The Mechanism of Thrombin Induced Prostaglandin I₂ (PGI₂) Enhancement in Human Umbilical Vein Endothelial Cells with Reference to the Gene Expression of PGI₂ Related Enzymes and Calcium Ion Kinetics

Abstract

Signal induction and molecular mechanisms of the thrombin induced prostaglandin I₂ (PGI₂) synthesis in human umbilical vein endothelial cells (HUVEC) were investigated in association with the intra Ca²⁺ and the gene expression of prostaglandin H₂ synthase (PGHS) and phospholipase A₂ (PLA₂) using the method of competitive polymerase chain reaction (PCR). Thrombin enhanced the synthesis of PGI₂ by time dependent manner. Thrombin stimulation caused an elevation of the cytosolic Ca²⁺, which in turn stimulate phospholipase A₂ (PLA₂), resulting in arachidonic acid cleavage from membrane phospholipids and its subsequent conversion into PGI₂ through PGHS pathway. The elevated cytosolic Ca²⁺ resulted from mainly the influx of extracellular Ca²⁺. Compatible to former reports, PGHS-1 mRNA was constitutively expressed, while PGHS-2 and PLA₂ mRNA was not. With the stimulation of thrombin, PLA₂ mRNA increased to 9-fold in 15 minutes, PGHS-1 mRNA to 3.4-fold in 180 minutes, PGHS-2 mRNA to 38-fold in 60 minutes. These results suggest that PGHS-1 is not only the constitutive enzyme but also the inducible enzyme and expression of PLA₂, PGHS-1, PGHS-2 mRNA cause to PGI₂ generation.