Fluid Shear Stress Activates Ca²⁺Influx Into Human Endothelial Cells via P2X4 Purinoceptors

<jats:p><jats:italic>Abstract</jats:italic>—Ca<jats:sup>2+</jats:sup>signaling plays an important role in endothelial cell (EC) responses to shear stress generated by blood flow. Our previous studies demonstrated that bovine fetal aortic ECs showed a shear stress–dependent Ca<jats:sup>2+</jats:sup>influx when exposed to flow in the presence of extracellular ATP. However, the molecular mechanisms of this process, including the ion channels responsible for the Ca<jats:sup>2+</jats:sup>response, have not been clarified. Here, we demonstrate that P2X4 purinoceptors, a subtype of ATP-operated cation channels, are involved in the shear stress–mediated Ca<jats:sup>2+</jats:sup>influx. Human umbilical vein ECs loaded with the Ca<jats:sup>2+</jats:sup>indicator Indo-1/AM were exposed to laminar flow of Hanks’ balanced salt solution at various concentrations of ATP, and changes in [Ca<jats:sup>2+</jats:sup>]<jats:sub>i</jats:sub>were monitored with confocal laser scanning microscopy. A stepwise increase in shear stress elicited a corresponding stepwise increase in [Ca<jats:sup>2+</jats:sup>]<jats:sub>i</jats:sub>at 250 nmol/L ATP. The shear stress–dependent increase in [Ca<jats:sup>2+</jats:sup>]<jats:sub>i</jats:sub>was not affected by phospholipase C inhibitor (U-73122) but disappeared after the chelation of extracellular Ca<jats:sup>2+</jats:sup>with EGTA, indicating that the Ca<jats:sup>2+</jats:sup>increase was due to Ca<jats:sup>2+</jats:sup>influx. Antisense oligonucleotides designed to knockout P2X4 expression abolished the shear stress–dependent Ca<jats:sup>2+</jats:sup>influx seen at 250 nmol/L ATP in human umbilical vein ECs. Human embryonic kidney 293 cells showed no Ca<jats:sup>2+</jats:sup>response to flow at 2 μmol/L ATP, but when transfected with P2X4 cDNA, they began to express P2X4 purinoceptors and to show shear stress–dependent Ca<jats:sup>2+</jats:sup>influx. P2X4 purinoceptors may have a “shear-transducer” property through which shear stress is perceived directly or indirectly and transmitted into the cell interior via Ca<jats:sup>2+</jats:sup>signaling.</jats:p>