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1.
Nat Commun ; 11(1): 3881, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753572

RESUMO

Cells typically respond to chemical or physical perturbations via complex signaling cascades which can simultaneously affect multiple physiological parameters, such as membrane voltage, calcium, pH, and redox potential. Protein-based fluorescent sensors can report many of these parameters, but spectral overlap prevents more than ~4 modalities from being recorded in parallel. Here we introduce the technique, MOSAIC, Multiplexed Optical Sensors in Arrayed Islands of Cells, where patterning of fluorescent sensor-encoding lentiviral vectors with a microarray printer enables parallel recording of multiple modalities. We demonstrate simultaneous recordings from 20 sensors in parallel in human embryonic kidney (HEK293) cells and in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and we describe responses to metabolic and pharmacological perturbations. Together, these results show that MOSAIC can provide rich multi-modal data on complex physiological responses in multiple cell types.


Assuntos
Técnicas Biossensoriais/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Microscopia de Fluorescência/métodos , Miócitos Cardíacos/metabolismo , Imagem Óptica/métodos , Potenciais de Ação/efeitos dos fármacos , Antagonistas Adrenérgicos beta/farmacologia , Técnicas Biossensoriais/instrumentação , Cálcio/química , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Peróxido de Hidrogênio/farmacologia , Concentração de Íons de Hidrogênio , Células-Tronco Pluripotentes Induzidas/citologia , Mitocôndrias/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Imagem Óptica/instrumentação , Oxidantes/farmacologia , Oxirredução/efeitos dos fármacos , Propanolaminas/farmacologia
2.
Life Sci ; 257: 118047, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32629001

RESUMO

AIM: The purpose of the study was to investigate what effects the sigma-1 receptor (S1R) could exert on the cardiac myocyte ion channels in a rodent model of depression and to explore the underlying mechanisms since depression is an independent risk factor for cardiovascular diseases including ventricular arrhythmias (VAs). MATERIALS AND METHODS: To establish the depression model in rats, chronic mild unpredictable stress (CMUS) for 28 days was used. The S1R agonist fluvoxamine was injected intraperitoneally from the second week to the last week for 21 days in total, and the effects were evaluated by patch clamp, western blot analysis, and Masson staining. KEY FINDINGS: We demonstrated that depression was improved after treatment with fluvoxamine. In addition, the prolongation of the corrected QT (QTc) interval under CMUS that increased vulnerability to VAs was significantly attenuated by stimulation of S1R due to the decreased amplitude of L-type calcium current (ICa-L) and the restoration of reduced transient outward potassium current (Ito) resulting from CMUS induction. The S1R also decelerated Ito inactivation and accelerated Ito recovery by activating Ca2+/calmodulin-dependent kinase II. Moreover, the stimulation of S1R ameliorated the structural remodeling as the substrate for maintenance of VAs. All these effects were abolished by the administration of S1R antagonist BD1047, which verified the roles for S1R. SIGNIFICANCE: Activation of S1R could decrease the vulnerability to VAs by inhibiting ICa-L and restoring Ito, in addition to ameliorating the CMUS-induced depressive symptoms and structural remodeling.


Assuntos
Depressão/metabolismo , Miócitos Cardíacos/metabolismo , Receptores sigma/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Arritmias Cardíacas/metabolismo , Depressão/fisiopatologia , Transtorno Depressivo/metabolismo , Transtorno Depressivo/fisiopatologia , Modelos Animais de Doenças , Fluvoxamina/metabolismo , Fluvoxamina/farmacologia , Ventrículos do Coração/efeitos dos fármacos , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Receptores sigma/metabolismo , Remodelação Ventricular/efeitos dos fármacos , Remodelação Ventricular/fisiologia
3.
Cardiovasc Ther ; 2020: 3480276, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32565909

RESUMO

Wenxin Keli (WXKL) is a traditional Chinese medicine drug approved for the treatment of cardiovascular diseases. This study aimed to identify WXKL-targeting genes involved in antiarrhythmic efficacy of WXKL. The Traditional Chinese Medicine Systems Pharmacology (TCMSP) technology platform was used to screen active compounds of WXKL and WXKL-targeting arrhythmia-related genes. A pig model of myocardial ischemia (MI) was established by balloon-expanding the endothelium of the left coronary artery. Pigs were divided into the model group and WXKL group (n = 6). MI, QT interval, heart rate, and arrhythmia were recorded, and the mRNA expression of target genes in myocardial tissues was detected by PCR. Eleven active ingredients of WXKL and eight WXKL-targeting arrhythmia-related genes were screened. Five pathways were enriched, and an "ingredient-gene-path" network was constructed. WXKL markedly decreased the incidence of arrhythmia in the MI pig model (P < 0.05). The QT interval was significantly shortened, and the heart rate was slowed down in the WXKL group compared with the model group (P < 0.05). In addition, the expression of sodium channel protein type 5 subunit alpha (SCN5A) and beta-2 adrenergic receptor (ADRB2) was downregulated, while muscarinic acetylcholine receptor M2 (CHRM2) was upregulated in the WXKL group (P < 0.05). In conclusion, WXKL may shorten the QT interval and slow down the heart rate by downregulating SCN5A and ADRB2 and upregulating CHRM2 during MI. These findings provide novel insight into molecular mechanisms of WXKL in reducing the incidence of ventricular arrhythmia.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Antiarrítmicos/farmacologia , Arritmias Cardíacas/prevenção & controle , Medicamentos de Ervas Chinesas/farmacologia , Frequência Cardíaca/efeitos dos fármacos , Isquemia Miocárdica/tratamento farmacológico , Potenciais de Ação/genética , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Frequência Cardíaca/genética , Masculino , Medicina Tradicional Chinesa , Isquemia Miocárdica/genética , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/fisiopatologia , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Mapas de Interação de Proteínas , Receptor Muscarínico M2/genética , Receptor Muscarínico M2/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Suínos , Porco Miniatura , Fatores de Tempo
4.
PLoS One ; 15(6): e0230465, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32559219

RESUMO

The slow afterhyperpolarising current, sIAHP, is a Ca2+-dependent current that plays an important role in the late phase of spike frequency adaptation. sIAHP is activated by voltage-gated Ca2+ channels, while the contribution of calcium from ryanodine-sensitive intracellular stores, released by calcium-induced calcium release (CICR), is controversial in hippocampal pyramidal neurons. Three types of ryanodine receptors (RyR1-3) are expressed in the hippocampus, with RyR3 showing a predominant expression in CA1 neurons. We investigated the specific role of CICR, and particularly of its RyR3-mediated component, in the regulation of the sIAHP amplitude and time course, and the activity-dependent potentiation of the sIAHP in rat and mouse CA1 pyramidal neurons. Here we report that enhancement of CICR by caffeine led to an increase in sIAHP amplitude, while inhibition of CICR by ryanodine caused a small, but significant reduction of sIAHP. Inhibition of ryanodine-sensitive Ca2+ stores by ryanodine or depletion by the SERCA pump inhibitor cyclopiazonic acid caused a substantial attenuation in the sIAHP activity-dependent potentiation in both rat and mouse CA1 pyramidal neurons. Neurons from mice lacking RyR3 receptors exhibited a sIAHP with features undistinguishable from wild-type neurons, which was similarly reduced by ryanodine. However, the lack of RyR3 receptors led to a faster and reduced activity-dependent potentiation of sIAHP. We conclude that ryanodine receptor-mediated CICR contributes both to the amplitude of the sIAHP at steady state and its activity-dependent potentiation in rat and mouse hippocampal pyramidal neurons. In particular, we show that RyR3 receptors play an essential and specific role in shaping the activity-dependent potentiation of the sIAHP. The modulation of activity-dependent potentiation of sIAHP by RyR3-mediated CICR contributes to plasticity of intrinsic neuronal excitability and is likely to play a critical role in higher cognitive functions, such as learning and memory.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Cálcio/metabolismo , Cálcio/farmacologia , Hipocampo/citologia , Células Piramidais/citologia , Células Piramidais/efeitos dos fármacos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Hipocampo/efeitos dos fármacos , Cinética , Camundongos , Células Piramidais/metabolismo , Ratos
5.
J Pharmacol Sci ; 143(4): 325-329, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32487451

RESUMO

The effects of class I antiarrhythmic drugs on the automaticity of isolated guinea pig pulmonary vein myocardia were investigated using microelectrode and voltage clamp methods. All of the drugs examined reduced the maximum rate of rise of automatic action potentials. The firing frequency and rate of diastolic depolarization were decreased by aprindine, flecainide and propafenone, but not by cibenzoline, disopyramide and pilsicainide, which correlated with blockade of the sodium current component induced by ramp depolarization mimicking the diastolic depolarization. In conclusion, class I antiarrhythmic drugs which block the diastolic sodium current component inhibit the automaticity of the pulmonary vein myocardium.


Assuntos
Antiarrítmicos/farmacologia , Veias Pulmonares/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Antiarrítmicos/classificação , Cobaias , Técnicas In Vitro , Microeletrodos , Técnicas de Patch-Clamp , Veias Pulmonares/metabolismo , Sódio/metabolismo
6.
Nat Commun ; 11(1): 2997, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561714

RESUMO

PIEZO2 is the essential transduction channel for touch discrimination, vibration, and proprioception. Mice and humans lacking Piezo2 experience severe mechanosensory and proprioceptive deficits and fail to develop tactile allodynia. Bradykinin, a proalgesic agent released during inflammation, potentiates PIEZO2 activity. Molecules that decrease PIEZO2 function could reduce heightened touch responses during inflammation. Here, we find that the dietary fatty acid margaric acid (MA) decreases PIEZO2 function in a dose-dependent manner. Chimera analyses demonstrate that the PIEZO2 beam is a key region tuning MA-mediated channel inhibition. MA reduces neuronal action potential firing elicited by mechanical stimuli in mice and rat neurons and counteracts PIEZO2 sensitization by bradykinin. Finally, we demonstrate that this saturated fatty acid decreases PIEZO2 currents in touch neurons derived from human induced pluripotent stem cells. Our findings report on a natural product that inhibits PIEZO2 function and counteracts neuronal mechanical sensitization and reveal a key region for channel inhibition.


Assuntos
Ácidos Graxos/administração & dosagem , Canais Iônicos/antagonistas & inibidores , Mecanotransdução Celular/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Propriocepção/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Algoritmos , Animais , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Canais Iônicos/genética , Canais Iônicos/metabolismo , Mecanotransdução Celular/genética , Mecanotransdução Celular/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Neurônios/fisiologia , Propriocepção/genética , Propriocepção/fisiologia , Ratos , Tato/efeitos dos fármacos , Tato/fisiologia
7.
PLoS One ; 15(6): e0234080, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32479525

RESUMO

In the present study we have characterized the biophysical properties of wild-type (WT) α1ß2 and α3ß2 GABAA receptors and probed the molecular basis for the observed differences. The activation and desensitization behavior and the residual currents of the receptors expressed in HEK293 cells were determined in whole-cell patch clamp recordings. Kinetic parameters of α1ß2 and α3ß2 activation differed significantly, with α1ß2 and α3ß2 exhibiting rise times (10-90%) of 24 ± 2 ms and 51 ± 7 ms, respectively. In contrast, the two receptors exhibited largely comparable desensitization behavior with decay currents that could be fitted to exponential functions with two or three components. Most notably, the two receptor compositions displayed different degrees of desentization, with the residual currents of α1ß2 and α3ß2 constituting 34 ± 2% and 21 ± 2% of the peak current, respectively. The respective contributions of the extracellular domains and the transmembrane/intracellular domains of the α-subunit to these physiological profiles were next assessed in recordings from cells expressing αß2 receptors comprising chimeric α-subunits. The rise times displayed by α1ECD/α3TMDß2 and α3ECD/α1TMDß2 receptors were intermediate to those of WT α1ß2 and WT α3ß2, and the distribution of the different components of the current decays exhibited by the two chimeric receptors followed the same pattern as the two WT receptors. The residual current exhibited by α1ECD/α3TMDß2 (23 ± 3%) was similar to that of α3ß2 but significantly different from that of α1ß2, whereas the residual current displayed by α3ECD/α1TMDß2 (27 ± 2%) was intermediate to and did not differ significantly from either of the WT receptors. This points to molecular differences in the transmembrane/intracellular domains of the α-subunit as the main determinants of the observed differences in receptor physiology between α1ß2 and α3ß2 receptors.


Assuntos
Receptores de GABA-A/metabolismo , Potenciais de Ação/efeitos dos fármacos , Células HEK293 , Humanos , Rim/fisiologia , Cinética , Técnicas de Patch-Clamp , Domínios Proteicos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores de GABA-A/genética , Ácido gama-Aminobutírico/farmacologia
8.
Toxicology ; 440: 152488, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32387285

RESUMO

Exposure to pyrethroids, a popular insecticide class that targets voltage-gated Na+ (Nav) channels, has been correlated to an increase in diagnosis of neurodevelopmental disorders, such as attention deficit hyperactive disorder (ADHD), in children. Dysregulation of medium spiny neurons (MSNs) firing in the nucleus accumbens (NAc) is thought to play a critical role in the pathophysiology of ADHD and other neurodevelopmental disorders. The Nav1.6 channel is the primary molecular determinant of MSN firing and is sensitive to modification by pyrethroids. Building on previous studies demonstrating that deltamethrin (DM), a commonly used pyrethroid, leads to use-dependent enhancement of sodium currents, we characterized the effect of the toxin on long-term inactivation (LTI) of the Nav1.6 channel, a parameter known to affect neuronal firing, and characterized changes in MSN intrinsic excitability. We employed whole-cell patch-clamp electrophysiology to measure sodium currents in HEK-293 cells stably expressing Nav1.6 channels and intrinsic excitability of MSNs in the brain slice preparation. We found that in response to repetitive stimulation acute exposure to 10 µM DM potentiated a build-up of residual sodium currents and modified availability of Nav1.6 by inducing LTI. In the NAc, DM modified MSN intrinsic excitability increasing evoked action potential firing frequency and inducing aberrant action potentials with low amplitude and depolarized voltage threshold, phenotypes that could be explained by DM induced changes on the Nav1.6 channel. These results provide a potential initial mechanism of toxicity of DM that could lead to disruption of the NAc circuitry overtime, increasing the risk of ADHD and other neurodevelopmental disorders.


Assuntos
Inseticidas/toxicidade , Canal de Sódio Disparado por Voltagem NAV1.6/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Nitrilos/toxicidade , Núcleo Accumbens/efeitos dos fármacos , Piretrinas/toxicidade , Bloqueadores dos Canais de Sódio/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Transtorno do Deficit de Atenção com Hiperatividade/metabolismo , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Núcleo Accumbens/citologia , Técnicas de Patch-Clamp , Sódio/metabolismo
9.
Mol Pharmacol ; 97(6): 355-364, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32241959

RESUMO

Voltage-gated potassium 11.1 (Kv11.1) channels play a critical role in repolarization of cardiomyocytes during the cardiac action potential (AP). Drug-mediated Kv11.1 blockade results in AP prolongation, which poses an increased risk of sudden cardiac death. Many drugs, like pentamidine, interfere with normal Kv11.1 forward trafficking and thus reduce functional Kv11.1 channel densities. Although class III antiarrhythmics, e.g., dofetilide, rescue congenital and acquired forward trafficking defects, this is of little use because of their simultaneous acute channel blocking effect. We aimed to test the ability of a combination of dofetilide plus LUF7244, a Kv11.1 allosteric modulator/activator, to rescue Kv11.1 trafficking and produce functional Kv11.1 current. LUF7244 treatment by itself did not disturb or rescue wild type (WT) or G601S-Kv11.1 trafficking, as shown by Western blot and immunofluorescence microcopy analysis. Pentamidine-decreased maturation of WT Kv11.1 levels was rescued by 10 µM dofetilide or 10 µM dofetilide + 5 µM LUF7244. In trafficking defective G601S-Kv11.1 cells, dofetilide (10 µM) or dofetilide + LUF7244 (10 + 5 µM) also restored Kv11.1 trafficking, as demonstrated by Western blot and immunofluorescence microscopy. LUF7244 (10 µM) increased IKv 11.1 despite the presence of dofetilide (1 µM) in WT Kv11.1 cells. In G601S-expressing cells, long-term treatment (24-48 hour) with LUF7244 (10 µM) and dofetilide (1 µM) increased IKv11.1 compared with nontreated or acutely treated cells. We conclude that dofetilide plus LUF7244 rescues Kv11.1 trafficking and produces functional IKv11.1 Thus, combined administration of LUF7244 and an IKv11.1 trafficking corrector could serve as a new pharmacological therapy of both congenital and drug-induced Kv11.1 trafficking defects. SIGNIFICANCE STATEMENT: Decreased levels of functional Kv11.1 potassium channel at the plasma membrane of cardiomyocytes prolongs action potential repolarization, which associates with cardiac arrhythmia. Defective forward trafficking of Kv11.1 channel protein is an important factor in acquired and congenital long QT syndrome. LUF7244 as a negative allosteric modulator/activator in combination with dofetilide corrected both congenital and acquired Kv11.1 trafficking defects, resulting in functional Kv11.1 current.


Assuntos
Antiarrítmicos/farmacologia , Canal de Potássio ERG1/efeitos dos fármacos , Compostos Orgânicos/farmacologia , Fenetilaminas/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Sulfonamidas/farmacologia , Potenciais de Ação/efeitos dos fármacos , Antiarrítmicos/química , Western Blotting , Simulação por Computador , Sinergismo Farmacológico , Canal de Potássio ERG1/fisiologia , Células HEK293 , Humanos , Microscopia de Fluorescência , Modelos Moleculares , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Compostos Orgânicos/química , Fenetilaminas/química , Bloqueadores dos Canais de Potássio/química , Sulfonamidas/química
10.
J Neurosci ; 40(18): 3504-3516, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32265260

RESUMO

The action potential (AP) waveform controls the opening of voltage-gated calcium channels and contributes to the driving force for calcium ion flux that triggers neurotransmission at presynaptic nerve terminals. Although the frog neuromuscular junction (NMJ) has long been a model synapse for the study of neurotransmission, its presynaptic AP waveform has never been directly studied, and thus the AP waveform shape and propagation through this long presynaptic nerve terminal are unknown. Using a fast voltage-sensitive dye, we have imaged the AP waveform from the presynaptic terminal of male and female frog NMJs and shown that the AP is very brief in duration and actively propagated along the entire length of the terminal. Furthermore, based on measured AP waveforms at different regions along the length of the nerve terminal, we show that the terminal is divided into three distinct electrical regions: A beginning region immediately after the last node of Ranvier where the AP is broadest, a middle region with a relatively consistent AP duration, and an end region near the tip of nerve terminal branches where the AP is briefer. We hypothesize that these measured changes in the AP waveform along the length of the motor nerve terminal may explain the proximal-distal gradient in transmitter release previously reported at the frog NMJ.SIGNIFICANCE STATEMENT The AP waveform plays an essential role in determining the behavior of neurotransmission at the presynaptic terminal. Although the frog NMJ is a model synapse for the study of synaptic transmission, there are many unknowns centered around the shape and propagation of its presynaptic AP waveform. Here, we demonstrate that the presynaptic terminal of the frog NMJ has a very brief AP waveform and that the motor nerve terminal contains three distinct electrical regions. We propose that the changes in the AP waveform as it propagates along the terminal can explain the proximal-distal gradient in transmitter release seen in electrophysiological studies.


Assuntos
Potenciais de Ação/fisiologia , Junção Neuromuscular/metabolismo , Neurotransmissores/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Feminino , Previsões , Masculino , Junção Neuromuscular/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Rana pipiens , Bloqueadores dos Canais de Sódio/farmacologia , Fatores de Tempo
11.
J Neurosci ; 40(18): 3591-3603, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32265261

RESUMO

The septo-hippocampal cholinergic system is critical for hippocampal learning and memory. However, a quantitative description of the in vivo firing patterns and physiological function of medial septal (MS) cholinergic neurons is still missing. In this study, we combined optogenetics with multichannel in vivo recording and recorded MS cholinergic neuron firings in freely behaving male mice for 5.5-72 h. We found that their firing activities were highly correlated with hippocampal theta states. MS cholinergic neurons were highly active during theta-dominant epochs, such as active exploration and rapid eye movement sleep, but almost silent during non-theta epochs, such as slow-wave sleep (SWS). Interestingly, optogenetic activation of these MS cholinergic neurons during SWS suppressed CA1 ripple oscillations. This suppression could be rescued by muscarinic M2 or M4 receptor antagonists. These results suggest the following important physiological function of MS cholinergic neurons: maintaining high hippocampal acetylcholine level by persistent firing during theta epochs, consequently suppressing ripples and allowing theta oscillations to dominate.SIGNIFICANCE STATEMENT The major source of acetylcholine in the hippocampus comes from the medial septum. Early experiments found that lesions to the MS result in the disappearance of hippocampal theta oscillation, which leads to speculation that the septo-hippocampal cholinergic projection contributing to theta oscillation. In this article, by long-term recording of MS cholinergic neurons, we found that they show a theta state-related firing pattern. However, optogenetically activating these neurons shows little effect on theta rhythm in the hippocampus. Instead, we found that activating MS cholinergic neurons during slow-wave sleep could suppress hippocampal ripple oscillations. This suppression is mediated by muscarinic M2 and M4 receptors.


Assuntos
Potenciais de Ação/fisiologia , Neurônios Colinérgicos/fisiologia , Hipocampo/fisiologia , Receptor Muscarínico M2/fisiologia , Receptor Muscarínico M4/fisiologia , Ritmo Teta/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Agonistas Colinérgicos/farmacologia , Neurônios Colinérgicos/química , Neurônios Colinérgicos/efeitos dos fármacos , Hipocampo/química , Hipocampo/efeitos dos fármacos , Masculino , Camundongos , Camundongos Transgênicos , Antagonistas Muscarínicos/farmacologia , Optogenética/métodos , Técnicas de Cultura de Órgãos , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/antagonistas & inibidores , Receptor Muscarínico M4/agonistas , Receptor Muscarínico M4/antagonistas & inibidores , Receptores Muscarínicos/fisiologia , Ritmo Teta/efeitos dos fármacos
12.
Neuron ; 106(5): 816-829.e6, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32229307

RESUMO

Sodium taste regulates salt intake. The amiloride-sensitive epithelial sodium channel (ENaC) is the Na+ sensor in taste cells mediating attraction to sodium salts. However, cells and intracellular signaling underlying sodium taste in taste buds remain long-standing enigmas. Here, we show that a subset of taste cells with ENaC activity fire action potentials in response to ENaC-mediated Na+ influx without changing the intracellular Ca2+ concentration and form a channel synapse with afferent neurons involving the voltage-gated neurotransmitter-release channel composed of calcium homeostasis modulator 1 (CALHM1) and CALHM3 (CALHM1/3). Genetic elimination of ENaC in CALHM1-expressing cells as well as global CALHM3 deletion abolished amiloride-sensitive neural responses and attenuated behavioral attraction to NaCl. Together, sodium taste is mediated by cells expressing ENaC and CALHM1/3, where oral Na+ entry elicits suprathreshold depolarization for action potentials driving voltage-dependent neurotransmission via the channel synapse. Thus, all steps in sodium taste signaling are voltage driven and independent of Ca2+ signals. This work also reveals ENaC-independent salt attraction.


Assuntos
Potenciais de Ação/fisiologia , Cálcio/metabolismo , Canais Epiteliais de Sódio/metabolismo , Sódio/metabolismo , Papilas Gustativas/citologia , Paladar/fisiologia , Potenciais de Ação/efeitos dos fármacos , Amilorida/farmacologia , Animais , Canais de Cálcio/metabolismo , Células Quimiorreceptoras/metabolismo , Células Quimiorreceptoras/fisiologia , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Camundongos , Neurônios Aferentes/metabolismo , Técnicas de Patch-Clamp , Transdução de Sinais/efeitos dos fármacos , Transmissão Sináptica , Papilas Gustativas/metabolismo , Papilas Gustativas/fisiologia
14.
Nat Commun ; 11(1): 1453, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32193397

RESUMO

The suprachiasmatic nucleus (SCN) circadian clock is critical for optimising daily cycles in mammalian physiology and behaviour. The roles of the various SCN cell types in communicating timing information to downstream physiological systems remain incompletely understood, however. In particular, while vasoactive intestinal polypeptide (VIP) signalling is essential for SCN function and whole animal circadian rhythmicity, the specific contributions of VIP cell output to physiological control remains uncertain. Here we reveal a key role for SCN VIP cells in central clock output. Using multielectrode recording and optogenetic manipulations, we show that VIP neurons provide coordinated daily waves of GABAergic input to target cells across the paraventricular hypothalamus and ventral thalamus, supressing their activity during the mid to late day. Using chemogenetic manipulation, we further demonstrate specific roles for this circuitry in the daily control of heart rate and corticosterone secretion, collectively establishing SCN VIP cells as influential regulators of physiological timing.


Assuntos
Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Neurônios GABAérgicos/metabolismo , Núcleo Supraquiasmático/fisiologia , Peptídeo Intestinal Vasoativo/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Bicuculina/farmacologia , Channelrhodopsins/química , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Relógios Circadianos/efeitos dos fármacos , Corticosterona/sangue , Corticosterona/metabolismo , Eletrodos Implantados , Feminino , Antagonistas de Receptores de GABA-A/farmacologia , Neurônios GABAérgicos/efeitos dos fármacos , Frequência Cardíaca/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Modelos Animais , Núcleo Supraquiasmático/citologia , Peptídeo Intestinal Vasoativo/antagonistas & inibidores , Peptídeo Intestinal Vasoativo/genética
15.
Circ Arrhythm Electrophysiol ; 13(4): e008130, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32202931

RESUMO

BACKGROUND: Rapid delayed rectifier K+ current (IKr) and late Na+ current (INaL) significantly shape the cardiac action potential (AP). Changes in their magnitudes can cause either long or short QT syndromes associated with malignant ventricular arrhythmias and sudden cardiac death. METHODS: Physiological self AP-clamp was used to measure INaL and IKr during the AP in rabbit and porcine ventricular cardiomyocytes to test our hypothesis that the balance between IKr and INaL affects repolarization stability in health and disease conditions. RESULTS: We found comparable amount of net charge carried by IKr and INaL during the physiological AP, suggesting that outward K+ current via IKr and inward Na+ current via INaL are in balance during physiological repolarization. Remarkably, IKr and INaL integrals in each control myocyte were highly correlated in both healthy rabbit and pig myocytes, despite high overall cell-to-cell variability. This close correlation was lost in heart failure myocytes from both species. Pretreatment with E-4031 to block IKr (mimicking long QT syndrome 2) or with sea anemone toxin II to impair Na+ channel inactivation (mimicking long QT syndrome 3) prolonged AP duration (APD); however, using GS-967 to inhibit INaL sufficiently restored APD to control in both cases. Importantly, INaL inhibition significantly reduced the beat-to-beat and short-term variabilities of APD. Moreover, INaL inhibition also restored APD and repolarization stability in heart failure. Conversely, pretreatment with GS-967 shortened APD (mimicking short QT syndrome), and E-4031 reverted APD shortening. Furthermore, the amplitude of AP alternans occurring at high pacing frequency was decreased by INaL inhibition, increased by IKr inhibition, and restored by combined INaL and IKr inhibitions. CONCLUSIONS: Our data demonstrate that IKr and INaL are counterbalancing currents during the physiological ventricular AP and their integrals covary in individual myocytes. Targeting these ionic currents to normalize their balance may have significant therapeutic potential in heart diseases with repolarization abnormalities. Visual Overview: A visual overview is available for this article.


Assuntos
Potenciais de Ação , Arritmias Cardíacas/metabolismo , Canais de Potássio de Retificação Tardia/metabolismo , Frequência Cardíaca , Miócitos Cardíacos/metabolismo , Potássio/metabolismo , Canais de Sódio/metabolismo , Sódio/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Antiarrítmicos/farmacologia , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/fisiopatologia , Arritmias Cardíacas/prevenção & controle , Canais de Potássio de Retificação Tardia/efeitos dos fármacos , Modelos Animais de Doenças , Insuficiência Cardíaca/complicações , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Frequência Cardíaca/efeitos dos fármacos , Cinética , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Coelhos , Canais de Sódio/efeitos dos fármacos , Suínos , Porco Miniatura
16.
Proc Natl Acad Sci U S A ; 117(14): 7990-8000, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32198206

RESUMO

Atrial fibrillation (AF) is prevalent in diabetes mellitus (DM); however, the basis for this is unknown. This study investigated AF susceptibility and atrial electrophysiology in type 1 diabetic Akita mice using in vivo intracardiac electrophysiology, high-resolution optical mapping in atrial preparations, and patch clamping in isolated atrial myocytes. qPCR and western blotting were used to assess ion channel expression. Akita mice were highly susceptible to AF in association with increased P-wave duration and slowed atrial conduction velocity. In a second model of type 1 DM, mice treated with streptozotocin (STZ) showed a similar increase in susceptibility to AF. Chronic insulin treatment reduced susceptibility and duration of AF and shortened P-wave duration in Akita mice. Atrial action potential (AP) morphology was altered in Akita mice due to a reduction in upstroke velocity and increases in AP duration. In Akita mice, atrial Na+ current (INa) and repolarizing K+ current (IK) carried by voltage gated K+ (Kv1.5) channels were reduced. The reduction in INa occurred in association with reduced expression of SCN5a and voltage gated Na+ (NaV1.5) channels as well as a shift in INa activation kinetics. Insulin potently and selectively increased INa in Akita mice without affecting IK Chronic insulin treatment increased INa in association with increased expression of NaV1.5. Acute insulin also increased INa, although to a smaller extent, due to enhanced insulin signaling via phosphatidylinositol 3,4,5-triphosphate (PIP3). Our study reveals a critical, selective role for insulin in regulating atrial INa, which impacts susceptibility to AF in type 1 DM.


Assuntos
Fibrilação Atrial/metabolismo , Remodelamento Atrial/fisiologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 1/complicações , Insulina/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Fibrilação Atrial/diagnóstico , Fibrilação Atrial/etiologia , Fibrilação Atrial/fisiopatologia , Remodelamento Atrial/imunologia , Células Cultivadas , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Modelos Animais de Doenças , Ecocardiografia , Eletrocardiografia , Átrios do Coração/citologia , Átrios do Coração/metabolismo , Átrios do Coração/patologia , Átrios do Coração/fisiopatologia , Humanos , Insulina/administração & dosagem , Insulina/genética , Canal de Potássio Kv1.5/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Miócitos Cardíacos/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Técnicas de Patch-Clamp , Potássio/metabolismo , Cultura Primária de Células , Sódio/metabolismo , Estreptozocina/toxicidade
17.
J Pharmacol Sci ; 143(1): 39-44, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32151542

RESUMO

We investigated effects of isoflurane and sevoflurane on sparfloxacin-induced QT-interval prolongation in guinea pigs under the monitoring of electrocardiogram and monophasic action potential (MAP), which was compared with those of halothane or non-inhaled anesthetics ketamine/xylazine. Intravenous administration of sparfloxacin at 3 and 10 mg/kg prolonged the QT interval and MAP duration together with bradycardic action under 4 different anesthetic conditions. The order of extent of prolongation of corrected QT interval after the administration of sparfloxacin was isoflurane ≈ sevoflurane ≈ halothane >> ketamine/xylazine, whereas that of the MAP90 at a pacing cycle length of 300 ms was halothane ≥ isoflurane ≈ sevoflurane >> ketamine/xylazine. These results suggest that isoflurane and sevoflurane as well as halothane could sensitize the heart to sparfloxacin-induced QT interval prolongation in guinea pigs.


Assuntos
Anestésicos Inalatórios/efeitos adversos , Isoflurano/efeitos adversos , Síndrome do QT Longo/induzido quimicamente , Sevoflurano/efeitos adversos , Potenciais de Ação/efeitos dos fármacos , Animais , Eletrocardiografia/efeitos dos fármacos , Fluoroquinolonas/administração & dosagem , Fluoroquinolonas/efeitos adversos , Cobaias , Halotano/efeitos adversos , Síndrome do QT Longo/fisiopatologia , Masculino
18.
Mol Pharmacol ; 97(4): 250-258, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32015008

RESUMO

Phenytoin is a hydantoin derivative that is used clinically for the treatment of epilepsy and has been reported to have antiarrhythmic actions on the heart. In a failing heart, the elevated diastolic Ca2+ leak from the sarcoplasmic reticulum can be normalized by the cardiac ryanodine receptor 2 (RyR2) inhibitor, dantrolene, without inhibiting Ca2+ release during systole or affecting Ca2+ release in normal healthy hearts. Unfortunately, dantrolene is hepatotoxic and unsuitable for chronic long-term administration. Because phenytoin and dantrolene belong to the hydantoin class of compounds, we test the hypothesis that dantrolene and phenytoin have similar inhibitory effects on RyR2 using a single-channel recording of RyR2 activity in artificial lipid bilayers. Phenytoin produced a reversible inhibition of RyR2 channels from sheep and human failing hearts. It followed a hyperbolic dose response with maximal inhibition of ∼50%, Hill coefficient ∼1, and IC50 ranging from 10 to 20 µM. It caused inhibition at diastolic cytoplasmic [Ca2+] but not at Ca2+ levels in the dyadic cleft during systole. Notably, phenytoin inhibits RyR2 from failing human heart but not from healthy heart, indicating that phenytoin may selectively target defective RyR2 channels in humans. We conclude that phenytoin could effectively inhibit RyR2-mediated release of Ca2+ in a manner paralleling that of dantrolene. Moreover, the IC50 of phenytoin in RyR2 is at least threefold lower than for other ion channels and clinically used serum levels, pointing to phenytoin as a more human-safe alternative to dantrolene for therapies against heart failure and cardiac arrythmias. SIGNIFICANCE STATEMENT: We show that phenytoin, a Na channel blocker used clinically for treatment of epilepsy, is a diastolic inhibitor of cardiac calcium release channels [cardiac ryanodine receptor 2 (RyR2)] at doses threefold lower than its current therapeutic levels. Phenytoin inhibits RyR2 from failing human heart and not from healthy heart, indicating that phenytoin may selectively target defective RyR2 channels in humans and pointing to phenytoin as a more human-safe alternative to dantrolene for therapies against heart failure and cardiac arrhythmias.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Cardiotônicos/farmacologia , Insuficiência Cardíaca/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Fenitoína/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Arritmias Cardíacas/tratamento farmacológico , Arritmias Cardíacas/patologia , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/uso terapêutico , Cardiotônicos/uso terapêutico , Dantroleno/farmacologia , Dantroleno/uso terapêutico , Relação Dose-Resposta a Droga , Vesículas Extracelulares , Insuficiência Cardíaca/patologia , Humanos , Bicamadas Lipídicas , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fenitoína/uso terapêutico , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Ovinos
19.
Nanotoxicology ; 14(5): 595-611, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32091294

RESUMO

Carbon black nanoparticles (CBNPs) can enter the central nervous system through blood circulation and olfactory nerves, affecting brain development or increasing neurological disease susceptibility. However, whether CBNPs exposure affects seizure is unclear. Herein, mice were exposed to two different doses of CBNPs (21 and 103 µg/animal) based on previous studies and the maximum exposure limitation (4 mg/m3) in occupational workplaces set by the Chinese government. In the pentylenetetrazol (PTZ) and kainic acid (KA) seizure models, high-dose CBNPs exposure increased seizure susceptibility in both models and increased spontaneous recurrent seizure (SRS) frequency in the KA model. In vivo local field potential (LFP) recording in KA model mice revealed that both low-dose and high-dose CBNPs exposure increased seizure-like event (SLE) frequency in the SRS interval but shortened SLE duration. Intriguingly, H&E staining and Nissl staining on brain tissue revealed that CBNPs exposure did not cause significant brain tissue morphology or neuronal damage. Detection of inflammatory factors, such as TNF-α, TGF-ß1, IL-1ß, and IL-6, in brain tissue showed that only high dose of CBNPs exposure increased the expression of cortical TGF-ß1. By using the primary cultured neurons, we observed that CBNPs exposure not only significantly decreased the expression of the neuronal marker MAP2 but also enhanced the levels of action potential frequency in the neurons. In general, CBNPs exposure can affect abnormal epileptic discharges during the seizure interval and enhance susceptibility to frequent seizures. Our findings suggest that minimizing CBNPs exposure may be a potential way to prevent or ease seizure.


Assuntos
Encéfalo/efeitos dos fármacos , Exposição por Inalação/efeitos adversos , Nanopartículas/toxicidade , Neurônios/efeitos dos fármacos , Convulsões/induzido quimicamente , Fuligem/toxicidade , Potenciais de Ação/efeitos dos fármacos , Animais , Encéfalo/imunologia , Encéfalo/patologia , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Exposição por Inalação/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/química , Neurônios/imunologia , Neurônios/patologia , Tamanho da Partícula , Técnicas de Patch-Clamp , Recidiva , Convulsões/patologia , Fuligem/química , Propriedades de Superfície
20.
Anesthesiology ; 132(5): 1034-1044, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32044799

RESUMO

BACKGROUND: General anesthetics-induced changes of electrical oscillations in the basal ganglia may render the identification of the stimulation targets difficult. The authors hypothesized that while sevoflurane anesthesia entrains coherent lower frequency oscillations, it does not affect the identification of the subthalamic nucleus and clinical outcome. METHODS: A cohort of 19 patients with Parkinson's disease with comparable disability underwent placement of electrodes under either sevoflurane general anesthesia (n = 10) or local anesthesia (n = 9). Microelectrode recordings during targeting were compared for neuronal spiking characteristics and oscillatory dynamics. Clinical outcomes were compared at 5-yr follow-up. RESULTS: Under sevoflurane anesthesia, subbeta frequency oscillations predominated (general vs. local anesthesia, mean ± SD; delta: 13 ± 7.3% vs. 7.8 ± 4.8%; theta: 8.4 ± 4.1% vs. 3.9 ± 1.6%; alpha: 8.1 ± 4.1% vs. 4.8 ± 1.5%; all P < 0.001). In addition, distinct dorsolateral beta and ventromedial gamma oscillations were detected in the subthalamic nucleus solely in awake surgery (mean ± SD; dorsal vs. ventral beta band power: 20.5 ± 6.6% vs. 15.4 ± 4.3%; P < 0.001). Firing properties of subthalamic neurons did not show significant difference between groups. Clinical outcomes with regard to improvement in motor and psychiatric symptoms and adverse effects were comparable for both groups. Tract numbers of microelectrode recording, active contact coordinates, and stimulation parameters were also equivalent. CONCLUSIONS: Sevoflurane general anesthesia decreased beta-frequency oscillations by inducing coherent lower frequency oscillations, comparable to the pattern seen in the scalp electroencephalogram. Nevertheless, sevoflurane-induced changes in electrical activity patterns did not reduce electrode placement accuracy and clinical effect. These observations suggest that microelectrode-guided deep brain stimulation under sevoflurane anesthesia is a feasible clinical option.


Assuntos
Anestésicos Inalatórios/administração & dosagem , Estimulação Encefálica Profunda/métodos , Neurônios/efeitos dos fármacos , Doença de Parkinson/terapia , Sevoflurano/administração & dosagem , Núcleo Subtalâmico/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Adulto , Idoso , Anestésicos Locais/administração & dosagem , Ritmo beta/efeitos dos fármacos , Ritmo beta/fisiologia , Estudos de Coortes , Eletroencefalografia/efeitos dos fármacos , Eletroencefalografia/métodos , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Neurônios/fisiologia , Doença de Parkinson/fisiopatologia , Núcleo Subtalâmico/fisiologia , Resultado do Tratamento
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