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

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 micromol/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.

Atherosclerosis and calcium signalling in endothelial cells

“…The cytosolic Ca 2 + concentration quickly returns to baseline and the internal stores recover towards their resting concentration. The finding of a cytosolic Ca 2 + transient is in agreement with experimental data (Dull and Davies, 1991;Yamamoto et al, 2000) and previous mathematical modelling results (Wiesner et al, 1996), and the model reproduces realistic response times and peak Ca 2 + concentrations.…”

“…oscillating), against ip, for fixed wall shear stress. The ATP concentration in the bloodstream is not known exactly, but is thought to be less than 2 µM (Yamamoto et al, 2000) (which is equivalent to a dimensionless value of 20), so Figure 7(a) shows behaviour for ATP concentration in the physiological range. For ip < 0.42, there is a single, stable fixed point, indicating that the system eventually settles down to a steady state ( this corresponds to the plateau phase in a stimulated cell).…”

The touching story of purinergic signaling in epithelial and endothelial cells

“…The P2X 4 receptor is the highest expressed P2 receptor in endothelium [79,80]. By using antisense oligonucleotides, the P2X 4 receptor was shown to be important for shear stress-dependent Ca 2+ influx via an ATP-dependent mechanism [81]. This indicates that ATP and P2 receptors may be of importance for shear stress-mediated effects which is in agreement with the well-established release of ATP from endothelial cells during shear stress [82].…”

My biorheology research guided by curiosity

“…When endothelial cells are exposed to shear stress, caveola (flask-like concave structures on the cell membrane) immediately release ATP [7], which opens the ATP-gated cation channel (P2X4), allowing the extracellu lar Ca 2+ to enter the cells [8]. The resultant local elevation of the Ca 2+ concentration is followed by release of Ca 2+ from the endoplasmic reticulum and a wavy spread of Ca 2+ level elevation throughout the cells [9].…”