Extracellular Concentrations Of Na Research Articles

Mechanism of Action of Nerve Growth Factor Induced Histamine Release from Mast Cells during the Inflammation Process

Mast cells and the biogenic amine, histamine, play a central role in inflammatory reactions. Nerve growth factor (NGF) is essetial for the survival of neurones and has significant role in inflammation and repair of tissues. It has been shown, that NGF induces degranulation of mast cells by interacting with tropomyosine-receptor kinase A (TrkA receptor). NGF can induce release of histamine and other mediators from mast cells or enhance secretion induced by different stimuli. The results of our and others previous studies show, that histamine release from mast cells induced by NGF is strongly dependent on the concentration of extracellular Na+ , Ca2+ ions. The extracellular Na+ ions can affect the activity of Na+ /Ca2+ exchanger of mast cells. Na+ , Ca2+ exchange mechanisms could be important in secretory process of mast cells. The studies of different signaling molecules in NGF induced histamine relese have shown that tyrosine kinase, phosphatidylinositol-3-kinase, protein kinase C and phospholipase C are also involved in the signal transduction process responsible for NGF induced histamine secretion from mast cells. These data indicate that NGF may contribute in allergic reactions and many other inflammatory states.

ВЛИЯНИЕ Na + НА НАГРУЖЕННЫЕ КАЛЬЦИЕМ МИТОХОНДРИИ СЕРДЦА КРЫСЫ И МИОКАРД ЛЯГУШКИ

A comparative study of the effect of calcium load on rat heart mitochondria [RHM(K+)] in which a part of the K+ ions in their matrix were replaced by Na+ ions [RHM(Na+)] was carried out. Calcium loading of RHM(K+) decreased their 2,4-dinitrophenol-uncoupled respiration and reduced the inner membrane potential (∆Ψmito). Swelling of these mitochondria increased in media with 25 mM potassium acetate or 125 mM NH4NO3. These effects of calcium loading were even greater in similar experiments with RHM(Na+). Inhibitors of the mitochondrial permeability transition pore (MPTP), ADP and cyclosporin A (CsA), abolished the above-mentioned effects of Ca2+ completely in experiments with RHM(K+) and only partially in experiments with RHM(Na+). A positive inotropic effect was observed with an increase in the extracellular concentration of Na+, however pre-incubation in a calcium-free solution led to a negative inotropic effect. Thus, the partial replacement of K+ by Na+ in the matrix made rat heart mitochondria more sensitive to Ca2+ and increased the probability of MPTP opening in their inner membrane. Along with an elevation of cytoplasmic [Na+]i, this can further increase calcium overload of cardiomyocytes, making their damage during ischemia/reperfusion more likely.

PH during non-synaptic epileptiform activity—computational simulations

The excitability of neuronal networks is strongly modulated by changes in pH. The origin of these changes, however, is still under debate. The high complexity of neural systems justifies the use of computational simulation to investigate mechanisms that are possibly involved. Simulated neuronal activity includes non-synaptic epileptiform events (NEA) induced in hippocampal slices perfused with high-K+ and zero-Ca2+, therefore in the absence of the synaptic circuitry. A network of functional units composes the NEA model. Each functional unit represents one interface of neuronal/extracellular space/glial segments. Each interface contains transmembrane ionic transports, such as ionic channels, cotransporters, exchangers and pumps. Neuronal interconnections are mediated by gap-junctions, electric field effects and extracellular ionic fluctuations modulated by extracellular electrodiffusion. Mechanisms investigated are those that change intracellular and extracellular ionic concentrations and are able to affect [H+]. Our simulations suggest that the intense fluctuations in intra and extracellular concentrations of Na+, K+ and Cl− that accompany NEA are able to affect the combined action of the Na+/H+ exchanger (NHE), /Cl− exchanger (HCE), H+ pump and the catalytic activity of intra and extracellular carbonic anhydrase. Cellular volume changes and extracellular electrodiffusion are responsible for modulating pH.

Changes in Erythrocytic Membrane Free Energy of Albino Rabbits Administered Ethanol Leaf Extract of Spondias mombin Linn

Aims: To determine the effect of ethanol extract of Spondias mombin Linn on membrane potential energy, the intracellular and extracellular concentrations of Na

Functional analysis of the human concentrative nucleoside transporter-1 variant hCNT1S546P provides insight into the sodium-binding pocket

SLC28 genes, encoding concentrative nucleoside transporter proteins (CNT), show little genetic variability, although a few single nucleotide polymorphisms (SNPs) have been associated with marked functional disturbances. In particular, human CNT1S546P had been reported to result in negligible thymidine uptake. In this study we have characterized the molecular mechanisms responsible for this apparent loss of function. The hCNT1S546P variant showed an appropriate endoplasmic reticulum export and insertion into the plasma membrane, whereas loss of nucleoside translocation ability affected all tested nucleoside and nucleoside-derived drugs. Site-directed mutagenesis analysis revealed that it is the lack of the serine residue itself responsible for the loss of hCNT1 function. This serine residue is highly conserved, and mutation of the analogous serine in hCNT2 (Ser541) and hCNT3 (Ser568) resulted in total and partial loss of function, respectively. Moreover, hCNT3, the only member that shows a 2Na(+)/1 nucleoside stoichiometry, showed altered Na(+) binding properties associated with a shift in the Hill coefficient, consistent with one Na(+) binding site being affected by the mutation. Two-electrode voltage-clamp studies using the hCNT1S546P mutant revealed the occurrence of Na(+) leak, which was dependent on the concentration of extracellular Na(+) indicating that, although the variant is unable to transport nucleosides, there is an uncoupled sodium transport.

Effects of Changes in Extracellular Concentration of Na+ and K+ ([Na+]o, [K+]o) on the Ca2+ Release Elicited by High Frequency Stimulation. Implications for Muscle Fatigue

Changes in [Na+]o and [K+]o occurring during high frequency stimulation has been proposed as a cause of muscle fatigue. We investigated this hypothesis by measuring the Ca2+ release elicited by short high frequency trains (100Hz, 10 pulses) in rested frog semitendinosus fibers exposed to various [Na+]o or [K+]o. Myoplasmic [Ca2+] changes (Ca2+ transients) elicited by action potentials (AP) were estimated from Ca2+-dependent OGB-5N fluorescence changes. Segments of fibers, stretched to 4.5-5μm, were mounted in an inverted double grease-gap chamber placed in an inverted microscope equipped for epifluorescence. Fibers were held at -100mV and stimulated with 0.5ms current pulses. Normal Ringer solution contained (mM): 115 NaCl, 2.5 KCl, 1.8 CaCl2, 10 MOPS, 10 dextrose, pH=7.2 with NaOH. [K+]o ([Na+]o) was increased (reduced) by equimolar replacement with Na+ (N-methyl-D-glucamine). Fibers were loaded (30min) with a solution containing (mM): 110 aspartate, 5 ATP-K2, 5 Na2-creatine-phosphate, 20 MOPS, 0.05-0.1 EGTA, 5 MgCl2, 0.2 OGB-5N, pH=7.2 with KOH. A complex interaction of the effects of changing [K+]o or [Na+]o on membrane potential, AP overshoot and duration, and Ca2+ release was found. Using normal Ringer, the amplitude of Ca2+ transients elicited by single pulses increased with depolarizations up to -65mV. Raising [K+]o had a dual effect on Ca2+ release. Ca2+ transient's amplitude increased between 2.5 to 10 mM, and decreased markedly for higher [K]o. Potentiation of Ca2+ release, but not depression, could be reverted by current injection. This suggests a depolarization independent effect of K+ ions on Ca2+ release. Halving [Na+]o did not affect Ca2+ release elicited by single stimulation, but impaired the release in response to repetitive stimulation. Further reduction of [Na+]o to 1/3 of normal highly reduced Ca2+ release.

Sodium self-inhibition of human epithelial sodium channel: selectivity and affinity of the extracellular sodium sensing site

The epithelial Na(+) channel (ENaC) is present in the apical membrane of "tight" epithelia in the distal nephron, distal colon, and airways. Its activity controls the rate of transepithelial sodium transport. Among other regulatory factors, ENaC activity is controlled by the concentration of extracellular Na(+), a phenomenon named self-inhibition. The molecular mechanism by which extracellular Na(+) concentration is detected is not known. To investigate the properties of the extracellular Na(+) sensing site, we studied the effects of extracellular cations on steady-state amiloride-sensitive outward currents in Na(+)-loaded oocytes expressing human ENaC and compared them with self-inhibition of inward current after fast solution changes. About half of the inhibition of outward Na(+) currents was due to self-inhibition itself and the rest might be attributed to conduction site saturation. Self-inhibition by extracellular Li(+) was similar to that of Na(+) except for slightly slower kinetics. Ionic selectivity of the inhibition for steady-state outward current was Na(+) > or = Li(+) > K(+). We estimated an apparent inhibitory constant (K(I)) of approximately 40 mM for extracellular Na(+) and Li(+) and found no evidence for a voltage dependence of the K(I). Protease treatment induced the expected increase of the amiloride-sensitive current measured in high-Na(+) concentrations which was due, at least in part, to abolition of self-inhibition. These results demonstrate that both self-inhibition and saturation play a significant role in the inhibition of ENaC by extracellular Na(+) and that Na(+) and Li(+) interact in a similar way with the extracellular cation sensing site.

Sensing Salt, Sending Signals

To survive, cells and organisms must regulate concentrations of Na + within a range compatible with physiological functions. Shimizu et al . show that Na + sensing occurs in part through an unusual mechanism in the mammalian brain in which neuronal activity is controlled by associated glial cells. Mammals appear to sense Na + concentrations in the blood through special brain structures known as circumventricular organs, in particular the subfornical organ (SFO), where a blood-brain barrier is not present, allowing cells to sense the chemical makeup of the blood. Sodium channels known as Na x channels are required for proper behavioral responses to excess Na + , but these channels are expressed on astrocytes rather than neurons. The authors used a yeast two-hybrid screen to detect proteins that interacted with the cytoplasmic domain of Na x . One of the interacting proteins was identified as the cell membrane sodium pump (Na + /K + -ATPase). In cultured cells expressing Na x , increased concentrations of extracellular Na + caused increased activity of the Na + /K + -ATPase. Suspecting that the association of Na x with the Na + /K + -ATPase might influence cellular energy metabolism, the authors showed that exposure of slices from the SFO of wild-type mice to excessive extracellular Na + caused an increase in glucose uptake that was absent in SFO slices from animals lacking Na x . This enhanced glucose use led to increased lactate secretion in the slices. The lactate in turn was found to increase the firing frequency of GABAergic neurons in the SFO slices. The effect on the neurons was reduced when activity of the transporter that carries lactate into the neurons was inhibited. Thus, the authors conclude that Na + sensing in the SFO results from glial cells that are the key sensors, which then signal to neighboring neurons by secretion of lactate. Commentary by Iadecola places the work in the context of other instances of lactate signaling in neurons and other physiological mechanisms that cooperate to keep Na + concentrations in check. H. Shimizu, E. Watanabe, T. Y. Hiyama, A. Nagakura, A. Fujikawa, H. Okado, Y. Yanagawa, K. Obata, M. Noda, Glial Na x channels control lactate signaling to neurons for brain [Na + ] sensing. Neuron 54 , 59-72 (2007). [Online Journal] C. Iadecola, Astrocytes take center stage in salt sensing. Neuron 54 , 3-5 (2007). [Online Journal]

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.

Intracellular and Extracellular Concentrations of Na+ Modulate Mg2+ Transport in Rat Ventricular Myocytes

Apparent free cytoplasmic concentrations of Mg2+ ([Mg2+]i) and Na+ ([Na+]i) were estimated in rat ventricular myocytes using fluorescent indicators, furaptra (mag-fura-2) for Mg2+ and sodium-binding benzofuran isophthalate for Na+, at 25°C in Ca2+-free conditions. Analysis included corrections for the influence of Na+ on furaptra fluorescence found in vitro and in vivo. The myocytes were loaded with Mg2+ in a solution containing 24mM Mg2+ either in the presence of 106mM Na+ plus 1mM ouabain (Na+ loading) or in the presence of only 1.6mM Na+ to deplete the cells of Na+ (Na+ depletion). The initial rate of decrease in [Mg2+]i from the Mg2+-loaded cells was estimated in the presence of 140mM Na+ and 1mM Mg2+ as an index of the rate of extracellular Na+-dependent Mg2+ efflux. Average [Na+]i, when estimated from sodium-binding benzofuran isophthalate fluorescence in separate experiments, increased from 12 to 31mM and 47mM after Na+ loading for 1 and 3h, respectively, and decreased to ∼0mM after 3h of Na+ depletion. The intracellular Na+ loading significantly reduced the initial rate of decrease in [Mg2+]i, on average, by 40% at 1h and by 64% at 3h, suggesting that the Mg2+ efflux was inhibited by intracellular Na+ with 50% inhibition at ∼40mM. A reduction of the rate of Mg2+ efflux was also observed when Na+ was introduced into the cells through the amphotericin B-perforated cell membrane (perforated patch-clamp technique) via a patch pipette that contained 130mM Na+. When the cells were heavily loaded with Na+ with ouabain in combination with intracellular perfusion from the patch pipette containing 130mM Na+, removal of extracellular Na+ caused an increase in [Mg2+]i, albeit at a very limited rate, which could be interpreted as reversal of the Mg2+ transport, i.e., Mg2+ influx driven by reversed Na+ gradient. Extracellular Na+ dependence of the rate of Mg2+ efflux revealed that the Mg2+ efflux was activated by extracellular Na+ with half-maximal activation at 55mM. These results contribute to a quantitative characterization of the Na+-Mg2+ exchange in cardiac myocytes.

Identification of potential regulatory sites of the Na+,K+-ATPase by kinetic analysis.

Kinetic models are presented that allow the Na(+),K(+)-ATPase steady-state turnover number to be estimated at given intra- and extracellular concentrations of Na(+), K(+), and ATP. Based on experimental transient kinetic data, the models utilize either three or four steps of the Albers-Post scheme, that is, E(2) --> E(1), E(1) --> E(2)P (or E(1) --> E(1)P and E(1)P --> E(2)P), and E(2)P --> E(2), which are the major rate-determining steps of the enzyme cycle. On the time scale of these reactions, the faster binding steps of Na(+), K(+), and ATP to the enzyme are considered to be in equilibrium. Each model was tested by comparing calculations of the steady-state turnover from rate constants and equilibrium constants for the individual partial reactions with published experimental data of the steady-state activity at varying Na(+) and K(+) concentrations. To provide reasonable agreement between the calculations and the experimental data, it was found that Na(+)/K(+) competition for cytoplasmic binding sites was an essential feature required in the model. The activity was also very dependent on the degree of K(+)-induced stimulation of the reverse reaction E(1) --> E(2). Taking into account the physiological substrate concentrations, the models allow the most likely potential sites of short-term Na(+),K(+)-ATPase regulation to be identified. These were found to be (a) the cytoplasmic Na(+) and K(+) binding sites, via changes in Na(+) or K(+) concentration or their dissociation constants, (b) ATP phosphorylation (as a substrate), via a change in its rate constant, and (c) the position of the E(2)<==>E(1) equilibrium.

Synthesis of functionalised macrocyclic compounds as Na+ and K+ receptors: a mild and high yielding nitration in water of mono and bis 2-methoxyaniline functionalised crown ethers

The syntheses of the bis-aromatic 15-crown-5 and 18-crown-6-ether derivatives 1 and 2, for the selective recognition of intra- and extracellular concentrations of Na+ and K+, from o-anisidine are described. These compounds were made with the aim of incorporating them into luminescent sensors. To facilitate their incorporation into such sensors, the compounds were nitrated. Whereas the use of conventional nitration reagents such as HNO3, HNO3–H2SO4 or NO2BF4 only gave low yields of the desired products, both mono- and bis-nitration products were formed in good yield by one-pot synthesis using NaNO2 in a mixture of water and acetic acid (10 ∶ 1). The use of X-ray crystallography proved the presence of the nitro-substituted products. An investigation into this mild nitration method revealed that the substitution pattern on the aromatic ring governed the ability of the compounds to undergo nitration.

Na self inhibition of human epithelial Na channel: temperature dependence and effect of extracellular proteases.

The regulation of the open probability of the epithelial Na+ channel (ENaC) by the extracellular concentration of Na+, a phenomenon called “Na+ self inhibition,” has been well described in several natural tight epithelia, but its molecular mechanism is not known. We have studied the kinetics of Na+ self inhibition on human ENaC expressed in Xenopus oocytes. Rapid removal of amiloride or rapid increase in the extracellular Na+ concentration from 1 to 100 mM resulted in a peak inward current followed by a decline to a lower quasi-steady-state current. The rate of current decline and the steady-state level were temperature dependent and the current transient could be well explained by a two-state (active-inactive) model with a weakly temperature-dependent (Q10act = 1.5) activation rate and a strongly temperature-dependant (Q10inact = 8.0) inactivation rate. The steep temperature dependence of the inactivation rate resulted in the paradoxical decrease in the steady-state amiloride-sensitive current at high temperature. Na+ self inhibition depended only on the extracellular Na+ concentration but not on the amplitude of the inward current, and it was observed as a decrease of the conductance at the reversal potential for Na+ as well as a reduction of Na+ outward current. Self inhibition could be prevented by exposure to extracellular protease, a treatment known to activate ENaC or by treatment with p-CMB. After protease treatment, the amiloride-sensitive current displayed the expected increase with rising temperature. These results indicate that Na+ self inhibition is an intrinsic property of sodium channels resulting from the expression of the α, β, and γ subunits of human ENaC in Xenopus oocyte. The extracellular Na+-dependent inactivation has a large energy of activation and can be abolished by treatment with extracellular proteases.

Dopamine-induced inhibition of Na+-K+-ATPase activity requires integrity of actin cytoskeleton in opossum kidney cells.

The present study evaluated the importance of the association between Na+-K+-ATPase and the actin cytoskeleton on dopamine-induced inhibition of Na+-K+-ATPase activity. The approach used measures the transepithelial transport of Na+ in monolayers of opossum kidney (OK) cells, when the Na+ delivered to Na+-K+-ATPase was increased at the saturating level by amphotericin B. The maximal amphotericin B (1.0 microg mL-1) induced increase in short-circuit current (Isc) was prevented by ouabain (100 microM) or removal of apical Na+. Dopamine (1 microM) applied from the apical side significantly decreased (29 +/- 5% reduction) the amphotericin B-induced increase in Isc, this being prevented by the D1-like receptor antagonist SKF 83566 (1 microM) and the protein kinase C (PKC) inhibitor chelerythrine (1 microM). Exposure of OK cells to cytochalasin B (1 microM) or cytochalasin D (1 microM), inhibitors of actin polymerization, from both cell sides reduced by 31 +/- 4% and 36 +/- 3% the amphotericin B-induced increase in Isc and abolished the inhibitory effect of apical dopamine (1 microM), but not that of the PKC activator phorbol-12,13-dibutyrate (PDBu; 100 nM). Colchicine (1 microM) failed to alter the inhibitory effects of dopamine. The relationship between Na+-K+-ATPase and the concentration of extracellular Na+ showed a Michaelis-Menten constant (Km) of 44.1 +/- 13.7 mM and a Vmax of 49.6 +/- 4.8 microA cm-2 in control monolayers. In the presence of apical dopamine (1 microM) or cytochalasin B (1 microM) Vmax values were significantly (P < 0.05) reduced without changes in Km values. These results are the first, obtained in live cells, showing that the PKC-dependent inhibition of Na+-K+-ATPase activity by dopamine requires the integrity of the association between actin cytoskeleton and Na+-K+-ATPase.

Gender Differences in Vascular Smooth Muscle Reactivity to Increases in Extracellular Sodium Salt

Hypertension is more common in men and postmenopausal women than in premenopausal women, and gender differences in sensitivity to high dietary Na(+) salt have been suggested; however, the vascular mechanisms involved are unclear. We investigated whether increases in the extracellular concentration of Na(+) ([Na(+)](e)) enhance the mechanisms of vascular smooth muscle contraction and whether the vascular effects of [Na(+)](e) exhibit gender differences. Isometric contraction and (45)Ca(2+) influx were measured in endothelium-denuded aortic strips that were isolated from intact male, intact female, castrated male, and ovariectomized (OVX) female Sprague-Dawley rats and incubated in Krebs' solution (2.5 mmol/L Ca(2+)) containing increasing [Na(+)](e) by the addition of 1, 3, 6, 10, 20, and 30 mmol/L NaCl. Increasing [Na(+)](e) for 30 minutes did not increase the resting tone or (45)Ca(2+) influx in any group of rats. Phenylephrine (Phe) caused concentration-dependent increases in contraction and (45)Ca(2+) influx. In vascular strips from intact males, increasing [Na(+)](e) by the addition of 1 to 6 mmol/L NaCl significantly increased the magnitude of Phe contraction and (45)Ca(2+) influx. Further increases in [Na(+)](e) by the addition of 10, 20, and 30 mmol/L NaCl increased Phe-induced (45)Ca(2+) influx but inhibited Phe contraction, possibly because of excessive increases in ionic strength. Preincubation with 2,4-dichlorobenzamil (10(-5) mol/L), inhibitor of the Na(+)-Ca(2+) exchanger, or KB-R7943 (10(-5) mol/L), selective inhibitor of the reverse mode of the Na(+)-Ca(2+) exchanger, abolished the increases in Phe contraction and (45)Ca(2+) influx at increasing [Na(+)](e) obtained by the addition of 1 to 6 mmol/L NaCl. Preincubation in Krebs' solution containing control [Na(+)](e) plus 1 to 6 mmol/L LiCl or N-methyl-D-glucamine did not increase Phe contraction. In intact females, the Phe contraction and Ca(2+) influx were less than those in intact males and were not enhanced with increases in [Na(+)](e). The enhancement of Phe contraction and Ca(2+) influx with increases in [Na(+)](e) were not significantly different between castrated male rats and intact male rats but were greater in OVX female rats than intact female rats. In OVX female rats or castrated male rats treated with 17beta-estradiol (but not 17alpha-estradiol) subcutaneous implants, no significant changes in Phe contraction or Ca(2+) influx with increases in [Na(+)](e) were observed. In OVX female or castrated male rats simultaneously treated with 17beta-estradiol plus the estrogen receptor antagonist ICI 182,780, the Phe contraction and Ca(2+) influx were enhanced with increases in [Na(+)](e). Thus, in intact male rats, small physiological increases in [Na(+)](e) enhance smooth muscle contraction to Phe by a mechanism involving Ca(2+) entry, possibly via the reverse mode of the Na(+)-Ca(2+) exchanger. This mechanism appears to be reduced in the presence of endogenous or exogenous estrogen and thereby protects female rats against excessive increases in vascular reactivity during high dietary Na(+) intake.

Modulation of Na+/H+ exchange activity by Cl-.

Na+/H+ exchanger (NHE) activity is exquisitely dependent on the intra- and extracellular concentrations of Na+ and H+. In addition, Cl- ions have been suggested to modulate NHE activity, but little is known about the underlying mechanism, and the Cl- sensitivity of the individual isoforms has not been established. To explore their Cl- sensitivity, types 1, 2, and 3 Na+/H+ exchangers (NHE1, NHE2, and NHE3) were heterologously expressed in antiport-deficient cells. Bilateral replacement of Cl- with nitrate or thiocyanate inhibited the activity of all isoforms. Cl- depletion did not affect cell volume or the cellular ATP content, which could have indirectly altered NHE activity. The number of plasmalemmal exchangers was unaffected by Cl- removal, implying that inhibition was due to a decrease in the intrinsic activity of individual exchangers. Analysis of truncated mutants of NHE1 revealed that the anion sensitivity resides, at least in part, in the COOH-terminal domain of the exchanger. Moreover, readdition of Cl- into the extracellular medium failed to restore normal transport, suggesting that intracellular Cl- is critical for activity. Thus interaction of intracellular Cl- with the COOH terminus of NHE1 or with an associated protein is essential for optimal activity.

Electrolyte Composition of Mink (Mustela vison) Erythrocytes and Active Cation Transporters of the Cell Membrane

Red blood cells from mink (Mustela vison) were characterized with respect to their electrolyte content and their cell membranes with respect to enzymatic activity for cation transport. The intra- and extracellular concentrations of Na+, K+, Cl-, Ca2+ and Mg2+ were determined in erythrocytes and plasma, respectively. Plasma and red cell water content was determined, and molal electrolyte concentrations were calculated. Red cells from male adult mink appeared to be of the low-K+, high-Na+ type as seen in other carnivorous species. The intracellular K+ concentration is slightly higher than the extracellular one and the plasma-to-cell chemical gradient for Na+ is weak, though even the molal concentrations may differ significantly. Consistent with the high intracellular Na+ and low K+ concentrations, a very low or no ouabain-sensitive Na+,K+-ATPase activity and no K+-activated pNPPase activity were found in the plasma membrane fraction from red cells. The Cl- and Mg2+ concentrations expressed per liter cell water were significantly higher in red cells than in plasma whereas the opposite was the case with Ca2+. The distribution of Cl- thus does not seem compatible with an inside-negative membrane potential in mink erythrocytes. In spite of a steep calcium gradient across the red cell membrane, neither a calmodulin-activated Ca2+-ATPase activity nor an ATP-activated Ca2+-pNPPase activity were detectable in the plasma membrane fraction. The origin of a supposed primary Ca2+ gradient for sustaining of osmotic balance thus seems uncertain.

Amphetamine increases the extracellular concentration of glutamate in striatum of the awake rat: involvement of high affinity transporter mechanisms

Using microdialysis it was found that intracerebral infusions of amphetamine increase the extracellular concentration of glutamate, and also of dopamine, aspartate, GABA, and taurine. The increases in glutamate produced by amphetamine was independent of calcium in the perfusion medium but was significantly attenuated by specific blockers of the high affinity transporters of this neurotransmitter. Amphetamine infusions also produced a decrease in the extracellular concentration of Na +, an increase in the extracellular concentration of lactate, and a decrease in haemoglobin in the area of perfusion. All these data suggest that amphetamine increases the extracellular concentration of glutamate and other neurotransmitters through a hypoxic mediated process. This study also shows that an alpha-noradrenergic receptor antagonist is able to attenuate the effects of amphetamine on the release of glutamate, dopamine, GABA and taurine, which further suggests a vasoconstrictor effect of amphetamine as a result of which hypoxia could develop.

Lithium regulation of protein phosphorylation in rat cerebral cortex slices in vitro.

The aim of the present study was to evaluate the direct effect of Li+ and Na+-Li+ exchange on protein phosphorylation in rat cerebral cortex slices incubated in Krebs Ringer medium. When Na+ concentration was varied in the incubation medium, either by replacement with Li+ or sucrose, a variable effect on [32P] phosphate incorporation into proteins was observed. Protein phosphorylation in cerebral cortex slices was very low in the absence of Na+, and some dependence of phosphorylating system of neural tissue on extra cellular concentration of Na+ was evident. Lithium was not able to replace sodium as far as protein phosphorylation in cortical slices is concerned. Ouabain was more effective in a Li+ containing medium in inhibiting protein phosphorylation, presumably due to improper functioning of the sodium pump.

Comparison of Methods for Measurement of Na+/Li+ Countertransport Across the Erythrocyte Membrane

About 30% of patients with insulin-dependent diabetes mellitus develop diabetic nephropathy. Since diabetic nephropathy contributes to a large extent to the high mortality of these patients, a risk marker for the development of this condition is desirable. Increase in Na+/Li+ countertransport across the red cell membrane has been suggested as such an early marker (1)(2)(3)(4), although this was not uniformly confirmed (5)(6)(7). In this study, the three main methods to load erythrocytes with Li+ were compared and tested for their measuring error and intrasubject variation of V max and K 0.5 for Na+. The “classic” LiCl loading [ 8 , 9] is the most “physiological” and noninvasive method. However, the loading procedure takes 3 h, which precludes the use of this method as a standard procedure. The Li2CO3 loading as described by Elving et al. (6) has the advantage that it takes only 30 min to load the cells with Li+. The Li+ enters the cell via the HCO3−/Cl− exchanger as a LiCO3− ion in exchange for a Cl− ion, which explains the fast Li+ loading. This method has been reported to give plots to which Michaelis–Menten kinetics apply (10). The nystatin method was developed by Canessa et al. (11) because at 150 mmol/L Na+ (the highest concentration that can be used at an osmolarity of 300 mosmol/L), the extracellular binding site for Na+ is often not saturated. With nystatin, an antifungal drug that penetrates the plasma membrane, the intra- and extracellular osmolarity can be raised to 600 mosmol/L, so extracellular concentrations of Na+ up to 300 mmol/L can be used. Because of this, K 0.5 values can in principle be measured more accurately than with the other methods. We …