MECHANISM OF SELECTIVE-INHIBITION OF HUMAN PROSTAGLANDIN G H SYNTHASE-1 AND SYNTHASE-2 IN INTACT-CELLS/

Selective inhibitors of prostaglandin synthase-2 (PGHS-2) possess pote nt anti-inflammatory, antipyretic, and analgesic properties but demons trate reduced side-effects (e.g. gastrotoxicity) when compared with no nselective inhibitors of PGHS-1 and -2. We investigated the mechanism of the differential inhibition of human PGHS-1 (hPGHS-1) and -2 (hPGHS -2) in intact cells by nonsteroidal anti-inflammatory drugs (NSAIDs) a nd examined factors that contribute to the increased potency of PGHS i nhibitors observed in intact cells versus cell-free systems. In intact Chinese hamster ovary (CHO) cell lines stably expressing the hPGHS is ozymes, both PGHS isoforms exhibited the same affinity for arachidonic acid. Exogenous and endogenous arachidonic acid were used as substrat es by both CHO [hPGHS-1] and CHO [hPGHS 2] cell lines. However, differ ences were observed in the ability of the hPGHS isoforms to utilize en dogenous arachidonic acid released intracellularly following calcium i onophore stimulation or released by human cytosolic phospholipase A(2) transiently expressed in the cells. Cell-based screening of PGHS inhi bitors demonstrated that the selectivities and potencies of PGHS inhib itors determined using intact cells are affected by substrate concentr ation and differ from that determined in cell free microsomal or purif ied enzyme preparations of PGHS isozymes. The mechanism of inhibition of PGHS isozymes by NSAIDs in intact cells involved a difference in th eir time-dependent inhibition. Indomethacin displayed time-dependent i nhibition of cellular hPGHS-1 and -2. In contrast, the selective PGHS- 2 inhibitor NS-398 exhibited time-independent inhibition of hPGHS-1 bu t time-dependent inhibition of hPGHS-2 in intact cells. Reversible inh ibition of cellular CHO [hPGHS-1] and CHO [hPGHS-2] was observed with the nonselective NSAIDs ibuprofen and indomethacin, whereas inhibition by the selective PGHS 2 inhibitor DuP-697 was reversible against hPGH S-1 but irreversible against hPGHS-2.