May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
Disparate Effects of EGF Receptor and PKC Stimulation on Capacitative Calcium Entry in Corneal Epithelial Cells
Author Affiliations & Notes
  • F. Zhang
    Biological Science, SUNY Optometry, New York, NY, United States
  • H. Yang
    Biological Science, SUNY Optometry, New York, NY, United States
  • P. Reinach
    Biological Science, SUNY Optometry, New York, NY, United States
  • Footnotes
    Commercial Relationships  F. Zhang, None; H. Yang, None; P. Reinach, None.
  • Footnotes
    Support  NIH Grant EY04795
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 3460. doi:
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      F. Zhang, H. Yang, P. Reinach; Disparate Effects of EGF Receptor and PKC Stimulation on Capacitative Calcium Entry in Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3460.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: We assessed in immortalized rabbit corneal epithelial cells (RCEC) whether EGF receptor (EGFR) stimulation elicits increases in capacitative calcium entry CCE that are similar to those mediated by protein kinase C stimulation. Methods: RCEC were loaded with 2 µM fura2- AM for 30 min. Single cell fluorescence imaging was performed on the stage of an inverted microscope. To activate CCE, intracellular calcium stores were initially depleted in Ca-free medium by inhibiting endoplasmic reticulum calcium pump activity with 5 µM cyclopiazonic acid. Calcium influx was characterized in BAPTA (5 µM) loaded cells based on the magnitude and rate of increase of [Ca2+ ]i resulting from adding back 5 mM calcium to the bathing medium. BAPTA loaded cells displayed more consistent calcium transients within a cell population. Results: Relative to its paired control, EGF (5 ng/ml) increased the slope of a transient rise more than 2.5-fold, which reached a level that was 20% higher. Direct stimulation of PKC with 10 µM PDBu increased the slope by the same amount as EGF, but the stabilized transient reached a value that was 79% greater than its paired control. Inhibition of PKC activation with 1 µM calphostin C decreased the slope by 75% and the stabilized transient reached a value that was 26% lower than its paired control. The inactive phorbol ester analogue, 4α phorbol didecanoate neither changed the slope nor the magnitude of the transient. With both PDBu and calphostin C, the slope of the calcium transient elicited by addback was indistinguishable from that obtained with calphostin C alone. The SOC blocker 2-APB (100 µM) only partially suppressed the slope and the magnitude of the transient resulting from calcium addback. Conclusions: Stimulation of PKC increased CCE more than EGF. One possible explanation for this disparity is that EGF stimulates different signaling pathways that may have opposing effects on CCE. An increase in store operated channel activity contributes to CCE elicited by either EGFR or PKC stimulation.

Keywords: calcium • signal transduction • cornea: epithelium 
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