Increase In Intracellular Sodium Research Articles

Simultaneous Increases in Intracellular Sodium and Tonicity Boost Antimicrobial Activity of Macrophages.

Inflamed and infected tissues can display increased local sodium (Na+) levels, which can have various effects on immune cells. In macrophages, high salt (HS) leads to a Na+/Ca2+-exchanger 1 (NCX1)-dependent increase in intracellular Na+ levels. This results in augmented osmoprotective signaling and enhanced proinflammatory activation, such as enhanced expression of type 2 nitric oxide synthase and antimicrobial function. In this study, the role of elevated intracellular Na+ levels in macrophages was investigated. Therefore, the Na+/K+-ATPase (NKA) was pharmacologically inhibited with two cardiac glycosides (CGs), ouabain (OUA) and digoxin (DIG), to raise intracellular Na+ without increasing extracellular Na+ levels. Exposure to HS conditions and treatment with both inhibitors resulted in intracellular Na+ accumulation and subsequent phosphorylation of p38/MAPK. The CGs had different effects on intracellular Ca2+ and K+ compared to HS stimulation. Moreover, the osmoprotective transcription factor nuclear factor of activated T cells 5 (NFAT5) was not upregulated on RNA and protein levels upon OUA and DIG treatment. Accordingly, OUA and DIG did not boost nitric oxide (NO) production and showed heterogeneous effects toward eliminating intracellular bacteria. While HS environments cause hypertonic stress and ionic perturbations, cardiac glycosides only induce the latter. Cotreatment of macrophages with OUA and non-ionic osmolyte mannitol (MAN) partially mimicked the HS-boosted antimicrobial macrophage activity. These findings suggest that intracellular Na+ accumulation and hypertonic stress are required but not sufficient to mimic boosted macrophage function induced by increased extracellular sodium availability.

Stimulation of Neurite Outgrowth in Cerebrocortical Neurons by Sodium Channel Activator Brevetoxin-2 Requires Both N-Methyl-D-aspartate Receptor 2B (GluN2B) and p21 Protein (Cdc42/Rac)-Activated Kinase 1 (PAK1).

N-methyl-D-aspartate (NMDA) receptors play a critical role in activity-dependent dendritic arborization, spinogenesis, and synapse formation by stimulating calcium-dependent signaling pathways. Previously, we have shown that brevetoxin 2 (PbTx-2), a voltage-gated sodium channel (VGSC) activator, produces a concentration-dependent increase in intracellular sodium [Na+]I and increases NMDA receptor (NMDAR) open probabilities and NMDA-induced calcium (Ca2+) influxes. The objective of this study is to elucidate the downstream signaling mechanisms by which the sodium channel activator PbTx-2 influences neuronal morphology in murine cerebrocortical neurons. PbTx-2 and NMDA triggered distinct Ca2+-influx pathways, both of which involved the NMDA receptor 2B (GluN2B). PbTx-2-induced neurite outgrowth in day in vitro 1 (DIV-1) neurons required the small Rho GTPase Rac1 and was inhibited by both a PAK1 inhibitor and a PAK1 siRNA. PbTx-2 exposure increased the phosphorylation of PAK1 at Thr-212. At DIV-5, PbTx-2 induced increases in dendritic protrusion density, p-cofilin levels, and F-actin throughout the dendritic arbor and soma. Moreover, PbTx-2 increased miniature excitatory post-synaptic currents (mEPSCs). These data suggest that the stimulation of neurite outgrowth, spinogenesis, and synapse formation produced by PbTx-2 are mediated by GluN2B and PAK1 signaling.

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.

Glibenclamide, a Sur1-Trpm4 antagonist, does not improve outcome after collagenase-induced intracerebral hemorrhage.

The sulfonylurea 1 transient receptor potential melastatin 4 (Sur1-Trpm4) receptor is selectively expressed after intracerebral hemorrhage (ICH). This upregulation contributes to increases in intracellular sodium. Water follows sodium through aquaporin channels, leading to cytotoxic edema. Even after edema is thought to have resolved, ionic dyshomeostasis persists, as does blood-brain barrier (BBB) damage. Glibenclamide, a hypoglycemic agent that inhibits Sur1-Trpm4, has been shown to reduce BBB damage and edema following infusion of autologous blood into the brain (ICH) as well as after other brain injuries. In order to further assess efficacy, we used the collagenase ICH model in rats to test whether glibenclamide reduces edema, attenuates ion dyshomeostasis, improves BBB damage, and reduces lesion volume. We tested a widely-used glibenclamide dose shown effective in other studies (10 μg/kg loading dose followed by 200 ng/hr for up to 7 days). Early initiation of glibenclamide did not significantly impact edema (72 hours), BBB permeability (72 hours), or lesion volume after ICH (28 days). Recovery from neurological impairments was also not improved by glibenclamide. These results suggest that glibenclamide will not improve outcome in ICH. However, the treatment appeared to be safe as there was no effect on bleeding or other physiological variables.

G protein-coupled receptor 37L1 regulates renal sodium transport and blood pressure.

G protein-coupled receptors (GPCRs) in the kidney regulate the reabsorption of essential nutrients, ions, and water from the glomerular filtrate. Abnormalities in renal epithelial ion transport play important roles in the pathogenesis of essential hypertension. The orphan G protein-coupled receptor 37L1 (GPR37L1), also known as endothelin receptor type B-like protein (ETBR-LP2), is expressed in several regions in the brain, but its expression profile and function in peripheral tissues are poorly understood. We found that GPR37L1 mRNA expression is highest in the brain, followed by the stomach, heart, testis, and ovary, with moderate expression in the kidney, pancreas, skeletal muscle, liver, lung, and spleen. Immunofluorescence analyses revealed the expression of GPR37L1 in specific regions within some organs. In the kidney, GPR37L1 is expressed in the apical membrane of renal proximal tubule cells. In human renal proximal tubule cells, the transient expression of GPR37LI increased intracellular sodium, whereas the silencing of GPR37LI decreased intracellular sodium. Inhibition of Na+/H+ exchanger isoform 3 (NHE3) activity abrogated the GPR37L1-mediated increase in intracellular sodium. Renal-selective silencing of Gpr37l1 in mice increased urine output and sodium excretion and decreased systolic and diastolic blood pressures. The renal-selective silencing of GPR37L1 decreased the protein expression of NHE3 but not the expression of Na+-K+-ATPase or sodium-glucose cotransporter 2. Our findings show that in the kidney, GPR37L1 participates in renal proximal tubule luminal sodium transport and regulation of blood pressure by increasing the renal expression and function of NHE3 by decreasing cAMP production. The role of GPR37L1, expressed in specific cell types in organs other than the kidney, remains to be determined.

High salt-induced hypertrophy of human vascular smooth muscle cells associated with a decrease in glycocalyx

All health organizations agree that, presently, the average daily salt (sodium chloride) consumption per person has attained almost double the recommended amount. A chronic high salt diet contributes to the increase in blood pressure and to the development of cardiovascular disease. Although our knowledge of hypertension, in general, is abundant, little is known about salt-sensitive hypertension.Here we tested the hypothesis that acute and/or chronic high salt mimicking that present in high-salt sensitive hypertensive patients may induce hypertrophy of human vascular smooth muscle cells (hVSMCs) and their nuclei that are associated with damage to the plasma membrane glycocalyx.Using quantitative 3D confocal microscopy coupled to immunofluorescence techniques, we tested the effects of acute (2–4 days) and chronic (6–16 days) treatments of hVSMCs without (145 mM) or with high (149 mM) extracellular sodium chloride. Our results showed that acute treatment with high salt significantly decreased the relative density of membrane glycocalyx without affecting the whole cell and nuclear volumes of hVSMCs. However, chronic treatments with high salts induced significant decreases in the relative density of glycocalyx accompanied by significant increases in the whole cell and nuclear volumes as well as in the protein/DNA ratio. The high salt-induced hVSMC hypertrophy was associated with a sustained increase in intracellular sodium and calcium.Our results clearly showed that, increasing salt concentration by as little as 4 mM immediately induced damage to the cell membrane glycocalyx leading to chronic Na+ and Ca2+ overloads and hVSMC hypertrophy. The latter may reduce the lumen of arteries leading to an increase in blood pressure. Future identification of the mechanisms that are implicated in a high salt-induced remodeling of hVSMCs may permit the development of new therapeutic interventions for the treatment of high salt-sensitive hypertension and the prevention of the associated cardiovascular diseases.

Sodium bicarbonate cotransporter NBCe2 gene variants increase sodium and bicarbonate transport in human renal proximal tubule cells.

RationaleSalt sensitivity of blood pressure affects >30% of the hypertensive and >15% of the normotensive population. Variants of the electrogenic sodium bicarbonate cotransporter NBCe2 gene, SLC4A5, are associated with increased blood pressure in several ethnic groups. SLC4A5 variants are also highly associated with salt sensitivity, independent of hypertension. However, little is known about how NBCe2 contributes to salt sensitivity, although NBCe2 regulates renal tubular sodium bicarbonate transport. We hypothesized that SLC4A5 rs10177833 and rs7571842 increase NBCe2 expression and human renal proximal tubule cell (hRPTC) sodium transport and may be a cause of salt sensitivity of blood pressure.ObjectiveTo characterize the hRPTC ion transport of wild-type (WT) and homozygous variants (HV) of SLC4A5.Methods and resultsThe expressions of NBCe2 mRNA and protein were not different between hRPTCs carrying WT or HV SLC4A5 before or after dopaminergic or angiotensin (II and III) stimulation. However, luminal to basolateral sodium transport, NHE3 protein, and Cl-/HCO3- exchanger activity in hRPTCs were higher in HV than WT SLC4A5. Increasing intracellular sodium enhanced the apical location of NBCe2 in HV hRPTCs (4.24±0.35% to 11.06±1.72% (P<0.05, N = 3, 2-way ANOVA, Holm-Sidak test)) as determined by Total Internal Reflection Fluorescence Microscopy (TIRFM). In hRPTCs isolated from kidney tissue, increasing intracellular sodium enhanced bicarbonate-dependent pH recovery rate and increased NBCe2 mRNA and protein expressions to a greater extent in HV than WT SLC4A5 (+38.00±6.23% vs HV normal salt (P<0.01, N = 4, 2-way ANOVA, Holm-Sidak test)). In hRPTCs isolated from freshly voided urine, bicarbonate-dependent pH recovery was also faster in those from salt-sensitive and carriers of HV SLC4A5 than from salt-resistant and carriers of WT SLC4A5. The faster NBCe2-specific bicarbonate-dependent pH recovery rate in HV SCL4A5 was normalized by SLC4A5- but not SLC4A4-shRNA. The binding of purified hepatocyte nuclear factor type 4A (HNF4A) to DNA was increased in hRPTCs carrying HV SLC4A5 rs7571842 but not rs10177833. The faster NBCe2-specific bicarbonate-dependent pH recovery rate in HV SCL4A5 was abolished by HNF4A antagonists.ConclusionNBCe2 activity is stimulated by an increase in intracellular sodium and is hyper-responsive in hRPTCs carrying HV SLC4A5 rs7571842 through an aberrant HNF4A-mediated mechanism.

Gene Variants in Sodium Bicarbonate Cotransporter NBCe2 Increase Sodium and Bicarbonate Transport in Human Renal Proximal Tubule

RationaleThe sodium bicarbonate cotransporter NBCe2 (encoded by SLC4A5) is involved in the regulation of renal tubular sodium bicarbonate transport. We hypothesized that human SLC4A5 single nucleotide polymorphisms (SNPs, rs10177833 and rs7571842) increase NBCe2 expression and renal proximal tubule (RPT) sodium transport and may be a cause of salt sensitivity of blood pressure.ObjectiveTo characterize the function of wild‐type (WT) and homozygous variants (HV) of SLC4A5.Methods and ResultsThe expressions of NBCe2 mRNA and protein were not different between RPT cells (RPTCs) carrying WT (n=4) or HV SLC4A5 (n=6) before or after dopaminergic or angiotensin (II and III) stimulation. However, total transcellular (luminal to basolateral) RPTC sodium transport, NHE3 protein expression, and Cl−/HCO3− exchanger activity were higher in HV than WT SLC4A5. Bicarbonate‐dependent pH recovery after an intracellular acid load, as well as NBCe2 mRNA and protein, was increased when intracellular sodium was elevated (5.2±1.1 mM) in HV SLC4A5 compared with WT SLC4A5 (pH recovery Vmax 0.22±0.02 and Km 6.39±0.81 in HV vs Vmax 0.28±0.02 and Km 4.30±0.53 in WT, P&lt;0.05, P&lt;0.05, n=3). The increased NBCe2‐specific bicarbonate‐dependent pH recovery in HV was verified in stable SLC4A5 shRNA cell lines (n=8/12/group). RPTCs carrying HV SLC4A5 had increased binding of HNF4A to SLC4A5 DNA (1.52±0.10‐fold, N=3, P&lt;0.05, t‐test), using purified HNF4A and ChIP and oligo binding assays. Additionally, the difference between HV and WT in bicarbonate‐dependent pH recovery was abolished by two HNF4A antagonists. RPTCs isolated from human urine of salt‐sensitive subjects who are HV for SLC4A5 had increased bicarbonate dependent pH recovery, relative to salt‐resistant subjects who are WT for SLC4A5 (n=4/group).ConclusionsNBCe2 activity is stimulated by an increase in intracellular sodium and is hyper‐responsive in RPTCs carrying HV SLC4A5 through an aberrant HNF4A‐mediated mechanism, which may be a key mechanism in the etiology of human salt sensitivity.

Gastrorenal communication: sniffing and tasting.

A video slideshow introduction to the Symposium by organiser Pedro A. Jose can be found here.

The sodium-bicarbonate cotransporter NBCe2 (slc4a5) expressed in human renal proximal tubules shows increased apical expression under high-salt conditions.

The electrogenic sodium bicarbonate cotransporter (NBCe2) is encoded by SLC4A5, variants of which have been associated with salt sensitivity of blood pressure, which affects 25% of the adult population. NBCe2 is thought to mediate sodium bicarbonate cotransport primarily in the renal collecting duct, but NBCe2 mRNA is also found in the rodent renal proximal tubule (RPT). The protein expression or function of NBCe2 has not been demonstrated in the human RPT. We validated an NBCe2 antibody by shRNA and Western blot analysis, as well as overexpression of an epitope-tagged NBCe2 construct in both RPT cells (RPTCs) and human embryonic kidney 293 (HEK293) cells. Using this validated NBCe2 antibody, we found NBCe2 protein expression in the RPT of fresh and frozen human kidney slices, RPTCs isolated from human urine, and isolated RPTC apical membrane. Under basal conditions, NBCe2 was primarily found in the Golgi, while NBCe1 was primarily found at the basolateral membrane. Following an acute short-term increase in intracellular sodium, NBCe2 expression was increased at the apical membrane in cultured slices of human kidney and polarized, immortalized RPTCs. Sodium bicarbonate transport was increased by monensin and overexpression of NBCe2, decreased by NBCe2 shRNA, but not by NBCe1 shRNA, and blocked by 2,2'-(1,2-ethenediyl)bis[5-isothiocyanato-benzenesulfonic acid]. NBCe2 could be important in apical sodium and bicarbonate cotransport under high-salt conditions; the implication of the ex vivo studies to the in vivo situation when salt intake is increased remains unclear. Therefore, future studies will examine the role of NBCe2 in mediating increased renal sodium transport in humans whose blood pressures are elevated by an increase in sodium intake.

Abstract 116: Characterization of a Sodium Responsive Human Sodium Bicarbonate Transporter NBCe2 in Human Proximal Tubule

The electrogenic sodium bicarbonate cotransporter (NBCe2) is encoded by SLC4A5, variants of which have been associated with salt sensitivity of blood pressure (BP), which affects 25% of the adult population. NBCe2 is thought to mediate sodium bicarbonate cotransport primarily in the renal collecting duct, but NBCe2 mRNA is also found in the rodent renal proximal tubule (RPT), another site of bicarbonate transport. The protein expression or function of NBCe2 has not been demonstrated in the human RPT. We validated an NBCe2 antibody by siRNA and Western blot, as well as overexpression of an epitope-tagged NBCe2 construct in both RPT cells (RPTCs) and HEK293 cells. We immune-localized (peptide pre-absorbable) NBCe2 protein in the RPT of fresh and frozen human kidney slices, RPTCs isolated from human urine, and in isolated RPTC apical membrane. NBCe2 was primarily found in the Golgi while NBCe1, another electrogenic member of the same family, was primarily found at the basolateral membrane, under basal conditions. Following a short-term increase in intracellular sodium, NBCe2 expression increased at the apical membrane in cultured slices of human kidney (1.75±0.15 fold increase over basal, N=3, p&lt;0.05) and polarized, immortalized RPTCs (0.08±0.015 vs 0.03±0.006 control, NBCe2/CD13 RFU, N=3, p&lt;0.05), while the basolateral localization of NBCe1 was decreased (30.7±5.4% over basal, N=3, p&lt;0.05). DIDS (electrogenic sodium bicarbonate inhibitor, 500 μM, 10 min) inhibitable bicarbonate dependent pH recovery in the presence of EIPA (NHE inhibitor,100 nM, 10 min) after ammonium chloride prepulse (20 mM, 5 min) was higher in the cells cultured under high salt conditions (26.7±7.2% over basal, N=3, p&lt;0.05). Thus, NBCe2 appears to be important in apical sodium and bicarbonate cotransport under high salt conditions. Future studies will examine the role of NBCe2 in mediating increased renal sodium transport in human salt sensitivity of BP.

Transcriptional Regulation of the Sodium-activated Potassium Channel SLICK (KCNT2) Promoter by Nuclear Factor-κB

Although recent studies have shown the sodium-activated potassium channel SLACK (KCNT1) can contribute to neuronal excitability, there remains little information on the physiological role of the closely related SLICK (KCNT2) channel. Activation of SLICK channels may be important during pathological states such as ischemia, in which an increase in intracellular sodium and chloride can perturb membrane potential and ion homeostasis. We have identified two NFκB-binding sites within the promoter region of the human SLICK (KCNT2) and orthologous rat Slick (Kcnt2) genes, suggesting that conditions in which NFκB transcriptional activity is elevated promote expression of this channel. NFκB binding to the rat Slick promoter was confirmed in vivo by ChIP analyses, and NFκB was found differentially bound to the two sites. We verified NFκB transcriptional regulation of SLICK/Slick by mutational analyses and studying gene expression by luciferase assay in P19 cells, where NFκB is constitutively active. For the rat gene, activation of the Slick promoter was found to be additive in single NFκB mutations and synergistic in double mutations. Unexpectedly, for the human gene, NFκB exhibited cooperativity in activating the SLICK promoter. The human SLICK promoter constructs were then tested under hypoxic conditions in PC-12 cells, where NFκB is not active. Only under hypoxic conditions could luciferase activity be detected; the double NFκB mutant construct failed to exhibit activity. Transcriptional regulation of Slick by NFκB was verified in primary neurons. The Slick transcript decreased 24 h after NFκB inhibition. Our data show SLICK expression is predominantly under the control of NFκB. Because neuronal NFκB activation occurs during stressful stimuli such as hypoxia and injury, our findings suggest that SLICK is a neuroprotective gene.

Interactive HIV-1 Tat and morphine-induced synaptodendritic injury is triggered through focal disruptions in Na⁺ influx, mitochondrial instability, and Ca²⁺ overload.

Synaptodendritic injury is thought to underlie HIV-associated neurocognitive disorders and contributes to exaggerated inflammation and cognitive impairment seen in opioid abusers with HIV-1. To examine events triggering combined transactivator of transcription (Tat)- and morphine-induced synaptodendritic injury systematically, striatal neuron imaging studies were conducted in vitro. These studies demonstrated nearly identical pathologic increases in dendritic varicosities as seen in Tat transgenic mice in vivo. Tat caused significant focal increases in intracellular sodium ([Na(+)]i) and calcium ([Ca(2+)]i) in dendrites that were accompanied by the emergence of dendritic varicosities. These effects were largely, but not entirely, attenuated by the NMDA and AMPA receptor antagonists MK-801 and CNQX, respectively. Concurrent morphine treatment accelerated Tat-induced focal varicosities, which were accompanied by localized increases in [Ca(2+)]i and exaggerated instability in mitochondrial inner membrane potential. Importantly, morphine's effects were prevented by the μ-opioid receptor antagonist CTAP and were not observed in neurons cultured from μ-opioid receptor knock-out mice. Combined Tat- and morphine-induced initial losses in ion homeostasis and increases in [Ca(2+)]i were attenuated by the ryanodine receptor inhibitor ryanodine, as well as pyruvate. In summary, Tat induced increases in [Na(+)]i, mitochondrial instability, excessive Ca(2+) influx through glutamatergic receptors, and swelling along dendrites. Morphine, acting via μ-opioid receptors, exacerbates these excitotoxic Tat effects at the same subcellular locations by mobilizing additional [Ca(2+)]i and by further disrupting [Ca(2+)]i homeostasis. We hypothesize that the spatiotemporal relationship of μ-opioid and aberrant AMPA/NMDA glutamate receptor signaling is critical in defining the location and degree to which opiates exacerbate the synaptodendritic injury commonly observed in neuroAIDS.

Targeted osmotic lysis of metastatic carcinomas by simultaneous blockade of sodium pumps and stimulation of voltage‐gated sodium channels (842.1)

Metastatic carcinomas (MCs) overexpress voltage‐gated sodium channels (VGSCs) by up to 1500‐fold. We reasoned that electrical stimulation of VGSCs with concomitant blockade of Na+, K+‐ATPase (sodium pumps) would produce a rapid increase in intracellular sodium, causing an influx of water. This would cause the MC cells to swell and burst. To test this hypothesis in vitro, we cultured MDA‐MB‐231 cells, which overexpress NaV1.5 VGSCs by 1400‐fold. Treatment with the sodium pump blocker ouabain then electrical stimulation of the cells produced lysis in as little as 16 sec. Ouabain or electric current alone did not lyse the cells. Treatment did not lyse MCF‐10a (normal breast) cells. To test the hypothesis in vivo, we established MDA‐MB‐231 allographs in nude mice. When tumor diameter reached approximately 1 cm, we administered 10 mg/kg ouabain s.c. or saline. 30 and 60 min later, tumors received 60 sec of 10V DC delivered through direct contact stimulation. This single‐treatment produced 70% cell lysis in tumors treated with both ouabain and stimulation. Tumors treated with saline alone and saline plus stimulation averaged 10% lysis. Ouabain alone produced 15% cell death. Treatment did not affect noncancerous tissue. In survival studies, single day treatment attenuated tumor growth by 45%. 3‐Day treatment attenuated growth by 60%. Thus, targeted osmotic lysis can be an effective treatment of MCs that overexpress VGSCs.

A Sodium-Pump-Mediated Afterhyperpolarization in Pyramidal Neurons

The sodium-potassium ATPase (i.e., the "sodium pump") plays a central role in maintaining ionic homeostasis in all cells. Although the sodium pump is intrinsically electrogenic and responsive to dynamic changes in intracellular sodium concentration, its role in regulating neuronal excitability remains unclear. Here we describe a physiological role for the sodium pump in regulating the excitability of mouse neocortical layer 5 and hippocampal CA1 pyramidal neurons. Trains of action potentials produced long-lasting (∼20 s) afterhyperpolarizations (AHPs) that were insensitive to blockade of voltage-gated calcium channels or chelation of intracellular calcium, but were blocked by tetrodotoxin, ouabain, or the removal of extracellular potassium. Correspondingly, the AHP time course was similar to the decay of activity-induced increases in intracellular sodium, whereas intracellular calcium decayed at much faster rates. To determine whether physiological patterns of activity engage the sodium pump, we replayed in vitro a place-specific burst of 15 action potentials recorded originally in vivo in a CA1 "place cell" as the animal traversed the associated place field. In both layer 5 and CA1 pyramidal neurons, this "place cell train" generated small, long-lasting AHPs capable of reducing neuronal excitability for many seconds. Place-cell-train-induced AHPs were blocked by ouabain or removal of extracellular potassium, but not by intracellular calcium chelation. Finally, we found calcium contributions to the AHP to be temperature dependent: prominent at room temperature, but largely absent at 35°C. Our results demonstrate a previously unappreciated role for the sodium-potassium ATPase in regulating the excitability of neocortical and hippocampal pyramidal neurons.

Sevoflurane Preconditioning Attenuates the Fall in Adenosine Triphosphate Levels, but Does Not Alter the Changes in Sodium and Potassium Levels during Hypoxia in Rat Hippocampal Slices

Sevoflurane preconditioning improves recovery after hypoxia. Sevoflurane administered before and during hypoxia improved recovery and attenuated the changes in intracellular sodium, potassium, and adenosine triphosphate (ATP) levels during hypoxia. In this study, the authors examine the effects of sevoflurane applied only before hypoxia on sodium, potassium, and ATP. Hippocampal slices from adult male Sprague-Dawley rats were pretreated with 4% sevoflurane, washed, and then subjected to hypoxia (n≥8 animals/group). The cornus ammonis 1 regions of the hippocampal slices were micro-dissected and sodium, potassium, and ATP concentrations measured. Pretreatment with sevoflurane for 15 or 60 min did not attenuate the increase in intracellular sodium or the decrease in intracellular potassium during hypoxia. After 60 min of preconditioning and 5 min of hypoxia, sodium increased 57% (vs. nonpreconditioned hypoxia 54% increase) and potassium decreased 31% (vs. 26%). These changes were not statistically significant versus untreated hypoxia. The 60-min sevoflurane preconditioning group had statistically significant higher ATP levels at 5 min of hypoxia (3.8 nmol/mg dry wt.) when compared to untreated hypoxic tissue (2.1 nmol/mg). There was no significant difference in ATP levels between the sevoflurane preconditioned and the untreated tissue before hypoxia (8.9 vs. 8.5 nmoles/mg, respectively). Preconditioning with sevoflurane for 60 min before hypoxia does not alter changes in intracellular sodium and potassium during hypoxia but does attenuate the fall in intracellular ATP levels during hypoxia. Thus, there are differences between anesthetic preconditioning and when anesthetics are present before and during hypoxia.

Increases in intracellular sodium activate transcription and gene expression via the salt-inducible kinase 1 network in an atrial myocyte cell line

Cardiac hypertrophy (CH) generally occurs as the result of the sustained mechanical stress caused by elevated systemic arterial blood pressure (BP). However, in animal models, elevated salt intake is associated with CH even in the absence of significant increases in BP. We hypothesize that CH is not exclusively the consequence of mechanical stress but also of other factors associated with elevated BP such as abnormal cell sodium homeostasis. We examined the effect of small increases in intracellular sodium concentration ([Na(+)](i)) on transcription factors and genes associated with CH in a cardiac cell line. Increases in [Na(+)](i) led to a time-dependent increase in the expression levels of mRNA for natriuretic peptide and myosin heavy chain genes and also increased myocyte enhancer factor (MEF)2/nuclear factor of activated T cell (NFAT) transcriptional activity. Increases in [Na(+)](i) are associated with activation of salt-inducible kinase 1 (snflk-1, SIK1), a kinase known to be critical for cardiac development. Moreover, increases in [Na(+)](i) resulted in increased SIK1 expression. Sodium did not increase MEF2/NFAT activity or gene expression in cells expressing a SIK1 that lacked kinase activity. The mechanism by which SIK1 activated MEF2 involved phosphorylation of HDAC5. Increases in [Na(+)](i) activate SIK1 and MEF2 via a parallel increase in intracellular calcium through the reverse mode of Na(+)/Ca(2+)-exchanger and activation of CaMK1. These data obtained in a cardiac cell line suggest that increases in intracellular sodium could influence myocardial growth by controlling transcriptional activation and gene expression throughout the activation of the SIK1 network.

Integrin-mediated Membrane Blebbing Is Dependent on Sodium-Proton Exchanger 1 and Sodium-Calcium Exchanger 1 Activity

Integrin signaling and membrane blebbing modulate cell adhesion, spreading, and migration. However, the relationship between integrin signaling and membrane blebbing is unclear. Here, we show that an integrin-ligand interaction induces both membrane blebbing and changes in membrane permeability. Sodium-proton exchanger 1 (NHE1) and sodium-calcium exchanger 1 (NCX1) are membrane proteins located on the bleb membrane. Inhibition of NHE1 disrupts membrane blebbing and decreases changes in membrane permeability. However, inhibition of NCX1 enhances cell blebbing; cells become swollen because of NHE1 induced intracellular sodium accumulation. Our study found that NHE1 induced sodium influx is a driving force for membrane bleb growth, while sodium efflux (and calcium influx) induced by NCX1 in a reverse mode results in membrane bleb retraction. Together, these findings reveal a novel function for NHE1 and NCX1 in membrane blebbing and permeability, and establish a link between membrane blebbing and integrin signaling.

Differential effects of Na+–K+ ATPase blockade on cortical layer V neurons

Sodium-potassium ATPase ('Na(+)-K(+) ATPase') contributes to the maintenance of the resting membrane potential and the transmembrane gradients for Na(+) and K(+) in neurons. Activation of Na(+)-K(+) ATPase may be important in controlling increases in intracellular sodium during periods of increased neuronal activity. Down-regulation of Na(+)-K(+) ATPase activity is implicated in numerous CNS disorders, including epilepsy. Although Na(+)-K(+) ATPase is present in all neurons, little is known about its activity in different subclasses of neocortical cells. We assessed the physiological properties of Na(+)-K(+) ATPase in fast-spiking (FS) interneurons and pyramidal (PYR) cells to test the hypothesis that Na(+)-K(+) ATPase activity would be relatively greater in neurons that generated high frequency action potentials (the FS cells). Whole-cell patch clamp recordings were made from FS and PYR neurons in layer V of rat sensorimotor cortical slices maintained in vitro using standard techniques. Bath perfusion of Na(+)-K(+) ATPase antagonists (ouabain or dihydro-ouabain) induced either a membrane depolarization in current clamp, or inward current under voltage clamp in both cell types. PYR neurons were divided into two subpopulations based on the amplitude of the voltage or current shift in response to Na(+)-K(+) ATPase blockade. The two PYR cell groups did not differ significantly in electrophysiological properties including resting membrane potential, firing pattern, input resistance and capacitance. Membrane voltage responses of FS cells to Na(+)-K(+) ATPase blockade were intermediate between the two PYR cell groups (P < 0.05). The resting Na(+)-K(+) ATPase current density in FS interneurons, assessed by application of blockers, was 3- to 7-fold larger than in either group of PYR neurons. Na(+)-K(+) ATPase activity was increased either through direct Na(+) loading via the patch pipette or by focal application of glutamate (20 mM puffs). Under these conditions FS interneurons exhibited the largest increase in Na(+)-K(+) ATPase activity. We conclude that resting Na(+)-K(+) ATPase activity and sensitivity to changes in internal Na(+) concentration vary between and within classes of cortical neurons. These differences may have important consequences in pathophysiological disorders associated with down-regulation of Na(+)-K(+) ATPase and hyperexcitability within cortical networks.

Induction of Vertebrate Regeneration by a Transient Sodium Current

Amphibians such as frogs can restore lost organs during development, including the lens and tail. To design biomedical therapies for organ repair, it is necessary to develop a detailed understanding of natural regeneration. Recently, ion transport has been implicated as a functional regulator of regeneration. Whereas voltage-gated sodium channels play a well known and important role in propagating action potentials in excitable cells, we have identified a novel role in regeneration for the ion transport function mediated by the voltage-gated sodium channel, Na(V)1.2. A local, early increase in intracellular sodium is required for initiating regeneration following Xenopus laevis tail amputation, and molecular and pharmacological inhibition of sodium transport causes regenerative failure. Na(V)1.2 is absent under nonregenerative conditions, but misexpression of human Na(V)1.5 can rescue regeneration during these states. Remarkably, pharmacological induction of a transient sodium current is capable of restoring regeneration even after the formation of a nonregenerative wound epithelium, confirming that it is the regulation of sodium transport that is critical for regeneration. Our studies reveal a previously undetected competency window in which cells retain their intrinsic regenerative program, identify a novel endogenous role for Na(V) in regeneration, and show that modulation of sodium transport represents an exciting new approach to organ repair.