Involvement of STIM1 in the proteinase‐activated receptor 1‐mediated Ca2+ influx in vascular endothelial cells

Thrombin increases the cytosolic Ca²⁺ concentrations and induces NO production by activating proteinase‐activated receptor 1 (PAR₁) in vascular endothelial cells. The store‐operated Ca²⁺ influx is a major Ca²⁺ influx pathway in non‐excitable cells including endothelial cells and it has been reported to play a role in the thrombin‐induced Ca²⁺ signaling in endothelial cells. Recent studies have identified stromal interaction molecule 1 (STIM1) to function as a sensor of the store site Ca²⁺ content, thereby regulating the store‐operated Ca²⁺ influx. However, the functional role of STIM1 in the thrombin‐induced Ca²⁺ influx and NO production in endothelial cells still remains to be elucidated. Fura‐2 and diaminorhodamine‐4M fluorometry was utilized to evaluate the thrombin‐induced changes in cytosolic Ca²⁺ concentrations and NO production, respectively, in porcine aortic endothelial cells transfected with small interfering RNA (siRNA) targeted to STIM1. STIM1‐targeted siRNA suppressed the STIM1 expression and the thapsigargin‐induced Ca²⁺ influx. The degree of suppression of the STIM1 expression correlated well to the degree of suppression of the Ca²⁺ influx. The knockdown of STIM1 was associated with a substantial inhibition of the Ca²⁺ influx and a partial reduction of the NO production induced by thrombin. The thrombin‐induced Ca²⁺ influx exhibited the similar sensitivity toward the Ca²⁺ influx inhibitors to that seen with the thapsigargin‐induced Ca²⁺ influx. The present study provides the first evidence that STIM1 plays a critical role in the PAR₁‐mediated Ca²⁺ influx and Ca²⁺‐dependent component of the NO production in endothelial cells. J. Cell. Biochem. 108: 499–507, 2009. © 2009 Wiley‐Liss, Inc.