Differential Ca2+ signaling by thrombin and protease-activated receptor-1-activating peptide in human brain microvascular endothelial cells

Yuri V. Kim, Francescopaolo Di Cello, Coryse S. Hillaire, Kwang Sik Kim

Research output: Contribution to journalArticlepeer-review

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 Ca 2+-ATPase inhibitor thapsigargin. Thrombin induced transient [Ca 2+]i increase, whereas PAR1-AP exhibited sustained [Ca2+]i rise. The PAR1-AP-induced sustained [Ca 2+]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.

Original languageEnglish (US)
Pages (from-to)C31-C42
JournalAmerican Journal of Physiology - Cell Physiology
Volume286
Issue number1 55-1
DOIs
StatePublished - Jan 2004

Keywords

  • Desensitization
  • Digital imaging
  • Store-operated calcium influx
  • Transendothelial electrical resistance

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

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