No evidence for a role in signal-transduction of Na+/K+-ATPase interaction with putative endogenous ouabain.

A cascade of events (signal-transduction), mainly seen in rat cardiac myocytes and renal cells, is thought to occur after ouabain interaction with a minor fraction of Na+/K+-ATPase. A higher intracellular Na+ concentration followed sodium pump inhibition by ouabain with a subsequent gradual increase or oscillations in intracellular Ca2+ concentration. Whether this increase in intracellular Ca2+ concentration is part of the cascade, a result of the cascade or a totally independent phenomenon are conflicting interpretations that are discussed. At best, however, the cascade is initiated by ouabain concentrations several orders of magnitude higher than the measured plasma concentrations of putative endogenous ouabain. The experimentally high ouabain concentration may be critical for another reason. Most tissues contain various isoforms of the catalytic alpha-peptide of Na+/K+-ATPase with an individual sublocalization and, in rats, with different ouabain-sensitivity. The almost ouabain-insensitive alpha1-isoform of Na+/K+-ATPase is essentially unaffected by the high ouabain concentration, whereas ouabain-sensitive alpha-isoforms, possibly confined to membrane structures near cytosolic microdomains and Na+/Ca2+ exchangers, may be totally blocked. Classifying endogenous ouabain as a physiological inducer of the signaling system on this background seems hazardous.     

Effects of nucleotides and Na + on ouabain activation of the phosphatase associated with Na + , K + -ATPase

Ouabain activation of the phosphatase associated with Na⁺,K⁺-ATPase is a time-dependent process which is stimulated by ATP and other nucleotides. Further stimulation by Na⁺ is observed under certain conditions. The stimulatory effect of ATP was found to be due to an increase in the affinity of the enzyme for ouabain. The time required for maximal ouabain activation to be achieved was decreased by ATP and further decreased by ATP + Na⁺.These conditions for maximal activation by ouabain are similar to those required for maximal ouabain binding and suggest that the same ouabain site is responsible for activation of Mg²⁺-dependent phosphatase and for inhibition of Na⁺,K⁺-ATPase and K⁺-phosphatase.     

Kinetics studies on the interaction between ouabain and (Na+,K+)-ATPase.

The association and dissociation rate constants for the interaction of [3H]-ouabain with partially purified rat brain (Na+,K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in vitro were estimated from the time course of the [3H]-ouabain binding observed in the presence of Na+, Mg2+ and ATP by a polynomial approximation-curve-fitting technique. The reduction of the association rate constant by K+ was greater than its reduction of the dissociation rate constant. Thus, the affinity of Na+,K+)-ATPase for ouabain was reduced by K+. The binding-site concentration was unaffected by K+. Consistent with these findings, the addition of KCl to an incubation mixture at the time when [3H]-ouabain binding to (Na+,K+)ATPase is close to equilibrium, caused an immediate decrease in bound ouabain concentration, apparently shifting towards a new, lower equilibrium concentration. Dissociation rate constants which were estimated following the termination of the ouabain-binding reaction were different from those estimated with above methods and may not be useful in predicting the ligand effects on equilibrium of the ouabain-enzyme interaction.     

Age-dependent effect of ouabain on renal Na+,K+-ATPase

AimsThis study examines the effect of chronic ouabain-treatment on renal Na⁺ handling in 12-week and 52-week old rats.Main methodsWistar Kyoto rats aged 5 weeks or 45 weeks were treated with ouabain or vehicle during 7 weeks. Blood pressure was measured in conscious animals throughout the study. After 7 weeks of treatment urinary electrolyte concentration, Na⁺,K⁺-ATPase activity and α₁-subunit expression were determined in 12-week and 52-week old rats.Key findingsIn 12-week and 52-week old rats ouabain produced a significant increase in systolic blood pressure. Although no differences were observed in Na⁺ excretion in these animals, 12-week old ouabain-treated rats had lower Na⁺,K⁺-ATPase activity in proximal tubules. However, 12-week old ouabain-treated rats had decreased fractional excretion of Na⁺. In proximal tubules of 52-week old rats Na⁺,K⁺-ATPase activity did not differ between vehicle and ouabain-treated groups.SignificanceOur results show that in Wistar Kyoto rats renal response to ouabain treatment may be age-dependent and that the hypertensive effect of ouabain is independent of the effect on renal Na⁺,K⁺-ATPase.     

Some total and partial reactions of Na+/K+-ATPase using ATP and acetyl phosphate as a substrate

Acetyl phosphate, as a substrate of (Na+ + K+)-ATPase, was further characterized by comparing its effects with those of ATP on some total and partial reactions carried out by the enzyme. In the absence of Mg2+ acetyl phosphate could not induce disocclusion (release) of Rb+ from E2(Rb); nor did it affect the acceleration of Rb+ release by non-limiting concentrations of ADP. In K+-free solutions and at pH 7.4 sodium ions were essential for ATP hydrolysis by (Na+ + K+)-ATPase; when acetyl phosphate was the substrate a hydrolysis (inhibited by ouabain) was observed in the presence and absence of Na+. In liposomes with (Na+ + K+)-ATPase incorporated and exposed to extravesicular (intracellular) Na+, acetyl phosphate could sustain a ouabain-sensitive Rb+ efflux; the levels of that flux were similar to those obtained with micromolar concentrations of ATP. When the liposomes were incubated in the absence of extravesicular Na+ a ouabain-sensitive Rb+ efflux could not be detected with either substrate. Native (Na+ + K+)-ATPase was phosphorylated at 0 degrees C in the presence of NaCl (50 mM for ATP and 10 mM for acetyl phosphate); after phosphorylation had been stopped by simultaneous addition of excess trans-1,2-diaminocyclohexane-N,N,N',N' tetraacetic acid and 1 M NaCl net synthesis of ATP by addition of ADP was obtained with both phosphoenzymes. The present results show that acetyl phosphate can fuel the overall cycle of cation translocation by (Na+ + K+)-ATPase acting only at the catalytic substrate site; this takes place via the formation of phosphorylated intermediates which can lead to ATP synthesis in a way which is indistinguishable from that obtained with ATP.     

Temperature-activity relationships of cation activation and ouabain inhibition of (Na+ + K+)-ATPase.

The nonlinear temperature-activity relationship of membrane preparations of (Na⁺ + K⁺)-ATPase gives rise to discontinuities in Arrhenius plots of this enzyme. The different apparent energies of activation of (Na⁺ + K⁺) — ATPase which are observed above and below the critical temperature of the system have been considered to result from different conformational forms of the enzyme protein. Because both activation of (Na⁺ + K⁺)-ATPase by cations, and its specific inhibition by cardiac glycosides may be influenced by the conformational form of the enzyme protein, we have reexamined the effect of temperature upon the activation energy of the system under the different experimental conditions of cation activation and ouabain inhibition.Our results indicate that the activation of (Na⁺ + K⁺)-ATPase by cations, is less influenced by change in temperature than is inhibition of the enzyme by ouabain. In addition, mild lipolysis by phospholipase-A had a marked effect upon the ouabain-dependent response of the enzyme to temperature, but not upon the cation-dependent response. The effect of phospholipase-A can be overcome by reincubation of the treated preparation with phosphatidyl serine.We conclude that the ouabain-dependent temperature effects of (Na⁺ + K⁺)-ATPase are more dependent upon the integrity and nature of the membrane lipids than are the cation-dependent responses. It is possible that phosphatidyl serine plays a unique role in this regard.     

Red cell ouabain binding sites, Na+K+-ATPase, and intracellular Na+ as individual characteristics

Healthy male volunteers were infused for three hours with either a dopamine hydrochloride solution at a rate of 4 ug/kg/min or with normal saline. Plasma amine oxidase and platelet MAO activity towards benzylamine both increased in response to intravenous dopamine. There was no increase in enzyme activity when dopamine was added to the platelet and plasma enzymes in vitro. This heretofore unreported increase in the oxidative deaminating capacity of the human organism may represent an adaptive physiologic response to the high circulating levels of dopamine and provides further evidence for a possible functional significance of these enzymes in man.     

Effect of ouabain on the Na+, K+-ATPase function and multiplication of transformed cells in culture

A study was made of the ouabain effect (10(-3] on cell proliferation and the dependence of ATP hydrolysis on Na/K-concentration in homogenates of mouse hepatoma (XXIIa) and of L-cells, both sensitive and resistant to etidium bromide. Na+, K+-ATPase activity was found in homogenates of cells from sparse cultures in the presence of ouabain, the activity being stimulated by the Na/K-ratio pecular for the maximum enzymatic activity in cells from the dense cultures. The effect of ouabain on the cell proliferation is similar to the effect of transition of sparse cultures to dense ones.     

Solvent effects on substrate and phosphate interactions with the (Na+ + K+)-ATPase

(Na+ + K+)-ATPase activity of a dog kidney enzyme preparation was markedly inhibited by 10-30% (v/v) dimethyl sulfoxide (Me2SO) and ethylene glycol (Et(OH)2); moreover, Me2SO produced a pattern of uncompetitive inhibition toward ATP. However, K+-nitrophenylphosphatase activity was stimulated by 10-20% Me2SO and Et(OH)2 but was inhibited by 30-50%. Me2SO decreased the Km for this substrate but had little effect on the Vmax below 30% (at which concentration Vmax was then reduced). Me2SO also reduced the Ki for Pi and acetyl phosphate as competitors toward nitrophenyl phosphate but increased the Ki for ATP, CTP and 2-O-methylfluorescein phosphate as competitors. Me2SO inhibited K+-acetylphosphatase activity, although it also reduced the Km for that substrate. Finally, Me2SO increased the rate of enzyme inactivation by fluoride and beryllium. These observations are interpreted in terms of the E1P to E2P transition of the reaction sequence being associated with an increased hydrophobicity of the active site, and of Me2SO mimicking such effects by decreasing water activity: (i) primarily to stabilize the covalent E2P intermediate, through differential solvation of reactants and products, and thereby inhibiting the (Na+ + K+)-ATPase reaction and acting as a dead-end inhibitor to produce the pattern of uncompetitive inhibition; inhibiting the K+-acetylphosphatase reaction that also passes through an E2P intermediate; but not inhibiting (at lower Me2SO concentrations) the K+-nitrophenylphosphatase reaction that does not pass through such an intermediate; and (ii) secondarily to favor partitioning of Pi and non-nucleotide phosphates into the hydrophobic active site, thereby decreasing the Km for nitrophenyl phosphate and acetyl phosphate, the Ki for Pi and acetyl phosphate in the K+-nitrophenylphosphatase reaction, accelerating inactivation by fluoride and beryllium acting as phosphate analogs, and, at higher concentrations, inhibiting the K+-nitrophenylphosphatase reaction by stabilizing the non-covalent E2.P intermediate of that reaction. In addition, Me2SO may decrease binding at the adenine pocket of the low-affinity substrate site, represented as an increased Ki for ATP, CTP and 3-O-methylfluorescein phosphate.     

Na+, K+-ATPase in HeLa cells after prolonged growth in low K+ or ouabain

Effects of long-term, subtotal inhibition of Na⁺-K⁺ transport, either by growth of cells in sublethal concentrations of ouabain or in low-K⁺ medium, are described for HeLa cells. After prolonged growth in 2 × 10^?8 M ouabain, the total number of ouabain molecules bound per cell increases by as much as a factor of three, mostly due to internalization of the drug. There is only about a 20% increase in ouabain-binding sites on the plasma membrane, representing amodest induction of Na⁺, K⁺-ATPase. In contrast, after long-term growth in low K⁺ there can be a twofold or greater increase in ouabain binding per cell, and in this case the additional sites are located in the plasma membrane. The increase is reversible. To assess the corresponding transport changes, we have separately estimated the contributions of increased intracellular [Na⁺] and of transport capacity (number of transport sites) to transport regulation. During both induction and reversal, short-term regulation is achieved primarily by changes in [Na⁺]_i. More slowly, long-term regulation is achieved by changes in the number of functional transporters in the plasma membrane as assessed by ouabain binding, V_max for transport, and specific phosphorylation. Parallel exposure of cryptic Na⁺, K⁺-ATPase activity with sodium dodecyl sulfate in the plasma membranes of both induced and control cells showed that the induction cannot be accounted for by an exposure of preexisting Na⁺, K⁺-ATPase in the plasma membrane. Analysis of the kinetics of reversal indicates that it may be due to a post-translational event.     

Stability of [3H]ouabain binding to the (Na+ + K+)-ATPase solubilized with C12E8

The (Na+ + K+)-ATPase from dog kidney and partially purified membranes from HK dog erythrocytes were labeled with [3H]ouabain, solubilized with C12E8 and analyzed by HPLC through a TSK-GEL G3000SW column in the presence of C12E8, Mg2+, HPO4(2-) and glycerol at 20-23 degrees C. The peaks of [3H]ouabain bound to the enzyme from dog kidney and HK dog erythrocyte membranes corresponded to each other with apparent molecular weights of 470 000-490 000. In addition, these bindings of [3H]ouabain to the (Na+ + K+)-ATPase were observed to be stable at 20-23 degrees C for at least 18 h after the solubilization.     

Study of ADP effect on brain Na+, K+-ATPase]

A mechanism of K-insensitive, ouabain-dependent liberation of Na+ from the cell during an increase in ADP intracellular concentration is studied. It is shown that the increase in the ADP/ATP ratio does not change the Na+, K+-ATPase affinity to K+ ions and does not result in the Na-activated, K-independent ATPase reaction. ADP protects ATPase from the inhibition by ouabain which is accounted for by a decrease in the concentration of a glycoside-sensitive form of the enzyme E2-P due to a turnover of the phosphokinase step of the reaction, but not due to the binding of free Mg2+ ions. The results obtained suggest that the increase in ADP concentration within the cell activates Na-Nan exchange along Na-transporting channels of the ionic pump.     

Inhibition of (Na+ + K+)-ATPase by ouabain: involvement of calcium and membrane proteins.

Treatment of plasma membrane isolated from murine plasmocytoma MOPC 173 with an EDTA-containing buffer resulted in a 300-fold increase in sensitivity of (Na+ + K+)-stimulated Mg2+-ATPase to ouabain. This phenomenon was associated with the solubilization by EDTA of phospholipid free proteins (approx. 30 000-34 000 daltons) from the cytoplasmic face of the plasma membrane and with removal of about 90% of the membrane bound Ca2+. The recovery of the original resistance to ouabain required specifically Ca2+ and was associated with a binding of the solubilized proteins to the membrane.     

Correlation between microsomal (Na+ + K+)-ATPase activity and [3H]ouabain binding to heart tissue homogenates.

ATP plus Mg2+ plus Na+ supported [3H]ouabain binding to canine left ventricular tissue homogenates and microsomal (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity from the same tissue were measured. A linear relationship was found between the initial velocity of [3H]ouabain binding to tissue homogenates and microsomal (Na+ + K+)-ATPase activity from the same tissue in the presence and absence of in vivo bound digoxin. In vivo bound digoxin reduced both measurements. With tissue from digoxin-free hearts, a linear relationship was also obtained between the initial velocity and the maximum level of [3H]ouabain binding to tissue homogenate. Binding of [3H]ouabain to whole tissue homogenate is a convenient method for estimating (Na+ + K+)-ATPase activity in small left ventricular biopsy samples.     

Estimation of ouabain specifically bound to dog renal (Na+ + K+)-ATPase in vivo.

Suspensions of viable renal cortical cells hydrolyzed a synthetic ester substrate (alpha-N-tosyl-L-arginine methyl ester, Tos-Arg-OMe) and generated kinins from a kininogen substrate. This kallikrein-like esterase activity increased linearly with cell number, or time of exposure to substrate. No radiolabelled substrate or product was found within the cells. Most of the activity appeared to be on cell surfaces as supernatant media had less than 20% of the Tos-Arg-OMe esterase activity on the cell suspensions. Cell surface Tos-Arg-OMe esterase activity was inhibited by aprotinin, benzamidine, pentamidine, and a tris-amidine derivative (alpha,alpha',alpha'-tris(3-amidinophenoxy)mesitylene). Preincubation of cells with phospholipase A2 increased renal cell surface esterase activity up to 76% while only slightly increasing supernatant activity. In contrast, preincubation with deoxycholate caused clearing of suspensions and a marked increase in supernatant esterase activity. Renal cell kininogenase (EC 3.4.21.8) activity was inhibited by preincubation with aprotinin, the tris-amidine derivative, or anti-rat urinary kallikrein antibody. Kallikrein elaborated by renal cells formed a single precipitin line with an antibody to rat urinary kallikrein but the two enzymes were not immunologically identical. We conclude that kallikrein's active sites are facing the external environment of renal cortical cells in suspension with access to substrates, inhibitors, and antibody.