Fate of histone messenger RNA in mengovirus-infected Ehrlich ascites tumor cells.

Histone mRNA was isolated from mengovirus-infected Ehrlich ascites tumor cells at various times postinfection and quantitated in a reticulocyte cell-free protein-synthesizing system. The amount of translatable histone mRNA decreases during the first hour postinfection by 30%, rises during the following 1-1.5 h by 10-15%, drops progressively in the further course of infection, and reaches 20% of the control at the end of the infectious cycle (8-9 h postinfection). On the basis of the relative histone mRNA contents, the histone-synthesizing potentials of mengovirus-infected Ehrlich ascites tumor cells are substantially higher throughout infection than actually expressed in vivo. This result indicates that the virus-induced shutoff of histone synthesis is not directly the consequence of inactivation or degradation of histone mRNA. Most of the histone mRNA recovered from mengovirus-infected Ehrlich ascites tumor cells is bound to ribosomes. Late in infection, certain mRNAs are co-isolated with histone mRNAs, very likely due to loss or shortening of poly(A) occurring after release of the mRNAs from polyribosomes.     

Coding capacity of poly(A)+mRNA isolated from mengovirus-infected Ehrlich ascites tumor cells.

Total poly (A)+mRNA was isolated from mengovirus-infected Ehrlich ascites tumor cells at various times postinfection and quantitated in a cell-free system derived from uninfected ascites cells. Basic proteins were separated from acidic proteins by carboxymethyl cellulose chromatography. At the end of the infectious cycle, 8h postinfection, the cellular contents of most mRNAs coding for basic ribosomal proteins are still between 70 and 90 percent of those measured at the beginning of infection or in uninfected cells. On the basis of this result, the rapid shutoff of host protein synthesis after mengovirus infection of Ehrlich ascites tumor cells cannot be the consequence of the inactivation of host template RNA.     

Coding capacity of poly(A)+ mRNA isolated from mengovirus-infected Ehrlich ascites tumor cells

Total poly(A)⁺ mRNA was isolated from mengovirus-infected Ehrlich ascites tumor cells at various times postinfection and quantitated in a cell-free system derived from uninfected ascites cells. Basic proteins were separated from acidic proteins by carboxymethyl cellulose chromatography. At the end of the infectious cycle, 8 h postinfection, the cellular contents of most mRNAs coding for basic ribosomal proteins are still between 70 and 90 per cent of those measured at the beginning of infection or in uninfected cells. On the basis of this result, the rapid shutoff of host protein synthesis after mengovirus infection of Ehrlich ascites tumor cells cannot be the consequence of the inactivation of host template RNA.     

ATP-producing and consuming processes of Ehrlich mouse ascites tumor cells in proliferating and resting phases.

The extents of ATP-yielding and consuming processes in Ehrlich mouse ascites tumor cells during the proliferating and resting growth phase were compared. In the resting phase the total ATP production was decreased by one-third. The ATP supply by oxidative phosphorylation was drastically reduced, whereas the rate of glycolysis stayed nearly constant. All ATP-consuming processes investigated, i.e., protein turnover, Na+/K(+)-ATPase, Ca2(+)-ATPase, and RNA synthesis, were decreased proportionally with the total ATP consumption.     

Balancing of mitochondrial and glycolytic ATP production and of the ATP-consuming processes of Ehrlich mouse ascites tumour cells in a high phosphate medium

A balance of energy budgeting of Ehrlich mouse ascites tumour cells including mitochondrial and glycolytic ATP production and about 80% of ATP consumption in a high phosphate medium is presented. In the share of glycolysis was about one-third of the total ATP production, more than twice that found in a low phosphate medium. The extent of a single energy reaction was assessed from the decrease of coupled oxygen consumption and lactate formation following the specific inhibition of this process. The inhibitory effects on coupled respiration and glycolysis were identical for the energy consuming processes measured: protein turnover, Na+/K(+)-ATPase, Ca2(+)-transport and RNA synthesis.     

The role of bound potassium ions in the hydrolysis of low concentrations of adenosine triphosphate by preparations of membrane fragments from ox brain cerebral cortex

1. The intrinsic Na(+), K(+), Mg(2+) and Ca(2+) contents of a preparation of membrane fragments from ox brain were determined by emission flame photometry. 2. Centrifugal washing of the preparation with imidazole-buffered EDTA solutions decreased the bound Na(+) from 90+/-20 to 24+/-12, the bound K(+) from 27+/-3 to 7+/-2, the bound Mg(2+) from 20+/-2 to 3+/-1 and the bound calcium from 8+/-1 to <1nmol/mg of protein. 3. The activities of the Na(+)+K(+)+Mg(2+)-stimulated adenosine triphosphatase and the Na(+)-dependent reaction forming bound phosphate were compared in the unwashed and washed preparations at an ATP concentration of 2.5mum (ATP/protein ratio 12.5pmol/mug). 4. The Na(+)-dependent hydrolysis of ATP as well as the plateau concentration of bound phosphate and the rate of dephosphorylation were decreased in the washed preparation. The time-course of formation and decline of bound phosphate was fully restored by the addition of 2.5mum-magnesium chloride and 2mum-potassium chloride. Addition of 2.5mum-magnesium chloride alone fully restored the plateau concentration of bound phosphate, but the rate of dephosphorylation was only slightly increased. Na(+)-dependent ATP hydrolysis was partly restored with 2.5mum-magnesium chloride; addition of K(+) in the range 2-10mum-potassium chloride then further restored hydrolysis but not to the control rate. 5. Pretreatment of the washed preparation at 0 degrees C with 0.5nmol of K(+)/mg of protein so that the final added K(+) in the reaction mixture was 0.1mum restored the Na(+)-dependent hydrolysis of ATP and the time-course of the reaction forming bound phosphate. 6. The binding of [(42)K]potassium chloride by the washed membrane preparation was examined. Binding in a solution containing 10nmol of K(+)/mg of protein was linear over a period of 20min and was inhibited by Na(+). Half-maximal inhibition of (42)K(+)-binding required a 100-fold excess of sodium chloride. 7. It was concluded (a) that a significant fraction of the apparent Na(+)-dependent hydrolysis of ATP observed in the unwashed preparation is due to activation by bound K(+) and Mg(2+) of the Na(+)+K(+)+Mg(2+)-stimulated adenosine triphosphatase system and (b) that the enzyme system is able to bind K(+) from a solution of 0.5mum-potassium chloride.     

The effect of inhibitors of ionic passive transport on the structure of a cell population of Ehrlich's ascites carcinoma

Triampur is a drug, containing triamterene and dichlotiazide, which inhibits passive influx of Na+ through cell membrane. Triampur was injected intraperitoneally to mice with the Ehrlich ascite carcinoma. The drug was injected in the exponential phase of tumor growth with 2 hour intervals within 24 hours. A subsequent sedimentation and fractionation of cells according to the cell cycle position displayed an additional peak in sedimentogram, that was absent in the control sedimentogram. This peak is due to cell blocking in G1-period; the pool of blocked cells contains 20-25% of the total cell population. If the drug was injected during 3 days and nights with 8-hour intervals, the pool of blocked cells amounted up to 40%. This effect is reversible: when the drug is cancelled, the peak of G1-blocked cells is reduced. Estimation of intracellular Na(+) and K(+) concentration and Na+, K(+) pump indicates a significant decrease in Na+ permeability in the blocked cells due to Triampur effect. The obtained data well compare with an idea of the importance of increasing a passive influx of Na+ for G1-S transition, and indicate a possibility to affect the structure of tumor cell population via the system of intracellular ionic homeostasis.     

Muscle K+, Na+, and Cl disturbances and Na+-K+ pump inactivation: implications for fatigue.

Membrane excitability is a critical regulatory step in skeletal muscle contraction and is modulated by local ionic concentrations, conductances, ion transporter activities, temperature, and humoral factors. Intense fatiguing contractions induce cellular K(+) efflux and Na(+) and Cl(-) influx, causing pronounced perturbations in extracellular (interstitial) and intracellular K(+) and Na(+) concentrations. Muscle interstitial K(+) concentration may increase 1- to 2-fold to 11-13 mM and intracellular K(+) concentration fall by 1.3- to 1.7-fold; interstitial Na(+) concentration may decline by 10 mM and intracellular Na(+) concentration rise by 1.5- to 2.0-fold. Muscle Cl(-) concentration changes reported with muscle contractions are less consistent, with reports of both unchanged and increased intracellular Cl(-) concentrations, depending on contraction type and the muscles studied. When considered together, these ionic changes depolarize sarcolemmal and t-tubular membranes to depress tetanic force and are thus likely to contribute to fatigue. Interestingly, less severe local ionic changes can also augment subtetanic force, suggesting that they may potentiate muscle contractility early in exercise. Increased Na(+)-K(+)-ATPase activity during exercise stabilizes Na(+) and K(+) concentration gradients and membrane excitability and thus protects against fatigue. However, during intense contraction some Na(+)-K(+) pumps are inactivated and together with further ionic disturbances, likely precipitate muscle fatigue.     

A kinetic study of the gill (Na+, K+)-ATPase, and its role in ammonia excretion in the intertidal hermit crab, Clibanarius vittatus

To better comprehend the role of gill ion regulatory mechanisms, the modulation by Na(+), K(+), NH(4)(+) and ATP of (Na(+), K(+))-ATPase activity was examined in a posterior gill microsomal fraction from the hermit crab, Clibanarius vittatus. Under saturating Mg(2+), Na(+) and K(+) concentrations, two well-defined ATP hydrolyzing sites were revealed. ATP was hydrolyzed at the high-affinity sites at a maximum rate of V=19.1+/-0.8 U mg(-1) and K(0.5)=63.8+/-2.9 nmol L(-1), obeying cooperative kinetics (n(H)=1.9); at the low-affinity sites, hydrolysis obeyed Michaelis-Menten kinetics with K(M)=44.1+/-2.6 mumol L(-1) and V=123.5+/-6.1 U mg(-1). Stimulation by Na(+) (V=149.0+/-7.4 U mg(-1); K(M)=7.4+/-0.4 mmol L(-1)), Mg(2+) (V=132.0+/-5.3 U mg(-1); K(0.5)=0.36+/-0.02 mmol L(-1)), NH(4)(+) (V=245.6+/-9.8 U mg(-1); K(M)=4.5+/-0.2 mmol L(-1)) and K(+) (V=140.0+/-4.9 U mg(-1); K(M)=1.5+/-0.1 mmol L(-1)) followed a single saturation curve and, except for Mg(2+), obeyed Michaelis-Menten kinetics. Under optimal ionic conditions, but in the absence of NH(4)(+), ouabain (K(I)=117.3+/-3.5 mumol L(-1)) and orthovanadate inhibited up to 67% of the ATPase activity. The inhibition studies performed suggest the presence of F(0)F(1), V- and P-ATPases, but not Na(+)-, K(+)- or Ca(2+)-ATPases as contaminants in the gill microsomal preparation. (Na(+), K(+))-ATPase activity was synergistically modulated by NH(4)(+) and K(+). At 20 mmol L(-1) K(+), a maximum rate of V=290.8+/-14.5 U mg(-1) was seen as NH(4)(+) concentration was increased up to 50 mmol L(-1). However, at fixed NH(4)(+) concentrations, no additional stimulation was found for increasing K(+) concentrations (V=135.2+/-4.1 U mg(-1) and V=236.6+/-9.5 U mg(-1) and for 10 and 30 mmol L(-1) NH(4)(+), respectively). This is the first report to detail ionic modulation of gill (Na(+), K(+))-ATPase in C. vittatus, revealing an asymmetrical, synergistic stimulation of the enzyme by K(+) and NH(4)(+), as yet undescribed for other (Na(+), K(+))-ATPases, and should provide a better understanding of NH(4)(+) excretion in pagurid crabs.     

Heart slice NMR

Nuclear magnetic resonance (NMR) spectroscopy of the heart is normally carried out using whole heart preparations under coronary perfusion. In such preparations, either radical changes in ionic composition of the perfusate or applications of numerous drugs would affect coronary microcirculation. This report communicates the first (31)P NMR spectroscopy study using a heart slice preparation (left ventricular slices) superfused with extracellular medium. The ratio of phosphocreatine concentration to ATP concentration was approximately 2.1. Also, intracellular pH and Mg(2+) concentration ([Mg(2+)](i)), estimated from the chemical shifts of inorganic phosphate and ATP, were comparable with those under retrograde perfusion. [Mg(2+)](i) was significantly increased by the removal of extracellular Na(+), supporting the essential role of Na(+)-coupled Mg(2+) transport in Mg(2+) homeostasis of the heart. Heart slice preparation could also be used to evaluate the potency of cardiac drugs, regardless of their possible effects on coronary microcirculation.     

X-ray microanalysis of the Malpighian tubules of the black field cricket Teleogryllus oceanicus: the roles of Na K ATPase and the Na K 2Cl cotransporter

Both main and distal segments of the Malpighian tubules were sensitive to ouabain and furosemide but in different ways. Oubain had no effect on secretion rate by the main segment but in the secreted fluid Na(+) concentration increased substantially whereas K(+) decreased. Similarly intracellular elemental Na concentration increased and K decreased. Furosemide decreased the secretion rate of the main segment by 80%. The Na(+) concentration in the secreted fluid increased markedly but K(+) was not affected. Intracellular elemental Na concentration also increased but K was unchanged. In the distal segments both ouabain and furosemide decreased secretion rate by 40% but although ouabain had no effect on the composition of the secreted fluid, furosemide caused a substantial reduction in the concentrations of Mg(2+) and Cl(-) and a substantial increase in Na(+) and K(+) concentrations. The evidence suggests that the main segment contains a Na K ATPase and possibly a Na K 2Cl cotransporter whereas the distal segment may contain a Na K ATPase and a furosemide sensitive Mg(2+) transporter. K(+) entry into the cells of the main segment may be partially effected by a Na K 2Cl cotransporter but may be primarily via Na K ATPase in the distal segment.     

Identification of Ca2+-pump-related phosphoprotein in plasma membrane vesicles of Ehrlich ascites carcinoma cells

Plasma membrane vesicles of Ehrlich ascites carcinoma cells have been isolated to a high degree of purity. In the presence of Mg2+, the plasma membrane preparation exhibits a Ca2+-dependent ATPase activity of 2 mumol Pi per h per mg protein. It is suggested that this (Ca2+ + Mg2+)-ATPase activity is related to the measured Ca2+ transport which was characterized by Km values for ATP and Ca2+ of 44 +/- 9 microM and 0.25 +/- 0.10 microM, respectively. Phosphorylation of plasma membranes with [gamma-32P]ATP and analysis of the radioactive species by polyacrylamide gel electrophoresis revealed a Ca2+-dependent hydroxylamine-sensitive phosphoprotein with a molecular mass of 135 kDa. Molecular mass and other data differentiate this phosphoprotein from the catalytic subunit of (Na+ + K+)-ATPase and from the catalytic subunit of (Ca2+ + Mg2+)-ATPase of endoplasmic reticulum. It is suggested that the 135 kDa phosphoprotein represents the phosphorylated catalytic subunit of the (Ca2+ + Mg2+)-ATPase of the plasma membrane of Ehrlich ascites carcinoma cells. This finding is discussed in relation to previous attempts to identify a Ca2+-pump in plasma membranes isolated from nucleated cells.     

Characterization of Na(+)-dependent Mg2+ efflux from Mg2(+)-loaded rat erythrocytes

Na(+)-dependent Mg2+ efflux from Mg2(+)-loaded rat erythrocytes was determined from the increase of extracellular Mg2+ concentration or decrease of intracellular Mg2+ content, as measured by means of atomic absorption spectrophotometry. Mg2+ efflux was specifically combined with the uptake of Na+ at a stoichiometric ratio of 2Na+:1Mg2+, indicating electroneutral Na+/Mg2+ antiport. Na+/Mg2+ antiport depended on intracellular ATP and was inhibited by amiloride and quinidine, but was insensitive to strophanthin. Net Mg2+ efflux was only occurring at increased concentration of intracellular Mg2+ ([Mg2+]i), and stopped when the physiological Mg2+ content was reached. Intracellular Mg2+ acted cooperatively with a Hill coefficient of 2.4, which may indicate gating of Na+/Mg2+ antiport at increased [Mg2+]i. At increased intracellular Na+ concentration, Na+ competed with intracellular Mg2+ for Mg2+ efflux and Na+ could leave the rat erythrocyte via this transport system. Na+/Mg2+ antiport was working asymmetrically with respect to extra- and intracellular Na+ and Mg2+, and did not perform net Mg2+ uptake.     

Nucleotides and Mg2+ ions differentially regulate K+ channels and non-selective cation channels present in cells forming the stomatal complex

Voltage-dependent inward-rectifying (K(in)) and outward-rectifying (K(out)) K(+) channels are capable of mediating K(+) fluxes across the plasma membrane. Previous studies on guard cells or heterologously expressed K(+) channels provided evidence for the requirement of ATP to maintain K(+) channel activity. Here, the nucleotide and Mg(2+) dependencies of time-dependent K(in) and K(out) channels from maize subsidiary cells were examined, showing that MgATP as well as MgADP function as channel activators. In addition to K(out) channels, these studies revealed the presence of another outward-rectifying channel type (MgC) in the plasma membrane that however gates in a nucleotide-independent manner. MgC represents a new channel type distinguished from K(out) channels by fast activation kinetics, inhibition by elevated intracellular Mg(2+) concentration, permeability for K(+) as well as for Na(+) and insensitivity towards TEA(+). Similar observations made for guard cells from Zea mays and Vicia faba suggest a conserved regulation of channel-mediated K(+) and Na(+) transport in both cell types and species.     

Studies on the microsomal sodium-plus-potassium ion-stimulated adenosine triphosphatase system in rat ventral prostate

A Mg(2+)+Na(+)+K(+)-stimulated adenosine triphosphatase (ATPase) preparation was isolated from rat ventral prostate by flotation of microsomal membranes in high-density sucrose solutions. The reaction medium for optimum Na(+)+K(+)-stimulated ATPase activity was found to be: Na(+), 115mm; K(+), 7-10mm; Mg(2+), 3mm; ATP, 3mm; tris buffer, pH7.4 at 38 degrees , 20mm. The average DeltaP(i) (Mg(2+)+Na(+)+K(+) minus Mg(2+)+Na(+)) was 9mumoles/mg. of protein/hr., representing a 30% increase over the Mg(2+)+Na(+)-stimulated ATPase activity. At high concentrations, K(+) was inhibitory to the enzyme activity. Half-maximal inhibition of Na(+)+K(+)-stimulated ATPase activity was elicited by ouabain at 0.1mm. The preparation exhibited phosphatase activity towards ribonucleoside triphosphates other than ATP. However, stimulation of P(i) release by Na(+)+K(+) was observed only with ATP as substrate. The apparent K(m) for ATP for Na(+)+K(+)-stimulated activity was about 0.3x10(-3)m. Ca(2+) inhibited only the Na(+)+K(+)-stimulated ATPase activity. Mg(2+) could be replaced by Ca(2+) but then no Na(+)+K(+) stimulation of ATPase activity was noticed. The addition of testosterone or dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) in vitro at 0.1-10mum under a variety of experimental conditions did not significantly increase the Na(+)+K(+)-stimulated ATPase activity. The enzyme preparations from prostates of orchidectomized rats, however, exhibited a drastic decrease in the specific activity of Na(+)+K(+)-stimulated ATPase; these changes were prevented in the orchidectomized rats by injection of testosterone propionate.     

On the mechanism of glycolysis stimulation by neutral detergents in 3T3 and Ehrlich ascites tumor cells

Glycolysis of 3T3 and Ehrlich ascites tumor cells was greatly enhanced by Nonidet P-40 or Triton X-100 at about 100 micrograms/mg cell protein. This enhanced glycolysis was partly sensitive to rutamycin and partly sensitive to ouabain, suggesting that the detergent released the control of the ATPase of the mitochondria and of the plasma membrane Na+K+-ATPase. Nonidet P-40 had no effect on glycolysis in cell-free extracts from Ehrlich ascites tumor cells to which soluble mitochondrial ATPase was added. Measuring ouabain-sensitive 22Na efflux and using ouabain-sensitive lactate production as a measure of ATP hydrolysis by the Na+K+ pump, it was shown that Nonidet P-40 greatly decreased the efficiency of the Na+K+ pump. Quercetin increased the efficiency of pumping in EAT cells both in the absence and presence of the detergent.     

The MgtC virulence factor of Salmonella enterica serovar Typhimurium activates Na(+),K(+)-ATPase

The mgtC gene of Salmonella enterica serovar Typhimurium encodes a membrane protein of unknown function that is important for full virulence in the mouse. Since mgtC is part of an operon with mgtB which encodes a Mg(2+)-transporting P-type ATPase, MgtC was hypothesized to function in ion transport, possibly in Mg(2+) transport. Consequently, MgtC was expressed in Xenopus laevis oocytes, and its effect on ion transport was evaluated using ion selective electrodes. Oocytes expressing MgtC did not exhibit altered currents or membrane potentials in response to changes in extracellular H(+), Mg(2+), or Ca(2+), thus ruling out a previously postulated function as a Mg(2+)/H(+) antiporter. However, addition of extracellular K(+) markedly hyperpolarized membrane potential instead of the expected depolarization. Addition of ouabain to block the oocyte Na(+),K(+)-ATPase completely prevented hyperpolarization and restored the normal K(+)-induced depolarization response. These results suggested that the Na(+),K(+)-ATPase was constitutively activated in the presence of MgtC resulting in a membrane potential largely dependent on Na(+),K(+)-ATPase. Consistent with the involvement of Na(+),K(+)-ATPase, oocytes expressing MgtC exhibited an increased rate of (86)Rb(+) uptake and had increased intracellular free [K(+)] and decreased free [Na(+)] and ATP. The free concentrations of Mg(2+) and Ca(2+) and cytosolic pH were unchanged, although the total intracellular Ca(2+) content was slightly elevated. These results suggest that the serovar Typhimurium MgtC protein may be involved in regulating membrane potential but does not directly transport Mg(2+) or another ion.     

The influence of cellular amino acids and the Na+ : K+ pump on the membrane potential of the Ehrlich ascites tumor cell.

The membrane potential of the Ehrlich ascites tumor cell was shown to be influenced by its amino acid content and the activity of the Na+ :K+ pump. The membrane potential (monitored by the fluorescent dye, 3,3'-dipropylthiodicarbocyanine iodide) varied with the size of the endogenous amino acid pool and with the concentration of accumulated 2-aminoisobutyrate. When cellular amino acid content was high, the cells were hyperpolarized; as the pool declined in size, the cells were depolarized. The hyperpolarization seen with cellular amino acid required cellular Na+ but not cellular ATP. Na+ efflux was more rapid from cells containing 2-aminoisobutyrate than from cells low in internal amino acids. These observations indicate that the hyperpolarization recorded in cells with high cellular amino acid content resulted from the electrogenic co-efflux of Na+ and amino acids. Cellular ATP levels were found to decline rapidly in the presence of the dye and hence the influence of the pump was seen only if glucose was added to the cells. When the cells contained normal Na+ (approx. 30mM), the Na+ :K+ pump was shown to have little effect on the membrane potential (the addition of ouabain had little effect on the potential). When cellular Na+ was raised to 60mM, the activity of the pump changed the membrane potential from the range -25 to -30 mV to -44 to -63 mV. This hyperpolarization required external K+ and was inhibited by ouabain.     

The Na+-K+ pump and the thermogenesis of brown fatty tissue]

Mechanisms of participation of Na+K(+)-pump in regulation of the brown adipose tissue are discussed. It is settled that the increase in ATP hydrolysis by Na(+)-K(+)-ATPase is not a dominating factor of thermogenesis activation. It is assumed that the Na(+)-K(+)-pump, through the intracellular K+ concentration, serves a chain relating proliferation and thermogenesis of adipocytes.