The Journal of Membrane Biology

, Volume 183, Issue 2, pp 93–101

Cell Signaling Pathways Mediating Epidermal Growth Factor Stimulation of Na:K:2Cl Cotransport Activity in Rabbit Corneal Epithelial Cells

  • H.  Yang
  • Z.  Wang
  • Y.  Miyamoto
  • P. S.  Reinach

DOI: 10.1007/s00232-001-0057-6

Cite this article as:
Yang, H., Wang, Z., Miyamoto, Y. et al. J. Membrane Biol. (2001) 183: 93. doi:10.1007/s00232-001-0057-6

Abstract.

We characterized the signaling and ion transport pathways that mediate epidermal growth factor receptor physiological control in SV40-immortalized rabbit corneal epithelial cells (tRCEC). Our evaluation employed single-cell fluorescence imaging to measure the intracellular [Na+]i in these cells loaded with the Na+ sensitive dye, SBFI. EGF (1 to 5 ng/ml) transiently increased [Na+]i from 10 mm to as much as 35 mm after 25 min, which was followed by a decline towards its control value. These increases waned at higher EGF concentrations up to 50 ng/ml. Both inhibition of EGF receptor-linked tyrosine kinase activity (50 μm RG-13022) and cPLA2 activity (10 μm AACOCF3) obviated EGF-induced increases in [Na+]i. In contrast, PGE2 (10 μg/ml) and cAMP (2 mm) increased [Na+]i by 25 mm. Inhibition of NKCC activity through exposure to either Cl-free Ringers or 300 μm furosemide in NaCl Ringers eliminated EGF-induced increases in [Na+]i. Similarly, EGF failed to increase [Na+]i following inhibition of: 1) PKA activity (10 μm H-89); 2) Erk1/2 (15 μm PD98059) or 3) p38 (15 μm SB203580) activity. Stimulation protein kinase C activity (0.1 μm PMA) transiently increased [Na+]i followed by a decline towards its baseline value. EGF-induced increases in [Na+]i were unaltered by inhibition of K+ conductance (100 μm 4-AP). Taken together, EGF stimulates Erk1/2; p38 and cPLA2 activity. Their stimulation increases PGE2 and cAMP levels resulting in PKA and NKCC activation.

Key words: Cornea — Epidermal growth factor — Na:K:2Cl — cAMP — Proliferation — Epithelium

Copyright information

© 2001 Springer-Verlag New York Inc.

Authors and Affiliations

  • H.  Yang
    • 1
  • Z.  Wang
    • 1
  • Y.  Miyamoto
    • 2
  • P. S.  Reinach
    • 1
  1. 1.State University of New York, College of Optometry, Department of Biological Sciences, 33 W. 42nd Street, New York, NY 10036, USAUS
  2. 2.Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo 1-1-1 Yayoi, Bunkyo, Tokyo, Japan 113-8657JP