Arachidonic acid metabolism in the human mast cell line HMC-1: 5-lipoxygenase gene expression and biosynthesis of thromboxane.
Metabolism of arachidonic acid was studied in the unique human mast cell line HMC-1. By HPLC and/or gas chromatography mass spectrometry (GC-MS), 19 oxygenated metabolites were identified, including monohydroxy acids, leukotrienes, prostaglandins, and thromboxane. Intact cells incubated with the calcium ionophore A23187 and arachidonic acid expressed 5-lipoxygenase activity and produced 5-hydroxyeicosatetraenoic acid (5-HETE) as the major metabolite (745 pmol/10(7) cells) followed by leukotriene (LT) C4 (245 pmol/10(7) cells) and 11-trans-LTC4 (74 pmol/10(7) cells). Low but clearly detectable levels of LTB4 were also observed. The total amounts of 5-LO products were comparable to those obtained with RBL-1 cells and corresponded to approx. 30% of the levels obtained with isolated human polymorphonuclear leukocytes. Time-course experiments revealed that HMC-1 cells contained the enzyme activities required to metabolize LTC4 into LTD4 and further into LTE4. The profile of prostanoids included, prostaglandin (PG) E2, PGF2 alpha, and PGD2, whereas 6-keto-PGF1 alpha, reflecting prostacyclin formation, could not be detected. Furthermore, we were able to unambiguously establish that HMC-1 cells could produce substantial amounts of thromboxane (TX) A2, measured as TXB2 (0.1-2.2 nmol/10(7) cells). Generation of TXA2 in such quantities, exceeding those of LTC4, suggests that mast cells may be an important source of thromboxane and points to a possible role for these cells in hemostasis and thrombosis. After approx. 10 passages in culture, 5-lipoxygenase activity in HMC-1 cells drastically declined concomitantly with changes in growth behavior and cell morphology. Analysis by Northern and Western blots revealed that loss of 5-lipoxygenase activity correlated well with a reduced 5-lipoxygenase gene expression at both a transcriptional and translational level. This loss of enzyme activity and gene expression may be related to a genetic abnormality propagated in HMC-1 cells, i.e., a 10;16 translocation, which thus involves the chromosome containing the 5-lipoxygenase gene.
Macchia
L
L
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Hamberg
M
M
Kumlin
M
M
Butterfield
J H
JH
Haeggström
J Z
JZ
D001094:Arachidonate 5-Lipoxygenase / Q000235:genetics* / Q000378:metabolism; D001095:Arachidonic Acids / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology; D002455:Cell Division; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D002999:Clone Cells; D003470:Culture Media; D004305:Dose-Response Relationship, Drug; D015777:Eicosanoids / Q000097:blood; D015870:Gene Expression; D006801:Humans; D007213:Indomethacin / Q000494:pharmacology; D007700:Kinetics; D015289:Leukotrienes / Q000096:biosynthesis; D013058:Mass Spectrometry; D008407:Mast Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D012643:Selenium; D013931:Thromboxanes / Q000096:biosynthesis*
D001095:Arachidonic Acids; D003470:Culture Media; D015777:Eicosanoids; D015289:Leukotrienes; D013931:Thromboxanes; D000001:Calcimycin; D001094:Arachidonate 5-Lipoxygenase; D012643:Selenium; D007213:Indomethacin