Comparative study of properties of Na+, K+-ATPase and Mg2+-ATPase of the myometrium plasma membrane

The comparative research of catalytic properties of two ATP-hydrolases of the sarcolemma of the smooth muscle of the uterus--ouabaine-sensitive Na+,K+-ATPase and ouabaine-resistent Mg2+-ATPase is carried out. The specific enzymatic activity of Na+,K+-ATPase and Mg2+-ATPase makes 10.2 +/- 0.7 and 18.1 +/- 1.2 mmol P/mg of protein for 1 hour, accordingly. The action of ouabaine on Na+,K+-ATPase is characterized by magnitude of quotient of inhibition I0.5=21.3 +/- 1.5 mkM. Processing of the sarcolemma fraction by digitonin in concentrations 0.001 +/- 0.1% promotes an activation of Na+,K+ATPase and Mg2+- ATPase, and in the first case much more efficiently than in the second. The kinetics of accumulation of the product of ATP-hydrolase reactions of phosphate satisfies the laws of the zero order reaction (incubation time--about 10 min). Na+,K+-ATPase is highly specific concerning the univalent cations--Na+, K+, however Li+ can partially substitute K+. Activity of Mg2+-ATPase is not specific concerning univalent cations. The dependence of Na+,K+-ATPase activity on pH in the range of 6.0-8.0 is characterized by the bell-shaped curve, at the same time the linear dependence on pH is peculiar to Mg2+-ATPase. The functioning of Na+,K+-ATPase is provided only by ATP, in the case of Mg2+-ATPase ATP can be successfully replaced with other nucleotidetriphosphates. It is supposed that the obtained experimental data can be beneficial in further research of membranous mechanisms underlying the cation exchange in the smooth muscles, in particular when studying the role of the plasma membrane in the maintenance of electromechanical coupling in them, and also in the regulation of ionic homeostasis in myocytes.     

The calixarenes C-97 and C-107 stimulate influence of ouabain on the Na+,K+-ATPase activity in plasmatic membrane of smooth muscle cells

In the experiments carried out with the suspension of the myometrium cell plasmatic membranes treated with 0.1% digitonin solution the authors investigated influence of the calix[4]arenes C-97 and C-107 (codes are shown) on ouabain effect on the Na+,K+-ATPase activity. It was shown that calixarenes in concentration 100 tiM inhibited by 97-98% the enzymatic Na+,K+-ATPase activity, while they did not practically influence on the basal Mg2+-ATPase activity, and suppressed much more effective than ouabain the sodium pump enzymatic activity: in the case of the action of the calixarenes the value of the apparent constant of inhibition I0.5 was < 0.1 microM while for ouabain it was 15-25 microM. The negative cooperative effect was typical of the inhibitory action of calixarenes, as well as ouabain: the value of Hills factor nH = 0.3-0.5 <1. The modelling compound M-3 (0.1 microM 4 microM)--a fragment of the calixarene C-107--did not practically influence the enzymatic activities as Na+,K+-ATPase and basal Mg2+-ATPase. Hence the influence of calixarene C-107 on the Na+, K+-ATPase activity is caused by cooperative action of two fragments M-3 and effect of calixarene bowl, rather than by simple action of the fragment M-3. Calixarenes C-97 and C-107, used in concentration corresponding to values of I0.5 (40 and 60 nM, accordingly), essentially stimulated inhibiting action of ouabain on the specific Na+, K+-ATPase activity in the memrane fraction. Under coaction of ouabain with calixarene C-97 or C-107 there was no additive effect of the action of these inhibitors on the Na+,K+-ATPase activity. Calixarene C-97 brought in the incubation medium in concentration of 10 nM not only led to inhibition of the Na+,K+-ATPase activity relative to control, but also simultaneously increased the affinity of the enzyme for the cardiac glycoside: the magnitudes of the apparent constant of inhibition I0.5 were 21.0 +/- 5.2 microM and 5.3 +/- 0.7 microM. It is concluded, that highly effective inhibitors of the Na+,K+-ATPase activity--calixarenes C-97 and C-107 can enhance the effect of the sodium pump conventional inhibitor--ouabain, increasing the affinity of the enzyme for the cardiac glycoside (on the example of calixarene C-97).     

Competitive membrane adsorption of Na+, K+, and Ca2+ in smooth muscle cells

Summary A theory for Na⁺, K⁺ and Ca²⁺ competitive adsorption to a charged membrane is used to explain a number of experimental observations in smooth muscle. Adsorption is described by Langmuir isotherms for mono- and divalent cations which in turn are coupled in a self-consistent way to the bulk solution through the diffuse double layer theory and the Boltzman equations. We found that the dissociation constants for binding of Na⁺, K⁺ and Ca²⁺ in guinea pig taenia coli areca. 0.009, 1.0, and 4×10^–8 m, respectively. Furthermore, the effect of a Ca²⁺ pump that maintains free surface Ca²⁺ concentration constant is investigated. A decrease in intracellular Na⁺ content results in an increased Ca²⁺ uptake; part of this uptake is due to an increase in surface-bound Ca²⁺ in an intracellular compartment which is in contact with the myofilaments. Variations in the amount of charge available to bind Ca²⁺ and the surface charge density are studied and their effect interpreted in terms of different pharmacological agents.     

Effects of sodium nitroprusside and nitrite on ouabaine-sensitive Na+, K(+)-ATPase activity of myometrium smooth muscle

Effective inhibiting effect of sodium nitroprusside and nitrite on Na+, K(+)-ATPase enzymatic activity of miometrium sarcolemma fraction was shown. Seeming Ki was of micromolar and submicromolar magnitudes. Investigations with sodium nitroprusside demonstrated an uncompetitive inhibition for ATP (growth of affinity for ATP and decrease of maximal velocity) and mixed inhibition for cations (decrease of maximal velocity and activation of constant for K+). Inhibitory effect of ouabain was reduced in the presence of sodium nitroprusside; ditiothreitol prevented enzyme inactivation by sodium nitroprusside. Kinetic analysis of experimental results using ouabain and ditiothreitol suggests chemical modification of enzyme sulfhydryl groups. Resistant component of Na+, K(+)-ATPase activity, which is sensitive to the action of detergent digitonine, was observed. In comparative investigations with postnucleus fraction stimulating actions of sodium nitroprusside, sodium nitrite, cGMP (more enhance) were shown. Methylene blue (soluble guanilate-cyclase inhibitor) prevented the activation of Na+, K(+)-ATPase activity by sodium nitrite. We suppose that the way of enzyme activation is prevalent in the condition of the moderate formation of nitric oxide and in the absence of hyper(over)production of reactive oxygen species.     

Ca++ accumulation and latent Na+, K+-ATPase activity of heterogeneous membrane fractions of vascular smooth muscle

Two different heterogeneous subcellular fractions of canine aorta were studied. Both contained ATP-dependent, oxalate stimulated ⁴⁵Ca sequestration, and ³H-ouabain binding. Neither showed any ouabain inhibited ATPase activity until treated with SDS. After such treatment, both fractions demonstrated significant ouabain-inhibited ATPase activity. Thus, even though transport ATPase was not readily demonstrable in control fractions, sarcolemmal fragments are still present as evidenced by both ³H-ouabain binding and detergent treatment.     

The calixarene C-107 increases the affinity of the Na+,K(+)-ATPase in plasmatic membrane of smooth muscle cells to the ouabain

In the experiments carried out with the suspension of the myometrium cell plasmatic membranes treated with 0.1% digitonin solution we investigated the influence of calixarene C-107 (5,17-diamino(2-pyridyl)methylphosphono-11,23-di-tret-butyl-26,28-dihydroxy-25,27-dipropoxyca-lix[4]arene) on the Na+,K(+)-ATPase activity. It was shown that this calixarene increased the affinity of the enzyme for the sodium pump conventional inhibitor - ouabain: the magnitudes of the seeming constant of inhibition I0.5 changed from 26.9 +/- 1.3 mM to 10.9 +/- 0.6 mM. However the ouabain itself did not influence on the affinity of the Na+,K(+)-ATPase for calixarene C-107.     

Effect of n-chloromercuribenzoate and albumin on Mg2+, Ca2+-ATPase activity of plasma membranes of smooth muscle cells

It is shown that Mg2+, Ca2+-ATPase activity of plasma membrane fragments from the rat small intestine myocytes is inhibited by p-chloromercuribenzoate and dithionitrobenzoate (50 and 90%, respectively). The effect of p-chloromercuribenzoate inhibition is removed by serum albumin promoting a rise in the ATPase activity of the plasma membranes and shifting the temperature maximum point up to 32 degrees C (in the norm the maximum is observed at 36 degrees C). According to the data presented, albumin changes the composition and phase state of the lipid surrounding of the membrane enzymes.     

Vanadate sensitivity of Na+, K+-ATPase from Schistosoma mansoni and its modulation by Na+, K+ and Mg2+

Vanadate inhibitory effects on Na+, K+-ATPases from carcass of Schistosoma mansoni and from lamb kidney outer medulla were compared in the presence of various concentrations of Na+, K+ and Mg2+. Depending on the ionic conditions, the schistosomal Na+, K+-ATPase was 2.4- to 175-fold less sensitive to vanadate than the lamb kidney enzyme. In 100 mM Na+, 3 mM K+ and 3 mM Mg2+, schistosomal Na+, K+-ATPase was surprisingly resistant to vanadate (I50 = 944 microM). The difference in vanadate sensitivity between schistosomal and lamb Na+, K+-ATPases may be due to a species difference in the efficacy of Na+, K+ and Mg2+ in promoting conformational changes between E1 and E2 forms of the enzyme.     

Potassium effects on contraction in arterial smooth muscle mediated by Na+, K+-ATPase

A local increase in the extracellular potassium concentration [K+]o, up to about 8 meq/liter either by topical application or intra-arterial infusion of K+ salts, causes arteriolar dilation and decreased resistance to blood flow in systemic vascular beds. Isolated vascular smooth muscle responds to a similar increase in [K+] in the bathing fluid with relaxation if the preparation has some initial active tension. Reduction in [K+] over physiological ranges produces arteriolar constriction and increased resistance to blood flow. K+ vasodilation is accompanied by hyperpolarization of the smooth muscle cell whereas the vasoconstriction is accompanied by depolarization. All these responses can be blocked by ouabain, a potent Na+, K+-ATPase inhibitor. It is therefore thought that K+ vasodilation results from stimulation of the electrogenic Na+-K+ pump whereas the vasoconstriction results from inhibition of this pump. A number of conditions that alter resistance also alter interstitial fluid [K+]. These include exercise, myocardial ischemia, epileptic convulsions, and evoked electrical activity of the somatomotor cortex. Certain findings, including those during administration of ouabain, suggest that changes in [K+] contribute significantly to some of the changes in resistance.     

Effect of organic solvents on catalytic and functional activity of transport Ca2+,Mg2+-ATPase from smooth muscle cell membrane

It was shown that organic solvents (dioxane, acetone, ethanol, dimethylsulfoxide) at concentrations of < 10% suppress the activity of transport Ca2+, Mg(2+)-ATPase solubilized from plasmatic membranes of smooth muscle cells and Mg(2+)-ATP-dependent accumulation of Ca2+ ions in inverted membrane vesicles. It was found that one of the reasons for the inhibition of enzymatic and transport activity of Ca2+, Mg(2+)-ATPase by the action of these solvents is an increase in the attractive force between oppositely charged active center of the enzyme and the product (products) of the ATP-hydrolase reaction, which is induced by a decrease in the dielectric permeability of incubation medium.     

Inhibition of Na+, K+-ATPase of intact mouse soleus muscle by Mg++

The effect of 10 mM MgCl₂ on the inhibition of respiration by ouabain was investigated with intact mouse soleus muscle preparations. Although ouabain caused a 19.7% inhibition of respiration of soleus muscle incubated in 1 mM MgCl₂ buffer, the response of respiration to ouabain was abolished upon incubation in buffer containing 10 mM MgCl₂. Initial respiration rates were significantly decreased in soleus muscle exposed to 10 mM, as contrasted to 1 mM, MgCl₂.     

Activation of AMP-activated protein kinase stimulates Na+,K+-ATPase activity in skeletal muscle cells

Contraction stimulates Na(+),K(+)-ATPase and AMP-activated protein kinase (AMPK) activity in skeletal muscle. Whether AMPK activation affects Na(+),K(+)-ATPase activity in skeletal muscle remains to be determined. Short term stimulation of rat L6 myotubes with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), activates AMPK and promotes translocation of the Na(+),K(+)-ATPase α(1)-subunit to the plasma membrane and increases Na(+),K(+)-ATPase activity as assessed by ouabain-sensitive (86)Rb(+) uptake. Cyanide-induced artificial anoxia, as well as a direct AMPK activator (A-769662) also increase AMPK phosphorylation and Na(+),K(+)-ATPase activity. Thus, different stimuli that target AMPK concomitantly increase Na(+),K(+)-ATPase activity. The effect of AICAR on Na(+),K(+)-ATPase in L6 myotubes was attenuated by Compound C, an AMPK inhibitor, as well as siRNA-mediated AMPK silencing. The effects of AICAR on Na(+),K(+)-ATPase were completely abolished in cultured primary mouse muscle cells lacking AMPK α-subunits. AMPK stimulation leads to Na(+),K(+)-ATPase α(1)-subunit dephosphorylation at Ser(18), which may prevent endocytosis of the sodium pump. AICAR stimulation leads to methylation and dephosphorylation of the catalytic subunit of the protein phosphatase (PP) 2A in L6 myotubes. Moreover, AICAR-triggered dephosphorylation of the Na(+),K(+)-ATPase was prevented in L6 myotubes deficient in PP2A-specific protein phosphatase methylesterase-1 (PME-1), indicating a role for the PP2A·PME-1 complex in AMPK-mediated regulation of Na(+),K(+)-ATPase. Thus contrary to the common paradigm, we report AMPK-dependent activation of an energy-consuming ion pumping process. This activation may be a potential mechanism by which exercise and metabolic stress activate the sodium pump in skeletal muscle.     

Effect of spermine on activity of purified Ca2+, Mg2+-ATPase from plasma membranes of myometrium cells

Effect of endogenous polyamine spermine, a relaxant of smooth muscle, on the activity of myometrium cell plasma membrane Ca2+, Mg(2+)-ATPase was studied. It was observed a tendency to activation of enzyme at the spermine concentrations 0.1-0.5 mM, the increase of the polyamine concentrations up to 10 mM inhibited. ATPase by 80% (I50 = 5.5 +/- 0.3 mM). Spermine inhibited enzyme decreasing its turnover rate and affinity for Ca2+. The ATPase affinity for Mg2+ increased in the presence of spermine. It was revealed, that the inhibitory effect of spermine is changed by the stimulatory effect under the increase of Ca2+ concentration (up to 2.6 microM), that correlates with the relaxing effect of this polyamine on the smooth muscle.     

Na+,K+-ATPase activity in cultured C6 glioma cells

Rat C₆ glioma cells were cultured for 4 days in MEM medium supplemented with 10% bovine serum and Na⁺,K⁺-ATPase activity was determined in homogenates of harvested cells. Approximately 50% of enzyme activity was attained at 1.5 mM K⁺ and the maximum (2.76±0.13 mol Pi/h/mg protein) at 5 mM K⁺. The specific activity of Na⁺,K⁺-ATPase was not influenced by freezing the homogenates or cell suspensions before the enzyme assay. Ten minutes' exposure of glioma cells to 10^–4 or 10^–5 M noradrenaline (NA) remained without any effect on NA⁺,K⁺-ATPase activity. Neither did the presence of NA in the incubation medium, during the enzyme assay, influence the enzyme activity. The nonresponsiveness of Na⁺,K⁺-ATPase of C₆ glioma cells to NA is consistent with the assumption that (+) form of the enzyme may be preferentially sensitive to noradrenaline. Na⁺,K⁺-ATPase was inhibited in a dose-dependent manner by vanadate and 50% inhibition was achieved at 2×10^–7 M concentration. In spite of the fact that Na⁺,K⁺-ATPase of glioma cells was not responsive to NA, the latter could at least partially reverse vanadate-induced inhibition of the enzyme. Although the present results concern transformed glial cells, they suggest the possibility that inhibition of glial Na⁺,K⁺-ATPase may contribute to the previously reported inhibition by vanadate of Na⁺,K⁺-ATPase of the whole brain tissue.     

Effect of eosin Y on Ca2+, Mg2+-ATPase of the smooth muscle sarcolemma

Eosin Y was studied with the aim to elucidate the mechanism of its inhibitory effect on the activity of Ca(2+)-transporting ATPase of myometrium cell plasma membrane. The inhibitor was studied for its effect on the maximal rate of the ATP-hydrolase reaction catalyzed by Ca2+, Mg(2+)-ATPase, on the enzyme affinity for the substrate and a possibility of enzyme activity protection under the inhibitor effect by the main reagents of ATP-hydrolase reaction. It was established that eosin Y decreased the turnover rate of this enzyme and his affinity for ATP. Preincubation of ATPase with ATP (or ATP plus MgCl2) had no effect on the extent of enzyme inhibition by eosin Y. This result proves that eosin Y and ATP do not compete for the site of binding on the enzyme.     

Variance analysis of Cd2+, Zn2+, and Mn2+ effect on membrane Na+,K(+)-ATPase activity of loach embryos

The evaluation of influences Cd2+, Zn2+, Mn2+ in concentrations 10(-6), 10(-5), 10(-4) M (factor of dose) on the Na+, K(+)-ATP-ase activity in the early period of development (60-330 min.) of loach embryos (time factor) using one- and two-factor analysis of variance has been performed. It has been detected, that the changes of enzyme activity are mainly caused by action of the explored cations and do not depend on time of embryos development. The most influence on activity in the indicated period of embryos development of loach renders Cd2+ in concentration 10(-4) M, relative value of its influence being 95.7% (p = 0.01). Substantial concentration dependence of the Na+, K(+)-ATP-ase activity is exposed to the action of each of cations. The values of the influence of their concentration changes during the studied period of development differ insignificantly for all cations and make 76.2-77.5% (p < 0.01).     

Effect of glycosphingolipids on erythrocyte Na+,K+-ATPase activity

The total fractions of gangliosides and cerebrosides isolated from the tissue of human brain were studied for their effect on the Na+, K+-ATPase activity of native erythrocytes and their membranes. It is shown that gangliosides depending on time of their preincubation with the enzyme preparation and concentration produce both the activating and inhibiting action and cerebrosides--only the inhibiting one. Gangliosides inhibit the transport ATPase activity noncompetitively with respect to ATP and Na+ and competitively--to K+, cerebrosides inhibit it noncompetitively with respect to all ATPase activators.     

Palytoxin Acts on Na+,K+-ATPase but not Nongastric H+,K+-ATPase

Palytoxin (PTX) opens a pathway for ions to pass through Na,K-ATPase. We investigate here whether PTX also acts on nongastric H,K-ATPases. The following combinations of cRNA were expressed in Xenopus laevis oocytes: Bufo marinus bladder H,K-ATPase α₂- and Na,K-ATPase β₂-subunits; Bufo Na,K-ATPase α₁- and Na,K-ATPase β₂-subunits; and Bufo Na,K-ATPase β₂-subunit alone. The response to PTX was measured after blocking endogenous Xenopus Na,K-ATPase with 10 μm ouabain. Functional expression was confirmed by measuring ⁸⁶Rb uptake. PTX (5 nm) produced a large increase of membrane conductance in oocytes expressing Bufo Na,K-ATPase, but no significant increase occurred in oocytes expressing Bufo H,K-ATPase or in those injected with Bufo β₂-subunit alone. Expression of the following combinations of cDNA was investigated in HeLa cells: rat colonic H,K-ATPase α₁-subunit and Na,K-ATPase β₁-subunit; rat Na,K-ATPase α₂-subunit and Na,K-ATPase β₂-subunit; and rat Na,K-ATPase β₁- or Na,K-ATPase β₂-subunit alone. Measurement of increases in ⁸⁶Rb uptake confirmed that both rat Na,K and H,K pumps were functional in HeLa cells expressing rat colonic HKα₁/NKβ₁ and NKα₂/NKβ₂. Whole-cell patch-clamp measurements in HeLa cells expressing rat colonic HKα₁/NKβ₁ exposed to 100 nm PTX showed no significant increase of membrane current, and there was no membrane conductance increase in HeLa cells transfected with rat NKβ₁- or rat NKβ₂-subunit alone. However, in HeLa cells expressing rat NKα₂/NKβ₂, outward current was observed after pump activation by 20 mm K⁺ and a large membrane conductance increase occurred after 100 nm PTX. We conclude that nongastric H,K-ATPases are not sensitive to PTX when expressed in these cells, whereas PTX does act on Na,K-ATPase.     

Effect of calmodulin on myometrial cell membrane Ca2+,Mg2+-ATPase activity

Myometrium cell plasma membrane Ca2+, Mg(2+)-ATPase purified by an affinity chromatography on calmodulin-sepharose 4B is calmodulin-dependent enzyme. Concentration of calmodulin required for half-maximal activation of enzyme was about 26 nM. By unlike to the enzymes originated from other tissues sensitivity to the calmodulin of the myometrial sarcolemma Ca(2+)-transporting ATPase was lower: calmodulin increased Vmax of ATPase about 1.25-fold, the apparent constant of the activation of enzyme by Ca2+ failed to alter independently on the phospholipid presenting at the enzyme isolation.