Guinea Pig Lung Membranes Research Articles

Modulation of affinity and density of LTB 4 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 B 4 (LTB 4) on guinea pig lung membranes (GPLM). Divalent cations Mg 2+ = Ca 2+ > Mn 2+) stimulated, whereas EDTA inhibited (IC 50 = 0.31 ± 0.08 mM) binding of [ 3H]LTB 4. 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 < 0.01 for control and EDTA-treated respectively), but no significant change in receptor affinity (K D = 0.67 ± 0.16 nM and 1.01 ± 0.19 nM. P > 0.05). Competition experiments with LTB 4 and the low-affinity (K 1 = 165 nM) competitive LTB 4-antagonist U75302, also demonstrated that EDTA caused a significant reduction (1.7 and 3.6 fold, P < 0.05 and P < 0.01, respectively), in affinity to both ligands. In the same experiments, the guanine nucleotide analog GppNHp also reduced the affinity for LTB 4 and U75302, similar to that observed with EDTA, suggesting that removal (Mg 2+), or addition (GppNHp), of allosteric modulators of G-protein(s), causes reduction in receptor affinity. Saturation experiments also demonstrated that GppNHp, or GTP(γS), caused a significant reduction (40–50%) in receptor density. A larger reduction in affinity for U75302 (3- to 3.6-fold) than for LTB 4 (1.7-fold) was induced by EDTA as well as GTP analogs. The data suggest that LTB 4 receptors on GPLM can undergo a transition between a G-protein coupled, high-affinity state, to an uncoupled state with fewer number of receptors, that possess lower affinity for ligands.

Comparison of the Bronchopulmonary and Pressor Activities of Endothelin Isoforms ET-1, ET-2, and ET-3 and Characterization of Their Binding Sites in Guinea Pig Lung

In anesthetized and ventilated guinea pigs intravenous injection of ET-1, ET-2, or ET-3 (1 or 2 nmol/kg) induced similar dose-dependent increases in pulmonary inflation pressure (PIP) associated with increases in mean arterial blood pressure (MBP). Pretreatment of the guinea pigs with 1 mg/kg intravenous indomethacin significantly inhibited the increase in PIP evoked by 2 nmol/kg of ET-1, ET-2, or ET-3. In contrast, the increase in MBP following injection of the various ET isotypes was not significantly affected by indomethacin. Injection of ET-1, ET-2, or ET-3 (40, 120, and 400 pmol) via the pulmonary artery of isolated and perfused guinea pig lungs induced dose-dependent increases in PIP and pulmonary perfusion pressure (PPP), thromboxane B2 (TXB2) release, and formation of lung edema. In keeping with the in vivo results, no marked differences were observed between the activities of ET-1, ET-2, and ET-3 on isolated and perfused guinea pig lungs. Indomethacin (5 microM) added to the perfusion medium significantly inhibited the alterations of PIP and PPP, TXB2 release, and edema formation evoked by 400 pmol ET-1, ET-2, or ET-3. High-affinity binding sites for ET-1, ET-2, and ET-3 exhibiting similar characteristics were identified on guinea pig lung membrane. Therefore ET-1, ET-2, and ET-3 exert comparable bronchopulmonary and pressor activities in the guinea pig and probably act via interaction with the same binding site. In addition, the ET-induced increase in PIP and pulmonary vasoconstriction are primarily mediated via the production of cyclooxygenase metabolites.

High-affinity receptors for bombesin-like peptides in normal guinea pig lung membranes

The binding of the radiolabelled bombesin analogue [ 125I-Tyr 4]bombesin to guinea-pig lung membranes was investigated. Binding of [ 125I-Tyr 4]bombesin was specific, saturable, reversible and linearly related to the protein concentration. Scatchard analysis of equilibrium binding data at 25°C indicated the presence of a single class of non-interacting binding sites for bombesin (B max = 7.7 fmol/mg protein). The value of the equilibrium dissociation constant (K D = 90 pM) agrees with a high-affinity binding site. Bombesin and structurally related peptides such as [Tyr 4]bombesin, neuromedin B and neuromedin C inhibited the binding of [ 125I-Tyr 4]bombesin in an order of potencies as follows : [Tyr 4]bombesin > bombesin ⩾ neuromedin C ⪢ neuromedin B. These results indicate that guinea-pig lung membranes possess a single class of bombesin receptors with a high affinity for bombesin and a lower one for neuromedin B.

Comparative in vitro effects of guinea pig VIP and common VIP on liver and lung membranes from guinea pig and rat and on human lymphoblastic SUP-T1 membranes

Guinea pig VIP differs from VIP of several mammals by its amino acids in positions 5, 9, 19 and 26. We tested a) its ability to occupy VIP receptors in liver and lung membranes of rat and guinea pig and in the human lymphoblastic SUP-T1 cell line and b) the ensuing adenylate cyclase stimulation. In liver and lung membranes from rat, guinea pig VIP was less potent than common VIP to occupy high and low affinity VIP receptors. In rat liver both VIP activated adenylate cyclase mostly through high affinity receptors. In rat lung, guinea pig VIP activated the enzyme mostly through high affinity receptors and was less efficient than common VIP acting through both classes of receptors. In guinea pig liver and lung membranes, binding inhibition curves were steeper than with rat preparations and adenylate cyclase appeared to be mostly activated through high affinity VIP receptors in liver and through both classes of receptors in lung. On human lymphoblastic SUP-T1 membranes both VIP were equally potent and efficient to inhibit tracer binding and activate adenylate cyclase.

Pharmacologic and pharmacokinetic profile of SK&F S-106203, a potent, orally active peptidoleukotriene receptor antagonist, in guinea-pig

In this report the pharmacologic and pharmacokinetic profile of the leukotriene receptor antagonist 3(S)-1(2-carboxyethyl)thio]-3-12-(8-phenyloctyl)phenyl] propanoic acid (SK&F S-106203) in guinea-pigs is described. In isolated guinea-pig tracheae SK&F S-106203 was a potent, competitive antagonist of leukotriene (LT) D 4-induced contractions (pA 2 = 7.6). SK&F S-106203 was also a potent antagonist of LTE 4-induced contractions (pK B = 7.3), but had little effect on those elicited by LTC 4 (pK B = 5.5). SK&F S-106203 (10 μM) had no effect on contractions produced by histamine, carbachol, KCI, U-44069, PGF 2α or PGD 2. In addition, SK&F S-106203 (10 μM) did not inhibit cyclic nucleotide phosphodiesterase (PDE) activity of several PDE isozymes. In guinea-pig lung membrane preparations, SK&F S-106203 was a potent antagonist of 3H-LTD 4 binding with a K i = 19.4 ± 2.1 nM (n = 5). The pharmacokinetic profile of SK&F S-106203 was determined in unanesthetized guinea-pigs. Following an i.v. (bolus) dose (25 mg/kg), SK&F S-106203 disappeared from plasma in a biphasic fashion with half-lives of 0.1 h (50% of the area under the plasma concentration-time curve, AUC) and 11 h. The AUC obtained for SK&F S-106203 following i.v. administration was 87.3 ± 7.5 μg-h/ml. Following an oral dose of SK&F S-106203 (100 mg/kg), the maximal plasma concentration (C max) and the time C max was achieved (T max were 21.62 ± 2.26 kg/ml and 4 ± 1 h, respectively; the AUC was 279.9 ± 41.8 μg-h/ml. Studies examining the effects of i.v. infusion of SK&F S-106203 revealed that marked inhibition of LTD 4-induced bronchospasm was produced with steady-state plasma levels of SK&F S-106203 < 1 μg/ml (< 2 μM). Oral (p.o.) pretreatment with 100 μmol/kg SK&F S-106203 for up to 24 h essentially abolished LTD 4-induced bronchospasm; this correlated with sustained plasma concentrations of > 2 μg/ml. The results indicate that in guinea-pig airways, SK&F S-106203 is a potent and selective LT receptor antagonist that is active via aerosol, oral and i.v. routes of administration. When given orally, SK&F S-106203 is highly bioavailable and has a very long duration of action which correlates with the pharmacokinetic profile of the compound. SK&F S-106203 may be useful therapy in asthma and other disorders in which the Us are thought to play a prominent pathophysiological role.

Characterization of the guinea pig lung membrane leukotriene D4 receptor solubilized in an active form. Association and dissociation with an islet-activating protein-sensitive guanine nucleotide-binding protein.

Membrane fractions from the guinea pig lung had high- and low-affinity binding sites for LTD4 with Kd values of 0.016 and 9.1 nM, respectively. In the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or by prior treatment of the membrane with islet-activating protein (IAP), the high-affinity site shifted to a low-affinity state. Consistently, a 41-kDa protein was ADP-ribosylated by treatment of the lung membranes with IAP, and this event was inhibited by the addition of GTP gamma S. We solubilized the LTD4 receptor from the lung membranes in an active form with 5 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and 10% glycerol. On a gel filtration column, the binding activity was eluted at the volume corresponding to a Mr of 70,000 or over 500,000 in the presence or absence of Mg2+ (5-20 mM), respectively, in solubilizing buffers. The Kd value of [3H]LTD4 binding to the 70-kDa protein was similar to the low-affinity binding constant of the membrane and was insensitive to GTP gamma S. The preparation solubilized in the absence of Mg2+ showed both high- and low-affinity binding sites for LTD4, and the addition of GTP gamma S shifted the high-affinity site to a low-affinity one. Thus, 1) the LTD4 receptor is coupled to an IAP-sensitive GTP-binding protein, 2) this GTP-binding protein is dissociable from the receptor by solubilizing the lung membrane with CHAPS and Mg2+, and 3) the receptor associated to or dissociated from a GTP-binding protein exhibited a high- or low-affinity state, respectively. These data provide an insight into the molecular mechanism of regulation of the LTD4 receptor signaling process by association and dissociation with an IAP-sensitive GTP-binding protein.

Characterization of Receptors for Platelet-activating Factor in Guinea Pig Lung Membranes

Platelet-activating factor (PAF) is a potent contractile agonist in guinea pig peripheral lung strips. Whether PAF acts via specific receptor sites in the lung has not been fully established. To determine if specific receptor sites could be demonstrated in guinea pig peripheral lung tissue, we performed direct radioligand binding studies in tissue homogenates with [3H]C16-PAF (1-O-hexadecyl-sn-glyceryl-3-phosphorylcholine) and the PAF antagonists [3H]WEB 2086 and [3H]RP52770. These studies demonstrated binding sites for [3H]C16-PAF of high affinity (Kd of 2.6 nM from saturation isotherms and 0.9 nM from kinetic experiments) and a mean density of 200 fmol/mg protein. Binding was inhibited to the same degree with unlabeled C16-PAF, WEB 2086, and RP52770, all with pseudo-Hill slopes of unity. [3H]WEB 2086 binding demonstrated a receptor density similar to that of [3H]C16-PAF (226 fmol/mg protein) and was inhibited to the same degree by unlabeled C16-PAF, C18-PAF, WEB 2086, and RP52770. Although antagonist inhibition yielded pseudo-Hill slopes near unity, agonist inhibition slopes were shallow, suggesting two types or states for the PAF receptors. Direct binding studies with [3H]RP52770 revealed a much larger density of binding sites (1,200 fmol/mg protein), and this binding was not inhibited with C16-PAF, C18-PAF, WEB 2086, or lyso-PAF. These results indicate that guinea pig lung has specific binding sites for [3H]C16-PAF, that WEB 2086 is an effective antagonist of C16- and C18-PAF binding at these sites, and that RP52770 binds to the PAF site but, in addition, binds to another site with a much greater density.

The inhibition of [ 3H]leukotriene D 4 binding to guinea-pig lung membranes. The correlation of binding affinity with activity on the guinea-pig ileum

Guinea-pig lung membranes contain high affinity (K D = 0.8 nM) binding sites for [ 3H]leukotriene D 4 ([ 3H]LTD 4). The binding is inhibited by leukotriene antagonists, such as ICI 198615 and SK & F 104353, in a manner consistent with the Law of Mass Action at a single site. It is also inhibited by a range of leukotriene analogues in a dose-related manner. Inhibition by some of these e.g. LTC 4 suggests that the [ 3H]LTD 4 binding sites are heterogeneous. The binding affinity of the leukotriene analogues is significantly correlated (P < 0.001) to their spasmogenic activity on guinea-pig ileum but not on guinea-pig lung strip. The binding affinity of the leukotriene antagonists is also correlated to their antagonist activity on guinea-pig ileum (P < 0.05) but not on guinea-pig lung. These results indicate that the [ 3H]LTD 4 binding site in guinea-pig lung is similar to the leukotriene receptor activated by LTD 4 and LTE 4 on guinea-pig ileum. The contractile response of guinea-pig lung strips to leukotrienes, in the presence of indomethacin, is mediated by a distinct type of leukotriene receptor.

Neomycin induces high‐affinity agonist binding of G‐protein‐coupled receptors

Neomycin, an inositol-phospholipid-binding aminoglycoside antibiotic, is known to interfere with signal transduction mechanisms involving phospholipase C as effector enzyme. In this study, we report that neomycin can also markedly influence agonist binding of G-protein-coupled receptors. In membranes of differentiated human leukemia cells (HL 60 cells), neomycin (0.1-10 mM) was found to induce high-affinity binding of the chemotactic tripeptide, N-formyl-methionylleucylphenylalanine (fMet-Leu-Phe), to its receptor sites in a manner similar to magnesium. Gentamycin and streptomycin, two other aminoglycoside antibiotics, were as potent and as effective as neomycin or magnesium in inducing high-affinity agonist receptor binding. Pretreatment of the cells with pertussis toxin reduced the effects of magnesium and neomycin on agonist receptor binding likewise. In contrast, magnesium but not neomycin largely enhanced the potency of guanine nucleotides, particularly of GTP and its analog, guanosine-5'-O-(3-thiotriphosphate), to reduce fMet-Leu-Phe receptor binding, while maximal inhibition of agonist receptor binding by guanine nucleotides was identical with magnesium and neomycin. Furthermore, neomycin could not replace magnesium in providing stimulation of HL 60 membrane high-affinity GTPase by fMet-Leu-Phe. In close agreement to these findings on the pertussis-toxin-sensitive Gi-protein-coupled formyl peptide receptors, neomycin in a manner similar to magnesium induced high-affinity agonist binding of Gs-protein-coupled beta-adrenoceptors. Similar to formyl peptide receptor binding, high-affinity binding of isoproterenol to beta-adrenoceptors in guinea pig lung membranes induced by magnesium and neomycin was inhibited by the GTP analog, guanosine-5'-O-(3-thiotriphosphate), to a similar maximal extent but with an about 100-fold higher potency in the presence of magnesium than in the presence of neomycin. The data presented thus indicate that neomycin and other aminoglycoside antibiotics can mimic the action of magnesium (or other divalent cations) in inducing high-affinity agonist binding of Gi- and Gs-protein-coupled receptors, but not in inducing subsequent G-protein activation by guanosine triphosphates. The data, furthermore, suggest that neomycin by this selective action will be a powerful tool to dissect the multiple sites of magnesium's action in the agonist receptor-G-protein interaction.

Characterization of autoantibodies to vasoactive intestinal peptide in asthma

Vasoactive intestinal peptide (VIP) is a potent relaxant of the airway smooth muscle. In this study, VIP-binding autoantibodies were observed in the plasma of 18% asthma patients and 16% healthy subjects. Immunoprecipitation studies and chromatography on DEAE-cellulose and immobilized protein G indicated that the plasma VIP-binding activity was largely due to IgG antibodies. Saturation analysis of VIP binding by the plasma suggested the presence of one or two classes of autoantibodies, distinguished by their apparent equilibrium affinity constants ( K a). The autoantibodies from asthma patients exhibited a larger VIP-binding affinity compared to those from healthy subjects ( K a 7.8 × 10 9 M −1 and 0.13 × 10 9 M −1, respectively; P < 0.005). The antibodies were specific for VIP, judged by their poor reaction with peptides bearing partial sequence homology with VIP (peptide histidine isoleucine, growth hormone releasing factor and secretin). IgG prepared from the plasma of an antibody-positive asthma patient inhibited the saturable binding of 125I-VIP by receptors in guinea pig lung membranes (by 39–59%; P < 0.001). These observations are consistent with a role for the VIP autoantibodies in the airway hyperresponsiveness of asthma.

Muscarinic receptor subtypes in human and guinea pig lung

Muscarinic receptor subtypes in human and guinea pig lung membranes were characterised using selective muscarinic antagonists. Competition experiments were carried out against [ 3H](−)-quinuclidinyl benzilate binding at 25°C in Tris-HCl buffer; non-specific binding was determined in the presence of 1 μM atropine. Of all the antagonists examined, only the M 1-selective antagonist pirenzepine exhibited a heterogeneous binding profile (n H < 1.0), best described by two-binding sites of high and low affinity. Binding of [ 3H]pirenzepine confirmed the presence of a high proportion of high affinity (M 1) receptors (60% of total) in human peripheral lung. The high potency of M 3-selective antagonists 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP) and hexahydrosiladifenidol suggested the presence of M 3 receptors, but the low potency of AF-DX 116 and methoctramine indicated that there was no significant population of M 2 receptors present. The existence of muscarinic receptor subtypes in lung may have important clinical implication but their cellular localisation remains to be determined.

Characterization of calcitonin gene-related peptide(CGRP) receptors in guinea pig lung.

Receptors for calcitonin gene-related peptide (CGRP) in the lung membranes of guinea pig were characterized by using a millititer plate precoated with polyethylenimine. Specific binding of 125I-CGRP was time-dependent, rapid, and reversible, and the binding increased linearly with increasing concentrations of membrane protein. Scatchard analysis revealed two classes of CGRP binding sites: high affinity sites with a KD value of 7.17 x 10(-11) M and a Bmax of 364 fmol/mg protein and low affinity sites with a KD value of 1.7 x 10(-8) M and a Bmax of 39594 fmol/mg protein. Furthermore, the specific binding of 125I-CGRP was dissociated in the presence of GTP or Gpp(NH)p, suggesting that CGRP receptors in guinea pig lung membranes were coupled to the guanine nucleotide regulatory protein. Scatchard analysis of CGRP binding in the presence of GTP revealed selective inhibition of the binding to high affinity binding sites. Unlabeled CGRP displaced the binding of 125I-CGRP to guinea pig lung membranes with an IC50 value of 3.1 x 10(-10) M. In contrast, salmon calcitonin and human calcitonin displaced the binding at 600-fold higher concentrations. We suggest that both low and high affinity binding sites for CGRP exist in the lung membranes of the guinea pig, and the high affinity binding sites for CGRP may be coupled to GTP binding regulatory protein.

Characterization of Calcitonin Gene-Related Peptide (CGRP) Receptors in Guinea Pig Lung

Receptors tor calcitonin gene-related peptide (CGRP) in the lung membranes of guinea pig were characterized by using a millititer plate precoated with polyethylenimine. Specific binding of 125I-CGRP was time-dependent, rapid, and reversible, and the binding increased linearly with increasing concentrations of membrane protein. Scatchard analysis revealed two classes of CGRP binding sites: high affinity sites with a KD value of 7.17×10-11 M and a Bmax of 364 fmol/mg protein and low affinity sites with a KD value of 1.7χ10-8 M and a Bmax of 39594 fmol/mg protein. Furthermore, the specific binding of 125I-CGRP was dissociated in the presence of GTP or Gpp(NH)p, suggesting that CGRP receptors in guinea pig lung membranes were coupled to the guanine nucleotide regulatory protein. Scatchard analysis of CGRP binding in the presence of GTP revealed selective inhibition of the binding to high affinity binding sites. Unlabeled CGRP displaced the binding of 125I-CGRP to guinea pig lung membranes with an IC50 value of 3.1 χ 10-10 M. In contrast, salmon calcitonin and human calcitonin displaced the binding at 600-fold higher concentrations. We suggest that both low and high affinity binding sites for CGRP exist in the lung membranes of the guinea pig, and the high affinity binding sites for CGRP may be coupled to GTP binding regulatory protein.

Pharmacology of L-660,711 (MK-571): a novel potent and selective leukotriene D4 receptor antagonist

L-660,711 (3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl) ((3-dimethyl amino-3-oxo propyl)thio)methyl)thio)propanoic acid is a potent and selective competitive inhibitor of [3H]leukotriene D4 binding in guinea pig (Ki value, 0.22 nM) and human (Ki value, 2.1 nM) lung membranes but is essentially inactive versus [3H]leukotriene C4 binding (IC50 value in guinea pig lung, 23 microM). Functionally it competitively antagonized contractions of guinea pig trachea and ileum induced by leukotriene (LT) D4 (respective pA2 values, 9.4 and 10.5) and LTE4 (respective pA2 values, 9.1 and 10.4) and contractions of human trachea induced by LTD4 (pA2 value, 8.5). L-660,711 (5.8 x 10(-8)M) antagonized contractions of guinea pig trachea induced by LTC4 in the absence (dose ratio = 28) but not in the presence of 45 mM L-serine borate (dose ratio less than 2). L-660,711 (1.9 x 10(-5)M) did not block contractions of guinea pig trachea induced by histamine, acetylcholine, 5-hydroxytryptamine, PGF2 alpha, U-44069, or PGD2. In the presence of atropine, mepyramine, and indomethacin, L-660,711 (1.9 x 10(-5)M) inhibited a small component of the response to antigen on guinea pig trachea but completely blocked anti-IgE-induced contractions of human trachea. L-660,711 (i.v.) antagonized bronchoconstriction induced in anesthetized guinea pigs by i.v. LTC4, LTD4, and LTE4 but did not block bronchoconstriction to arachidonic acid, U-44069, 5-hydroxytryptamine, histamine, or acetylcholine. Intraduodenal L-660,711 antagonized LTD4 (0.2-12.8 micrograms/kg)-induced bronchoconstriction in guinea pigs, and p.o. L-660,711 blocked LTD4- and Ascaris-induced bronchoconstriction in conscious squirrel monkeys and ovalbumin-induced bronchoconstriction in conscious sensitized rats treated with methysergide (3 micrograms/kg). The pharmacological profile of L-660,711 indicates that it is a potent, selective, orally active leukotriene receptor antagonist which is well suited to determine the role played by LTD4 and LTE4 in asthma and other pathophysiologic conditions.

Photoaffinity labeling the beta-adrenergic receptor with an iodoazido derivative of norepinephrine.

An iodinated photosensitive derivative of norepinephine, N-(p-azido-m-iodophenethylamidoisobutyl)-norepinephrine (NAIN), has been synthesized and characterized. NAIN stimulated adenylate cyclase activity in guinea pig lung membranes in a manner similar to (-)-isoproterenol and was inhibited by (-)-alprenolol. NAIN was shown to compete with [125I]iodocyanobenzylpindolol for the beta-adrenergic receptor in guinea pig lung membranes with an affinity which was dependent on the presence of guanyl nucleotides. Carrier-free radioiodinated NAIN ([125I]NAIN) was used at 2 nM to photoaffinity label the beta-adrenergic receptor in guinea pig lung membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of (-)-alprenolol (1 microM) protectable [125I]NAIN labeling showed the same molecular mass polypeptide (65 kDa) that was specifically derivatized with the antagonist photolabel [125I]iodoazidobenzylpindolol. Specific labeling of the beta-adrenergic receptor with [125I]NAIN was dependent on the presence of MgCl2 and the absence of guanyl nucleotide. Guanosine-5'-O-(3-thiotriphosphate (100 microM) abolished specific labeling by [125I]NAIN. N-Ethylmaleimide (2 mM) in the presence of [125I]NAIN protected against the magnesium and guanyl nucleotide effect. These data show that NAIN is an agonist photolabel for the beta-adrenergic receptor.

A novel leukotriene D 4/E 4 antagonist, SR2640 (2-[3-(2-quinolylmethoxy)phenylamino]benzoic acid)

We have studied the leukotriene antagonistic activity of a novel compound, SR2640 (2-[3-(2-quinolylmethoxy)phenylamino]benzoic acid), in vitro and in vivo. SR2640 inhibited LTD 4 − but not histamine-induced contractions of guinea-pig ileum and trachea in a concentration-dependent manner. Schild plot analysis of tracheal LTD 4 antagonism yielded a pA 2 value of 8.7 and a slope not different from unity. SR2640 concentration dependently inhibited the binding of 0.4 nM [ 3H]LTD 4 to guinea-pig lung membranes with an IC 50 value of 23 nM. The curve was parallel to that of unlabelled LTD 4 (IC 50 = 2.2 nM). SR2640 was equally effective in antagonizing LTD 4 and LTE 4, but was much less potent in reducing LTC 4-induced ileum contractions. In vivo, SR2640 in the dose range 0.03–1.00 mg/kg shifted the dose-response curve for guinea-pig bronchoconstriction induced by intravenous LTD 4 administration to the right at a rate proportional to the dose of SR2640, without reducing the maximum attinable obstruction: the slope of the Schild plot was 0.99. SR2640 (1 mg/kg) also caused a significant inhibition of antigen-induced bronchoconstriction in anaesthetized guinea-pigs pretreated with pyrilamine, indomethacin, propranolol and suxamethonium. In conclusion, SR2640 appears to be a potent and selective competitive LTD 4/LTE 4 antagonist, and may be useful in elucidating the role of leukotrienes in human asthma.

Modulation of guinea-pig lung adenylate cyclase by ovalbumin sensitization

1. The regulation of lung adenylate cyclase was investigated in guinea pigs sensitized with high dose (300–500 μg kg −) ovalbumin to raise IgG(I) and low dose (2.8–4.0 μg kg −) to raise to IgG/ IgE (II) antibodies. 2. Basal activity of sensitized II (IgG/IgE antibodies) lung adenylate cyclase was approximately double that of the control values. 3. Guanosine 5′-triphosphate (GTP) was a potent activator of adenylate cyclase from the sensitized II (IgG/IgE) lung membranes. 4. The sensitivity of lung membrane adenylate cyclase to stimulation by β-adrenoceptor agonists or VIP was reduced whereas no significant difference was observed with histamine or bradykinin on the sensitized II membranes. 5. Possible mechanisms involved in the increase in basal activity and in the decreased sensitivity to β-adrenoceptor- and VIP-mediated stimulation of adenylate cyclase by ovalbumin sensitization of guineapig lung membranes are discussed.

Autoradiographic localization of leukotriene C 4 and D 4 binding sites in guinea-pig lung

Binding of [3H] leukotriene C4 and D4 to guinea-pig lung sections was charaterised and binding sites were localized by autoradiography. Both leukotrienes bound to guinea-pig lung sections and membranes with high affinity and with similar charateristics to binding in a membrane preparation. Autoradiography revealed that the distribution of LTC4 and D4 binding sites was markedly different. Smooth muscle and epithelium of central and peripheral airways were densely labelled with [3H]LTC4; vascular smooth muscle and alveolar walls were also labelled. With [3H]LTD4, however, there was no detectable labelling of airways or vessels but subtantial labelling of alveolar walls. This lends futher support that LTC4 and LTD4 binding sites differ and may not be identical with functional receptors.

L-648,051, a potent and specific aerosol active leukotriene D4 antagonist.

L-648,051 (I) is a potent, competitive and selective antagonist of the leukotriene D4 receptor in guinea pig and human lung tissue. Its activity on isolated smooth muscle preparations and its superiority relative to other agents (L-649,923 (II) or FPL 55712) in reversing ongoing contraction to LTD4 was somewhat unexpected in light of the results of receptor binding studies (Ki = 6.2 microM for L-648,051 versus 0.4 microM for L-649,923 and 2.0 microM for FPL 55712 on guinea pig lung membranes). The reasons for this observed superiority are unknown but may relate to the rate at which various antagonists equilibrate with the leukotriene receptors on various tissues. The physical properties (polarity) of L-648,051 may contribute to this enhanced rate of equilibration. The rapid metabolism and elimination of L-648,051 in vivo should be an advantage for a topical agent by minimizing systemic exposure. The potential role for L-648,051 as a novel aerosol therapy for asthma is now being investigated.

Concerning the assays for autonomic nerve receptors. Effects of incubation temperature and time on alterations in the number of receptors.

Alterations in autonomic nerve receptors play an important role in various pathological conditions, including bronchial asthma. Nevertheless, receptor assay conditions such as incubation temperature and incubation time are not consistent among various investigators. This study was designed to clarify the effects of incubation temperature and time on alterations in the number of autonomic nerve receptors. Guinea pig lung membranes were divided into five groups which were incubated under different incubation temperatures and over different incubation times. After incubation, the following experiments were performed. Beta-, alpha-1-adrenergic, and muscarinic acetylcholine receptor assays were performed by direct binding technique using L-3H-dihydroalprenolol, 3H-bunazosin, and L-3H-quinuclidinyl benzilate, respectively. Elevation of incubation temperature and prolongation of incubation time caused a significant decrease in the number of beta-adrenergic receptors and an increase in the number of alpha-1-adrenergic receptors. The number of muscarinic acetylcholine receptors did not change significantly in spite of changes in incubation temperature and time. Adenylate cyclase activity was measured by following the synthesis of cyclic adenosine monophosphate from nonradioactive adenosine triphosphate. Isoproterenol-stimulated adenylate cyclase activity decreased significantly in correspondence with the elevation of incubation temperature and prolongation of the incubation time. Contents of free fatty acids in lung membranes were measured by high-performance liquid chromatography. Free fatty acid contents increased significantly in accordance with elevation of incubation temperature and prolongation of incubation time which reflected on the degradation of membrane phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)