Cytoplasmic potassium-sodium balance in the cardiac muscle cell of young and old rats in oxygen-substrate deficiency

Age-related changes in the concentrations of the main cations potassium and sodium in the cardiac muscle cell of Wistar rats were studied. The cytoplasmic concentrations of potassium and sodium were measured by electron probe microanalysis. The results obtained showed differences both in concentrations of the cations between young and old reference animals, and in the cardiomyocyte response to the state of acute hypoxic deenergization modeled on a perfused heart. The data obtained are consistent with the hypothesis of the presence of genetically determined age-related changes in the conductance of potassium channels, which occur in old animals against the background of short supply of oxygen and substrate to tissues.     

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.     

Nuclear magnetic resonance studies of the location and function of plant nutrients in vivo

The cytoplasmic and vacuolar pools of ammonium, inorganic phosphate and potassium can be studied non-invasively in plant tissues using high resolution nuclear magnetic resonance spectroscopy. The techniques that allow these pools to be discriminated in vivo are described and their application to plants is reviewed with reference to the phosphorus, nitrogen and potassium nutrition of root tissues.     

Ultracytochemical study of Ca++-ATPase and K+-NPPase activities in retinal photoreceptors of the guinea pig

Summary Ca⁺⁺-ATPase activity was demonstrated histochemically at light- and electron-microscopic levels in inner and outer segments of retinal photoreceptor cells of the guinea pig with the use of a newly developed one-step lead-citrate method (Ando et al. 1981). The localization of ouabain-sensitive, K⁺-dependent p-nitrophenylphosphatase (K⁺-NPPase) activity, which represents the second dephosphorylative step of the Na⁺-K⁺-ATPase system, was studied by use of the one-step method newly adapted for ultracytochemistry (Mayahara et al. 1980). In retinal photoreceptor cells fixed for 15 min in 2% paraformaldehyde the electron-dense Ca⁺⁺-ATPase reaction product accumulated significantly on the inner membranes of the mitochondria but not on the plasmalemma or other cytoplasmic elements of the inner segments. The membranes of the outer segments remained unstained except the membrane arrays in close apposition to the retinal pigment epithelium. The cytochemical reaction was Ca⁺⁺- and substrate-dependent and showed sensitivity to oligomycin. When Mg⁺⁺-ions were used instead of Ca⁺⁺-ions, a distinct reaction was also found on mitochondrial inner membranes.In contrast to the localization of the Ca⁺⁺ -ATPase activity, the K⁺-NPPase activity was demonstrated only on the plasmalemma of the inner segments, but not on the mitochondria, other cytoplasmic elements or the outer segment membranes. This reaction was almost completely abolished by ouabain or by elimination of K⁺ from the incubation medium.Fellow of the Alexander von Humboldt Foundation, Bonn, Federal Republic of Germany     

Ammonium ion transport—a cause of cell death

Ammonium can be transported into the cell by ion pumps in the cytoplasmic membrane. Ammonia then diffuse out through the cell membrane. A futile cycle is created that results in cytoplasmic acidification and extracellular alkalinisation. Ammonium transport can be quantified by measuring the extracellular pH changes occurring in a cell suspension (in PBS) after addition of ammonium. By using this technique, in combination with specific inhibitors of various ion pumps, it was shown that ammonium ions are transported across the cytoplasmic membrane by the Na⁺K⁺2Cl^--cotransporter in both hybridoma and myeloma cells. Further, the Na⁺/H⁺ exchanger, which regulates intracellular pH by pumping out protons, was shown to be active during ammonium exposure. The viability of hybridoma cells suspended in PBS and exposed to NH _inf4 ^sup+ for only 90 min, was reduced by 11% (50% necrosis and 50% apoptosis). A control cell suspension did not loose viability during this time. Turning off the activity of the Na⁺/H⁺ exchanger (by amiloride) during ammonium exposure decreased viability further, while inhibiting transport itself (by bumetanide) restored viability to the same level as for the control experiment with bumetanide alone. These results show that one effect of ammonia/ammonium on cell physiology is specifically related to the inward transport of ammonium ions by membrane bound ion pumps.Abbreviations q _pH specific rate of pH increase (pH units per min and 10⁶ cells per ml)     

The response of the filamentous cyanobacterium Spirulina platensis to salt stress

The responses of the filamentous cyanobacterium Spirulina platensis to increased NaCl concentrations (0.25–1.0 M) in addition to the concentration of sodium in the growth medium were studied. A two stage response to the salt stress was observed. This consisted of a relatively short shock stage, followed by adaptation process. It was shown that upon exposure to high salt concentrations of 0.5 M and above, immediate inhibition of photosynthesis and respiration, and complete cessation of growth occurred. After a time lag, the energy-yielding processes exhibited restored activity. At 0.5 and 1.0M NaCl photosynthesis reached 80% and 50% that of the control, while respiration was enhanced by 140 and 200%, respectively. The time lags were longer when the cells were exposed to higher NaCl concentrations. The resumption of growth and the establishment of new steady state growth rates were found to be correlated to the recovery in respiration. The relationship between the growth rates after adaptation and the increased NaCl concentrations was found to be inversely linear. The cellular sodium content was maintained at a constant low level, regardless of the external NaCl concentration, while potassium content declined linearly vs. the external NaCl concentration. The carbohydrate content of the cells rose exponentially with the increase in NaCl concentration.Publication No. 34 from the Micro-Algal Biotechnology Lab.     

Sodium-translocating adenosine triphosphatase inStreptococcus faecalis

Sodium-transloating ATPase in the fermentative bacteriumStreptococcus faecalis exchanges sodium for potassium ions. Sodium ions stimulate its activity, but K⁺ ions have no significant effect at present. Although the molecular nature of the sodium ATPase is not clear, the enzyme is distinct from other ion-motive ATPases (E₁E₂ type and F₁F₀ type) as judged by its resistance to vanadate as well as dicyclohexylcarbodiimde. The sodium ATPase is induced when cells are grown on media rich in sodium, particularly under conditions that limit the generation of a proton potential or block the constitutive sodium/proton antiporter, indicating that an increase in the cytoplasmic sodium level serves as the signal. The enzyme is not induced in response to K⁺ deprivation. The sodium ATPase may have evolved to cope with a sodium-rich environment under conditions that limit the magnitude of the proton potential.     

Simultaneous influx of ammonium and potassium into maize roots: kinetics and interactions

The interaction between ammonium and potassium during influx was examined in roots of dark-grown decapitated corn seedlings (Zea mays L., cv. Pioneer 3369A). Influx was measured during a 10-min exposure to either (¹⁵NH₄)₂SO₄ ranging from 10 to 200 M NH ₄ ⁺ with and without 200 M K(⁸⁶Rb)Cl or to K(⁸⁶Rb)Cl ranging from 10 to 200 M K⁺ with and without 200 M NH ₄ ⁺ as (¹⁵NH₄)₂SO₄. The simple Michaelis-Menten model described the data well only for potassium influx in the presence of ambient ammonium. For the other three instances, the data were improved by assuming that a second influx mechanism became operative as the low-concentration phase approached saturation. Two distinct mechanisms are thus indicated for both ammonium and potassium influx within the range of 10 to 200 M.The influx mechanism operating at low concentrations showed greater affinity for potassium than for ammonium, even though the capacity for ammonium transport was twice as large as that for potassium. It is suggested that this phase involved a common transport system for the two ions and that localized low acidity next to the internal surface, following H⁺ extrusion, favored ammonium deprotonation and dissociation from the transport system-ammonium complex. Parallel decreases in V _max and increases in K_m of the low-concentration saturable phase occurred for ammonium influx when ambient potassium was present and for potassium influx when ambient ammonium was present. The data support a mixed-type inhibition in each case. Simultaneous measurement of potassium and ammonium influx showed that they were highly negatively correlated at the lower concentrations, indicating that the extent to which influx of the inhibited ion was restricted was associated with influx of the inhibitor ion. Presence of ambient ammonium eliminated the second phase of potassium influx. In contrast, the presence of ambient potassium decreased the concentration at which the second phase of ammonium influx was initiated but did not restrict the rate.Paper no. 11131 of the Journal Series of the North Carolina Agricultural Research ServiceThe use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the products named, nor criticism of similar ones not mentioned     

Cytosolic calcium,oxygen consumption and the intracellular pH of stimulated neutrophils

The cytoplasmic pH undergoes a biphasic change when neutrophils are activated. The role of Ca²⁺ in initiating these changes was investigated. No correlation was found between the increased cytosolic [Ca²⁺] and the stimulation of the Na⁺/H⁺ antiport. Similarly, the cytoplasmic acidification elicited by activation in Na⁺-free media was found to be unrelated to [Ca²⁺]. Reversal of Na⁺/H⁺ exchange was also ruled out as the source of the acidification. Data using a variety of soluble activators indicate that metabolic acid generation is largely responsible for the observed drop in cytoplasmic pH.     

Kidney Na+,K(+)-ATPase is associated with moesin

Na+,K(+)-ATPase is a ubiquitous plasmalemmal membrane protein essential for generation and maintenance of transmembrane Na+ and K+ gradients in virtually all animal cell types. Activity and polarized distribution of renal Na+,(+)-ATPase appears to depend on connection of ankyrin to the spectrin-based membrane cytoskeleton as well as on association with actin filaments. In a previous study we showed copurification and codistribution of renal Na+,K(+)-ATPase not only with ankyrin, spectrin and actin, but also with two further peripheral membrane proteins, pasin 1 and pasin 2. In this paper we show by sequence analysis through mass spectrometry as well as by immunoblotting that pasin 2 is identical to moesin, a member of the FERM (protein 4.1, ezrin, radixin, moesin) protein family, all members of which have been shown to serve as cytoskeletal adaptor molecules. Moreover, we show that recombinant full-length moesin as well as its FERM domain bind to Na+,K(+)-ATPase and that this binding can be inhibited by an antibody specific for the ATPase activity-containing cytoplasmic loop (domain 3) of the Na+,K(+)-ATPase alpha-subunit. This loop has been previously shown to be a site essential for ankyrin binding. These observations indicate that moesin might not only serve as direct linker molecule of Na+,K(+)-ATPase to actin filaments but also modify ankyrin binding at domain 3 of Na+,K(+)-ATPase in a way similar to protein 4.1 modifying the binding of ankyrin to the cytoplasmic domain of the erythrocyte anion exchanger (AE1).     

Kinetics of K+-p-Nitrophenyl Phosphatase Stimulation by a Brain Soluble Fraction

We have already described the separation of two brain soluble fractions by Sephadex G-50, one of which stimulates (peak I) and the other inhibits (peak II) Na⁺, K⁺-ATPase and K⁺-p-nitrophenylphosphatase (K⁺-p-NPPase) activities. Here we examine the features of synaptosomal membrane p-NPPase activity in the presence and absence of brain peak I. It was observed that stimulation of Mg²⁺, K⁺-p-NPPase activity by peak I was concentration dependent, The ability of peak I to stimulate p-NPPase activity was lost by heat treatment followed by brief centrifugation. Pure serum albumin also stimulated enzyme activity. K⁺-p-NPPase stimulation by peak I proved dependent on K⁺ concentration but independent of Mg²⁺ and substrate p-nitrophenylphosphate concentrations. Since our determinations were performed in a non-phosphorylating condition reflecting the Na⁺, K⁺-ATPase Na⁺ site, it is suggested that peak I may stimulate the Na⁺-dependent enzyme phosphorylation known to take place from the internal cytoplasmic side.     

Osmotic tolerance and intracellular ion concentrations of bovine sperm are affected by cryopreservation

In this study, the effects of cryopreservation on osmoregulation and ion homeostasis in bovine sperm were studied. We determined: (1) the osmotic tolerance limits and cell volume response upon exposure to anisotonic conditions, (2) the intracellular pH and potassium concentration, and (3) expression and localization of proteins encoding for potassium and chloride ion channels. A flow cytometric approach was used for simultaneous assessment of cell volume and viability of propidium iodide stained sperm in anisotonic media. Osmotic tolerance was found to be decreased after cryopreservation, especially in the 120 to 60 mOsm/kg osmotic range. The critical osmolality at which half of the sperm population survived increased from 55 to 89 mOsm/kg. The osmotic cell volume response for viable sperm was similar before and after cryopreservation, with an osmotic inactive volume of about 70%. The intracellular pH, determined by recording changes in carboxyfluorescein fluorescence of sperm in media with different pH before and after addition of digitonin, decreased from 6.28 in diluted sperm to 6.16 after cryopreservation. The intracellular potassium concentration, determined using the potassium ionophore nigericin and incubation in media with various potassium concentrations, increased from 154 mM to 183 mM before and after cryopreservation, respectively. The levels of the chloride and potassium ion channel proteins chloride channel 3 protein (CLC-3) and two pore domain potassium channel 2 protein (TASK-2), as detected using Western blot analysis, were not affected by cryopreservation. Immunolocalization studies showed that CLC-3 is present in the acrosome and midpiece as well as in the upper and lower tail. In conclusion, cryopreserved sperm exhibit reduced tolerance to hypotonic stress, a decreased intracellular pH, and increased intracellular potassium level.     

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.     

Modulations of rabbit erythrocyte ATPase activities induced by in vitro and in vivo exposure to ethanol

Alcohol intake is associated with numerous degenerative disorders, and the detrimental effects of alcohol may be due to its influence on plasma membrane and cellular transport systems. The aim of the present study was to compare in vitro and in vivo effects of ethanol on rabbit erythrocyte ATPase activities and correlate them with ethanol-induced oxidative stress. Age-matched male rabbits were given 5% ethanol in 2% sucrose solution, for 6 weeks ad libitum; control animals were given tap water. Daily intake of ethanol was 5 g/kg body weight; this experimental regimen resulted in an average serum ethanol concentration of 16.77 ± 2.00 mM/l. After this period, blood was collected, serum ethanol concentration was determined and erythrocyte membranes were prepared according to the method of Post et al. Activities of Na⁺/K⁺- and Mg²⁺-ATPases were determined. Thiobarbituric acid-reactive substance (TBARS) assay was used to detect levels of lipid peroxidation, a major indicator of oxidative stress. In vitro ethanol inhibits both Na⁺/K⁺-ATPase and Mg²⁺-ATPase, but Na⁺/K⁺-ATPase is more sensitive to the ethanol-induced inhibition. Increasing concentration of ethanol increased TBARS production, but significant difference was attained only at 5 and 12.5 mM of ethanol. Chronic ethanol consumption induced significant increase in Na⁺/K⁺- and Mg²⁺-ATPase activity, and TBARS production. Our results suggest that increased ATPase activity induced by chronic ethanol consumption is due to oxidative, induced modification of membrane phospholipids and proteins, which are responsible for inhibition of ATPase activity. Increased production of TBARS induced by in vitro exposure to ethanol is not the only factor that influences ATPases activity. Further research is needed to elucidate this relationship.     

Infrared spectroscopic study of the structural and functional properties of the Na/H antiporter MjNhaP1 from Methanococcus jannaschii

In this study, structural, functional, and mechanistic properties of the Na⁺/H⁺ antiporter MjNhaP1 from Methanococcus jannaschii were analyzed by infrared spectroscopic techniques. Na⁺/H⁺ antiporters are generally responsible for the regulation of cytoplasmic pH and Na⁺ concentration. MjNhaP1 is active in the pH range between pH 6 and pH 6.5; below and above it is inactive.The secondary structure analysis on the basis of ATR-IR spectra provides the first insights into the structural changes between inactive (pH 8) and active (pH 6) state of MjNhaP1. It results in decreased ordered structural elements with increasing the pH-value i.e. with inactivation of the protein. Analysis of temperature-dependent FTIR spectra indicates that MjNhaP1 in the active state exhibits a much higher unfolding temperature in the spectral region assigned to α-helical segments. In contrast, the temperature-induced structural changes for β-sheet structure are similar for inactive and active state. Consequently, this structure element is not the part of the activation region of the protein. The surface accessibility of the protein was analyzed by following the extent of H/D exchange. Due to higher content of unordered structural elements a higher accessibility for amide protons is observed for the inactive as compared to the active state of MjNhaP1. Altogether, the results present the active state of MjNhaP1 as the state with ordered structural elements which exhibit high thermal stability and increased hydrophobicity.     

Investigations of the inhibitory effect of propranolol,chlorpromazine, quinine,and dicyclohexylcarbodiimide on the swelling of yeast mitochondria in potassium acetate. Evidences for indirect effects mediated by the lipid phase

The mode of action of propranolol, chlorpromazine, and quinine, three cationic drugs inhibiting swelling of yeast mitochondria in potassium acetate, was investigated by looking at their effect on fluorescent probes of the polar heads and of the nonpolar moiety of the membranes, under inhibitory conditions of swelling. As expected, propranolol and chlorpromazine exhibited specificity for anionic phospholipids since they increased the binding of the anionic probe 1-anilino 8-naphthalenesulfonate (ANS). Although propranolol did not release 1,6-diphenyl-1,3,5-hexatriene (DPH) from the hydrophobic moiety of the membrane, it increased the excimer/ monomer fluorescence ratio of 10-(1-pyrene)decanoate, suggesting that it induced a limitation in the movements of the aliphatic chains of phospholipids. Opposite to propranolol, chlorpromazine removed DPH from the membrane, suggesting that it bound essentially to the hydrophobic moiety. However, chloramphenicol, which was also able to remove DPH but did not increase the binding of ANS, did not inhibit swelling. Inhibition by chlorpromazine therefore appeared to be related to its binding to the hydrophobic moiety of anionic phospholipids. Quinine had no effect on membrane properties: at inhibitory concentrations of swelling in potassium acetate, it did not inhibit swelling in ammonium phosphate (mediated by the phosphate/H⁺ cotransporter), whereas propranolol and chlorpromazine did, suggesting a more specific effect of quinine on (a) protein(s) involved in the K⁺/H⁺ exchange. Dicyclohexylcarbodiimide (DCCD), which irreversibly inhibits the swelling in potassium acetate, bound to ethanolamine heads; despite this effect, DCCD had no major consequences on the binding of the probes. Consequently, propranolol and chlorpromazine are of no help for characterizing protein(s) catalyzing the K⁺/H⁺ exchange, although their effect on lipids seems to involve limited zones of the inner mitochondrial membrane. Quinine and DCCD, although they also bind to lipids, may inhibit the activity by acting on a limited number of proteins.     

Ouabain Binding and Potassium Transport in Young and Old Populations of Human Red Cells

Human red blood cells were separated according to density by centrifugation through mixtures of phthalate esters. The densest 20% of the erythrocyte population (old cells) had reduced volume and water content compared to the lightest 20% of the cells (young cells). Corpuscular hemoglobin content was unchanged. Young cells had 50% more potassium (K⁺) than old cells, but their total intracellular concentration was only slightly higher; old cells had a small increase in sodium (Na⁺) concentration. Active K⁺ transport of young cells was 37% higher than that of old cells. [³H] + Ouabain binding revealed that this difference was the result of more K⁺ pump sites on young cells, which bound 530 ouabain molecules per cell at 100% K⁺ pump inhibition, as compared to 400 for old cells; unseparated cells bound 450-500 molecules. The relative rates of ouabain binding were identical for the two cell types. Old cells exhibited a greater passive permeability to K⁺, haying a rate coefficient for ouabain-insensitive K⁺ influx 1.8 times that of young cells. There is evidence to suggest that in the face of reduced pump activity this augmented K⁺ “leak” might enhance the osmotic stability of the old cells and function to lengthen their life span.     

Cytoplasmic K/Na balance in heart muscle cells of young and old rats under oxygen and substrate deficiency

Age-related changes in the content of the major cellular cations of potassium and sodium in heart muscle cells of Wistar rats have been studied. The cytoplasmic concentration of potassium and sodium was determined by the electron probe microanalysis. The results revealed differences in both the concentration of the elements in young and old control animals and the responses of a cardiomyocyte to the state of acute hypoxic deenergization modelled on a perfused heart. The data are consistent with the hypothesis about the presence of genetically-related age changes in the conductance of potassium channels, which in old animals are realized against the background of deficient supply of tissues with oxygen and substrate.     

Regulation of cytoplasmic pH in rat sublingual mucous acini at rest and during muscarinic stimulation

The regulation of intracellular pH (pHi) in rat sublingual mucous acini was monitored using dual-wavelength microfluorometry of the pH-sensitive dye BCECF (2',7'-biscarboxyethyl-5(6)-carboxyfluorescein). Acini attached to coverslips and continuously superfused with HCO3(-)-containing medium (25 mM NaHCO3/5% CO2; pH 7.4) have a steady-state pHi of 7.25 +/- 0.02. Acid loading of acinar cells using the NH4+/NH3 prepulse technique resulted in a Na(+)-dependent, MIBA-inhibitable (5-(N-methyl-N-isobutyl) amiloride, Ki approximately 0.42 microM) pHi recovery, the kinetics of which were not influenced by the absence of extracellular Cl-. The rate and magnitude of the pHi recovery were dependent on the extracellular Na+ concentration, indicating that Na+/H+ exchange plays a critical role in maintaining pHi above the pH predicted for electrochemical equilibrium. When the NH4+/NH3 concentration was varied, the rate of pHi recovery was enhanced as the extent of the intracellular acidification increased, demonstrating that the activity of the Na+/H+ exchanger is regulated by the concentration of intracellular protons. Switching BCECF-loaded acini to a Cl(-)-free medium did not significantly alter resting pHi, suggesting the absence of Cl-/HCO3- exchange activity. Muscarinic stimulation resulted in a rapid and sustained cytosolic acidification (t 1/2 < 30 sec; 0.16 +/- 0.02 pH unit), the magnitude of which was amplified greater than two-fold in the presence of MIBA (0.37 +/- 0.05 pH unit) or in the absence of extracellular Na+ (0.34 +/- 0.03 pH unit). The agonist-induced intracellular acidification was blunted in HCO3(-)-free media and was inhibited by DPC (diphenylamine-2-carboxylate), an anion channel blocker. In contrast, the acidification was not influenced by removal of extracellular Cl-. The Ca2+ ionophore, ionomycin, mimicked the effects of stimulation, whereas preloading acini with BAPTA (bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid) to chelate intracellular Ca2+ blocked the agonist-induced cytoplasmic acidification. The above results indicate that during muscarinic stimulation an intracellular acidification occurs which: (i) is partially buffered by increased Na+/H+ exchange activity; (ii) is most likely mediated by HCO3- efflux via an anion channel; and (iii) requires an increase in cytosolic free [Ca2+].