Sequential activation of RhoA and FAK/paxilin leads to ATP release and actin reorganization in human endothelium

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Abstract

We have investigated the cellular mechanisms of mechanical stress-induced immediate responses in human umbilical vein endothelial cells (HUVECs). Hypotonic stress (HTS) induced ATP release, which evoked a Ca2+ transient, followed by actin reorganization within a few minutes, in HUVECs. Disruption of the actin cytoskeleton did not suppress HTS-induced ATP release, and inhibition of the ATP-mediated Ca2+ response did not affect actin reorganization, thereby indicating that these two responses are not interrelated. ATP release and actin reorganization were also induced by lysophosphatidic acid (LPA . HTS and LPA induced membrane translocation of RhoA, which occurs when RhoA is activated, and tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin. Tyrosine kinase inhibitors (herbimycin A or tyrphostin 46) inhibited both HTS- and LPA-induced ATP release and actin reorganization, but did not affect RhoA activation. In contrast, Rho-kinase inhibitor (Y27632) inhibited all of the HTS- and LPA-induced responses. These results indicate that the activation of the RhoA/Rho-kinase pathway followed by tyrosine phosphorylation of FAK and paxillin leads to ATP release and actin reorganization in HUVECs. Furthermore, the fact that HTS and LPA evoke exactly the same intracellular signals and responses suggests that even these immediate mechanosensitive responses are in fact not mechanical stress-specific.

Original languageEnglish
Pages (from-to)479-488
Number of pages10
JournalJournal of Physiology
Volume558
Issue number2
DOIs
Publication statusPublished - Jul 15 2004

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Focal Adhesion Protein-Tyrosine Kinases
Osmotic Pressure
Endothelium
Human Umbilical Vein Endothelial Cells
Paxillin
rho-Associated Kinases
Mechanical Stress
Adenosine Triphosphate
Tyrosine
Actins
Phosphorylation
Actin Cytoskeleton
Protein-Tyrosine Kinases
ATP-G-actin
Membranes

All Science Journal Classification (ASJC) codes

  • Physiology

Cite this

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title = "Sequential activation of RhoA and FAK/paxilin leads to ATP release and actin reorganization in human endothelium",
abstract = "We have investigated the cellular mechanisms of mechanical stress-induced immediate responses in human umbilical vein endothelial cells (HUVECs). Hypotonic stress (HTS) induced ATP release, which evoked a Ca2+ transient, followed by actin reorganization within a few minutes, in HUVECs. Disruption of the actin cytoskeleton did not suppress HTS-induced ATP release, and inhibition of the ATP-mediated Ca2+ response did not affect actin reorganization, thereby indicating that these two responses are not interrelated. ATP release and actin reorganization were also induced by lysophosphatidic acid (LPA . HTS and LPA induced membrane translocation of RhoA, which occurs when RhoA is activated, and tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin. Tyrosine kinase inhibitors (herbimycin A or tyrphostin 46) inhibited both HTS- and LPA-induced ATP release and actin reorganization, but did not affect RhoA activation. In contrast, Rho-kinase inhibitor (Y27632) inhibited all of the HTS- and LPA-induced responses. These results indicate that the activation of the RhoA/Rho-kinase pathway followed by tyrosine phosphorylation of FAK and paxillin leads to ATP release and actin reorganization in HUVECs. Furthermore, the fact that HTS and LPA evoke exactly the same intracellular signals and responses suggests that even these immediate mechanosensitive responses are in fact not mechanical stress-specific.",
author = "Masakazu Hirakawa and Masahiro Oike and Yuji Karashima and Yushi Ito",
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T1 - Sequential activation of RhoA and FAK/paxilin leads to ATP release and actin reorganization in human endothelium

AU - Hirakawa, Masakazu

AU - Oike, Masahiro

AU - Karashima, Yuji

AU - Ito, Yushi

PY - 2004/7/15

Y1 - 2004/7/15

N2 - We have investigated the cellular mechanisms of mechanical stress-induced immediate responses in human umbilical vein endothelial cells (HUVECs). Hypotonic stress (HTS) induced ATP release, which evoked a Ca2+ transient, followed by actin reorganization within a few minutes, in HUVECs. Disruption of the actin cytoskeleton did not suppress HTS-induced ATP release, and inhibition of the ATP-mediated Ca2+ response did not affect actin reorganization, thereby indicating that these two responses are not interrelated. ATP release and actin reorganization were also induced by lysophosphatidic acid (LPA . HTS and LPA induced membrane translocation of RhoA, which occurs when RhoA is activated, and tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin. Tyrosine kinase inhibitors (herbimycin A or tyrphostin 46) inhibited both HTS- and LPA-induced ATP release and actin reorganization, but did not affect RhoA activation. In contrast, Rho-kinase inhibitor (Y27632) inhibited all of the HTS- and LPA-induced responses. These results indicate that the activation of the RhoA/Rho-kinase pathway followed by tyrosine phosphorylation of FAK and paxillin leads to ATP release and actin reorganization in HUVECs. Furthermore, the fact that HTS and LPA evoke exactly the same intracellular signals and responses suggests that even these immediate mechanosensitive responses are in fact not mechanical stress-specific.

AB - We have investigated the cellular mechanisms of mechanical stress-induced immediate responses in human umbilical vein endothelial cells (HUVECs). Hypotonic stress (HTS) induced ATP release, which evoked a Ca2+ transient, followed by actin reorganization within a few minutes, in HUVECs. Disruption of the actin cytoskeleton did not suppress HTS-induced ATP release, and inhibition of the ATP-mediated Ca2+ response did not affect actin reorganization, thereby indicating that these two responses are not interrelated. ATP release and actin reorganization were also induced by lysophosphatidic acid (LPA . HTS and LPA induced membrane translocation of RhoA, which occurs when RhoA is activated, and tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin. Tyrosine kinase inhibitors (herbimycin A or tyrphostin 46) inhibited both HTS- and LPA-induced ATP release and actin reorganization, but did not affect RhoA activation. In contrast, Rho-kinase inhibitor (Y27632) inhibited all of the HTS- and LPA-induced responses. These results indicate that the activation of the RhoA/Rho-kinase pathway followed by tyrosine phosphorylation of FAK and paxillin leads to ATP release and actin reorganization in HUVECs. Furthermore, the fact that HTS and LPA evoke exactly the same intracellular signals and responses suggests that even these immediate mechanosensitive responses are in fact not mechanical stress-specific.

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