Leukotriene B3, leukotriene B4 and leukotriene B5; Binding to leukotriene B4 receptors on rat and human leukocyte membranes

Specific high-affinity binding sites for [³H]-leukotriene B₄ have been identified on membrane preparations from rat and human leukocytes. The rat and human leukocyte membrane preparations show linearity of binding with increasing protein concentration, saturable binding and rapid dissociation of binding by excess unlabelled leukotriene B₄. Dissociation constants of 0.5 to 2.5 nM and maximum binding of 5000 fmoles/mg protein were obtained for [³H] leukotriene B₄ binding to these preparations. Displacement of [³H]-leukotriene B₄ by leukotriene B₄ was compared with displacement by leukotriene B₃ and leukotriene B₅ which differ from leukotriene B₄ only by the absence of a double bond at carbon 14 or the presence of an additional double bond at carbon 17, respectively. Leukotriene B₃ was shown to be equipotent to leukotriene B₄ in ability to displace [³H]-leukotriene B₄ from both rat and human leukocyte membranes while leukotriene B₅ was 20–50 fold less potent. The relative potencies for the displacement of [³]-leukotriene B₄ by leukotrienes B₃, B₄ and B₅ on rat and human leukocyte membranes were shown to correlate well with their potencies for the induction of the aggregation of rat leukocytes and the chemokinesis of human leukocytes.     

Activation of leukocyte movement and displacement of [3H]leukotriene B4 from leukocyte membrane preparations by (12R)- and (12S)-hydroxyeicosatetraenoic acid

Both (12R)- and (12S)-hydroxyeicosatetraenoic acid were demonstrated to produce aggregation of rat leukocytes and enhance human leukocyte chemokinesis. (12R)-Hydroxyeicosatetraenoic acid was 10-20-fold more potent than (12S)-hydroxyeicosatetraenoic acid but at least 500-fold less potent than leukotriene B4 in these assays. These relative potencies are correlated with the potencies of (12R)- and (12S)-hydroxyeicosatetraenoic acid for competition of [3H]leukotriene B4 binding to rat and human leukocyte membrane preparations.     

Affinity labeling of the membrane protein-binding component of human polymorphonuclear leukocyte receptors for leukotriene B4.

A radiolabeled N-(3-aminopropyl)-leukotriene B4 amide ([3H]LTB4-APA) analog of the potent leukocyte chemotactic factor leukotriene B4 (LTB4) binds to receptors for LTB4 in plasma membrane-enriched preparations from human blood polymorphonuclear leukocytes (PMNL) and intact PMNL with respective mean dissociation constants of 2.3 nM and 69 nM at 4 degrees C. The [3H]LTB4-APA bound to plasma membrane-enriched preparations from PMNL was covalently cross-linked to membrane proteins with disuccinimidyl suberate. Solubilization and resolution by SDS-PAGE of proteins from [3H]LTB4-APA-labeled PMNL membranes revealed predominant labeling of a 60-kDa protein. Labeling of the PMNL membrane protein was inhibited by LTB4 and its analogs at concentrations similar to those inhibiting the binding of [3H]LTB4 to its receptor, with an identical rank order of potency of LTB4 greater than 20-hydroxy-LTB4 greater than LTB4-APA = 5(S),12(R)-dihydroxy-eicosa-14-cis-6,8,10-trans-tetraenoic acid much greater than LTD4 = LTC4. GTP suppressed the labeling of the 60-kDa PMNL membrane protein to an extent consistent with the decrease in receptor affinity for LTB4 induced by GTP. The stereospecificity of the affinity cross-linking reaction and the regulation by GTP support the identification of an approximately 60-kDa protein as the binding component of the PMNL receptor for LTB4.     

Analogs of leukotriene B4: effects of modification of the hydroxyl groups on leukocyte aggregation and binding to leukocyte leukotriene B4 receptors

The syntheses and agonist and binding activities of 5(S)-hydroxy- 6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (12-deoxy LTB4), 5(S), 12(S)-dihydroxy-6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (12-epi LTB4), 12(R)-hydroxy-6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (5-deoxy LTB4), 5(R), 12(S)-dihydroxy-6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (5-epi LTB4), 6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (5, 12-deoxy LTB4) are described. These leukotriene B4 analogs were all able to aggregate rat leukocytes and compete with [3H]-leukotriene B4 for binding to rat and human leukocyte leukotriene B4 receptors with varying efficacy. The analog in which the 12-hydroxyl group was removed was severely reduced both in agonist action (aggregation) and binding. The epimeric 12-hydroxyl analog demonstrated better agonist and binding properties than the analog without a hydroxyl at this position. In contrast, in the case of the 5-hydroxyl the epimeric hydroxyl analog had greatly reduced agonist and binding activities while the 5-deoxy analog demonstrated potency only several fold less than leukotriene B4 itself. The dideoxy leukotriene B4 analog was more than a thousand fold less active than leukotriene B4 as an agonist and in binding to the leukotriene B4 receptor. These results show that binding to the leukocyte leukotriene B4 receptor requires a hydroxyl group at the 12 position in either stereochemical orientation but that the presence of a hydroxyl at the 5 position is less important. However, the epimeric C5 leukotriene B4 analog clearly interacts unfavourably with the binding site of the leukotriene B4 receptor.     

Characteristics of the epitope of leukotriene B4 recognized by a highly specific mouse monoclonal antibody

Mouse monoclonal IgG2b antibodies to leukotriene B4 bind [3H]leukotriene B4 with an affinity one-thirtieth to one-third that of different rabbit antibodies to leukotriene B4. The concentrations of related ligands required to inhibit by 50% the binding of [3H]leukotriene B4 define cross-reactivities of approximately 100% for carboxyl-derivatives of leukotriene B4, 10% for 12(S)-leukotriene B4 and 8 cis-leukotriene B4, which were not distinguished from leukotriene B4 by polyclonal antibodies, 3-5% for the two isomers of 6 trans-leukotriene B4, 5% for 20-OH-leukotriene B4 and 20-COOH-leukotriene B4, and less than 1% for other leukotrienes, mono-hydroxy-eicosatetraenoic acids, and the two leukotriene B4-like isomers of 8, 15-di-hydroxy-eicosatetraenoic acid. Thus the monoclonal combining site is highly specific for the di-hydroxy-triene portion of leukotriene B4.     

Selective modulation by guanine nucleotides of the high affinity subset of plasma membrane receptors for leukotriene B4 on human polymorphonuclear leukocytes

Isolated human polymorphonuclear (PMN) leukocyte plasma membranes express high affinity (mean Kd = 0.12 nM) and low affinity (mean Kd = 50 nM) receptors for the chemotactic factor leukotriene B4 (5(S),12(R)-dihydroxy-eicosa-6,14 cis-8,10 trans-tetraenoic acid; LTB4) that are similar to those on intact PMN leukocytes. A portion of high affinity LTB4-R on PMN leukocyte membranes were converted to the low affinity state by GTP (mean +/- SE = 28.6 +/- 14.0%) and nonhydrolyzable GTP analogues, such as 5'-guanylylimidodiphosphate (GMP-PNP), in a concentration-dependent, nucleotide-specific, and reversible manner, without altering the intrinsic binding affinities of either class. [3H]GMP-PNP bound specifically to one class of receptors (mean Kd = 13 nM) on PMN leukocyte membranes. The interdependence of the LTB4-binding membrane protein and guanine nucleotide-binding protein was suggested by the capacity of LTB4 to enhance by a maximum of 150% the binding of [3H]GMP-PNP to PMN leukocyte membranes by increasing the number, but not altering the affinity, of receptors for GMP-PNP. Pertussis toxin, but not cholera toxin, reversed the enhancement of binding of [3H]GMP-PNP produced by LTB4. Guanine nucleotide-binding proteins and high affinity LTB4-R thus exhibit a mutual regulation that differs mechanistically from that of peptide chemotactic factor receptors on PMN leukocytes.     

Recognition of human polymorphonuclear leukocyte receptors for leukotriene B4 by rabbit anti-idiotypic antibodies to a mouse monoclonal antileukotriene B4

Rabbit anti-idiotypic IgG antibodies to the combining site of a mouse monoclonal IgG2b antibody to leukotriene B4 (LTB4) cross-reacted with human polymorphonuclear (PMN) leukocyte receptors for LTB4. Anti-idiotypic IgG and Fab both inhibited the binding of [3H]LTB4, but not [3H]N-formylmethionyl-leucylphenylalanine (fMLP), to PMN leukocytes with similar concentration-effect relationships, whereas neither nonimmune rabbit IgG nor Fab had any inhibitory activity. At a concentration of anti-idiotypic IgG that inhibited by 50% the binding of [3H] LTB4 to PMN leukocytes, the antibodies preferentially recognized high affinity receptors. Anti-idiotypic IgG and Fab inhibited PMN leukocyte chemotactic responses to LTB4, but not fMLP, with concentration-effect relationships resembling those characteristic of the inhibition of binding of [3H] LTB4, without altering the LTB4-induced release of beta-glucuronidase. Chemotaxis and increases in the cytoplasmic concentration of calcium equal in magnitude to those elicited by optimal concentrations of LTB4 were attained at respective concentrations of anti-idiotypic IgG equal to and 1/25 the level required for inhibition of binding of [3H]LTB4 by approximately 50%. Thus, the anti-idiotypic antibodies bound to PMN leukocyte receptors for LTB4 with a specificity, preference for high affinity sites, and capacity to alter PMN leukocyte functions that were similar to LTB4.     

Peptidoleukotrienes: distinct receptors for leukotriene C4 and D4 in the guinea-pig lung

Using [3H]-leukotriene C4 ([3H]-LTC4) and [3H]-leukotriene D4 ([3H]-LTD4), specific peptidoleukotriene receptors have been identified in membranes derived from guinea-pig lung. In the presence of 0.1 mM guanyl-5'-yl-imidodiphosphate, which completely inhibits [3H]-LTD4 binding, [3H]-LTC4 binding was protein- and temperature-dependent, reached equilibrium within 15 minutes at 20 degrees C and was reversible. Guanine nucleotides had no effect on the [3H]-LTC4 binding. Competition studies with [3H]-LTC4, peptidoleukotrienes C4, D4, E4 and the peptidoleukotriene antagonist FPL 55712 revealed an order of potency of leukotriene C4 much greater than E4 greater than D4 greater than FPL 55712. [3H]-LTD4 competition studies indicated an order of potency of LTD4 greater than LTE4 greater than LTC4 much greater than FPL 55712. Bioconversion of [3H]-LTC4, as determined by high performance liquid chromatography, was less than 3 percent. The data suggest the guinea-pig lung may contain biochemically distinct receptors for LTC4 and LTD4.     

Molecular and cellular properties of human polymorphonuclear leukocyte receptors for leukotriene B4

The distinctive characteristics of human polymorphonuclear (PMN) leukocyte receptors for leukotriene B4 (LTB4) have been elucidated by studies of binding of [3H]LTB4, the structure of protein constituents of the receptors isolated from plasma membranes, and the effects of antireceptor antibodies. A high-affinity class of 4400 receptors with a KD of 0.4 nM mediates chemotaxis and increased adherence of PMN leukocytes, whereas a low-affinity class of 270,000 receptors with a KD of 61 nM mediates the release of lysosomal enzymes and increases in oxidative metabolism. The low-affinity receptors are composed of a 60,000-dalton protein-binding unit. The high-affinity receptors are composed of the same binding unit in association with a 40,000-dalton guanine nucleotide-binding protein. That antireceptor antibodies as well as LTB4 distinguish the two classes of receptors with different functional consequences suggests the possibility of unique approaches to the regulation of leukocyte function at the receptor level.     

Specific binding of leukotriene B4 to receptors on human polymorphonuclear leukocytes

Leukotriene B4 (5(S),12(R)-di-hydroxy-eicosa-6,14-cis-8,10-trans-tetraenoic acid [LTB4]) is a product of the 5-lipoxygenation of arachidonic acid, which elicits human PMN leukocyte chemotactic responses in vitro that are 50% of the maximal level at concentrations of 3 X 10(-9) M to 10(-8) M and are maximal at 2 X 10(-8) M to 10(-7) M. The specific binding of highly purified [3H]LTB4 to human PMN leukocytes was assessed both by extracting the unbound and weakly bound [3H]LTB4 with acetone at -78 degrees C and by centrifuging the PMN leukocytes through cushions of phthalate oil to separate the unbound from bound [3H]LTB4. The levels of total binding of [3H]LTB4 and of nonspecific binding of [3H]LTB4, in the presence of a 1500-fold molar excess of nonradioactive LTB4, were approximately two times higher with the phthalate oil method. Scatchard plots of the concentration dependence of the specific binding (total - nonspecific binding) of [3H]LTB4 to PMN leukocytes were linear for the acetone extraction and phthalate oil methods and revealed dissociation constants of 10.8 X 10(-9) M and 13.9 X 10(-9) M, respectively, and mean of 2.6 X 10(4) and 4.0 X 10(4) receptors per PMN leukocyte. The 5(S),12(S)-all-trans-di-HETE analog of LTB4 and 5-HETE competitively inhibited by 50% the binding of [3H]LTB4 to PMN leukocytes at respective concentrations that evoked half-maximal chemotactic responses, whereas neither N-formyl-methionyl-leucyl-phenylalanine nor chemotactic fragments of C5 inhibited the binding. Human erythrocytes exhibited no specific binding sites for [3H]LTB4. Human PMN leukocytes possess a subset of receptors for LTB4 that are distinct from those specific for peptide chemotactic factors.     

Leukotriene A3. A poor substrate but a potent inhibitor of rat and human neutrophil leukotriene A4 hydrolase

Analysis of leukotriene B4 production by purified rat and human neutrophil leukotriene (LT) A4 hydrolases in the presence of 5(S)-trans-5,6-oxido-7,9-trans-11-cis-eicosatrienoic acid (leukotriene A3) demonstrated that this epoxide is a potent inhibitor of LTA4 hydrolase. Insignificant amounts of 5(S), 12(R)-dihydroxy-6-cis-8,10-trans-eicosatrienoic acid (leukotriene B3) were formed by incubation of rat neutrophils with leukotriene A3 or by the purified rat and human LTA4 hydrolases incubated with leukotriene A3. Leukotriene A3 was shown to be a potent inhibitor of leukotriene B4 production by rat neutrophils and also by purified rat and human LTA4 hydrolases. Covalent coupling of [3H]leukotriene A4 to both rat and human neutrophil LTA4 hydrolases was shown, and this coupling was inhibited by preincubation of the enzymes with leukotriene A4. Preincubation of rat neutrophils with leukotriene A3 also prevented labeling of LTA4 hydrolase by [3H]leukotriene A4. This result indicates that leukotriene A3 prevents covalent coupling of the substrate leukotriene A4 and inhibits the production of leukotriene B4 by blocking the binding of leukotriene A4 to the enzyme.     

Binding and metabolism of leukotriene B4 by neutrophils and their subcellular organelles

The subcellular distribution of leukotriene (LT)B4 binding and metabolizing sites was investigated in human neutrophils. Cells were disrupted by nitrogen cavitation and fractionated by Percoll density gradient centrifugation to yield cytoplasm, membranes, azurophilic granules, and specific granules. Only membrane fractions contained high affinity [3H]LTB4 binding sites. Binding of radiolabeled ligand to membranes was rapid, reversible, and saturable; it was blocked by a series of LTB4 analogues at concentrations corresponding to their respective potencies in 1) blocking [3H]LTB4 binding to whole cells and 2) stimulating neutrophil degranulation responses. In contrast, [3H]LTB4 was metabolized by fractions enriched with markers for cytoplasm plus endoplasmic reticulum. The metabolic activity was sedimented by ultracentrifugation, enhanced by NADPH, and inhibited at 4 degrees C. The cell-free system, like intact cells, metabolized [3H]LTB4 to omega-oxidized product rapidly and quantitatively at 37 degrees C but was inactive at 4 degrees C. Whole cells converted radiolabel to 20-hydroxy (approximately 30% of product) and 20-carboxy (approximately 70% of product) derivatives; the cell-free system formed principally 20-hydroxy-[3H]LTB4. These products were less bioactive than LTB4. Nevertheless, metabolism of LTB4 played little role in limiting the cells' response to the ligand: neutrophils completed degranulation and became desensitized to LTB4 within 3-5 min of exposure. Within this time frame, they oxidized less than 30% of the stimulus, and the extracellular fluid of these neutrophil suspensions was fully capable of activating fresh cells. We conclude that neutrophils transmit bioactions of LTB4 via a specific receptor integrally associated with their plasmalemma and/or endoplasmic reticulum. They inactivate the stimulus via a particulate omega-oxidase. At the level of the individual cell, receptor down-regulation, rather than ligand metabolism, appears to limit functional responses such as degranulation.     

Ligand-induced formation of the leukotriene B4 receptor-G protein complex of human polymorphonuclear leukocytes.

The components of the polymorphonuclear leukocyte (PMNL) receptor for leukotriene B4 (LTB4) were examined by Sephacryl S-300 exclusion chromatography of PMNL membrane proteins, which were solubilized before and after the binding of [3H] LTB4. When the PMNL membranes were solubilized in 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and filtered on Sephacryl S-300 prior to addition of [3H] LTB4, the binding activity was associated with a 65 kD protein. In contrast, the radioactivity of [3H] LTB4 bound to PMNL membranes prior to solubilization was recovered predominantly with a 140 kD protein. When PMNL membranes had been pretreated with pertussis toxin, but not cholera toxin, before the addition of LTB4 and subsequent solubilization, radioactivity was recovered predominantly with the 65 kD protein. The addition of guanylylimidodiphosphate (GMP-PNP), a nonhydrolyzable derivative of guanosine triphosphate (GTP), to PMNL membrane receptors bearing [3H] LTB4 either prior to or after CHAPS solubilization reduced the yield of the 140 kD presumed LTB4 receptor protein-G protein complex. That the maximum specific binding of [35S] guanosine-5'-0-3-thiotriphosphate (GTP-gammaS) to LTB4-binding proteins in the Sephacryl S-300 effluent corresponded to the 140 kD protein supported the presence of a G protein in the LTB4 receptor complex.     

Human endothelial cells stimulate leukotriene synthesis and convert granulocyte released leukotriene A4 into leukotrienes B4, C4, D4 and E4

Incubation of human endothelial cells with leukotriene A4 resulted in the formation of leukotrienes B4, C4, D4 and E4. Endothelial cells did not produce leukotrienes after stimulation with the ionophore A23187 and/or exogenously added arachidonic acid. However, incubation of polymorphonuclear leukocytes with ionophore A23187 together with endothelial cells led to an increased synthesis of cysteinyl-containing leukotrienes (364%, mean, n = 11) and leukotriene B4 (52%) as compared to leukocytes alone. Thus, the major part of leukotriene C4 recovered in mixed cultures was attributable to the presence of endothelial cells. Similar incubations of leukocytes with fibroblasts or smooth muscle cells did not cause an increased formation of leukotriene C4 or leukotriene B4. The increased biosynthesis of cysteinyl-containing leukotrienes and leukotriene B4 in coincubation of leukocytes and endothelial cells appeared to be caused by two independent mechanisms. First, cell interactions resulted in an increased production of the total amount of leukotrienes, suggesting a stimulation of the leukocyte 5-lipoxygenase pathway, induced by a factor contributed by endothelial cells. Secondly, when endothelial cells prelabeled with [35S]cysteine were incubated with either polymorphonuclear leukocytes and A23187, or synthetic leukotriene A4, the specific activity of the isolated cysteinyl-containing leukotrienes were similar. Thus, transfer of leukotriene A4 from stimulated leukocytes to endothelial cells appeared to be an important mechanism causing an increased formation of cysteinyl-containing leukotrienes in mixed cultures of leukocytes and endothelial cells. In conclusion, the present study indicates that the vascular endothelium, when interacting with activated leukocytes, modulates both the quantity and profile of liberated leukotrienes.     

A radioimmunoassay for leukotriene B4

A radioimmunoassay for leukotriene B4 has been developed. The assay is sensitive; 5 pg LTB4 caused significant inhibition of binding of [3H]-LTB4 and 50% displacement occurred with 30 pg. The specificity of the assay has been critically examined; prostaglandins, thromboxane B2 and arachidonic acid do not exhibit detectable cross-reactions (less than 0.03%). However, some non-cyclic dihydroxy- and monohydroxy-eicosatetraenoic acids do cross-react slightly (e.g. diastereomers of 5,12-dihydroxy-6,8,10-trans-14-cis-eicosatetraenoic and 12-hydroxy-5,8,10,14-eicosatetraenoic acids cross-react 3.3% and 2.0% respectively). The assay has been used to monitor the release of LTB4 from human neutrophils in response to the divalent cation ionophore, A23187. The immunoreactive material released during these incubations was confirmed as LTB4 by reverse phase high pressure liquid chromatography following solvent extraction and silicic acid chromatography.     

Hepatic uptake and metabolic disposition of leukotriene B4 in rats.

1. In isolated perfused rat liver and in vivo, up to 25% of [3H]leukotriene B4 was eliminated from the circulation via hepatic uptake and biliary excretion within 1 h. Total body recovery of 3H amounted to about 60% of infused [3H]leukotriene B4. 2. Hepatobiliary excretion of leukotriene B4 and its metabolites exceeded renal elimination by about 4-fold and depended, in contrast with excretion of cysteinyl leukotriene E4, upon continuous taurocholate supply. 3. Analyses of bile, liver and recirculated perfusate using h.p.l.c. indicated that the liver metabolized leukotriene B4 extensively to omega-carboxyleukotriene B4 and its beta-oxidized derivatives, and no unmetabolized leukotriene B4 appeared in bile. These results substantiate the important contribution of the hepatobiliary system with respect to the metabolic fate of leukotriene B4.     

The development of a sensitive and specific radioreceptor assay for leukotriene B4

To establish a simple and sensitive quantitation of leukotriene B4 (LTB4), we developed a radioreceptor assay (RRA) using a highly specific [3H]leukotriene B4[( 3H]LTB4) binding to a guinea pig spleen homogenate. The assay detected LTB4 levels as low as 0.12 pmol per tube. Fifty percent inhibition of bound [3H]LTB4 was obtained by 2.5 nM of unlabeled LTB4. [3H]LTB4 competition studies indicated that 20-hydroxy-LTB4 was 8 times, 6-trans-LTB4 was 640 times and 20-carboxy-LTB4 was 1000 times less effective than LTB4. The peptide leukotrienes C4, D4 and E4 showed no effect on [3H]LTB4 binding. Recovery rates averaged 97% after ethanol extraction and evaporation of known amounts of LTB4. The intra-assay coefficients of variation for three samples were 2.4%, 7.2% and 8.4%, respectively. This assay was validated by measuring LTB4 released from human granulocytes stimulated with calcium ionophore A23187. The LTB4 level was maximal at 10 min (156.8 +/- 36.2 pmol/3 x 10(6) cells) and decreased rapidly after 15 min. This radioreceptor assay for leukotriene B4 is highly sensitive and is comparable to the reported sensitivity by radioimmunoassay. The method is simpler and less expensive than other methods such as high pressure liquid chromatography and is suitable for routine measurement of leukotriene B4.     

Detergent solubilization of human neutrophil leukotriene B4 receptors

Specific leukotriene B4 (LTB4) receptors in human neutrophils were solubilized by treatment of "receptor fraction" membranes with the zwitterionic detergent (3-[(3-cholamidopropyl)-dimethylammonio]1-propane sulfonate (CHAPS). The soluble receptors were assayed by polyethylene glycol (PEG) precipitation coupled with Millipore filtration. The solubilized receptors retained all of the characteristics of the receptor sites in intact neutrophils. The binding of LTB4 was rapid, reversible and stereospecific. Mathematical modeling analysis revealed biphasic binding of [3H] LTB4 indicating two classes of binding sites. The high affinity binding site had a dissociation constant of 1.93 nM and Bmax of 281 fmoles/mg protein; the low affinity binding site had a dissociation constant of 78.92 nM and Bmax of 2522 fmoles/mg protein. Competitive binding experiments with structural analogs of LTB4 demonstrate that the interaction between LTB4 and its binding site is stereospecific and correlates with the relative biological activity of the analogs. These data suggest that it may be possible to purify the LTB4 receptor from human neutrophil membranes.     

Identification of leukotriene D4 specific binding sites in the membrane preparation isolated from guinea pig lung

A radioligand binding assay has been established to study leukotriene specific binding sites in the guinea pig and rabbit tissues. Using high specific activity [3H]-leukotriene D4 [( 3H]-LTD4), in the presence or absence of unlabeled LTD4, the diastereoisomer of LTD4 (5R,6S-LTD4), leukotriene E4 (LTE4) and the end-organ antagonist, FPL 55712, we have identified specific binding sites for [3H]-LTD4 in the crude membrane fraction isolated from guinea pig lung. The time required for [3H]-LTD4 binding to reach equilibrium was approximately 20 to 25 min at 37 degrees C in the presence of 10 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl. The binding of [3H]-LTD4 to the specific sites was saturable, reversible and stereospecific. The maximal number of binding sites (Bmax), derived from Scatchard analysis, was approximately 320 +/- 200 fmol per mg of crude membrane protein. The dissociation constants, derived from kinetic and saturation analyses, were 9.7 nM and 5 +/- 4 nM, respectively. The specific binding sites could not be detected in the crude membrane fraction prepared from guinea pig ileum, brain and liver, or rabbit lung, trachea, ileum and uterus. In radioligand competition experiments, LTD4, FPL 55712 and 5R,6S-LTD4 competed with [3H]-LTD4. The metabolic inhibitors of arachidonic acid and SKF 88046, an antagonist of the indirectly-mediated actions of LTD4, did not significantly compete with [3H]-LTD4 at the specific binding sites. These correlations indicated that these specific binding sites may be the putative leukotriene receptors in the guinea-pig lung.     

Direct photoaffinity labeling of leukotriene binding sites

Due to their conjugated double bonds the leukotrienes themselves are photolabile compounds and may therefore be used directly for photoaffinity labeling of leukotriene binding sites. Cryofixation eliminates unspecific labeling taking place in solution by photoisomers and photodegradation products of leukotrienes. After fixation of receptor ligand interactions by shock-freezing of the samples, irradiation-induced highly reactive excited states and/or intermediates can form covalent bonds with the respective binding site in the frozen state. After cryofixation of a solution of albumin incubated with [3H8]leukotriene E4, irradiation at 300 nm resulted in time-dependent incorporation of radioactivity into the protein. Photoaffinity labeling of rat as well as of human blood serum with [3H8]leukotriene E4 after cryofixation revealed that only one polypeptide with an Mr of 67,000 was labeled. This polypeptide was identified as albumin. Photoaffinity labeling of rat liver membrane subfractions enriched with sinusoidal membranes resulted in the labeling of a polypeptide with an apparent Mr of 48,000, whereas no polypeptide was predominantly labeled in the subfraction enriched with canalicular membranes. Photoaffinity labeling of isolated hepatocytes disclosed different leukotriene E4 binding polypeptides. In the particulate fraction of hepatocytes a polypeptide with an apparent Mr of 48,000 was labeled predominantly, whereas in the soluble fraction several polypeptides were labeled to a similar extent. One of these, with an apparent Mr of 25,000, was identified as subunit 1 of glutathione transferases by immunoprecipitation. The method of direct photoaffinity labeling in the frozen state after cryofixation using leukotrienes as photoactivatable compounds, as exemplified by leukotriene E4, may be most useful for the identification and characterization of various leukotriene binding sites, including receptors, leukotriene-metabolizing enzymes, and transport systems.