Subcellular Localization and Kinetic Characterization of a Gill (Na+, K+)-ATPase from the Giant Freshwater Prawn Macrobrachium rosenbergii

The stimulation by Mg²⁺, Na⁺, K⁺, NH₄ ⁺, and ATP of (Na⁺, K⁺)-ATPase activity in a gill microsomal fraction from the freshwater prawn Macrobrachium rosenbergii was examined. Immunofluorescence labeling revealed that the (Na⁺, K⁺)-ATPase α-subunit is distributed predominantly within the intralamellar septum, while Western blotting revealed a single α-subunit isoform of about 108 kDa M _r. Under saturating Mg²⁺, Na⁺, and K⁺ concentrations, the enzyme hydrolyzed ATP, obeying cooperative kinetics with V _M = 115.0 ± 2.3 U mg^?1, K _0.5 = 0.10 ± 0.01 mmol L^?1. Stimulation by Na⁺ (V _M = 110.0 ± 3.3 U mg^?1, K _0.5 = 1.30 ± 0.03 mmol L^?1), Mg²⁺ (V _M = 115.0 ± 4.6 U mg^?1, K _0.5 = 0.96 ± 0.03 mmol L^?1), NH₄ ⁺ (V _M = 141.0 ± 5.6 U mg^?1, K _0.5 = 1.90 ± 0.04 mmol L^?1), and K⁺ (V _M = 120.0 ± 2.4 U mg^?1, K _M = 2.74 ± 0.08 mmol L^?1) followed single saturation curves and, except for K⁺, exhibited site–site interaction kinetics. Ouabain inhibited ATPase activity by around 73 % with K _I = 12.4 ± 1.3 mol L^?1. Complementary inhibition studies suggest the presence of F₀F₁–, Na⁺-, or K⁺-ATPases, but not V(H⁺)- or Ca²⁺-ATPases, in the gill microsomal preparation. K⁺ and NH₄ ⁺ synergistically stimulated enzyme activity (≈25 %), suggesting that these ions bind to different sites on the molecule. We propose a mechanism for the stimulation by both NH₄ ⁺, and K⁺ of the gill enzyme.     

Active K+ transport in Mycoplasma mycoides var. Capri. Relationships between K+ distribution,electrical potential and ATPase activity

The addition of 5 · 10^?5 M or less of dicyclohexylcarbodiimide to Mycoplasma mycoides var. Capri preferentially influences K⁺ influx rather than efflux and reduces by 30–40% the activity of the membrane-bound Mg²⁺-ATPase. Adding valinomycin to metabolizing cells does not markedly affect K⁺ distribution but induces a rapid and complete loss of intracellular K⁺ in non-metabolizing cells. Uncoupling agents such as dinitrophenol, carbonyl cyanide $\text{p-}\text{trifluoromethoxyphenylhydrazone}$, dissipate the K⁺ concentration gradient only when combined with valinomycin.Variations in the merocyanine fluorescence intensity indicate that a transmembrane electrical potential (Δψ) is generated on cell energization. This Δψ, not affected by valinomycin or uncouplers when used alone, is collapsed by a mixture of both. No change in fluorescence intensity can be detected when glucose is added to dicyclohexylcarbodiimide treated organisms.These experiments suggest that the membrane-bound Mg-ATPase activity controls K⁺ distribution in these organisms through the generation of a transmembrane electrical potential difference.     

Direct formation of axes in new plantlets of <Emphasis Type="Italic">Kappaphycus alvarezii</Emphasis> (Doty) Doty,as influenced by the use of AMPEP K<Superscript>+</Superscript>, spindle inhibitors,and plant growth hormones

In order to induce the direct formation of erect dark brown vegetative shoots of Kappahycus alvarezii, under micro-propagation conditions, the agricultural bio-stimulant “Acadian Marine Plant Extract Powder” (or AMPEP) K⁺ (a Canadian commercial extract of the brown seaweed Ascophyllum nodosum with enhanced levels of potassium) was used singly or in combination with colchicine or oryzalin at different concentrations plus (synthetic) plant growth regulators (i.e., PGR, IAA, and kinetin) over 45 days of incubation. Amongst the treatments tested, 5 mg L^?1 AMPEP K⁺ with PGR produced the longest direct axis shoots (9.6 ± 0.33 mm), followed by 0.1 mg L^?1 AMPEP K⁺ + 1.0 mg L^?1 oryzalin with PGR (8.7 ± 0.00 mm), 10 mg L^?1 AMPEP K⁺ + 0.5 mg L^?1 colchicine with PGR (8.6 ± 1.20 mm), and 0.5 mg AMPEP K⁺ only (5.3 ± 1.8 mm). The shortest, direct axis shoots measured were the following: 8.3 ± 0.33 mm with 0.1, 1 and 10 mg L^?1 AMPEP K⁺ with PGR, 6.0 ± 0.58 mm with 0.5 mg L^?1 AMPEP K⁺ + 0.1 mg L^?1 oryzalin with PGR, 5.3 ± 0.89 mm with 0.5 mg L^?1 AMPEP K⁺ + 0.1 mg L^?1 colchicine with PGR, and finally, 2.7 ± 0.33 mm with 0.1 mg L^?1 AMPEP K⁺ only. The percentage of direct shoots formed ranged from 93 to 100 % and 87 to 100 % in AMPEP K⁺ with PGR and AMPEP K⁺ only, respectively, while 62–100 % and 87–100 % were observed in AMPEP K⁺ + colchicine with PGR and AMPEP K⁺ + oryzalin with PGR, respectively. The use of AMPEP K⁺ with the PGR resulted in the longest and highest percentage formation of direct axis shoots. These observations, taken together, suggested the efficacy of AMPEP K⁺ used as a protocol to induce direct, erect shoots for the micro-propagation of K. alvarezii plantlets under laboratory conditions.     

Energetics of sodium-dependent α-aminoisobutyric acid transport in the moderate halophile Vibrio costicola

The energetics of α-aminoisobutyric acid transport were examined in Vibrio costicola grown in a medium containing the NaCl content (1 M) optimal for growth. Respiration rate, the membrane potential (Δψ) and α-aminoisobutyric acid transport had similar pH profiles, with optima at 8.5–9.0. Cells specifically required Na⁺ ions to transport α-aminoisobutyric acid and to maintain the highest Δψ (150–160 mV). Sodium was not required to sustain high rates of O₂-uptake. Δψ (and α-aminoisobutyric acid transport) recovered fully upon addition of Na⁺ to Na⁺-deficient cells, showing that Na⁺ is required in formation or maintenance of the transmembrane gradients of ions. Inhibitions by protonophores, monensin, nigericin and respiratory inhibitors revealed a close correlation between the magnitudes of Δψ and α-aminoisobutyric acid transport. Also, dissipation of Δψ with triphenylmethylphosphonium cation abolished α-aminoisobutyric acid transport without affecting respiration greatly. On the other hand, alcohols which stimulated respiration showed corresponding increases in α-aminoisobutyric acid transport, without affecting Δψ. Similarly, N,N′-dicyclohexylcarbodiimide (10 μM) stimulated respiration and α-aminoisobutyric acid transport and did not affect Δψ, but caused a dramatic decline in intracellular ATP content. From these, and results obtained with artificially established energy sources (Δψ and Na⁺ chemical potential), we conclude that Δψ is obligatory for α-aminoisobutyric acid transport, and that for maximum rates of transport an Na⁺ gradient is also required.     

Cell density during differentiation can alter the phenotype of bone marrow-derived macrophages

Background

Bone marrow-derived macrophages (BMDMs) are widely used primary cells for studying macrophage function. However, despite numerous protocols that are currently available, lack of a notable consensus on generating BMDMs may obscure the reliability in comparing findings from different studies or laboratories.

Findings

In this study, we addressed the effect of cell density on the resulting macrophage population. With reference to previously published methods, bone marrow cells from wild type C57BL/6 mice were plated at either 4?×?10⁵ cells or 5?×?10⁶ cells per 10 cm and cultured in 20% L-cell conditioned media for 7 days, after which they were analyzed for cell surface markers, production of proinflammatory cytokines, and responsiveness to polarizing signals. Reproducibly, cells plated at lower density gave a pure population of CD11b⁺F4/80⁺ macrophages (97.28?±?0.52%) with majority being Ly-6C^-Ly-6G^- and c-Fms⁺, while those plated at higher density produced less CD11b⁺F4/80⁺ cells and a considerably higher proportion of CD11b⁺F4/80⁺CD11c⁺ (68.72?±?2.52%) and Ly-6C^-Ly-6G⁺ (71.10?±?0.90%) cells. BMDMs derived from higher plating density also secreted less proinflammatory cytokines such as IL-6, IL-12 and TNF-α and were less phagocytic, and had a different pattern of expression for M1- and M2-related genes upon LPS or IL-4 stimulation.

Conclusions

Overall, our findings indicate that altering cell density during BMDM differentiation can give rise to distinct macrophage populations that could vary the outcome of a functional study.

     

Analysis of potassium conductance in squid giant axons

Assuming a model of facilitated ionic transport across axonal membranes proposed by McIlroy (1975) and extended by McIlroy and Hahn (1978), it is shown that if the selectivity coefficient, π_K, of the potassium conducting system ?59 the permeabilityP _Ks, of the periaxonal barrier of the squid giant axon for K⁺ ions?(1.2±0.44)×10^?4 cm sec^?1 and the thickness of the periaxonal space ?477±168 Å. Using a value (10^?4 cm sec^?1) ofP _Ks in the foregoing range the experimental curves for the steady state membrane ionic conductance versus measured membrane potential difference (p.d.), ?, of Gilbert and Ehrenstein (1969) are corrected for the effect of accumulation of K⁺ in the periaxonal space. This correction is most marked for the axon immersed in a natural ionic environment, whose conductance curve is shifted ?70mV along the voltage axis in the hyperpolarization direction. By assuming that the physico-chemical connection between a depolarization of the axonal membrane and the consequent membrane conductance changes is a Wien dissociative effect of the membrane's electric field on a weak electrolyte situated in the axolemma, the position of the peaks of the corrected conductance versus ? curves can be identified with zero membrane electric field and hence with zero p.d.across the axolemma. A set of values for the double-layer p.d.s at the axonal membrane interfaces with the external electrolytes in the vicinity of the K⁺ conducting pores can therefore be deduced for the various external electrolytes employed by Gilbert and Ehrenstein. A model of these double-layer p.d.s in which the membrane interfaces are assumed to possess fixed monovalent negatively charged sites, at least in the neighbourhood of the K⁺ conducting pores, is constructed. It is shown that, using the previously deduced values for the doublelayer p.d.s, such a model has a consistent, physically realistic solution for the distance between the fixed charged sites and for the dissociation constants of these sites in their interaction with the ions of the extramembrane electrolytes.     

The Equilibria of Lipid–K<Superscript>+</Superscript> Ions in Monolayer at the Air/Water Interface

The effect of K⁺ ion interaction with monolayers of phosphatidylcholine (lecithin, PC) or cholesterol (Ch) was investigated at the air/water interface. We present surface tension measurements of lipid monolayers obtained using a Langmuir method as a function of K⁺ ion concentration. Measurements were carried out at 22°C using a Teflon trough and a Nima 9000 tensiometer. Interactions between lecithin and K⁺ ions or Ch and K⁺ ions result in significant deviations from the additivity rule. An equilibrium theory to describe the behavior of monolayer components at the air/water interface was developed in order to obtain the stability constants and area occupied by one molecule of lipid–K⁺ ion complex (LK⁺). The stability constants for lecithin–K⁺ ion (PCK⁺) complex, \( K_{{{\text{PCK}}^{ + } }} = { 3}. 2 6\times 10^{ 2} {\text{dm}}^{ 3} \,{\text{mol}}^{ - 1} \), and for cholesterol–K⁺ ion (ChK⁺) complex, \( K_{{{\text{ChK}}^{ + } }} = { 1}.00 \times 10^{ 3} {\text{dm}}^{ 3} \,{\text{mol}}^{ - 1} \), were calculated by inserting the experimental data. The value of area occupied by one PCK⁺ complex is 60 Å² molecule^?1, while the area occupied by one ChK⁺ complex is 40.9 Å² molecule^?1. The complex formation energy (Gibbs free energy) values for the PCK⁺ and ChK⁺ complexes are ?14.18 ± 0.71 and ?16.92 ± 0.85 kJ mol^?1, respectively.     

Alterations in erythrocyte membrane fluidity and Na+/K+-ATPase activity in chronic alcoholics

Ethanol disorders biological membranes causing perturbations in the bilayer and also by altering the physicochemical properties of membrane lipids. But, chronic alcohol consumption also increases nitric oxide (NO) production. There was no systemic study was done related to alcohol-induced production of NO and consequent formation of peroxynitrite mediated changes in biophysical and biochemical properties, structure, composition, integrity and function of erythrocyte membranes in chronic alcoholics. Hence, keeping all these conditions in mind the present study was undertaken to investigate the role of over produced nitric oxide on red cell membrane physicochemical properties in chronic alcoholics. Human male volunteers aged 44 ± 6 years with similar dietary habits were divided into two groups, namely nonalcoholic controls and chronic alcoholics (~125 g of alcohol at least five times per week for the past 10–12 years). Elevated nitrite and nitrate levels in plasma and lysate, changes in erythrocyte membrane individual phospholipid composition, increased lipid peroxidation, protein carbonyls, cholesterol and phospholipids ratio (C/P ratio) and anisotropic value (γ) with decreased sulfhydryl groups and Na⁺/K⁺-ATPase activity in alcoholics was evident from this study. RBC lysate NO was positively correlated with C/P ratio (r = 0.547) and anisotropic (γ) value (r = 0.428), Na⁺/K⁺-ATPase activity was negatively correlated with RBC lysate NO (r = ?0.372) and anisotropic (γ) value (r = ?0.624) in alcoholics. Alcohol-induced overproduction of nitric oxide reacts with superoxide radicals to produce peroxynitrite, which appears to be responsible for changes in erythrocyte membrane lipids and the activity of Na⁺/K⁺-ATPase.     

Engineering of formate dehydrogenase: synergistic effect of mutations affecting cofactor specificity and chemical stability

Formate dehydrogenases (FDHs) are frequently used for the regeneration of cofactors in biotransformations employing NAD(P)H-dependent oxidoreductases. Major drawbacks of most native FDHs are their strong preference for NAD⁺ and their low operational stability in the presence of reactive organic compounds such as α-haloketones. In this study, the FDH from Mycobacterium vaccae N10 (MycFDH) was engineered in order to obtain an enzyme that is not only capable of regenerating NADPH but also stable toward the α-haloketone ethyl 4-chloroacetoacetate (ECAA). To change the cofactor specificity, amino acids in the conserved NAD⁺ binding motif were mutated. Among these mutants, MycFDH A198G/D221Q had the highest catalytic efficiency (k _cat/K _m) with NADP⁺. The additional replacement of two cysteines (C145S/C255V) not only conferred a high resistance to ECAA but also enhanced the catalytic efficiency 6-fold. The resulting quadruple mutant MycFDH C145S/A198G/D221Q/C255V had a specific activity of 4.00?±?0.13 U?mg^?1 and a K _{m, NADP} ⁺ of 0.147?±?0.020 mM at 30 °C, pH 7. The A198G replacement had a major impact on the kinetic constants of the enzyme. The corresponding triple mutant, MycFDH C145S/D221Q/C255V, showed the highest specific activity reported to date for a NADP⁺-accepting FDH (v _max, 10.25?±?1.63 U?mg^?1). However, the half-saturation constant for NADP⁺ (K _{m, NADP} ⁺ , 0.92?±?0.10 mM) was about one order of magnitude higher than the one of the quadruple mutant. Depending on the reaction setup, both novel MycFDH variants could be useful for the production of the chiral synthon ethyl (S)-4-chloro-3-hydroxybutyrate [(S)-ECHB] by asymmetric reduction of ECAA with NADPH-dependent ketoreductases.     

The relation between membrane ionic current and ATP synthesis in chromatophores from Rhodopseudomonas capsulata

(1) Under conditions in which membrane potential (Δψ) was the sole contributor to the proton-motive force, the steady-state rate of ATP synthesis in chromatophores increased disproportionately when Δψ was increased: the rate had an approximately sixth-power dependence on Δψ. (2) Simultaneous measurements showed that the dissipative ionic current (J_DIS) across the chromatophore membrane had a related dependence on Δψ, i.e., the membrane conductance increased markedly as Δψ increased. (3) For comparable Δψ values, J_DIS was greater in phosphorylating than in non-phosphorylating chromatophores. For comparable actinic light intensities, Δψ was smaller in phosphorylating than in non-phosphorylating chromatophores. (4) At either low pH or in the presence of venturicidin, oligomycin or dicyclohexylcarbodiimide to inhibit ATP synthesis, J_DIS was substantially depressed, particularly at high Δψ. Even under these conditions the membrane conductance was dependent on Δψ. (5) Also in intact cells, J_DIS was depressed in the presence of venturicidin. Points 1–5 are interpreted in terms of a Δψ -driven H⁺ flux through the F₀ channel of the ATPase synthase. The high-power dependence of the F₀ conductance on Δψ determines the dependence of the rate of ATP synthesis on Δψ. The Δψ -dependent conductance of F₀ dominates the electrical properties of the membrane. In chromatophores the ionic current accompanying ATP synthesis was more than 50% of the total membrane ionic current at maximal Δψ. (6) The rate of cyclic electron transport was calculated from J_DIS. This led to an estimate of 0.77 ± 0.22 for the $\text{ATP}\text{2}\text{e}{}^{\mathrm{?}}$ ratio and of 3.5 ± 1.3 for the $\text{H}{}^{\mathrm{+}}\text{ATP}$ ratio. (7) Severe inhibition of the electron-transport rate by decreasing the light intensity led to an almost proportionate decrease in the rate of ATP synthesis. The chromatophores were able to maintain proportionality by confining electron-transport phosphorylation to a narrow range of Δψ. This is a consequence of the remarkable conductance properties of the membrane.     

Molecular cloning and characterization of two novel fructose-specific transporters from the osmotolerant and fructophilic yeast Candida magnoliae JH110

Sugar transport is very critical in developing an efficient and rapid conversion process of a mixture of sugars by engineered microorganisms. By using expressed sequence tag data generated for the fructophilic yeast Candida magnoliae JH110, we identified two fructose-specific transporters, CmFSY1 and CmFFZ1, which show high homology with known fructose transporters of other yeasts. The CmFSY1 and CmFFZ1 genes harbor no introns and encode proteins of 574 and 582 amino acids, respectively. Heterologous expression of the two fructose-specific transporter genes in a Saccharomyces cerevisiae, which is unable to utilize hexoses, revealed that both transporters are functionally expressed and specifically transport fructose. These results were further corroborated by kinetic analysis of the fructose transport that showed that CmFsy1p is a high-affinity fructose–proton symporter with low capacity (K _M?=?0.13?±?0.01 mM, V _max?=?2.1?±?0.3 mmol h^?1 [gdw]^?1) and that CmFfz1p is a low-affinity fructose-specific facilitator with high capacity (K _M?=?105?±?12 mM, V _max?=?8.6?±?0.7 mmol h^?1 [gdw]^?1). These fructose-specific transporters can be used for improving fructose transport in engineered microorganisms for the production of biofuels and chemicals from fructose-containing feedstock.     

Membrane potential and delta pH dependency of reverse electron transport-associated hydrogen peroxide production in brain and heart mitochondria

Succinate-driven reverse electron transport (RET) is one of the main sources of mitochondrial reactive oxygen species (mtROS) in ischemia-reperfusion injury. RET is dependent on mitochondrial membrane potential (Δψ_m) and transmembrane pH difference (ΔpH), components of the proton motive force (pmf); a decrease in Δψ_m and/or ΔpH inhibits RET. In this study we aimed to determine which component of the pmf displays the more dominant effect on RET-provoked ROS generation in isolated guinea pig brain and heart mitochondria respiring on succinate or α-glycerophosphate (α-GP). Δψ_m was detected via safranin fluorescence and a TPP⁺ electrode, the rate of H₂O₂ formation was measured by Amplex UltraRed, the intramitochondrial pH (pH_in) was assessed via BCECF fluorescence. Ionophores were used to dissect the effects of the two components of pmf. The K⁺/H⁺ exchanger, nigericin lowered pH_in and ΔpH, followed by a compensatory increase in Δψ_m that led to an augmented H₂O₂ production. Valinomycin, a K⁺ ionophore, at low [K⁺] increased ΔpH and pH_in, decreased Δψ_m, which resulted in a decline in H₂O₂ formation. It was concluded that Δψ_m is dominant over ?pH in modulating the succinate- and α-GP-evoked RET. The elevation of extramitochondrial pH was accompanied by an enhanced H₂O₂ release and a decreased ?pH. This phenomenon reveals that from the pH component not ?pH, but rather absolute value of pH has higher impact on the rate of mtROS formation. Minor decrease of Δψ_m might be applied as a therapeutic strategy to attenuate RET-driven ROS generation in ischemia-reperfusion injury.     

Effect of external ATP on the plasma membrane permeability and (Na++K+)-ATPase activity of mouse neuroblastoma cells

1. Addition of 3.5 mM ATP to mouse neuroblastoma Neuro-2A cells results in a selective enhancement of the plasma membrane permeability for Na⁺ relative to K⁺, as measured by cation flux measurements and electro-physiological techniques. 2. Addition of 3.5 mM ATP to Neuro-2A cells results in a 70% stimulation of the rate of active K⁺ -uptake by these cells, partly because of the enhanced plasma membrane permeability for Na⁺. Under these conditions the pumping activity of the Neuro-2A (Na⁺+K⁺)-ATPase is optimally stimulated with respect to its various substrate ions. 3. External ATP significantly enhances the affinity of the Neuro-2A (Na⁺+K⁺)-ATPase for ouabain, as measured by direct [³H]ouabain-binding studies and by inhibition studies of active K⁺ uptake. In the presence of 3.5 mM ATP and the absence of external K⁺ both techniques indicate an apparent dissociation constant for ouabain of 2·10^?6 M. Neuro-2A cells contain (3.5±0.7)·10⁵ ouabain-binding sites per cell, giving rise to an optimal pumping activity of (1.7±0.4)·10^?20 mol K⁺/min per copy of (Na⁺+K⁺)-ATPase at room temperature.     

An electrophysiological study of the membrane properties of the immature and mature oocyte of the Batstar,Patiria miniata

Immature oocyte membrane properties of a starfish, Patiria miniata, were investigated by microelectrode techniques. The resting membrane potential in artificial seawater (ASW) was ?78.5 ± 6.7 mV (n = 61, inside negative). This was mainly accounted for by a selective permeability to potassium ions. Potassium ion-selective microelectrodes were used to measure intracellular K⁺ ion activity, which was 350 mM. The sodium to potassium permeability ratio was 0.02 ± 0.01 (n = 4). The current-voltage relation was nonlinear. The I–V curve included both areas of inward and outward rectification. The dependence of inward rectification upon the K⁺ ion electrochemical gradient was demonstrated. The membrane was capable of a regenerative action potential due to permeability changes for Ca²⁺ and Na⁺ ions. The Ca and Na components of the action potential were identified. The Ca component was reversibly suppressed by cobalt and irreversibly blocked by D-600. The Na component was tetrodotoxin (TTX) insensitive. The excitable response of P. miniata oocytes is similar to that described by Miyazaki et al. (1975a) for those of the starfish Asterina pectinifera.Immature oocytes were stimulated to mature with 10^?5M 1-methyladenine (1-MA) during continuous monitoring of the membrane potential. The resting potential in ASW became more inside negative during maturation. This change of the passive membrane property of the oocyte may be accounted for by the increased selectivity to K⁺ ions. The specific membrane resistance near the resting potential increased from 4.2 ± 1.4 to 21 ± 8.7 kΩ·cm² (n = 15) during maturation, while the specific membrane capacitance decreased slightly from 2 ± 0.5 to 1.7 ± 0.6 μF/cm² (n = 5). Maturation had little effect upon the active membrane properties.     

Functional Identification of H<Superscript>+</Superscript>-ATPase and Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Antiporter in the Plasma Membrane Isolated from the Root Cells of Salt-Accumulating Halophyte <Emphasis Type="Italic">Suaeda altissima</Emphasis>

A membrane fraction enriched in plasma membrane (PM) vesicles was isolated from the root cells of a salt-accumulating halophyte Suaeda altissima (L.) Pall. by means of centrifugation in discontinuous sucrose density gradient. The PM vesicles were capable of generating ΔpH at their membrane and the transmembrane electric potential difference (Δψ). These quantities were measured with optical probes, acridine orange and oxonol VI, sensitive to ΔpH and Δψ, respectively. The ATP-dependent generation of ΔpH was sensitive to vanadate, an inhibitor of P-type ATPases. The results contain evidence for the functioning of H⁺-ATPase in the PM of the root cells of S. altissima. The addition of Na⁺ and Li⁺ ions to the outer medium resulted in dissipation of ΔpH preformed by the H⁺-ATPase, which indicates the presence in PM of the functionally active Na⁺/H⁺ antiporter. The results are discussed with regard to involvement of the Na⁺/H⁺ antiporter and the PM H⁺-ATPase in loading Na⁺ ions into the xylem of S. altissima roots.     

A study of the current-voltage relationships of electrogenetic active and passive membrane elements in riccia fluitans

Potassium- and proton-dependent membrane potential, conductance, and current-voltage characteristics (I–V curves) have been measured on rhizoid cells of the liverwort Riccia fluitans. The potential difference (E_m) measured with microelectrodes across plasmalemma and tonoplast is depolarized to the potassium-sensitive diffusion potential (E_D) in the presence of 1 mM NaCN, 1 mM NaN₃, or at temperatures below 6°C. Whereas the temperature change from 25°C to 5°C decreases the membrane conductance (g_m) from 0.71 to 0.43 S ? m^?2, 1 mM NaCN increases g_m by about 25%. The membrane displays potassium-controlled rectification which gradually disappears at temperatures below 5°C. The potassium pathway can be described by an equivalent circuit of a diode and an ohmic resistor in parallel. In the potential interval of E_D ± 100 mV the measured I-V curves roughly fit the theoretical curves obtained from a modified diode equation. ⁸⁶Rb⁺(K⁺)-influx is voltage sensitive: In the presence of 1 mM NaCN, ⁸⁶Rb⁺-influx follows a hyperbolic function corresponding to a low conductance at low [K⁺]_o and high conductance at high [K⁺]_o. On the contrary ⁸⁶Rb⁺-influx is linear with [K⁺]_o when pump activity is normal. It is believed that there are two K⁺-transport pathways in the Riccia membrane, one of which is assigned to the low conductance (0.2 S · m^?2), the other to a temperature-dependent facilitated diffusion system with a higher conductance (7.7 S · m^?2). The electrogenic pump essentially acts as a current source and consumes about 39% of the cellular ATP-turnover. In the presence of 30 μM CCCP the saturation current of 0.1 A · m^?2 is doubled to about 0.2 A · m^?2, and the electromotive force of ?360 mV switches to ?250 mV. It is suggested that this may be due to a change in stoichiometry from one to two transported charges per ATP hydrolyzed.     

Two-Probe Microfluorometry Estimation of Transmembrane Potential (ΔΨ p) Slight Changes in Individual Hepatocytes Under Short-Term Hormone Action

Early events in individual hepatocytes of rat activated by adrenaline (10^?6M) and phenylephrine (10^?5M) have been investigated by quantitative image microfluorometry and microspectrofluorometry. Cationic DiOC₂ and anionic SqSC₄ probes have been used for image analysis and transmembrane potential (ΔΨ _p) estimation in real-time studies. Fluorescence spectra resulting from the accumulation of dyes in single cells were recorded. Based on the mean fluorescence intensity, the magnitude of ΔΨ _p was calculated by Nernst equation adapted for lipophilic cationic probes. DiOC₂ has revealed that both hormones induce biphasic hyperpolarization of hepatocytes membrane with α-agonist phenylephrine causing ΔΨ _p changes at higher amplitude. The first increase of ΔΨ _p within 2 and 5 min (ΔΔΨ _p = ?8.6 ± 4.2 mV) apparently related to Na⁺/K⁺-ATPase activation by the Ca²⁺-mobilizing hormone. The second peak of hyperpolarization (ΔΔΨ _p = ?13.2 ± 3.2 mV) between 25 and 30 min, after a transient decrease of ΔΨ _p (ΔΔΨ _p = 10.9 ± 4.3 mV) over 15 min experiment, probably is mediated by phenylephrine stimulating action on K⁺-channels. K⁺ channel blocker (Ba²⁺ or 4-aminopyridine) as well as elevating of extracellular K⁺ prevented the hyperpolarization. Modulation of PLD-dependent signal transduction pathway by 0.4 % butanol had a weak influence on the first increase of ΔΨ _p but it abolished the second phase of hyperpolarization. That points to PLD involvement in the ΔΨ _p fluctuations mediated by K⁺-channels in response to phenylephrine. Based on SqSC₄, fluorescent parameters estimation of relative changes of ΔΨ _p revealed similar character of time dependence with two phases of hyperpolarization. Synchronic fluctuation of ΔΨ _p determined by oppositely charged probes demonstrate that the quantitative microfluorometry allows to evaluate slight ΔΨ _p changes separately from ΔΨ _m in non-excitable individual cells at the short-term hormone action.     

Potassium currents in cultured rabbit retinal pigment epithelial cells

Membrane potential and ionic currents were studied in cultured rabbit retinal pigment epithelial (RPE) cells using whole-cell patch clamp and perforated-patch recording techniques. RPE cells exhibited both outward and inward voltage-dependent currents and had a mean membrane capacitance of 26±12 pF (sd, n=92). The resting membrane potential averaged ?31±15 mV (n=37), but it was as high as ?60 mV in some cells. When K⁺ was the principal cation in the recording electrode, depolarization-activated outward currents were apparent in 91% of cells studied. Tail current analysis revealed that the outward currents were primarily K⁺ selective. The most frequently observed outward K⁺ current was a voltage- and time-dependent outward current (I _K) which resembled the delayed rectifier K⁺ current described in other cells. I _K was blocked by tetraethylammonium ions (TEA) and barium (Ba²⁺) and reduced by 4-aminopyridine (4-AP). In a few cells (3–4%), depolarization to ?50 mV or more negative potentials evoked an outwardly rectifying K⁺ current (I _Kt) which showed more rapid inactivation at depolarized potentials. Inwardly rectifying K⁺ current (I _KI) was also present in 41% of cells. I _KI was blocked by extracellular Ba²⁺ or Cs⁺ and exhibited time-dependent decay, due to Na⁺ blockade, at negative potentials. We conclude that cultured rabbit RPE cells exhibit at least three voltage-dependent K⁺ currents. The K⁺ conductances reported here may provide conductive pathways important in maintaining ion and fluid homeostasis in the subretinal space.     

Functional differentiation of a human ganglion cell derived neuroblastoma cell line SH-SY5Y induced by a phorbol ester (TPA)

When the human neuroblastoma cell line SH-SY5Y is exposed to 12-o-tetradecanoyl-phorbol-13-acetate (TPA) the cells grow long processes indicative of neural differentiation. Concomitantly there is an increase in the resting membrane potential from ?44 ± 2 mV found in untreated cells to ?63 ± 4 mV after induction. The TPA treated cells are depolarized when the external potassium concentration is increased to 46 mM and upon addition of veratridine. In contrast to the untreated cells depolarization in differentiated cells leads to an increase in the rate of Ca²⁺ influx. This increase in Ca²⁺ influx is blocked by the Ca²⁺ channel antagonist, verapamil, while the Na⁺ channel blocker tetrodotoxin only marginally inhibits the K⁺ depolarization-induced Ca²⁺ influx.The results suggest that the induction of morphological differentiation in this cell line is associated with the appearance of features of excitable cells.     

Decolorization of azo dyes by Geobacter metallireducens

Geobacter metallireducens was found to be capable of decolorizing several azo dyes with different structures to various extents. Pyruvate, ethanol, acetate, propionate, and benzoate could support 66.3?±?2.6?93.7?±?2.1 % decolorization of 0.1 mM acid red 27 (AR27) in 40 h. The dependence of the specific decolorization rate on AR27 concentration (25 to 800 μM) followed Michaelis–Menten kinetics (K _m?=?186.9?±?1.4 μΜ, V _max?=?0.65?±?0.02 μmol?mg protein^?1 h^?1). Enhanced AR27 decolorization was observed with the increase of cell concentrations ranging from 7.5 to 45 mgL^?1. AR27 decolorization by G. metallireducens was retarded by the presence of goethite, which competed electrons with AR27 and was reduced to Fe(II). The addition of low concentrations of humic acid (1?100 mgL^?1) or 2-hydroxy–1,4-naphthoquinone (0.5?50 μM) could improve the decolorization performance of G. metallireducens. High-performance liquid chromatography analysis suggested reductive pathway to be responsible for decolorization. This was the first study on azo dye decolorization by Geobacter strain and might improve our understanding of natural attenuation and bioremediation of environments polluted by azo dyes.