Digestive Diseases and Sciences

, Volume 63, Issue 3, pp 619–627 | Cite as

Neuronal Nitric Oxide Synthase Is a Novel Biomarker for the Interstitial Cells of Cajal in Stress-Induced Diarrhea-Dominant Irritable Bowel Syndrome

  • Da Eun Jang
  • Ji Hyun Bae
  • Yoo Jin Chang
  • Yoon Hoo Lee
  • Ki Taek Nam
  • Il Yong Kim
  • Je Kyung Seong
  • Yong Chan Lee
  • Su Cheong YeomEmail author
Original Article



Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder involving changes in normal bowel movements. The pathophysiology of IBS is not clearly understood owing to the lack of identifiable pathological abnormalities and reliable biomarkers.


The aim of this study was to discover the novel and reliable biomarker for IBS.


In this study, neonatal maternal separation (NMS) stress model was used for the IBS mouse model. Further assessment was conducted with whole gastrointestinal transit test, quantitative RT-PCR, histological examination, and western blot.


Male pups developed symptoms similar to those of human IBS with diarrhea (IBS-D), such as low-grade inflammation, stool irregularity, and increased bowel motility. NMS stress influenced to the interstitial cells of Cajal (ICC) and induced altered bowel motility, resulting in IBS-D-like symptoms. In addition, we found neuronal nitric oxide synthase (nNOS) to be a novel biomarker for ICC under NMS stress. nNOS expression was only observed in the ICC of the submucosal plexus of IBS-D mice, and the inhibition of nNOS changed the phenotype from IBS-D to IBS with constipation.


Our study demonstrates that early-life stress can influence to ICC and modulate bowel activity and that nNOS might be used as a biomarker for ICC stimulation in IBS.


Interstitial cells of Cajal irritable bowel syndrome Neonatal maternal separation Neuronal nitric oxide synthase 



This study was supported by the Korea Food and Drug Administration Grant (13182KFDA660 and 14182KFDA978) funded by the Korea government.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10620_2018_4933_MOESM1_ESM.pdf (212 kb)
Supplementary material 1 (PDF 212 kb)


  1. 1.
    Canavan C, West J, Card T. The epidemiology of irritable bowel syndrome. Clin Epidemiol. 2014;6:71–80.PubMedPubMedCentralGoogle Scholar
  2. 2.
    Piche M, Bouin M, Arsenault M, Poitras P, Rainville P. Decreased pain inhibition in irritable bowel syndrome depends on altered descending modulation and higher-order brain processes. Neuroscience. 2011;195:166–175.CrossRefPubMedGoogle Scholar
  3. 3.
    Manning AP, Thompson WG, Heaton KW, Morris AF. Towards positive diagnosis of the irritable bowel. Br Med J. 1978;2:653–654.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Thompson WG, Longstreth GF, Drossman DA, Heaton KW, Irvine EJ, Muller-Lissner SA. Functional bowel disorders and functional abdominal pain. Gut. 1999;45:I43–I47.Google Scholar
  5. 5.
    Shih YC, Barghout VE, Sandler RS, et al. Resource utilization associated with irritable bowel syndrome in the United States 1987–1997. Dig Dis Sci. 2002;47:1705–1715.CrossRefPubMedGoogle Scholar
  6. 6.
    Philpott H, Gibson P, Thien F. Irritable bowel syndrome—an inflammatory disease involving mast cells. Asia Pac Allergy. 2011;1:36–42.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Moloney RD, Johnson AC, O’Mahony SM, Dinan TG, Greenwood-Van Meerveld B, Cryan JF. Stress and the microbiota-gut-brain axis in visceral pain: relevance to irritable bowel syndrome CNS. Neurosci Ther. 2016;22:102–117.CrossRefGoogle Scholar
  8. 8.
    Venkova K, Johnson AC, Myers B, Greenwood-Van Meerveld B. Exposure of the amygdala to elevated levels of corticosterone alters colonic motility in response to acute psychological stress. Neuropharmacology. 2010;58:1161–1167.CrossRefPubMedGoogle Scholar
  9. 9.
    Fichna J, Storr MA. Brain-Gut interactions in IBS. Front Pharmacol. 2012;3:127.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Tramullas M, Dinan TG, Cryan JF. Chronic psychosocial stress induces visceral hyperalgesia in mice. Stress. 2012;15:281–292.CrossRefPubMedGoogle Scholar
  11. 11.
    Piche T, Barbara G, Aubert P, et al. Impaired intestinal barrier integrity in the colon of patients with irritable bowel syndrome: involvement of soluble mediators. Gut. 2009;58:196–201.CrossRefPubMedGoogle Scholar
  12. 12.
    Moloney RD, O’Leary OF, Felice D, Bettler B, Dinan TG, Cryan JF. Early-life stress induces visceral hypersensitivity in mice. Neurosci Lett. 2012;512:99–102.CrossRefPubMedGoogle Scholar
  13. 13.
    Smith SM, Vale WW. The role of the hypothalamic-pituitary-adrenal axis in neuroendocrine responses to stress. Dialogues Clin Neurosci. 2006;8:383–395.PubMedPubMedCentralGoogle Scholar
  14. 14.
    Jones MP, Dilley JB, Drossman D, Crowell MD. Brain-gut connections in functional GI disorders: anatomic and physiologic relationships. Neurogastroenterol Motil Off J Eur Gastrointest Motil Soc. 2006;18:91–103.CrossRefGoogle Scholar
  15. 15.
    Lomax AE, Sharkey KA, Furness JB. The participation of the sympathetic innervation of the gastrointestinal tract in disease states. Neurogastroenterol Motil Off J Eur Gastrointest Motil Soc. 2010;22:7–18.Google Scholar
  16. 16.
    Spiller R, Lam C. An update on post-infectious irritable bowel syndrome: role of genetics, immune activation, serotonin and altered. J Neurogastroenterol Motil. 2012;18:258–268.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Ren TH, Wu J, Yew D, et al. Effects of neonatal maternal separation on neurochemical and sensory response to colonic distension in a rat model of irritable bowel syndrome. Am J Physiol Gastrointest Liver Physiol. 2007;292:G849–G856.CrossRefPubMedGoogle Scholar
  18. 18.
    Barreau F, Ferrier L, Fioramonti J, Bueno L. New insights in the etiology and pathophysiology of irritable bowel syndrome: contribution of neonatal stress models. Pediatric Res. 2007;62:240–245.CrossRefGoogle Scholar
  19. 19.
    O’Mahony SM, Hyland NP, Dinan TG, Cryan JF. Maternal separation as a model of brain-gut axis dysfunction. Psychopharmacology. 2011;214:71–88.CrossRefPubMedGoogle Scholar
  20. 20.
    Dorner G, Tonjes R, Hecht K, et al. Pyridostigmine administration in newborn rats prevents permanent mental ill-effects produced by maternal deprivation. Endokrinologie.. 1981;77:101–104.PubMedGoogle Scholar
  21. 21.
    Matthews K, Wilkinson LS, Robbins TW. Repeated maternal separation of preweanling rats attenuates behavioral responses to primary and conditioned incentives in adulthood. Physiol Behav. 1996;59:99–107.CrossRefPubMedGoogle Scholar
  22. 22.
    Mayer EA, Bradesi S, Chang L, Spiegel BM, Bueller JA, Naliboff BD. Functional GI disorders: from animal models to drug development. Gut. 2008;57:384–404.CrossRefPubMedGoogle Scholar
  23. 23.
    Barreau F, Ferrier L, Fioramonti J, Bueno L. Neonatal maternal deprivation triggers long term alterations in colonic epithelial barrier and mucosal immunity in rats. Gut. 2004;53:501–506.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Chung EK, Zhang X, Li Z, Zhang H, Xu H, Bian Z. Neonatal maternal separation enhances central sensitivity to noxious colorectal distention in rat. Brain Res. 2007;1153:68–77.CrossRefPubMedGoogle Scholar
  25. 25.
    Coutinho SV, Plotsky PM, Sablad M, et al. Neonatal maternal separation alters stress-induced responses to viscerosomatic nociceptive stimuli in rat. Am J Physiol Gastrointest Liver Physiol. 2002;282:G307–G316.CrossRefPubMedGoogle Scholar
  26. 26.
    Spandidos A, Wang X, Wang H, Seed B. PrimerBank: a resource of human and mouse PCR primer pairs for gene expression detection and quantification. Nucleic Acids Res.. 2010;38:D792–D799.CrossRefPubMedGoogle Scholar
  27. 27.
    Zhou M, Jia P, Chen J, et al. Laxative effects of Salecan on normal and two models of experimental constipated mice. BMC Gastroenterol.. 2013;13:52.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Mehri D, Monsef-Esfahani H, Gharibzadeh S, Jafari K, Faghihi M. Effects of black tea extract and its thearubigins on whole gut transit time in mice: involvement of 5-HT3 receptors. Jundishapur J Nat Pharm Prod. 2008;2008:39–44.Google Scholar
  29. 29.
    Hu S, Xu W, Miao X, et al. Sensitization of sodium channels by cystathionine beta-synthetase activation in colon sensory neurons in adult rats with neonatal maternal deprivation. Exp Nneurol. 2013;248:275–285.CrossRefGoogle Scholar
  30. 30.
    Kiss A, Mravec B, Palkovits M, Kvetnansky R. Stress-induced changes in tyrosine hydroxylase gene expression in rat hypothalamic paraventricular, periventricular, and dorsomedial nuclei. Ann NY Acad Sci. 2008;1148:74–85.CrossRefPubMedGoogle Scholar
  31. 31.
    Tank AW, Xu L, Chen X, Radcliffe P, Sterling CR. Post-transcriptional regulation of tyrosine hydroxylase expression in adrenal medulla and brain. Ann NY Acad Sci. 2008;1148:238–248.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Zhang R, Zou N, Li J, et al. Elevated expression of c-fos in central nervous system correlates with visceral hypersensitivity in irritable bowel syndrome (IBS): a new target for IBS treatment. Int J Colorectal Dis. 2011;26:1035–1044.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Eshraghian A, Eshraghian H. Interstitial cells of Cajal: a novel hypothesis for the pathophysiology of irritable bowel syndrome. Can J Gastroenterol. 2011;25:277–279.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Sung R, Kim YC, Yun HY, et al. Interstitial cells of Cajal (ICC)-like-c-kit positive cells are involved in gastritis and carcinogenesis in human stomach. Oncol Rep.. 2011;26:33–42.PubMedGoogle Scholar
  35. 35.
    Sanders KM, Ward SM, Koh SD. Interstitial cells: regulators of smooth muscle function. Physiol Rev. 2014;94:859–907.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Takahashi T. Pathophysiological significance of neuronal nitric oxide synthase in the gastrointestinal tract. J Gastroenterol. 2003;38:421–430.CrossRefPubMedGoogle Scholar
  37. 37.
    Gaynes BN, Drossman DA. The role of psychosocial factors in irritable bowel syndrome baillieres. Best Pract Res Clin Gastroenterol. 1999;13:437–452.CrossRefGoogle Scholar
  38. 38.
    Dinan TG, Quigley EM, Ahmed SM, et al. Hypothalamic-pituitary-gut axis dysregulation in irritable bowel syndrome: plasma cytokines as a potential biomarker? Gastroenterology. 2006;130:304–311.CrossRefPubMedGoogle Scholar
  39. 39.
    Zhang M, Leung FP, Huang Y, Bian ZX. Increased colonic motility in a rat model of irritable bowel syndrome is associated with up-regulation of L-type calcium channels in colonic smooth muscle cells. Neurogastroenterol Motil Off J Eur Gastrointest Motil Soc. 2010;22:e162–e170.Google Scholar
  40. 40.
    Lu X, Zhang S, Yang C, et al. Effect of TongXie-YaoFang on Cl(−) and HCO3(−) transport in diarrhea-predominant irritable bowel syndrome rats. Evid Based Complement Alternat Med. 2016;2016:7954982.PubMedPubMedCentralGoogle Scholar
  41. 41.
    Cashman MD, Martin DK, Dhillon S, Puli SR. Irritable bowel syndrome: a clinical review. Curr Rheumatol Rev.. 2016;12:13–26.CrossRefPubMedGoogle Scholar
  42. 42.
    Ohman L, Simren M. Pathogenesis of IBS: role of inflammation, immunity and neuroimmune interactions. Nat Rev Gastroenterol Hepatol. 2010;7:163–173.CrossRefPubMedGoogle Scholar
  43. 43.
    Mulak A, Tache Y, Larauche M. Sex hormones in the modulation of irritable bowel syndrome. World J Gastroenterol. 2014;20:2433–2448.CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Zhu LJ, Liu MY, Li H, et al. The different roles of glucocorticoids in the hippocampus and hypothalamus in chronic stress-induced HPA axis hyperactivity. PLoS ONE. 2014;9:e97689.CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Streutker CJ, Huizinga JD, Driman DK, Riddell RH. Interstitial cells of Cajal in health and disease. Part II: ICC and gastrointestinal stromal tumours. Histopathology. 2007;50:190–202.CrossRefPubMedGoogle Scholar
  46. 46.
    Lee MY. Does decreased c-kit expression in myenteric interstitial cells of Cajal cause decreased spontaneous contraction in murine proximal colon? J Neurogastroenterol Motil. 2015;21:1–3.CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Radenkovic G, Savic V, Mitic D, Grahovac S, Bjelakovic M, Krstic M. Development of c-kit immunopositive interstitial cells of Cajal in the human stomach. J Cell Mol Med.. 2010;14:1125–1134.PubMedGoogle Scholar
  48. 48.
    Wang XY, Chen JH, Li K, Zhu YF, Wright GW, Huizinga JD. Discrepancies between c-kit positive and Ano1 positive ICC-SMP in the W/Wv and wild-type mouse colon; relationships with motor patterns and calcium transients. Neurogastroenterol Motil Off J Eur Gastrointest Motil Soc. 2014;26:1298–1310.CrossRefGoogle Scholar
  49. 49.
    Tjong YW, Ip SP, Lao L, et al. Role of neuronal nitric oxide synthase in colonic distension-induced hyperalgesia in distal colon of neonatal maternal separated male rats. Neurogastroenterol Motil Off J Eur Gastrointest Motil Soc. 2011;23:666-e278.Google Scholar
  50. 50.
    Iino S, Horiguchi K, Nojyo Y. Interstitial cells of Cajal are innervated by nitrergic nerves and express nitric oxide-sensitive guanylate cyclase in the guinea-pig gastrointestinal tract. Neuroscience. 2008;152:437–448.CrossRefPubMedGoogle Scholar
  51. 51.
    Choi KM, Gibbons SJ, Roeder JL, et al. Regulation of interstitial cells of Cajal in the mouse gastric body by neuronal nitric oxide. Neurogastroenterol Motil Off J Eur Gastrointest Motil Soc. 2007;19:585–595.CrossRefGoogle Scholar
  52. 52.
    Saur D, Paehge H, Schusdziarra V, Allescher HD. Distinct expression of splice variants of neuronal nitric oxide synthase in the human gastrointestinal tract. Gastroenterology. 2000;118:849–858.CrossRefPubMedGoogle Scholar
  53. 53.
    Watanabe Y, Ando H, Seo T, et al. Attenuated nitrergic inhibitory neurotransmission to interstitial cells of Cajal in the lower esophageal sphincter with esophageal achalasia in children. Pediatr Int Off J Jpn Pediatric Soc. 2002;44:145–148.CrossRefGoogle Scholar
  54. 54.
    Muller M, Colcuc S, Drescher DG, et al. Murine genetic deficiency of neuronal nitric oxide synthase (nNOS(-/-)) and interstitial cells of Cajal (W/W(v)): Implications for achalasia? J Gastroenterol Hepatol. 2014;29:1800–1807.CrossRefPubMedGoogle Scholar
  55. 55.
    Powell AK, Bywater RA. Murine intestinal migrating motor complexes: longitudinal components. Neurogastroenterol Motil Off J Eur Gastrointest Motil Soc. 2003;15:245–256.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Graduate School of International Agricultural TechnologySeoul National UniversityPyeongchangRepublic of Korea
  2. 2.Designed Animal and Transplantation Research Institute, Institute of Greenbio Research and TechnologySeoul National UniversityPyeongchangRepublic of Korea
  3. 3.Severance Biomedical Science Institute, Brain Korea 21 PLUS Project for Medical ScienceYonsei University College of MedicineSeoulRepublic of Korea
  4. 4.Department of Veterinary ScienceSeoul National UniversitySeoulRepublic of Korea
  5. 5.Yonsei UniversitySeoulRepublic of Korea

Personalised recommendations