Differential Ca signaling by thrombin and protease-activated receptor-1-activating peptide in human brain microvascular endothelial cells

Kim, Yuri V., Francescopaolo Di Cello, Coryse S. Hillaire, and Kwang Sik Kim. Differential Ca signaling by thrombin and protease-activated receptor-1-activating peptide in human brain microvascular endothelial cells. Am J Physiol Cell Physiol 286: C31–C42, 2004. First published August 27, 2003; 10.1152/ajpcell.00157. 2003.—Thrombin and related protease-activated receptors 1, 2, 3, and 4 (PAR1–4) play a multifunctional role in many types of cells including endothelial cells. Here, using RT-PCR and immunofluorescence staining, we showed for the first time that PAR1–4 are expressed on primary human brain microvascular endothelial cells (HBMEC). Digital fluorescence microscopy and fura 2 were used to monitor intracellular Ca concentration ([Ca ]i) changes in response to thrombin and PAR1-activating peptide (PAR1-AP) SFFLRN. Both thrombin and PAR1-AP induced a dose-dependent [Ca ]i rise that was inhibited by pretreatment of HBMEC with the phospholipase C inhibitor U-73122 and the sarco(endo)plasmic reticulum Ca -ATPase inhibitor thapsigargin. Thrombin induced transient [Ca ]i increase, whereas PAR1-AP exhibited sustained [Ca ]i rise. The PAR1-AP-induced sustained [Ca ]i rise was significantly reduced in the absence of extracellular calcium or in the presence of an inhibitor of store-operated calcium channels, SKF-96365. Restoration of extracellular Ca to the cells that were initially activated by PAR1-AP in the absence of extracellular Ca resulted in significant [Ca ]i rise; however, this effect was not observed after thrombin stimulation. Pretreatment of the cells with a low thrombin concentration (0.1 nM) prevented [Ca ]i rise in response to high thrombin concentration (10 nM), but pretreatment with PAR1-AP did not prevent subsequent [Ca ]i rise to high PAR1-AP concentration. Additionally, treatment with thrombin decreased transendothelial electrical resistance in HBMEC, whereas PAR1-AP was without significant effect. These findings suggest that, in contrast to thrombin, stimulation of PAR1 by untethered peptide SFFLRN results in stimulation of store-operated Ca influx without significantly affecting brain endothelial barrier functions.