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 Ca2+concentration ([Ca2+]i) changes in response to thrombin and PAR1-activating peptide (PAR1-AP) SFFLRN. Both thrombin and PAR1-AP induced a dose-dependent [Ca2+]irise that was inhibited by pretreatment of HBMEC with the phospholipase C inhibitor U-73122 and the sarco(endo)plasmic reticulum Ca2+-ATPase inhibitor thapsigargin. Thrombin induced transient [Ca2+]iincrease, whereas PAR1-AP exhibited sustained [Ca2+]irise. The PAR1-AP-induced sustained [Ca2+]irise 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 Ca2+to the cells that were initially activated by PAR1-AP in the absence of extracellular Ca2+resulted in significant [Ca2+]irise; however, this effect was not observed after thrombin stimulation. Pretreatment of the cells with a low thrombin concentration (0.1 nM) prevented [Ca2+]irise in response to high thrombin concentration (10 nM), but pretreatment with PAR1-AP did not prevent subsequent [Ca2+]irise 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 Ca2+influx without significantly affecting brain endothelial barrier functions.
Stimulation of phospholipase C in cultured microvascular endothelial cells from human frontal lobe by histamine, endothelin and purinoceptor agonists
