Hydrogen peroxide-induced calcium influx in lung microvascular endothelial cells involves TRPV4

Karthik Suresh, Laura Servinsky, Jose Reyes, Syeda Baksh, Clark Undem, Michael Caterina, David B Pearse, Larissa Shimoda

Research output: Contribution to journalArticle

Abstract

In acute respiratory distress syndrome, both reactive oxygen species (ROS) and increased intracellular calcium ([Ca2[1]]i) are thought to play important roles in promoting endothelial paracellular permeability, but the mechanisms linking ROS and [Ca2[1]]i in microvascular endothelial cells are not known. In this study, we assessed the effect of hydrogen peroxide (H2O2) on [Ca2[1]]i in mouse and human lung microvascular endothelial cells (MLMVEC and HLMVEC, respectively). We found that in both MLMVECs and HLMVECs, exogenously applied H2O2 increased [Ca2[1]]i through Ca2[1] influx and that pharmacologic inhibition of the calcium channel transient receptor potential vanilloid 4 (TRPV4) attenuated the H2O2-induced Ca2[1]influx. Additionally, knockdown of TRPV4 in HLMVEC also attenuated calcium influx following H2O2 challenge. Administration of H2O2 or TRPV4 agonists decreased transmembrane electrical resistance (TER), suggesting increased barrier permeability. To explore the regulatory mechanisms underlying TRPV4 activation by ROS, we examined H2O2-induced Ca2[1] influx in MLMVECs and HLMVECs with either genetic deletion, silencing, or pharmacologic inhibition of Fyn, a Src family kinase. In both MLMVECs derived from mice deficient for Fyn and HLMVECs treated with either siRNA targeted to Fyn or the Src family kinase inhibitor SU-6656 for 24 or 48 h, the H2O2-induced Ca2[1] influx was attenuated. Treatment with SU-6656 decreased the levels of phosphorylated, but not total, TRPV4 protein and had no effect on TRPV4 response to the external agonist, GSK1016790A. In conclusion, our data suggest that application of exogenous H2O2 increases [Ca2[1]]i and decreases TER in microvascular endothelial cells via activation of TRPV4 through a mechanism that requires the Src kinase Fyn.

Original languageEnglish (US)
Pages (from-to)L1467-L1477
JournalAmerican Journal of Physiology - Lung Cellular and Molecular Physiology
Volume309
Issue number12
DOIs
StatePublished - 2015

Fingerprint

TRPV Cation Channels
Hydrogen Peroxide
Endothelial Cells
Calcium
Lung
src-Family Kinases
Reactive Oxygen Species
Electric Impedance
Permeability
Adult Respiratory Distress Syndrome
Calcium Channels
Small Interfering RNA

Keywords

  • Ards
  • Calcium
  • Lung injury
  • Ros
  • TRPV4

ASJC Scopus subject areas

  • Pulmonary and Respiratory Medicine
  • Physiology (medical)
  • Cell Biology
  • Physiology

Cite this

Hydrogen peroxide-induced calcium influx in lung microvascular endothelial cells involves TRPV4. / Suresh, Karthik; Servinsky, Laura; Reyes, Jose; Baksh, Syeda; Undem, Clark; Caterina, Michael; Pearse, David B; Shimoda, Larissa.

In: American Journal of Physiology - Lung Cellular and Molecular Physiology, Vol. 309, No. 12, 2015, p. L1467-L1477.

Research output: Contribution to journalArticle

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AU - Suresh, Karthik

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AU - Undem, Clark

AU - Caterina, Michael

AU - Pearse, David B

AU - Shimoda, Larissa

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AB - In acute respiratory distress syndrome, both reactive oxygen species (ROS) and increased intracellular calcium ([Ca2[1]]i) are thought to play important roles in promoting endothelial paracellular permeability, but the mechanisms linking ROS and [Ca2[1]]i in microvascular endothelial cells are not known. In this study, we assessed the effect of hydrogen peroxide (H2O2) on [Ca2[1]]i in mouse and human lung microvascular endothelial cells (MLMVEC and HLMVEC, respectively). We found that in both MLMVECs and HLMVECs, exogenously applied H2O2 increased [Ca2[1]]i through Ca2[1] influx and that pharmacologic inhibition of the calcium channel transient receptor potential vanilloid 4 (TRPV4) attenuated the H2O2-induced Ca2[1]influx. Additionally, knockdown of TRPV4 in HLMVEC also attenuated calcium influx following H2O2 challenge. Administration of H2O2 or TRPV4 agonists decreased transmembrane electrical resistance (TER), suggesting increased barrier permeability. To explore the regulatory mechanisms underlying TRPV4 activation by ROS, we examined H2O2-induced Ca2[1] influx in MLMVECs and HLMVECs with either genetic deletion, silencing, or pharmacologic inhibition of Fyn, a Src family kinase. In both MLMVECs derived from mice deficient for Fyn and HLMVECs treated with either siRNA targeted to Fyn or the Src family kinase inhibitor SU-6656 for 24 or 48 h, the H2O2-induced Ca2[1] influx was attenuated. Treatment with SU-6656 decreased the levels of phosphorylated, but not total, TRPV4 protein and had no effect on TRPV4 response to the external agonist, GSK1016790A. In conclusion, our data suggest that application of exogenous H2O2 increases [Ca2[1]]i and decreases TER in microvascular endothelial cells via activation of TRPV4 through a mechanism that requires the Src kinase Fyn.

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