The mechanism of thrombin-induced prostacyclin synthesis in human endothelial cells with reference to the gene transcription of prostacyclin-related enzymes and Ca2+ kinetics.
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.
Sato
T
T
Second Department of Medicine, Kyoto Prefectural University of Medicine, Japan.
Sawada
S
S
Tsuda
Y
Y
Komatsu
S
S
Akamatsu
N
N
Kono
Y
Y
Higaki
T
T
Imamura
H
H
Tada
Y
Y
Yamasaki
S
S
Tamagaki
T
T
Nakagawa
K
K
Tsuji
H
H
Nakagawa
M
M
D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D011464:Epoprostenol / Q000096:biosynthesis*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000032:analysis; D010741:Phospholipases A / Q000235:genetics*; D054467:Phospholipases A2; D016133:Polymerase Chain Reaction; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology*; D013997:Time Factors; D014158:Transcription, Genetic / Q000235:genetics*; D014471:Umbilical Veins / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism
D012333:RNA, Messenger; D015544:Inositol 1,4,5-Trisphosphate; D011464:Epoprostenol; D011451:Prostaglandin-Endoperoxide Synthases; D010741:Phospholipases A; D054467:Phospholipases A2; D013917:Thrombin; D002118:Calcium