Sato T, Sawada S, Tsuda Y, Komatsu S, Akamatsu N, Kono Y, Higaki T, Imamura H, Tada Y, Yamasaki S, Tamagaki T, Nakagawa K, Tsuji H, Nakagawa M
Second Department of Medicine, Kyoto Prefectural University of Medicine, Japan.
J Pharmacol Toxicol Methods. 1999 Aug;41(4):173-82. doi: 10.1016/s1056-8719(99)00039-8.
This study was designed to evaluate the effect of thrombin on prostacyclin (PGI2) production in cultured human vascular endothelial cells in association with intracellular Ca2+ and with the gene expression of prostaglandin H2 synthase (PGHS) and phospholipase A2 (PLA2) using competitive polymerase chain reaction. Thrombin enhanced the PGI2 synthesis dependent with time. Additionally, thrombin increased the intracellular Ca2+, which stimulates PLA2, resulting in arachidonic acid cleavage from membrane phospholipids and its subsequent conversion into PGI2 through the PGHS pathway. The elevation of intracellular Ca2+ was a result of Ca2+ influx and Ca2+ release from its intracellular storage sites. In this study, PGHS-1 mRNA was constitutively expressed, whereas PGHS-2 mRNA was not. With the stimulation of thrombin, cytosolic PLA2 (cPLA2) mRNA increased 9-fold at 15 min, PGHS-1 mRNA increased 3.4-fold at 180 min, and PGHS-2 mRNA increased 38-fold at 60 min. These results suggest that the elevation of intracellular Ca2+ and the expression of cPLA2, PGHS-1, and PGHS-2 mRNA cause PGI2 generation.
本研究旨在利用竞争性聚合酶链反应，评估凝血酶对培养的人血管内皮细胞中前列环素（PGI2）生成的影响，并探讨其与细胞内Ca2+以及前列腺素H2合酶（PGHS）和磷脂酶A2（PLA2）基因表达的关系。凝血酶可随时间增强PGI2的合成。此外，凝血酶可增加细胞内Ca2+，后者刺激PLA2，导致膜磷脂中的花生四烯酸裂解，并随后通过PGHS途径转化为PGI2。细胞内Ca2+的升高是Ca2+内流及其从细胞内储存位点释放的结果。在本研究中，PGHS-1 mRNA呈组成性表达，而PGHS-2 mRNA未表达。在凝血酶刺激下，胞质型PLA2（cPLA2）mRNA在15分钟时增加9倍，PGHS-1 mRNA在180分钟时增加3.4倍，PGHS-2 mRNA在60分钟时增加38倍。这些结果表明，细胞内Ca2+的升高以及cPLA2、PGHS-1和PGHS-2 mRNA的表达导致了PGI2的生成。
