The mode of activation by potassium of potassium-dependent and ouabain-sensitive phosphatase activity.

A new simple procedure has been developed for the purification of plasma membranes from rabbit kidney microsomes which yields a three- to fourfold increase in the specific activity of Na⁺-K⁺-adenosine triphosphatase (ATPase). The procedure differs from previous methods with deoxycholate or other detergents and does not change the molecular activity of the ATPase. The K⁺-dependent p-nitrophenylphosphatase activity of the native Na⁺-K⁺-ATPase is controlled more effectively by Mg²⁺ in the presence of K⁺ at concentrations higher than that of Mg²⁺, and by K⁺ in the presence of Mg²⁺ at concentrations higher than that of K⁺. The enzyme in its Mg²⁺-regulating state, which shows K⁺-saturation curves with a Hill coefficient of 1, is less sensitive to ouabain (I_0.5 = 90 μM) and corresponds to the enzyme conformation reported previously which is inhibited by the concurrent presence of Na⁺ and ATP or of Na⁺ and oligomycin (I_0.5 is the midpoint of the saturation curve). The enzyme in its K⁺-regulating state, which shows K⁺-saturation curves with a Hill coefficient of 2, is more sensitive to ouabain inhibition (I₀₅ = 8 μM) and corresponds to the enzyme conformation which is stimulated by the concurrent presence of Na⁺ and ATP or of Na⁺ and oligomycin. There appear to be two conformations of the enzyme that are regulated by Mg²⁺ binding on the inhibitory sites of the enzyme.     

RAPID EFFECTS OF VERATRIDINE, TETRODOTOXIN, GRAMICIDIN D, VALINOMYCIN AND NaCN ON THE NA+, K+ AND ATP CONTENTS OF SYNAPTOSOMES

Abstract— The effects of brief exposures of a number of depolarizing agents on ²⁴Na⁺ influx and on the Na⁺, K⁺ and ATP contents of synaptosomes were studied using a Millipore filtration technique to terminate the reaction. When synaptosomes were incubated in normal medium, there was a rapid influx of ²⁴Na⁺ and a gain in Na’contents; neither the ²⁴Na⁺ influx nor the Na⁺ gain were blocked by tetrodotoxin suggesting that this Na⁺ entry did not involve Na⁺-channels. Veratridine markedly increased the rate of ²⁴Na⁺ influx into synaptosomes and also increased the Na⁺ content and decreased the K⁺ content of synaptosomes within the first 10s of exposure. The normal ion contents were reversed by 1 min. The effects of veratridine on Na⁺ influx and on synaptosomal ion contents were prevented by tetrodotoxin and required Na⁺ in the medium. The ionophores gramicidin D and valinomycin also rapidly reversed the Na⁺ and K⁺ contents of synaptosomes, but these effects could not be blocked by tetrodotoxin. The reducing effect of gramicidin D on synaptosomal K⁺ content required Na’in the medium, whereas valinomycin caused a fall in the K⁺ content of synaptosomes in a Na⁺-free medium. Veratridine and gramicidin D, at concentrations known to reverse the synaptosomal ion contents, did not affect synaptosomal ATP levels. In contrast, valinomycin and NaCN caused an abrupt fall in synaptosomal ATP levels. The above findings suggest that veratridine quickly alters synaptosomal Na⁺ and K⁺ contents by opening Na ⁺-channels in the presynaptic membrane, and provide direct evidence for the existence of Na⁺-channels in synaptosomes. In contrast, gramicidin D and valinomycin appear to act independently of Na ⁺-channels, possibly by their ionophoric effects and, in the case of valinomycin, by diminishing synaptosomal ATP contents and hence diminishing Na⁺-pump activity. The rapid reversals of Na⁺ and K⁺ contents by these drugs could affect the resting membrane potentials, Na⁺-Ca²⁺ exchange across the synaptosomal membrane, and the release, synthesis and uptake of neurotransmitters by synaptosomes.     

Properties and nature of (Na+ + Mg2+)-dependent adenosine triphosphatase in the gills of the eel, angvilla anguilla (L.)

The specific activity of (Na⁺ + Mg²⁺)-dependent ATPase is three times greater in the microsomes of sea-water eels than in freshwater eels; the specific activity is one quarter of that of (Na⁺ + K⁺ + Mg²⁺)-dependent ATPase in both cases.(Na⁺ + Mg²⁺)-dependent ATPase is optimally active in a medium containing 8 mM NaCl, 4 mM MgCI₂, 4 mM ATP, pH 8.8 and at 30 °C; the enzyme is inhibited by ouabain, by NaCl concentrations > 100 mM and by treatment with urea.It is concluded that the (Na⁺ + Mg²⁺)-dependent ATPase activity of gills arises from the presence of a (Na⁺ + K⁺ + Mg²⁺)-dependent ATPase.     

Quantitative aspects of the interaction between ouabain and (Na + K)-activated ATPase in vitro

The inhibitory effect of ouabain on (Na⁺ + K⁺)-activated ATPase (Mg²⁺-dependent, (Na⁺ + K⁺)-activated ATP phosphohydrolase, EC 3.6.1.3) obtained from rat brain microsomal fraction was re-examined using a modified method to estimate the inhibited reaction velocity. This method involves a preincubation of a ouabain-enzyme mixture in the presence of Na⁺, Mg²⁺ and ATP to bring the ouabain-enzyme reaction to near equilibrium. The (Na⁺ + K⁺)-activated ATPase reaction was subsequently started by the addition of a KCl solution.     

The Extrusion of Sodium Ions from Presynaptic Nerve Endings of Rat Cerebral Cortex

(Na⁺-K⁺) ATPase is present in synaptosomal preparations and it is assumed to represent the sodium-potassium pump. 10 μm -noradrenaline activates (Na⁺-K⁺) ATPase approximately 100%, but 50 μm -noradrenaline does not stimulate the rate of ²²Na extrusion from synaptosomes. The results suggest that it is unlikely that the noradrenaline stimulation of (Na⁺-K⁺) ATPase is part of a feedback mechanism whereby released noradrenaline can influence the activity of the presynaptic sodium pump.     

RELEASE OF ATP FROM A SYNAPTOSOMAL PREPARATION BY ELEVATED EXTRACELLULAR K+ AND BY VERATRIDINE

A technique was developed which permitted the release of ATP from synaptosomes by elevated extracellular K⁺ or by veratridine to be directly and continuously monitored. The released ATP interacted with firefly luciferin and luciferase in the incubation medium to produce light which could be detected by a photomultiplier. The assay system was specific for ATP, in that similar concentrations of adenosine, AMP or ADP did not produce chemiluminescence. Moreover, the maximum peak of light emission correlated linearly with the concentrations of ATP present in the medium, so that semiquantitative estimates of ATP release could be made. Elevating the extracellular K⁺ concentration produced a graded release of ATP from synaptosomes. Rb⁺ also released ATP but Na⁺, Li⁺ and choline did not. The response to elevated K⁺ was not blocked by tetrodotoxin (TTX), indicating that this effect was not mediated by the opening of Na⁺-channels in synaptosomal membranes. Veratridine (50 μM) caused a graded release of ATP which was larger and more prolonged than that caused by elevated K⁺. The release of ATP by veratridine was blocked by TTX indicating that the opening of Na⁺-channels was involved. Neither veratridine nor elevated K⁺ released ATP from microsomal or mitochondrial fractions, showing that the release of ATP probably did not originate from microsomal, vesicular or mitochondrial contaminants of the synaptosomal preparation. Release of ATP by elevated K⁺ was diminished in a medium lacking CaCl₊ or when EGTA was added to chelate Ca²⁺. In contrast, release by veratridine appeared to be augmented in Ca²⁺-free media or in the presence of EGTA. The K⁺-induced release of ATP, which is Ca²⁺ dependent, closely resembles the exocytotic release of putative neurotransmitters from presynaptic nerve-terminals. On the other hand, the apparent lack of a Ca²⁺ requirement for veratridine's action suggests that this process could originate from other sites, or involve mechanisms other than conventional neurotransmitter release processes.     

Effect of Na+, K+, Mg2+ and ATP on the Relative Electrophoretic Mobility of Sugar Beet Membrane Particles with NaK ATPase Activity

Electrophoretic measurements on membrane coated particles were performed with a Zytopherometer. Tris-HCl buffer 0.2 M pH 7.0 at 37°C with addition of different combinations of Na⁺, K⁺, Mg²⁺ and ATP was used as test medium. The membranes were of two types, an untreated preparation with low NaK ATPase activity and a deoxycholate treated preparation with high NaK ATPase activity. There was no marked difference in reaction between the two types of membranes. To both types of membranes Mg²⁺ gave a strong positive and ATP a slight negative addition to the membrane charge. In the presence of ATP Na⁺ gave a higher charge contribution than did K⁺ or a combination of Na⁺ and K⁺. This implies that K⁺ gives a higher affinity for ATP than Na⁺ does and or that ATP mediates a higher affinity for Na⁺ than for K⁺.     

Development of Na+-stimulated glucose oxidation in synaptosomes

Abstract— d -[¹⁴C]Glucose was oxidized to ¹⁴CO₂ by synaptosomes prepared from adult rat brain. Added Na⁺ stimulated glucose oxidation by 179%, but K⁺ and choline were without effect. Li⁺ stimulated glucose oxidation by 64%. Ouabain largely prevented the stimulatory effect of Na⁺ on glucose oxidation but had no effect in the absence of Na⁺. 2-Deoxy-d -glucose competitively inhibited glucose oxidation differently at two different ranges of deoxyglucose and glucose concentrations; the K_i was 0.54 and 16 mm , respectively. In the presence or absence of Na⁺ 2,4-DNP-stimulated glucose oxidation by 370% while iodoacetate inhibited glucose oxidation by 87–95%. There was a striking increase in Na⁺-stimulated glucose oxidation with development but glucose oxidation in the absence of Na⁺ did not change dramatically with age. Taken together the data suggest synaptosomes exhibit coupled respiration which can be modulated by Na⁺. In addition, the appearance of Na⁺-stimulated glucose oxidation with maturation probably is linked to the development of Na⁺-K⁺-ATPase acitivity in the synaptosomal membrane.     

Sensitivity of ATPase activities to fire ant venom and abdomen preparations.

The sensitivity of fire ant, $\text{Solenopsis richteri}$ (Forel), head homogenate ATPase to its venom and to a cyclohexane extract of whole fire ants were investigated. Na⁺K⁺ and oligomycin-sensitive Mg²⁺ ATPase activities were inhibited by both preparations. Oligomycin-insensitive Mg²⁺ ATPase activity was inhibited by low concentrations but showed strong stimulation at high concentrations of the venom preparations. Lineweaver-Burk plots of enzyme data in the presence or absence of inhibitor indicated that the inhibitor action was non-competitive with ATP for Na⁺K⁺ and oligomycin-sensitive Mg²⁺ ATPase activities. However, the oligomycin-insensitive Mg²⁺ ATPase activity showed a mixed type response to the inhibitor. Tests on pure samples of known venom components indicate that they cause the observed effects on the ATPase activities.     

Effect of the diazonium salt of sulfanilic acid on human erythrocyte ATPase activity

External treatment of human erythrocytes with the diazonium salt of sulfanilic acid does not inhibit the Mg²⁺-dependent ATPase but does markedly inhibit the Ca²⁺-stimulated ATPase activity. Inhibition of the (Na⁺ + K⁺)-dependent activity is dependent upon the concentration of diazonium salt used. Treatment of membrane fragments does not irreversibly inhibit the (Na⁺ + K⁺)-dependent ATPase even though the diazonium salt binds covalently to membrane components. However, the Mg²⁺-dependent and Ca²⁺-stimulated ATPase activities are irreversibly inhibited. ATP and Mg-ATP will completely protect the (Na⁺ + K⁺)-dependent ATPase when present during treatment of membrane fragments with the diazonium salt, but only Mg-ATP will protect the Mg²⁺-dependent ATPase from inhibition. The Ca²⁺-stimulated ATPase activity is not protected.     

Ca2+-Dependent activities of (Na+ + K+)-ATPase

Experiments on the effects of varying concentrations of Ca²⁺ on the Mg²⁺ + Na⁺-dependent ATPase activity of a highly purified preparation of dog kidney (Na⁺ + K⁺)-ATPase showed that Ca²⁺ was a partial inhibitor of this activity. When Ca²⁺ was added to the reaction mixture instead of Mg²⁺, there was a ouabain-sensitive Ca²⁺ + Na⁺-dependent ATPase activity the maximal velocity of which was 30 to 50% of that of Mg²⁺ + Na⁺-dependent activity. The apparent affinities of the enzyme for Ca²⁺ and CaATP seemed to be higher than those for Mg²⁺ and MgATP. Addition of K⁺, along with Ca²⁺ and Na⁺, increased the maximal velocity and the concentration of ATP required to obtain half-maximal velocity. The maximal velocity of the ouabain-sensitive Ca²⁺ + Na⁺ + K⁺-dependent ATPase was about two orders of magnitude smaller than that of Mg²⁺ + Na⁺ + K⁺-dependent activity. In agreement with previous observations, it was shown that in the presence of Ca²⁺, Na⁺, and ATP, an acid-stable phosphoenzyme was formed that was sensitive to either ADP or K⁺. The enzyme also exhibited a Ca²⁺ + Na⁺-dependent ADP-ATP exchange activity. Neither the inhibitory effects of Ca²⁺ on Mg²⁺-dependent activities, nor the Ca²⁺-dependent activities were influenced by the addition of calmodulin. Because of the presence of small quantities of endogenous Mg²⁺ in all reaction mixtures, it could not be determined whether the apparent Ca²⁺-dependent activities involved enzyme-substrate complexes containing Ca²⁺ as the divalent cation or both Ca²⁺ and Mg²⁺.     

Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase Activity in the Posterior Gills of the Blue Crab, <Emphasis Type="Italic">Callinectes ornatus</Emphasis> (Decapoda,Brachyura): Modulation of ATP Hydrolysis by the Biogenic Amines Spermidine and Spermine

We investigated the effect of the exogenous polyamines spermine, spermidine and putrescine on modulation by ATP, K⁺, Na⁺, NH₄ ⁺ and Mg²⁺ and on inhibition by ouabain of posterior gill microsomal Na⁺,K⁺-ATPase activity in the blue crab, Callinectes ornatus, acclimated to a dilute medium (21‰ salinity). This is the first kinetic demonstration of competition between spermine and spermidine for the cation sites of a crustacean Na⁺,K⁺-ATPase. Polyamine inhibition is enhanced at low cation concentrations: spermidine almost completely inhibited total ATPase activity, while spermine inhibition attained 58%; putrescine had a negligible effect on Na⁺,K⁺-ATPase activity. Spermine and spermidine affected both V and K for ATP hydrolysis but did not affect ouabain-insensitive ATPase activity. ATP hydrolysis in the absence of spermine and spermidine obeyed Michaelis–Menten behavior, in contrast to the cooperative kinetics seen for both polyamines. Modulation of V and K by K⁺, Na⁺, NH₄ ⁺ and Mg²⁺ varied considerably in the presence of spermine and spermidine. These findings suggest that polyamine inhibition of Na⁺,K⁺-ATPase activity may be of physiological relevance to crustaceans that occupy habitats of variable salinity.     

Cardiac sarcolemmal Na+-Ca2+ exchange and Na+-K+ ATPase activities and gene expression in alloxan-induced diabetes in rats

To determine the sequence of alterations in cardiac sarcolemmal (SL) Na⁺-Ca²⁺ exchange, Na⁺-K⁺ ATPase and Ca²⁺-transport activities during the development of diabetes, rats were made diabetic by an intravenous injection of 65 mg/kg alloxan. SL membranes were prepared from control and experimental hearts 1-12 weeks after induction of diabetes. A separate group of 4 week diabetic animals were injected with insulin (3 U/day) for an additional 4 weeks. Both Na⁺-K⁺ ATPase and Ca²⁺-stimulated ATPase activities were depressed as early as 10 days after alloxan administration; Mg²⁺ ATPase activity was not depressed throughout the experimental periods. Both Na⁺-Ca²⁺ exchange and ATP-dependent Ca²⁺-uptake activities were depressed in diabetic hearts 2 weeks after diabetes induction. These defects in SL Na⁺-K⁺ ATPase and Ca-transport activities were normalized upon treatment of diabetic animals with insulin. Northern blot analysis was employed to compare the relative mRNA abundances of _--subunit of Na⁺-K⁺ ATPase and Na⁺-Ca²⁺ exchanger in diabetic ventricular tissue vs. control samples. At 6 weeks after alloxan administration, a significant depression of the Na⁺-K⁺ ATPase _-- subunit mRNA was noted in diabetic heart. A significant increase in the Na⁺-Ca²⁺ exchanger mRNA abundance was observed at 3 weeks which returned to control by 5 weeks. The results from the alloxan-rat model of diabetes support the view that SL membrane abnormalities in Na⁺-K⁺ ATPase, Na⁺Ca²⁺ exchange and Ca²⁺-pump activities may lead to the occurrence of intracellular Ca²⁺ overload during the development of diabetic cardiomyopathy but these defects may not be the consequence of depressed expression of genes specific for those SL proteins.     

Intrasynaptosomal Free Mg2+ Concentration Measured with the Fluorescent Indicator Mag-Fura-2: Modulation by Na+ Gradient and by Extrasynaptosomal ATP

Abstract: Synaptosomes can be loaded with mag-fura-2 without significant perturbation of their ATP content by incubation for 10 min at 37°C with 10 µM mag-fura-2 acetoxymethyl ester in Hanks'-HEPES buffer (pH 7.45). The intrasynaptosomal free Mg²⁺ concentration ([Mg²⁺]_i) was found to be dependent on external Mg²⁺ concentration, increasing from 0.8 to 1.25 mM when the concentration of Mg²⁺ in the incubation medium increased from 1 to 8 mM. Dissipation of the Na⁺ gradient across the plasma membrane of synaptosomes by treatment with the Na⁺ ionophore monensin (0.2 mM) or with veratridine (0.2 mM) and ouabain (0.6 mM) produced a moderate increase of [Mg²⁺]_i, from 1.0 to 1.2–1.3 mM in an incubation medium containing 5 mM Mg²⁺. Plasma membrane depolarization by incubation of synaptosomes in a medium containing 68 mM KCl and 68 mM NaCl had no effect on [Mg²⁺]_i. Reversal of the Na⁺ gradient by incubation of synaptosomes in a medium in which external Na⁺ was replaced by choline increased [Mg²⁺]_i up to 1.6 and 2.2 mM for extrasynaptosomal Mg²⁺ concentrations of 1 and 8 mM, respectively. We conclude that a Na⁺/Mg²⁺ exchange operates in the plasma membrane of synaptosomes. In the presence of Mg²⁺ in the incubation medium, extrasynaptosomal ATP, but not ADP or adenosine, increased [Mg²⁺]_i from 1.1 ± 0.1 up to 1.6 ± 0.1 mM. The nonhydrolyzable ATP analogue adenosine 5′-(βγ-imido)triphosphate antagonized the effect of ATP, but had no effect by itself on [Mg²⁺]_i. It is concluded that Mg²⁺ transport across the plasma membrane of synaptosomes is modulated by the activity of an ecto-ATPase or an ecto-protein kinase.     

Lead ion activates phosphorylation of electroplax Na+- and K+-dependent adenosine triphosphatase ((NaK)-ATPase) in the absence of sodium ion.

PbCl₂ in micromolar concentrations stimulates phosphorylation of electroplax microsomal protein in the absence of Na⁺. Other divalent cations showed little or no such effect. The (Mg²⁺ + Pb²⁺)- and (Mg²⁺ + Na⁺)-dependent membrane-bound protein kinase activities in electroplax particulate preparations exhibit properties in common, including their acid stability, ouabain sensitivity, ATP specificity, and molecular size. It is concluded that the (Mg²⁺ + Pb²⁺)-dependent phosphoprotein is part of the Na⁺-, K⁺-dependent adenosine triphosphatase [(NaK)ATPase]. The Pb²⁺-dependent product, in contrast to the Na⁺-dependent one, is insensitive to K⁺ and the hydrolysis of ATP is thus inhibited.     

Partial characterization of an endogenous factor which modulates the effect of catecholamines on synaptosomal Na+, K+-ATPase

We have previously presented evidence for the existence of a brain soluble factor which mediates the stimulation of synaptosomal ATPases by catecholamines. The stimulation of synaptosomal ATPases by dopamine plus brain soluble fraction was not modified if the soluble fraction was heated for 5 min at 95°C. One day after preparation, the soluble factor inhibited the Na⁺, K⁺-ATPase, but not the Mg²⁺-ATPase activity, and subsequent addition of noradrenaline stimulated the ATPases activities. The inhibitory effect of a 24 h soluble fraction disappeared if the soluble fraction was dialyzed; in this case, noradrenaline did not activate the enzyme activities. Gel filtration in Sephadex G-50 permitted separating a subfraction which inhibited ATPase activity (peak II) from another which stimulated ATPase activity (peak I). Peak I stimulated both Na⁺, K⁺, and Mg²⁺ ATPases. Peak II inhibited only Na⁺, K⁺-ATPase, and when stored acidified, it mediated ATPases stimulation by noradrenaline.Special Issue dedicated to Prof. Eduardo De Robertis.     

Studies on plasma membranes. XXI. Inhibition of liver plasma membrane enzymes by tumour-promoting phorbolester, mitotic inhibitors and cytochalasin B

The tumour promotor tetradecanoyl phorbol acetate (TPA) inhibited the Mg²⁺-, Ca²⁺- and (Na⁺-K⁺)ATPases of rat-liver plasma membranes. A nonpromoting phorbolester derivative was without effect. Colchicine and/or vinblastine inhibited the (Na⁺-K⁺)ATPase, glucagon-stimulated adenylate cyclase, and cyclic adenosine-3, 5-monophosphate (c-AMP) phosphodiesterase, but were without significant effect on the Mg²⁺- or Ca²⁺-ATPase. Cytochalasin B inhibited the (Na⁺-K⁺)ATPase. The results furnish the first direct evidence that these drugs may interact with plasma membranes. The mechanism of the enzyme inhibitions is briefly discussed.     

The Effect of Ouabain on the Release of [14C]Acetylcholine and Other Substances from Synaptosomes

Abstract: The effect of ouabain and dihydroouabain on Na⁺-K⁺ ATPase, ⁸⁶Rb uptake and the release of [¹⁴C]ACh (acetylcholine) from synaptosomal preparations of guinea pigs was compared. At low concentrations of glycoside (<50 μm ) there was a good correlation between the potency of ouabain and of dihydroouabain in inhibiting Na⁺-K⁺ ATPase and in causing the release of [^l4C]ACh in a nondepolarising medium. Ouabain (200 μM) increased the release of [¹⁴C]ACh evoked by 25 mm -KCl, but not that evoked by 100μm -veratrine. The enhancement of release was independent of the presence of calcium. It was observed that in addition to [¹⁴C]ACh release, choline efflux was also stimulated by ouabain, independently of the presence of Ca²⁺. Experiments with hemicholinium-3 showed that the ouabain-induced increase in choline efflux was not due to an inhibition of reuptake. The effect of ouabain on intrasynaptosomal K⁺ concentration was measured in order to investigate the degree of depolarisation it caused. The decrease in K⁺ was found to be similar in magnitude and time course to that caused by veratrine. It was shown that ouabain-induced depolarisation caused an increased efflux of another positive ion (dibenzyldimethylammonium chloride) and retention of a negatively charged ion (chloride), as would be expected from the operation of the electrochemical potential gradient changing as a result of depolarisation. It is suggested that ouabain acts to stimulate ACh release from synaptosomes as follows: following blockage of the Na⁺-K⁺ ATPase there is rapid depolarisation which, if Ca²⁺ is present, provokes the normal Ca²⁺-dependent transmitter release process to occur. In addition, depolarisation accelerates the leakage of positive ions down their electrochemical potential gradient, but causes a retention of negative ions. Such an action does not depend on the presence of Ca²⁺, nor is it specific to transmitters.