The binding of [3H]leukotriene C4 to guinea-pig lung membranes. The lack of correlation of LTC4 functional activity with binding affinity

European Journal of Pharmacology, 143 (1987) 323-334

323

Elsevier

EJP 50010

The binding of [3Hlleukotriene C 4 to guinea-pig lung membranes. The lack of correlation of LTC 4 functional activity with binding affinity Peter N o r m a n *, T r e v o r S. A b r a m , H a r o l d C. K l u e n d e r a n d N i g e l J. C u t h b e r t

1 Phillip

J. G a r d i n e r

Miles Laboratories Ltd., Stoke Court, Stoke Poges, Slough SL2 4LY, U.K. and I Miles Laboratories Inc., Elkhart, IN, U.S.A. Received 27 May 1987, revised MS received 11 August 1987, accepted 18 August 1987

High affinity binding sites for [3H]leukotriene C 4 ([3H]LTC4) have been identified and characterised in guinea-pig lung membranes. [3H]LTC4 bound to these membranes with a pharmacological specificity totally distinct to that previously observed for [3H]LTD4 binding in guinea-pig lung. Scatchard analysis of saturation binding data showed a single class of binding sites, with a dissociation constant (KD) of 52.6 _+4.9 nM and a density (Bmax) of 30 + 12 pmol/mg membrane protein. The binding was inhibited with high affinity by a variety of glutathione-containing leukotriene analogues. Most notable was the inhibition by 1,2,3,4-tetranor L T C 4 ( K i = 118 nM) and S-decylglutathione (K i = 154 nM) since neither of these compounds were contractile agonists on guinea-pig parenchymal strips or guinea-pig ileum nor were they antagonists of LTC4-induced contractions of these smooth muscle preparations. These results indicate that the observed binding of [3H]LTC4 to guinea-pig lung membranes is not to a contractile receptor. Leukotriene receptors; Leukotrienes (C4, D 4, E 4, F4), Slow reacting substance of anaphylaxis (SRS-A); Glutathione; Lung; (Guinea-pig)

1. Introduction

Slow reacting substance of anaphylaxis (SRS-A) is generated in lung by a variety of stimuli and is thought to be an important mediator of immediate hypersensitivity reactions such as b r o n c h o constriction in allergic asthma (Brocklehurst, 1960). The major constituents of SRS-A have recently been identified as leukotriene C 4 (LTC4), leukotriene D 4 (LTD4) and leukotriene E a (LTE 4) (Lewis et al., 1980a; Morris et al., 1980; Lewis et al., 1980b). All three of these sulphido-leukotrienes are derived from arachidonic acid by the sequential action of a series of enzymes commencing with 5-1ipoxygenation (Taylor and Clarke, 1986). All three of these leukotrienes are potent

* To whom all correspondence should be addressed.

spasmogens on many smooth muscle preparations, generally being considerably more potent than standard reference spasmogens such as histamine (Drazen et al., 1980). In nearly all the tissues where they are active LTC 4 and L T D 4 are equipotent and have equal maximal efficiencies suggesting that they act at the same receptor(s). However, the leukotriene antagonist FPL55712 is considerably more effective against L T D 4 than LTC 4 on both guinea-pig trachea and ileum suggesting that the leukotrienes may act at different receptors (Snyder and Krell, 1984; Weichman and Tucker, 1985; Cuthbert and Gardiner, 1987). The availability of radiolabelled leukotrienes permits an alternative investigation of the existence of distinct leukotriene receptors using ligand binding studies. Using radiolabelled L T D 4 ([3H]LTD4) we, and others (Norman et al., 1984; Mong et al., 1984, Cheng and Townley, 1984a)

0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

324 have shown that there is a discreet, apparently selective, binding site for LTD 4. It has been shown that [3H]LTE4 also binds to this site (Mong et al., 1985b; Cheng and Townley, 1984b). Although the binding of [3H]leukotriene C4 to guinea-pig lung membranes has been described (Cheng and Townley, 1984a; Hogaboom et al., 1983), this binding has not been thoroughly characterised. The following paper describes our initial studies towards such a characterisation.

2. Materials and methods

2.1. Chemicals and reagents LTC4, LTC 4 methyl ester (methyl-5(S)-hydroxy-6(R)-S-glutathionyleicosatetraenoate), 7-cisLTC 1, deamino-LTC4, LTD4, LTE 4 and L T F4 were all synthesised in our laboratories as the 5(S), 6(R)-isomers from 2-deoxy-D-ribose broadly using the method described by Rokach et al. (1981). 4(S)5(R)-2-nor-LTD 1 ( S K & F 101132) was prepared as described (Gleason et al., 1983). 1,2,3,4Tetranor LTC 4 (2-(RS)-S-glutathionyl-3(E)5(E) 7(Z),10(Z)-hexadecatetraenol) was synthesised as described below. Leukotriene B4 (LTB4) was synthesised from L-arabinose. All the leukotrienes and leukotriene analogues were purified by reversed-phase high performance liquid chromatography (HPLC) and were > 95% pure. [3H]LTC4 (30-50 C i / m m o l ) was supplied by Dr. M. Bye (Amersham International) and was also shown to be > 95% pure by HPLC. FPL55712 was also synthesised in our laboratory. LY171883 was a gift from Eli Lilly. S K & F 88046 was synthesised by Dr. U. Behner (Bayer AG, Wuppertal). Other drugs were obtained from the company indicated: propranolol (ICI), phenoxybenzamine and cimetidine (SK&F), mepyramine (M&B), captopril (Squibb), phentolamine (Ciba), methysergide (Sandoz), prostacylin (Wellcome). U46619 was purchased from Upjohn, P G F 2 was purchased from Ono. Diltiazem, nifedipine, verapamil, Rev 5901, AH19437, BM13177 and EP045 were obtained from Dr. H. Boeshagen (Bayer AG, Wuppertal) 5-(S)-hydroxyeicosatetra-

enoic acid was purchased from Biomol Laboratories. Atropine, glutathione, histamine, indomethacin, L-cysteine, guanylimidodiphosphate, L-glutamic acid, S-decylglutathione, S-propylglutathione, polypep and Tris were purchased from Sigma Chemical Co. All salts used were of Analar grade.

2.2. Membrane preparation Male Dunkin-Hartley guinea-pigs were killed by cervical dislocation. The lungs were removed, dissected free of major bronchi, and finely minced with scissors. This was homogenised in 10 volumes of ice-cold buffer (0.32 M sucrose, 0.154 M sodium chloride in 50 mM Tris HC1 pH 7.4) using a Polytron PT-10 for a total of 1 min using 10 bursts at a setting of 8. (All operations were carried out at 0-4°C.) The homogenate was centrifuged (1000 × g for 10 min) to remove undislocated cells and nuclei and the supernatant then centrifuged at 50 000 x g for 20 min to pellet a crude plasma membrane fraction. These were washed by resuspending in 50 mM Tris HC1 pH 7.4 and again centrifuging at 50000 x g for 20 min. The pellets were then resuspended in 2 volumes 50 mM Tris HC1 pH 7.4, aliquoted into plastic tubes, rapidly frozen and stored at - 7 0 ° C for up to 3 months. (Preliminary investigations revealed that binding was not affected by freezing of the membranes.) The protein concentration was determined by the method of Bradford (1976) using bovine serum albumin as a standard.

2.3. Radioligand binding assay Binding assays were performed at 2 0 ° C in a buffer containing 40 mM L-serine-borate (Tate and Meister, 1978), 10 mM L-cysteine and 1% polypep in 50 mM Tris HC1 pH 7.4 in a final volume of 250 /~1 unless otherwise indicated. Assays were initiated by addition of 50 /~1 lung membranes (20-75~g) protein and terminated after 30 min by addition of 3.5 ml ice-cold Tris HC1 pH 7.4 and rapid filtration through Whatman G F / B filters which were washed twice with 3.5 ml ice-cold buffer. Inhibition experiments were carried out by ad-

325 ding increasing concentrations of test drug to 2 nM [3H]LTC4. Non-specific binding was defined as the difference between total binding and binding in the presence of 5/~M LTC 4. Specific binding was typically 80-90% total binding. Saturation assays were performed by incubation of increasing concentrations (1-70 nM) [3H]LTC4 in the presence, or absence, of 5 /~M LTC 4. Kinetic experiments were performed by addition of lung membranes to 5-10 ml [3H]LTC4 in the absence, or presence, of 5/~M LTC 4. Aliquots of 200 /H were removed, and filtered as above, at appropriate time intervals. All assays were carried out in triplicate and were repeated at least three times. The washed filters were transferred to scintillation vials and 0.5 ml NCS tissue solubiliser (Amersham) and 10 ml Unisolve-1 (Koch-Light) were added. After a period of 3 h, 100 /~1 glacial acetic acid was added and the radioactivity in the vials was determined on a LKB 1217 liquid scintillation spectrometer using the external standard ratio method.

branes (250 t~g/ml) were incubated with [3H]LTC4 (5 nM) for 10-60 min at 23°C in buffer containing 1% polypep, 50 mM Tris HC1 and concentrations of L-serine-borate between 0 and 50 mM. Incubations were terminated by addition of 2 ml ice-cold Tris and centrifugation for 5 min at 5 000 × g. The supernatant was filtered and injected directly onto the HPLC. Aliquots of 100/~1 were removed prior to injection and the radioactivity present was measured by liquid scintillation spectrometry. The retention times were compared with authentic LTC 4, LTD4, LTE4, [3H]LTC4 and [3H]LTD4. A reverse-phase Cls (RP-18 10 /~m) /~-Bondapak column (0.39 × 30 cm) was used with an eluent of acetonitrile:water:acetic acid pH 5.6 (37.5 : 62.5 : 0.1 (v/v)) at a flow rate of 1 ml/min. The eluent from the column was mixed with scintillation fluid (Unisolve-1) at 3 m l / m i n and passed through a radiochemical detector (NE Isoflo 1). Radiochemical peaks were quantified using an N E Isomess 2000 software package. The absorbance at 280 nm was measured to detect unlabelled leukotrienes.

2.4. Analysis of data Saturation binding data was analysed directly using the L I G A N D programme of Munson and Rodbard (1980). Non-specific binding was treated as an independent parameter in this analysis. The data was analysed for binding to one or two class(es) of specific sites and one non-specific site. The significance of the more complex model was assessed by use of the partial F test. The data was also analysed, after subtraction of the non-specific binding, by the method of Scatchard (1949) and estimates of the BmaX and K D obtained by unweighted linear regression. Competition binding experiments were also analysed after subtraction of non-specific binding. ICs0 values were obtained from the indirect Hill plot by linear regression. Competition curves were also analysed by a simple curve fitting programme for binding to one or two sites as described by Richardson and Humrich (1984).

2.5. Metabolism of [-~H]LTC4 by guinea-pig lung Reverse phase HPLC was used to study the metabolism of [3H]LTC4. Guinea-pig lung mem-

2.6. Preparation of racemic 1,2,3,4-tetranor-LTC 4 Glycidaldehyde was treated with 4-oxobut2(E)-enyltriphenylphosphorane (Berenguer et al., 1971) in methylene chloride and afforded 6,7oxido-2(E), 4(E)-heptadienal (50% yield) following iodine double bond isomerisation and silica chromatography. (UV MeOH: max. 272 nm.) This product was then treated with 3(Z)-nonenyltriphenylphosphonium tosylate (Ernest et al., 1982), in the presence of butyllithium, in tetrahydrofuran and hexamethylphosphoramide to give 1,2-oxido-3(E), 5(E), 7(Z), 10(Z)-hexadecatetraene (78% yield) following chromatography on silica (UV cyclohexane: max. 290.9, 279.3, 270 nm). Epoxide opening, in the usual manner, with fully blocked glutathione smoothly generated the title compound following reverse phase HPLC purification (Zorbax acetonitrile/water 70/30) and deprotection using aqueous lithium hydroxide in tetrahydrofuran. (UV H20" max. 280.6 nm; FAB MS M -a 538.)

326 2.7. Spasmogenic activity on guinea-pig ileum and perenchymal strips

3. Results 3.1. Metabolic studies

Male Dunkin-Hartley guinea-pigs (400-500 g) were killed by cervical dislocation and the lungs and ileum removed. Lung parenchymal strips 2 c m x 3 mm × 3 mm were prepared using the technique described by Lulich et al. (1976). Longitudinal strips of guinea-pig ileum were cut each being 5 cm in length. A thread or cotton tie were secured at opposite ends of the preparation to enable suspension in the tissue bath but with the lumen open for Tyrode access. Each tissue was suspended in a 10 ml tissue-bath and connected to an Harvard isotonic transducer with a load of 0.5 g for ileum and 1 g for lung strips. The tissues were bathed in modified Tyrode solution of the following composition (mM): NaC1 137, KC1 2.7, CaC12 2.4, MgC12 2.1, NaH2PO4 0.5, N a H C O 3 11.9, D-glucose 9.2 and indomethacin 0.003. The tissue baths were maintained at 3 7 ° C and continuously aerated with 95% 02-5% CO2. After 60 rain equilibration the tissues maximal contractant activity was determined by the addition of 100 /~M histamine. This was washed out and once a steady baseline had been obtained cumulative concentration-response curves were generated for the indicated agonists by progressively increasing their bath concentration according to the method of Van Rossum (1963). Concentration-response curves to test agonists were constructed using paired tissues the second tissue being used for the reference agonist (LTC4). Once the bath concentration of test agonist had reached 10 /~M the tissue was allowed to equilibrate for 15 rain and a concentration-response curve to the reference agonist (LTC4) was then generated to test for partial agonism or antagonism. Observations were normalised against the maximal LTC4 response and expressed as a mean of the total number of observations. The ECs0 (concentration producing a 50% maximal response) values for the agonist were calculated using least squares regression analysis.

[3H]Leukotriene C 4 undergoes rapid and virtually quantitative conversion to [3H]leukotriene D 4 when incubated with guinea-pig lung membranes as demonstrated by the radiochromatogram in fig. la. Incubation in the presence of the y-glutamyltranspeptidase (EC 2.3.2.2.) inhibitor L-serine borate at concentrations in excess of 10 mM essentially suppresses this conversion. Accordingly all binding experiments were performed in the presence of 40 mM L-serine borate. These investigations were carried out using low substrate concentrations (2-5 nM) but it has been reported that even high concentrations (200 nM) of LTC 4 undergo rapid conversion to LTD 4 when incubated with chopped lung tissue (Aharony et al., 1985). 3.2. Kinetic studies Incubation of guinea-pig lung membranes with [3H]LTC4 at 2 0 ° C indicated that specific binding generally reached equilibrium after 10-15 rain and was stable for at least 60 min. Addition of excess unlabelled LTC 4 (5 /tM) induced a rapid reversal of specific binding with complete dissociation being observed within 5 min (fig. 2). The rapid changes in levels of bound [3H]LTC4 are principally due to a very high dissociation rate constant (k 1) and consequently it is difficult to accurately determine the rate constant of the reaction. However, the pseudo first-order rate constant was calculated for each experiment and the association rate constant (kl) was then calculated using the Bmax obtained from saturation analysis k 1=(4.44___1.02)×10 6 mol-1 m i n - I ( n = 4 ) . The dissociation rate constant was calculated to be k_ 1 = 0.58 + 0.14 m i n - 1 (n = 4) giving a kinetically determined dissociation constant K i ) = (k 1/kl) of 130 nM. This is slightly lower than the value determined from equilibrium experiments (see below). 3.3. Saturation studies In the light of the time course studies all equilibrium experiments were carried out for an in-

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Fig. 1. Radiochromatograms of [3H]LTC4 incubated with guinea-pig lung membranes. [3H]LTC4, 5 nM, was incubated with lung membranes (250 /~g/ml) in 50 m M Tris HC1 (pH 7.4) in a volume of 1 ml. After 30 min 2 ml ice-cold buffer was added and the sample was centrifuged at 5 000 x g for 5 min. Of the filtered supernatant 1 ml was injected onto a C]8 /x-Bondapak column eluted with acetonitrile : water : acetic acid (37.5:62.5:0.1) at a flow rate of l m l / m i n . The eluent was passes through a radiochemical detector (NE Isoflo 1) after mixing with scintillation fluid (3 m l / m i n ) . This detected tritium with 5% efficiency. (a) In the absence of L-serine borate. (b) In the presence of 50 m M L-serine borate. The retention times of authentic LTC 4, L T D 4 and LTE 4 are indicated. The chromatograms are from a single representative experiment.

cubation time of 30 min. To determine the affinity and density of the [3H]LTC4 binding sites guineapig lung membranes were incubated with increasing concentrations of [3H]LTC4. As shown in fig. 3a, specific binding proved to be a curvilinear function of ligand concentration whereas nonspecific binding was linear with respect to ligand concentration. Because of the relatively low affinity of the ligand it was not possible to clearly demonstrate saturability of the binding site.

Fig. 2. The kinetics of [3H]LTC4 binding to guinea-pig lung membranes. Guinea-pig lung membranes (100 # g / m l ) were incubated with 5 nM [3H]LTC4 in 50 m M Tris-HC1 (pH 7.4) containing 40 m M L-serine borate in a volume of 10 ml. A parallel incubation was carried out in the presence of 5 I~M LTC 4 to determine non-specific binding. The amount of membrane bound [3H]LTC4 in 200 ~tl aliquots was determined as described in Materials and methods. Specific binding was calculated by subtracting non-specific from total binding. The specific binding is shown as the mean of a triplicate determination from a single representative experiment. Samples were incubated from 0 to 60 min at 20 ° C solely with [3H]LTC4 (B) or with [3H]LTC4 for 30 min followed by addition of 5 /LM LTC 4 (D).

Transformation of the data according to Scatchard (1949) gave a linear plot as shown in fig. 3b. The only way in which the binding of the ligand can be examined with near saturating concentrations is by performing displacement experiments with increasing concentrations of unlabelled ligand. Experiments of this type when analysed by L I G A N D confirmed the presence of a moderate affinity site. Some of these experiments suggested the presence of a lower affinity site ( K D = 400 nM-2 ~tM), but this was not always observed. These experiments confirmed that the higher affinity site was present in very high concentrations (Bmax = 30 + 12 p m o l / m g protein, n = 9) which are substantially higher than the reported concentrations of various neurotransmitter receptors found in guinea-pig lungs. 3. 4. Inhibition studies

We examined the effects on [3H]LTC4 binding of increasing concentrations of a variety of related

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Fig. 3. Saturation experiment and Scatchard Analysis of [3H]LTC4 specific binding m e m b r a n e protein (100 /.tg/ml) was incubated with [3H]LTC4 (from 6 to 66 nM) in the presence of 50 m M Tris HCI buffer (pH 7.4) containing 40 m M L-serine borate and 1% polypep in a volume of 250/~l at 20 ° C for 30 min. Non-specific binding was determined in the presence of 5 # M LTC 4. (a) Specific binding (11) and non-specific binding (O) are shown as the means_+ S.E.M. of triplicate determinations of a representative experiment. The straight line was calculated by linear regression. The saturation isotherm was plotted according to the equation Bound = ([3H]LTC4] + Bmax)/([[ 3H]LTC4] + K D) where Bmax = 32.7 p m o l / m g protein and K D = 39.2 nM. (b) The same data plotted according to Scatchard. Results from a representative experiment are shown.

c o m p o u n d s a n d k n o w n l e u k o t r i e n e antagonists. All four s u l p h i d o p e p t i d e leukotrienes d i s p l a c e d [3H]LTC4 b i n d i n g in a d o s e d e p e n d e n t m a n n e r as d i d 5 - ( S ) - H E T E a n d LTB 4. L T C 4 was m u c h m o r e p o t e n t than the o t h e r leukotrienes as illustrated in fig. 4a. G l u t a t h i o n e was f o u n d to be w e a k l y effective in d i s p l a c i n g [3H]LTC4 binding. T h e o b s e r v e d r a n k o r d e r of d i s p l a c e m e n t was L T C 4 >> L T F 4 > L T D 4 > 5 - H E T E = L T E 4 = LTB 4 > glutathione. W e f o u n d that all of the l e u k o t r i e n e a n t a g o n i s t s that we e x a m i n e d also p r o d u c e d a c o n c e n t r a t i o n related displacement of [3H]LTC4 binding with the following r a n k o r d e r of p o t e n c y although n o n e was p a r t i c u l a r l y effective, S K & F 101132 > F P L 5 5 7 1 2 > S K & F 88046 > LY 171883 (fig. 4b). A l l the results o b t a i n e d are s u m m a r i s e d in table 1. W e synthesised several a n a l o g u e s of L T C 4 to assist us in our c h a r a c t e r i s a t i o n of l e u k o t r i e n e receptors. A c c o r d i n g l y we e x a m i n e d the effect of these c o m p o u n d s on [3H]LTC4 binding. T y p i c a l d i s p l a c e m e n t curves are shown in fig. 5. T h e C 1m o n o m e t h y l ester of LTC4 was f o u n d to be o n l y slightly less effective than L T C 4 whilst d e a m i n o L T C 4 w a s m u c h less effective being c o m p a r a b l e t o L T F 4. R e m o v a l of three d o u b l e b o n d s to give 7-cis-LTC 1 p r o d u c e d a c o m p o u n d that was equieffective with L T C 4. These results i n d i c a t e d that

TABLE 1 The inhibition of 2 n M [3H]LTC4 binding to guinea-pig lung membranes. Competition experiments were performed as described in Materials and methods. The data was transformed into the log-logit form (indirect Hill plot) and least-squares linear regression gave the Hill Slope (nil) and the IC50 as the intercept on the abscissa. The K i was calculated using the method of Cheng and Prusoff (1973) K i = I C s 0 / ( I + [ 3 H LTC 4 ] / K D)Compound

n

K i (/iM)

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4 4 3

20.2_+2.0 35.5_+5.9 3.88_+0.79

3 3 3

21.3 _+1.8 15.0 _+10.0 662_+235

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Fig. 4. Pharmacological specificity of [3H]LTC4 binding to guinea-pig membranes. [3 H]LTC4 (2 nM) was incubated with membrane protein (100 ~g/ml) in 50 mM Tris HCI (pH 7.4) containing 40 mM L-serine borate and 1% polypep and in the presence of increasing concentrations of (a) LTC 4 (11), LTF4 (El), LTD 4 (0), LTE4 (~), and glutathione (A). (b) SK&F 101132 (11), FPL55712 (vl), SK&F 88046 (A) and LY171883 (0). In a volume of 250 ~1 at 20 o C for 30 min. Non-specific binding was defined as binding in the presence of 5 ~M LTC 4. The percent specific binding was calculated and plotted against the concentration of displacing agent used. (L-cysteine, 10 mM, was added to the incubation buffer for experiments with LTD 4 and SK&F101132.) All points were determined in triplicate. All curves are plotted for inhibition of binding to a single site in accord with the Law of Mass Action except for glutathione (2 sites). Displacement curves for 5(S)-HETE and LTB4 were omitted for the sake of clarity. All results shown are from representative experiments.

this site was h i g h l y s e l e c t i v e for g l u t a t h i o n e c o n taining leukotrienes. We therefore prepared 1,2,3,4-tetranor-LTC 4 and purchased S-propylglutathione and S-decylglutathione. We found S-prop y l g l u t a t h i o n e to b e o n l y slightly m o r e e f f e c t i v e t h a n g l u t a t h i o n e b u t , to o u r surprise, b o t h o f the other, more lipophilic, compounds had similar p o t e n c i e s to t h e L T C 4 m e t h y l ester in d i s p l a c i n g b o u n d [DH]LTC4.

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Fig. 5. The structural specificity of [3H]LTC4 binding to guinea-pig lung membranes. [3H]LTC4 (2 nM) was incubated with membrane protein (100 ~g/ml) in 50 mM Tris HCI (pH 7.4) containing 40 mM L-serine borate plus 1% polypep and in the presence of increasing concentrations of 7-cis-LTC 1 (O), LTC4-Me ester (©), tetranor LTC4 ([2) S-decyl glutathione ( 0 ) , deamin°-LTC4 (A) and S-propylglutathione (~), in a volume of 250 btl at 20°C for 30 min. Non-specific binding was defined as binding in the presence of 5 /.tM LTC 4. The percent specific binding was calculated and plotted against the concentration of displacing agent used. All points were determined in triplicate. All curves are plotted for inhibition of binding to a single site in accord with the Law of Mass Action except for S-propylglutathione (2 sites). All results shown are from representative experiments.

3.5. Pharmacological characterisation of the binding site I n a d d i t i o n to e x a m i n i n g the effects o f s t r u c t u r ally r e l a t e d c o m p o u n d s , we also e x a m i n e d the effects on [3H]LTC4 binding of a range of unrel a t e d d r u g s o f w i d e l y d i f f e r i n g s t r u c t u r e s a n d actions. All o f these w e r e f o u n d to p r o d u c e n o s i g n i f i c a n t d i s p l a c e m e n t of [DH]LTC4 b i n d i n g w h e n t e s t e d at c o n c e n t r a t i o n s up to 100 t~M, i.e. w e l l a b o v e t h e i r n o r m a l t h e r a p e u t i c dose. T h o s e c o m p o u n d s tested a r e listed in t a b l e 2. Since metal ions and guanine nucleotides have b e e n f o u n d to h a v e p r o n o u n c e d effects o n the b i n d i n g o f a g o n i s t s ( C o o p e r , 1983) we e x a m i n e d

330 200

TABLE 2 Pharmacologically active agents that do not displace [3H]LTC4 binding. These compounds failed to produce a significant inhibition of 2 nM [3H]LTC4 binding at drug concentrations up to 100 /xM. Student's t-test was used to assess whether binding in the presence of drug differed significantly from that in its absence. Drug Atropine Phentolamine Yohimbine Propranolol Histamine Mepyramine Cimetidine Methysergide Phenoxybenzamine Prostaglandin F 2 Prostacyclin Indomethacin Captopril Diltiazem ) Nifedipine ~ Verapamil } L-Glutamic acid REV 5901 U46619 AH 19437 ~ EP 045 BM 13177

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a /fl-Antagonist az-Antagonist fl-Antagonist H 1 / H 2 agonist H l antagonist H z antagonist 5-HT antagonist a-Antagonist Vasoconstrictor Vasodilator Cyclo-oxygenase inhibitor Angiotensin II inhibitor

--6

-5

-4

-3

-2

-1

LOS[N] Fig. 6. The regulation of [3H]LTC4 binding by nucleotides and cations. [3H]LTC4, 2 nM, was incubated with guinea-pig lung membranes (100 ~g/ml) in 50 mM Tris HCI (pH 7.4) containing 40 mM L-serine borate and 1% polypep in the presence of increasing concentrations of GppNHP (11), Ca 2+ (E2) or Na ÷ (A) in a volume of 250 /H at 20 °C for 30 min. Non-specific binding was determined under identical conditions in the presence of 5 btM LTC4.

Calcium antagonists Neurotransmitter Lipoxygenase inhibitor Thromboxane mimetic Thromboxane antagonists

the effects of increasing concentrations of calcium and sodium ions and the non-hydrolysable GTP analogue guanylimidodiphosphate (GppNHP) on [ 3 H ] L T C 4 b i n d i n g . A s s h o w n i n fig. 6, G p p N H P had no effect on [3H]LTC4 binding whilst both calcium and sodium produced a dose-dependent potentiation of specific binding.

3. 6. Spasmogenic actiuity of leukotrienes on guineapig ileum and lung strip All those compounds listed in table 1 (except 5-HETE, LTB 4 and glutathione) were examined for spasmogenic activity on guinea-pig ileum and l u n g strip. T h o s e c o m p o u n d s t h a t p r o d u c e d l i t t l e or no responses were then examined for antagonism of LTC4-induced contractions. For t h o s e c o m p o u n d s t h a t w e r e f o u n d to b e a g o n i s t s , t h e p D 2 ( n e g a t i v e l o g a r i t h m o f t h e ECs0 ) w a s

calculated. In the case of the antagonists the pK B values were calculated. All the results are summ a r i s e d i n t a b l e 3. TABLE 3 The contractile activity of leukotrienes on guinea-pig tissue. The agonist activity was determined as described in the Materials and methods and the EDs0 calculated. The negative logarithm is the pD 2. Those compounds with no contractile activity were assessed for their ability to shift the dose-response curve to LTC 4. Where a shift was observed the dissociation constant (KB) was calculated. Those compounds which displayed no antagonist activity are shown as having a pK B < 4. Compound

Ileum pD 2

LTC 4 LTD 4 LTE a LTF4 LTC4-Me Deamino-LTC 4 7-cis- LTC ] Tetranor-LTC4 S-Decylglutathione S-Propylglutathione FPL55712 LY 171883 SK&F 88046 SK&F 101132

Parenchyma pK B

7.70 8.40 7.92 6.15 7.00 5.72 7.35

pD 2

pK B

8.10 7.40 6.40 6.06 6.55 5.72 5.00 <4 <4 <4 5.0 5.5 <4 <4

<4 <4 <4 <4 <4 <4 <4

331

a)

3.7. Correlation of binding affinity and smooth muscle activity

12 ml

T h e o c c u p a n c y of this L T C 4 b i n d i n g site was c a l c u l a t e d a n d c o m p a r e d with the o b s e r v e d cont r a c t a n t response of guinea-pig lung p a r e n c h y m a to L T C 4 in the presence of i n d o m e t h a c i n a n d L-serine borate. A s shown in fig. 7, there is a g o o d a g r e e m e n t b e t w e e n the b i n d i n g site o c c u p a n c y a n d s p a s m o g e n i c activity which w o u l d suggest a very small r e c e p t o r reserve in this tissue if the o b s e r v e d specific b i n d i n g sites c o r r e s p o n d to L T C 4 receptors. T h e b i n d i n g affinities of the c o m p o u n d s listed in table 1 were c o n v e r t e d to their negative logar i t h m s a n d c o m p a r e d with the p D z / p K B values d e t e r m i n e d for the s a m e c o m p o u n d s on g u i n e a - p i g ileum a n d p a r e n c h y m a . T h o s e c o m p o u n d s for which no f u n c t i o n a l activity was o b s e r v e d were a r b i t r a r i l y assigned a p K B of 4 for the p u r p o s e s of the c o r r e l a t i o n analysis. T h e values are illustrated g r a p h i c a l l y in fig. 8 a n d least-squares linear regression c o n f i r m e d the initial i m p r e s s i o n that there was n o c o r r e l a t i o n b e t w e e n the b i n d i n g affinities a n d f u n c t i o n a l activity on either tissue.

i.

7C 6(

70 60 ~.

5(

5O 4O

4(

+ -10

~"

10 -9

-,

-4°

LO6tLTC4]

Fig. 7. Comparison of the calculated LTC 4 binding site occupancy with the contractile response of guinea-pig

parenchymal strips to LTC4. The contractile response of guinea-pig parenchymal strips was determined by construction of cumulative dose-response curves to LTC4 in the presence of 45 mM L-serine borate and 1 mM indomethacin. The responses were normalised relative to that of 1/~M LTC4 defined as 100% and are shown as the mean+ S.E. of six preparations. The plotted curve shows the calculated binding site occupancy( = [LTC4] x 100/([LTC4] + KD)) using the mean K D ( = 52.6 nm) determined in these studies.

m,

m, m7 ms

IN2 m,

n3

14

m, n,

DIz

10121311

3 4 5 6 7 8 PKt

m, [']11

U7

4

AA

10 13

A

14

4 5 6 7 PKt

9Aide

8 9

Fig. 8. The lack of correlation of binding affinity with functional activity. The binding affinity (pKi) of the following compounds was plotted against their functional activity (pD 2

or pKB) on (a) guinea-pig parenchymal strip and (b) guinea-pig ileum. Agonists (m), antagonists (O), inactive compounds (zx). (1) LTC4, (2) LTD4, (3) LTE 4, (4) LTF4, (5) LTC4-Me, (6) deamino-LTC4, (7) 7-cis-LTCt, (8) tetranor-LTC4, (9) S-decylglutathione, (10) S-propylglutathione, (11) FPL55712, (12) LY171883, (13) SK&F88046, (14) SK&F101132.

4. D i s c u s s i o n

O u r results d e m o n s t r a t e that guinea-pig lung c o n t a i n s a highly selective b i n d i n g site for l e u k o t r i e n e s to which L T C 4 b i n d s with high affinity. C o m p a r i s o n of the characteristics of this b i n d ing site with those r e p o r t e d in the s a m e p r e p a r a tion for [3H]LTD4 ( P o n g a n d D e Haven, 1983; N o r m a n et al., 1984) reveals that they are distinct entities with a m u c h greater c o n c e n t r a t i o n of the [3H]LTC4 site. F u r t h e r evidence of the distinct n a t u r e of these site is p r o v i d e d b y a u t o r a d i o g r a p h i c localisation studies which show that the [3H]LTC4 sites are p r i n c i p a l l y localised to s m o o t h muscle a n d a i r w a y e p i t h e l i u m whereas the [3H]LTD4 sites a p p e a r e d to b e m o r e p e r i p h e r a l in l o c a t i o n ( N o r m a n et al., 1987). This clearly suggested that the [3H]LTC4 b i n d ing sites we have c h a r a c t e r i s e d c o r r e s p o n d to p h y s i o l o g i c a l l y relevant r e c e p t o r s since one w o u l d a n t i c i p a t e that r e c e p t o r s for a p o t e n t c o n t r a c t i l e a g o n i s t of a i r w a y tissue w o u l d b e f o u n d on a i r w a y s m o o t h muscle. T h e a g r e e m e n t b e t w e e n the c a l c u l a t e d b i n d i n g site o c c u p a n c y a n d the ob-

332 served contractile response (fig. 7) would appear to confirm this hypothesis. The low affinity of FPL55712 for the observed binding site agrees with its ineffectiveness (at 10 ttM) at antagonising the direct response of parenchymal strips to leukotrienes (Weichman et al., 1982). This latter observation has prompted speculation that the guinea-pig lung contains a distinct leukotriene receptor from those present in guinea-pig ileum and trachea. However, LTC 4 and L T D 4 are essentially equipotent and equieffective in contracting guinea-pig parenchymal strips. Such an observation suggests that they probably act at the same receptor and one would therefore expect to find c o m p a r a b l e binding affinities for the two leukotrienes. The dramatic difference in binding affinities observed in this study hardly agrees with the observed responses, and would imply the existence of distinct receptors for LTC 4 and L T D 4. If there are distinct receptors for L T C 4 and L T D 4 in guinea-pig lung one would anticipate that compounds which displace [3 H]LTC4 binding with high affinity would possess comparable biological activity. However, we found no correlation between functional activity and binding affinity (fig. 8). The greatest discrepancies were with the compounds tetranor-LTC 4 and S-decylglutathione both of which were devoid of spasmogenic activity on the lung strip and failed to antagonise leukotriene-induced contractions despite binding affinities (Ki) of 118 and 154 nM. Whilst one could speculate that the latter compound, being highly modified, might not reach the receptor because of transport problems the former is only slightly altered from the natural compound and should therefore not have a problem of receptor access. The conclusion from such results must be that the binding site under study is not a contractile receptor. The high degree of specificity that this site displays for glutathione-containing molecules suggests that it may have some functional role and in view of the localisation of these sites it suggests that it plays a role in either the transport, or metabolism, of LTC 4. Clearly the glutathione specificity of the site points to the latter role but the observed affinities do not agree with the

Michaelis constants (KM) observed for the metabolism of LTC 4 by y-glutamyltranspeptidase (Orning and Hammarstrom, 1984). These studies were carried out with known enzymes from organs in which leukotrienes have not been shown to possess physiological effects and it may be the case that organs in which LTC 4 is physiologically active contain an iso-enzyme of y-glutamyltranspeptidase specific for LTC 4. This lack of correlation of binding affinity with functional activity is far from unique in the history of binding studies (Laduron, 1984). It further serves to illustrate the point that binding affinity must be compared with, and correlate to, functional activity before a binding site can reasonably be described as a functional receptor. It has recently been reported (Mong et al., 1985a) that one can inhibit [3H]LTC4 binding with high affinity by LTC 4 analogues yet such compounds were devoid of functional activity. Our studies confirm their results using slightly different LTC 4 analogues. The ease of performance of radioligand binding assays and availability of new radioligands has encouraged the description of a large number of 'receptors' solely on the basis of binding experiments. We have shown here that with a new ligand we can find a novel, selective, binding site but we have conclusively demonstrated that this is not to a contractile receptor, although much of our data suggested that we were observing binding to a receptor. If we compare our studies of [3H]LTC4 binding with those reported by other groups to tissues such as guinea-pig brain, human lung and rat lung, we find that the reported ligand affinity varies markedly between 2 and 100 nM but the binding capacity is always very high (5-100 p m o l / m g protein); furthermore the binding is potentiated by sodium and calcium ions but is not affected by guanine nucleotides and, in inhibition e x p e r i m e n t s , L T C 4 is always much more potent than other leukotrienes at displacing [3H]LTC4 binding. This suggests that the binding site is essentially similar in every case and is unlikely to be a contractile receptor. This provides an explanation for the presence of such binding sites in a tissue which produces little, or no, contractile

333 r e s p o n s e t o l e u k o t r i e n e s , n a m e l y r a t l u n g ( P o n g et al., 1 9 8 3 ; B r u n e t et al., 1985). It is p e r t i n e n t t o p o i n t o u t t h a t a n u m b e r o f reviews have appeared which cite binding studies w i t h [ 3 H ] L T C 4 a n d [ 3 H ] L T D 4 as a b s o l u t e p r o o f that there are at least two distinct leukotriene r e c e p t o r s h i g h l y s p e c i f i c f o r L T C 4 a n d L T D 4 (e.g. L e w i s a n d A u s t e n , 1984). W e h a v e s h o w n t h a t t h i s e v i d e n c e is i n v a l i d a n d f u r t h e r m o r e w e w o u l d s u g g e s t t h a t it is i n t u i t i v e l y u n l i k e l y t h a t t w o s u c h structurally similar agonists would possess highly d i s t i n c t r e c e p t o r s (cf. a d r e n a l i n e a n d n o r a d r e n a line). W e w o u l d , h o w e v e r , a g r e e t h a t t h e r e a r e t w o or more distinct leukotriene receptors on the basis o f f u n c t i o n a l s t u d i e s w i t h a n t a g o n i s t s s u c h as F P L 5 5 7 1 2 a n d L Y 171883. T o f u r t h e r d e l i n e a t e the leukotriene receptors requires the synthesis of n o v e l l e u k o t r i e n e a n t a g o n i s t s w h i c h w o r k o n tiss u e s w h e r e F P L 5 5 7 1 2 is i n e f f e c t i v e , s u c h as t h e g u i n e a - p i g l u n g s t r i p . W e feel t h a t b i n d i n g s t u d i e s , carefully performed can aid such receptor classification, although not necessarily with the currently available ligands, and that studies of the biochemical r e s p o n s e s t o t h e s e c o n t r a c t i l e a g o n i s t s (e.g. phosphatidylinositol turnover) could further clarify the nature of these receptors.

Acknowledgements The authors wish to express their appreciation to Mrs. Mandy Raynham for the preparation of the manuscript and to Dr. A. Richardson for supplying a curve-fining programme.

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