title: Differential Ca2+ signaling by thrombin and protease-activated receptor-1-activating peptide in human brain microvascular endothelial cells.
abstract: 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+]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.
authors.affiliation: Division of Pediatric Infectious Diseases, Johns Hopkins University School of Medicine, 600 N. Wolfe Street, Park 256, Baltimore, MD 21287, USA.
authors.forename: Yuri V
authors.initials: YV
authors.lastname: Kim
authors.forename: Francescopaolo
authors.initials: F
authors.lastname: Di Cello
authors.forename: Coryse S
authors.initials: CS
authors.lastname: Hillaire
authors.forename: Kwang Sik
authors.initials: KS
authors.lastname: Kim
mesh_terms: D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002199:Capillary Permeability / Q000187:drug effects; D002478:Cells, Cultured; D002560:Cerebrovascular Circulation / Q000187:drug effects / Q000502:physiology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D045604:Extracellular Fluid / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008833:Microcirculation / Q000187:drug effects / Q000502:physiology; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology*; D009994:Osmolar Concentration; D010739:Phospholipase D / Q000037:antagonists & inhibitors; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D013917:Thrombin / Q000494:pharmacology*
chemical_list: D004791:Enzyme Inhibitors; D009842:Oligopeptides; C462999:PAR-1-activating peptide; D010739:Phospholipase D; D013917:Thrombin; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium