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 Ca ²⁺ 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 Ca ²⁺ content, thereby regulating the store-operated Ca²⁺ influx. However, the functional role of STIM1 in the thrombin-induced Ca ²⁺ 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 Ca ²⁺ influx exhibited the similar sensitivity toward the Ca ²⁺ 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 Ca ²⁺-dependent component of the NO production in endothelial cells.