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) C-4 (245 pmol/10(7) cells) and Il-trans-LTC(4) (74 pmol/10(7) cells). Low but clearly detectable levels of LTB(4) were also observed. The total amounts of 5-LO products were comparable to those obtained with RBL-I 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 LTC(4) into LTD(4) and further into LTE(4). The profile of prostanoids included, prostaglandin (PG) E(2), PGF(2 alpha), and PGD(2), whereas 6-keto-PGF(1 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) A(2), measured as TXB(2) (0.1-2.2 nmol/10(7) cells). Generation of TXA(2) in such quantities, exceeding those of LTC(4), 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.