title: Hydrogen peroxide-induced calcium influx in lung microvascular endothelial cells involves TRPV4.
abstract: In acute respiratory distress syndrome, both reactive oxygen species (ROS) and increased intracellular calcium ([Ca(2+)]i) are thought to play important roles in promoting endothelial paracellular permeability, but the mechanisms linking ROS and [Ca(2+)]i in microvascular endothelial cells are not known. In this study, we assessed the effect of hydrogen peroxide (H2O2) on [Ca(2+)]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 [Ca(2+)]i through Ca(2+) influx and that pharmacologic inhibition of the calcium channel transient receptor potential vanilloid 4 (TRPV4) attenuated the H2O2-induced Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 [Ca(2+)]i and decreases TER in microvascular endothelial cells via activation of TRPV4 through a mechanism that requires the Src kinase Fyn.
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine and.
authors.forename: Karthik
authors.initials: K
authors.lastname: Suresh
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine and.
authors.forename: Laura
authors.initials: L
authors.lastname: Servinsky
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine and.
authors.forename: Jose
authors.initials: J
authors.lastname: Reyes
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine and.
authors.forename: Syeda
authors.initials: S
authors.lastname: Baksh
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine and.
authors.forename: Clark
authors.initials: C
authors.lastname: Undem
authors.affiliation: Department of Neurosurgery, Johns Hopkins School of Medicine, Baltimore, Maryland.
authors.forename: Michael
authors.initials: M
authors.lastname: Caterina
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine and.
authors.forename: David B
authors.initials: DB
authors.lastname: Pearse
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine and lshimod1@jhmi.edu.
authors.forename: Larissa A
authors.initials: LA
authors.lastname: Shimoda
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008168:Lung / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D055806:Microvessels / Q000187:drug effects / Q000378:metabolism*; D010539:Permeability / Q000187:drug effects; D051076:Proto-Oncogene Proteins c-fyn / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D050916:TRPV Cation Channels / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: C530602:N-(1-((4-(2-(((2,4-dichlorophenyl)sulfonyl)amino)-3-hydroxypropanoyl)-1-piperazinyl)carbonyl)-3-methylbutyl)-1-benzothiophene-2-carboxamide; D017382:Reactive Oxygen Species; D013449:Sulfonamides; D050916:TRPV Cation Channels; D006861:Hydrogen Peroxide; D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases; D007930:Leucine; D002118:Calcium
keywords: ARDS; ROS; TRPV4; calcium; lung injury