Skip to main content

Advertisement

SpringerLink
Log in

Melittin as an Epithelial Permeability Enhancer I: Investigation of Its Mechanism of Action in Caco-2 Monolayers

  • Research Paper
  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

Abstract

Purpose

Melittin is an amphipathic antimicrobial peptide which has been shown to enhance the permeability of mannitol and reduce transepithelial electrical resistance (TER) across Caco-2 monolayers. The aim of this work was to further examine the potential of melittin as a paracellular permeability enhancer and to investigate the mechanism of interaction with tight junction proteins in Caco-2.

Materials and Methods

The permeability of a range of fluorescent markers of differing molecular weights across monolayers was examined and immunofluorescence and western blotting analysis of tight junction proteins were also carried out. The mechanism of TER reduction was also examined using cell signalling inhibitors.

Results

Apical but not basolateral addition of melittin increased the permeability of a range FITC-dextrans (4–70 kDa) across monolayers. Melittin effects were reversible and no cytotoxicity was evident in polarized Caco-2 epithelia at the concentrations used. Altered expression of ZO-1, E-cadherin and F-actin was also detected. The phospholipase A2 inhibitors, aristolochic acid and indomethacin and the cyclooxygenase inhibitor, piroxicam, partially attenuated melittin-induced TER reduction, suggesting that part of the mechanism by which melittin opens tight junctions involves prostaglandin signalling.

Conclusions

Apically-added melittin opens tight junctions, causing dramatic TER reductions with significant increases in flux of dextrans. These effects appear mediated in part via PLA2 and involve alterations in specific tight junction proteins.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. R. J. Mrsny. Modification of epithelial tight junction integrity to enhance transmucosal absorption. Crit. Rev. Ther. Drug Carr. Syst. 22:331–418 (2005).

    Article  CAS  Google Scholar 

  2. M. J. Cano-Cebrian, T. Zornoza, L. Granero, and A. Polache. Intestinal absorption enhancement via the paracellular route by fatty acids, chitosans and others: a target for drug delivery. Curr. Drug. Deliv. 2:9–22 (2005).

    Article  PubMed  CAS  Google Scholar 

  3. N. A. Motlekar, K. S. Srivenugopal, M. S. Wachtel, and B. B. Youan. Oral delivery of low-molecular-weight heparin using sodium caprate as absorption enhancer reaches therapeutic levels. J. Drug Target. 13:573–583 (2005).

    Article  PubMed  CAS  Google Scholar 

  4. P. D. Ward, T. K. Tippin, and D. R. Thakker. Enhancing paracellular permeability by modulating epithelial tight junctions. Pharm. Sci. Technol. Today 3:346–358 (2000).

    Article  PubMed  CAS  Google Scholar 

  5. T. Lindmark, J. D. Soderholm, G. Olaison, G. Alvan, G. Ocklind, and P. Artursson. Mechanism of absorption enhancement in humans after rectal administration of ampicillin in suppositories containing sodium caprate. Pharm. Res. 14:930–935 (1997).

    Article  PubMed  CAS  Google Scholar 

  6. T. W. Leonard, J. Lynch, M. J. McKenna, and D. J. Brayden. Promoting absorption of drugs in humans using medium-chain fatty acid-based solid dosage forms: GIPET. Expert Opin. Drug Deliv. 3:685–692 (2006).

    Article  PubMed  CAS  Google Scholar 

  7. N. S. Harhaj and D. A. Antonetti. Regulation of tight junctions and loss of barrier function in pathophysiology. Int. J. Biochem. Cell Biol. 36:1206–1237 (2004).

    Article  PubMed  CAS  Google Scholar 

  8. D. A. Mann and A. D. Frankel. Endocytosis and targeting of exogenous HIV-1 Tat protein. EMBO J. 10:1733–1739 (1991).

    PubMed  CAS  Google Scholar 

  9. M. S. Dilber, A. Phelan, A. Aints, A. J. Mohamed, G. Elliott, C. I. Smith, and P. O’Hare. Intercellular delivery of thymidine kinase prodrug activating enzyme by the herpes simplex virus protein, VP22. Gene Ther. 6:12–21 (1999).

    Article  PubMed  CAS  Google Scholar 

  10. A. Fasano and S. Uzzau. Modulation of intestinal tight junctions by Zonula occludens toxin permits enteral administration of insulin and other macromolecules in an animal model. J. Clin. Invest. 99:1158–1164 (1997).

    PubMed  CAS  Google Scholar 

  11. I. E. Andras, H. Pu, M. A. Deli, A. Nath, B. Hennig, and M. Toborek. HIV-1 Tat protein alters tight junction protein expression and distribution in cultured brain endothelial cells. J. Neurosci. Res. 74:255–265 (2003).

    Article  PubMed  CAS  Google Scholar 

  12. D. Allende, S. A. Simon, and T. J. McIntosh. Melittin-induced bilayer leakage depends on lipid material properties: evidence for toroidal pores. Biophys. J. 88:1828–1837 (2005).

    Article  PubMed  CAS  Google Scholar 

  13. L. Yang, T. A. Harroun, T. M. Weiss, L. Ding, and H. W. Huang. Barrel-stave model or toroidal model? A case study on melittin pores. Biophys. J. 81:1475–1485 (2001).

    Article  PubMed  Google Scholar 

  14. P. Liu, P. Davis, H. Liu, and T. R. Krishnan. Evaluation of cytotoxicity and absorption enhancing effects of melittin—a novel absorption enhancer. Eur. J. Pharm. Biopharm. 48:85–87 (1999).

    Article  PubMed  CAS  Google Scholar 

  15. S. Maher and S. McClean. Investigation of the cytotoxicity of eukaryotic and prokaryotic antimicrobial peptides in intestinal epithelial cells in vitro. Biochem. Pharmacol. 71:1289–1298 (2006).

    Article  PubMed  CAS  Google Scholar 

  16. M. T. Tosteson, S. J. Holmes, M. Razin, and D. C. Tosteson. Melittin lysis of red cells. J. Membr. Biol. 87:35–44 (1985).

    Article  PubMed  CAS  Google Scholar 

  17. P. M. Hwang and H. J. Vogel. Structure-function relationships of antimicrobial peptides. Biochem. Cell. Biol. 76:235–246 (1998).

    Article  PubMed  CAS  Google Scholar 

  18. W. S. Foster and H. W. Jarrett. Melittin-silica, a high-pressure affinity chromatography resin for calmodulin. J. Chromatogr. 403:99–107 (1987).

    Article  PubMed  CAS  Google Scholar 

  19. K. J. Baker, J. M. East, and A. G. Lee. Mechanism of inhibition of the Ca(2+)-ATPase by melittin. Biochemistry 34:3596–3604 (1995).

    Article  PubMed  CAS  Google Scholar 

  20. J. Cuppoletti and D. H. Malinowska. Interaction of polypeptides with the gastric (H+ + K+)ATPase: melittin, synthetic analogs, and a potential intracellular regulatory protein. Mol. Cell. Biochem. 114:57–63 (1992).

    Article  PubMed  CAS  Google Scholar 

  21. K. R. Gravitt, N. E. Ward, and C. A. O’Brian. Inhibition of protein kinase C by melittin: antagonism of binding interactions between melittin and the catalytic domain by active-site binding of MgATP. Biochem. Pharmacol. 47:425–427 (1994).

    Article  PubMed  CAS  Google Scholar 

  22. H. K. Paudel, Y. H. Xu, H. W. Jarrett, and G. M. Carlson. The model calmodulin-binding peptide melittin inhibits phosphorylase kinase by interacting with its catalytic center. Biochemistry 32:11865–11872 (1993).

    Article  PubMed  CAS  Google Scholar 

  23. B. H. Knowles and R. W. Farndale. Activation of insect cell adenylate cyclase by Bacillus thuringiensis delta-endotoxins and melittin. Toxicity is independent of cyclic AMP. Biochem. J. 253:235–241 (1988).

    PubMed  CAS  Google Scholar 

  24. M. Diener and W. Rummel. Phospholipase A2 and mediation of the activation of short-circuit current in the rat colonic mucosa. Naunyn-Schmiedeberg’s Arch. Pharmacol. 343:652–658 (1991).

    Article  CAS  Google Scholar 

  25. P. Artursson and J. Karlsson. Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells. Biochem. Biophys. Res. Commun. 175:880–885 (1991).

    Article  PubMed  CAS  Google Scholar 

  26. T. Lindmark, N. Schipper, L. Lazorova, A. G. de Boer, and P. Artursson. Absorption enhancement in intestinal epithelial Caco-2 monolayers by sodium caprate: assessment of molecular weight dependence and demonstration of transport routes. J. Drug Target. 5:215–223 (1998).

    Article  PubMed  CAS  Google Scholar 

  27. T. Suzuki and H. Hara. Difructose anhydride III and sodium caprate activate paracellular transport via different intracellular events in Caco-2 cells. Life Sci. 79:401–410 (2006).

    Article  PubMed  CAS  Google Scholar 

  28. C. J. Watson, M. Rowland, and G. Warhurst. Functional modeling of tight junctions in intestinal cell monolayers using polyethylene glycol oligomers. Am. J. Physiol., Cell Physiol. 281:C388–C397 (2001).

    PubMed  CAS  Google Scholar 

  29. M. Tomita, M. Hayashi, and S. Awazu. Absorption-enhancing mechanism of EDTA, caprate, and decanoylcarnitine in Caco-2 cells. J. Pharm. Sci. 85:608–611 (1996).

    Article  PubMed  CAS  Google Scholar 

  30. S. Maher, L. Feighery, D. J. Brayden, and S. McClean. Melittin as a permeability enhancer II: in vitro investigations in human mucus secreting intestinal monolayers and rat colonic mucosae. Pharm. Res. (2007, in press). DOI 10.1007/s11095-007-9246-z

  31. D. Brayden, E. Creed, A. O’Connell, H. Leipold, R. Agarwal, and A. Leone-Bay. Heparin absorption across the intestine: effects of sodium N-[8-(2-hydroxybenzoyl)amino]caprylate in rat in situ intestinal instillations and in Caco-2 monolayers. Pharm. Res. 14:1772–1779 (1997).

    Article  PubMed  CAS  Google Scholar 

  32. L. Garrity-Ryan, B. Kazmierczak, R. Kowal, J. Comolli, A. Hauser, and J. N. Engel. The arginine finger domain of ExoT contributes to actin cytoskeleton disruption and inhibition of internalization of Pseudomonas aeruginosa by epithelial cells and macrophages. Infect. Immun. 68:7100–7113 (2000).

    Article  PubMed  CAS  Google Scholar 

  33. J. M. Anderson, C. M. Van Itallie, M. D. Peterson, B. R. Stevenson, E. A. Carew, and M. S. Mooseker. ZO-1 mRNA and protein expression during tight junction assembly in Caco-2 cells. J. Cell Biol. 109:1047–1056 (1989).

    Article  PubMed  CAS  Google Scholar 

  34. N. G. Schipper, S. Olsson, J. A. Hoogstraate, A. G. deBoer, K. M. Varum, and P. Artursson. Chitosans as absorption enhancers for poorly absorbable drugs 2: mechanism of absorption enhancement. Pharm. Res. 14:923–929 (1997).

    Article  PubMed  CAS  Google Scholar 

  35. L. Illum. Chitosan and its use as a pharmaceutical excipient. Pharm. Res. 15:1326–1331 (1998).

    Article  PubMed  CAS  Google Scholar 

  36. T. Lindmark, Y. Kimura, and P. Artursson. Absorption enhancement through intracellular regulation of tight junction permeability by medium chain fatty acids in Caco-2 cells. J. Pharmacol. Exp. Ther. 284:362–369 (1998).

    PubMed  CAS  Google Scholar 

  37. A. Fasano. Modulation of intestinal permeability: an innovative method of oral drug delivery for the treatment of inherited and acquired human diseases. Mol. Genet. Metab. 64:12–18 (1998).

    Article  PubMed  CAS  Google Scholar 

  38. G. Hecht, L. Pestic, G. Nikcevic, A. Koutsouris, J. Tripuraneni, D. D. Lorimer, G. Nowak, V. Guerriero, Jr., E. L. Elson, and P. D. Lanerolle. Expression of the catalytic domain of myosin light chain kinase increases paracellular permeability. Am. J. Physiol. 271:C1678–C1684 (1996).

    PubMed  CAS  Google Scholar 

  39. A. Fasano, C. Fiorentini, G. Donelli, S. Uzzau, J. B. Kaper, K. Margaretten, X. Ding, S. Guandalini, L. Comstock, and S. E. Goldblum. Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization, in vitro. J. Clin. Invest. 96:710–720 (1995).

    Article  PubMed  CAS  Google Scholar 

  40. K. Koumanov, A. Momchilova, and C. Wolf. Bimodal regulatory effect of melittin and phospholipase A2-activating protein on human type II secretory phospholipase A2. Cell Biol. Int. 27:871–877 (2003).

    Article  PubMed  CAS  Google Scholar 

  41. T. Sawai, N. Usui, J. Dwaihy, R. A. Drongowski, A. Abe, A. G. Coran, and C. M. Harmon. The effect of phospholipase A2 on bacterial translocation in a cell culture model. Pediatr. Surg. Int. 16:262–266 (2000).

    Article  PubMed  CAS  Google Scholar 

  42. R. Martin-Venegas, S. Roig-Perez, R. Ferrer, and J. J. Moreno. Arachidonic acid cascade and epithelial barrier function during Caco-2 cell differentiation. J. Lipid Res. 47:1416–1423 (2006).

    Article  PubMed  CAS  Google Scholar 

  43. A. Ohata, M. Usami, and M. Miyoshi. Short-chain fatty acids alter tight junction permeability in intestinal monolayer cells via lipoxygenase activation. Nutrition 21:838–847 (2005).

    Article  PubMed  CAS  Google Scholar 

  44. C. Celik-Ozenci, I. Ustunel, T. Erdogru, Y. Seval, E. T. Korgun, M. Baykara, and R. Demir. Ultrastructural and immunohistochemical analysis of rat uroepithelial cell junctions after partial bladder outlet obstruction and selective COX-2 inhibitor treatment. Acta Histochem. 107:443–451 (2006).

    Article  PubMed  Google Scholar 

  45. W. G. Jiang, R. P. Bryce, D. F. Horrobin, and R. E. Mansel. Regulation of tight junction permeability and occludin expression by polyunsaturated fatty acids. Biochem. Biophys. Res. Commun. 244:414–420 (1998).

    Article  PubMed  CAS  Google Scholar 

  46. J. R. Turner, J. M. Angle, E. D. Black, J. L. Joyal, D. B. Sacks, and J. L. Madara. PKC-dependent regulation of transepithelial resistance: roles of MLC and MLC kinase. Am. J. Physiol. 277:C554–C562 (1999).

    PubMed  CAS  Google Scholar 

  47. J. R. Turner, B. K. Rill, S. L. Carlson, D. Carnes, R. Kerner, R. J. Mrsny, and J. L. Madara. Physiological regulation of epithelial tight junctions is associated with myosin light-chain phosphorylation. Am. J. Physiol. 273:C1378–C1385 (1997).

    PubMed  CAS  Google Scholar 

  48. L. Pang, M. Nie, L. Corbett, R. Donnelly, S. Gray, and A. J. Knox. Protein kinase C-epsilon mediates bradykinin-induced cyclooxygenase-2 expression in human airway smooth muscle cells. FASEB J. 16:1435–1437 (2002).

    PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Programme for Research in Third Level Institutions (PRTLI) administered by HEA, Ireland. LF was funded by the Health Research Board (Ireland). The authors express their gratitude to James Reilly, ITT Dublin for advice on statistical analysis of the data.

Author information

Authors and Affiliations

  1. Institute of Technology Tallaght Dublin, Belgard Road, Tallaght, Dublin, Ireland

    Sam Maher & Siobhán McClean

  2. National Institute of Cellular Biotechnology, Dublin, Ireland

    Sam Maher & Siobhán McClean

  3. School of Agricultural, Food Science and Veterinary Medicine, University College Dublin, Dublin 4, Ireland

    Linda Feighery & David J. Brayden

Authors
  1. Sam Maher
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Linda Feighery
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David J. Brayden
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Siobhán McClean
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Siobhán McClean.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Maher, S., Feighery, L., Brayden, D.J. et al. Melittin as an Epithelial Permeability Enhancer I: Investigation of Its Mechanism of Action in Caco-2 Monolayers. Pharm Res 24, 1336–1345 (2007). https://doi.org/10.1007/s11095-007-9288-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11095-007-9288-2

Key words

Search

Navigation