15-Hydroxyeicosatetraenoic acid (15-HETE) receptors. Involvement in the 15-HETE-induced stimulation of the cryptic 5-lipoxygenase in PT-18 mast/basophil cells.
The mechanisms of stimulation of the inactive 5-lipoxygenase in mast/basophil PT-18 cells by microM 15-hydroxyeicosatetraenoic acid (15-HETE) was investigated. Treatment of PT-18 cells with pM 15-[3H]HETE at 4 degrees for 3 h resulted in the cell association of 10% of the ligand: two-thirds was incorporated into cellular lipids and a third was bound to specific 15-HETE cellular binding sites. Binding data analysis indicated a single class of 15-HETE binding sites with a Kd of 162 nM and a Bmax of 7.1 x 10(5) sites/cell. Unlabeled 15-HETE, 12-HETE, and 5,15-diHETE inhibited the binding of 15-[3H]HETE to cells, whereas LTB4 and PGF2 alpha were relatively ineffective. 2.4 microM 15-HETE (unlabeled) prevented 50% 15-[3H]HETE incorporation. Examination of the effects of 15-HETE methyl ester, 12-HETE, 5,15-diHETE, and pertussis toxin on both the 15-HETE-induced 5-lipoxygenase activation and 15-HETE cell association processes indicated a preponderant correlation of this activation process with specific 15-HETE binding rather than 15-HETE incorporation into phospholipids. In addition, 5,15-diHETE itself stimulated the inactive 5-lipoxygenase and eight times more [3H]diHETE was bound to cells than became incorporated into cellular lipids. The results support the involvement of low affinity 15-HETE receptors, rather than 15-HETE incorporation into cellular lipids, in the 15-HETE-induced stimulation of the 5-lipoxygenase in PT-18 cells.
Vonakis
B M
BM
Department of Biochemistry and Molecular Biology, George Washington University Medical Center, Washington, DC 20037.
Vanderhoek
J Y
JY
D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D001491:Basophils / Q000187:drug effects / Q000201:enzymology*; D001667:Binding, Competitive; D002460:Cell Line; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism / Q000494:pharmacology*; D007536:Isomerism; D007700:Kinetics; D008407:Mast Cells / Q000187:drug effects / Q000201:enzymology*; D037342:Pertussis Toxin; D010743:Phospholipids / Q000096:biosynthesis; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D018055:Receptors, Eicosanoid; D013329:Structure-Activity Relationship; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
C078191:15-hydroxyeicosatetraenoic acid receptor; D006893:Hydroxyeicosatetraenoic Acids; D010743:Phospholipids; D011956:Receptors, Cell Surface; D018055:Receptors, Eicosanoid; D010566:Virulence Factors, Bordetella; C025984:15-hydroxy-5,8,11,13-eicosatetraenoic acid; C034922:5,15-dihydroxy-6,8,11,13-eicosatetraenoic acid; D001094:Arachidonate 5-Lipoxygenase; D037342:Pertussis Toxin