Sodium Influx Research Articles

Tracing oral Na+ and K+ in sweat during exercise and recovery in horses.

What is the central question of this study? What are the mechanisms by which equine sweat glands transport sodium, potassium and water into sweat? What is the main finding and its importance? The flux of sodium into sweat does not have an active transport component, the flux of potassium into sweat is partially dependent on an active transport mechanism, and there is no evidence for paracellular transport. In two series of experiments, this study used radioactive sodium (Na+ ) and potassium (K+ ) to trace the net flux, and calculate the unidirectional fluxes, of these ions from extracellular fluid into sweat of horses during exercise and recovery. The effect of an oral electrolyte supplement (PNW) on the sweating responses and ion fluxes was also examined. Compared to 8litres of water (controls), provision of 8litres of PNW resulted in significantly increased sweating duration (P<0.001). Two hours before exercise, 99 Tc-labelled diethylene-triamine-pentaacetate (DTPA) was administered i.v. to determine if there was paracellular flux of this molecule in sweat glands during the period of sweating. One hour before beginning moderate-intensity exercise, horses were nasogastrically administered either 24 Na (1-3 litres) or 42 K (8 litres) with water (control) or an electrolyte supplement. Both radiotracers appeared in sweat within 10min of exercise onset, and the sweat specific activity of both ions increased during exercise (P<0.001), approaching plasma specific activities. There was no appearance of 99 Tc-DTPA in sweat. The activities of 24 Na and 42 K, together with the concentrations Na+ , K+ and Cl- , argued against significant paracellular flux of these ions into the lumen of sweat glands. The flux analysis for 24 Na indicated a small intracellular pool within sweat gland cells, and no evidence for an active transport component. The flux analysis for 42 K indicated a relatively large intracellular equilibration pool within sweat gland cells, with evidence for an active transport component. The results are discussed with respect to the current understanding of sweat gland epithelial cell ion transport mechanisms at both the basal and the apical membranes. It appears likely that the majority of ions appearing in sweat pass through sweat gland epithelial cells by transcellular mechanisms that include ion transporting pathways as well as apical vesicular exocytosis.

Continuous monitoring of plant sodium transport dynamics using clinical PET

BackgroundThe absorption, translocation, accumulation and excretion of substances are fundamental processes in all organisms including plants, and have been successfully studied using radiotracers labelled with 11C, 13N, 14C and 22Na since 1939. Sodium is one of the most damaging ions to the growth and productivity of crops. Due to the significance of understanding sodium transport in plants, a significant number of studies have been carried out to examine sodium influx, compartmentation, and efflux using 22Na- or 24Na-labeled salts. Notably, however, most of these studies employed destructive methods, which has limited our understanding of sodium flux and distribution characteristics in real time, in live plants. Positron emission tomography (PET) has been used successfully in medical research and diagnosis for decades. Due to its ability to visualise and assess physiological and metabolic function, PET imaging has also begun to be employed in plant research. Here, we report the use of a clinical PET scanner with a 22Na tracer to examine 22Na-influx dynamics in barley plants (Hordeum vulgare L. spp. Vulgare—cultivar Bass) under variable nutrient levels, alterations in the day/night light cycle, and the presence of sodium channel inhibitors.Results3D dynamic PET images of whole plants show readily visible 22Na translocation from roots to shoots in each examined plant, with rates influenced by both nutrient status and channel inhibition. PET images show that plants cultivated in low-nutrient media transport more 22Na than plants cultivated in high-nutrient media, and that 22Na uptake is suppressed in the presence of a cation-channel inhibitor. A distinct diurnal pattern of 22Na influx was discernible in curves displaying rates of change of relative radioactivity. Plants were found to absorb more 22Na during the light period, and anticipate the change in the light/dark cycle by adjusting the sodium influx rate downward in the dark period, an effect not previously described experimentally.ConclusionsWe demonstrate the utility of clinical PET/CT scanners for real-time monitoring of the temporal dynamics of sodium transport in plants. The effects of nutrient deprivation and of ion channel inhibition on sodium influx into barley plants are shown in two proof-of-concept experiments, along with the first-ever 3D-imaging of the light and dark sodium uptake cycles in plants. This method carries significant potential for plant biology research and, in particular, in the context of genetic and treatment effects on sodium acquisition and toxicity in plants.

Interleukin 8 Elicits Rapid Physiological Changes in Neutrophils That Are Altered by Inflammatory Conditions

A sufficient response of neutrophil granulocytes stimulated by interleukin (IL)-8 is vital during systemic inflammation, for example, in sepsis or severe trauma. Moreover, IL-8 is clinically used as biomarker of inflammatory processes. However, the effects of IL-8 on cellular key regulators of neutrophil properties such as the intracellular pH (pH_i) in dependence of ion transport proteins and during inflammation remain to be elucidated. Therefore, we investigated in detail the fundamental changes in pH_i, cellular shape, and chemotactic activity elicited by IL-8. Using flow cytometric methods, we determined that the IL-8-induced cellular activity was largely dependent on specific ion channels and transporters, such as the sodium-proton exchanger 1 (NHE1) and non-NHE1-dependent sodium flux. Exposing neutrophils in vitro to a proinflammatory micromilieu with N-formyl-Met-Leu-Phe, LPS, or IL-8 resulted in a diminished response regarding the increase in cellular size and pH. The detailed kinetics of the reduced reactivity of the neutrophil granulocytes could be illustrated in a near-real-time flow cytometric measurement. Last, the LPS-mediated impairment of the IL-8-induced response in neutrophils was confirmed in a translational, animal-free human whole blood model. Overall, we provide novel mechanistic insights for the interaction of IL-8 with neutrophil granulocytes and report in detail about its alteration during systemic inflammation.

Canopy modification of base cations deposition in a subtropical broadleaved forest: Spatial characteristics, canopy budgets and acid neutralizing capacity

Atmospheric deposition of base cations in contact with the canopy alters their chemical characteristics and plays a vital role in offsetting soil acidification and nutrient imbalance by replenishing the base cationic pool in forest ecosystems. However, the effects of site characteristics on deliveries of base cations fluxes via throughfall (TF) and stemflow (SF) across topographic gradients within a watershed remain less understood. Here, we examined the concentrations and fluxes of base cations (potassium (K+), calcium (Ca2+), magnesium (Mg2+) and sodium (Na+)), acid neutralizing capacities (ANC) and alkalinity (ALK) in bulk precipitation (BP), TF and SF in an upper slope plot (UP) and a lower slope plot (LP) in a subtropical broadleaved montane forest, Southeast China. In addition, a canopy budget model was applied to distinguish the relative contributions of dry deposition (DD) and canopy exchange (CE) to net TF + SF (NTF) flux. Results showed that annual volume weighted mean concentrations of base cations in TF and SF were significantly higher than those in BP in both UP and LP. Annual fluxes of TF or SF base cations had no significant differences between UP and LP, resulting in a comparable of the NTF fluxes. However, the relative contributions of DD and CE to NTF were different at the two slope plots. NTF K+ flux was dominated by CE regardless of slope position. Unlike K+, NTF Ca2+ and Mg2+ fluxes were dominated by DD in UP, while CE in LP. In addition, ANC and ALK of TF and SF were significantly higher compared to BP, suggesting rainwater beneath the canopy became less acidic and may mitigate in part the risk of soil acidification, especially in the dormant season. This study can provide insights into the spatial variability of chemical characteristics of base cations, and improve our understanding of the quantities and controls of external and internal nutrient inputs occurring within the canopy in forest watershed.

Cation Flux and Electrolyte Composition of Frozen-Deglycerolized Blood

Cation flux and the extracellular and erythrocyte concentrations of sodium, potassium, calcium, magnesium, chloride, and acid soluble phosphorus were measured in fresh blood collected in various anticoagulant solutions, ACD blood stored at 4 C, and in frozen-deglycerolized blood stored at -80 to -120 C. Considerable variations in concentrations of extracellular and erythrocyte electrolytes were encountered due to certain characteristics of the anticoagulants employed and to the effects of storage, temperature, pH, and extracellular electrolyte concentration on erythrocyte electrolyte concentration. In contrast to ACD blood stored at 4 C, frozen-deglycerolized blood 1 day to 32 months old had slightly elevated erythrocyte concentrations of sodium and chloride and normal erythrocyte concentrations of potassium and phosphorus. Both ACD and frozen-deglycerolized blood had normal sodium and potassium fluxes. These data show that prolonged freezing and glycerolization of erythrocytes do not significantly impair the active transport processes responsible for maintenance of cellular electrolyte concentration.

Characterization of Procoagulant COAT Platelets in Patients with Glanzmann Thrombasthenia.

Patients affected by the rare Glanzmann thrombasthenia (GT) suffer from defective or low levels of the platelet-associated glycoprotein (GP) IIb/IIIa, which acts as a fibrinogen receptor, and have therefore an impaired ability to aggregate platelets. Because the procoagulant activity is a dichotomous facet of platelet activation, diverging from the aggregation endpoint, we were interested in characterizing the ability to generate procoagulant platelets in GT patients. Therefore, we investigated, by flow cytometry analysis, platelet functions in three GT patients as well as their ability to generate procoagulant collagen-and-thrombin (COAT) platelets upon combined activation with convulxin-plus-thrombin. In addition, we further characterized intracellular ion fluxes during the procoagulant response, using specific probes to monitor by flow cytometry kinetics of cytosolic calcium, sodium, and potassium ion fluxes. GT patients generated higher percentages of procoagulant COAT platelets compared to healthy donors. Moreover, they were able to mobilize higher levels of cytosolic calcium following convulxin-plus-thrombin activation, which is congruent with the greater procoagulant activity. Further investigations will dissect the role of GPIIb/IIIa outside-in signalling possibly implicated in the regulation of platelet procoagulant activity.

(Invited) Technology of Gallium Nitride Crystal Growth - Challenges and Perspectives

It is well known that gallium nitride (GaN) is the basic material for fabricating optoelectronic devices as Light Emitting Diodes (LEDs) and Laser Diodes (LDs). GaN is also an attractive choice for electronic high-frequency and high-power devices. This is due to its wide band gap, high electron velocity, high breakdown field strength and high thermal conductivity. Most of the mentioned GaN-based devices are built on foreign substrates (sapphire, silicon, or silicon carbide). For many years, there was no mass production of native substrates. This happened because GaN crystal growth is quite a challenging process. The compound melts congruently at high temperature and extremely high nitrogen pressure (>6 GPa). Thus, today, it is impossible to grow GaN from the melt. The crystals should be obtained by other techniques requiring lower pressure and temperature. Crystallization from gas phase, solution, or any combination thereof must be included. For more than three decades, crystallization of bulk GaN has remained a big challenge for the nitride community. Work on developing bulk GaN has mainly been motivated by the LD market. All commercially available LD epi-structures are built on GaN wafers. Today, however, not only the LDs, but also the electronic industry and the demand for vertical high power transistors and diodes seem to be the driving force for crystallizing bulk GaN. Thus, GaN substrates of relatively high quality have recently become available. Today, GaN is crystallized by three main methods: i/ sodium flux (Na-flux); ii/ halide vapor phase epitaxy (HVPE); and iii/ basic or acidic ammonothermal growth. Perspectives of all the mentioned above technologies will be presented in this lecture. Special attention will be paid to the basic ammonothermal method and HVPE. It will be shown that growth from the vapor phase needs a new philosophy. The crystallization processes on foreign seeds (sapphire and gallium arsenide) reached their maximum possibilities. Despite many tricks during crystal growth runs the structural quality as well as yield of the obtained GaN crystals and wafers are very low and cannot be improved. The only way is GaN-on-GaN crystallization. Recently, significant progress has been presented in the basic ammonothermal method. High structural quality crystals of lateral size larger than 2 inches and thickness exceeding 6 mm were obtained in one crystal growth process. Use of ammonothermally grown GaN as seed for HVPE will be presented. All challenges and difficulties to grow bulk GaN from the vapor phase will be demonstrated. Scenarios for the further development of bulk GaN crystallization will be shown. Important parameters of GaN substrates for epitaxy of optoelectronic and electronic device structures will be discussed. A new way of better controlling the electrical properties of GaN crystals and wafers will be proposed.

Current methods for GaN synthesis and the limitations

The application of GaN has greatly enhanced the performance of optoelectrical and microelectronic devices. However, the industrial application still faces huge challenges due to the difficulty during the synthesis process. This paper introduces several current methods for GaN synthesis and talks about their advantages and limitations. Four methods are introduced: high pressure nitrogen solution growth process, ammonothermal method, sodium flux process and hydride vapor phase epitaxy. As a result, they are only suitable for specific use like scientific research and can’t be used for massive production. This paper reviews main GaN synthesis methods and points out the limitations they have to overcome for commercial applications.

Stimulation of Epithelial Sodium Channels in Endothelial Cells by Bone Morphogenetic Protein-4 Contributes to Salt-Sensitive Hypertension in Rats.

Previous studies have shown that high salt induces artery stiffness by causing endothelial dysfunction via increased sodium influx. We used our unique split-open artery technique combined with protein biochemistry and in vitro measurement of vascular tone to test a hypothesis that bone morphogenetic protein 4 (BMP4) mediates high salt-induced loss of vascular relaxation by stimulating the epithelial sodium channel (ENaC) in endothelial cells. The data show that high salt intake increased BMP4 both in endothelial cells and in the serum and that exogenous BMP4 stimulated ENaC in endothelial cells. The data also show that the stimulation is mediated by p38 mitogen-activated protein kinases (p38 MAPK) and serum and glucocorticoid-regulated kinase 1 (Sgk1)/neural precursor cell expressed developmentally downregulated gene 4-2 (Nedd4-2) (Sgk1/Nedd4-2). Furthermore, BMP4 decreased mesenteric artery relaxation in a benzamil-sensitive manner. These results suggest that high salt intake stimulates endothelial cells to express and release BMP4 and that the released BMP4 reduces artery relaxation by stimulating ENaC in endothelial cells. Therefore, stimulation of ENaC in endothelial cells by BMP4 may serve as another pathway to participate in the complex mechanism of salt-sensitive (SS) hypertension.

Sodium-Calcium Exchanger Reverse Mode Sustains Dichotomous Ion Fluxes Required for Procoagulant COAT Platelet Formation.

Procoagulant collagen-and-thrombin (COAT)-activated platelets represent a subpopulation of activated platelets, which retain a coat of prohemostatic proteins and express phosphatidylserine on their surface. Dichotomous intracellular signaling generating procoagulant platelet activity instead of traditional aggregating endpoints is still not fully elucidated. It has been demonstrated that secondary messengers such as calcium and sodium play a critical role in platelet activation. Therefore, we developed a flow cytometric analysis to investigate intracellular ion fluxes simultaneously during generation of aggregating and procoagulant platelets. Human platelets were activated by convulxin-plus-thrombin. Cytosolic calcium, sodium, and potassium ion fluxes were visualized by specific ion probes and analyzed by flow cytometry. We observed high and prolonged intracellular calcium concentration, transient sodium increase, and fast potassium efflux in COAT platelets, whereas aggregating non-COAT platelets rapidly decreased their calcium content, maintaining higher cytosolic sodium, and experiencing lower and slower potassium depletion. Considering these antithetical patterns, we investigated the role of the sodium-calcium exchanger (NCX) during convulxin-plus-thrombin activation. NCX inhibitors, CBDMB and ORM-10103, dose-dependently reduced the global calcium mobilization induced by convulxin-plus-thrombin activation and dose-dependently prevented formation of procoagulant COAT platelets. Our data demonstrate that both NCX modes are used after convulxin-plus-thrombin-induced platelet activation. Non-COAT platelets use forward-mode NCX, thus pumping calcium out and moving sodium in, while COAT platelets rely on reverse NCX function, which pumps additional calcium into the cytosol, by extruding sodium. In conclusion, we described for the first time the critical and dichotomous role of NCX function during convulxin-plus-thrombin-induced platelet activation.

Comparison of Anti-oncotic Effect of TRPM4 Blocking Antibody in Neuron, Astrocyte and Vascular Endothelial Cell Under Hypoxia.

In stroke and other neurological diseases, Transient Receptor Potential Melastatin 4 (TRPM4) has been reported to cause oncotic cell death which is due to an excessive influx of sodium ions. Following stroke, hypoxia condition activates TRPM4 channel, and the sodium influx via TRPM4 is further enhanced by an increased TRPM4 expression. However, the effect of TRPM4 inhibition on oncotic cell death, particularly during the acute stage, remains largely unknown. Recently, we have developed a polyclonal antibody M4P that specifically inhibits TRPM4 channel. M4P blocks the channel via binding to a region close to the channel pore from extracellular space. Using M4P, we evaluated the acute effect of blocking TRPM4 in neurons, astrocytes, and vascular endothelial cells. In a rat stroke model, M4P co-localized with neuronal marker NeuN and endothelial marker vWF, whereas few GFAP positive astrocytes were stained by M4P in the ipsilateral hemisphere. When ATP was acutely depleted in cultured cortical neurons and microvascular endothelial cells, cell swelling was induced. Application of M4P significantly blocked TRPM4 current and attenuated oncosis. TUNEL assay, PI staining and western blot on cleaved Caspase-3 revealed that M4P could ameliorate apoptosis after 24 h hypoxia exposure. In contrast, acute ATP depletion in cultured astrocytes failed to demonstrate an increase of cell volume, and application of M4P or control IgG had no effect on cell volume change. When TRPM4 was overexpressed in astrocytes, acute ATP depletion successfully induced oncosis which could be suppressed by M4P treatment. Our results demonstrate that comparing to astrocytes, neurons, and vascular endothelial cells are more vulnerable to hypoxic injury. During the acute stage of stroke, blocking TRPM4 channel could protect neurons and vascular endothelial cells from oncotic cell death.

The Production and Composition of Chinese Lead-Barium Glass through Experimental Laboratory Replication

Lead-barium glass appeared during the late Warring States period in China (457 - 221 BCE) and was considered as a Chinese invention due to the unique presence of high barium. There is no indication as to how it was made. A set of twenty-four different combinations of possible raw materials were melted to test and systematically evaluate the possible recipes for lead-barium glass. Specifically, the function and source of sodium in lead-barium glass were explored. The results show that sodium source is crucial for lead-barium glass forming in this particular system. This paper explores an alternative formulation since sodium flux is not reported in historical Chinese sources, in which the Na2O is provided by including soda-lime glass in the mixture. This particular formulation provides a potential explanation for the source of the sodium in lead-barium glass.

The Renal Risk Variants of Apolipoprotein L-1 Lead to an Influx of Sodium and Calcium That Drive Cytotoxicity

The Renal Risk Variants of Apolipoprotein L-1 Lead to an Influx of Sodium and Calcium That Drive Cytotoxicity

Time Course of Tissue Sodium Flux in Maintenance Hemodialysis (MHD) Patients

Time Course of Tissue Sodium Flux in Maintenance Hemodialysis (MHD) Patients

Investigating the mechanism of vanadium toxicity in freshwater organisms

Vanadium (V) could present a risk for aquatic organisms from the Alberta oil sands region, if present in high concentrations. An industry pilot project has used petroleum coke (PC) as a sorbent to remove organic toxicants from oil sands process-affected water (OSPW), but it also caused V to leach from PC into the OSPW, reaching concentrations of up to 7 mg V/L (a level known to be toxic to aquatic organisms). Vanadium is a transition metal with several oxidation states, which could potentially elicit its toxicity through either ion imbalance or oxidative stress. This study investigated the effect of V on Daphnia magna and Oncorhynchus mykiss. Daphinds and O. mykiss were exposed to concentrations of V up to their respective calculated median lethal concentration (LC50): 3 mg V/L for D. magna and 7 mg V/L for O. mykiss. For both organisms, the influence of V on sodium flux and whole body sodium was evaluated. Its effect on whole body calcium and the oxidative stress responses in O. mykiss at the gill and liver levels was also studied. Results suggested that 3.1 mg V/L for D. magna and 6.8 mg V/L for O. mykiss caused an overall increase in sodium influx in both the daphnids and rainbow trout. However, concentrations of V ranging between 0.2 and 4 mg V/L for D. magna and 1.8 and 6 mg V/L for O. mykiss reduced whole body sodium in both organisms and whole body calcium in O. mykiss. Concentrations above 3.6 mg V/L caused significant lipid peroxidation in the gills and liver of rainbow trout, while 1.9 mg V/L produced a substantial decrease in the fish gill GSH:GSSG ratio, but no change in the ratio between these thiols in the liver. Concentrations of 6.62 mg V/L sharply increased catalase activity in the liver but not in the gills. Neither liver nor gill superoxide dismutase was altered by V. Overall, results suggest that both ion imbalance and oxidative stress are part of the mechanism of toxicity of V in D. magna and O. mykiss and that further research is warranted to fully elucidate the mechanism(s) of V toxicity in aquatic organisms.

Growth of bulk GaN crystals

Perspectives about growth of bulk gallium nitride crystals, fabricating high structural quality gallium nitride wafers and the market demand for them are presented. Three basic crystal growth technologies, halide vapor phase epitaxy, sodium flux, and ammonothermal, are described. Their advantages and disadvantages, recent development, and possibilities are discussed. The main difficulty with crystallization of thick GaN is determined. Some new solutions for bulk growth are proposed. It is shown that only crystallization on high structural quality native seeds will ensure proper progress. New ideas for fabricating gallium nitride crystals and wafers with a better control of their structural properties and point defect concentration are proposed.

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice.

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol's critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

Pre-clinical to Clinical Translational Failures and Current Status of Clinical Trials in Stroke Therapy: A Brief Review

In stroke (cerebral ischemia), despite continuous efforts both at the experimental and clinical level, the only approved pharmacological treatment has been restricted to tissue plasminogen activator (tPA). Stroke is the leading cause of functional disability and mortality throughout worldwide. Its pathophysiology starts with energy pump failure, followed by complex signaling cascade that ultimately ends in neuronal cell death. Ischemic cascade involves excessive glutamate release followed by raised intracellular sodium and calcium influx along with free radicals’ generation, activation of inflammatory cytokines, NO synthases, lipases, endonucleases and other apoptotic pathways leading to cell edema and death. At the pre-clinical stage, several agents have been tried and proven as an effective neuroprotectant in animal models of ischemia. However, these agents failed to show convincing results in terms of efficacy and safety when the trials were conducted in humans following stroke. This article highlights the various agents which have been tried in the past but failed to translate into stroke therapy along with key points that are responsible for the lagging of experimental success to translational failure in stroke treatment.

Flow Cytometric Monitoring of Dynamic Cytosolic Calcium, Sodium, and Potassium Fluxes Following Platelet Activation.

The interaction of platelet agonists with their respective membrane receptors triggers intracellular signaling, among which cytosolic ion fluxes play an important role in activation processes. While the key contribution of intercellular free calcium is accepted, sodium and potassium roles in platelet activation have been less investigated in recent studies. Here, we implemented a novel flow-cytometric method to monitor over time cytosolic free calcium, sodium, and potassium ion fluxes upon platelet activation and we demonstrate the feasibility of real-time visualization of ion kinetics, in particular with a focus on sodium and potassium. Platelets were loaded with selective ion indicators, Fluo-3 (Ca2+ ), ION NaTRIUM Green-2 (Na+ ), and ION Potassium Green-2 (K+ ). Fluorescence was monitored by flow cytometry. After measurement of a stable baseline, platelets were activated and ion indicator fluorescence was acquired over time, up to 10 min. Platelets were activated with either thromboxane analogue U46619, ADP, thrombin, TRAP6 (PAR-1 agonist), AYPGKF (PAR-4 agonist), convulxin (collagen receptor GPVI agonist), or combinations thereof. We evaluated preanalytical parameters (in particular dye loading time and concentration) to implement an accurate method. Subsequently, we characterized cytosolic calcium, sodium, and potassium kinetics in response to platelet agonists. We observed different patterns of agonist synergism. In conclusion, the present work highlights the use of cytosolic ion monitoring by flow cytometry to investigate characteristic calcium, sodium, and potassium mobilization patterns following platelet activation. This easy technique opens a new way to analyze signaling in different platelet subpopulations and it should prove useful for investigating platelet pathophysiology. © 2020 International Society for Advancement of Cytometry.

Intercellular Sodium Regulates Repolarization in Cardiac Tissue with Sodium Channel Gain of Function

In cardiac myocytes, action potentials are initiated by an influx of sodium (Na+) ions via voltage-gated Na+ channels. Na+ channel gain of function (GOF), arising in both inherited conditions associated with mutation in the gene encoding the Na+ channel and acquired conditions associated with heart failure, ischemia, and atrial fibrillation, enhance Na+ influx, generating a late Na+ current that prolongs action potential duration (APD) and triggering proarrhythmic early afterdepolarizations (EADs). Recent studies have shown that Na+ channels are highly clustered at the myocyte intercalated disk, facilitating formation of Na+ nanodomains in the intercellular cleft between cells. Simulations from our group have recently predicted that narrowing the width of the intercellular cleft can suppress APD prolongation and EADs in the presence of Na+ channel mutations because of increased intercellular cleft Na+ ion depletion. In this study, we investigate the effects of modulating multiple extracellular spaces, specifically the intercellular cleft and bulk interstitial space, in a novel computational model and experimentally via osmotic agents albumin, dextran 70, and mannitol. We perform optical mapping and transmission electron microscopy in a drug-induced (sea anemone toxin, ATXII) Na+ channel GOF isolated heart model and modulate extracellular spaces via osmotic agents. Single-cell patch-clamp experiments confirmed that the osmotic agents individually do not enhance late Na+ current. Both experiments and simulations are consistent with the conclusion that intercellular cleft narrowing or expansion regulates APD prolongation; in contrast, modulating the bulk interstitial space has negligible effects on repolarization. Thus, we predict that intercellular cleft Na+ nanodomain formation and collapse critically regulates cardiac repolarization in the setting of Na+ channel GOF.