Differential Ca2+ signaling by thrombin and protease-activated receptor-1-activating peptide in human brain microvascular endothelial cells |
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Authors: | Kim Yuri V Di Cello Francescopaolo Hillaire Coryse S Kim Kwang Sik |
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Institution: | Division of Pediatric Infectious Diseases, Johns Hopkins University School of Medicine, 600 N. Wolfe Street, Park 256, Baltimore, MD 21287, USA. |
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Abstract: | Thrombin and related protease-activated receptors 1, 2, 3, and 4 (PAR14) 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 PAR14 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+]i rise 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+]i increase, whereas PAR1-AP exhibited sustained Ca2+]i rise. The PAR1-AP-induced sustained Ca2+]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 Ca2+ to the cells that were initially activated by PAR1-AP in the absence of extracellular Ca2+ resulted in significant Ca2+]i rise; however, this effect was not observed after thrombin stimulation. Pretreatment of the cells with a low thrombin concentration (0.1 nM) prevented Ca2+]i rise in response to high thrombin concentration (10 nM), but pretreatment with PAR1-AP did not prevent subsequent Ca2+]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 Ca2+ influx without significantly affecting brain endothelial barrier functions. store-operated calcium influx; desensitization; transendothelial electrical resistance; digital imaging |
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