Changes of intracellular Na+ concentration in erythrocytes caused by pulsed electrical field

Changes of sodium ionic concentration of human erythrocytes applied to pulsed electrical field (PEF) were studied by using shift reagent and NMR spectroscopy. The results show that the concentration of intracellular Na⁺ increases with the increasing intensity of PEF when the erythrocytes are applied to PEF with higher intensities. The relationship between intracellular Na concentrations and the intensities of PEF does not follow linear or exponen-tial behavior. As the intensities increase, the intracellular Na⁺concentrations increase even faster by an exponential curve. However under effects of PEF at lower intensities, intracellular Na⁺ concentration decreases. Ouabain can in-hibit the decrease of intracellular Na concentration, and the inhibition increases with the increasing concentration of ouabain, suggesting that Na⁺ , K⁺ -ATPase on cell membrane can be activated by PEF at lower intensities. Direct measurement of activities of the enzyme by using Malachite green method has confirmed this observation. Cell perme-abilities to ions, activation of enzymes by electrical fields and transmission of physical signals like PEF across cell mem-branes are discussed.     

Changes of intracellular Na~ concentration in erythrocytes caused by pulsed electrical field

Changes of sodium ionic concentration of human erythrocytes applied to pulsed electrical field (PEF) were studied by using shift reagent and NMR spectroscopy. The results show that the concentration of intracellular Na increases with the increasing intensity of PEF when the erythrocytes are applied to PEF with higher intensities. The relationship between intracellular Na concentrations and the intensities of PEF does not follow linear or exponen-tial behavior. As the intensities increase, the intracellular Na concentrations increase even faster by an exponential curve. However under effects of PEF at lower intensities, intracellular Na concentration decreases. Ouabain can in-hibit the decrease of intracellular Na concentration, and the inhibition increases with the increasing concentration of ouabain, suggesting that Na , K -ATPase on cell membrane can be activated by PEF at lower intensities. Direct measurement of activities of the enzyme by using Malachite green method has confirmed this observatio     

Effect of ouabain,amiloride, and antidiuretic hormone on the sodium-transport pool in isolated epithelia from frog skin (Rana temporaria)

Summary When tracer Na⁺ is added to the solution bathing the apical side of isolated epithelia the observed transepithelial tracer influx increases with time until a steady state is reached. The build-up of the tracer flux follows a single exponential course. The halftime for this build-up under control conditions was 0.92 ±0.06 min, and in the presence of ouabain 4.51±0.7 min. It is shown that the calculated Na⁺-transport pool is located in the cells. The Na⁺-transport pool under control conditions was 35.6 ±3.4 nmol/cm², which corresponds to an intracellular Na⁺ concentration of 7.9mm. Activation of the active Na⁺ transport by addition of antidiuretic hormone resulted in a highly significant increase in the Na⁺ transport pool, and inhibition of the transcellular Na⁺ transport with amiloride resulted in a decrease in the Na⁺-transport pool.Furthermore, the active Na⁺ transport increased along anS-shaped curve with increasing intracellular Na⁺ concentration (Na⁺-transport pool). The Na⁺ pump was found to be half saturated at an intracellular Na⁺ concentration of 12.5mm.     

Interactions between growth, Cl− and Na+ uptake, and water relations of plants in saline environments. I. Slightly vacuolated cells

Abstract. Slightly vacuolated cells, i.e. microalgae and meristematic cells of vascular plants, maintain low Cl^? and Na⁺ concentrations even when exposed to a highly saline environment. The factors regulating the internal ion concentration are the relative rate of volume expansion, the membrane permeability to ions, the electrical potential, and the active ion fluxes. For ion species which are not actively transported, a formula is developed which relates the internal concentration to the rate of expansion of cell volume, the permeability of membranes to that ion, and the electrical potential. For example, when the external concentration of Cl^? is high, and Cl^? influx is probably mainly passive, the formula predicts that rapid growth keeps the internal Cl^? concentration lower than that in a non-growing cell with the same electrical potential; this effect is substantial if the plasmalemma has a low permeability to Cl^?. For ion species which are actively transported, the rate of pumping must be considered. For instance Na⁺ concentrations are kept low mainly by an efficient Na⁺ extrusion pump which works against the electric field across the membrane. The requirement for Na⁺ extrusion is related to the external Na⁺ concentration, the rate of expansion of cell volume, the membrane permeability, and the electrical potential. It is possible that microalgae have a more positive electrical potential than many other plant cells; if so, requirements for high rates of active Na⁺ extrusion will be lower. The required rates of Na⁺ extrusion are lower during rapid growth, provided that the permeability of the plasmalemma to Na⁺ is low. The energy required for the regulation of Cl^? and Na⁺ concentrations is low, especially in rapidly expanding cells where Na⁺ extrusion requires only 1–2% of the energy normally produced in respiration. The exclusion of these ions, however, must be accompanied by the synthesis of enough organic compounds to provide adequate osmotic solutes for the increases in volume accompanying growth. This process reduces the substrates available for respiration and synthesis of cell constituents, but the reduction is not prohibitively large—even for cells growing in 750 mol m^?3 NaCl, the carbohydrate accumulated as osmotic solute is only 10% of that consumed in respiration.     

Participation of protein phosphatases in regulation of sodium transport across the erythrocyte membrane of the lamprey Lampetra fluviatilis

Erythrocytes of lamprey Lampetra fluviatilis were incubated in standard isotonic medium at 20°C with ²²Na to determine the unidirectional Na⁺ influx. Cell incubation in the presence of various protein phosphatase inhibitors (NaF, cantharidin, calyculin A) led to a considerable increase of Na⁺ transport into erythrocytes. The stimulation of Na⁺ influx into erythrocytes rose with increase of concentration of calyculin A within the range of 10–100 nM. The calyculin A concentration producing a 50% activation of Na⁺ transport amounted to 41.5 nM. Under optimal experimental conditions, the Na⁺ influx increased from control level of 5–8 to 20–40 mmol/l cells/h under effect of protein phosphatase blockers. The Na⁺ transport induced by these inhibitors was completely suppressed on addition of amiloride to the incubation medium. The treatment of lamprey erythrocytes with protein phosphatase inhibitors was accompanied by a small (～12%), but statistically significant decrease of intracellular Na⁺ content. A small decrease of intracellular K⁺ content in erythrocyte was observed only under the effect of NaF. The obtained data allow making the conclusion that protein phosphatases of the PP1 and PP2A types play a significant role in regulation of Na⁺ transport across the lamprey erythrocyte membrane in both directions.     

Cytochalasin B induces changes in cytoplasmic Na+/K+ balance of two-cell mouse embryos

Changes in intracellular elemental (Na, K) concentrations caused by cytochalasin B were measured by electron probe microanalysis. Cytochalasin B is applied to transfer somatic cell nuclei into early embryo cells. This chemical causes a cytoskeleton rearrangement that may activate potassium channels, which, in turn, results in a cytoplasmic Na⁺/K⁺ imbalance. Our study showed that cytochalasin B reduced the intracellular sodium concentration. After the exposure of the mouse embryo with Dulbecco’s solution free from chemical, the Na⁺/K⁺ balance in cytoplasm reached the initial level. Possible mechanisms of registered changes in intracellular Na⁺ concentration are discussed.     

Effects of growth conditions on the ion composition of Bifidobacterium bifidum subsp. pennsylvanicum

The cation content of Bifidobacterium bifidum subsp. pennsylvanicum was markedly influenced by the washing procedure of the cells, by the growth phase and the temperature, and by the composition of the culture medium. Optimal retention of cations was achieved by washing with 0.25 M MgCl₂ at 20 C. The intracellular Na⁺ concentration rose during growth in normal medium to a constant value in the stationary phase, the K⁺ concentration rose in the exponential phase, but fell in the stationary phase. Cells from 29-C cultures contained more Na⁺ and less K⁺ in the stationary phase than did cells from 37-C cultures, but the total cation content was the same at 29 and 37 C.Intracellular Na⁺ and K⁺ concentrations were dependent on the concentrations in the medium and on its osmolarity. The intracellular Na⁺/K⁺ ratio varied from 0.04 to 2.3. The concentrations of Na⁺, K⁺ and phosphate in the medium hardly affected growth. Mg²⁺-deficiency of the medium markedly decreased the concentration of Mg²⁺ within the cell; its concentration in the cell sap was greatly affected, but the amount of sedimentable, bound Mg²⁺ only slightly. The content of K⁺ within the cell decreased in Mg²⁺-deficient medium, but the concentration of Na⁺ did not. Omission of Tween 80 as well as its substitution by Tween 20 caused a decrease of intracellular K⁺. Cells from Tween 40 and Tween 60 cultures additionally contained markedly less Na⁺.The present investigations have been carried out with financial support from the Netherlands Organization for the Advancement of Pure Research (ZWO) through the Netherlands Foundation for Chemical Research (SON).     

Alteration of cellular ionic constituents by external ionic conditions,and its significance in the development ofDictyostelium discoideum

Effects of external ionic conditions ofD. discoideum cells were examined in relation to intracellular ionic concentrations, the activity of pyruvate kinase and the amount of ATP. Main components of metal cations in heat extracts of vegetative cells were K⁺, Na⁺, Mg²⁺ and Ca²⁺ whose concentrations in a cell were about 35.0, 3.6, 10.6 and 2.3 mM, respectively. External Na⁺ at the concentration more than 50 mM inhibited the formation of cell aggregates in the presence of 10^?4M Ca²⁺. Such an inhibitory effect of Na⁺ was completely nullified by the addition of more than 10 mM K⁺. External Na⁺ caused a rapid decrease in intracellular K⁺, but an increase in intracellular Na⁺. Furthermore, it was found that the cells containing a high concentration of Na⁺ can develop normally in the presence of exogenous 10 mM K⁺, where intracellular K⁺ was maintaned at about 30 mM, irrespective of a high concentration of intracellular Na⁺ (about 30 mM). These suggest that the Na⁺-inhibition of the development is caused by a decrease in intracellular K⁺, but not by an increase in intracellular Na⁺. Pyruvate kinase extracted from the organism required K⁺ for its activation. The vegetative cells incubated in 50 mM Na⁺ contained only about 10 mM K⁺ which is insufficient for the enzyme activation. However, the amount of ATP in the cells containing less K⁺ was similar to that in those with much K⁺. These results are discussed in relation to the activity of glycolysis.     

Activation of the Na,K-pump by isoproterenol, methylxanthines, and iodoacetate in erythrocytes of the frogRana temporaria

Our preliminary studies have shown that the Na,K-pump in frog erythrocytes is activated by isoproterenol (ISP), phosphodiesterase blocker (3-isobutyl-methylxantine, IBMX), and by iodoacetate (MIA). The aim of the present study was to determine a mechanism responsible for the effect of MIA on the Na,K-pump activity in frog red blood cells as well as the role of G proteins and intracellular messengers in modulation of active K⁺ transport induced by ISP. An additive stimulation of active K⁺ (⁸⁶Rb) transport in frog erythrocytes was found after exposure of the cells to MIA in a combination with ISP or IBMX. The treatment of the red blood cells with 1 mM MIA for 1 or 2 h was associated with a significant decrease in intracellular Na⁺ concentration, on average, by 13 and 20%, respectively, suggesting a direct action of MIA on the Na,K-pump. Incubation of cells in the presence of dibutyryl-cAMP (1 mM) or adenylate cyclase activator forskolin (0.1 mM) caused stimulation of the active K⁺ influx by 21.8 and 27.9%, respectively. AlF ₄ ^- and cholera toxin able to increase cell cAMP levels via G protein interactions had no effect on the total and IPS-induced K⁺ influx in frog erythrocytes. The treatment of the red blood cells with sodium nitroprusside that increases cGMP concentration in cells also had no effect on the K⁺ influx. The stimulatory influence of ISP on the Na,K-pump was reduced with increase of the intracellular Na⁺ concentration. ISP increased affinity of the Na,K-pump to Na⁺ (the Mihaelis constant K_M = 34.4 ± 5.1 in control and 25.3 ± 2.8 mM in the presence of ISP,p < 0.01), but did not change maximal velocity (8.1 ± 0.6 and 7.7 ± 0.3 mmol/1/h in the control and ISP-treated cells, respectively). The results obtained indicate the presence of several different signal pathways involved in regulation of the Na,K-pump activity in frog erythrocytes.     

Kinetic properties of sodium transport pathways in the river lamprey <Emphasis Type="Italic">Lampetra fluviatilis</Emphasis> erythrocytes

To activate Na⁺/H⁺ exchange, intracellular pH (pH_i) of erythrocytes of the river lamprey Lampetra fluviatilis were changed from 6 and 8 using nigericin. The Na⁺/H⁺ exchanger activity was estimated from the values of amiloride-sensitive components of Na⁺ (²²Na) inflow or of H⁺ outflow from erythrocytes. Kinetic parameters of the carrier functioning were determined by using Hill equation. Dependence of Na⁺ and H⁺ transport on pH_i value is described by hyperbolic function with the Hill coefficient value (n) close to 1. Maximal rate of ion transport was within the limits of 9–10 mmol/l cells/min, and the H⁺ concentration producing the exchanger 50% activation amounted to 0.6–1.0 μM. Stimulation of H⁺ outcome from acidified erythrocytes (pH_i 5.9) with increase of H⁺ concentration in the incubation medium is described by Hill equation with n value of 1.6. Concentration Na⁺ for the semimaximal stimulation of H⁺ outcome amounted to 10 mM. The obtained results indicate the presence in lamprey erythrocytes of only binding site for H⁺ from the cytoplasm side and the presence of positive cooperativity in Na⁺-binding from the extracellular side of the Na⁺/H⁺ exchanger. Na⁺ efflux from cells in the Na⁺-free medium did not change at a 10-fold increase of H⁺ concentration in the incubation medium. The presented data indicate differences of kinetic properties of the lamprey erythrocyte Na⁺/H⁺ exchanger and of this carrier isoforms in mammalian cells. In intact erythrocytes the dependence of the amiloride-sensitive Na⁺ inflow on its concentration in the medium is described by Hill equitation with n 1.6. The Na⁺ concentration producing the 50% transport activation amounted to 39 mM and was essentially higher as compared with that in acidified erythrocytes. These data confirm conception of the presence of two amiloride-sensitive pathways of Na+ transport in lamprey erythrocytes.     

High salt‐treatment‐induced Na+ extrusion and low salt‐treatment‐induced Na+ accumulation in suspension‐cultured cells of the mangrove plant,Bruguiera sexangula

A suspension‐cultured cell strain of the mangrove plant (Bruguiera sexangula) was established from a callus culture and maintained in an amino acid medium in the absence of NaCl. NaCl non‐adapted cells were transferred to media containing 0–200 mm NaCl. The initial growth rate decreased gradually with increasing salt concentrations. However, at up to 150 mm NaCl, cell number growth at the highest point was almost the same as that at lower salt concentrations. Cells even continued to grow in the presence of 200 mm NaCl. Cells incubated in a medium containing 50 mm NaCl for 3 weeks accumulated Na⁺, while those incubated in 150 mm NaCl for 2 d showed only a transient increase in Na⁺ and Cl^– concentrations. In the latter treatment, the intracellular concentration of Na⁺ returned to the original low level within 2 weeks. It took a longer time for Cl^– to return to its original level. As a result, the Na⁺ and Cl^– concentrations in cells cultured with 50 mm NaCl were much larger than those in cells cultured with 150 mm NaCl. The intracellular distribution of ions after transfer to the medium containing 150 mm NaCl was analysed by isolating the vacuoles. Treatment with amiloride, an inhibitor of the Na⁺/H⁺ antiporter, suppressed the recovery of Na⁺ to the original level in the cells. Treatment with 150 mm NaCl for 3 d stimulated the activities of both the vanadate‐dependent H⁺‐ATPase and the Na⁺/H⁺ antiporter in the plasma membrane fraction.     

Comparison between a Stable NaCl-Selected Nicotiana Cell Line and the Wild Type : K, Na, and Proline Pools as a Function of Salinity

An NaCl-resistant line has been developed from suspension-cultured tobacco cells (Nicotiana tabacum/gossii) by stepwise increases in the NaCl concentration in the medium. Resistance showed stability through at least 24 generations in the absence of added NaCl.

Above an external NaCl concentration of 35 millimolar, proline concentration in the selected cells rose steeply with external NaCl, particularly so above 100 millimolar NaCl. Proline accumulation in the wild type was far slighter. Selected cells which had been grown for 24 generations in the absence of added NaCl accumulated proline strongly on re-exposure to NaCl medium, indicating stability of this character. Proline accumulation was fully reversible with a half-time of about 6 hours. When selected cells were transferred sequentially to lower and lower NaCl concentrations, their proline content fell to the level corresponding to the new NaCl concentration. The NaCl-selected cells responded to water stress (i.e. added mannitol) by accumulating markedly more proline than did the wild type.

The addition of Ca²⁺ to the growing and rinsing media minimized Na⁺ and K⁺ binding in the Donnan free space of cell walls and thus allowed assessment of intracellular Na⁺ and K⁺. In both cell types, internal Na⁺ content rose steadily as a function of external NaCl concentration. In the course of 7 days in NaCl media, the wild type cells lost a considerable part of their K⁺ content, the extent of the loss increasing with rise in external NaCl concentration. The selected cells, by contrast, lost no K⁺ at external NaCl concentrations below 50 millimolar external NaCl, and at higher concentrations lost less than the wild type.

     

Determination of Ion Content and Ion Fluxes in the Halotolerant Alga Dunaliella salina

A method to determine intracellular cation contents in Dunaliella by separation on cation-exchange minicolumns is described. The separation efficiency of cells from extracellular cations is over 99.9%; the procedure causes no apparent perturbation to the cells and can be applied to measure both fluxes and internal content of any desired cation. Using this technique it is demonstrated that the intracellular averaged Na⁺, K⁺, and Ca²⁺ concentrations in Dunaliella salina cultured at 1 to 4 molar NaCl, 5 millimolar K⁺, and 0.3 millimolar Ca²⁺ are 20 to 100 millimolar, 150 to 250 millimolar, and 1 to 3 millimolar, respectively. The intracellular K⁺ concentration is maintained constant over a wide range of media K⁺ concentrations (0.5-10 millimolar), leading to a ratio of K⁺ in the cells to K⁺ in the medium of 10 to 1,000. Severe limitation of external K⁺, induces loss of K⁺ and increase in Na⁺ inside the cells. The results suggest that Dunaliella cells possess efficient mechanisms to eliminate Na⁺ and accumulate K⁺ and that intracellular Na⁺ and K⁺ concentrations are carefully regulated. The contribution of the intracellular Na⁺ and K⁺ salts to the total osmotic pressure of cells grown at 1 to 4 molar NaCl, is 5 to 20%.     

Homeostasis of Sodium and Potassium Ions in Erythrocytes of the Lamprey Lampetra fluviatilis: Effect of Ion Transport and Metabolic Blockers, and Ionophores

After incubation of lamprey Lampetra fluviatilis erythrocytes in the standard medium for 90–120 min, intracellular Na⁺ and K⁺ content remained unchanged (28.7 ± 1.1 and 66.3 ± 1.5 mmol/l cells, respectively, n = 33). The erythrocyte ion content also did not change after treatment of the cells with ion transport inhibitors, Ba^{2 +} and amiloride. Addition of 0.1 mM ouabain to the incubation medium led to a decrease of K⁺ content by 8.4 ± 1.2 and to an increase of Na⁺ content by 2.4 ± 0.8 mmol/l/2 h. Similar reciprocal changes in the cellular ion composition were observed after treatment of the erythrocytes by oxidative metabolism inhibitors (rotenone and CCCP—carbonyl cyanide m-chlorophenyl-hydrazone). The metabolic blockers produced more significant ion composition changes in comparison with ouabain. An increase of intracellular Na⁺ content under effect of CCCP was completely inhibited by amiloride. It can be suggested that inhibition of oxidative metabolism is accompanied by a cell acidification and Na⁺/H⁺ exchange activation. Erythrocyte acidification by a K⁺/H⁺ ionophore led to a rapid cellular Na⁺ accumulation, which indicates the presence of a Na⁺/H⁺ exchanger with high activity. The K⁺ ionophore valinomycin produced a relatively small K⁺ loss from the lamprey erythrocytes to indicate a low anion conductance of the cells. The data obtained indicate an important role of oxidative metabolism in the monovalent ion homeostasis in the lamprey red blood cells.     

ALBUMIN CAN REVERSE THE RELEASE OF POTASSIUM FROM HUMAN ERYTHROCYTES TREATED WITH THE NON-IONIC DETERGENT,BRIJ 58

Exchange of erythrocyte intracellular (i/c) K⁺for extracellular (e/c) Na⁺in human erythrocytes treated with sub-CMC concentrations of the non-ionic detergent Brij 58 can be stopped by reincubation in serum or albumin containing solutions. The progressive equilibration of the K⁺contents of detergent-treated human erythrocytes with the incubation medium was reversed by an albumin-mediated withdrawal of detergent molecules from the cell. Re-establishment of near normal [K⁺] in terms of K⁺/kg water proceeds in two ways: (i) a metabolism-dependent net accumulation of K⁺ions; and (ii) a metabolism-independent shrinkage of erythrocytes, this being the more significant factor.     

Activation of Na+,H+-Exchange in Erythrocytes of the River Lamprey Lampetra fluviatilis

To study H⁺ transport, the lamprey red blood cells were acidified to pH 6.0 by a pretreatment with an ionophore, nigericin. Incubation of the acidified cells in NaCl-medium at pH 8.0 was accompanied by a rapid H⁺ efflux from the erythrocytes. There was a tenfold decrease of the H⁺ efflux rate on addition to NaCl-medium of dimethylamiloride or on replacing Na⁺ in the medium (KCl-medium, pH 8.0). A high rate of Na+ influx into the acidified erythrocytes occurred only in the presence of H⁺ gradient (pH medium 8.0), but not in its absence (pH medium 6.0). The Na⁺-dependent H⁺ efflux from the cells and H⁺-dependent Na⁺ influx into the cells were quantitatively similar (about 700 mmol/l cells/h). A rapid elevation of the intracellular Na⁺ concentration as measured by flame photometry was also observed during incubation of the acidified cells in NaCl-medium (pH 8.0). The H⁺-dependent Na⁺ influx and an increase of the Na⁺ content in the acidified cells were significantly inhibited by amiloride. The data obtained for the first time prove with certainty the presence of the Na⁺/H⁺ exchanger in erythrocytes of the river lamprey.     

Osmoadaptation in Representatives of Haloalkaliphilic Bacteria from Soda Lakes

The adaptation of microorganisms to life in brines allows two strategies: the accumulation of organic osmoregulators in the cell (as in many moderate halophiles, halomonads in particular) or the accumulation of inorganic ions at extremely high intracellular concentrations (as, for example, in haloanaerobes). To reveal the regularities of osmoregulation in haloalkaliphiles developing in soda lakes, Halomonas campisalis Z-7398-2 and Halomonas sp. AIR-2 were chosen as representatives of halomonads, and Natroniella acetigena, as a representative of haloanaerobes. It was established that, in alkaliphilic halomonads, the intracellular concentrations of inorganic ions are insufficient for counterbalancing the environmental osmotic pressure and balance is attained due to the accumulation of organic osmoregulators, such as ectoine and betaine. On the contrary, the alkaliphilic haloanaerobe N. acetigena employs K⁺, Na⁺, and Cl^? ions for osmoregulation. High intracellular salt concentrations increasing with the content of Na⁺ in the medium were revealed in this organism. At a concentration of 1.91 M Na⁺ in the medium, N. acetigena accumulated 0.83 M K⁺, 0.91 M Na⁺, and 0.29 M Cl^? in cells, and, with an increase in the Na⁺ content in the medium to 2.59 M, it accumulated 0.94 M K⁺, 1.98 M Na⁺, and 0.89 M Cl^?, which counterbalanced the external osmotic pressure and provided for cell turgor. Thus, it was shown that alkaliphilic microorganisms use osmoregulation strategies similar to those of halophiles and these mechanisms are independent of the mechanism of pH homeostasis.     

Mode of transport and possible mechanism of action of l-phenylalanine benzyl ester as an anti-sickling agent

l-Phenylalanine benzyl ester (Phe-Bz) and a number of ester analogues prevent sickling of erythrocytes from sickle cell disease patients. The compounds tested exhibit anti-sickling activity in the concentration range 0.5–3.0 mM. A general feature of these compounds is the presence of two aromatic rings in their molecular structure. The anti-sickling agents rapidly enter the erythrocyte and are hydrolysed to their component molecules. Incubation of human erythrocytes with 3.0 mM l-phenylalanine for 30 min at 37°C results in accumulation of 2.0 mmol l-phenyalanine/l cells, while incubation of erythrocytes with 3.0 mM Phe-Bz under similar conditions results in the production of 4.0 mmol l-phenylalanine/l cells and an equivalent amount of benzyl alcohol. Both l-phenylalanine and benzyl alcohol are inhibitors of the gelation of deoxyhaemoglobin S (deoxy-HbS) in vitro. Moreover, Phe-Bz and related anti-sickling agents fluidize the lipid bilayer of the erythrocyte membrane, inhibiting several transport systems, including those for l-phenylalanine, uridine and sulphate ions, as well as the Na⁺ pump and the Na⁺/K⁺ cotransporter, but increasing the passive influx and efflux of both cations and anions. The accumulation of Phe-Bz hydrolysis products within the erythrocyte together with the effects of Phe-Bz on cation permeability result in the influx of water causing the cell to swell. Thus, treatment of erythrocytes with 3.0 mM Phe-Bz at 37°C for 30 min causes an increase in mean cell volume of 14.8%, decreasing the mean intracellular haemoglobin concentration from 34 to 29.6 g%. The increase in cell volume caused by Phe-Bz and its analogues together with the direct effects of their hydrolysis products on HbS probably act in concert to bring about the anti-sickling effect.     

Effects of salt stress and exogenous Ca2+ on Na+ compartmentalization,ion pump activities of tonoplast and plasma membrane in Nitraria tangutorum Bobr. leaves

Nitraria tangutorum Bobr. is a typical halophyte with superior tolerance to salinity. However, little is known about its physiological adaptation mechanisms to the salt environment. In the present study, N. tangutorum seedlings were treated with different concentrations of NaCl (100, 200, 300 and 400 mmol L^?1) combined with five levels of Ca²⁺ (0, 5, 10, 15 and 20 mmol L^?1) to investigate the effects of salt stress and exogenous Ca²⁺ on Na⁺ compartmentalization and ion pump activities of tonoplast and plasma membrane (PM) in leaves. Na⁺ and Ca²⁺ treatments increased the fresh weight and dry weight of N. tangutorum seedlings. The absorption of Na⁺ in roots, stems and leaves was substantially increased with the increases of NaCl concentration, and Na⁺ was mainly accumulated in leaves. Exogenous Ca²⁺ reduced Na⁺ accumulation in roots but promoted Na⁺ accumulation in leaves. The absorption and transportation of Ca²⁺ in N. tangutorum seedlings were inhibited under NaCl treatments. Exogenous Ca²⁺ promoted Ca²⁺ accumulation in the plant. Na⁺ contents in apoplast and symplast of leaves were also significantly increased, and symplast was the main part of Na⁺ intracellular compartmentalization. The tonoplast H⁺-ATPase and H⁺-PPase activities were significantly promoted under salt stress (NaCl concentrations ≤300 mmol L^?1). PM H⁺-ATPase activities gradually increased under salt stress (NaCl concentrations ≤200 mmol L^?1) followed by decreases with NaCl concentration increasing. The tonoplast H⁺-ATPase, H⁺-PPase and PM H⁺-ATPase activities increased first with the increasing exogenous Ca²⁺ concentration, reached the maximums at 15 mmol L^?1 Ca²⁺, and then decreased. The tonoplast and PM Ca²⁺-ATPase activities showed increasing trends with the increases of NaCl and Ca²⁺ concentration. These results suggested that certain concentrations of exogenous Ca²⁺ effectively enhanced ion pump activities of tonoplast and PM as well as promoted the intracellular Na⁺ compartmentalization to improve the salt tolerance of N. tangutorum.     

Mode of transport and possible mechanism of action of l-phenylalanine benzyl ester as an anti-sickling agent

l-Phenylalanine benzyl ester (Phe-Bz) and a number of ester analogues prevent sickling of erythrocytes from sickle cell disease patients. The compounds tested exhibit anti-sickling activity in the concentration range 0.5–3.0 mM. A general feature of these compounds is the presence of two aromatic rings in their molecular structure. The anti-sickling agents rapidly enter the erythrocyte and are hydrolysed to their component molecules. Incubation of human erythrocytes with 3.0 mM l-phenylalanine for 30 min at 37°C results in accumulation of 2.0 mmol l-phenyalanine/l cells, while incubation of erythrocytes with 3.0 mM Phe-Bz under similar conditions results in the production of 4.0 mmol l-phenylalanine/l cells and an equivalent amount of benzyl alcohol. Both l-phenylalanine and benzyl alcohol are inhibitors of the gelation of deoxyhaemoglobin S (deoxy-HbS) in vitro. Moreover, Phe-Bz and related anti-sickling agents fluidize the lipid bilayer of the erythrocyte membrane, inhibiting several transport systems, including those for l-phenylalanine, uridine and sulphate ions, as well as the Na⁺ pump and the Na⁺/K⁺ cotransporter, but increasing the passive influx and efflux of both cations and anions. The accumulation of Phe-Bz hydrolysis products within the erythrocyte together with the effects of Phe-Bz on cation permeability result in the influx of water causing the cell to swell. Thus, treatment of erythrocytes with 3.0 mM Phe-Bz at 37°C for 30 min causes an increase in mean cell volume of 14.8%, decreasing the mean intracellular haemoglobin concentration from 34 to 29.6 g%. The increase in cell volume caused by Phe-Bz and its analogues together with the direct effects of their hydrolysis products on HbS probably act in concert to bring about the anti-sickling effect.