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 · 10⁵ sites/cell. Ouabain inhibition of (Na⁺, K⁺)-pump function was monitored by the cellular uptake of ^B6Rb over 5 min. The larger fraction of ^B6Rb uptake was ouabain sensitive and the ouabain concentration giving half-maximal inhibition was 2 · 10^?7 M. The cellular distribution of the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ 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.     

[3H]ouabain binding and 86rubidium uptake during the cell growth cycle in L5178Y murine lymphoblasts

There was a parallel alteration in [³H]ouabain binding and ⁸⁶Rb⁺ uptake during the cell growth cycle in L5178Y murine lymphoblasts. The initial rate of Rb⁺ uptake and [³H]ouabain binding was highest at the stationary phase of the cell growth cycle. The possible relationship between changes in cation transport and membrane properties to the cell growth cycle is discussed.     

Direct photoaffinity labeling of the primary region of the ouabain binding site of (Na+ + K+)-ATPase with [3H]ouabain, [3H]digitoxin and [3H]digitoxigenin.

The tritiated cardiotonic steroids, ouabain, digitoxin, and digitoxigenin are shown to photolabel the large polypeptide but not the glycoprotein or proteolipid component of the (Na+ + K+)-ATPase when they are bound to the inhibitory site and exposed to light of 220 or 254 nm. The extent of photolabeling is low, less than 1%, and is limited by photocross-linking of the enzyme. The mechanism of photoincorporation does not appear to be either photolysis of the lactone ring in ouabain or photolysis of tryptophan or tyrosine residues in the polypeptide.     

Characterization of the stimulation of neuronal Na+, K+-ATPase activity by low concentrations of ouabain

Low concentrations (< 10^?7 M) of ouabain stimulate the activity of Na⁺, K⁺-ATPase in whole homogenates of rat brain. The magnitude of this stimulation varies from 5 to 70%. The concentrations of ouabain which induces maximal stimulation is also highly variable and ranges between 10^?9 to 10^?7 M. The ouabain stimulation disappears following 1:50 dilution and 2 h preincubation or freezing and thawing of the membranes or their treatment with deoxycholate. “Aging” of a preparation of ATPase also results in loss of its ability to be stimulated by ouabain but ouabain inhibition is preserved. No stimulation of enzyme activity by ouabain is observed in rat brain microsomal fraction. The β-adrenergic blocker propranolol does not inhibit the ouabain induced stimulation of ATPase activity. It is suggested that the stimulation of Na⁺, K⁺-ATPase activity by low concentrations of cardiac glycosides if a result of either the displacement of an endogenous ouabain-like compound from the enzyme or an indirect effect by changing membrane surrounding environment of the Na⁺, K⁺-ATPase.     

Solvent effects on ouabain binding to the (Na+,K+)-ATPase of rat brain

The effects of the solvents deuterated water (²H₂O) and dimethyl sulfoxide (Me₂SO) on [³H]ouabain binding to (Na⁺,K⁺)-ATPase under different ligand conditions were examined. These solvents inhibited the type I ouabain binding to the enzyme (i.e., in the presence of Mg²⁺+ATP+Na⁺). In contrast, both solvents stimulated type II (i.e., Mg²⁺+P_i-, or Mn²⁺-dependent) binding of the drug. The solvent effects were not due to pH changes in the reaction. However, pH did influence ouabain binding in a differential manner, depending on the ligands present. For example, changes in pH from 7.05 to 7.86 caused a drop in the rate of binding by about 15% in the presence of Mg²⁺+Na⁺+ATP, 75% in the Mg²⁺+P_i system, and in the presence of Mn²⁺ an increase by 24% under similar conditions. Inhibitory or stimulatory effects of solvents were modified as various ligands, and their order of addition, were altered. Thus, ²H₂O inhibition of type I ouabain binding was dependent on Na⁺ concentration in the reaction and was reduced as Na⁺ was elevated. Contact of the enzyme with Me₂SO, prior to ligands for type I binding, resulted in a greater inhibition of ouabain binding than that when enzyme was exposed to Na⁺+ATP first and then to Me₂SO. Likewise, the stimulation of type II binding was greater when appropriate ligands acted on enzyme prior to addition of the solvent. Since Me₂SO and ²H₂O inhibit type I ouabain binding, it is proposed that this reaction is favored under conditions which promote loss of H₂O, and E₁ enzyme conformation; the stimulation of type II ouabain binding in the presence of the solvents suggests that this type of binding is favored under conditions which promote the presence of H₂O at the active enzyme center and E₂ enzyme conformation. This postulation of a role of H₂O in modulating enzyme conformations and ouabain interaction with them is in concordance with previous observations.     

The effect of sodium on inorganic phosphate- and p-nitrophenyl phosphate-facilitated ouabain binding to (Na+ + K+)-activated ATPase

The effect of the hydrolysis product P_i and the artificial substrate p-nitrophenyl phosphate (p-nitrophenyl-P) on ouabain binding to (Na⁺ + K⁺)-activated ATPase was investigated.The hypothesis that (Mg²⁺ + p-nitrophenyl-P)-supported ouabain binding might be due to P_i release and thus (Mg²⁺ + P_i)-supported could not be confirmed.The enzyme · ouabain complexes obtained with different substrates were characterized according to their dissociation rates after removal of the ligands facilitating binding. The character of the enzyme · ouabain complex is determined primarily by the monovalent ion present during ouabain binding, but, qualitatively at least, it is immaterial whether binding was obtained with p-nitrophenyl phosphate or P_i.The presence or absence of Na⁺ during binding has a special influence upon the character of the enzyme · ouabain complex. Without Na⁺ and in the presence of Tris ions the complex obtained with (Mg²⁺ + P_i) and that obtained with (Mg²⁺ + p-nitrophenyl-P) behaved in a nearly identical manner, both exhibiting a slow decay. High Na⁺ concentration diminished the level of P_i-supported ouabain binding, having almost no effect on p-nitrophenyl phosphate-supported binding. Both enzyme · ouabain complexes, however, now resembled the form obtained with (Na⁺ + ATP), as judged from their dissociation rates and the K⁺ sensitivity of their decay. The complexes obtained at a high Na⁺ concentration underwent a very fast decay which could be slowed considerably after adding a low concentration of K⁺ to the resuspension medium. The most stable enzyme · ouabain complex was obtained in the presence of Tris ions only, irrespective of whether p-nitrophenyl phosphate or P_i facilitated complex formation. The presence of K⁺ gave rise to a complex whose dissociation rate was intermediate between those of the complexes obtained in the presence of Tris and a high Na⁺ concentration.It is proposed that the different ouabain dissociation rates reflect different reactive state of the enzyme. The resemblance between the observations obtained in phosphorylation and ouabain binding experiments is pointed out.     

Photoincorporation of [3H]-chloropromazine into a solubilized bovine striatal preparation

Photolysis at 300 nm of [³H]-chlorpromazine (CPZ) in the presence of a digitonin-solubilized bovine striatal homogenate results in the formation of a high molecular mass component (>200,000 daltons) which does not arise upon photolysis of either [³H]-CPZ or the solubilized preparation separately. Additional components of low molecular mass are observed in both photolyzed and unphotolyzed preparations. The high molecular mass component to which [³H]-CPZ binds irreversibly contains a significant fraction of the initial [³H] activity. The formation of the high molecular mass component is inhibited by 35 ± 9% when the original photolysis is performed in the presence of 500 nM (+)-butaclamol, an active antagonist of specific binding to postsynaptic dopamine receptors, compared to photolysis in the presence of 500 nM (?)-butaclamol, which has negligible specific affinity for these dopamine receptors. EGTA at a concentration of 5mM has no effect on the photolabeling by [³H]-CPZ. SDS-polyacrylamide gel electrophoretic separation of the high molecular mass component shows a major band at ～61,000 daltons which is not evidenced in the low molecular mass fraction. These observations suggest that upon photoexcitation CPZ may photocrosslink a complex of membrane proteins which includes a neuronal DA receptor.     

Intracellular disposition of [3H]-cocaine, [3H]-norcocaine, [3H]-benzoylecgonine and [3H]-benzoylnorecgonine in the brain of rats

[³H]-cocaine, [³H]-norcocaine, [³H]-benzoylecgonine and [³H]-benzoylnorecgonine were administered i.c. in equi-potent pharmacologic doses and the intracellular disposition and metabolism of each drug determined. Norcocaine and cocaine rapidly entered and egressed from the brain so that 4.8–6.1% of the radioactivity present in brain at one minute was observed at 30 minutes. The highest levels of subcellular radioactivity were generally found in the microsomal plus supernatant, followed by the nuclear and shocked mitochondrial fractions. No apparent localization of the radioactivity occured in synaptic membranes. The brain/plasma (B/P) ratio curves for cocaine and norcocaine were similar; however, the norcocaine values were considerably higher at each time interval. Benzoylecgonine and benzoylnorecgonine had higher comparative B/P ratios than cocaine or norcocaine and persisted in brain for a longer period of time so that 0.6–2.1% of the radioactivity present in brain at 1 hour was detected at 24 hours. Cocaine and norcocaine were extensively metabolized to the benzoylmetabolites. Benzoylecgonine was metabolized to benzoylnorecgonine and benzoylnorecgonine was unmetabolized. The brain disposition data and B/P ratios agreed quite well with the overall pharmacologic action of cocaine and its metabolites.     

Estimation of ouabian specifically bound to dog renal (Na+ + K+)-ATPase in vivo

It is not known whether ouabain injected into the kidney in vivo is bound exclusively to the (Na⁺ + K⁺)-ATPase and whether the reduction of sodium pumping capacity is large enough to account for the reduction in sodium reabsorption. In the present study on dogs the total amount of parenchymal ouabain was therefore estimated and the specific renal binding compared to the reduction in (Na⁺ + K⁺)-ATPase activity. Ouabain, 120 nmol/kg body weight, was injected into the renal artery in vivo reducing the (Na⁺ + K⁺)-ATPase activity by 3lmost 80%. After nephrectomy, tissue ouabain could be quantified by radioimmunoassay after heating the homogenate to 70°C for 30 min; negligible amounts were detectable without heating. No correlation between ouabain binding and tissue volume, protein content, DNA content or Mg²⁺-ATPase content could be found when comparing the following four fractions of the kidney: outer cortex, inner cortex, outer medulla and papilla. For the whole kidney, mean parenchymal tissue concentration of ouabain equalled 0.58 ± 0.03 μmol/100 g wet tissue. Only 21.3 ± 1.2% of the ouabain was confined to the outer medulla corresponding to 54 ± 4 nmol giving a tissue concentration of 1.08 ± 0.05 μmol/100 g wet tissue. The renal ouabain concentrations were highly correlated to the reduction in (Na⁺ + K⁺)-ATPase activity, giving a ratio between the reduction in hydrolysis rate and bound ouabain (turnover number) of 6105 min^?1 which is close to the value of 7180 min^?1 found by in vitro Scatchard analysis. No ouabain seems to be bound to other tissue components than the (Na⁺ + K⁺)-ATPase and the present method is therefore a simple way of measuring the number of inhibited (Na⁺ + K⁺)-ATPase molecules after in vivo injection of ouabain.     

ACTIVE POTASSIUM TRANSPORT AND [Na++ K+]ATPase ACTIVITY IN CULTURED GLIOMA AND NEUROBLASTOMA CELLS

—The ouabain-sensitive K⁺ uptake and ATPase activities of cultured glioma and neuroblastoma cells were studied. Both cell lines showed ouabain-sensitive K⁺ uptake which correlated with the level of [Na⁺+ K⁺]ATPase activity found in the respective total cell homogenate. The glioma cells had a 2.1-fold higher rate of K⁺ uptake than neuroblastoma cells, and a 2.4-fold higher [Na⁺+ K⁺]ATPase activity. In the presence of ouabain neuroblastoma cells released K⁺ and took up Na⁺ in a 1:1 ratio. These results are compared and contrasted with similar studies on brain tissue and isolated cells. It is suggested that the cultured cell lines may serve as good models for the cation transport properties of their tissue counterparts.     

[3H]GABA binding in the cerebellum of the reeler murine mutant

In the cerebellum of the reeler mutant mouse, characterized morphologically by depletion of the granule cell population and abnormal synapse formation, increased GABA concentration and alterations in [³H]GABA binding have been observed. This study shows decreased affinity of the Na⁺-independent, high affinity GABA binding component of synaptosomal membranes and an increased affinity of the Na⁺-dependent, high affinity GABA binding component in reeler cerebellar homogenate and synaptic membranes. In contrast to the changes in affinity, the number of both Na⁺-dependent and Na⁺-independent binding sites was not significantly altered. The decreased affinity of the Na⁺-independent GABA binding and the increased affinity of the Na⁺-dependent binding, evidenced only in cerebellar tissue, were interpreted to indicate, respectively, hypo- and hypersensitivity of the postsynaptic and presynaptic elements of cerebellar GABAergic synapses, induced by the depressed excitatory granule cell input and/or the increased mossy fiber contact with the ectopic Purkinje cells.     

Purification and characterization of an (Na++K+)-ATPase proteolipid labeled with a photoaffinity derivative of ouabain

Highly purified lamb kidney (Na⁺+K⁺)-ATPase was photoaffinity labeled with the tritiated 2-nitro-5-azidobenzoyl derivative of ouabain (NAB-ouabain). The labeled (Na⁺+K⁺)-ATPase was mixed with unlabeled carrier enzyme. Two proteolipid (γ1 and γ2) fractions were then isolated by chromatography on columns of Sepharose CL-6B and Sephadex LH-60. The two fractions were interchangeable when rechromatographed on the LH-60 column, suggesting that γ1 is an aggregated form of γ2. The total yield was 0.8–1.5 mol of γ component per mol of catalytic subunit recovered. This indicates that the γ component is present in stoichiometric amounts in the (Na⁺+K⁺)-ATPase. The proteolipids that were labeled with NAB-ouabain copurified with the unlabeled proteolipids.     

Improved purification and partial characterization of (Na+, K+)-ATPase from cardiac muscle

A method is described for purification of (N⁺, K⁺)-ATPase which yields approximately 60 mg of enzyme from 800 g of cardiac muscle with specific activities ranging from 340 to 400 μmol inorganic phosphate/mg protein per h (units/mg). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated the presence of a major 94 000 dalton polypeptide and four or five lesser components, one of which was a glycoprotein with an apparent molecular weight of 58 000. The enzyme preparation bound 600–700 pmol of [³H]ouabain/mg protein when incubated in the presence of either Mg²⁺ plus P_i or Mg²⁺ plus ATP plus Na⁺, and incorporated more than 600 pmol ³²P/mg protein when incubated with γ-³²P-labeled ATP in the presence of Mg²⁺ and Na⁺. The preparation is approximately 35% pure.     

A reconstituted Na++K+ pump in liposomes containing purified (Na++K+-ATPase from kidney medulla

Liposomes containing either purified or microsomal (Na⁺,K⁺)-ATPase preparations from lamb kidney medulla catalyzed ATP-dependent transport of Na⁺ and K⁺ with a ratio of approximately 3Na⁺ to 2K⁺, which was inhibited by ouabain. Similar results were obtained with liposomes containing a partially purified (Na⁺,K⁺-ATPase from cardiac muscle. This contrasts with an earlier report by Goldin and Tong (J. Biol. Chem. 249, 5907–5915, 1974), in which liposomes containing purified dog kidney (Na⁺,K⁺)-ATPase did not transport K⁺ but catalyzed ATP-dependent symport of Na⁺ and Cl^?. When purified by our procedure, dog kidney (Na⁺,K⁺)-ATPase showed some ability to transport K⁺ but the ratio of Na⁺ : K⁺ was 5 : 1.     

Binding of [3H]cytochalasin B and [3H]colchicine to isolated liver plasma membranes

The binding to isolated hepatocyte plasma membranes of radioactively labelled inhibitors of microfilamentous and microtubular protein function ([³H]cytochalasin B and [³H]colchicine, respectively) was studied as one means of assessing the degree of association of these proteins with cell surface membranes. [³H]Cytochalasin B which behaved identically to the unlabelled compound with respect to binding to these membranes was prepared by reduction of cytochalasin A with NaB³H₄. The binding was rapid, readily reversible, proportional to the amount of membrane and relatively insentive to changes of pH or ionic strength. At 10^?6 M [³H]cytochalasin B, glucose or p-chloromercuribenzoate, an inhibitor of glucose transport inhibited binding by about 20%; treatment of membranes with 0.6 M KI which depolymerizes F actin to G actin caused about 60% inhibition of binding. These two types of inhibition were additive indicating two separate classes of binding sites, one associated with sugar transport and one with microfilaments. Filamentous structures with the diameter of microfilaments (50 Å) were seen in electron micrographs of thin sections of the membranes. At concentrations greater than 10^?5 M [³H]cytochalasin B, binding was proportional to drug concentration, characteristic of non-specific adsorption or partitioning. Intracellular membranes of the hepatocyte also bound [³H]cytochalasin B, those of the smooth endoplasmic reticulum to a greater extent than plasma membranes.[³H]Colchicine bound to plasma membranes in proportion to the amount of membrane and at a rate compatible with binding to tubulin. However, other properties of the binding including effects of temperature, drug concentration and antisera against tubulin were different from those of binding to tubulin. Hence, no evidence was obtained for association of microtubular elements with these membranes. Despite this there appeared to be an interdependence between microtubule and microfilament inhibitors: vinblastine sulfate stimulated [³H]cytochalasin B binding and cytochalasin B stimulated ³H colchicine binding. [³H]Colchicine also bound to intracellular membranes, especially smooth microsomes.     

Interaction of Ca2+ with (Na+ + K+)-ATPase: Properties of the Ca2+-stimulated phosphatase activity

Ca²⁺ inhibited the Mg²⁺-dependent and K⁺-stimulated p-nitrophenylphosphatase activity of a highly purified preparation of dog kidney (Na⁺ + K⁺)-ATPase. In the absence of K⁺, however, a Mg²⁺-dependent and Ca²⁺-stimulated phosphatase was observed, the maximal velocity of which, at pH 7.2, was about 20% of that of the K⁺-stimulated phosphatase. The Ca²⁺-stimulated phosphatase, like the K⁺-stimulated activity, was inhibited by either ouabain or Na⁺ or ATP. Ouabain sensitivity was decreased with increase in Ca²⁺, but the K_0.5 values of the inhibitory effects of Na⁺ and ATP were independent of Ca²⁺ concentration. Optimal pH was 7.0 for Ca²⁺-stimulated activity, and 7.8–8.2 for the K⁺-stimulated activity. The ratio of the two activities was the same in several enzyme preparations in different states of purity. The data indicate that (a) Ca²⁺-stimulated phosphatase is catalyzed by (Na⁺ + K⁺)-ATPase; (b) there is a site of Ca²⁺ action different from the site at which Ca²⁺ inhibits in competition with Mg²⁺; and (c) Ca²⁺ stimulation can not be explained easily by the action of Ca²⁺ at either the Na⁺ site or the K⁺ site.     

Effect of ouabain upon diuretic-sensitive K+ transport in cultured cells evidence for separate modes of operation of the transporter

(1) Unidirectional K⁺ (⁸⁶Rb) influx and efflux were measured in subconfluent layers of MDCK renal epithelial cells and HeLa carcinoma cells. (2) In both MDCK and HeLa cells, the furosemide-inhibitable and chloride-dependent component of K⁺ influx/efflux was stimulated 2-fold by a 30 min incubation in 1 · 10^?3 M ouabain. (3) Measurements of net K⁺ loss and Na⁺ gain in ouabain-treated cells at 1 h failed to show any diuretic sensitive component, confirming the exchange character of the diuretic-sensitive fluxes. (4) Prolonged incubations for 2.5 h in ouabain revealed a furosemide- and anion-dependent K⁺ (Cl^?) outward net flux uncoupled from net Na⁺ movement. Net K⁺ (Cl^?) outward flux was half-maximally inhibited by 2 μM furosemide. (5) After 2.5 h ouabain treatment, the anion and cation dependence of the diuretic-sensitive K⁺ influx/efflux were essentially unchanged when compared to untreated controls.     

Modulation of striatal [3H]-glutamic acid release by dopaminergic drugs

Potassium induced release of [³H]-glutamic acid which had been recently taken up into striatal tissue in vitro was demonstrated and shown to be predominantly Ca²⁺-dependent. Dopamine and several DA agonists produced marked inhibition of this K⁺ -induced release which could be antagonised by butyrophenones. The effect of DA could not be mimicked by high concentrations of dibutyryl cAMP. These results are consistent with the existence of a D2-like DA receptor on striatal glutamatergic terminals capable of modulating the transmitter release.     

Ouabain binding sites and the (Na+,K+)-ATPase of brain microsomal membranes

Beef brain microsomes bound approximately 180–220 pmoles of [³H]ouabain per mg of protein in the presence of either MgCl₂ and inorganic phosphate or ATP, MgCl₂ and NaCl. The ouabain-binding capacity and the ouabain-membrane complex were more stable than the (Na⁺,K⁺)-ATPase activity to treatment with agents known to affect the membrane integrity, such as, NaClO₄, sodium dodecyl sulfate, $\text{p-}\text{chloromercuribenzoate}$, urea. ultrasonication, heating, pH and phospholinase C.The presence of binding sites that were normally inaccessible to ouabain in brain microsomes was demonstrated. These sites appeared after disruption of microsomes with 2 M NaClO₄ as evidenced by increased binding of [³H]ouabain. These sites may be buried during the subcellular fractionation procedure and could be accessible in the intact cell.