Modulation of Ca2+ transients in cultured endothelial cells in response to fluid flow through alphav integrin.

In order to determine whether integrin dynamics is associated with intracellular Ca(2+) concentration (Ca(2+)) mobilization in ECs in response to hemodynamic forces, changes in Ca(2+) in fluo-4-loaded cultured bovine aortic endothelial cells (BAECs) under fluid flow conditions were visualized employing laser scanning confocal microscopy. Following the onset of flow stimulus, transient increases in Ca(2+) occurred several times in individual BAECs during the 30-min observation period. The frequency of these Ca(2+) transients was clearly reduced by the application of an integrin antagonist (GRGDSP peptide). Furthermore, treatment of cells with an integrin activator (Mn(2+)) resulted in reduction of peak Ca(2+) levels and elevated frequency, which was markedly rescued upon GRGDSP administration. In contrast, an actin de-polymerizing agent (cytochalasin D) exerted no inhibitory effects; rather, cytochalasin D more likely facilitated Ca(2+) transients. Moreover, Ca(2+) transients, which were suppressed by short interference RNA-induced silencing of alphav integrin, exhibited greater frequently in cells cultured on vitronectin substratum in comparison with those cultured on fibronectin or collagen substratum. Either removal of extracellular Ca(2+), application of an inhibitor of endoplasmic reticulum Ca(2+)-ATPase (thapsigargin) or non-selective cation channel blocker (La(3+)) inhibited the Ca(2+) transients. Additionally, Ca(2+) transients were attenuated by extracellular signal-regulated kinase (ERK) kinase inhibitor (U0126); in contrast, Ca(2+) transients were unaffected by tyrosine kinase inhibitor (genistein) or phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002). Therefore, our findings revealed that alphav integrin dynamics modulates the frequency of flow-induced Ca(2+) transients in BAECs in an ERK-dependent fashion.