Kim Yuri V, Di Cello Francescopaolo, Hillaire Coryse S, Kim Kwang Sik
Division of Pediatric Infectious Diseases, Johns Hopkins University School of Medicine, 600 N. Wolfe Street, Park 256, Baltimore, MD 21287, USA.
Am J Physiol Cell Physiol. 2004 Jan;286(1):C31-42. doi: 10.1152/ajpcell.00157.2003. Epub 2003 Aug 27.
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.
凝血酶及相关蛋白酶激活受体1、2、3和4（PAR1 - 4）在包括内皮细胞在内的多种细胞类型中发挥着多功能作用。在此，我们通过逆转录聚合酶链反应（RT - PCR）和免疫荧光染色，首次证明PAR1 - 4在原代人脑海微血管内皮细胞（HBMEC）上表达。利用数字荧光显微镜和fura 2监测凝血酶和PAR1激活肽（PAR1 - AP）SFFLRN刺激后细胞内钙离子浓度（[Ca2 +]i）的变化。凝血酶和PAR1 - AP均诱导剂量依赖性的[Ca2 +]i升高，该升高被用磷脂酶C抑制剂U - 73122和肌浆网Ca2 + - ATP酶抑制剂毒胡萝卜素预处理HBMEC所抑制。凝血酶诱导[Ca2 +]i短暂升高，而PAR1 - AP则表现出[Ca2 +]i持续升高。在无细胞外钙或存在储存 - 操作性钙通道抑制剂SKF - 96365的情况下，PAR1 - AP诱导的[Ca2 +]i持续升高显著降低。将细胞外Ca2 +恢复到最初在无细胞外Ca2 +条件下被PAR1 - AP激活的细胞中，导致显著的[Ca2 +]i升高；然而，凝血酶刺激后未观察到这种效应。用低浓度凝血酶（0.1 nM）预处理细胞可防止对高浓度凝血酶（10 nM）产生的[Ca2 +]i升高，但用PAR1 - AP预处理不能防止随后对高浓度PAR1 - AP产生的[Ca2 +]i升高。此外，凝血酶处理降低了HBMEC的跨内皮电阻，而PAR1 - AP则无显著影响。这些发现表明，与凝血酶不同，无束缚肽SFFLRN刺激PAR1会导致储存 - 操作性Ca2 +内流增加，而不会显著影响脑内皮屏障功能。
