Dissociation of Na+,K+-ATPase inhibition from digitalis inotropy.

Recent findings from our laboratory as well as those of other laboratories do not support the postulation that the mechanism of the positive inotropic action of digitalis is due to inhibition of NA,K-ATPase. Using short-acting digitalis steroids and drug washout experiments, in isolated myocardial preparations, it has been demonstrated that Na,K-ATPase isolated from such preparations is still significantly inhibited, whereas the positive inotropic effect is no longer present. Also, based on kinetic measurements the two exponential rate constants observed for drug half-life, a rapid and slow phase, were found to be associated, respectively, with the very short inotropic half-life and the very long enzyme inhibition half-life. In addition, a dissociation of the transient inotropic effects of digitalis was observed from the long lasting cardiotoxic effects of digitalis during drug washout. Moreover, a temporal correlation was noted between the persistent inhibitory effects of digitalis on Na,K-ATPase and the persistent cardiotoxic effects of digitalis. Therefore, it is concluded that inhibition of Na,K-ATPase is not responsible for the positive inotropic action of digitalis, but may be the mechanism, at least in part, for certain cardiotoxic effects of digitalis.     

Involvement of Na+, K+-ATPase inhibition in K562 cell differentiation induced by bufalin

Human leukemia K562 cell differentiation induction by naturally occurring bufadienolides purified from the Chinese drug Senso and synthetic bufalin derivatives was examined by a nitro blue tetrazolium reduction assay. Bufalin showed the strongest activity among all the bufadienolides tested in this study. The degree of the induction of nitro blue diformazan positive cells by the bufadienolides correlated well with their inhibitory activities against Na⁺, K⁺ -ATPase prepared from K562 cells in vitro. N⁺, K⁺ -ATPases from a variant K562 clone (ouabain resistant, OuaR) and murine leukemia cell line M1-T22, which were insensitive to the bufadienolides in terms of growth inhibition and cell differentiation, appeared to be refractory to bufalin in vitro. A binding study of ³H-bufalin and ³H-ouabain revealed that saturated levels of both ligands associated with K562 cells were virtually similar; however, affinity of ³H-bufalin was considerably higher than ³H-ouabain. The saturated level of ³H-bufalin observed in the OuaR cells was approximately half of that observed in K562 cells without a change in its affinity. Association of ³H-bufalin with K562 cells was completely blocked by pretreatment of the cells with cold ouabain at concentrations saturating the binding sites. These results suggest that bufalin acts on the cells by binding to sites on the cell membrane which also bind ouabain. It is thus proposed that N⁺, K⁺ -ATPase inhibition is closely related to the initiation process in the induction of K562 cell differentiation induced by bufalin. © 1994 Wiley-Liss, Inc.     

Modulation of cardiac glycoside inhibition of (Na+ + K+)-ATPase by membrane lipids. Difference between species

The role of lipids in the modulation of the ouabain-sensitivity of membrane (Na+ + K+)-ATPase from different species has been studied using a reconstitution procedure which promotes lipid exchange during detergent depletion by Sephadex chromatography. Hybrid reconstitution of delipidated (Na+ + K+)-ATPase preparations from bovine brain into the lipids obtained from crab nerve enzyme preparations significantly reduces the sensitivity of the brain enzyme to inhibition by ouabain. Conversely, reconstitution of crab nerve enzyme into the lipids from bovine brain enzyme preparations increases the sensitivity of the crab enzyme to ouabain inhibition. These opposing effects demonstrate the role of membrane lipids in modulating the enzyme-inhibition relationship in preparations from these different species.     

Localization of Na+, K+-ATPase in brain.

Enzyme activity, representing the sites of K+-stimulated p-nitrophenylphosphatase, a component of the sodium, potassium-stimulated-adenosinetriphosphatase system, has been localized in the somatosensory cortex of the rat brain. The reaction product is most obviously associated with fibers that are thought to be axons and dendrites. Large dendrite-like fibers appear to arise in layer 5 of the cortex and arborize in layers 1 through 4. Smaller, reactive fibers are found throughout the cortical layers. Neuron cell bodies did not exhibit substantial enzymatic activity. It did not appear that glia contributed significantly to the activity in cerebral cortex.     

Adenovirus-dependent changes in cell membrane permeability: role of Na+, K+-ATPase.

Adenovirus-dependent release of choline phosphate from KB cells at pH 6.0 was partially blocked by ouabain. In K+-containing medium, maximum inhibition of release was obtained by 10(-5) M ouabain and half-maximal inhibition was achieved by about 0.5 X 10(-6)M ouabain. Ouabain did not block either the binding or the uptake of adenovirus by KB cells. Without K+, about 25% of cell-associated choline phosphate was released by adenovirus, whereas with 1 mM K+ about 50% was released. This activation by K+ was blocked by 0.1 mM ouabain. HeLa cells behaved like KB cells, but a mutant of HeLa cells resistant to ouabain (D98-OR) released much lower amounts of choline phosphate in response to human adenovirus type 2 (Ad2). Wild-type D98-OR cells bound nearly the same amount of adenovirus as did normal HeLa cells. Ad2 also increased the activity of Na+,K+-ATPase in KB cells, with maximum activation at 50 micrograms of Ad2 per ml. In D98-OR cells, Ad2 failed to activate Na+,K+-ATPase activity. Ad2-dependent lysis of endocytic vesicles (receptosomes) was assayed by measuring Ad2-dependent enhancement of epidermal growth factor-Pseudomonas exotoxin toxicity. This action of adenovirus was increased when K+ was present in the medium. Under the conditions used, K+ had no effect on the amount of Ad2 or epidermal growth factor taken up by the cells. On the basis of these results, it is suggested that Ad2-dependent cellular efflux of choline phosphate and adenovirus-dependent lysis of receptosomes may require Na+,K+-ATPase activity.     

Uncoupling of ATP binding to Na+,K+-ATPase from its stimulation of ouabain binding: studies of the inhibition of Na+,K+-ATPase by a monoclonal antibody

The effects of a monoclonal antibody, prepared against the purified lamb kidney Na+,K+-ATPase, on the enzyme's Na+,K+-dependent ATPase activity were analyzed. This antibody, designated M10-P5-C11, is directed against the catalytic subunit of the "native" holoenzyme. It inhibits greater than 90% of the ATPase activity and acts as a noncompetitive or mixed inhibitor with respect to the ATP, Na+, and K+ dependence of enzyme activity. It inhibits the Na+- and Mg2+ATP-dependent phosphoenzyme intermediate formation. In contrast, it has no effect on K+-dependent p-nitrophenylphosphatase (pNPPase) activity, the interconversion of the phosphoenzyme intermediates, and ADP-sensitive or K+-dependent dephosphorylation. It does not alter ATP binding to the enzyme nor the covalent labeling of the enzyme at the presumed ATP site by fluorescein 5'-isothiocyanate (FITC), but it prevents the ATP-induced stimulation in the rate of cardiac glycoside [3H]ouabain binding to the Na+,K+-ATPase. M10-P5-C11 binding appears to inhibit enzyme function by blocking the transfer of the gamma-phosphoryl of ATP to the phosphorylation site after ATP binding to the enzyme has occurred. In the presence of Mg2+ATP, it also prevents the ATP-induced transmembrane conformational change that enhances cardiac glycoside binding. This uncoupling of ATP binding from its stimulation of ouabain binding and enzyme phosphorylation demonstrates the existence of an enzyme-Mg2+ATP transitional intermediate preceding the formation of the Na+-dependent ADP-sensitive phosphoenzyme intermediate. These results are also consistent with a model of the Na+,K+-ATPase active site being composed of two distinct but interacting regions, the ATP binding site and the phosphorylation site.     

Na+,K+-ATPase: 40 years of investigations

Nobel Prize of 1997 in chemistry was awarded to three scientists fruitfully working in bioenergetics. J. Walker and P. Boyer were awarded the Prize for studies of structure and mechanism of functioning of the H+-transporting (mitochondrial) adenosine triphosphatase. The decision of the Nobel Committee was not unexpected, since these works were very impressive. Special attention was drawn to the fact that the investigations of Walker, the recognized specialist in protein structure, made possible the experimental confirmation of regularities in the mitochondrial ATPase functioning discovered by P. Boyer. The third member of this triumph of bioenergetics is Jens-Christian Skou who described the Na+,K+-activated ATPase in 1957 and then characterized the enzyme properties in detail. Forty years of his scientific biography were devoted to this enzyme. Along with accumulation of scientific knowledge, that constituted the fundamental contribution to bioenergetics (J.Skou is rightfully considered as one of founders of this branch in the present-day biology), the world-wide known school of scientists was established, and starting from 1974, members of this school organize regular conferences on this enzyme.     

Inhibition of cardiac Na+, K+-ATPase activity by dynorphin-A and ethylketocyclazocine

The effect of various opioids on Na+, K+ -ATPase partially purified from rat heart was examined. Dynorphin-A (1-13), dynorphin-A (1-17) and ethylketocyclazocine (EKC), which are k-type opiate agonists, markedly inhibited the enzyme activity in a dose-dependent manner; IC50 values were 12 microM, 21 microM and 0.38 mM, respectively. Morphine (mu-type agonist), methionine- and leucine-enkephalin (delta-type agonist) at the concentration of 1 mM did not affect the enzyme activity. The effect of dynorphin-A (1-13) and EKC was not antagonized by naloxone. Dynorphin-A (1-13) mainly decreased Vmax value without the change of Km value in the activation of Na+, K+-ATPase by ATP, Na+ and K+. Dynorphin-A(1-13) inhibited the partial reactions of Na+, K+-ATPase at the different degree of the potency; the inhibition of K+-stimulated phosphatase was greater than that of Na+-dependent phosphorylation. The present study suggests that dynorphin-A and EKC have an effect on cardiovascular system which is mediated by the inhibition of Na+, K+-ATPase in the heart.     

Demonstration of an endogenous activator for the Na+, K+-ATPase system

A heat-labile, non-dialysable and protease-sensitive endogenous activator (NaAF) capable of stimulating the Na⁺, K⁺-ATPase system has been demonstrated. The activator (NaAF) activity was partially enriched (about 10 fold) by dialysis (30 kDa cutoff) under negative pressure and pH 4.8 precipitation. The NaAF has been found to occur in the cytosolic fractions of tissues such as the kidney and brain from two different species (rabbit and pig) tested so far. Also, the factor from one tissue stimulates with equal efficacy the Na⁺, K⁺-ATPase systems of other tissues regardless of the species; thus demonstrating universal nature of the activator. Some degree of cross-reactivity was noted between the activating effects of this activator (for the Na⁺,K⁺-ATPase) and that for the H⁺,K⁺-ATPase recently described (J. Biol. Chem. 262:5664–5670, 1987). The purified NaAF obtained from sephacryl S-300 column chromatography activates the pure renal medullary Na⁺,K⁺-ATPase in a dose dependent manner.A preliminary account of this work was published in Fed. Proc. 46(4): 4466, 1987     

Na+,K+-ATPase: half-of-the-subunits cross-linking reactivity suggests an oligomeric structure containing a minimum of four catalytic subunits

When Na⁺,K⁺-ATPase was reacted with Cu²⁺ and o-phenanthroline under conditions where the formation of a cross-linked dimer of the catalytic subunit (α,α-dimer) is dependent on the prior phosphorylation of the enzyme by ATP, it was found that (a) only half of the α-subunit content is phosphorylated, and only half is cross-linked; and (b) a phosphorylated α-subunit is cross-linked to an unphosphorylated α-subunit. It is suggested that the functional unit of the membrane-bound enzyme contains at least four α-subunits, and that ligand-induced half-of-the-sites reactivity may be exerted across two different intersubunit domains of the tetramer.     

Vanadate inhibition of rat cerebral cortex Na+, K+-ATPase during postnatal development

The Na⁺, K⁺-ATPase activity and its response to vanadate inhibition was investigated in cerebral cortex homogenates of 7-, 12- and 18-day-old rats. The enzyme was inhibited by vanadate in a dose-dependent manner in all these age groups. Furthermore, there was a different sensitivity towards vanadate during postnatal development; the concentration of V⁺⁵ needed for 50% inhibiton of Na⁺, K⁺-ATPase was 1.1×10^–6M, 2×10^–7M and 4.4×10^–7M for 7-, 12- and 18-day-old rats, respectively. It is suggested that the different sensitivity of Na⁺, K⁺-ATPase towards vanadate inhibition during postnatal development might be due to age-dependent changes in the ratio of various cell types.Special Issue dedicated to Dr. O. H. Lowry.     

Microscopical methods for the localization of Na+,K+-ATPase

Summary Na⁺, K⁺-ATPase plays a central role in the ionic and osmotic homeostasis of cells and in the movements of electrolytes and water across epithelial boundaries. Microscopic localization of the enzyme is, therefore, of crucial importance in establishing the subcellular routes of electrolyte flow across structurally complex and functionally polarized epithelia. Recently developed approaches to the localization of Na⁺, K⁺-ATPase are reviewed. These methods rely on different properties of the enzyme and encompass cytochemical localization of the K⁺-dependent nitrophenylphosphatase component of the enzyme, autoradiographic localization of tritiated ouabain binding sites, and immunocytochemical localization of the holoenzyme and of its catalytic subunit. The rationales for each of these techniques are outlined as are the critieria that have been established to validate each method. The observed localization of Na⁺, K⁺-ATPase in various tissues is discussed, particularly as it relates to putative and hypothetical mechanisms that are currently thought to mediate reabsorptive and secretory electrolyte transport.     

Arrestins and spinophilin competitively regulate Na+,K+-ATPase trafficking through association with a large cytoplasmic loop of the Na+,K+-ATPase

The activity and trafficking of the Na(+),K(+)-ATPase are regulated by several hormones, including dopamine, vasopressin, and adrenergic hormones through the action of G-protein-coupled receptors (GPCRs). Arrestins, GPCR kinases (GRKs), 14-3-3 proteins, and spinophilin interact with GPCRs and modulate the duration and magnitude of receptor signaling. We have found that arrestin 2 and 3, GRK 2 and 3, 14-3-3 epsilon, and spinophilin directly associate with the Na(+),K(+)-ATPase and that the associations with arrestins, GRKs, or 14-3-3 epsilon are blocked in the presence of spinophilin. In COS cells that overexpressed arrestin, the Na(+),K(+)-ATPase was redistributed to intracellular compartments. This effect was not seen in mock-transfected cells or in cells expressing spinophilin. Furthermore, expression of spinophilin appeared to slow, whereas overexpression of beta-arrestins accelerated internalization of the Na(+),K(+)-ATPase endocytosis. We also find that GRKs phosphorylate the Na(+),K(+)-ATPase in vitro on its large cytoplasmic loop. Taken together, it appears that association with arrestins, GRKs, 14-3-3 epsilon, and spinophilin may be important modulators of Na(+),K(+)-ATPase trafficking.     

Na+,K+-ATPase enzyme units in skeletal muscle from lean and obese mice.

[³H]-Ouabain binding to muscle preparations was utilized to estimate the number of Na⁺,K⁺-ATPase enzyme units in hindlimbs from 8 week old lean and obese mice. Specific [³H]-ouabain binding per mg particulate protein was 36% lower in obese mice; whereas, the affinity of the binding sites for ouabain was similar in obese and lean mice. Since obese mice had less muscle than lean mice, the number of Na⁺,K⁺-ATPase enzyme units in hindlimbs from obese mice was less than half the number observed in lean mice.     

(Na+,K+)-ATPase: a new assay of Na+-ATPase reveals covert anti-pump antibodies

A new assay is described for rat (Na⁺,K⁺)-ATPase [EC 3.6.1.3] prepared from renal medullary or crude liver membranes. With ATP at 1 μm, initial rates of ouabain-sensitive decreases in substrate concentrations are followed by measuring diminished ATP-driven luciferin-luciferase light production. Under these conditions, using highly purified enzyme preparations, Na⁺ and K⁺ ions stimulate and inhibit initial ATP hydrolysis rates, respectively. Therefore, it is likely that the assay measures Na⁺-ATPase partial reactions of the pump. A monospecific polyclonal rabbit anti-rat pump antiserum blocks Na⁺-dependent ATPase measured with the luciferase-linked ATPase assay, whereas conventional assays of purified pump activity at 3.0 mm ATP fail to reveal immunochemical blockade.     

Selective inhibition by ionophore A23187 of the enzyme isomerization in the catalytic cycle of Na+, K+ -ATPase

The effect of an ionophore A23187 on the purified Na+,K+-ATPase from the outer medulla of pig kidney was investigated. When the enzyme was pretreated with A23187 in the presence of Na+ and K+, the ATPase activity was inhibited almost completely. When the pretreatment was performed in the presence of Na+ and absence of K+, formation of the phosphoenzyme (EP) from ATP was only slightly retarded. The steady state level of EP thus formed was not altered, but EP decomposition was strongly inhibited. Under these conditions, the accumulated EP was sensitive to ADP and insensitive to K+. On the other hand, when the pretreatment was performed in the absence of Na+ and presence of K+, EP formation following simultaneous addition of Na+ and ATP was extremely slow, but the steady state level of EP was not substantially altered. When the pretreatment was performed in the absence of Na+ and presence of K+, EP formation from Pi was unaffected, and the EP formed was in rapid equilibrium with Pi of the medium. These results demonstrate that A23187 selectively inhibits isomerization of the enzyme between the high Na+ and low K+ affinity form and the low Na+ and high K+ affinity form in the catalytic cycle, whether or not the enzyme is phosphorylated. This inhibition is quite similar to the A23187-induced inhibition of the enzyme isomerization in the catalytic cycle of the Ca2+ -ATPase from sarcoplasmic reticulum (Hara, H., and Kanazawa, T. (1986)J. Biol. Chem.261, 16584-16590). These findings suggest that some common mechanism, which is involved in the enzyme isomerization, between these two transport ATPases is strongly disturbed by A23187.     

Investigations of alkaline phosphatase Ca+2-ATPase and Na+, K+-ATPase during beta-APN-induced initial bone mineralization inhibition

Tibias of 6-day-old white Leghorn chick embryos treated with beta-aminopropionitrile (beta-APN; 0.1 mg/egg/day) for 4 days and injected with 3H-proline or 3H-tetracycline on the 11th day were analyzed for incorporation of 3H-proline and 3H-tetracycline. The incorporation of 3H-proline was comparable in the controls and beta-APN-treated embryos. However, the incorporation of 3H-tetracycline was significantly lower in beta-APN-treated embryos. The bone ash contents were also lower in the latter group. Alkaline phosphatase and Ca+2-ATPase were found to be significantly lower in beta-APN-treated embryonic bones. There was, however, no difference in the activity of Na+, K+-ATPase. The histochemical examination showed the alkaline phosphatase to be present on osteoblasts and matrix vesicle plasma membranes at the periosteal surface. The chick embryonic liver tissue showed no significant differences in the activities of any of the above enzymes. The results suggest that beta-APN-induced inhibition of the bone mineralization may be due to the bone-specific inhibition of alkaline phosphatase and Ca+2-ATPase.     

Identification and function of a cytoplasmic K+ site of the Na+, K+ -ATPase

A cytoplasmic nontransport K(+)/Rb(+) site in the P-domain of the Na(+), K(+)-ATPase has been identified by anomalous difference Fourier map analysis of crystals of the [Rb(2)].E(2).MgF(4)(2-) form of the enzyme. The functional roles of this third K(+)/Rb(+) binding site were studied by site-directed mutagenesis, replacing the side chain of Asp(742) donating oxygen ligand(s) to the site with alanine, glutamate, and lysine. Unlike the wild-type Na(+), K(+)-ATPase, the mutants display a biphasic K(+) concentration dependence of E(2)P dephosphorylation, indicating that the cytoplasmic K(+) site is involved in activation of dephosphorylation. The affinity of the site is lowered significantly (30-200-fold) by the mutations, the lysine mutation being most disruptive. Moreover, the mutations accelerate the E(2) to E(1) conformational transition, again with the lysine substitution resulting in the largest effect. Hence, occupation of the cytoplasmic K(+)/Rb(+) site not only enhances E(2)P dephosphorylation but also stabilizes the E(2) dephosphoenzyme. These characteristics of the previously unrecognized nontransport site make it possible to account for the hitherto poorly understood trans-effects of cytoplasmic K(+) by the consecutive transport model, without implicating a simultaneous exposure of the transport sites toward the cytoplasmic and extracellular sides of the membrane. The cytoplasmic K(+)/Rb(+) site appears to be conserved among Na(+), K(+)-ATPases and P-type ATPases in general, and its mode of operation may be associated with stabilizing the loop structure at the C-terminal end of the P6 helix of the P-domain, thereby affecting the function of highly conserved catalytic residues and promoting helix-helix interactions between the P- and A-domains in the E(2) state.