HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: 286/1/C31    most recent 00157.2003v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (19) Citing Articles via Google Scholar Google Scholar Articles by Kim, Y. V. Articles by Kim, K. S. Search for Related Content PubMed PubMed Citation Articles by Kim, Y. V. Articles by Kim, K. S.

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

This article has been cited by other articles:

				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]