Intracellular Sodium Research Articles

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.

Isolation and characterization of Salt Overly Sensitive family genes in spinach

The Salt Overly Sensitive (SOS) pathway regulates intracellular sodium ion homeostasis as a salt-stress response in plants. This pathway involves three main genes designated as SOS1, SOS2 and SOS3, which are members of the Na+ /H+ exchanger (NHX), CBL-interacting protein kinase (CIPK) and Calcineurin B-like (CBL) gene families, respectively. To identify and characterize SOS genes in spinach (Spinacia oleracea), a species of the Amaranthaceae family, we conducted genome-wide identification and phylogenetic analyses of NHX, CIPK and CBL genes from four Amaranthaceae species, Arabidopsis and rice. Most Amaranthaceae genes exhibited orthologous relationships with Arabidopsis and/or rice, except a clade of Vac-type Amaranthaceae NHX genes. Phylogenetic analyses also revealed gene gain/loss events in Amaranthaceae species and the intron-less to intron-rich evolution of CIPK genes. A bacterial protein-rooted CIPK tree allowed naming most of the phylogenetic clades based on their evolutionary history. Single S. oleracea (So) SOS1, SOS2 and SOS3 proteins were identified. Direct protein-protein interaction was observed between SoSOS2 and SoSOS3 but not between SoSOS2 and SoSOS1 based on yeast two-hybrid assay. This may suggest distinct modes of action of spinach SOS proteins compared to Arabidopsis SOS proteins. Unlike SoSOS1 and SoSOS2, which were expressed at similar or higher levels in leaves than roots, SoSOS3 expression was significantly higher in roots than leaves, suggesting its greater importance in roots. The expression of SoSOS3 was upregulated in both roots and leaves under salinity compared to the control; however, SoSOS1 was only upregulated in roots. Thus, this study demonstrated the conservation of SOS pathway genes in spinach and also highlighted the complexity of SOS signaling in Amaranthaceae species.

Transient Receptor Potential Cation Channels and Calcium Dyshomeostasis in a Mouse Model Relevant to Malignant Hyperthermia.

Until recently, the mechanism for the malignant hyperthermia crisis has been attributed solely to sustained massive Ca release from the sarcoplasmic reticulum on exposure to triggering agents. This study tested the hypothesis that transient receptor potential cation (TRPC) channels are important contributors to the Ca dyshomeostasis in a mouse model relevant to malignant hyperthermia. This study examined the mechanisms responsible for Ca dyshomeostasis in RYR1-p.G2435R mouse muscles and muscle cells using calcium and sodium ion selective microelectrodes, manganese quench of Fura2 fluorescence, and Western blots. RYR1-p.G2435R mouse muscle cells have chronically elevated intracellular resting calcium and sodium and rate of manganese quench (homozygous greater than heterozygous) compared with wild-type muscles. After exposure to 1-oleoyl-2-acetyl-sn-glycerol, a TRPC3/6 activator, increases in intracellular resting calcium/sodium were significantly greater in RYR1-p.G2435R muscles (from 153 ± 11 nM/10 ± 0.5 mM to 304 ± 45 nM/14.2 ± 0.7 mM in heterozygotes P < 0.001] and from 251 ± 25 nM/13.9 ± 0.5 mM to 534 ± 64 nM/20.9 ± 1.5 mM in homozygotes [P < 0.001] compared with 123 ± 3 nM/8 ± 0.1 mM to 196 ± 27 nM/9.4 ± 0.7 mM in wild type). These increases were inhibited both by simply removing extracellular Ca and by exposure to either a nonspecific (gadolinium) or a newly available, more specific pharmacologic agent (SAR7334) to block TRPC6- and TRPC3-mediated cation influx into cells. Furthermore, local pretreatment with SAR7334 partially decreased the elevation of intracellular resting calcium that is seen in RYR1-p.G2435R muscles during exposure to halothane. Western blot analysis showed that expression of TRPC3 and TRPC6 were significantly increased in RYR1-p.G2435R muscles in a gene-dose-dependent manner, supporting their being a primary molecular basis for increased sarcolemmal cation influx. Muscle cells in knock-in mice expressing the RYR1-p.G2435R mutation are hypersensitive to TRPC3/6 activators. This hypersensitivity can be negated with pharmacologic agents that block TRPC3/6 activity. This reinforces the working hypothesis that transient receptor potential cation channels play a critical role in causing intracellular calcium and sodium overload in malignant hyperthermia-susceptible muscle, both at rest and during the malignant hyperthermia crisis.

Fluorometric Na+ Evaluation in Single Cells Using Flow Cytometry: Comparison with Flame Emission Assay.

Sodium is a key playerin the fundamental cell functions. Fluorescent probes are indispensable tools for monitoring intracellular sodium levels in single living cells. Since the fluorescence of sodium-sensitive dyes in cells is significantly different from that in an aqueous solution, the fluorescence signal is calibrated in situ indirectly using ionophores for equalizing external and intracellular ion concentration. Attempts to compare data obtained using fluorescent probes and by direct flame emission analysis are sparse and results are inaccurate. We determined the intracellular sodium concentration in U937 cells by flow cytometry using the Na+-sensitive probe Asante Natrium Green-2 (ANG), and by standard flame emission photometry combined with the cellular water determination by cell density in Percoll gradient. The intracellular Na+concentrations was modifiedusing known ionophores or, alternatively, by blocking the sodium pump with ouabain or by causing cell apoptosis with staurosporine. It is revealed that both methods are comparable when intracellular sodium concentration was modified by ouabain-mediated blockage of the sodium pump or staurosporine-induced apoptosis. The ANG fluorescence of cells treated with ionophores is approximately two times lower than that in cells with the same Na+ concentration but not treated with ionophores. Although the mechanism is still unknown, this effect should be taken into account when a quantitative assessment of the concentration of intracellular sodium is required. The sodium sensitive dye ANG-2 is a sensitive and useful probe for determination changes in Na+ content and concentration both in single cells and subcellular microparticles. The ANG fluorescence determined in the studied cells in the absence of ionophores, cannot be used as a measure of the real intracellular concentration of Na+ if calibration was carried out in the presence of ionophores.

Heritable arrhythmias associated with abnormal function of cardiac potassium channels.

Cardiomyocytes express a surprisingly large number of potassium channel types. The primary physiological functions of the currents conducted by these channels are to maintain the resting membrane potential and mediate action potential repolarization under basal conditions and in response to changes in the concentrations of intracellular sodium, calcium, and ATP/ADP. Here, we review the diversity and functional roles of cardiac potassium channels under normal conditions and how heritable mutations in the genes encoding these channels can lead to distinct arrhythmias. We briefly review atrial fibrillation and J-wave syndromes. For long and short QT syndromes, we describe their genetic basis, clinical manifestation, risk stratification, traditional and novel therapeutic approaches, as well as insights into disease mechanisms provided by animal and cellular models.

Silver nanoparticles and silver ions cause inflammatory response through induction of cell necrosis and the release of mitochondria in vivo and in vitro.

Owing to the excellent antibacterial and antiviral activity, silver nanoparticles have a widespread use in the food and pharmaceutical industries. With the increase in the production and use of the related products, the potential hazard of silver nanoparticles has aroused public attention. The main purpose of this study is to explore the toxicity of silver nanoparticles and induction of lung inflammation in vitro and in vivo. Here, we validated that small amounts of silver ions dissolved from silver nanoparticles caused the depolarization of plasma membrane, resulting in an overload of intracellular sodium and calcium, and eventually led to the cell necrosis. The blockers of calcium or sodium channels inversed the toxicity of silver ions. Then, we instilled silver nanoparticles or silver nitrate (50μg per mouse) into the lungs of mice, and this induced pulmonary injury and mitochondrial content release, led to the recruitment of neutrophils to the lung tissue via p38 MAPK pathway. Altogether, these data show that released silver ions from nanoparticles induced cell necrosis through Na+ and Ca2+ influx and triggered pulmonary inflammation through elevating mitochondrial-related contents released from these necrotic cells.

Evidence for macromolecular crowding as a direct apoptotic stimulus.

Potassium loss and persistent shrinkage have both been implicated in apoptosis but their relationship and respective roles remain controversial. We approached this problem by clamping intracellular sodium and potassium in HeLa or MDCK cells using a combination of ionophores. Although ionophore treatment caused significant cell swelling, the initial volume could be restored and further reduced by application of sucrose. The swollen cells treated with ionophores remained viable for at least 8 h without any signs of apoptosis. Application of sucrose and the resulting shrinkage caused volume-dependent intrinsic apoptosis with all its classical features: inversion of phosphatidylserine, caspase activation and Bcl-2-dependent release of cytochrome c from mitochondria. In other experiments, apoptosis was induced by addition of the protein kinase inhibitor staurosporine at various degrees of swelling. Our results show that: (1) persistent shrinkage can cause apoptosis regardless of intracellular sodium or potassium composition or of the state of actin cytoskeleton; (2) strong potassium dependence of caspase activation is only observed in swollen cells with a reduced density of cytosolic proteins. We conclude that macromolecular crowding can be an important factor in determining the transition of cells to apoptosis.

The nature of cell volume sensor in shrinkage‐induced apoptosis

Potassium loss and persistent shrinkage have been both implicated in apoptosis, but their relationship and respective roles remain obscure. We approached this problem by clamping intracellular sodium and potassium in HeLa, MDBK or MDCK cells by a combination of ionophores and ouabain. Although ionophores cause significant cell swelling, the initial volume can be restored and further reduced by applying external sucrose. We showed that swollen cells treated with ionophores remain viable for at least 4–8 hours without developing any signs of apoptosis. Application of sucrose and the resulting shrinkage caused volume‐dependent intrinsic apoptosis with all its classical features: inversion of phosphatidylserine, caspase activation, cytochrome c release from mitochondria. Furthermore, overexpression of Bcl‐2 prevents mitochondrial loss of cytochrome c in shrunken cells. All these responses occurred to approximately the same degree regardless of whether the cells were kept in a high potassium/zero sodium or in a high sodium/zero potassium buffer, with only a slight enhancement under potassium‐free conditions. However, apoptosis induced by protein kinase inhibitor staurosporine in ionophore‐treated and swollen cells was strongly inhibited by as little as 5 mM potassium. Addition of 50 mM sucrose to restore the initial cell volume eliminated potassium dependence of staurosporine‐induced caspase activation. We have eliminated potassium as a stimulus in shrinkage induced apoptosis and are left with the choice between macromolecular crowding and cytoskeleton, which according to our unconfirmed preliminary data may be cytoskeleton. Thus, our results show that: (1) persistent shrinkage (presumably mediated through macromolecular crowding) acts as a direct proapoptotic signal independently of potassium; (2) strong potassium dependence of caspase activation is only observed in swollen cells with reduced cytoplasmic density.

23 Na-MRI as a Noninvasive Biomarker for Cancer Diagnosis and Prognosis.

The influx of sodium (Na+) ions into a resting cell is regulated by Na+ channels and by Na+/H+ and Na+/Ca2+ exchangers, whereas Na+ ion efflux is mediated by the activity of Na+/K+‐ATPase to maintain a high transmembrane Na+ ion gradient. Dysfunction of this system leads to changes in the intracellular sodium concentration that promotes cancer metastasis by mediating invasion and migration. In addition, the accumulation of extracellular Na+ ions in cancer due to inflammation contributes to tumor immunogenicity. Thus, alterations in the Na+ ion concentration may potentially be used as a biomarker for malignant tumor diagnosis and prognosis. However, current limitations in detection technology and a complex tumor microenvironment present significant challenges for the in vivo assessment of Na+ concentration in tumor. 23Na‐magnetic resonance imaging (23Na‐MRI) offers a unique opportunity to study the effects of Na+ ion concentration changes in cancer. Although challenged by a low signal‐to‐noise ratio, the development of ultrahigh magnetic field scanners and specialized sodium acquisition sequences has significantly advanced 23Na‐MRI. 23Na‐MRI provides biochemical information that reflects cell viability, structural integrity, and energy metabolism, and has been shown to reveal rapid treatment response at the molecular level before morphological changes occur. Here we review the basis of 23Na‐MRI technology and discuss its potential as a direct noninvasive in vivo diagnostic and prognostic biomarker for cancer therapy, particularly in cancer immunotherapy. We propose that 23Na‐MRI is a promising method with a wide range of applications in the tumor immuno‐microenvironment research field and in cancer immunotherapy monitoring.Level of Evidence2Technical EfficacyStage 2

Role of Deranged Energy Deprivation Signaling in the Pathogenesis of Cardiac and Renal Disease in States of Perceived Nutrient Overabundance.

Sodium-glucose cotransporter 2 inhibitors reduce the risk of serious heart failure and adverse renal events, but the mechanisms that underlie this benefit are not understood. Treatment with SGLT2 inhibitors is distinguished by 2 intriguing features: ketogenesis and erythrocytosis. Both reflect the induction of a fasting-like and hypoxia-like transcriptional paradigm that is capable of restoring and maintaining cellular homeostasis and survival. In the face of perceived nutrient and oxygen deprivation, cells activate low-energy sensors, which include sirtuin-1 (SIRT1), AMP-activated protein kinase (AMPK), and hypoxia inducible factors (HIFs; especially HIF-2α); these enzymes and transcription factors are master regulators of hundreds of genes and proteins that maintain cellular homeostasis. The activation of SIRT1 (through its effects to promote gluconeogenesis and fatty acid oxidation) drives ketogenesis, and working in concert with AMPK, it can directly inhibit inflammasome activation and maintain mitochondrial capacity and stability. HIFs act to promote oxygen delivery (by stimulating erythropoietin and erythrocytosis) and decrease oxygen consumption. The activation of SIRT1, AMPK, and HIF-2α enhances autophagy, a lysosome-dependent degradative pathway that removes dangerous constituents, particularly damaged mitochondria and peroxisomes, which are major sources of oxidative stress and triggers of cellular dysfunction and death. SIRT1 and AMPK also act on sodium transport mechanisms to reduce intracellular sodium concentrations. It is interesting that type 2 diabetes mellitus, obesity, chronic heart failure, and chronic kidney failure are characterized by the accumulation of intracellular glucose and lipid intermediates that are perceived by cells as indicators of energy overabundance. The cells respond by downregulating SIRT1, AMPK, and HIF-2α, thus leading to an impairment of autophagic flux and acceleration of cardiomyopathy and nephropathy. SGLT2 inhibitors reverse this maladaptive signaling by triggering a state of fasting and hypoxia mimicry, which includes activation of SIRT1, AMPK, and HIF-2α, enhanced autophagic flux, reduced cellular stress, decreased sodium influx into cells, and restoration of mitochondrial homeostasis. This mechanistic framework clarifies the findings of large-scale randomized trials and the close association of ketogenesis and erythrocytosis with the cardioprotective and renoprotective benefits of these drugs.

Autophagy stimulation and intracellular sodium reduction as mediators of the cardioprotective effect of sodium-glucose cotransporter 2 inhibitors.

In five large-scale trials involving >40 000 patients, sodium-glucose cotransporter 2 (SGLT2) inhibitors decreased the risk of serious heart failure events by 25-40%. This effect cannot be explained by control of hyperglycaemia, since it is not observed with antidiabetic drugs with greater glucose-lowering effects. It cannot be attributed to ketogenesis, since it is not causally linked to ketone body production, and the benefit is not enhanced in patients with diabetes. The effect cannot be ascribed to a natriuretic action, since SGLT2 inhibitors decrease natriuretic peptides only modestly, and they reduce cardiovascular death, a benefit that diuretics do not possess. Although SGLT2 inhibitors increase red blood cell mass, enhanced erythropoiesis does not favourably influence the course of heart failure. By contrast, experimental studies suggest that SGLT2 inhibitors may reduce intracellular sodium, thereby preventing oxidative stress and cardiomyocyte death. Additionally, SGLT2 inhibitors induce a transcriptional paradigm that mimics nutrient and oxygen deprivation, which includes activation of adenosine monophosphate-activated protein kinase, sirtuin-1, and/or hypoxia-inducible factors-1α/2α. The interplay of these mediators stimulates autophagy, a lysosomally-mediated degradative pathway that maintains cellular homeostasis. Autophagy-mediated clearance of damaged organelles reduces inflammasome activation, thus mitigating cardiomyocyte dysfunction and coronary microvascular injury. Interestingly, the action of hypoxia-inducible factors-1α/2α to both stimulate erythropoietin and induce autophagy may explain why erythrocytosis is strongly correlated with the reduction in heart failure events. Therefore, the benefits of SGLT2 inhibitors on heart failure may be mediated by a direct cardioprotective action related to modulation of pathways responsible for cardiomyocyte homeostasis.

Identification of Sodium Sensitive Site and Chloride Sensitive Site on the C-Terminus of Rat KCNT1 Channel

KCNT1 channel is a sodium and chloride activated potassium channel that is widely expressed in nervous system. Our previous study had identified D818 mutants can dramatically decrease the sodium sensitivity of KCNT1 channel. But the full co-ordinate sodium sensitive site and chloride site of this channel remain unknown. In this abstract, we reported that identification of a sodium binding site of this channel by fully screening 109 negatively charged amino acids mutants on the C-terminus of Slack channel. Results show several mutants of these negative charged channel increased Kd of sodium sensitivity to more than 1 M while several mutants did not show currents. Subsequently, we screened mutants that did not show currents on the background of mutant M335A because the mutant M335A of KCNT1 channel can be activated without intracellular sodium. Thus, we got either macroscopic current or single channel current from all mutants. Combined with electrophysiology experimental data and molecular dynamic simulation, we found that one subunit of Slack channel could bind two sodium ions. One dominant sodium sensitive site is located on RCK1 domain of the Slack channel. Another weak sodium binding pocket that is composed of 6 spatially closed negative charged residues. In the meantime, by molecular dynamic prediction and alignment with C.elegan Slo2 channel, we screened mutants of some positive charged residues on the C-terminus of the rat KCNT1 channel. One chloride binding site was identified from RCK1 domain of KCNT1 channel. In conclusion, our results dissected sodium and chloride sensitive sites of KCNT1 channel.

SGLT2 Inhibitors Play a Salutary Role in Heart Failure via Modulation of the Mitochondrial Function

Three cardiovascular outcome trials of sodium glucose cotransporter 2 (SGLT2) inhibitors, including the EMPA-REG OUTCOME trial, CANVAS Program, and DECLARE TIMI 58 trial, revealed that SGLT2 inhibitors were superior to a matching placebo in reducing cardiovascular events, including mortality and hospitalization for heart failure, in patients with type 2 diabetes. However, the detailed mechanism underlying the beneficial effects that SGLT2 inhibitors exert on cardiovascular diseases remains to be elucidated. We herein review the latest findings of the salutary mechanisms of SGLT2 inhibitors in cardiomyocytes, especially focusing on their mitochondrial function-mediated beneficial effects. The administration of SGLT2 inhibitors leads to the elevation of plasma levels of ketone bodies, which are an efficient energy source in the failing heart, by promoting oxidation of the mitochondrial coenzyme Q couple and enhancing the free energy of cytosolic ATP hydrolysis. SGLT2 inhibitors also promote sodium metabolism-mediated cardioprotective effects. These compounds could reduce the intracellular sodium overload to improve mitochondrial energetics and oxidative defense in the heart through binding with NHE and/or SMIT1. Furthermore, SGLT2 inhibitors could modulate mitochondrial dynamics by regulating the fusion and fission of mitochondria. Together with ongoing large-scale clinical trials to evaluate the efficacy of SGLT2 inhibitors in patients with heart failure, intensive investigations regarding the mechanism through which SGLT2 inhibitors promote the restoration in cases of heart failure would lead to the establishment of these drugs as potent anti-heart failure drugs.

Stomach gastrin is regulated by sodium via PPAR-α and dopamine D1 receptor

Gastrin, secreted by stomach G cells in response to ingested sodium, stimulates the renal cholecystokinin B receptor (CCKBR) to increase renal sodium excretion. It is not known how dietary sodium, independent of food, can increase gastrin secretion in human G cells. However, fenofibrate (FFB), a peroxisome proliferator-activated receptor-α (PPAR-α) agonist, increases gastrin secretion in rodents and several human gastrin-secreting cells, via a gastrin transcriptional promoter. We tested the following hypotheses: (1.) the sodium sensor in G cells plays a critical role in the sodium-mediated increase in gastrin expression/secretion, and (2.) dopamine, via the D1R and PPAR-α, is involved. Intact human stomach antrum and G cells were compared with human gastrin-secreting gastric and ovarian adenocarcinoma cells. When extra- or intracellular sodium was increased in human antrum, human G cells, and adenocarcinoma cells, gastrin mRNA and protein expression/secretion were increased. In human G cells, the PPAR-α agonist FFB increased gastrin protein expression that was blocked by GW6471, a PPAR-α antagonist, and LE300, a D1-like receptor antagonist. LE300 prevented the ability of FFB to increase gastrin protein expression in human G cells via the D1R, because the D5R, the other D1-like receptor, is not expressed in human G cells. Human G cells also express tyrosine hydroxylase and DOPA decarboxylase, enzymes needed to synthesize dopamine. G cells in the stomach may be the sodium sensor that stimulates gastrin secretion, which enables the kidney to eliminate acutely an oral sodium load. Dopamine, via the D1R, by interacting with PPAR-α, is involved in this process.

Amiodarone exacerbates brain injuries after hypoxic\u2013ischemic insult in mice

BackgroundSodium ion transportation plays a crucial role in the pathogenesis of hypoxic–ischemic brain injury. Amiodarone, a Vaughan-Williams class III antiarrhythmic drug, has been widely used to treat life-threatening arrhythmia and cardiac arrest worldwide. In addition to its inhibitory effects on the potassium channel, amiodarone also blocks various sodium ion transporters, including the voltage-gated sodium channel, sodium pump, and Na+/Ca+ exchanger. Considering these pharmacological profile, amiodarone may affect the influx–efflux balance of sodium ion in the hypoxic–ischemic brain. Previous studies suggest that the blockade of the voltage-gated sodium channel during hypoxic–ischemic brain injury exerts neuroprotection. On the contrary, the blockade of sodium pump or Na+/Ca+ exchanger during hypoxia–ischemia may cause further intracellular sodium accumulation and consequent osmotic cell death. From these perspectives, the effects of amiodarone on sodium ion balance on the hypoxic–ischemic brain can be both protective and detrimental depending on the clinical and pathophysiological conditions. In this study, we therefore investigated the effect of amiodarone on hypoxic–ischemic brain injury using a murine experimental model.ResultsCompared with the control group mice, mice that received amiodarone after induction of 40-min hypoxic–ischemic brain injury exhibited lower survival rates over 7 days and worse neurological function. After 25-min hypoxic–ischemic brain injury, amiodarone treated mice exhibited larger infarct volumes (16.0 ± 6.9 vs. 24.2 ± 6.8 mm3, P < 0.05) and worse neurological function. In addition, the brains harvested from the amiodarone-treated mice contained larger amounts of sodium (194.7 ± 45.1 vs. 253.5 ± 50.9 mEq/kg dry weight, P < 0.01) and water (259.3 ± 8.9 vs. 277.2 ± 12.5 mg, P < 0.01). There were no significant differences in hemodynamic parameters between groups.ConclusionsAmiodarone exacerbated brain injuries and neurological outcomes after hypoxic–ischemic insults. Severe brain sodium accumulation and brain edema were associated with the detrimental effects of amiodarone. Amiodarone at the clinical dose can exacerbate brain injury after hypoxic–ischemic insult by affecting sodium ion transportation and facilitate intracellular sodium accumulation in the brain.

Relation between activity-induced intracellular sodium transients and ATP dynamics in mouse hippocampal neurons.

Employing quantitative Na+ -imaging and Förster resonance energy transfer-based imaging with ATeam1.03YEMK (ATeam), we studied the relation between activity-induced Na+ influx and intracellular ATP in CA1 pyramidal neurons of the mouse hippocampus. Calibration of ATeam in situ enabled a quantitative estimate of changes in intracellular ATP concentrations. Different paradigms of stimulation that induced global Na+ influx into the entire neuron resulted in decreases in [ATP] in the range of 0.1-0.6mm in somata and dendrites, while Na+ influx that was locally restricted to parts of dendrites did not evoke a detectable change in dendritic [ATP]. Our data suggest that global Na+ transients require global cellular activation of the Na+ /K+ -ATPase resulting in a consumption of ATP that transiently overrides its production. For recovery from locally restricted Na+ influx, ATP production as well as fast intracellular diffusion of ATP and Na+ might prevent a local drop in [ATP]. Excitatory neuronal activity results in the influx of Na+ through voltage- and ligand-gated channels. Recovery from accompanying increases in intracellular Na+ concentrations ([Na+ ]i ) is mainly mediated by the Na+ /K+ -ATPase (NKA) and is one of the major energy-consuming processes in the brain. Here, we analysed the relation between different patterns of activity-induced [Na+ ]i signalling and ATP in mouse hippocampal CA1 pyramidal neurons by Na+ imaging with sodium-binding benzofurane isophthalate (SBFI) and employing the genetically encoded nanosensor ATeam1.03YEMK (ATeam). In situ calibrations demonstrated a sigmoidal dependence of the ATeam Förster resonance energy transfer ratio on the intracellular ATP concentration ([ATP]i ) with an apparent KD of 2.6mm, indicating its suitability for [ATP]i measurement. Induction of recurrent network activity resulted in global [Na+ ]i oscillations with amplitudes of ∼10mm, encompassing somata and dendrites. These were accompanied by a steady decline in [ATP]i by 0.3-0.4mm in both compartments. Global [Na+ ]i transients, induced by afferent fibre stimulation or bath application of glutamate, caused delayed, transient decreases in [ATP]i as well. Brief focal glutamate application that evoked transient local Na+ influx into a dendrite, however, did not result in a measurable reduction in [ATP]i . Our results suggest that ATP consumption by the NKA following global [Na+ ]i transients temporarily overrides its availability, causing a decrease in [ATP]i . Locally restricted Na+ transients, however, do not result in detectable changes in local [ATP]i , suggesting that ATP production, together with rapid intracellular diffusion of both ATP and Na+ from and to unstimulated neighbouring regions, counteracts a local energy shortage under these conditions.

Elevation of Intracellular Na+ Contributes to Expression of Early Response Genes Triggered by Endothelial Cell Shrinkage.

Prolonged hyperosmotic shrinkage evokes expression of osmoprotective genes via nuclear factor NFAT5-mediated pathway and activates Na+ influx via hypertonicity-induced cation channels (HICC). In human umbilical vein endothelial cells (HUVEC) elevation of intracellular sodium concentration ([Na+]i) triggers transcription of dozens of early response genes (ERG). This study examined the role of monovalent cations in the expression of Na+i-sensitive ERGs in iso- and hyperosmotically shrunken HUVEC. Cell volume was measured by 3D reconstruction of cell shape and as 14C-urea available space. Intracellular Na+ and K+ content was measured by flame atomic absorption spectrometry. ERG transcription was estimated by RT-PCR. Elevation of medium osmolality by 150 mM mannitol or cell transfer from hypo- to isosmotic medium decreased cell volume by 40-50%. Hyperosmotic medium increased [Na+]i by 2-fold whereas isosmotic shrinkage had no impact on this parameter. Hyperosmotic but not isosmotic shrinkage increased up-to 5-fold the content of EGR1, FOS, ATF3, ZFP36 and JUN mRNAs. Expression of these ERGs triggered by hyperosmotic shrinkage and Na+,K+-ATPase inhibition by 0.1 µM ouabain exhibited positive correlation (R2=0.9383, p=0.0005). Isosmotic substitution of NaCl by N-methyl-D-glucamine abolished an increment of [Na+]i and ERG expression triggered by mannitol addition. Augmented expression of ERGs in hyperosmotically shrunken HUVEC is mediated by elevation of [Na+]i.

SLC8 family of sodium/calcium exchangers (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

The sodium/calcium exchangers (NCX) use the extracellular sodium concentration to facilitate the extrusion of calcium out of the cell. Alongside the plasma membrane Ca2+-ATPase (PMCA) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA), as well as the sodium/potassium/calcium exchangers (NKCX, SLC24 family), NCX allow recovery of intracellular calcium back to basal levels after cellular stimulation. When intracellular sodium ion levels rise, for example, following depolarisation, these transporters can operate in the reverse direction to allow calcium influx and sodium efflux, as an electrogenic mechanism. Structural modelling suggests the presence of 9 TM segments, with a large intracellular loop between the fifth and sixth TM segments.

Abstract P2047: Human Renal Proximal Tubule Cells Cultured Under High Salt Conditions Increase Angiotensin Type 2 Receptor Abundance In Secreted Exosomes

The dopamine type 1 (D 1 R) and angiotensin type 2 (AT 2 R) are both natriuretic receptors that regulate renal sodium homeostasis. Aberrant D 1 R and AT 2 R mechanisms are implicated in hypertension, salt-sensitive hypertension, and in normotensive salt sensitives. Increased AT 2 R recruitment to the plasma membrane has been shown to occur when the D 1 R is stimulated by dopamine and increases with increased extracellular and intracellular sodium. RPTCs secrete exosomes which are plasma membrane vesicles between 50 and 90 nm in diameter and which encapsulate proteins and microRNA from the cell. We hypothesized that exosomal AT 2 R may reflect plasma membrane abundance of the RPTCs responding to the exposure to increased sodium. This study examined the abundance of the AT 2 R found on the surface of these exosomes in response to changing salt concentrations. Immortalized hRPTC were treated in serum free media cultured overnight with either an additional 50 mM sodium chloride or choline chloride as an osmotic equivalent, and exosomes were purified and concentrated from cell culture supernatant. The exosomes were then captured with magnetic lectin affinity beads and immunostained with fluorescent AT 2 R that increased in high salt conditions compared to vehicle control treated cells (VEH 71,884±7099 RFU, n=17, 190 mM Sodium, 94,180±5487 RFU, n=16, p= 0.018, t-test). The increase levels of AT 2 R are appropriate for their natriuretic activity. Future work will examine this response as it relates to salt sensitivity and inverse salt sensitivity of blood pressure as we previously reported using miRNA.

23Na MRI of human skeletal muscle using long inversion recovery pulses

23Na inversion recovery (IR) imaging allows for a weighting toward intracellular sodium in the human calf muscle and thus enables an improved analysis of pathophysiological changes of the muscular ion homeostasis. However, sodium signal-to-noise ratio (SNR) is low, especially when using IR sequences. 23Na has a nuclear spin of 3/2 and therefore experiences a strong electrical quadrupolar interaction. This results in very short relaxation times as well as in possible residual quadrupolar splitting. Consequently, relaxation effects during a radiofrequency pulse can no longer be neglected and even allow for increasing SNR as has previously been shown for human brain and knee. The aim of this work was to increase the SNR in 23Na IR imaging of the human calf muscle by using long inversion pulses instead of the usually applied short pulses. First, the influence of the inversion pulse length (1 to 20 ms) on the SNR as well as on image contrast was simulated for different model environments and verified by phantom measurements. Depending on the model environment (agarose 4% and 8%, xanthan 2% and 3%), SNR values increased by a factor of 1.15 up to 1.35, while NaCl solution was successfully suppressed. Thus, image contrast between the non-suppressed model compartments changes with IR pulse length. Finally, in vivo measurements of the human calf muscle of ten healthy volunteers were conducted at 3 Tesla. On average, a 1.4-fold increase in SNR could be achieved by increasing the inversion pulse length from 1 ms to 20 ms, leaving all other parameters – including the scan time – constant. This enables 23Na IR MRI with improved spatial resolution or reduced acquisition time.