Abstract
Recently, we have shown that ionophore activation of human leukocytes results in leukotriene synthesis and a translocation of 5-lipoxygenase from the cytosol to cellular membrane. This membrane translocation was postulated to be an important early activation step for the enzyme. 3-[1-(p-Chlorobenzyl)-5-(isopropyl)-3-tert-butylthioindol-2-yl]-2, 2- dimethylpropanoic acid (MK886) is a potent and specific inhibitor of leukotriene biosynthesis in vivo and in intact cells, but has no direct effect on 5-lipoxygenase activity in cell-free systems. In this report, we show that MK886 can both prevent and reverse the membrane translocation of 5-lipoxygenase, in conjunction with the inhibition of leukotriene synthesis. Similar compounds of the indole class could also inhibit the membrane translocation of 5-lipoxygenase in a rank order of potency that correlated with their potencies for leukotriene synthesis inhibition. In contrast L-656,224, a direct 5-lipoxygenase inhibitor, had no effect on the translocation of the enzyme. Attempts to demonstrate the effects of MK886 on the association of 5-lipoxygenase with membrane in cell-free preparations failed due to a nonspecific Ca2+-dependent sedimentation of the enzyme. The mechanism of action of MK-886 is therefore to block translocation, prevent subsequent activation of 5-lipoxygenase, and hence block cellular leukotriene biosynthesis.
Highlights
We have shown that ionophore activation of human leukocytes results in leukotriene synthesis and a translocation of 5-lipoxygenase from the cytosol to cellular membrane
The exact mechanism for 5-lipoxygenase activation remains unclear, we recently reported that treatment of human leukocytes with ionophore A23187 results in leukotriene synthesis accompanied by the loss of 5-lipoxygenase protein and activity from the cell cytosol (100,000 X g supernatant) and the accumulation of inactive enzyme in cell membranes (100,000 x g pellet) [11]
Leukotriene synthesis was measured, and the cells were homogenized for the preparation of 100,000 X g supernatants and pellets
Summary
Human Leukocyte Suspensions-Human leukocyte concentrates (buffy coat) were obtained from local blood collection centers. Incubation Conditions-For most experiments, 20-ml aliquots of the cell suspensions were prepared, and inhibitors were added as a stock solution (0.2 or 1.0 mM) in ethanol to give the desired final concentration. After a IO-min incubation with ionophore, cell samples were placed on ice, and 800 ~1 of a solution (100 mM) of EDTA was added (4 mM final concentration). The cells were homogenized by sonication for the analysis of 5-lipoxygenase activity by direct enzyme assay and immunoreactive 5-lipoxygenase protein by the immunoblot technique. Ot&r Methods-Procedures for the preparation of cell homogenates and subcellular fractions, the determination of cellular leukotriene synthesis, the assay for 5-lipoxygenase activity, and general protein assay have been thoroughly described elsewhere [11]. Statiitical significance of the differences between sample populations was determined by the paired t test, with significance accepted at p < 0.05
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