Characterization of specific subcellular 15-hydroxyeicosatetraenoic acid (15-HETE) binding sites on rat basophilic leukemia cells.
15-Hydroxyeicosatetraenoic acid [15-(S)-HETE], a major arachidonic acid metabolite produced from the 15-lipoxygenase pathway, has been characterized as an antiinflammatory cellular mediator since it can inhibit the in vivo and in vitro formation of the proinflammatory leukotrienes via the 5-lipoxygenase pathway in various cells. 15-HETE has been confirmed to inhibit the 5-lipoxygenase in rat basophilic leukemia cell (RBL-1) homogenates with an I50 = 7.7 microM. The I50 of the 12-HETE isomer was 6 microM whereas prostaglandin F2 alpha was ineffective. In order to examine the mechanistic basis underlying the inhibitory action of 15-HETE, association assays of [3H]-15-HETE with RBL-1 subcellular fractions were carried out. The presence of the zwitterionic detergent CHAPS enhanced specific [3H]-15-HETE binding in the membrane fractions three-fold and specific 15-HETE binding was distributed among the nuclear (32%)-, granule (19%)-, plasma membrane (35%)-, and cytosol (14%)-enriched fractions. Studies using combined granule and plasma membrane enriched-, CHAPS treated-fractions showed that [3H]-15-HETE binding was time-dependent, specific and reversible, sensitive to pertussis toxin treatment, and indicated a single class of binding sites with a Kd = 460 +/- 160 nM and Bmax = 5.0 +/- 1.1 nM. Competition experiments showed that the order of 15-HETE or analogs in inhibiting the binding of [3H]-15-HETE was: 15(S)-HETE > or = 12-(S)-HETE = 5-(S)-HETE > 15-(R)-HETE > arachidonic acid. Prostaglandin F2 alpha and lipoxin B4 were ineffective as competitors. The similar profiles of the binding assays and inhibition of the 5-lipoxygenase suggest that 15-HETE binding sites may mediate this inhibitory action of 15-HETE.
Kang
L T
LT
Department of Biochemistry and Molecular Biology, George Washington University Medical Center, Washington, D.C., USA.
Vanderhoek
J Y
JY
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D000001:Calcimycin / Q000494:pharmacology; D002460:Cell Line; D002793:Cholic Acids; D015237:Dinoprost / Q000494:pharmacology; D006893:Hydroxyeicosatetraenoic Acids / Q000494:pharmacology*; D015471:Leukemia, Basophilic, Acute; D016859:Lipoxygenase Inhibitors; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology; D014407:Tumor Cells, Cultured
D002793:Cholic Acids; D006893:Hydroxyeicosatetraenoic Acids; D016859:Lipoxygenase Inhibitors; D000001:Calcimycin; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; C025984:15-hydroxy-5,8,11,13-eicosatetraenoic acid; D015237:Dinoprost; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate