KINETICS OF PROSTACYCLIN SYNTHESIS IN PGHS-1-OVEREXPRESSED ENDOTHELIAL-CELLS

The availability of a human endothelial cell overexpressed with prosta glandin H synthase-1 (PGHS-1) by retrovirus-mediated gene transfer mad e it possible to quantify the kinetics of prostacyclin [prostaglandin (PG) I-2] synthesis and PGHS-1 turnover. Prostacyclin synthesis in res ponse to arachidonate (AA) and ionophore A-23187 fit a single exponent ial kinetics. The rate constants for AA- and ionophore-treated cells w ere 0.064 min(-1) [half-life (t(1/2)) of 11 min] and 0.032 min(-1) (t( 1/2) = 22 min), respectively. The rate constant of PGI(2) synthesis fr om PGH(2) was 0.13 min(-1). Using kinetic analysis coupled with comput er modeling, the PGHS-1 half-life was determined to be 10.8 min. PGI(2 ) production under successive treatments with AA or ionophore was redu ced by only similar to 30% after each treatment. The decline of PGI(2) synthesis corresponded to the reduction of PGHS-1 mass. The half-life of PGI(2) synthesis from this analysis was at least an order of magni tude higher than that estimated from the single-dose experiment. These findings indicate that similar to 30% of PGHS-1 was degraded during e ach catalysis-induced autoinactivation and that the extent and duratio n of PGI(2) synthesis are governed by the level of PGHS-1 mass.