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RECEPTORS AND SIGNAL TRANSDUCTION

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

¹Division of Pediatric Infectious Diseases and ²Department of Neurology, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21287

Submitted 22 April 2003 ; accepted in final form 21 August 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 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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				D. J. Grab, G. Perides, J. S. Dumler, K. J. Kim, J. Park, Y. V. Kim, O. Nikolskaia, K. S. Choi, M. F. Stins, and K. S. Kim Borrelia burgdorferi, Host-Derived Proteases, and the Blood-Brain Barrier Infect. Immun., February 1, 2005; 73(2): 1014 - 1022. [Abstract] [Full Text] [PDF]