Brain leukotriene C4 binding sites are S-alkylglutathione binding sites

Leukotriene C4 binding to mouse brain membranes was readily displaced by S-alkylglutathione derivatives, with the affinity of the test compound increasing as the alkyl chain length increases. S-decylglutathione was as potent as leukotriene C4. These data suggest that brain membrane leukotriene C4 binding sites are S-alkylglutathione binding sites, possibly microsomal glutathione transferase.     

Post soluble binding to the leukotriene D4 receptor from guinea pig lung membranes

Guinea pig lung membranes were extracted with 1% digitonin and yielded a preparation that contained soluble leukotriene D4 (LTD4) receptor. Specific binding of the high affinity radiolabeled receptor antagonist [3H]ICI-198615 to the soluble LTD4 receptor was time dependent and reversible. The dissociation constant (Kd) and the density (Bmax) of [3H]ICI-198615 binding to the soluble LTD4 receptor was 0.2 +/- 0.08 nM and 380 +/- 40 fmol/mg of protein, respectively. Radioligand competition studies showed several classes of structurally diverse, functionally defined, receptor antagonists competed with [3H]ICI-198615 binding to the soluble receptor. The rank order of potency and specificity of these antagonists in binding to the soluble receptor were equivalent to those determined from the membrane-bound receptor binding assay and from the smooth muscle contraction assay. Binding of LTD4 to the soluble receptor was observed, in the competition assay, only in the low affinity state (Ki = 2 microM). Size-exclusion chromatography of the soluble LTD4 receptor showed that the apparent molecular weight of the LTD4 receptor in digitonin micelle was approximately 300,000.     

Regulation of ligand binding to leukotriene D4 receptors: effects of cations and guanine nucleotides

High affinity, stereoselective specific binding sites for [3H]leukotriene D4 [( 3H]LTD4) have been demonstrated in guinea pig lung membranes. Purine nucleotides quantitatively reduced [3H]LTD4 specific binding with a rank order potency of guanosine-5'-O-3-thiotriphosphate (GTP gamma S) = guanyl-5'-yl-imido-diphosphate [Gpp(NH)p] greater than GTP greater than ATP greater than GDP. In the presence of 1 microM Gpp(NH)p, the maximum number (Bmax) of [3H]LTD4 specific binding sites was reduced to 41 +/- 10 percent of the control level (950 +/- 150 fmol/mg membrane protein). In the presence of 3 microM Gpp(NH)p, the rate of association of [3H]LTD4 to the specific sites was estimated to have increased 2.5-fold. The rate of dissociation of [3H]LTD4 from the specific sites was also increased significantly in the presence of 50 microM Gpp(NH)p. The divalent cations, Ca2+ and Mg2+ (10 mM), increased the Bmax 2-fold and had minimal effects on the dissociation constant (Kd) of [3H]LTD4 specific binding. Sodium ions, at a concentration of 50 mM, reduced the Bmax, and had minimal effects on the Kd of [3H]LTD4 specific binding. These data indicate that guanine nucleotides, Na+, Mg2+ and Ca2+ regulate [3H]LTD4 binding to its receptors in guinea pig lung.     

Identification of multiple nitrobenzylthioinosine binding sites in guinea pig platelets: Comparison with binding in guinea pig erythrocytes

The novel existence of multiple binding sites for the potent nucleoside transporter Probe, [³H]nitrobenzylthioinosine, was identified in guinea pig platelet membranes and the binding characteristics compared to those of guinea pig erythrocyte membranes. Scatchard analysis of the binding in platelets reveled two high affinity binding sites with affinity constant (K_D) of 0.94 ± 0.07 nM and 83 ± 13 nM with corresponding maximal binding capacities (B_max) of 21 ± 7 and 110 ± 25 fmol/mg protein, respectively. In comparison, guinea pig erythrocyte membranes revealed a homogeneous population of the binding sites with K_D of 0.17 ± 0.04 nM and a B_max value of 73 ± 11 fmol/mg protein. Biphasic semi-log plots of the binding site heterogeneity in erythrocytes not reveled by Scatchard plots. Determination of the potencies of selected drugs in inhibiting the binding showed evidence of differential interacitons with the binding sites by various agents which may be exploited pharmacologically. © 1993 Wiley-Liss, Inc.     

High-affinity dextromethorphan binding sites in guinea pig brain. I. Initial characterization

Tritiated dextromethorphan ([3H]DM) binds to two distinct sites in guinea pig brain, a high-affinity site (Kd = 13-20 nM) and a low-affinity site (Kd greater than 200 nM). Binding of [3H] DM to the high-affinity site is rapid, reversible, saturable, proportional to tissue concentration, and pH-dependent. The sites have a protein-like component, since preincubating brain homogenate in the presence of proteolytic enzymes and protein-modifying reagents significantly reduces binding. There is also a progressive loss of binding when brain homogenate is heated to temperatures in excess of 37 degrees. Millimolar concentrations of lithium, calcium, magnesium, and manganese decrease DM binding while sodium, in concentrations as high as 100 mM, has little effect; calcium in micromolar concentrations slightly enhances binding. The pons-medulla and cerebellum contain the highest density of sites. Subcellular localization studies have shown that high-affinity sites are confined almost exclusively to the microsomal fraction. Binding of DM to brain microsomes does not appear to be related to drug-metabolizing enzymes. The characteristics of DM binding suggest that DM sites are not a subclass of opiate receptors. Studies using tritiated dextrorphan as radioligand failed to reveal a high-affinity binding site for in brain.     

Identification of specific binding sites for leukotriene C4 in membranes from human lung

Leukotriene C4 (LTC4), one of the major components of the slow-reacting substance of anaphylaxis (SRS-A), is a potent constrictor of bronchial smooth muscle in many species including humans. Here we report the identification and characterization of specific binding sites for LTC4 in membranes from human lung parenchyma. At 4 degrees, 3H-LTC4 binding is specific, saturable (Bmax = 32-41 pmoles/mg prot.), rapid (equilibrium being attained within 15 min), reversible and of high affinity (Kd = 3.6-7 X 10(-8) M). The binding sites are sensitive to heat and probably possess a protein moiety, being inactivated upon trypsinization. CaCl2 affects both the association and the dissociation rate and dose-dependently enhances the binding of 3H-LTC4 at equilibrium; maximal enhancement (4-fold) occurred at 10(-2)M CaCl2. Unlabelled LTC4 is able to complete with 3H-LTC4 for its binding sites with an IC50 of 7.8 X 10(-8) M. The addition of 10(-2) M CaCl2 increases the potency of LTC4 in inhibiting the binding (2.2-fold); both the competition curves are monophasic, indicating the existence of a homogeneous class of binding sites. In the presence of CaCl2, LTD4, LTE4 and the SRS-A antagonist FPL 55712 can inhibit 3H-LTC4 specific binding, being, however, less potent than LTC4 (IC50 S = 2.2 X 10(-6), 2.4 X 10(-5) M, for LTD4, LTE4 and FPL 55712, respectively). FPL 55712 displayed a competitive mechanism; its affinity, however, was lower if absorption to glass was not prevented. The present studies indicate that specific binding sites for 3H-LTC4 exist in human lung parenchyma, and that a receptor-mediated process might be involved in the bronchoconstriction induced by LTC4.     

Computer-assisted modeling of multiple dextromethorphan and sigma binding sites in guinea pig brain.

Computer-assisted, simultaneous analysis of self- and cross-displacement experiments demonstrated the existence of several binding sites in guinea pig brain for dextromethorphan, (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ((+)-3-PPP), and 1,3-di-o-tolyl guanidine (DTG). Dextromethorphan binds with high affinity to two sites (R1 Kd 50-83 and R2 Kd 8-19 nM) and with low affinity to two additional sites (R3 and R4). (+)-3-PPP binds to one high-affinity (R1 Kd 24-36 nM), to one intermediate-affinity (R3 Kd 210-320 nM), and to two (R2 and R4) low-affinity sites. DTG binds with almost identical high affinity to two different sites (R1 Kd 22-24 and R3 Kd 13-16 nM). These results confirm that dextromethorphan, (+)-3-PPP, and DTG bind to the common DM1/sigma 1 site (R1). The binding of DTG to two different sites with identical affinities precludes the use of this compound as a specific marker for sigma receptors. Besides, haloperidol displaces labeled ligands from both high-affinity DTG sites (R1 and R3) with high affinity. Thus, haloperidol sensitivity should not be used as the single criterion to identify a putative receptor. The resolution of these novel sites also may provide new insights into the multiple effects of antipsychotic drugs. In addition, this investigation has important implications regarding the methods that must be applied to characterize multiple binding sites and their relations with putative receptors.     

Effect of age on leukotriene B4 production in guinea pig whole blood.

We evaluated the effect of age on eicosanoid production in guinea pig blood. Heparinized blood from 7-10-day, 6-week, or 6-month-old guinea pigs was incubated with 150 microM arachidonic acid (AA) for 5 min, followed by stimulation with A23187 (20 micrograms/mL) for an additional 10 min at 37 degrees. The reaction was terminated by centrifugation, and the production of plasma leukotriene (LT) B4 and C4, prostaglandin E2 (PGE2), and thromboxane B2 (TXB2) was determined by enzyme-linked immunoassay (ELISA). LTC4, PGE2, and TXB2 formation were unaffected by age. In marked contrast, production of LTB4 was increased 4- to 5-fold as age increased from 7-10 days (9.51 +/- 2.07 ng/mL) or 6 weeks (8.83 +/- 1.81 ng/mL) to 6 months (40.57 +/- 9.66 ng/mL). To determine the effect of age on the total eicosanoid product profile, blood was stimulated in the presence of [14C]AA, and plasma metabolites were separated by reverse-phase high pressure liquid chromatography (RP-HPLC) and quantitated using on-line radiochemical detection. In addition to increased LTB4 production, a modest increase in 12-hydroxyeicosatetraenoic acid (12-HETE) production was also observed in the 6-month-old animals. Previous studies have demonstrated interference of 12-HETE in the immunoassay of LTB4. Therefore, to validate the authenticity of the plasma leukotriene ELISA measurements, samples were precipitated with methanol and fractionated by RP-HPLC. The fractions co-eluting with [3H]LTB4 or [3H]LTC4 were dried under vacuum and reconstituted in ELISA buffer, and leukotrienes were quantitated. As seen previously, following HPLC purification LTB4 production remained significantly elevated in the 6-month-old guinea pigs, whereas LTC4 production was unaffected by age. To further document the selectivity of this effect on LTB4 production, we evaluated the effect of increasing age on cyclooxygenase or phospholipase A2 (PLA2) activity. Neither cyclooxygenase nor PLA2 activity was elevated as animals matured. In conclusion, the capacity of whole blood to produce LTB4, but not LTC4, TXB2, or PGE2, was elevated markedly in older animals.     

Characterization of the binding of [3H](+)-pentazocine to sigma recognition sites in guinea pig brain.

The selective sigma compound (+)-pentazocine was radiolabeled and its binding characteristics in guinea pig brain membranes were investigated. [3H](+)-Pentazocine bound to a single high-affinity site with a KD of 2.9 nM and a Bmax of 1998 fmol/mg protein. Saturation was achieved at a ligand concentration of 15 nM. Maximal specific binding was observed at 37 degrees C and was greater than 90% of total binding. Equilibrium was reached by 120 min and dissociation was complete by 420 min, with a t1/2 of 121 min. Li+, Ca2+ and Mg2+ inhibited binding at high concentrations, and binding was insensitive to adenyl and guanyl nucleotides. Stereoselectivity was observed for the inhibition of binding by benzomorphans, 3-(3-hydroxyphenyl)-N-propylpiperidine and butaclamol, and the (+) enantiomers and alpha diastereomers of pentazocine and cyclazocine were more potent than their corresponding (-) enantiomers and beta diastereomers. The rank order of potency for the sigma reference agents to displace [3H](+)-pentazocine binding was similar to that reported using the [3H]sigma ligands dextromethorphan, 1,3-di(2-tolyl)guanidine and (+)-3-(3-hydroxyphenyl)-N-propylpiperidine. Haloperidol, (+)-pentazocine, (+)-3-(3-hydroxyphenyl)-N-propylpiperidine and rimcazole were competitive inhibitors of binding to the [3H](+)-pentazocine-defined sigma recognition site, suggesting that these different structural classes of compounds all bind to a single molecular entity.     

Opioid binding sites in the guinea pig and rat kidney: radioligand homogenate binding and autoradiography.

The specific binding of the selective mu-, delta-, and kappa-opioid ligands [3H][D-Ala2,MePhe4,Gly-ol5]enkephalin ([3H] DAGOL), [3H][D-Pen2,D-Pen5]enkephalin ([3H]DPDPE), and [3H]U69593, respectively, to crude membranes of the guinea pig and rat whole kidney, kidney cortex, and kidney medulla was investigated. In addition, the distribution of specific 3H-opioid binding sites in the guinea pig and rat kidney was visualized by autoradiography. Homogenate binding and autoradiography demonstrated the absence of mu- and kappa-opioid binding sites in the guinea pig kidney. No opioid binding sites were demonstrable in the rat kidney. In the guinea pig whole kidney, cortex, and medulla, saturation studies demonstrated that [3H]DPDPE bound with high affinity (KD = 2.6-3.5 nM) to an apparently homogeneous population of binding sites (Bmax = 8.4-30 fmol/mg of protein). Competition studies using several opioid compounds confirmed the nature of the delta-opioid binding site. Autoradiography experiments demonstrated that specific [3H]DPDPE binding sites were distributed radially in regions of the inner and outer medulla and at the corticomedullary junction of the guinea pig kidney. Computer-assisted image analysis of saturation data yielded KD values (4.5-5.0 nM) that were in good agreement with those obtained from the homogenate binding studies. Further investigation of the delta-opioid binding site in medulla homogenates, using agonist ([3H]DPDPE) and antagonist ([3H]diprenorphine) binding in the presence of Na+, Mg2+, and nucleotides, suggested that the delta-opioid site is linked to a second messenger system via a GTP-binding protein. Further studies are required to establish the precise localization of the delta binding site in the guinea pig kidney and to determine the nature of the second messenger linked to the GTP-binding protein in the medulla.     

Histamine, leukotriene D4 and platelet-activating factor in guinea pig passive cutaneous anaphylaxis.

The involvement of histamine, leukotriene D4 (LTD4) and platelet-activating factor (PAF) in cutaneous anaphylaxis was investigated in a guinea pig model. When given alone, the H1 receptor antagonist chlorpheniramine, the LTD4/E4 antagonist LY171883 and the PAF antagonist WEB2086 were unable to inhibit increased microvascular plasma protein leakage in passive cutaneous anaphylaxis (PCA) reactions, as monitored by the extravasation of intravenously injected 125I-albumin. Furthermore the H2 receptor antagonist cimetidine and the serotonin antagonist methysergide were unable to reduce PCA responses when given alone or in combination with chlorpheniramine. In marked contrast, combinations of antagonists were able to reduce plasma leakage significantly. A combination of chlorpheniramine, LY171883 and WEB2086 virtually abolished plasma leakage during the PCA response, but did not influence the plasma protein leakage induced by intradermal injection of bradykinin. These results demonstrate that these allergic reactions involve several mediators and that the inability of an individual mediator antagonist to reduce responses does not necessarily rule out a role for that mediator.     

Modulation of (+)-[3H]pentazocine binding to guinea pig cerebellum by divalent cations.

The ability of cations to modulate the binding of the sigma 1 receptor-selective ligand (+)-[3H]pentazocine to guinea pig cerebellum was investigated. Di- and trivalent cations biphasically inhibited (+)-[3H]pentazocine binding, revealing multiple affinity states. The rank order of potency of these cations (based on the high affinity component of inhibition) was Zn2+ > Co2+ > La3+ = Ni2+ = Cd2+ = Mn2+ = Gd2+ > Ba2+ = Sr2+ > Mg2+ > Ca2+. The inhibition of 1,3-[3H]di(2-tolyl)guanidine binding to the sigma 2 receptor by these cations differed qualitatively and quantitatively from their effects on (+)-[3H]pentazocine binding. Although monovalent cations decreased the Kd for (+)-[3H]pentazocine binding, divalent cations split (+)-[3H]pentazocine binding into low and high affinity components. The Bmax of the high affinity component decreased with increasing divalent cation concentrations. Both mono- and divalent cations significantly reduced the rate of association of (+)-[3H]pentazocine with the sigma 1 receptor without altering the dissociation rate. (+)-[3H]Pentazocine binding was not altered by guanine nucleotides or by treatment with cholera or pertussis toxins. However, nonselective cation channel blockers (cinnarizine, hydroxyzine, prenylamine, amiodarone, and proadifen) potently inhibited (+)-[3H]pentazocine binding. These results indicate that physiologically relevant concentrations of divalent cations allosterically modulate (+)-[3H]pentazocine binding to the sigma 1 receptor, to reveal multiple affinity states. These sites do not represent sigma 1 to sigma 2 subtype interconversion or ternary complex formation with guanine nucleotide-binding proteins. However, the rank order of cation potency and the inhibition of binding by cation channel blockers is consistent with a potential role for sigma receptors as constituents of cation channels.     

Sotalol potentiates the leukotriene C4-induced contractions and thromboxane A2 release of guinea pig lung parenchymal strips

The leukotriene C4 (LTC4)-induced contraction and thromboxane A2 (TxA2) release of the guinea pig lung parenchymal (GPLP) strip are both inhibited by the beta 2-adrenergic agent salbutamol. The effect of LTC4 is restored to nearly normal by the beta-adrenergic antagonist sotalol. The latter substance alone also induces a contraction in the GPLP strip and potentiates the contractions and the TxA2 release of LTC4. During the sotalol-induced contractions, no TxA2 release occurs. An antihistaminic, mepyramine had no effect on the sotalol-induced contraction. When sotalol is added repeatedly to a GPLP strip, only the first time a contraction occurs.     

Computer-assisted modeling of multiple dextromethorphan and sigma binding sites in guinea pig brain

Computer-assisted, simultaneous analysis of self- and cross-displacement experiments demonstrated the existence of several binding sites in guinea pig brain for dextromethorphan, (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ((+)-3-PPP)- and 1,3-di-o-totyl guanidine (DTG). Dextromethorphan binds with high affinity to two sites (R₁ K_d 50–83 and R₂ K_d 8–19 nM) and with low affinity to two additional sites (R₃ and R₄). (+)-3-PPP binds to one high-affinity (R₁ K_d 24–36 nM), to one intermediate-affinity (R₃ K_d 210–320 nM), and to two (R₂ and R₄) low-affinity sites. DTG binds with almost identical high affinity to two different sites (R₁ K_d 22–24 and R₃ K_d 13–16 nM). These results confirm that dextromethorphan, (+)-3-PPP, and DTG bind to the common DM₁/σ₁ site (R₁). The binding of DTG to two different sites with identical affinities precludes the use of this compound as a specific marker for σ receptors. Besides, haloperidol displaces labeled ligands from both high-affinity DTG sites (R₁ and R₃) with high affinity. Thus, haloperidol sensitivity should not be used as the single criterion to identify a putative receptor. The resolution of these novel sites also may provide new insights into the multiple effects of antipsychotic drugs. In addition, this investigation has important implications regarding the methods that must be applied to characterize multiple binding sites and their relations with putative receptors.     

Characterization of the cloned guinea pig leukotriene B4 receptor: comparison to its human orthologue

A cDNA clone coding for the guinea pig leukotriene B₄ (BLT) receptor has been isolated from a lung cDNA library. The guinea pig BLT receptor has an open reading frame corresponding to 348 amino acids and shares 73% and 70% identity with human and mouse BLT receptors, respectively. Scatchard analysis of membranes prepared from guinea pig and human BLT receptor-transfected human embryonic kidney (HEK) 293 EBNA (Epstein–Bar Virus Nuclear Antigen) cells showed that both receptors displayed high affinity for leukotriene B₄ (K_d value of 0.4 nM) and were expressed at high levels (B_max values ranging from 9 to 12 pmol/mg protein). The rank order of potency for leukotrienes and related analogs in competition for [³H]leukotriene B₄ specific binding at the recombinant guinea pig BLT receptor is leukotriene B₄>20-OH-leukotriene B₄>12(R)-HETE ((5Z,8Z,10E,12(R)14Z)-12-hydroxyeicosatetraen-1-oic acid)>12(S)-HETE ((5Z,8Z,10E,12(S)14Z)-12-Hydroxyeicosatetraen-1-oic acid)>20-COOH-leukotriene B₄>U75302 (6-(6-(3-hydroxy-1E,5Z-undecadienyl)-2-pyridinyl)-1,5-hexanediol)leukotriene C₄=leukotriene D₄=leukotriene E₄. For the human receptor the rank order of 12(S)-HETE, 20-COOH-leukotriene B₄ and U75302 was reversed. Xenopus melanophore and HEK aequorin-based reporter gene assays were used to demonstrate that the guinea pig and human BLT receptors can couple to both the cAMP inhibitory and intracellular Ca²⁺ mobilization signaling pathways. However, in the case of the aequorin-expressing HEK cells (designated AEQ17-293) transfected with either the guinea pig or human BLT receptor, expression of G_α16 was required to achieve a robust Ca²⁺ driven response. Leukotriene B₄ was a potent agonist in functional assays of both the guinea pig and human BLT receptors. U-75302 a leukotriene B₄ analogue which possesses both agonistic and antagonistic properties behaved as a full agonist of the guinea pig and human BLT receptors in AEQ17-293 cells and not as an antagonist. The recombinant guinea pig BLT receptor will permit the comparison of the intrinsic potencies of leukotriene B₄ receptor antagonists used in guinea pig in vivo models of allergic and inflammatory disorders.     

Functional interactions between nucleotide binding domains and leukotriene C4 binding sites of multidrug resistance protein 1 (ABCC1)

Multidrug resistance protein 1 (MRP1) is a member of the "C" branch of the ATP-binding cassette transporter superfamily. The NH(2)-proximal nucleotide-binding domain (NBD1) of MRP1 differs functionally from its COOH-proximal domain (NBD2). NBD1 displays intrinsic high-affinity ATP binding and little ATPase activity. In contrast, ATP binding to NBD2 is strongly dependent on nucleotide binding by NBD1, and NBD2 is more hydrolytically active. We have demonstrated that occupancy of NBD2 by ATP or ADP markedly decreased substrate binding by MRP1. We have further explored the relationship between nucleotide and substrate binding by examining the effects of various ATP analogs and ADP trapping, as well as mutations in conserved functional elements in the NBDs, on the ability of MRP1 to bind the photoactivatable, high-affinity substrate cysteinyl leukotriene C(4) (LTC(4))(.) Overall, the results support a model in which occupancy of both NBD1 and NBD2 by ATP results in the formation of a low-affinity conformation of the protein. However, nonhydrolyzable ATP analogs (beta,gamma-imidoadenosine 5'-triphosphate and adenylylmethylene diphosphonate) failed to substitute for ATP or adenosine 5'-O-(thiotriphosphate) (ATPgammaS) in decreasing LTC(4) photolabeling. Furthermore, mutations of the signature sequence in either NBD that had no apparent effect on azido-ATP binding abrogated the formation of a low-affinity substrate binding state in the presence of ATP or ATPgammaS. We suggest that the effect of these mutations, and possibly the failure of some ATP analogs to decrease LTC(4) binding, may be attributable to an inability to elicit a conformational change in the NBDs that involves interactions between the signature sequence and the gamma-phosphate of the bound nucleotide.     

Regulation of membrane histamine H1-receptor binding sites by guanine nucleotides, mono- and divalent cations

Agonist interaction with histamine H1-receptor in [3H] mepyramine bovine aortic membranes labeled with [3H] mepyramine is selectively regulated by cations and guanine nucleotides. GTP and his nonhydrolisable analog Gpp(NH)p' markedly decrease histamine affinity for [3H] mepyramine binding sites. The effect of GTP is reversed in the presence of divalent cation, magnesium. Calcium and sodium ions have little effect on histamine binding whereas magnesium ions decrease the affinity of histamine for the radioantagonist binding sites about tenfold. GTP has little effect on [3H] mepyramine binding and the interaction of H1-antagonist triprolidine with histamine H1-receptors. The above results indicate that the effect of guanine nucleotides, mono and divalent cations involves the effect on membrane signal transducing mechanism probably GTP-binding protein(s) cation regulatory site(s) rather than receptor binding site directly.     

Modulation of affinity and density of LTB4 receptors on guinea pig lung membranes by divalent cations and guanine nucleotides.

We investigated the mechanism by which guanine nucleotides and divalent cations modulate the affinity and apparent density of high-affinity receptors for Leukotriene B4 (LTB4) on guinea pig lung membranes (GPLM). Divalent cations (Mg2+ = Ca2+ greater than Mn2+) stimulated, whereas EDTA inhibited (IC50 = 0.31 +/- 0.08 mM) binding of [3H]LTB4. Saturation analysis demonstrated that omission of divalent cations caused a two-fold reduction in apparent site density, (B max = 297 +/- 24 fmol/mg protein vs. 149 +/- 21 fmol/mg protein, P less than 0.01, for control and EDTA-treated respectively), but no significant change in receptor affinity (KD = 0.67 +/- 0.16 nM and 1.01 +/- 0.19 nM, P greater than 0.05). Competition experiments with LTB4 and the low-affinity (Ki = 165 nM) competitive LTB4-antagonist U75302, also demonstrated that EDTA caused a significant reduction (1.7 and 3.6-fold, P less than 0.05 and P less than 0.01, respectively), in affinity to both ligands. In the same experiments, the the guanine nucleotide analog GppNHp also reduced the affinity for LTB4 and U75302, similar to that observed with EDTA, suggesting that removal (Mg2+), or addition (GppNHp), of allosteric modulators of G-protein(s), causes reduction in receptor affinity. Saturation experiments also demonstrated that GppNHp, or GTP(gamma S), caused a significant reduction (40-50%) in receptor density. A larger reduction in affinity for U75302 (3- to 3.6-fold) than for LTB4 (1.7-fold) was induced by EDTA as well as GTP analogs.(ABSTRACT TRUNCATED AT 250 WORDS)