, Volume 6, Issue 5, pp 325–334 | Cite as

Expression and Activity Patterns of Nitric Oxide Synthases and Antioxidant Enzymes Reveal a Substantial Heterogeneity Between Cardiac and Vascular Aging in the Rat

  • Bernd van der Loo
  • Markus Bachschmid
  • Ralf Labugger
  • Stefan Schildknecht
  • Juliane Kilo
  • Raphael Hahn
  • Miriam Palacios-Callender
  • Thomas F. Lüscher
Research Article


We investigated the effects of aging and ischemia–reperfusion (I/R) injury on the expression and activity of nitric oxide (NO) synthases and superoxide dismutase (SOD) isoforms. To this end we perfused excised hearts from young (6 months old) and old (31–34 months old) rats according to the Langendorff technique. The isolated hearts were, after baseline perfusion for 30 min, either subjected to 20 min of global no-flow ischemia followed by 40 min of reperfusion or were control-perfused (60 min normoxic perfusion). Both MnSOD and Cu,ZnSOD expression remained unchanged with increasing age and remained unaltered by I/R. However, SOD activity decreased from 7.55 ± 0.1 U/mg protein in young hearts to 5.94 ± 0.44 in old hearts (P<0.05). Furthermore, I/R led to a further decrease in enzyme activity (to 6.35 ± 0.41 U/mg protein; P<0.05) in myocardium of young, but not in that of old animals. No changes in myocardial protein-bound 3-nitrotyrosine levels could be detected. Endothelial NOS (eNOS) expression and activity remained unchanged in aged left ventricles, irrespective of I/R injury. This was in steep contrast to peripheral (renal and femoral) arteries obtained from the same animals where a marked age-associated increase of eNOS protein expression could be demonstrated. Inducible NOS expression was undetectable either in the peripheral arteries or in the left ventricle, irrespective of age. In particular when associated with an acute pathology, which is furthermore limited to a certain time frame, changes in the aged myocardium with respect to enzymes crucially involved in maintaining the redox homeostasis, seem to be much less pronounced or even absent compared to the vascular aging process. This may point to heterogeneity in the molecular regulation of the cardiovascular aging process.


antioxidant enzymes cardiovascular aging ischemia–reperfusion nitric oxide oxidative stress stunning superoxide dismutase 



superoxide dismutase




nitric oxide


endothelial nitric oxide synthase (NOS III)


inducible nitric oxide synthase (NOS II)






left ventricular developed pressure














copper, zinc


cyclic guanosine monophosphate




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  1. Antunes, F, Han, D, Cadenas, E 2002Relative contributions of heart mitochondria glutathione peroxidase and catalase to H2O2 detoxification in in vivo conditionsFree Radic Biol Med3312601267CrossRefPubMedGoogle Scholar
  2. Assem, M, Teyssier, JR, Benderitter, M, Terrand, J, Laubriet, A, Javouhey, A, David, M, Rochette, L 1997Pattern of superoxide dismutase enzymatic activity and RNA changes in rat heart ventricles after myocardial infarctionAm J Pathol151549555PubMedGoogle Scholar
  3. Baker, MA, Cerniglia, GJ, Zaman, A 1990Microtiter plate assay for the measurement of glutathione and glutathione disulfide in large numbers of biological samplesAnal Biochem190360365CrossRefPubMedGoogle Scholar
  4. Barton, M, Cosentino, F, Brandes, RP, Moreau, P, Shaw, S, Luscher, TF 1997Anatomic heterogeneity of vascular aging. Role of nitric oxide and endothelinHypertension30817824PubMedGoogle Scholar
  5. Beckman, JS, Ischiropoulos, H, Zhu, L, Woerd, M, Smith, C, Chen, J, Harrison, J, Martin, JC, Tsai, M 1992Kinetics of superoxide dismutase- and iron-catalyzed nitration of phenolics by peroxynitriteArch Biochem Biophys298438445CrossRefPubMedGoogle Scholar
  6. Brahmajothi, MV, Campbell, DL 1999Heterogeneous basal expression of nitric oxide synthase and superoxide dismutase isoforms in mammalian heart. Implications for mechanisms governing indirect and direct nitric oxide-related effectsCirc Res85575587PubMedGoogle Scholar
  7. Camici, PG, Rimold, O 1999Myocardial hibernation versus repetitive stunning in patientsCardiol Rev73943PubMedCrossRefGoogle Scholar
  8. Cernadas, MR, Sanchez Miguel, L, Garcia-Duran, M, Gonzalez-Fernandez, F, Millas, I, Monton, M, Rodrigo, J, Rico, L, Fernandez, P, Frutos, T, Rodriguez-Feo, JA, Guerra, J, Caramelo, C, Casado, S, Lopez-Farre,  1998Expression of constitutive and inducible nitric oxide synthases in the vascular wall of young and aging ratsCirc Res83279286PubMedGoogle Scholar
  9. Csiszar, A, Ungvari, Z, Edwards, JG, Kaminski, P, Wolin, MS, Koller, A, Kaley, G 2002Aging-induced phenotypic changes and oxidative stress impair coronary arteriolar functionCirc Res9011591166CrossRefPubMedGoogle Scholar
  10. Finkel, T, Holbrook, NJ 2000Oxidants, oxidative stress and the biology of ageingNature408239247CrossRefPubMedGoogle Scholar
  11. Folkow, B, Svanborg, A 1993Physiology of cardiovascular agingPhysiol Rev73725764PubMedGoogle Scholar
  12. Goettsch, W, Lattmann, T, Amann, K, Szibor, M, Morawietz, H, Munter, K, Muller, SP, Shaw, S, Barton, M 2001Increased expression of endothelin-1 and inducible nitric oxide synthase isoform II in aging arteries in vivo: implications for atherosclerosisBiochem Biophys Res Comm280908913CrossRefPubMedGoogle Scholar
  13. Goldstein, S, Czapski, G, Lind, J, Merenyi, G 2000Tyrosine nitration by simultaneous generation of NO and O 2 under physiological conditions. How the radicals do the jobJ Biol Chem27530313036PubMedCrossRefGoogle Scholar
  14. Klainguti, M, Aigner, S, Kilo, J, Eppenberger, HM, Mandinova, A, Aebi, U, Schaub, MC, Shaw, SG, Luscher, TF, Atar, D 2000Lack of nuclear apoptosis in cardiomyocytes and increased endothelin-1 levels in a rat heart model of myocardial stunningBasic Res Cardiol95308315CrossRefPubMedGoogle Scholar
  15. Knowles, RG, Salter, M 1994

    Measurement of NOS activity by conversion of radiolabeled arginine to citrulline using ion exchange separation

    Titheradge, MA eds. Methods in Molecular BiologyHumana Press IncTotowa, MJ, USA6773
    Google Scholar
  16. Kuzuya, M, Kuzuya, F 1993Probucol as an antioxidant and antiatherogenic drugFree Radic Biol Med146777CrossRefPubMedGoogle Scholar
  17. Lakatta, EG 2003Arterial and cardiac aging: major shareholders in cardiovascular disease enterprises. Cellular and molecular clues to heart and arterial agingCirculation107490497PubMedCrossRefGoogle Scholar
  18. Lakatta, EG 2001Heart aging. A fly in the ointment?Circ Res88984986PubMedCrossRefGoogle Scholar
  19. Liu, P, Hock, CE, Nagele, R, Sun, FF, Wong, PY 1997Formation of nitric oxide, superoxide, and peroxynitrite in myocardial ischemia-reperfusion injury in ratsAm J Physiol272H2327H2336PubMedGoogle Scholar
  20. Lopez, MF, Melov, S 2002Applied proteomics: mitochondrial proteins and effect on functionCirc Res90380389CrossRefPubMedGoogle Scholar
  21. Lüscher, TF, Noll, G 1995The endothelium in coronary vascular controlHeart Dis3110Google Scholar
  22. Mac Millan-Crow, LA, Crow, JP, Kerby, JD, Beckman, JS, Thompson, JA 1996Nitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allograftsProc Natl Acad Sci USA931185311858CrossRefGoogle Scholar
  23. Matz, RL, Sotomayor, MA, Schott, C, Stoclet, JC, Andriantsitohaina, R 2000Vascular bed heterogeneity in age-related endothelial dysfunction with respect to NO and eicosanoidsBr J Pharmacol131303311CrossRefPubMedGoogle Scholar
  24. McCord, JM, Fridovich, I 1969Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein)J Biol Chem24460496055PubMedGoogle Scholar
  25. Radi, R 2004Nitric oxide, oxidants, and protein tyrosine nitrationProc Natl Acad Sci USA10140034008CrossRefPubMedGoogle Scholar
  26. Radi, R, Beckman, JS, Bush, KM, Freeman, BA 1991Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxideJ Biol Chem26642444250PubMedGoogle Scholar
  27. Reiter, CD, Teng, RJ, Beckman, JS 2000Superoxide reacts with nitric oxide to nitrate tyrosine at physiological pH via peroxynitriteJ Biol Chem2753246032466CrossRefPubMedGoogle Scholar
  28. Sia, YT, Parker, TG, Liu, P, Tsoporis, JN, Adam, A, Rouleau, JL 2002Improved post-myocardial survival with probucol in rats: effects on left ventricular function, morphology, cardiac oxidative stress and cytokine expressionJ Am Coll Cardiol39148156CrossRefPubMedGoogle Scholar
  29. Stadtman, ER 1992Protein oxidation and agingScience25712201224PubMedCrossRefGoogle Scholar
  30. Subramanian, R, Volovsek, A, Ho, YS 1993Lack of change in MnSOD during ischemia/reperfusion of isolated rat heartJ Mol Cell Cardiol2511791186CrossRefPubMedGoogle Scholar
  31. Tschudi, M, Barton, M, Bersinger, NA, Moreau, P, Cosentino, F, Noll, G, Malinski, T, Luscher, TF 1996Effect of age on kinetics of nitric oxide release in rat aorta and pulmonary arteryJ Clin Invest98899905PubMedCrossRefGoogle Scholar
  32. Loo, B, Bachschmid, M, Spitzer, V, Brey, L, Ullrich, V, Luscher, TF 2003Decreased plasma and tissue levels of vitamin C in a rat model of aging: implications for antioxidative defenseBiochem Biophys Res Comm303483487PubMedCrossRefGoogle Scholar
  33. Loo, B, Labugger, R, Skepper, JN, Bachschmid, M, Kilo, J, Powell, JM, Palacios-Callender, M, Erusalimsky, JD, Quaschning, T, Malinski, T, Gygi, D, Ullrich, V, Luscher, TF 2000Enhanced peroxynitrite formation is associated with vascular agingJ Exp Med19217311743PubMedCrossRefGoogle Scholar
  34. Loo, B, Lüscher, TF 2002

    Endothelium-mediated signaling and vascular aging

    Hagen, T eds. Mechanisms of Cardiovascular Aging. Advances in Cell Aging and GerontologyElsevier ScienceAmsterdam, The Netherlands127144
    Google Scholar
  35. Zeiher, AM, Drexler, H, Saurbier, B, Just, H 1993Endothelium-mediated coronary blood flow modulation in humans. Effects of age, atherosclerosis, hypercholesterolemia, and hypertensionJ Clin Invest92652662PubMedCrossRefGoogle Scholar
  36. Zieman, SJ, Gerstenblith, G, Lakatta, EG, Rosas, GO, Vandegaer, K, Ricker, KM, Hare, JM 2001Upregulation of the nitric oxide-cGMP pathway in aged myocardium. Physiological response to l-arginineCirc Res8897102PubMedGoogle Scholar
  37. Zou, MH, Leist, M, Ullrich, V 1999Selective nitration of prostacyclin synthase and defective vasorelaxation in atherosclerotic bovine cornary arteriesAm J Pathol15413591365PubMedGoogle Scholar
  38. Zou, MH, Martin, C, Ullrich, V 1997Tyrosine nitration as a mechanism of selective inactivation of prostacyclin synthase by peroxynitriteBiol Chem378707713PubMedCrossRefGoogle Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  • Bernd van der Loo
    • 1
  • Markus Bachschmid
    • 2
  • Ralf Labugger
    • 3
  • Stefan Schildknecht
    • 2
  • Juliane Kilo
    • 1
  • Raphael Hahn
    • 2
  • Miriam Palacios-Callender
    • 4
  • Thomas F. Lüscher
    • 1
    • 3
  1. 1.Division of Cardiology, Cardiovascular CentreUniversity Hospital ZurichZürichSwitzerland
  2. 2.Department of BiologyUniversity of KonstanzKonstanzGermany
  3. 3.Division of Cardiovascular Research, Institute of PhysiologyUniversity Zurich-IrchelSwitzerland
  4. 4.Wolfson Institute for Biomedical ResearchUniversity College LondonUK

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