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1.
Life Sci ; 255: 117814, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32439300

RESUMO

AIMS: Amiodarone (AMIO) is currently used in medical practice to reverse ventricular tachycardia. Here we determine the effects of AMIO in the electromechanical properties of isolated left ventricle myocyte (LVM) from mice and guinea pig and in a cellular model of Long QT Syndrome Type 3 (LQTS-3) using anemone neurotoxin 2 (ATX II), which induces increase of late sodium current in LVM. MAIN METHODS AND KEY FINDINGS: Using patch-clamp technique, fluorescence imaging to detect cellular Ca2+ transient and sarcomere detection systems we evaluate the effect of AMIO in healthy LVM. AMIO produced a significant reduction in the percentage of sarcomere shortening (0.1, 1 and 10 µM) in a range of pacing frequencies, however, without significant attenuation of Ca2+ transient. Also, 10 µM of AMIO caused the opposite effect on action potential repolarization of mouse and guinea pig LVM. When LVM from mouse and guinea pig were paced in a range of pacing frequencies and exposed to ATX (10 nM), AMIO (10 µM) was only able to abrogate electromechanical arrhythmias in LVM from guinea pig at lower pacing frequency. SIGNIFICANCE: AMIO has negative inotropic effect with opposite effect on action potential waveform in mouse and guinea pig LVM. Furthermore, the antiarrhythmic action of AMIO in LQTS-3 is species and frequency-dependent, which indicates that AMIO may be beneficial for some types of arrhythmias related to late sodium current.


Assuntos
Amiodarona/farmacologia , Antiarrítmicos/farmacologia , Doença do Sistema de Condução Cardíaco/tratamento farmacológico , Síndrome do QT Longo/tratamento farmacológico , Miócitos Cardíacos/efeitos dos fármacos , Amiodarona/administração & dosagem , Animais , Antiarrítmicos/administração & dosagem , Doença do Sistema de Condução Cardíaco/fisiopatologia , Relação Dose-Resposta a Droga , Cobaias , Ventrículos do Coração/citologia , Síndrome do QT Longo/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Sarcômeros/efeitos dos fármacos , Sarcômeros/metabolismo , Canais de Sódio/efeitos dos fármacos , Canais de Sódio/metabolismo , Especificidade da Espécie
2.
Am J Physiol Heart Circ Physiol ; 318(6): H1436-H1440, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32383994

RESUMO

Cardiac arrhythmias significantly contribute to mortality in Duchenne muscular dystrophy (DMD), a degenerative muscle disease triggered by mutations in the gene encoding for the intracellular protein dystrophin. A major source for the arrhythmias in patients with DMD is impaired ventricular impulse conduction, which predisposes for ventricular asynchrony, decreased cardiac output, and the development of reentrant mechanisms. The reason for ventricular conduction impairments and the associated arrhythmias in the dystrophic heart has remained unidentified. In the present study, we explored the hypothesis that dystrophin-deficient cardiac Purkinje fibers have reduced Na+ currents (INa), which would represent a potential mechanism underlying slowed ventricular conduction in the dystrophic heart. Therefore, by using a Langendorff perfusion system, we isolated Purkinje fibers from the hearts of adult wild-type control and dystrophin-deficient mdx mice. Enhanced green fluorescent protein (eGFP) expression under control of the connexin 40 gene allowed us to discriminate Purkinje fibers from eGFP-negative ventricular working cardiomyocytes after cell isolation. Finally, we recorded INa from wild-type and dystrophic mdx Purkinje fibers for comparison by means of the whole cell patch clamp technique. We found substantially reduced INa densities in mdx compared with wild-type Purkinje fibers, suggesting that dystrophin deficiency diminishes INa. Because Na+ channels in the Purkinje fiber membrane represent key determinants of ventricular conduction velocity, we propose that reduced INa in Purkinje fibers at least partly explains impaired ventricular conduction and the associated arrhythmias in the dystrophic heart.NEW & NOTEWORTHY Dystrophic cardiac Purkinje fibers have abnormally reduced Na+ current densities. This explains impaired ventricular conduction in the dystrophic heart.


Assuntos
Arritmias Cardíacas/metabolismo , Doença do Sistema de Condução Cardíaco/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Ramos Subendocárdicos/metabolismo , Canais de Sódio/metabolismo , Potenciais de Ação/fisiologia , Animais , Arritmias Cardíacas/complicações , Arritmias Cardíacas/fisiopatologia , Doença do Sistema de Condução Cardíaco/complicações , Doença do Sistema de Condução Cardíaco/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/complicações , Distrofia Muscular de Duchenne/fisiopatologia , Sódio/metabolismo
3.
Environ Toxicol ; 35(7): 774-782, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32061153

RESUMO

This study aims to investigate the protective effects of the Bauhinia championii (BC) against ischemia/reperfusion (I/R)-induced injury in an isolated heart model. Langendorff-perfused C57BL/6JNarl mice hearts were performed with 30 minutes ischemia and 60 minutes reperfusion by left anterior descending artery ligation. Before reperfusion, boiling water extracts of BC (10 mg/L) was pretreated for 15 minutes. During reperfusion, BC significantly decreased the occurrence of ventricular arrhythmias by lead II electrocardiogram (ECG). Electrophysiological effect of BC was further determined in isolated ventricular myocytes by whole-cell patch clamp technique. The underlying mechanism may result from its Na+ channel blocking activity characterized with reduced rise slope of action potential and Na+ current density. Moreover, BC dramatically reduced I/R-caused infarct size, which was accessed by 2,3,5-triphenyltetrazolium chloride (TTC) assay. Since BC decreased I/R-induced myoglobin release and oxidation of Ca2+ -calmodulin-dependent protein kinase, inhibition of myocardial necroptosis may account for the protective effects of BC on myocytes lose. This study indicated that BC may prevent I/R induced ventricular arrhythmias and myocyte death by blocking Na+ channels and decreasing necroptosis, respectively. Since most of the available antiarrhythmic remedies have unwanted adverse actions, BC could be a novel candidate for the treatment of myocardial infarction and ventricular arrhythmia.


Assuntos
Bauhinia/química , Coração/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Extratos Vegetais/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Animais , Eletrocardiografia , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Necroptose/efeitos dos fármacos , Técnicas de Patch-Clamp , Componentes Aéreos da Planta/química , Extratos Vegetais/isolamento & purificação , Bloqueadores dos Canais de Sódio/isolamento & purificação , Canais de Sódio/metabolismo
4.
Am J Physiol Regul Integr Comp Physiol ; 318(4): R772-R780, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32101460

RESUMO

Femoral artery occlusion in rats has been used to study human peripheral artery disease (PAD). Using this animal model, a recent study suggests that increases in levels of tumor necrosis factor-α (TNF-α) and its receptor lead to exaggerated responses of sympathetic nervous activity and arterial blood pressure as metabolically sensitive muscle afferents are activated. Note that voltage-dependent Na+ subtype NaV1.8 channels (NaV1.8) are predominately present in chemically sensitive thin fiber sensory nerves. The purpose of this study was to examine the role played by TNF-α in regulating activity of NaV1.8 currents in muscle dorsal root ganglion (DRG) neurons of rats with PAD induced by femoral artery occlusion. DRG neurons from control and occluded limbs of rats were labeled by injecting the fluorescent tracer DiI into the hindlimb muscles 5 days before the experiments. A voltage patch-clamp mode was used to examine TTX-resistant (TTX-R) NaV currents. Results were as follows: 72 h of femoral artery occlusion increased peak amplitude of TTX-R [1,922 ± 139 pA in occlusion (n = 11 DRG neurons) vs. 1,178 ± 39 pA in control (n = 10), means ± SE; P < 0.001 between the 2 groups] and NaV1.8 currents [1,461 ± 116 pA in occlusion (n = 11) and 766 ± 48 pA in control (n = 10); P < 0.001 between groups] in muscle DRG neurons. TNF-α exposure amplified TTX-R and NaV1.8 currents in DRG neurons of occluded muscles in a dose-dependent manner. Notably, the amplification of TTX-R and NaV1.8 currents induced by TNF-α was attenuated in DRG neurons with preincubation with respective inhibitors of the intracellular signaling pathways p38-MAPK, JNK, and ERK. In conclusion, our data suggest that NaV1.8 is engaged in the role of TNF-α in amplifying muscle afferent inputs as the hindlimb muscles are ischemic; p38-MAPK, JNK, and ERK pathways are likely necessary to mediate the effects of TNF-α.


Assuntos
Membro Posterior/inervação , Músculo Esquelético/inervação , Canal de Sódio Disparado por Voltagem NAV1.8/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Tetrodotoxina/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Artéria Femoral , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Canal de Sódio Disparado por Voltagem NAV1.8/genética , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/fisiologia , Canais de Sódio
5.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 37(2): 178-181, 2020 Feb 10.
Artigo em Chinês | MEDLINE | ID: mdl-32034750

RESUMO

OBJECTIVE: To explore the genetic etiology of a child with autism, mental retardation and epilepsy. METHODS: Conventional G-banding chromosomal analysis was carried out. Chromosomal variation was also detected by single nucleotide polymorphism microarray (SNP array). Pathogenic mutations were screened by high-throughput sequencing and validated by Sanger sequencing. Pathologic significance of the candidate mutations was analyzed through search of database and literature review. RESULTS: No karyotypic abnormality was found with the child and his parents, while SNP array has detected a 460 kb deletion in the 14q11.2 region in the child. High-throughput and Sanger sequencing revealed a novel mutation of the NALCN gene in the child, in addition with a hemizygous mutation of the COL4A5 gene in the child and his mother. CONCLUSION: The 14q11.2 microdeletion and NALCN mutation may contribute to the autism, mental retardation and epilepsy in this child.


Assuntos
Deleção Cromossômica , Deficiência Intelectual , Criança , Bandeamento Cromossômico , Cromossomos Humanos Par 14 , Testes Genéticos , Humanos , Canais Iônicos , Cariotipagem , Proteínas de Membrana , Mutação , Canais de Sódio
6.
Pest Manag Sci ; 76(6): 2251-2259, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31981401

RESUMO

BACKGROUND: Recent outbreaks of dengue and Zika have emphasized the importance to effectively control Aedes aegypti, which vectors the viruses causing these diseases. Pyrethroid insecticides are primarily used to control adult A. aegypti, especially during disease outbreaks. However, pyrethroid resistance in A. aegypti is an increasing problem. Mutations in the voltage-sensitive sodium channel (Vssc) are a common mechanism of pyrethroid resistance. The F1534C mutation is common and distributed globally in A. aegypti populations, but previous studies disagree about the role of this mutation in conferring resistance to pyrethroid insecticides. RESULTS: We isolated a congenic strain (1534C:ROCK) which was closely related to a susceptible strain Rockefeller (ROCK), but was homozygous for the 1534C Vssc allele. We determined resistance levels against eight insecticides that target the VSSC: six pyrethroids, DDT and DCJW (the bioactivated metabolite of indoxacarb). The resistance levels ranged from 7- to 16-fold, and resistance was inherited as an incompletely recessive trait. We also found a novel 367I+1520I+1534C allele, in addition to the 1534C and 1520I+1534C alleles, in mosquitoes from Thailand. The T1520I mutation did not increase pyrethroid resistance beyond what was conferred by the F1534C mutation alone. CONCLUSION: The F1534C Vssc mutation is common in A. aegypti populations and confers 7- to 16-fold resistance to pyrethroids, DDT, and DCJW in Aedes aegypti. These resistance levels are considerably less than previously reported for the S989P+V1016G mutations. Our results provide useful information for resistance management, specifically the levels of resistance conferred by the most common Vssc mutation in A. aegypti. © 2020 Society of Chemical Industry.


Assuntos
Aedes , Animais , Inseticidas , Mosquitos Vetores , Mutação , Piretrinas , Canais de Sódio
7.
Life Sci ; 244: 117333, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31962132

RESUMO

AIMS: Detect the antiarrhythmic effect of crotonoside (Cro). MAIN METHODS: We used whole-cell patch-clamp techniques to detect the effects of Cro on action potentials (APs) and transmembrane ion currents in isolated rabbit left ventricular myocytes. We also verified the effect of Cro on ventricular arrhythmias caused by aconitine in vivo. KEY FINDINGS: Cro reduced the maximum depolarization velocity (Vmax) of APs and shortened the action potential duration (APD) in a concentration-dependent manner, but it had no significant effect on the resting membrane potential (RMP) or action potential amplitude (APA). It also inhibited the peak sodium current (INa) and L-type calcium current (ICaL) in a concentration-dependent manner with half-maximal inhibitory concentrations (IC50) of 192 µmol/L and 159 µmol/L, respectively. However, Cro had no significant effects on the inward rectifier potassium current (IK1) or rapidly activating delayed rectifier potassium current (IKr). Sea anemone toxin II (ATX II) increased the late sodium current (INaL), but Cro abolished this effect. Moreover, Cro significantly abolished ATX II-induced early afterdepolarizations (EADs) and high extracellular Ca2+ concentration (3.6 mmol/L)-induced delayed afterdepolarizations (DADs). We also verified that Cro effectively delayed the onset time and reduced the incidence of ventricular arrhythmias caused by aconitine in vivo. SIGNIFICANCE: These results revealed that Cro effectively inhibits INa, INaL, and ICaL in ventricular myocytes. Cro has antiarrhythmic potential and thus deserves further study.


Assuntos
Guanina/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Antiarrítmicos/metabolismo , Antiarrítmicos/farmacologia , Arritmias Cardíacas/fisiopatologia , Cálcio/metabolismo , Canais de Cálcio/efeitos dos fármacos , China , Feminino , Guanina/metabolismo , Ventrículos do Coração/metabolismo , Técnicas de Patch-Clamp/métodos , Coelhos , Sódio/metabolismo , Canais de Sódio/efeitos dos fármacos
8.
Nat Commun ; 11(1): 512, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980605

RESUMO

Mechanisms for human sinoatrial node (SAN) dysfunction are poorly understood and whether human SAN excitability requires voltage-gated sodium channels (Nav) remains controversial. Here, we report that neuronal (n)Nav blockade and selective nNav1.6 blockade during high-resolution optical mapping in explanted human hearts depress intranodal SAN conduction, which worsens during autonomic stimulation and overdrive suppression to conduction failure. Partial cardiac (c)Nav blockade further impairs automaticity and intranodal conduction, leading to beat-to-beat variability and reentry. Multiple nNav transcripts are higher in SAN vs atria; heterogeneous alterations of several isoforms, specifically nNav1.6, are associated with heart failure and chronic alcohol consumption. In silico simulations of Nav distributions suggest that INa is essential for SAN conduction, especially in fibrotic failing hearts. Our results reveal that not only cNav but nNav are also integral for preventing disease-induced failure in human SAN intranodal conduction. Disease-impaired nNav may underlie patient-specific SAN dysfunctions and should be considered to treat arrhythmias.


Assuntos
Arritmias Cardíacas/fisiopatologia , Sistema de Condução Cardíaco/fisiopatologia , Neurônios/metabolismo , Nó Sinoatrial/fisiopatologia , Canais de Sódio/metabolismo , Potenciais de Ação/fisiologia , Adulto , Idoso , Alcoolismo/genética , Arritmias Cardíacas/genética , Doença Crônica , Simulação por Computador , Feminino , Átrios do Coração/metabolismo , Átrios do Coração/fisiopatologia , Sistema de Condução Cardíaco/metabolismo , Insuficiência Cardíaca/genética , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Cardiovasculares , Imagem Óptica , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Nó Sinoatrial/metabolismo , Canais de Sódio/genética , Estresse Fisiológico , Adulto Jovem
9.
Cell ; 180(1): 122-134.e10, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31866066

RESUMO

Voltage-gated sodium channel Nav1.5 generates cardiac action potentials and initiates the heartbeat. Here, we report structures of NaV1.5 at 3.2-3.5 Å resolution. NaV1.5 is distinguished from other sodium channels by a unique glycosyl moiety and loss of disulfide-bonding capability at the NaVß subunit-interaction sites. The antiarrhythmic drug flecainide specifically targets the central cavity of the pore. The voltage sensors are partially activated, and the fast-inactivation gate is partially closed. Activation of the voltage sensor of Domain III allows binding of the isoleucine-phenylalanine-methionine (IFM) motif to the inactivation-gate receptor. Asp and Ala, in the selectivity motif DEKA, line the walls of the ion-selectivity filter, whereas Glu and Lys are in positions to accept and release Na+ ions via a charge-delocalization network. Arrhythmia mutation sites undergo large translocations during gating, providing a potential mechanism for pathogenic effects. Our results provide detailed insights into Nav1.5 structure, pharmacology, activation, inactivation, ion selectivity, and arrhythmias.


Assuntos
Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/ultraestrutura , Animais , Linhagem Celular , Células HEK293 , Coração/fisiologia , Humanos , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp/métodos , Ratos , Sódio/metabolismo , Canais de Sódio/química , Relação Estrutura-Atividade , Canais de Sódio Disparados por Voltagem/metabolismo , Canais de Sódio Disparados por Voltagem/ultraestrutura
10.
J Matern Fetal Neonatal Med ; 33(2): 253-257, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30033781

RESUMO

Background: Amniotic fluid (AF) is a complex structure with a changing content by gestation. Lower genomic expression of Na channels in airways was shown to be associated with respiratory distress syndrome (RDS). The aim of this study was to determine the possible role of amniotic fluid pH and electrolytes for prediction of neonatal respiratory morbidities.Methods: This was a prospective controlled cohort study. During C-section, 1 ml of AF was aspirated before incision of membranes. AF pH and electrolytes were analyzed by blood gas analyzer. Maternal and neonatal demographic features and clinical outcomes, respiratory morbidities were all recorded.Results: AF Na and K values were significantly higher in all infants with respiratory morbidities compared with those who did not develop respiratory findings. AF Na value was significantly higher in preterm neonates with RDS as well as in term neonates with transient tachypnea of the newborn (TTN). AF pH did not show any significant difference for prediction of respiratory morbidities in term and preterm infants.Conclusion: This is the first study that reported the value of AF Na and K levels for prediction of respiratory morbidities in term and preterm infants. However, further studies including larger number of infants are required to confirm the role of AF analysis to predict neonatal respiratory morbidities. Randomized controlled trial (RCT) number: NCT02813954.


Assuntos
Líquido Amniótico/química , Eletrólitos/metabolismo , Taquipneia Transitória do Recém-Nascido/sangue , Gasometria , Feminino , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Masculino , Potássio/sangue , Gravidez , Estudos Prospectivos , Canais de Sódio/metabolismo
11.
PLoS Comput Biol ; 15(12): e1007455, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31790388

RESUMO

Cortical spreading depression (SD) is a spreading disruption of ionic homeostasis in the brain during which neurons experience complete and prolonged depolarizations. SD is the basis of migraine aura and is increasingly associated with many other brain pathologies. Here, we study the role of glutamate and NMDA receptor dynamics in the context of an ionic electrodiffusion model. We perform simulations in one (1D) and two (2D) spatial dimension. Our 1D simulations reproduce the "inverted saddle" shape of the extracellular voltage signal for the first time. Our simulations suggest that SD propagation depends on two overlapping mechanisms; one dependent on extracellular glutamate diffusion and NMDA receptors and the other dependent on extracellular potassium diffusion and persistent sodium channel conductance. In 2D simulations, we study the dynamics of spiral waves. We study the properties of the spiral waves in relation to the planar 1D wave, and also compute the energy expenditure associated with the recurrent SD spirals.


Assuntos
Depressão Alastrante da Atividade Elétrica Cortical/fisiologia , Ácido Glutâmico/metabolismo , Modelos Neurológicos , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Encéfalo/metabolismo , Biologia Computacional , Simulação por Computador , Humanos , Canais Iônicos/metabolismo , Neurônios/metabolismo , Dinâmica não Linear , Potássio/metabolismo , Canais de Sódio/metabolismo
12.
Genes Dev ; 33(23-24): 1739-1750, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31753914

RESUMO

Proliferating cells, typically considered "nonexcitable," nevertheless, exhibit regulation by bioelectric signals. Notably, voltage-gated sodium channels (VGSC) that are crucial for neuronal excitability are also found in progenitors and up-regulated in cancer. Here, we identify a role for VGSC in proliferation of Drosophila neuroblast (NB) lineages within the central nervous system. Loss of paralytic (para), the sole gene that encodes Drosophila VGSC, reduces neuroblast progeny cell number. The type II neuroblast lineages, featuring a population of transit-amplifying intermediate neural progenitors (INP) similar to that found in the developing human cortex, are particularly sensitive to para manipulation. Following a series of asymmetric divisions, INPs normally exit the cell cycle through a final symmetric division. Our data suggests that loss of Para induces apoptosis in this population, whereas overexpression leads to an increase in INPs and overall neuroblast progeny cell numbers. These effects are cell autonomous and depend on Para channel activity. Reduction of Para expression not only affects normal NB development, but also strongly suppresses brain tumor mass, implicating a role for Para in cancer progression. To our knowledge, our studies are the first to identify a role for VGSC in neural progenitor proliferation. Elucidating the contribution of VGSC in proliferation will advance our understanding of bioelectric signaling within development and disease states.


Assuntos
Proliferação de Células/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/citologia , Drosophila/genética , Células-Tronco Neurais/citologia , Canais de Sódio/genética , Canais de Sódio/metabolismo , Animais , Apoptose , Contagem de Células , Linhagem da Célula/genética , Expressão Gênica , Técnicas de Silenciamento de Genes
14.
Elife ; 82019 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-31765298

RESUMO

Voltage-dependent sodium and calcium channels in pain-initiating nociceptor neurons are attractive targets for new analgesics. We made a permanently charged cationic derivative of an N-type calcium channel-inhibitor. Unlike cationic derivatives of local anesthetic sodium channel blockers like QX-314, this cationic compound inhibited N-type calcium channels more effectively with extracellular than intracellular application. Surprisingly, the compound is also a highly effective sodium channel inhibitor when applied extracellularly, producing more potent inhibition than lidocaine or bupivacaine. The charged inhibitor produced potent and long-lasting analgesia in mouse models of incisional wound and inflammatory pain, inhibited release of the neuropeptide calcitonin gene-related peptide (CGRP) from dorsal root ganglion neurons, and reduced inflammation in a mouse model of allergic asthma, which has a strong neurogenic component. The results show that some cationic molecules applied extracellularly can powerfully inhibit both sodium channels and calcium channels, thereby blocking both nociceptor excitability and pro-inflammatory peptide release.


Assuntos
Canais de Cálcio Tipo N/genética , Inflamação Neurogênica/tratamento farmacológico , Dor/tratamento farmacológico , Canais de Sódio/genética , Animais , Bupivacaína/farmacologia , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Modelos Animais de Doenças , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/patologia , Humanos , Lidocaína/análogos & derivados , Lidocaína/farmacologia , Camundongos , Inflamação Neurogênica/genética , Inflamação Neurogênica/patologia , Nociceptores , Dor/genética , Dor/patologia , Bloqueadores dos Canais de Sódio/farmacologia
15.
Am J Physiol Renal Physiol ; 317(6): F1462-F1474, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31566436

RESUMO

The goal of the present study was to investigate the functional implications of sexual dimorphism in the pattern of transporters along the rodent nephron as reported by Veiras et al. (J Am Soc Nephrol 28: 3504-3517, 2017). To do so, we developed sex-specific computational models of water and solute transport along the superficial nephrons from male and female rat kidneys. The models account for the sex differences in the abundance of apical and basolateral transporters, single nephron glomerular filtration rate, and tubular dimensions. Model simulations predict that ~70% and 60% of filtered Na+ is reabsorbed by the proximal tubule of male and female rat kidneys, respectively. The lower fractional Na+ reabsorption in female kidneys is due primarily to their smaller transport area, lower Na+/H+ exchanger activity, and lower claudin-2 abundance, culminating in significantly larger fractional delivery of water and Na+ to the downstream nephron segments in female kidneys. Conversely, the female distal nephron exhibits a higher abundance of key Na+ transporters, including Na+-K+-Cl- cotransporters, Na+-Cl- cotransporters, and epithelial Na+ channels. The higher abundance of transporters accounts for the enhanced water and Na+ transport along the female, relative to male, distal nephron, resulting in similar urine excretion between the sexes. Consequently, in response to a saline load, the Na+ load delivered distally is greater in female rats than male rats, overwhelming transport capacity and resulting in higher natriuresis in female rats.


Assuntos
Proteínas de Transporte/metabolismo , Néfrons/metabolismo , Animais , Claudinas/metabolismo , Feminino , Taxa de Filtração Glomerular , Túbulos Renais/metabolismo , Masculino , Modelos Biológicos , Ratos , Caracteres Sexuais , Sódio/metabolismo , Sódio/urina , Canais de Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Água/metabolismo
16.
Pflugers Arch ; 471(11-12): 1383-1396, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31654198

RESUMO

The epithelial Na+ channel (ENaC) is essential for Na+/K+ homeostasis and blood pressure control. Its activity is regulated by proteases in rodents. To gain more information on proteolytic ENaC regulation in humans, we tested the hypotheses that (1) human kidney α- and γ-ENaC subunits are furin-cleaved, glycosylated, and altered by medication that change plasma aldosterone; (2) prostasin-cleaved γ-ENaC is increased in proteinuria, and (3) cleaved ENaC moieties prevail at the membranes and in urinary extracellular vesicles (uEVs). We developed three monoclonal antibodies (mAbs) targeting (1) the neo-epitope generated after furin cleavage in γ-ENaC (mAb-furin); (2) the intact prostasin cleavage-site in γ-ENaC (mAb-intactRKRK), and (3) the α-ENaC subunit (mAb-alpha). Nephrectomy tissue and uEVs were used for immunoblotting and -histochemistry. In human kidney tissue, mAb-furin detected a ≈ 65-70 kDa protein, compatible with furin-cleaved γ-ENaC; mAb-intactRKRK detected full-length (≈ 90-100 kDa) and furin-cleaved (≈ 70-75 kDa) γ-ENaC. mAb-alpha detected a ≈ 50 kDa protein compatible with furin-cleaved α-subunit. Furin-cleaved γ-ENaC was detected predominantly within membrane fractions and deglycosylation shifted full-length γ-ENaC migration ~ 20 kDa. While γ-ENaC uEV levels were below the detection limit, α-ENaC migrated as intact (≈ 75 kDa) and furin-cleaved (≈ 50 kDa) in uEVs. Kidney levels of α- and γ-ENaC in diuretic- (n = 3) and ACE-inhibitor-treated (n = 4) patients were not different from controls (n = 4). Proteinuric patients (n = 6) displayed similar level of furin-cleaved γ-ENaC as controls (n = 4). Cleaved α-ENaC abundance was significantly lower in the kidneys from proteinuria patients. In conclusion, the study demonstrates ENaC cleavage as an event in human kidney that could contribute to physiological regulation and pathophysiological activation of ENaC.


Assuntos
Canais Epiteliais de Sódio/metabolismo , Epitélio/metabolismo , Furina/metabolismo , Rim/metabolismo , Subunidades Proteicas/metabolismo , Canais de Sódio/metabolismo , Aldosterona/metabolismo , Animais , Diuréticos/farmacologia , Epitélio/efeitos dos fármacos , Glicosilação , Humanos , Rim/efeitos dos fármacos , Camundongos , Proteinúria/metabolismo , Serina Endopeptidases/metabolismo , Sódio/metabolismo
17.
Neuron ; 104(5): 947-959.e5, 2019 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-31629603

RESUMO

Insect olfactory receptors operate as ligand-gated ion channels that directly transduce odor stimuli into electrical signals. However, in the absence of any known intermediate transduction steps, it remains unclear whether and how these ionotropic inputs are amplified in olfactory receptor neurons (ORNs). Here, we find that amplification occurs in the Drosophila courtship-promoting ORNs through Pickpocket 25 (PPK25), a member of the degenerin/epithelial sodium channel family (DEG/ENaC). Pharmacological and genetic manipulations indicate that, in Or47b and Ir84a ORNs, PPK25 mediates Ca2+-dependent signal amplification via an intracellular calmodulin-binding motif. Additionally, hormonal signaling upregulates PPK25 expression to determine the degree of amplification, with striking effects on male courtship. Together, these findings advance our understanding of sensory neurobiology by identifying an amplification mechanism compatible with ionotropic signaling. Moreover, this study offers new insights into DEG/ENaC activation by highlighting a novel means of regulation that is likely conserved across species.


Assuntos
Proteínas de Drosophila/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Comportamento Sexual Animal/fisiologia , Olfato/fisiologia , Canais de Sódio/metabolismo , Animais , Corte , Drosophila melanogaster , Masculino
18.
Genes (Basel) ; 10(10)2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31561430

RESUMO

Transcriptional responses to the appropriate temporal pattern of action potential firing are essential for long-term adaption of neuronal properties to the functional activity of neural circuits and environmental experience. However, standard transcriptome analysis methods can be too limited in identifying critical aspects that coordinate temporal coding of action potential firing with transcriptome response. A Pearson correlation analysis was applied to determine how pairs of genes in the mouse dorsal root ganglion (DRG) neurons are coordinately expressed in response to stimulation producing the same number of action potentials by two different temporal patterns. Analysis of 4728 distinct gene-pairs related to calcium signaling, 435,711 pairs of transcription factors, 820 pairs of voltage-gated ion channels, and 86,862 pairs of calcium signaling genes with transcription factors indicated that genes become coordinately activated by distinct action potential firing patterns and this depends on the duration of stimulation. Moreover, a measure of expression variance revealed that the control of transcripts abundances is sensitive to the pattern of stimulation. Thus, action potentials impact intracellular signaling and the transcriptome in dynamic manner that not only alter gene expression levels significantly (as previously reported) but also affects the control of their expression fluctuations and profoundly remodel the transcriptional networks.


Assuntos
Potenciais de Ação , Redes Reguladoras de Genes , Neurônios/metabolismo , Transcriptoma , Animais , Sinalização do Cálcio , Células Cultivadas , Gânglios Espinais/citologia , Camundongos , Neurônios/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canais de Sódio/genética , Canais de Sódio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
19.
BMC Biol ; 17(1): 63, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31412898

RESUMO

BACKGROUND: Voltage-gated sodium (Nav) channels have traditionally been considered a trademark of excitable cells. However, recent studies have shown the presence of Nav channels in several non-excitable cells, such as astrocytes and macrophages, demonstrating that the roles of these channels are more diverse than was previously thought. Despite the earlier discoveries, the presence of Nav channel-mediated currents in the cells of retinal pigment epithelium (RPE) has been dismissed as a cell culture artifact. We challenge this notion by investigating the presence and possible role of Nav channels in RPE both ex vivo and in vitro. RESULTS: Our work demonstrates that several subtypes of Nav channels are found in human embryonic stem cell (hESC)-derived and mouse RPE, most prominently subtypes Nav1.4, Nav1.6, and Nav1.8. Whole cell patch clamp recordings from the hESC-derived RPE monolayers showed that the current was inhibited by TTX and QX-314 and was sensitive to the selective blockers of the main Nav subtypes. Importantly, we show that the Nav channels are involved in photoreceptor outer segment phagocytosis since blocking their activity significantly reduces the efficiency of particle internalization. Consistent with this role, our electron microscopy results and immunocytochemical analysis show that Nav1.4 and Nav1.8 accumulate on phagosomes and that pharmacological inhibition of Nav channels as well as silencing the expression of Nav1.4 with shRNA impairs the phagocytosis process. CONCLUSIONS: Taken together, our study shows that Nav channels are present in RPE, giving this tissue the capacity of fast electrical signaling. The channels are critical for the physiology of RPE with an important role in photoreceptor outer segment phagocytosis.


Assuntos
Fagocitose/genética , Epitélio Pigmentado da Retina/fisiologia , Transdução de Sinais/genética , Canais de Sódio/fisiologia , Animais , Células-Tronco Embrionárias Humanas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp
20.
PLoS Comput Biol ; 15(8): e1006938, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31469828

RESUMO

The mechanism(s) of action of most commonly used pharmacological blockers of voltage-gated ion channels are well understood; however, this knowledge is rarely considered when interpreting experimental data. Effects of blockade are often assumed to be equivalent, regardless of the mechanism of the blocker involved. Using computer simulations, we demonstrate that this assumption may not always be correct. We simulate the blockade of a persistent sodium current (INaP), proposed to underlie rhythm generation in pre-Bötzinger complex (pre-BötC) respiratory neurons, via two distinct pharmacological mechanisms: (1) pore obstruction mediated by tetrodotoxin and (2) altered inactivation dynamics mediated by riluzole. The reported effects of experimental application of tetrodotoxin and riluzole in respiratory circuits are diverse and seemingly contradictory and have led to considerable debate within the field as to the specific role of INaP in respiratory circuits. The results of our simulations match a wide array of experimental data spanning from the level of isolated pre-BötC neurons to the level of the intact respiratory network and also generate a series of experimentally testable predictions. Specifically, in this study we: (1) provide a mechanistic explanation for seemingly contradictory experimental results from in vitro studies of INaP block, (2) show that the effects of INaP block in in vitro preparations are not necessarily equivalent to those in more intact preparations, (3) demonstrate and explain why riluzole application may fail to effectively block INaP in the intact respiratory network, and (4) derive the prediction that effective block of INaP by low concentration tetrodotoxin will stop respiratory rhythm generation in the intact respiratory network. These simulations support a critical role for INaP in respiratory rhythmogenesis in vivo and illustrate the importance of considering mechanism when interpreting and simulating data relating to pharmacological blockade.


Assuntos
Modelos Neurológicos , Sistema Respiratório/efeitos dos fármacos , Sistema Respiratório/inervação , Bloqueadores dos Canais de Sódio/farmacologia , Animais , Biologia Computacional , Simulação por Computador , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Centro Respiratório/efeitos dos fármacos , Centro Respiratório/fisiologia , Sistema Respiratório/metabolismo , Riluzol/farmacologia , Canais de Sódio/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Tetrodotoxina/farmacologia
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