Intracellular Sodium Research Articles

Potassium deficiency decreases the capacity for urea synthesis and markedly increases ammonia in rats.

Our study provides novel findings of experimental hypokalemia reducing urea cycle functionality and thereby severely increasing plasma ammonia. This is pathophysiologically interesting because plasma ammonia increases during hypokalemia by a hitherto unknown mechanism, which may be particular important in relation to the unexplained link between hypokalemia and hepatic encephalopathy. Potassium deficiency decreases gene expression, protein synthesis, and growth. The urea cycle maintains body nitrogen homeostasis including removal of toxic ammonia. Hyperammonemia is an obligatory trait of liver failure, increasing the risk for hepatic encephalopathy, and hypokalemia is reported to increase ammonia. We aimed to clarify the effects of experimental hypokalemia on the in vivo capacity of the urea cycle, on the genes of the enzymes involved, and on ammonia concentrations. Female Wistar rats were fed a potassium-free diet for 13 days. Half of the rats were then potassium repleted. Both groups were compared with pair- and free-fed controls. The following were measured: in vivo capacity of urea-nitrogen synthesis (CUNS); gene expression (mRNA) of urea cycle enzymes; plasma potassium, sodium, and ammonia; intracellular potassium, sodium, and magnesium in liver, kidney, and muscle tissues; and liver sodium/potassium pumps. Liver histology was assessed. The diet induced hypokalemia of 1.9 ± 0.4 mmol/L. Compared with pair-fed controls, the in vivo CUNS was reduced by 34% (P < 0.01), gene expression of argininosuccinate synthetase 1 (ASS1) was decreased by 33% (P < 0.05), and plasma ammonia concentrations were eightfold elevated (P < 0.001). Kidney and muscle tissue potassium contents were markedly decreased but unchanged in liver tissue. Protein expressions of liver sodium/potassium pumps were unchanged. Repletion of potassium reverted all the changes. Hypokalemia decreased the capacity for urea synthesis via gene effects. The intervention led to marked hyperammonemia, quantitatively explainable by the compromised urea cycle. Our findings motivate clinical studies of patients with liver disease.

The Functional Properties, Physiological Roles, Channelopathy and Pharmacological Characteristics of the Slack (KCNT1) Channel.

The KCNT1 gene encodes the sodium-activated potassium channel that is abundantly expressed in the central nervous system of mammalians and plays an important role in reducing neuronal excitability. Structurally, the KCNT1 channel is absent of voltage sensor but possesses a long C-terminus including RCK1 and RCK2domain, to which the intracellular sodium and chloride bind to activate the channel. Recent publications using electron cryo-microscopy (cryo-EM) revealed the open and closed structural characteristics of the KCNT1 channel and co-assembly of functional domains. The activation of the KCNT1 channel regulates various physiological processes including nociceptive behavior, itch, spatial learning. Meanwhile, malfunction of this channel causes important pathophysiological consequences, including Fragile X syndrome and a wide spectrum of seizure disorders. This review comprehensively describes the structure, expression patterns, physiological functions of the KCNT1 channel and emphasizes the channelopathy of gain-of-function KCNT1 mutations in epilepsy.

Study and Classification of Cell Bio-Impedance Signature for Identification of Malignancy Using Artificial Neural Network

This article presents development of a microcapacitive sensor-based bio-impedance classification system for identification of malignant cells in a known volume (400 $\mu \text{L}$ ) of biological tissue sample. Malignancy induces various physical changes in an affected cell, among which increased intracellular water and sodium ion content are a couple of prominent ones. In this work, the change in electrical properties of a cell [here white blood cell (WBC)] that is membrane and cytoplasm permittivity due to afore-mentioned reasons is used for distinguishing malignant from normal cells based on their deviation in bio-impedance signature. This change in bio-impedance signature of the WBC is measured by exciting the mentioned microcapacitive sensor by a 2 V p-p signal. The developed system consists of an on-board impedance analyzer to carry out bio-impedance spectroscopy over a frequency range of 1–100 kHz. The bio-impedance data are then obtained from the sensor for different concentrations of cultured human malignant and normal white blood cells. The difference in impedance values between concentrations is then used as a discerning factor for identification the degree of malignancy, for which an artificial neural network (ANN) is employed. A detailed study considering detection efficiency for this ANN revealed classification accuracy up to 95.0%, upon which this ANN model is transferred to a portable single board computer (SBC) along with other associated circuitry that could carry out the same classification at a nominal power consumption of 516.45 mW.

Molecular Modification of Transient Receptor Potential Canonical 6 Channels Modulates Calcium Dyshomeostasis in a Mouse Model Relevant to Malignant Hyperthermia.

Pharmacologic modulation has previously shown that transient receptor potential canonical (TRPC) channels play an important role in the pathogenesis of malignant hyperthermia. This study tested the hypothesis that genetically suppressing the function of TRPC6 can partially ameliorate muscle cation dyshomeostasis and the response to halothane in a mouse model relevant to malignant hyperthermia. This study examined the effect of overexpressing a muscle-specific nonconducting dominant-negative TRPC6 channel in 20 RYR1-p.R163C and 20 wild-type mice and an equal number of nonexpressing controls, using calcium- and sodium-selective microelectrodes and Western blots. RYR1-p.R163C mouse muscles have chronically elevated intracellular calcium and sodium levels compared to wild-type muscles. Transgenic expression of the nonconducting TRPC6 channel reduced intracellular calcium from 331 ± 34 nM (mean ± SD) to 190 ± 27 nM (P < 0.0001) and sodium from 15 ± 1 mM to 11 ± 1 mM (P < 0.0001). Its expression lowered the increase in intracellular Ca2+ of the TRPC6-specific activator hyperforin in RYR1-p.R163C muscle fibers from 52% (348 ± 37 nM to 537 ± 70 nM) to 14% (185 ± 11 nM to 210 ± 44 nM). Western blot analysis of TRPC3 and TRPC6 expression showed the expected increase in TRPC6 caused by overexpression of its dominant-negative transgene and a compensatory increase in expression of TRPC3. Although expression of the muscle-specific dominant-negative TRPC6 was able to modulate the increase in intracellular calcium during halothane exposure and prolonged life (35 ± 5 min vs. 15 ± 3 min; P < 0.0001), a slow, steady increase in calcium began after 20 min of halothane exposure, which eventually led to death. These data support previous findings that TRPC channels play an important role in causing the intracellular calcium and sodium dyshomeostasis associated with RYR1 variants that are pathogenic for malignant hyperthermia. However, they also show that modulating TRPC channels alone is not sufficient to prevent the lethal effect of exposure to volatile anesthetic malignant hyperthermia-triggering agents.

Abstract 15315: SGLT2 Inhibitor Ertugliflozin Decreases Elevated Intracellular Sodium, Improves Myocardial Energetics and Enhances Function in Metabolic Heart Disease

Background: Sodium-glucose co-transporter 2 (SGLT2) inhibitors are antidiabetic drugs of great interest in cardiology due to their improvement of heart failure outcomes independent of diabetes. As SGLT2 is not expressed in cardiomyocytes, the mechanism of such benefit remains unclear. Elevated myocardial intracellular sodium [Na + ] i has been found in heart failure and SGLT2 inhibition lowers [Na + ] i in isolated cardiomyocytes. Elevated [Na + ] i was shown to decrease mitochondrial calcium via mitochondrial Na/Ca exchanger (NCx MITO ), resulting in decreased mitochondrial ATP synthesis. We have previously shown that mice fed a diet high in fat and sugar (HFHS) develop metabolic heart disease (MHD) characterized by decreased mitochondrial ATP synthesis with decreased phosphocreatine (PCr), worsened diastolic function and contractile reserve. We hypothesize that the SGLT2 inhibitor ertugliflozin (ERTU) decreases the elevated [Na + ] i to improve energetics and contractile function in MHD. Methods and Results: Isolated hearts from mice after 6 months of HFHS vs. control diet (CD), +/- ERTU in the last month, were studied using 31 P and 23 Na NMR spectroscopy to measure PCr/ATP ratio and [Na + ] i , respectively. As expected, HFHS hearts showed lower PCr/ATP, diastolic dysfunction (↑LVEDP) and lack of contractile reserve (↓RPP) during high work protocol compared to CD hearts. Myocardial [Na + ] i was elevated more than 2-fold in HFHS compared to CD. One month of ERTU treatment decreased [Na + ] i and improved energetics and contractile function in HFHS to levels similar to or better than CD. Perfusion with CGP 37157, which inhibits NCx MITO , improved PCr/ATP in HFHS hearts. Conclusion: Lowering of myocardial [Na + ] i by ertugliflozin contributes to improved energetics and function in MHD. These results suggest targeting [Na + ] i as an effective strategy to improve cardiac dysfunction in MHD and other forms of heart disease associated with elevated myocardial [Na + ] i.

Dehydrated Hereditary Stomatocytosis in a Multi-Generational American Family with a KCNN4 Gardos Channel Mutation

INTRODUCTION: Dehydrated hereditary stomatocytosis (DHSt) is an autosomal dominant red blood cell membrane disorder characterized by hemolytic anemia and splenomegaly. DHSt has an estimated incidence of 1:50,000 births, and the degree of anemia varies within and between families. Although transfusion support during childhood is not uncommon, continued requirement into adulthood is rare. The most frequent cause of DHSt is a gain-of-function mutation of the PIEZO1 gene, leading to delayed channel inactivation that results in a monovalent cation leak and an increase in intracellular calcium (Ca2+). Many of these patients develop recurrent thromboses post splenectomy. Other DHSt patients have mutations in KCNN4, which encodes the Gardos channel, with mutations causing increased Ca2+ sensitivity and potassium efflux. To our knowledge, 42 patients from ten families have been described with four distinct KCNN4 mutations: Arg352His, Val282Met or Val282Glu, and a 28bp deletion encompassing the exon-intron 7 junction. We report herein the eighth family with the Arg352His locus mutation. CASE REPORT: Five subjects from a single family were enrolled in this study (affected proband, unaffected husband, two affected children, and an unaffected grandchild). The proband has had hemolytic anemia since childhood. She had undergone splenectomy at age three and cholecystectomy at eight. Her anemia persisted, with a mean hemoglobin (Hgb) of 10.5g/dL and a reticulocyte count (retic ct) of 12.6%, with no need for transfusion support or iron chelation therapy (Table 1). Extensive testing revealed slightly decreased osmotic fragility and mildly elevated intracellular sodium concentration, of 19.4 mEq/L. Her daughter has mild splenomegaly and anemia, with a mean Hgb of 11.1g/dL and retic ct of 6.7%, whereas her son has more severe disease, with a mean Hgb of 9.3g/dL and a retic ct of &amp;gt;22%. He underwent splenectomy as a teenager for immune thrombocytopenia, and has required chelation therapy with deferasirox since age 31, when his ferritin rose to &amp;gt;1000ng/mL. Similar to results reported by others, splenectomy did not alter the severity of hemolysis in either the proband or her son, and neither developed thrombotic complications 57 and 7 years post splenectomy, respectively. The proband's daughter has a child with a normal Hgb, and is presumed unaffected. Stomatocytes were rarely seen on the peripheral blood smears of the proband and her children. METHODS: A clinically available 39 gene hemolytic anemia panel on the proband failed to identify the mutation underlying this disorder. We therefore performed whole exome sequencing on all five family members. We prioritized the analysis of 23 additional genes that are included in hemolytic anemia panels from two other reference laboratories and are involved in disorders of red blood cell membrane or cytoskeletal proteins of potential clinical relevance to the study population. RESULTS: A single missense mutation, Arg352His within KCNN4, was identified in all three affected individuals. This heterozygous mutation was present in the proband and her affected two children, and absent in her unaffected grandchild and husband. CONCLUSIONS: To our knowledge, this Pennsylvania family is only the eleventh described to have DHSt secondary to a KCNN4 mutation. This disorder is likely much more prevalent than reported, due to the rarity of stomatocytes on peripheral blood smears, the omission of the KCNN4 gene from hemolytic panels offered by some reference laboratories, and variable clinical presentation. KCNN4 mutations should be investigated if other causes are not identified in patients with lifelong hemolytic anemia suspected of having a red cell membrane protein or cytoskeletal disorder. Appropriate diagnosis may allow severely affected patients to be considered for treatment with the experimental Gardos channel inhibitor senicapoc. Additionally, mutational diagnosis is especially important when considering the adverse outcomes post splenectomy in PIEZO1 as compared to KCNN4 mutations. Disclosures Eyster: SPARK:Research Funding;Sanofi:Research Funding;Novo Nordisk:Research Funding;Baxalta/Shire:Research Funding.

Study on mechanism of Tibetan medicine Zuomua Decoction in treatment of hypertension based on network pharmacology and molecular docking technology

Hypertension is a kind of chronic cardiovascular system disease caused by a series of factors and carriers dysfunction, which belongs to the category of Tibetan medicine &quot;Chalong disease&quot;, and has a high rate of disability and mortality. Zuomua Decoction is a classical Tibetan medicine for Chalong disease, but its mechanism is not clear. Therefore, in this paper we explored the multi-components, multi-targets and multi-channels mechanism of Zuomua Decoction in the treatment of hypertension based on network pharmacology and molecular docking technology. First of all, the chemical components of Zuomua Decoction were obtained in the retrieval of traditional Chinese medicine systems pharmacology database(TCMSP), China National Knowledge Infrastructure(CNKI) and Wanfang database. The potential targets of Zuomua Decoction were predicted by BATMAN-TCM database, and the targets of hypertension were obtained by using DisGeNET database. The intersection of these two targets set was taken to obtain the potential targets of Zuomua Decoction in the treatment of hypertension, and then the chemical compositions-targets network was constructed. Secondly, the intersection targets were imported into STRING database to obtain the interaction relationship of intersection targets, and the protein interaction network of Zuomua Decoction in the treatment of hypertension was constructed in Cytoscape. Topological, GO, and KEGG enrichment analysis were used to construct the key targets-signal pathways-biological processes network diagram and explore the mechanism of Zuomua Decoction in the treatment of hypertension. Finally, the key targets were selected to construct the pharmacodynamic identification models to verify the effect mode of Zomua Decoction in treating hypertension. The results showed that there were 61 chemical components and 90 potential targets in the compounds-targets network. We obtained 21 key targets, 154 signal pathways, and 382 biological processes in topological, GO, and KEGG enrichment analysis of the protein interaction network, and in the comprehensive analysis, it was found that Zuomua Decoction could reduce blood pressure by regulating renin angiotension aldosterone system, balancing the concentration of intracellular calcium and sodium ions and regulating vasoconstriction and relaxation. ACE, AGTR1, and ADRB2 were used as the carriers for molecular docking study on the components of Zuoma Decoction, and the results showed that the chemical components of Zuomua Decoction had a good binding activity with key targets. The purpose of this study is to provide ideas for the in-depth study of Zuoma Decoction in the treatment of hypertension, and provide scientific basis for its clinical rational application.

Cellular mechanisms of early tachycardia-induced ventricular dysfunction in the human heart

Abstract Background Tachycardia-induced cardiomyopathy (TCM) is a reversible form of ventricular dysfunction caused by persistent tachycardia. Characterization of TCM is mainly based on artificially RV paced animal models. Moreover, the underlying mechanisms and time course from compensation to failure remain unclear. This study aimed to investigate early cellular remodeling of tachycardia-induced myocardial dysfunction in human myocardium. Methods and results To elucidate early cellular electrophysiological targets mediating the transition to TCM, we chronically paced (120bpm vs 60bpm control) human induced pluripotent stem cell cardiomyocytes (hiPS-CM) for up to 7d. As a major substrate of cellular myocardial dysfunction, we investigated the influence of chronic tachycardia on cellular Ca cycling. After 24h of persistent tachycardia we detected a significant decrease in Ca transient (CaT) amplitude and reduced diastolic Ca levels (Fura-2). Meanwhile, Ca elimination time (RT80) was unchanged compared to control (n=44/42 cells / 8 diff.). Caffeine application was performed to evaluate sarcoplasmic reticulum (SR) Ca load. We found a shortening of caffeine-induced CaT relaxation time, whereas SR Ca load was unchanged (n=12/13 /8). Further illustrating the transition to TCM, CaT amplitude was progressively decreased after 7d of chronic tachycardia. In contrast to 24h of tachycardia, 7d persistent stimulation resulted in slowed relaxation (RT80, n=75/65 /7). These findings could be explained by a significant reduction of SERCA activity (Ksys-Kcaff) and SR Ca load (n=14/12 / 7). Diastolic Ca concentration remained reduced (n=75/65 /7), in total suggesting a shift to transsarcolemmal Ca elimination. Sodium measurements (SBFI) revealed a significant increase of intracellular sodium concentration (n=69/69 /5) after 7d of tachycardia. In patch clamp experiments we detected a prolongation of action potential duration as early as 24h after onset of tachycardia (n=26/21 /4), which persisted throughout 7d of pacing (n=8/12 /3). Resting membrane potential and action potential amplitude were not changed. Finally, we investigated tachycardia-mediated effects on pre-existing human heart failure (HF). 8h tachycardic stimulation (120bpm) of human HF ventricular trabeculae compromised systolic force, while diastolic tension and relaxation time were markedly increased compared to control (60bpm) (n=7/6 trabeculae /6 human hearts). The extensive molecular characterization of involved ion channels and pathways mediating transition to TCM is currently under investigation. Conclusion This study demonstrates that a persistent tachycardia adversely alters cardiomyocyte excitation-contraction coupling via early electrophysiological cellular remodeling. In pre-existing HF persistent tachycardia strongly aggravates ventricular dysfunction. Our first translational investigation in human myocardium may help to understand the pathophysiology of an underrated and very prevalent disease. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Else-Kröner-Fresenius-Stiftung

Cardioprotective actions of the sodium-activated potassium channel Slack (aka Slo2.2, KNa1.1 or KCNT1)

Abstract Background Sodium-activated potassium channels (KNa) Slack (sequence like a calcium-activated potassium channel, aka Slo 2.2, KNa1.1 or KCNT1) are widely expressed throughout neuronal tissue, whereas their presence and function in the cardiovascular system is not well understood. Due to high intracellular sodium concentrations ([Na+]i) necessary to induce half-maximal channel activation, we hypothesized that Slack function is attributed to pathophysiological conditions such as myocardial ischemia. Purpose To elucidate the putative functions of Slack in the murine heart and in cardiomyocytes (CMs) and to explore whether the ischemia and reperfusion (I/R)-induced cardiac damage is affected by endogenous Slack channel activity. Methods I/R injury was evaluated in global and CM-specific Slack knockout mice (Slack gKO, CM Slack KO) and compared to litter-matched controls (Slack gWT, CM Slack CTR) by applying an in vivo model of acute myocardial infarction (MI). Infarct size (IS) was assessed at baseline, after ischemic pre- (iPre) and postconditioning (iPost) and in response to cinaciguat (CIN), a cGMP-elevating agent. Moreover, Slack expression and function in CMs was studied by biochemical and electrophysiological means and by utilizing the newly developed FRET-based K+ probe GEPII 1.0. Results IS in Slack gKO mice was increased in comparison to gWT littermates. In addition, the cardioprotection afforded by iPost was attenuated in the absence of Slack. To test if the increased vulnerability to I/R injury of the Slack gKO mouse model was originating from Slack activity in CMs, we subjected CM-specific Slack CTR and KO mutants to an identical MI procedure. IS measurements confirmed increased cardiac damage at baseline and reduced cardioprotective effects afforded by iPre and iPost in CM Slack KO mice. Interestingly, CIN (i.p., 30 min prior to I/R) reduced IS to a similar extent in both genotypes, suggesting that Slack functions in a cGMP-independent manner. Whole-cell patch clamp experiments on CMs demonstrated a reduction of the KNa-inhibitors clofilium- and chinidine-sensitive K+ outward currents in Slack gKO CM. Extracellular potassium ([K+]ex) accumulation measured with GEPII 1.0 was lower in Slack gKO versus gWT CM pools exposed to membrane permeabilizing agent digitonin. Accordingly, [K+]ex evoked by the Slack activators niclosamide and bithionol was lower in the absence of functional Slack in CMs. Conclusion The presented findings establish an important role of Slack channels for cardioprotective signalling mechanisms during I/R in vivo and for mediating beneficial effects of mechanical conditioning on IS. Corroborating in vitro studies on adult CMs exhibit an impaired [K+]ex dynamic in response to genetic or pharmacological modulation of Slack activity. Thus, we conclude that Slack-dependent K+ signalling pathways in CMs may represent a promising drug target that renders the heart muscle less vulnerable to the I/R-induced damage. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Work in the authors' laboratories is supported by grants from the Deutsche Forschungsgemeinschaft (DFG) (to R.L.) and the DFG Research Unit 2060, “cGMP Signaling in Cell Growth and Survival” (to R.L. and P.R.).

Structure and elevator mechanism of the mammalian sodium/proton exchanger NHE9

Na+/H+ exchangers (NHEs) are ancient membrane‐bound nanomachines that work to regulate intracellular pH, sodium levels and cell volume. NHE activities contribute to the control of the cell cycle, cell proliferation, cell migration and vesicle trafficking. NHE dysfunction has been linked to many diseases, and they are targets of pharmaceutical drugs. Despite their fundamental importance to cell homeostasis and human physiology, structural information for the mammalian NHEs was lacking. Here, we report the cryogenic electron microscopy structure of NHE isoform 9 (SLC9A9) from Equus caballus at 3.2 Å resolution, an endosomal isoform highly expressed in the brain and associated with autism spectrum (ASD) and attention deficit hyperactivity (ADHD) disorders. Despite low sequence identity, the NHE9 architecture and ion‐binding site are remarkably most similar to distantly related bacterial Na+/H+ antiporters with 13 transmembrane segments. Collectively, we reveal the conserved architecture of the NHE ion‐binding site, their elevator‐like structural transitions, the functional implications of autism disease mutations and the role of phosphoinositide lipids to promote homodimerization that, together, have important physiological ramifications.

Intracellular Sodium Changes in Cancer Cells Using a Microcavity Array-Based Bioreactor System and Sodium Triple-Quantum MR Signal

The sodium triple-quantum (TQ) magnetic resonance (MR) signal created by interactions of sodium ions with macromolecules has been demonstrated to be a valuable biomarker for cell viability. The aim of this study was to monitor a cellular response using the sodium TQ signal during inhibition of Na/K-ATPase in living cancer cells (HepG2). The cells were dynamically investigated after exposure to 1 mM ouabain or K+-free medium for 60 min using an MR-compatible bioreactor system. An improved TQ time proportional phase incrementation (TQTPPI) pulse sequence with almost four times TQ signal-to-noise ratio (SNR) gain allowed for conducting experiments with 12–14 × 106 cells using a 9.4 T MR scanner. During cell intervention experiments, the sodium TQ signal increased to 138.9 ± 4.1% and 183.4 ± 8.9% for 1 mM ouabain (n = 3) and K+-free medium (n = 3), respectively. During reperfusion with normal medium, the sodium TQ signal further increased to 169.2 ± 5.3% for the ouabain experiment, while it recovered to 128.5 ± 6.8% for the K+-free experiment. These sodium TQ signal increases agree with an influx of sodium ions during Na/K-ATPase inhibition and hence a reduced cell viability. The improved TQ signal detection combined with this MR-compatible bioreactor system provides a capability to investigate the cellular response of a variety of cells using the sodium TQ MR signal.

Viscoelasticity and Volume of Cortical Neurons under Glutamate Excitotoxicity and Osmotic Challenges

Neural activity depends on the maintenance of ionic and osmotic homeostasis. Under these conditions, the cell volume must be regulated to maintain optimal neural function. A disturbance in the neuronal volume regulation often occurs in pathological conditions such as glutamate excitotoxicity. The cell volume, mechanical properties, and actin cytoskeleton structure are tightly connected to achieve the cell homeostasis. Here, we studied the effects of glutamate-induced excitotoxicity, external osmotic pressure, and inhibition of actin polymerization on the viscoelastic properties and volume of neurons. Atomic force microscopy was used to map the viscoelastic properties of neurons in time-series experiments to observe the dynamical changes and a possible recovery. The data obtained on cultured rat cortical neurons were compared with the data obtained on rat fibroblasts. The neurons were found to be more responsive to the osmotic challenges but less sensitive to the inhibition of actin polymerization than fibroblasts. The alterations of the viscoelastic properties caused by glutamate excitotoxicity were similar to those induced by the hypoosmotic stress, but, in contrast to the latter, they did not recover after the glutamate removal. These data were consistent with the dynamic volume changes estimated using ratiometric fluorescent dyes. The recovery after the glutamate-induced excitotoxicity was slow or absent because of a steady increase in intracellular calcium and sodium concentrations. The viscoelastic parameters and their changes were related to such parameters as the actin cortex stiffness, tension, and cytoplasmic viscosity.

Role of the Intracellular Sodium Homeostasis in Chemotaxis of Activated Murine Neutrophils.

The importance of the intracellular Ca2+ concentration ([Ca2+]i) in neutrophil function has been intensely studied. However, the role of the intracellular Na+ concentration ([Na+]i) which is closely linked to the intracellular Ca2+ regulation has been largely overlooked. The [Na+]i is regulated by Na+ transport proteins such as the Na+/Ca2+-exchanger (NCX1), Na+/K+-ATPase, and Na+-permeable, transient receptor potential melastatin 2 (TRPM2) channel. Stimulating with either N-formylmethionine-leucyl-phenylalanine (fMLF) or complement protein C5a causes distinct changes of the [Na+]i. fMLF induces a sustained increase of [Na+]i, surprisingly, reaching higher values in TRPM2−/− neutrophils. This outcome is unexpected and remains unexplained. In both genotypes, C5a elicits only a transient rise of the [Na+]i. The difference in [Na+]i measured at t = 10 min after stimulation is inversely related to neutrophil chemotaxis. Neutrophil chemotaxis is more efficient in C5a than in an fMLF gradient. Moreover, lowering the extracellular Na+ concentration from 140 to 72 mM improves chemotaxis of WT but not of TRPM2−/− neutrophils. Increasing the [Na+]i by inhibiting the Na+/K+-ATPase results in disrupted chemotaxis. This is most likely due to the impact of the altered Na+ homeostasis and presumably NCX1 function whose expression was shown by means of qPCR and which critically relies on proper extra- to intracellular Na+ concentration gradients. Increasing the [Na+]i by a few mmol/l may suffice to switch its transport mode from forward (Ca2+-efflux) to reverse (Ca2+-influx) mode. The role of NCX1 in neutrophil chemotaxis is corroborated by its blocker, which also causes a complete inhibition of chemotaxis.

Frontiers of Sodium MRI Revisited: From Cartilage to Brain Imaging.

Sodium magnetic resonance imaging (23Na‐MRI) is a highly promising imaging modality that offers the possibility to noninvasively quantify sodium content in the tissue, one of the most relevant parameters for biochemical investigations. Despite its great potential, due to the intrinsically low signal‐to‐noise ratio (SNR) of sodium imaging generated by low in vivo sodium concentrations, low gyromagnetic ratio, and substantially shorter relaxation times than for proton (1H) imaging, 23Na‐MRI is extremely challenging. In this article, we aim to provide a comprehensive overview of the literature that has been published in the last 10–15 years and which has demonstrated different technical designs for a range of 23Na‐MRI methods applicable for disease diagnoses and treatment efficacy evaluations. Currently, a wider use of 3.0T and 7.0T systems provide imaging with the expected increase in SNR and, consequently, an increased image resolution and a reduced scanning time. A great interest in translational research has enlarged the field of sodium MRI applications to almost all parts of the body: articular cartilage tendons, spine, heart, breast, muscle, kidney, and brain, etc., and several pathological conditions, such as tumors, neurological and degenerative diseases, and others. The quantitative parameter, tissue sodium concentration, which reflects changes in intracellular sodium concentration, extracellular sodium concentration, and intra–/extracellular volume fractions is becoming acknowledged as a reliable biomarker. Although the great potential of this technique is evident, there must be steady technical development for 23Na‐MRI to become a standard imaging tool. The future role of sodium imaging is not to be considered as an alternative to 1H MRI, but to provide early, diagnostically valuable information about altered metabolism or tissue function associated with disease genesis and progression.Level of Evidence1Technical Efficacy Stage1

Sodium Fluctuations in Astroglia and Their Potential Impact on Astrocyte Function.

Astrocytes are the main cell type responsible for the regulation of brain homeostasis, including the maintenance of ion gradients and neurotransmitter clearance. These processes are tightly coupled to changes in the intracellular sodium (Na+) concentration. While activation of the sodium-potassium-ATPase (NKA) in response to an elevation of extracellular K+ may decrease intracellular Na+, the cotransport of transmitters, such as glutamate, together with Na+ results in an increase in astrocytic Na+. This increase in intracellular Na+ can modulate, for instance, metabolic downstream pathways. Thereby, astrocytes are capable to react on a fast time scale to surrounding neuronal activity via intracellular Na+ fluctuations and adjust energy production to the demand of their environment. Beside the well-documented conventional roles of Na+ signaling mainly mediated through changes in its electrochemical gradient, several recent studies have identified more atypical roles for Na+, including protein interactions leading to changes in their biochemical activity or Na+-dependent regulation of gene expression. In this review, we will address both the conventional as well as the atypical functions of astrocytic Na+ signaling, presenting the role of transporters and channels involved and their implications for physiological processes in the central nervous system (CNS). We will also discuss how these important functions are affected under pathological conditions, including stroke and migraine. We postulate that Na+ is an essential player not only in the maintenance of homeostatic processes but also as a messenger for the fast communication between neurons and astrocytes, adjusting the functional properties of various cellular interaction partners to the needs of the surrounding network.

Functional modulation of atrio-ventricular conduction by enhanced late sodium current and calcium-dependent mechanisms in Scn5a1798insD/+ mice.

Aims SCN5A mutations are associated with arrhythmia syndromes, including Brugada syndrome, long QT syndrome type 3 (LQT3), and cardiac conduction disease. Long QT syndrome type 3 patients display atrio-ventricular (AV) conduction slowing which may contribute to arrhythmogenesis. We here investigated the as yet unknown underlying mechanisms.Methods and resultsWe assessed electrophysiological and molecular alterations underlying AV-conduction abnormalities in mice carrying the Scn5a1798insD/+ mutation. Langendorff-perfused Scn5a1798insD/+ hearts showed prolonged AV-conduction compared to wild type (WT) without changes in atrial and His-ventricular (HV) conduction. The late sodium current (INa,L) inhibitor ranolazine (RAN) normalized AV-conduction in Scn5a1798insD/+ mice, likely by preventing the mutation-induced increase in intracellular sodium ([Na+]i) and calcium ([Ca2+]i) concentrations. Indeed, further enhancement of [Na+]i and [Ca2+]i by the Na+/K+-ATPase inhibitor ouabain caused excessive increase in AV-conduction time in Scn5a1798insD/+ hearts. Scn5a1798insD/+ mice from the 129P2 strain displayed more severe AV-conduction abnormalities than FVB/N-Scn5a1798insD/+ mice, in line with their larger mutation-induced INa,L. Transverse aortic constriction (TAC) caused excessive prolongation of AV-conduction in FVB/N-Scn5a1798insD/+ mice (while HV-intervals remained unchanged), which was prevented by chronic RAN treatment. Scn5a1798insD/+-TAC hearts showed decreased mRNA levels of conduction genes in the AV-nodal region, but no structural changes in the AV-node or His bundle. In Scn5a1798insD/+-TAC mice deficient for the transcription factor Nfatc2 (effector of the calcium-calcineurin pathway), AV-conduction and conduction gene expression were restored to WT levels.ConclusionsOur findings indicate a detrimental role for enhanced INa,L and consequent calcium dysregulation on AV-conduction in Scn5a1798insD/+ mice, providing evidence for a functional mechanism underlying AV-conduction disturbances secondary to gain-of-function SCN5A mutations.

Relaxometry and quantification in sodium MRI of cerebral gliomas: A FET-PET and MRI small-scale study.

Sodium MRI is a promising method for assessing the metabolic properties of brain tumours. In a recent study, a strong relationship between semi-quantitative abnormalities in sodium MRI and the mutational status of the isocitrate dehydrogenase enzyme (IDH) with untreated cerebral gliomas was observed. Here, sodium relaxometry in brain tumour tissue was investigated in relation to molecular markers in order to reveal quantitative sodium tissue parameters and the differences between healthy tissue and brain tumour. The previous semi-quantitative approach is extended by use of suitable relaxometry methods accompanied by numerical simulation to achieve detailed quantitative analysis of intra- and extracellular sodium concentration using an enhanced SISTINA sequence at 4 T. Using optimised techniques, biexponential sodium relaxation times in tumour (T*2f , T*2s ) and in healthy contralateral brain tissue (T*2f,CL , T*2s,CL ) were estimated in 10 patients, along with intracellular sodium molar fractions (χ, χCL ), volume fractions (η, ηCL ) and concentrations (ρin , ρin,CL ). The total sodium tissue concentrations (ρT , ρT,CL ) were also estimated. The ratios T*2f /T*2f,CL (P = .05), η/ηCL (P = .02) and χ/χCL (P = .02) were significantly lower in IDH mutated than in IDH wildtype gliomas (n = 4 and n = 5 patients, respectively). The Wilcoxon rank-sum test was used to compare sodium MRI parameters in patients with and without IDH mutation. Thus, quantitative analysis of relaxation rates, intra- and extracellular sodium concentrations, intracellular molar and volume fractions based on enhanced SISTINA confirmed a relationship between abnormalities in sodium parameters and the IDH mutational status in cerebral gliomas, hence catering for the potential to provide further insights into the status of the disease.

Epicortical Brevetoxin Treatment Promotes Neural Repair and Functional Recovery after Ischemic Stroke

Emerging literature suggests that after a stroke, the peri-infarct region exhibits dynamic changes in excitability. In rodent stroke models, treatments that enhance excitability in the peri-infarct cerebral cortex promote motor recovery. This increase in cortical excitability and plasticity is opposed by increases in tonic GABAergic inhibition in the peri-infarct zone beginning three days after a stroke in a mouse model. Maintenance of a favorable excitatory–inhibitory balance promoting cerebrocortical excitability could potentially improve recovery. Brevetoxin-2 (PbTx-2) is a voltage-gated sodium channel (VGSC) gating modifier that increases intracellular sodium ([Na+]i), upregulates N-methyl-D-aspartate receptor (NMDAR) channel activity and engages downstream calcium (Ca2+) signaling pathways. In immature cerebrocortical neurons, PbTx-2 promoted neuronal structural plasticity by increasing neurite outgrowth, dendritogenesis and synaptogenesis. We hypothesized that PbTx-2 may promote excitability and structural remodeling in the peri-infarct region, leading to improved functional outcomes following a stroke. We tested this hypothesis using epicortical application of PbTx-2 after a photothrombotic stroke in mice. We show that PbTx-2 enhanced the dendritic arborization and synapse density of cortical layer V pyramidal neurons in the peri-infarct cortex. PbTx-2 also produced a robust improvement of motor recovery. These results suggest a novel pharmacologic approach to mimic activity-dependent recovery from stroke.

Intracellular ion concentrations and cation-dependent remodelling of bacterial MreB assemblies

Here, we measured the concentrations of several ions in cultivated Gram-negative and Gram-positive bacteria, and analyzed their effects on polymer formation by the actin homologue MreB. We measured potassium, sodium, chloride, calcium and magnesium ion concentrations in Leptospira interrogans, Bacillus subtilis and Escherichia coli. Intracellular ionic strength contributed from these ions varied within the 130–273 mM range. The intracellular sodium ion concentration range was between 122 and 296 mM and the potassium ion concentration range was 5 and 38 mM. However, the levels were significantly influenced by extracellular ion levels. L. interrogans, Rickettsia rickettsii and E. coli MreBs were heterologously expressed and purified from E. coli using a novel filtration method to prepare MreB polymers. The structures and stability of Alexa-488 labeled MreB polymers, under varying ionic strength conditions, were investigated by confocal microscopy and MreB polymerization rates were assessed by measuring light scattering. MreB polymerization was fastest in the presence of monovalent cations in the 200–300 mM range. MreB filaments showed high stability in this concentration range and formed large assemblies of tape-like bundles that transformed to extensive sheets at higher ionic strengths. Changing the calcium concentration from 0.2 to 0 mM and then to 2 mM initialized rapid remodelling of MreB polymers.

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.