dinoprost and Leukemia--Basophilic--Acute

The ubiquitous hydroxylated fatty acids derived from arachidonic acid (HETEs) or linoleic acid (HODEs) exhibit diverse biological effects including chemotaxis, cell proliferation, and modulation of several enzymatic pathways, including the 5-lipoxygenase leading to the inflammatory leukotrienes. It was observed that 12(S)- and 15(S)-HETE and 13(S)-HODE (12- and 15-lipoxygenase-derived metabolites, respectively) inhibited the 5-lipoxygenase present in rat basophilic leukemia (RBL-1) cell homogenates whereas the 15(R) chiral enantiomer and the nonhydroxylated linoleic, oleic, and stearic acids were either less potent or ineffective. In examining the mechanism of this inhibition, the relative effectiveness of several fatty acids in displacing [3H]15-HETE bound to cytosol preparations were compared and the results indicated that these (S) hydroxy fatty acids and 5(S)-HETE were significantly more potent than either the 15(R) enantiomer, 15(S)-HETE methyl ester, arachidonic acid, or prostaglandin F2alpha. In order to identify the protein(s) that specifically binds HETEs, 15(S)-HETE biotin hydrazide was used as a probe to detect any HETE-protein complexes as this compound both inhibited the 5-lipoxygenase and interfered with the binding of [3H]15-HETE to cytosol preparations. SDS-PAGE analysis and chemiluminescent detection revealed that the major cytosolic proteins that bound this biotinylated probe had molecular masses of 43 and 51 kD. Fatty acid competition experiments indicated that the order of effectiveness in displacing this probe from these proteins was 13(S)-HODE > 5(S)-HETE approximately equal to 15(S)-HETE > > stearic acid approximately equal to arachidonic acid approximately equal to 15(R)-HETE. Amino acid sequence analysis showed that the 43 kD protein was actin. These findings suggest the possibility that actin may play a major role in the biological effects of monohydroxylated metabolites derived from cellular 5-, 12-, and 15-lipoxygenases.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Actins; Amino Acid Sequence; Animals; Arachidonate 15-Lipoxygenase; Biotinylation; Carrier Proteins; Cytosol; Dinoprost; Fatty Acid-Binding Protein 7; Fatty Acid-Binding Proteins; Hydroxyeicosatetraenoic Acids; Kinetics; Leukemia, Basophilic, Acute; Ligands; Linoleic Acid; Linoleic Acids; Molecular Sequence Data; Myelin P2 Protein; Neoplasm Proteins; Nerve Tissue Proteins; Oleic Acid; Rats; Stearic Acids; Stereoisomerism; Structure-Activity Relationship; Tumor Cells, Cultured

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.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Binding Sites; Binding, Competitive; Calcimycin; Cell Line; Cholic Acids; Dinoprost; Hydroxyeicosatetraenoic Acids; Leukemia, Basophilic, Acute; Lipoxygenase Inhibitors; Rats; Subcellular Fractions; Tumor Cells, Cultured

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonate Lipoxygenases; Arthritis, Experimental; Carrageenan; Cyclooxygenase Inhibitors; Dinoprost; Edema; Inflammation; Leukemia, Basophilic, Acute; Leukotriene B4; Lipoxygenase Inhibitors; Mycobacterium; Phenols; Pyrazoles; Rats; Stomach Ulcer; Tumor Cells, Cultured; Zymosan