Skip to main content

Advertisement

SpringerLink
Log in

Histamine-induced Myosin Light Chain Phosphorylation Breaks Down the Barrier Integrity of Cultured Corneal Epithelial Cells

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

Abstract

Purpose

To investigate changes in the phosphorylation of myosin light chain (MLC) in response to histamine and its effect on the barrier integrity of corneal epithelial cells.

Materials and Methods

Experiments were performed in bovine corneal epithelial cells (BCEC). RT-PCR and Western blotting were employed to characterize expression of H1 receptors and MLC kinase (MLCK). Phosphorylation of MLC was assessed by urea-glycerol gel electrophoresis and Western blotting. Barrier integrity was determined as permeability to horseradish peroxidase (HRP; 44 kDa) across monolayers grown on porous filters.

Results

Expression of both H1 receptors and MLCK was found in BCEC. Exposure to histamine induced significant MLC phosphorylation concomitant with an increase in HRP permeability. In addition, organization of the cortical actin found in resting cells was disrupted. In contrast to histamine, ATP (a P2Y receptor agonist) induced dephosphorylation of MLC. Pre-exposure to ATP reduced the effect of histamine on HRP permeability and disruption of cortical actin.

Conclusion

MLC phosphorylation, a biochemical pre-requisite for increased contractility of the actin cytoskeleton, led to histamine-induced breakdown of the barrier integrity in the corneal epithelial cells. This is attributed to weakening of the tethering forces at the tight junctions by the centripetal forces produced by increased actin contractility.

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
Fig. 8
Fig. 9
Fig. 10

Similar content being viewed by others

References

  1. B. J. McLaughlin, R. B. Caldwell, Y. Sasaki, and T. O. Wood. Freeze-fracture quantitative comparison of rabbit corneal epithelial and endothelial membranes. Curr. Eye Res. 4:951–961 (1985).

    PubMed  CAS  Google Scholar 

  2. Y. Wang, M. Chen, and J. M. Wolosin. ZO-1 in corneal epithelium; stratal distribution and synthesis induction by outer cell removal. Exp. Eye Res. 57:283–292 (1993).

    Article  PubMed  CAS  Google Scholar 

  3. J. M. Diamond. Twenty-first Bowditch lecture. The epithelial junction: bridge, gate, and fence. Physiologist. 20:10–18 (1977).

    PubMed  CAS  Google Scholar 

  4. B. Gumbiner. Structure, biochemistry, and assembly of epithelial tight junctions. Am. J. Physiol. 253:C749–C758 (1987).

    PubMed  CAS  Google Scholar 

  5. M. A. Behzadian, X. L. Wang, L. J. Windsor, N. Ghaly, and R. B. Caldwell. TGF-beta increases retinal endothelial cell perme?>ability by increasing MMP-9: possible role of glial cells in endothelial barrier function. Investig. Ophthalmol. Vis. Sci. 42:853–859 (2001).

    CAS  Google Scholar 

  6. F. Hollande, E. M. Blanc, J. P. Bali, R. H. Whitehead, A. Pelegrin, G. S. Baldwin, and A. Choquet. HGF regulates tight junctions in new nontumorigenic gastric epithelial cell line. Am. J. Physiol.: Gasterointest. Liver Physiol. 280:G910–G921 (2001).

    CAS  Google Scholar 

  7. S. V. Walsh, A. M. Hopkins, and A. Nusrat. Modulation of tight junction structure and function by cytokines. Adv. Drug Deliv. Rev. 41:303–313 (2000).

    Article  PubMed  CAS  Google Scholar 

  8. B. P. McNamara, A. Koutsouris, C. B. O’Connell, J. P. Nougayrede, M. S. Donnenberg, and G. Hecht. Translocated EspF protein from enteropathogenic Escherichia coli disrupts host intestinal barrier function. J. Clin. Invest. 107:621–629 (2001).

    PubMed  CAS  Google Scholar 

  9. X. Yi, Y. Wang, and F. S. Yu. Corneal epithelial tight junctions and their response to lipopolysaccharide challenge. Investig. Ophthalmol. Vis. Sci. 41:4093–4100 (2000).

    CAS  Google Scholar 

  10. M. Itoh, M. Furuse, K. Morita, K. Kubota, M. Saitou, and S. Tsukita. Direct binding of three tight junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins. J. Cell Biol. 147:1351–1363 (1999).

    Article  PubMed  CAS  Google Scholar 

  11. A. Nusrat, J. R. Turner, and J. L. Madara. Molecular physiology and pathophysiology of tight junctions. IV. Regulation of tight junctions by extracellular stimuli: nutrients, cytokines, and immune cells. Am. J. Physiol.: Gasterointest. Liver Physiol. 279:G851–G857 (2000).

    CAS  Google Scholar 

  12. J. G. Garcia, H. W. Davis, and C. E. Patterson. Regulation of endothelial cell gap formation and barrier dysfunction: role of myosin light chain phosphorylation. J. Cell. Physiol. 163:510–522 (1995).

    Article  PubMed  CAS  Google Scholar 

  13. J. R. Turner. ‘Putting the squeeze’ on the tight junction: understanding cytoskeletal regulation. Semin. Cell Dev. Biol. 11:301–308 (2000).

    Article  PubMed  CAS  Google Scholar 

  14. S. M. Dudekand and J. G. Garcia. Cytoskeletal regulation of pulmonary vascular permeability. J. Appl. Physiol. 91:1487–1500 (2001).

    Google Scholar 

  15. H. Lumand and A. B. Malik. Regulation of vascular endothelial barrier function. Am. J. Physiol. 267:L223–L241 (1994).

    Google Scholar 

  16. M. Satpathy, P. Gallagher, M. Lizotte-Waniewski, and S. P. Srinivas. Thrombin-induced phosphorylation of the regulatory light chain of myosin II in cultured bovine corneal endothelial cells. Exp. Eye Res. 79:477–486 (2004).

    Article  PubMed  CAS  Google Scholar 

  17. R. Yuhan, A. Koutsouris, S. D. Savkovic, and G. Hecht. Enteropathogenic Escherichia coli-induced myosin light chain phosphorylation alters intestinal epithelial permeability. Gastroenterology. 113:1873–1882 (1997).

    Article  PubMed  CAS  Google Scholar 

  18. Y. Zolotarevsky, G. Hecht, A. Koutsouris, D. E. Gonzalez, C. Quan, J. Tom, R. J. Mrsny, and J. R. Turner. A membrane-permeant peptide that inhibits MLC kinase restores barrier function in in vitro models of intestinal disease.[erratum appears in Gastroenterology 2002 Oct;123(4):1412]. Gastroenterology 123:163–172 (2002).

    Article  PubMed  CAS  Google Scholar 

  19. L. Shen, E. D. Black, E. D. Witkowski, W. I. Lencer, V. Guerriero, E. E. Schneeberger, and J. R. Turner. Myosin light chain phosphorylation regulates barrier function by remodeling tight junction structure. J. Cell Sci. 119:2095–2106 (2006).

    Article  PubMed  CAS  Google Scholar 

  20. K. E. Kammand and J. T. Stull. Dedicated myosin light chain kinases with diverse cellular functions. J. Biol. Chem. 276:4527–4530 (2001).

    Article  Google Scholar 

  21. A. P. Somlyo and A. V. Somlyo. Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. Physiol. Rev. 83:1325–1358 (2003).

    PubMed  CAS  Google Scholar 

  22. A. B. Moy, J. Van Engelenhoven, J. Bodmer, J. Kamath, C. Keese, I. Giaever, S. Shasby, and D. M. Shasby. Histamine and thrombin modulate endothelial focal adhesion through centripetal and centrifugal forces. J. Clin. Invest. 97:1020–1027 (1996).

    Article  PubMed  CAS  Google Scholar 

  23. G. P. van Nieuw Amerongen, R. Draijer, M. A. Vermeer, and V. W. van Hinsbergh. Transient and prolonged increase in endothelial permeability induced by histamine and thrombin: role of protein kinases, calcium, and RhoA. Circ. Res. 83:1115–1123 (1998).

    PubMed  Google Scholar 

  24. S. P. Srinivas, M. Satpathy, Y. Guo, and V. Anandan. Histamine-induced phosphorylation of the regulatory light chain of myosin II disrupts the barrier integrity of corneal endothelial cells. Investig. Ophthalmol. Vis. Sci. 47:4011–4018 (2006).

    Article  Google Scholar 

  25. A. Leonardi. The central role of conjunctival mast cells in the pathogenesis of ocular allergy. Curr. Allergy Asthma Rep. 2:325–331 (2002).

    Article  PubMed  Google Scholar 

  26. L. Bieloryand and S. Ghafoor. Histamine receptors and the conjunctiva. Curr. Opin. Allergy Clin. Immunol. 5:437–440 (2005).

    Article  Google Scholar 

  27. T. Noll, M. Schafer, U. Schavier-Schmitz, and H. M. Piper. ATP induces dephosphorylation of myosin light chain in endothelial cells. Am. J. Physiol., Cell Physiol. 279:C717–C723 (2000).

    PubMed  CAS  Google Scholar 

  28. M. Satpathy, P. Gallagher, Y. Jin, and S. P. Srinivas. Extracellular ATP opposes thrombin-induced myosin light chain phosphorylation and loss of barrier integrity in corneal endothelial cells. Exp. Eye Res. 81:183–192 (2005).

    Article  PubMed  CAS  Google Scholar 

  29. S. P. Srinivas, M. Satpathy, P. Gallagher, E. Lariviere, and W. Van Driessche. Adenosine induces dephosphorylation of myosin II regulatory light chain in cultured bovine corneal endothelial cells. Exp. Eye Res. 79:543–551 (2004).

    Article  PubMed  CAS  Google Scholar 

  30. A. P. Somlyo and A. V. Somlyo. Signal transduction and regulation in smooth muscle.[erratum appears in Nature 1994 Dec 22–29;372(6508):812]. Nature 372:231–236 (1994).

    Article  PubMed  CAS  Google Scholar 

  31. V. Lazarand and J. G. Garcia. A single human myosin light chain kinase gene (MLCK; MYLK). Genomics 57:256–267 (1999).

    Article  Google Scholar 

  32. K. Kimura, M. Ito, M. Amano, K. Chihara, Y. Fukata, M. Nakafuku, B. Yamamori, J. Feng, T. Nakano, K. Okawa, A. Iwamatsu, and K. Kaibuchi. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase)[see comment]. Science 273:245–248 (1996).

    Article  PubMed  CAS  Google Scholar 

  33. K. M. Crawford, D. K. MacCallum, and S. A. Ernst. Histamine H1 receptor-mediated Ca2+ signaling in cultured bovine corneal endothelial cells. Investig. Ophthalmol. Vis. Sci. 33:3041–3049 (1992).

    CAS  Google Scholar 

  34. K. M. Crawford, D. K. MacCallum, and S. A. Ernst. Agonist-induced Ca2+ mobilization in cultured bovine and human corneal endothelial cells. Curr. Eye Res. 12:303–311 (1993).

    PubMed  CAS  Google Scholar 

  35. N. A. Sharif, T. K. Wiernas, W. E. Howe, B. W. Griffin, E. A. Offord, and A. M. Pfeifer. Human corneal epithelial cell functional responses to inflammatory agents and their antagonists. Investig. Ophthalmol. Vis. Sci. 39:2562–2571 (1998).

    CAS  Google Scholar 

  36. A. Kittel, E. Kaczmarek, J. Sevigny, K. Lengyel, E. Csizmadia, and S. C. Robson. CD39 as a caveolar-associated ectonucleotidase. Biochem. Biophys. Res. Commun. 262:596–599 (1999).

    Article  PubMed  CAS  Google Scholar 

  37. A. J. Marcus, M. J. Broekman, J. H. Drosopoulos, N. Islam, D. J. Pinsky, C. Sesti, and R. Levi. Metabolic control of excessive extracellular nucleotide accumulation by CD39/ecto-nucleotidase-1: implications for ischemic vascular diseases. J. Pharmacol. Exp. Ther. 305:9–16 (2003).

    Article  PubMed  CAS  Google Scholar 

  38. T. Hashikawa, M. Takedachi, M. Terakura, T. Saho, S. Yamada, L. F. Thompson, Y. Shimabukuro, and S. Murakami. Involvement of CD73 (ecto-5′-nucleotidase) in adenosine generation by human gingival fibroblasts. J. Dent. Res. 82:888–892 (2003).

    Article  PubMed  CAS  Google Scholar 

  39. M. Eto, T. Ohmori, M. Suzuki, K. Furuya, and F. Morita. A novel protein phosphatase-1 inhibitory protein potentiated by protein kinase C. Isolation from porcine aorta media and characterization. J. Biochem. 118:1104–1107 (1995).

    PubMed  CAS  Google Scholar 

  40. M. Eto, S. Senba, F. Morita, and M. Yazawa. Molecular cloning of a novel phosphorylation-dependent inhibitory protein of protein phosphatase-1 (CPI17) in smooth muscle: its specific localization in smooth muscle. FEBS Lett. 410:356–360 (1997).

    Article  PubMed  CAS  Google Scholar 

  41. S. M. Bloemers, S. Verheule, M. P. Peppelenbosch, M. J. Smit, L. G. Tertoolen, and S. de Laat. Sensitization of the histamine H1 receptor by increased ligand affinity. J. Biol. Chem. 273:2249–2255 (1998).

    Article  PubMed  CAS  Google Scholar 

  42. T. Noll, H. Holschermann, K. Koprek, D. Gunduz, W. Haberbosch, H. Tillmanns, and H. M. Piper. ATP reduces macromolecule permeability of endothelial monolayers despite increasing [Ca2+]i. Am. J. Physiol. 276:H1892–H1901 (1999).

    PubMed  CAS  Google Scholar 

  43. J. Qiao, F. Huang, and H. Lum. PKA inhibits RhoA activation: a protection mechanism against endothelial barrier dysfunction. Am. J. Physiol., Lung Cell. Mol. Physiol. 284:L972–L980 (2003).

    CAS  Google Scholar 

  44. G. Burnstock. P2 purinoceptors: historical perspective and classification. Ciba Found. Symp. 198:1–28; discussion 29–34 (1996).

    PubMed  CAS  Google Scholar 

  45. K. Enomoto, K. Furuya, S. Yamagishi, T. Oka, and T. Maeno. The increase in the intracellular Ca2+ concentration induced by mechanical stimulation is propagated via release of pyrophosphorylated nucleotides in mammary epithelial cells. Pflugers Arch. Gesamte Physiol. Menschen Tiere. 427:533–542 (1994).

    Article  CAS  Google Scholar 

  46. 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 

Download references

Acknowledgements

Supported by VISTAKON Research Grant, American Optometric Foundation, 2004 (SPS) and NEI 14415 (SPS).

Author information

Authors and Affiliations

  1. School of Optometry, Indiana University, 800 East Atwater Avenue, Bloomington, Indiana, 47405, USA

    Ying Guo, Charanya Ramachandran, Minati Satpathy & Sangly P. Srinivas

Authors
  1. Ying Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Charanya Ramachandran
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Minati Satpathy
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sangly P. Srinivas
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sangly P. Srinivas.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Guo, Y., Ramachandran, C., Satpathy, M. et al. Histamine-induced Myosin Light Chain Phosphorylation Breaks Down the Barrier Integrity of Cultured Corneal Epithelial Cells. Pharm Res 24, 1824–1833 (2007). https://doi.org/10.1007/s11095-007-9309-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11095-007-9309-1

Key words

Search

Navigation