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.     

Lack of immunological cross reactivity between the transport enzymes (Na+ + K+)-ATPase and (K+ + H+)-ATPase

Goat antisera against (Na⁺ + K⁺)-ATPase and its isolated subunits and against (K⁺ + H⁺)-ATPase have been prepared in order to test for immune cross-reactivity between the two enzymes, whose catalytic subunits show great chemical similarity. None of the (Na⁺ + K⁺)-ATPase antisera cross-reacted with (K⁺ + H⁺)-ATPase or inhibited its enzyme activity. The same was true for the (K⁺ + H⁺)-ATPase antiserum with regard to (Na⁺ + K⁺)-ATPase and its subunits and its enzyme activity. So not withstanding the chemical similarity of their subunits, there is no immunological cross-reactivity between these two plasma membrane ATPases.Number LIII in the series Studies on (Na⁺ + K⁺)-Activated ATPase.     

Interactions of K+ ATP channel blockers with Na+/K+-ATPase

Two K⁺ _ATP channel blockers, 5-hydroxydecanoate (5-HD) and glyburide, are often used to study cross-talk between Na⁺/K⁺-ATPase and these channels. The aim of this work was to characterize the effects of these blockers on purified Na⁺/K⁺-ATPase as an aid to appropriate use of these drugs in studies on this cross-talk. In contrast to known dual effects (activating and inhibitory) of other fatty acids on Na⁺/K⁺-ATPase, 5-HD only inhibited the enzyme at concentrations exceeding those that block mitochondrial K⁺ _ATP channels. 5-HD did not affect the ouabain sensitivity of Na⁺/K⁺-ATPase. Glyburide had both activating and inhibitory effects on Na⁺/K⁺-ATPase at concentrations used to block plasma membrane K⁺ _ATP channels. The findings justify the use of 5-HD as specific mitochondrial channel blocker in studies on the relation of this channel to Na⁺/K⁺-ATPase, but question the use of glyburide as a specific blocker of plasma membrane K⁺ _ATP channels, when the relation of this channel to Na⁺/K⁺-ATPase is being studied.     

The influence of potassium ion (K+) on digoxin-induced inhibition of porcine cerebral cortex Na+ / K+-ATPase

The in vitro influence of potassium ion modulations, in the concentration range 2 mM-500 mM, on digoxin-induced inhibition of porcine cerebral cortex Na+ / K+-ATPase activity was studied. The response of enzymatic activity in the presence of various K+ concentrations to digoxin was biphasic, thereby, indicating the existence of two Na+ / K+-ATPase isoforms, differing in the affinity towards the tested drug. Both isoforms showed higher sensitivity to digoxin in the presence of K+ ions below 20 mM in the medium assay. The IC50 values for high/low isoforms 2.77 x 10(-6) M / 8.56 x 10(-5) M and 7.06 x 10(-7) M / 1.87 x 10(-5) M were obtained in the presence of optimal (20 mM) and 2 mM K+, respectively. However, preincubation in the presence of elevated K+ concentration (50-500 mM) in the medium assay prior to Na+ / K+-ATPase exposure to digoxin did not prevent the inhibition, i.e. IC50 values for both isoforms was the same as in the presence of the optimal K+ concentration. On the contrary, addition of 200 mM K+ into the medium assay after 10 minutes exposure of Na+ / K+-ATPase to digoxin, showed a time-dependent recovery effect on the inhibited enzymatic activity. Kinetic analysis showed that digoxin inhibited Na+ / K+-ATPase by reducing maximum enzymatic velocity (Vmax) and Km, implying an uncompetitive mode of interaction.     

Activation of (Na+ + K+)-ATPase

Enzymes catalyze essential chemical reactions needed for living processes. (Na+ +K+)-ATPase (NKA) is one of the key enzymes that control intracellular ion homeostasis and regulate cardiac function. Little is known about activation of NKA and its biological impact. Here we show that native activity of NKA is markedly elevated when protein-protein interaction occurs at the extracellular DVEDSYGQQWTYEQR (D-R) region in the alpha-subunit of the enzyme. The apparent catalytic turnover of NKA is approximately twice as fast as the controls for both ouabain-resistant and ouabain-sensitive enzymes. Activation of NKA not only markedly protects enzyme function against denaturing, but also directly affects cellular activities by regulating intracellular Ca2+ transients and inducing a positive inotropic effect in isolated rat cardiac myocytes. Immunofluorescent labeling indicates that the D-R region of NKA is not a conventional digitalis-binding site. Our findings uncover a novel activation site of NKA that is capable of promoting the catalytic function of the enzyme and establish a new concept that activating of NKA mediates cardiac contraction.     

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.     

Regulation of Glial Na+/K+-ATPase by Serotonin: Identification of Participating Receptors

The purpose of the present study was the characterization of the receptors participating in the regulatory mechanism of glial Na⁺/K⁺-ATPase by serotonin (5-HT) in rat brain. The activity of the Na⁺ pump was measured in four brain regions after incubation with various concentrations of serotoninergic agonists or antagonists. A concentration-dependent increase in enzyme activity was observed with the 5-HT_1A agonist R (+)-2-dipropylamino-8-hydroxy-1,2,3, 4-tetrahydronaphthalene hydrobromide (8-OH-DPAT) in homogenates or in glial membrane enriched fractions from cerebral cortex and in hippocampus. Spiperone, a 5-HT_1A antagonist, completely inhibited the response to 8-OH-DPAT but had no effect on Na⁺/K⁺-ATPase activity in cerebellum where LSD, a 5-HT₆ agonist, elicited a dose-dependent response similar to that of 5-HT. In brainstem, a lack of reponse to 5-HT and other agonists was confirmed. Altogether, these results show that serotonin modulates glial Na⁺/K⁺-ATPase activity in the brain, apparently not through only one type of 5-HT receptor. It seems that the receptor system involved is different according to the brain region. In cerebral cortex, the response seems to be mediated by 5-HT_1A as well as in hippocampus but not in cerebellum where 5-HT₆ appears as the receptor system involved.     

In search of synaptosomal Na+, K+-ATPase regulators

The arrival of the nerve impulse to the nerve endings leads to a series of events involving the entry of sodium and the exit of potassium. Restoration of ionic equilibria of sodium and potassium through the membrane is carried out by the sodium/potassium pump, that is the enzyme Na⁺,K⁺-ATPase. This is a particle-bound enzyme that concentrates in the nerve ending or synaptosomal membranes. The activity of Na⁺,K⁺-ATPase is essential for the maintenance of numerous reactions, as demonstrated in the isolated synaptosomes. This lends interest to the knowledge of the possible regulatory mechanisms of Na⁺,K⁺-ATPase activity in the synaptic region. The aim of this review is to summarize the results obtained in the author's laboratory, that refer to the effect of neurotransmitters and endogenous substances on Na⁺,K⁺-ATPase activity. Mention is also made of results in the field obtained in other laboratories. Evidence showing that brain Na⁺,K⁺-ATPase activity may be modified by certain neurotransmitters and insulin have been presented. The type of change produced by noradrenaline, dopamine, and serotonin on synaptosomal membrane Na⁺,K⁺-ATPase was found to depend on the presence or absence of a soluble brain fraction. The soluble brain fraction itself was able to stimulate or inhibit the enzyme, an effect that was dependent in turn on the time elapsed between preparation and use of the fraction. The filtration of soluble brain fraction through Sephadex G-50 allowed the separation of two active subfractions: peaks I and II. Peak I increased Na⁺,K⁺- and Mg²⁺-ATPases, and peak II inhibited Na⁺,K⁺-ATPase. Other membrane enzymes such as acetylcholinesterase and 5′-nucleotidase were unchanged by peaks I or II. In normotensive anesthetized rats, water and sodium excretion were not modified by peak I but were increased by peak II, thus resembling ouabain effects.³H-ouabain binding was unchanged by peak I but decreased by peak II in some areas of the CNS assayed by quantitative autoradiography and in synaptosomal membranes assayed by a filtration technique. The effects of peak I and II on Na⁺,K⁺-ATPase were reversed by catecholamines. The extent of Na⁺,K⁺-ATPase inhibition by peak II was dependent on K⁺ concentration, thus suggesting an interference with the K⁺ site of the enzyme. Peak II was able to induce the release of neurotransmitter stored in the synaptic vesicles in a way similar to ouabain. Taking into account that peak II inhibits only Na⁺,K⁺-ATPase, increases diuresis and natriuresis, blocks high affinity³H-ouabain binding, and induces neurotransmitter release, it is suggested that it contains an ouabain-like substance.     

Characterization of Na+K+-ATPase in bovine sperm

Existing as a ubiquitous transmembrane protein, Na⁺K⁺-ATPase affects sperm fertility and capacitation through ion transport and a recently identified signaling function. Functional Na⁺K⁺-ATPase is a dimer of α and β subunits, each with isoforms (four and three, respectively). Since specific isoform pairings and locations may influence or indicate function, the objective of this study was to identify and localize subunits of Na⁺K⁺-ATPase in fresh bull sperm by immunoblotting and immunocytochemistry using antibodies against α1 and 3, and all β isoforms. Relative quantity of Na⁺K⁺-ATPase in head plasma membranes (HPM's) from sperm of different bulls was determined by densitometry of immunoblot bands, and compared to bovine kidney. Sperm and kidney specifically bound all antibodies at kDa equivalent to commercial controls, and to additional lower kDa bands in HPM. Immunofluorescence of intact sperm confirmed that all isoforms were present in the head region of sperm and that α3 was also uniformly distributed post-equatorially. Permeabilization exposing internal membranes typically resulted in an increase in fluorescence, indicating that some antibody binding sites were present on the inner surface of the HPM or the acrosomal membrane. Deglycosylation of β1 reduced the kDa of bands in sperm, rat brain and kidney, with the kDa of the deglycosylated bands differing among tissues. Two-dimensional blots of β1 revealed three distinct spots. Based on the unique quantity, location and structure Na⁺K⁺-ATPase subunits in sperm, we inferred that this protein has unique functions in sperm.     

Inhibition of Na+/K+-ATPase and Mg2+-ATPase by metal ions and prevention and recovery of inhibited activities by chelators

Kinetics and inhibition of Na⁺/K⁺-ATPase and Mg²⁺-ATPase activity from rat synaptic plasma membrane (SPM), by separate and simultaneous exposure to transition (Cu²⁺, Zn²⁺, Fe²⁺ and.Co²⁺) and heavy metals (Hg²⁺and Pb²⁺) ions were studied. All investigated metals produced a larger maximum inhibition of Na⁺/K⁺-ATPase than Mg²⁺-ATPase activity. The free concentrations of the key species (inhibitor, MgATP^{2 ?} , MeATP^{2 ?} ) in the medium assay were calculated and discussed. Simultaneous exposure to the combinations Cu²⁺/Fe²⁺ or Hg²⁺/Pb²⁺caused additive inhibition, while Cu²⁺/Zn²⁺ or Fe²⁺/Zn²⁺ inhibited Na⁺/K⁺-ATPase activity synergistically (i.e., greater than the sum metal-induced inhibition assayed separately). Simultaneous exposure to Cu²⁺/Fe²⁺ or Cu²⁺/Zn²⁺ inhibited Mg²⁺-ATPase activity synergistically, while Hg²⁺/Pb²⁺ or Fe²⁺/Zn²⁺ induced antagonistic inhibition of this enzyme. Kinetic analysis showed that all investigated metals inhibited Na⁺/K⁺-ATPase activity by reducing the maximum velocities (V_max) rather than the apparent affinity (K_m) for substrate MgATP^2-, implying the noncompetitive nature of the inhibition. The incomplete inhibition of Mg²⁺-ATPase activity by Zn²⁺, Fe²⁺ and Co²⁺ as well as kinetic analysis indicated two distinct Mg²⁺-ATPase subtypes activated in the presence of low and high MgATP^{2 ?} concentration. EDTA, L-cysteine and gluthathione (GSH) prevented metal ion-induced inhibition of Na⁺/K⁺-ATPase with various potencies. Furthermore, these ligands also reversed Na⁺/K⁺-ATPase activity inhibited by transition metals in a concentration-dependent manner, but a recovery effect by any ligand on Hg²⁺-induced inhibition was not obtained.     

Isolation and characterization of monoclonal antibodies to (Na + K)-ATPase

Four stable hybridoma cell lines secreting antibodies specific to the membrane (Na⁺ + K⁺)-dependent ATPase isolated from lamb kidney medulla have been produced by fusing mouse myeloma cells with spleen cells from immunized mice. These cell lines produce IgG γ₁ heavy chain and κ light chain antibodies which are directed against the catalytic or α-subunit of the (Na⁺ + K⁺)-ATPase enzyme. Binding studies, using antibodies that were produced by growing hybridomas in vivo and purified by affinity column chromatography, suggest a somewhat higher affinity of these antibodies for the isolated α-subunit than for the ‘native’ holoenzyme. In addition, these monoclonal antibodies show no reactivity with either the glycoprotein (β) subunit of the lamb enzyme nor the (Na⁺ + K⁺)-ATPase from rat kidney, an ouabain-insensitive organ. Cotitration binding experiments have shown that the antibodies from two cell lines originally isolated independently from the same culture plate well population of fused cells bind to the same determinant site and are probably the same antibody. Cotitration and competition binding studies with two other antibodies have revealed two additional distinct antibody binding sites which appear to have little overlap with the first site. One of the three different antibodies isolated caused a partial inhibition of the (Na⁺ + K⁺)-ATPase activity. This antibody appears to be directed against a specific functionally important site of the α-subunit and is a competitive inhibitor of ATP binding. Under optimum conditions of ATPase activity, this inhibitory effect is not altered by the presence of the other two antibodies.     

Na+/K+-ATPase inhibition during cardiac myocyte swelling: Involvement of intracellular pH and Ca2+

Previous studies in chick embryo cardiac myocytes have shown that the inhibition of Na⁺/K⁺-ATPase with ouabain induces cell shrinkage in an isosmotic environment (290 mOsm). The same inhibition produces an enhanced RVD (regulatory volume decrease) in hyposmotic conditions (100 mOsm). It is also known that submitting chick embryo cardiomyocytes to a hyperosmotic solution induces shrinkage and a concurrent intracellular alkalization. The objective of this study was to evaluate the involvement of intracellular pH (pH_i), intracellular Ca²⁺ ([Ca²⁺]_i) and Na⁺/K⁺-ATPase inhibition during hyposmotic swelling. Changes in intracellular pH and Ca²⁺ were monitored using BCECF and fura-2, respectively. The addition of ouabain (100 M) under both isosmotic and hyposmotic stimuli resulted in a large increase in [Ca²⁺]_i (200%). A decrease in pH_i (from 7.3 ± 0.09 to 6.4 ± 0.08, n = 6; p < 0.05) was only observed when ouabain was applied during hyposmotic swelling. This acidification was prevented by the removal of extracellular Ca²⁺. Inhibition of Na⁺/H²⁺ exchange with amiloride (1 mM) had no effect on the ouabain-induced acidification. Preventing the mitochondrial accumulation of Ca²⁺ using CCCP (10 M) resulted in a blockade of the progressive acidification normally induced by ouabain. The inhibition of mitochondrial membrane K⁺/H⁺ exchange with DCCD (1 mM) also completely prevented the acidification. Our results suggest that intracellular acidification upon cell swelling is mediated by an initial Ca²⁺ influx via Na⁺/Ca²⁺ exchange, which under hyposmotic conditions activates the K⁺ and Ca²⁺ mitochondrial exchange systems (K⁺/H⁺ and Ca²⁺/H⁺).Deceased     

Amino group modification of (Na++K+)-ATPase

The effects of three amino group reagents on the activity of (Na⁺+K⁺)-ATPase³ and its component K⁺-stimulatedp-nitrophenylphosphatase activity from rabbit kidney outer medulla have been studied. All three reagents cause inactivation of the enzyme. Modification of amino groups with trinitrobenzene sulfonic acid yields kinetics of inactivation of both activities, which depend on the type and concentration of the ligands present. In the absence of added ligands, or with either Na⁺ of Mg²⁺ present, the enzyme inactivation process follows complicated kinetics. In the presence of K⁺, Rb⁺, or Tl⁺, protection occurs due to a change of the kinetics of inactivation toward a first-order process. ATP protects against inactivation at a much lower concentration in the absence than in the presence of Mg²⁺ (P ₅₀ 6 µM vs. 1.2 mM). Under certain conditions (100 µM reagent, 0.2 M triethanolamine buffer, pH 8.5) modification of only 2% of the amino groups is sufficient to obtain 50% inhibition of the ATPase activity. Modification of amino groups with ethylacetimidate causes a nonspecific type of inactivation of (Na⁺+K⁺)-ATPase. Mg²⁺ and K⁺ have no effects, and ATP only a minor effect, on the degree of modification. The K⁺-stimulatedp-nitrophenylphosphatase activity is less inhibited than the (Na⁺+K⁺)-ATPase activity. Half-inhibition of the (Na⁺+K⁺)-ATPase is obtained only after 25% modification of the amino groups. Modification of amino groups with acetic anhydride also causes nonspecific inactivation of (Na⁺+K⁺)-ATPase. Mg²⁺ has no effect, and ATP has only a slight protecting effect. The K⁺-stimulatedp-nitrophenylphosphatase activity is inhibited in parallel with the (Na⁺+K⁺)-ATPase activity. Half-inactivation of the (Na⁺+K⁺)-ATPase activity is obtained after 20% modification of the amino groups.This article is No. 52 in the series Studies on (Na⁺+K⁺)-Activated ATPase.     

ATP-binding is stabilized by a stacking interaction within the binding site of Na+/K+ -ATPase

Site-directed mutagenesis was applied to modify phenylalanines (Phe(475)Trp, Phe(548)Tyr, and both) to generate mutants on the basis of molecular modeling of the ATP-binding domain of Na(+)/K(+)-ATPase, in order to characterize the forces that stabilize ATP in its binding pocket. Each of the mutants was examined by Raman difference spectroscopy, i.e., as a difference between the spectrum of the domain with and without bound ATP. It was shown that Phe(475) plays a key role in stabilizing ATP-binding by a stacking interaction. Phe(548) co-stabilizes ATP on the opposite site of the binding pocket and its type of interaction with ATP-binding differs from that of Phe(475).     

Interaction of some Pd(II) complexes with Na+/K+-ATPase: Inhibition,kinetics, prevention and recovery

The aim of this work was to investigate the influence of [PdCl₄]^{2 ?} , [PdCl(dien)]⁺ and [PdCl(Me₄dien)]⁺ complexes on Na⁺/K⁺-ATPase activity. The dose-dependent inhibition curves were obtained in all cases. IC₅₀ values determined by Hill analysis were 2.25 × 10^{? 5} M, 1.21 × 10^{? 4} M and 2.36 × 10^{? 4} M, respectively. Na⁺/K⁺-ATPase exhibited typical Michelis-Menten kinetics in the presence of Pd(II) complexes. Kinetic parameters (V_max, K_m) derived using Eadie–Hofstee transformation indicated a noncompetitive type of Na⁺/K⁺-ATPase inhibition. The inhibitor constants (K_i) were determined from Dixon plots. The order of complex affinity for binding with Na⁺/K⁺-ATPase, deducted from K_i values, was [PdCl₄]^{2 ?} >[PdCl(dien)]⁺>[PdCl(Me₄dien)]⁺. The results indicated that the potency of Pd(II) complexes to inhibit Na⁺/K⁺-ATPase activity depended strongly on ligands of the related compound. Furthermore, the ability of SH-donor ligands, l-cysteine and glutathione, to prevent and recover the Pd(II) complexes-induced inhibition of Na⁺/K⁺-ATPase was examined. The addition of 1 mM l-cysteine or glutathione to the reaction mixture before exposure to Pd(II) complexes prevented the inhibition by increasing the IC₅₀ values by one order of magnitude. Moreover, the inhibited enzymatic activity was recovered by addition of SH-donor ligands in a concentration-dependent manner.     

Ouabain-induced Internalization and Lysosomal Degradation of the Na+/K+-ATPase

Internalization of the Na⁺/K⁺-ATPase (the Na⁺ pump) has been studied in the human lung carcinoma cell line H1299 that expresses YFP-tagged α1 from its normal genomic localization. Both real-time imaging and surface biotinylation have demonstrated internalization of α1 induced by ≥100 nm ouabain which occurs in a time scale of hours. Unlike previous studies in other systems, the ouabain-induced internalization was insensitive to Src or PI3K inhibitors. Accumulation of α1 in the cells could be augmented by inhibition of lysosomal degradation but not by proteosomal inhibitors. In agreement, the internalized α1 could be colocalized with the lysosomal marker LAMP1 but not with Golgi or nuclear markers. In principle, internalization could be triggered by a conformational change of the ouabain-bound Na⁺/K⁺-ATPase molecule or more generally by the disruption of cation homeostasis (Na⁺, K⁺, Ca²⁺) due to the partial inhibition of active Na⁺ and K⁺ transport. Overexpression of ouabain-insensitive rat α1 failed to inhibit internalization of human α1 expressed in the same cells. In addition, incubating cells in a K⁺-free medium did not induce internalization of the pump or affect the response to ouabain. Thus, internalization is not the result of changes in the cellular cation balance but is likely to be triggered by a conformational change of the protein itself. In physiological conditions, internalization may serve to eliminate pumps that have been blocked by endogenous ouabain or other cardiac glycosides. This mechanism may be required due to the very slow dissociation of the ouabain·Na⁺/K⁺-ATPase complex.     

Effect of tissue specificity of brain soluble fractions on Na+, K+-ATPase activity

Previous evidence from this laboratory indicated that catecholamines and brain endogenous factors modulate Na⁺, K⁺-ATPase activity of the synaptosomal membranes. The filtration of a brain total soluble fraction through Sephadex G-50 permitted the separation of two fractions-peaks I and II-which stimulated and inhibited Na⁺, K⁺-ATPase, respectively (Rodríguez de Lores Arnaiz and Antonelli de Gomez de Lima, Neurochem. Res.11, 1986, 933). In order to study tissue specificity a rat kidney total soluble was fractionated in Sephadex G-50 and kidney peak I and II fractions were separated; as control, a total soluble fraction prepared from rat cerebral cortex was also processed. The UV absorbance profile of the kidney total soluble showed two zones and was similar to the profile of the brain total soluble. Synaptosomal membranes Na⁺, K⁺- and Mg²⁺-ATPases were stimulated 60–100% in the presence of kidney and cerebral cortex peak I; Na⁺, K⁺-ATPase was inhibited 35–65% by kidney peak II and 60–80% by brain peak II. Mg²⁺-ATPase activity was not modified by peak II fractions. ATPases activity of a kidney crude microsomal fraction was not modified by kidney peak I or brain peak II, and was slightly increased by kidney peak II or brain peak I. Kidney purified Na⁺, K⁺-ATPase was increased 16–20% by brain peak I and II fractions. These findings indicate that modulatory factors of ATPase activity are not exclusive to the brain. On the contrary, there might be tissue specificity with respect to the enzyme source.     

Identification of cisplatin-binding sites on the large cytoplasmic loop of the Na+/K+-ATPase

Cisplatin is the most widely used chemotherapeutic drug for the treatment of various types of cancer; however, its administration brings also numerous side effects. It was demonstrated that cisplatin can inhibit the Na⁺/K⁺-ATPase (NKA), which can explain a large part of the adverse effects. In this study, we have identified five cysteinyl residues (C452, C456, C457, C577, and C656) as the cisplatin binding sites on the cytoplasmic loop connecting transmembrane helices 4 and 5 (C45), using site-directed mutagenesis and mass spectrometry experiments. The identified residues are known to be susceptible to glutathionylation indicating their involvement in a common regulatory mechanism.     

Cationic nanocarriers induce cell necrosis through impairment of Na+/K+-ATPase and cause subsequent inflammatory response

Nanocarriers with positive surface charges are known for their toxicity which has limited their clinical applications. The mechanism underlying their toxicity, such as the induction of inflammatory response, remains largely unknown. In the present study we found that injection of cationic nanocarriers, including cationic liposomes, PEI, and chitosan, led to the rapid appearance of necrotic cells. Cell necrosis induced by cationic nanocarriers is dependent on their positive surface charges, but does not require RIP1 and Mlkl. Instead, intracellular Na⁺ overload was found to accompany the cell death. Depletion of Na⁺ in culture medium or pretreatment of cells with the Na⁺/K⁺-ATPase cation-binding site inhibitor ouabain, protected cells from cell necrosis. Moreover, treatment with cationic nanocarriers inhibited Na⁺/K⁺-ATPase activity both in vitro and in vivo. The computational simulation showed that cationic carriers could interact with cation-binding site of Na⁺/K⁺-ATPase. Mice pretreated with a small dose of ouabain showed improved survival after injection of a lethal dose of cationic nanocarriers. Further analyses suggest that cell necrosis induced by cationic nanocarriers and the resulting leakage of mitochondrial DNA could trigger severe inflammation in vivo, which is mediated by a pathway involving TLR9 and MyD88 signaling. Taken together, our results reveal a novel mechanism whereby cationic nanocarriers induce acute cell necrosis through the interaction with Na⁺/K⁺-ATPase, with the subsequent exposure of mitochondrial damage-associated molecular patterns as a key event that mediates the inflammatory responses. Our study has important implications for evaluating the biocompatibility of nanocarriers and designing better and safer ones for drug delivery.     

K+-conductance and electrogenic Na+/K+ transport of cultured bovine pigmented ciliary epithelium

Summary Using intracellular microelectrode technique, we investigated the changes in membrane voltage (V) of cultured bovine pigmented ciliary epithelial cells induced by different extracellular solutions. (1)V in 213 cells under steady-state conditions averaged –46.1±0.6 mV (sem). (2) Increasing extracellular K⁺ concentration ([K⁺] _o ) depolarizedV. Addition of Ba²⁺ could diminish this response. (3) Depolarization on doubling [K⁺] _o was increased at higher [K⁺] _o (or low voltage). (4) Removing extracellular Ca²⁺ decreasedV and reduced theV amplitude on increasing [K⁺] _o . (5)V was pH sensitive. Extra-and intracellular acidification depolarizedV; alkalinization induced a hyperpolarization.V responses to high [K⁺] _o were reduced at acidic extracellular pH. (6) Removing K _o ⁺ depolarized, K _o ⁺ readdition after K⁺ depletion transiently hyperpolarizedV. These responses were insensitive to Ba²⁺ but were abolished in the presence of ouabain or in Na⁺-free medium. (7) Na⁺ readdition after Na⁺ depletion transiently hyperpolarizedV. This reaction was markedly reduced in the presence of ouabain or in K⁺-free solution but unchanged by Ba²⁺. It is concluded that in cultured bovine pigmented ciliary epithelial cells K⁺ conductance depends on Ca²⁺, pH and [K⁺] _o (or voltage). An electrogenic Na⁺/K⁺-transport is present, which is stimulated during recovery from K⁺ or Na⁺ depletion. This transport is inhibited by ouabain and in K⁺-or Na⁺-free medium.