Hypergravity induces ATP release and actin reorganization via tyrosine phosphorylation and RhoA activation in bovine endothelial cells

Abstract

Mechanical stresses regulate physiological and pathological functions of vascular endothelial cells. We examined, in this study, the effects of hypergravity on endothelial functions. Hypergravity (3 G) applied by low speed centrifuge immediately induced a membrane translocation of small G-protein RhoA and tyrosine phosphorylation of 125 kDa FAK in bovine aortic endothelial cells (BAECs). Hypergravity also induced a transient reorganization of actin fibers in 3 min, which was inhibited by Rho-kinase inhibitor (Y27632) and tyrosine kinase inhibitors (herbimycin A and tyrphostin 46). Furthermore, the extracellular ATP concentration ([ATP]o) was increased by 2 G and 3 G hypergravity in 5 min, and the inhibitors of Rho-kinase, tyrosine kinase, and volume-regulated anion channels (VRAC; verapamil, tamoxifen and fluoxetine) significantly suppressed [ATP]o elevation. Application of 3 G hypergravity for 1 h increased the nuclear uptake of BrdU, which was inhibited by Rho-kinase inhibitor and VARC inhibitors. Furthermore, intermittent application of 3 G hypergravity for 1 or 2 h/day stimulated endothelial migration in 5 days, and this was inhibited by suramin, a P2 antagonist. Collectively, these results indicate that hypergravity induces ATP release and actin reorganization via RhoA activation and FAK phosphorylation, thereby activating cell proliferation and migration in BAECs. These also suggest that gravity can be regarded as an extracorporeal signal that could significantly affect endothelial functions.

New insights regarding the regulation of chemotaxis by nucleotides, adenosine, and their receptors

“…ATP/P2 Motility [150] Exposure to hypergravity induces the release of ATP and migration of bovine aortic endothelial cells in a RhoA activation/FAK phosphorylation-dependent manner [75]. Studies of HUVECs reveal that extracellular ATP and UTP both induce FAK phosphorylation and promote endothelial migration through the activation of P2Y 2 and P2Y 4 receptors [76].…”

Hypergravity and Hypobaric Hypoxic Conditions Promote Endothelial Cell and Platelet Activation

“…However, the exposure duration to these conditions had some effects on cell viability and density. Koyama et al (2009) quantified an increase in ATP release after exposure to mild hypergravity (2G/3G for 5 min), and suggested this may alter proliferation but did not quantify this. This was confirmed by others, using slightly different exposures and measurement techniques (Versari et al, 2007).…”

“…Koyama et al (2009) show a transient change in the actin cytoskeleton in response to short-term hypergravity stimulation. While their data show some subtle qualitative differences, our method of quantification would not necessarily detect these differences (Fig.…”

ATP Release Mechanisms of Endothelial Cell–Mediated Stimulus-Dependent Hyperalgesia

“…, connexin hemichannels, pannexin channels, maxi-anion channels, volume regulated anion channels and P2X7 receptors). Several of these mechanisms have already been demonstrated to be present in endothelial cells 40, 41 and, importantly from a practical standpoint, there is a relatively well-developed pharmacology available for the study of these mechanisms. Therefore, in the present study we evaluated the role of these ATP release mechanisms in ET-1 induced SDH.…”