Identification of leukotriene D4 specific binding sites in the membrane preparation isolated from guinea pig lung

PROSTAGLANDINS

IDENTIFICATION OF LEUKOTRIENED SPECIFIC BINDING SITES IN THE MEMBRANE PREPARATION ISOL;tTED FROM GUINEAPIG LUNG

Jeffrey

Seymour Mong, Hsiao-Ling WuI Mike A. Clark, M. Stadel, John G. Gleason and Stanley T. Crooke Deft. of Molecular Pharmacology, Dept. of Medicinal Chemistry Smith Kline and French Laboratories 1500 Spring Garden Street Philadelphia, Pa. 19101

Abstract A radioligand binding assay has been established to study leukotriene specific binding si es in the guinea pig and rabbit tissues. Using high s ecific activity [5HI-leukotriene D4 ([3H]-LTD4), in the presence or (5R,6S-LTD4), the diastereoisomer of LTD a 1sence of unlabeled LTD leukotriene E4 (LTE4) an %' the end-organ antagonist, F 8 L 55712, we have identified specific binding sites for [3H]-LTD4 in the crude membrane The time required for [3H]-LTD4 fraction isolated from guinea pig lung. binding to reach equilibrium was approximately 20 to 25 min at 37°C in the presence of 0 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl. The binding of [4 HI-LTD4 to the specific sites was saturable, reversible and The maximal number of binding sites (Bmaxl, derived from stereospecific. Scatchard analysis, was approximately 320+200 fmol per mg of crude membrane The dissociation constants, derived from kinetic and saturation protein. analyses, were 9.7 nM and 5+4 nM, respectively. The specific binding sites could not be detected in the crude membrane fraction prepared from guinea pig ileum, brain and liver, or rabbit lung, trachea, ileum and uterus. In radioligand con etition experiments, LTD4, FPL 55712 and 5R,6S-LTD4 The metabolic Inhibitors of arachidonic acid competed with [t; HI-LTD4. and SKF 88046, an antagonist of the indirectly-mediated actions of LTD4, did not significantly compete with [3H]-LTD4 at the specific binding sites. These correlations indicated that these specific binding sites may be the putative leukotriene receptors in the guinea-pig lung.

The abbreviations used are: 1, LTC4, diastereoisomeric leukotriene C4, 5R- hydroxy-6S and 5S-hydroxy-6R-S-glutathionyl-7E,9E,llZ, 14Z-cis-eicosatetraenoic acid; 2, LTD4, or 5S,6R-LTD4 the natural form LTD 9E,llZ,14Z-cis-elcosa 0'etraenoic acid; 5S-hydroxy-6R-S-Lcystelnylglycyl-7E, 3, 5R?6S-LTD4, the unnatural form LTD4, 5R-hydroxy-6S-S-Lcystelnylglycyl-7E, llZ,14Z-cis- eicosatetraenolc acid; 4, LTE4, diastereo- isomeric LTE4, 5R-hydroxy-6S and 5S-hydroxy-6R-S-L-cysteinyl7E,9E,llZ, 14Z-cis-eicosatetraenoic acid; 5,SKF 88046, N,N'-bis [7-(3-chlorophenylaminosulfonyl)-1,2,3,4,-tetrahydroisoquinolyl]disulfonylimide; 6, HPLC, high pressure liquid chromatography; 7, Phenidone, l-phenyl-3- pyrazolidone; 8, NDGA, nor-dihydroquaiaretic acid,2,3-bis (3,4-dihydroxybenzyll-butane.

DECEMBER

1984 VOL. 28 NO. 6

805

PROSTAGLANDINS

Introduction D4 and E4 are the major constituents of slow Leukotrienes (LTsl C reacting substance o 8' anaphylaxls (SRS-Al involved in immediate type hypersensitivity (l-81. The LTs have been demonstrated to induce prolonged contraction of airway smooth muscle, and thus, may play a role in bronchopulmonary diseases (9-11). Two mechanisms have been proposed to account for the spasmogenic activity of LTs. LTs may induce secondary prostaglandin metabolites (e.g., thromboxanes) which may induce smooth muscle contraction (11-141, or LTs may interact with subcellular components, i.e., receptors, that are involved in the transduction of the signal into the cells of the target-organs (15). A third possibility is that the release of prostaglandins induced by leukotriene may also be coupled to leukotriene interactions with their receptors; i.e., they may serve as "second messengers". A series of structural analogs of LTs has been developed and the results obtained from the structure-activity studies (15-17) indicate that LT-induced smooth muscle contraction is highly specific and can be antagonized by the end-organ antagonist FPL 55712 (la), thus suggesting the possible existence of LT receptors in the target organs (15, 19, However, only limited evidence is currently available to 20). substantiate the hypothesis that there are specific binding sites or receptors in the tissues responsive to LTs. Consequently, we have initiated studies to identify and characterize the putative binding sites of the LTs in the target organs.

In the present study, we have established a radioligand filter binding assay and directly demonstrated high affinity, saturable and stereospecific binding sites, in the target organs. This assay method offers efficient, accurate and quantitative measurement of the affinity It may also prove to be and density of the specific binding sites. valuable for the characterization of leukotriene-receptor binding and regulation as well as for the design of leukotriene antagonists. Materials

and Methods

Chemicals, Reagents and Radioligand. Diastereoisomeric mixture leukotriene C4 (LTC4; 5S,6R-LTC4 and 5R,6S-LTC4lI (4-6, 21, 22), (5S,6R) leukotriene D4 2 (natural form LTD4), 5R,6S-LTD4 3 (unnatural form LTD4), diastereoisomenc mixture LTE4 4 (LTE4; 5S,6R-LTE4, and 5R,6S-LTE4) and SKF 880465 were prepared by total synthesis and were 99.0 percent pure as defined by high pressure liquid chromatography6 (HPLC) analysis (23). These compounds were stored under argon in liquid nitrogen or at -70°C. Under these conditions. they were stable for several months. Two lots of [14, 15-3H]-LTD4, (C3H]-LTD4, lot numbers 1796-247, 1796-297) obtained from hew England Nuclear Co. (Boston, MA) were reported to be greater than 95 percent and 97 percent pure with specific ac ivities of 40.5 Ci mnol and 37 Ci/m mol, respectively. The 114, 15- 5H]-LTC4 ([IHI-LTC4, 41 Ci/mmol, lot number 1619-160), also obtained from New England Nuclear Co., was

806

DECEMBER

1984 VOL. 28 NO. 6

PROSTAGLANDINS

reported to be greater than 95 percent pure and free from [PHI-LTD~ The purity and identity of these radioligands were and [3H]-LTE4. also analyzed by HPLC methods in our laboratory and were greater than 90 percent pure. The leukotriene antagonist FPL 55712 was a gift from Fison Ltd. (Leicestershire, England). Phenidone and NDGA and (nordihydroguaiaretic acid), soybean trypsin inhibitor bacitracin, benzamidine, phenylmethylsulfonyl fluoride were purchased from Sigma Sodium meclofenamate was a gift from Company (St. Louis, MO). Warner-Lambert Company (Ann Arbor, MI), Indomethacin was a gift from Merck Company, Inc. (Rahway, NJ) Crude Membrane Preparation. Female albino guinea pigs (Hartley strain, 600-800 gram body weight) were sacrificed by decapitation. The lungs and other organs were removed, minced into small pieces and rinsed in phosphate-buffered saline. The tissues (15 grams) were resuspended in 50 ml of homogenization buffer (0.25 M sucrose, 10 mM Tris-HCl pH 7.5) containing protease inhibitors (soybean trypsin inhibitor (5ug/ml), bacitracin (100 pg/mll, benzamidine (1 mM) and phenylmethylsulfonyl fluoride (10 uM) to avoid proteolysis during the processes of The tissues were homogenized with a homogenization and centrifugation. Brinkman PT-20 polytron for a total of 1 minute with 10 set pulses at a The homogenates were then centrifuged (1000 x g for setting of 6 at O'C. 10 min) to remove tissue clumps, unbroken cells and nuclei. The supernatant was recentrifuged at 30,000 x g for 30 min to yield pellets which were referred to as crude membrane fractions. This crude membrane fraction was resuspended in the 50 ml of homogenization buffer and The crude membrane fraction was re-pelleted (30,000 X g for 30 mini. then resuspended in the 20 ml of incubation buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl), homogenized in a Teflon homogenizer and The pellets were resuspended in recentrifuged at 30,000 x g for 30 min. the incubation buffer with a Teflon homoqenizer at a concentration of 10 to 20 mg/ml of protein in the suspension. The concentrations of proteins were determined using the method of Bradford (24). The membranes isolated from rabbit tissues (female Flemish giants) were prepared by procedures identical to those described above. Crude membranes were prepared from Receptor-Ligand Binding Assays. freshly isolated or liquid nitrogen preserve tissues from guinea pigs or The incubation mixture contained [ 4HI-LTD4 in the presence rabbits. or absence of LTD4 (the natural form 5S,6R-LTD4) or other competing ligands and membrane preparations in theoincubation buffer. Incubations were performed from zero to 50 min at 37 C in the presence of argon. For saturation (and Scatchard) analyses, quadruplicate aliquots of 100 ~1 samples were taken from single incubation mixture (500 ~1) and analyzed. For kinetic studies, duplicate 100 ~1 aliquots were taken from the incubation mixtures (1000 ~1) and analyzed. Glass containers and pipettes were silanized using prosil-28 (PCR Research Chemicals). Free ligands were separated from membrane-bound ligands by the Aliquots taken from the reaction mixture following filtration technique. were diluted in a reservoir containing 4 ml of the reaction buffer at

DECEMBER

1984VOL 28N0.6

807

PROSTAGLANDINS

O°C. The membrane bound ligands were retained on the filter paper (Whatman GF/C filter paper) and the free ligands were allowed to pass through under reduced pressure. The filter papers were then washed 4 times with the incubation buffer at 0°C. The length of time involved in filtration and washi g was approximately 10 to 15 sec. Dissociation of the membrane bound C3 HI-LTD4, under these conditions, was minimal (unpublished results). The radioactivity retained on the filter was determined in a Beckman LS-7800 liquid scintillation counter with an efficiency of 50-60 percent.

those employed in the binding studies. After incubation, the particulate material in the reaction mixture was sedimented by centrifugation at 30,000 x g for 10 min at 4OC. The radioactivity of the supernatant was determined by counting a 50 ~1 aliquot. The supernatant was filtered and then injected for HPLC analysis. The pellets were resuspended in 0.5 ml of the incubation buffer and 1 ml of acetone was added to extract the leukotrienes. The membrane was then centrifuged at 10,000 x g for 10 min at 4'C. The pellets were re-extracted with acetone and the rupernatants from the first and second extractions pooled. The acetone was then evaporated under argon. The radioactivity of an aliquot of the aqueous solution was determined to ensure greater than 99 percent recovery. The aqueous solution was then subjected to HPLC analysis. Reverse phase Cl8 LiChosorb column (0.46 x 25 cm) was used with acetonitrile/lO mM phosphate buffer (pH 6.7) as mobile phase at a flow rate of 2 ml/min for the separation of LTC4, LTD4 and LTE4. The radioactivity of 1 ml fractions were determined by liquid scintillation counting. The recovery of radioactivity from each chromatogram was 100+5 percent of the total radioactive material injected into the column. -

Results Kinetics of [3H]-LTD4 Binding to Membranes from Guinea Pig Lung_. IJHI-LTDp binding to the crude membrane fraction isolated from guinea pig lung'appeared to be rapid. As shown in fig 1, the amount of radioligand bound to the membrane particulates approached equilibrium within 20 to 25 min at 37°C. Under the experimental conditions employed, the binding was stable for at least 40 to 50 min. When tge binding was performed in the presence of 2.5 PM unlabeled LTD4, the [ HI-LTD4 binding again approached equilibrium within 15 to 20 min although at a reduced level (fig. 1). These experiments suggest that crude membranes isolated from guinea pig lung contain low capacity specific binding sites for [3H]-LTD4. The amount of [3H]-LTD4 binding, in the presence

DECEMBER

1984 VOL. 28 NO. 6

PROSTAGLANDINS of the excess LTD4, (approximately 30 to 40 percent of total binding) The data shown in fig. 1, were represents the non-specific binding. replotted to calculate the observed rate of association (Kobs). The Kobs was calculated to be 0.226 min-I (fig. 1, inset).

4 8 TlME(MINUTES)

0

1 o-

-P

E 1

I

I

I

10

20

30

TlME(MINUTES) Fig. 1

Binding of C3H]-LTD4 to the crude membrane preparation isolated from guinea-pig tissues. Crude membrane preparation w s isolated as described in Materials and Methods. The [9 HI-LTD4 (2.5 nM) was incubated with the membrane protein (2 mg/ml) in a final volume of 1 ml at 37°C (-a-). The incubation buffer contained 150 mM NaCl, 10 mM Tris-HCl buffer (pH 7.5) and 2 mg of membrane protein isolated from guinea pig lung. The LTD4 (2.5 uM) was also included in identical but separate reaction mixtures (-o-) in addition to all the reagents and components. Duplicate aliquots of 100 ~1 of reaction mixture were taken at given points of time and analyzed by the vacuum filtration method. The data from 4 separate experiments were pooled and shown. The standard deviation was calculated Inset, the observed association rate plot. from 4 experiments. B, specific binding of [3H]-LT04 at each indicated point of time; Beq, Specific binding of [3H]-LTD4 at equilibrium.

DECEMBER

1984 VOL. 28 NO. 6

809

PROSTAGLANDINS

Since the concentration of [3H]-LTD4 was in excess of the number binding sites, the extent of specific binding, in theory, should

of be

proportional to the concentration of membrane protein added. The results shown in fig. 2 demonstrate that the amount of specific binding (derived by subtracting the nonspecific from the total binding) was directly proportional to the amount of membrane protein present in the reaction mi xture (fig. 2, inset).

(mg/ml)PROTElN

I 0

10

20

30

40

50

n ) or 4.8 mg/ml ( o 1 of crude membrane protein in fhe presence of 150 mM NaCl and 10 mM Tris-HCL (pH 7.5) at 37 C from zero to The specific binding, obtained by subtracting the 50 min. nonspecific from the total b'nding, was converted into fmol of binding sites assuming the [3H]-LTD4 has a specific activity of 40 Ci/mmol (1 fmol = 88 DPM). Inset, protein dependency of The amount of specific binding was the specific binding. plotted against the concentration of protein added in the incubation mixture.

Dissociation

810

of specifically

bound

C3H]-LTD4.

The binding

DECEMBER

of

1984 VOL. 28 NO. 6

PROSTAGLANDINS

L3Hl-LTD4 to specific sites was reversible. In the experiment shown in fig. 3, the [ HI-LTD4 was allowed to bind to the membrane preparation for 30 min. Concentrated LTD4 (1 mM1 was added at the indicated times to a final concentration of 2.5 WM (unlabeled LTD4). The reaction mixtures were incubated for an additional 30 min at 37°C. The membrane associated radioactivity apparently decreased, within 30 min, to a level that was not distinguishable from the level of nonspecific binding. These results suggest that C3Hl-LTD4 binding was reversible and was dissociated from the specific binding sites in the presence of excess amount of the unlabeled LTD4. The data were replotted (fig. 3, inset) to calculate the rate of dissociation (Koff I. Under the experimental conditions, the dissociation

rate constant

was 0.181 min-l.

TIME (MINUTES)

F'ig. 3

Dissociation of [3H]-LTD4 specificdll:, 'zund to the membrane. [3Hl-LTD4 (2.5 nM1 was incubated with quinea-pig lung membranes in 10 mM Tris-HCL buffs (pH 7.51, 150 mM NaCl at 37OC from zero to 30 min ( a 1. A small volume of concentrated LTD4 (1 mM1 was added in the reaction mixtures to make the LTD4 concentration 2.5 WM. The unlabeled LTD4 was included in a separate reaction mixtures to maintain at a concentration of 2.5 PM from zero to 50 min in order to assess the level of non-specific binding. The results shown are the pooled data from The Dissociation rate plot from one 3 experiments. Inset. experiment is shown. B, specific binding of C3Hl-LTDj at indicated point of time. Beq, specific binding of [ HI-LTD4 at equilibrium. The associa ion rate can be calculated from the equation Kon = (Kobs-K,ffI/[ 5 H-LTD41.

DECEMBER

1984 VOL. 28 NO. 6

811

PROSTAGLANDINS The Kinetics of Specific Binding. The rate constants for association (Ken) and dissociation [K ) tor specific binding can be calculated from the data shown in fios. Offand 3 (25). The dissociation constant (Kn) can also be calculated from the association and dissociation rate consta!ts to be 9.7 nM which is in good agreement with the KD determined from saturation analysis (see below). Saturation and Scatchard Analyses. Saturation and Scatchard analyses of the [3H]-LTD4 specific binding data were performed to determine the dissociation constant and the maximum number of specific binding sites (&la 1 figs. 4a and 4b show data from a typica v in the crude membrane fraction. experiment. In this experiment, ths non-specific binding increased linearly with increasing cokentrations of [ HI-LTD4 in the reaction mixture. The total [3H]-LTD4 bincing (including the specific and the non-sseclflc components) also increased with increasing concentration of [ HI-LTD4. The specific component of the binding reached a plateau at approximately 280 fmol binding sites per mg of protein. This experiment suggests that the non-specific component represents high capacity;!non-saturable) and low affinity binding. The specific component apparently represents low-capacity, (saturable) and high affinity binding. The data in fig. 4a were reanalyzed by the method of Scatchard (26), to evaluate the KD and B . As indicated in fig. 4b, the linearity of the plot suggested tffzf there was, under the experimental conditions, a single class of LTD4 specific bindin sites. The KD and Bma , extrapolated from the slope and the ordina ! e intercept, were estima Eed to be 9 nM and 430 fmol binding sites per mg protein respectively in this experiment. The mean KD and &lax derived from four experiments were 5 -+ 4 nM, and 320 -+ 200 fmol/mg protein respectively.

0

4

8

CONC.

812

12

OF [3H]-LTD,

16

20

(nM)

DECEMBER

1984 VOL. 28 NO. 6

PROSTAGLANDINS

$

,280 -

5 k

240 -

z 0 E t n z

200 160-

2 (13 120-

m

404

0 I

0

200 [W-LTD,

I

I

I

I

4

8

12

16

400

600

BOUND I

20

600 (PM) I

24

CONC.OF[sH]-LTD,(nM)

Fig. 4

Saturation and Scatchard analyses of [3H]-LTD4 specific binding sites. (a) Saturation Analysis. The crude membrane preparation isolated from guinea pig lung (2 mg/ml) was incubated with c3H]-LTD4 (from 1 to 20 nM) in 10 mM Tris-HCL buffer (pH 7.5) containing 150 mM NaCl ( -e- ). The non-specific binding was estimated by inc uding the unlabeled LTD4 at concentrations 1000 fold of the [3HI-LTDa (_o_) under the identical conditions. The specific binding at each concentration of [3H]-LTD4 was determined by subtracting the non-specific binding from the total binding after equilibrium was achieved. Each point represents-mean derived from quadruplicate aliquots, a representative experiment is shown. (b) Scatchard Analysis. B, the concentration of specifically bound. B/F, specifically bound [3H]-LTDq/Free [3H]-LTD4.

To evaluate the specificity of Specificity of Leukotriene Binding Sites. the leukotriene binding sites, radioligand competition studies were performed in the presence or absence of a variety of unlabeled leukotriene agonists and antagonists. The competition curves of each of these agonists and antagonists were parallel (fig. 5) suggesting these agents bound to the same class of binding sites. As shown in fig 5, LTD4 and LTE4 competed with [3H]-LTD4 (3.5 nM) at concentrations

DECEMBER

1984 VOL. 28 NO. 6

813

PROSTAGLANDINS

approximately 230 and 400 times less efficient than the natural 5S,6RLTD4, respectively. LTC4 also competed with 13Hl-LTD4 for the specific binding sites. However, these experiments were performed in the presence of 80 mM serine-borate, 5 mM cysteine and 5 mM glycine (27) to prevent the rapid bioconversion of LTC4 by the membrane fraction isolated from guinea-pig lung (29). The relative potency in competition for the binding sites was 5S,6R-LTD4 > LTE4 > > with [PHI-LTD LTC4 > FPL557 4 2 2 5R,6S-LTD4 > SKF 88046. The inhibitors of cyclooxygenase and lipoxygenase, e.g., nordihydroguaiaretic acid (NDGA), phenidone, the leukotriene metabolism inhibitor serine-borate complex and the indirect inhibitor of SRS-A, SKF 88046, did not display significant competitiveness. The inhibition constants (Ki) were calculated (28) and shown in Table 1.

Fig. 5

814

Competition of C3Hl-LTD4 specific binding by leukotriene agonists and antagonists. The [3Hl-LTD4 (3.5 nM) was incubated with the membranes isolated from guinea pig lung in 10 mM Tris-HCL buffer (pH 7.5) containing 150 mM NaCl and the following agents for competition analysis; unlabeled 5S,6RLTD4 (-o-1, 5R,6S-LTD4 (-+I, FPL 57712 (-0-j LTE (-m-1, diastereoisomeric mixture of LTC4 t-h-1 and the S1 S-A indirect antagonist SKF 88046 (-A.-). Competition of LTC4 with C3Hl-LTD4 was carried out in the presence of 80 mM serine-borate 5 mM cysteine and 5 mM glycine to prevent bioconversion of LTC4. Each point represents duplicate determinations, the results from 2 experiments were shown. Complete inhibition (0 percent specific binding) was defined as the amount of binding in the presence of 3.5 PM LTD4.

DECEMBER

1984 VOL. 28 NO. 6

PROSTAGLANDINS

Table

1.

Inhibition

Competitor 5S,6R-LTD4 5R,6S-LTD4 FPL 55712 LTE4 SKF 88046 NDGA Phenidone Serine-borate Cysteine Glycine a

constants

for leukotriene

Inhibition Constant 10 + 2.2 3750 T 1100 2,300 45 + 10 80,nOO >1,000,000 >1,000,000 >1,000,000 >1,000,000 >1,000,000

agonists

and antagonistsa

(Ki,nM) f : 3 2 3 ; 3 3

Competition experiments were performed as described in fig. 5. Ki values were calculated according to the formula: Ki = IC O/(1 + [3H-LTD4]/KD) (28) and are reported as mean + .?.E. (n = 3) or mean (n = 2). [3H-LTD4] is the concentration of free [3H]-LTO4 in the assay tube, and KD is the calculated equilibrium dissociation constant.

Species and Tissue Variability of the LTD4 Specific Binding Sites. The charactenstics of [JH]-LTD binding to crude membrane fractions prepared from a variety of :issues obtained from guinea pigs and rabbits The results shown in table 2 indicate that specific binding were compared. sites were detected in crude membrane fractions isolated from guinea pig lung, heart, trachea and uterus. However, in the rabbit, only the membrane isolated from the heart showed specific binding sites. Specific binding sites were not detected in the minimally or nonresponsive tissues such as guinea pig brain, rabbit lung, trachea or uterus membrane preparations. Metabolism of [3H]-LTD4 by the Crude Membrane Fraction. High pressure liquid chromatog-was employed to examine the degree of bioconversion of In these experiments, the leukotriene-like materials (free, [3H]-LTD4. unbound ligand) from the supernatant or extracted from the crude membrane fraction (bound ligandl, under present conditions, were fractionated by HPLC, (Materials and Methods). It was found that in the control samples (membrane free incubation for 30 min), the majority of the radioactivity (greater than 70 percent) eluted with a retention time equal to that of the unlabeled LT04 standard (fig. 6a). The majority of the membrane bound, extractable radioactivity (approximately 70 percent of total) also eluted as Approximately 5 percent and 1 percent C3H]-LTD4 (fig. 6b). radioactivity eluted with a retention time identical to those of LTE4 and The chromatogram of the radioactive material obtained LTC4 respectively. from the supernatant (fig. 6c) was similar to that from the membrane bound fractions, i.e., the majority of the radioactive material eluted as LTD4 Only 8 percent and 1 percent eluted as LTE4 and LTC4, (80 percent). These results demonstrate that, under the experimental respectively. only a small percentage of C3H]-LTD4 conditions employed in this stud was converted to [3H]-LTC4 and [??HI-LTE4. The ma'ority of the radioactive material bound to the membranes was [J HI-LTD4.

DECEMBER

1984 VOL. 28 NO. 6

815

PROSTAGLANDINS

LTC, II

4ooa

1

LTC,

LTC,

LTD,

1

LTE4

LTD,

LTEl

80%

1 8%

1%

zz

LTE4

70%

11

3000 -

6

LTD,

2000 1000 -

2 4

6 8 1012141618

FRACTION Fig. 6

816

2022 24 262830

NUMBER

High Pressure Liquid Chromatogram of Leukotrienes. [3Hl-LTO4 was incubated without (6a) or with (6b,6c) the crude membrane (2 mg/ml) isolated from guinea pig lung in 10 mM Tris-HCL pH 7.5 The leukotrienes containing 150 mM NaCl at 37°C for 30 min. extracted from the membrane pellet (6b) and from the supernatant (6~) were then subjected to HPLC analysis as described in Acetonitrile/lO mM phosphate buffer was Materials and Methods. Acetonitrile at 25 to 30 percent was used as the mobile phase. used in the first 10 min, and 35 percent acetonitrile was used for Under these next 10 min 0.5 ml fractions were collected. conditions, the elution time of the LTC4, LTD4 and LlE4 standards are 2.8, 5.6 and 7.6 min, respectively.

DECEMBER

1984 VOL. 28 NO. 6

PROSTAGLANDINS

Table 2. Tissue binding sitesp

and species

rabbitb lung trachea ileum uterus brain heart liver

variability

fmol/mg NDL ND ND ND NTd 136 + 30 ND -

of the [~H]-LTo~

Guinea

specific

Pig fmol/mg 191 6 65 5 1: ND 44 + 11 ND 166 + 30 ND -

a.

The presence or absence of [3H]-LTD4 specific binding sites were determined by kinetic experiments using the methods described in The Legend of fig 1. 3.5 nM [JH]-LTD4 was incubated with the membranes isolated from rabbit and guinea-pig tissues (Materials and Methods) in the presence of 10 mM Tris buffero(pH 7.51, 150 mM NaCl and 2 mg of protein from zero to 40 min at 37 C.

b.

Female flemish giant rabbits with body weight used in this study.

C.

ND, not detected, the steady state level of nonspecific binding was either greater than or equivalent to the teady-state level of total binding. Hence, no specific binding of [3 HI-LTD4 can be determined.

d.

NT, not tested.

approximately

1.5 Kg were

Discussion

In the guinea pig, LTD4 induced contraction is apparently mediated through direct and indirect mechanisms (12, 14). The indirect component of LTD4 induced tissue contraction can be blocked by cyclooxygenase inhibitors, e.g., indomethacin and meclofenamic acid, suggesting that LTD4 may induce prostaglandin synthesis and, thus, induce tissue contraction. The direct effects of LTD4 have been postulated to be receptor mediated because of the following observations: (A) The structure-activity relationships observed in organ-bath studies suggest that the peptide structures of leukotrienes determined the biological activity and the lipid domain seemed to be slightly less important (15); (6) The LTE4, and the 5R,6S-LTD4, the stereoisomer of LTD4, prepared synthetically, are full agonists yet are 5 and 500 times less active than the natural 5S,6R-LTD4 (14, 16, 17); (Cl The time of onset of the LTD -induced tissue contraction is from 1 to 5 min and the concentration of L 4 D4 required is in the nanomolar range (12, 14, 16); (D) and The leukotriene end-organ antagonist FPL55712, can block the LTD4 induced smooth muscle contraction. These observations also suggest that the postulated receptors display high affinity, rapid onset of occupancy and stereospecificity for the ligand.

DECEMBER

1984 VOL. 28 NO. 6

817

PROSTAGLANDINS

The results of the present study suggest that in the crude membrane fractions prepared from guinea pig lung tissue, there are high affinity (KD = 5+4 nM), saturable (320+200 fmol binding sites/mg protein) and stereosaective binding sites for LTD4. The binding of [3Hl-LTD4 is rapid (15 to 20 min to reach a plateau phase). The Ki of LTE4 and 5R,6S-LTD4 are 5 and 400 times, respectively, of that of the The LTD4 antagonist FPL55712, in the range of 5S,6R-LTD4. pharmacological concentrations (1 to 10 PM), effectively competed with c3H]-LTD4 for the specific binding sites. Thus, the rank order potency and stereospecificity of the agonists and antagonists in organ-bath studies appear to correlate with the radioligand competition studies (fig. 5, table 11. The results obtained from this study and others (29. 301, therefore, provide direct evidence for the existence of the postulated leukotriene receptors in the crude membrane fractions isolated from guinea pig lung. The LTD4 specific binding sites have also been found in the membrane fractions isolated from tissues that are highly responsive to LTs, e.g., guinea pig trachea (141, uterus (31) and heart (32. 33). The specific binding sites were not detected in the minimally or non-responsive tissues of guinea pig or rabbit, e.g., liver, brain of guinea pig, lung, trachea and uterus of rabbit (12, 34). These results correlate with the pharmacological activities of leukotrienes in these tissues and provide additional evidence that the LTD4 binding sites are physiologically relevant receptors. The only exception to this correlation is in the ileum of guinea pig, in which no detectable specific binding was identified. It is possible that the specific binding sites cannot be detected in this tissue due to proteolytic degradation or presence of high levels of nonspecific binding. Alternatively, the conditions employed in this study may not be suitable for the detection of soecific bindinq sites in this tissue due to the fact that smooth muscle pre arations isolated from ileum respond to LTD4 differently from the luno (19 ‘; . It is also oossible that the membrane isolated from guinea-pig ileum may contain a high level of an enzyme that promotes biotransformation or degradation of c3H]-LTD4. At present, however, no clear explanation for this discrepancy is apparent. The HPLC profile of the membrane bound radioactive leukotrienes was examined to exclude the possibility of bioconyersion of th labeling ligand. (C3H]-LTD may be metabolized into [ HI-LTC4 or [5 HI-LTE4 via y-glutamy 4 transpeptidase and dipeptidase, respectively). The majority of the radioactive material bound to the membrane in the supernatant (fig. 6) eluted with a retention time equal to that of the LTD4 standard. Only insignificant amounts of bioconversion occurred under the experimental conditions. These results suggest that the specific binding sites detected under the experimental conditions reflect primarily the [3H]-LTD4 The recovery of [3 HI-LTD4 from the supernatant, membrane binding sites. extract and from the membrane-free incubation (fig. 6a did not exceed 80 This is probably due to instability of the 5 3HI-LTD4 during percent. incubation, extraction and chromatography since the [ HI-LTD4 employed

818

DECEMBER

1984 VOL. 28 NO. 6

PROSTAGLANDINS

for these studies was more than 90 percent pure (Materials and Methods). Recent evidence suggests that many of the enzymes involved in arachidonic acid metabolism and leukotriene biosynthesis may reside in the "particulate" or membrane fraction of the RBL-1 cells (35). Thus. the Dossibilitv exists that a fraction or all of the specific binding sites demonstrated in this study may be contributed by these enzymes. This is unlikely, however, for the following reasons: (A) The characteristics, KD and B, x of the [3H]-LTD4 specific binding sites were essentially unaltere! when the binding studies were performed in the presence of inhibitors for arachidonic acid and leukotriene metabolism, e.g., HOGA serine-borate, cysteine, and phenidone. The fact that (B) the bulk of [3~1-~~~ in the reaction mixture remained in an unmetabolized form, i.e., [PHI-LTD~ (fig. 61, suggests that these enzymes may not be highly active or may not be present (C) The end-organ antagonist FPL 55712, but not the in large quantity. lipoxygenase-cyclooxygenase inhibitors (table 1) competed with [3H]-LT04 for the binding sites at a concentration comparable to that used in the organ-bath studies (1 to 10 PM). These observations lead us to conclude that the LTD4 specific binding characterized in this study is not likely to be due to binding to these enzymes. (20) have suggested that there may be two distinct receptors Krell etc., Using a similar approach, Fleisch et al. (19) suggested that for LTD4. the putative LTD4 receptor in the guinea pig iTeiiiii appeared to be distinctly different from the receptors in the lung or trachea. Recently, [3H1-LTC4 specific binding sites have been identified in the longitudinal smooth muscle of guinea-pig ileum (361, uterus (371, rat lung (38)r and human fetal lung membranes (39). The Kj and Bm of these binding sites were estimated to be 20 to 40 nM an 20 to 86 pmol/mg protein

FPL55712 > 5R,6S-LTD4. distinctly different populationsof specific binding sites for LTC4 and This suggestion is also substantiated by the observation that in LTD4. the presence of high concentration of serine-borate (45 mM; which inhibits the conversion of LTC4 to LTD4). The LTC4 induced smooth muscle contractile activity in guinea pig trachea was not antaoonized by FPL 557 12 (40). It is likely-that-the LTb4-receptor is subject to-a variety of regulatory processes and affected by a variety of cofactors; e.g., monovalent, divalent cations, guanine nucleotide, pH, chelating agents, reducing or sulfhydryl group containing agents (29, 41-44). Questions concerning cofactors, receptor homogeneity/heterogeneity, affinity states, receptor subtypes, second messengers and regulation can new be analyzed biochemically. The leukotriene receptor binding assay described in the present studies offers an efficient, accurate and quantitative method to determine the affinity and density of the specific binding sites. It may also prove to be a valuable tool for the design of leukotriene antagonists.

DECEMBER

1984 VOL. 28 NO. 6

819

PROSTAGLANDINS

ACKNOWLEDGEENTS We are indebted to Ms. J. Seaman for typographical assistance; Drs. B. Weichman, C. Perchonock for chemicals and reagents; Drs. C. H. tiuang, G.K. Hogaboom, E. Suen, J. Chan, R. Shorr, and M. Wasserman for their suggestions and critiques.

REFERENCES Brocklehurst, W.E. The release of histamine and formation of a 1) slow-reacting substance (SRS-A) during anaphylactic shock. J. Physiol . 151: 416. 1960. Lewis R.A.,usten, K.F., Drazen, J.M., Clark, D.A., Marfat, A. and 2) E.J. Corey. Slow reacting substances of anaphylaxis: Identification of leukotrienes C and D from human and rat sources. Proc. Natl. Acad. Sci. U.S.A. 77: 3710. 1980. Morris, H.R., Taylor, G.W., Piper, P.J., Samhoun, M.H. and J.R. 3) Tippins. Slow reacting substances (SRS): The structure identification of SRS from rat basophilic leukemia (RBL-1) cells. Prostaglandins 19: 185. 1980. Murphy, R.C., Hzmarstrom, S. and B. Samuelsson. Leukotriene C, a 4) slow reacting substance (SRS) from mouse mastocytoma cells. Proc. Natl. Acad. Sci. U.S.A. 76: 4275. 1979. Hammarstrom, S., Samuelszn, B., Clark, D.A., Goto, G., Marfat, A., 5) Mioskowski? C. and E.J. Corey. Stereochemistry of leukotriene C-l. Biochem. Biophys. Res. Commun. 92: 946. 1980. S. and R.C. Murphy. Samuelsson, B., Borgeat, P., HaKarstrom, 6) Introduction of a nomenclature; leukotrienes. Prostaglandins, -17: 785. 1979 Leukotriene D: A Orning, L., Wammarstrom, S. and B. Samuelsson. 7) slow reacting substance from rat basophilic leukemic cells. Proc. Natl. Acad. Sci. U.S.A. 77: 2014. 1980. Corey, E.J., Arai, K, anrC. Mioskowski. Total synthesis of (+,-I 8) 5,6-oxido-7,trans-11,14 cis-eicosapentanoic acid, a possible J. Amer. Chem. Sot. 101: 6748. 1979. precussor of SRS-A. B. Samuelsson. Dahlen, S.E., Hedqvist, P., Hammarstrom, cand 9) Leukotrienes are potent constrictors of human bronchi. Nature -288: 484. 1980. 10) Hanna, C.J., Bach, M.K., Pane, P.D. and R.R. Schellenberg. Slow-reacting substances (leukotrienes) contract human airway and pulmonary vascular smooth muscle in vitro. Nature 290: 343. 1981. Dixon, rand E.V 11) Holroyde, M.D., Altougan, R.E.C.,Ton., Elliott Selective inhibition of bronchoconstriction induced by leukotriene C and leukotriene D in: Advances in Prostaglandin, I Thrcmboxane and Leukotriene Research 9: Leukotrienes and other (B. Samuelsson and R. Paoletti eds.) Raven Lipoxygeanse Products. Press, N.Y. 1982. p. 237.

820

DECEMBER

1984 VOL. 28 NO. 6

PROSTAGLANDINS

12)

13)

14)

15)

16)

17)

18)

19)

20)

211

221 231 24)

25)

261

Piper, P.J. and M.N. Samhoun. The mechanism of action of leukotrienes C4 and 04 in guinea pig isolated perfused lung and parenchymal strips of guinea pig, rabbit and rat. Prostaglandins 21: 793. 1981. Fijlco, G., Hasson, G. and E. Grastrom. Leukotriene C4 stimulates TxB2 formation in isolated sensitized guinea pig lung. Biochem. Pharm. 30: 2492. 1981. WeichmaK B.M. Muccitelli, R.M., Osborne, R.R., Holden, D.A., Gleason, J.G. and M.A. Wasserman. In vitro and in vivo mechanisms of leukotriene-mediated bronchoconstriction in guineapig. J. Pharmacol. Exp. Therap. 222: 202. 1982. Lewis, R.A., Drazen, J.MrFigueredo, J.C., Corey, E.J. and K.F. Austen. A review of recent contribution on biologically active products of arachidonate conversion. Int. J. Immunopharm. -4: 85. 1982 Drazen, J.M., Austen, K.F., Lewis, R.A., Clark, D.A., Goto, G., Marfat, A. and E.J. Corey Comparative airway and vascular activities of leukotriene C-l and D in vivo and -in vitro. Proc. Natl. Acad. Sci. U.S.A. 78: 3887. 1986. Baker, S.R., Boot, J.R.,Jamieson, W.B., Osborne, D.J. And W.J.F. Comparative in vitro pharmacology of leukotriene D4 and Sweatman. Biochem. B?-om Res. Comm. 103: 1258. 1981. its isomers. Augstein, J., Farmer, J.B., Lee, T.B., Shea= P. and M.L. Selective inhibitor of slow reacting substance of Tattersall. anaphylaxis. Nature New Biol. 245: 215. 1973. Spaethe. Slow reacting Fleisch, J.H., Haisch, K.D. andml. substance of anaphylaxis (SRS-Sl release from guinea pig lung during antigen or ionophore induced contraction. J. Pharmacol. Exp. Therap. 221: 146. 1982. Krell, Rx, Tsai, B.S., Berdoulay, A., Barone, M. and R.I. Giles. Heterogeneity of leukotriene receptors in guinea-pig trachea. Prostaglandins, 25: 171. 1983. Corey, E.J., ClaX, D.A., Goto, G., Marfat, A., Mioskowski, B., Samuelsson, B. and S. Hammarstrom Stereospecific total synthesis of a "slow reacting substance" of anaphylaxis, leukotriene C-l. J. Amer. Chem. Sot. 102: 1436. 1980. Nomenclature for leukotrienes. Samuelsson, B. and. Hammarstrom. Prostaglandins, 19: 645. 1980. D. and C. Kinzig. Convergent synthesis of Gleason? J.G., Bean, Tetrahedron Lett. 21: 1129. 1980. leukotriene A methylester. A rapid and sensitive method for the quantitation of Bradford, M.M. microgram quantities of protein utilizing the principle of Anal. Biochem. 72: 248. 1976. protein-dye binding. iE Adrenergic Pharmacology. Williams, L.T. and R.J. Lefkowitz. [L.T. Willaims and R.J. Lefkowitz, eds.). Raven Press, N.Y., 1978, p 27. Scatchard, G. The attraction of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. -51: 660. 1949.

DECEMBER

1984 VOL. 28 NO. 6

821

PROSTAGLANDINS 27) 28)

29) 30)

31) 32) 331

34)

35)

36)

37)

38) 39)

40)

41)

42)

43) 44)

Tate, S.S. and A. Meister. Serine-borate conplex as a transition-state inhibitor of y-glutamyl transpeptidase. Proc. Natl. Acad. Sci. U.S.A. 75: 4806. 1978. Cheng, Y. and N.H.Prusoff; Relationship between the inhibition constant (Ki) and the concentration of inhibitor which cause 50 percent inhibition (150) of an enzymatic reaction. Biochem. Pharmacol. 22: 3099. 1973. Binding of leukotriene Bruns, R., Roomsen, W.J. and T.A. Pu sley. Regulation by ions and and D4 to membranes from guinea pig 9 ung.

nucleotides. Life Sciences 33: 645. 1983. Pong, S.S. and R. DeHaven. CEracterization of leukotriene D4 receptor in guinea pig lung. Proc. Natl. Acad. Sci. U.S.A. -80: 7415. 1983. Weichman, B.M. and S.S. Tucker. Contraction of guinea pig uterus by synthetic leukotrienes. Prostaglandins 24: 245. 1982. Letts, L,G. and P,J. Piper. The actionsyf leukotriene C and D4 on guinea-pig isolated hearts. Br. J. Pharmacol. 76: 4 69. 1982. Burke, J.A., Levi, R., Buo, Z.G. and E.G. Corey. Leukotrienes C4 D4 and E4: Effects on human and guinea-pig cardiac preparations in vitro. J. Pharmacol. and Exp. Therap. 221: 235. 1982. Edqvist, P., Dahlen, S., Gustofsson, L., mmarstrom, S., and 8. Samuelsson. Biological profile of leukotriene C4 and D4. Acta, Physiol. Stand. 7: 331. 1980. Jakschik, B.A., Harper,T. and R.C. Murphy. Leukotriene C4 and D4 formation by particulate enzymes. J. Biol. Chem. -257: 5346. 1980. The binding Welt n, A.F., Nicosia, S., Crowley, H.J. and D. Olivia. (LTC4) to guinea pi ileal lon itudinal of [s HI-leukotriene C Fe i . Proc. (Amer. Sot. 8. muscle membranes. 101. Chem. 3 _* 42. 2091. 1983. Levinson, S.L. Binding of [3Hl-leukotriene C4 to specific receptor sites in guinea pig uterine membrane preparations. Pharmacologist, 25: 201. 1983. Pong, S., DeHaveriT R.N., Kuehl, F.A. and R.W. Egan. Leukotriene C4 binding to rat lung membranes. J. Biol. Chem. 258: 9616. 1983. Lewis, M.A., Mong, S., Vesella, R.L., Hogaboom, G.K Wu, H.L. and S.T. Crooke. Identification of specific binding sites for leukotriene C4 in human fetal lung. Prostaglandins, (in press). 1984. Snyder, D.W., Barone, M., Morrissette, M.P., Bernstein, P.R. and R.D. Krell. Antagonist of the contractile activity of leukotriene (LT) C4 isolated guinea pig trachea (GPT) by FP\ 55712 is Pharmacologist 25: 205. 1983. dependent upon me abolism to LTD4. Hoffman, B.B., Mulliken-Kilpatrick, D. and R.J. Lef%&tz. Heterogeneity of radioligand binding to alpha-adrenergic receptor: Analysis of guanine nucleotide regulation of agonist binding in 1980. relation to receptor subtypes. J. Biol. Chem. 255: 4645. Magnesium Williams, L.T., Mullikin, D. and R.J. Lefkowitz. dependence of agonist binding to adenylate cyclase-coupled hormone J. Biol. Chem. 253: 2984. 1978. receptors. Sibley, D.W. and I. Creese.Tegulation of ligand binding to pituitary D-2 dopaminergic receptor. J. Biol. Chem. 258: 4957. 1983. Stiles? G.L., Hoffman, B.B., Hubbard, M., Caron! M.G.yd R.J. Guanine nucleotides and CII adrenergic receptor. Lefkcwitz. Biochem. Pharmacol. 32: 69. 1983. Editor:

822

C4

Priscilla

J. Piper

Received: Accepted:

6-28-84 9-24-84

DECEMBER

1984 VOL. 28 NO. 6