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Laurate Permeates the Paracellular Pathway for Small Molecules in the Intestinal Epithelial Cell Model HT-29/B6 via Opening the Tight Junctions by Reversible Relocation of Claudin-5

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An Erratum to this article was published on 23 December 2014

ABSTRACT

Purpose

To mechanistically analyze effects of the medium-chain fatty acid laurate on transepithelial permeability in confluent monolayers of the intestinal epithelial cell line HT-29/B6, in context with an application as an absorption enhancer improving transepithelial drug permeation.

Methods

Transepithelial resistance and apparent permeability for paracellular flux markers was measured using Ussing-type chambers. Two-path impedance spectroscopy was employed to differentiate between transcellular and paracellular resistance, and confocal imaging and Western blotting was performed.

Results

Laurate resulted in a substantial and reversible decrease in transepithelial resistance by 50% which was attributed to a decrease in paracellular resistance. Simultaneously, an increase in permeability for fluorescein (330 Da) was detected, while permeabilities for 4 kDa FITC-dextran and sulpho-NHS-SS-biotin (607 Da) remained unaltered. Confocal laser-scanning microscopy revealed a marked reduction of claudin-5, while other tight junction proteins including tricellulin, a protein preventing the paracellular passage of macromolecules, were not affected.

Conclusions

Laurate induces an increase in paracellular permeability for molecules up to a molecular mass of 330 Da by retrieval of claudin-5 from tight junctions without affecting tricellular contacts and the paracellular passage of macromolecules. We hereby provide, for the first time, a mechanistical explanation of laurate-induced permeability enhancement on molecular level.

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Abbreviations

FITC:

Fluorescein isothiocyanate

MCFA:

Medium-chain fatty acids

NHS:

n-hydroxysuccinimide

TER:

Transepithelial resistance

TJ:

Tight junctions

References

  1. Döring F, Walter J, Will J, Föcking M, Boll M, Amasheh S, et al. Delta-aminolevulinic acid transport by intestinal and renal peptide transporters and its physiological and clinical implications. J Clin Invest. 1998;101(12):2761–7.

    Article  PubMed Central  PubMed  Google Scholar 

  2. Döring F, Will J, Amasheh S, Clauss W, Ahlbrecht H, Daniel H. Minimal molecular determinants of substrates for recognition by the intestinal peptide transporter. J Biol Chem. 1998;273(36):23211–8.

    Article  PubMed  Google Scholar 

  3. Guo A, Hu P, Balimane PV, Leibach FH, Sinko PJ. Interactions of a non-peptidic drug, valacyclovir, with the human intestinal peptide transporter (hPEPT1) expressed in a mammalian cell line. J Pharmacol Exp Ther. 1999;289(1):448–54.

    CAS  PubMed  Google Scholar 

  4. Rosenthal R, Heydt MS, Amasheh M, Stein C, Fromm M, Amasheh S. Analysis of absorption enhancers in epithelial cell models. Ann N Y Acad Sci. 2012;1258:86–92.

    Article  CAS  PubMed  Google Scholar 

  5. Aungst BJ. Absorption enhancers: applications and advances. AAPS J. 2012;14(1):10–8.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  6. Markov AG, Veshnyakova A, Fromm M, Amasheh M, Amasheh S. Segmental expression of claudin proteins correlates with tight junction barrier properties in rat intestine. J Comp Physiol B. 2010;180(4):591–8.

    Article  CAS  PubMed  Google Scholar 

  7. Furuse M, Hata M, Furuse K, Yoshida Y, Haratake A, Sugitani Y, et al. Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin-1-deficient mice. J Cell Biol. 2002;156(6):1099–111.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Günzel D, Yu AS. Claudins and the modulation of tight junction permeability. Physiol Rev. 2013;93(2):525–69.

    Article  PubMed Central  PubMed  Google Scholar 

  9. Furuse M, Fujita K, Hiiragi T, Fujimoto K, Tsukita S. Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol. 1998;141(7):1539–50.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  10. Furuse M, Hirase T, Itoh M, Nagafuchi A, Yonemura S, Tsukita S, et al. Occludin: a novel integral membrane protein localizing at tight junctions. J Cell Biol. 1993;23(6):1777–88.

    Article  Google Scholar 

  11. Ikenouchi J, Furuse M, Furuse K, Sasaki H, Tsukita S, Tsukita S. Tricellulin constitutes a novel barrier at tricellular contacts of epithelial cells. J Cell Biol. 2005;171(6):939–45.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  12. Raleigh DR, Marchiando AM, Zhang Y, Shen L, Sasaki H, Wang Y, et al. Tight junction-associated MARVEL proteins marveld3 tricellulin and occludin have distinct but overlapping functions. Mol Biol Cell. 2010;21(7):1200–13.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  13. Rosenthal R, Günzel D, Finger C, Krug SM, Richter JF, Schulzke JD, et al. The effect of chitosan on transcellular and paracellular mechanisms in the intestinal epithelial barrier. Biomaterials. 2012;33(9):2791–800.

    Article  CAS  PubMed  Google Scholar 

  14. Krug SM, Amasheh M, Dittmann I, Christoffel I, Fromm M, Amasheh S. Sodium caprate as an enhancer of macromolecule permeation across tricellular tight junctions of intestinal cells. Biomaterials. 2013;34(1):275–82.

    Article  CAS  PubMed  Google Scholar 

  15. Lindmark T, Kimura Y, Artursson P. Absorption enhancement through intracellular regulation of tight junction permeability by medium chain fatty acids in Caco-2 cells. J Pharmacol Exp Ther. 1998;284(1):362–9.

    CAS  PubMed  Google Scholar 

  16. Lindmark T, Söderholm JD, Olaison G, Alván G, Ocklind G, Artursson P. Mechanism of absorption enhancement in humans after rectal administration of ampicillin in suppositories containing sodium caprate. Pharm Res. 1997;14(7):930–5.

    Article  CAS  PubMed  Google Scholar 

  17. Lindmark T, Nikkilä T, Artursson P. Mechanisms of absorption enhancement by medium chain fatty acids in intestinal epithelial Caco-2 cell monolayers. J Pharmacol Exp Ther. 1995;275(2):958–64.

    CAS  PubMed  Google Scholar 

  18. Maher S, Leonard TW, Jacobsen J, Brayden DJ. Safety and efficacy of sodium caprate in promoting oral drug absorption: from in vitro to the clinic. Adv Drug Deliv Rev. 2009;61(15):1427–49.

    Article  CAS  PubMed  Google Scholar 

  19. Francois CA, Connor SL, Wander RC, Connor WE. Acute effects of dietary fatty acids on the fatty acids of human milk. Am J Clin Nutr. 1998;67(2):301–8.

    CAS  PubMed  Google Scholar 

  20. Moltó-Puigmartí C, Castellote AI, Carbonell-Estrany X, López-Sabater MC. Differences in fat content and fatty acid proportions among colostrum, transitional, and mature milk from women delivering very preterm, preterm, and term infants. Clin Nutr. 2011;30(1):116–23.

    Article  PubMed  Google Scholar 

  21. Zenger V, Laurate C. US Food and Drug Administration. High laurate canola oil. BNF No.25. 1995. http://www.fda.gov/Food/FoodScienceResearch/Biotechnology/Submissions/ucm161141.htm. Accessed 28 February 2014.

  22. Kreusel KM, Fromm M, Schulzke JD, Hegel U. Cl- secretion in epithelial monolayers of mucus-forming human colon cells (HT-29/B6). Am J Physiol. 1991;261(4):C574–82.

    CAS  PubMed  Google Scholar 

  23. Krug SM, Fromm M, Günzel D. Two-path impedance spectroscopy for measuring paracellular and transcellular epithelial resistance. Biophys J. 2009;97(8):2202–11.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  24. Amasheh S, Meiri N, Gitter AH, Schöneberg T, Mankertz J, Schulzke JD, et al. Claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells. J Cell Sci. 2002;115(24):4969–76.

    Article  CAS  PubMed  Google Scholar 

  25. Amasheh S, Schmidt T, Mahn M, Florian P, Mankertz J, Tavalali S, et al. Contribution of claudin-5 to barrier properties in tight junctions of epithelial cells. Cell Tissue Res. 2005;321(1):89–96.

    Article  CAS  PubMed  Google Scholar 

  26. Amasheh M, Luettig J, Amasheh S, Zeitz M, Fromm M, Schulzke JD. Effects of quercetin on the colonic cell culture model HT-29/B6 and rat intestine in vitro. Ann N Y Acad Sci. 2012;1258:100–7.

    Article  CAS  PubMed  Google Scholar 

  27. Amasheh S, Milatz S, Krug SM, Bergs M, Amasheh M, Schulzke JD, et al. Na+ absorption defends from paracellular back-leakage by claudin-8 upregulation. Biochem Biophys Res Commun. 2009;378(1):45–50.

    Article  CAS  PubMed  Google Scholar 

  28. Krug SM, Amasheh S, Richter JF, Milatz S, Günzel D, Westphal JK, et al. Tricellulin forms a barrier to macromolecules in tricellular tight junctions without affecting ion permeability. Mol Biol Cell. 2009;20(16):3713–24.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  29. Anderson JM, Van Itallie CM. Physiology and function of the tight junction. Cold Spring Harb Perspect Biol. 2009;1(2):a002584.

    Article  PubMed Central  PubMed  Google Scholar 

  30. Piontek J, Winkler L, Wolburg H, Müller SL, Zuleger N, Piehl C, et al. Formation of tight junction: determinants of homophilic interaction between classic claudins. FASEB J. 2008;22:146–58.

    Article  CAS  PubMed  Google Scholar 

  31. Morita K, Sasaki H, Furuse M, Tsukita S. Endothelial claudin: claudin-5/TMVCF constitutes tight junction strands in endothelial cells. J Cell Biol. 1999;147(1):185–94.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  32. Nitta T, Hata M, Gotoh S, Seo Y, Sasaki H, Hashimoto N, et al. Size-selective loosening of the blood-brain barrier in claudin-5-deficient mice. J Cell Biol. 2003;161(3):653–60.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  33. Amasheh S, Fromm M, Günzel D. Claudins of intestine and nephron - a correlation of molecular tight junction structure and barrier function. Acta Physiol. 2011;201(1):133–40.

    Article  CAS  Google Scholar 

  34. Rahner C, Mitic LL, Anderson JM. Heterogeneity in expression and subcellular localization of claudins 2, 3, 4, and 5 in the rat liver, pancreas, and gut. Gastroenterology. 2001;120(2):411–22.

    Article  CAS  PubMed  Google Scholar 

  35. Poliak S, Matlis S, Ullmer C, Scherer SS, Peles E. Distinct claudins and associated PDZ proteins form different autotypic tight junctions in myelinating Schwann cells. J Cell Biol. 2002;159(2):361–72.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  36. Wang F, Daugherty B, Keise LL, Wei Z, Foley JP, Savani RC, et al. Heterogeneity of claudin expression by alveolar epithelial cells. Am J Respir Cell Mol Biol. 2003;29(1):62–70.

    Article  CAS  PubMed  Google Scholar 

  37. Sirotkin H, Morrow B, Saint-Jore B, Puech A, Das Gupta R, Patanjali SR, et al. Identification, characterization, and precise mapping of a human gene encoding a novel membrane-spanning protein from the 22q11 region deleted in velo-cardio-facial syndrome. Genomics. 1997;42(2):245–51.

    Article  CAS  PubMed  Google Scholar 

  38. Kojima S, Rahner C, Peng S, Rizzolo LJ. Claudin 5 is transiently expressed during the development of the retinal pigment epithelium. J Membr Biol. 2002;186(2):81–8.

    Article  CAS  PubMed  Google Scholar 

  39. Amer B, Nebel C, Bertram HC, Mortensen G, Hermansen K, Dalsgaard TK. Novel method for quantification of individual free fatty acids in milk using an in-solution derivatisation approach and gas chromatography-mass spectrometry. Int Dairy J. 2013;32:199–203.

    Article  CAS  Google Scholar 

  40. Hering NA, Andres S, Fromm A, van Tol EA, Amasheh M, Mankertz J, et al. Transforming growth factor-β, a whey protein component, strengthens the intestinal barrier by upregulating claudin-4 in HT-29/B6 cells. J Nutr. 2011;141(5):783–9.

    Article  CAS  PubMed  Google Scholar 

  41. Markov AG, Kruglova NM, Fomina YA, Fromm M, Amasheh S. Altered expression of tight junction proteins in mammary epithelium after discontinued suckling in mice. Pflugers Arch. 2012;463(2):391–8.

    Article  CAS  PubMed  Google Scholar 

  42. Del Vecchio G, Tscheik C, Tenz K, Helms HC, Winkler L, Blasig R, et al. Sodium caprate transiently opens claudin-5-containing barriers at tight junctions of epithelial and endothelial cells. Mol Pharm. 2012;9(9):2523–33.

    Article  PubMed  Google Scholar 

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ACKNOWLEDGMENTS AND DISCLOSURES

We thank Detlef Sorgenfrei, In-Fah M. Lee, and Anja Fromm for their expert technical assistance. This work was supported by grants of the Deutsche Forschungsgemeinschaft (Grants DFG FOR 721/2, DFG SFB 852), and the St. Petersburg State University (Grant SPbGU 1.37.118.201).

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Correspondence to Salah Amasheh.

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Dittmann, I., Amasheh, M., Krug, S.M. et al. Laurate Permeates the Paracellular Pathway for Small Molecules in the Intestinal Epithelial Cell Model HT-29/B6 via Opening the Tight Junctions by Reversible Relocation of Claudin-5. Pharm Res 31, 2539–2548 (2014). https://doi.org/10.1007/s11095-014-1350-2

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  • DOI: https://doi.org/10.1007/s11095-014-1350-2

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