Receptors for the 5-Oxo Class of Eicosanoids in Neutrophils

Abstract

5-Hydroxy-and 5-oxo-eicosatetraenoate (5-HETE and 5-oxoETE) activate polymorphonuclear neutrophils (PMNs) through a common, receptor-like recognition system. To define this system, we examined the interaction of these eicosanoids with human PMNs. -20). The data imply that 5-HETE acts through a unique recognition system. However, evidence relating this system to receptor-like binding sites has proven difficult to obtain. Although PMNs process 5-HETE through various metabolic pathways (6 -8, 12, 21-23), one route, esterification, has presented an obstacle for receptor studies. The reaction occurs in PMNs or their isolated plasma membranes at 4 or 37°C and results in the acylation of 5-[ 3 H]HETE to membrane glycerolipids: PMNs and plasmalemma accumulate 5-HETE almost exclusively in esterified form without evidence of receptor binding (10, 21). Because esterification is a ubiquitous means for processing fatty acids, other cell types are apt also to esterify 5-HETE and thereby obscure the receptor binding of the compound. Despite this difficulty, however, putative receptors for 5-HETE merit study. PMNs, eosinophils, and monocytes dehydrogenate 5-HETE to 5-oxoETE (6,(13)(14)(15)25). 5-OxoETE is ϳ10-fold stronger than 5-HETE in stimulating PMNs and monocytes (9 -13, 19). It is even more active on eosinophils (14 -17), eliciting the chemotaxis response of this cell at concentrations 10,000-, 1000-, and Ͼ10-fold lower than LTB 4 , 5-HETE, or other chemotactic factors, respectively (18). 5-OxoETE down-regulates these cells to itself and 5-HETE but not to LTB 4 or other stimuli (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). Possibly, therefore, putative 5-HETE receptors and their preferred ligand, 5-oxoETE, participate in recruiting eosinophils to sites of allergic reactivity (18). These receptors might also mediate the remodeling of bone, growth of prostate cancer cells, contraction of uterus, and transmission of nerve impulses (26 -29). We report here on studies identifying PMN membrane sites that bind 5-oxoETE with the specificity and other properties anticipated for receptors of the 5-oxo class of eicosanoids. EXPERIMENTAL PROCEDURESBuffers and Other Reagents-Cells and membranes were suspended in a modified Hanks' balanced salt solution (9) containing 1.4 mM CaCl 2 and 0.7 mM MgCl 2 unless indicated otherwise. Stimuli, glycerolipids, and other reagents were obtained commercially (8, 9). Triacsin C was purchased from Biomol (Plymouth Meeting, PA).

The Coupling of 5-Oxo-Eicosanoid Receptors to Heterotrimeric G Proteins

“…GTP␥S inhibited the binding of both radioligands, whereas ATP␥S had no such effect (Table II). These results parallel those observed with partially purified plasma membranes from PMN (11). Since FMLP, C5a, PAF, and IL-8 did not displace 5-oxoETE binding to plasma membranes (11), and since LXA 4 and LXB 4 did not alter 5-oxoETE binding to mixed membranes (Fig.…”

“…These results parallel those observed with partially purified plasma membranes from PMN (11). Since FMLP, C5a, PAF, and IL-8 did not displace 5-oxoETE binding to plasma membranes (11), and since LXA 4 and LXB 4 did not alter 5-oxoETE binding to mixed membranes (Fig. 6), the 5-oxoETE binding site appears distinct from those for other granulocyte-stimulating eicosanoids and CF.…”

International Union of Pharmacology XLIV. Nomenclature for the Oxoeicosanoid Receptor

“…O'Flaherty andcoworkers (1995, 1996) reported that LTB 4 and 5-HETE had similar stimulatory activity in human neutrophils but the latter mediator had little affinity for BLT receptors and was resistant to BLT antagonists. These observations were extended by monitoring 3 H-labeled 5-oxo-ETE binding in PMN plasma membranes and demonstrating that specific binding sites for this radiolabel were not effected by other receptor ligands (O'Flaherty et al, 1998(O'Flaherty et al, , 2000. These results suggested a putative receptor for the oxoeicosanoids with the most potent native ligand being 5-oxo-ETE.…”

Identification of a Novel Human Eicosanoid Receptor Coupled to Gi/o

“…This eicosanoid is known as a potent chemotactic factor of eosinophils and neutrophils (37)(38)(39)(40). O'Flaherty et al (41,42), and O'Flaherty and Rossi (43) had revealed that the chemotactic activity would be mediated through a pertussis toxin-sensitive GPCR. As a separate line of interesting studies on 5-oxo-ETE and the derivatives, Ghosh and Myers (44,45) had identified 5-oxo-ETE and 5-HETE as mitogenic as well as anti-apoptotic factors in prostate cancer cells.…”