Hirano Katsuya, Nomoto Namie, Hirano Mayumi, Momota Fumi, Hanada Akiko, Kanaide Hideo
Division of Molecular Cardiology, Research Institute of Angiocardiology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
J Pharmacol Exp Ther. 2007 Aug;322(2):668-77. doi: 10.1124/jpet.107.121038. Epub 2007 May 9.
Proteinase-activated receptors 1 and 4 (PAR(1) and PAR(4)) are the major receptors mediating thrombin-induced NO production in endothelial cells. The intracellular signaling following their activation still remains to be elucidated. The present study provides the first evidence for the distinct Ca(2+) requirement for the NO production between PAR(1) and PAR(4). The activation of PAR(1) by the activating peptide (PAR(1)-AP) elevated cytosolic Ca(2+) concentrations (Ca(2+)) and activated NO production in porcine aortic and human umbilical vein endothelial cells, whereas it had little effect on bovine aortic endothelial cells. PAR(4) activation by PAR(4)-AP consistently induced NO production without an appreciable Ca(2+) elevation in three types of endothelial cells. The PAR(1)-mediated NO production was significantly inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), whereas the PAR(4)-mediated NO production was resistant. NO production following the PAR(1) and PAR(4) activation was significantly inhibited by pertussis toxin, but it was resistant to a Galpha(q/11) inhibitor, YM254890 [(1R)-1-[(3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-3-[(1R)-1-methoxyethyl]-4,9,10,12,16,22-hexamethyl-15-methylene-2,5,8,11,14,17,20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosan-6-yl]-2-methylpropyl rel-(2S,3R)-2-acetamido-3-hydroxy-4-methylpentanoate]. However, YM254890 abrogated the PAR(1)-mediated Ca(2+) signal. PAR(4)-mediated NO production was substantially inhibited by the inhibitors of phosphotidylinositol-3 kinase (PI3K) and Akt, as well as by the dominant negative mutant of Akt. The PAR(1)-mediated NO production was relatively resistant to inhibitors of PI3K. An immunoblot analysis revealed a transient increase in the phosphorylation of Akt and endothelial NO synthase following the PAR(4) stimulation. In conclusion, PAR(1) and PAR(4) engage distinct signal transduction mechanisms to activate NO production in vascular endothelial cells. PAR(4) preferably activates Galpha(i/o) and induced NO production in a manner mostly independent of Ca(2+) but dependent on the PI3K/Akt pathway, whereas PAR(1) activates both the Ca(2+)-dependent and -independent mechanisms.
蛋白酶激活受体1和4（PAR(1)和PAR(4)）是介导凝血酶诱导内皮细胞产生一氧化氮（NO）的主要受体。它们激活后的细胞内信号传导仍有待阐明。本研究首次提供证据表明，PAR(1)和PAR(4)在产生NO方面对Ca(2+)的需求不同。激活肽（PAR(1)-AP）激活PAR(1)可提高猪主动脉和人脐静脉内皮细胞的胞质Ca(2+)浓度（Ca(2+)）并激活NO生成，而对牛主动脉内皮细胞影响不大。PAR(4)-AP激活PAR(4)在三种类型的内皮细胞中均能持续诱导NO生成，且Ca(2+)无明显升高。1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸（BAPTA）可显著抑制PAR(1)介导的NO生成，而PAR(4)介导的NO生成则不受影响。百日咳毒素可显著抑制PAR(1)和PAR(4)激活后的NO生成，但对Gα(q/11)抑制剂YM254890 [（1R）-1-[(3S,6S,9S,12S,18R,21S,22R)-21-乙酰氨基-18-苄基-3-[(1R)-1-甲氧基乙基]-4,9,10,12,16,22-六甲基-15-亚甲基-2,5,8,11,14,17,20-七氧代-1,19-二氧杂-4,7,10,13,16-五氮杂环二十二碳-6-基]-2-甲基丙基rel-(2S,3R)-2-乙酰氨基-3-羟基-4-甲基戊酸酯]有抗性。然而，YM254890可消除PAR(1)介导的Ca(2+)信号。PAR(4)介导的NO生成受到磷脂酰肌醇-3激酶（PI3K）和Akt抑制剂以及Akt显性负突变体的显著抑制。PAR(1)介导的NO生成对PI3K抑制剂相对不敏感。免疫印迹分析显示，PAR(4)刺激后Akt和内皮型NO合酶的磷酸化短暂增加。总之，PAR(1)和PAR(4)通过不同的信号转导机制激活血管内皮细胞中的NO生成。PAR(4)优先激活Gα(i/o)并以主要独立于Ca(2+)但依赖于PI3K/Akt途径的方式诱导NO生成，而PAR(1)则激活Ca(2+)依赖性和非依赖性机制。
