Blocker Of Na Research Articles

The role of the hydrogen sulfide donor in adenosine diphosphate-induced platelet aggregation in patients with coronary heart disease

Introduction. Platelet activation is the initial stage of thrombotic complications in pathological conditions, primarily in atherosclerotic cardiovascular diseases. Endogenous hydrogen sulfide (H2S) is an antiplatelet agent, but the specific ways to realize its effects are not studied enoughAim: To study the effect of H2S on adenosine diphosphate (ADP)-induced platelet aggregation in patients with coronary heart disease (CHD) with blockers of Na+,K+,2Cl- cotransporter (NKCC), an anion exchanger, and a phosphodiesterase (PDE) inhibitor of cyclic nucleotides.Material and Methods. The study included 22 patients with CHD. The control group included 14 healthy volunteers. Platelet aggregation was determined by turbidimetric method. The degree of aggregation (%) and the size of aggregates (rel. units) were measured. ADP (2 μM) was an aggregation inductor. In some cases the incubation medium contained the hydrogen sulfide donor NaHS (1–100 μM) and aggregation modifierResults. The H2S donor at a concentration of 100 μM reduced the parameters of ADP-dependent platelet aggregation in healthy volunteers and increased it in patients with coronary artery disease. NKCC and anion exchanger blockers, as well as a PDE inhibitor, reduced ADP-dependent platelet aggregation in healthy volunteers. The combined action of these agents and NaHS enhanced the inhibitory effects of the applied modifiers. The results obtained for platelet aggregation in patients with coronary artery disease differed in different directionConclusion. The obtained data indicate that the antiaggregation effect of H2S is realized through the effect on the NKCC and anion exchanger, as well as due to the effect on the links of the signaling system mediated by cyclic nucleotides.

The neuropeptide RhoprCCHamide2 inhibits serotonin-stimulated transcellular Na+ transport across the anterior midgut of the vector of Chagas disease, Rhodnius prolixus.

Rhodnius prolixus is a blood-feeding insect vector of Trypanosoma cruzi, a protozoan parasite that causes Chagas disease. During each blood meal, the animals ingest large volumes of blood, that may be up to 12 times the unfed body mass. These blood meals impose a significant osmotic stress for the animals due to the hyposmotic condition of the ingested blood compared with the insect's hemolymph. Thus the insect undergoes a massive postprandial diuresis that allows for the excretion of the plasma fraction of the blood in less than two hours. Diuresis is performed by the excretory system, consisting of the Malpighian tubules and gut, under the control of diuretic and anti-diuretic factors. We investigated the ion transport machinery triggered by stimulation with the diuretic factor serotonin in the anterior midgut (i.e.crop) and the effect of the diuretic modulator RhoprCCHamide2. Ussing chamber assays revealed that serotonin-stimulated increase in transepithelial short-circuit current (Isc) was more sensitive to the blockage with amiloride than 5-N-ethyl-N-isopropyl amiloride (EIPA), suggesting the involvement of Na+ channels. Incubation in Na+-free, but not Cl--free saline, blocked the effect of serotonin on Isc. Moreover, treatment with Na+-K+-2Cl- cotransporter (NKCC) and Na+-Cl- cotransporter (NCC) blockers had no effect on fluid secretion but was blocked by amiloride. Blockage of Na+/K+-ATPase with ouabain inhibited Isc but the H+-ATPase inhibitor bafilomycin had no effect. The neuropeptide RhoprCCHamide2 diminished serotonin-stimulated Isc across the crop. The results suggest that Na+ undergoes active transport via an apical amiloride-sensitive Na+ channel and a basolateral ouabain-sensitive Na+/K+-ATPase, while Cl- is transported through a passive paracellular pathway.

Na+/K+-ATPase as a Target of Cardiac Glycosides for the Treatment of SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), identified for the first time in Wuhan, China, causes coronavirus disease 2019 (COVID-19), which moved from epidemic status to becoming a pandemic. Since its discovery in December 2019, there have been countless cases of mortality and morbidity due to this virus. Several compounds such as chloroquine, hydroxychloroquine, lopinavir-ritonavir, and remdesivir have been tested as potential therapies; however, no effective treatment is currently recommended by regulatory agencies. Some studies on respiratory non-enveloped viruses such as adenoviruses and rhinovirus and some respiratory enveloped viruses including human respiratory syncytial viruses, influenza A, parainfluenza, SARS-CoV, and SARS-CoV-2 have shown the antiviral activity of cardiac glycosides, correlating their effect with Na+/K+-ATPase (NKA) modulation. Cardiac glycosides are secondary metabolites used to treat patients with cardiac insufficiency because they are the most potent inotropic agents. The effects of cardiac glycosides on NKA are dependent on cell type, exposure time, and drug concentration. They may also cause blockage of Na+ and K+ ionic transport or trigger signaling pathways. The antiviral activity of cardiac glycosides is related to cell signaling activation through NKA inhibition. Nuclear factor kappa B (NFκB) seems to be an essential transcription factor for SARS-CoV-2 infection. NFκB inhibition by cardiac glycosides interferes directly with SARS-CoV-2 yield and inflammatory cytokine production. Interestingly, the antiviral effect of cardiac glycosides is associated with tyrosine kinase (Src) activation, and NFκB appears to be regulated by Src. Src is one of the main signaling targets of the NKA α-subunit, modulating other signaling factors that may also impair viral infection. These data suggest that Src-NFκB signaling modulated by NKA plays a crucial role in the inhibition of SARS-CoV-2 infection. Herein, we discuss the antiviral effects of cardiac glycosides on different respiratory viruses, SARS-CoV-2 pathology, cell signaling pathways, and NKA as a possible molecular target for the treatment of COVID-19.

Tridax procumbens leaf extract elicits relaxation of rat mesenteric artery by activation of Na+ -K+ ATPase / Na+ - Ca2+ exchanger

Background: Tridax procumbens leaf extract has been reported to show antihypertensive actions through its vasodilatory effects. Although several mechanisms of action have been reported, the sub-cellular mechanism of action of the extract is yet to be fully elucidated. Aim and Objectives: In this study, we investigate the involvement of Na+ - K+ -pump and Na+- Ca2+ exchanger in the vasorelaxant activities of T. procumbens extract (TPE) in isolated rat superior mesenteric arteries. Materials and Methods: Superior mesenteric artery isolated from healthy, young adult Wistar rats (250–300 g) and pre-contracted with phenylephrine (PE) (10-7M) were treated with various concentrations of aqueous extract of TPE (0.5–9 mg/mL). The changes in arterial tension were recorded and interaction between TPE with the selective blocker of Na+ - K+ ATPase (ouabain) and putative Na+-Ca2+ exchanger inhibitor (NiCl2) was evaluated. Results: The result indicated that TPE significantly inhibit the cumulative concentration response curves to PE (10-9 - 10-5 M) in a concentration-dependent manner. Pre-treatment with both ouabain and nickel chloride (NiCl2), abolished the vasorelaxant responses of TPE. Furthermore, TPE reduced the relaxant effect with decreased extracellular Na+ concentration. TPE-induced vasorelaxation was completely abolished in a Na+ -free solution, a condition that eliminates the influence of the forward mode of the exchanger. Conclusion: The results provide direct hypothetical evidence that the activation of Na+ - K+ ATPase and stimulation of the forward mode of the Na+-Ca2+ exchanger contribute to the vasorelaxation induced by TPE in mesenteric artery.

Effects of Ion Exchange Process on Catalyst Activity and Plasma-Assisted Reactor Toward Cracking of Palm Oil into Biofuels

Biofuels can be produced through a conventional catalytic cracking system and/or a hybrid catalytic-plasma cracking system. This paper was focused on studying effect of Na+ ion exchange to HY-Zeolite catalyst on catalyst performance to convert palm oil into biofuels over a conventional continuous fixed bed catalytic cracking reactor and comparing the catalytic cracking performance when carried out in a continuous hybrid catalytic-plasma reactor. The catalysts were characterized by X-ray Diffraction (XRD) and Bruneuer-Emmet-Teller (BET) surface area methods. The biofuels product were analyzed using Gas Chromatography-Mass Spectrometry (GC-MS) to determine the hydrocarbons composition of biofuels product. From the results, ion exchange process of Na+ into HY-Zeolite catalyst decreases the catalyst activity due to decreasing the number of active sites caused by blocking of Na+ ion. The selectivity to gasoline ranges achieved 34.25% with 99.11% total conversion when using HY catalyst over conventional continuous fixed bed reactor system. Unfortunately, the selectivity to gasoline ranges decreased to 13.96% and the total conversion decrease slightly to 98.06% when using NaY-Zeolite catalyst. As comparison when the cracking reaction was carried out in a hybrid catalytic-plasma reactor using a spent residual catalytic cracking (RCC) catalyst, the high energetics electron from plasma can improve the reactor performance, where the conversion and yield were increased and the selectivity to lower ranges of hydrocarbons was increased. However, the last results were potential to be intensively studied with respect to relation between reactor temperature and plasma-assisted catalytic reactor parameters. Copyright © 2019 BCREC Group. All rights reserved

A micro-fabricated in vitro complex neuronal circuit platform

Developments in micro-manufacture as well as biofabrication technologies are driving our ability to create complex tissue models such as ‘organ-on-a-chip’ devices. The complexity of neural tissue, however, requires precisely specific cellular connectivity across many neuronal populations, and thus there have been limited reports of complex ‘brain-on-a-chip’ technologies modelling specific cellular circuit function. Here we describe the development of a model of in vitro brain circuitry designed to accurately reproduce part of the complex circuitry involved in neurodegenerative diseases; using segregated co-culture of specific basal ganglia (BG) neuronal subtypes to model central nervous system circuitry. Lithographic methods and chemical modification were used to form structured micro-channels, which were populated by specifically cultured neuronal sub-types to represent parts of the inter-communicating neural circuit. Cell morphological assessment and immunostaining showed connectivity, which was supported by electrophysiology measurements. Electrical activity of cells was measured using patch-clamp, showing voltage dependant Na+ and K+ currents, and blocking of Na+ current by TTX, and calcium imaging showing TTX-sensitive slow Ca2+ oscillations resulting from action potentials. Monitoring cells across connected ports post-TTX addition demonstrated both upstream and downstream changes in activity, indicating network connectivity. The model developed herein provides a platform technology that could be used to better understand neurological function and dysfunction, contributing to a growing urgency for better treatments of neurodegenerative disease. We anticipate the use of this advancing technology for the assessment of pharmaceutical and cellular therapies as a means of pre-clinical assessment, and further for the advancement of neural engineering approaches for tissue engineering.

The Blocking Effect of Clay in Groundwater Systems: A Case Study in an Inland Plain Area.

In order to increase understanding of the hydrogeochemical effects that influence changes in the quality of salt water, we investigated the distribution of saline and fresh water in an inland plain area and, in particular, the scarcity of fresh water resources. Taking the inland plain in Jiyang County as a specific case study, samples of undisturbed clay and underground saline water from different depths were collected to examine hydrogeological changes. A wide variety of methods was used to analyze the blocking effect of clay on the chemical characteristics of the groundwater. These include real-time monitoring for field water quality, tests for isothermal adsorption, a factor analysis model, physiochemical analysis, and correlation analysis. Our results show that the optimal adsorption isotherm of clay for Na+, Ca2+ and Mg2+ in groundwater conform to the established Henry and Langmuir equations for adsorption isotherms. The influence of clay mineral types and content on the blockage of Na+, Ca2+ and Mg2+ in groundwater samples were evident at different depths, with the clay adsorption capacity increasing in line with increases in the clay mineral content. Clay at different depths was found to have the strongest blocking effect on Na+ in groundwater, being systematically greater than its effect on Ca2+ and Mg2+. It is believed that the blocking effect of clay has an important influence on the hydrochemical zoning of groundwater in inland plains and the formation of saline water in groundwater systems. This study therefore provides concrete evidence in support of this supposed effect.

Büchi's model based analysis of local anesthetic action in procaine hydrochloride: Vibrational spectroscopic approach

The drug action of ester type local anesthetic (LA) procaine hydrochloride (PRC HCl) is activated by blocking Na+ ion flow when it binds to the ion channel in the ligand gated sodium ion channel protein. Büchi's model, explains binding action of ester type LA drug with receptor in terms of charge transfer, dipole-dipole, hydrogen bonding and van der Waals interactions through lipophilic, ester and hydrophilic moieties. The present work investigates molecular structural and vibrational spectral features of para amino benzoate group, ester part and tertiary amino group respectively belonging to lipophilic, ester and hydrophilic moieties, accountable for the binding of drug to sodium channel. The electron transport mechanism through the ring responsible for structural deviation from benzenoid to quinonoid form and consequent dipolar nature of carbonyl group have been investigated, based on the analysis of XRD, DFT computed molecular structure, 8a ring mode and NBO charges. The characteristic UV absorption peaks and vibrational marker bands of LA drugs have been identified and the charge transfer interaction responsible for lipophilic binding has been investigated. The blocking of Na+ in the ion channel has been probed using attractive and repulsive energy profile. The molecular polarizability has been computed to substantiate the correlation between the structure activity relationship of LA drug molecule and molecular polarizability. The low toxicity of PRC HCl was evaluated using in vitro cytotoxicity study, confirming it as a potential short acting local anesthetic.

Retinal pH and Acid Regulation During Metabolic Acidosis

ABSTRACTPurpose: Changes in retinal pH may contribute to a variety of eye diseases. To study the effect of acidosis alone, we induced systemic metabolic acidosis and hypothesized that the retina would respond with altered expression of genes involved in acid/base regulation.Methods: Systemic metabolic acidosis was induced in Long–Evans rats for up to 2 weeks by adding NH4Cl to the drinking water. After 2 weeks, venous pH was 7.25 ± 0.08 (SD) and [HCO3−] was 21.4 ± 4.6 mM in acidotic animals; pH was 7.41 ± 0.03 and [HCO3−] was 30.5 ± 1.0 mM in controls. Retinal mRNAs were quantified by quantitative reverse transcription polymerase chain reaction. Protein was quantified with Western blots and localized by confocal microscopy. Retinal [H+]o was measured in vivo with pH microelectrodes in animals subjected to metabolic acidosis and in controls.Results: NH4Cl in drinking water or given intravenous was effective in acidifying the retina. Cariporide, a blocker of Na+/H+ exchange, further acidified the retina. Metabolic acidosis for 2 weeks led to increases of 40–100% in mRNA for carbonic anhydrase isoforms II (CA-II) and XIV (CA-XIV) and acid-sensing ion channels 1 and 4 (ASIC1 and ASIC4) (all p < 0.005). Expression of anion exchange protein 3 (AEP-3) and Na+/H+ exchanger (NHE)-1 also increased by ≥50% (both p < 0.0001). Changes were similar after 1 week of acidosis. Protein for AEP-3 doubled. NHE-1 co-localized with vascular markers, particularly in the outer plexiform layer. CA-II was located in the neural parenchyma of the ganglion cell layer and diffusely in the rest of the inner retina.Conclusions: The retina responds to systemic acidosis with increased expression of proton and bicarbonate exchangers, carbonic anhydrase, and ASICs. While responses to acidosis are usually associated with renal regulation, these studies suggest that the retina responds to changes in local pH presumably to control its acid/base environment in response to systemic acidosis.

Functional Comparison of Cultures of IPSC-Derived Human Neurons and Rodent Neurons

Functional Comparison of Cultures of IPSC-Derived Human Neurons and Rodent Neurons

Computer modeling defines the system driving a constant current crucial for homeostasis in the mammalian cochlea by integrating unique ion transports

The cochlear lateral wall—an epithelial-like tissue comprising inner and outer layers—maintains +80 mV in endolymph. This endocochlear potential supports hearing and represents the sum of all membrane potentials across apical and basolateral surfaces of both layers. The apical surfaces are governed by K+ equilibrium potentials. Underlying extracellular and intracellular [K+] is likely controlled by the “circulation current,” which crosses the two layers and unidirectionally flows throughout the cochlea. This idea was conceptually reinforced by our computational model integrating ion channels and transporters; however, contribution of the outer layer’s basolateral surface remains unclear. Recent experiments showed that this basolateral surface transports K+ using Na+, K+-ATPases and an unusual characteristic of greater permeability to Na+ than to other ions. To determine whether and how these machineries are involved in the circulation current, we used an in silico approach. In our updated model, the outer layer’s basolateral surface was provided with only Na+, K+-ATPases, Na+ conductance, and leak conductance. Under normal conditions, the circulation current was assumed to consist of K+ and be driven predominantly by Na+, K+-ATPases. The model replicated the experimentally measured electrochemical properties in all compartments of the lateral wall, and endocochlear potential, under normal conditions and during blocking of Na+, K+-ATPases. Therefore, the circulation current across the outer layer’s basolateral surface depends primarily on the three ion transport mechanisms. During the blockage, the reduced circulation current partially consisted of transiently evoked Na+ flow via the two conductances. This work defines the comprehensive system driving the circulation current.

Extracellular phosphates enhance activities of voltage-gated proton channels and production of reactive oxygen species in murine osteoclast-like cells.

Osteoclasts are highly differentiated bone-resorbing cells and play a significant role in bone remodelling. In the resorption pit, inorganic phosphate (Pi) concentrations increase because of degradation of hydroxyapatite. We studied effects of extracellular Pi on voltage-gated H+ channels in osteoclast-like cells derived from a macrophage cell line (RAW264). Extracellular Pi (1.25-20mM) increased the H+ channel currents dose dependently and reversibly. The Pi-induced increases were attenuated by removal of extracellular Na+ and by phosphonoformic acid, a blocker of Na+-dependent Pi transporters. Pi increased the maximal conductance, decreased activation time constant, increased deactivation time constant, and shifted the conductance-voltage relationship to more negative voltages. The most marked change was enhanced gating which was mainly caused by elevation of intracellular Pi levels. The Pi-induced enhanced gating was partially inhibited by protein kinase C (PKC) inhibitors, GF109203X and staurosporine, indicating that PKC-mediated phosphorylation was involved in part. The increase in the maximal conductance was mainly due to accompanying decrease in intracellular pH. These effects of Pi were not affected by intracellular Mg2+, bafilomycin A1 (V-ATPase inhibitor) and removal of intracellular ATP. Extracellular Pi also upregulated reactive oxygen species (ROS). Diphenyleneiodonium chloride, an inhibitor of NADPH oxidases, decreased ROS production and partially attenuated the enhanced gating. In the cells during later passages where osteoclastogenesis declined, H+ channel activities and ROS production were both modest. These results suggest that, in osteoclasts, ambient Pi is a common enhancer for H+ channels and ROS production and that potentiation of H+ channels may help ROS production.

INFLUENCE HYDROGEN SULFIDE ON CA2 + -DEPENDENT POTASSIUM PERMEABILITY OF THE MEMBRANE OF HUMAN ERYTHROCYTES

The purpose of the research was to study the influence of hydrogen sulfide donor NaHS on red blood cells membrane potential changes caused by the activation of Ca2+ dependent K+ channels in the presence of calcium ionophore or artificial electron donor system ascorbate phenazine methosulfate. Materials and methods. We used the packed erythrocytes obtained from venous blood of 25 healthy volunteers at the age of 20-27 years. Registration of the membrane potential of erythrocytes in the presence of Ca2+ ionophore (A23187) or artificial electron donor system ascorbate – FMS was performed with potentiometric method based on the fact that in the presence of the protonophore distribution of H + depends on the membrane potential Em like as Em = RT/F (pHi – pH0 ). Here pHi and pH0 are pH of the incubation medium and cytoplasm, respectively. An amplitude of hyperpolarizing response (HR) was calculated as an integral characteristic of Ca2+-dependent permeability for K+. Results. The addition of NaHS at concentrations from 0.2 mM to 0.005 mM into the cells incubation medium caused a change in the amplitude of hyperpolarizing response of erythrocyte membranes induced by both methods. In the presence of 0.005 mM NaHS the amplitude of A23187-dependent HR increased significantly, whereas the amplitude of the redox-dependent HR decreased. Suppression of A23187-dependent HR in the presence of higher concentrations of NaHS was more pronounced than the suppression of the amplitude of redox-dependent response. The amplitude of A23187-dependent HR increased under the joint action of hydrogen sulfide and the blocker of Na+, K+, 2Cl- -cotransport (NKCC) bumetanide, and the amplitude of redox-dependent HR decreased in comparison with the parameters obtained in the absence of a blocker. Conclusion. It is found that hydrogen sulfide exerts a modulatory effect on K (Ca2+) - channels of erythrocyte membrane. The effect of H2 S depends on the method of activation of the studied channels. A23187-dependent HR was more sensitive to H2S in comparison to redox-induced HR of erythrocyte membrane. The effect of NaHS on the amplitude of the HR in the presence of NKCC blocker bumetanide also depended on the method of stimulation of channels.

Effect of flecainide derivatives on sarcoplasmic reticulum calcium release suggests a lack of direct action on the cardiac ryanodine receptor.

Background and PurposeFlecainide is a use‐dependent blocker of cardiac Na+ channels. Mechanistic analysis of this block showed that the cationic form of flecainide enters the cytosolic vestibule of the open Na+ channel. Flecainide is also effective in the treatment of catecholaminergic polymorphic ventricular tachycardia but, in this condition, its mechanism of action is contentious. We investigated how flecainide derivatives influence Ca2 +‐release from the sarcoplasmic reticulum through the ryanodine receptor channel (RyR2) and whether this correlates with their effectiveness as blockers of Na+ and/or RyR2 channels.Experimental ApproachWe compared the ability of fully charged (QX‐FL) and neutral (NU‐FL) derivatives of flecainide to block individual recombinant human RyR2 channels incorporated into planar phospholipid bilayers, and their effects on the properties of Ca2 + sparks in intact adult rat cardiac myocytes.Key ResultsBoth QX‐FL and NU‐FL were partial blockers of the non‐physiological cytosolic to luminal flux of cations through RyR2 channels but were significantly less effective than flecainide. None of the compounds influenced the physiologically relevant luminal to cytosol cation flux through RyR2 channels. Intracellular flecainide or QX‐FL, but not NU‐FL, reduced Ca2 + spark frequency.Conclusions and ImplicationsGiven its inability to block physiologically relevant cation flux through RyR2 channels, and its lack of efficacy in blocking the cytosolic‐to‐luminal current, the effect of QX‐FL on Ca2 + sparks is likely, by analogy with flecainide, to result from Na+ channel block. Our data reveal important differences in the interaction of flecainide with sites in the cytosolic vestibules of Na+ and RyR2 channels.

Block of Na+/Ca2+ exchanger by SEA0400 in human right atrial preparations from patients in sinus rhythm and in atrial fibrillation

The Na+/Ca2+ exchanger (NCX) plays a major role in myocardial Ca2+ homoeostasis, but is also considered to contribute to the electrical instability and contractile dysfunction in chronic atrial fibrillation (AF). Here we have investigated the effects of the selective NCX blocker SEA0400 in human right atrial cardiomyocytes from patients in sinus rhythm (SR) and AF in order to obtain electrophysiological evidence for putative antiarrhythmic activity of this new class of drugs. Action potentials were measured in right atrial trabeculae using conventional microelectrodes. Human myocytes were enzymatically isolated. Rat atrial and ventricular cardiomyocytes were used for comparison. Using perforated-patch, NCX was measured as Ni2+-sensitive current during ramp pulses. In ruptured-patch experiments, NCX current was activated by changing the extracellular Ca2+ concentration from 0 to 1mM in Na+-free bath solution (100mM Na+ intracellular, “Hilgemann protocol”). Although SEA0400 was effective in rat cardiomyocytes, 10µM did not influence action potentials and contractility, neither in SR nor AF. SEA0400 (10μM) also failed to affect human atrial NCX current measured with perforated patch. With the “Hilgemann protocol” SEA0400 concentration-dependently suppressed human atrial NCX current, and its amplitude was larger in AF than in SR cardiomyocytes. Our results confirm higher NCX activity in AF than SR. SEA0400 fails to block Ni2+-sensitive current in human atrial cells unless unphysiological conditions are used. We speculate that block of NCX with SEA0400 depends on intracellular Na+ concentration.

Intracellular ion signaling influences myelin basic protein synthesis in oligodendrocyte precursor cells

Myelination in the central nervous system depends on axon-oligodendrocyte precursor cell (OPC) interaction. We suggest that myelin synthesis may be influenced by [Na+]i and [Ca2+]i signaling in OPCs. Experiments were performed in mouse cultured OPCs at day in vitro (DIV) 2–6 or acute slices of the corpus callosum at postnatal days (P) 10–30. Synthesis of Myelin Basic Protein (MBP), an “executive molecule of myelin”, was used as readout of myelination. Immunohistological data revealed that MBP synthesis in cultured OPCs starts around DIV4. Transient elevations of resting [Ca2+]i and [Na+]i levels were observed in the same temporal window (DIV4-5). At DIV4, but not at DIV2, both extracellular [K+] ([K+]e) elevation (+5mM) and partial Na+,K+-ATPase (NKA) inhibition elicited [Na+]i and [Ca2+]i transients. These responses were blocked with KB-R7943 (1μM), a blocker of Na+-Ca2+ exchanger (NCX), indicating an involvement of NCX which operates in reverse mode. Treatment of OPCs with culture medium containing elevated [K+] (+5mM, 24h) or ouabain (500nM, 24h) increased resting [Ca2+]i and facilitated MBP synthesis. Blockade of NCX with KB-R7943 (1μM, 12h) reduced resting [Ca2+]i and decreased MBP synthesis. Similar to the results obtained in OPC cultures, OPCs in acute callosal slices demonstrated an increase in resting [Ca2+]i and [Na+]i levels during development. NCX blockade induced [Ca2+]i and [Na+]i responses in OPCs at P20-30 but not at P10. We conclude that local [Na+]i and/or membrane potential changes can modulate Ca2+ influx through NCX and in turn MBP synthesis. Thus neuronal activity-induced changes in [K+]e may via NCX and NKA modulate myelination.

The Bacterial Flagellar Type III Export Gate Complex Is a Dual Fuel Engine That Can Use Both H+ and Na+ for Flagellar Protein Export.

The bacterial flagellar type III export apparatus utilizes ATP and proton motive force (PMF) to transport flagellar proteins to the distal end of the growing flagellar structure for self-assembly. The transmembrane export gate complex is a H+–protein antiporter, of which activity is greatly augmented by an associated cytoplasmic ATPase complex. Here, we report that the export gate complex can use sodium motive force (SMF) in addition to PMF across the cytoplasmic membrane to drive protein export. Protein export was considerably reduced in the absence of the ATPase complex and a pH gradient across the membrane, but Na+ increased it dramatically. Phenamil, a blocker of Na+ translocation, inhibited protein export. Overexpression of FlhA increased the intracellular Na+ concentration in the presence of 100 mM NaCl but not in its absence, suggesting that FlhA acts as a Na+ channel. In wild-type cells, however, neither Na+ nor phenamil affected protein export, indicating that the Na+ channel activity of FlhA is suppressed by the ATPase complex. We propose that the export gate by itself is a dual fuel engine that uses both PMF and SMF for protein export and that the ATPase complex switches this dual fuel engine into a PMF-driven export machinery to become much more robust against environmental changes in external pH and Na+ concentration.

Effect of Flecainide Derivatives on Sarcoplasmic Reticulum Ca2+ Release Confirms a Lack of Direct Action on the Cardiac Ryanodine Receptor

Flecainide is a use-dependent blocker of the cardiac Na+ channel. It has been shown to be effective in the treatment of catecholaminergic polymorphic ventricular tachycardia (CPVT), however, its mechanism of action is contentious. The mechanisms involved in block have been elucidated by using fully charged (QX-FL) and neutral (NU-FL) derivatives of flecainide, establishing that Na+ channel block requires entry of the cationic form of the molecule into the cytosolic vestibule of the open channel. We investigated how flecainide derivatives influence RyR2-mediated Ca2+-release from the sarcoplasmic reticulum and whether this correlates with their effectiveness as blockers of Na+ channels and/or RyR2. We compared the effects of flecainide, QX-FL and NU-FL applied extracellulary or via intra-pipette, on the properties of Ca2+ sparks in intact adult rat cardiac myocytes with their ability to block recombinant human RyR2. Intracellular flecainide or QX-FL reduced Ca2+ spark frequency whereas NU-FL did not. Both QX-FL and NU-FL were partial blockers of the non-physiological cytosolic to luminal flux of cations through RyR2 (resulting in different residual currents) but were significantly less effective than flecainide. None of the compounds influenced luminal to cytosol cation flux through RyR2. Given QX-FL inability to block physiologically-relevant cation flux through RyR2, and its comparative lack of efficacy in blocking the cytosolic-to-luminal current through the channel, the effect of QX-FL on Ca2+ sparks is likely, by analogy with flecainide, to result from Na+ channel block. Our data reveal important differences in the nature of molecular interaction of flecainide with sites in the cytosolic vestibules of Na+ and RyR2 channels. Research supported by the British Heart Foundation.

Human-induced pluripotent stem cell-derived cardiomyocytes from cardiac progenitor cells: effects of selective ion channel blockade.

Human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are likely to revolutionize electrophysiological approaches to arrhythmias. Recent evidence suggests the somatic cell origin of hiPSCs may influence their differentiation potential. Owing to their cardiomyogenic potential, cardiac-stromal progenitor cells (CPCs) are an interesting cellular source for generation of hiPSC-derived cardiomyocytes. The effect of ionic current blockade in hiPSC-derived cardiomyocytes generated from CPCs has not been characterized yet. Human-induced pluripotent stem cell-derived cardiomyocytes were generated from adult CPCs and skin fibroblasts from the same individuals. The effect of selective ionic current blockade on spontaneously beating hiPSC-derived cardiomyocytes was assessed using multi-electrode arrays. Cardiac-stromal progenitor cells could be reprogrammed into hiPSCs, then differentiated into hiPSC-derived cardiomyocytes. Human-induced pluripotent stem cell-derived cardiomyocytes of cardiac origin showed higher upregulation of cardiac-specific genes compared with those of fibroblastic origin. Human-induced pluripotent stem cell-derived cardiomyocytes of both somatic cell origins exhibited sensitivity to tetrodotoxin, a blocker of Na+ current (INa), nifedipine, a blocker of L-type Ca2+ current (ICaL), and E4031, a blocker of the rapid component of delayed rectifier K+ current (IKr). Human-induced pluripotent stem cell-derived cardiomyocytes of cardiac origin exhibited sensitivity to JNJ303, a blocker of the slow component of delayed rectifier K+ current (IKs). In hiPSC-derived cardiomyocytes of cardiac origin, INa, ICaL, IKr, and IKs were present as tetrodotoxin-, nifedipine-, E4031-, and JNJ303-sensitive currents, respectively. Although cardiac differentiation efficiency was improved in hiPSCs of cardiac vs. non-cardiac origin, no major functional differences were observed between hiPSC-derived cardiomyocytes of different somatic cell origins. Further studies are warranted to characterize electrophysiological properties of hiPSC-derived cardiomyocytes generated from CPCs.

AMPA receptor activation causes preferential mitochondrial Ca2+ load and oxidative stress in motor neurons

It is well established that motor neurons are highly vulnerable to glutamate induced excitotoxicity. The selective vulnerability of these neurons has been attributed to AMPA receptor mediated excessive rise in cytosolic calcium and consequent mitochondrial Ca2+ loading. Earlier we have reported that in motor neurons a generic rise in [Ca2+]i does not always lead to mitochondrial Ca2+ loading and membrane depolarization but it occurs upon AMPA receptor activation. The mechanism of such specific mitochondrial involvement upon AMPA receptor activation is not known. The present study examines the mitochondrial Ca2+ regulation and oxidative stress in spinal cord neurons upon AMPA subtype of glutamate receptor activation. Stimulating the spinal neurons with AMPA exhibited a sharp rise in [Ca2+]m in both motor and other spinal neurons that was sustained up to the end of recording time of 30min. The rise in [Ca2+]m was substantially higher in motor neurons than in other spinal neurons which could be due to the differential mitochondrial homeostasis in two types of neurons. To examine this possibility, we measured AMPA induced [Ca2+]m loading in the presence of mitochondrial inhibitors. In both cell types the AMPA induced [Ca2+]m loading was blocked by mitochondrial calcium uniporter blocker ruthenium red. In motor neurons it was also inhibited substantially by CGP37157 and cyclosporine-A, the blockers of Na+/Ca2+ exchanger and mitochondrial permeability transition pore (MPTP) respectively, whereas no effect of these agents was observed in other spinal neurons. Thus in motor neurons the Ca2+ sequestration by mitochondria occurs through mitochondrial calcium uniporter as well as due to reversal of Na+/Ca2+ exchanger, in contrast the latter pathway does not contribute in other spinal neurons. The ROS formation was inhibited by nitric oxide synthase (NOS) inhibitor L-NAME in both types of neurons, however the mitochondrial complex-I inhibitor rotenone suppressed the ROS formation only in motor neurons. It appears that activation of cytoplasmic nNOS leads to ROS formation in both types of spinal neurons but mitochondria is the major source of ROS in motor neurons. Spinal neurons exhibited a significant time dependent fall in glutathione (GSH) level. The GSH level in motor neurons did not recover even at 24h after AMPA exposure, whereas the other spinal neurons exhibited a tendency to maintain the GSH after a certain level suggesting that the oxidative stress is arrested in other spinal neurons but it continues to increase in motor neurons. Thus our results demonstrate that upon AMPA receptor stimulation the motor neurons employ some additional pathways for regulation of mitochondrial calcium and oxidative stress as compared to other spinal neurons. It is suggested that such differential signaling mechanisms in motor neurons could be crucial for their selective vulnerability to excitotoxicity.