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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.
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
3.
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
4.
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
5.
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
6.
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
7.
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
8.
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
9.
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
10.
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
11.
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
12.
J Pharmacol Toxicol Methods ; 100: 106605, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31255744

RESUMO

INTRODUCTION: Cardiac late Na+ current (INaL) contributes to ventricular action potential duration. Pathological increase in INaL is arrhythmogenic, and inhibition of INaL offers protection against ventricular repolarization disturbance. Recently, two INaL datasets generated by different laboratories that assessed current inhibition by a panel of clinical drugs as a part of the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative were published. The results revealed a surprising degree of data variability despite of the use of a standardized voltage protocol. This study investigated whether remaining procedural differences related to experimental methods and data analysis associated with these datasets can produce differences in INaL pharmacology. METHODS: Whole cell voltage clamp recordings were performed on cells expressing NaV1.5 α- and ß1-subunits to study: 1) the impact of gating modifiers used to augment INaL (ATX-II vs. veratridine), internal solution composition (with vs. without ATP and GTP), and recording temperature (23 °C vs 37 °C) on stability of INaL measured across the duration of a patch clamp experiment; 2) mechanisms of each gating modifier on Na+ channels; and 3) effects of six drugs (lidocaine, mexiletine, chloroquine, ranolazine, ritonavir, and verapamil) on INaL induced by either gating modifier. RESULTS: Stability of INaL is affected by the choice of gating modifier, presence of nucleotides in the internal solution, and recording temperature. ATX-II and veratridine produced different changes in Na+ channel gating, inducing mechanistically distinct INaL. Drug potencies on inhibiting INaL were dependent on the choice of gating modifier and current region where drug effects were measured. DISCUSSION: INaL pharmacology can be impacted by all experimental factors examined in this study. The effect of gating modifier and current region used to quantify drug inhibition alone led to 30× difference in half inhibitory concentration (IC50) for ritonavir, demonstrating that substantial difference in drug inhibition can be produced. Drug potencies on inhibiting INaL derived from different patch clamp studies may thus not be generalizable. For INaL pharmacology to be useful for in silico modeling or interpreting drug-induced changes in cardiac action potentials or ECG, standardizing INaL experimental procedures including data analysis methods is necessary to minimize data variability.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Arritmias Cardíacas/induzido quimicamente , Ventrículos do Coração/efeitos dos fármacos , Canais de Sódio/efeitos dos fármacos , Arritmias Cardíacas/diagnóstico , Simulação por Computador , Ventrículos do Coração/metabolismo , Humanos , Nucleotídeos/metabolismo , Técnicas de Patch-Clamp , Canais de Sódio/metabolismo , Temperatura
13.
Cardiovasc J Afr ; 30(5): 268-274, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31361295

RESUMO

INTRODUCTION: Our previous experiments showed that the transient sodium current (INa) was abnormally increased in early ischaemia and atorvastatin could inhibit INa. The aim of this study was to observe the time-dependent effects of simulated ischaemia on INa and characterise the direct effects of atorvastatin on ischaemic INa. METHODS: Left ventricular myocytes were isolated from Wistar rats and randomly divided into two groups: a control group (normal to simulated ischaemia) and a statin group (normal to simulated ischaemia with 5 µmol/l atorvastatin). The INa was recorded under normal conditions (as baseline) by whole-cell patch clamp and recorded from three to 21 minutes in the next phase of simulated ischaemic conditions. RESULTS: In the control group, normalised INa (at -40 mV) was increased to the peak (1.15 ± 0.08 mA) at three minutes of ischaemia compared with baseline (0.95 ± 0.04 mA, p < 0.01), it subsequently returned to baseline levels at nine and 11 minutes of ischaemia (0.98 ± 0.12 and 0.92 ± 0.12 mA, respectively), and persistently decreased with prolonged ischaemic time. In the statin group, there were no differences between baseline and the early stages of ischaemia (0.97 ± 0.04 mA at baseline vs 0.92 ± 0.12 mA in ischaemia for three minutes, p > 0.05). CONCLUSION: Our results suggest that, in the early stages of ischaemia, changes in INa in ventricular myocytes are time-dependent, showing an initial increase followed by a decrease, while atorvastatin inhibited the transient increase in INa and made the change more gradual.


Assuntos
Antiarrítmicos/farmacologia , Arritmias Cardíacas/prevenção & controle , Atorvastatina/farmacologia , Ventrículos do Coração/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Isquemia Miocárdica/tratamento farmacológico , Miócitos Cardíacos/efeitos dos fármacos , Canais de Sódio/efeitos dos fármacos , Sódio/metabolismo , Animais , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Feminino , Frequência Cardíaca/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Ventrículos do Coração/fisiopatologia , Cinética , Masculino , Potenciais da Membrana , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/fisiopatologia , Miócitos Cardíacos/metabolismo , Ratos Wistar , Canais de Sódio/metabolismo
14.
J Agric Food Chem ; 67(28): 7793-7809, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31274315

RESUMO

Indoxacarb, a commercialized oxadiazine insecticide, nearly irreversibly blocks open/inactivated, but not resting sodium channels. The structure-activity relationships showed that the substituents at the position of the chiral atom in the oxadiazine ring are very important to the biological activity of oxadiazine insecticide. Here we synthesized a series of tricyclic oxadiazine 4a-methyl ester derivatives. The chiral atom in the oxadiazine ring has been epimerized and substituted with either pyrethric acid or cinnamic acid derivatives. Benzene ring in the tricyclic moiety was substituted with a chlorine, fluorine, or bromine atom, and nitrogen-linked benzene ring was substituted with a trifluoromethyl or trifluoromethoxy group. Toxicity of these compounds against Spodoptera litura F. was evaluated. Diastereoisomers of most toxic compounds J7 and J9 with pyrethric acid moiety were separated by flash column chromatography. The more polar diastereoisomers, J7-L-Rf and J9-L-Rf, and compounds J24 and J26 with cinnamic acid moiety exhibited highest insecticidal activities. We further used Monte Carlo energy minimizations to dock compound J7 and J24 in the NavMs-based homology model of the open cockroach sodium channel. In the low-energy binding modes, the compound interacted with residues in the inner pore and domain interfaces, which previously were proposed to contribute to receptors of pyrethroids and sodium channel blocker insecticides. Our results define compound J7 and J24 as a potentially useful optimized hit for the development of multiple sites sodium channel blocker or modulator.


Assuntos
Inseticidas/química , Inseticidas/toxicidade , Oxazinas/química , Oxazinas/toxicidade , Bloqueadores dos Canais de Sódio/química , Bloqueadores dos Canais de Sódio/toxicidade , Animais , Baratas/efeitos dos fármacos , Baratas/metabolismo , Descoberta de Drogas , Ésteres/química , Ésteres/farmacologia , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Modelos Moleculares , Canais de Sódio/química , Canais de Sódio/metabolismo , Spodoptera/efeitos dos fármacos , Spodoptera/metabolismo , Relação Estrutura-Atividade
15.
PLoS One ; 14(7): e0218738, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31260485

RESUMO

C. elegans neuronal system constitutes the ideal framework for studying simple, yet realistic, neuronal activity, since the whole nervous system is fully characterized with respect to the exact number of neurons and the neuronal connections. Most recent efforts are devoted to investigate and clarify the signal processing and functional connectivity, which are at the basis of sensing mechanisms, signal transmission, and motor control. In this framework, a refined modelof whole neuron dynamics constitutes a key ingredient to describe the electrophysiological processes, both at thecellular and at the network scale. In this work, we present Hodgkin-Huxley-based models of ion channels dynamics black, built on data available both from C. elegans and from other organisms, expressing homologous channels. We combine these channel models to simulate the electrical activity oftwo among the most studied neurons in C. elegans, which display prototypical dynamics of neuronal activation, the chemosensory AWCON and the motor neuron RMD. Our model properly describes the regenerative responses of the two cells. We analyze in detail the role of ion currents, both in wild type and in in silico knockout neurons. Moreover, we specifically investigate the behavior of RMD, identifying a heterogeneous dynamical response which includes bistable regimes and sustained oscillations. We are able to assess the critical role of T-type calcium currents, carried by CCA-1 channels, and leakage currents in the regulation of RMD response. Overall, our results provide new insights in the activity of key C. elegans neurons. The developed mathematical framework constitute a basis for single-cell and neuronal networks analyses, opening new scenarios in the in silico modeling of C. elegans neuronal system.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Modelos Neurológicos , Neurônios Motores/fisiologia , Rede Nervosa/fisiologia , Células Receptoras Sensoriais/fisiologia , Transmissão Sináptica/fisiologia , Animais , Caenorhabditis elegans/citologia , Proteínas de Caenorhabditis elegans/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Simulação por Computador , Expressão Gênica , Transporte de Íons , Neurônios Motores/citologia , Rede Nervosa/citologia , Canais de Potássio/genética , Canais de Potássio/metabolismo , Células Receptoras Sensoriais/citologia , Análise de Célula Única/métodos , Canais de Sódio/genética , Canais de Sódio/metabolismo
16.
Circ Arrhythm Electrophysiol ; 12(7): e007294, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31248280

RESUMO

BACKGROUND: Phthalates are used as plasticizers in the manufacturing of flexible, plastic medical products. Patients can be subjected to high phthalate exposure through contact with plastic medical devices. We aimed to investigate the cardiac safety and biocompatibility of mono-2-ethylhexyl phthalate (MEHP), a phthalate with documented exposure in intensive care patients. METHODS: Optical mapping of transmembrane voltage and pacing studies were performed on isolated, Langendorff-perfused rat hearts to assess cardiac electrophysiology after MEHP exposure compared with controls. MEHP dose was chosen based on reported blood concentrations after an exchange transfusion procedure. RESULTS: Thirty-minute exposure to MEHP increased the atrioventricular node (147 versus 107 ms) and ventricular (117 versus 77.5 ms) effective refractory periods, compared with controls. Optical mapping revealed prolonged action potential duration at slower pacing cycle lengths, akin to reverse use dependence. The plateau phase of the action potential duration restitution curve steepened and became monophasic in MEHP-exposed hearts (0.18 versus 0.06 slope). Action potential duration lengthening occurred during late-phase repolarization resulting in triangulation (70.3 versus 56.6 ms). MEHP exposure also slowed epicardial conduction velocity (35 versus 60 cm/s), which may be partly explained by inhibition of Nav1.5 (874 and 231 µmol/L half-maximal inhibitory concentration, fast and late sodium current). CONCLUSIONS: This study highlights the impact of acute MEHP exposure, using a clinically relevant dose, on cardiac electrophysiology in the intact heart. Heightened clinical exposure to plasticized medical products may have cardiac safety implications-given that action potential triangulation and electrical restitution modifications are a risk factor for early after depolarizations and cardiac arrhythmias.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Arritmias Cardíacas/induzido quimicamente , Dietilexilftalato/análogos & derivados , Equipamentos e Provisões/efeitos adversos , Sistema de Condução Cardíaco/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Plastificantes/toxicidade , Animais , Arritmias Cardíacas/diagnóstico , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Simulação por Computador , Dietilexilftalato/toxicidade , Desenho de Equipamento , Sistema de Condução Cardíaco/metabolismo , Sistema de Condução Cardíaco/fisiopatologia , Humanos , Preparação de Coração Isolado , Masculino , Modelos Cardiovasculares , Ratos Sprague-Dawley , Período Refratário Eletrofisiológico/efeitos dos fármacos , Medição de Risco , Canais de Sódio/efeitos dos fármacos , Canais de Sódio/metabolismo , Fatores de Tempo , Imagens com Corantes Sensíveis à Voltagem
17.
PLoS Negl Trop Dis ; 13(6): e0007432, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31158225

RESUMO

BACKGROUND: Multiple mutations in the voltage-gated sodium channel have been associated with knockdown resistance (kdr) to DDT and pyrethroid insecticides in a major human disease vector Aedes aegypti. One mutation, V1016G, confers sodium channel resistance to pyrethroids, but a different substitution in the same position V1016I alone had no effect. In pyrethroid-resistant Ae. aegypti populations, V1016I is often linked to another mutation, F1534C, which confers sodium channel resistance only to Type I pyrethroids including permethrin (PMT), but not to Type II pyrethroids including deltamethrin (DMT). Mosquitoes carrying both V1016G and F1534C exhibited a greater level of pyrethroid resistance than those carrying F1534C alone. More recently, a new mutation T1520I co-existing with F1534C was detected in India. However, whether V1016I or T1520I enhances pyrethroid resistance of sodium channels carrying F1534C remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: V1016I, V1016G, T1520I and F1534C substitutions were introduced alone and in various combinations into AaNav1-1, a sodium channel from Aedes aegypti. The mutant channels were then expressed in Xenopus oocytes and examined for channel properties and sensitivity to pyrethroids using the two-electrode voltage clamping technique. The results showed that V1016I or T1520I alone did not alter the AaNav1-1 sensitivity to PMT or DMT. However, the double mutant T1520I+F1534C was more resistant to PMT than F1534C, but remained sensitive to DMT. In contrast, the double mutant V1016I+F1534C was resistant to DMT and more resistant to PMT than F1534C. Furthermore, V1016I/G and F1534C channels, but not T1520I, were resistant to dichlorodiphenyltrichloroethane (DDT). Cryo-EM structures of sodium channels suggest that T1520I allosterically deforms geometry of the pyrethroid receptor site PyR1 in AaNav1-1. The small deformation does not affect binding of DDT, PMT or DMT, but in combination with F1534C it increases the channel resistance to PMT and DDT. CONCLUSIONS/SIGNIFICANCE: Our data corroborated the previously proposed sequential selection of kdr mutations in Ae. aegypti. We proposed that mutation F1534C first emerged in response to DDT/pyrethroids providing a platform for subsequent selection of mutations V1016I and T1520I that confer greater and broader spectrum of pyrethroid resistance.


Assuntos
Aedes/genética , DDT/farmacologia , Evolução Molecular , Resistência a Inseticidas , Inseticidas/farmacologia , Piretrinas/farmacologia , Canais de Sódio/genética , Aedes/efeitos dos fármacos , Substituição de Aminoácidos , Animais , Células Cultivadas , Expressão Gênica , Mosquitos Vetores/efeitos dos fármacos , Mosquitos Vetores/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Canais de Sódio/metabolismo , Xenopus
18.
Front Neural Circuits ; 13: 41, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31213991

RESUMO

Heterogeneity of neural properties within a given neural class is ubiquitous in the nervous system and permits different sub-classes of neurons to specialize for specific purposes. This principle has been thoroughly investigated in the hindbrain of the weakly electric fish A. leptorhynchus in the primary electrosensory area, the Electrosensory Lateral Line lobe (ELL). The pyramidal cells (PCs) that receive inputs from tuberous electroreceptors are organized in three maps in distinct segments of the ELL. The properties of these cells vary greatly across maps due to differences in connectivity, receptor expression, and ion channel composition. These cells are a seminal example of bursting neurons and their bursting dynamic relies on the presence of voltage-gated Na+ channels in the extensive apical dendrites of the superficial PCs. Other ion channels can affect burst generation and their expression varies across ELL neurons and segments. For example, SK channels cause hyperpolarizing after-potentials decreasing the likelihood of bursting, yet bursting propensity is similar across segments. We question whether the depolarizing mechanism that generates the bursts presents quantitative differences across segments that could counterbalance other differences having the opposite effect. Although their presence and role are established, the distribution and density of the apical dendrites' Na+ channels have not been quantified and compared across ELL maps. Therefore, we test the hypothesis that Na+ channel density varies across segment by quantifying their distribution in the apical dendrites of immunolabeled ELL sections. We found the Na+ channels to be two-fold denser in the lateral segment (LS) than in the centro-medial segment (CMS), the centro-lateral segment (CLS) being intermediate. Our results imply that this differential expression of voltage-gated Na+ channels could counterbalance or interact with other aspects of neuronal physiology that vary across segments (e.g., SK channels). We argue that burst coding of sensory signals, and the way the network regulates bursting, should be influenced by these variations in Na+ channel density.


Assuntos
Dendritos/metabolismo , Peixe Elétrico/metabolismo , Células Piramidais/metabolismo , Rombencéfalo/metabolismo , Canais de Sódio/metabolismo , Animais , Sistema da Linha Lateral/metabolismo
19.
Sci Adv ; 5(6): eaax2650, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31223657

RESUMO

Dinoflagelates and cyanobacteria produce saxitoxin (STX), a lethal bis-guanidinium neurotoxin causing paralytic shellfish poisoning. A number of metazoans have soluble STX-binding proteins that may prevent STX intoxication. However, their STX molecular recognition mechanisms remain unknown. Here, we present structures of saxiphilin (Sxph), a bullfrog high-affinity STX-binding protein, alone and bound to STX. The structures reveal a novel high-affinity STX-binding site built from a "proto-pocket" on a transferrin scaffold that also bears thyroglobulin domain protease inhibitor repeats. Comparison of Sxph and voltage-gated sodium channel STX-binding sites reveals a convergent toxin recognition strategy comprising a largely rigid binding site where acidic side chains and a cation-π interaction engage STX. These studies reveal molecular rules for STX recognition, outline how a toxin-binding site can be built on a naïve scaffold, and open a path to developing protein sensors for environmental STX monitoring and new biologics for STX intoxication mitigation.


Assuntos
Proteínas de Transporte/metabolismo , Saxitoxina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Linhagem Celular , Cianobactérias/metabolismo , Humanos , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/farmacologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Rana catesbeiana , Células Sf9 , Canais de Sódio/metabolismo , Tireoglobulina/metabolismo , Transferrina/metabolismo
20.
Int J Mol Sci ; 20(9)2019 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-31035589

RESUMO

BACKGROUND: Astaxanthin (ATX) is a lipophilic compound found in many marine organisms. Studies have shown that ATX has many strong biological properties, including antioxidant, antiviral, anticancer, cardiovascular, anti-inflammatory, neuro-protective and anti-diabetic activities. However, no research has elucidated the effect of ATX on ionic channels. ATX can be extracted from shrimp by-products. Our work aims to characterize ATX cell targets to lend value to marine by-products. METHODS: We used the Xenopus oocytes cell model to characterize the pharmacological target of ATX among endogenous Xenopus oocytes' ionic channels and to analyze the effects of all carotenoid-extract samples prepared from shrimp by-products using a supercritical fluid extraction (SFE) method. RESULTS: ATX inhibits amiloride-sensitive sodium conductance, xINa, in a dose-dependent manner with an IC50 of 0.14 µg, a maximum inhibition of 75% and a Hill coefficient of 0.68. It does not affect the potential of half activation, but significantly changes the kinetics, according to the slope factor values. The marine extract prepared from shrimp waste at 10 µg inhibits xINa in the same way as ATX 0.1 µg does. When ATX was added to the entire extract at 10 µg, inhibition reached that induced with ATX 1 µg. CONCLUSIONS: ATX and the shrimp Extract inhibit amiloride-sensitive sodium channels in Xenopus oocytes and the TEVC method makes it possible to measure the ATX inhibitory effect in bioactive SFE-Extract samples.


Assuntos
Produtos Biológicos/farmacologia , Descoberta de Drogas , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Oócitos/fisiologia , Amilorida/farmacologia , Animais , Descoberta de Drogas/métodos , Canais de Sódio/metabolismo , Xenopus laevis
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