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 2ϩ ]i) are thought to play important roles in promoting endothelial paracellular permeability, but the mechanisms linking ROS and [Ca 2ϩ ]i in microvascular endothelial cells are not known. In this study, we assessed the effect of hydrogen peroxide (H2O2) on [Ca 2ϩ ]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 2ϩ ]i through Ca 2ϩ influx and that pharmacologic inhibition of the calcium channel transient receptor potential vanilloid 4 (TRPV4) attenuated the H2O2-induced Ca 2ϩ 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 2ϩ 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 H 2O2-induced Ca 2ϩ 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 2ϩ ]i and decreases TER in microvascular endothelial cells via activation of TRPV4 through a mechanism that requires the Src kinase Fyn.

Dual contribution of TRPV4 antagonism in the regulatory effect of vasoinhibins on blood-retinal barrier permeability: diabetic milieu makes a difference

“…These data are consistent with the observations that intravenous administration of TRPV4 activators can lead to circulatory collapse, which at least is partly explained by increased capillary permeability and fluid loss in the lungs 46,70,89,90 . Also, 4α-PDD exposure decreases TER in microvascular endothelial cells, including primary culture of retinal capillary endothelial cells, which is in agreement with previous studies 19,34,44,91 . Further analysis should include trpv4 −/− mice to define the precise role of endogenous TRPV4 channels in BRB transport.…”

“…These differences may be due to pharmacological considerations but they also suggest that 4α-PDD may activate additional pathways than DETANONOate to reorganize actin. The canonical phospholipase A2 and ROS pathways may be involved as previously described 91,101,109 .…”

Markers and Biomarkers of Endothelium: When Something Is Rotten in the State

“…TRPM2 is a transducer that converts oxidative stress into calcium signaling and plays an important role in ROS-coupled diseases [115]. On the other hand, exogenous H 2 O 2 increases [Ca 2+ ] i and decreases transmembrane electrical resistance in lung microvascular ECs via activation of TRPV4 through a mechanism that requires the Src kinase Fyn [116]. Nevertheless, we have recently estimated that H 2 O 2 in low noncytotoxic concentrations causes elevation of [Ca 2+ ] cyt in cultured HUVECs through calcium release from the two-pore channels of endolysosomal vesicles [117].…”

Redox regulation of transient receptor potential channels in the endothelium

“…Desensitization appears to be a consequence of reduced interaction between TRPV4 and calmodulin 122 , a previously described Ca 2+ -dependent mechanism of TRPV4 inactivation 118 . In cultured pulmonary endothelial cells, H 2 O 2 induces Ca 2+ influx through a mechanism that is dependent of TRPV4 activity 123 . Although not directly evaluated in the study, it is possible that in the short-term (1 hour of exposure) H 2 O 2 may act directly on the TRPV4 channel, whereas on the long-term (24 hours of exposure to H 2 O 2 ) TRPV4 activity is regulated by the Src kinase Fyn 123 .…”

“…In cultured pulmonary endothelial cells, H 2 O 2 induces Ca 2+ influx through a mechanism that is dependent of TRPV4 activity 123 . Although not directly evaluated in the study, it is possible that in the short-term (1 hour of exposure) H 2 O 2 may act directly on the TRPV4 channel, whereas on the long-term (24 hours of exposure to H 2 O 2 ) TRPV4 activity is regulated by the Src kinase Fyn 123 . Interestingly, Ca 2+ entry through TRPV4 channels may elevate ROS production in the coronary endothelium, providing a positive feedback mechanism to stimulate flow-induced dilation 121 .…”