Generation of leukotrienes by human monocytes pretreated with cytochalasin B and stimulated with formyl-methionyl-leucyl-phenylalanine.
The N-formylated tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) initiated the generation of immunoreactive C-6 sulfidopeptide leukotrienes and of leukotriene B4 (LTB4) in a dose-dependent manner from monolayers of human monocytes pretreated for 10 min with 5 micrograms/ml of cytochalasin B. The EC50 for the immunoreactive C-6 sulfidopeptide leukotrienes was 10(-8) M FMLP and for immunoreactive LTB4 was 5 X 10(-8) M FMLP. The maximal response to FMLP occurred within 10 min, and the sum of the two classes of leukotrienes generated was about 1/6 that obtained from monocytes stimulated with calcium ionophore A23187. The requirement for cytochalasin B in order for FMLP, but not the calcium ionophore, to stimulate leukotriene generation is compatible with the ability of cytochalasin B to augment in other cells certain stimulus-specific transmembrane responses that are not dependent on the integrity of the cytoskeleton. Resolution by reverse phase high performance liquid chromatography of the products released from monocytes pretreated with cytochalasin B and stimulated with FMLP or calcium ionophore yielded a single peak of immunoreactive LTB4 eluting at the same retention time as the synthetic standard; immunoreactive C-6 sulfidopeptide leukotrienes eluted at the retention times of leukotriene C4 (LTC4) and leukotriene D4 (LTD4). [3H]LTB4 was not metabolically altered by monocytes pretreated with cytochalasin B and activated with FMLP in comparison with cells treated with buffer alone, whereas [3H]LTC4 was partially converted to [3H]LTD4. The leukotriene-generating response of monolayers of human monocytes pretreated with cytochalasin B to FMLP is receptor-mediated, as indicated by the inactivity of the structural analog N-acetyl-methionyl-leucyl-phenylalanine and by the capacity of the FMLP receptor antagonist carbobenzoxyphenylalanyl-methionine to inhibit the agonist action of FMLP in a dose-response fashion.
D000001:Calcimycin / Q000494:pharmacology; D002478:Cells, Cultured; D003571:Cytochalasin B / Q000494:pharmacology*; D004151:Dipeptides / Q000494:pharmacology; D004306:Dose-Response Relationship, Immunologic; D006801:Humans; D007700:Kinetics; D007975:Leukotriene B4 / Q000096:biosynthesis* / Q000378:metabolism; D009000:Monocytes / Q000276:immunology / Q000378:metabolism*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism / Q000494:pharmacology*; D010587:Phagocytosis / Q000187:drug effects; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000187:drug effects; D013997:Time Factors
D004151:Dipeptides; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; C035192:carbobenzoxyphenylalanylmethionine; D007975:Leukotriene B4; D000001:Calcimycin; D003571:Cytochalasin B; D009240:N-Formylmethionine Leucyl-Phenylalanine