European Journal of Nutrition

, Volume 53, Issue 3, pp 813–821 | Cite as

Long-term dietary l-arginine supplementation increases endothelial nitric oxide synthase and vasoactive intestinal peptide immunoexpression in rat small intestine

  • Ksenija Velickovic
  • Milica Markelic
  • Igor Golic
  • Vesna Otasevic
  • Ana Stancic
  • Aleksandra Jankovic
  • Milica Vucetic
  • Biljana Buzadzic
  • Bato Korac
  • Aleksandra KoracEmail author
Original Contribution


Background and aims

Nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) are important intestinal neurotransmitters that coexist in the gut enteric nervous system and play an important role in intestinal physiology (e.g., absorption, motility, fluid secretion and smooth muscle relaxation). It is also known that cold exposure alters several aspects of gastrointestinal physiology and induces hyperphagia to meet increased metabolic demands, but there are no data regarding NO and VIP involvement in intestinal response during acclimation to cold. The objective of this study was to determine the influence of long-term l-arginine supplementation on the expression of the three isoforms of nitric oxide synthase (NOS) and VIP in small intestine of rats acclimated to room temperature or cold.


Animals (six per group) acclimated to room temperature (22 ± 1 °C) and cold (4 ± 1 °C), respectively, were treated with 2.25 % l-arginine, a substrate for NOSs, or with 0.01 % N ω-nitro-l-arginine methyl ester, an inhibitor of NOSs, for 45 days. The topographical distribution of VIP and NOSs expression in small intestine was studied by immunohistochemistry, and ImageJ software was used for semiquantitative densitometric analysis of their immunoexpression.


Long-term dietary l-arginine supplementation increases VIP and NOSs immunoexpression at room temperature while at cold increases the endothelial NOS, inducible NOS and VIP but decrease neuronal NOS in rat small intestine.


Our results demonstrate that long-term dietary l-arginine supplementation modulates NOSs and VIP immunoexpression in rat small intestine with respect to ambient temperature, pointing out the eNOS as a predominant NOS isoform with an immunoexpression pattern similar to VIP.


Arginine Small intestine Nitric oxide synthase Vasoactive intestinal peptide 



Nitric oxide


Nitric oxide synthase


Endothelial NOS


Inducible NOS


Neuronal NOS


Vasoactive intestinal peptide


N ω-nitro-l-arginine methyl ester


Interstitial cells of Cajal



This research was supported by the Grant from Serbian Ministry of Education, Science and Technological Development, #173055.

Conflict of interest

The authors declare that they have no conflict of interests.


  1. 1.
    Wu G, Morris SM Jr (1998) Arginine metabolism: nitric oxide and beyond. Biochem J 336:1–17Google Scholar
  2. 2.
    Wu X, Ruan Z, Gao Y, Yin Y, Zhou X, Wang L, Geng M, Hou Y, Wu G (2010) Dietary supplementation with l-arginine or N-carbamylglutamate enhances intestinal growth and heat shock protein-70 expression in weanling pigs fed a corn- and soybean- meal-based diet. Amino Acids 39:831–839CrossRefGoogle Scholar
  3. 3.
    Koppelmann T, Pollak Y, Mogilner J, Bejar J, Coran AG, Sukhotnik I (2012) Dietary l-arginine supplementation reduces Methotrexate-induced intestinal mucosal injury in rat. BMC Gastroenterol 12:41CrossRefGoogle Scholar
  4. 4.
    Yao K, Guan S, Li T, Huang R, Wu G, Ruan Z, Yin Y (2011) Dietary l-arginine supplementation enhances intestinal development and expression of vascular endothelial growth factor in weanling piglets. Br J Nutr 105:703–709CrossRefGoogle Scholar
  5. 5.
    Taboada MC, Rodriguez B, Millan R, Miguez I (2006) Role of dietary l-arginine supplementation on serum parameters and intestinal enzyme activities in rats fed an excess-fat diet. Biomed Pharmacother 60:10–13CrossRefGoogle Scholar
  6. 6.
    Janero DR (2001) Nutritional aspects of nitric oxide: human health implications and therapeutic opportunities. Nutrition 17:896–903CrossRefGoogle Scholar
  7. 7.
    Petrovic V, Korac A, Buzadzic B, Korac B (2005) The effects of l-arginine and l-NAME supplementation on redox-regulation and thermogenesis in interscapular brown adipose tissue. J Exp Biol 208:4263–4271CrossRefGoogle Scholar
  8. 8.
    Beck PL, Xavier R, Wong J, Ezedi I, Mashimo H, Mizoguchi A, Mizoguchi E, Bhan AK, Podolsky DK (2004) Paradoxical roles of different nitric oxide synthase isoforms in colonic injury. Am J Physiol Gastrointest Liver Physiol 286:137–147CrossRefGoogle Scholar
  9. 9.
    Petersson J, Schreiber O, Steege A, Patzak A, Hellsten A, Phillipson M, Holm L (2007) eNOS involved in colitis-induced mucosal blood flow increase. Am J Physiol Gastrointest Liver Physiol 293:1281–1287CrossRefGoogle Scholar
  10. 10.
    Castro M, Munoz JM, Arruebo MP, Murillo MD, Arnal C, Bonafonte JI, Plaza MA (2012) Involvement of neuronal nitric oxide synthase (nNOS) in the regulation of migrating motor complex (MMC) in sheep. Vet J 192:352–358CrossRefGoogle Scholar
  11. 11.
    Hansen MB (2003) Neurohumoral control of gastrointestinal motility. Physiol Res 52:1–30Google Scholar
  12. 12.
    Bredt DS, Hwang PM, Snyder SH (1990) Localization of nitric oxide synthase indicating a neural role for nitric oxide. Nature 347(6295):768–770CrossRefGoogle Scholar
  13. 13.
    Suthamnatpong N, Hata F, Kanada A, Takeuchi T, Yagasaki O (1993) Mediators of nonadrenergic, noncholinergic inhibition in the proximal, middle and distal regions of rat colon. Br J Pharmacol 108:348–355CrossRefGoogle Scholar
  14. 14.
    Furness JB, Bornstein JC, Murphy R, Pompolo S (1992) Roles of peptides in transmission in the enteric nervous system. Trends Neurosci 15:66–71CrossRefGoogle Scholar
  15. 15.
    Dick JM, Van Molle W, Brouckaert P, Lefebvre RA (2002) Relaxation by vasoactive intestinal polypeptide in the gastric fundus of nitric oxide synthase-deficient mice. J Physiol 538:133–143CrossRefGoogle Scholar
  16. 16.
    Grider JR (1993) Interplay of VIP and nitric oxide in regulation of the descending relaxation phase of peristalsis. Am J Physiol 264:334–340Google Scholar
  17. 17.
    Murthy KS, Grider JR, Jin JG, Makhlouf GM (1996) Interplay of VIP and nitric oxide in the regulation of neuromuscular function in the gut. Ann NY Acad Sci 805:355–362CrossRefGoogle Scholar
  18. 18.
    Grider JR, Murthy KS (2008) Autoinhibition of endothelial nitric oxide synthase (eNOS) in gut smooth muscle by nitric oxide. Regul Pept 151:75–79CrossRefGoogle Scholar
  19. 19.
    Kaushik S, Kaur J (2003) Chronic cold exposure affects the antioxidant defense system in various rat tissues. Clin Chim Acta 333:69–77CrossRefGoogle Scholar
  20. 20.
    Kaushik S, Kaur J (2005) Effect of chronic cold stress on intestinal epithelial cell proliferation and inflammation in rats. Stress 8:191–197CrossRefGoogle Scholar
  21. 21.
    Soderholm JD, Perdue MH (2001) Stress and gastrointestinal tract. II. Stress and intestinal barrier function. Am J Physiol Gastrointest Liver Physiol 280:7–13Google Scholar
  22. 22.
    Irwin N, Francis JM, Flatt PR (2011) Alterations of glucose-dependent insulinotropic polypeptide (GIP) during cold acclimation. Regul Pept 167:91–96CrossRefGoogle Scholar
  23. 23.
    Saha SK, Ohinata H, Kuroshima A (1996) Effects of acute and chronic inhibition of nitric oxide synthase on brown adipose tissue thermogenesis. Jpn J Physiol 46:375–382CrossRefGoogle Scholar
  24. 24.
    Petrovic V, Buzadzic B, Korac A, Vasilijevic A, Jankovic A, Micunovic K, Korac B (2008) Antioxidative defence alterations in skeletal muscle during prolonged acclimation to cold: role of l-arginine/NO-producing pathway. J Exp Biol 211:114–120CrossRefGoogle Scholar
  25. 25.
    He J, Sun X, Qian K-Q, Liu X, Wang Z, Chen Y (2009) Protection of cerulein-induced pancreatic fibrosis by pancreas-specific expression of Smad7. Biochim Biophys Acta 1792:56–60CrossRefGoogle Scholar
  26. 26.
    Qu XW, Wang H, De Plaen IG, Rozenfeld RA, Hsueh W (2001) Neuronal nitric oxide synthase (NOS) regulates the expression of inducible NOS in rat small intestine via modulation of nuclear factor kappa B. FASEB J 15:439–446CrossRefGoogle Scholar
  27. 27.
    Teshfam M, Brujeni GN, Hassanpour H (2006) Evaluation of endothelial and inducible nitric oxide synthase mRNA expression in the lung of broiler chickens with developmental pulmonary hypertension due to cold stress. Br Poult Sci 47:223–229CrossRefGoogle Scholar
  28. 28.
    Mourad FH, Barada KA, Abdel-Malak N, Bou Rached NA, Khoury CI, Saade NE, Nassar CF (2003) Interplay between nitric oxide and vasoactive intestinal polypeptide in inducing fluid secretion in rat jejunum. J Physiol 550:863–871CrossRefGoogle Scholar
  29. 29.
    Nishizaki K, Nakao K, Ishii H, Yamanaka H, Tokunaga A, Nakagawa K, Yamamura T, Noguchi K (2003) Induction of neuronal nitric oxide synthase by sympathetic denervation is mediated via alpha 2-adrenoceptors in the jejunal myenteric plexus. Brain Res 965:121–129CrossRefGoogle Scholar
  30. 30.
    Takahashi T (2003) Pathophysiological significance of neuronal nitric oxide synthase in the gastrointestinal tract. J Gastroenterol 38:421–430CrossRefGoogle Scholar
  31. 31.
    Xie QW, Cho HJ, Calaycay J, Mumford RA, Swiderek KM, Lee TD, Ding A, Troso T, Nathan C (1992) Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science 256:225–228CrossRefGoogle Scholar
  32. 32.
    Porras M, Martin MT, Torres R, Vergara P (2006) Cyclical upregulated iNOS and long-term downregulated nNOS are the bases for relapse and quiescent phases in a rat model of IBD. Am J Physiol Gastrointest Liver Physiol 290:423–430CrossRefGoogle Scholar
  33. 33.
    Wang XY, Paterson C, Huizinga JD (2003) Cholinergic and nitrergic innervation of ICC-DMP and ICC-IM in the human small intestine. Neurogastroenterol Motil 15:531–543CrossRefGoogle Scholar
  34. 34.
    Streutker CJ, Huizinga JD, Driman DK, Riddell RH (2007) Interstitial cells of Cajal in health and disease. Part I: normal ICC structure and function with associated motility disorders. Histopathology 50:176–189CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Ksenija Velickovic
    • 1
  • Milica Markelic
    • 1
  • Igor Golic
    • 1
  • Vesna Otasevic
    • 2
  • Ana Stancic
    • 2
  • Aleksandra Jankovic
    • 2
  • Milica Vucetic
    • 2
  • Biljana Buzadzic
    • 2
  • Bato Korac
    • 2
  • Aleksandra Korac
    • 1
    Email author
  1. 1.Faculty of BiologyUniversity of BelgradeBelgradeSerbia
  2. 2.Institute for Biological Research “Sinisa Stankovic”University of BelgradeBelgradeSerbia

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