Biomedical subjects
Publications and source records attributed to K M Metters.
The structure-activity relationship of a series of styrylpyridine analogs of MK-0476 (montelukast, Singulair) is described. This work has led to the identification of a number of potent and orally active cysLT1 receptor (LTD4 receptor) antagonists including 2ab (L-733,321) as an optimized candidate.
A novel photoaffinity probe for the leukotriene D4 receptor (LTD4) is described. L-745310, which is structurally related to the potent LTD4 antagonist MK-0476 (Singulair), was found to selectively label a 43-kDa protein in guinea-pig lung membrane previously identified as the LTD4 receptor.
1. A human embryonic kidney cell line [HEK 293(EBNA)] stably expressing the human recombinant prostaglandin D2 (PGD2) receptor (hDP) has been characterized with respect to radioligand binding and signal transduction properties by use of prostanoids and prostanoid analogues. Radioligand binding studies included saturation analyses, the effects of nucleotide analogues, the initial rate of ligand-receptor association and equilibrium competition assays. In addition, adenosine 3':5'-cyclic monophosphate (cyclic AMP) generation in response to ligand challenge was also measured, as this is the predominant hDP signalling pathway. 2. L-644,698 ((4-(3-(3-(3-hydroxyoctyl)-4-oxo-2-thiazolidinyl) propyl) benzoic acid) (racemate)) was identified as a novel ligand having high affinity for hDP with an inhibitor constant (Ki) of 0.9 nM. This Ki value was comparable to the Ki values obtained in this study for ligands that have previously shown high affinity for DP: PGD2 (0.6 nM), ZK 110841 (0.3 nM), BW245C (0.4 nM), and BW A868C (2.3 nM). 3. L-644,698 was found to be a full agonist with an EC50 value of 0.5 nM in generating cyclic AMP following activation of hDP. L-644,698 is, therefore, comparable to those agonists with known efficacy at the DP receptor (EC50): PGD2 (0.5 nM), ZK 110841 (0.2 nM) and BW245C (0.3 nM). 4. L-644,698 displayed a high degree of selectivity for hDP when compared to the family of cloned human prostanoid receptors: EP1 (> 25,400 fold), EP2 (approximately 300 fold), EP3-III (approximately 4100 fold), EP4 (approximately 10000 fold), FP (> 25,400 fold), IP (> 25,400 fold) and TP (> 25,400 fold). L-644,698 is, therefore, one of the most selective DP agonists as yet described. 5. PGJ2 and delta12-PGJ2, two endogenous metabolites of PGD2, were also tested in this system and shown to be effective agonists with Ki and EC50 values in the nanomolar range for both compounds. In particular, PGJ2 was equipotent to known DP specific agonists with a Ki value of 0.9 nM and an EC50 value of 1.2 nM.
We have characterized the rat prostanoid EP1, EP2, EP3alpha and EP4 receptor subtypes cloned from spleen, hepatocyte and/or kidney cDNA libraries. Comparison of the deduced amino acid sequences of the rat EP receptors with their respective homologues from mouse and human showed 91% to 98% and 82% to 89% identity, respectively. Radioreceptor binding assays and functional assays were performed on EP receptor expressing human embryonic kidney (HEK) 293 cells. The KD values obtained with prostaglandin E2 for the prostanoid receptor subtypes EP1, EP2, EP3alpha and EP4 were approximately 24, 5, 1 and 1 nM, respectively. The rank order of affinities for various prostanoids at the prostanoid receptor subtypes EP2, EP3alpha and EP4 receptor subtypes was prostaglandin E2 = prostaglandin E1 > iloprost > prostaglandin F2alpha > prostaglandin D2 > U46619. The rank order at the prostanoid EP1 receptor was essentially the same except that iloprost had the highest affinity of the prostanoids tested. Of the selective ligands, butaprost was selective for prostanoid EP2, M&B28767 and sulprostone were selective for EP3alpha and enprostil displayed dual selectivity, interacting with both prostanoid receptor subtypes EP1 and EP3alpha. All four receptors coupled to their predominant signal transduction pathways in HEK 293 cells. Notably, using a novel aequorin luminescence assay to monitor prostanoid EP1 mediated increases in intracellular calcium, both iloprost and sulprostone were identified as partial agonists. Finally, by Northern blot analysis EP3 transcripts were most abundant in liver and kidney whereas prostanoid EP2 receptor mRNA was expressed in spleen, lung and testis and prostanoid EP1 receptor mRNA transcripts were predominantly expressed in the kidney. The rat prostanoid EP1 probes also detected additional and abundant transcripts present in all the tissues examined. These were found to be related to the expression of a novel protein kinase gene and not the prostanoid EP1 gene [Batshake, B., Sundelin, J., 1996. The mouse genes for the EP1 prostanoid receptor and the novel protein kinase overlap. Biochem. Biophys. Res. Commun. 227. 1329-1333].
A cDNA encoding a functional human prostanoid DP (hDP) receptor has been constructed from a genomic clone and a fragment cloned by 3'-rapid amplification of cDNA ends-polymerase chain reaction. The hDP receptor consists of 359 amino acid residues with a predicted molecular mass of 40,276 and has the putative heptahelical transmembrane domains characteristics of G-protein-coupled receptors. The deduced amino acid sequence of the hDP receptor, when compared with all other members of the prostanoid receptor family, shows the highest degree of identity with the hIP and hEP2 receptors, followed by the hEP4 receptor. Radioreceptor binding studies using membranes prepared from mammalian COS-M6 cells transiently transfected with an expression vector containing the DP receptor cDNA showed that the rank order of affinities for prostaglandins and prostaglandin analogs, in competition for [3H]prostaglandin D2 (PGD2) specific binding sites, was as predicted for the DP receptor, with PGD2 >> PGE2 > PGF2 alpha = iloprost > U46619. The signal transduction pathway of the cloned hDP receptor was studied by transfecting the hDP expression vector in HEK 293(EBNA) cells. Activation of the hDP receptor with PGD2 resulted in an elevation of intracellular cAMP and in mobilization of Ca2+, but did not lead to generation of inositol 1,4,5-trisphosphate. Northern blot analysis of human tissue showed that the hDP receptor was a very discrete tissue distribution and was detectable only in retina and small intestine. In summary, we have cloned and expressed a functional cDNA for the hDP receptor.
The current challenge in research on leukotriene receptors is to clone these molecules. Traditional protein purification approaches have not been successful in providing sequence information. Solubilization of cys-LT1 has been achieved but results in the dissociation of G-proteins and the loss of high affinity binding (Mong et al., 1986b; Mong and Sarau, 1990), while cys-LT2 activity cannot be monitored by other than functional assays and there have not been any purification attempts. Partial purification of B-LT has been reported but has not been continued to homogeneity (Sherman et al., 1992; Votta et al., 1990; Miki et al., 1990). Nor have attempts to clone these receptors through either homology screening or expression cloning been successful. The cloning of the prostanoid receptors, described in detail elsewhere in this volume, has shown that these receptors belong to a distinct family within the G-protein-coupled receptor superfamily. It is probable, therefore, that the leukotriene receptors will also belong to a separate group within this superfamily since phylogenic comparisons have shown that receptors displaying high affinity for structurally related ligands exist as discrete families. Recently, a human cDNA encoding an orphan FMLP-related receptor cloned from HL60 cells of myeloid lineage was identified as the receptor for another eicosanoid, lipoxin A (Fiore et al., 1994). FMLP has a similar profile of biological actions to LTB4. Moreover, LTD4 showed a high degree of cross-reactivity with this receptor with an affinity only 20-fold less that of lipoxin A, although LTB4 was inactive. It remains to be determined whether the leukotriene receptors will fall into this class of receptors. The cloning of the leukotriene receptors will allow identification of the different receptor types and subtypes and potentially splice variants. Evaluation of currently developed antagonists at these receptor types could also open the way for novel therapies for inflammatory conditions.
The human peripheral cannabinoid (CB2) receptor has been cloned by reverse transcription-polymerase chain reaction from human spleen RNA and expressed, to study both ligand binding characteristics and signal transduction pathways. Receptor binding assays used the aminoalkylindole [3H]Win 55212-2 and membranes from transiently transfected COS-M6 cells. Saturation analysis showed that [3H]Win 55212-2 specific binding to the CB2 receptor was of high affinity, with a Kd of 2.1 +/- 0.2 nM (four experiments), and a high level of expression was attained, with a maximal number of saturable binding sites of 24.1 +/- 4.4 pmol/mg of protein (four experiments). The rates of association and dissociation for [3H]Win 55212-2 specific binding were both rapid when measured at 30 degrees. [3H]Win 55212-2 specific binding to the CB2 receptor was moderately enhanced by divalent and monovalent cations but was only slightly inhibited by guanosine-5'-O-(3-thio)-triphosphate. Competition for [3H]Win 55212-2 specific binding to the CB2 receptor was stereoselective, with the following rank order of potency for the more active stereoisomers: HU-210 > (-)-CP-55940 approximately Win 55212-2 >> (-)delta 9-THC > anandamide. The signaling pathway of the human CB2 receptor was investigated in a CB2-CHO-K1 stable cell line. CB2 receptor activation by cannabinoid agonists inhibited forskolin-induced cAMP production in a concentration-dependent and stereoselective manner but did not increase either cAMP production or Ca2+ mobilization in fura-2/acetoxymethyl ester-loaded CB2-CHO-K1 cells. The CB2 receptor-mediated inhibition of forskolin-induced cAMP production was abolished by pretreatment of the cells with 10 ng/ml pertussis toxin. These results demonstrate that the CB2 receptor is functionally coupled to inhibition of adenylyl cyclase activity via a pertussis toxin-sensitive G protein.
Dimethyl sulfoxide-differentiated U937 (dU937) cells express high affinity G-protein-coupled receptors for leukotriene (LT)D4 and LTB4 and, as described here, specific binding sites for LTC4. The specific binding of [3H]LTC4 was of low affinity (KD = 26 nM) and high abundance (Bmax = 33 pmol/mg of protein), as compared to LTD4 and LTB4 receptors. In addition, although [3H]LTC4 specific binding was enhanced by divalent cations, it was not inhibited by nonhydrolyzable GTP analogs. [3H]LTC4 specific binding to dU937 cell membranes does not have, therefore, the characteristics of binding to a G-protein-coupled receptor. Competition for [3H]LTC4 specific binding to dU937 cell membranes by leukotrienes and related analogs, including N-methylated LTC4, as well as glutathione, suggested a dependence on the presence of an arachidonic acid backbone, although varying degrees of saturation were well tolerated, and that the glutathione moiety of LTC4 in particular was important in determining affinity. The possibility that [3H]LTC4 specific binding was to a member of the glutathione S-transferase (GST) family of enzymes, such as LTC4 synthase, cytosolic GST, or microsomal GST, was therefore investigated. [3H]LTC4 specific binding sites could be separated from LTC4 synthase and cytosolic GSTs by differential detergent solubilization, but cofractionated with microsomal GST during solubilization and subsequent anion exchange chromatography. In membranes that were depleted of LTC4 synthase and cytosolic GSTs, 125I-azido-LTC4 (a photoaffinity probe based on LTC4) specifically photolabeled in a cation-dependent manner a 17-kDa polypeptide that was comparable in mass to the microsomal GST polypeptide. Furthermore, [3H]LTC4 bound specifically to purified human microsomal GST with the same characteristics as to the endogenous dU937-cell membrane specific binding sites. The principal [3H]LTC4 specific binding site present in dU937 cells, therefore, is not a G-protein-coupled receptor, LTC4 synthase, or cytosolic GSTs, but is microsomal GST. Finally, the 1:3 stoichiometry of [3H]LTC4 specific binding to purified microsomal GST is consistent with the enzyme functioning as a homotrimer.
A cDNA clone coding for a functional human prostanoid IP receptor has been isolated from a lung cDNA library. The human IP receptor consists of 386 amino acid residues with a predicted molecular mass of 40,961, and has the seven putative transmembrane domains characteristic of G-protein-coupled receptors. Challenge of Xenopus oocytes co-expressing the IP receptor and the cystic fibrosis transmembrane conductance regulator (cAMP-activated Cl- channel) with the stable prostacyclin analog iloprost resulted in specific inward Cl- currents, demonstrating that the cDNA encoded a functional IP prostanoid receptor coupled to elevation in cAMP. Radioreceptor binding studies using membranes prepared from mammalian COS cells transfected with the IP receptor cDNA showed that the rank order of potency for prostaglandins and prostaglandin analogs in competition for [3H]iloprost specific binding sites was as predicted for the IP receptor, with iloprost >> carbacyclin >> prostaglandin (PG) E2 > PGF 2 alpha = PGD2 = U46619. Northern blot analysis showed that IP mRNA was most abundantly expressed in kidney, with lesser amounts detected in lung and liver. In summary, we have cloned and expressed a cDNA for the human prostanoid IP receptor that is functionally coupled to a signaling pathway involving stimulation of intracellular cAMP production.
A cDNA clone encoding the human prostaglandin (PG) E2 receptor EP2 subtype has been isolated from a human lung cDNA library. The 1.9-kilobase pair cDNA, hEP2, encodes for a 488-amino acid protein with a predicted molecular mass of 53,115 and has the seven putative transmembrane domains characteristic of G protein-coupled receptors. The specific binding of [3H]PGE2 to COS cell membranes transfected with the hEP2 cDNA was of high affinity with an equilibrium dissociation constant (Kd) of 1 nM and the rank order of potency for prostaglandins in competition for [3H]PGE2 specific binding was PGE1 = PGE2 >> iloprost > PGF2 alpha > PGD2. In competition studies using more selective prostanoid-receptor agonist and antagonists, the [3H]PGE2 specific binding was competed by MB28767, an EP3 agonist, but not by the EP1-preferring antagonists AH6809 and SC19220, or by the EP2 agonist butaprost. Electrophysiological studies of Xenopus oocytes co-injected with hEP2 and cystic fibrosis transmembrane conductance regulator (cAMP-activated Cl- channel) cDNAs detected PGE2-specific inward Cl- currents, demonstrating that the hEP2 cDNA encoded a functional receptor which produced an increase in cAMP levels. Thus, we have cloned the human EP2 receptor subtype which is functionally coupled to increase in cAMP. Northern blot analysis showed that hEP2 is expressed as a 3.8-kilobase mRNA in a number of human tissues with the highest expression levels present in the small intestine.
Functional cDNA clones coding for three isoforms of the human prostaglandin E receptor EP3 subtype have been isolated from kidney and uterus cDNA libraries. The three isoforms, designated hEP3-I, hEP3-II and hEP3-III, have open reading frames corresponding to 390, 388 and 365 amino acids, respectively. They differ only in the length and amino acid composition of their carboxy-terminal regions, beginning at position 360. The human EP3 receptor has seven predicted transmembrane spanning domains and therefore belongs to the G-protein-coupled receptor family. The rank order of potency for prostaglandins and related analogs in competition for [3H]PGE2 specific binding to membranes prepared from transfected COS cells was comparable for all three isoforms, and as predicted for the EP3 receptor, with PGE2 = PGE1 >> PGF2 alpha = iloprost > PGD2 >> U46619. In addition, the EP3-selective agonist MB28767 was a potent competing ligand with an IC50 value of 0.3 nM, whereas the EP1-selective antagonist AH6909 gave IC50 values of 2-7 microM and the EP2-selective agonist butaprost was inactive. In summary, we have cloned three isoforms of the human EP3 receptor having comparable ligand binding properties.
A cDNA clone coding for a functional human prostanoid FP receptor has been isolated from a uterus cDNA library. The human FP receptor consists of 359 amino acid residues with a predicted molecular mass of 40,060, and has the seven putative transmembrane domains characteristic of G-protein-coupled receptors. Challenge of Xenopus oocytes expressing the FP receptor with 10 nM of either prostaglandin (PG) F2 alpha or the selective FP-receptor agonist fluprostenol resulted in an elevation in intracellular Ca2+. Radioreceptor binding studies using membranes prepared from mammalian COS cells transfected with the FP receptor cDNA showed that the rank order of potency for prostaglandins and prostaglandin analogs in competition for [3H]PGF2 alpha specific binding sites was as predicted for the FP receptor, with PGF2 alpha approximately fluprostenol > PGD2 > PGE2 > U46619 > iloprost. In summary, we have cloned the human prostanoid FP receptor which is functionally coupled to the Ca2+ signalling pathway.
Agonist-induced changes in intracellular calcium ion concentration ([Ca++]i) were examined in human monocytic leukemia THP-1 cells loaded with fura 2/acetoxymethyl ester (fura 2/AM). Leukotriene (LT)D4 induced a concentration-dependent biphasic response consisting of a transient phase (up to 5-fold peak increase) followed by a sustained phase, showing characteristics of a receptor-operated calcium channel. Homologous desensitization to LTD4 was observed. The responses to LTD4 were reduced by 80 to 90% in calcium-free buffer. The responses to LTD4 in a calcium-free buffer were dependent upon the duration of prior exposure of the cells to a calcium-free environment. The response at 30 or 60 min after exposure to calcium-free buffer was greater than that at earlier time points (time-dependent sensitization). Similar responses were obtained with THP-1 cells exposed to EDTA-containing buffer. It is speculated that such time-dependent sensitization is a result of changes at the receptor level. The responses to LTD4 were blocked by two specific LTD4 antagonists, MK-0571 and ICI-204,219, in a concentration-dependent manner. When given after addition of LTD4, MK-0571 or ICI-204,219 reversed the sustained phase of the LTD4-induced response, suggesting that maintenance of the response requires persistent activation of the LTD4 receptor. ICI-204,219 was 5 to 10 times more potent than MK-0571 (IC50 values of 1.1 and 9.3 nM, respectively), in agreement with results from radioligand binding studies reported separately.
Specific binding sites for [3H]leukotriene (LT)D4 and [3H]LTC4 have been identified in sheep lung parenchymal membranes. [3H] LTD4 specific binding was of high affinity (KD = 0.56 nM), saturable (Bmax = 43 fmol/mg of protein), stimulated by divalent cations and inhibited by nonhydrolyzable GTP analogs. LTs and LTD4-receptor antagonists competed for [3H]LTD4 specific binding with the rank order of potency predicted for the LTD4 receptor: LTD4 > ONO-1078 > ICI 204,219 > MK-571 > LTE4 > LTC4 > BAY u9773 >> LTB4. In contrast, [3H]LTC4 specific binding was of lower affinity (KD = 27 nM), abundant (Bmax = 87 pmol/mg of protein) and although stimulated by divalent cations was unaffected by GTP analogs. LTs and LTC4 analogs competed for [3H]LTC4 specific binding with the following rank order of potency: LTC2 > LTC3 > LTC4 > LTC5 >> N-methyl-LTC4 >> LTD4 approximately LTB4 approximately LTB4. [3H]LTD4 specific binding to sheep lung membranes has, therefore, the characteristics of being to a G-protein-coupled LTD4 receptor, whereas the profile of [3H]LTC4 specific binding strongly suggests that these sites are not LT-receptor related. Photolabeling of sheep lung membranes using [125I]azido-LTC4, a photoactivable LTC4 analog, resulted in the selective photolabeling of two polypeptides migrating at 30 kDa and 19 kDa. The selective photolabeling of the 19 kDa polypeptide could be modulated in an identical manner to [3H]LTC4 specific binding. This protein is, therefore, a candidate for being the principal [3H]LTC4 specific site in sheep lung membranes and has a comparable molecular mass to microsomal glutathione S-transferase, recently shown to be the predominant LTC4 binding protein in cellular membranes.
A functional cDNA clone coding for the human prostaglandin E receptor EP1 subtype has been isolated from a human erythroleukemia cell cDNA library probed by low-stringency hybridization using a polymerase chain reaction fragment of the human thromboxane receptor. The human EP1 receptor is comprised of 402 amino acids with a predicted molecular mass of 41,858 and has the topography common to all G-protein-coupled receptors with seven predicted transmembrane spanning domains. Prostaglandin (PG) E2 challenge of Xenopus oocytes injected with EP1 cDNA resulted in an increase in intracellular Ca2+. In addition, the rank order of potency for prostaglandins in competition for [3H]PGE2 specific binding to membranes prepared from EP1 cDNA transfected COS cells was PGE2 > PGE1 > PGF2 alpha > PGD2. Furthermore, the EP1 receptor-selective antagonists AH 6809 and SC19220 were more potent than the EP2 receptor-selective agonist butaprost in these competition binding assays. In summary, therefore, we have cloned the human EP1 receptor subtype which is functionally coupled to an increase in intracellular Ca2+.
The human acute monocytic leukemia cell line THP-1 has been identified, by radioligand binding, as expressing the leukotriene D4 receptor at a high level (4000 binding sites per cell), without the need for further cell differentiation. [3H]Leukotriene D4-specific binding to THP-1 cell membranes was of high affinity (KD = 0.47 nM) and saturable, enhanced by divalent cations but inhibited by both monovalent cations and non-hydrolyzable GTP analogs. The cysteinyl leukotrienes competed for [3H]leukotriene D4-specific binding with the following rank order of potency: leukotriene D4 >> leukotriene E4 > leukotriene C4. In addition, leukotriene D4-receptor antagonists from two structural classes, the quinolines MK-571 and L-697,008, and the indole ICI 204,219, displayed nanomolar potency in [3H]leukotriene D4 competition assays. These data show that [3H]leukotriene D4-specific binding to THP-1 cell membranes fulfils the criteria for binding to a leukotriene D4 receptor regulated through interaction with a G protein. Several novel features of the THP-1 leukotriene D4 receptor were investigated. Culture of THP-1 cells in the presence of tunicamycin, an inhibitor of N-glycosylation, resulted in a 6-fold decrease in the number of detectable [3H]leukotriene D4-specific binding sites. Target-size analysis by radiation inactivation estimated a molecular mass of 65 kDa for the [3H]leukotriene D4 specific binding site(s) present in THP-1 cell membranes. Together, these results suggest that the human THP-1 cell leukotriene D4 receptor is a glycosylated protein with a molecular mass of approx. 65 kDa within the membrane environment.
The leukotriene (LT)D4 receptor has been defined as a G-protein-coupled receptor. In order to characterize this receptor, an iodinated, photoactivatable azido derivative of LTD4 (125I-azido-LTD4) has been synthesized for use as a photoaffinity probe. The characteristics of 125I-azido-LTD4 specific binding to guinea pig lung membranes were directly comparable to those of [3H]LTD4 specific binding to this tissue. 125I-Azido-LTD4 specific binding was saturable and of high affinity, enhanced by divalent cations and inhibited by sodium ions, but not potassium ions. 125I-Azido-LTD4 specific binding was also strongly inhibited by the nonhydrolyzable GTP analog, GTP gamma S, with ATP gamma S being 100-fold less potent, suggesting this inhibition was due to selective interaction with a G-protein. The cysteinyl leukotrienes competed for 125I-azido-LTD4 specific binding to guinea pig lung membranes with the following rank order of potency: LTD4 > LTE4 > LTC4, while the non-cysteinyl LTB4 was virtually inactive. Two structurally different LTD4 receptor antagonists, MK-571 and ICI 204,219, also competed for 125I-azido-LTD4 specific binding with nanomolar potency, whereas the leukotriene synthesis inhibitor, MK-886, was 10,000-fold less active. These data are in agreement with 125I-azido-LTD4 binding specifically to a G-protein-coupled LTD4 receptor. Photolysis of 125I-azido-LTD4 under equilibrium binding conditions resulted in the selective radiolabeling of a 45-kDa guinea pig lung membrane protein. The photolabeling of this 45-kDa protein was saturable, modulated by cations and inhibited by nucleotide analogs in an analogous way to 125I-azido-LTD4 specific binding. In addition, the photolabeling of this protein was inhibited in a concentration-dependent manner by all competing ligands, with the same rank order of potency and IC50 values as determined in the 125I-azido-LTD4 binding assay. It is proposed, therefore, that this novel 45-kDa protein is the guinea pig lung LTD4 receptor.
The leukotriene D4 receptor has been fully characterized by radioligand binding in membrane preparations from dimethyl sulphoxide-differentiated U937 cells, a human monocyte leukemia cell line, and, in parallel experiments, compared with leukotriene D4 receptor found in human lung and guinea-pig lung preparations. [3H]Leukotriene D4 specific binding in differentiated U937 cell membranes is of high affinity (KD = 0.35 nM), saturable (Bmax = 287 fmol/mg protein), with differentiation resulting in a 3-5-fold increase in the number of detectable binding sites. [3H]Leukotriene D4-specific binding in differentiated U937 cell membranes displays several features of G-protein-coupled receptors, being inhibited by GTP analogues and sodium ions, but increased by divalent cations. These characteristics are shared with [3H]leukotriene D4-specific binding in human and guinea-pig lung preparations. However, differences between these leukotriene D4 receptor types were observed. [3H]Leukotriene D4 equilibrium binding to differentiated U937 cell membranes could be dissociated to non-specific binding levels by 1000-fold excess of competing ligand, whereas binding to guinea-pig lung membranes was only partially dissociated under these conditions. In addition, differences in potency were demonstrated in competition studies with leukotriene E4 and leukotriene C4, although leukotriene D4 and the leukotriene D4-receptor antagonists MK-571 and ICI 204,219 were equipotent in competing for [3H]leukotriene D4-specific binding in all three membranes preparations. In conclusion, the leukotriene D4 receptor in differentiated U937 cell membranes resembles that in human lung, validating the use of this cell line as a suitable source of receptor in the development of potent specific antagonists.