The sodium cycle: A novel type of bacterial energetics

The progress of bioenergetic studies on the role of Na⁺ in bacteria is reviewed. Experiments performed over the past decade on several bacterial species of quite different taxonomic positions show that Na⁺ can, under certain conditions, substitute for H⁺ as the coupling ion. Various primary Na⁺ pumps ( generators) are described, i.e., Na⁺-motive decarboxylases, NADH-quinone reductase, terminal oxidase, and ATPase. The formed is shown to be consumed by Na⁺ driven ATP-synthase, Na⁺ flagellar motor, numerous Na⁺, solute symporters, and the methanogenesis-linked reverse electron transfer system. InVibrio alginolyticus, it was found that , generated by NADH-quinone reductase, can be utilized to support all three types of membrane-linked work, i.e., chemical (ATP synthesis), osmotic (Na⁺, solute symports), and mechanical (rotation of the flagellum). InPropionigenum modestum, circulation of Na⁺ proved to be the only mechanism of energy coupling. In other species studied, the Na⁺ cycle seems to coexist with the H⁺ cycle. For instance, inV. alginolyticus the initial and terminal steps of the respiratory chain are Na⁺ - and H⁺-motive, respectively, whereas ATP hydrolysis is competent in the uphill transfer of Na⁺ as well as of H⁺. In the alkalo- and halotolerantBacillus FTU, there are H⁺ - and Na⁺-motive terminal oxidases. Sometimes, the Na⁺-translocating enzyme strongly differs from its H⁺-translocating homolog. So, the Na⁺-motive and H⁺-motive NADH-quinone reductases are composed of different subunits and prosthetic groups. The H⁺-motive and Na⁺-motive terminal oxidases differ in that the former is ofaa ₃-type and sensitive to micromolar cyanide whereas the latter is of another type and sensitive to millimolar cyanide. At the same time, both Na⁺ and H⁺ can be translocated by one and the sameP. modestum ATPase which is of the F₀F₁-type and sensitive to DCCD. The sodium cycle, i.e., a system composed of primary generator(s) and consumer(s), is already described in many species of marine aerobic and anaerobic eubacteria and archaebacteria belonging to the following genera:Vibrio, Bacillus, Alcaligenes, Alteromonas, Salmonella, Klebsiella, Propionigenum, Clostridium, Veilonella, Acidaminococcus, Streptococcus, Peptococcus, Exiguobacterium, Fusobacterium, Methanobacterium, Methanococcus, Methanosarcin, etc. Thus, the sodium world seems to occupy a rather extensive area in the biosphere.     

Role of the furosemide-sensitive Na+/K+ transport system in determining the steady-state Na+ and K+ content and volume of human erythrocytesin vitro andin vivo

Summary To study the physiological role of the bidirectionally operating, furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes, the effect of furosemide on red cell cation and hemoglobin content was determined in cells incubated for 24 hr with ouabain in 145mm NaCl media containing 0 to 10mm K⁺ or Rb⁺. In pure Na⁺ media, furosemide accelerated cell Na⁺ gain and retarded cellular K⁺ loss. External K⁺ (5mm) had an effect similar to furosemide and markedly reduced the action of the drug on cellular cation content. External Rb⁺ accelerated the Na⁺ gain like K⁺, but did not affect the K⁺ retention induced by furosemide. The data are interpreted to indicate that the furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes mediates an equimolar extrusion of Na⁺ and K⁺ in Na⁺ media (Na⁺/K⁺ cotransport), a 1:1 K⁺/K⁺ (K⁺/Rb⁺) and Na⁺/Na⁺ exchange progressively appearing upon increasing external K⁺ (Rb⁺) concentrations to 5mm. The effect of furosemide (or external K⁺/Rb⁺) on cation contents was associated with a prevention of the cell shrinkage seen in pure Na⁺ media, or with a cell swelling, indicating that the furosemide-sensitive Na⁺/K⁺ transport system is involved in the control of cell volume of human erythrocytes. The action of furosemide on cellular volume and cation content tended to disappear at 5mm external K⁺ or Rb⁺. Thein vivo red cell K⁺ content was negatively correlated to the rate of furosemide-sensitive K⁺ (Rb⁺) uptake, and a positive correlation was seen between mean cellular hemoglobin content and furosemide-sensitive transport activity. The transport system possibly functions as a K⁺ and waterextruding mechanism under physiological conditiosin vivo. The red cell Na⁺ content showed no correlation to the activity of the furosemide-sensitive transport system.     

Ouabain resistance of the epithelial cell line (Ma104) is not due to lack of affinity of its pumps for the drug

Na⁺, K⁺-pumps of most eukaryotic animal cells bind ouabain with high affinity, stop pumping, and consequently loose K⁺, detach from each other and from the substrate, and die. Lack of affinity for the drug results in ouabain resistance. In this work, we report that Ma104 cells (epithelial from Rhesus monkey kidney) have a novel form of ouabain-resistance: they bind the drug with high affinity (Km about 4×10^–8 m), they loose their K⁺ and stop proliferating but, in spite of these, up to 100% of the cells remain attached in 1.0 m ouabain, and 53% in 1.0 mm. When 4 days later ouabain is removed from the culture medium, cells regain K⁺ and resume proliferation. Strophanthidin, a drug that attaches less firmly than ouabain, produces a similar phenomenon, but allows a considerably faster recovery. This reversal may be associated to the fact that, while in ouabain-sensitive MDCK cells Na⁺, K⁺-ATPases blocked by the drug are retrieved from the plasma membrane, those in Ma104 cells remain at the cell-cell border, as if they were cell-cell attaching molecules. Cycloheximide (10 g/ml) and chloroquine (10 m) impair this recovery, suggesting that it also depends on the synthesis and insertion of a crucial protein component, that may be different from the pump itself. Therefore ouabain resistance of Ma104 cells is not due to a lack of affinity for the drug, but to a failure of its Na⁺, K⁺-ATPases to detach from the plasma membrane in spite of being blocked by ouabain.We wish to thank Dr. E. Rodríguez-Boulán for the generous supply of Ma104 cells, as well as acknowledge the generous economic support of the National Research Council (CONACYT) of Mexico. Confocal experiments were performed in the Confocal Microscopy Unit of the Physiology Department, CINVESTAV.     

Sodium ATPase and Sodium/proton Antiporter Are Not Obligatory for Sodium Homeostasis of Enterococcus hirae at Acid pH

Enterococcus hirae grows in a broad pH range from 5 to 11. An E. hirae mutant 7683 lacking the activities of two sodium pumps, Na⁺-ATPase and Na⁺/H⁺ antiporter, does not grow in high Na⁺ medium at pH above 7.5. We found that 7683 grew normally in high Na⁺ medium at pH 5.5. Although an energy-dependent sodium extrusion at pH 5.5 was missing, the intracellular levels of Na⁺ and K⁺ were normal in this mutant. The Na⁺ influx rates of 7683 and two other strains at pH 5.5 were much slower than those at pH 7.5. These results suggest that Na⁺ elimination of this bacterium at acid pH is achieved by a decrease in Na⁺ entry and a normal K⁺ uptake.     

Internal radiotherapy of painful bone metastases

Internal radiotherapy is effective in the treatment of metastatic bone pain and can improve quality of life. A number of controlled studies using various agents have shown a mean response rate in pain relief of 70–80% of treated patients. Some investigators prefer radionuclides which emit low beta particles for the treatment of bone pain, because the assumption of lower bone marrow toxicity of this agents. However, neither dosimetric data for radiation absorbed dose to the bone marrow nor clinical blood count depression have shown any significant differences between these agents. Other researchers suggest enhanced antitumoral effects using high-energy beta emitters and propose aggressive first-line treatment in the early disease stage instead of using these radiopharmaceuticals only in end-stage patients suffering intractable bone pain. Another approach consists of including other treatment modalities such as autologous stem cell rescue or in combination with chemo or bisphosphonate therapy to a radionuclide treatment scheme. Future research should focus more on the curative effects of combination with radiosensitizer, for example, chemotherapy, or repeated treatments with bone seeking agents.     

Metabolism of NAD+ in Nuclei of Saccharomyces cerevisiae during Stimulation of Its Biosynthesis by Nicotinamide

The activities of nuclear enzymes involved in NAD⁺ metabolism in Saccharomyces cerevisiae strain 913a-1 and its mutant 110 previously selected as an NAD⁺ producer were investigated. The presence of extracellular nicotinamide increased the total NAD⁺ pool in the cells and increased [³H]nicotinic acid incorporation; however, NAD⁺ concentration in isolated nuclei decreased slightly. The stimulating effect of nicotinamide on intracellular synthesis of NAD⁺ correlated with increases in ADP-ribosyl transferase, NAD⁺-pyrophosphorylase, and NAD⁺ ase activities.     

Intracellular calcium content of human erythrocytes: Relation to sodium transport systems

Summary To study the possible role of intracellular Ca (Ca _i ) in controlling the activities of the Na⁺–K⁺ pump, the Na⁺–K⁺ cotransport and the Na⁺/Li⁺ exchange system of human erythrocytes, a method was developed to measure the amount of Ca embodied within the red cell. For complete removal of Ca associated with the outer aspect of the membrane, it proved to be essential to wash the cells in buffers containing less than 20nm Ca. Ca was extracted by HClO₄ in Teflon^® vessels boiled in acid to avoid Ca contaminations and quantitated by flameless atomic absorption. Ca _i of fresh human erythrocytes of apparently healthy donors ranged between 0.9 and 2.8 mol/liter cells. The mean value found in females was significantly higher than in males. The interindividual different Ca contents remained constant over periods of more than one year. Sixty to 90% of Ca _i could be removed by incubation of the cells with A23187 and EGTA. The activities of the Na⁺–K⁺ pump, of Na⁺–K⁺ cotransport and Na⁺/Li⁺ exchange and the mean cellular hemoglobin content fell with rising Ca _i ; the red cell Na⁺ and K⁺ contents rose with Ca _i . Ca depletion by A23187 plus EGTA as well as chelation of intracellular Ca²⁺ by quin-2 did not significantly enhance the transport rates. It is concluded that the large scatter of the values of Ca _i of normal human erythrocytes reported in the literature mainly results from a widely differing removal of Ca associated with the outer aspect of the membrane.     

Effects of Tetraethylammoniumchloride (TEA), Vanadate,and Alkali Ions on the Lateral Leaflet Movement Rhythm of Desmodium motorium (Houtt.) Merr

The period (～3-5 min) of the ultradian rhythm of the lateral leaflet movement of Desmodium motorium is strongly lengthened (≤30-40%) by the K⁺ channel blocker tetraethylammoniumchloride (20, 30, and 40 mM) and vanadate (0.5 and 1 mM), which is an effective inhibitor of the plasma membrane-bound H⁺ pump. The alkali ions K⁺, Na⁺, Rb⁺, and Cs⁺ (10-40 mM) shorten the period only slightly (≤ 10–15%). Li⁺ (5-30 mM), however, increases the period of the leaflet rhythm drastically (≤80%). We concluded that the plasmalemma-H⁺-ATP-ase-driven K⁺ transport through K⁺ channels is an essential component of the ultradian oscillator of Desmodium, as has been proposed for the circadian oscillator.     

Effect of tissue specificity of brain soluble fractions on Na+, K+-ATPase activity

Previous evidence from this laboratory indicated that catecholamines and brain endogenous factors modulate Na⁺, K⁺-ATPase activity of the synaptosomal membranes. The filtration of a brain total soluble fraction through Sephadex G-50 permitted the separation of two fractions-peaks I and II-which stimulated and inhibited Na⁺, K⁺-ATPase, respectively (Rodríguez de Lores Arnaiz and Antonelli de Gomez de Lima, Neurochem. Res.11, 1986, 933). In order to study tissue specificity a rat kidney total soluble was fractionated in Sephadex G-50 and kidney peak I and II fractions were separated; as control, a total soluble fraction prepared from rat cerebral cortex was also processed. The UV absorbance profile of the kidney total soluble showed two zones and was similar to the profile of the brain total soluble. Synaptosomal membranes Na⁺, K⁺- and Mg²⁺-ATPases were stimulated 60–100% in the presence of kidney and cerebral cortex peak I; Na⁺, K⁺-ATPase was inhibited 35–65% by kidney peak II and 60–80% by brain peak II. Mg²⁺-ATPase activity was not modified by peak II fractions. ATPases activity of a kidney crude microsomal fraction was not modified by kidney peak I or brain peak II, and was slightly increased by kidney peak II or brain peak I. Kidney purified Na⁺, K⁺-ATPase was increased 16–20% by brain peak I and II fractions. These findings indicate that modulatory factors of ATPase activity are not exclusive to the brain. On the contrary, there might be tissue specificity with respect to the enzyme source.     

Importance of the seryl and threonyl residues of the fifth transmembrane domain to the substrate specificity of yeast plasma membrane Na+/H+ antiporters

The Zygosaccharomyces rouxii Na⁺/H⁺ antiporter Sod2-22p is a member of the subfamily of yeast plasma membrane Nha/ Sod antiporters that do not recognize potassium as their substrate. A functional study of two ZrSod2-22p mutated versions that improved the tolerance of a S. cerevisiae alkali-metal-cation sensitive strain to high extracellular concentration of KCl identified two polar non-charged amino-acid residues in the fifth transmembrane domain, Thr141 and Ser150, as being involved in substrate recognition and transport in yeast Nha/Sod antiporters. A reciprocal substitution of amino-acid residues with a hydroxyl group at these positions, T141S or S150T, produced a broadened cation selectivity of the antiporter for K⁺, in addition to Na⁺ and Li⁺. Site-directed mutagenesis of Ser150 showed that while the replacement of Ser150 with a small hydrophobic (valine) or negatively charged (aspartate) amino acid did not produce a significant change in ZrSod2-22p substrate specificity, the introduction of a positive charge at this position stopped the activity of the antiporter. This data demonstrates that the amino-acid composition of the fifth transmembrane domain, mainly the presence of amino acids containing hydroxyl groups in this part of the protein, is critical for the recognition and transport of substrates and could participate in conformational movements during the binding and/or cation transport cycle in yeast plasma membrane Na⁺/H⁺ antiporters.     

Characterization of Syrian hamster gastric mucosal H+,K+-ATPase

Employing a simple one-step sucrose gradient fractionation method, gastric mucosal membrane of Syrian hamster was prepared and demonstrated to be specifically enriched in H⁺,K⁺-ATPase activity. The preparation is practically devoid of other ATP hydrolyzing activity and contains high K⁺-stimulated ATPase, activity of at least 4–5 fold compared to basal ATPase activity. The H⁺,K⁺-ATPase showed hydroxylamine-sensitive phosphorylation and K⁺-dependent dephosphorylation of the phospho-enzyme, characteristic inhibition by vanadate, omeprazole and SCH 28080, and nigericin-reversible K⁺-dependent H⁺-transport — properties characteristic of gastric proton pump One notable difference with H⁺,K⁺-ATPase of other species has been the observation of valinomycin-independent H⁺ transport in such membrane vesicles. It is proposed that such H⁺,K⁺-ATPase-rich hamster gastric mucosal membrane preparation might provide a unique model to study physiological aspects of H⁺,K⁺-ATPase-function in relation to HCl secretion.     

Formation Mechanism of the Free Radical Product and Its Precursor by the Reaction of Dehydro-L-ascorbic Acid with Amino Acid

During the formation of radical A (2) and its precursor (tris(2-deoxy-2-L-ascorbyl)amine, 1) by the reaction of dehydroascorbic acid (DHA) with amino acid, ascorbic acid (AsA) and the reduced red pigment (3) were newly identified, in addition to scorbamic acid (SCA) and the red pigment (4), as intermediate products. The addition of AsA to the DHA-amino acid reaction, as well as to the DHA-SCA reaction, greatly increased the formation of 3 and 1. The reaction of AsA with 4 gave rapidly 3, followed by the gradual production of 1. From these results, a reaction pathway is proposed that 3 formed by the reduction of 4 with AsA is a key intermediate and its condensation with DHA followed by reduction with AsA might produce 2 and 1.     

Activity of Ion Transporters and Salt Tolerance in Barley

The authors attempted to relate the cultivar-specific salt tolerance in barley (Hordeum distichum L.) to the efficiency of ion transporters in the plasmalemma and tonoplast. The study involved plasmalemma and tonoplast membrane vesicles isolated from roots and leaves of the 7-day-old barley seedlings exposed to elevated NaCl concentrations. Two barley cultivars were employed: salt-tolerant cv. Elo and salt-susceptible cv. Belogorskii. The vesicles were used to measure the transport activity of plasmalemma and tonoplast proton pumps and the cation/anion exchange. The data obtained in the experiments demonstrated that the changes in the activity of ion transporters under salt stress conditions correlated with the barley cultivar-specific tolerance to elevated NaCl concentrations.     

Metabolic control of the K+ channel of human red cells

Summary The effects of cAMP, ATP and GTP on the Ca²⁺-dependent K⁺ channel of fresh (1–2 days) or cold-stored (28–36 days) human red cells were studied using atomic absorption flame photometry of Ca²⁺-EGTA loaded ghosts which had been resealed to monovalent cations in dextran solutions. When high-K⁺ ghosts were incubated in an isotonic Na⁺ medium, the rate constant of Ca²⁺-dependent K⁺ efflux was reduced by a half on increasing the theophylline concentration to 40mm. This effect was observed in ghosts from both fresh and stored cells, but only if they were previously loaded with ATP. The inhibition was more marked when Mg²⁺ was added together with ATP, and it was abolished by raising free Ca²⁺ to the micromolar level. Like theophylline, isobutyl methylxanthine (10mm) also affected K⁺ efflux. cAMP (0.2–0.5mm), added both internally and externally (as free salt, dibutyryl or bromide derivatives), had no significant effect on K⁺ loss when the ghost free-Ca²⁺ level was below 1 m, but it was slightly inhibitory at higher concentrations. The combined presence of cAMP (0.2mm) plus either theophylline (10mm), or isobutyl methylxanthine (0.5mm), was more effective than cAMP alone. This inhibition showed a strict requirement for ATP plus Mg²⁺ and it, was not overcome by raising internal Ca²⁺. Ghosts from stored cells seemed more sensitive than those from fresh cells, to the combined action of cAMP and methylxanthines. Loading ATP into ghosts from fresh or stored cells markedly decreased K⁺ loss. Although this effect was observed in the absence of added Mg²⁺ (0.5mm EDTA present), it was potentiated upon adding 2mm Mg²⁺. The K⁺ efflux from ATP-loaded ghosts was not altered by dithio-bis-nitrobenzoic acid (10mm) or acridine orange (100 m), while it was increased two-to fourfold by incubating with MgF₂ (10mm), or MgF₂ (10mm)+theophylline (40mm), respectively. By contrast, a marked efflux reduction was obtained by incorporating 0.5mm GTP into ATP-containing ghosts. The degree of phosphorylation obtained by incubating membranes with (-³²P)ATP under various conditions affecting K⁺ channel activity, was in direct correspondence to their effect on K⁺ efflux. The results suggest that the K⁺ channel of red cells is under complex metabolic control, via cAMP-mediated and nonmediated mechanisms, some which require ATP and presumably, involve phosphorylation of the channel proteins.     

Mechanisms of liquid flux across pulmonary alveolar epithelial cell monolayers

Summary Active transport of sodium by pulmonary alveolar epithelial cells (AEC) is believed to be an important component of edema clearance in the normal and injured lung. Data supporting this premise have come from measurements of sodium movement across AEC monolayers or from perfused lung model systems. However, direct measurement of fluid flux across AEC monolayers has not been reported. In the present work, AEC were studied with an experimental system for the measurement of fluid flux (Jv) across functionally intact cell monolayers. Primary adult rat type II alveolar epithelial cells were cultured on 0.8 μm nuleopore filters previously coated with gelatin and fibronectin. Intact monolayers were verified by high electrical resistance (> 1000 Θ) at 4–5 d of primary culture. At the same time interval, transmission electron microscopy revealed cells with type I cell-like morphology throughout the monolayer. These were characterized by both adherens and tight junctional attachments. Fluid flux across the monolayers was measured volumetrically over a period of 2 h in the presence of HEPES-buffered DMEM containing 3% fatty acid-free bovine serum albumin. Flux (Jv) was inhibited 39% by 1 × 10⁻⁴ M ouabain (P < 0.01) and 27% by 5 × 10⁻⁴ M amiloride (P < 0.05). These data support the concept that AEC Na⁺/K⁺-ATPase and Na⁺ transport systems are important determinants of AEC transepithelial fluid movement in vitro.     

In-gel activity staining of oxidized nicotinamide adenine dinucleotide kinase by blue native polyacrylamide gel electrophoresis

Oxidized nicotinamide adenine dinucleotide (NAD(+)) kinase (NADK, E.C. 2.7.1.23) plays an instrumental role in cellular metabolism. Here we report on a blue native polyacrylamide gel electrophoretic technique that allows the facile detection of this enzyme. The product, oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)), formed following the reaction of NADK with NAD(+) and adenosine 5'-triphosphate was detected with the aid of glucose-6-phosphate dehydrogenase or NADP(+)-isocitrate dehydrogenase, iodonitrotetrazolium chloride, and phenazine methosulfate. The bands at the respective activity sites were excised and subjected to native and denaturing two-dimensional electrophoresis for the determination of protein levels. Hence this novel electrophoretic method allows the easy detection of NADK, a critical enzyme involved in pyridine homeostasis. Furthermore, this technique allowed the monitoring of the activity and expression of this kinase in various biological systems.