Differential Ca2+ signaling by thrombin and protease-activated receptor-1-activating peptide in human brain microvascular endothelial cells
Differential Ca2+ signaling by thrombin and protease-activated receptor-1-activating peptide in human brain microvascular endothelial cells
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DOI：
10.1152/ajpcell.00157.2003
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发表时间：
2004-01-01
影响因子：
5.5
通讯作者：
Kim, KS
中科院分区：
文献类型：
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作者：
Kim, YV;Di Cello, F;Kim, KS
凝血酶和相关的蛋白酶激活受体1、2、3和4(PAR1-4)在包括内皮细胞在内的多种细胞中发挥着多功能的作用。在这里，利用RT-PCR和免疫荧光染色，我们首次显示PAR1-4在原代培养的人脑微血管内皮细胞(HBMEC)上表达。用数字荧光显微镜和Fura-2检测凝血酶和PAR1激活肽(PAR1-AP)SF-FLRN对细胞内钙离子浓度([Ca+](I))的影响。凝血酶和PAR1-AP均引起剂量依赖性的[Ca~(2+)](I)升高，这种升高可被磷脂酶C抑制剂U-73122和肌浆网钙-ATPase抑制剂thapsigargin所抑制。凝血酶诱导的一过性[Ca~(2+)](I)升高，而PAR1-AP呈现持续性的[Ca~(2+)](I)升高。在无细胞外钙或存在钙通道阻滞剂SKF-96365的情况下，PAR1-AP诱导的持续的[Ca~(2+)](I)升高显著降低。在无细胞外Ca~(2+)的情况下，恢复PAR1-AP激活的细胞外Ca~(2+)使[Ca~(2+)](I)显著升高，但在凝血酶刺激后未观察到这种作用。低浓度凝血酶(0.1 nM)可阻止高凝血酶浓度(10 NM)引起的细胞内[Ca~(2+)](I)升高，但PAR1-AP不能阻止细胞内[Ca~(2+)](I)随后升高。此外，凝血酶可降低HBMEC的跨内皮电阻，而PAR1-AP对此无明显影响。这些发现表明，与凝血酶相比，游离多肽SFFLRN刺激PAR1可刺激钙离子内流，而不显著影响脑内皮屏障功能。
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) SF-FLRN. 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.