On the expression and regulation of 5-lipoxygenase in human lymphocytes.
The expression of arachidonate 5-lipoxygenase (arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) and the 5-lipoxygenase-activating protein (FLAP) genes in human tonsillar B cells and lymphoblastoid B-cell lines was demonstrated at the transcriptional level by reverse transcription-PCR analysis. Also, five lymphoblastoid T-cell lines were investigated and found to express the FLAP gene but not the 5-lipoxygenase gene, suggesting that the transcriptional regulation of these two genes is different. Western blot analysis of the cytosolic proteins from a lymphoblastoid B-cell line with an antiserum raised against purified human leukocyte 5-lipoxygenase revealed an immunoreactive band that comigrated with recombinant human 5-lipoxygenase. Intact B cells produced very low amounts of leukotriene B4 and 5-hydroxyeicosatetraenoic acid upon stimulation with the calcium ionophore A23187 and arachidonic acid, in comparison to the amounts formed by sonicates of these cells. However, preincubation of intact lymphoblastoid B cells with the glutathione-depleting agents azodicarboxylic acid bis(dimethylamide) or 1-chloro-2,4-dinitrobenzene prior to the addition of the calcium ionophore A23187 and arachidonic acid led to similar amounts of leukotriene B4 as were formed by sonicated cells. In contrast, the glutathione synthesis inhibitor buthionine sulfoximine diminished the cellular level of glutathione by greater than 90% but did not influence the production of leukotriene B4 or 5-hydroxyeicosatetraenoic acid in intact cells. These results demonstrate that certain drugs affecting the redox status can stimulate the cryptic 5-lipoxygenase activity in intact lymphoblastoid B cells but that the mechanism of this activation is unclear and appears not to be directly related to intracellular glutathione levels.
Jakobsson
P J
PJ
Department of Physiological Chemistry, Karolinska Institutet, Stockholm, Sweden.
Steinhilber
D
D
Odlander
B
B
Rådmark
O
O
Claesson
H E
HE
Samuelsson
B
B
D058927:5-Lipoxygenase-Activating Proteins; D001094:Arachidonate 5-Lipoxygenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001402:B-Lymphocytes / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology; D001483:Base Sequence; D000001:Calcimycin / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D003958:Diamide / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007975:Leukotriene B4 / Q000378:metabolism; D008213:Lymphocyte Activation; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D014066:Palatine Tonsil / Q000201:enzymology; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013601:T-Lymphocytes / Q000201:enzymology*
D058927:5-Lipoxygenase-Activating Proteins; C548592:ALOX5AP protein, human; D002352:Carrier Proteins; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D003958:Diamide; D007975:Leukotriene B4; D000001:Calcimycin; D001094:Arachidonate 5-Lipoxygenase; D005978:Glutathione