15-Hydroxyeicosatetraenoic acid (15-HETE) receptors: Involvement in the 15-HETE-induced stimulation of the cryptic 5-lipoxygenase in PT-18 mast/basophil cells

Becky Marie Vonakis, Jack Y. Vanderhoek

Research output: Contribution to journalArticle

Abstract

The mechanism of stimulation of the inactive 5-lipoxygenase in mast/basophil PT-18 cells by μM 15-hydroxyeicosatetraenoic acid (15-HETE) was investigated. Treatment of PT-18 cells with pM 15-[3H]HETE at 4° 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 DM and a Bmax of 7.1 × 105 sites/cell. Unlabeled 15-HETE 12-HETE, and 5,15-diHETE inhibited the binding of 15-[3H]HETE to cells, whereas LTB4 and PGF were relatively ineffective. 2.4 μM 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-h-poxygenase in PT-18 cells.

Original languageEnglish (US)
Pages (from-to)23625-23631
Number of pages7
JournalJournal of Biological Chemistry
Volume267
Issue number33
StatePublished - Nov 25 1992
Externally publishedYes

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Hydroxyeicosatetraenoic Acids
Arachidonate 5-Lipoxygenase
Basophils
Mast Cells
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid
Lipids
15-hydroxyeicosatetraenoic acid receptor
Binding Sites
Chemical activation
Dinoprost
Leukotriene B4
Association reactions
Pertussis Toxin
Phospholipids
Esters

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "15-Hydroxyeicosatetraenoic acid (15-HETE) receptors: Involvement in the 15-HETE-induced stimulation of the cryptic 5-lipoxygenase in PT-18 mast/basophil cells",
abstract = "The mechanism of stimulation of the inactive 5-lipoxygenase in mast/basophil PT-18 cells by μM 15-hydroxyeicosatetraenoic acid (15-HETE) was investigated. Treatment of PT-18 cells with pM 15-[3H]HETE at 4° 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 DM and a Bmax of 7.1 × 105 sites/cell. Unlabeled 15-HETE 12-HETE, and 5,15-diHETE inhibited the binding of 15-[3H]HETE to cells, whereas LTB4 and PGF2α were relatively ineffective. 2.4 μM 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-h-poxygenase in PT-18 cells.",
author = "Vonakis, {Becky Marie} and Vanderhoek, {Jack Y.}",
year = "1992",
month = "11",
day = "25",
language = "English (US)",
volume = "267",
pages = "23625--23631",
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T1 - 15-Hydroxyeicosatetraenoic acid (15-HETE) receptors

T2 - Involvement in the 15-HETE-induced stimulation of the cryptic 5-lipoxygenase in PT-18 mast/basophil cells

AU - Vonakis, Becky Marie

AU - Vanderhoek, Jack Y.

PY - 1992/11/25

Y1 - 1992/11/25

N2 - The mechanism of stimulation of the inactive 5-lipoxygenase in mast/basophil PT-18 cells by μM 15-hydroxyeicosatetraenoic acid (15-HETE) was investigated. Treatment of PT-18 cells with pM 15-[3H]HETE at 4° 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 DM and a Bmax of 7.1 × 105 sites/cell. Unlabeled 15-HETE 12-HETE, and 5,15-diHETE inhibited the binding of 15-[3H]HETE to cells, whereas LTB4 and PGF2α were relatively ineffective. 2.4 μM 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-h-poxygenase in PT-18 cells.

AB - The mechanism of stimulation of the inactive 5-lipoxygenase in mast/basophil PT-18 cells by μM 15-hydroxyeicosatetraenoic acid (15-HETE) was investigated. Treatment of PT-18 cells with pM 15-[3H]HETE at 4° 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 DM and a Bmax of 7.1 × 105 sites/cell. Unlabeled 15-HETE 12-HETE, and 5,15-diHETE inhibited the binding of 15-[3H]HETE to cells, whereas LTB4 and PGF2α were relatively ineffective. 2.4 μM 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-h-poxygenase in PT-18 cells.

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