Kinetics of prostacyclin synthesis in PGHS-1-overexpressed endothelial cells

The availability of a human endothelial cell overexpressed with prostaglandin H synthase-1 (PGHS-1) by retrovirus-mediated gene transfer made it possible to quantify the kinetics of prostacyclin [prostaglandin (PG)I2] synthesis and PGHS-1 turnover. Prostacyclin synthesis in response to arachidonate (AA) and ionophore A-23187 fit a single exponential kinetics. The rate constants for AA- and ionophore-treated cells were 0.064 min-1 [half-life (t1/2) of 11 min] and 0.032 min-1 (t1/2 = 22 min), respectively. The rate constant of PGI2 synthesis from PGH2 was 0.13 min-1. Using kinetic analysis coupled with computer modeling, the PGHS-1 half-life was determined to be 10.8 min. PGI2 production under successive treatments with AA or ionophore was reduced by only approximately 30% after each treatment. The decline of PGI2 synthesis corresponded to the reduction of PGHS-1 mass. The half-life of PGI2 synthesis from this analysis was at least an order of magnitude higher than that estimated from the single-dose experiment. These findings indicate that approximately 30% of PGHS-1 was degraded during each catalysis-induced autoinactivation and that the extent and duration of PGI2 synthesis are governed by the level of PGHS-1 mass.