Lionel Lelievre

Two active Na+/K+-ATPases of high affinity for ouabain in adult rat brain membranes.

The degree of heterogeneity of active Na+/K(+)-ATPases has been investigated in terms of ouabain sensitivity. A mathematical analysis of the dose-response curves (inhibition of Na+/K(+)-ATPase) at equilibrium is consistent with the putative existence of three inhibitory states for ouabain two of high (very high plus high) and one of low affinity. The computed IC50 values are: 23.0 +/- 0.15 nM, 460 +/- 4.0 nM and 320 +/- 4.6 microM, respectively. The relative abundance of the three inhibitory states was estimated as: 39%, 36% and 20%, respectively. Direct measurements of [3H]ouabain-binding at equilibrium carried out on membrane preparations with ATP, Mg2+ and Na+ also revealed two distinct high affinity-binding sites, the apparent Kd values of which were 17.0 +/- 0.2 nM (very high) and 80 +/- 1 nM (high), respectively. Dissociation processes were studied at different ouabain concentrations according to both reversal of enzyme inhibition and [3H]ouabain release. The reversal of enzyme inhibition occurred at three different rates, depending upon the ouabain doses used (10 nM, 2 and 100 microM). When the high-affinity sites were involved (ouabain doses lower than 2 microM) the dissociation process was biphasic. A similar biphasic pattern was also detected by [3H]ouabain-release. The time-course of [3H]ouabain dissociation (0.1 microM) was also biphasic. These data indicate that the three catalytic subunits of rat brain Na+/K(+)-ATPase alpha 1, alpha 2 and alpha 3 (Hsu, Y.-M. and Guidotti, G. (1989) Biochemistry 28, 569-573) are able to hydrolyse ATP and exhibit different affinities for cardiac glycosides.

Rat cardiac hypertrophy. Altered sodium-calcium exchange activity in sarcolemmal vesicles.

The sodium‐calcium exchange activity has been studied in sarcolemmal vesicles isolated from rat ventricles hypertrophied by pressure overload. 4 weeks after aortic stenosis the degree of hypertrophy varied from 30 to 70%. The Na+‐dependent 45Ca2+ influx and efflux were up to 50% decreased and the sensitivity to Ca2+ was 13‐fold lower in vesicles from hypertrophied heart as compared to those from normal heart. However, the Na+,K+‐ATPase activity, the orientation of the vesicles and the passive Ca2+ permeability were found to be similar in the two heart groups. These results indicate that the sarcolemmal Na+/Ca2+ exchange activity could be qualitatively and/or quantitatively changed in hypertrophied rat heart.

Inhibition of (Na+ + K+)-ATPase by ouabain: Involvement of calcium and membrane proteins

Treatment of plasma membrane isolated from murine plasmocytoma MOPC 173 with an EDTA-containing buffer resulted in a 300-fold increase in sensitivity of (Na+ + K+)-stimulated Mg2+-ATPase to ouabain. This phenomenon was associated with the solubilization by EDTA of phospholipid free proteins (approx. 30 000-34 000 daltons) from the cytoplasmic face of the plasma membrane and with removal of about 90% of the membrane bound Ca2+. The recovery of the original resistance to ouabain required specifically Ca2+ and was associated with a binding of the solubilized proteins to the membrane.

Plasma membranes from fibroblastic cells in culture isolation, morphological and enzymatic identification

Abstract Plasma membranes were isolated from murine plasmocytoma cells in culture, by a procedure involving lysis in hypoosmotic medium leaving the nuclei intact, and separation of surface membranes from the lysate constituents on a discontinuous sucrose gradient. The purity of the fractions was assessed by electron microscopy and by assaying enzymes for cross-contaminants. Phosphohydrolases, including the (Na+ + K+)-stimulated Mg2+-ATPase (EC 3.6.1.3) and 5′-nucleotidase (EC 3.1.3.5), were concentrated in the plasma membrane-rich fractions. These fractions were essentially free from NADH: cytochrome c reductase, lysosomes and mitochondrial membrane enzymes.

Respective involvements of high- and low- affinity digitalis receptors in the inotropic response of isolated rat heart to ouabain

High- and low-affinity digitalis receptors coexist in rat cardiac sarcolemma. In this study, their relative involvement in the inotropic effect of ouabain was evaluated on an isolated Langendorff rat heart preparation working under isovolumic conditions at a low external calcium concentration (0.25 mM). This involvement was estimated according to both the development of the inotropic response to ouabain (10(-8)-10(-4)M) and the time course of the washing out of the biological effect. In each phenomenon considered, and whatever the index of inotropy chosen, the high-affinity digitalis receptor (EC50: 1-2 X 10(-8) M) contributed to 25-40% of the maximal inotropy (evoked by 10(-4) M ouabain). Low-affinity receptors (EC50: 1-2 X 10(-5) M) accounted for 60-75%. These apparent affinities were identical to those previously determined in sarcolemma isolated from rat heart perfused with 0.25 mM Ca2+. The biphasic effect of ouabain was related to both the inhibition of high- and low-sensitivity Na+, K+-ATPase forms and the corresponding number of ouabain-binding sites occupied. These results support the concept that the Na+, K+-ATPase highly sensitive to ouabain as revealed by lowering calcium is the in vivo manifestation of the high-sensitivity inotropic component.

Involvement of tropomyosin in the sensitivity of Na+ + K+ ATPase to ouabain

Abstract The Na+/K+ ATPase sensitivity to ouabain was shown to be increased by 300 to 1000-fold after treatment of the plasma membrane by EDTA. Addition of proteins detached from the plasma membrane with Ca2+ ions to EDTA treated membranes reconstituted the original Na+/K+ ATPase resistance to ouabain inhibition. Tropomyosin with Ca2+ ions (not with Mg2+ ions) induced the same effect. When suboptimal doses of tropomyosin were used for such a reconstitution, the dose-response curve indicated a full reconstitution of a given percentage of enzyme molecules. This observation led us to assume a direct or indirect effect of tropomyosin on Na+/K+ ATPase functions.

An organophosphorus compound, Vx, selectively inhibits the rat cardiac Na+,K+‐ATPase α1 isoform Biochemical basis of the cardiotoxicity of Vx

Serine‐specific reagents, anticholinesterase organophosphorus compounds like Vx provoke, in the micromolar range, digitalis‐like ventricular arrythmias of non‐cholinergic origin in rodent hearts. The sensitivities of the two rat cardiac Na+,K+‐ATPase isoforms (α1 and α2) to Vx (0.1–100 μM) were measured in sarcolemma vesicles. At 1 μM Vx, the inhibition of the total activity averaged 18% but never exceeded 75% with 100 μM. When the α2 isoform activity was inhibited by 0.1 μM ouabain, α1 was 35% inhibited by 1 μM Vx, i.e. a 16±4% inhibition of the total acitivity. The cardiac α1 being related to the digitalis‐induced toxicity, its selective inhibition by a micromolar dose of Vx fully accounts for the cardiotoxicity of Vx. Inasmuch as Vx had no effect on the rat kidney α1, differentially inactivated the cardiac isozymes and specifically reacted with serine residues, the putative binding‐site(s) of the organophosphorus compound on the Na+,K+‐ATPase molecules has been considered.

Relationship between intracellular K+ concentrations and K+ fluxes in growing and contact-inhibited cells.

The K+ content and the K+ flux were measured in the cell lines ME2 and MF2 isolated from plasmocytoma MOPC 173. Both cell lines were shown to have the seem K+ content and the same K+ steady state flux per unit of surface area. In ME2 cells, no modification of the exchange movement was observed during contact inhibition. However, contact-inhibited cells exhibited an increased resistance to depletion, characterized by a lower K+ net movement. The (Na+ plus K+)-ATPase measured in homogenates is poorly correlated to in vivo cation fluxes both because of the enhancement due, presumably, to the drop of K+ concentration on the cytoplasmic face of the membrane and because of losses during preparation which can be conspicuous, especially in contact-inhibited cells. The K+ net flux is considerable increased when the intracellular K+ level is reduced after preincubation of the cells in a K+ -free medium. Thus, internal K+ seems to regulate the K+ influx.

Three Na+,K+-ATPase forms in rat heart as revealed by K+/ouabain antagonism

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Localization of the catalytic site of 5′-nucleotidase at the inner surface of murine plasmocytoma plasma membranes

Many ‘plasma membrane-marker enzymes’ have already been described. The physiologic significance of some of them is still enigmatic, although (Na* t K’)-ATPase or adenylate cyclase, for instance, are well characterized. 5’-Nucleotidase is now generally accepted to be located on the plasma membrane, i.e., to be markedly enriched in this fraction. However, the position of the catalytic site-of this enzyme on the outer or inner face of the membrane has not been disputed in terms of cell type and plasma membrane areas. Comparing enzymatic activities of intact cells with that of disrupted cells, some authors have demonstrated that S’nucleotidase is an ecto-enzyme in various cells [ 1,2] . However, Shultz and Thompson [3] noticed an enhanced AMP hydrolysis in cell homogenate when compared to whole cells (at least 8times). So they considered the hydrolytic site being located at the cytoplasmic face. Using antibodies which inhibited the enzymatic activity assayed in plasma membranes or in purified enzyme, Curd and Evans [4] conclude that S’nucleotidase is located on the outer surface of mouse liver cells. When cytochemical procedures, involving the trapping of inorganic phosphate as lead phosphate, are used to localize this activity, the reaction product was found by many authors to be localized on the external side of plasma membranes both in isolated cell fractions and in situ