The specific binding of the platelet-activating factor (PAF) receptor antagonist WEB 2086 and the benzodiazepine flunitrazepam to rat hepatocytes

Thieno-triazolodiazepines WEB 2086 and BN 50739 have been described as the potent PAF receptor antagonists. Binding of radiolabeled [³H]WEB 2086 has been widely employed to characterize PAF receptors in different cells. In a search for a PAF receptor in isolated rat hepatocytes, we discovered that the binding of [³H]WEB to rat hepatocytes was highly specific but had a relatively low affinity with a K_d of 113 nM and B_max of 0.65 pmol/10⁶ cells in freshly isolated cell suspension and K_d of 1.65 μM and B_max of 2.0 pmol/plate in cultured hepatocytes. No consistent specific binding of [³H]PAF itself was found in the same cell preparations. The binding of [³H]flunitrazepam in the presence of the peripheral type of benzodiazepine receptor antagonist Ro 5-4864 was saturated and exhibited a K_i of 3.8 nM and B_max of 3.5 pmol/plate. The central type of benzodiazepine receptor antagonist clonazepam also competed for the [³H]flunitrazepam binding, however with a much lower affinity. Various antagonists inhibited the binding of [³H]WEB 2086 with a rank order BN 50739⪢Ro 5-4864≥clonazepam. Interestingly, bicuculline, a specific antagonist of GABA(A) recognition sites, also significantly reduced the binding of [³H]WEB 2086. The binding of [³H]flunitrazepam was inhibited with a rank potency BN 50739⪢WEB 2086. Taken together, these findings suggest that the specific binding of PAF receptor antagonists WEB 2086 and BN 50739 in rat hepatocytes does not involve PAF receptors and occurs via peripheral benzodiazepine and, possibly GABA(A) receptor sites.     

Attenuation of amiodarone induced lung fibrosis and phospholipidosis in hamsters, by treatment with the platelet activating factor receptor antagonist, WEB 2086

Therapeutic use of amiodarone (AMD), a Class III antiarrhythmic drug is complicated by the development of lung fibrosis (LF) and phospholipidosis (PL). In the present study, the effectiveness of a PAF antagonist, WEB 2086, against AMD induced LF and PL has been tested in hamsters. The animals were randomly divided into four groups: (1) saline + H(2)O; (2) WEB + H(2)O; (3) saline + AMD; and (4) WEB + AMD. Saline or WEB (10 mg/kg i.p.) was given 2 days prior to intratracheal instillation of water or AMD (1.5 mumol/0.25 ml/100 g BW) and thereafter daily throughout the study. Twenty-eight days after intratracheal instillation, the animals were killed and the lungs processed for various assays. The amount of lung hydroxyproline, an index of LF, in saline + H(2)O, WEB + H(2)O, saline + AMD, and WEB + AMD groups were 959 +/- 46, 1035 +/- 51, 1605 +/- 85 and 1374 +/- 69 mug/lung, respectively. Total lung PL, an index of phospholipidosis, in the corresponding groups were 8.4 +/- 0.4, 8.3 +/- 0.3, 11.7 +/- 0.3 and 9.9 mug/lung. Lung malondialdehyde, an index of lipid peroxidation and superoxide dismutase activity in saline + H(2)O WEB + H(2)O, saline + AMD, and WEB + AMD were 93.0 +/- 4.3, 93.0 +/- 2.7, 138.9 +/- 6.0 and 109.0 +/- 3.8 nmol/lung and 359.7 +/- 13.9, 394.0 +/- 22.8, 497.5 +/- 19.7 and 425.5 +/- 4.9 units/lung, respectively. Administration of AMD alone caused significant increases in all the above indexes of lung toxicity, and treatment with WEB 2086 minimized the AMD induced toxicity as reflected by significant decreases in these indexes. Histopathological studies revealed a marked reduction in the extent and severity of lung lesions in the WEB + AMD group compared with the saline + AMD group. Treatment with WEB 2086 also reduced the acute mortality from 35% in saline + AMD group to 22% in WEB + AMD group. It was concluded that PAF is involved in the AMD induced lung fibrosis and phospholipidosis and that the PAF receptor antagonist may, therefore, be potentially useful in reducing AMD induced lung toxicity.     

Characterization of N-formyl-methionyl-leucyl-phenylalanine receptors on human neutrophils. Effects of isolation and temperature on receptor expression and functional activity

The study of polymorphonuclear neutrophil (PMN) surface receptor expression provides a means for the assessment of PMN function and state of cellular activation. In this study, we characterized binding of the chemotactic peptide FMLP to whole PMN, with particular attention to those variables that may account for the wide variation reported in the literature. These included avoidance of oxidized FMLP as a radioligand contaminant, determination of the optimal cold ligand concentration necessary for achieving minimal nonspecific binding throughout the range of radioligand concentrations used in saturation experiments (greater than or equal to 5 x 10(-5) M), avoidance of radioligand concentrations that equal or exceed receptor saturation and are not suitable for Scatchard analysis (greater than or equal to 60 to 80 nM), and avoidance of inadvertent receptor mobilization due to room temperature PMN isolation techniques and cell warming. PMN isolated and maintained at 4 degrees C expressed a single, high affinity population of FMLP receptors (approximately 6000 receptors per cell) with a KD of 15.5 nM. These characteristics, and in particular the single-affinity nature of the expressed FMLP receptor site, were derived from saturation experiments and confirmed with agonist competition studies. PMN subjected to room temperature isolation or 37 degrees C warming exhibited a 2.5-fold increase in FMLP receptor expression (approximately 15,000 receptors per cell) without changes in receptor affinity. These latter PMN, in correlation with increased receptor expression, had increased initial, maximal rates of FMLP-induced superoxide generation (10.2 vs 6.3 nmol/min/10(6) PMN for cells isolated and maintained at 4 degrees C) as a manifestation of their functional activation. The avoidance of inadvertent cellular activation during PMN isolation is essential to studies of PMN function, activation and the role of FMLP receptor expression/mobilization in these processes.     

Pharmacological actions of Y-24180, a new specific antagonist of platelet activating factor (PAF): II. Interactions with PAF and benzodiazepine receptors

The inhibitory effect of Y-24180, 4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6,9-dimethyl-6H-t hieno [3,2-f][1,2,4]triazolo [4,3-a][1,4]diazepine, on platelet activating factor (PAF)-induced platelet aggregation and the specific binding of 3H-PAF to platelets was compared with other thienodiazepine derivatives, WEB 2086 and etizolam. Y-24180 inhibited PAF-induced rabbit platelet aggregation in vitro (IC50 3.84 nM), but had little effect on adenosine diphosphate- or arachidonic acid-induced aggregation. WEB 2086 and etizolam also showed an inhibitory effect of PAF-induced aggregation (IC50 values are 456 and 6730 nM, respectively). In PAF-induced human platelet aggregation, Y-24180 (IC50 0.84 nM) was more potent than WEB 2086 (IC50 4.21 nM) and etizolam (IC50 998 nM). Y-24180, WEB 2086 and etizolam displaced 3H-PAF binding from the washed-platelets of rabbits with an IC50 value of 3.50, 9.35 and 29.5 nM, respectively. In rabbits, pretreatment with Y-24180 and WEB 2086 antagonized PAF-induced platelet aggregation dose-dependently. The significant inhibitory effect of Y-24180 (1 mg/kg, p.o.) lasted 72 hr after a single dose oral administration. WEB 2086 (10 mg/kg, p.o.) also antagonized the ex vivo response induced by PAF 1 hr after administration, but no significant effect was observed 3 hr after administration. Y-24180 displaced 3H-diazepam binding from the synaptosomal membranes of rat cerebral cortex with a Ki value of 3.68 microM. The affinity of Y-24180 for benzodiazepine(BZP) receptors was lower than those of WEB 2086 and etizolam and was about 1000 times lower than that for PAF receptors in platelets.     

Pharmacological actions of Y-24180, a new specific antagonist of Platelet Activating Factor (PAF): II. Interactions with PAF and benzodiazepine receptors

The inhibitory effect of Y-24180, 4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine, on platelet activating factor (PAF)-induced platelet aggregation and the specific binding of ³H-PAF to platelets was compared with other thienodiazepine derivatives, WEB 2086 and etizolam. Y-24180 inhibited PAF-induced rabbit platelet aggregation in vitro (IC₅₀ 3.84 nM), but had little effect on adenosine diphosphate- or arachidonic acid-induced aggregation. WEB 2086 and etizolam also showed an inhibitory effect of PAF-induced aggregation (IC₅₀ values are 456 and 6730 nM, respectively). In PAF-induced human platelet aggregation, Y-24180 (IC₅₀ 0.84 nM) was more potent than WEB 2086 (IC₅₀ 4.21 nM) and etizolam (IC₅₀ 998 nM). Y-24180, WEB 2086 and etizolam displaced ³H-PAF binding from the washed-platelets of rabbits with an IC₅₀ value of 3.50, 9.35 and 29.5 nM, respective- ly. In rabbits, pretreatment with Y-24180 and WEB 2086 antagonized PAF-induced platelet aggregation dose-dependently. The significant inhibitory effect of Y-24180(1 mg/kg, p.o.) lasted 72 hr after a single dose oral administration. WEB 2086 (10 mg/kg, p.o.) also antagonized the ex vivo response induced by PAF 1 hr after administration, but no significant effect was observed 3 hr after administration. Y-24180 displaced ³H-diazepam binding from the synaptosomal membranes of rat cerebral cortex with a Ki value of 3.68 μ M. The affinity of Y-24180 for benzodiazepine(BZP) receptors was lower than those of WEB 2086 and etizolam and was about 1000 times lower than that for PAF receptors in platelets.     

Characterization of specific binding sites for PAF in the iris and ciliary body of rabbit

The protective effect exerted by BN 52021 a specific PAF-receptor antagonist in experimentally induced ocular inflammatory disorders led us to investigate the possible presence of specific receptors for PAF in rabbit iris and ciliary body. Two classes of PAF binding sites were found in isolated iris and ciliary process of pigmented rabbit eyes: a high affinity site Kd1 congruent to 4.9 +/- 0.47 nM, Bmax1 congruent to 3.17 +/- 0.50 pmoles/mg protein, a low affinity sites Kd2 congruent to 11.6 +/- 0.33 nM, Bmax2 congruent to 12.46 +/- 2.3 pmoles/mg protein for iris. The specific binding was not affected by lyso-PAF the biologically inactive precursor and metabolite of PAF, up to 10(-6) M; inhibition by unlabelled PAF demonstrated a biphasic curve partially antagonized by BN 52021. The present results demonstrate the presence of specific binding sites for PAF in rabbit eyes which could mediate the action of this mediator in eye inflammatory processes and explain the protective effect observed with BN 52021.     

Characterization of a specific adenosine receptor on human lymphocytes.

We have examined the mechanism of action of adenosine, a naturally occurring nucleoside that has profound effects on lymphocyte function. Adenosine (0.01 micrometer to 10 micrometer) increased lymphocytes cAMP levels in a dose-dependent fashion with a maximal (10 micrometer) increase of about 4-fold, whereas adenine, guanosine, and inosine had no effect on lymphocyte cAMP levels at concentrations of 100 micrometer. Adenosine appears to act on the cell surface since 1) 2-chloroadenosine, a poorly metabolized adenosine analogue, was as active as adenosine and 2) dipyridamole, which markedly inhibited [3H]-adenosine uptake by human lymphocytes, did not affect adenosine-induced accumulation of cAMP. The specificity of the adenosine effect was established by showing that the methylxanthine derivatives, theophylline and 3-isobutyl-1-methylxanthine (IBMX), specifically block the accumulation of cAMP in lymphocytes induced by adenosine. Theophylline is a competitive inhibitor of the effect of adenosine, with an estimated dissociation constant of theophylline-receptor complex of about 6.3 X 10(-7) M. The results suggest that adenosine increases the intracellular cAMP content of lymphocytes as a result of its interaction with a specific membrane receptor which results in the activation of adenylate cyclase.     

Differences between agonist and antagonist binding following beta-adrenergic receptor desensitization.

The specific beta-adrenergic agonist radioligand (+/-)-[3H]hydroxybenzylisoproterenol ([3H]HBI) was used to investigate alterations in the beta-adrenergic receptors of frog erythrocytes occurring during the process of agonist-induced, receptor-specific desensitization. There was close agreement between the percentage fall in [3H]HBI binding and that in catecholamine-stimulated adenylate cyclase activity following periods of preincubation of up to 7 h with 0.1 mM (-)-isoproterenol. Desensitization was maximal by 5 h, resulting in a 69% reduction in [3H]HBI binding and a 67% reduction in isoproterenol-stimulated adenylate cyclase activity. In contrast, binding of the beta-adrenergic antagonist (-)-[3H]dihydroalprenolol was significantly less affected by desensitization (p is less than 0.05 at 2 1/2, 5, and 7 h), showing a maximum reduction in binding of only 35% in these experiments. The consistent close agreement of reduction in agonist binding with that in hormone-stimulated adenylate cyclase activity, together with the significant difference observed between agonist and antagonist binding, implies that an alteration occurs during desensitization which preferentially interferes with agonist binding, while antagonist binding is less affected. The locus of this agonist-specific alteration may be the receptor binding site or a site involved in receptor-enzyme coupling. Agonist binding studies may now be used to assess more completely the desensitized state of beta-adrenergic receptors in systems in which marked desensitization of beta-adrenergic responses is associated with little or no reduction in antagonist binding.     

Characterization of ligand binding,DNA binding and phosphorylation of progesterone receptor by two novel progesterone receptor antagonist ligands

In order to gain a better understanding of the distinctive mechanisms of the various types of antiprogestins, we have characterized in vitro ligand binding, specific DNA binding and phosphorylation of progesterone receptor (PR) from T47D cells after treatment of cells with progestins (progesterone, R5020) and antiprogestins (RU486, ZK98299, Org 31806 and Org 31710). Treatment of the cells with R5020 or PR antagonists, with the exception of ZK98299, resulted in a quantitative upshift of PR-A and PR-B indicative of ligand/DNA-induced phosphorylation of PR. Treatment of cells with RU486, Org 31710 or Org 31806, but not R5020 or ZK98299 resulted in detectable PR-progesterone response element complexes (PR-PREc) as assessed by gel mobility shift assay. Although treatment of cells with ZK98299, a type I PR antagonist, did not induce phosphorylation, the antiprogestins, Org 31806 and Org 31710, in a manner identical to RU486, did. Our data suggest that Org 31806 and Org 31710 affect propertie s of PR from T47D cells that are similar to RU486. (Mol Cell Biochem 175: 205–212, 1997)     

Characterization of a novel, non-peptidyl antagonist of the human glucagon receptor

We have identified a series of potent, orally bioavailable, non-peptidyl, triarylimidazole and triarylpyrrole glucagon receptor antagonists. 2-(4-Pyridyl)-5-(4-chlorophenyl)-3-(5-bromo-2-propyloxyphenyl)p yrr ole (L-168,049), a prototypical member of this series, inhibits binding of labeled glucagon to the human glucagon receptor with an IC50 = 3. 7 +/- 3.4 nM (n = 7) but does not inhibit binding of labeled glucagon-like peptide to the highly homologous human glucagon-like peptide receptor at concentrations up to 10 microM. The binding affinity of L-168,049 for the human glucagon receptor is decreased 24-fold by the inclusion of divalent cations (5 mM). L-168,049 increases the apparent EC50 for glucagon stimulation of adenylyl cyclase in Chinese hamster ovary cells expressing the human glucagon receptor and decreases the maximal glucagon stimulation observed, with a Kb (concentration of antagonist that shifts the agonist dose-response 2-fold) of 25 nM. These data suggest that L-168,049 is a noncompetitive antagonist of glucagon action. Inclusion of L-168, 049 increases the rate of dissociation of labeled glucagon from the receptor 4-fold, confirming that the compound is a noncompetitive glucagon antagonist. In addition, we have identified two putative transmembrane domain residues, phenylalanine 184 in transmembrane domain 2 and tyrosine 239 in transmembrane domain 3, for which substitution by alanine reduces the affinity of L-168,049 46- and 4. 5-fold, respectively. These mutations do not alter the binding of labeled glucagon, suggesting that the binding sites for glucagon and L-168,049 are distinct.     

Localization of agonist and antagonist binding domains of the human neurokinin-1 receptor.

To identify the molecular determinants of ligand-receptor interactions, the extracellular domain of the human neurokinin-1 receptor was systematically substituted with the corresponding sequences from the other two neurokinin receptor subtypes. Three residues within the first extracellular segment and 2 residues of the second segment are required for the optimal binding of all three natural peptide agonists. The divergent nature of 4 of the 5 residues supports the hypothesis that the peptide binding site on the neurokinin-1 receptor is not highly conserved in the other two receptor subtypes. In contrast, substitution of part of the third extracellular segment and the fourth extracellular segment with the corresponding amino acids of the human neurokinin-3 receptor results in an increase in neurokinin B affinity without affecting substance P binding, suggesting that the two peptides do not interact with the same set of functional groups on the receptor. Among the four extracellular regions, only parts of the third and fourth segments affect the binding of the quinuclidine antagonist L-703,606, and these two regions may partially account for the neurokinin-1 receptor subtype specificity of this non-peptide antagonist. These studies demonstrate that both the extracellular and transmembrane domains of the neurokinin-1 receptor are involved in the binding of substance P and related peptides.     

Activation of NADPH oxidase in human neutrophils. Synergism between fMLP and the neutrophil products PAF and LTB4

The induction of the respiratory burst in human neutrophils by combinations of fMLP and either PAF or LTB4 was studied. Pretreatment with PAF (0.0001 to 10 uM), which by itself did not elicit the burst, greatly enhanced the rate and extent of fMLP-induced superoxide production. A synergism of a different kind was observed with the reversed stimulus sequence: Pretreatment with fMLP made the neutrophils capable to respond to PAF with superoxide production. A moderate enhancement of the fMLP response was also obtained following pretreatment with LTB4. The response of the cells to LTB4, however, was not influenced by fMLP, and no synergism was observed between the two neutrophil products PAF and LTB4. The results of this study demonstrate a marked synergism between fMLP and PAF and suggest that PAF may function as an amplifier of the respiratory burst response of stimulated neutrophils.     

Characterization of receptor for granulocyte colony-stimulating factor on human circulating neutrophils

We have made a mutein of human G-CSF with more stable, and potent biological activity. Using 125 I-labeled mutein human G-CSF, high affinity binding sites were identified on human circulating neutrophils. Receptor number per cell was 560 with a Kd of 250 pM. The human G-CSF receptor was identified as a single subunit protein of Mr approximately 150,000.     

Characterization of the modes of binding between human sweet taste receptor and low-molecular-weight sweet compounds

One of the most distinctive features of human sweet taste perception is its broad tuning to chemically diverse compounds ranging from low-molecular-weight sweeteners to sweet-tasting proteins. Many reports suggest that the human sweet taste receptor (hT1R2-hT1R3), a heteromeric complex composed of T1R2 and T1R3 subunits belonging to the class C G protein-coupled receptor family, has multiple binding sites for these sweeteners. However, it remains unclear how the same receptor recognizes such diverse structures. Here we aim to characterize the modes of binding between hT1R2-hT1R3 and low-molecular-weight sweet compounds by functional analysis of a series of site-directed mutants and by molecular modeling-based docking simulation at the binding pocket formed on the large extracellular amino-terminal domain (ATD) of hT1R2. We successfully determined the amino acid residues responsible for binding to sweeteners in the cleft of hT1R2 ATD. Our results suggest that individual ligands have sets of specific residues for binding in correspondence with the chemical structures and other residues responsible for interacting with multiple ligands.     

Different antagonist binding properties of human and rat histamine H3 receptors

Different histamine H3-receptor antagonists have been tested in displacement studies at human and rat H3 receptors in stably transfected cells. Based on an actual rhodopsin structure, models for receptor antagonist interaction were developed for receptors of both species. Similarities and discrepancies in binding profiles can be explained, but not quantified by hydrophilic interactions with Asp114 and an important lipophilic binding pocket modified by two nearby amino acids.     

Characterization of binding of a specific antagonist, [3H]-SQ 29,548, to soluble thromboxane A2/prostaglandin H2 (TP) receptors in human platelet membranes.

Binding of [3H]-SQ 29,548 was characterized to soluble thromboxane A2/prostaglandin H2 (TP) receptors from human platelet membranes as a means of examining ligand-receptor interactions outside the lipophilic environment of the cell membrane. Kinetic determination revealed a rate of ligand-receptor association of 1.4 x 10(7) +/- 0.2 M-1 x min-1 and a rate of dissociation of 0.5 +/- 0.07 min-1. The resultant equilibrium affinity constant was 36.3 +/- 5.8 nM. Saturation binding analysis revealed a single class of [3H]-SQ 29,548 binding sites with an affinity constant of 39.7 +/- 4.3 nM and a B(max) of 1735.7 +/- 69.1 fmol/mg protein. Specific [3H]-SQ 29,548 binding was inhibited by specific TP receptor antagonists and agonists in a rank order of potency similar to that seen in platelet membranes: SQ 33,961 much greater than SQ 29,548 greater than BM 13,505 greater than or equal to U 46619 greater than BM 13,177. PGD2, PGE2 and PGI2 did not appreciably inhibit the specific binding of [3H]-SQ 29,548. These data indicate that [3H]-SQ 29,548 binding to soluble human platelet TP receptors was specific, saturable, and reversible.     

Anion binding and controlled aggregation of human interleukin-1 receptor antagonist

Highly concentrated human recombinant interleukin-1 receptor antagonist (IL-1ra) aggregates at elevated temperature without perturbation in its secondary structure. The protein aggregation can be suppressed depending on the buffer ionic strength and the type of anion present in the sample solution. Phosphate is an approximately 4-fold weaker suppressant than either citrate or pyrophosphate on the basis of the measured protein aggregation rates. This is in agreement with the strength of protein-anion interactions at the IL-1ra single anion-binding site as judged by the estimated dissociation constant values of 2.9 mM, 3.8 mM, and 13.7 mM for pyrophosphate, citrate, and phosphate, respectively. The strength of binding also correlates with the anion size and with the number of ionized groups available per molecule at a given pH. Affinity probing of IL-1ra with methyl acetyl phosphate (MAP) in combination with proteolytic digestion and mass spectral analysis show that an anion-binding site location on the IL-1ra surface is contributed by lysine-93 and lysine-96 of the loop 84-98 as well as by lysine-6 of the unstructured N-terminal region 1-7. The replacement of lysine-93 with alanine by site-directed mutagenesis results in dramatically suppressed IL-1ra aggregation. Furthermore, when the unstructured N-terminal region of IL-1ra is removed by limited proteolysis, a 2-fold increase in the time course of the aggregation lag phase is observed for the truncated protein. An anion-controlled mechanism of IL-1ra aggregation is proposed by which the anion competition for the protein cationic site prevents formation of intermolecular cation-pi interactions and, thus, interferes with the protein asymmetric self-association pathway.     

Correlation between trypsin binding to a specific receptor and prostacyclin production in cultured vascular endothelial cells

The correlation between the binding and processing of trypsin and its effect on prostacyclin (PGI2) production in cultured adult bovine aortic endothelial (ABAE) cells was studied. ABAE cells demonstrated an ability to produce PGI2 in a dose-response manner to trypsin at the range of 0.1-2.0 micrograms/ml with a saturation at a concentration of 1 microgram/ml. Likewise, 125I-trypsin binding to the cultured cells increased in a dose-response way and reached saturation at a concentration of about 1 microgram/ml; 125I-trypsin was bound to a specific high-affinity cell-surface receptor with a dissociation constant (Kd) of 1.5 X 10(-8) M and each of the confluent ABAE cells has about 1.2 X 10(5) such receptors sites. The cell-surface receptor for trypsin displayed specific characteristics and an excess amount of unlabeled trypsin successfully abolished 125I-trypsin binding while thrombin in excess failed to compete for 125I-trypsin binding. Only a small fraction of the cell-surface-bound 125I-trypsin was internalized and subsequently degraded by ABAE cells as compared to the process of 125I-trypsin internalization by human skin fibroblasts (HSF). This study demonstrated that the stimulatory effect of trypsin on prostacyclin production and release by ABAE cells might be mediated by a specific cell-surface receptor for trypsin on these cells distinct from the thrombin receptor.     

Analysis of the relation between receptor binding affinity and antagonist efficacy of antiglucocorticoids

The biological potencies of four antiglucocorticoids, RU486 (RU), dexamethasone-oxetanone (DOX), R5020, and progesterone have been studied with respect to dexamethasone induction of tyrosine aminotransferase (TAT) in rat hepatoma tissue culture (HTC) cells. Their inhibitory effects in whole-cell competition binding studies (at 37 degrees C) and in TAT induction studies were analyzed by Dixon plots and Schild plots, respectively. We show that: In both cases, there is an actual competition of each antiglucocorticoid with the agonist dexamethasone for the same binding site; the two Kd values derived from the two plots are almost identical for each antiglucocorticoid; RU486 can be distinguished from the three other antiglucocorticoids by its high biological efficacy and its high affinity for the glucocorticoid receptor in whole cells at 37 degrees C (identical to its affinity in cytosol at 0 degree C). These results imply that: There is a linear correlation between the antagonist efficacies of antiglucocorticoids and their affinities for the glucocorticoid receptor in whole cells at 37 degrees C; the antagonistic action is solely mediated by competition with the agonist for the receptor binding site; this is verified by the fact that in all cases, in the presence or absence of antiglucocorticoids, a specific TAT induction level was always related to the same level of receptor saturation by the agonist in whole cells; the phenomena responsible for the high antagonist efficacy of RU486 are also responsible for its high affinity in whole cells at 37 degrees C.