Osmoregulation in Dunaliella tertiolecta: effects of salt stress,and the external pH on the internal pH

The intracellular pH of the halotolerant green algae Dunaliella tertiolecta, was determined by the distribution of 5,5-dimethyl-2(¹⁴C)-oxalolidine-2,5-dione (DMO) between the cell and the surrounding medium. 5,5-dimethyl-2(¹⁴C)oxalolidine-2,4-dione was not metabolized by the algal cells. The intracellular pH of Dunaliella tertiolecta was 6.8 in the dark and 7.4 in the light. During a salt stress, after two hours, the intracellular pH was increased by 0.2 pH units in both light and dark. The salt stressed cells maintained a constant pH of about 7.5 over the pH range of 6.5 to 8.5. Because of the relatively low permeability coefficient of the plasma membrane for DMO, this technique does not permit rapid pH determinations during the induction period after a salt stress. The magnitude of the salt induced pH changes measured 2 h after the salt stress implies a minor importance of this alkalization in this time range, but does not exclude a larger importance of pH changes for osmoregulation during the induction period.Abbreviations Chl chlorophyll - DMO 5,5-dimethyl-2(¹⁴C)oxalolidine-2,4-dione - PCV packed cell volume - SDS sodium dodecyl sulfate     

Generation and maintenance of suspension cultures from cotyledons and their organogenic potential of two mangrove species, Sonneratia alba and S. caseolaris

Liquid cultures were successfully generated from cotyledons of two Sonneratia species, S. alba and S. caseolaris in Murashige and Skoog (MS) medium containing 0.1 μmol L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Adventitious roots differentiated from cotyledons of S. alba. Proliferated cells were subcultured and a large volume of suspension cells was subsequently established in 100-mL flasks. All the cytokinins tested inhibited cell proliferation. After three years of culture, the potential to differentiate was tested as indicated by greening of the cells. Greening occurred when suspension cells were transferred to solid MS medium with and without 0.1 μmol L⁻¹ 2,4-D. Greening was stimulated by low concentrations of the weak auxins indolebutyric acid (IBA) and naphthaleneacetic acid (NAA) while 2,4-D stimulated late-stage greening. Abscisic acid (ABA) inhibited greening. Gibberellic acid (GA₃) at 1.0 μmol L⁻¹ stimulated callus greening and was not inhibitory even when tested at high concentrations. Cytokinins were inhibitory in combination with 0.1 μmol L⁻¹ of either IBA or NAA. The cause of different effects of plant hormones on growth and differentiation was discussed. Small-scale liquid media and 24-well culture plates of solid media methods developed in this paper are suitable for the optimization of hormonal conditions for cell proliferation and differentiation.     

Opposite effects of synthetic auxins, 2,4-dichlorophenoxyacetic acid and 1-naphthalene acetic acid on growth of true ginseng cell culture and synthesis of ginsenosides

Effects of synthetic auxins (2,4-D and NAA) on growth of true ginseng (Panax ginseng C.A. Mey) suspension culture and ginsenoside synthesis were investigated. Cell suspensions were grown for 6–8 subcultures on media supplemented with various phytohormones. In all media supplemented with 2,4-D and cytokinins (benzyladenine or kinetin), the cell culture showed sustained growth both in the presence and absence of casein hydrolysate. The average growth index, determined from fresh weight increment over one subculture, equaled to 5.16 ± 0.90, and the maximum mitotic index was 2%. These cell populations having cell volume of 10–17 × 10⁴ μm³ were composed mostly (up to 60–80%) of 5-to 10-cell aggregates with unimodal distribution of nuclear DNA. These cell suspensions were suitable for isolation of protoplasts. The total average content of ginsenosides in the cell culture grown in the presence of 2,4-D constituted 0.18% of dry matter. In media supplemented with NAA, the cell growth was retarded irrespective of the cytokinin species and presence or absence of casein hydrolysate. The growth index (the ratio of final to initial fresh weights) was on average 2.15 ± 0.37, and the mitotic index did not exceed 0.13%. These suspensions, characterized by cell volume of 22–50 × 10⁴ μm³, were composed of large aggregates (> 50 cells). The attempts to isolate protoplasts from these suspensions were unsuccessful. About 25% of cells cultured in the presence of NAA had doubled nuclear DNA content by the end of the subculture. The total content of ginsenosides in cell cultures grown with NAA was on average 4.46% of cell dry matter. The results indicate that ginsenoside synthesis depends on the extent of differentiation in the population of true ginseng cells grown in suspension culture. A certain extent of cytodifferentiation in the cell culture was observed in the presence of NAA, whereas 2,4-D supported only cell proliferation in vitro.     

Inhibition of leukotriene B4 biosynthesis by a novel phosphodiesterase inhibitor

TVX 2706, a 3-ethyl-1-(3-nitrophenyl)-2,4-[1H, 3H] quanzazolidione was found to exert strong antiinflammatory properties in vivo. This antiinflammatory potency obviously depends on a pronounced inhibition of phosphodiesterase (PDE) activity, shown in cell culture systems as well as in homogenates of rat PMNL, that causes a marked elevation of intracellular cAMP. In the present study, we have examined the effect of TVX 2706 on the inhibition of leukotriene B₄ (LTB₄) biosynthesis in rat PMNL by the aid of HPLC. TVX 2706 causes an inhibition of LTB₄ generation in an biphasic manner, obviously characteristic for this substance. Within the range of ^1x10^?4M (100%) to 5×10^?6M (40%) the inhibition is concentration-dependent, but all lower concentration down to 5×10^?5 M reduced LTB₄ synthesis to 30–40% of control values on a dose independent manner. No other substance tested until now, produces this characteristics, reproducible pattern of marked leukotriene inhibition. Our results suggest, that inhibition of LTB₄ biosynthesis may be induced by elevated intracellular CAMP levels. In accordance with the biphasic cellular response there is a proved different inhibitory activity of TVX 2706 on high and low affinity PDE that could be responsible for this substance specific effect. Although the exact mode of action of TVX 2706 remains unexplained, all in vivo and in vitro results prove TVX 2706 to be a very potent antiinflammatory substance with an interesting pharmacological profile.     

Absolute measurement of cell expansion in plant cell suspension cultures

A method is described for the measurement of intracellular volume (V_i) in cell cultures. In principle, any stable compound that neither penetrates the plasma membrane nor binds to the cells can be used to trace the total extracellular (apoplastic) volume and hence to estimate the intracellular volume. No suitable coloured or UV-absorbing compound could be found among those tested; the main problems were binding to the cell surface and/or instability in the medium. However, [¹⁴C]mannitol was an acceptable apoplastic marker, by use of which we showed that 21–47% of total packed cell volume (PCV) was intracellular, and 14–33% of total settled cell volume (SCV) was intracellular. Therefore, measurements of PCV and SCV misrepresent cell expansion to a variable extent. Cultures of Acer, Rosa, Spinacia and Zea achieved final symplastic volumes of only 9, 14, 6 and 6%, respectively, of the total suspension culture volume.     

RAPID ALDOSTERONE-INDUCED CELL VOLUME INCREASE OF ENDOTHELIAL CELLS MEASURED BY THE ATOMIC FORCE MICROSCOPE

Atomic force microscopy (AFM) is a useful technique for imaging the surface of living cells in three dimensions. The authors applied AFM to obtain morphological information of individual cultured endothelial cells of bovine aorta under stationary and strain conditions and to simultaneously measure changes in cell volume in response to aldosterone. This mineralocorticoid hormone is known to have acute, non-genomic effects on intracellular pH, intracellular electrolytes and inositol-1,4,5-triphosphate production. In this study whether endothelial cells under tension change their volume in response to aldosterone was tested. Such changes were already shown in human leukocytes measured by Coulter counter. In contrast to leukocytes that are more or less spherical and live in suspension, endothelial cells exhibit a complex morphology and adhere to a substrate. Thus, measurements of discrete cell volume changes in endothelial cells under physiological condition is only feasible with more sophisticated techniques. By using AFM we could precisely measure the absolute cell volume of individual living endothelial cells. Before the addition of aldosterone the cell volume of mechanically stressed endothelial cells mimicking arterial blood pressure was 1827±172fl. Cell volume was found to increase by 28% 5min after hormone exposure. Twenty-five minutes later cell volume was back to normal despite the continuous presence of aldosterone in the medium. Amiloride, a blocker of the plasma membrane Na⁺/H⁺exchanger prevented the initial aldosterone-induced volume increase. Taken together, AFM disclosed a transient swelling of endothelial cells induced by the activation of an aldosterone sensitive plasma membrane Na⁺/H⁺exchanger.     

Strong buffering capacity of insect cells. Implications for the baculovirus expression system

Insect cells are widely used for expression of a variety of different proteins by using the baculovirus expression system. The applicability of this system depends on production of proteins which have biological properties similar to their native counterparts. One application has been the expression of viral capsid proteins and their assembly into empty capsid structures to provide new viral immunogens which retain complex antigenic sites. An important parameter for efficient folding and assembly of proteins into viral procapsids may be the intracellular pH, particularly for acid-labile particles such as foot-and-mouth disease virus (FMDV). Benzoic acid was used as an effective indicator of intracellular pH in insect cells and 3-O-methyl glucose to measure cell volumes. We have determined the intracellular volume of theSpodoptera frugiperda IPLB-Sf21 insect cells 0.50±0.08 pL per cell. Using the distribution of [¹⁴C]-benzoic acid, we show that the intracellular pH remains constant at pH 7.0 when the cells are grown in media with pH values ranging from 6.2 to 6.8 and, moreover, is not affected by baculovirus infection. These results suggest that insect cells are suitable to express and produce acid-labile structures via the baculovirus expression system and that assembly of proteins and viral procapsids could occur.     

Lymphocyte response to phytohemagglutinin: Intracellular volume and intracellular [K+]

The effect of phytohemagglutinin (PHA) on lymphocytes was examined with respect to free intracellular water volume and intracellular [K⁺]. At a cell concentration of 30 × 10⁶ lymphocytes/ml in modified Hank's Buffered Salt Solution (HBSS) in the presence of 10% human AB serum, addition of PHA at 3 mg/ml resulted in a 24–27% decrease in free intracellular water space within 30 to 60 minutes and a return to control level after three hours. A larger change in intracellular water (44%) was observed under similar conditions in the absence of serum. The absolute intracellular K⁺ content did not change after PHA addition, but the cell water volume decrease arising from PHA addition resulted in a 29% increase in intracellular [K⁺] at 60 minutes. The decrease in lymphocyte water volume induced by PHA was also observed for concanavalin A which stimulates lymphocyte proliferation, but not for wheat germ lectin, an agglutinating agent which is not mitogenic. Thus, volume regulation may be closely associated with the mitogenicity of these compounds.     

19F NMR studies of changes in membrane potential and intracellular volume during dexamethasone-induced apoptosis in human leukemic cell lines

The induction of apoptosis in leukemic cells by dexamethasone is well known, but the mechanism of this type of cell death and of dexamethasone resistance by some variants is still poorly understood. Apoptotic cell death is preceded by many changes in cellular properties, such as glucose metabolism, cell size, cell density, and others. In this study, ¹⁹F-NMR has been used to characterize changes in cell membrane potential and intracellular accessible volume during dexamethasone induced apoptosis. One dex-sensitive (CEM-C7) and three dex-resistant variants (CEM-C1, CEM-ICR27, and CEM-4R4) were examined. We have observed separate intracellular and extracellular resonances for trifluoroacetate and trifluoroacetamide added to suspended leukemic cells. From the equilibrium distribution of these fluoro-compounds between intra and extracellular spaces, the changes in membrane potential and intracellular accessible volume were calculated. The membrane potential for CEM-C7 cells was found to significantly decrease in the presence of dexamethasone (9-mV decrease within 18 h of dexamethasone treatment), while that of CEM-ICR27 was found in some samples to increase on dexamethasone incubation. The membrane potential for CEM-C1 decreased slightly, while that of CEM-4R4 was not appreciably affected by dexamethasone. The reduction of membrane potential seems to be an early step in the mechanism of dexamethasone induced apoptosis. Although the intracellular volume varied with cell type and dexamethasone incubation (for CEM-C7), the fractional intracellular volume (α = V_in/V_cell was found to be the same (0.82 ± 0.06) for all the cell lines in the presence and absence of dexamethasone. © 1993 Wiley-Liss, Inc.     

Substrate Utilization by Suspension Cultures and Somatic Embryos of Daucus carota L. Measured by C NMR

The uptake and utilization of sucrose by embryogenic suspension cultures of carrot (Daucus carota L.) growing in the presence of 2,4-D and by somatic embryos derived from these cultures was monitored using ¹³C nuclear magnetic resonance. The exogeneously supplied sucrose was completely hydrolyzed before cell entry; glucose was taken up preferentially when the cells were cultured in the presence of 2,4-D, while glucose and fructose were utilized at similar rates by somatic embryos in the absence of 2,4-D. Both suspension cells and somatic embryos accumulated high intracellular levels predominantly of glucose and sucrose, the latter being resynthesized intracellularly from the constitutive hexoses. Initially, fructose was converted mainly into glucose and sucrose rather than being catabolized directly through glycolysis or the pentose phosphate pathway. Carbohydrate supply that exceeded cellular demand resulted in intracellular accumulation of mono- or disaccharides. The capacity of cultured carrot cells to produce somatic embryos appeared to be positively correlated with high intracellular levels of glucose.     

Determination of intracellular osmotic volume and sodium concentration in dunaliella

A new method to measure intracellular volume in Dunaliella was developed, where lithium ions are used as monitors of the extracellular volume. Li⁺ is shown to be impenetrable to the intracellular volume, insignificantly absorbed to the algae, and is rapidly and evenly distributed within the extracellular volume. The method is suggested to be free of several limitations and consistent errors present in several previously employed techniques.

Using the new technique it is shown that both Dunaliella salina and Dunaliella bardawil adjust to a constant cellular volume when grown in a medium containing salt concentrations ranging from 0.5 molar to 4 molar NaCl. That volume is 90 femtoliter per cell for D. salina and 600 femtoliter per cell for D. bardawil. Nonosmotic volume accounts for about 10% of the total cell volume.

The intracellular sodium concentration, as determined with the new technique, was under all experimental conditions tested below 100 millimolar. This was true both for cells grown on 0.5 to 4 molar NaCl, and during the osmoregulatory process. It is thus concluded that intracellular NaCl is a minor contributor to the overall intracellular osmotic pressure in Dunaliella.

     

Ca2+ signaling, intracellular pH and cell volume in cell proliferation

Mitogens control progression through the cell cycle in non-transformed cells by complex cascades of intracellular messengers, such as Ca²⁺ and protons, and by cell volume changes. Intracellular Ca²⁺ and proton concentrations are critical for linking external stimuli to proliferation, motility, apoptosis and differentiation. This review summarizes the role in cell proliferation of calcium release from intracellular stores and the Ca²⁺ entry through plasma membrane Ca²⁺ channels. In addition, the impact of intracellular pH and cell volume on cell proliferation is discussed.     

Auxin requirements of sycamore cells in suspension culture

Sycamore (Acer pseudoplatanus L.) cell suspension cultures (strain OS) require 2,4-dichlorophenoxyacetic acid (2,4-D) in their culture medium for normal growth. If the 2,4-D is omitted, rates of cell division are dramatically reduced and cell lysis may occur. Despite this `auxin requirement,' it has been shown by gas chromatography-mass spectrometry that the cells synthesize indol-3yl-acetic acid (IAA). Changes in free 2,4-D and IAA in the cells during a culture passage have been monitored.

There is a rapid uptake of 2,4-D by the cells during the lag phase leading to a maximum concentration per cell (125 nanograms per 10⁶ cells) on day 2 followed by a decline to 45 nanograms per 10⁶ cells by day 9 (middle of linear phase). The initial concentration of IAA (0.08 nanograms per 10⁶ cells) rises slowly to a peak of 1.4 nanograms per 10⁶ cells by day 9 then decreases rapidly to 0.2 nanograms per 10⁶ cells by day 15 (early declining phase) and 0.08 nanograms per 10⁶ cells by day 23 (early stationary phase).

     

Synthesis and evaluation of piperazine and homopiperazine analogues of JS-K,an anti-cancer lead compound

Here we report a number of novel JS-K structural analogues with sub-micromolar anti-proliferative activities against human leukemia cell lines HL-60 and U937; JS-K is the anti-cancer lead compound O²-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate. The ability of these compounds to generate intracellular nitric oxide correlated well with their observed anti-proliferative effects: analogues that had potent inhibitory activity against leukemia cells formed elevated levels of intracellular nitric oxide.     

The influence of the chloride currents on action potential firing and volume regulation of excitable cells studied by a kinetic model

In excitable cells, the generation of an action potential (AP) is associated with transient changes of the intra- and extracellular concentrations of small ions such as Na⁺, K⁺ and Cl⁻. If these changes cannot be fully reversed between successive APs cumulative changes of trans-membrane ion gradients will occur, impinging on the cell volume and the duration, amplitude and frequency of APs. Previous computational studies focused on effects associated with excitation-induced changes of potassium and sodium. Here we present a model based study on the influence of chloride on the fidelity of AP firing and cellular volume regulation during excitation. Our simulations show that depending on the magnitude of the basal chloride permeability two complementary types of responsiveness and volume variability exist: (i) At high chloride permeability (typical for muscle cells), large excitatory stimuli are required to elicit APs; repetitive stimuli of equal strength result in almost identical spike train patterns (Markovian behavior), however, long excitation may lead to after discharges due to an outward directed current of intracellular chloride ions which accumulate during excitation; cell volume changes are large. (ii) At low chloride permeability (e.g., neurons), small excitatory stimuli are sufficient to elicit APs, repetitive stimuli of equal strength produce spike trains with progressively changing amplitude, frequency and duration (short-term memory effects or non-Markovian behavior); cell volume changes are small. We hypothesize that variation of the basal chloride permeability could be an important mechanism of neuronal cells to adapt their responsiveness to external stimuli during learning and memory processes.     

Intracellular Biopotentials During Static Extracellular Stimulation

Two properties of the intracellular potentials and electric fields resulting from static extracellular stimulation are obtained for arbitrarily shaped cells. First, the values of intracellular potential are shown to be bounded by the maximum and minimum values of extracellular potential on the surface of the cell. Second, the volume average of the magnitude of intracellular electric field is shown to have an upper bound given by the ratio of the magnitude of the largest extracellular potential difference on the surface of the cell to a generalized length constant λ = [σ_intraV_cell/(σ_memb A_cell)]^1/2, where V_cell and A_cell are the volume and surface area of the cell, σ_intra is the intracellular conductivity (reciprocal ohms per centimeter), and σ_memb is the membrane conductivity (reciprocal ohms per square centimeter). The use of the upper bound on the volume average of the magnitude of intracellular electric field as an estimate for intracellular isopotentiality is discussed and the use of the generalized length constant for electrically describing arbitrary cells is illustrated for cylindrical- and spheroidal-shaped cells.     

Sulfate flux in high sodium cat red cells

The transport of radioactive sulfate in cat red cells has been studied. The rate constant for ³⁵SO₄ inward movement under steady-state conditions is 0.24 ± 0.02/hr. This movement was found to be sensitive to osmotic changes in cell volume and to the nature of anions in the incubation medium; it increases with increasing cell volume and decreases with decreasing cell volume. The anions SCN, NO₃, and I were found to inhibit the uptake of ³⁵SO₄. Furthermore, 1-fluoro-2,4-dinitrobenzene at a concentration of 1 mM inhibits (>90%) this uptake. The inward movement of erythritol-¹⁴C shows qualitatively the same dependence on cell volume as ³⁵SO₄, but it is insensitive to the nature of the anion present in the bathing medium. It was also found that the usually observed inhibition of radioactive Na uptake by SCN in cat red cells can be reversed when cell volume is increased.     

Use of salicylic acid to measure the apparent intracellular pH in the ehrlich ascites-tumor cell and Escherichia coli

The distribution of salicylic acid between the intracellular and extracellular phases has been used to estimate the intracellular pH in the Ehrlich cell and Escherichia coli. The validity of the method was established by: (i) comparison of the results obtained with salicylic acid with those obtained with 5,5-dimethyloxazolidine-2,4-dione; (ii) by following changes of the apparent intracellular pH under circumstances in which such changes are predictable, e.g., the addition of weak acids or proton conductors to the incubation medium during incubation at acidic pH; (iii) by comparison of the apparent intracellular pH changes with the uptake of H⁺ by the cells estimated from the changes of the medium pH. Optimal results are obtained with this indicator when the extracellular pH is below 5.5, because in this case the indicator is to a sufficient extent in its penetrating form, so that its movement can reflect intracellular pH changes occurring in less than 30 s. When the intracellular pH falls below 5.2 measurable binding of salicylic acid to the intracellular material of the Ehrlich cell takes place, but above this pH no binding has been found.The Ehrlich cell and cells of Escherichia coli behaved similarly under various experimental circumstances tested, but striking differences were found in the inherent permeability of the membrane to H⁺ and in the changes in this parameter by lowering the temperature to 2°C.     

A Method to Simultaneously Detect Changes in Intracellular Ca<Superscript>2+</Superscript> Concentration and Cell Volume

A method to simultaneously assess the changes in intracellular calcium concentration and cell volume in single cells was developed using the Ca²⁺-sensitive fluorescent probe Fura-2 and a three-dimensional image-surface reconstruction technique, respectively. Studies with this method showed that Fura-2 loading had no significant effect on the kinetics of A549 human epithelial cell swelling in a hypotonic solution, as well as the volume restoration kinetics. Significant changes in intracellular Ca²⁺ concentration were not observed in the examined volume modulation range. The results suggest that Ca²⁺-mediated signaling pathways are not involved in the autoregulation of the cell volume in A549 cells exposed to hypotonic conditions.     

Cell volume regulation following hypotonic shock in hepatocytes isolated from Sparus aurata

The response of isolated hepatocytes of Sparus aurata to hypotonic shock was studied by the aid of videometric and light scattering methods. The isolated cells exposed to a rapid change (from 370 to 260 mOsm/kg) of the osmolarity of the bathing solution swelled but thereafter underwent a decrease of cell volume tending to recovery the original size. This homeostatic response RVD (regulatory volume decrease) was inhibited in the absence of extracellular Ca²⁺ and in the presence of TMB8, an inhibitor of Ca²⁺ release from intracellular stores. It is likely that Ca²⁺ entry through verapamil sensitive Ca²⁺-channels, probably leading to a release of Ca²⁺ from intracellular stores, is responsible for RVD since the blocker impaired the ability of the cell to recover its volume after the hypotonic shock. RVD tests performed in the presence of various inhibitors of different transport mechanisms, such as BaCl₂, quinine, glybenclamide and bumetanide as well as in the presence of a KCl activator, NEM, led us to suggest that the recovery of cell volume in hypotonic solution is accomplished by an efflux of K⁺ and Cl^? through conductive pathways paralleled by the operation of the KCl cotransport, followed by an obliged water efflux from the cells.