Fluid Shear Stress Activates Ca 2+ Influx Into Human Endothelial Cells via P2X4 Purinoceptors

Abstract

Abstract —Ca 2+ 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 2+ 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 2+ 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 2+ influx. Human umbilical vein ECs loaded with the Ca 2+ indicator Indo-1/AM were exposed to laminar flow of Hanks’ balanced salt solution at various concentrations of ATP, and changes in [Ca 2+ ] i were monitored with confocal laser scanning microscopy. A stepwise increase in shear stress elicited a corresponding stepwise increase in [Ca 2+ ] i at 250 nmol/L ATP. The shear stress–dependent increase in [Ca 2+ ] i was not affected by phospholipase C inhibitor (U-73122) but disappeared after the chelation of extracellular Ca 2+ with EGTA, indicating that the Ca 2+ increase was due to Ca 2+ influx. Antisense oligonucleotides designed to knockout P2X4 expression abolished the shear stress–dependent Ca 2+ influx seen at 250 nmol/L ATP in human umbilical vein ECs. Human embryonic kidney 293 cells showed no Ca 2+ 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 2+ 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 2+ signaling.