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Calcium signaling induced by adhesion mediates protein tyrosine phosphorylation and is independent of pHi
Author(s) -
TrinkausRandall V.,
Kewalramani R.,
Payne J.,
CornellBell A.
Publication year - 2000
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/1097-4652(200009)184:3<385::aid-jcp14>3.0.co;2-7
Subject(s) - thapsigargin , tyrosine phosphorylation , bapta , cytosol , intracellular , phosphorylation , microbiology and biotechnology , extracellular , chemistry , adhesion , contact inhibition , cell adhesion , biophysics , biochemistry , biology , cell , enzyme , organic chemistry
Our goal was to evaluate early signaling events that occur as epithelial cells make initial contact with a substrate and to correlate them with phosphorylation. The corneal epithelium was chosen to study signaling events that occur with adhesion because it represents a simple system in which the tissue adheres to a basal lamina, is avascular, and is bathed by a tear film in which changes in the local environment are hypothesized to alter signaling. To perform these experiments we developed a novel adhesion assay to capture the changes in intracellular Ca 2+ and pH that occur as a cell makes its initial contact with a substrate. The first transient cytosolic Ca 2+ peak was detected only as the cell made contact with the substrate and was demonstrated using fluorimetric assays combined with live cell imaging. We demonstrated that this transient Ca 2+ peak always preceded a cytoplasmic alkalization. When the intracellular environment was modified, the initial response was altered. Pretreatment with 1,2‐bis( o ‐aminophenoxy)ethane‐ N,N,N ′ N ′‐tetraacetic acid (BAPTA), an intracellular chelator, inhibited Ca 2+ mobilization, whereas benzamil altered the duration of the oscillations. Thapsigargin caused an initial Ca 2+ release followed by a long attenuated response. An inositol triphosphate analog induced a large initial response, whereas heparin inhibited Ca 2+ oscillations. Inhibitors of tyrosine phosphorylation did not alter the initial mobilization of cytosolic Ca 2 but clearance of cytosolic Ca 2+ was inhibited. Exposing corneal epithelial cells to BAPTA, benzamil, or thapsigargin also attenuated the phosphorylation of the focal adhesion protein paxillin. However, although heparin inhibited Ca 2+ oscillations, it did not alter phosphorylation of paxillin. These studies demonstrate that the initial contact that a cell makes with a substrate modulates the intracellular environment, and that changes in Ca 2+ mobilization can alter later signaling events such as the phosphorylation of specific adhesion proteins. These findings may have implications for wound repair and development. J. Cell. Physiol. 184:385–399, 2000. © 2000 Wiley‐Liss, Inc.