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To investigate the precise mechanism of xenon (Xe), pharmacologically isolated AMPA/KA and NMDA receptor-mediated spontaneous (s) and evoked (e) excitatory postsynaptic currents (s/eEPSCAMPA/KA and s/eEPSCNMDA) were recorded from mechanically isolated single spinal sacral dorsal commissural nucleus (SDCN) neurons attached with glutamatergic nerve endings (boutons) using conventional whole-cell patch-clamp technique. We analysed kinetic properties of both s/eEPSCAMPA/KA and s/eEPSCNMDA by focal single- and/or paired-pulse electrical stimulation to compare them. The s/eEPSCNMDA showed smaller amplitude, slower rise time, and slower 1/e decay time constant (τDecay) than those of s/eEPSCAMPA/KA. We previously examined how Xe modulates s/eEPSCAMPA/KA, therefore, examined the effects on s/eEPSCNMDA in the present study. Xe decreased the frequency and amplitude of sEPSCNMDA, and decreased the amplitude but increased the failure rate and paired-pulse ratio of eEPSCNMDA without affecting their τDecay. It was concluded that Xe might suppress NMDA receptor-mediated synaptic transmission via both presynaptic and postsynaptic mechanisms.

Asunto(s)

Potenciales Postsinápticos Excitadores , Neuronas , Receptores de N-Metil-D-Aspartato , Xenón , Animales , Receptores de N-Metil-D-Aspartato/metabolismo , Xenón/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/fisiología , Ratas , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Médula Espinal/fisiología , Sinapsis/efectos de los fármacos , Sinapsis/fisiología , Ratas Sprague-Dawley , Técnicas de Placa-Clamp , Receptores AMPA/metabolismo , Receptores AMPA/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Masculino

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@#Objective To investigate the effect of microparticles(MPs)derived from bone marrow mesenchymal stem cells(BMSCs) on myocardial hypertrophy and its mechanism.Methods The osteogenic differentiation and adipogenic differentiation of mesenchymal stem cells(MSCs) were induced. After isolation and purification,the morphological characteristics were observed by transmission electron microscope,and the MPs surface antigen was identified by flow cytometry. Myocardial hypertrophy model was induced by using isoprenaline(ISO)in rats,which were measured for the cardiac structure and function by echocardiography,and then detected for various indexes of the heart and isolated left ventricle. Single ventricular myocytes of rats were acutely isolated and divided into control group(Control group),cardiomyocyte hypertrophy group(ISO group),MPs group(MPs group),and MPs supernatant group(Supernatant group). The mRNA expressions of atrial natriuretic peptide(ANP)and B-type natriuretic peptide(BNP)were detected by qRTPCR. The expression levels of calmodulin-dependent protein kinaseⅡ（CaMKⅡ）and phosphorylated calmodulin-dependent protein kinaseⅡ（p-CaMKⅡ）were detected by ELISA. The L-type calcium current(LCa-L)in single ventricular myocyte of various groups was recorded by whole-cell patch clamp.Results The bone nodules of MSCs osteogenic differentiation turned red after alizarin red staining,and lipid droplets of adipogenic differentiation turned red after oil red O staining;Under transmission electron microscope,MPs membrane had a complete structure,a clear outline and a diameter of about200 nm;The positive rates of CD29 and CD90 on the surface of MPs were(98. 24 ± 0. 82)% and(97. 69 ± 1. 83)%,respectively. Compared with Control group,the left ventricular end diastolic dimension(LVEDD)reduced signifi-cantly(t =5. 065,P < 0. 05),while the interventricular septum end-diastolic dimension(IVSd),left ventricular posterior wall dimension(LVPWd),heart weight to body weight ratio(HW/BW),and heart weight to tibial length ratio(HW/Tibia)significantly increased in ISO group(t = 4. 013,2. 368,4. 392,5. 043 and 6. 120,respectively,each P < 0. 05),indicating that the hypertrophic model was successfully established. The expression levels of ANP and BNP mRNA in hypertrophic cardiomyocytes of rats in ISO group were significantly higher than those in Control group(t = 25. 120 and18. 261,respectively,each P < 0. 01);While the expression levels of ANP and BNP mRNA in MPs group significantly reduced after incubation with 48 μg/mL MPs for 48 h compared with ISO group(t = 12. 110 and 3. 526,respectively,each P < 0. 05);The expression levels of CaMK Ⅱand p-CaMKⅡ in ISO group were significantly higher than those in Control group(t = 3. 278 and 4. 181,respectively,each P < 0. 05),while the expression of p-CaMK Ⅱ in MPs group decreased significantly(t = 5. 420,P < 0. 05);The calcium current density in ISO group was significantly higher than that in Control group(t = 15. 261,P < 0. 01),while that in MPs group was significantly lower than that in ISO group(t =6. 216,P < 0. 05).Conclusion MSC-MPs can significantly inhibit ISO-induced cardiomyocyte hypertrophy in rats,which is related to its down-regulation of cardiomyocyte CaMKⅡ and inhibition of L-type calcium channel.

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The cotton bollworm, Helicoverpa armigera is one of the worldwide pests. Electrophysiological properties of voltage-gated sodium channels in central neurons of sensitive and pyrethroid resistant H. armigera were investigated using whole-cell patch clamp technique. The modification effects of pyrethroid insecticides deltamethrin and tefluthrin on sodium channels were also compared. The V0.5 of voltage dependence of activation of resistant H. armigera sodium channels (resistant channels) exhibited an obvious depolarizing shift by 13.52 mV compared to that of sensitive H. armigera sodium channels (sensitive channels). In contrast, the V0.5 of the voltage dependence of steady-state inactivation of the resistant channels showed a significant hyperpolarizing shift by 7.59 mV in comparison with that of the sensitive channels. The time course of recovery from inactivation for the resistant channels was prolonged significantly, by 0.17 ms, compared with that for the sensitive channels. We also assessed the use-dependent effects of deltamethrin and tefluthrin on sensitive sodium channels. Repetitive depolarization remarkably increased the extent of the sensitive channel modification by 10 µM deltamethrin by ~4.61-fold but had no effect on the extent of sensitive channel modifications by 10 µM tefluthrin. These results provide more direct evidence for the presence of nerve insensitivity in resistant H. armigera strains in North of China. The sodium channels of the resistant H. armigera differ from those of the sensitive H. armigera in the fundamental electrophysiological properties, and correspondingly, have a different response to the modification of pyrethroids. Both deltamethrin and tefluthrin have effects on the closed state of the sensitive sodium channels, but deltamethrin has higher affinity to the open state of these channels.

Asunto(s)

Insecticidas , Mariposas Nocturnas , Piretrinas , Animales , China , Ciclopropanos , Hidrocarburos Fluorados , Insecticidas/toxicidad , Neuronas , Nitrilos , Piretrinas/toxicidad

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AIMS: Ethanol is known to induce NO release and coronary vasorelaxation. Evidence suggests that K+ channels, especially a Ca2+-activated K+ channel (KCa), may regulate endothelial NO production. We aimed to investigate the ethanol effect on K+ currents in human coronary artery endothelial cells (HCAECs), identify the K+ channel type/subtype and signaling pathway involved, and demonstrate the relevance to ethanol-induced NO release. MAIN METHODS: Ionic currents of cultured HCAECs were studied using whole-cell patch clamp technique. NO production were measured using the fluorescent probe, 2,3-diaminonaphthalene. KEY FINDINGS: We found that ethanol significantly potentiated HCAEC current (maximal increase to 155.68 ±â€¯18.93%, 20 mM ethanol, +80 mV; mean ±â€¯SEM, n = 9). Ethanol-induced current was significantly inhibited by blockers of IKCa or SKCa (intermediate- or small-conductance KCa), but not by blocking other K+ channels. When other known HCAEC channels were inhibited except IKCa, 20 mM ethanol significantly increased IKCa current to 198 ±â€¯25.11% (n = 6), but it could not enhance SKCa current that was similarly isolated. Moreover, ethanol-induced NO release was prevented by blocking IKCa channel, adenosine A2A receptor (A2AR), Gs protein, or protein kinase A (PKA). SIGNIFICANCE: This study was the first to demonstrate that acute ethanol exposure could activate endothelial IKCa channel, via A2AR-Gs-PKA signaling, leading to increased whole-cell current and NO release, which could be an important mechanism underlying ethanol-induced NO release and vasodilation.

Asunto(s)

Células Endoteliales/efectos de los fármacos , Etanol/farmacología , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Óxido Nítrico/metabolismo , Línea Celular , Vasos Coronarios/citología , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Humanos , Transporte Iónico/efectos de los fármacos , Técnicas de Placa-Clamp

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OBJECTIVE: The electrical properties of olfactory cells (OCs) are typically examined using animals such as newts, mice, and frogs, with few studies on human OCs. This study investigated the electrical properties of human cells from olfactory epithelium (hCOEs) obtained from subjects of olfactory epithelium showing no clinical symptoms during endoscopic sinus surgery. METHODS: hCOEs were isolated by collagenase treatment for whole-cell patch clamp recording. The identity of the cells was confirmed by immunohistochemistry with an antibody against olfactory maker protein. Under the voltage clamp with the whole-cell recording configuration, the voltage-gated currents of isolated hCOEs were recorded when the membrane potential was depolarized from a holding potential of -100 mV in a stepwise manner between -90 mV and + 40 mV. RESULTS: Only one of 14 hCOE samples expressed a transient inward current at the depolarizing voltage step that was activated by depolarization beyond -40 mV and reached a peak at -30 mV. Delayed and sustained outward currents (444 ± 106 pA at + 40 mV pulse; n = 20) were suppressed by tetraethyl ammonium (n = 3), which is consistent with the properties of newt OCs. CONCLUSIONS: Most hCOEs did not exhibit the transient inward current observed in animal models. These findings provide insight into the physiological basis of the unique aspects of human olfactory signal transduction.

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Potenciales de la Membrana/fisiología , Mucosa Olfatoria/fisiología , Neuronas Receptoras Olfatorias/fisiología , Animales , Humanos , Mucosa Olfatoria/citología , Técnicas de Placa-Clamp , Salamandridae

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In multi-cellular organisms, cells communicate with each other utilizing chemical messengers. For many of these messenger molecules, the membrane is an insurmountable barrier. Yet, they act by binding to surface proteins often triggering a cascade of reactions inside the cell. Accordingly, studying ligand-receptor interactions at the cellular surface is key to understanding important aspects of membrane biology. However, despite a multitude of approaches to study membrane features, there is a need for developing techniques that can measure ligand binding with high temporal resolution and on a single cellular level. We recently developed a label-free approach to study ligand binding in real time. This methodology capitalizes on changes of the membrane's surface potential induced by the adsorption of a charged ligand. The resulting apparent alteration of membrane capacitance is measurable by capacitance recordings. Herein, we describe the implementation of the same using recordings obtained from HEK293 cells stably expressing the human serotonin transporter (SERT), which were challenged with the inhibitor cocaine.

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New dihydropyrimidines bearing various lipophilic pharmacophores and functionalities at position 3 were designed and synthesized. The basic framework of the new compounds was designed to maintain the main structural requirements for calcium channel blocking activity of the known dihydropyridines and dihydropyrimidines calcium channel blockers. The newly synthesized compounds were evaluated as antagonists for CaV1.2 and CaV3.2 using the whole-cell patch clamp technique. Seven compounds (4b, 4c, 6c, 9, 13c, 13e and 17b) showed promising dual calcium channel blocking activity and three compounds (13b, 14b and 17a) were selective against Cav3.2. Their drug-likeness has been assessed using Molinspiration and Molsoft softwares. Their physicochemical properties and pharmacokinetic profiles recommend that they can be considered as drug-like candidates.

Asunto(s)

Bloqueadores de los Canales de Calcio/farmacología , Pirimidinas/farmacología , Animales , Bloqueadores de los Canales de Calcio/síntesis química , Bloqueadores de los Canales de Calcio/química , Bloqueadores de los Canales de Calcio/farmacocinética , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo T/metabolismo , Línea Celular , Diseño de Fármacos , Humanos , Estructura Molecular , Técnicas de Placa-Clamp , Pirimidinas/síntesis química , Pirimidinas/química , Pirimidinas/farmacocinética , Ratas , Solubilidad , Relación Estructura-Actividad

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Taurine-magnesium coordination compound (TMCC) exhibits antiarrhythmic effects in cesium-chloride-and ouabain-induced arrhythmias; however, the mechanism underlying these effects on arrhythmia remains poorly understood. Here, we investigated the effects of TMCC on aconitine-induced arrhythmia in vivo and the electrophysiological effects of this compound in rat ventricular myocytes in vitro. Aconitine was used to induce arrhythmias in rats, and the dosages required to produce ventricular premature contraction (VPC), ventricular tachycardia (VT), ventricular fibrillation (VF), and cardiac arrest (CA) were recorded. Additionally, the sodium current (INa) and L-type calcium current (ICa,L) were analyzed in normal and aconitine-treated ventricular myocytes using whole-cell patch-clamp recording. In vivo, intravenous administration of TMCC produced marked antiarrhythmic effects, as indicated by the increased dose of aconitine required to induce VPC, VT, VF, and CA. Moreover, this effect was abolished by administration of sodium channel opener veratridine and calcium channel agonist Bay K8644. In vitro, TMCC inhibited aconitine-induced increases in INa and ICa,L. These results revealed that TMCC inhibited aconitine-induced arrhythmias through effects on INa and ICa,L.

Asunto(s)

Aconitina , Antiarrítmicos/uso terapéutico , Arritmias Cardíacas/inducido químicamente , Arritmias Cardíacas/tratamiento farmacológico , Canales Iónicos/efectos de los fármacos , Compuestos de Magnesio/uso terapéutico , Taurina/uso terapéutico , Animales , Canales de Calcio Tipo L/efectos de los fármacos , Fenómenos Electrofisiológicos/efectos de los fármacos , Femenino , Paro Cardíaco/inducido químicamente , Paro Cardíaco/prevención & control , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/efectos de los fármacos , Masculino , Miocitos Cardíacos/efectos de los fármacos , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Canales de Sodio/efectos de los fármacos

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A new meroterpenoid, named acetoxydehydroaustin A (1) and the known meroterpenoid austin (2) were isolated from the plant pathogenic fungus Verticillium albo-atrum. Their structures were established based on general spectroscopic techniques and the relative configuration of compound 1 was determined by single-crystal X-ray diffraction analysis. We first investigated and identified their significant electrophysiological effects on the gating kinetics of voltage-gated sodium channels in central neurons acutely dissociated from Helicoverpa armigera using whole-cell patch clamp technique. Similar to the effects of pyrethroids on sodium late currents, both compounds produced concentration-dependent modification of sodium channels, prolonging the kinetics of channel inactivation to generate large persistent late currents during depolarization. However, different from the effects of tefluthrin and deltamethrin on sodium channels, two meroterpenoids did not induce tail currents during deactivation. Compounds 1 and 2 also caused depolarizing shifts in the voltage dependence of channel activation. The V0.5 shifted about 5.02mV and 6.32mV in the depolarizing direction by 50µM 1 and 50µM 2. The V0.5 of voltage-dependent inactivation shifted about 11.42mV and 11.62mV respectively in the hyperpolarizing direction by 50µM 1 and 100µM 2. In addition, they prolonged the time course of recovery from fast-inactivation for sodium channels. The effects of two compounds on the voltage-dependent gating substantially increased the size of sodium window currents. The overlapped area of window currents increased about 89.69% and 44.51% respectively by 10µM compound 1 and 10µM compound 2. These findings show that both compounds have effects on sodium channel activation, inactivation and window currents. The voltage-gated sodium channels in central neurons of H. armigera are the target sites of two meroterpenoid natural products.

Asunto(s)

Insecticidas/farmacología , Mariposas Nocturnas/efectos de los fármacos , Neuronas/efectos de los fármacos , Terpenos/farmacología , Verticillium/química , Canales de Sodio Activados por Voltaje/efectos de los fármacos , Animales , Cristalografía por Rayos X , Fermentación , Insecticidas/química , Modelos Moleculares , Estructura Molecular , Mariposas Nocturnas/citología , Mariposas Nocturnas/metabolismo , Neuronas/metabolismo , Técnicas de Placa-Clamp , Análisis Espectral/métodos , Terpenos/química

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Objective To research the changes in hippocampal voltage-gated sodium channel of Lithium chloride-Pilocarpine epileptic rat models,including Ⅰ sodium channel α subunit protein (Nav1.1),mRNA of Ⅰ sodium channel alpha subunit protein gene and function of sodium channel.Methods Epileptic rat models of Lithium chloride-Pilocarpine were established.Nav1.1 expression in the hippocampus of experimental rats was detected by immunohistochemical staining method,and the changes in voltage-gated sodium channel function (the current-voltage curves,activation and inactivation curves and the recovery curve) of hippocampus nerve cells were detected by whole cell patch-clamp technique.Results (1) The Lithium chloride-Pilocarpine rat models were successfully reproduced.Three stages of behavior (acute,latent and chronic) of rat models were observed.The blank control group was free of seizure.(2) Immunohistochemistry results:neurons in CA1 and DG regions of hippocampal of epileptic rats were normal,and there was no obvious change in the expression of Nav1.1.In CA3 area,the degeneration and necrosis of neurons were obvious.Staining of Nav1.1 became superficial and even disappeared in these areas,but the normal tissues were enhanced around degenerative and necrotic neurons.Compared with the blank control group,the expression of Nav1.1 in the model group was higher(0.235 ±0.008 vs.O.210 ±0.002),and there was statistically significant difference (t'=-7.426,P ＜ 0.05).(3) The whole-cell patch-clamp technique showed that the sodium current density of the model group increased significantly compared with that of the blank group [(-319.70 ± 28.24) pA/pF vs.(-229.06 ± 26.01) pA/pF,t =8.178,P ＜ 0.05],the threshold value of activation curve decreased (4.15 ± 0.80 vs.4.50 ±0.85,t =11.020,P ＜ 0.05),the threshold value of inactivation curve increased (7.47 ± 0.53 vs.6.24 ±0.31,t =6.940,P ＜ 0.05),and the recovery time after inactivation shortened [(1.36 ± 0.15) ms vs.(1.86 ± 0.21)ms,t =6.712,P ＜ 0.05],and there were all statistically significant differences.Conclusion Repeated seizures can lead to increase Nav1.1 compensatory expression of,and significantly increase sodium channel current density,while the threshold value of activation curve decreases,the threshold value of inactivation curve rises,and the recovery time after inactivation is shortened,which eventually leads to increased neuron excitability and is more likely to cause seizures.

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Aim To observe the effect of antibody NCX-3F10 on the main ion current of rat ventricular myocytes and its effect on arrhythmias induced by ischemia/reperfusion(I/R).Methods ① The whole-cell patch clamp technique was employed to record the Na+/Ca2+ exchange current(INa/Ca) and other major ion currents in rat ventricular myocytes.② The rat models of arrhythmia induced by ischemia/reperfusion were established by ligating the left coronary artery to in vivo and in vitro.Then the effects of antibody on the arrhythmia were observed.③ The IonOptix ion imaging system was used to observe the effect of antibody on calcium transients in single ventricular myocytes.Results ① The antibody NCX-3F10 dose-dependently inhibited INa/Ca from 5 to 40 mg·L-1.The IC50 for outward and inward currents was 11.15 and 11.69 mg·L-1, and the maximum inhibitory rates were 61% and 62%, respectively.The antibody also had an inhibitory effect on calcium current(ICa-L), and had no significant effect on inward rectifier potassium current(IK1), transient outward potassium current(Ito) and sodium current(INa).② In the isolated rat heart group I/R, 100% rats showed ventricular tachycardia, and 88.89% rats had ventricular fibrillation.After administration of antibody NCX-3F10(10 mg·L-1) 5 min before reperfusion, the incidence of ventricular tachycardia decreased to 44.43%(P<0.05), and the duration of ventricular tachycardia and ventricular fibrillation was also shortened remarkably(P<0.05).③ In the anesthetized rats after administration of antibody NCX-3F10(50 μg·kg-1) 5 min before reperfusion, the incidence and duration of ventricular tachycardia,the incidence and duration of ventricular fibrillation, and total number of ventricular premature beats were significantly decreased(P<0.05).④ From 5 to 40 mg·L-1, NCX-3F10 antibody decreased calcium transient amplitude in rat single ventricular myocytes dose-dependently(P<0.05).Conclusions The NCX-3F10 antibody shows significant arrhythmic effects on ischemia-reperfusion induced arrhythmia in rats both in vitro and in vivo, the underlying mechanism of which is related to NCX and L-type calcium current inhibition and calcium overload reduction by the NCX antibody.

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Swelling-activated chloride currents (ICl.swell) are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C (PKC) in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate (PDBu) enhanced ICl.swell in a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.

Asunto(s)

Canales de Cloruro/metabolismo , Cloruros/metabolismo , Miocitos Cardíacos/metabolismo , Proteína Quinasa C/metabolismo , Antracenos/farmacología , Cloruros/agonistas , Cloruros/antagonistas & inhibidores , Medios de Cultivo/metabolismo , Medios de Cultivo/farmacología , Relación Dosis-Respuesta a Droga , Potenciales Evocados/efectos de los fármacos , Potenciales Evocados/fisiología , Atrios Cardíacos/citología , Atrios Cardíacos/efectos de los fármacos , Atrios Cardíacos/metabolismo , Humanos , Soluciones Hipotónicas/metabolismo , Soluciones Hipotónicas/farmacología , Indoles/farmacología , Transporte Iónico/efectos de los fármacos , Maleimidas/farmacología , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Técnicas de Placa-Clamp , Forbol 12,13-Dibutirato/farmacología , Cultivo Primario de Células

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OBJECTIVE To investigate the effect of new baicalin(BC) metal ions(Co2+,Cu2+, and Ni2+)complexes(BMCs)on ion channels Kv1.4 and Cav3.2. METHODS HEK293 or CHO cells loaded with various ion channels(hERG,Kv1.2,Kv1.3,Kv1.4,Kv1.5,Kv1.6,Kv1.7,Kv1.8,Kir1.1, Kir2.1,KCNQ and Cav3.2)were obtained by stable transfection method. Whole-cell patch-clamp tech?nique was used to record current changes of each ion channel induced by BC and BMC in 10μmoL · L-1. The effect of different concentrations(0.3,1,3,10 and 30μmoL · L-1)of BC-Co and BC-Cu on Kv1.4 and Cav3.2 current was detected by whole-cell patch-clamp technique. RESULTS A model of HEK293 cells or CHO cells that stably expressed various ion channels was obtained. BMCs (BC-Co,BC-Cu and BC-Ni)had some impact on various ion channels,especially on Kv1.4 and Cav3.2. The inhibitory rate induced by BC-Co,BC-Cu and BC-Ni(10 μmol · L-1)was 91%,76% and-10%,respectively,for Kv1.4;and 43%,57%and-14%,respectively,for Cav3.2. IC50 of BC-Co was 1.69 and 0.81μmoL·L-1 for Kv1.4 and Cav3.2. IC50 of BC-Cu was 1.66 and 0.58μmoL · L-1 for Kv1.4 and Cav3.2. CONCLUSION BC-Cu and BC-Co concentration-dependently inhibit Kv1.4 and Cav3.2 ion channels.

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Aim To investigate the effect of zacopride ( Zac) on cardiac arrhythmia in isoproterenol ( ISO)-in-duced myocardial hypertrophic rats and the underlying electrophysiological mechanisms .Methods ① Fifty-one rats were randomly divided into control group ( n=17 ) , ISO group ( n=17 ) and ISO +Zac group ( n =17 ) .Rat model with cardiac arrhythmia and hypertro-phy was established by intraperitoneal ISO ( 5 mg?kg -1 ) injection.②ECGs were recorded to observe the effects of Zac on arrhythmia in model rats .③ Whole-cell patch clamp was applied to record inwardly rectifi-er potassium current(IK1), resting membrane potential ( RMP ) and amplicated delayed afterdepolarizations (DADs).Results ① Echocardiographic examination showed that , left ventricular end-diastolic dimension (LVEDD) and left ventricular end-systolic dimension (LVESD) significantly decreased in rats in ISO group compared with control group , whereas left ventricular posterior wall end-diastolic thickness ( LVPWd) and in-terventricular septum end-diastolic thickness ( IVSd ) increased ( P<0.05 ) , suggesting rat model of isoprot-erenol-induced myocardial hypertrophy was successfully established .② ECGs showed that 88.89% of rats in ISO group had ventricular premature beats ( VPBs ) , which significantly decreased to 11.11% after the ap-plication of Zac ( P <0.05 ) .③ Values of RMP de-creased from ( -71.05 ±1.27 ) mV in control group to (-69.38 ±1.21 ) mV in ISO group ( P<0.05 ) . After Zac administration , RMP significantly increased to ( -73.86 ±1.33 ) mV compared with control and ISO group(P<0.05).④DADs and TA incidence sig-nificantly decreased from 88.24% in ISO group to 11.76%in ISO+Zac group ( P<0.05 ) .⑤ Compared with control group , IK1 density was markedly reduced in ISO group, whereas Zac could effectively rescue IK1 suppression to normal level .Conclusions Zac, as a selective IK1 channel agonist , can significantly inhibit cardiac arrhythmia in isoproterenol-induced myocardial hypertrophic rats , which is mainly attributed to in-creased RMP by enhancing IK1 and subsequent suppres-sion of DADs.

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Swelling-activated chloride currents (ICl.swell) are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C (PKC) in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate (PDBu) enhanced ICl.swell in a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.

Asunto(s)

Humanos , Antracenos , Farmacología , Canales de Cloruro , Metabolismo , Cloruros , Metabolismo , Medios de Cultivo , Metabolismo , Farmacología , Relación Dosis-Respuesta a Droga , Potenciales Evocados , Fisiología , Atrios Cardíacos , Biología Celular , Metabolismo , Soluciones Hipotónicas , Metabolismo , Farmacología , Indoles , Farmacología , Transporte Iónico , Maleimidas , Farmacología , Miocitos Cardíacos , Biología Celular , Metabolismo , Técnicas de Placa-Clamp , Forbol 12,13-Dibutirato , Farmacología , Cultivo Primario de Células , Proteína Quinasa C , Metabolismo

RESUMEN

AIMS: Ginsenosides, active components in ginseng, have been shown to increase nitric oxide (NO) production in aortic endothelial cells. This effect was reversed by tetraethylammonium (TEA) inhibition of endothelial Ca(2+)-activated K(+) (KCa) channels. The objectives of this study, therefore, were to test 1) whether vasorelaxing ginsenoside Re could affect KCa current, an important regulator of NO production, in human coronary artery endothelial cells (HCAECs); and 2) whether small-conductance KCa (SKCa) channel was the channel subtype involved. MAIN METHODS: Ionic currents of cultured HCAECs were studied using whole-cell patch clamp technique. KEY FINDINGS: Ginsenoside Re dose-dependently increased endothelial outward currents, with an EC50 of 408.90±1.59nM, and a maximum increase of 36.20±5.62% (mean±SEM; p<0.05). Apamin, an SKCa channel inhibitor, could block this effect, while La(3+), a nonselective cation channel (NSC) blocker, could not. When NSC channel, inward-rectifier K(+) channel, intermediate-, and large-conductance KCa channels were simultaneously blocked, ginsenoside Re could still increase outward currents significantly (35.49±4.22%; p<0.05); this effect was again abolished by apamin. Repeating the experiments when Cl(-) channel was additionally blocked gave similar results. Finally, we demonstrated that ginsenoside Re could hyperpolarize HCAECs; this effect was reversed by apamin. These data clearly indicate that ginsenoside Re increased HCAEC outward current via SKCa channel activation, and NSC channel was not involved. SIGNIFICANCE: This is the first report to demonstrate that ginsenoside Re could increase SKCa channel activity in HCAECs. This can be a mechanism mediating ginseng's beneficial actions on coronary vessels.

Asunto(s)

Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Ginsenósidos/farmacología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Vasodilatadores/farmacología , Apamina/farmacología , Línea Celular , Vasos Coronarios/citología , Humanos , Lantano/farmacología , Panax/química , Técnicas de Placa-Clamp , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/agonistas , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/antagonistas & inhibidores

RESUMEN

Endocannabinoid anandamide (N-arachidonoyl ethanolamide; AEA) has been shown to cause negative inotropic and antiarrhythmic effects in ventricular myocytes. In this study, using whole-cell patch clamp technique, we have investigated the effects of AEA on cardiac Na(+)/Ca(2+) exchanger (NCX1)-mediated currents. AEA suppressed NCX1 with an IC50 value of 4.7 µM. Both inward and outward components of exchanger currents were suppressed by AEA equally. AEA inhibition was mimicked by the metabolically stable analogue, methanandamide (metAEA, 10 µM) while it was not influenced by inhibition of fatty acid amide hydrolase with 1 µM URB597 incubation. The effect of AEA, was not altered in the presence of cannabinoid receptor 1 and 2 antagonists AM251 (1 µM) and AM630 (1 µM), respectively. In addition, inhibition by AEA remained unchanged after pertussis toxin (PTX, 2 µg/ml) treatment or following the inclusion of GDP-ß-S (1 mM) in pipette solution. Currents mediated by NCX1 expressed in HEK-293 cells were also inhibited by 10 µM AEA a partially reversible manner. Confocal microscopy images indicated that the intensity of YFP-NCX1 expression on cell surface was not altered by AEA. Collectively, the results indicate that AEA directly inhibits the function of NCX1 in rat ventricular myocytes and in HEK-293 cells expressing NCX1.

Asunto(s)

Ácidos Araquidónicos/farmacología , Endocannabinoides/farmacología , Miocitos Cardíacos/efectos de los fármacos , Alcamidas Poliinsaturadas/farmacología , Intercambiador de Sodio-Calcio/metabolismo , Animales , Benzamidas/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Carbamatos/farmacología , Células Cultivadas , Células HEK293 , Humanos , Masculino , Microscopía Confocal , Miocitos Cardíacos/fisiología , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Intercambiador de Sodio-Calcio/genética

RESUMEN

Aim To provide an electrophysiological basis for hippocampus involved emotional changes caused by tinnitus, the effects of salicylate on electro-physiological characteristics of the voltage-gated calci-um channels in hippocampal neurons were performed. Method The effects of salicylate on voltage-gated cal-cium channels in rat hippocampal neurons were stud-ied, using the whole-cell voltage clamp method. Re-sults Salicylate blocked the voltage-gated calcium channels ( ICa ) in a concentration-dependent manner (0.1~10 mmol · L-1 ) . The half-inhibition concen-tration ( IC50 ) values for the blocking action of salicy-late on ICa were 1.64 mmol·L-1 . With 1 mmol·L-1 salicylate applicalted into bath solution, the steady-state activation curve of calcium channel was shifted by about 9 mV in the hyperpolarizing direction, and its steady-state inactivation curve was not changed. Con-clusion Salicylate inhibits ICa in rat hippocampal neu-rons and significantly affects the activation kinetics of ICa ,which could be related to emotional changes caused by tinnitus.

RESUMEN

Aim To investigate the effects of TMCC on abnormal L-type calcium current (ICa,L) in rat ventric-ular cardiomyocytes during hypoxia-reoxygenation to find out the mechanism of antiarrhythmic effect. Methods Whole-cell patch clamp was used to record ICa,L in the ventricular cardiomyocytes during hypoxia-reoxygenation in rat under amiodarone and different concentrations of TMCC. Results In hypoxia-reoxy-genation model, peak ICa,L increased from ( 3. 35 ± 0. 50 ) pA/pF to ( 5. 69 ± 0. 25 ) pA/pF ( n =6 , P 0. 05),(4. 41 ± 0. 22) pA/pF, (3. 82 ± 0. 21)pA/pF(n=6, P<0. 01) by TMCC(100, 200, 400 μmol·L-1 ) and amidodarone 24. 24 μmol·L-1 restored peak ICa,L to(3. 66 ± 0. 27)pA/pF (n=6,P<0. 01 ) . Compared to control group, hypoxia-reoxy-genation turned ICa,L steady-state activation curves to left and inactivation curves to right, which quickened activation and slowed inactivation, TMCC ( 200, 400μmol · L-1 ) and amiodarone could restore the left shift activation curves and right shift inactivation curves. Conclusion TMCC can concentration-de-pendently restore the increase of calcium current due to hypoxia-reoxygenation by promoting inactivation process and inhibiting activation process, and the effect is equal to that of amiodarone. TMCC blocks ICa,L of the ventricular cardiomyocytes, which may be one of its antiarrhythmic mechanisms.

RESUMEN

Myocardial ischemic injury activates cardiac sympathetic afferent fibers and elicits a sympathoexcitatory reflex by exciting sympathetic efferent action, with resultant augmentation of myocardial oxygen consumption, leading to a vicious cycle of exaggerating myocardial ischemia. P2X7 receptor participates in the neuronal functions and the neurological disorders. This study examined the role of P2X7 receptor of superior cervical ganglia (SCG) in sympathoexcitatory reflex. Our results showed that the expression of P2X7 receptor at both mRNA and protein in SCG was increased after myocardial ischemic injury. P2X7 receptor agonists at the same concentration activated much larger amplitudes of the currents in the SCG neurons of myocardial ischemic rats than those in control rats. P2X7 receptor antagonist (brilliant blue G, BBG) significantly inhibited P2X7 receptor agonist-activated currents in the SCG neurons. Excessive phosphorylation of MAPK ERK1/2 upon the activation of P2X7 receptor might be a mechanism mediating the signal transduction after myocardial ischemic injury. Therefore, the sensitized P2X7 receptor in SCG was involved in the nociceptive transmission of sympathoexcitatory reflex induced by myocardial ischemic injury.

Asunto(s)

Isquemia Miocárdica/metabolismo , Receptores Purinérgicos P2X7/fisiología , Ganglio Cervical Superior/metabolismo , Regulación hacia Arriba , Animales , Western Blotting , Sistema de Señalización de MAP Quinasas , Masculino , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Purinérgicos P2X7/metabolismo