Modulation of Ca2+ transients in cultured endothelial cells in response to fluid flow through alpha v integrin

摘要

In order to determine whether integrin dynamics is associated with intracellular Ca2+ concentration ([Ca2+](i)) mobilization in ECs in response to hemodynamic forces, changes in [Ca2+](i) 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 [Ca2+](i) occurred several times in individual BAECs during the 30-min observation period. The frequency of these [Ca2+](i) transients was clearly reduced by the application of an integrin antagonist (GRGDSP peptide). Furthermore, treatment of cells with an integrin activator (Mn2+) resulted in reduction of peak [Ca2+](i) 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 [Ca2+](i) transients. Moreover, [Ca2+](i) transients, which were suppressed by short interference RNA-induced silencing of alpha v integrin, exhibited greater frequently in cells cultured on vitronectin substratum in comparison with those cultured on fibronectin or collagen substratum. Either removal of extracellular Ca2+, application of an inhibitor of endoplasmic reticulum Ca2+-ATPase (thapsigargin) or non-selective cation channel blocker (La3+) inhibited the [Ca2+](i) transients. Additionally, [Ca2+](i) transients were attenuated by extracellular signal-regulated kinase (ERK) kinase inhibitor (U0126); in contrast, [Ca2+](i) transients were unaffected by tyrosine kinase inhibitor (genistein) or phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002). Therefore, our findings revealed that alpha v integrin dynamics modulates the frequency of flow-induced [Ca2+](i) transients in BAECs in an ERK-dependent fashion. (c) 2007 Elsevier Inc. All rights reserved.