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1.
PLoS One ; 15(7): e0236288, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32702049

RESUMO

Although voltage-gated Ca2+ channels (VGCC) are a major Ca2+ entry pathway in vascular smooth muscle cells (VSMCs), several other Ca2+-influx mechanisms exist and play important roles in vasoreactivity. One of these is store-operated Ca2+ entry (SOCE), mediated by an interaction between STIM1 and Orai1. Although SOCE is an important mechanism of Ca2+ influx in non-excitable cells (cells that lack VGCC); there is debate regarding the contribution of SOCE to regulate VSMC contractility and the molecular components involved. Our previous data suggest acid-sensing ion channel 1a (ASIC1a) is a necessary component of SOCE and vasoconstriction in small pulmonary arteries. However, it is unclear if ASIC1a similarly contributes to SOCE and vascular reactivity in systemic arteries. Considering the established role of Orai1 in mediating SOCE in the systemic circulation, we hypothesize the involvement of ASIC1a in SOCE and resultant vasoconstriction is unique to the pulmonary circulation. To test this hypothesis, we examined the roles of Orai1 and ASIC1a in SOCE- and endothelin-1 (ET-1)-induced vasoconstriction in small pulmonary and mesenteric arteries. We found SOCE is coupled to vasoconstriction in pulmonary arteries but not mesenteric arteries. In pulmonary arteries, inhibition of ASIC1a but not Orai1 attenuated SOCE- and ET-1-induced vasoconstriction. However, neither inhibition of ASIC1a nor Orai1 altered ET-1-induced vasoconstriction in mesenteric arteries. We conclude that SOCE plays an important role in pulmonary, but not mesenteric, vascular reactivity. Furthermore, in contrast to the established role of Orai1 in SOCE in non-excitable cells, the SOCE response in pulmonary VSMCs is largely mediated by ASIC1a.


Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Cálcio/metabolismo , Artérias Mesentéricas/fisiologia , Artéria Pulmonar/fisiologia , Vasoconstrição , Canais Iônicos Sensíveis a Ácido/genética , Animais , Canais de Cálcio Tipo L/metabolismo , Endotelina-1/farmacologia , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Ligação Proteica/efeitos dos fármacos , Artéria Pulmonar/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Wistar , Molécula 1 de Interação Estromal/metabolismo
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.
Proc Natl Acad Sci U S A ; 117(13): 7447-7454, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32165542

RESUMO

Acid-sensing ion channels (ASICs) are proton-gated cation channels that contribute to neurotransmission, as well as initiation of pain and neuronal death following ischemic stroke. As such, there is a great interest in understanding the in vivo regulation of ASICs, especially by endogenous neuropeptides that potently modulate ASICs. The most potent endogenous ASIC modulator known to date is the opioid neuropeptide big dynorphin (BigDyn). BigDyn is up-regulated in chronic pain and increases ASIC-mediated neuronal death during acidosis. Understanding the mechanism and site of action of BigDyn on ASICs could thus enable the rational design of compounds potentially useful in the treatment of pain and ischemic stroke. To this end, we employ a combination of electrophysiology, voltage-clamp fluorometry, synthetic BigDyn analogs, and noncanonical amino acid-mediated photocrosslinking. We demonstrate that BigDyn binding results in an ASIC1a closed resting conformation that is distinct from open and desensitized states induced by protons. Using alanine-substituted BigDyn analogs, we find that the BigDyn modulation of ASIC1a is primarily mediated through electrostatic interactions of basic amino acids in the BigDyn N terminus. Furthermore, neutralizing acidic amino acids in the ASIC1a extracellular domain reduces BigDyn effects, suggesting a binding site at the acidic pocket. This is confirmed by photocrosslinking using the noncanonical amino acid azidophenylalanine. Overall, our data define the mechanism of how BigDyn modulates ASIC1a, identify the acidic pocket as the binding site for BigDyn, and thus highlight this cavity as an important site for the development of ASIC-targeting therapeutics.


Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Dinorfinas/metabolismo , Canais Iônicos Sensíveis a Ácido/genética , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Neuropeptídeos/fisiologia , Oócitos/metabolismo , Prótons , Xenopus laevis
4.
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
5.
Sci Rep ; 10(1): 2168, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034189

RESUMO

Unipolar brush cells (UBCs) are excitatory granular layer interneurons in the vestibulocerebellum. Here we assessed motor coordination and balance to investigate if deletion of acid-sensing ion channel 5 (Asic5), which is richly expressed in type II UBCs, is sufficient to cause ataxia. The possible cellular mechanism underpinning ataxia in this global Asic5 knockout model was elaborated using brain slice electrophysiology. Asic5 deletion impaired motor performance and decreased intrinsic UBC excitability, reducing spontaneous action potential firing by slowing maximum depolarization rate. Reduced intrinsic excitability in UBCs was partially compensated by suppression of the magnitude and duration of delayed hyperpolarizing K+ currents triggered by glutamate. Glutamate typically stimulates burst firing subsequent to this hyperpolarization in normal type II UBCs. Burst firing frequency was elevated in knockout type II UBCs because it was initiated from a more depolarized potential compared to normal cells. Findings indicate that Asic5 is important for type II UBC activity and that loss of Asic5 contributes to impaired movement, likely, at least in part, due to altered temporal processing of vestibular input.


Assuntos
Canais Iônicos Sensíveis a Ácido/genética , Potenciais de Ação , Ataxia Cerebelar/metabolismo , Neurônios/metabolismo , Animais , Ataxia Cerebelar/genética , Ataxia Cerebelar/patologia , Ácido Glutâmico/metabolismo , Masculino , Camundongos , Neurônios/fisiologia , Potássio/metabolismo
6.
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
7.
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
8.
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
9.
Channels (Austin) ; 13(1): 367-373, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31526163

RESUMO

To elucidate the pathological significance of acid-sensing ion channels (ASICs) in intervertebral disc degeneration (IVDD), the database of Medline, Web of Science, and EmBase were carefully screened. Search terms used in each database varied slightly to optimize results. Data relating to the correlation between ASICs and IVDD was systematically collected and integrated into the review. 11 basic science studies, containing the related information, were finally identified for inclusion. Intervertebral disc degeneration (IVDD) is a common disease in middle-aged and elderly people, which has a great impact on patients' quality of life. Many research teams have attempted to elucidate the pathogenesis of this degenerative disease, and have made considerable progress. Acid-sensing ion channels (ASICs) were once reported to be able to regulate the apoptosis process of chondrocytes in joint cartilage, which has been transplanted into the IVDD-related research. ASIC1a functions as the mediator for cells in nucleus pulposus (NP) and endplate (EP), with whose activation the apoptosis process would be accelerated. Moreover, ASIC1a's activation could also regulate the anabolism in chondrocytes of EP, facilitating the degeneration. ASIC3 would only promote the degeneration in NP, possibly via its pro-inflammatory effect. The distribution of ASICs in NP, EP, annulus fibrosus, and the particular functions of ASIC1a and ASIC3 remind us about the pathological significance of ASICs in IVDD, which could be a promising therapeutic target in future treatment for IVDD.


Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Degeneração do Disco Intervertebral/metabolismo , Canais Iônicos Sensíveis a Ácido/química , Canais Iônicos Sensíveis a Ácido/genética , Apoptose , Condrócitos/citologia , Condrócitos/metabolismo , Humanos , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/fisiopatologia , Simulação de Acoplamento Molecular , Núcleo Pulposo/metabolismo
10.
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
11.
Sci Rep ; 9(1): 12123, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31431652

RESUMO

Fibromyalgia (FM) is characterized by chronic widespread pain. The pathogenesis of FM remains unclear. No specific biomarkers are available. Animal models of FM may provide an opportunity to explore potential biomarkers in a relative homogenous disease condition. Here, we probed the metabolomics profiles of serum and urine in a mouse model of FM induced by intermittent cold stress (ICS). We focused on the role of acid-sensing ion channel 3 (ASIC3) in the metabolomics profiling because ICS treatment induced chronic widespread muscle pain lasting for 1 month in wild-type (Asic3+/+) but not Asic3-knockout (Asic3-/-) mice. Serum and urine samples were collected from both genotypes at different ICS stages, including before ICS (basal level) and post-ICS at days 10 (middle phase, P10) and 40 (recovery phase, P40). Control naïve mice and ICS-induced FM mice differed in 1H-NMR- and LC-MS-based metabolomics profiling. On pathway analysis, the leading regulated pathways in Asic3+/+ mice were taurine and hypotaurine, cysteine and methionine, glycerophospholipid, and ascorbate and aldarate metabolisms, and the major pathways in Asic3-/- mice involved amino acid-related metabolism. Finally, we developed an algorithm for the impactful metabolites in the FM model including cis-aconitate, kynurenate, taurine, pyroglutamic acid, pyrrolidonecarboxylic acid, and 4-methoxyphenylacetic acid in urine as well as carnitine, deoxycholic acid, lysoPC(16:0), lysoPC(20:3), oleoyl-L-carnitine, and trimethylamine N-oxide in serum. Asic3-/- mice were impaired in only muscle allodynia development but not other pain symptoms in the ICS model, so the ASIC3-dependent metabolomics changes could be useful for developing diagnostic biomarkers specific to chronic widespread muscle pain, the core symptom of FM. Further pharmacological validations are needed to validate these metabolomics changes as potential biomarkers for FM diagnosis and/or treatment responses.


Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Fibromialgia/metabolismo , Canais Iônicos Sensíveis a Ácido/genética , Algoritmos , Animais , Biomarcadores/sangue , Biomarcadores/urina , Dor Crônica/metabolismo , Dor Crônica/terapia , Temperatura Baixa , Modelos Animais de Doenças , Progressão da Doença , Feminino , Fibromialgia/terapia , Metabolômica , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mialgia/metabolismo , Mialgia/terapia , Estresse Fisiológico
12.
Circ Res ; 125(10): 907-920, 2019 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-31451088

RESUMO

RATIONALE: Precise regulation of cerebral blood flow is critical for normal brain function. Insufficient cerebral blood flow contributes to brain dysfunction and neurodegeneration. Carbon dioxide (CO2), via effects on local acidosis, is one of the most potent regulators of cerebral blood flow. Although a role for nitric oxide in intermediate signaling has been implicated, mechanisms that initiate CO2-induced vasodilation remain unclear. OBJECTIVE: Acid-sensing ion channel-1A (ASIC1A) is a proton-gated cation channel that is activated by extracellular acidosis. Based on work that implicated ASIC1A in the amygdala and bed nucleus of the stria terminalis in CO2-evoked and acid-evoked behaviors, we hypothesized that ASIC1A might also mediate microvascular responses to CO2. METHODS AND RESULTS: To test this hypothesis, we genetically and pharmacologically manipulated ASIC1A and assessed effects on CO2-induced dilation of cerebral arterioles in vivo. Effects of inhalation of 5% or 10% CO2 on arteriolar diameter were greatly attenuated in mice with global deficiency in ASIC1A (Asic1a-/-) or by local treatment with the ASIC inhibitor, psalmotoxin. Vasodilator effects of acetylcholine, which acts via endothelial nitric oxide synthase were unaffected, suggesting a nonvascular source of nitric oxide may be key for CO2 responses. Thus, we tested whether neurons may be the cell type through which ASIC1A influences microvessels. Using mice in which Asic1a was specifically disrupted in neurons, we found effects of CO2 on arteriolar diameter were also attenuated. CONCLUSIONS: Together, these data are consistent with a model wherein activation of ASIC1A, particularly in neurons, is critical for CO2-induced nitric oxide production and vasodilation. With these findings, ASIC1A emerges as major regulator of microvascular tone.


Assuntos
Canais Iônicos Sensíveis a Ácido/deficiência , Circulação Cerebrovascular/fisiologia , Hipercapnia/metabolismo , Vasodilatação/fisiologia , Canais Iônicos Sensíveis a Ácido/genética , Animais , Dióxido de Carbono/farmacologia , Circulação Cerebrovascular/efeitos dos fármacos , Hipercapnia/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Óxido Nítrico/metabolismo , Vasodilatação/efeitos dos fármacos
13.
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
14.
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
15.
J Neurosci ; 39(29): 5773-5793, 2019 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101759

RESUMO

Chronic pain is a serious debilitating disease for which effective treatment is still lacking. Acid-sensing ion channel 1a (ASIC1a) has been implicated in nociceptive processing at both peripheral and spinal neurons. However, whether ASIC1a also contributes to pain perception at the supraspinal level remains elusive. Here, we report that ASIC1a in ACC is required for thermal and mechanical hypersensitivity associated with chronic pain. ACC-specific genetic deletion or pharmacological blockade of ASIC1a reduced the probability of cortical LTP induction and attenuated inflammatory thermal hyperalgesia and mechanical allodynia in male mice. Using cell type-specific manipulations, we demonstrate that ASIC1a in excitatory neurons of ACC is a major player in cortical LTP and pain behavior. Mechanistically, we show that ASIC1a tuned pain-related cortical plasticity through protein kinase C λ-mediated increase of membrane trafficking of AMPAR subunit GluA1 in ACC. Importantly, postapplication of ASIC1a inhibitors in ACC reversed previously established nociceptive hypersensitivity in both chronic inflammatory pain and neuropathic pain models. These results suggest that ASIC1a critically contributes to a higher level of pain processing through synaptic potentiation in ACC, which may serve as a promising analgesic target for treatment of chronic pain.SIGNIFICANCE STATEMENT Chronic pain is a debilitating disease that still lacks effective therapy. Ion channels are good candidates for developing new analgesics. Here, we provide several lines of evidence to support an important role of cortically located ASIC1a channel in pain hypersensitivity through promoting long-term synaptic potentiation in the ACC. Our results indicate a promising translational potential of targeting ASIC1a to treat chronic pain.


Assuntos
Canais Iônicos Sensíveis a Ácido/biossíntese , Giro do Cíngulo/metabolismo , Isoenzimas/deficiência , Neuralgia/metabolismo , Plasticidade Neuronal/fisiologia , Medição da Dor/métodos , Proteína Quinase C/deficiência , 6-Ciano-7-nitroquinoxalina-2,3-diona/administração & dosagem , Canais Iônicos Sensíveis a Ácido/genética , Animais , Células Cultivadas , Giro do Cíngulo/efeitos dos fármacos , Isoenzimas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microinjeções/métodos , Neuralgia/genética , Neuralgia/prevenção & controle , Plasticidade Neuronal/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Medição da Dor/efeitos dos fármacos , Proteína Quinase C/genética
16.
J Cell Mol Med ; 23(6): 3940-3950, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30938088

RESUMO

Acid-sensing ion channel 1a (ASIC1a) allows Na+ and Ca2+ flow into cells. It is expressed during inflammation, in tumour and ischaemic tissue, in the central nervous system and non-neuronal injury environments. Endoplasmic reticulum stress (ERS) is caused by the accumulation of misfolded proteins that interferes with intracellular calcium homoeostasis. Our recent reports showed ASIC1a and ERS are involved in liver fibrosis progression, particularly in hepatic stellate cell (HSC) activation. In this study, we investigated the roles of ASIC1a and ERS in activated HSC. We found that ASIC1a and ERS-related proteins were up-regulated in carbon tetrachloride (CCl4 )-induced fibrotic mouse liver tissues, and in patient liver tissues with hepatocellular carcinoma with severe liver fibrosis. The results show silencing ASIC1a reduced the expression of ERS-related biomarkers GRP78, Caspase12 and IREI-XBP1. And, ERS inhibition by 4-PBA down-regulated the high expression of ASIC1a induced by PDGF, suggesting an interactive relationship. In PDGF-induced HSCs, ASIC1a was activated and migrated to the cell membrane, leading to extracellular calcium influx and ERS, which was mediated by PI3K/AKT pathway. Our work shows PDGF-activated ASIC1a via the PI3K/AKT pathway, induced ERS and promoted liver fibrosis progression.


Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Cálcio/metabolismo , Carcinoma Hepatocelular/metabolismo , Estresse do Retículo Endoplasmático/genética , Neoplasias Hepáticas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Canais Iônicos Sensíveis a Ácido/genética , Animais , Tetracloreto de Carbono/toxicidade , Carcinoma Hepatocelular/genética , Caspases/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/metabolismo , Neoplasias Hepáticas/genética , Masculino , Proteínas de Membrana/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
17.
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
18.
J Gen Physiol ; 151(7): 944-953, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31010811

RESUMO

Acid-sensing ion channels (ASICs), sensory molecules that continuously monitor the concentration of extracellular protons and initiate diverse intracellular responses through an influx of cations, are assembled from six subtypes that can differentially combine to form various trimeric channel complexes and elicit unique electrophysiological responses. For instance, homomeric ASIC1a channels have been shown to exhibit prolonged desensitization, and acid-evoked currents become smaller when the channels are repeatedly activated by extracellular protons, whereas homomeric or heteromeric ASIC2a channels continue to respond to repetitive acidic stimuli without exhibiting such desensitization. Although previous studies have provided evidence that both the desensitization of ASIC1a and rapid resensitization of ASIC2a commonly require domains that include the N terminus and the first transmembrane region of these channels, the biophysical basis of channel gating at the amino acid level has not been clearly determined. Here, we confirm that domain-swapping mutations replacing the N terminus of ASIC2a with that of ASIC2b result in de novo prolonged desensitization in homomeric channels following activation by extracellular protons. Such desensitization of chimeric ASIC2a mutants is due neither to internalization nor to degradation of the channel proteins. We use site-directed mutagenesis to narrow down the relevant portion of the N terminus of ASIC2a, identifying three amino acid residues within the N terminus (T25, T39, and I40) whose mutation is sufficient to phenocopy the desensitization exhibited by the chimeric mutants. A similar desensitization is observed in heteromeric ASICs containing the mutant subunit. These results suggest that T25, T39, and I40 of ASIC2a are key residues determining the rapid resensitization of homomeric and heteromeric ASIC2a channels upon proton activation.


Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Ativação do Canal Iônico , Mutação de Sentido Incorreto , Canais Iônicos Sensíveis a Ácido/química , Canais Iônicos Sensíveis a Ácido/genética , Animais , Células HEK293 , Humanos , Camundongos , Domínios Proteicos , Prótons
19.
Genes Brain Behav ; 18(7): e12531, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30375184

RESUMO

Pavlovian fear conditioning has been shown to depend on acid-sensing ion channel-1A (ASIC1A); however, it is unknown whether conditioning to rewarding stimuli also depends on ASIC1A. Here, we tested the hypothesis that ASIC1A contributes to Pavlovian conditioning to a non-drug reward. We found effects of ASIC1A disruption depended on the relationship between the conditional stimulus (CS) and the unconditional stimulus (US), which was varied between five experiments. In experiment 1, when the CS preceded the US signaling an upcoming reward, Asic1a-/- mice exhibited a deficit in conditioning compared to Asic1a+/+ mice. Alternatively, in experiment 2, when the CS coinitiated with the US and signaled immediate reward availability, the Asic1a-/- mice exhibited an increase in conditioned responses compared to Asic1a+/+ mice, which contrasted with the deficits in the first experiment. Furthermore, in experiments 3 and 4, when the CS partially overlapped in time with the US, or the CS was shortened and coinitiated with the US, the Asic1a-/- mice did not differ from control mice. The contrasting outcomes were likely because of differences in conditioning because in experiment 5 neither the Asic1a-/- nor Asic1a+/+ mice acquired conditioned responses when the CS and US were explicitly unpaired. Taken together, these results suggest that the effects of ASIC1A disruption on reward conditioning depend on the temporal relationship between the CS and US. Furthermore, these results suggest that ASIC1A plays a critical, yet nuanced role in Pavlovian conditioning. More research will be needed to deconstruct the roles of ASIC1A in these fundamental forms of learning and memory.


Assuntos
Canais Iônicos Sensíveis a Ácido/genética , Condicionamento Clássico , Recompensa , Animais , Medo , Feminino , Deleção de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL
20.
Curr Drug Targets ; 20(1): 111-121, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30124148

RESUMO

The H+-gated (proton) currents are widely present in brain sensory neuronal system and various studies identified the structural units and deciphered the physiological and pathological function of ion channels. The normal neuron requires an optimal pH to carry out its functions. In acidosis, the ASICs (Acid-sensing Ion Channels) are activated in both the CNS (central nervous system) and PNS (peripheral nervous system). ASICs are related to degenerin channels (DEGs), epithelial sodium cation channels (ENaCs), and FMRF-amide (Phe-Met-Arg-Phe-NH2)-gated channels (FaNaC). Its activation leads physiologically to pain perception, synaptic plasticity, learning and memory, fear, ischemic neuronal injury, seizure termination, neuronal degeneration, and mechanosensation. It detects the level of acid fluctuation in the extracellular environment and responds to acidic pH by increasing the rate of membrane depolarization. It conducts cations like Na+ (Sodium) and Ca2+ (Calcium) ions across the membrane upon protonation. The ASICs subtypes are characterized by differing biophysical properties and pH sensitivities. The subtype ASIC1 is involved in various CNS diseases and therefore focusing on its specific functional properties will guide in drug design methods. The review highlights the cASIC1 (Chicken ASIC1) crystal structures, involvement in physiological environment and limitations of currently available inhibitors. In addition, it details the mutational data available to design an inhibitor against hASIC1 (Human ASIC1).


Assuntos
Bloqueadores do Canal Iônico Sensível a Ácido/química , Canais Iônicos Sensíveis a Ácido/química , Doenças do Sistema Nervoso Central/tratamento farmacológico , Desenho de Fármacos , Neurônios/efeitos dos fármacos , Bloqueadores do Canal Iônico Sensível a Ácido/farmacologia , Bloqueadores do Canal Iônico Sensível a Ácido/uso terapêutico , Canais Iônicos Sensíveis a Ácido/genética , Canais Iônicos Sensíveis a Ácido/metabolismo , Animais , Doenças do Sistema Nervoso Central/genética , Doenças do Sistema Nervoso Central/patologia , Cristalografia por Raios X , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Neurônios/patologia , Mutação Puntual , Domínios Proteicos/efeitos dos fármacos , Domínios Proteicos/genética , Relação Estrutura-Atividade
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