Figure 1.

Thrombin induces COX-2 expression and PGE₂ generation in human cardiomyocytes. (A) Human cardiomyocytes were incubated with various concentrations of thrombin for the indicated time intervals. The cell lysates were subjected to Western blot analysis. (B) The cells were incubated with thrombin (3 U·mL⁻¹) for the indicated time intervals. The levels of COX-2 mRNA were analysed by RT-PCR and real-time PCR. Human cardiomyocytes were co-transfected with a COX-2 promoter luciferase reporter gene with a β-galactosidase plasmid and then incubated with thrombin (3 U·mL⁻¹) for indicated time intervals (black bar). The COX-2 luciferase activity was detected and normalized to β-galactosidase activity. (C) The conditioned media were collected for PGE₂ generation analysis by an EIA kit. The basal value of PGE₂ is ranged from 2–4 ng·mL⁻¹. (D–E) Human cardiomyocytes were pretreated with actinomycin D (Act. D) or cycloheximide CHI for 1 h and then incubated with thrombin (3 U·mL⁻¹) for (D) 16 h or (E) 4 h. The COX-2 protein and mRNA levels was determined by Western blot (D) and real-time PCR (E). Data are expressed as mean ± SEM (n = 3). *P < 0.05; ^#P < 0.01, significantly different from cells incubated with vehicle alone (A–C) or thrombin alone (D–E).