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.     

The inhibitory activity of a brain extract on synaptosomal Na+,K+-ATPase is sensitive to carboxypeptidase A and to chelating agents

In the present study some properties of an inhibitory extract of synaptosomal membrane Na⁺,K⁺-ATPase were investigated. This extract (peak II) was prepared by gel filtration in Sephadex G-50 of a soluble fraction of the rat cerebral cortex. Ultrafiltration of peak II through Amicon membranes indicated that the inhibitor has a low MW (<1000). The inhibitory activity was not modified by heating in neutral pH at 95°C for 20 min but it was destroyed by charring in acid pH at 200°C for 120 min. The inhibitory activity decreased by incubation of peak II with carboxypeptidase A. These findings suggest that the factor responsible for the inhibition of Na⁺,K⁺-ATPase activity is probably a polypeptide. On the other hand, the inhibition was reverted by the chelators EDTA and EGTA, indicating the participation of an ionic compound as well. The increase of Mg²⁺ concentration during the enzyme assay did not increase the inhibition, indicating that the ion involved might not be vanadate. It is suggested that both a polypeptide and an ionic compound coparticipate in the inhibitory effect of peak II on Na⁺,K⁺-ATPase activity.     

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.     

Differential Properties Between an Endogenous Brain Na+, K+-ATPase Inhibitor and Ouabain

By means of a Sephadex G-50 column and anionic exchange HPLC a cerebral cortex soluble fraction (II-E) which highly inhibits neuronal Na⁺-K⁺-ATPase activity has been previously obtained. Herein, II-E properties are compared with those of the cardenolide ouabain, the selective and specific Na⁺, K⁺-ATPase inhibitor. It was observed that alkali treatment destroyed II-E but not ouabain inhibitory activity. II-E presented a maximal absorbance at 265 nm both at pH 7 and pH 2 which diminished at pH 10. Ouabain showed a maximum at 220 nm which was not altered by alkalinization. II-E was not retained in a C-18 column, indicating its hydrophilic nature, whereas ouabain presented a 26-min retention time in reverse phase HPLC. Therefore, it is concluded that the inhibitory factor present in II-E is structurally different to ouabain.     

Kinetics of Na+, K+-ATPase Inhibition by a Rat Brain Endogenous Factor (II-E)

Previous work from this laboratory led to the isolation by gel filtration and anionic exchange HPLC of a rat brain fraction named II-E, which highly inhibits synaptosomal membrane Na⁺, K⁺-ATPase activity. In this study we evaluated the kinetics of such inhibition and found that inhibitory potency was independent of Na⁺(1.56–200 mM), K⁺(1.25–40 mM), or ATP (1–8 mM) concentration. Hanes-Woolf plots indicated that II-E decreases Vmax but does not alter K_Mvalue, and suggested uncompetitive inhibition for Na⁺, K⁺or ATP. However, II-E became a stimulator at 0.5 mM ATP concentration. It is postulated that this brain factor may modulate ionic transport at synapses, thus participating in central neurotransmission.     

Is cysteine residue important in FITC-sensitive ATP-binding site of P-type ATPases? A commentary to the state of the art

Treatment of P-type ATPases (from mammalian sources) by fluorescein isothiocyanate (ITC) revealed the ITC label on a lysine residue that was than considered as essential for binding of ATP in the ATP-binding site of these enzymes. On the other hand, experiments with site directed mutagenesis excluded the presence of an essential lysine residue that would be localized in the ATP binding sites of ATPases. Other previous studies, including those of ourselves, indicated that the primary site of isothiocyanate interaction may be the sulflhydryl group of a cysteine residue and this may be essential for binding of ATP. In addition considerable knowledge accumulated since yet also about the differences in stability of reaction product of isothiocyanates with SH- or NH₂- groups. Based upon evaluation of the data available up to now, in present paper the following tentative roles for lysine and cysteine residues located in the ATP-binding site of P-type ATPases are proposed: The positively charged micro-domain of the lysine residue may probably attract the negatively charged phosphate moiety of the ATP molecule whereas the cysteine residue may probably be responsible for recognition and binding of ATP by creation of a proton bridge with the amino group in position 6 on the adenosine ring of ATP.     

The Identification of the Sodium Pump

The identification of the sodium potassium pump as a Na⁺, K⁺-ATPase is described.     

Structural features of cation transport ATPases

Several cation transport ATPases, sharing the common feature of a phosphorylated intermediate in the process of ATP utilization, are compared with respect to their subunit composition and amino acid sequence. The main component of these enzymes is a polypeptide chain of MW slightly exceeding 100,000, comprising an extramembranous globular head which is connected through a stalk to a membrane-bound region. With reference to the Ca²⁺ ATPase of sarcoplasmic reticulum, it is proposed that the catalytic (ATP binding and phosphorylation) domain resides in the extramembranous globular head, while cation binding occurs in the membrane region. Therefore, these two functional domains are separated by a distance of approximately 50 Å. Alignment of amino acid sequences reveals extensive homology in the isoforms of the same ATPases, but relatively little homology in different cation ATPases. On the other hand, all cation ATPases considered in this analysis retain a consensus sequence of high homology, spanning the distance between the phosphorylation site and the preceding transmembrane helix. It is proposed that this sequence provides long-range functional linkage between catalytic and cation-binding domains. Thereby, translocation of bound cation occurs through a channel formed by transmembrane helices linked to the phosphorylation site. Additional sequences at the carboxyl terminal provide regulatory domains in certain ATPases.     

The Study of Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Activity of Rat Brain during Crush Syndrome

Crush syndrome (CS) results from severe traumatic damage to the organism that is characterized by stress, acute homeostatic failure of the tissues, and myoglobinuria with severe intoxication. This leads to an acute impairment of kidneys and heart. The peripheral and central nervous systems are also the subject of significant changes in CS. Na⁺, K⁺-ATPase is a critical enzyme in neuron that is essential for the regulation of neuronal membrane potential, cell volume as well as transmembrane fluxes of Ca⁺⁺ and Excitatory Amino Acids. In the present study, Na⁺, K⁺-ATPase activity of rat brain regions [Olfactory lobes (OL), Cerebral cortex (CC), Cerebellum (CL), and Medulla oblongata (MO)] during CS was investigated. Experimental model of CS in albino rats was induced by 2-h of compression followed by 2, 24, and 48-h of decompression of femoral muscle tissue. In this study, we have observed elevation in Na⁺, K⁺-ATPase activity above normal/control levels in all parts of brain (OL: 34.4%; CC: 1.0%; CL: 3.3% and MO: 45%) during 2-h compression in comparison to controls.     

An Endogenous Na+, K+-ATPase Inhibitor Enhances Phosphoinositide Hydrolysis in Neonatal but Not in Adult Rat Brain Cortex

The effect of an endogenous Na⁺, K⁺-ATPase inhibitor, termed endobain E, on phosphoinositide hydrolysis was studied in rat brain cortical prisms and compared with that of ouabain. As already shown for ouabain, a transient effect was obtained with endobain E; maximal accumulation of inositol phosphates induced by endobain E was 604 ± 138% and 186 ± 48% of basal values in neonatal and adult rats, respectively. The concentration-response plot for the interaction between endobain E and phosphoinositide turnover differed from that of ouabain, thus suggesting the involvement of distinct mechanisms. In the presence of endobain E plus ouabain at saturating concentrations, no additive effect was recorded, suggesting that both substances share at least a common step in their activation mechanism of inositol phosphates metabolism or that they enhance phosphatidylinositol 4,5-biphosphate breakdown from the same membrane precursor pool, until its exhaustion. Experiments with benzamil, a potent blocker of Na⁺/Ca²⁺ exchanger, showed that it partially and dose-dependently inhibited endobain E effect. These results indicate that the endogenous Na⁺, K⁺-ATPase inhibitor endobain E, like ouabain, is able to stimulate phosphoinositide turnover transiently during postnatal brain development.     

Preconditioning Prevents the Inhibition of Na+,K+-ATPase Activity after Brain Ischemia

Application of single transient forebrain ischemia (ISC) in adult Wistar rats, lasting 2 or 10 min, caused inhibition of Na⁺,K⁺-ATPase activity in cytoplasmic membrane fractions of hippocampus and cerebral cortex immediately after the event. In the 2-min ISC group followed by 60 min of reperfusion, the enzyme inhibition was maintained in the cortex, while there was an increase in hippocampal enzyme activity; both effects were over 1 day after the event. However, in the 10-min ISC group enzyme inhibition had been maintained for 7 days in both cerebral structures. Interestingly, ischemic preconditioning (2-min plus 10-min ISC, with a 24-hour interval in between) prevented the inhibitory effect of ischemia/reperfusion on Na⁺,K⁺-ATPase activity observed either after a single insult of 2 min or 10 min ischemia. We suggest that the maintenance of Na⁺,K⁺-ATPase activity afforded by preconditioning be related to cellular neuroprotection.     

An endogenous factor which interacts with synaptosomal membrane Na+, K+-ATPase activation by K+

In previous papers, the isolation of brain soluble fractions able to modify neuronal Na⁺, K⁺-ATPase activity has been described. One of those fractions-peak I-stimulates membrane Na⁺, K⁺-ATPase while another-peak II-inhibits this enzyme activity, and has other ouabain-like properties. In the present study, synaptosomal membrane Na⁺, K⁺-ATPase was analyzed under several experimental conditions, using ATP orp-nitrophenylphosphate (p-NPP) as substrate, in the absence and presence of cerebral cortex peak II. Peak II inhibited K⁺-p-NPPase activity in a concentration dependent manner. Double reciprocal plots indicated that peak II uncompetitively inhibits K⁺-p-NPPase activity regarding substrate, Mg²⁺ and K⁺ concentration. Peak II failed to block the known K⁺-p-NPPase stimulation caused by ATP plus Na⁺. At various K⁺ concentrations, percentage K⁺-p-NPPase inhibition by peak II was similar regardless of the ATP plus Na⁺ presence, indicating lack of correlation with enzyme phosphorylation. Na⁺, K⁺-ATPase activity was decreased by peak II depending on K⁺ concentration. It is postulated that the inhibitory factor(s) present in peak II interfere(s) with enzyme activation by K⁺.     

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.     

The interaction between calcium and the activation of Na+, K+-ATPase by noradrenaline

Summary The interaction of noradrenaline, various cation chelators and calcium on Na⁺, K⁺-ATPase from rat cerebral cortex plasma membranes was studied. It was shown that chelation of inhibitory cations by EGTA, EDTA and dipyridyl activated Na⁺, K⁺-ATPase to the same extent as noradrenaline but at higher concentrations; increasing concentrations of EGTA depressed the activation by noradrenaline; calcium in the form of a calcium-EGTA buffer depressed Na⁺, K⁺-ATPase at physiological concentrations; the inhibition of Na⁺, K⁺-ATPase by calcium is dependent on the magnesium concentration in the assay and the inhibition by calcium was partially reversed by noradrenaline.     

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.     

Mg2+/Ca2+-ATPase Activity Is Not Enriched in Synaptic Vesicles Isolated from Rat Cerebral Cortex

Neuronal ATPases comprise a wide variety of enzymes which are not uniformly distributed in different membrane preparations. Since purified vesicle fractions have Mg²⁺/Ca²⁺-ATPase, the purpose of the present study was to know whether such enzyme activities have a preferential concentration in a synaptic vesicle fraction in order to be used as markers for these organelles. Resorting to a procedure developed in this Institute, we fractionated the rat cerebral cortex by differential centrifugation following osmotic shock of a crude mitochondrial fraction and separated a purified synaptic vesicle fraction over discontinuous sucrose gradients. Mg²⁺/Ca²⁺-ATPase activities and ultrastructural studies of isolated fractions were carried out. It was observed that similar specific activities for Mg²⁺/Ca²⁺-ATPases were found in all fractions studied which contain synaptic vesicles and/or membranes. Although the present results confirm the presence of Mg²⁺ and Ca²⁺-ATPase activities in synaptic vesicles preparations, they do not favor the contention that Mg²⁺/Ca²⁺-ATPase is a good marker for synaptic vesicles.     

The effects of captopril and losartan on erythrocyte membrane Na+/K+- ATPase activity in experimental diabetes mellitus

Diabetes mellitus induces a decrease in sodium potassium-adenosine triphosphatase (Na⁺/K⁺- ATPase) activity in several tissues in the rat and red blood cells (RBC) and nervous tissue in human patients. This decrease in Na⁺/K⁺- ATPase activity is thought to play a role in the development of long term complications of the disease. Angiotensin enzyme inhibitors (ACEi) and angiotensin-II receptor antagonists (ARBs) reduce proteinuria and retard the progression of renal failure in patients with IDDM and diabetic rats. We investigated the effects of captopril and losartan, which are used in the treatment of diabetic nephropathy, on Na⁺/K⁺- ATPase activity. Captopril had an inhibitory effect on red cell plasma membrane Na⁺/K⁺ ATPase activity, but losartan did not. Our study draws attention to the inhibitory effect of captopril on Na⁺/K⁺ ATPase activity. Micro and macro vascular complications are preceeding mortality and morbidity causes in diabetes mellitus. There is a strong relationship between the decrease in Na⁺/K⁺ ATPase activity and hypertension. The non-sulphydryl containing ACEi and ARBs must be the choice of treatment in hypertensive diabetic patients and diabetic nephropathy.     

Modulations of rabbit erythrocyte ATPase activities induced by in vitro and in vivo exposure to ethanol

Alcohol intake is associated with numerous degenerative disorders, and the detrimental effects of alcohol may be due to its influence on plasma membrane and cellular transport systems. The aim of the present study was to compare in vitro and in vivo effects of ethanol on rabbit erythrocyte ATPase activities and correlate them with ethanol-induced oxidative stress. Age-matched male rabbits were given 5% ethanol in 2% sucrose solution, for 6 weeks ad libitum; control animals were given tap water. Daily intake of ethanol was 5 g/kg body weight; this experimental regimen resulted in an average serum ethanol concentration of 16.77 ± 2.00 mM/l. After this period, blood was collected, serum ethanol concentration was determined and erythrocyte membranes were prepared according to the method of Post et al. Activities of Na⁺/K⁺- and Mg²⁺-ATPases were determined. Thiobarbituric acid-reactive substance (TBARS) assay was used to detect levels of lipid peroxidation, a major indicator of oxidative stress. In vitro ethanol inhibits both Na⁺/K⁺-ATPase and Mg²⁺-ATPase, but Na⁺/K⁺-ATPase is more sensitive to the ethanol-induced inhibition. Increasing concentration of ethanol increased TBARS production, but significant difference was attained only at 5 and 12.5 mM of ethanol. Chronic ethanol consumption induced significant increase in Na⁺/K⁺- and Mg²⁺-ATPase activity, and TBARS production. Our results suggest that increased ATPase activity induced by chronic ethanol consumption is due to oxidative, induced modification of membrane phospholipids and proteins, which are responsible for inhibition of ATPase activity. Increased production of TBARS induced by in vitro exposure to ethanol is not the only factor that influences ATPases activity. Further research is needed to elucidate this relationship.     

Kinetics of K+-p-Nitrophenyl Phosphatase Stimulation by a Brain Soluble Fraction

We have already described the separation of two brain soluble fractions by Sephadex G-50, one of which stimulates (peak I) and the other inhibits (peak II) Na⁺, K⁺-ATPase and K⁺-p-nitrophenylphosphatase (K⁺-p-NPPase) activities. Here we examine the features of synaptosomal membrane p-NPPase activity in the presence and absence of brain peak I. It was observed that stimulation of Mg²⁺, K⁺-p-NPPase activity by peak I was concentration dependent, The ability of peak I to stimulate p-NPPase activity was lost by heat treatment followed by brief centrifugation. Pure serum albumin also stimulated enzyme activity. K⁺-p-NPPase stimulation by peak I proved dependent on K⁺ concentration but independent of Mg²⁺ and substrate p-nitrophenylphosphate concentrations. Since our determinations were performed in a non-phosphorylating condition reflecting the Na⁺, K⁺-ATPase Na⁺ site, it is suggested that peak I may stimulate the Na⁺-dependent enzyme phosphorylation known to take place from the internal cytoplasmic side.