51

European Journal of Pharmacology, 194 (1991) 51-61 © 1991 Elsevier Science Publishers B.V. 0014-2999/91/$03.50 ADONIS 0014299991001791

EJP 51730

Characterization of the leukotriene D 4 receptor in hyperreactive rat lung K a t h l e e n M. Metters, Elizabeth A. F r e y and A n t h o n y W. F o r d - H u t c h i n s o n Department of Pharmacology, Merck Frosst Centre~or Therapeutic Research, P.O. Box 1005, Pointe Claire, Dorval, Quebec, Canada H9R 4P8 Received 2 November 1990, accepted 27 November 1990

A [3H]leukotriene D4 radioreceptor binding assay has been established in rat lung and has been used to fully characterize the leukotriene D4 receptor in lung membranes from an inbred strain of rats displaying non-specific bronchial hyperreactivity. [3H]leukotriene D4 specific binding in this tissue is of high affinity (K D 0.12 nM), saturable (Bmax 42 fmol/mg protein), inhibited by both guanine nucleotide analogues and sodium ions and increased by divalent cations. In addition, K i values show that agonists, but not antagonists, compete for [3H]leukotriene D4 binding in rat lung with the same potency as they compete for [3H]leukotriene D4 binding in guinea-pig lung, the classical tissue for leukotriene D4 receptor studies. Finally, [3H]leukotriene D4 binding in hyperreactive rat lung has been compared with [3H]leukotriene D4 binding in lung tissue from Fischer rats, which are a less responsive strain. Leukotriene I34; Leukotriene D4 receptors; Bronchial hyperreactivity; (Rat)

1. Introduction

It has been known for almost a decade that the major constituents of the slow-reacting substance of anaphylaxis are the peptidyl leukotrienes, leukotriene (LT) C 4, L T D 4 and LTE4, Samuelsson (1983). These compounds have been proposed as mediators of immediate hypersensitivity reactions, including the bronchoconstriction experienced during human bronchial asthma (Piper, 1984). Early reports provoked particular interest in L T D 4, describing this substance as being the most a b u n d a n t peptidyl leukotriene produced during anaphylaxis in the guinea pig and the most spasmogenic on respiratory smooth muscle (Lewis et al., 1980; Morris et al., 1980; Keppler et al., 1987). It is now widely accepted that the physiological actions of L T D 4 are mediated by interaction of this ligand with specific L T D 4 receptors. This receptor has been extensively characterized in guinea-pig lung preparations where LTD 4 binding is found to be saturable and of high affinity, inhibited by both non-hydrolysable G T P analogues and sodium ions, but increased by divalent cations, observations consistent with the proposal that the LTD 4 receptor is regulated through interaction with a G T P binding protein (G-protein) (Bruns et al., 1983; Pang and DeHaven, 1983; Mong et al., 1984; 1985b).

Correspondence to: K.M. Metters, Department of Pharmacology, Merck Frosst Centre for Therapeutic Research, P.O. Box 1005, Pointe Claire, Dorval, Quebec, Canada H9R 4P8.

Considerable effort has been expended in the development of specific LTD 4 receptor antagonists for use as therapeutic agents in the treatment of diseases such as bronchial asthma. In order to assess candidate LTD 4 receptor antagonists, several animal models have been established, including L T D 4 or ascaris-challenge of either conscious squirrel monkeys or sheep and antigen challenge of sensitized rats or guinea pigs (Hamel et al., 1986; Abraham et al., 1986; Piechuta et al., 1987; Patterson and Kelly, 1974). An inbred line of hyperreactive rats has been developed which provides a reproducible model for assessing the biological activity of candidate LTD 4 receptor antagonists (Holme and Piechuta, 1981; Piechuta et al., 1987). These hyperreactive rats are homogeneous in their response to antigen challenge, producing prolonged and reproducible periods of dyspnea, as well as other symptoms associated with asthma. The hypothesis that LTD 4 plays a role in mediating the antigen-induced bronchoconstriction observed in the hyperreactive rat has been supported by evidence from a series of experiments in which these animals have been directly compared with Fischer rats, which do not develop dyspnea when challenged with antigen. Hyperreactive rats display a striking fall in dynamic compliance, but no significant change in pulmonary resistance, following aerosol application of LTD 4, whereas Fischer rats do not. Consistent with this, in vitro assays have shown that parenchyma from hyperreactive rats is considerably more responsive to L T D 4 than similar preparations from Fischer rats, whereas rat

52 trachea is unresponsive to leukotrienes (Brunet et al., 1983). In addition, lung tissues from sensitized hyperreactive rats release significantly higher amounts of peptidyl leukotrienes, when challenged with antigen, than the corresponding Fischer rat tissues (Brunet et al., 1985). This finding is consistent with in vivo experiments showing elevated levels of leukotrienes in bile following antigen provocation (Foster et al., 1988). Finally, the involvement of LTD 4 in mediating the antigen-induced bronchoconstriction observed in this model has been demonstrated by showing that the duration of antigen-induced dyspnea in the hyperreactive rat can be inhibited by an antiserotonin agent, methysergide in combination with either an LTD 4 receptor antagonist or a leukotriene biosynthesis inhibitor (Piechuta et al., 1987). Although rat models have been used to assess LTD 4 receptor antagonists (Piechuta et al., 1987; Jones et al., 1989), this receptor has not yet been fully characterized in rat lung. In this report we describe the development of an [3H]LTD4 radioreceptor binding assay using rat lung membrane preparations and show that [3H]LTD4 binding in this tissue fulfils the criteria necessary for binding to an authentic LTD 4 receptor. Furthermore, we have used this assay to compare L T D 4 receptors in lung preparations from both hyperreactive and Fischer rats, in order to evaluate whether the differences displayed by these strains in response to either antigen or leukotriene challenge, are reflected at the receptor level.

2. Materials and methods

2.1. Materials Inbred hyperreactive rats were bred under pathogen-free conditions at Taconic Farms (Germantown, NY); Fischer rats were from Charles River; [3H]LTD4 (180 C i / m m o l ) and [3H]LTC4 (39.3 C i / m m o l ) were from New England Nuclear; LTC 4, LTD4, LTE4, 5R,6S-LTD4, MK-571 (Jones et al., 1989), ICI 198,615 (L-669,020), ICI 204,219 (L-691,013) and YM 17690 (L-682,111) were synthesized by the Department ot Medicinal Chemistry at The Merck Frosst Centre for Therapeutic Research; SKF 104353 was a generous gift from Smith, Kline and French Laboratories; Cilastatin (MK-791) was from Merck, Sharp and Dohme; Acivicin was from The Upjohn Company; G T P ' t S and GMPPNP were from Boehringer Mannheim; DL-cysteine, glycine, D- and L-penicillamine, L-serine, boric acid, trypsin inhibitor (soybean), bacitracin, benzamidine and phenylmethylsulfonylfluoride were from the Sigma Chemical Company. All other reagents were of analytical grade.

2.2. Methods 2.2.1. Rat lung membrane preparation Rats (male; 250-300g) were killed and lung tissues were removed. All subsequent procedures were performed either on ice or at 4 ° C . Connective tissue and large blood vessels were dissected away and the lung tissue was finely minced prior to homogenisation in 10 volumes of 10 mM HEPES p H 7.4 containing 0.25% ( w / v ) sucrose, 2 mM EDTA, 5 # g / m l trypsin inhibitor (soybean), 100 /zg/ml bacitracin, 1 mM benzamidine and 10 /~M phenylmethylsulfonylfluoride, using 5 s bursts of a polytron (Brinkman Instruments). The homogenate was centrifuged at 1000 × g for 10 min and the supernatant was decanted and centrifuged for a further 10 min at 10 000 × g. The resulting supernatant was subjected to a final centrifugation step for 40 min at 100000 × g and the final supernatant was discarded prior to storage of the membrane pellets at - 8 0 ° C. 2.2.2. [ 3H]Leukotriene radioreceptor binding assay [3H]Leukotriene radioreceptor binding assays were routinely performed in a final volume of 500 ~1 of 10 mM HEPES pH 7.4 containing 20 mM CaC12 and 0.01% ( w / v ) bovine serum albumin. For the [3H]LTD4 binding assay incubations also included 20 m M Lpenicillamine, 50 mM serine-borate (only when LTC4 was used as a competing ligand), 150 pM [3H]LTD4 (180 Ci/mmol, - 15 000 cpm) and either 140 #g or 25 /~g of rat or guinea-pig lung membrane protein, respectively. For the [3H]LTC4 binding assay incubations also included 50 mM serine-borate, 1 nM [3H]LTC4 (39.3 Ci/mmol, - 22 000 cpm) and 10 ~g of rat lung membrane protein. Non-specific binding was determined in the presence of either 1 /~M L T D 4 or 10 /~M of the LTD 4 receptor antagonist MK-571 (Jones et al., 1989) for [3H]LTD4 binding assays, or 1 /~M LTC 4 for [3H]LTC4 binding assays. Incubations were conducted for 60 min at 2 2 ° C prior to separation of the bound and free radioligand by rapid filtration through Whatman G F / B filter paper presoaked at 4 ° C in 10 mM HEPES pH 7.4 containing 0.01% ( w / v ) bovine serum albumin. Filters were then washed with 16 ml of 10 mM HEPES pH 7.4 containing 0.01% ( w / v ) bovine serum albumin, at 4 ° C, before residual radioactivity bound to the filters was determined by liquid scintillation counting in 10 ml of Biofluor (New England Nuclear). The counting efficiency was typically 45-50% for [3H]leukotriene. All buffers were adjusted with 10 M KOH. Modifications made to this general method for individual experiments are described in detail in the appropriate figure legends. In all experiments specific binding was defined as the difference between total binding and non-specific binding, determined in the presence of an excess (1000-fold) of the appropriate unlabelled competing ligand. Under

53

these experimental conditions, lung membrane preparations bound approximately 5-7.5% of the [3H]leukotriene added to the incubation. Specific binding was linear with respect to both lung membrane protein concentration and radioligand concentration. [3H]LTD4 specific binding routinely represented 74 or 92% of the total [3H]LTD4 binding to rat or guinea-pig lung preparations, respectively, whereas [3H]LTC4 specific binding represented 90% of total [3H]LTC4 binding to rat lung membranes.

2.2.3. Reverse phase high performance fiquid chromatography Reverse phase high performance liquid chromatography (RP-HPLC) was conducted to assess the stability of [3H]LT under the conditions employed for radioreceptor binding assays. The experimental procedure was as follows: receptor binding assays were performed in a final incubation volume of 1.5 ml of 10 mM HEPES pH 7.4 containing 20 mM CaC12, 0.01% (w/v) bovine serum albumin, 150 pM [3H]LT (180 Ci/mmol), 420/xg of rat membrane protein and 0-20 mM of the appropriate enzyme inhibitor, except for serine-borate where the concentration range was 0-50 mM. The inhibitors tested were acivicin and serine-borate in [3H]LTC4 incubations and DL-cysteine, glycine, cilastatin and D- and L-penicillamine in [3H]LTD4 incubations, reference table 1. Following a 60 min incubation at 22 ° C, samples were filtered through Whatman G F / B filters and the filtrate collected (unbound ligand). Filters were then washed with 16 ml of ice-cold 10 mM HEPES pH 7.4 containing 0.01% (w/v) bovine serum albumin, prior to elution of the bound ligand with 2 ml of 50% (v/v)

TABLE 1 Inhibition of leukotriene metabolism by rat lung membranes. [3H]LTC4 and [3H]LTD4 were incubated with rat lung membrane in the presence of increasing concentrations of enzyme inhibitor and analysed by R P - H P L C as described in Methods. The percentage conversion of [3H]LTC4 or [3H]LTD4 at each inhibitor concentration was determined from the corresponding R P - H P L C profile. The IC~o value has been defined as the concentration of inhibitor needed to inhibit 50% conversion of radioligand under these incubation conditions, n.i. indicates that no inhibition was observed up to 20 m M inhibitor. These are representative data from two experimental observations giving similar values. Inhibitor

IC5o (mM)

LTC~ to LTD~ conversion Serine-borate Acivicin

5.0 2.3

LTD~ to LTE~ conversion L-Penicillamine D-Penicillamine DL-Cysteine Glycine Cilastatin

3.6 > 20 9.6 n.i. n.i.

MeOH. Bound and unbound ligand fractions were dried under vacuum, resuspended in 100 /~1 of RP-HPLC buffer (54 : 28 : 18 : 1 (v/v) H20-CH3CN-MeOH-HOAc adjusted to pH 5.6 with 10 M NaOH) containing leukotriene standards (50 ~tg/ml of LTC4, LTD4, LTE 4 and N-acetyl-LTE4) and analysed by RP-HPLC using a Nova Pak C18 column (0.39 × 15 cm; Waters Assoc.) eluted isocratically at a flow rate of 1 ml / min with RP-HPLC buffer. [3H]Leukotriene peaks were monitored by on-line liquid scintillation counting (Berthold HPLC radioactivity monitor LB 506 C1) following mixing with 4 volumes of Aquasol (New England Nuclear) and identified by co-elution of non-radioactive leukotrienes monitored by optical density at 280 nm. The optical density signal generated spectrophotometrically was converted and displayed in/~V by the Berthold HPLC radioactivity monitor, where 1 absorbance unit corresponded to 1 V.

Protein determination Protein determinations were conducted using the Pierce BCA reagent kit with bovine serum albumin as standard.

3. Results

3.1. Leukotriene metabolism Initial experiments employing RP-HPLC analysis showed that both [3H]LTC4 and [3H]LTD4 were completely degraded to [3H]LTE4 following incubation with rat lung membrane preparations under conditions successfully used for guinea-pig lung [3H]LTD4 receptor binding assays. Several proposed inhibitors of the 3'glutamyl-transpe.ptidase responsible for LTC4 conversion to LTD4 (Orning and Hammarstr~Sm, 1980) and the dipeptidase responsible for LTD4 conversion to LTE 4 (Hammarstr~Sm et al., 1985) have been assessed for their effectiveness in preventing this degradation. The inhibitors chosen were acivicin (0rning et al., 1981) and serine-borate (Tate and Meister, 1978) for yglutamyl-transpeptidase and DL-cysteine (Sok et al., 1980), glycine (Mong et al., 1984), cilastatin (K~511er et al., 1985) and both D- and L-penicillamine (Huber and Keppler, 1987) for the dipeptidase. Leukotriene receptor binding assays for [3H]LTC4 and [3H]LTD4 were performed in the presence of increasing concentrations of the appropriate enzyme inhibitor, as described in Methods. Bound and unbound radioligand fractions were separated by filtration and the profile of radiolabelled material analysed by RPHPLC. Analogous profiles were obtained for each inhibitor and representative results, in this case for the inhibition of [3H]LTD4 metabolism by DL-cysteine, are shown in fig. 1. Analysis of the unbound fractions

54 A. LT STANDARDS 5000 I

t~--LTC4

o .[

r'~._-.~_o~_~.J / c. 10ram CYSTEJNE

~:~

50 l

'~'-,..-~._

LTD4---~/I

/

50

LTE4

D. 20raM CYSTEINE

LTD4___~ If/1 LTE4

0

5

10

15

20

25

30

RETENTION TIME (MIN) Fig. 1. RP-HPLC analysis of leukotriene metabolism. RP-HPLC was conducted to assess the stability of [3H]LT under the conditions employed for radioreceptor binding assays as described in Methods. Representative results for the inhibition of [3H]LTD4 metabolism by DL-cysteine are shown above with the retention times of the leukotriene standards, LTC4, LTD4, LTE4 and N-acetyl-LTE4 standards (A) and the profile of radiolabelled material following incubation of [3H]LTD4 with rat lung membrane in the absence (B) and presence of 10 mM (C) and 20 mM (D) of DL-cysteine.

showed that in the absence of inhibitor [3H]LTD4 was completely converted to radiolabelled material which co-eluted with the L T E 4 standard, panels A and B. However, in the presence of 10 m M DL-cysteine, 52% of the [3H]LTD4 remained as authentic ligand, co-eluting with the L T D 4 standard, with only 38% present in the LTE 4 standard position, panel C. Increasing the DL-cysteine concentration to 20 m M inhibited approximately 90% of the [3H]LTD4 conversion, panel D. Since both conversion of [3H]LTD4 and the appearance of the radiolabelled [3H]LTE4-1ike product were inhibited in a concentration dependent manner, it was concluded that the [3H]LTD4 degradation observed under these assay conditions was due to metabolism from [ 3H]LTD4 to [ 3H]LTE4. Radiolabelled material was only recovered in the bound fractions from incubations containing either 10 m M or 20 m M DL-cysteine where [3H]LTD4 degradation was inhibited. Under these conditions, [3H]LTD4 was the only product recovered, coeluting with the L T D 4 standard. In constrast, there was no detectable [3H]LTE4 recovered in the bound frac-

tion from incubations performed in the absence of inhibitor, when analysis of the unbound fraction showed that the [3H]LTD4 had been,completely converted to [3H]LTE4. These data indicate that [3H]LTE4 was not bound in significant amounts under these assay conditions. The results for the series of inhibitors investigated are summarized in table 1. Both acivicin and serineborate inhibited [3H]LTC4 metabolism, with IC50 values of 2.3 m M and 5 m M respectively. L-Penicillamine was the most effective inhibitor of [3H]LTD4 metabolism, having an IC50 of 3.6 mM. This was in constrast to D-penicillamine which inhibited only 12% of [3H]LTD4 conversion at 20 raM, the highest concentration tested. DL-cysteine was approximately 3-fold less potent than L-penicillamine with an IC50 of 9.6 mM, whilst no inhibition of [3H]LTD4 metabolism was observed using either cilastatin or glycine at concentrations up to 20 mM. Based on these results 50 m M serine borate and 20 m M L-penicillamine were routinely included in leukotriene receptor binding assay incubations since, under these conditions, metabolism of both LTC 4 and L T D 4, respectively, was completely inhibited. Similar R P - H P L C analysis of guinea-pig lung [3H]LTD4 receptor binding assay incubations showed that radioligand degradation was not a problem in the presence of this tissue. Therefore guinea-pig lung [3H]LTD4 binding assays were performed in the absence and presence of 20 m M L-penicillamine to assess whether this inhibitor had an effect on [3H]LTD4 receptor binding constants. Scatchard analysis demonstrated that the addition of 20 m M of L-penicillamine to the incubation medium had no effect on the dissociation constant (K D = 0.1 nM in both cases) or the m a x i m u m number of specific binding sites (Bmax = 477 f m o l / m g protein in the absence and 428 f m o l / m g protein in the presence of inhibitor), for the specific binding of [3H]LTD4 to this tissue. These values are representative data from two experimental observations.

3.2. Rates of association and dissociation of [3H]LTD4 binding to rat lung The rate of association of [3H]LTD4 binding to rat lung m e m b r a n e was slow, reaching equilibrium over a 60 min incubation period (fig. 2). Addition of a large excess of unlabelled competing agonist (1/~M LTD4) or antagonist (10 # M MK-571), upon establishment of equilibrium, dissociated approximately 60% of [3H] L T D 4 binding. The inclusion of 50 m M NaC1, or 300 FM GTP'~S, with the competing ligand, provoked complete dissociation of [3H]LTD4 binding to non-specific levels, while the effect of either 50 m M NaC1, or 300 /~M GTPTS, alone was intermediate. F r o m these data [3H]LTD4 receptor equilibrium binding assays were conducted with a 60 min incubation at 22 ° C.

55 1500

TOTAL

e~

o z ¢3

1000

MK-571 NaCI MK-571/NaCI NON-SPECIFIC

z

500 I._l "10

0

I

I

I

50

100

150

effects of both non-hydrolysable GTP analogues and cations on [3H]LTD4 binding to rat lung membrane was examined in more detail. The non-hydrolysable GTP analogues, GTPyS and GMP-PNP, were found to inhibit [3H]LTD4 specific binding to rat lung membrane with IC50 values of 20 #M and 325 /~M, respectively (fig. 4). Scatchard plot analysis of [3H]LTD4 specific binding saturation curves performed in the absence and presence of 300 #M GTPyS showed that the principle effect of GTP analogues was on the number of binding sites and not the K D. With the inclusion of 300 /xM GTPyS in the

INCUBATION TIME (MIN)

Fig. 2. Rates of association and dissociation of [3H]LTD4 binding to rat lung membrane. The rate of association of total (O) and nonspecific (A) binding were monitored by sampling from homogenous receptor binding incubations of 20 ml and 10 ml, respectively. Incubations were performed in the absence (e) and presence (A) of 10 #M MK-571 and 500 #i aliquots were removed and filtered at the required time intervals. After 60 min the rate of dissociation of total binding was monitored, by sampling, following the addition of either 10 # M MK-571 (zx), 50 mM NaCI ( o ) or 10 #M MK-571 with 50 mM NaCI (1:3). Essentially the same results were obtained replacing 10 #M MK-571 with 1 #M LTD4, or 50 mM NaC1 with 300 /~M GTPyS. These are representative data from four experimental observations giving similar results.

A. SATURATION CURVE A

c1_z I.-LU

~O

I-

100

=E O ~ zE O o

=E

75 50

SPEC,FtC

25

250

3.3. Scatchard and Hill plot analysis of [3H]LTD4 binding to rat lung [3H]LTD4 specific binding to rat lung membrane is saturable, as demonstrated by incubating tissue with an increasing concentration (0.01-1 nM) of [3H]LTD4 in the absence and presence of unlabelled competing ligand (fig. 3A). Analysis by Scatchard plot showed that [3H]LTD4 specific binding approximates to a single binding site model with a K o of 0.12 nM and a Bmax of 42 fmol/mg of protein (fig. 3B). Transformation of these data by Hill plot gave a straight line with a Hill coefficient of 0.995, close to unity, indicating that [3H]LTD4 binds in an independent manner to a site, or sites, with one apparent affinity in this tissue (Fig. 3C). Scatchard plot analysis of [3H]LTD4 specific binding to guinea-pig lung membrane, conducted under the same incubation conditions, resulted in a K o of 0.096 nM and a Bmax of 428 fmol/mg of protein, showing that a profound difference between these tissues is in LTD4 receptor density, which is approximately 10-fold higher in guinea-pig lung.

3.4. Modulation of [~H]LTD4 binding to rat lung by GTP analogues and cations As previously described, the addition of either 300 #M GTPTS or 50 mM NaC1 to [3H]LTD4 receptor equilibrium binding incubations provoked dissociation of the ligand from the receptor (Fig. 2). Therefore, the

560

B. SCATCHARD PLOT

.i ,. n-

750

FREE[3H]-LTD4(pM) 1

1 0 0'0

C. HILL PLOT

~0 6

r~ z 4

-1

rnO 02

'l ' 10

20

30

40

50

BOUND[3H] LTD4 (fmol/mg PROTEIN)

Fig. 3. Scatchard analysis of [3H]LTD4 binding to rat lung membrane. Receptor binding assays were performed over the concentration range 0.01-1 nM [3H]LTD4, as described in Methods. At each radioligand concentration non-specific binding was determined using 10 # M MK-571. (A) shows total (e), non-specific ( o ) and the deduced specific binding saturation curve (A), under these conditions. (B) and (C) show the analysis of the saturation curve by Scatchard and Hill plot. Scatchard plot analysis was performed using Accufit Saturation-One Site data analysis software (Beckman) based on the linear transformation of Scatchard (1949) and Rosenthal (1967). The equilibriurp dissociation constant of the radioligand (KD) is equal to the negative reciprocal of the slope and the maximum number of binding sites (Bmax ) is equal to the intercept on the X-axis. Hill plot analysis was performed by expressing log B/(B,~ax-B ) as a function of the log of free radioligand concentration, where Bm~ represents the maximum specific binding and B represents the specific binding at each radioligand concentration. These are representative data from four experimental observations. K D and Bmax values are given in table 3.

56 i n c u b a t i o n m e d i u m the Bma × d e c r e a s e d 3-fold, from 38.6 to 13.2 f m o l / m g o f m e m b r a n e protein, while c o m p a r a ble K o values of 0.1 a n d 0.16 n M were o b t a i n e d , i n d i c a t i n g that the affinity of the r e m a i n i n g b i n d i n g sites for [3H]LTD4 was n o t d r a m a t i c a l l y changed. Similar results were o b t a i n e d with the inclusion of 0-100 m M NaC1 in the i n c u b a t i o n m e d i u m . NaC1 inh i b i t e d [3H]LTD4 specific b i n d i n g with an IC~0 of 36 m M . This decrease can be a t t r i b u t e d to the effects o f s o d i u m ions since KC1 i n c r e a s e d [3H]LTD4 specific b i n d i n g over the s a m e c o n c e n t r a t i o n range when inc u b a t e d with rat lung m e m b r a n e u n d e r the s a m e assay conditions. In contrast, specific b i n d i n g of [3H]LTD4 to rat lung m e m b r a n e was m a r k e d l y increased in the p r e s e n c e of d i v a l e n t cations, with CaC12 a n d MgC12 having essentially i d e n t i c a l effects. [3H]LTD4 specific b i n d i n g inc r e a s e d a p p r o x i m a t e l y 38-fold over a d i v a l e n t c a t i o n c o n c e n t r a t i o n range up to 30 m M , with higher c a t i o n c o n c e n t r a t i o n s p r o v i n g i n h i b i t o r y , as shown in fig. 5. H a l f m a x i m a l s t i m u l a t i o n was achieved at 1 m M of either CaCI 2 or MgC12.

o z o

_z

m O LL UJ IZ.

1000 750 /

MgCI2 Cacl2

500

u~

,¢

~ ,

/

~ NaCI

:3'

250

o

~

o

-

0

1

10

100

CATION CONCENTRATION (mM) Fig. 5. Effect of cations on [3H]LTD4 binding to rat lung membrane. Receptor binding assays were performed as described in Methods in the presence of 0-100 mM KC1 (B), NaCI (n), CaCI 2 (O) and MgCI z (©). The 20 mM CaCI 2 routinely used in binding assays was omitted except in the case of NaCI where it was necessary to provide a sufficient level of specific binding to distinguish sodium ion effects. At each cation concentration non-specific binding was determined using 10 /LM MK-571. Results are expressed as specific binding in cpm as a function of cation concentration. These are representative data from two experimental observations giving similar values.

3.5. Competition for [3H]LTD4 specific binding to rat lung by LTD 4 receptor agonists and antagonists Selected agonists a n d a n t a g o n i s t s have been used to c o m p e t e for [3H]LTD4 specific b i n d i n g in rat lung (fig. 6 a n d table 2). T h e r a n k o r d e r o f p o t e n c y d i s p l a y e d b y the p e p t i d y l leukotrienes, L T C 4, L T D 4, L T E 4 a n d the L T D 4 e n a n t i o m e r , 5R,6S-LTD4, closely p a r a l l e l e d their p o t e n c y in c o m p e t i n g for [3H]LTD4 b i n d i n g in guineapig lung. A s expected, L T D 4 was the m o s t p o t e n t c o m -

O. 800

(.9 Z Z

600

m

400 ~ 7 1 ~ M P _ P N L) uJ Q.

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~r I-._1

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P

200 GTPyS /(

o

1/

0

I

[

I

I

1

10

100

1000

GTP ANALOGUE (pM) Fig. 4. Effect of GTP analogues on [3H]LTD4 binding to rat lung membrane. Receptor binding assays were performed as described in Methods in the presence of 0-1 mM GTP~,S (O) or GMP-PNP (©). At each GTP analogue concentration non-specific binding was determined using 10 ~tM MK-571. Results are expressed as specific binding in cpm as a function of GTP analogue concentration. These are representative data from two experimental observations giving similar values.

p e t i n g ligand, h a v i n g a K i of a r o u n d 0.13 n M for b o t h species. T h e L T D 4 e n a n t i o m e r 5 R , 6 S - L T D 4 also disp l a y e d similar K i values for b o t h species, b e i n g app r o x i m a t e l y 70-fold less p o t e n t t h a n L T D 4. This result clearly illustrates the stereoselectivity of the L T D 4 rec e p t o r for the e n d o g e n o u s ligand. L T E 4 a n d L T C 4 gave c o m p a r a b l e K i values in b o t h rat lung a n d g u i n e a - p i g lung, with L T E 4 b e i n g m o r e p o t e n t t h a n LTC4, an o b s e r v a t i o n in a g r e e m e n t w i t h the p r o p o s a l that L T E 4 b e h a v e s as a p a r t i a l a g o n i s t at L T D 4 r e c e p t o r s ( M o n g et al., 1985a). In a d d i t i o n , the b e n z a m i d e d e r i v a t i v e Y M 17690, which is a n o n - a n a l o g o u s L T D 4 r e c e p t o r a g o n i s t ( T o m i o k a et al., 1987), was also effective in c o m p e t i n g for [3H]LTD4 b i n d i n g in b o t h species, a l t h o u g h the K i value o b t a i n e d for rat lung was 0.38 n M , a p p r o x i m a t e l y 6-fold less t h a n the K i of 0.06 n M o b s e r v e d for guineapig lung. I n m a r k e d constrast, we f o u n d that the L T D 4 receptor a n t a g o n i s t s M K - 5 7 1 , I C I 198,615 a n d I C I 204,219 were b e t w e e n 20- a n d 60-fold less p o t e n t in c o m p e t i n g for l i g a n d b i n d i n g in the rat lung t h a n in the g u i n e a - p i g lung. M K - 5 7 1 gave a K i value of 53 n M in the rat, as c o m p a r e d with 1.14 n M in the g u i n e a pig. Similarly, two r e l a t e d I C I r e c e p t o r a n t a g o n i s t s , I C I 198,615 ( A h a r o n y et al., 1987) a n d I C I 204,219 h a d K i values of 14.8 a n d 11.8 n M in the rat as c o m p a r e d with 0.69 a n d 0.2 n M in the guinea pig, i n d i c a t i n g the s a m e d i s p a r i t y . T h e e x c e p t i o n to this t r e n d was S K F 104353 ( G l e a s o n et al., 1987), which was less p o t e n t in the g u i n e a - p i g lung t h a n either of the o t h e r two a n t a g o n i s t s a n d disp l a y e d c o m p a r a b l e K i values in b o t h species tested.

57

3.6. Competition for [3H]LTC4 specific binding to rat lung by LTD 4 receptor agonists and antagonists [3H]LTC4 specific binding to rat lung membrane preparations has been previously reported (Pong et al., 1983). In order to ensure that [3H]LTD4 was not binding to this site in this study, experiments were conducted where LTC 4, LTD 4, MK-571 and ICI 198,615 were used to compete for [3H]LTC4 specific binding to rat lung membranes. As shown in fig. 7, only LTC 4 was an effective competing ligand, displaying an IC50 of 15 nM. Inhibition of [3H]LTC4 binding by LTD 4 and MK-571 was only observed at 1-10 /~M, the highest ligand concentrations tested, while ICI 198,615 proved totally unable to compete for [3H]LTC4 binding.

TABLE 2 Competition curves. Selected agonists and antagonists have been used to compete for [3H]LTD4 binding in lung membrane preparations from inbred hyperreactive and Fischer rat strains, fig. 6. The K i values obtained (-+S.D.) are shown in table 2 and compared with similar values from guinea-pig lung derived in parallel experiments. The number of experiments are indicated in parentheses. Where n = 2 values were generally within 20% of each other, n.d. = not determined. Competing

K i (nM)

ligand

Hyperreactive

Fischer

Guinea pig

32 +_6 0.14+0.02 4.3 _+2.3 9.4 _+3.8 0.38

(3) (9) (3) (3) (2)

59 (2) 0.15-+0.02(4) 5.6 (2) 12.0 (2) n.d.

19 -+3 (3) 0.11_+0.01 (5) 1.3 -+0.34(3) 6.2 -+1.1 (3) 0.06 (2)

53 _+13 14.8 _+ 1.3 11.8 3.1 -+1.2

(3) (3) (2) (3)

39 12.1 n.d. n.d.

Agonist LTC 4 LTD 4 LTE 4 5R,6S-LTD 4 YM-17690

Antagonist MK-571 ICI 198,615 ICI 204,219 SKF 104353

(2) (2) (2)

1.14-+0.5 0.69±0.22 0.20 2.5 _+0.47

(3) (3) (2) (3)

A. AGONISTS

leO 0

C

i ,0 '" o. tn

0.01 0.1 ~B. ANTAGONISTS lOO

~E

1

10

100

1000

ICI 198,615

80 60

K-571

,0 1.01

\ 0.1

1

10

100

1000

COMPETING LIGAND (nM)

Fig. 6. Competition for [3H]LTD4 specific binding to rat lung membrane by LTD 4 receptor (A) agonists and (B) antagonists. Receptor binding assays were performed as described in the Methods in the presence of 0.01 nM-10 #M of LTD 4 (o), 5R,6S LTD 4 (,x), LTC 4 (&), LTE 4 (O), MK-571 (~), ICI 198,615 (11) and SKF 104353 (13). Competition experiments were analysed and ICs0 values derived using the Kinetic, EBDA, Ligand, Lowry program from Biosoft (McPherson, 1985). The corresponding K i values were derived from the equation K i = IC5o/1 +(L/Ko) where K i is equal to the apparent equilibrium dissociation constant of the unlabelled competing ligand, K o is equal to the equilibrium dissociation constant of the radioligand, IC50 equals the concentration of unlabelled competing ligand that inhibits 50% of radioligand binding and L is equal to the concentration of free radioligand. These are representative data from at least three experimental observations. The K i values are given in table 2.

3. 7. Comparison of [3H]LTD4 binding in lung from hyperreactioe and Fischer rat strains Since hyperreactive and Fischer rat strains have differing profiles with respect to both responsiveness to LTD 4 challenge and production of leukotrienes upon antigen challenge, the possibility that these differences may be reflected at the level of the LTD 4 receptor was investigated. [3H]LTD4 binding to lung preparations from Fischer rats was found to have directly comparable characteristics to [3H]LTD4 binding to lung preparations from hyperreactive rats, with respect to association and dissociation profiles and modulation by GTP analogues, sodium ions and divalent cations (data not shown). In addition, no marked differences in the ability of either agonists or antagonists to compete for [3H]LTD4 binding in lung membranes from either hyperreactive or Fischer rats was observed (table 2). Moreover, Scatchard plot analysis of [3H]LTD4 specific binding to lung membranes from these strains gave almost identical K D values of 0.12 nM for hyperreactive and 0.13 nM for Fischer rats (table 3). However, the value for Bma x increased approximately 30% from 30 to 42 f m o l / m g of protein when comparing Fischer rats TABLE 3 Scatchard analysis of [3H]LTD4 binding in lung membranes from hyperreactive and Fischer rat strains. Scatchard analysis was performed as outlined in fig. 3. The number of experiments is indicated in parentheses. Values shown are _+S.D. Rat strain

K D (nM)

Bmax ( f m o l / m g protein)

Hyperreactive Fischer

0.12 + 0.01 (4) 0.13_+0.06 (3)

42 + 3 (4) 30_+3 (3)

58 ICI 198,615

o z

100

_z m

_o tl.i o.. o9 :E

_~

x

80

60

40

< :E

' N

MK-571

\

20

/

o

")~ 0

I 1

I 10

I 100

1000

I 10000

COMPETING LIGAND (nM)

Fig. 7. Competition for [3H]LTC4 specific binding to rat lung membrane by LTC 4, L T D 4, MK-571 and ICI 198,615. Receptor binding assays were performed as described in the Methods in the presence of 0.1 nM-10 ~ M of LTD 4 (©), LTC4 (A), MK-571 (n) and ICI 198,615 (I). Data were analysed and ICso values were determined as described in fig. 6. These are representative data from two experimental observations giving similar results.

with the hyperreactive strain. This slight increase in the maximum number of binding sites was routinely observed, using several different tissue preparations, and was always in the same order, demonstrating that hyperreactive rats have, in general, a higher L T D 4 receptor density than Fischer rats (table 3).

4.

Discussion

An LTD 4 radioreceptor binding assay has been established in rat lung. This assay depends on the use of metabolic inhibitors to prevent the conversion of [ 3 H ] L T D 4 to [3H]LTE4 which occurs in the presence of rat lung membranes. The inhibitor chosen was L-penicillamine which proved the most potent inhibitor of the enzymes(s) responsible for LTD 4 metabolism in this preparation. Since rat lung LTD 4 radioreceptor binding assays could not be performed in the absence of this inhibitor the effect of L-penicillamine on [3H]LTD4 receptor binding in this tissue could not be demonstrated directly. However, since radioligand degradation did not effect guinea-pig lung [3H]LTD4 receptor binding this tissue was used as the most appropriate control to assess whether L-penicillamine had an effect on [3H]LTD4 receptor binding constants. Parallel guineapig lung [3H]LTD4 binding assays were performed in the absence and presence of 20 mM L-penicillamine and demonstrated that this inhibitor did not alter the K D o r Bmax of [3H]LTD4 receptor binding in this tissue. In addition, the K i values for competition by both the agonist LTD 4 and the antagonist MK-571 were unaffected by this compound (data not shown). It was

concluded, therefore, that since the inclusion of Lpenicillamine did not modulate [3H]LTD4 receptor binding in guinea-pig lung binding incubations this inhibitor should not modulate [3H]LTD4 receptor binding in rat lung binding incubations. In addition, whenever LTC 4 was present in the binding assay medium LTC 4 to LTD 4 conversion was prevented by the addition of serine-borate. This complex has been extensively used as a n inhibitor of LTC 4 metabolism. Previous studies have shown that LTD 4 receptor mediated events are G T P dependent and occur via the formation of a high affinity agonist-receptor complex coupled to a G-protein. In the case of the L T D 4 receptor it has been proposed that upon receptor activation a minimum of two different G-proteins can interact with the receptor-ligand complex, linking with at least three intracellular signal transduction systems, to mediate the physiological response (Crooke et al., 1989). Extensive radiolabeling studies of G-protein-coupled receptors have resulted in a series of generalized observations true for most systems investigated. Routinely, radiolabelled agonists are found to bind specifically to a homogeneous population of sites, with binding being inhibited by both G T P analogues and monovalent cations, but increased by divalent cations. These observations have been explained by the ternary complex model where the receptor may exist in two affinity states, the free receptor, which has a low affinity for the agonist and the receptor-G-protein complex, which has high affinity for the agonist. Compounds like stable G T P analogues and sodium ions, which dissociate this receptor-G-protein complex, shift the receptor into the low affinity state, thereby inhibiting agonist binding, whereas divalent ca-

59 tions stabilize the ligand-receptor-G-protein ternary complex (Lefkowitz et al., 1983). Specific binding of the agonist [3H]LTD4 to guineapig lung, currently the most comprehensively studied tissue, has been shown to agree with these criteria, both in our hands (data not shown) and in reports from several groups, being inhibited by GMP-PNP, G T P y S and NaC1 and enhanced by CaC12, MgC12 and MnCI 2 (Bruns et al., 1983; Pong and DeHaven, 1983; Mong et al., 1985b). The data presented in this report demonstrate that [3H]LTD4 binding to rat lung also displays these characteristics. [3H]LTD4 binding to rat lung membrane preparations is saturable and of high affinity, displaying a K D of 0.12 nM, directly comparable to the value of 0.095 nM obtained for guinea-pig lung under identical conditions. Analysis by both Scatchard and Hill plots confirm that the agonist [3H]LTD4 binds to a homogeneous population of sites in this tissue. Furthermore, specific binding was found to be strongly inhibited by GTP'rS, G M P - P N P and sodium ions, but dramatically enhanced by divalent cations. However, a profound difference between the two species is observed in the maximum number of [3H]LTDa-receptor binding sites which, in rat lung, is 10-fold less than the level in guinea-pig lung, providing one explanation for the lack of success in examining LTD 4 receptors in this tissue. It has been necessary to employ [3H]LTD4 of higher specific activity (180 Ci/mmol) than previously available (38.4 C i / m m o l ) to establish a successful LTD 4 receptor binding assay in rat lung. Measuring the rates of association and dissociation of [3H]LTD4 specific binding to rat lung also gave profiles in agreement with those observed in guinea-pig lung. The rates of association and, most strikingly, dissociation are slow. This is compatible with the slow onset of action and reversal of LTD 4 induced smooth muscle contractions (Jones et al., 1982). Complete dissociation could only be provoked in the presence of either G T P analogues or sodium ions, presumably by provoking dissociation of the G-protein from the receptor, with the subsequent loss of high affinity agonist binding, evidence consistent with the ternary complex theory. The pharmacological profile of [3H]LTD4 binding in rat lung was further characterized by elucidating the K i values for a range of known L T D 4 receptor agonists and antagonists. LTC4, LTD 4, LTE 4 and the enantiomer 5R,6S LTD 4 had K i values directly comparable to those found in guinea-pig lung, again providing strong evidence that [3H]LTD4 is binding to the LTD 4 receptor in rat lung. In particular, the results with the two enantiomers of L T D 4 indicate that the receptor has stereoselectivity for the physiological ligand. Marked differences between [3H]LTD4 binding in rat and guinea-pig lung were, however, observed in antagonist competition binding assays. Here, there was a 15- to

60-fold difference in the ability of both MK-571 and two ICI compounds to compete for [3H]LTD4 binding, with the K i values for rat lung being consistently higher than in guinea-pig lung. The MK-571 and ICI compounds belong to different structural classes, indicating that this disparity is probably not exclusive to one class of compounds. However, SKF 104353, which belongs to a third structural class, did not show this preference for guinea-pig LTD 4 receptors. One possible explanation for this anomaly is that both MK-571 and the ICI compounds bind to an additional site(s) on the guineapig lung receptor which is not involved in the binding of the natural agonist. This additional site(s) may not be present as part of the rat lung receptor, thereby reducing the affinity of these antagonists in this species. In constrast, SKF 104353 is structurally related to the leukotrienes and may therefore interact with the same binding determinants on the LTD 4 receptor as the natural agonist. Hence the affinity of this antagonist may remain the same for both rat lung and guinea-pig lung LTD 4 receptors. However, a full interpretation for these observations awaits more information concerning the LTD 4 receptor at the molecular and biochemical level. The possibility that [3H]LTD4 was behaving as an agonist at the previously documented rat lung LTC 4 binding site (Pong et al., 1983) was excluded by competing for [3H]LTC4 specific binding to rat lung membranes with the agonist L T D 4 and the antagonists MK571 and ICI 198,615. All three ligands were shown to be ineffective in competing for [3H]LTC4 binding in this issue as compared with their ability to compete for [3H]LTD4 binding, demonstrating that [3H]LTD4 is not binding to the LTC4 binding site in the radioreceptor assay described in this report. This provides additional evidence that [3H]LTD4 is binding to an authentic LTD 4 receptor in this study. Experiments directly comparing lung membranes from hyperreactive and Fischer rat strains failed to unveil any difference between these animals with respect to the K D or K i values they displayed in the [3H]LTD4 radioreceptor binding assay. There was a consistently higher BmaXvalue observed in hyperreactive animals, indicating that this strain has a higher receptor density. However the difference is not remarkable and the physiological significance of this is unclear. Therefore, it has been concluded that the limited response of Fischer rats to bronchial challenge with either antigen and LTD 4, as compared to hyperreactive rats, cannot be rationalized at the L T D 4 receptor level. In summary, the successful establishment of a [3H]L T D 4 radioreceptor binding assay in rat lung membranes has allowed the characterization of the rat L T D 4 receptor in this tissue. This LTD 4 receptor displays all the essential characteristics attributed to LTD 4 receptors in lung tissues from other species studied (Bruns et

60 al., 1983; P o n g a n d D e H a v e n , 1983; M o n g et al., 1984; 1985b;

L e w i s et al., 1985; M o n g et al., 1988). I n a d -

d i t i o n , it h a s b e e n s h o w n t h a t t h e L T D 4 r e c e p t o r is a p p a r e n t l y i d e n t i c a l in h y p e r r e a c t i v e a n d F i s c h e r r a t s t r a i n s , w h i c h h a v e d i f f e r e n t r e s p o n s e s to l e u k o t r i e n e s .

Acknowledgements The authors wish to thank Haydn Williams for the synthesis of ICI 198,615, ICI 204,219 and YM-17690, Aiden Foster for criticism of the manuscript and Barbara Pearce for secretarial assistance.

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Tomioka, K., T. Yamada, K. Teramura, M. Terai, K. Hidaka, T. Mase, H. Hara and K. Murase, 1987, Isolated tissue and binding studies of YM-17690, a novel and non-analogous leukotriene agonist, J. Pharm. Pharmacol. 39, 819.