Skip to main content
SpringerLink
Log in

Quantification of the total Na,K-ATPase concentration in atria and ventricles from mammalian species by measuring3H-ouabain binding to intact myocardial samples. Stability to short term ischemia reperfusion

  • Original Contributions
  • Published:
Basic Research in Cardiology Aims and scope Submit manuscript

Summary

Na,K-ATPase concentration was measured by vanadata facilitated3H-ouabain binding to intact samples taken from various parts of porcine and canine myocardium. In porcine and canine heart3H-ouabain binding site concentration in ventricles was 1.4–2.5 times larger than in atria. Evaluation of3H-ouabain binding kinetics revealed no major difference between atria and ventricles: Equilibrium was obtained after the same incubation time in right atrium (RA) as in left ventricle (LV), both in porcine and canine heart. Unspecific uptake and retention of3H-ouabain was for porcine heart RA and LV 1.5 and 1.4, respectively, and for canine heart RA and LV, both 1.2% filling (i.c., volume (ml) of incubation medium3H-radioactivity taken up per mass unit (g wet wt.) of tissue multiplied by 100). The apparent dissociation constant (K d ) was 1.4×10−8 and 1.9×10−8 in porcine RA and LV and 2.6×10−8 and 6.1×10−8 mol/l in canine RA and LV. Loss of specifically bound3H-ouabain during the washout procedure occurred with a half-life time (T1/2) of 16.7 in RA and LV of porcine heart and 91.2 and 151.6h in RA and LV of canine heart. Duly corrected for these errors of the method-factor 1.16 and 1.13, respectively, for porcine RA and LV, and factor 1.11 and 1.13 for canine RA and LV, total3H-ouabain binding site concentration was found to be 553±74 and 1037±45 pmol/g wet wt. (means±SEM, n=5) in porcine RA and LV, and 569±37 and 1410±40 pmol/g wet wt. (means ±SEM, n=5) in the canine RA and LV. These values were confirmed by measurements of3H-digoxin binding to the porcine heart. The present quantification of myocardial Na, K-ATPase gives values up to 154 times higher than measurements based upon Na,K-ATPase activities in membrane fractions where the recovery of Na,KK-ATPase may be less than 1% due to loss during purification. A higher Na,K-ATPase concentration is found in small animals than in large animals. A relationship between higher concentration of Na, K-ATPase and larger pressure work in ventricles compared to atria is suggested. Myocardial3H-ouabain binding sites were found to be stable for 20 min of ischemia, followed by 1h of reperfusion, supporting the concept that myocyte injury induced by short term ischemia may be reversible and that reperfusion may result in normalization.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Beller GA, Conroy J, Smith TW (1976) Ischemia-induced alteration in myocardial (Na+-K+)-ATPase and cardiac glycoside binding. J Clin Invest 57:341–350

    PubMed  Google Scholar 

  2. Charlemangne D, Mayoux E, Poyard M, Oliviero P, Geering K (1987) Identification isoforms of the catalytic subunit of Na,K-ATPase in myocytes from adult rat heart. J Biol Chem 262:8941–8943

    PubMed  Google Scholar 

  3. Chemnitius JM, Sasaki Y, Burger W, Bing RJ (1985) The effect of ischemia and reperfusion on sarcolemmal function in perfused canine hearts. J Mol Cell Cardiol 17:1139–1150

    PubMed  Google Scholar 

  4. Clausen T, Everts ME, Kjeldsen K (1987) Quantification of the maximum capacity for active sodium-potassium transport in rat skeletal muscle. J Physiol Lond 388:163–181

    PubMed  Google Scholar 

  5. Clausen T, Hansen O (1974) Ouabain binding and Na+-K+-transport in rat muscle cells and adipocytes. Biochim Biophys Acta 345:387–404

    Google Scholar 

  6. Clausen T, Sellin LC, Thesleff S (1981) Quantitative changes in ouabain binding after denervation and during reinnervation of mouse skeletal muscle. Acta Physiol Scand 111:373–375

    PubMed  Google Scholar 

  7. Godin DV, Bhimji S (1987) Effects of allopurinol on myocardial ischemic injury induced by coronary artery ligation and reperfusion. Biochem Pharmacol 36:2101–2107

    PubMed  Google Scholar 

  8. Hansen O (1979) Facilitation of ouabain binding to (Na++K+)-ATPase by vanadate at in vivo concentrations. Biochim Biophys Acta 568:265–269

    PubMed  Google Scholar 

  9. Hansen O (1984) Interaction of cardiac glycosides with (Na+ +K+-activated ATPase. A biochemical link to digitalis-induced inotropy. Pharmacol Rev 36:143–163

    PubMed  Google Scholar 

  10. Hansen O, Clausen T (1988) Quantitative determination of Na+-K+-ATPase and other sarcolemmal components in muscle cells. Am J Physiol 254:C1-C7

    PubMed  Google Scholar 

  11. Hansen O, Skou JC (1973) A study on the influence of the concentration of Mg++, Pi, K+, Na+, and tris on (Mg++ +Pi)-supported g-strophanthin binding to (Na++K+)-activated ATPase from ox brain. Biochim Biophys Acta 311:51–66

    PubMed  Google Scholar 

  12. Harper JS, Lochner A (1989) Sarcolemmal integrity during ischaemia and reperfusion of the isolated rat heart. Basic Res Cardiol 84:208–226

    PubMed  Google Scholar 

  13. Jones LR, Besch HR (1984) Isolation of canine cardiac sarcolemmal vesicles. Meth Pharmacol 5:1–12

    Google Scholar 

  14. Jones LR, Maddock SW, Besch HR (1980) Unmasking effect of alamethicin on the (Na+, K+-ATPase, beta-adrenergic receptor-coupled adenylate cyclase, and cAMP-dependent protein kinase activities of cardiac sarcolemmal vesicles. J Biol Chem 255:9971–9980

    PubMed  Google Scholar 

  15. Kim M-S, Akera T (1987) O2 free radicals: Cause of ischemia-reperfusion injury to cardiac Na+-K+-ATPase. Am J Physiol 252:H252–H257

    PubMed  Google Scholar 

  16. Kim DH, Akera T, Kennedy RH (1983) Ischemia-induced enhancement of digitalis sensitivity in isolated guinea pig heart. J Pharmacol Exp Ther 226:335–342

    PubMed  Google Scholar 

  17. Kjeldsen K (1986) Complete quantification of the total concentration of rat skeletal muscle Na++K+-dependent ATPase by measurements of (3H)ouabain binding. Biochem J 240:725–730

    PubMed  Google Scholar 

  18. Kjeldsen K (1987) Regulation of3H-ouabain binding sites in mammalian skeletal muscle.-Effects of age, K-depletion, thyroid status and hypertension. Dan Med Bull 34:15–46

    Google Scholar 

  19. Kjeldsen K (1988) Homogeneity of3H-ouabain binding sites in rat soleus muscle. Biochem J 249:481–485

    PubMed  Google Scholar 

  20. Kjeldsen K, Bjerregaard P, Richter EA, Thomsen PEB, Nørgaard A (1988) Na+, K+-ATPase concentration in rodent and human heart and skeletal muscle. Apparent relationship to muscle performance. Cardiovasc Res 22:95–100

    PubMed  Google Scholar 

  21. Kjeldsen K, Grøn P (1990) Age dependent change in myocardial cardiac glycoside receptor (Na, K-ATPase) concentration in children. J Cardiovase Pharmacol 15:332–337

    Google Scholar 

  22. Kjeldsen K, Nørgaard A, Clausen T (1984) The age-dependent changes in the number of3H-ouabain binding sites in mammalian skeletal muscle. Pflügers Arch 402:100–108

    Google Scholar 

  23. Kjeldsen K, Nørgaard A, Hansen O, Clausen T (1985) Significance of skeletal muscle digitalis receptors for3H-ouabain distribution in the guinea pig. J Pharmacol Exp Ther 234:720–727

    PubMed  Google Scholar 

  24. Kjeldsen K, Richter EA, Galbo H, Lortie G, Clausen T (1986) Training increases the concentration of3H-ouabain binding sites in rat skeletal muscle. Biochim Biophys Acta 860:708–712

    PubMed  Google Scholar 

  25. Kloner RA, Braunwald E (1980) Observation on experimental ischaemia. Cardiovasc Res 14:371–395

    PubMed  Google Scholar 

  26. Knochel JP, Blachley JD, Johnson JH, Carter NW (1985) Muscle cell electrical hyperpolarization and reduced exercise hyperkalemia in physically conditioned dogs. J Clin Invest 75:740–745

    PubMed  Google Scholar 

  27. Maixent JM, Charlemagne D, Chapelle B, Lelievre LG (1987) Two Na, K-ATPase isoenzymes in canine cardiac myocytes. J Biol Chem 262:6842–6848

    PubMed  Google Scholar 

  28. Matsui H, Schwartz A (1966) Purification and properties of a highly active ouabain-sensitive Na+, K+-dependent adenosinetriphosphatase from cardiac tissue. Biochim Biophys Acta 128:380–390

    PubMed  Google Scholar 

  29. Nørgaard A, Baandrup U, Larsen JS, Kjeldsen K (1987) Heart Na, K-ATPase activity in cardi hamsters as estimated from K-dependent 3-O-MFPase activity in crude homogenates. J Moll Cell Cardiol 19:589–594

    Google Scholar 

  30. Nørgaard A, Bagger JP, Bjerregaard P, Baandrup U, Kjeldsen K, Thomsen FEB (1988) Relation of left ventricular function and Na, K-pump concentration in suspected idiopathic dilated cardiomyopathy. Am J Cardiol 61:1312–1315

    PubMed  Google Scholar 

  31. Nørgaard A, Kjeldsen K, Hansen O (1985) K+-dependent 3-O-methylfluorescein phosphatase activity in crude homogenates of rodent heart ventricle: Effect of K+ depletion and changes in thyroid status. Eur J Pharmacol 113:373–382

    PubMed  Google Scholar 

  32. Nørgaard A, Kjeldsen K, Hansen O, Clausen T, Larsen CG, Larsen FG (1986) Quantification of the3H-ouabain binding site concentration in human myocardium: a postmortem study. Cardiovasc Res 20:428–435

    PubMed  Google Scholar 

  33. Panagia V, Singh JN, Anand-Srivastava MB, Pierce GN, Jasmin G, Dhalla NS (1984) Sarcolemmal alterations during the development of genetically determined cardiomyopathy. Cardiovascular Res 18:567–572

    Google Scholar 

  34. Pang DC, Weglicki WB (1977) Cardiac sarcolemma of the hamster. Enrichment of the (Na++K+)-ATPase. Biochim Biophys Acta 465:411–414

    Google Scholar 

  35. Schwatz A, Wood JM, Allen JC, Bornet EP, Entman ML, Goldstein MA, Sordahl LA, Suzuki M (1973) Biochemical and morphological correlates of cardiac ischemia. Am J Cardiol 32:46–61

    PubMed  Google Scholar 

  36. Sejersted OM, Andersen FR, Ilebekk A (1985) Potassium balance and ouabain binding sites in intact porcine hearts during isoproterenol infusion. Advances in myocardiology 8:83–95

    Google Scholar 

  37. Thomlins B, Harding SE, Kirby MS, Poole-Wilson PA, Williams AJ (1986) Contamination of a cardiac sarcolemmal preparation with endothelial plasma membrane. Biochim Biophys Acta 856:137–143

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Medicine B, Division of Cardiology, Rigshospitalet, University of Copenhagen School of Medicine, Blegdamsvej 9, 2100, Copenhagen, Denmark

    T. A. Schmidt, J. H. Svendsen, S. Haunsø & K. Kjeldsen

Authors
  1. T. A. Schmidt
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J. H. Svendsen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. S. Haunsø
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. K. Kjeldsen
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schmidt, T.A., Svendsen, J.H., Haunsø, S. et al. Quantification of the total Na,K-ATPase concentration in atria and ventricles from mammalian species by measuring3H-ouabain binding to intact myocardial samples. Stability to short term ischemia reperfusion. Basic Res Cardiol 85, 411–427 (1990). https://doi.org/10.1007/BF01907133

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01907133

Key words

Search

Navigation