ROLE OF 5-LIPOXYGENASE-ACTIVATING PROTEIN IN THE REGULATION OF 5-LIPOXYGENASE ACTIVITY IN HUMAN NEUTROPHILS

Using intact and fractionated human polymor-phonuclear leukocytes (PMN L), we provide evidence that the enantioselective leukotriene synthesi s inhibitor (LSI) BAY X 1005, (R)-2-[4-(quinolin-2-yl-methoxy)phenyl]- 2 cyclopentyl acetic acid binds specifically to a high-affinity bindin g site, which is most likely identical to FLAP. BAY X 1005 blocks the translocation of 5-lipoxygenase (5-LOX) in PMNL stimulated by the calc ium ionophore A23187 or chemotactic stimuli such as PAF, C5a or fMLP a s does MK-886. In contrast to the direct 5-LOX inhibitors (LOI) A-6407 7 and AA-861, the degree of leukotriene synthesis inhibition declines with increasing duration of A23187-induced leukocyte activation in the presence of BAY X 1005 and MK-886. Kinetic studies performed with BAY X 1005 showed that this effect was not accompanied by a significant t ranslocation of 5-LOX from the cytosol to the microsomal fraction. Bec ause FLAP has been implicated in the transfer of arachidonic acid to 5 -LOX and A23187 is a potent activator of leukocyte phospholipase Az, w e hypothesized that the observed loss of leukotriene synthesis inhibit ion may be due to competition of BAY X 1005 binding by endogenously re leased arachidonic acid. Accordingly, binding of BAY X 1005 to FLAP in intact and fractionated cells is dose-dependently inhibited by arachi donic acid and other unsaturated long-chain fatty acids, but not by sa turated fatty acids. Therefore, we conclude that BAY X 1005 or MK-886 inhibit leukotriene biosynthesis by binding to FLAP, thereby preventin g 5-LOX translocation and substrate transfer to the enzyme.