An analysis of the relationship between 5-lipoxygenase product generation and the secretion of preformed mediators from mouse bone marrow-derived mast cells.
The quantitative relationships between the secretion of a granule-associated mediator, beta-hexosaminidase, and the oxidative metabolism of arachidonic acid by the 5-lipoxygenase pathway were analyzed for a homogeneous population of T cell-dependent, bone marrow-derived, murine mast cells. The mast cells were either sensitized with a monoclonal IgE and challenged with specific antigen, or to bypass a transmembrane signal, were stimulated with calcium ionophore A23187. The released products of the 5-lipoxygenase pathway were quantitated by integrated ultraviolet absorbance after resolution by reverse phase-high performance liquid chromatography in the case of 5-hydroxy-eicosatetraenoic acid (5-HETE), and by separate radioimmunoassays for leukotriene C4 (LTC4) and leukotriene B4 (LTB4). The activation-release response of the cells was perturbated by the introduction of three pharmacologic agents, each directed to different steps in the 5-lipoxygenase pathway of arachidonic acid metabolism, and the action of each agent was determined for separate cell samples while present and after its removal by washing. 5,6-Dehydroarachidonic acid (5,6-DHA), an irreversible inhibitor of 5-lipoxygenase, prevented formation of 5-HETE from exogenous [14C]arachidonic acid and from membrane-derived arachidonic acid in a dose-related fashion when sensitized mast cells, preincubated with drug, were washed before antigen activation. Release of 5-HETE, LTC4, and LTB4 was inhibited by 5,6-DHA in a corresponding dose-related fashion, with a minimal preincubation period of 1 to 5 min before the cells were washed and subjected to antigen-dependent activation. In contrast, the inhibitory effect of 5,6-DHA on beta-hexosaminidase release was lost after three washes and was not evident after one wash unless the preincubation period was extended to 15 min. The capacity of 5,6-DHA to prevent leukotriene generation without altering beta-hexosaminidase release was also observed with ionophore-activated mast cells. Preincubation of sensitized cells with diethylcarbamazine (DEC), followed by a wash before antigen-dependent activation, produced inhibition of leukotriene generation, no effect on beta-hexosaminidase release, and augmentation of 5-HETE release at the maximum dose studied; thus, DEC interrupts the pathway distal to the formation of 5-hydroperoxy-eicosatetraenoic acid (5-HPETE) from arachidonic acid by 5-lipoxygenase. Preincubation of sensitized cells with incremental amounts of the prostacyclin analog U-60,257, followed by washing and antigen challenge, inhibited LTC4 release without altering the release of LTB4 or beta-hexosaminidase.(ABSTRACT TRUNCATED AT 400 WORDS)
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001091:Arachidonate Lipoxygenases; D001095:Arachidonic Acids / Q000037:antagonists & inhibitors; D001854:Bone Marrow Cells; D004049:Diethylcarbamazine / Q000494:pharmacology; D006596:Hexosaminidases / Q000378:metabolism*; D006893:Hydroxyeicosatetraenoic Acids; D007073:Immunoglobulin E / Q000502:physiology; D007975:Leukotriene B4 / Q000037:antagonists & inhibitors; D008084:Lipoxygenase / Q000378:metabolism*; D016859:Lipoxygenase Inhibitors; D008297:Male; D008407:Mast Cells / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D013189:SRS-A / Q000037:antagonists & inhibitors; D001619:beta-N-Acetylhexosaminidases
D001095:Arachidonic Acids; D006893:Hydroxyeicosatetraenoic Acids; D016859:Lipoxygenase Inhibitors; D013189:SRS-A; D007975:Leukotriene B4; D007073:Immunoglobulin E; C022022:5-hydroxy-6,8,11,14-eicosatetraenoic acid; C025421:eicosa-8,11,14-trien-5-ynoic acid; D001091:Arachidonate Lipoxygenases; D008084:Lipoxygenase; D006596:Hexosaminidases; D001619:beta-N-Acetylhexosaminidases; D015126:8,11,14-Eicosatrienoic Acid; D004049:Diethylcarbamazine