Intracellular Concentrations Of Na Research Articles

Accumulation of Li+ Ions Triggers Changes in FOS, JUN, EGR1, and MYC Transcription in the LiCl-Treated Human Umbilical Vein Endothelial Cells (HUVEC).

Changes in intracellular concentrations of Na+ and K+ are shown to alter gene expression. Another monovalent cation, Li+, is well known as a medicine for treatment of psychiatric disorders, but mechanism of its action is obscure. Thus, it is important to evaluate the effect of Li+ on gene expression in endothelial cells. Here we studied influence of the increased intracellular Na+ or Li+ concentrations on transcription of Na+i/K+i-sensitive genes. Treatment of the human endothelial cells (HUVEC) with LiCl for 1.5h resulted in accumulation of Li+ in the cells. This was followed by increase in the FOS and EGR1 mRNAs levels and decrease in the JUN and MYC mRNA levels. Treatment of HUVEC with the Na+-ionophore monensin led to accumulation of Na+ and loss of K+ ions. However, monensin had no significant effect on gene expression. Incubation of HUVEC with elevated extracellular NaCl concentration increased intracellular K+ concentration and transcription of the ATF3 gene, while transcription of the JUN gene decreased. These results indicate that Na+ and Li+ ions have different effects on the gene expression profile in the cells that is likely associated with the fact that they affect differently the intracellular monovalent cations ratio.

Epi-reevesioside F inhibits Na+/K+-ATPase, causing cytosolic acidification, Bak activation and apoptosis in glioblastoma.

Epi-reevesioside F, a new cardiac glycoside isolated from the root of Reevesia formosana, displayed potent activity against glioblastoma cells. Epi-reevesioside F was more potent than ouabain with IC50 values of 27.3±1.7 vs. 48.7±1.8 nM (P < 0.001) and 45.0±3.4 vs. 81.3±4.3 nM (P < 0.001) in glioblastoma T98 and U87 cells, respectively. However, both Epi-reevesioside F and ouabain were ineffective in A172 cells, a glioblastoma cell line with low Na+/K+-ATPase α3 subunit expression. Epi-reevesioside F induced cell cycle arrest at S and G2 phases and apoptosis. It also induced an increase of intracellular concentration of Na+ but not Ca2+, cleavage and exposure of N-terminus of Bak, loss of mitochondrial membrane potential, inhibition of Akt activity and induction of caspase cascades. Potassium supplements significantly inhibited Epi-reevesioside F-induced effects. Notably, Epi-reevesioside F caused cytosolic acidification that was highly correlated with the anti-proliferative activity. In summary, the data suggest that Epi-reevesioside F inhibits Na+/K+-ATPase, leading to overload of intracellular Na+ and cytosolic acidification, Bak activation and loss of mitochondrial membrane potential. The PI3-kinase/Akt pathway is inhibited and caspase-dependent apoptosis is ultimately triggered in Epi-reevesioside F-treated glioblastoma cells.

Serum and intracellular levels of ionized sodium, potassium, and magnesium in type 2 diabetic subjects

Background and Aims: Alterations of ionized sodium, potassium, and magnesium in the serum or within erythrocytes have been reported in diabetes mellitus (DM) subjects, both as causes and consequences. There is also increasing evidence that electrolyte imbalances are early biochemical events responsible for long-term diabetic complications. Considerable variations in the electrolyte metabolism may exist in populations depending on the genetic constitution, nutritional status, and environmental situation. The present study was undertaken to investigate the serum and erythrocyte levels of Na + , K + , and Mg 2+ , and also to explore their relationship to glycemic status in a group of Bangladeshi type 2 diabetic patients without any complications. Materials and Methods: There were 30 newly-diagnosed type 2 diabetic subjects [age in years 45.17 ± 1.66; body mass index (BMI) 27.26 ± 1.59, M ± SD] who were studied with 30 age- and BMI-matched control subjects (age 46.30 ± 2.41; BMI 26.50 ± 1.78). Serum and intracellular concentration of Na + , K + , and Mg 2+ were estimated by the ion sensitive electrode (ISE) method using an Auto Analyzer (Nova Biomedical Corporation, 200 Prospect Street, Waltham, MA 02254-9141, USA). Serum glucose and lipid profile were measured by enzymatic colorimetric method. Results: The serum levels of ions (mmol/L, M ± SD) in the control subjects were as follows: Na + - 145 ± 1, K + - 3.78 ± 0.25, and Mg 2+ - 0.47 ± 0.02. In diabetic subjects, significantly lower value of Na + (143 ± 2, P 2+ (0.44 ± 0.03, P + (4.19 ± 0.41, P + and Mg 2+ showed negative correlation ( P + showed positive correlation ( P + was higher [(values in mmol/L, M ± SD): 11.20 ± 5.40 in diabetic subjects vs. 10.40 ± 4.77 in control subjects, P + [(values in mmol/L, M ± SD): 139 ± 4.8 in control subjects vs. 133.06 ± 3.71 in diabetic subjects, P 2+ [(values in mmol/L, M ± SD): 1.82 ± 0.22 in control subjects vs. 1.75 ± 0.20 in diabetic subjects, P + ( P + and Mg 2+ ( P Conclusions: These data confirm the existence of hyponatremia, hyperkalemia, and hypomagnesemia, paralleled by a reverse change of Na + and K + in erythrocytes of type 2 diabetic subjects. The study also demonstrates that hyperglycemia-induced effects on cellular transport process play a major role in mediating the electrolyte imbalances in diabetes.

Functional Interaction Between Na/K-ATPase and NMDA Receptor in Cerebellar Neurons.

NMDA receptors play a crucial role in regulating synaptic plasticity and memory. Activation of NMDA receptors changes intracellular concentrations of Na(+) and K(+), which are subsequently restored by Na/K-ATPase. We used immunochemical and biochemical methods to elucidate the potential mechanisms of interaction between these two proteins. We observed that NMDA receptor and Na/K-ATPase interact with each other and this interaction was shown for both isoforms of α subunit (α1 and α3) of Na/K-ATPase expressed in neurons. Using Western blotting, we showed that long-term exposure of the primary culture of cerebellar neurons to nanomolar concentrations of ouabain (a cardiotonic steroid, a specific ligand of Na/K-ATPase) leads to a decrease in the levels of NMDA receptors which is likely mediated by the α3 subunit of Na/K-ATPase. We also observed a decrease in enzymatic activity of the α1 subunit of Na/K-ATPase caused by NMDA receptor activation. This effect is mediated by an increase in intracellular Ca(2+). Thus, Na/K-ATPase and NMDA receptor can interact functionally by forming a macromolecular complex which can be important for restoring ionic balance after neuronal excitation. Furthermore, this interaction suggests that NMDA receptor function can be regulated by endogenous cardiotonic steroids which recently have been found in cerebrospinal fluid or by pharmacological drugs affecting Na/K-ATPase function.

Transepithelial glucose transport and Na+/K+ homeostasis in enterocytes: an integrative model

The uptake of glucose and the nutrient coupled transcellular sodium traffic across epithelial cells in the small intestine has been an ongoing topic in physiological research for over half a century. Driving the uptake of nutrients like glucose, enterocytes must have regulatory mechanisms that respond to the considerable changes in the inflow of sodium during absorption. The Na-K-ATPase membrane protein plays a major role in this regulation. We propose the hypothesis that the amount of active Na-K-ATPase in enterocytes is directly regulated by the concentration of intracellular Na(+) and that this regulation together with a regulation of basolateral K permeability by intracellular ATP gives the enterocyte the ability to maintain ionic Na(+)/K(+) homeostasis. To explore these regulatory mechanisms, we present a mathematical model of the sodium coupled uptake of glucose in epithelial enterocytes. Our model integrates knowledge about individual transporter proteins including apical SGLT1, basolateral Na-K-ATPase, and GLUT2, together with diffusion and membrane potentials. The intracellular concentrations of glucose, sodium, potassium, and chloride are modeled by nonlinear differential equations, and molecular flows are calculated based on experimental kinetic data from the literature, including substrate saturation, product inhibition, and modulation by membrane potential. Simulation results of the model without the addition of regulatory mechanisms fit well with published short-term observations, including cell depolarization and increased concentration of intracellular glucose and sodium during increased concentration of luminal glucose/sodium. Adding regulatory mechanisms for regulation of Na-K-ATPase and K permeability to the model show that our hypothesis predicts observed long-term ionic homeostasis.

Ouabain switches Na+ transport to Na+/Na+ equivalent exchange in normal and apoptotic lymphoid cells

A theory of change of the ionic fluxes in the lymphoid cells in their transition from normal to apoptosis, we have developed previously, is applied to the analysis of Na+/Na+ exchange fluxes in human lymphoid cells U937 exposed to ouabain. We solve a system of equations describing changes in the intracellular concentrations of Na+, K+ and Cl-, membrane potential and cell volume. It is shown that the Na+ input (I(Na/Na)) flux and output flux through the Na+/Na+ tract increased 4 times in 8 hours after disconnecting Na+/K+ -ATPase for normal cell U937. These fluxes increased 2.6 times for apoptotic cells. The value of I(Na/Na) after 8 hours off pump by ouabain is 97% of the total Na+ input for both cell types. It is concluded that ouabain not only inhibits the Na+/K+ -ATPase, but also increases Na+ exchange fluxes through the Na+/Na+ tract, thereby switching sodium transport across the membrane of lymphoid cells to Na+/Na+ equivalent exchange.

Identity, expression and functional role of the sodium-activated potassium current in vestibular ganglion afferent neurons

Vestibular afferent neurons (VANs) transmit information from the vestibular end organs to the central nuclei. This information is encoded within the firing pattern of these cells and is heavily influenced by the K+ conductances expressed by vestibular neurons. In the present study, we describe the presence of a previously unidentified Na+-activated K+ conductance (KNa) in these cells. We observed that the blocking of Na+ channels by tetrodotoxin (TTX) or the substitution of choline for Na+ in the extracellular solution during voltage clamp pulses resulted in the reduction of a sustained outward current that was dependent on the Na+ current. Furthermore, increases in the intracellular concentration of Na+ that were made by blocking the Na+/K+ ATPase with ouabain increased the amplitude of the outward current, and reduction of the intracellular Cl− concentration reduced the TTX-sensitive outward current. The substitution of Li+ for Na+ in the extracellular solution significantly reduced the amplitude of the outward current in voltage clamp pulses and decreased the afterhyperpolarization (AHP) of the action potentials in current clamp experiments. These electrophysiological results are consistent with the presence of mRNA transcripts for the KNa subunits Slick and Slack in the vestibular ganglia and in the sensory epithelium, which were detected using reverse-transcription polymerase chain reaction (RT-PCR). These results are also consistent with the immunolabeling of Slick and Slack protein in isolated vestibular neurons, in the vestibular ganglion and in the vestibular sensory epithelium. These results indicate that KNa channels are expressed in VANs and in their terminals. Furthermore, these data indicate that these channels may contribute to the firing pattern of vestibular neurons.

Inhibition of Na+‐H+ exchange as a mechanism of rapid cardioprotection by resveratrol

BACKGROUND AND PURPOSE Resveratrol is a polyphenol abundantly found in grape skin and red wine. In the present study, we investigated whether resveratrol exerts protective effects against cardiac ischaemia/reperfusion and also explored its mechanisms. EXPERIMENTAL APPROACH Infarct size and functional recovery in rat isolated perfused hearts subjected to no-flow global ischaemia followed by reperfusion were measured. Cultured neonatal rat cardiomyocytes were exposed to H(2)O(2) (100 µmol·L(-1)) to induce cell injury. Intracellular ion concentrations were measured using specific dyes. Western blotting was used to quantify protein expression levels. KEY RESULTS In rat isolated perfused hearts, treatment with resveratrol (20 and 100 µmol·L(-1)) 15 min before ischaemia considerably improved left ventricular functional recovery and infarct size. In cultured neonatal rat cardiomyocytes, resveratrol significantly attenuated the increase in reactive oxygen species (ROS) and loss of mitochondrial inner membrane potential. Resveratrol also suppressed the increase in intracellular concentrations of Na(+) ([Na(+)](i)) and Ca(2+) ([Ca(2+)](i)) after H(2)O(2) application; however, it did not suppress the ouabain-induced [Ca(2+) ](i) increase. By measuring changes in intracellular pH recovery after acidification, we also confirmed that acid-induced activation of the Na(+)-H(+) exchanger (NHE) was prevented by pretreatment with resveratrol. Furthermore, resveratrol inhibited the H(2)O(2)-induced translocation of PKC-α from the cytosol to the cell membrane; this translocation is believed to activate NHE. CONCLUSION AND IMPLICATIONS Resveratrol exerts cardioprotection by reducing ROS and preserving mitochondrial function. The PKC-α-dependent inhibition of NHE and subsequent attenuation of [Ca(2+)](i) overload may be a cardioprotective mechanism.

Influence of intracellular Na+, K+ and Cl- on the salt tolerance in suspension cell cultures of Medicago media

In the process of selection for salt tolerance, it is important to understand the physiological basis of ion management executed by the cells through the exclusion, accumulation or maintenance of ratios of specific ions. Intracellular accumulation of Na + , K + and Cl- ions in the cells in vitro was studied as a factor in salt tolerance in suspension cultures of Medicago media cv. Rambler. Cells selected for NaCl tolerance (R151-S) and the non-selected R151 (normal) cells were grown in MS medium containing 0, 100, 250, 400 and 550 mM NaCl, Na + , Cl - or K + for 5 weeks and examined for relative growth rate per week (RGR/week) and intracellular concentration of Na + , Cl - and K + ions. R151-S cells were found to be significantly more tolerant to the elevated Na + and Cl- levels than R151. When the effects of Na + and Cl - ions were compared, R151-S appeared to be more tolerant of higher concentrations of Cl - as compared to the equimolar concentrations of Na + . As the concentration of NaCl in the medium increased, the intracellular concentrations of Na + increased, though not at a proportional rate. Also, when the external Cl- concentration increased, there was no significant change in the intracellular accumulation of Na + . On the contrary, intracellular Na + decreased when the external K + was increased. In the cells adapted to high concentration of NaCl, the concentration of intracellular K + was lower than the non-adapted cells. The adequate intracellular K + concentration for optimum growth in M. media suspension cultures was found to be 3000 µM/g dry weight. Excess or deficiency of intracellular K + caused severe reduction in the growth rates in all cell lines. Key words : Salt tolerance, Medicago media, suspension cell culture, intracellular ions, organic relative growth rate.

Inhibition of voltage‐gated Na+ current by nanosecond pulsed electric field (nsPEF) is not mediated by Na+ influx or Ca2+ signaling

In earlier studies, we found that permeabilization of mammalian cells with nsPEF was accompanied by prolonged inhibition of voltage-gated (VG) currents through the plasma membrane. This study explored if the inhibition of VG Na(+) current (I(Na)) resulted from (i) reduction of the transmembrane Na(+) gradient due to its influx via nsPEF-opened pores, and/or (ii) downregulation of the VG channels by a Ca(2+)-dependent mechanism. We found that a single 300 ns electric pulse at 1.6-5.3 kV/cm triggered sustained Na(+) influx in exposed NG108 cells and in primary chromaffin cells, as detected by increased fluorescence of a Sodium Green Dye. In the whole-cell patch clamp configuration, this influx was efficiently buffered by the pipette solution so that the increase in the intracellular concentration of Na(+) ([Na](i)) did not exceed 2-3 mM. [Na](i) increased uniformly over the cell volume and showed no additional peaks immediately below the plasma membrane. Concurrently, nsPEF reduced VG I(Na) by 30-60% (at 4 and 5.3 kV/cm). In control experiments, even a greater increase of the pipette [Na(+)] (by 5 mM) did not attenuate VG I(Na), thereby indicating that the nsPEF-induced Na(+) influx was not the cause of VG I(Na) inhibition. Similarly, adding 20 mM of a fast Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) into the pipette solution did not prevent or attenuate the inhibition of the VG I(Na) by nsPEF. These findings point to possible Ca(2+)-independent downregulation of the VG Na(+) channels (e.g., caused by alteration of the lipid bilayer) or the direct effect of nsPEF on the channel.

Cellular location of K, Na, Cd and Zn in the moss Pseudoscleropodium purum in an extensive survey

The aim of the present study was to determine if use of the Sequential Elution Technique (SET) improves interpretation of data on the total concentrations of elements in the moss Pseudoscleropodium purum, obtained in a regional network, by providing information about the cellular location, bioavailability and temporal representativeness of different elements. In total, 147 sites located at the vertices of a 15 × 15 km network were sampled. The total, extra- and intracellular concentrations of K, Na, Cd and Zn were determined and the spatial structure was analysed by use of robust variograms. The percentage of intracellular K was high (85%) compared with that of the extracellular K (15%), whereas those of the extra and intracellular Na and Cd were similar, both close to 50%. Zinc comprised a higher percentage of extracellular metal (61%). Spatial structure was clearly observed in both the total and intracellular concentrations of Na and in the total and extracellular concentrations of Zn. These results demonstrated that use of the SET improves interpretation of the total concentrations of elements, with differences between the elements that are metabolically regulated and those that possibly are not. Despite problems regarding particles deposited on the moss surface, the extracellular concentration of elements may provide information about the potential risks associated with possible solubilization of these particles and their posterior absorption. However, the intracellular concentration, which is not influenced by the presence of particles on the moss surface, provides more realistic toxicological information, as it may be related to metabolic processes in the moss. We therefore recommend prioritizing the use of the intracellular fraction when using mosses in environmental monitoring networks.

Mutational analysis of NHAoc/NHA2 in Saccharomyces cerevisiae

Background NHAoc/NHA2 is highly and selectively expressed in osteoclasts and plays a role(s) in normal osteoclast differentiation, apoptosis and bone resorptive function in vitro. Extensive mutational analysis of a bacterial homologue, NhaA, has revealed a number of amino acid residues essential for its activity. Some of these residues are evolutionarily conserved and have been shown to be essential not only for activity of NhaA in bacteria, but also of NHAoc/NHA2 in eukaryotes. Methods The salt-sensitive Saccharomyces cerevisiae strain BW31a was used for heterologous expression of mutants of NHAoc/NHA2. Membrane expression of NHAoc/NHA2 was confirmed by confocal microscopy. Intracellular concentration of Na+ (a measure of Na+ antiporter activity) was estimated by atomic absorption spectroscopy. The growth phenotypes of cells expressing NHAoc/NHA2 mutants were studied on YNB agar supplemented with NaCl and by growth curves in YNB broth. Results Mutations in amino acid residues V161 and F357 reduced the ability of transfected BW31a cells to remove intracellular sodium and to grow in NaCl-containing medium. Yeast expressing the double mutant F357 F437 cannot grow in 0.4 M NaCl, suggesting that these residues are also essential for antiporter activity. Conclusions Evolutionarily conserved amino acids are required for full antiporter function. General Significance Mutations in these amino acid residues may impact NHAoc activity and therefore osteoclast function in vitro and in vivo.

Minding the store of Ca2+ during ischaemia/reperfusion

Cardiac ischaemia puts a tremendous stress on cardiac myocytes, and abrupt reperfusion causes dramatic changes during the first seconds to minutes that profoundly affect cell survival vs. cell death.1–3 Myocyte ionic changes can contribute to these critical effects. During ischaemia, the concentration of intracellular Na+ ([Na+] i ) rises because of a combination of increased Na+ entry via Na+/H+ exchange and tetrodotoxin-sensitive Na+ channels and reduced extrusion via Na+/K+-ATPase (as ATP and phosphocreatine are gradually depleted and ΔGATP declines). The rise in [Na+] i contributes to cellular Ca2+ gain via shifts in Na+/Ca2+ exchange, and thus elevation of diastolic [Ca2+] i . Contractile force and pressure development are abolished in the first minute or so of ischaemia because of rapidly developing acidosis. More gradually, phosphate rises and eventually rigor crossbridges form as [ATP] is dissipated. Reperfusion causes an abrupt normalization of extracellular pH, which drives a rapid rise in [Na+] i via Na+/H+ exchange. This is thought to drive a rapid further rise in [Ca2+] i (via Na+/Ca2+ exchange), which along with pH normalization causes a rise in diastolic force. Spontaneous Ca2+ transients, oscillations, and arrhythmias are also observed upon reperfusion, and agents that inhibit Na+ and Ca2+ loading can improve the long-term recovery after reperfusion.4 Despite intensive study of ischaemia/reperfusion, remarkably little is known about the dynamic changes in sarcoplasmic reticulum (SR) Ca … *Corresponding author. Tel: +1 530 752 6517; Fax: +1 530 752 7710, Email: dmbers{at}ucdavis.edu

Sodium and potassium uptake of Ulva – Application of marine macro-algae for space agriculture

The treatment of sodium, Na, and potassium, K, presents a challenge in space agriculture material recycling, as humans require Na and plants cannot grow at high Na concentrations. Hence, we are proposing the use of marine macro-algae to harvest K and other minerals from composted human waste. Ulva was selected for this feasibility study, since it tolerates a wide range of salinity levels. Growth capability of Ulva was examined under various total salinity levels and proportions of Na and K in the incubation medium. A homeostatic feature of Ulva was found in its intra-cellular concentration of Na and K, and in the intra-cellular ratio between Na and K (at 0.58 ± 0.30, lower than that of human metabolic waste). Intracellular concentration of K in Ulva is 20 times higher than seawater. Because of these characteristics, Ulva is a good candidate species for space agriculture.

Intracellular Sodium Regulates Proteolytic Activation of the Epithelial Sodium Channel

Na(+) transport across epithelia is mediated in part by the epithelial Na(+) channel ENaC. Previous work indicates that Na(+) is an important regulator of ENaC, providing a negative feedback mechanism to maintain Na(+) homeostasis. ENaC is synthesized as an inactive precursor, which is activated by proteolytic cleavage of the extracellular domains of the alpha and gamma subunits. Here we found that Na(+) regulates ENaC in part by altering proteolytic activation of the channel. When the Na(+) concentration was low, we found that the majority of ENaC at the cell surface was in the cleaved/active state. As Na(+) increased, there was a dose-dependent decrease in ENaC cleavage and, hence, ENaC activity. This Na(+) effect was dependent on Na(+) permeation; cleavage was increased by the ENaC blocker amiloride and by a mutation that decreases ENaC activity (alpha(H69A)) and was reduced by a mutation that activates ENaC (beta(S520K)). Moreover, the Na(+) ionophore monensin reversed the effect of the inactivating mutation (alpha(H69A)) on ENaC cleavage, suggesting that intracellular Na(+) regulates cleavage. Na(+) did not alter activity of Nedd4-2, an E3 ubiquitin ligase that modulates ENaC cleavage, but Na(+) reduced ENaC cleavage by exogenous trypsin. Our findings support a model in which intracellular Na(+) regulates cleavage by altering accessibility of ENaC cleavage sites to proteases and provide a molecular explanation for the earlier observation that intracellular Na(+) inhibits Na(+) transport via ENaC (Na(+) feedback inhibition).

Heading in the Wrong Direction?

The neurotransmitter dopamine is cleared from synapses by presynaptic dopamine transporters, which thus play a key role in regulating dopaminergic signaling. Mazei-Robison et al ., who previously identified a mutant form of the dopamine transporter in two brothers with attention deficit hyperactivity disorder (ADHD), investigated the effects of this mutation on transporter function. When transfected into human embryonic kidney (HEK) 293T cells, the total and surface abundance of the mutant transporter [in which an alanine at residue 559 is substituted with valine (hDAT A559V)] was similar to that of the transfected wild-type human dopamine transporter (hDAT), as was dopamine uptake. Amperometric recordings of cells preloaded with dopamine, however, revealed that, whereas cells expressing hDAT showed little dopamine efflux, hDAT A559V cells exhibited pronounced dopamine efflux currents. Whole-cell patch clamp analysis combined with experiments in which the intracellular concentrations of Na + or dopamine were varied indicated that hDAT A559V showed an exaggerated increase in dopamine efflux in response to depolarization compared with hDAT and an increased sensitivity to intracellular Na + . Intriguingly, amphetamine, which is used to treat ADHD, enhanced hDAT-mediated dopamine efflux (as it typically does) but inhibited hDAT A559V–mediated efflux. The authors thus propose that alterations in dopamine efflux through hDAT may play a role in ADHD and perhaps other brain disorders associated with altered dopamine signaling. M. S. Mazei-Robison, E. Bowton, M. Holy, M. Schmudermaier, M. Freissmuth, H. H. Sitte, A. Galli, R. D. Blakely, Anomalous dopamine release associated with a human dopamine transporter coding variant. J. Neurosci . 28 , 7040-7046 (2008). [Abstract] [Full Text]

Neuron is the primary target of Ca 2+ paradox-type insult-induced cell injury in neuron/astrocyte co-cultures

“Ca 2+ paradox” is the phenomenon whereby the intracellular concentration of Ca 2+ paradoxically increases during reperfusion with normal Ca 2+-containing media after brief exposure to a Ca 2+-free solution. The present study aims to characterize the Ca 2+ paradox induced cell injury in neuron/astrocyte co-cultures. Prior exposure of the co-cultures to a low Ca 2+ solution for 60 min significantly injured only neurons after reperfusion with a normal Ca 2+ medium for 24 h, but astrocytes remained intact. An analysis of the Ca 2+ paradox-induced changes in the intracellular concentration of Na + revealed that the concentration in astrocytes increased significantly during the reperfusion episode, resulting in a reversal of the operation of the astrocytic Na +-dependent glutamate transporter GLT-1. These results suggested that Ca 2+ paradox-induced accumulation of Na + in astrocytes was crucially involved in the excitotoxic neuronal injury resulting from the reversed astrocytic GLT-1 during the reperfusion episode. Previous studies have suggested that Ca 2+ paradox-induced injury in the brain occurs first in astroglial cells and only later in neurons resulting from the prior damage of astrocytes. Here we show that if “Ca 2+ paradox” occurs in the brain, neurons would be the primary target of Ca 2+ paradox-induced cell injury in the central nervous system.

Dependence of the intracellular concentrations of univalent ions and hydrogenase activity on the salt composition and pH of the medium in the haloalkaliphilic sulfate-reducing bacterium Desulfonatronum thiodismutans

It has been shown that the intracellular concentrations of Na+, K+, and Cl- ions in Desulfonatronum thiodismutans depend on the extracellular concentration of Na' ions. An increase in the extracellular concentration of Na+ results in the accumulation of K+ ions in cells, which points to the possibility that these ions perform an osmoprotective function. When the concentration of the NaCI added to the medium was increased to 4%, the concentration gradient of Cl- ions changed insignificantly. It was found that D. thiodismutans contains two forms of hydrogenase--periplasmic and cytoplasmic. Both enzymes are capable of functioning in solutions with high ionic force; however they exhibit different sensitivities to Na+, K+, and Li+ salts and pH. The enzymes were found to be resistant to high concentrations of Na+ and K+ chlorides and Na+ bicarbonate. The cytoplasmic hydrogenase differed significantly from the periplasmic one in having much higher salt tolerance and lower pH optimum. The activity of these enzymes depended on the nature of both the cationic and anionic components of the salts. For instance, the inhibitory effect of NaCl was less pronounced than that of LiCl, whereas Na+ and Li+ sulfates inhibited the activity of both hydrogenase types to an equal degree. The highest activity of these enzymes was observed at low Na+ concentrations, close to those typical of cells growing at optimal salt concentrations.

Calcium flux in turtle ventricular myocytes

The relative contribution of the sarcoplasmic reticulum (SR), the L-type Ca(2+) channel and the Na(+)/Ca(2+) exchanger (NCX) were assessed in turtle ventricular myocytes using epifluorescent microscopy and electrophysiology. Confocal microscopy images of turtle myocytes revealed spindle-shaped cells, which lacked T-tubules and had a large surface area-to-volume ratio. Myocytes loaded with the fluorescent Ca(2+)-sensitive dye Fura-2 elicited Ca(2+) transients, which were insensitive to ryanodine and thapsigargin, indicating the SR plays a small role in the regulation of contraction and relaxation in the turtle ventricle. Sarcolemmal Ca(2+) currents were measured using the perforated-patch voltage-clamp technique. Depolarizing voltage steps to 0 mV elicited an inward current that could be blocked by nifedipine, indicating the presence of Ca(2+) currents originating from L-type Ca(2+) channels (I(Ca)). The density of I(Ca) was 3.2 +/- 0.5 pA/pF, which led to an overall total Ca(2+) influx of 64.1 +/- 9.3 microM/l. NCX activity was measured as the Ni(+)-sensitive current at two concentrations of intracellular Na(+) (7 and 14 mM). Total Ca(2+) influx through the NCX during depolarizing voltage steps to 0 mV was 58.5 +/- 7.7 micromol/l and 26.7 +/- 3.2 micromol/l at 14 and 7 mM intracellular Na(+), respectively. In the absence of the SR and L-type Ca(2+) channels, the NCX is able to support myocyte contraction independently. Our results indicate turtle ventricular myocytes are primed for sarcolemmal Ca(2+) transport, and most of the Ca(2+) used for contraction originates from the L-type Ca(2+) channel.

Open probability of the epithelial sodium channel is regulated by intracellular sodium

The regulation of epithelial Na(+) channel (ENaC) activity by Na(+) was studied in Xenopus oocytes using two-electrode voltage clamp and patch-clamp recording techniques. Here we show that amiloride-sensitive Na(+) current (I(Na)) is downregulated when ENaC-expressing cells are exposed to high extracellular [Na(+)]. The reduction in macroscopic Na(+) current is accompanied by an increase in the concentration of intracellular Na(+) ([Na(+)](i)) and is only slowly reversible. At the single-channel level, incubating oocytes in high-Na(+) solution reduces open probability (P(o)) approximately twofold compared to when [Na(+)] is kept low, by increasing mean channel closed times. However, increasing P(o) by introducing a mutation in the beta-subunit (S518C) which, in the presence of [2-(trimethylammonium) ethyl] methane thiosulfonate (MTSET), locks the channel in an open state, could not alone abolish the downregulation of macroscopic current measured with exposure to high external [Na(+)]. Inhibition of the insertion of new channels into the plasma membrane using Brefeldin A revealed that surface channel lifetime is also markedly reduced under these conditions. In channels harbouring a beta-subunit mutation, R564X, associated with Liddle's syndrome, open probability in both high- and low-Na(+) conditions is significantly higher than in wild-type channels. Increasing the P(o) of these channels with an activating mutation abrogated the difference in macroscopic current observed between groups of oocytes incubated in high- and low-Na(+) conditions. These findings demonstrate that reduction of ENaC P(o) is a physiological mechanism limiting Na(+) entry when [Na(+)](i) is high.