Calcium signaling induced by adhesion mediates protein tyrosine phosphorylation and is independent of pHi

Our goal was to evaluate early signaling events that occur as epithelial ce lls make initial contact with a substrate and to correlate them with phosph orylation. The corneal epithelium was chosen to study signaling events that occur with adhesion because it represents a simple system in which the tis sue adheres to a basal lamina, is avascular, and is bathed by a rear Film i n which changes in the local Environment are hypothesized to alter signalin g. To perform these experiments we developed a novel adhesion assay to capt ure the changes in intracellular Ca2+ and pH that occur as a cell makes its initial contact with a substrate. The first transient cytosolic Ca2+ peak was detected only as the cell made contact with the substrate and was demon strated using fluorimetric assays combined with live cell imaging. We demon strated that this transient Ca2+ peak always preceded a cytoplasmic alkaliz ation. When the intracellular environment was modified, the initial respons e was altered. Pretreatment with 1,2-bis(o-aminophenoxy)ethane-N,N,N'N'-tet raacctic acid (BAPTA), an intracellular chelator, inhibited Ca2+ mobilizati on, whereas benzamil altered the duration of the oscillations. Thapsigargin caused an initial Ca2+ release followed by a long attenuated response. An inositol triphosphate analog induced a large initial response, whereas hepa rin inhibited Ca2+ oscillations. Inhibitors of tyrosine phosphorylation did not alter the initial mobilization of cytosolic Ca-2 but clearance of cyto solic Ca2+ was inhibited. Exposing corneal epithelial cells to BAPTA, benza mil, or thapsigargin also attenuated the phosphorylation of the focal adhes ion protein paxillin. However, although heparin inhibited Ca2+ oscillations , it did not alter phosphorylation of paxillin. These studies demonstrate t hat the initial contact that a cell makes with a substrate modulates the in tracellular environment, and that changes in Ca2+ mobilization can alter la ter signaling events such as the phosphorylation of specific adhesion prote ins. These findings may have implications for wound repair and development. J. Cell. Physiol. 184:385-399, 2000. (C) 2000 Wiley-Liss, Inc.