A possible mechanism for low affinity of silkworm Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase for K<Superscript>+</Superscript>

The affinity for K⁺ of silkworm nerve Na⁺/K⁺-ATPase is markedly lower than that of mammalian Na⁺/K⁺-ATPase (Homareda 2010). In order to obtain clues on the molecular basis of the difference in K⁺ affinities, we cloned cDNAs of silkworm (Bombyx mori) nerve Na⁺/K⁺-ATPase α and β subunits, and analyzed the deduced amino acid sequences. The molecular masses of the α and β subunits were presumed to be 111.5 kDa with ten transmembrane segments and 37.7 kDa with a single transmembrane segment, respectively. The α subunit showed 75% identity and 93% homology with the pig Na⁺/K⁺-ATPase α1 subunit. On the other hand, the amino acid identity of the β subunit with mammalian counterparts was as low as 30%. Cloned α and β cDNAs were co-expressed in cultured silkworm ovary-derived cells, BM-N cells, which lack endogenous Na⁺/K⁺-ATPase. Na⁺/K⁺-ATPase expressed in the cultured cells showed a low affinity for K⁺ and a high affinity for Na⁺, characteristic of the silkworm nerve Na⁺/K⁺-ATPase. These results suggest that the β subunit is responsible for the affinity for K⁺ of Na⁺/K⁺-ATPase.     

Role of heme oxygenase in heme-mediated inhibition of rat brain Na+−K+-ATPase: Protection by tin-protoporphyrin

Hemoglobin has been shown to inhibit brain Na⁺–K⁺-ATPase through an iron-dependent mechanism. Both hemoglobin and iron cause spontaneous peroxidation of brain lipids. Release of iron from the heme molecule in animal tissues is dependent on the activity of heme oxygenase. We hypothesized that inhibition of heme catabolism by heme oxygenase prevents the iron-mediated inhibition of Na⁺–K⁺-ATPase and might subsequently reduce the tissue damage. Therefore, we studied the effect of heme and tin-protoporphyrin, an inhibitor of heme oxygenase, on the activity of partially purified Na⁺–K⁺-ATPase from rat brain in the presence and absence of purified hepatic heme oxygenase. Heme alone at a concentration of 30 M did not inhibit Na⁺–K⁺-ATPase. However, in the presence of heme oxygenase, heme inhibited Na⁺–K⁺-ATPase by 75%. Pretreatment of rats with SnCl₂, a known inducer of heme oxygenase, reduced the basal activity of the brain Na⁺–K⁺-ATPase by 50%. Inhibition of heme oxygenase by tin-protoporphyrin (30 M) prevented the inhibition of Na⁺–K⁺-ATPase which occurred in the presence of heme and heme oxygenase. It is concluded that suppression of heme oxygenase by tin-protoporphyrin might be a therapeutic approach to management of hemoglobin-associated brain injury following CNS hemorrhage.     

Some in vivo and in vitro effects of ethacrynic acid on renal Na+,K+ -ATPase

Binding of [¹⁴C]ethaerynic acid [EA]at concentrations of EA from 10^?4m to 10^?2m to a membrane preparation containing Na⁺,K⁺-ATPase activity in vitro occurred in a nonsaturable manner; binding was stimulated by Na⁺ or K⁺, but was not affected by Mg²⁺ and/or ATP. [¹⁴C]EA significantly bound to a microsomal preparation with low Na⁺,K⁺-ATPase activity as well as to a heat-denatured enzyme; this binding reaction was not stimulated by Na⁺. These observations suggest that EA binds non-specifically or to nonspecific sites on membrane preparations. Nonselective binding of [¹⁴C]EA to subcellular particles after fractionation of slices also suggested the presence of nonspecific EA binding sites in vivo. In vitro [³H]ouabain binding to medullary and cortical Na⁺,K⁺-ATPase preparations was partially reduced by pretreatment with EA. On the other hand, [¹⁴C]EA binding to Na⁺,K⁺-ATPase was not affected by pretreatment of the preparation with ouabain (10^?6m to 5 × 10^?4m). EA reduced the sensitivity of [³H]ouabain binding to the enzyme preparation to Na⁴ and K⁺.EA was infused (0.1, 1.0, and 10 mg/min) into one renal artery of hydropenic dogs. A prompt natriuresis in the infused kidney occurred. Similar changes were observed in the contralateral kidney 20 min after starting the infusion. Both kidneys were removed 30 min after the beginning of the infusion, and Na⁺,K⁺-ATPase was isolated from the cortex and the medulla. Enzyme activity from cortex and medulla of either kidney was not significantly different from enzyme activity from cortex and medulla of control, uninfused dogs, regardless of dose of EA or method of enzyme isolation. Furthermore, in vitro binding of [³H]ouabain to Na⁺,K⁺-ATPase membrane preparations from cortex and medulla was the same for experimental and control kidneys. In vitro incubation of 2 × 10^?3m EA with a membrane preparation caused the same inhibition of ATPase activity when the enzyme was isolated either from control or EA-infused dogs. The inhibition could not be reversed by recentrifugation or rehomogenization of the enzyme. Our results do not support the concept that Na⁺,K⁺-ATPase is a pharmacological receptor for ethacrynic acid.     

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.     

Evidence That Antioxidants Prevent the Inhibition of Na+,K+-ATPase Activity Induced by Octanoic Acid in Rat Cerebral Cortex in Vitro

The objective of the present study was to investigate the in vitro effects of octanoic acid, which accumulates in medium-chain acyl-CoA dehydrogenase (MCAD) deficiency and in Reye syndrome, on key enzyme activities of energy metabolism in the cerebral cortex of young rats. The activities of the respiratory chain complexes I–IV, creatine kinase, and Na⁺, K⁺-ATPase were evaluated. Octanoic acid did not alter the electron transport chain and creatine kinase activities, but, in contrast, significantly inhibited Na⁺, K⁺-ATPase activity both in synaptic plasma membranes and in homogenates prepared from cerebral cortex. Furthermore, decanoic acid, which is also increased in MCAD deficiency, and oleic acid strongly reduced Na⁺, K⁺-ATPase activity, whereas palmitic acid had no effect. We also examined the effects of incubating glutathione and trolox (-tocopherol) alone or with octanoic acid on Na⁺, K⁺-ATPase activity. Tested compounds did not affect Na⁺, K⁺-ATPase activity by itself, but prevented the inhibitory effect of octanoic acid. These results suggest that inhibition of Na⁺, K⁺-ATPase activity by octanoic acid is possibly mediated by oxidation of essential groups of the enzyme. Considering that Na⁺, K⁺-ATPase is critical for normal brain function, it is feasible that the significant inhibition of this enzyme activity by octanoate and also by decanoate may be related to the neurological dysfunction found in patients affected by MCAD deficiency and Reye syndrome.     

Serotonin Modulation of Low-Affinity Ouabain Binding in Rat Brain Determined by Quantitative Autoradiography

Previous results showed that Na⁺/K⁺-ATPase may have a functional relationship with the neurotransmitter serotonin which activates the glial sodium pump in the rat brain. Both the reaction rate (V) of Na⁺/K⁺-ATPase activity and [³H]ouabain binding were significantly increased in the presence of serotonin. It is not known, however, which isoform is involved in the Na⁺/K⁺-ATPase response to serotonin and its regional distribution. Quantitative autoradiography of [³H]ouabain binding to rat brain slices was employed at different [³H]ouabain concentrations in order to gain information on both the distribution and the possible isoform involved. The results showed that 1500 nM [³H]ouabain binding was sensitive to serotonin 10^–3 M and significantly increased in the following brain regions: frontal cortex, areas CA1, CA2, and CA3 of the hippocampus, presubiculum, zona incerta, caudate putamen and the amygdaloid area, confirming and extending previous results. An effect of serotonin on brain but not kidney tissue at high, 1500 nM, and the lack of effect at low, 50 nM [³H]ouabain concentrations, strongly suggests the participation of the 2 isoform in the response of the pump to the neurotransmitter. Glial cells showed stimulation of ouabain binding by serotonin at ouabain concentrations above 350 nM. The present results open interesting questions related to the brain regions involved and the K⁺ handling by the glial 2 isoform of the pump.     

The Administration of Levocabastine,a NTS2 Receptor Antagonist,Modifies Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Properties

Neurotensin behaves as a neuromodulator or as a neurotransmitter interacting with NTS1 and NTS2 receptors. Neurotensin in vitro inhibits synaptosomal membrane Na⁺, K⁺-ATPase activity. This effect is prevented by administration of SR 48692 (antagonist for NTS1 receptor). The administration of levocabastine (antagonist for NTS2 receptor) does not prevent Na⁺, K⁺-ATPase inhibition by neurotensin when the enzyme is assayed with ATP as substrate. Herein levocabastine effect on Na⁺, K⁺-ATPase K⁺ site was explored. For this purpose, levocabastine was administered to rats and K⁺-p-nitrophenylphosphatase (K⁺-p-NPPase) activity in synaptosomal membranes and [³H]-ouabain binding to cerebral cortex membranes were assayed in the absence (basal) and in the presence of neurotensin. Male Wistar rats were administered with levocabastine (50 μg/kg, i.p., 30 min) or the vehicle (saline solution). Synaptosomal membranes were obtained from cerebral cortex by differential and gradient centrifugation. The activity of K⁺-p-NPPase was determined in media laking or containing ATP plus NaCl. In such phosphorylating condition enzyme behaviour resembles that observed when ATP hydrolyses is recorded. In the absence of ATP plus NaCl, K⁺-p-NPPase activity was similar for levocabastine or vehicle injected (roughly 11 μmole hydrolyzed substrate per mg protein per hour). Such value remained unaltered by the presence of 3.5 × 10^?6 M neurotensin. In the phosphorylating medium, neurotensin decreased (32 %) the enzyme activity in membranes obtained from rats injected with the vehicle but failed to alter those obtained from rats injected with levocabastine. Levocabastine administration enhanced (50 %) basal [³H]-ouabain binding to cerebral cortex membranes but failed to modify neurotensin inhibitory effect on this ligand binding. It is concluded that NTS2 receptor blockade modifies the properties of neuronal Na⁺, K⁺-ATPase and that neurotensin effect on Na⁺, K⁺-ATPase involves NTS1 receptor and -at least partially- NTS2 receptor.     

Reduction of Na(+),K(+)-ATPase activity in hippocampus of rats subjected to chemically induced hyperhomocysteinemia

Hyperhomocysteinemia occurs in homocystinuria, an inherited metabolic disease clinically characterized by thromboembolic episodes and a variable degree of neurological dysfunction whose pathophysiology is poorly known. In this study, we induced elevated levels of homocysteine (Hcy) in blood (500 M), comparable to those of human homocystinuria, and in brain (60 nmol/g wet tissue) of young rats by injecting subcutaneously homocysteine (0.3-0.6 mol/g of body weight) twice a day at 8-hr intervals from the 6th to the 28th postpartum day. Controls received saline in the same volumes. Na⁺,K⁺-ATPase and Mg²⁺-ATPase activities were determined in the hippocampus of treated Hcy- and saline-treated rats. Chronic administration of Hcy significantly decreased (40%) Na⁺,K⁺-ATPase activity but did not alter Mg²⁺-ATPase activity. Considering that Na⁺,K⁺-ATPase plays a crucial role in the central nervous system, our results suggest that the brain dysfunction found in homocystinuria may be related to the reduction of brain Na⁺,K⁺-ATPase activity.     

Inhibition of ATPases Enzyme Activities on Brain Disturbing Normal Oestrous Cycle

Wistar rats treated with -methyl- DL-p -tyrosine methylester showed significant level of inhibition in the activity of Na⁺, K⁺-ATPase, Mg²⁺-ATPase and Ca²⁺-ATPase enzymes in different regions of the brain. The enzyme activity was assayed in cerebral hemispheres, hypothalamus, thalamus, hippocampus, amygdala and septum at proestrous (12 h), estrous (25 h), metestrous (38 h) and diestrous periods (92 h) of the rat. The Na⁺, K⁺-ATPase activity was significantly inhibited in most of the brain regions after treated with -methyl- DL-p -tyrosine methylester (MPT) and this indicated that MPT affected the active transport system and nerve impulse transmission. Mg²⁺-ATPase and Ca²⁺-ATPase was also significantly (P < 0.001) reduced in different regions of the brain. The results revealed that MPT affected active transport system and nerve impulse transmission by inhibiting Na⁺, K⁺-ATPase and Ca²⁺-ATPase. It has induced energy crisis by inhibiting Mg²⁺-ATPase and all these cumulative effects of MPT have adversely affected the female Wistar rats. These effects have been manifested in the form of aberrations in the behavior of MPT treated female rats, which have shown their inability to perform their normal sexual activity.     

Regulation of the Na+/K+-ATPase by insulin: Why and how?

The sodium-potassium ATPase (Na⁺/K⁺-ATPase or Na⁺/K⁺-pump) is an enzyme present at the surface of all eukaryotic cells, which actively extrudes Na⁺ from cells in exchange for K⁺ at a ratio of 3:2, respectively. Its activity also provides the driving force for secondary active transport of solutes such as amino acids, phosphate, vitamins and, in epithelial cells, glucose. The enzyme consists of two subunits ( and ) each expressed in several isoforms. Many hormones regulate Na⁺/K⁺ -ATPase activity and in this review we will focus on the effects of insulin. The possible mechanisms whereby insulin controls Na⁺/K⁺-ATPase activity are discussed. These are tissue- and isoform-specific, and include reversible covalent modification of catalytic subunits, activation by a rise in intracellular Na⁺ concentration, altered Na⁺ sensitivity and changes in subunit gene or protein expression. Given the recent escalation in knowledge of insulin-stimulated signal transduction systems, it is pertinent to ask which intracellular signalling pathways are utilized by insulin in controlling Na⁺/K⁺-ATPase activity. Evidence for and against a role for the phosphatidylinositol-3-kinase and mitogen activated protein kinase arms of the insulin-stimulated intracellular signalling networks is suggested. Finally, the clinical relevance of Na⁺/K⁺-ATPase control by insulin in diabetes and related disorders is addressed.     

The activity of erythrocyte and brain Na+/K+ and Mg2+-ATPases in rats subjected to acute homocysteine and homocysteine thiolactone administration

Hyperhomocysteinemia is associated with various pathologies including cardiovascular disease, stroke, and cognitive dysfunctions. Systemic administration of homocysteine can trigger seizures in animals, and patients with homocystinuria suffer from epileptic seizures. Available data suggest that homocysteine can be harmful to human cells because of its metabolic conversion to homocysteine thiolactone, a reactive thioester. A number of reports have demonstrated a reduction of Na⁺/K⁺-ATPase activity in cerebral ischemia, epilepsy and neurodegeneration possibly associated with excitotoxic mechanisms. The aim of this study was to examine the in vivo effects of d,l-homocysteine and d,l-homocysteine thiolactone on Na⁺/K⁺- and Mg²⁺-ATPase activities in erythrocyte (RBC), brain cortex, hippocampus, and brain stem of adult male rats. Our results demonstrate a moderate inhibition of rat hippocampal Na⁺/K⁺-ATPase activity by d,l-homocysteine, which however expressed no effect on the activity of this enzyme in the cortex and brain stem. In contrast,d,l-homocysteine thiolactone strongly inhibited Na⁺/K⁺-ATPase activity in cortex, hippocampus and brain stem of rats. RBC Na⁺/K⁺-ATPase and Mg²⁺-ATPase activities were not affected by d,l-homocysteine, while d,l-homocysteine thiolactone inhibited only Na⁺/K⁺-ATPase activity. This study results show that homocysteine thiolactone significantly inhibits Na⁺/K⁺-ATPase activity in the cortex, hippocampus, and brain stem, which may contribute at least in part to the understanding of excitotoxic and convulsive properties of this substance.     

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.     

The effects of nitric oxide synthesis on the Na+,K+-ATPase activity in guinea pig kidney exposed to lipopolysaccharides

Endotoxins (lipopolysaccharides; LPS) are known to cause multiple organ failure, including renal dysfunction. LPS triggers the synthesis and release of cytokines and the vasodilatör nitric oxide (NO). A major contributor to the increase in NO production is LPS-stimulated expression of inducible nitric oxide synthase (iNOS). This occurs in vasculature and most organs including the kidney. During endotoxemia, NO and superoxide react spontaneously to form the potent and versatile oxidant peroxynitrite (ONOO^–) and the formation of 3-nitrotyrosine (nTyr)-protein adducts is a reliable biomarker of ONOO^– generation. Therefore, the present study was aimed at investigating the role of endogenous nitric oxide in regulating Na⁺,K⁺-ATPase activity in the kidney, and at investigating the possible contribution of reactive nitrogen species (RNS) by measuring of iNOS activity. In addition, the present study was aimed at investigating the relationship between nTyr formation with iNOS and Na⁺,K⁺-ATPase activities. Previously in our study, nTyr was not detectable in kidney of normal control animals but was detected markedly in LPS exposed animals. In this study, kidney Na⁺,K⁺-ATPase activity were maximally inhibited 6 h after LPS injection (P:0.000) and LPS treatment significantly increased iNOS activity of kidney (P:0.000). The regression analysis revealed a very close correlation between Na⁺,K⁺-ATPase activity and nTyr levels of LPS treated animals (r = –0.868, P = 0.001). Na⁺,K⁺-ATPase activity were also negatively correlated with iNOS activity (r = –0.877, P = 0.001) in inflamed kidney. These data suggest that NO and ONOO^– contribute to the development of oxidant injury. Furthermore, the source of NO may be iNOS. iNOS are expressed by the kidney, and their activity may increase following LPS administration. In addition, NO and ONOO^– formation inhibited Na⁺,K⁺-ATPase activity. This results also have strongly suggested that bacterial LPS disturbs activity of membrane Na⁺,K⁺-ATPase that may be an important component leading to the pathological consequences such as renal dysfunction in which the production of RNS are increased as in the case of LPS challenge. (Mol Cell Biochem 271: 107–112, 2005)     

Different properties of two brain extracts separated in sephadex G-50 that modify synaptosomal ATPase activities

We have previously reported that Na⁺,K⁺-ATPase of nerve ending membranes is stimulated by catecholamines only in the presence of a brain soluble fraction. The filtration of this soluble fraction through Sephadex G-50 permitted the separation of two extracts of maximal UV absorbance (peaks I and II) which showed different effects on ATPases. Peak I stimulated both Na⁺,K⁺-ATPase and Mg²⁺-ATPase activities and peak II inhibited Na⁺,K⁺-ATPase activity. We have now studied the activity of ATPases in the presence of the whole eluate obtained from the Sephadex G-50 column. It was observed that maximal effects on ATPases were obtained with peaks I and II. Peak I and peak II fractions were unable to modify the activity of acetylcholinesterase or 5-nucleotidase present in the synaptosomal membranes. The stimulatory effect of peak I on ATPases was concentration dependent (up to 1100), it was stable at different pHs and it was reverted by catecholamines. The inhibitory effect of peak II on Na⁺,K⁺-ATPase was concentration dependent (up to 150,000), it was stable only at acid pH, and it was partially reverted by catecholamines. These findings indicate that the factors responsible for the effects of peaks I and II have different properties and that their actions on ATPases show enzyme specifity.     

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.     

Na+, K+-ATPase Isozyme Diversity; Comparative Biochemistry and Physiological Implications of Novel Functional Interactions

Na⁺, K⁺-ATPase is ubiquitously expressed in the plasma membrane ofall animal cells where it serves as the principal regulator of intracellularion homeostasis. Na⁺, K⁺-ATPase is responsible for generating andmaintaining transmembrane ionic gradients that are of vital importance forcellular function and subservient activities such as volume regulation, pHmaintenance, and generation of action potentials and secondary activetransport. The diversity of Na⁺, K⁺-ATPase subunit isoforms andtheir complex spatial and temporal patterns of cellular expression suggestthat Na⁺, K⁺-ATPase isozymes perform specialized physiologicalfunctions. Recent studies have shown that the subunit isoformspossess considerably different kinetic properties and modes of regulationand the subunit isoforms modulate the activity, expression and plasmamembrane targeting of Na⁺, K⁺-ATPase isozymes. This review focuseson recent developments in Na⁺, K⁺-ATPase research, and in particular reportsof expression of isoforms in various tissues and experiments aimed atelucidating the intrinsic structural features of isoforms important forNa⁺, K⁺-ATPase function.     

Conformational changes of membrane-bound (Na+−K+)-ATPase as revealed by trypsin digestion

Summary To distinguish ligand-induced structural states of the (Na⁺–K⁺)-ATPase, the purified membrane-bound enzyme isolated from rat kidneys was digested with trypsin in the presence of various combinations of Na⁺, K⁺, Mg⁺⁺ and ATP. It was found that first the large and then the small polypeptide chain of the (Na⁺–K⁺)-ATPase was degraded, indicating that the lysine and arginine residues of the large chain are more exposed than are those of the small one. The (Na⁺–K⁺)-ATPase activity was inactivated in parallel with the degradation of the large polypeptide chain. After the degradation of the large polypeptide chain, about 75% of the (Na⁺–K⁺)-ATPase protein remained bound to the membrane, demonstrating that the split protein segments were only partially released.It was found that the combinations of ATP, Mg⁺⁺, Na⁺ and K⁺ present during trypsin digestion influenced the time course and degree of degradation of the (Na⁺–K⁺)-ATPase protein. The degradations of the large and the small polypeptide chain were affected in parallel. Thus, certain ATP and ligand combinations influenced neither the degradation of the large nor the degradation of the small polypeptide chain, whereas by other combinations of ATP and ligands the degree of susceptibility of both polypeptide chains to trypsin was equally increased or reduced.In the absence of ATP the time course of trypsin digestion of the (Na⁺–K⁺)-ATPase was the same, whether Na⁺ or K⁺ was present. With low ATP concentrations (e.g., 0.1mm), however, binding of Na⁺ or K⁺ led to different degradation patterns of the enzyme. If a high concentration of ATP (e.g., 10mm) was present, Na⁺ and K⁺ also influenced the degradation pattern of the (Na⁺–K⁺)-ATPase, but differentially compared to that at low ATP concentrations, since the effects of Na⁺ and K⁺ were reversed. Furthermore, it was found that the degradation of the small chain was only influenced by certain combinations of ATP, Mg⁺⁺, Na⁺ and K⁺ if the large chain was intact when the ligands were added to the enzyme.The described results demonstrate structural alterations of the (Na⁺–K⁺)-ATPase complex which are supposed to include a synchronous protrusion or retraction of both (Na⁺–K⁺)-ATPase subunits. The data further suggest that ATP and other ligands primarily alter the structure of the large (Na⁺–K⁺)-ATPase subunit. This structural alteration is presumed to lead to a synchronous movement of the small subunit of the enzyme. The structural state of the (Na⁺–K⁺)-ATPase is regulated by binding of Na⁺ or K⁺ to the enzyme-ATP complex. The effects of Na⁺ and K⁺ on the (Na⁺–K⁺)-ATPase structure are modulated by the ATP binding to high affinity and to low affinity ATP binding sites.     

Iron-Induced Inhibition of Na+, K+-ATPase and Na+/Ca2+ Exchanger in Synaptosomes: Protection by the Pyridoindole Stobadine

The effect of oxidative stress, induced by Fe²⁺-EDTA system, on Na⁺,K⁺-ATPase, Na⁺/Ca²⁺ exchanger and membrane fluidity of synaptosomes was investigated. Synaptosomes isolated from gerbil whole forebrain were incubated in the presence of 200 M FeSO₄-EDTA per mg of protein at 37°C for 30 min. The oxidative insult reduced Na⁺,K⁺-ATPase activity by 50.7 ± 5.0 % and Na⁺/Ca²⁺ exchanger activity measured in potassium and choline media by 47.1 ± 7.2 % and 46.7 ± 8.6 %, respectively. Membrane fluidity was also significantly reduced as observed with the 1,6-diphenyl-1,3,5-hexatriene probe. Stobadine, a pyridoindole derivative, prevented the decrease in membrane fluidity and in Na⁺/Ca²⁺ exchanger activity. The Na⁺,K⁺-ATPase activity was only partially protected by this lipid antioxidant, indicating a more complex mechanism of inhibition of this protein. The results of the present study suggest that the Na⁺/Ca²⁺ exchanger and the Na⁺,K⁺-ATPase are involved in oxidation stress-mediated disturbances of intracellular ion homeostasis and may contribute to cell injury.     

THE ADENOSINE DIPHOSPHATE-ADENOSINE TRIPHOSPHATE EXCHANGE REACTION OF EXTRACTED CEREBRAL MICROSOMES

—Microsomal fractions prepared from guinea pig cerebral cortex manifested ADP-ATP exchange activity, 40–99 per cent of which was extractable by dilute salt solutions. All of the (Na⁺, K⁺)-ATPase activity remained in the particulate material. The unextracted ADP-ATP exchange activity was stimulated six to seven fold by a non-ionic detergent (Lubrol W). When pre-extracted microsomes were sedimented in a sucrose density gradient, the ADP-ATP exchange activity was more widely distributed than (Na⁺, K⁺)-ATPase or adenylate kinase activities. The ADP-ATP exchange activity of microsomes extracted with NaI was stimulated by Na⁺ ions when the Mg²⁺ concentration in the reaction mixture was low (0·2 mm ). The Na⁺ stimulation of exchange activity was more variable than was the stimulation of phosphate formation by Na⁺ plus K⁺. The Na⁺-stimulated ADP-ATP exchange reaction of extracted microsomes may be a component of the (Na⁺, K⁺)-ATPase system, which has not been freed from adenylate kinase or possibly other contributing enzyme systems.