TY - JOUR
T1 - Arachidonic acid metabolism in the human mast cell line HMC-1
T2 - 5-lipoxygenase gene expression and biosynthesis of thromboxane
AU - Macchia, Luigi
AU - Hamberg, Mats
AU - Kumlin, Maria
AU - Butterfield, Joseph H.
AU - Haeggström, Jesper Z.
N1 - Funding Information:
The authorsa re greatlyi ndebtedt o Pedro Otavio de CamposL ima, MD, for valuabled iscussiona nd advice. We also thankD r. Ying-Yi Zhangf or the generougs ift of 5-LO antiseruman dlate Prof. H~tkanP erssonfo r providing an a-actinc DNA. This work was supportedb y the SwedishM edicalRe-searchC ouncil( grantsn o 03X-217,0 3X-10350a, nd 03X-05170)a nd Petrusa nd AugustaH edlundss tiftelse. L.M. was supportedb y the Italian National Research Council( CNR, P.F. FATMA, S.P. 2, grantn o 9300751.41; grant no. 93.04492.CT04a; nd grant no. AI93.00135.04) and by ItalienskaK ulturinstitute"tC .M. Lerici" of Stockholm.
PY - 1995/6/27
Y1 - 1995/6/27
N2 - 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/107 cells) followed by leukotriene (LT) C4 (245 pmol/107 cells) and 11-trans-LTC4 (74 pmol/107 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, PGF2α, and PGD2, whereas 6-keto-PGF1α, reflecting prostacyclin formation, could not be detected. Futhermore, 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/107 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.
AB - 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/107 cells) followed by leukotriene (LT) C4 (245 pmol/107 cells) and 11-trans-LTC4 (74 pmol/107 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, PGF2α, and PGD2, whereas 6-keto-PGF1α, reflecting prostacyclin formation, could not be detected. Futhermore, 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/107 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.
KW - 5-Lipoxygenase
KW - Arachidonic acid metabolism
KW - Gene expression
KW - Icosanoid
KW - Mast cell
KW - Thromboxane
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U2 - 10.1016/0005-2760(95)00048-H
DO - 10.1016/0005-2760(95)00048-H
M3 - Article
C2 - 7599181
AN - SCOPUS:0029033682
SN - 0005-2760
VL - 1257
SP - 58
EP - 74
JO - Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism
JF - Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism
IS - 1
ER -