Lipoxin recognition sites. Specific binding of labeled lipoxin A4 with human neutrophils.
Lipoxin A4 stimulates rapid lipid remodeling and a pertussis toxin-sensitive release of arachidonic acid in polymorphonuclear leukocytes (PMN) (Nigam, S., Fiore, S., Luscinskas, F.W., and Serhan, C.N. (1990) J. Cell. Physiol. 143, 512-523) and has been shown to inhibit leukocyte responses in several systems. To examine the basis underlying these actions, we have prepared [11,12-3H]lipoxin A4 (LXA4) and characterized its interactions with human PMN. Time course studies (0-90 min) with intact PMN demonstrated cell association of 3H label which was specific and reversible. PMN bound [3H]LXA4 with a Kd of 0.5 +/- 0.3 nM, representing approximately 1,830 sites/PMN, and the Hill plot value of 1.9 suggests cooperative binding. [3H]LXA4 binding was stereoselective since neither leukotriene B4 (LTB4), lipoxin B4 (LXB4), (6S)-LXA4, 11-trans-LXA4, nor SKF 104353 competed for [3H]LXA4-specific binding while LTD4 and LTC4 partially competed. Subcellular fractionation revealed that specific binding with [3H]LXA4 was associated with membrane (42.1%)-, granule (34.5%)-, and nuclear (23.3%)-enriched fractions, a distribution distinct from that of [14,15-3H] LTB4 binding. [11,12-3H]LXA4-specific binding was modulated by guanosine analogs, suggesting the involvement of G proteins. A fluorescent LXA4 derivative (methyl-7-methoxycoumarin-LXA4) competed with [3H]LXA4 binding to intact PMN and showed specific and reversible binding as monitored by flow cytometric analysis. These results indicate that PMN possess specific recognition sites for LXA4 which may mediate its actions.
Fiore
S
S
Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115.
Ryeom
S W
SW
Weller
P F
PF
Serhan
C N
CN
D001665:Binding Sites; D001667:Binding, Competitive; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000097:blood* / Q000138:chemical synthesis; D007700:Kinetics; D007975:Leukotriene B4 / Q000097:blood; D044045:Lipoxins; D009504:Neutrophils / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D014316:Tritium
D006893:Hydroxyeicosatetraenoic Acids; D044045:Lipoxins; C040527:lipoxin A4; D014316:Tritium; D007975:Leukotriene B4; D019204:GTP-Binding Proteins