Advertisement

Journal of Gastroenterology

, Volume 54, Issue 11, pp 994–1006 | Cite as

Cystic fibrosis transmembrane conductance regulator modulates enteric cholinergic activities and is abnormally expressed in the enteric ganglia of patients with slow transit constipation

  • Ka Ming Yeh
  • Olle Johansson
  • Huy Le
  • Karan Rao
  • Irit Markus
  • Dayashan Shevy Perera
  • David Zachary Lubowski
  • Denis Warwick King
  • Li Zhang
  • Hongzhuan Chen
  • Lu LiuEmail author
Original Article—Alimentary Tract

Abstract

Background

Cystic fibrosis transmembrane conductance regulator (CFTR) was recently found in the enteric nervous system, where its role is unclear. We aimed to identify which enteric neuronal structures express CFTR, whether CFTR modulates enteric neurotransmission and if altered CFTR expression is associated with slow transit constipation (STC).

Methods

Immunofluorescence double labeling was performed to localize CFTR with various neuronal and glial cell markers in the human colon. The immunoreactivity (IR) of CFTR and choline acetyltransferase (ChAT) on myenteric plexus of control and STC colon was quantitatively analyzed. In control colonic muscle strips, electrical field stimulation (EFS) evoked contractile responses and the release of acetylcholine (ACh) was measured in the presence of the CFTR channel inhibitor, CFTR(inh)-172.

Results

CFTR-IR was densely localized to myenteric ganglia, where it was co-localized with neuronal markers HuC/D and β-tubulin, and glial marker S-100 but little with glial fibrillary acidic protein. Vesicular ACh transport was almost exclusively co-localized with CFTR, but neurons expressing nitric oxide synthase were CFTR negative. Significant reductions of CFTR-IR (P < 0.01) and ChAT-IR (P < 0.05) were observed on myenteric ganglia of STC compared to control. Pre-treatment of colonic muscle strips with CFTR(inh)-172 (10 µM) significantly inhibited EFS-evoked contractile responses (P < 0.01) and ACh release (P < 0.05).

Conclusions

Co-localization of CFTR-IR with cholinergic markers, inhibition of EFS-induced colonic muscle contractility and ACh release by CFTR(inh)-172 suggest that CFTR modulates enteric cholinergic neurotransmission. The downregulation of CFTR and ChAT in myenteric ganglia of STC correlated with the impaired contractile responses to EFS.

Keywords

CFTR Motility disorder Slow transit constipation 

Notes

Acknowledgments

This study was supported by a project Grant from the National Health and Medical Research Council of Australia (APP1048885), and by the UNSW and Shanghai Jiao Tong University Collaborative Seed Grant. We would like to offer our special thanks to Stelina Drimousis and Erica Diezmos in specimen collection and preparation.

Compliance with ethical standards

Conflict of interest

There are no conflicts of interest.

Supplementary material

535_2019_1610_MOESM1_ESM.docx (4.7 mb)
Supplementary file1 (DOCX 4784 kb)
535_2019_1610_MOESM2_ESM.docx (157 kb)
Supplementary file2 (DOCX 157 kb)

References

  1. 1.
    Gustafsson JK, Ermund A, Ambort D, et al. Bicarbonate and functional CFTR channel are required for proper mucin secretion and link cystic fibrosis with its mucus phenotype. J Exp Med. 2012;209:1263–72.PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    Char JE, Wolfe MH, Cho HJ, et al. A little CFTR goes a long way: CFTR-dependent sweat secretion from G551D and R117H–5T cystic fibrosis subjects taking ivacaftor. PLoS ONE. 2014;9:e88564.PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Oca F, Dreux S, Gerard B, et al. Amniotic Fluid Digestive Enzyme Analysis Is Useful for Identifying CFTR Gene Mutations of Unclear Significance. Clin Chem. 2009;55:2214–7.CrossRefGoogle Scholar
  4. 4.
    Sorio C, Buffelli M, Angiari C, et al. Defective CFTR expression and function are detectable in blood monocytes: development of a new blood test for cystic fibrosis. PLoS ONE. 2011;6:e22212.PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Johansson J, Vezzalini M, Verze G, et al. Detection of CFTR protein in human leukocytes by flow cytometry. Cytometry A. 2014;85:611–20.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Verloo P, Kocken CH, Van der Wel A, et al. Plasmodium falciparum-activated chloride channels are defective in erythrocytes from cystic fibrosis patients. J Biol Chem. 2004;279:10316–22.CrossRefGoogle Scholar
  7. 7.
    Su M, Guo Y, Zhao Y, et al. Expression of cystic fibrosis transmembrane conductance regulator in paracervical ganglia this paper is one of a selection of papers published in this special issue entitled “Second International Symposium on Recent Advances in Basic, Clinical, and Social Medicine” and has undergone the journal's usual peer review process. Biochem Cell Biol. 2010;88:747–55.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Riordan JR, Rommens JM, Kerem B, et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science. 1989;245:1066–73.CrossRefGoogle Scholar
  9. 9.
    Dalli J, Rosignoli G, Hayhoe RP, et al. CFTR inhibition provokes an inflammatory response associated with an imbalance of the annexin A1 pathway. Am J Pathol. 2010;177:176–86.PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Malats N, Casals T, Porta M, et al. Cystic fibrosis transmembrane regulator (CFTR) ΔF508 mutation and 5T allele in patients with chronic pancreatitis and exocrine pancreatic cancer. Gut. 2001;48:70–4.PubMedCentralCrossRefPubMedGoogle Scholar
  11. 11.
    McLatchie LM, Young JS, Fry CH. Regulation of ACH release from guinea pig bladder urothelial cells: potential role in bladder filling sensations. Br J Pharmacol. 2014;171:3394–403.PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Ochodnicky P, Michel MB, Butter JJ, et al. Bradykinin modulates spontaneous nerve growth factor production and stretch-induced ATP release in human urothelium. Pharmacol Res. 2013;70:147–54.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Lubowski DZ, Chen FC, Kennedy ML, et al. Results of colectomy for severe slow transit constipation. Dis Colon Rectum. 1996;39:23–9.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Wong SW, Lubowski DZ. Slow-transit constipation: evaluation and treatment. ANZ J Surg. 2007;77:320–8.CrossRefGoogle Scholar
  15. 15.
    Preston DM, Lennardjones JE. Severe chronic constipation of young-women - idiopathic slow transit constipation. Gut. 1986;27:41–8.PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Bassotti G, Villanacci V, Maurer CA, et al. The role of glial cells and apoptosis of enteric neurones in the neuropathology of intractable slow transit constipation. Gut. 2006;55:41–6.PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Tomita R, Tanjoh K, Fujisaki S, et al. Regulation of the enteric nervous system in the colon of patients with slow transit constipation. Hepatogastroenterology. 2002;49:1540–4.PubMedPubMedCentralGoogle Scholar
  18. 18.
    Liu L, Shang F, Morgan MJ, et al. Cyclooxygenase-dependent alterations in substance P-mediated contractility and tachykinin NK1 receptor expression in the colonic circular muscle of patients with slow transit constipation. J Pharmacol Exp Ther. 2009;329:282–9.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Jung HK, Kim DY, Moon IH. Effects of gender and menstrual cycle on colonic transit time in healthy subjects. Korean J Intern Med. 2003;18:181.PubMedCentralCrossRefPubMedGoogle Scholar
  20. 20.
    Björnsson ES, Chey WD, Hooper F, et al. Impaired gastrocolonic response and peristaltic reflex in slow-transit constipation: role of 5-HT3 pathways. Am J Physiol Gastrointest Liver Physiol. 2002;283:G400–G407407.CrossRefGoogle Scholar
  21. 21.
    Frattini JC, Nogueras JJ. Slow transit constipation: a review of a colonic functional disorder. Clin Colon Rectal Surg. 2008;21:146–52.PubMedCentralCrossRefPubMedGoogle Scholar
  22. 22.
    Wong SW, Lubowski DZJ. Slow‐transit constipation: evaluation and treatment. ANZ J Surg. 2007;77:320–8.CrossRefGoogle Scholar
  23. 23.
    Strong TV, Boehm K, Collins FS. Localization of cystic fibrosis transmembrane conductance regulator mRNA in the human gastrointestinal tract by in situ hybridization. J Clin Invest. 1994;93:347–54.PubMedCentralCrossRefPubMedGoogle Scholar
  24. 24.
    Beyder A, Farrugia G. Ion channelopathies in functional GI disorders. Am J Physiol Gastrointest Liver Physiol. 2016;311:G581–G586586.PubMedCentralCrossRefPubMedGoogle Scholar
  25. 25.
    Cil O, Phuan PW, Lee S, et al. CFTR activator increases intestinal fluid secretion and normalizes stool output in a mouse model of constipation. Cell Mol Gastroenterol Hepatol. 2016;2:317–27.PubMedCentralCrossRefPubMedGoogle Scholar
  26. 26.
    Sharma S, Sharma T, Dhingra R, et al. Linaclotide-a novel secretagogue in the treatment of irritable bowel syndrome with constipation and chronic idiopathic constipation. Mini Rev Med Chem. 2013;13:1685–90.CrossRefGoogle Scholar
  27. 27.
    Xue R, Gu H, Qiu Y, et al. Expression of cystic fibrosis transmembrane conductance regulator in ganglia of human gastrointestinal tract. Sci Rep. 2016;6:30926.PubMedCentralCrossRefPubMedGoogle Scholar
  28. 28.
    Varghese F, Bukhari AB, Malhotra R, et al. IHC Profiler: an open source plugin for the quantitative evaluation and automated scoring of immunohistochemistry images of human tissue samples. PLoS ONE. 2014;9:e96801.PubMedCentralCrossRefPubMedGoogle Scholar
  29. 29.
    Dhooghe B, Noel S, Bouzin C, et al. Correction of chloride transport and mislocalization of CFTR protein by vardenafil in the gastrointestinal tract of cystic fibrosis mice. PLoS ONE. 2013;8:e77314.PubMedCentralCrossRefPubMedGoogle Scholar
  30. 30.
    Doucet L, Mendes F, Montier T, et al. Applicability of different antibodies for the immunohistochemical localization of CFTR in respiratory and intestinal tissues of human and murine origin. J Histochem Cytochem. 2003;51:1191–9.CrossRefGoogle Scholar
  31. 31.
    Valdivieso AG, Marin MC, Clauzure M, et al. Measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometry. Anal Biochem. 2011;418:231–7.CrossRefGoogle Scholar
  32. 32.
    Walsh DE, Harvey BJ, Urbach V. CFTR regulation of intracellular calcium in normal and cystic fibrosis human airway epithelia. J Membr Biol. 2000;177:209–19.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Billet A, Luo Y, Balghi H, et al. Role of tyrosine phosphorylation in the muscarinic activation of the cystic fibrosis transmembrane conductance regulator (CFTR). J Biol Chem. 2013;288:21815–233.PubMedCentralCrossRefPubMedGoogle Scholar
  34. 34.
    Billet A, Hanrahan JW. The secret life of CFTR as a calcium-activated chloride channel. J Physiol. 2013;591:5273–8.PubMedCentralCrossRefPubMedGoogle Scholar
  35. 35.
    Thiagarajah JR, Song Y, Haggie PM, et al. A small molecule CFTR inhibitor produces cystic fibrosis-like submucosal gland fluid secretions in normal airways. FASEB J. 2004;18:875–7.CrossRefGoogle Scholar
  36. 36.
    Leveque M, Penna A, Le Trionnaire S, et al. Phagocytosis depends on TRPV2-mediated calcium influx and requires TRPV2 in lipids rafts: alteration in macrophages from patients with cystic fibrosis. Sci Rep-Uk. 2018;8:4310.CrossRefGoogle Scholar
  37. 37.
    Zhao YM, Migita K, Sun J, et al. MRP transporters as membrane machinery in the bradykinin-inducible export of ATP. N-S Arch Pharmacol. 2010;381:315–20.CrossRefGoogle Scholar
  38. 38.
    Braunstein GM, Roman RM, Clancy JP, et al. Cystic fibrosis transmembrane conductance regulator facilitates ATP release by stimulating a separate ATP release channel for autocrine control of cell volume regulation. J Biol Chem. 2001;276:6621–30.CrossRefGoogle Scholar
  39. 39.
    Wedel T, Roblick UJ, Ott V, et al. Oligoneuronal hypoganglionosis in patients with idiopathic slow-transit constipation. Dis Colon Rectum. 2002;45:54–62.CrossRefGoogle Scholar
  40. 40.
    Park HJ, Kamm MA, Abbasi AM, et al. Immunohistochemical study of the colonic muscle and innervation in idiopathic chronic constipation. Dis Colon Rectum. 1995;38:509–13.CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Broadhead MJ, Bayguinov PO, Okamoto T, et al. Ca2+ transients in myenteric glial cells during the colonic migrating motor complex in the isolated murine large intestine. J Physiol. 2012;590:335–50.CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Boesmans W, Lasrado R, Vanden Berghe P, et al. Heterogeneity and phenotypic plasticity of glial cells in the mammalian enteric nervous system. GLIA. 2015;63:229–41.CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    MacEachern SJ, Patel BA, McKay DM, et al. Nitric oxide regulation of colonic epithelial ion transport: a novel role for enteric glia in the myenteric plexus. J Physiol. 2011;589:3333–48.PubMedCentralCrossRefPubMedGoogle Scholar
  44. 44.
    De Giorgio R, Giancola F, Boschetti E, et al. Enteric glia and neuroprotection: basic and clinical aspects. Am J Physiol Gastrointest Liver Physiol. 2012;303:G887–G89393.CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Gulbransen BD, Sharkey KA. Novel functional roles for enteric glia in the gastrointestinal tract. Nat Rev Gastroenterol Hepatol. 2012;9:625–32.CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Neunlist M, Van Landeghem L, Mahé MM, et al. The digestive neuronal–glial–epithelial unit: a new actor in gut health and disease. Nat Rev Gastroenterol Hepatol. 2013;10:90–100.CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Bassotti G, Chiarioni G, Imbimbo BP, et al. Impaired colonic motor response to cholinergic stimulation in patients with severe chronic idiopathic (slow transit type) constipation. Dig Dis Sci. 1993;38:1040–5.CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Wedel T, Spiegler J, Soellner S, et al. Enteric nerves and interstitial cells of Cajal are altered in patients with slow-transit constipation and megacolon. Gastroenterology. 2002;123:1459–67.CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    De Lisle RC. Altered transit and bacterial overgrowth in the cystic fibrosis mouse small intestine. Am J Physiol Gastrointest Liver Physiol. 2007;293:G104–G111111.CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Lynch SV, Goldfarb KC, Wild YK, et al. Cystic fibrosis transmembrane conductance regulator knockout mice exhibit aberrant gastrointestinal microbiota. Gut Microbes. 2013;4:41–7.PubMedCentralCrossRefPubMedGoogle Scholar
  51. 51.
    De Lisle RC, Meldi L, Flynn M, et al. Altered eicosanoid metabolism in the cystic fibrosis mouse small intestine. J Pediatr Gastroenterol Nutr. 2008;47:406–16.CrossRefGoogle Scholar
  52. 52.
    De Lisle RC, Sewell R, Meldi L. Enteric circular muscle dysfunction in the cystic fibrosis mouse small intestine. Neurogastroenterol Motil. 2010;22:341–e87.CrossRefGoogle Scholar
  53. 53.
    De Lisle RC, Borowitz D. The cystic fibrosis intestine. Cold Spring Harb Perspect Med. 2013;3:a009753.PubMedCentralCrossRefPubMedGoogle Scholar
  54. 54.
    Cil O, Phuan PW, Son JH, et al. Phenylquinoxalinone CFTR activator as potential prosecretory therapy for constipation. Transl Res. 2017;182(14–26):e4.Google Scholar

Copyright information

© Japanese Society of Gastroenterology 2019

Authors and Affiliations

  • Ka Ming Yeh
    • 1
  • Olle Johansson
    • 2
  • Huy Le
    • 1
  • Karan Rao
    • 1
  • Irit Markus
    • 1
  • Dayashan Shevy Perera
    • 3
  • David Zachary Lubowski
    • 3
  • Denis Warwick King
    • 3
  • Li Zhang
    • 4
  • Hongzhuan Chen
    • 5
  • Lu Liu
    • 1
    Email author
  1. 1.Department of Pharmacology, Faculty of Medicine, School of Medical SciencesUniversity of New South WalesSydneyAustralia
  2. 2.Faculty of Medicine and Health SciencesLinköping UniversityLinköpingSweden
  3. 3.Sydney Colorectal AssociatesSydneyAustralia
  4. 4.School of Biotechnology and Biomolecular SciencesUniversity of New South WalesSydneyAustralia
  5. 5.School of MedicineShanghai Jiao Tong UniversityShanghaiChina

Personalised recommendations