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Continuous exposure to l-arginine induces oxidative stress and physiological tolerance in cultured human endothelial cells

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Abstract

The therapeutic benefits of l-arginine (ARG) supplementation in humans, often clearly observed in short-term studies, are not evident after long-term use. The mechanisms for the development of ARG tolerance are not known and cannot be readily examined in humans. We have developed a sensitive in vitro model using a low glucose/low arginine culture medium to study the mechanisms of ARG action and tolerance using two different human endothelial cells, i.e., Ea.hy926 and human umbilical venous endothelial cells. Cultured cells were incubated with different concentrations of ARG and other agents to monitor their effects on endothelial nitric oxide synthase (eNOS) expression and function, as well as glucose and superoxide (O ·−2 ) accumulation. Short-term (2 h) exposure to at least 50 μM ARG moderately increased eNOS activity and intracellular glucose (p < 0.05), with no change in eNOS mRNA or protein expression. In contrast, 7-day continuous ARG exposure suppressed eNOS expression and activity. This was accompanied by increase in glucose and O ·−2 accumulation. Co-incubation with 100 μM ascorbic acid, 300 U/ml polyethylene glycol-superoxide dismutase (PEG-SOD), 100 μM l-lysine or 30 μM 5-chloro-2-(N-2,5-dichlorobenenesulfonamido)-benzoxazole (a fructose-1,6-bisphosphatase inhibitor) prevented the occurrence of cellular ARG tolerance. Short-term co-incubation of ARG with PEG-SOD improved cellular nitrite accumulation without altering cellular ARG uptake. These studies suggest that ARG-induced oxidative stress may be a primary causative factor for the development of cellular ARG tolerance.

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References

  • Bednarz K (2004) Am Ende der Welt: eine Reise durch Feuerland und Patagonien. 1. Aufl. edn. Rowohlt, Berlin

  • Bednarz B, Jaxa-Chamiec T, Maciejewski P, Szpajer M, Janik K, Gniot J, Kawka-Urbanek T, Drozdowska D, Gessek J, Laskowski H (2005) Efficacy and safety of oral l-arginine in acute myocardial infarction. Results of the multicenter, randomized, double-blind, placebo-controlled ARAMI pilot trial. Kardiol Pol 62(5):421–427

    PubMed  Google Scholar 

  • Du XL, Edelstein D, Dimmeler S, Ju Q, Sui C, Brownlee M (2001) Hyperglycemia inhibits endothelial nitric oxide synthase activity by posttranslational modification at the Akt site. J Clin Invest 108(9):1341–1348

    PubMed  CAS  Google Scholar 

  • Gitzelmann R (1995) Galactose-1-phosphate in the pathophysiology of galactosemia. Eur J Pediatr 154(7 Suppl 2):S45–S49

    Google Scholar 

  • Gitzelmann R, Bosshard NU (1995) Partial deficiency of galactose-1-phosphate uridyltransferase. Eur J Pediatr 154(7 Suppl 2):S40–S44

    Google Scholar 

  • Granner D, Pilkis S (1990) The genes of hepatic glucose metabolism. J Biol Chem 265(18):10173–10176

    PubMed  CAS  Google Scholar 

  • Ha YH, Milner JA (1979) Urea cycle operation in the arginine deficient rat. Biochem Med 22(2):149–155

    Article  PubMed  CAS  Google Scholar 

  • Hallemeesch MM, Lamers WH, Deutz NE (2002) Reduced arginine availability and nitric oxide production. Clin Nutr 21(4):273–279

    Article  PubMed  CAS  Google Scholar 

  • Hardy TA, May JM (2002) Coordinate regulation of l-arginine uptake and nitric oxide synthase activity in cultured endothelial cells. Free Radic Biol Med 32(2):122–131

    Article  PubMed  CAS  Google Scholar 

  • Hasselblatt M, Krampe H, Jacobs S, Sindram H, Armstrong VW, Hecker M, Ehrenreich H (2006) Arginine challenge unravels persistent disturbances of urea cycle and gluconeogenesis in abstinent alcoholics. Alcohol Alcohol 41(4):372–378

    PubMed  CAS  Google Scholar 

  • Kakoki M, Kim HS, Edgell CJ, Maeda N, Smithies O, DL M (2006) Amino acids as modulators of endothelium-derived nitric oxide. Am J Physiol Renal Physiol 291:297–304

    Article  Google Scholar 

  • Liu P, Hock CE, Nagele R, Wong PY (1997) Formation of nitric oxide, superoxide, and peroxynitrite in myocardial ischemia-reperfusion injury in rats. Am J Physiol 272(5 Pt 2):H2327–H2336

    Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193(1):265–275

    PubMed  CAS  Google Scholar 

  • Luiking YC, Engelen MP, Deutz NE (2010) Regulation of nitric oxide production in health and disease. Curr Opin Clin Nutr Metab Care 13(1):97–104

    Article  PubMed  CAS  Google Scholar 

  • Mabile L, Meilhac O, Escargueil-Blanc I, Troly M, Pieraggi MT, Salvayre R, Negre-Salvayre A (1997) Mitochondrial function is involved in LDL oxidation mediated by human cultured endothelial cells. Arterioscler Thromb Vasc Biol 17(8):1575–1582

    Article  PubMed  CAS  Google Scholar 

  • Maxwell AJ, Zapien MP, Pearce GL, MacCallum G, Stone PH (2002) Randomized trial of a medical food for the dietary management of chronic, stable angina. J Am Coll Cardiol 39(1):37–45

    Article  PubMed  CAS  Google Scholar 

  • Mehta S, Stewart DJ, Langleben D, Levy RD (1995) Short-term pulmonary vasodilation with l-arginine in pulmonary hypertension. Circulation 92(6):1539–1545

    Article  PubMed  CAS  Google Scholar 

  • Nishikawa T, Edelstein D, Du XL, Yamagishi S, Matsumura T, Kaneda Y, Yorek MA, Beebe D, Oates PJ, Hammes HP, Giardino I, Brownlee M (2000) Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature 404(6779):787–790

    Article  PubMed  CAS  Google Scholar 

  • Ohara Y, Peterson TE, Harrison DG (1993) Hypercholesterolemia increases endothelial superoxide anion production. J Clin Invest 91(6):2546–2551

    Article  PubMed  CAS  Google Scholar 

  • Oka RK, Szuba A, Giacomini JC, Cooke JP (2005) A pilot study of l-arginine supplementation on functional capacity in peripheral arterial disease. Vasc Med 10(4):265–274

    Article  PubMed  Google Scholar 

  • Pilkis SJ, Granner DK (1992) Molecular physiology of the regulation of hepatic gluconeogenesis and glycolysis. Annu Rev Physiol 54:885–909

    Article  PubMed  CAS  Google Scholar 

  • Popovic PJ, Zeh HJ 3rd, Ochoa JB (2007) Arginine and immunity. J Nutr 137(6 Suppl 2):1681S–1686S

    Google Scholar 

  • Rajapakse NW, Mattson DL (2009) Role of l-arginine in nitric oxide production in health and hypertension. Clin Exp Pharmacol Physiol 36(3):249–255

    Article  PubMed  CAS  Google Scholar 

  • Romero JR, Suzuka SM, Nagel RL, Fabry ME (2002) Arginine supplementation of sickle transgenic mice reduces red cell density and Gardos channel activity. Blood 99(4):1103–1108

    Article  PubMed  CAS  Google Scholar 

  • Sala R, Rotoli BM, Colla E, Visigalli R, Parolari A, Bussolati O, Gazzola GC, Dall’Asta V (2002) Two-way arginine transport in human endothelial cells: TNF-alpha stimulation is restricted to system y(+). Am J Physiol Cell Physiol 282(1):C134–C143

    PubMed  CAS  Google Scholar 

  • Schulman SP, Becker LC, Kass DA, Champion HC, Terrin ML, Forman S, Ernst KV, Kelemen MD, Townsend SN, Capriotti A, Hare JM, Gerstenblith G (2006) l-arginine therapy in acute myocardial infarction: the vascular interaction with age in myocardial infarction (VINTAGE MI) randomized clinical trial. JAMA 295(1):58–64

    Article  PubMed  CAS  Google Scholar 

  • Shin S, Fung SM, Mohan S, Fung HL (2011a) Simultaneous bioanalysis of l-arginine, l-citrulline, and dimethylarginines by LC-MS/MS. J Chromatogr B Anal Technol Biomed Life Sci 879(7–8):467–474

    Article  CAS  Google Scholar 

  • Shin S, Mohan S, Fung HL (2011b) Intracellular l-arginine concentration does not determine NO production in endothelial cells: implications on the Arginine Paradox. Biochem Biophys Res Commun (in press). doi:10.1016/j.bbrc.2011.09.112

  • Srinivasan S, Hatley ME, Bolick DT, Palmer LA, Edelstein D, Brownlee M, Hedrick CC (2004) Hyperglycaemia-induced superoxide production decreases eNOS expression via AP-1 activation in aortic endothelial cells. Diabetologia 47(10):1727–1734

    Article  PubMed  CAS  Google Scholar 

  • Tesfamariam B, Cohen RA (1992) Free radicals mediate endothelial cell dysfunction caused by elevated glucose. Am J Physiol 263(2 Pt 2):H321–H326

    Google Scholar 

  • Trabelsi F, Helie R, Bergeron R, Lavoie JM (1995) Effect of inhibition of gluconeogenesis on arginine-induced insulin secretion. Physiol Behav 57(4):797–802

    Article  PubMed  CAS  Google Scholar 

  • Vichinsky E (2002) New therapies in sickle cell disease. Lancet 360(9333):629–631. doi:10.1016/S0140-6736(02)09776-3

    Article  PubMed  Google Scholar 

  • Wilson AM, Harada R, Nair N, Balasubramanian N, Cooke JP (2007) l-arginine supplementation in peripheral arterial disease: no benefit and possible harm. Circulation 116(2):188–195

    Article  PubMed  CAS  Google Scholar 

  • Wu G, Morris SM Jr (1998) Arginine metabolism: nitric oxide and beyond. Biochem J 336(Pt 1):1–17

    PubMed  CAS  Google Scholar 

  • Zani BG, Bohlen HG (2005) Transport of extracellular l-arginine via cationic amino acid transporter is required during in vivo endothelial nitric oxide production. Am J Physiol Heart Circ Physiol 289(4):H1381–H1390

    Article  PubMed  CAS  Google Scholar 

  • Zhao HT, Kalivendi S, Zhang H, Joseph J, Nithipatikom K, Vasquez-Vivar J, Kalyanaraman B (2003) Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide. Free Radic Biol Med 34(11):1359–1368

    Article  PubMed  CAS  Google Scholar 

  • Zhou M, Martindale RG (2007) Arginine in the critical care setting. J Nutr 137(6 Suppl 2):1687S–1692S

    Google Scholar 

Download references

Acknowledgments

We thank Daniel Brazeau and Kathleen Boje for access to their genomic and cell culture facility, respectively. This work was supported in part by NIH grant HL081580.

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Correspondence to Ho-Leung Fung.

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Mohan, S., Wu, CC., Shin, S. et al. Continuous exposure to l-arginine induces oxidative stress and physiological tolerance in cultured human endothelial cells. Amino Acids 43, 1179–1188 (2012). https://doi.org/10.1007/s00726-011-1173-y

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  • DOI: https://doi.org/10.1007/s00726-011-1173-y

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