The Effect of Endogenous Modulator Endobain E on NMDA Receptor Is Interfered by Zn22+ but Is Independent of Modulation by Spermidine

A brain endogenous factor, termed endobain E, allosterically decreases [3H]dizocilpine binding to NMDA receptor. Such effect depends on receptor activation by the coagonists glutamate and glycine and is interfered by channel blockers, suggesting its interaction with the inner surface of the associated channel. To further analyze endobain E effect on NMDA receptor, in the current study competitive [3H]dizocilpine binding assays to brain membranes were performed with Zn2+ to block the associated channel, as well as with spermidine (SPD), which exerts positive allosteric modulation of NMDA receptor. Partially or nonadditive effects on [3H]dizocilpine binding were recorded, respectively, in the presence of endobain E at a concentration that inhibits binding 25% plus IC25 Zn2+ or endobain E at a concentration that inhibits binding 50% plus IC50 Zn2+. With an endobain E concentration that decreases 25% ligand binding, SPD potentiated binding over a wide concentration range but failed to modify endobain E effect. Similarly, [3H]dizocilpine binding reduction over a wide endobain E concentration range remained unaltered by high SPD concentrations. Additive effects were observed with endobain E at a concentration that decreases binding 25% plus IC25 SPD site antagonists arcaine or ifenprodil. Zn2+ experiments indicated that endobain E effect is interfered by channel blockade produced by this ion. Although endobain E effect is dependent on NMDA receptor activation by glutamate and glycine, it proves independent of the positive modulation exerted by SPD. Thus the endogenous modulator seems not to interact at NMDA receptor polyamine site, favoring the hypothesis that endobain E binds inside the associated channel.     

Endobain E, a brain endogenous factor, is present and modulates NMDA receptor in ischemic conditions

We have isolated from rat cerebral cortex an endogenous Na(+), K(+)-ATPase inhibitor, termed endobain E, which modulates glutamatergic N-methyl-d-aspartate (NMDA) receptor. This endogenous factor allosterically decreases [(3)H]dizocilpine binding to NMDA receptor, most likely acting as a weak channel blocker. In the present study we investigated whether endobain E is present in the cerebral cortex of rats subjected to ischemia and modulates NMDA receptor exposed to the same conditions. Ischemia-reperfusion was carried out by bilateral occlusion of common carotid arteries followed by a 15-min reperfusion period. Elution profile of brain soluble fraction showed that endobain E is present in cerebral cortex of ischemia-reperfusion rats. On assaying its effect on synaptosomal membrane Na(+), K(+)-ATPase activity and [(3)H]dizocilpine binding to cerebral cortex membranes prepared from animals without treatment, it was found that the endogenous modulator isolated from ischemia-reperfusion rats was able to inhibit both enzyme activity and ligand binding. On the other hand, endobain E prepared from rats without treatment also decreased binding to cerebral cortex or hippocampal membranes obtained from animals exposed to ischemia-reperfusion. Since ischemia decreases tissue pH and NMDA receptor activity varies according to proton concentration, pH influence on endobain E effect was tested. Endobain E ( approximately 80 mg original tissue) decreased [(3)H]dizocilpine binding 25% at pH 7.4 or 8.0 but 90% at pH 6.5. These results demonstrate that endobain E is present and also able to modulate NMDA receptor in the short-term period that follows cerebral ischemia and that its effect depends on proton concentration, suggesting greater NMDA receptor modulation by endobain E at low pH, typical of ischemic tissues.     

Endobain E, a brain Na+, K+ -ATPase inhibitor, decreases norepinephrine uptake in rat hypothalamus

The ability of an endogenous brain Na+, K+ -ATPase inhibitor, termed endobain E, to increase [3H]norepinephrine release in rat hypothalamus was previously reported. Endobain E effect on neurotransmitter uptake was studied by assaying [3H]norepinephrine uptake in rat hypothalamus preparations, to observe uptake inhibition, which reached 60% with endobain E equivalent to 100 mg fresh cerebral cortex, an effect achieved with 40 or 400 microM ouabain. Results support the proposal that endobain E behaves as an ouabain-like substance. Taken jointly results obtained on neurotransmitter release and uptake, the suggestion that endobain E may enhance norepinephrine availability in the synaptic gap and thus lead to an increase in noradrenergic activity is advanced.     

Modulation of Aspartate Release by Ascorbic Acid and Endobain E,an Endogenous Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Inhibitor

The isolation of a soluble brain fraction which behaves as an endogenous ouabain-like substance, termed endobain E, has been described. Endobain E contains two Na⁺, K⁺-ATPase inhibitors, one of them identical to ascorbic acid. Neurotransmitter release in the presence of endobain E and ascorbic acid was studied in non-depolarizing (0 mM KCl) and depolarizing (40 mM KCl) conditions. Synaptosomes were isolated from cerebral cortex of male Wistar rats by differential centrifugation and Percoll gradient. Synaptosomes were preincubated in HEPES-saline buffer with 1 mM d-[³H]aspartate (15 min at 37°C), centrifuged, washed, incubated in the presence of additions (60 s at 37°C) and spun down; radioactivity in the supernatants was quantified. In the presence of 0.5–5.0 mM ascorbic acid, d-[³H]aspartate release was roughly 135–215% or 110–150%, with or without 40 mM KCl, respectively. The endogenous Na⁺, K⁺-ATPase inhibitor endobain E dose-dependently increased neurotransmitter release, with values even higher in the presence of KCl, reaching 11-times control values. In the absence of KCl, addition of 0.5–10.0 mM commercial ouabain enhanced roughly 100% d-[³H]aspartate release; with 40 mM KCl a trend to increase was recorded with the lowest ouabain concentrations to achieve statistically significant difference vs. KCl above 4 mM ouabain. Experiments were performed in the presence of glutamate receptor antagonists. It was observed that MPEP (selective for mGluR5 subtype), failed to decrease endobain E response but reduced 50–60% ouabain effect; LY-367385 (selective for mGluR1 subtype) and dizocilpine (for ionotropic NMDA glutamate receptor) did not reduce endobain E or ouabain effects. These findings lead to suggest that endobain E effect on release is independent of metabotropic or ionotropic glutamate receptors, whereas that of ouabain involves mGluR5 but not mGluR1 receptor subtype. Assays performed at different temperatures indicated that in endobain E effect both exocytosis and transporter reversion are involved. It is concluded that endobain E and ascorbic acid, one of its components, due to their ability to inhibit Na⁺, K⁺-ATPase, may well modulate neurotransmitter release at synapses.     

Effects of Monovalent and Divalent Cations on 3-(+)[125I]Iododizocilpine Binding to the N-Methyl-d-Aspartate Receptor of Rat Brain Membranes

We have investigated the binding of 3-[125I]iododizocilpine ([125I]iodo-MK-801) to the N-methyl-D-aspartate (NMDA) receptor in well-washed rat brain membranes. [125I]Iododizocipline binding was displaced by the following: dizocilpine greater than thienylphencyclidine greater than phencyclidine greater than ketamine. Binding of [125I]iododizocilpine was enhanced by glutamate, glycine, and spermidine, whose actions could be reversed by CGS-19755, 7-chlorokynurenate, and arcaine, respectively. [125I]Iododizocilpine binding was also enhanced by a number of divalent cations, including Ba2+, Ca2+, Mg2+, Mn2+, and Sr2+, and several monovalent cations, including Na+ and K+. These cations enhanced [125I]iododizocilpine binding by an action at the polyamine site. In addition, the inhibitory effects associated with high concentrations of these cations was markedly reduced compared to those found in previous studies with [3H]dizocilpine. Analysis of the ability of spermidine, Mg2+, and Sr2+ to alter the inhibition of [125I]iododizocilpine by arcaine gave pA2 values of 5.41, 4.47, and 4.93, corresponding to EC50 concentrations of 3.9, 34.7, and 12.0 microM, respectively, suggesting that physiological concentrations of Mg2+ may occupy the polyamine site. These results demonstrate that [125I]iododizocilpine is a useful probe for the NMDA receptor. Moreover, its high specific activity and relative insensitivity to the inhibitory actions of divalent cations should make [125I]iododizocilpine a valuable ligand for the study of NMDA receptors in intact cellular systems.     

[3H]Ouabain binding and Na+, K+-ATPase in resealed human red cell ghosts

The interaction of the cardiac glycoside [3H]ouabain with the Na+, K+ pump of resealed human erythrocyte ghosts was investigated. Binding of [3H]ouabain to high intracellular Na+ ghosts was studied in high extracellular Na+ media, a condition determined to produce maximal ouabain binding rates. Simultaneous examination of both the number of ouabain molecules bound per ghost and the corresponding inhibition of the Na+, K+-ATPase revealed that one molecule of [3H]ouabain inhibited one Na+, K+-ATPase complex. Intracellular magnesium or magnesium plus inorganic phosphate produced the lowest ouabain binding rate. Support of ouabain binding by adenosine diphosphate (ADP) was negligible, provided synthesis of adenosine triphosphate (ATP) through the residual adenylate kinase activity was prevented by the adenylate kinase inhibitor Ap5A. Uridine 5'-triphosphate (UTP) alone did not support ouabain binding after inhibition of the endogenous nucleoside diphosphokinase by trypan blue and depletion of residual ATP by the incorporation of hexokinase and glucose. ATP acting solely at the high- affinity binding site of the Na+, K+ pump (Km approximately 1 microM) promoted maximal [3H]ouabain binding rates. Failure of 5'-adenylyl-beta- gamma-imidophosphate (AMP-PNP) to stimulate significantly the rate of ouabain binding suggests that phosphorylation of the pump was required to expose the ouabain receptor.     

Affinity labeling of the digitalis receptor with p-nitrophenyltriazene-ouabain, a highly specific alkylating agent

Three derivatives of ouabain have been synthesized which alkylate the digitalis receptor. These derivatives were formed through reductive amination of p-nitrophenyltriazene (NPT) ethylenediamine to the periodate-oxidized rhamnose moiety of ouabain. The non-covalent binding of the ouabain derivatives (NPT-ouabain, designated I, II, and III) was followed (i) by their ability to inhibit the activity of sodium- and potassium-activated ATPase ((Na+,K+)-ATPase) purified from the electric organ of Electrophorus electricus, (ii) by the binding of [3H]NPT-ouabain I to the enzyme, and (iii) by the inhibition of [3H]ouabain binding with unlabeled NPT-ouabain I. Covalent modification of the digitalis site of (Na+,K+)-ATPase occurs after long periods of time. At pH 7.5 (25 degrees C) the best alkylating derivative, NPT-ouabain I, gives maximum covalent labeling after 6 h. Only the large polypeptide chain (Mr = 93,000) of the purified enzyme is specifically labeled with [3H]NPT-ouabain I while the glycoprotein chain (Mr = 47,000) is not significantly labeled. Labeling of a microsomal fraction of the electric organ with [3H]NPT-ouabain I gave the same type of gel pattern as that observed with the purified enzyme. [3H]NPT-ouabain I was also used to label the digitalis receptor in highly purified axonal membranes and in cardiac membranes prepared from embryonic chick heart. Although the (Na+,K+)-ATPase in both types of membranes has a low affinity for ouabain, [3H]NPT-ouabain I proved to be a very efficient affinity label for the digitalis receptor. In the complex mixture of polypeptides found in these membrane preparations, only a single polypeptide chain having a Mr = 93,000 is specifically labeled by [3H]NPT-ouabain I.     

Na+, K+-ATPase of the canine mesenteric artery

Na+,K+-ATPase, the enzymatic moiety that operates as the electrogenic sodium-potassium pump of the cell plasma membrane, is inhibited by cardiac glycosides, and this specific interaction of a drug with an enzyme has been considered to be responsible for digitalis-induced vascular smooth muscle contraction. Although studies aimed at localization, isolation, and measurement of the Na+,K+-ATPase activity (or Na+, K- pump activity) indicate its presence in vascular smooth muscle sarcolemma, its characterization as the putative vasopressor receptor site for cardiac glycosides has depended on pharmacological studies of vascular response in vivo and on isolated artery contractile responses in vitro. More recently, radioligand-binding studies using [3H]ouabain have aided in the characterization of drug-enzyme interaction. Such studies indicate that in canine superior mesenteric artery (SMA), Na+,K+-ATPase is the only specific site of interaction of ouabain with resultant inhibition of the enzyme. The characteristics of [3H]ouabain binding to this site are similar to those of purified or partially purified Na+,K+-ATPase of other tissues, which suggests that if Na+,K+-ATPase inhibition is causally related to digitalis-mediated effects on vascular smooth muscle contraction, then therapeutic concentrations of cardiac glycosides could act to cause SMA vasoconstriction. The additional finding from radioligand-binding studies that Na+,K+-ATPase exists in much smaller quantities (density of sites per cell) in SMA than in either heart or kidney may have implications concerning its physiological, biochemical or pharmacological role in modulating vascular muscle tone.     

Modification of (Na+,K+)-ATPase function by purified antibodies to the holoenzyme. Effects on enzyme activity and (3H)ouabain binding.

Antibodies (abys) raised to (Na+,K+)-ATPase were purified by elution methods and shown to be cross-reactive with anti-gamma-globulin and the original antigen. Abys were isolated from two different antisera and the effects on (Na+,K+)-ATPase hydrolytic activity and [3H]ouabain binding were measured. The antisera fractions differed as to their maximum level of inhibition of hydrolytic activity and maximal [3H]ouabain binding, but both fractions caused inhibition of maximal [3H]ouabain binding to the same quantitative extent as inhibition of hydrolytic activity. Variable effects on the rate of [3H]ouabain binding were noted which were highly dependent on ligand conditions. During the "turnover state conditions" of the enzyme, the abys stimulated the rate of [3H]ouabain binding to the (Na+,K+)-ATPase. We conclude that effects of aby-(Na+,K+)-ATPase interaction depend upon degree of purity of aby, specificity, aby/enzyme ratios, and ligand conditions.     

Quantification of Rat Cerebral Cortex Na+,K+-ATPase: Effect of Age and Potassium Depletion

Na+,K(+)-ATPase concentration in rat cerebral cortex was studied by vanadate-facilitated [3H]ouabain binding to intact samples and by K(+)-dependent 3-O-methylfluorescein phosphatase activity determinations in crude homogenates. Methodological errors of both methods were evaluated. [3H]Ouabain binding to cerebral cortex obtained from 12-week-old rats measured incubating samples in buffer containing [3H]ouabain, and ouabain at a final concentration of 1 x 10(-6) mol/L gave a value of 11,351 +/- 177 (n = 5) pmol/g wet weight (mean +/- SEM) without any significant variation between the lobes. Evaluation of affinity for ouabain was in agreement with a heterogeneous population of [3H]ouabain binding sites. K(+)-dependent 3-O-methylfluorescein phosphatase activity in crude cerebral homogenates of age-matched rats was 7.24 +/- 0.14 (n = 5) mumol/min/g wet weight, corresponding to a Na+,K(+)-ATPase concentration of 12,209 +/- 236 pmol/g wet weight. It was concluded that the present methods were suitable for quantitative studies of cerebral cortex Na+,K(+)-ATPase. The concentration of rat cerebral cortex Na+,K(+)-ATPase showed approximately 10-fold increase within the first 4 weeks of life to reach a plateau of approximately 11,000-12,000 pmol/g wet weight, indicating a larger synthesis of Na+,K+ pumps than tissue mass in rat cerebral cortex during the first 4 weeks of development. K+ depletion induced by K(+)-deficient fodder for 2 weeks resulted in a slight tendency toward a reduction in K+ content (6%, p > 0.5) and Na+,K(+)-ATPase concentration (3%, p > 0.4) in cerebral cortex, whereas soleus muscle K+ content and Na+,K(+)-ATPase concentration were decreased by 30 (p < 0.02) and 32% (p < 0.001), respectively. Hence, during K+ depletion, cerebral cortex can maintain almost normal K+ homeostasis, whereas K+ as well as Na+,K+ pumps are lost from skeletal muscles.     

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

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

The colonic H+,K+-ATPase functions as a Na+-dependent K+(NH4+)-ATPase in apical membranes from rat distal colon.

Recent studies have suggested that the colonic H+,K+-ATPase (HKalpha2) can secrete either Na+ or H+ in exchange for K+. If correct, this view would indicate that the transporter could function as either a Na+ or a H+ pump. To investigate this possibility a series of experiments was performed using apical membranes from rat colon which were enriched in colonic H+,K+-ATPase protein. An antibody specific for HKalpha2 was employed to determine whether HKalpha2 functions under physiological conditions as a Na+-dependent or Na+-independent K+-ATPase in this same membrane fraction. K+-ATPase activity was measured as [gamma-32P]ATP hydrolysis. The Na+-dependent K+-ATPase accounted for approximately 80% of overall K+-ATPase activity and was characterized by insensitivity to Sch-28080 but partial sensitivity to ouabain. The Na+-independent K+-ATPase activity was insensitive to both Sch-28080 and ouabain. Both types of K+-ATPase activity substituted NH4+ for K+ in a similar manner. Furthermore, our results demonstrate that when incubated with native distal colon membranes, the blocking antibody inhibited dramatically Na+-dependent K+-ATPase activity. Therefore, these data demonstrate that HKalpha2 can function in native distal colon apical membranes as a Na+-dependent K+-ATPase. Elucidation of the role of the pump as a transporter of Na+ versus H+ or NH4+ versus K+ in vivo will require additional studies.     

Involvement of (Na+ + K+)-ATPase in binding and actions of palytoxin on human erythrocytes

Palytoxin (about 1 pM) increases the permeability of human erythrocytes. We now report its radiolabeling with 125I, followed by affinity purification on porcine kidney membranes. The resulting ligand binds fast and reversibly to intact erythrocytes. The Kd from velocity and equilibrium measurements is 2 X 10(-11) M, and the number of binding sites about 200 per cell. Binding is promoted by divalent cations (Ca2+ greater than Sr2+ greater than Ba2+) and by borate. It is inhibited by K+ (IC50 2 mM), ouabain (IC50 3 X 10(-9) M) and ouabagenin (IC50 6 X 10(-6) M). Conversely, [3H]ouabain is displaced by the substances and concentrations mentioned, and also by palytoxin (Ki 3 X 10(-11) M). Dog erythrocytes, which are known to possess a very low (Na+ + K+)-ATPase activity, are resistant to and lack specific binding sites for palytoxin. Binding of 125I-palytoxin, like that of [3H]ouabain, depends on the state of (Na+ + K+)-ATPase. ATP depletion decreases binding of both ligands to erythrocytes. Binding of 125I-palytoxin and [3H]ouabain to red cell stroma is partially restored by ATP. In contrast to [3H]ouabain, binding of 125I-palytoxin to red cell stroma is not promoted by Mg2+ and Pi. The data show that (a) all known promoters and inhibitors of palytoxin action on human red cells do so by enhancing or decreasing its binding, (b) (Na+ + K+)-ATPase serves as a receptor for palytoxin, and (c) the antagonism by ouabain is competitive at the receptor level. They support our previous hypothesis that palytoxin increases human erythrocyte permeability by formation of pores through (Na+ + K+)-ATPase or its close vicinity.     

Na+, K+ and Ca2+ antagonize the glutamate- and glycine-induced decrease of [3H]MK-801 binding observed in the presence of Mg2+ at low pH.

NMDA receptors are glutamate-regulated ion channels that are of great importance for many physiological and pathophysiological conditions in the mammalian central nervous system. We have previously shown that, at low pH, glutamate decreases binding of the open-channel blocker [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten, 5,10-imine ([3H]MK-801) to NMDA receptors in the presence of 1 mM Mg2+ but not in Krebs buffer. Here, we investigated which cations that block the glutamate-induced decrease in Krebs buffer, using [3H]MK-801 binding assays in membrane preparations from the rat cerebral cortex. At pH 6.0, Na+, K+, and Ca2+ antagonized the glutamate-induced decrease with cross-over values, which is a measure of the antagonist potencies of the cations, of 81, 71, and 26 mM, respectively, in the absence of added glycine. Thus, in Krebs buffer only the concentration of Na+ (126 mM) is sufficiently high to block the glutamate-induced decrease observed at low pH. In the presence of 1 mM Mg2+ and 10 mM Ca2+ at pH 7.4, the cross-over values for Na+, K+, and Ca2+ were 264, 139, and 122 mM, respectively, in the absence of added glycine. This is the same rank order of potency as observed at pH 6.0, suggesting that the less H+-sensitive and the less Ca2+-sensitive, glutamate-induced decreases in [3H]MK-801 binding represent the same entity. The glycine site antagonists 7-chlorokynurenate (10 microM) and 7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(H)-quinoline (L-701,324; 1 microM) antagonized the glutamate-induced decrease in [3H]MK-801 binding observed in presence of Mg2+ at pH 6.0, suggesting that glycine is required together with glutamate to induce the decrease observed at low pH. These results suggest that in addition to a previously described high-affinity binding site for H+ and Ca2+ there exist a low-affinity binding site for H+, Ca2+, Na+, and K+ on NMDA receptors. The latter site may under physiological conditions be blocked by Na+ or K+, depending on the extra/intracellular localization of the modulatory site. Both the high-affinity and low-affinity cation sites mediate antagonistic effects on the glutamate- and glycine-induced decrease of the affinity of the [3H]MK-801 binding site, which may correspond to similar changes in the affinity of the voltage-sensitive Mg2+-block site inside the NMDA receptor channel pore, which in turn may affect current and Ca2+ influx through activated NMDA receptor channels.     

Autoradiographic localisation of [3H]ouabain bound to cultured epithelial cell monolayers of MDCK cells

[3H]Ouabain binding to intact MDCK (cultured monolayers of dog kidney) cells of 60 serial passages is dependent upon ouabain concentration, time and medium K+. By utilising high K+ incubations to estimate non-specific [3H]ouabain-binding, the concentration of ouabain giving half maximal specific binding was estimated to be 1.0 . 10(-7) M and the total maximum binding to be 2.33 . 10(5) sites/cell. Ouabain inhibition of (Na+, K+)-pump function was monitored by the cellular uptake of 86Rb over 5 min. The larger fraction of 86Rb uptake was ouabain sensitive and the ouabain concentration giving half-maximal inhibition was 2 . 10(-7) M. The cellular distribution of the (Na+ + K+)-ATPase was investigated using [3H]ouabain autoradiography of intact freeze-dried epithelial monolayers of MDCK cells grown upon millipore filter supports. Binding of [3H]ouabain is localised over the lateral cellular membranes. Autoradiographic silver grain density is close to background levels over both the apical and basal (attachment) membranes.     

Phe783, Thr797, and Asp804 in transmembrane hairpin M5-M6 of Na+,K+-ATPase play a key role in ouabain binding

Ouabain is a glycoside that binds to and inhibits the action of Na+,K+-ATPase. Little is known, however, about the specific requirements of the protein surface for glycoside binding. Using chimeras of gastric H+,K+-ATPase and Na+,K+-ATPase, we demonstrated previously that the combined presence of transmembrane hairpins M3-M4 and M5-M6 of Na+,K+-ATPase in a backbone of H+,K+-ATPase (HN34/56) is both required and sufficient for high affinity ouabain binding. Since replacement of transmembrane hairpin M3-M4 by the N terminus up to transmembrane segment 3 (HNN3/56) resulted in a low affinity ouabain binding, hairpin M5-M6 seems to be essential for ouabain binding. To assess which residues of M5-M6 are required for ouabain action, we divided this transmembrane hairpin in seven parts and individually replaced these parts by the corresponding sequences of H+,K+-ATPase in chimera HN34/56. Three of these chimeras failed to bind ouabain following expression in Xenopus laevis oocytes. Altogether, these three chimeras contained 7 amino acids that were specific for Na+,K+-ATPase. Individual replacement of these 7 amino acids by the corresponding amino acids in H+,K+-ATPase revealed a dramatic loss of ouabain binding for F783Y, T797C, and D804E. As a proof of principle, the Na+,K+-ATPase equivalents of these 3 amino acids were introduced in different combinations in chimera HN34. The presence of all 3 amino acids appeared to be required for ouabain action. Docking of ouabain onto a three-dimensional-model of Na+,K+-ATPase suggests that Asp804, in contrast to Phe783 and Thr797, does not actually form part of the ouabain-binding pocket. Most likely, the presence of this amino acid is required for adopting of the proper conformation for ouabain binding.     

Localization and Characterization of Binding Sites with High Affinity for [3H]Ouabain in Cerebral Cortex of Rabbit Brain Using Quantitative Autoradiography

[3H]Ouabain binding was studied in sections of rabbit somatosensory cortex by quantitative autoradiography and in rabbit brain microsomal membranes using a conventional filtration assay. KD values of 8-12 nM for specific high-affinity binding of [3H]ouabain were found by both methods. High-affinity binding was not uniformly distributed in somatosensory cortex and was localized predominantly to laminae 1, 3, and 4. [3H]Ouabain binding in tissue sections was stimulated by the ligands Mg2+/Pi or Mg2+/ATP/Na+ and was inhibited by K+ (IC50 = 0.7-0.9 mM), N-ethylmaleimide, 5,5'-dithiobis(2-nitrobenzoic acid), and erythrosin B. We conclude that [3H]ouabain is reversibly and specifically bound with high affinity in rabbit brain tissue sections under conditions that favor phosphorylation of Na+,K+-ATPase. Quantitative autoradiography is a powerful tool for assessing the affinity and number of specific ouabain binding sites in brain tissue.     

Age-related changes in [3H] ouabain binding to synaptic plasma membranes isolated from mouse brains.

Age-related changes in ouabain binding to synaptic plasma membranes isolated from cerebral cortices of C57BL/6 mice were investigated to examine whether the density of Na+, K(+)-ATPase decreases with advancing age. Specific binding of [3H]ouabain did not change until around 20 months of age, but a 22% decrease in binding was found in the late senescent stage (29 months). Scatchard analysis of the binding revealed that the maximum number of binding sites (Bmax) was lower in aged mice, while the binding affinity (Kd) for ouabain receptor remained unchanged with aging. These results indicate that the density of Na+,K(+)-ATPase enzyme sites in the plasma membranes of brain synapses decreases in aged mice. Since the activity of Na+,K(+)-ATPase has been found to start declining at a much earlier stage [Tanaka, Y. & Ando, S. (1990) Brain Res. 506, 46-52; Ando, S. & Tanaka, Y. (1990) Gerontology 36, 10-14] than that at which the decrease of Bmax is manifested, at least two mechanisms may underlie the age-related decrease of the enzyme activity. We speculate that the lipid microenvironment which regulates the enzyme activity starts to change at the early stage of senescence, followed by the decrease in the enzyme content in the later stage, that is, both changes cooperatively diminish the Na+,K(+)-ATPase activity in senescence.     

Amplification of sodium- and potassium-activated adenosinetriphosphatase in HeLa cells by ouabain step selection

A multistep selection for ouabain resistance was used to isolate a clone of HeLa S3 cells that overproduces the plasma membrane sodium, potassium activated adenosinetriphosphatase (Na+,K+-ATPase). Measurements of specific [3H]ouabain-binding to the resistant clone, C+, and parental HeLa cells indicated that C+ cells contain 8-10 X 10(6) ouabain binding sites per cell compared with 8 X 10(5) per HeLa cell. Plasma membranes isolated from C+ cells by a vesiculation procedure and analyzed for ouabain-dependent incorporation of [32P]phosphate into a 100,000-mol-wt peptide demonstrated a ten- to twelvefold increase in Na+,K+-ATPase catalytic subunit. The affinity of the enzyme for ouabain on the C+ cells was reduced and the time for half maximal ouabain binding was increased compared with the values for the parental cells. The population doubling time for cultures of C+ cells grown in dishes was increased and C+ cells were unable to grow in suspension. Growth of C+ cells in ouabain-free medium resulted in revertant cells, C-, with biochemical and growth properties identical with HeLa. Karyotype analysis revealed that the ouabain-resistant phenotype of the C+ cells was associated with the presence of minute chromosomes which are absent in HeLa and C- cells. This suggests that a gene amplification event is responsible for the Na+,K+-ATPase increase in C+ cells.     

Constitution and properties of axonal membranes of crustacean nerves.

The purification of axonal membranes of crustaceans was followed by measuring enrichment in [3H]tetrodotoxin binding capacity and in Na+, K+-ATPase activity. A characteristic of these membranes is their high content of lipids and their low content of protein as compared to other types of plasmatic membranes. The axonal membrane contains myosin-like, actin-like, tropomyosin-like, and tubulin-like proteins. It also contains Na+, K+-ATPase and acetylcholinesterase. The molecular weights of these two enzymes after solubilization are 280,000 and 270,000, respectively. The molecular weights of the catalytic subunits are 96,000 for ATPase and 71,000 for acetylcholinesterase. We confirmed the presence of a nicotine binding component in the axonal membrane of the lobster but we have been unable to find [3H]nicotine binding to crab axonal membranes. The binding to axonal membranes og of the sodium channel, has been studied in detail. The dissociation constant for the binding of [3H]tetrodotoxin to the axonal membrane receptor is 2.9 nM at pH 7.4. The concentration of the tetrodotoxin receptor in crustacean membranes is about 10 pmol/mg of membrane protein, 7 times less than the acetylcholinesterase, 30 times less than the Na+, K+-ATPase, and 30 times less than the nicotine binding component in the lobster membrane. A reasonable estimate indicates that approximately only one peptide chain in 1000 constitutes the tetrodotoxin binding part of the sodium channel in the axonal membrane. Veratridine, which acts selectively on the resting sodium permeability, binds to the phospholipid part of the axonal membrane. [3H]Veratridine binding to membranes parallels the electrophysiological effect. Veratridine and tetrodotoxin have different receptor sites. Although tetrodotoxin can repolarize the excitable membrane of a giant axon depolarized by veratridine, veratridine does not affect the binding of [3H]tetrodotoxin to purified axonal membranes. Similarly, tetrodotoxin does not affect the binding of [3H]veratridine to axonal membranes. Scorpion neurotoxin I, a presynaptic toxin which affects both the Na+ and the K+ channels, does not interfere with the binding of [3H]tetrodotoxin or [3H]veratridine to axonal membranes. Tetrodotoxin, veratridine, and scorpion neurotoxin I, which have in common the perturbation of the normal functioning of the sodium channel, act upon three different types of receptor sites.