Ca 2+ ionophore A23187-stimulated secretion of azurophil granules in human polymorphonuclear leukocytes is largely mediated by endogenously formed leukotriene B 4

Abstract

The mode of action of the new leukotriene synthesis inhibitor BAY X1005 (( R)-2-[4-(quinolin-2-yl-methoxy)phenyl]-2-cyclopentyl acetic acid) and structurally-related quinoline derivatives is reflected by the binding to a high-affinity binding site presumably identical to FLAP (five lipoxygenase activating protein). In addition to FLAP, we have identified a second BAY X1005 (low-affinity) binding site localized in the granule fraction of human PMNL (polymorphonuclear leukocytes). Based on the hypothesis that the corresponding target protein might be involved in the regulation of granule release, the influence of the leukotriene synthesis inhibitors BAY X1005 and MK-886 and the direct 5-LOX (5-lipoxygenase, EC 1.13.11.34) inhibitor A-64077 on the A23187- and fMLP ( N-formyl-methionyl-leucyl-phenylalanin)-stimulated release of β-glucuronidase (as a marker for azurophil granules) and vitamin B 12-binding protein (as a marker for specific granules) was investigated. In contrast to MK-886, neither BAY X1005 nor A-64077 significantly affected fMLP-stimulated granule release. This was also true for the A23187-stimulated release of specific granules; however, under the same conditions the A23187-stimulated release of azurophil granules was almost totally inhibited by all three compounds. No obvious relationship between the corresponding ic 50 values and the ability of these compounds to compete for BAY X1005 binding at the low-affinity binding site existed. Instead, by extending these studies to additional inhibitors, a correlation between the ic 50 values for inhibition of A23187-stimulated (i) β-glucuronidase release and (ii) LTB 4 (leukotriene B 4) synthesis was found ( r = 0.969, N = 7). This relationship was independent of the mode of action of the compounds, namely direct 5-LOX inhibition or indirect 5-LOX inhibition mediated via binding to FLAP. These results suggest that 5-LOX metabolites may be involved in A23187-stimulated azurophil granule release. Of the two main biologically active 5-LOX metabolites synthesized under these conditions (LTB 4 and 5-hydroxyeicosatetraenoic acid), only LTB 4 stimulated β-glucuronidase release to nearly the same extent as A23187. In addition, this metabolite significantly enhanced A23187-stimulated β-glucuronidase release, but only at A23187 concentrations (⩾ 0.25 μmol L ) which by themselves were not sufficient to trigger LTB 4 formation. Moreover, the inhibition of A23187-stimulated β-glucuronidase release by BAY X1005 or A-64077 was totally reversed by the addition of LTB 4. Taken together, these results indicate that: (i) the BAY X1005 low-affinity binding site does not play a crucial role in the granule release process; (ii) compounds interfering with 5-LOX activity inhibit the release of azurophil granules upon A23187 stimulation; and (iii) this effect is largely due to the inhibition of endogenously synthesized LTB 4.