Pharmaceutical Research

, Volume 25, Issue 8, pp 1798–1806 | Cite as

Synergistic Effect of Amlodipine and Atorvastatin in Reversing LDL-Induced Endothelial Dysfunction

  • R. Preston Mason
  • Ruslan Kubant
  • Gehan Heeba
  • Robert F. Jacob
  • Charles A. Day
  • Yehudi S. Medlin
  • Philipp Funovics
  • Tadeusz Malinski
Research Paper



Statins and certain calcium channel blockers may improve nitric oxide (NO) release and endothelial function through various mechanisms, but their combined effects are not well understood.


The separate versus combined effects of amlodipine (AML) and atorvastatin (AT) on NO and peroxynitrite (ONOO) were measured in human umbilical vein endothelial cells (HUVEC) in the presence and absence of low-density lipoprotein (LDL) using electrochemical nanosensors.


The combination of AML (5 μmol/l) and AT (3-6 μmol/l) directly stimulated NO release that was about twofold greater than the sum of their separate effects (p < 0.05). This synergistic activity is attributed to enhanced endothelial NO synthase (eNOS) function and decreased cytotoxic ONOO. LDL (100 mg/dl) caused a dysfunction of HUVEC manifested by a 60% reduction in NO and an almost twofold increase in ONOO. Treatment with AML/AT partially reversed the effects of LDL on endothelial function, including a 90% increase in NO and 50% reduction in ONOO. Small-angle X-ray diffraction analysis indicates that AML and AT are lipophilic and share an overlapping molecular location in the cell membrane that could facilitate electron transfer for antioxidant mechanisms.


These findings indicate a synergistic effect of AML and AT on an increase in NO concentration, reduction of nitroxidative stress. Also, AML/AT partially restored the NO level of LDL-induced dysfunctional endothelium. Their combined effects may be enhanced by antioxidant properties related to their intermolecular actions in the cell membrane and an increase in the expression and coupling of endothelial nitric oxide synthase.

Key Words

endothelium LDL nitric oxide oxidative stress 


  1. 1.
    B. S. Oemar, M. R. Tschudi, N. Godoy, V. Brovkovich, T. Malinski, and T. F. Luscher. Reduced endothelial nitric oxide synthase expression and production in human atherosclerosis. Circulation 97(25):2494–2498 (1998).PubMedGoogle Scholar
  2. 2.
    D. G. Harrison, P. C. Freiman, M. L. Armstrong, M. L. Marcus, and D. D. Heistad. Alterations of vascular reactivity in atherosclerosis. Circ. Res. 61:74–80 (1987).Google Scholar
  3. 3.
    J. K. Liao. Endothelium and acute coronary syndromes. Clin. Chem. 44:1799–1808 (1998).PubMedGoogle Scholar
  4. 4.
    G. Kojda and D. G. Harrison. Interactions between NO and reactive oxygen species: pathophysiological importance in atherosclerosis, hypertension, diabetes and heart failure. Cardiovasc. Res. 43:562–571 (1999).PubMedCrossRefGoogle Scholar
  5. 5.
    M. R. Tschudi, M. Barton, N. A. Bersinger, P. Moreau, F. Cosentino, G. Noll, T. Malinski, and T. F. Luscher. Effect of age on kinetics of nitric oxide release in rat aorta and pulmonary artery. J. Clin. Invest. 98(4):899–905 (1996).PubMedCrossRefGoogle Scholar
  6. 6.
    O. A. Paniagua, M. B. Bryant, and J. A. Panza. Role of endothelial nitric oxide in shear stress-induced vasodilation in human microvasculature. Diminished activity in hypertensive and hypercholesterolemic patients. Circulation 103:1752–1758 (2001).PubMedGoogle Scholar
  7. 7.
    J. A. Panza, A. A. Quyyumi, J. E. Brush, and S. E. Epstein. Abnormal endothelium-dependent vascular relaxation in patients with essential hypertension. N. Engl. J. Med. 323:22–27 (1990).PubMedCrossRefGoogle Scholar
  8. 8.
    S. Taddei, A. Virdis, P. Mattei, and A. Salvetti. Vasodilation to acetylcholine in primary and secondary forms of human hypertension. Hypertension 21:929–933 (1993).PubMedGoogle Scholar
  9. 9.
    M. Rodriguez-Porcel, L. O. Lerman, J. Herrmann, T. Sawamura, C. Napoli, and A. Lerman. Hypercholesterolemia and hypertension have synergistic deleterious effects on coronary endothelial function. Arterioscler. Thromb. Vasc. Biol. 23:885–891 (2003).PubMedCrossRefGoogle Scholar
  10. 10.
    S. John and R. E. Schmieder. Impaired endothelial function in arterial hypertension and hypercholesterolemia: Potential mechanisms and differences. J. Hypertens. 18:363–374 (2000).PubMedCrossRefGoogle Scholar
  11. 11.
    J. D. Neaton and D. Wentworth. Serum cholesterol, blood pressure, cigarette smoking, and death from coronary heart disease. Overall findings and differences by age for 316,099 white men. Multiple Risk Factor Intervention Trial Research Group. Arch. Intern. Med. 152:56–64 (1992).PubMedCrossRefGoogle Scholar
  12. 12.
    F. Thomas, K. Bean, L. Guize, S. Quentzel, P. Argyriadis, and A. Benetos. Combined effects of systolic blood pressure and serum cholesterol on cardiovascular mortality in young (<55 years) men and women. Eur. Heart J. 23:528–535 (2002).PubMedCrossRefGoogle Scholar
  13. 13.
    U. Landmesser, H. Cai, S. Dikalov, L. McCann, J. Hwang, H. Jo, S. M. Holland, and D. G. Harrison. Role of p47(phox) in vascular oxidative stress and hypertension caused by angiotensin II. Hypertension 40:511–515 (2002).PubMedCrossRefGoogle Scholar
  14. 14.
    U. Landmesser, S. Dikalov, S. R. Price, L. McCann, T. Fukai, S. M. Holland, W. E. Mitch, and D. G. Harrison. Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J. Clin. Invest. 111:1201–1209 (2003).PubMedGoogle Scholar
  15. 15.
    Y. Ohara, T. E. Peterson, and D. G. Harrison. Hypercholesterolemia increases endothelial superoxide anion production. J. Clin. Invest. 91(6):2546–2551 (1993).PubMedCrossRefGoogle Scholar
  16. 16.
    J. K. Liao, W. S. Shin, W. Y. Lee, and S. L. Clark. Oxidized low-density lipoprotein decreases the expression of endothelial nitric oxide synthase. J. Biol. Chem. 270:319–324 (1995).PubMedCrossRefGoogle Scholar
  17. 17.
    L. Vergnani, S. Hatrik, F. Ricci, A. Passaro, N. Manzoli, G. Zuliani, V. Brovkovych, R. Fellin, and T. Malinski. Effect of native and oxidized low-density lipoprotein on endothelial nitric oxide and superoxide production: key role of l-arginine availability. Circulation 101(11):1261–1266 (2000).PubMedGoogle Scholar
  18. 18.
    R. P. Mason and R. F. Jacob. Membrane microdomains and vascular biology: Emerging role in atherogenesis. Circulation 107:2270–2273 (2003).PubMedCrossRefGoogle Scholar
  19. 19.
    D. G. Harrison. Cellular and molecular mechanisms of endothelial cell dysfunction. J. Clin. Invest. 100:2153–2157 (1997).PubMedCrossRefGoogle Scholar
  20. 20.
    X. Zhang and T. H. Hintze. Amlodipine releases nitric oxide from canine coronary microvessels: An unexpected mechanism of action of a calcium channel-blocking agent. Circulation 97:576–580 (1998).PubMedGoogle Scholar
  21. 21.
    T. J. Anderson, I. T. Meredith, A. C. Yeung, B. Frei, A. P. Selwyn, and P. Ganz. The effect of cholesterol-lowering and antioxidant therapy on endothelium-dependent coronary vasomotion. N. Engl. J. Med. 332:488–493 (1995).PubMedCrossRefGoogle Scholar
  22. 22.
    G. B. Mancini, G. C. Henry, C. Macaya, B. J. O'Neill, A. L. Pucillo, R. G. Carere, T. J. Wargovich, H. Mudra, T. F. Luscher, M. I. Klibaner, H. E. Haber, A. C. Uprichard, C. J. Pepine, and B. Pitt. Angiotensin-converting enzyme inhibition with quinapril improves endothelial vasomotor dysfunction in patients with coronary artery disease. The TREND (Trial on Reversing Endothelial Dysfunction) Study. Circulation 94(3):240–243 (1996).Google Scholar
  23. 23.
    S. Wolfrum, K. S. Jensen, and J. K. Liao. Endothelium-dependent effects of statins. Arterioscler. Thromb. Vasc. Biol. 23:729–736 (2003).PubMedCrossRefGoogle Scholar
  24. 24.
    R. P. Mason, M. F. Walter, and R. F. Jacob. Effects of HMG–CoA reductase inhibitors on endothelial function: Role of microdomains and oxidative stress. Circulation 109:II34–II41 (2004).PubMedGoogle Scholar
  25. 25.
    L. Kalinowski, I. T. Dobrucki, and T. Malinski. Cerivastatin potentiates nitric oxide release and eNOS expression through inhibition of isoprenoids synthesis. J. Physiol. Pharmacol. 53:585–595 (2002).PubMedGoogle Scholar
  26. 26.
    R. P. Mason, L. Kalinowski, R. F. Jacob, A. M. Jacoby, and T. Malinski. Nebivolol reduces nitroxidative stress and restores nitric oxide bioavailability in endothelium of black Americans. Circulation 112:3795–3801 (2005).PubMedCrossRefGoogle Scholar
  27. 27.
    R. P. Mason, P. Marche, and T. H. Hintze. Novel vascular biology of third-generation L-type calcium channel antagonists: Ancillary actions of amlodipine. Arterioscler. Thromb. Vasc. Biol. 23:2155–2163 (2003).PubMedCrossRefGoogle Scholar
  28. 28.
    R. P. Mason, M. F. Walter, C. A. Day, and R. F. Jacob. Intermolecular differences for HMG-CoA reductase inhibitors contribute to distinct pharmacologic and pleiotropic actions. Am. J. Cardiol. 96[suppl]:11F–23F (2005).PubMedCrossRefGoogle Scholar
  29. 29.
    S. E. Nissen, E. M. Tuzcu, P. Libby, P. D. Thompson, M. Ghali, D. Garza, L. Berman, H. Shi, E. Buebendorf, and E. J. Topol. Effect of antihypertensive agents on cardiovascular events in patients with coronary disease and normal blood pressure: the CAMELOT study: a randomized controlled trial. JAMA 292:2217–2226 (2004).PubMedCrossRefGoogle Scholar
  30. 30.
    P. S. Sever, B. Dahlof, and N. R. Poulter. Prevention of coronary and stroke events with atorvastatin in hypertensive patients who have average or lower-than-average cholesterol concentrations, in the Anglo-Scandinavian Cardiac Outcomes Trial-Lipid Lowering Arm (ASCOT-LLA): a multicentre randomised controlled trial. Lancet 361:1149–1158 (2003).PubMedCrossRefGoogle Scholar
  31. 31.
    H. M. Colhoun, D. J. Betteridge, P. N. Durrington, G. A. Hitman, Neil HAW, S. J. Livingstone, M. J. Thomason, M. I. Mackness, V. Charlton-Menys, and J. H. Fuller. Primary prevention of cardiovascular disease with atorvastatin in type 2 diabetes in the Collaborative Atorvastatin Diabetes Study (CARDS): Multicentre randomised placebo-controlled trial. Lancet 364:685–696 (2004).PubMedCrossRefGoogle Scholar
  32. 32.
    P. Sever, B. Dahlof, N. Poulter, H. Wedel, G. Beevers, M. Caulfield, R. Collins, S. Kjeldsen, A. Kristinsson, G. McInnes, J. Mehlsen, M. Niemenem, E. O'Brien, and J. Ostergren. Potential synergy between lipid-lowering and blood-pressure-lowering in the Anglo-Scadinavian Cardiac Outcomes Trail. Eur. Heart J. 27:2982–2988 (2006).PubMedCrossRefGoogle Scholar
  33. 33.
    D. J. M. Delsing, J. W. Jukema, M. A. van de Wiel, J. J. Emeis, A. van der Laarse, L. M. Havekes, and H. M. G. Princen. Differential effects of amlodipine and atorvastatin treatment and their combination on atherosclerosis in ApoE*3-Leiden transgenic mice. J. Cardiovasc. Pharmacol. 42:63–70 (2003).PubMedCrossRefGoogle Scholar
  34. 34.
    L. Kalinowski, L. W. Dobrucki, V. Brovkovich, and T. Malinski. Increased nitric oxide bioavailability in endothelial cells contributes to the pleiotropic effect of cerivastatin. Circulation 105:933–938 (2002).PubMedCrossRefGoogle Scholar
  35. 35.
    J. Xue, X. Ying, J. Chen, Y. Xian, and L. Jin. Amperometric ultramicrosensors for peroxynitrite detection and its application toward single myocardial cells. Anal. Chem. 72:5313–5321 (2000).PubMedCrossRefGoogle Scholar
  36. 36.
    V. Lvovich and A. Scheeline. Amperometric sensors for simultaneous superoxide and hydrogen peroxide detection. Anal. Chem. 69:454–462 (1997).CrossRefGoogle Scholar
  37. 37.
    T. Malinski, and Z. Taha. Nitric oxide release from a single cell measured in situ by a porphyrinic-based microsensor. Nature 358:676–678 (1992).PubMedCrossRefGoogle Scholar
  38. 38.
    P. Vallance, S. Patton, K. Bhagat, R. MacAllister, M. Radomski, S. Moncada, and T. Malinski. Direct measurement of nitric oxide in human beings. Anal. Chem. 346:153–154 (1995).Google Scholar
  39. 39.
    J. E. Bennett and T. Malinski. Conductive polymeric porphyrin films: application in the electrocatalytic oxidation of hydrazine. Chem. Mater. 3:490–495 (1991).CrossRefGoogle Scholar
  40. 40.
    A. D. Bangham, M. M. Standish, and J. C. Watkins. Diffusion of univalent ions across the lamellae of swollen phospholipids. J. Mol. Biol. 13:238–252 (1965).PubMedGoogle Scholar
  41. 41.
    D. W. Chester, L. G. Herbette, R. P. Mason, A. F. Joslyn, D. J. Triggle, and D. E. Koppel. Diffusion of dihydropyridine calcium channel antagonists in cardiac sarcolemmal lipid multibilayers. Biophys. J. 52(6):1021–1030 (1987).PubMedCrossRefGoogle Scholar
  42. 42.
    R. P. Mason, G. E. Gonye, D. W. Chester, and L. G. Herbette. Partitioning and location of Bay K 8644, 1,4-dihydropyridine calcium channel agonist, in model and biological membranes. Biophys. J. 55(4):769–778 (1989).PubMedGoogle Scholar
  43. 43.
    R. P. Mason and R. F. Jacob. X-ray diffraction analysis of membrane structure changes with oxidative stress. In D. Armstrong (ed.), Methods in Molecular Biology: Ultrastructural and Molecular Biology Protocols. Vol 193. Humana Press Inc., Totowa, NJ, 2002, pp. 71–80.Google Scholar
  44. 44.
    T. N. Tulenko, M. Chen, P. E. Mason, and R. P. Mason. Physical effects of cholesterol on arterial smooth muscle membranes: Evidence of immiscible cholesterol domains and alterations in bilayer width during atherogenesis. J. Lipid. Res. 39:947–956 (1998).PubMedGoogle Scholar
  45. 45.
    L. G. Herbette, T. MacAlister, T. F. Ashavaid, and R. A. Colvin. Structure-function studies of canine cardiac sarcolemmal membranes. II. Structural organization of the sarcolemmal membrane as determined by electron microscopy and lamellar X-ray diffraction. Biochim. Biophys. Acta. 812(3):609–623 (1985).PubMedCrossRefGoogle Scholar
  46. 46.
    K. K. Koh, M. J. Quon, S. H. Han, W. J. Chung, J. Y. Ahn, Y. H. Seo, M. H. Kang, T. H. Ahn, I. S. Choi, and E. K. Shin. Additive beneficial effects of losartan combined with simvastatin in the treatment of hypercholesterolemic, hypertensive patients. Circulation 110:3687–3692 (2004).PubMedCrossRefGoogle Scholar
  47. 47.
    H. E. Andrews, K. R. Bruckdorfer, R. C. Dunn, and M. Jacobs. Low-density lipoproteins inhibit endothelium-dependent relaxation in rabbit aorta. Nature 327(6119):237–239 (1987).PubMedCrossRefGoogle Scholar
  48. 48.
    K. A. Pritchard, L. Groszek, D. M. Smalley, W. C. Sessa, M. Wu, P. Villalon, M. S. Wolin, and M. B. Stemerman. Native low-density lipoprotein increases endothelial cell nitric oxide synthase generation of superoxide anion. Circ. Res. 77(3):510–518 (1995).PubMedGoogle Scholar
  49. 49.
    D. W. Stepp, J. Ou, A. W. Ackerman, S. Welak, D. Klick, K. A. Pritchard Jr. Native LDL and minimally oxidized LDL differentially regulate superoxide anion in vascular endothelium in situ. Am J Physiol, Heart Circ Physiol. 283(2):H750–H759 (2002).Google Scholar
  50. 50.
    F. Vidal, C. Colome, J. Martinez-Gonzalez, and L. Badimon. Atherogenic concentrations of native low-density lipoproteins down-regulate nitric-oxide-synthase mRNA and protein levels in endothelial cells. Eur. J. Biochem. 252(3):378–384 (1998).PubMedCrossRefGoogle Scholar
  51. 51.
    J. Martinez-Gonzalez, B. Raposo, C. Rodriguez, and L. Badimon. 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibition prevents endothelial NO synthase downregulation by atherogenic levels of native LDLs: balance between transcriptional and posttranscriptional regulation. Arterioscler. Thromb. Vasc. Biol. 21(5):804–809 (2001).PubMedGoogle Scholar
  52. 52.
    Z. Ou, J. Ou, A. W. Ackerman, K. T. Oldham, K. A. Pritchard Jr. L-4F, an apolipoprotein A-1 mimetic, restores nitric oxide and superoxide anion balance in low-density lipoprotein-treated endothelial cells. Circulation 107(11):1520–1524 (2003).PubMedCrossRefGoogle Scholar
  53. 53.
    U. Laufs, V. La Fata, J. Plutzky, and J. K. Liao. Upregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitors. Circulation 97(12):1129–1135 (1998).PubMedGoogle Scholar
  54. 54.
    A. H. Wagner, T. Kohler, U. Ruckschloss, I. Just, and M. Hecker. Improvement of nitric oxide-dependent vasodilation by HMG–CoA reductase inhibitors through attenuation of endothelial superoxide anion formation. Arterioscler. Thromb. Vasc. Biol. 20:61–69 (2000).PubMedGoogle Scholar
  55. 55.
    S. Wassmann, U. Laufs, K. Muller, C. Konkol, K. Ahlbory, A. T. Baumer, W. Linz, M. Bohm, and G. Nickenig. Cellular antioxidant effects of atorvastatin in vitro and in vivo. Arterioscler. Thromb. Vasc. Biol. 22:300–305 (2002).PubMedCrossRefGoogle Scholar
  56. 56.
    O. Feron, C. Dessy, J. P. Desager, and J. L. Balligand. Hydroxy-methylgluataryl-coenzyme A reductase inhibition promotes endothelial nitric oxide synthase activation through a decrease in caveolin abundance. Circulation 103:113–118 (2001).PubMedGoogle Scholar
  57. 57.
    R. P. Mason, M. F. Walter, M. W. Trumbore, E. G. Olmstead Jr., and P. E. Mason. Membrane antioxidant effects of the charged dihydropyridine calcium antagonist amlodipine. J. Mol. Cell. Cardiol. 31:275–281 (1999).PubMedCrossRefGoogle Scholar
  58. 58.
    F. Franzoni, G. Santoro, F. Regoli, Y. Plantinga, F. R. Femia, A. Carpi, and F. Galetta. An in vitro study of the peroxyl and hydroxyl radical scavenging capacity of the calcium antagonist amlodipine. Biomed. Pharmacother. 58:423–426 (2004).PubMedGoogle Scholar
  59. 59.
    M. McIntyre, C. A. Hamilton, D. D. Rees, J. L. Reid, and A. F. Dominiczak. Sex differences in the abundance of endothelial nitric oxide in a model of genetic hypertension. Hypertension 30:1517–1524 (1997).PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • R. Preston Mason
    • 1
    • 2
  • Ruslan Kubant
    • 3
  • Gehan Heeba
    • 3
  • Robert F. Jacob
    • 2
  • Charles A. Day
    • 2
  • Yehudi S. Medlin
    • 2
  • Philipp Funovics
    • 3
    • 4
  • Tadeusz Malinski
    • 3
  1. 1.Department of MedicineBrigham and Women’s Hospital, Harvard Medical SchoolBostonUSA
  2. 2.Elucida ResearchBeverlyUSA
  3. 3.Department of Chemistry and BiochemistryOhio UniversityAthensUSA
  4. 4.University Clinic of Orthopedic Surgery, Medical University of ViennaViennaAustria

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