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

Thrombin increases the cytosolic Ca(2+) concentrations and induces NO production by activating proteinase-activated receptor 1 (PAR(1)) in vascular endothelial cells. The store-operated Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) influx. The degree of suppression of the STIM1 expression correlated well to the degree of suppression of the Ca(2+) influx. The knockdown of STIM1 was associated with a substantial inhibition of the Ca(2+) 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 Ca(2+) influx. The present study provides the first evidence that STIM1 plays a critical role in the PAR(1)-mediated Ca(2+) influx and Ca(2+)-dependent component of the NO production in endothelial cells. (c) 2009 Wiley-Liss, Inc.