Abstract 5845: TRPV4 Mediates Fluid Shear Stress-induced Ca2+ Influx in Endothelial Cells
The transient receptor potential vallinoid type 4 (TRPV4) channel, a mechanosensitive Ca2+-permeable channel expressed in endothelial cells of several species and vascular beds, has been implicated in the release of vasoactive factors and vasorelaxation in response to fluid shear stress. It remains unclear how shear stress activates TRPV4 channels and whether flow-induced dilation is mediated by TRPV4-dependent Ca2+ influx. Therefore, we investigated the potential role of TRPV4 in flow-induced Ca2+ entry using both native endothelial cells and over-expression cell systems. The Ca2+ entry was measured by a fura-2/Mn2+ quenching assay. In human coronary arterial endothelial cells (HCAEC), fluid shear stress (10 dyne/cm2) induced a rapid Ca2+ influx (the rate of Mn2+ quench of 0.087±0.008 vs. 0.035±0.007 under basal conditions; n=5) and this response was largely abolished by the TRPV4 channel blocker ruthenium red (0.040±0.004; n=3). Ruthenium red has no significant effect on the Ca2+ release from endoplasmic reticulum in response to bradykinin. The TRPV4-specific channel agonist, GSK 1016790A (5 nM), also elicited a robust Ca2+ entry in these endothelial cells. HEK 293 cells were transfected with a human TRPV4 transgene tagged with green fluorescent protein (GFP). The expression and membrane localization of TRPV4 channels in transfected cells were confirmed by Western blot analysis, confocal fluorescence imaging and positive Ca2+ responses to the TRPV4 agonist 4 α-phorbol-12, 13-didecanoate (4 αPDD). The transfected HEK 293 cells exhibited a marked increase in shear stress-induced Ca2+ entry (the rate of Mn2+ quench of 0.19±0.04 vs. 0.06±0.01 in control non-transfected cells; n=4 –5), which was blocked by ruthenium red (0.08±0.01). Shear stress also elicited an increase in global cytosolic Ca2+ in TRPV4-transfected HEK 293 but not in control non-transfected cells. Together, these data indicate that shear stress activates TRPV4 channels and consequently induces Ca2+ entry in both endothelial cells and cells over-expressed with these channel proteins. It is suggested that this flow-sensitive Ca2+ influx via TRPV4 channels in endothelial cells plays a critical role in mediating endothelial shear responses such as flow-mediated dilations.
This research has received full or partial funding support from the American Heart Association, National Center.