A calcium-independent 5-lipoxygenase system in mast/basophil PT-18 cells.
Mammalian 5-lipoxygenase systems exist in inactive or cryptic states and have to be stimulated in order to metabolize exogenous [14C]arachidonic acid to 5-HETE and leukotrienes. In most cells, both the activation process and the 5-lipoxygenase activity are calcium-dependent. However, the cryptic 5-lipoxygenase system in the murine PT-18 mast/basophil cell line, which can be stimulated by 15-hydroxyeicosatetraenoic acid (15-HETE), is unusual. Studies with fura-2 loaded PT-18 cells indicate that increases in cytosolic calcium do not appear to correlate with enhanced 5-lipoxygenase product formation. Thus, both the calcium ionophore ionomycin and arachidonic acid increase cytosolic calcium levels but have very little effect on [14C]5-HETE formation, whereas 15-HETE induces large increases in [14C]5-HETE production but no concomitant enhancement in cytosolic calcium is observed. Chelation of extracellular calcium by 3 mM EGTA resulted in a 30-40% inhibition of [14C]5-HETE formation induced by 15 HETE, whereas 3 mM EGTA has no appreciable effect on a crude PT-18 5-lipoxygenase homogenate. These results indicate that in PT-18 cells, calcium does not appear to play an important role in either the 15-HETE-induced activation process, or the enzymatic activity of the cryptic 5-lipoxygenase system.
Vonakis
B M
BM
Department of Biochemistry and Molecular Biology-George Washington University, School of Medicine and Health Sciences, Washington, D.C. 20037.
Vanderhoek
J Y
JY
D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D001091:Arachidonate Lipoxygenases / Q000378:metabolism*; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism / Q000494:pharmacology; D001491:Basophils / Q000187:drug effects / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D005707:Gallic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D006893:Hydroxyeicosatetraenoic Acids / Q000096:biosynthesis / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D008407:Mast Cells / Q000187:drug effects / Q000201:enzymology*; D051379:Mice
D001095:Arachidonic Acids; D002121:Calcium Channel Blockers; D006893:Hydroxyeicosatetraenoic Acids; D016718:Arachidonic Acid; C022022:5-hydroxy-6,8,11,14-eicosatetraenoic acid; D004533:Egtazic Acid; D015759:Ionomycin; C006014:8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate; D005707:Gallic Acid; C025984:15-hydroxy-5,8,11,13-eicosatetraenoic acid; D001091:Arachidonate Lipoxygenases; D001094:Arachidonate 5-Lipoxygenase; D002118:Calcium