Effects of mitogens on sodium-potassium transport, H-ouabain binding, and adenosine triphosphatase activity in lymphocytes

The effect of various mitogens was studied on sodium (Na⁺) potassium (K⁺) transport, ³H-ouabain binding, and adenosine triphosphatase (ATPase) activity in human and sheep peripheral lymphocytes. Concanavalin A (ConA), phytohemagglutinin (PHA), horse anti-lymphocyte serum (ALS), and anti-IgG antisera, in order of decreasing potency, stimulated in particular the ouabain-sensitive K⁺ pump influx, while the cardiac glycoside-insensitive K⁺ leak flux was only slightly affected. Sheep lymphocytes primed in vivo with human IgG as antigen also responded with K⁺ pump flux activation when exposed to the antigen in vitro. Both PHA and ConA also stimulated active Na⁺ efflux in human lymphocytes. Apparently these mitogens activate the Na⁺K⁺ pump system in the lymphocyte membrane—an assumption supported by the finding of a significant activation of the ouabain-sensitive Na⁺K⁺-ATPase. From rate studies of ³H-ouabain binding carried out at 37 °C in presence and absence of sodium azide, and at 0 °C, it is concluded that PHA alters the rate of ouabain uptake to these cells. Thus PHA may alter the affinity of the pump for ouabain, equivalent to an increased cation turnover per pump site. However, our findings do not completely discount the possibility that PHA also increases the total number of ouabain molecules bound and therefore of Na⁺K⁺ pumps.     

Accumulation of Iron in the Rabbit Erythroid Cell As Affected by Ouabain, Sodium and Potassium Ions, and Temperature

Reticulocytosis was induced in rabbits with phenylhydrazine. The accumulation of a small part of ⁵⁹Fe in blood cells of these animals was inhibited by ouabain and related to changes in extracellular sodium and potassium concentrations. Sodium increases movement from the cell surface into the cell, whereas potassium and ouabain decrease this movement. ⁵⁹Fe movement was found to be temperature-dependent. Thus, the Na-K ATPase system appears to be important in the movement of iron from the cell membrane (stroma) to the cell interior, but influences only a small part of the total iron transport.     

Properties of (Na+ + K+)-activated ATPase in rat liver plasma membranes enriched with bile canaliculi

Liver plasma membranes enriched in bile canaliculi were isolated from rat liver by a modification of the technique of Song et al. (J. Cell Biol. (1969) 41, 124–132) in order to study the possible role of ATPase in bile secretion. Optimum conditions for assaying (Na⁺ + K⁺)-activated ATPase in this membrane fraction were defined using male rats averaging 220 g in weight. (Na⁺ + K⁺)-activated ATPase activity was documented by demonstrating specific cation requirements for Na⁺ and K⁺, while the divalent cation, Ca²⁺, and the cardiac glycosides, ouabain and scillaren, were inhibitory. (Na⁺ + K⁺)-activated ATPase activity averaged 10.07 ± 2.80 μmol P_i/mg protei per h compared to 50.03 ± 11.41 for Mg²⁺-activated ATPase and 58.66 ± 10.07 for 5′-nucleotidase. Concentrations of ouabain and scillaren which previously inhibited canalicular bile secretion in the isolated perfused rat liver produced complete inhibition of (Na⁺ + K⁺)-activated ATPase without any effect on Mg²⁺-activated ATPase. Both (Na⁺ + K⁺)-activated ATPase and Mg²⁺-activated ATPase demonstrated temperature dependence but differed in temperature optima. Temperature induced changes in specific activity of (Na⁺ + K⁺)-activated ATPase directly paralleled previously demonstrated temperature optima for bile secretion. These studies indicate that (Na⁺ + K⁺)-activated ATPase is present in fractions of rat liver plasma membranes that are highly enriched in bile canaliculi and provide a model for further study of the effects of various physiological and chemical modifiers of bile secretion and cholestasis.     

The modification of the unidirectional calcium fluxes of sarcoplasmic reticulum vesicles by monovalent cation ionophores

Calcium uptake by rabbit skeletal muscle sarcoplasmic reticulum vesicles in phosphate-containing media exhibits time-dependent changes that arise from changing rates of calcium influx and efflux. The monovalent cation ionophore gramicidin, added before the start of the calcium uptake reaction, delayed the spontaneous calcium release that normally occurred after approx. 6 min in such reactions; the rate of calcium efflux was inhibited while calcium influx was little affected. Under these conditions, Ca²⁺-activated ATPase activity could remain unaltered.Gramicidin stimulated calcium uptake irrespective of the presence of a K⁺ gradient across the vesicle membrane. Valinomycin stimulated calcium uptake in a manner similar to that for gramicidin even in an NaCl-containing medium lacking potassium. Thus, dissipation of a transmembrane K⁺ gradient is unlikely to account for the effects of these ionophores on the spontaneous changes in calcium flux rates.Addition of gramicidin to partially calcium-filled vesicles inhibited the phase of spontaneous calcium reuptake because both calcium influx and efflux were inhibited. Addition of gramicidin to partially calcium-filled vesicles in the presence of a water-soluble protein, such as bovine serum albumin, creatine kinase or pyruvate kinase, markedly stimulated calcium uptake. This stimulatory effect was due primarily to inhibition of calcium efflux, calcium influx being minimally influenced by the ionophore.After cleavage of the 100 000 dalton ATPase to 50 000 dalton fragments, which was not associated with changes in Ca²⁺-activated ATPase activity or initial calcium uptake rate, gramicidin increased rather than decreased calcium content when added to vesicles after the initial maximum in calcium content. Thus, the ability of monovalent cation ionophores to block calcium efflux from calcium-filled vesicles may reflect their interaction with a portion of the Ca²⁺-activated ATPase protein.     

Solubilization of a divalent cation dependent ATPase from dog heart sarcolemma

Heart sarcolemma has been shown to contain an ATPase hydrolizing system which is activated by millimolar concentrations of divalent cations such as Ca²⁺ or Mg²⁺. Although Ca²⁺-dependent ATPase is released upon treating sarcolemma with trypsin, a considerable amount of the divalent cation dependent ATPase activity was retained in the membrane. This divalent cation dependent ATPase was solubilized by sonication of the trypsin-treated dog heart sarcolemma with 1% Triton X-100. The solubilized enzyme was subjected to column chromatography on a Sepharose-6B column, followed by ion-exchange chromatography on a DEAE cellulose column. The enzyme preparation was found to be rather labile and thus the purity of the sample could not be accurately assessed. The solubilized ATPase preparations did not show any cross-reactivity with dog heart myosin antiserum or with Na⁺ + K⁺ ATPase antiserum. The enzyme was found to be insensitive to inhibitors such as ouabain, verapamil, oligomycin and vanadate. The enzyme preparation did not exhibit any Ca²⁺-stimulated Mg²⁺ dependent ATPase activity. Furthermore, the low affinity of the enzyme for Ca^2– (Ka = 0.3 mM) rules out the possibility of its involvement in the Ca²⁺ pump mechanism located in the plasma membrane of the cardiac cell.     

Active cation transport and Na+K+Mg ATPase of the monotreme erythrocytes

The erythrocytes of the echidna (Tachyglossus aculeatus) and platypus (Ornithorhynchus anatinus), which are practically devoid of intracellular ATP content (1), were examined for active Rb⁸⁶ influx and for the presence of Na+K+Mg ATPase. We found that intact erythrocytes of both species possess the ability to actively transport cations. Ouabain sensitive Rb⁸⁶ influx in the echidna was approximately 0.17 μmoles/ml cells × hr, whereas the platypus exhibited a higher value of 0.43 μmoles/ml cells × hr. Surprisingly, ouabain sensitive Na+K+Mg ATPase activity of isolated membranes was high amounting to some 15 to 25 fold higher than the human erythrocyte counterpart determined under identical conditions. These findings suggest that a trace amount of ATP is sufficient to maintain active cation transport across the monotreme cell membranes.     

A novel fluorescence imaging approach to monitor salt stress‐induced modulation of ouabain‐sensitive ATPase activity in sunflower seedling roots

Seedlings exposed to salt stress are expected to show modulation of intracellular accumulation of sodium ions through a variety of mechanisms. Using a new methodology, this work demonstrates ouabain (OU)‐sensitive ATPase activity in the roots of sunflower seedlings subjected to salt stress (120 mM NaCl). 9‐Anthroylouabain (a derivative of ouabain known to inhibit Na⁺,K⁺‐ATPase activity in animal systems, EC 3.6.3.9) has been used as a probe to analyze OU‐sensitive ATPase activity in sunflower (Helianthus annuus) seedling roots by spectrofluorometric estimation and localization of its spatial distribution using confocal laser scanning microscopy. Salt stress for 48 h leads to a significant induction of OU‐sensitive ATPase activity in the meristematic region of the seedling roots. Calcium ions (10 mM) significantly inhibit enzyme activity and a parallel accumulation of sodium ions in the cytosol of the columella cells, epidermis and in the cells of the meristematic region of the roots is evident. As a rapid response to NaCl stress, the activity of OU‐sensitive ATPase gets localized in the nuclear membrane of root protoplasts and it gets inhibited after treatment with calcium ions. Nuclear membrane localization of the OU‐sensitive ATPase activity highlights a possible mechanism to efflux sodium ions from the nucleus. Thus, a correlation between OU‐sensitive ATPase activity, its modulation by calcium ions and accumulation of sodium ions in various regions of the seedling roots, has been demonstrated using a novel approach in a plant system.     

Ouabain-insensitive salt and water movements in duck red cells. I. Kinetics of cation transport under hypertonic conditions

Duck red cells in hypertonic media experience rapid osmotic shrinkage followed by gradual reswelling back toward their original volume. This uptake of salt and water is self limiting and demands a specific ionic composition of the external solution. Although ouabain (10(-4)M) alters the pattern of cation accumulation from predominantly potassium to sodium, it does not affect the rate of the reaction, or the total amount of salt or water taken up. To study the response without the complications of active Na-K transport, ouabain was added to most incubations. All water accumulated by the cells can be accounted for by net salt uptake. Specific external cation requirements for reswelling include: sufficient sodium (more than 23 mM), and elevated potassium (more than 7 mM). In the absence of external potassium cells lose potassium without gaining sodium and continue to shrink instead of reswelling. Adding rubidium to the potassium- free solution promotes an even greater loss of cell potassium, yet causes swelling due to a net uptake of sodium and rubidium followed by chloride. The diuretic furosemide (10(-3)M) inhibits net sodium uptake which depends on potassium (or rubidium), as well as inhibits net sodium uptake which depends on sodium. As a result, cell volume is stabilized in the presence of this drug by inhibition of shrinkage, at low, and of swelling at high external potassium. The response has a high apparent energy of activation (15-20 kcal/mol). We propose that net salt and water movements in hypertonic solutions containing ouabain are mediated by direct coupling or cis-interaction, between sodium and potassium so that the uphill movement of one is driven by the downhill movement of the other in the same direction.     

Characterization of ATPase activity associated with corn leaf plasma membranes

A Mg²⁺-dependent, cation-stimulated ATPase was associated with plasma membranes isolated from corn leaf mesophyll protoplasts. Potassium was the preferred monovalent cation for stimulating the ATPase above the Mg²⁺-activated level. The enzyme was substrate-specific for ATP, was inhibited by N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, p-chloromercuribenzoate, and orthovanadate, but was insensitive to oligomycin or sodium azide. A K_m of 0.28 millimolar Mg²⁺-ATP was determined for the K⁺-ATPase, and the principal effect of potassium was on the V_max for ATP hydrolysis. Since potassium stimulation was not saturated at high concentrations, a nonspecific role was proposed for potassium stimulation. A nonspecific phosphatase was also found to be associated with corn leaf plasma membranes. However, it could not be determined positively whether this activity represented a separate enzyme.     

SO2-dependent cation competition and compartmentalization in Norway spruce needles

Ion contents in needles from Norway spruce trees [Picea abies (L.) Karst.] growing in Würzburg and in the SO₂-polluted Erzgebirge mountains were analysed to quantify cations which accumulate together with sulphate. In Würzburg there was a positive correlation of potassium (0.680 ± 0.300 Eq Eq^?1 SO₄^?2), magnesium (0.415 ± 0.111 Eq Eq^?1 SO₄^?2) and zinc (0.059 ± 0.006 Eq Eq^?1 SO₄^2?). In the Erzgebirge, potassium was also the stoichiometrically most important cation (0–887 ± 0–180 Eq K⁺ Eq^?1 SO₄^2?). All other correlations examined were weak or statistically non-significant. At both sites the calcium content of spruce needles did not depend on the sulphate content. The lack of a role for Ca²⁺ in neutralizing sulphate is a consequence of the presence of free oxalic acid in needles. Soluble oxalic acid precipitates Ca²⁺, which thereby becomes unavailable as a counterion for SO₄^2?. The activity coefficients of Ca²⁺ and oxalate^2?, and the solubility product of Ca-oxalate, were determined from in vivo data. It is concluded that the chronic accumulation of atmospheric sulphate in spruce needle vacuoles depletes available potassium and thereby strongly interferes with spruce growth and canopy turnover. This leads to impaired spruce vitality, even at sites where acute SO₂ disease symptoms are absent.     

The apparent discrepancy of ouabain inhibition of cation transport and of lymphocyte proliferation is explained by time-dependency of ouabain binding

Mitogenesis of human blood lymphocytes in culture is inhibited by concentrations of ouabain that are approximately one order of magnitude lower than those that block Na and K transport. For example, the 50% inhibition (ID₅₀) of Na-K transport, 280 nM, is seven-fold greater than the ID₅₀ for RNA synthesis, DNA synthesis, or blastogenesis, ?40 nM. Yet, inhibition of transport and consequent reduction in cell K is considered responsible for the effects of ouabain on mitogenesis. Since synthetic processes are assessed at least 24 hours after lymphocyte stimulation, this discrepancy could be explained by either 1) a progressive increase in K leak, or 2) a progressive inhibition of Na-K transport by ouabain during 24 hours of PHA treatment. We found that the lymphocyte membrane leak rate of K increased immediately after PHA treatment but did not increase further from 4 to 24 hours. In contrast, the ouabain sensitivity of ⁴²K uptake was markedly increased with time: ID₅₀ for ⁴²K uptake of 35 nM at 24 hours as compared to 280 nM at 30 minutes. Measurement of ouabain binding revealed a seven-fold increase in the lymphocyte-associated ouabain after 24 hours compared to binding at 1 hour. These data indicate that the dose response of ouabain inhibition of active K transport and lymphocyte proliferation are closely correlated if one considers the slow membrane binding of ouabain at low concentrations.     

Further studies of the selective inhibition by ouabain of antigen-induced proliferation of human lymphocytes

Cultures of human lymphocytes incubated for 48 hr in the presence of 2 × 10^?7M solutions of the cardiotonic steroid ouabain lose the proliferative response to antigens (SL-0, SK-SD) but can still proliferate when stimulated by nonspecific mitogens (PHA, Con A, pokeweed mitogen). The two-way mixed lymphocyte reaction was also irreversibly lost if cells of both donors were subjected to ouabain pretreatment. Neither cell counts nor cell viability (determined by dye exclusion) were significantly affected by the ouabain treatment. Pretreatment of a suspension of macrophages with the cardiac glycoside did not diminish their capacity to restore the proliferative response to antigen of macrophage-depleted lymphocyte suspensions; on the other hand, untreated macrophages could not restore the proliferative response of cultures of ouabain-pretreated lymphocytes. The ouabain treatment did not change the proportion of cells able to bind fluorescent anti-immunoglobulin nor did it modify the proportion of lymphocytes forming rosettes with either untreated, or antibody coated, red cells. Increased concentration of K⁺ in the medium, either during or after the ouabain treatment, did not reduce the ouabain effect. We conclude that the selective loss of certain lymphocyte functions caused by ouabain pretreatment was due to an effect on the lymphocyte and not on the macrophage; the effect was not due to the elimination of a relatively large fraction of the cells nor to a generalized disappearance of membrane antigens and receptors.     

Characterization of temperature-sensitive mutants of animal cells

An early increase in lymphocyte plasma membrane K⁺ transport is essential for PHA stimulated lymphocytes to divide. Little is known about the specific source and amount of energy required to support the increased transport by activated lymphocytes. Since ouabain, a cardiac glycoside, specifically inhibits the transport ATPase, we have measured the decrement in glycolysis and tricarboxylic acid cycle activity when untreated and PHA treated lymphocytes were exposed to ouabain. This metabolic decrement represents the portion of metabolism associated with monovalent cation transport and closely related processes. Since TCA cycle activity accounted for only 0.2% of glucose consumption, aerobic glycolysis was the major source of energy, i.e., ATP, for increased transport. Approximately one-third of the total lactate production in both control and PHA stimulated lymphocytes was ouabain-sensitive. Ouabain sensitive lactate production in control, 105 μmol/10¹⁰ cells/hour, increased 1.8-fold to 193 μmol/10¹⁰ cells/hour after PHA treatment. Active K⁺ influx in similar cell populations increased from 40 μmol/10¹⁰ cells/hour to 74 μmol/10¹⁰ cells/hour (1.9-fold) after PHA treatment. The increment in ouabain-sensitive energy production and K⁺ transport were closely correlated and, therefore, 0.38 moles of K⁺ are transported for each mole of ATP generated in both control and PHA treated cells. The increased requirement for transport related energy is provided by increasing the ouabain-sensitive ATP production rather than altering the efficiency of ATP transduction.     

The effects of sodium and potassium on ouabain binding by human erythrocytes

Ouabain binding by the human erythrocyte membrane is reversible, exhibits a high degree of chemical specificity, and can be detected at ouabain concentrations as low as 1 x 10^-10 M. The relation between ouabain binding and ouabain concentration can be described by a rectangular hyperbola permitting determination of the maximal binding (B _max) and the ouabain concentration at which ouabain binding is half-maximal (K_B). Reducing the external sodium concentration increased K_B, while reducing the external potassium concentration decreased K_B. Neither cation altered B _max The reciprocal of K_B was a linear function of the sodium concentration at sodium concentrations ranging from 0 to 150 mM. Conversely, the relation between the reciprocal of K_B and the external potassium concentration was nonlinear, and raising the potassium concentration above 4 mM produced no further increase in K_B. These results are compatible with a model which postulates that the erythrocyte membrane contains a finite number of receptors each composed of a glycoside-binding site and a cation-binding site. When sodium occupies the cation-binding site, the affinity of the glycoside site for ouabain is increased; when potassium occupies the cation-binding site the affinity of the glycoside site for ouabain is decreased.     

Monovalent ion stimulated adenosine triphosphatase from oat roots

Monovalent ion stimulated ATPase activity from oat (Avena sativa) roots has been found to be associated with various membrane fractions (cell wall, mitochondrial and microsomal) of oat roots. The ATPase requires Mg²⁺ (or Mn⁺²) but is further stimulated by K⁺ and other monovalent ions. The monovalent ions are ineffective in the absence of the divalent activating cation. The ATPase has been described with respect to monovalent ion specificity, temperature, pH, substrate specificity, and Mg²⁺ and K⁺ concentrations. It was further shown that oligomycin inhibits a part of the total ATPase activity and on the basis of the oligomycin sensitivity it appears that at least 2 membrane associated ATPases are being measured. The mitochondrial fraction is most sensitive to oligomycin and the microsomal fraction is least sensitive to oligomycin. The oligomycin insensitive ATPase appears to be stimulated more by K⁺ than the oligomycin sensitive ATPase.     

Differences in selected zinc metabolism parameters in obese and normal-weight hypertensive patients following treatment with spironolactone

Twenty-three hypertensive outpatients aged 18–53 yr (average: 39.8±10.4 yr) were classified into two groups according to body mass index (BMI). Six patients exceeded the BMI limit, set at 30 kg/m². All were treated with 100 mg/d spironolactone and were subject to before and after measurements of their arterial pressure, efflux rate constants of zinc from lymphocytes (total ERCt-Zn and ouabain-dependent ERCos-Zn), serum zinc (Zn-s), lymphocyte zinc (Zn-l), serum aldosterone (Ald-s), plasma renin activity (PRA), serum sodium (Na-s), and potassium (K-s). After 7 d of spironolactone treatment, the ERCt-Zn change in normal-weight patients was +0.78±0.57, and −0.22±0.69 in obese patients. In the same manner, the change of ERCos-Zn was +0.59±0.94 and −0.025±0.32 in normal and obese patients, respectively. Serum Zn was increased in normal-weight patients but remained unchanged in the obese. The initial lymphocyte zinc values were significantly lower in obese patients, but increased up to normal values after spironolactone treatment.     

Interactions of erythrosin B (U.S. F,D & C Red 3) with rat cortical membranes

Erythrosin B inhibits Na, K-ATPase in rat brain tissue as demonstrated by studying glycoside binding, ATPase activity and ion fluxes. The potency of the noncompetitive inhibition of [³H]-ouabain binding by erythrosin B is influenced by glycoside concentration, monovalent cation concentration, and incubation time. [¹⁴C]- Erythrosin B binds to synaptic membranes prepared from rat cortex. Erythrosin B and some of its structural analogs inhibit both [³H]-ouabain and [¹⁴C]-erythrosin B binding, but ouabain and other glycosides do not inhibit the binding of [¹⁴C]-erythrosin B. Subcellular distributions of [³H]-ouabain and [¹⁴C]- erythrosin B binding in fractionated cortical tissue preparations are equivalent and parallel ATPase activity. The dissimilar response of [³H]-ouabain binding and [¹⁴C]-erythrosin B binding to changes in tissue preparation, incubation temperature, and partial solubilization of binding sites by deoxycholate (DOC) Suggests two separate binding sites for erythrosin and ouabain to rat cortical membranes.     

Studies on the System Regulating Proton Movement across the Chloroplast Envelope : Effects of ATPase Inhibitors, Mg, and an Amine Anesthetic on Stromal pH and Photosynthesis

Studies were undertaken to further characterize the spinach (Spinacea oleracea) chloroplast envelope system, which facilitates H⁺ movement into and out of the stroma, and, hence, modulates photosynthetic activity by regulating stromal pH. It was demonstrated that high envelope-bound Mg²⁺ causes stromal acidification and photosynthetic inhibition. High envelope-bound Mg²⁺ was also found to necessitate the activity of a digitoxinand oligomycin-sensitive ATPase for the maintenance of high stromal pH and photosynthesis in the illuminated chloroplast. In chloroplasts that had high envelope Mg²⁺ and inhibited envelope ATPase activity, 2-(diethylamino)-N-(2,6-dimethylphenyl)acetamide was found to raise stromal pH and stimulate photosynthesis. 2-(Diethylamino)-N-(2,6-dimethylphenyl)acetamide is an amine anesthetic that is known to act as a monovalent cation channel blocker in mammalian systems. We postulate that the system regulating cation and H⁺ fluxes across the plastid envelope includes a monovalent cation channel in the envelope, some degree of (envelope-bound Mg²⁺ modulated) H⁺ flux linked to monovalent cation antiport, and ATPase-dependent H⁺ efflux.     

Interactions of ouabain and vanadate with (Na+,K+)ATPase and isolated cardiac muscle

The interactions of ouabain and vanadate with (Na⁺,K⁺)ATPase were investigated at different potassium concentrations. Also, the contractile effects of a mixture of these two inhibitors were compared to those produced by ouabain or vanadate alone. The results from the enzyme and contractile studies suggested that inhibition of sarcolemmal (Na⁺,K⁺)ATPase was involved in mediating the positive inotropic effect of vanadate.     

Nonselective cation channel activated by patch excision from lobster olfactory receptor neurons

Summary A nonselective cation channel activated by patch excision was characterized in inside-out patches from spiny lobster olfactory receptor neurons. The channel, which was permeable to Na⁺, K⁺ and Cs⁺, had a conductance of 320 pS and was weakly voltage dependent in the presence of micromolar divalent cations. Millimolar internal divalent cations caused a voltage-and concentration-dependent block of Na⁺ permeation. Analysis of the voltage dependence indicated that the proportion of the membrane's electric field sensed by Mg²⁺ was >1, suggesting that the channel contains a multi-ion pore. Internal divalent cations also reduced the frequency of channel opening in a concentration-dependent, but not voltage-dependent, manner, indicating that different cation binding sites affect gating and conductance. While block of gating prevented determining if internal divalent cations permeate the channel, a channel highly permeable to external divalent cations was observed upon patch excision to the inside-out configuration. The monovalent and divalent cation conductances shared activation by patch excision, weak voltage dependence, and steady-state activity, suggesting that they are the same channel. These data extend our understanding of this type of channel by demonstrating permeation by monovalent cations, detailing Mg²⁺ block of Na^– permeation, and demonstrating the channel's presence in arthropods.