LXR␣ and -␤ are nuclear receptors that regulate the metabolism of several important lipids, including cholesterol and bile acids. Previously, we have proposed that LXRs regulate these pathways through their interaction with specific, naturally occurring oxysterols, including 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 24(S),25-epoxycholesterol. Using a ligand binding assay that incorporates scintillation proximity technology to circumvent many of the problems associated with assaying extremely hydrophobic ligands, we now demonstrate that these oxysterols bind directly to LXRs at concentrations that occur in vivo. To characterize further the structural determinants required for potent LXR ligands, we synthesized and tested a series of related compounds for binding to LXRs and activation of transcription. These studies revealed that position-specific monooxidation of the sterol side chain is requisite for LXR high-affinity binding and activation. Enhanced binding and activation can also be achieved through the use of 24-oxo ligands that act as hydrogen bond acceptors in the side chain. In addition, introduction of an oxygen on the sterol B-ring results in a ligand with LXR␣-subtype selectivity. These results support the hypothesis that naturally occurring oxysterols are physiological ligands for LXRs and show that a rational, structure-based approach can be used to design potent LXR ligands for pharmacologic use.Nonsteroidal nuclear hormone receptors are ligand-activated transcription factors that regulate an array of signaling pathways. Several members of this protein family, including the vitamin D receptor, retinoic acid receptor, and peroxisome proliferator-activated receptor, are primary regulators in nutrient metabolism and are known to be associated with a variety of metabolic disorders (1). These receptors function by forming obligate heterodimers with the retinoid X receptor (RXR) and binding specific DNA sequences (response elements) within the promoters of the genes they regulate. Upon binding ligand, the heterodimerized receptor undergoes a conformational change that results in enhanced transcription of the target gene. Thus far, all known native ligands for nuclear receptors are small hydrophobic compounds, many of which are the products of lipid metabolism (1).The liver X receptors (LXRs) were originally identified as orphan members of the nuclear receptor superfamily because their ligands were unknown. Like other receptors in the family, LXRs heterodimerize with RXR and bind to specific response elements (LXREs) characterized by direct repeats separated by four nucleotides (2-4). Two genes (␣ and ␤) are known to encode LXR proteins. LXR␣ is expressed most highly in the liver and to a lesser extent in the kidney, small intestine, spleen, and adrenal gland (2, 4). In contrast to the restricted expression pattern of LXR␣, LXR␤ is ubiquitously expressed (5). In a comprehensive screen for LXR ligands we observed that the naturally occurring oxysterols 24(S)-hydroxycholesterol [24(S)-H...

The pharmacologic activities of leukotrienes C-i and D (LTC-1 and LTD), constituents of slow reacting substance of anaphylaxis (SRS-A), were evaluated in vitro on airway contractile tissues and in vivo on pulmonary mechanical function, mean systemic arterial pressure, and cutaneous microcirculation. In vitro both LTC-Land LTD were potent and selective peripheral airway agonists, being more active than histamine; furthermore, LTD was active on peripheral airways at concentrations 1/100th those of LTC-1. The concentration-effect relationship for LTD and the profile of antagonism by FPL 55712 are consistent with the activity. of this molecule at two separate peripheral airway receptors. Ii vivo, LTC1 and LTD were nearly equally active in their effects on pulmonary mechanics, and the pattern of alterations was consistent with the predominant site of action being in the lung periphery. Furthermore, both agents had a direct systemic arterial hypotensive effect and were vasoactive on the cutaneous microcirculation. Thus, these compounds are likely to be major mediators of the pathologic alterations in immediate type hypersensitivity reactions in which peripheral airway constriction and hypotension are prominent features.Slow reacting substance of anaphylaxis (SRS-A) (1), which is an activity generated during immediate type hypersensitivity reactions (2), has been found to be composed of leukotrienes C-i (LTC-1) and D (LTD) (3). Native SRS-A has a unique profile of contractile activity in vitro in smooth muscle preparations (4). Both partially and highly purified native SRS-A produced by an anaphylactic reaction in the rat peritoneal cavity (SRS-Arat) exhibit a preferential contractile activity for guinea pig pulmonary parenchymal strips compared to musculus trachealis (5), and LTC-1 and LTD exhibit the same differential effect, at concentrations less than 0.1% of those required for histamine to be active (3). Partially purified SRS-Arat augments vascular permeability when injected into guinea pig skin (6). When administered intravenously into the unanesthetized guinea pig, it produces an alteration in pulmonary mechanics consistent with peripheral rather than central airway action (7). We have now demonstrated that intravenous infusion of LTC-1 and LTD alters pulmonary mechanics in unanesthetized and anesthetized guinea pigs in a manner similar to native partially purified SRS-Arat and that, in addition, these newly described products of arachidonate metabolism (8-10) can differ in their actions on the cutaneous microvasculature and on mean systemic arterial pressure.MATERIALS AND METHODS LTC-1 and LTD were prepared as described (3, 10), sealed in ampoules in 10% methanol under argon, and stored frozen until the day of use. Histamine diphosphate was obtained from Sigma. FPL 55712, a specific SRS-A antagonist, was a gift from P. Sheard (Fisons Pharmaceuticals, Ltd., U.K.).Tracheal spirals and parenchymal strips were prepared for recording isometric contractile activity (5) and allowed to relax to baseline tensi...

5-Lipoxygenase pathway-derived products of arachidonic acid released by human eosinophils activated in vitro have been measured by using radioimmunoassays specific for leukotriene B4 (LTB4) and for sulfidopeptide leukotrienes including leukotriene C4 (LTC4). Eosinophil-enriched leukocytes (mean, 85% eosinophils) from five hypereosinophilic donors activated with 5.0 pAM ionophore A23187 for 15 min at 37C in the presence of 50 mM L-serine released 69 ± 28 and 1.5 + 0.8 (mean ± SEM) ng of LTC4 and LTB4, respectively, per 106 cells; ratios of LTC4 to LTB4 ranged from 16 to 149. Eosinophils stimulated with ionophore (2.5 ,M) or phorbol myristate acetate (1 ,ug per ml) metabolized exogenously added LTC4 to products that coeluted on reverse-phase high-performance liquid chromatography with synthetic S-diastereoisomeric LTC4 sulfoxides and 6-trans-LTB4 diastereoisomers, and this metabolic inactivation was inhibited by Lserine or catalase. Ionophore-activated eosinophils purified from three normal donors also preferentially generated LTC4 (38 ± 3 ngper 106cells) relative to LTB4 (6.0 ± 3.1 ng per 106cells), whereas neutrophils from the same donors released LTB4 (48 ± 21 ng per 106 cells) in a >7-fold excess to LTC4. The predominant production by human eosinophils of LTC4 with its potent smooth muscle spasmogenic and vasoactive properties may contribute to the pathobiology of allergic and other diseases associated with eosinophilia.Human polymorphonuclear leukocytes, activated with diverse stimuli, oxidatively metabolize arachidonic acid by the 5-lipoxygenase-dependent pathway to 5,6-trans-oxido-7,9-trans-11, 14-cis-icosatetraenoic acid (leukotriene A4, LTA4) (1), which in turn is converted enzymatically to (5S,6R)-5,6-dihydroxy-6,14-cis-8, 10-trans-icosatetraenoic acid (leukotriene B4, LTB4) or to (5S,6R)-5-hydroxy-6-S-glutathionyl-7,9-trans-11, 14-cis-icosatetraenoic acid (leukotriene C4, LTC4) (2-4). LTB4 is a potent chemoattractant and aggregating stimulus for both neutrophilic and eosinophilic polymorphonuclear leukocytes (5, 6), and LTC4 is exquisitely active as a spasmogenic and vasoactive substance when administered locally to human airways and skin, respectively (7,8).Human polymorphonuclear leukocytes, predominantly neutrophils, when stimulated with the calcium ionophore A23187 produce LTB4 in marked preference to the sulfidopeptide leukotrienes, LTC4 and its peptide cleavage products (5S,6R)-5-hydroxy-6-S-cysteinylglycyl-7,9-trans -11, 14 -cis-icosatetraenoic acid (leukotriene D4, LTD4) and (5S,6R)-5-hydroxy-6-Scysteinyl-7,9-trans-11,14-cis-icosatetraenoic acid (leukotriene E4, LTE4) (2,4,9,10 diastereoisomers, and the S-diastereoisomeric sulfoxides of LTC4 were prepared as described (11)(12)(13)(14).Cell Purification. Human neutrophils were prepared from citrate-anticoagulated blood of normal volunteer donors by dextran sedimentation of erythrocytes, centrifugation on cushions of Ficoll/Hypaque, and hypotonic lysis of erythrocytes (15). Human eosinophils were obtained from the citrate-anticoagulated b...