Intracellular Sodium Concentration Research Articles

Role of the cardiac Na+/H+exchanger in [Ca2+]iand [Na+]ihandling during intracellular acidosis. Effect of cariporide (Hoe 642)

Intracellular acidosis is one of the alterations occurring in cardiac ischemia and has been discussed to be important in altering excitation–contraction coupling. The aim of this study was to determine how intracellular acidosis may affect intracellular sodium and calcium handling. Cardiomyocytes were isolated from the hearts of adult male guinea-pigs by standard techniques and superfused with modified Tyrode’s solution at room temperature, either HEPES buffered containing 10 mM NaHCO3or HEPES buffered without NaHCO3, in order to examine a possible interaction with the sodium bicarbonate symport. The whole cell voltage clamp technique was used utilizing 3 M Ω pipettes filled with (mM): Cs aspartate 120, CsCl 20, MgCl21, NaCl 5, Mg-ATP 2, HEPES 10 and either 100 μ M Fura-2 or 100 μ M SBFI. The pH of the pipette solution was either 7.2 or 6.5. Cells were kept at a holding potential of −80 mV and after a pre-pulse to −40 mV the membrane was continuously clamped to potentials from −30 to +80 mV in 10 mV steps. Intracellular Ca2+or Na+were estimated using the Fura-2 or SBFI technique (impermeable salt), respectively. The cardiac Na+/H+exchanger was inhibited using the Na+/H+- exchange inhibitor cariporide (Hoe 642) (1 μ M), when indicated. In NaHCO3-free experiments we found an increase in intracellular sodium reflected by a rise in the SBFI ratio of 0.326 ± 0.01 upon intracellular acidification, in contrast to cells perfused at pH = 7.2 (no significant increase in intracellular Na+) (P< 0.05). There was no difference in intracellular calcium handling between cells perfused with solutions of pH = 7.2 or 6.5 (Fura-2 Δ ratio: 0.79 ± 0.10 vs 0.82 ± 0.07, n.s.). The l -type calcium current also remained unchanged. Blockade of the Na+/H+exchanger by Hoe 642 had no influence on cells perfused at pH = 7.2 but inhibited the increase in intracellular Na+at pH = 6.5 (0.023 ± 0.026 in the presence of Hoe 642 vs 0.326 ± 0.01 without Hoe 642, P< 0.05) without affecting [Ca2+]ior thel -type calcium current. In cells superfused with a Tyrode solution containing NaHCO3, the increase in intracellular sodium concentration was even more pronounced. Under these conditions Hoe 642 also antagonized this increase in intracellular sodium but without reaching the control level. We conclude that under these experimental conditions intracellular acidification causes an increase in [Na+]iwithout changing intracellular Ca2+or the l -type calcium current. In addition in bicarbonate-buffered systems the acidosis-induced increase in sodium is enhanced which may involve the Na+/HCO3−symport. The effect of cariporide (Hoe 642) in intracellular acidosis seems to be based on antagonization of the rise in intracellular sodium rather than calcium in this model.

Simultaneous intracellular calcium and sodium flux imaging in human vanilloid receptor 1 (VR1)-transfected human embryonic kidney cells: a method to resolve ionic dependence of VR1-mediated cell death.

The vanilloid receptor 1 (VR1) is a ligand-gated, nonselective cation channel important for the sensory processing of painful stimuli. Activation of VR1 leads to increases in intracellular concentrations of calcium and sodium. Prolonged activation of VR1 in mammalian expression systems leads to cell death. The mechanism of VR1-mediated toxicity may have relevance to pathophysiological processes that can occur in neurons. Therefore, we have evaluated the relative contributions of intracellular calcium and sodium changes to VR1-mediated toxicity in human embryonic kidney 293 cells stably transfected with the human VR1 channel. The data demonstrate that VR1 receptor agonists capsaicin and resiniferatoxin lead to a sustained increase in intracellular calcium and sodium in a concentration-dependent manner, followed by cell death. Pretreatment with VR1 receptor antagonists capsazepine or ruthenium red block both the calcium and sodium responses to agonists, and block agonist-induced cell death in a concentration-dependent manner. However, addition of antagonists several minutes after agonists selectively reverses the agonist-induced increase in intracellular calcium, but does not reverse the elevated intracellular sodium concentration. Nonetheless, antagonists retain protective efficacy against capsaicin toxicity when added several minutes after capsaicin, conditions in which the cells still manifest elevated intracellular sodium, but not elevated intracellular calcium. In addition, a transient VR1-mediated increase in intracellular calcium that returns to baseline within minutes, induced by a rapid drop in pH, from pH 7.5 to pH 6.3, also does not lead to cell death. Collectively, these data demonstrate that the most important intracellular ionic change for mediating VR1-dependent toxicity is a sustained increase of calcium.

Hematin is one of the cytotoxic factors in pancreatitis-associated ascitic fluid that causes hepatocellular injury

Background. We recently demonstrated that pancreatitis-associated ascitic fluid (PAAF) contains cytotoxic factor(s), inducing apoptosis in hepatocytes, and that PAAF induces hepatic adenosine triphosphate depletion, hepatocellular acidosis, and accumulation of hepatic intracellular sodium. Because ascitic fluid and serum from patients with hemorrhagic pancreatitis contain a lot of hematin, we aimed to test the hypothesis that hematin can induce hepatocellular injury, and then we compared its cytotoxicity with that of PAAF. Methods. In vivo effects of intraperitoneal injection of hematin into the liver of healthy rats were evaluated with in situ nick-end labeling, blood biochemical analysis, and nuclear magnetic resonance spectroscopy. In vitro cytotoxic and apoptosis-inducing activities of hematin on rat primary culture hepatocytes were investigated with a cellular proliferation assay kit and DNA fragmentation enzyme-linked immunosorbent assay, respectively. Furthermore, PAAF was fractionated with Sephacryl S-300 gel column chromatography, and cytotoxic activities of its fractions on a human hepatoma cell line (HuH-7) were compared with those of hematin. Results. Intraperitoneal injection of hematin into healthy rats caused apoptosis in the hepatocytes and elevated serum glutamate oxaloacetic transaminase and lactate dehydrogenase levels. Intraperitoneal injection of hematin also caused a significant decrease in the hepatic β-adenosine triphosphate/inorganic phosphate ratio, severe hepatic intracellular acidosis, and a significant increase of hepatic intracellular sodium (Na+) concentration, similar to the effects of PAAF. In vitro, hematin decreased hepatocyte viability and increased the DNA fragmentation of hepatocytes, similar to the effects of 10% PAAF. Albumin reversed the cytotoxic effects of hematin and PAAF on HuH-7 cells nearly completely and partially, respectively. Fractionation of PAAF and hematin by gel column chromatography revealed that the first peak of cytotoxic activity of PAAF corresponded to that of hematin and that the cytotoxic activity was reversed by albumin nearly completely. Conclusions. These results suggest that hematin is one of the cytotoxic factors in PAAF that causes hepatocellular injury and that cellular injuries caused by hematin may be involved in the development of multiple organ failure associated with severe acute pancreatitis. (Surgery 2002;131:66-74.)

Functional analysis of the Na+/H+ antiporter encoding genes of the cyanobacterium Synechocystis PCC 6803.

The role of putative Na+/H+ antiporters encoded by nhaS1 (slr1727), nhaS3 (sll0689), nhaS4 (slr1595), and nhaS5 (slr0415) in salt stress response and internal pH regulation of the cyanobacterium Synechocystis PCC 6803 was investigated. For this purpose the mutants (single, double, and triple) impaired in genes coding for Na+/H+ antiporters were constructed using the method of interposon mutagenesis. PCR analyses of DNA demonstrated that mutations in nhaS1, nhaS4, and nhaS5 genes were segregated completely and the mutants contained only inactivated copies of the corresponding genes. Na+/H+ antiporter encoded by nhaS3 was essential for viability of Synechocystis since no completely segregated mutants were obtained. The steady-state intracellular sodium concentration and Na+/H+ antiporter activities were found to be the same in the wild type and all mutants. No differences were found in the growth rates of wild type and mutants during their cultivation in liquid media supplemented with 0.68 M or 0.85 M NaCl as well as in media buffered at pH 7.0, 8.0, or 9.0. The expression of genes coding for Na+/H+ antiporters was studied. No induction of any Na+/H+ antiporter encoding gene expression was found in wild type or single mutant cells grown under high salt or at different pH values. Nevertheless, in cells of double and triple mutants adapted to high salt or alkaline pH some of the remaining Na+/H+ antiporter encoding genes showed induction. These results might indicate that some of Na+/H+ antiporters can functionally replace each other under stress conditions in Synechocystis cells lacking the activity of more than one antiporter.

Na + Influx Inhibits Neuronal Ca 2+ Channels

Experiments on isolated rat brain neurons with an elevated intracellular sodium concentration (due to tetanic stimulation) demonstrated the existence of earlier unknown negative modulation of calcium channels by intracellular sodium.

Measured muscle sodium content in biopsy specimens is a reflection of true intracellular content

Determined on the basis of small skeletal muscle biopsies, muscle sodium content has a very high coefficient of variation. Furthermore, at least some of the measured sodium must originate from the extracellular space. In order to assess the applicability of the measurement of intracellular sodium on small muscle biopsy specimens, the measured sodium content was related to the content of dry solids in 25 muscle biopsy samples, and compared with the theoretical content of sodium with varying extra- and intracellular water content in biopsy samples. Four of the 25 measurements were clearly outliers. Disregarding these outliers, it was found that muscle sodium content varied with intracellular water content, whereas the theoretical effect of addition of extracellular water could not account for the observed values. The difference depended upon the specified conditions, but the slope of the theoretical regression line (- 25.92 mmol · (kg dry weight) -1 · % -1, which was closest to the observed slope, -8.55 mmol · (kg dry weight) -1 · % -1 , differed substantially (p < 0.0001). No association was found between the primarily intracellular ions muscle potassium and muscle magnesium on the one hand and either muscle sodium or muscle water content on the other. The measured sodium content in muscle biopsy specimens, which are freeze-dried and dissected, seems to reflect the true intracellular sodium content to some extent. The total content of sodium seems to be closely related to the content of water within the skeletal muscle cells.

The ketogenic diet may have mood-stabilizing properties

The ketogenic diet, originally introduced in the 1920s, has been undergoing a recent resurgence as an adjunctive treatment for refractory epilepsy, particularly in children. In this difficult-to-treat population, the diet exhibits remarkable efficacy with two-thirds showing significant reduction in seizure frequency and one-third becoming nearly seizure-free. There are several reasons to suspect that the ketogenic diet may also have utility as a mood stabilizer in bipolar illness. These include the observation that several anticonvulsant interventions may improve outcome in mood disorders. Furthermore, beneficial changes in brain-energy profile are noted in subjects on the ketogenic diet. This is important since global cerebral hypometabolism is a characteristic of the brains of depressed or manic individuals. Finally, the extracellular changes that occur in ketosis would be expected to decrease intracellular sodium concentrations, a common property of all effective mood stabilizers. Trials of the ketogenic diet in relapse prevention of bipolar mood episodes are warranted.

NO decreases thick ascending limb chloride absorption by reducing Na+-K+-2Cl−cotransporter activity

First published August 9, 2001; 10.1152/ajprenal.0075.2001.—We have reported that nitric oxide (NO) inhibits thick ascending limb (THAL) chloride absorption ( JCl−). NaCl transport in the THAL depends on apical Na+-K+-2Cl−cotransporters, apical K+channels, and basolateral Na+-K+-ATPase. However, the transporter inhibited by NO is unknown. We hypothesized that NO decreases THAL JCl−by inhibiting the Na+-K+-2Cl−cotransporter. THALs from Sprague-Dawley rats were isolated and perfused. Intracellular sodium ([Na+]i) and chloride concentrations ([Cl−]i) were measured with sodium green and SPQ, respectively. The NO donor spermine NONOate (SPM) decreased [Na+]ifrom 13.5 ± 1.2 to 9.6 ± 1.6 mM ( P &lt; 0.05) and also decreased [Cl−]i( P &lt; 0.01). We next tested whether NO decreases Na+-K+-2Cl−cotransporter activity by measuring the initial rate of Na+transport. In the presence of SPM in the bath, initial rates of Na+entry were 49.6 ± 6.0% slower compared with control rates ( P &lt; 0.05). To determine whether NO inhibits apical K+channel activity, we measured the change in membrane potential caused by an increase in luminal K+from 1 to 25 mM using a potential-sensitive fluorescent dye. In the presence of SPM, increasing luminal K+concentration depolarized THALs to the same extent as it did in control tubules. We then tested whether a change in apical K+permeability could affect NO-induced inhibition of THAL JCl−. In the presence of luminal valinomycin, which increases K+permeability, addition of SPM decreased THAL JCl−by 41.2 ± 10.4%, not significantly different from the inhibition observed in control tubules. We finally tested whether NO alters the affinity or maximal rate of Na+-K+-ATPase by measuring oxygen consumption rate (Qo2) in THAL suspensions in the presence of nystatin in varying concentrations of Na+. In the presence of 10.5 mM Na+, nystatin increased Qo2to 119.1 ± 19.2 and 125.6 ± 23.4 nmol O2· mg protein−1· min−1in SPM- and furosemide-treated tubules, respectively. In the presence of 145 mM extracellular Na+, nystatin increased Qo2by 104 ± 7 and 94 ± 20% in NO- and furosemide-treated tubules, respectively. We concluded that NO decreases THAL JCl−by inhibiting Na+-K+-2Cl−cotransport rather than inhibiting apical K+channels or the sodium pump.

Inadvertent infusion of a high dose of potassium chloride via a thoracic epidural catheter

Inadvertent infusion of a high dose of potassium chloride via a thoracic epidural catheter

Inhibition of Na +/Ca 2+ exchange by KB-R7943, a novel selective antagonist, attenuates phosphoethanolamine and free fatty acid efflux in rat cerebral cortex during ischemia-reperfusion injury

Reversal of the Na +/Ca 2+ exchanger (NCX) occurs during ischemia–reperfusion injury as a result of changes in intracellular pH and sodium concentration. Inhibition of NCXs has been shown to be neuroprotective in vitro. In this study, we evaluated the effects of KB-R7943 (50 μM), a specific inhibitor of the reverse mode of NCX, applied topically onto rat cerebral cortex prior to and during ischemia. Amino acid and free fatty acid levels in cortical superfusates, withdrawn at 10-min intervals from bilateral cortical windows, were analyzed by high-performance liquid chromatography. During a 20-min period of ischemia in control animals, there were significant increases in all amino acids and in all FFAs. Following reperfusion, all FFAs remained significantly elevated. Application of KB-R7943 (50 μM) significantly inhibited effluxes of phosphoethanolamine, but had no effect on glutamate, aspartate, taurine or GABA levels. KB-R7943 also resulted in significant reductions in levels of myristic, docosahexaenoic and arachidonic acid during ischemia and in reperfusion levels of arachidonic and docosahexaenoic acids. These data indicate that inhibition of Na +/Ca 2+ exchange likely prevented the activation of phospholipases that usually occurs following an ischemic insult as evidenced by its attenuation of phosphoethanolamine and free fatty acid efflux. The inhibition of phospholipases may be an essential component of the neuroprotective benefits of Na +/Ca 2+ exchange inhibitors in ischemia–reperfusion injury and may provide a basis for their possible use in therapeutic strategies for stroke.

ATP from glycolysis is required for normal sodium homeostasis in resting fast-twitch rodent skeletal muscle

Myocellular sodium homeostasis is commonly disrupted during critical illness for unknown reasons. Recent data suggest that changes in intracellular sodium content and the amount of ATP provided by glycolysis are closely related. The role of glycolysis and oxidative phosphorylation in providing fuel to the Na(+)-K(+) pump was investigated in resting rat extensor digitorum longus muscles incubated at 30 degrees C for 1 h. Oxidative inhibition with carbonyl cyanide m-chlorophenylhydrazone, known as CCCP (0.2 microM), or by hypooxygenation did not alter myocellular sodium or potassium content ([Na(+)](i), [K(+)](i), respectively), whereas treatment with iodoacetic acid (0.3 mM), which effectively blocked glycolysis, dramatically increased [Na(+)](i) and the [Na(+)](i)/[K(+)](i) ratio. Experiments using ouabain and measurements of myocellular high-energy phosphates indicate that Na(+)-K(+)-ATPase activity is only impaired when glycolysis is inhibited. The data suggest that normal glycolysis is required to regulate intracellular sodium in fast-twitch skeletal muscles, because it is the predominant source of the fuel for the Na(+)-K(+)-ATPase.

Alloxan decreases intracellular potassium content of the isolated frog skin epithelium

Alloxan has been widely used to provoke diabetes mellitus. This compound induces necrosis of the β-pancreatic cells and the renal tubules. However, the mechanism of this action has not been fully established. There is some evidence that this drug may act by an alteration of several ionic transport mechanisms. Nevertheless, there is scant information on the effect of alloxan on these ionic transport mechanisms of the membrane in epithelial cells. We reported that this drug induces a decrease in sodium transport in the frog skin. In order to obtain information about the mechanism involved in the sodium transport diminution provoked by alloxan, in this study the function of Na +–K +ATPase enzyme on transepithelial sodium transport altered by alloxan is explored. We measured changes in the short circuit current and in the intracellular content of sodium and potassium under conditions of maximally stimulated enzyme activity. Short circuit current was not modified by the treatment with alloxan during the period of highest activity of the enzyme, suggesting a site of action independent of this ATPase. Cell potassium was reduced in alloxan-treated epithelia, without significant changes in Na + content. This finding points out the existence of an alteration induced by alloxan of some modulator mechanisms of the intracellular K + concentration. The treatment of the frog skin with cesium chloride, a K + channel blocker, prevented the decrease of Na + transport produced by alloxan. This result suggests an action of this diabetogenic drug on the K + channels of the frog skin epithelium.

Analysis of high intracellular [Na+]-induced release of [3H]noradrenaline in rat hippocampal slices.

Our aim was to investigate the mechanisms involved in the high intracellular sodium-induced transmitter release in the CNS through the characterisation of the veratridine-evoked (40 microM) noradrenaline release from rat hippocampal slices. The response to veratridine was completely inhibited by tetrodotoxin (1 microM), indicating that the effect is due to the activation of sodium channels. Omission of Ca2+ from the superfusion fluid inhibited the veratridine-evoked release by 72%, showing that the majority of release results from external Ca2+-dependent exocytosis. The residual Ca2+-independent release was not blocked by the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester (100 microM) suggesting that intracellular Ca2+ stores are not involved in this component of veratridine effect. The noradrenaline uptake blockers, desipramine (10 microM) and nisoxetine (10 microM), inhibited the external Ca2+-independent release by 50 and 46%, respectively, indicating that the release partly originates from the reversal of transporters (carrier-mediated release). In contrast to uptake blockers, lowering the temperature, another possibility to inhibit transporter function, completely inhibited the effect of veratridine in the absence of Ca2+. Further experiments revealed that low temperature (20 and 12 degrees C) reduces the veratridine-induced increase of intracellular sodium concentration ([Na+]i) in rat cortical synaptosomes (68 and 78% inhibition, respectively). The clinical relevance of our data is that during ischemia a massive release of transmitters occurs mainly due to the elevation of [Na+]i, which contributes to the development of ischemic brain injury. Our results show that low temperature may be a better therapeutic approach to the treatment of ischemia because it has a dual action on this process. Firstly, it inhibits the function of uptake transporters and hence reduces the carrier-mediated outflow of transmitters. Secondly, it inhibits the sodium influx and therefore prevents the unwanted elevation of [Na+]i. Our data also suggest that veratridine stimulation can be a suitable model for ischemic conditions.

Hypoglycemia enhances ionotropic but reduces metabotropic glutamate responses in substantia nigra dopaminergic neurons.

It is widely accepted that energy deprivation causes a neuronal death that is mainly determined by an increase in the extracellular level of glutamate. Consequently an excessive membrane depolarization and a rise in the intracellular concentration of sodium and calcium are produced. In spite of this scenario, the function of excitatory and inhibitory amino acids during an episode of energy failure has not been studied yet at a cellular level. In a model of cerebral hypoglycemia in the rat substantia nigra pars compacta, we measured neuronal responses to excitatory amino acid agonists. Under single-electrode voltage-clamp mode at -60 mV, the application of the ionotropic glutamate receptor agonists N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid, kainate, and the metabotropic group I agonist (S)-3,5-dihydroxyphenilglycine (DHPG) produced reversible inward currents in the dopaminergic cells. In addition, an outward current was caused by the superfusion of the metabotropic GABA(B) agonist baclofen. Glucose deprivation enhanced the inward responses caused by each ionotropic glutamate agonist. In contrast, hypoglycemia depressed the DHPG-induced inward current and the baclofen-induced outward current. These effects of hypoglycemia were reversible. To test whether a failure of the Na(+)/K(+) ATPase pump could account for the modification of the agonist-induced currents during hypoglycemia, we treated the midbrain slices with strophanthidin (1-3 microM). Strophanthidin enhanced the inward currents caused by glutamate agonists. However, it did not modify the GABA(B)-induced outward current. Our data suggest that glucose deprivation enhances the inward current caused by the stimulation of ionotropic glutamate receptors while it dampens the responses caused by the activation of metabotropic receptors. Thus a substantial component of the augmented neuronal response to glutamate, during energy deprivation, is very likely due to the failure of Na(+) and Ca(2+) extrusion and might ultimately favor excitotoxic processes in the dopaminergic cells.

Changes in cell volume and internal sodium concentration in HeLa cells during exponential growth and following lonidamine treatment

Cell volumes decreased in HeLa cells as a function of time after seeding during exponential growth. Cell volume distributions revealed the presence of two cell populations in all stages of growth. When cells approached confluence, the ratio of the two populations abruptly shifted towards that characterised by the smallest volume. Percentages of G1-, S- and G2 + M-phase cells were also measured and it was found that G1 frequency increased as a function of cell density during exponential growth. Intracellular sodium concentration, [Na]i was monitored by 23Na NMR in the presence of 5 mM dysprosium (III) tripolyphosphate. [Na]i increased from 22.8 to 59.0 mM in cells from the second to the seventh day after seeding. Treatment with lonidamine, an antitumoral drug that it is known to slow down cell growth by affecting aerobic glycolysis, produced a complete block of cell progression after a few days of treatment. The progression of cell volume distributions towards smaller volumes and the increase in internal sodium concentration as a function of time after seeding were also affected by the drug. These phenomena were related to the existence of a subpopulation of mitotically inactive G1-phase cells during exponential growth, pointing out that a density-dependent cellular mechanism regulates the cell cycling in HeLa cells.

Sodium-lithium countertransport activity is linked to chromosome 5 in baboons.

The genes involved in the regulation of cellular sodium transport characteristics, which are correlated with some forms of essential hypertension, have not yet been identified. We are studying the genes and environmental factors that affect red blood cell sodium-lithium countertransport (SLC) activity and intracellular sodium (ICNa) concentration in 634 baboons that comprise 11 pedigrees of 2 and 3 generations each. To detect and locate possible quantitative trait loci (QTLs) that affect SLC activity and ICNa concentration, we performed a genome screen by using a maximum likelihood-based variance-components linkage analysis program (SOLAR). SLC and ICNa phenotypes as well as genotypes on 281 microsatellite loci were available for all pedigreed animals. Both SLC and ICNa traits were highly heritable (residual heritability 0.593+/-0.083 [P<0.0001] and 0.739+/-0.082 [P<0.0001], respectively). We obtained evidence that a possible QTL for SLC activity is located on the baboon homologue of human chromosome 4 between D4S2456 and D4S2365 with a maximum multipoint lod score of 9.3 (P<10(-)(10)) near D4S1645. This QTL accounts for approximately two thirds of the total additive genetic variation in SLC activity in baboons. Although ICNa concentration was highly heritable, we found no evidence for linkage to a QTL with use of this methodology. Thus, we have evidence that a gene located on the baboon homologue of human chromosome 4 (baboon chromosome 5) affects cell sodium transport in baboons.

Short-term regulation of the proximal tubule Na+,K+-ATPase: increased/decreased Na+,K+-ATPase activity mediated by protein kinase C isoforms.

In different species and tissues, a great variety of hormones modulate Na+,K+-ATPase activity in a short-term fashion. Such regulation involves the activation of distinct intracellular signaling networks that are often hormone- and tissue-specific. This minireview focuses on our own experimental observations obtained by studying the regulation of the rodent proximal tubule Na+,K+-ATPase. We discuss evidence that hormones responsible for regulating kidney proximal tubule sodium reabsorption may not affect the intrinsic catalytic activity of the Na+,K+-ATPase, but rather the number of active units within the plasma membrane due to shuttling Na+,K+-ATPase molecules between intracellular compartments and the plasma membrane. These processes are mediated by different isoforms of protein kinase C and depend largely on variations in intracellular sodium concentrations.

Biology of the 2Na+/1H+ antiporter in invertebrates.

The functional expression of membrane transport proteins that are responsible for exchanging sodium and protons is a ubiquitous phenomenon. Among vertebrates the Na+/H+ antiporter occurs in plasma membranes of polarized epithelial cells and non-polarized cells such as red blood cells, muscle cells, and neurons, and in each cell type the transporter exchanges one sodium for one hydrogen ion, is inhibited by amiloride, and regulates intracellular pH and sodium concentration within tight limitations. In polarized epithelial cells this transporter occurs in two isoforms, each of which is restricted to either the brush border or basolateral cell membrane, and perform somewhat different tasks in the two locations. In prokaryotic cells, sodium/proton exchange occurs by an electrogenic 1Na+/2H+ antiporter that is coupled to a primary active proton pump and together these two proteins are capable of tightly regulating the intracellular concentrations of these cations in cells that may occur in environments of 4 M NaCl or pH 10-12. Invertebrate epithelial cells from the gills, gut, and kidney also exhibit electrogenic sodium/proton exchange, but in this instance the transport stoichiometry is 2Na+/1H+. As with vertebrate electroneutral Na+/H+ exchange, the invertebrate transporter is inhibited by amiloride, but because of the occurrence of two external monovalent cation binding sites, divalent cations are able to replace external sodium and also be transported by this system. As a result, both calcium and divalent heavy metals, such as zinc and cadmium, are transported across epithelial brush border membranes in these animals and subsequently undergo a variety of biological activities once accumulated within these cells. Absorbed epithelial calcium in the crustacean hepatopancreas may participate in organismic calcium balance during the molt cycle and accumulated heavy metals may undergo complexation reactions with intracellular anions as a detoxification mechanism. Therefore, while the basic process of sodium/proton exchange may occur in invertebrate cells, the presence of the electrogenic 2Na+/1H+ antiporter in these cells allows them to perform a wide array of functions without the need to develop and express additional specialized transport proteins. J. Exp. Zool. 289:232-244, 2001.

Heavy metal cytotoxicity studied by electron probe X-ray microanalysis of cultured rat hepatocytes

Cytotoxicity of the heavy metals gold, mercury, thallium and lead was studied by measuring the intracellular element distribution of cultured rat hepatocytes by energy dispersive electron probe X-ray microanalysis of freeze-dried cryosections in a scanning transmission electron microscope. Exposure of the cells to aqueous solutions containing heavy metal ions in concentrations reaching a critical concentration caused increase of intracellular sodium and chloride content accompanied or followed by decrease of intracellular potassium content. Thus, the intracellular potassium/sodium ratio drastically decreased from control values of approximately 10 to values below 1 before changes of cell morphology became visible. In experiments with gold or mercury the decrease of the potassium/sodium ratio was preceded by transient cytoplasmic increase of sulfur and phosphorus. Heavy metal concentrations exceeding the critical concentration also caused an increase of cytoplasmic calcium concentration and finally decay of the cell structure. Cytotoxicity of heavy metals was found to increase in the order Pb, Au, Tl, Hg. Cytotoxic effects by Au, Tl or Hg in moderate concentrations were reduced by simultaneous addition of Zn or Pb to the culture medium. The results obtained prove electron probe X-ray microanalysis of cryosections as a sensitive probe of cell viability.

Fatty Acid Cytotoxicity to Bovine Lens Epithelial Cells: Investigations on Cell Viability, ecto-ATPase, Na+, K+-ATPase and Intracellular Sodium Concentrations

Unsaturated non-esterified fatty acids have been shown to be cytotoxic in micromolar concentrations to bovine lens epithelial cells, in the following order: arachidonic acid>linoleic acid>oleic acid=linolenic acid. As unsaturated free fatty acids are known to be Na+, K+-ATPase inhibitors, the aim of the study was to investigate whether or not the fatty acid cytotoxicity is correlated with effects on Na+, K+-ATPase activity and function in bovine lens epithelial cells. Furthermore, we also examined the effects of linoleic acid on an ecto-ATPase activity which could be demonstrated on the outside of primarily cultured bovine lens epithelial cells. It has already been shown that 10μ mol l−1linoleic acid was cytotoxic but did not impair the ecto-ATPase activity of intact cells nor the Na+, K+-ATPase in enriched membrane fractions. Na+, K+-ATPase activity was slightly activated with 10μ mol l−1linoleic acid and inhibited by about 50% with 100μ mol l−1. Using the sodium-binding benzofuran isophthalate, measurements of intracellular sodium concentrations were carried out. In serum-starved bovine lens epithelial cells the basal [Na+]inwas clearly lower than 5mmol l−1. When the function of the Na+, K+-ATPase was interrupted by omitting K+-ions from the medium, [Na+]inincreased at a rate of 0.318mmol l−1min−1. Linoleic acid intensified that increase strongly in a concentration dependent manner. However, in K+-containing medium the linoleic acid-induced increase of [Na+]inwas completely prevented. Therefore, the high linoleioc acid cytotoxicity cannot be mediated by linoleic acid effects on Na+, K+-ATPase activity and function in bovine lens epithelial cells.