 channels has been reported to provide beneficial effects in chronic intractable cough. The present chapter covers these intriguing channels in great detail, highlighting their diverse mechanisms and broad potential for therapeutic utility.


Assuntos
Bloqueadores do Canal Iônico Sensível a Ácido/uso terapêutico , Bloqueadores dos Canais de Cálcio/uso terapêutico , Neuralgia/tratamento farmacológico , Manejo da Dor , Canais de Receptores Transientes de Potencial/antagonistas & inibidores , Canais Iônicos Sensíveis a Ácido , Canais de Cálcio , Humanos , Inflamação
8.
Invest Ophthalmol Vis Sci ; 60(14): 4701-4710, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31725169

RESUMO

Purpose: Usher syndrome (USH) is a rare disorder characterized by retinitis pigmentosa (RP) and sensorineural hearing loss. Several genes are responsible for the disease, but not all cases are explained by mutations in any of these, supporting the fact that there remain other unknown genes that have a role in the syndrome. We aimed to find the genetic cause of presumed USH patients lacking pathogenic mutations in the known USH genes. Methods: Whole exome sequencing was performed on a priori USH-diagnosed subjects from nine unrelated families, which had shown negative results for an USH-targeted panel in a previous study. Results: We identified possible pathogenic variants in six of the studied families. One patient harbored mutations in REEP6 and TECTA, each gene tentatively causative of one of the two main symptoms of the disease, mimicking the syndrome. In three patients, only the retinal degeneration causative mutations were detected (involving EYS, WDR19, and CNGB1 genes). Another family manifested a dementia-linked retinal dystrophy dependent on an allele dosage in the GRN gene. Last, another case presented a homozygous mutation in ASIC5, a gene not yet associated with USH. Conclusions: Our findings demonstrate that pending cases should be clinically and genetically carefully assessed, since more patients than expected may be either related phenocopies or affected by a more complex disease encompassing additional symptoms rather than classical USH.


Assuntos
Proteínas do Olho/genética , Proteínas de Membrana/genética , Síndromes de Usher/genética , Sequenciamento Completo do Genoma , Canais Iônicos Sensíveis a Ácido/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Proteínas do Citoesqueleto/genética , Análise Mutacional de DNA , Proteínas da Matriz Extracelular/genética , Feminino , Proteínas Ligadas por GPI/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Linhagem , Fenótipo , Progranulinas/genética , Retinite Pigmentosa/diagnóstico , Retinite Pigmentosa/genética , Síndromes de Usher/diagnóstico
9.
Am J Physiol Renal Physiol ; 317(5): F1305-F1310, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31566429

RESUMO

We investigated the involvement of brain-derived neurotrophic factor (BDNF) in bladder and urethral dysfunction using spinal cord-injured mice. We evaluated bladder and urethral function of female mice with 4-wk spinal cord injury (SCI) by filling cystometry and electromyography (EMG) of the external urethral sphincter (EUS) under a conscious condition. Anti-BDNF antibodies (10 µg·kg-1·h-1) were administered in some mice for 1 wk before the evaluation. Bladder and spinal (L6-S1) BDNF protein levels were examined by ELISA. Transcript levels of transient receptor potential channels or acid-sensing ion channels (Asic) in L6-S1 dorsal root ganglia were evaluated by RT-PCR. Voided volume and voiding efficiency were significantly increased without any changes in nonvoiding contractions, and the duration of reduced EMG activity during the voiding phase was significantly prolonged in anti-BDNF antibody-treated SCI mice. Compared with spinal cord-intact mice, SCI mice showed increased concentrations of bladder and spinal BDNF. Anti-BDNF antibody treatment decreased bladder and spinal BDNF protein concentrations of SCI mice. Asic2 and Asic3 transcripts were significantly increased after SCI but decreased after anti-BDNF antibody administration. These results indicate that upregulated expression of bladder and spinal BDNF is involved in the emergence of inefficient voiding in SCI mice. Thus, BDNF-targeting treatment could be an effective modality for the treatment of voiding problems, including inefficient voiding and detrusor sphincter dyssynergia after SCI.


Assuntos
Anticorpos , Fator Neurotrófico Derivado do Encéfalo/antagonistas & inibidores , Traumatismos da Medula Espinal/complicações , Transtornos Urinários/etiologia , Canais Iônicos Sensíveis a Ácido/genética , Canais Iônicos Sensíveis a Ácido/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/imunologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Feminino , Regulação da Expressão Gênica/imunologia , Camundongos , DNA Polimerase Dirigida por RNA , Reação em Cadeia da Polimerase em Tempo Real , Medula Espinal/metabolismo , Regulação para Cima , Bexiga Urinária/metabolismo
10.
Biomed Res Int ; 2019: 4714279, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31531354

RESUMO

Little is known about the function of acid-sensing ion channels (ASICs) in bone cells or osteoporotic vertebral fractures (OVF). This study delineated ASICs expression in adult human bone marrow-mesenchymal stem cells- (BM-MSC-) derived osteoblasts and in OVF bone cells. Adult BM-MSC-derived osteoblasts were isolated and cultured in different pH values. Osteogenic markers as alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OC) mRNA were assessed. Western blots method was applied to analyze ASICs protein expression in different pH values. Amiloride was added into the osteogenic media to analyze the Na+/K+ ATPase change. We harvested the vertebral cancellous bone through a bone biopsy needle in 26 OVF patients when performing percutaneous vertebroplasty. Six vertebral bone specimens obtained from 4 patients with high-energy vertebral fractures were used as the control. The reverse transcription polymerase chain reaction was performed to analyze the quantitative mRNA expression of ASICs. Osteogenic markers as ALP, OPN, and OC mRNA were higher expressed in increasing pH values throughout osteoblastogenesis. ASIC proteins were higher expressed in lower pH media, especially ASIC3, and ASIC4. The highest protein expression at days 7, 14, and 21 was ASIC2, ASIC4, and ASIC3, respectively. Expression of Na+/K+ ATPase was significantly decreased in cultured osteoblasts by addition of amiloride into the pH 6.9 osteogenic media. ASIC2 mRNA was most highly expressed with a 65.93-fold increase in the biopsied vertebral bone cells in OVF compared with the control. In conclusion, we found osteoblastogenesis was reduced in an acidic environment, and ASIC2, ASIC3, and ASIC4 were most highly expressed in turn during osteoblastogenesis within acidic media. ASIC2 was the most abundantly expressed gene in human bone cells in OVF compared with the control. ASIC2 could be crucial in the pathogenesis of osteoporosis and could serve as a therapeutic target for antiosteoporotic therapies.


Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Osso e Ossos/metabolismo , Fraturas por Osteoporose/metabolismo , Fraturas da Coluna Vertebral/metabolismo , Coluna Vertebral/metabolismo , Animais , Medula Óssea/metabolismo , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Osteócitos/metabolismo , Osteopontina/metabolismo , RNA Mensageiro/metabolismo
11.
Philos Trans R Soc Lond B Biol Sci ; 374(1785): 20190291, 2019 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-31544616

RESUMO

Nociceptors, i.e. sensory neurons tuned to detect noxious stimuli, are found in numerous phyla of the Animalia kingdom and are often polymodal, responding to a variety of stimuli, e.g. heat, cold, pressure and chemicals, such as acid. Owing to the ability of protons to have a profound effect on ionic homeostasis and damage macromolecular structures, it is no wonder that the ability to detect acid is conserved across many species. To detect changes in pH, nociceptors are equipped with an assortment of different acid sensors, some of which can detect mild changes in pH, such as the acid-sensing ion channels, proton-sensing G protein-coupled receptors and several two-pore potassium channels, whereas others, such as the transient receptor potential vanilloid 1 ion channel, require larger shifts in pH. This review will discuss the evolution of acid sensation and the different mechanisms by which nociceptors can detect acid. This article is part of the Theo Murphy meeting issue 'Evolution of mechanisms and behaviour important for pain'.


Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Evolução Biológica , Nociceptividade , Nociceptores/metabolismo , Dor , Animais , Humanos , Dor/etiologia , Dor/fisiopatologia
12.
Artigo em Chinês | MEDLINE | ID: mdl-31550769

RESUMO

Acid-sensing ion channels are a class of extracellular H(+) activated cation channels, belonging to the amiloride-sensitive epithelial Na(+) channel/degenerin (ENaC/DEG) superfamily. During extracellular acidification, the channels are activated and produce corresponding action potential. Acid-sensing ion channels are extensively expressed in the peripheral and central nervous system. It plays an important in synaptic plasticity, mechanical sensation, injury sensation related to acidosis of local tissues, acid reception and retinal regulation. This article reviews the expression, biological characteristics and functions of acid-sensing ion channels in cochlea, vestibular tissue and auditory center, so as to improve the understanding of physiology and pathophysiology of auditory system.


Assuntos
Canais Iônicos Sensíveis a Ácido , Córtex Auditivo , Cóclea , Vestíbulo do Labirinto , Canais Iônicos Sensíveis a Ácido/genética , Canais Iônicos Sensíveis a Ácido/metabolismo , Córtex Auditivo/fisiologia , Cóclea/fisiologia , Humanos , Sensação , Vestíbulo do Labirinto/fisiologia
13.
Stroke ; 50(10): 2902-2911, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31412757

RESUMO

Background and Purpose- Sex differences in the incidence and outcome of stroke have been well documented. The severity of stroke in women is, in general, significantly lower than that in men, which is mediated, at least in part, by the protective effects of ß-estradiol. However, the detailed mechanisms underlying the neuroprotection by ß-estradiol are still elusive. Recent studies have demonstrated that activation of ASIC1a (acid-sensing ion channel 1a) by tissue acidosis, a common feature of brain ischemia, plays an important role in ischemic brain injury. In the present study, we assessed the effects of ß-estradiol on acidosis-mediated and ischemic neuronal injury both in vitro and in vivo and explored the involvement of ASIC1a and underlying mechanism. Methods- Cultured neurons and NS20Y cells were subjected to acidosis-mediated injury in vitro. Cell viability and cytotoxicity were measured by methylthiazolyldiphenyl-tetrazolium bromide and lactate dehydrogenase assays, respectively. Transient (60 minutes) focal ischemia in mice was induced by suture occlusion of the middle cerebral artery in vivo. ASIC currents were recorded using whole-cell patch-clamp technique while intracellular Ca2+ concentration was measured with fluorescence imaging using Fura-2. ASIC1a expression was detected by Western blotting and quantitative real-time polymerase chain reaction. Results- Treatment of neuronal cells with ß-estradiol decreased acidosis-induced cytotoxicity. ASIC currents and acid-induced elevation of intracellular Ca2+ were all attenuated by ß-estradiol treatment. In addition, we showed that ß-estradiol treatment reduced ASIC1a protein expression, which was mediated by increased protein degradation, and that estrogen receptor α was involved. Finally, we showed that the level of ASIC1a protein expression in brain tissues and the degree of neuroprotection by ASIC1a blockade were lower in female mice, which could be attenuated by ovariectomy. Conclusions- ß-estradiol can protect neurons against acidosis-mediated neurotoxicity and ischemic brain injury by suppressing ASIC1a protein expression and channel function. Visual Overview- An online visual overview is available for this article.


Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Estradiol/farmacologia , Neurônios/efeitos dos fármacos , Acidente Vascular Cerebral/metabolismo , Acidose/complicações , Animais , Isquemia Encefálica/complicações , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Acidente Vascular Cerebral/etiologia , Acidente Vascular Cerebral/patologia
14.
Biomed Pharmacother ; 117: 109061, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31202172

RESUMO

This study aimed to explore the effects of hypoxia and acid-sensing ion channel 3 (ASIC3) on nucleus pulposus cells from cell behavior to molecular mechanism. Primary rabbit nucleus pulposus cells were isolated and identified by HE, toluidine blue and immunohistochemical staining of collagen II. 2% O2 and 48 h were screened as optimal oxygen concentration and effect time, respectively, by determining cell apoptosis and mRNA expression of ASIC3, hypoxia inducible factor-1α (HIF-1α) and aquaporin 3. FLuo-3 AM labeling showed that the Ca2+ concentration in cells increased under hypoxia condition. shRNA-ASIC3 and ASIC3 expression vector were transfected into cells. Subsequently, cells were divided into six groups: Control, 2% O2, shRNA-NC+2% O2, shRNA-ASIC3 + 2% O2, Vector+2% O2 and ASIC3 + 2% O2. Flow cytometry, CCK-8 assay, transmission electron microscopy, immunofluorescent labeling, RT-PCR and western blot demonstrated that hypoxia and ASIC3 over-expression inhibited the proliferation, arrested cell cycle in G1 phase, promoted the apoptosis, initiated the autophagy and up-regulated the expression of ASIC3, HIF-1α, light chain 3, p-ERK1/2 and p-MAPK. However, ASIC3 silencing could significantly relieve these phenomena. Co-immunoprecipitation assay found ASIC3 was interacted with HIF-1α&ERK1/2. Evaluation of the effect of HIF-1αsilencing on ASIC3 expression showed that the high expression of ASIC3 induced by hypoxia was reduced significantly by HIF-1α silencing. In conclusion, hypoxia and ASIC3 changed the behavior of nucleus pulposus cells by activating the MAPK pathway. HIF-1α and ASIC3 could regulate each other in nucleus pulposus cells.


Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Núcleo Pulposo/patologia , Canais Iônicos Sensíveis a Ácido/genética , Animais , Apoptose/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Cálcio/metabolismo , Ciclo Celular , Hipóxia Celular , Sobrevivência Celular , Células Cultivadas , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Sistema de Sinalização das MAP Quinases , Núcleo Pulposo/ultraestrutura , Oxigênio/farmacologia , Fosforilação , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , Fatores de Tempo
15.
Dokl Biochem Biophys ; 485(1): 111-114, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31201627

RESUMO

In this study, we performed the docking of ligands of the ASIC1a ion channel, which exert potentiating or inhibitory effects by stabilizing the open and closed states, respectively. It is shown for the first time that the direction of effect may depend on the three-dimensional structure of the ligand. Potentiators and inhibitors differently interact with the amino acid residues of the so-called "acidic pocket," where the binding of protons takes place. These results open up an opportunity for theoretical design of new pharmaceuticals.


Assuntos
Canais Iônicos Sensíveis a Ácido/química , Simulação de Acoplamento Molecular , Sítios de Ligação , Humanos
16.
Synapse ; 73(10): e22120, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31180161

RESUMO

Extracellular pH changes may constitute significant signals for neuronal communication. During synaptic transmission, changes in pH in the synaptic cleft take place. Its role in the regulation of presynaptic Ca2+ currents through multivesicular release in ribbon-type synapses is a proven phenomenon. In recent years, protons have been recognized as neurotransmitters that participate in neuronal communication in synapses of several regions of the CNS such as amygdala, nucleus accumbens, and brainstem. Protons are released by nerve stimulation and activate postsynaptic acid-sensing ion channels (ASICs). Several types of ASIC channels are expressed in the peripheral and central nervous system. The influx of Ca2+ through some subtypes of ASICs, as a result of synaptic transmission, agrees with the participation of ASICs in synaptic plasticity. Pharmacological and genetical inhibition of ASIC1a results in alterations in learning, memory, and phenomena like fear and cocaine-seeking behavior. The recognition of endogenous molecules, such as arachidonic acid, cytokines, histamine, spermine, lactate, and neuropeptides, capable of inhibiting or potentiating ASICs suggests the existence of mechanisms of synaptic modulation that have not yet been fully identified and that could be tuned by new emerging pharmacological compounds with potential therapeutic benefits.


Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Encéfalo/fisiologia , Prótons , Transmissão Sináptica/fisiologia , Animais , Humanos
17.
Elife ; 82019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31045491

RESUMO

ASICs are proton-gated sodium channels expressed in neurons. Structures of chicken ASIC1 in three conformations have advanced understanding of proton-mediated gating; however, a molecular mechanism describing desensitization from open and pre-open states (steady-state desensitization or SSD) remains elusive. A distinct feature of the desensitized state is an 180o rotation of residues L415 and N416 in the ß11- ß12 linker that was proposed to mediate desensitization; whether and how it translates into desensitization has not been explored yet. Using electrophysiological measurements of injected Xenopus oocytes, we show that Q276 in ß9 strand works with L415 and N416 to mediate both types of desensitization in ASIC1a, ASIC2a and ASIC3. Q276 functions as a valve that enables or restricts rotation of L415 and N416 to keep the linker compressed, its relaxation lengthens openings and leads to sustained currents. At low proton concentrations, the proposed mechanism working in only one of three subunits of the channel is sufficient to induce SSD.


Assuntos
Canais Iônicos Sensíveis a Ácido/química , Canais Iônicos Sensíveis a Ácido/metabolismo , Sódio/metabolismo , Canais Iônicos Sensíveis a Ácido/genética , Substituição de Aminoácidos , Animais , Concentração de Íons de Hidrogênio , Mutagênese Sítio-Dirigida , Oócitos , Técnicas de Patch-Clamp , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Xenopus
18.
Biomed Pharmacother ; 115: 108915, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31055237

RESUMO

Pyrazoles are potent medicinal scaffolds and exhibit a wide spectrum of biological activities, such as analgesic, anti-inflammatory and antipyretic. In this paper we report on research we have performed with the aim of continuing the biological evaluation of the regio-isomeric pyrazole compounds, LQFM-020 (fluorine, para position), LQFM-021 (fluorine, meta position), and LQFM-039 (fluorine, ortho position) in models of pain induced by acidified saline, capsaicin, and formalin. We also investigated the mechanisms of action of these compounds via electrophysiological analyses using the two-electrode voltage-clamp technique and heterologous expression in Xenopus laevis oocytes. This enabled us to study different potassium channel subtypes: the ASIC-1α channel, TRPV-1, and µMOR receptors. Our results indicate that LQFM-020, LQFM-021, and LQFM-039 (15, 30 or 60 mg.kg-1) compounds inhibited the nociceptive response induced by acidified saline in a dose-dependent manner. The dose of 30 mg.kg-1 inhibited the nociceptive response induced by capsaicin by 53.3%, 51.4%, and 52.1%, respectively. In addition, we found that naloxone reverses the antinociceptive effect produced by the compounds in both phases of the formalin test. In electrophysiological analyses, we observed that the LQFM-020, LQFM-021, and LQFM-039 compounds did not modulate voltage-gated K + channel subtypes. In contrast, all the compounds tested inhibited the ASIC-1α channel at pH 4.5, with IC50-values of 96.1, 91.6, and 235.2 µM, respectively. All compounds also inhibited the TRPV-1 channel with IC50-values of 139.1, 212.5, and 159.1 µM, respectively. In contrast to the ASIC-1α and TRPV-1 targets, all compounds showed agonist activity on the µMOR receptor with an EC50-value of 117.4, 98.9, and 86.3 µM, respectively. We thus conclude that the ASIC-1α, TRPV-1, and µMOR channels are targets that are directly involved in the antinociceptive effect of LQFM-020, LQFM-021, and LQFM-039. Furthermore, the modifications of the fluorine positions in the phenyl analogs do not change the analgesic effect. However, LQFM-039 showed lower interaction with ASIC-1α channel.


Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Analgésicos/farmacologia , Nociceptividade/efeitos dos fármacos , Pirazóis/farmacologia , Receptores Opioides mu/metabolismo , Canais de Cátion TRPV/metabolismo , Potenciais de Ação/efeitos dos fármacos , Analgésicos/química , Animais , Masculino , Camundongos , Estrutura Molecular , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Medição da Dor , Técnicas de Patch-Clamp , Pirazóis/química , Xenopus laevis
19.
Int J Mol Sci ; 20(7)2019 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-30959896

RESUMO

Acid-sensing ion channel 3 (ASIC3) is an important member of the acid-sensing ion channels family, which is widely expressed in the peripheral nervous system and contributes to pain sensation. ASICs are targeted by various drugs and toxins. However, mechanisms and structural determinants of ligands' action on ASIC3 are not completely understood. In the present work we studied ASIC3 modulation by a series of "hydrophobic monoamines" and their guanidine analogs, which were previously characterized to affect other ASIC channels via multiple mechanisms. Electrophysiological analysis of action via whole-cell patch clamp method was performed using rat ASIC3 expressed in Chinese hamster ovary (CHO) cells. We found that the compounds studied inhibited ASIC3 activation by inducing acidic shift of proton sensitivity and slowed channel desensitization, which was accompanied by a decrease of the equilibrium desensitization level. The total effect of the drugs on the sustained ASIC3-mediated currents was the sum of these opposite effects. It is demonstrated that drugs' action on activation and desensitization differed in their structural requirements, kinetics of action, and concentration and state dependencies. Taken together, these findings suggest that effects on activation and desensitization are independent and are likely mediated by drugs binding to distinct sites in ASIC3.


Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Aminas/química , Aminas/farmacologia , Guanidina/análogos & derivados , Guanidina/farmacologia , Animais , Células CHO , Cricetulus , Eletrofisiologia , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas
20.
Life Sci ; 227: 193-200, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-31026454

RESUMO

AIMS: The recent identification of acid sensing ion channels (ASICs) in vascular beds suggests their possible involvement in modulating vasomotor tone. Therefore, we investigated the gene expression profiles of ASIC subtypes in the middle cerebral artery (MCA) of Wistar rats and the functional implication of ASICs in acidosis-induced relaxation as well as maintenance of resting tension. MAIN METHODS: Real time PCR was employed to study the pattern of ASIC mRNA expression in the MCA wall in comparison with (i) matching brain tissue samples and (ii) arteries cultured for 24 h and 48 h. The functional implication regarding vasomotor response to acidosis and maintenance of resting tension was assessed using in vitro myography. KEY FINDINGS: A robust mRNA expression of ASIC-1, -2 and -4 was found in brain tissue samples and to a lower extent in freshly isolated MCA. In the MCA wall, short term culture induced a down-regulation of ASIC-1 and -2 expression without any remarkable change in ASIC-4 expression. Acidosis induced a pH-related relaxation of freshly isolated MCA ring segments, being more pronounced after short term culture. Incubation with the ASIC blocker amiloride moderately enhanced acidosis-induced relaxation, in cultured MCAs somewhat stronger than in freshly isolated vessels. In addition, amiloride resulted in a decrease of resting tension, albeit only in freshly isolated MCA. SIGNIFICANCE: Our results comprehensively describe ASIC subtype composition in the rat MCA in physiological and pathological conditions and strongly suggest the involvement of ASICs in the modulation of vasomotor responses under conditions of normal or decreased pH values.


Assuntos
Canais Iônicos Sensíveis a Ácido/genética , Canais Iônicos Sensíveis a Ácido/fisiologia , Artérias Cerebrais/fisiologia , Acidose/metabolismo , Animais , Células Cultivadas , Concentração de Íons de Hidrogênio , Masculino , Artéria Cerebral Média/metabolismo , Artéria Cerebral Média/patologia , Ratos , Ratos Wistar , Transcriptoma/genética , Sistema Vasomotor
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