title: Calcium ionophore and chemotactic peptide stimulation of peptidoleukotriene synthesis in DMSO-differentiated HL60 cells.
abstract: The human promyelocytic leukemia cell line HL60 can be differentiated to mature granulocytes upon exposure to DMSO (1.3%, 6 days). The ability of these cells to metabolize arachidonic acid via the 5-lipoxygenase pathway to form 5-HETE, LTB4, and 5,12-diHETEs, has been previously documented. However, the production of peptidoleukotrienes by DMSO-differentiated HL60 cells has not been previously reported. Arachidonic acid metabolites produced via 5-lipoxygenase were identified by reverse-phase, high-performance liquid chromatography, immunoreactivity specific for peptidoleukotriene, glutamyl transpeptidase transformation, characteristic UV spectra, and GC mass spectra. Leukotriene synthesis in the DMSO-differentiated HL60 cell is maximal at 5 min when stimulated with the calcium ioniphore, A23187 (1 microM), in the presence of calcium. These cells produce 12.94 +/- 1.8 ng/10(6) cells of LTC4 and 3.8 +/- 0.4 ng/10(6) cells of LTB4. LTC4 and LTB4 are also synthesized in the undifferentiated cell when stimulated with 1 microM A23187 and 1 mM Ca2+, but in much smaller quantities, i.e., 1.91 +/- 0.42 ng/10(6) cells of LTC4 and 0.41 ng +/- 0.06/10(6) cells of LTB4. The synthetic chemotactic peptide, f-Met-Leu-Phe, also elicits formation of LTC4 and LTB4 in a dose-dependent manner in the presence of exogenously added calcium. Maximal stimulation of DMSO-differentiated cells with f-Met-Leu-Phe produces 2.5 +/- 0.2 ng of LTC4 and 1.45 +/- 0.2 ng of LTB4 per 10(6) cells. The observation that DMSO-differentiated HL60 cells produce LTC4, as well as other 5-lipoxygenase products, increases the utility of this cell line for unraveling the regulation of leukotriene biosynthesis by granulocytes.
mesh_terms: D001095:Arachidonic Acids / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000378:metabolism* / Q000473:pathology; D007975:Leukotriene B4 / Q000096:biosynthesis*; D008297:Male; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils; D013189:SRS-A / Q000096:biosynthesis*
chemical_list: D001095:Arachidonic Acids; D013189:SRS-A; D007975:Leukotriene B4; D000001:Calcimycin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D002118:Calcium; D004121:Dimethyl Sulfoxide