Reprinted irom Na/K-ATPase and Rclalcd Transport ATPascs Structure, Mechanism, and Régulation Volume 834 of ihe Aimais ofthe New York Academy of Sciences November3, 1997
Transposing Results from an Artiiicial Minicell to a Real Cell Expérimental Evidence for a Working Hypothesis Linking Na,K-ATPase Permeability States to Spécifie Altérations of Cell Life" BEATRICE M. ANNER* Department of Pharmacology Geneva University Médical School, CMU CH-1211 Geneva 4, Switzerland The Na,K-ATPase is an ancestral and ubiquitous membrane transport System; ils présence is indissociable from cell life because it establishes and maintains a steep reciprocal Na/K gradient across thé cell membrane, a fundamental biological mechanism for biophysical, osmotic, and signaling purposes. In view of ils importance, spécifie altérations in thé functioning and structural integrity ofthe Na,K-ATPase will predictably be echoed by distinct changes of cell life. Three différent classes of compounds produce well-defined altérations in thé Na,K-ATPase turnover: (1) thé cardioactive steroids (e.g., ouabain) block ATPase activity with negatively coopérative dose-effect curves spanning several orders of magnitude,1 (2) thé highly toxic nonpeptidic and hydrophilic palytoxin from coral inhibits thé Na,K-ATPase reversibly by a mechanism antagonized by cardioactive steroids,2'3 and (3) mercury and silver inactivate it with narrow dose-effect curves.4'5 To assess thé effects of thèse three différent patterns of Na,K-ATPase inhibition on its permeability, thé system must be inserted into tight artificial phospholipid vesicles (liposomes) where transmembrane gradients can be built up or dissipated. In a newly developed liposome system, artificial minicells, in which thé Na,K-ATPase functions as in cells, thé ouabain-blocked Na,K-ATPase expresses a conformation tight to Rb ions,6 whereas thé forms carrying palytoxin, mercury, or silver form Na,K-ATPasemediated leakage pathways across thé otherwise tight phospholipid bilayer.3-5'7 The same inhibitors were then applied to isolated peripheral human lymphocytes. The présent work constitutes a synthesis of thé data obtained with isolated, reconstituted, or cellular Na,K-ATPase. RESULTS Ouabain Keeps thé Pump Tight and Préserves Lymphocyte Life Artificial minicells consist of predominantly single-walled liposomes containing purified Na,K-ATPase molécules inserted randomly in their membranes (FiG. 1A); "This study was supported by Swiss National Science Foundation grants 31-30317.90 and 31-37552.93 *Tel: 41 22 702 54 61; fax: 41 22 70254 52; e-mail:
[email protected]
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B
FIGURE 1. Na,K-ATPase reconstituted with egg-phosphatidylcholine by thé cholate-dialysis procédure forms a homogeneous population of predominantly single-walled vesicles containing noninteracting randomly oriented14 Na,K-ATPase molécules (A, arrows). By including a réservoir of ATP molécules and Na ions during dialysis, artificial minicells are forrned in which thé Na,K-ATPase functions as in ceils. Peripheral human lymphocytes were isolated and incubated without (B) or with 1 mM ouabain for 3 hours (C) or with 3 nM palytoxin for 15 minutes (D) and processed for électron microscopy as described. IU2J6 Acell représentative for each incubation condition is shown.
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they contain a réservoir of ATP and Na for activating thé right-side-oriented Na,KATPase upon extemal Rb or K addition.8 When ouabain is added to artificial minicells which are in thé process of Rb accumulation, thé uptake is interrupted1 and thé Rb ions remain entrapped throughout a 15-minute gel-filtration step despite an inside-out Rb gradient,8 indicating that no Rb ions leak across thé E2-ouabain form. The gel filtration procédure used for removal of external isotope is a sensitive indicator of pump tightness.9 Ouabain added before Rb fully prevents Rb accumulation by thé artificial minicells8 with dose-effect curves corresponding to thé unreconstituted form.1-4'10 In analogy to artificial minicells, ouabain inhibits entirely thé [8flRb]K uptake by isolated human lymphocytes in nanomolar concentrations (TABLE 1). Yet, despite thé potent inhibition of active cellular K uptake, resulting in increased intracellular Na and decreased K concentrations, survival of thé cells is not affected as assessed by exclusion of ethidium homodimer or trypan blue" or by uptake and intracellular hydrolysis of calcein.12 In conformity to thé viability measurements, thé morphology of thé ouabain-treated cells is unaltered13 (Fio. 1C) compared to that of control cells (Fie. 1B).
Palytoxin Induces Pump-Leak Conversion and Lymphocyte Death In sharp contrast to ouabain, palytoxin opens a leakage pathway in thé Na,KATPase molécule of artificial minicells membranes, whereas Na,K-ATPase-free phospholipid bilayers remain tight in thé présence of palytoxin.3 Ouabain antagonizes thé palytoxin-induced leak.3 Conversely, ouabain is poorly effective when thé artificial minicells contain thé ouabain-resistant rénal a 1 Na,K-ATPase, supporting thé concept that spécifie ouabain binding and not some unspecified palytoxin-ouabain interaction underlies thé ouabain-antagonized palytoxin action.3 Palytoxin applied to isolated human lymphocytes causes rapid cell swelling and bursting." The fine structure of thé cell gave away to a progressively amorphous aspect (Fie. 1D). In peripheral lymphocytes, ouabain antagonizes thé pore-forming effect of palytoxin" as in artificial minicells3 and in other cell Systems.2
TABLE 1. Na,K-ATPase in Artificial Minicells and Human Lymphocytes Average diameter Surface Na,K-ATPase molécules, average number14-15 Na,K-ATPase molécules, n/um2 Ouabain- sensitive 86Rb-uptake Ouabain sensitivity (1C50) Ouabain-palytoxin antagonism Na,K-ATPase-ouabain form Na,K-ATPase-palytoxin form Na,K-ATPase-mercury form Na,K-ATPase-silver form
Artificial Minicells
Human Lymphocytes
lOOnm 3.14 X 10-' 4 m 2 10 320 Yes1'8 50nM-100MM 1A1 °
10 um 3.14 x 10-'° m2 30,000 95 Yes11'12 25 nM16
Yes3
Yes"-12
Tight6 Leaky'
Cell survival11'12 Celldeath11 Celldeath n Cell death13
Leaky7 Leaky5
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ANNALS NEW YORK ACADEMY OF SCIENCES SUMMARY AND CONCLUSION
The présent work sets in parallel data obtained with thé Na,K-ATPase in artificial vesicular membranes (artificial minicells) and in peripheral human lymphocytes ex vivo. The Na,K-ATPase was purified and reconstituted into single-walled, tight liposomes filled with a réservoir of ATP and Na in which thé Na.K-ATPase functions as in cells, that is, thé receptor is accessible on thé liposome surface (artificial minicells). In this System, an E2-ouabain state imperméable to Rb ions and an Na,K-ATPase-palytoxin state leaky for Rb ions were characterized. The tight E^ouabain form préserves thé viability of isolated lymphocytes, whereas thé leaky Na,K-ATPase-palytoxin induces rapid cell bursting and death by a ouabain-sensitive mechanism. Thus, thé effect of Na,K-ATPase inhibitors on lymphocyte survival can be predicted from permeability measurements in artificial minicells as verified also with thé leak-inducing metals mercury7-13 and silver.5'13
REFERENCES 1. ANNER, B. M., H. G. REY, M. MOOSMAYER, I. MESZOELY & G. T. HAUPERT, JR. 1990. Hypothalamic Na,K-ATPase inhibitor characterized in two-sided liposomes containing pure rénal Na.K-ATPase. Am. J. Physiol. 258: F144-F153. 2. HABERMANN, E. 1989. Palytoxin acts through Na\K+-ATPase. Toxicon 27: 1171-1187. 3. ANNER, B. M. & M. MOOSMAYER. 1994. Na,K-ATPase characterised in artificial membranes. 2. Successive measurement of ATP-driven Rb-accumulation, ouabain-blocked Rb-flux and palytoxin-induced Rb-efflux. Mol. Membr. Biol. H: 247-254. 4. ANNER, B. M., M. MOOSMAYER & E. IMESCH. 1992. Mercury blocks Na,K-ATPase by a ligand-dependent and réversible mechanism. Am. J. Physiol. 262: F830-F836. 5. HUSSAIN, S., E. MENEGHINI, M. MOOSMAYER, D. LACOTTE & B. M. ANNER. 1994. Potent and réversible interaction of silver with pure Na,K-ATPase and Na,K-ATPaseliposomes. Biochim. Biophys. Acta 1190: 4Q2-4Q8. 6. ANNER, B. M., M. MOOSMAYER & E. IMESCH. 1994. Na,K-ATPase characterised in artifical membranes. 1. Prédominant conformations and ion fluxes associated with active and inhibited states. Mol. Membr. Biol. 11: 237-245. 7. IMESCH, E. & B. M. ANNER. 1992. Mercury weakens thé membrane anchoring of Na,K-ATPase. Am. J. Physiol. 262: F830-F836. 8. REY, H. G., M. MOOSMAYER & B. M. ANNER. 1987. Characterization of Na,K-ATPaseliposomes. III. Controlled activation and inhibition of symmetric pumps by timed asymmetric ATP, RbCl and cardiac glycoside addition. Biochim. Biophys. Acta 900: 2737. 9. ANNER, B. M. 1983. Hypothesis: A bar model for thé pump and channel function of thé reconstituted Na,K-ATPase. 1983. FEBS Lett. 158: 7-11. 10. ANNER, B. M., E. IMESCH & M. MOOSMAYER. 1991. Defective Na transport of murine ouabain-resistant alpha-1 Na,K-ATPase. Soc. Gen. Physiol. 46/2: 479-482. 11. FALCIOLA, J., B. VOLET, R. M. ANNER, M. MOOSMAYER, D. LACOTTE &B. M. ANNER. 1994. Rôle of cell membrane Na.K-ATPase for survival of human lymphocytes in vitro. Biosci, Rep. 14: 189-204. 12. FALCIOLA, J. C. 1995. Survie de Lymphocytes Isolés du Sang Humain: Rôle de la Na,K-ATPase. M.D. thesis, Geneva University, Geneva, Switzerland. 13. HUSSAIN, S., B. VOLET, C. BURRUS, R. ANNER, D. LACOTTE, M. MOOSMAYER & B. M. ANNER. 1995. Uptake of silver by isolated human lymphocytes in présence of L-cysteine or 7V-acetyl-cysteine. In Vitro Toxicol. 8: 377-388. 14. ANNER, B. M., H. P. TING-BEALL & J. D. ROBERTSON. 1984. Characterization of (Na + K)ATPase liposomes. I. Effect of enzyme concentration and modification on liposome
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size, intramembrane particle formation and Na,K-transport. Biochim. Biophys. Acta 773:253-261. 15. BERNTOP, E., K. BERNTOP, D. NELSON & N. HENNINGSEN. 1985. pHJOuabain binding and sodium content in lymphocyte sub-populations and thé démonstration of increased binding in type 2 diabètes mellitus. Scand. J. Clin. Invest. 45: 27-36. 16. ANNER, B. M., D. LACOTTE, R. M. ANNER & M. MOOSMAYER. 1994. Interaction of hypothalamic Na,K-ATPase inhibitor with isolated human peripheral blood mononuclear cells. Biosci. Rep. 14: 231-242.
