Characterization of a leukotriene D4 receptor in guinea pig lung.

Proc. Nati. Acad. Sci. USA Vol. 80, pp. 7415-7419, December 1983

Biochemistry

Characterization of a leukotriene D4 receptor in guinea pig lung (FPL 55712/ionic interaction/asthma/GTP)

SHENG-SHUNG PONG AND ROBERT N. DEHAVEN Merck Institute for Therapeutic Research, Rahway, NJ 07065

Communicated by Roy Vagelos, August 26, 1983

ABSTRACT [3H]Leukotriene D4 was found to bind, in a saturable manner and with exceedingly high affinity, to a membrane preparation from guinea pig lung. Measurement of saturation at equilibrium yielded Kd values of 5.46 ± 0.31 X 10-11 M at 200C and 2.12 ± 0.37 x 10-10 M at 00C while the numbers of binding sites (Bm.) were 384 ± 34 and 302 ± 44 fmol/mg of protein at 20 and at 0C, respectively. The time courses of both association and dissociation were slow but the rate of dissociation was accelerated by either NaCl or GTP. Binding was enhanced by Ca 2+ , Mn2" and inhibited by Na' but not by LiW or K+, sugMg2e, and gesting that the binding of leukotriene D4 may be regulated by ions. Leukotriene E4, but not leukotriene C4, had a high affinity for the putative receptor, consistent with the pharmacological evidence that the actions of leukotrienes D4 and E4 are mediated by a receptor distinct from that for leukotriene C4. Affinities of stereoisomers and related compounds for the leukotriene D4 binding sites closely paralleled their contractile activities in guinea pig lung parenchymal strips. In addition, the antagonist of slow-reacting substance of anaphylaxis, FPL 55712, inhibited the binding of [3H]leukotriene D4 with a K; value of 1 x 10-7 M, which is in agreement with reported Kb values obtained in pharmacological studies.

EXPERIMENTAL PROCEDURES Materials. [3H]LTD4 (40 Ci/mmol; 1 Ci = 37 GBq) was obtained from David Ahern of New England Nuclear. Synthetic LTB4, LTC4, LTD4, (5S,6S)-LTD4, (5R,6R)-LTD4, (5R,6S)LTD4, and LTE4 were provided by Yves Girard at Merck-Frosst (Kirkland, PQ, Canada). FPL 55712 was obtained from Fisons Pharmaceuticals (Loughborogh, England) and prostaglandins, from Ono Pharmaceutical (Osaka, Japan). GTP (trilithium salt) was purchased from Calbiochem-Behring, and all other chemicals were obtained from commercial sources. Preparation of Guinea Pig Lung Membranes. Male Hartley guinea pigs (250-500 g) were sacrificed by decapitation and the lungs were removed and homogenized for 15-30 sec in 10 vol (wt/vol) of 50 mM Tris-HCI buffer (pH 7.4) with a Brinkman Polytron homogenizer set at 6. The homogenate was centrifuged at 1,000 x g for 10 min at 0C, and the supernatant was then centrifuged at 45,000 X g for 10 min. The resulting pellets were stored at -80°C for up to 1 month with no significant loss of specific [3H]LTD4 binding. [3H]LTD4 Binding Assay. Unless stated otherwise, binding assays were performed by adding 50 1.d of lung membranes (50150 jig of protein) to 0.95 ml of 0.37 nM [3H]LTD4 (17,000 cpm)/ 50 mM Tris-HCl, pH 7.4/20 mM CaCl2 and incubating the mixture for 150 min at 0°C or for 40 min at 20°C. Bound radioactivity was separated from free by vacuum filtration through Whatman GF/B filters and these were then rapidly washed with three 5-ml portions of ice-cold buffer. The amount of radioactivity remaining on the filters was determined in Aquasol-2. Specific binding was defined as the difference between total binding and binding in the presence of 100 nM LTD4 (nonspecific binding). Nonspecific binding was 10-20% of total binding. Assays were carried out in duplicate or triplicate and experiments were repeated at least three times. Metabolism of [3H]LTD4. To determine whether [3H]LTD4 was metabolized during the binding assay, incubation mixtures were filtered and the filtrate was collected (unbound fraction). The radioactivity retained on the filter (bound fraction) was further washed three times with cold Tris-HCl buffer, extracted four times at 0°C with 1-ml portions of methanol/0.5% acetic acid, and concentrated under a stream of nitrogen. The recovery of radioactivity from the filter was 82%. The unbound fraction and the extracted bound fraction were analyzed by reversed-phase HPLC on a series of two uBondapak C18 columns (3.9 x 30 cm each; Waters Associates) protected by a 3-cm column packed with Lichroprep RP-18 (Bodman Chemicals, Media, PA). Peaks of radioactivity were identified by coelution with LTC4, LTD4, and LTE4 standards monitored by UV absorption at 280 nm in elution systems of CH3CN/H20/CH3COOH (35:65:0.1) adjusted to pH 5.8 with 10% NH40H and CH30H/

Slow-reacting substance of anaphylaxis is generated in lung by various stimuli, including immunological challenge, and is considered to be an important mediator of immediate hypersensitivity reactions, such as bronchoconstriction in allergic asthma (1, 2). The major constituents of slow-reacting substance of anaphylaxis have been identified as 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11, 14-cis-icosatetraenoic acid (leukotriene C4, LTC4) and the 6(R)-S-cysteinylglycyl (leukotriene D4, LTD4) and 6(R)-S-cysteinyl (leukotriene E4, LTE4) analogs (3-6). Among these peptidoleukotrienes, LTD4 is the most spasmogenic on respiratory smooth muscle and is the predominant substance produced during anaphylaxis in guinea pig (4-6). The stereospecific and highly potent effects of peptidoleukotrienes on smooth muscles are consistent with interaction with a specific receptor(s). The observation that the antagonist of slow-reacting substance of anaphylaxis, FPL 55712 (7), inhibits contractions of guinea pig lung parenchymal strips induced by LTD4 more effectively than those induced by LTC4 has led to a proposal that the LTC4 receptor may be distinct from the LTD4 receptor (8, 9). Recently, we have characterized a putative LTC4 receptor in rat lung membranes for which LTD4 has a low affinity (10). In the work described here, we have used [3H]LTD4 to detect a specific high-affinity binding site for LTD4 in a membrane fraction from guinea pig lung that is sensitive to low concentrations of FPL 55712. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviations: LTB4, LTC4, LTD4, and LTE4, leukotriene B4, C4, D4, and E4, respectively. 7415

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Biochemistry: Pong and DeHaven

Proc. Natl. Acad. Sci. USA 80 (1983)

H20/CH3COOH (70:30:0.1) adjusted to pH 5.4 with 10% NH40H. Recovery of the radioactivity from the column was 40-100%. More than 90% of the radioactivity in the bound and unbound fractions was recovered in a single major peak and it was identified as [3H]LTD4, indicating that during incubations at 0 and 20TC there was no significant metabolism of [3H]LTD4. Saturation Experiments. Saturation experiments, performed by incubating a fixed concentration of tissue with various concentrations of [3H]LTD4, were analyzed in three ways: (i) by plotting specific [3H]LTD4 binding vs. concentration of free [3H]LTD4 in which the data were fit by iterative analysis to the equation Y = {(a - d)/[I + (X/c)b]} + d (11), where Y is the amount bound; X, the concentration of free [3H]LTD4; a, the minimum specific binding; d, the maximum number of binding sites (Bm.); c, the Kd; and b, the slope of the corresponding logit-log plot, (ii) by Scatchard analysis using the computer programs developed by Munson and Rodbard (12), which provide weighted least-squares estimates of binding parameters, and (iii) by a Hill plot to determine the presence or absence of cooperativity and an additional estimation of the Kd. RESULTS To determine the optimal conditions for the binding assay, we studied the dependence of binding on time, temperature, and protein concentration, as well as the stability of the ligand under various conditions. The time and temperature dependences of specific [3H]LTD4 binding are summarized in Fig. 1. The rate of binding was increased by increasing the temperature to 200C; Binding at 37°C reached maximum at 10-20 min but this was followed by a gradual decrease, suggesting metabolism of the ligand or instability of the receptor (data not shown). Based on these experiments, we chose 0°C for 150 min or 20°C for 40 min for routine assays. The calculated association rate constant values were 3.21 0.21 108 M-'min-1 at 20°C (n = 4) and 4.18 0.82 107 M'1 min-' at 0°C (n = 3). Under these assay conditions, specific binding increased linearly with tissue concentrations up to 250 Ag of protein/ml and no significant metabolism of [3H]LTD4 was detected. Incubation of membranes at 600C for 4 min completely abolished specific binding without affecting nonspecific binding in a subsequent binding assay. The dissociation of the [3H]LTD4-receptor complex initiated by the addition of 400 nM unlabeled LTD4 is shown in Fig. 2. Semilog plots show a biphasic dissociation at 20°C in which 1530% of the binding had a t,,2 of 48.6 6.5 min (n = 5) but the ±

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FIG. 1. Time and temperature dependence of specific ['H]LTD4 binding to guinea pig lung membranes. ['H1LTD4 (0.37 n1M) was incubated with 133 ug of membrane protein for different times at 0°C (o) and 20°C (o). The lower equilibrium binding at 0°C is a result of the higher Kd at this temperature (see Fig. 4).

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FIG. 2. Time and temperature dependence of [3H]LTD4 dissociation. [3H]LTD4 (0.37 nM) was incubated with 147 ug of membrane protein at 000 for 150 min or at 200C for 40 min. At the end of this incubation (zero time) 0.4 MM unlabeled LTD4 was added and the incubation was continued for the indicated times at the same temperature. The amounts of [3H)LTD4 bound at zero time in control values were 189 and 246 fnol/mg of protein at 00 and 200C, respectively. For the NaCl or GTP experiments, 50 mM NaCl or 200 gM GTP was added together with the unlabeled LTD4.

remainder had a t112 of >4 hr. At 00C, the til2 of dissociation was >6 hr. The rate of dissociation was accelerated by 50 mM NaCl or 200 AtM GTP with which 85-90% of the bound [3H]LTD4 was dissociated by 3 hr at 00C. KC1 (50 mM) had little effect on the rate of dissociation under the same conditions (data not shown). The effects of the chloride salts of Ca2 , Mg2+, and Mn2+ on the specific binding of [3H]LTD4 are shown in Fig. 3A. Each of these ions increased the binding by 150-200%. Mn2+ was slightly more effective than the other two ions. Half-maximal stimulation by divalent cations was achieved at approximately 1-2 mM and maximal stimulation at 10-20 mM. The potassium salt of EDTA at concentrations up to 20 mM had no effect on specific binding in the absence of divalent cations. In contrast to the effect of divalent cations, NaCl completely abolished specific binding with IC50 values of 2.3 mM at 0C (Fig. 3B) and 35 mM at 20'C. LiCI and KC1 were much less effective, with IC50 values of >100 mM. Saturability of specific [3H]LTD4 binding was shown by incubating tissue with increasing concentrations of [3H]LTD4 (Fig. 4A). The data are consistent with the law of mass action regarding a simple bimolecular interaction between ligand and receptor. Computer-assisted Scatchard analysis (Fig. 4B) of specific binding showed a single binding site with dissociation constants (Kd) of 2.12 ± 0.37 x 10-10 M at 0°C (n = 6) and 5.46 ± 0.31 x 10"1 M at 20°C (n = 3). The maximum number of specific binding sites (Bm.) was not significantly different at 0°C (Bmax = 302 ± 44 fmol/mg of protein, n = 6) than at 20°C (Bmax = 384 + 34 fmol/mg of protein, n = 3). When the data were analyzed by a Hill plot (Fig. 4C), straight lines with Hill coefficients (nH) of 0.89 at 0°C and 0.97 at 200C were obtained, indicating no significant cooperativity among binding sites. The specificity of [3H]LTD4 binding was established by determining the ability of related compounds to compete with [3H]LTD4 for the binding sites (Fig. 5A). To compare the relative affinities of each compound for the LTD4 receptor, ap-

Biochemistry: Pong and DeHaven

Proc. Natl. Acad. Sci. USA 80 (1983)

7417

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FIG. 3. Effects of cations on specific [3H]LTD4 binding. Lung membranes (147,ug ofprotein) were incubated at O0 with 0.37 nM [3H]LTD4 in the presence of CaCl2 (A), Mg9l2 (C), or MnCl2 (0) (A) or of NaCl (o), KCl (C), or LiOl (A) (B).

parent Ki values were determined from the IC50 values for each compound and the Kd for [3H]LTD4 binding described in Fig. 4, using the equation Ki = IC50/(1 + [LTD4]/Kd). Unlabeled LTD4 displaced [3H]LTD4 with a Ki value of 3.2 x 10-1o M at

FIG. 4. Saturation isotherms for [3H]LTD4 binding at 00 and 2000. Specific binding was measured over a radioligand concentration range of 4 pM to 1 nM. Lung membranes (70 jig of membrane protein) were incubated with ligand for 150 min at 000 (m) or for 40 min at 2000 (0). Data from a typical saturation isotherm were analyzed in three ways. (A) Klotz plot. (B) Scatchard plot. (C) Hill plot. B, bound; F, free.

00C, which is close to the Kd value determined by Scatchard analysis. LTE4 (Ki = 7.8 x 10-1 M) had an affinity of 5=0% of that of LTD4. LTB4 (Ki > 1 x 10-5 M) was inactive. The stereoisomers, (5R,6S)-LTD4 (Ki = 2.2 X 10-8 M), (5R,6R)-LTD4 (K = 3.8 x 10-8 M), and (5S,6S)-LTD4 (Ki = 3.13 X 10-7 M)

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FIG. 5. Inhibition of [3H]LTD4 binding to guinea pig lung membranes by various compounds. Lung membranes were added to mixtures containing the agents and [3H]LTD4 (0.37 nM) and incubated for 150 min at 000. Results are expressed as percent of inhibition of [3H]LTD4 specifically bound. (A) Without serine-borate complex. (B) With 10 mM serine-borate complex and LTC4 (0) or LTD4 (0).

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Biochemistry: Pong and DeHaven

had affinities of :1.5%, 0.87%, and 0.10% of that of natural (SS,6R)-LTD4. To further characterize the pharmacological properties of LTD4 binding, compounds known to be active in the contraction of smooth muscle were tested. FPL 55712 inhibited [3H]LTD4 binding with a Ki of 1.5 X 10-7 M at 0C (Fig. 5A) and 1.1 X 10-7 M at 200C (data not shown). Indomethacin had a Ki of 3 x 10-5 M. Histamine, arecoline, timolol, isoproterenol, yohimbine, and prazosin were inactive at 500 ,AM. When tested at concentrations up to 100 ttM, reduced and oxidized glutathione, oleic acid, icosenoic acid, and prostaglandins Al, El, E2, and F2a, did not inhibit [3H]LTD4 binding. Inhibition constants for 8,11,14-icosatriynoic acid, 5,8,11,14-icosatetraynoic acid, arachidonic acid, and linolenic acid were 18, 10, 4, and 3 X 10-6 M, respectively. The latter two compounds also reduced nonspecific binding; therefore, their effects are probably due to nonspecific interactions. Although little metabolism of LTD4 occurred under the conditions of binding either at 00C or 20TC, complete conversion of LTC4 to LTD4 was observed at 0C during the [3H]LTD4 competition assays, resulting in an inhibition curve identical to that for LTD4. However, 10 mM serine-borate complex, an inhibitor of y-glutamyl transpeptidase (13), completely prevented the conversion of LTC4 to LTD4 (data not shown) and shifted the inhibition curve for LTC4 (Kj = 3.1 X 10-8 M) by two orders of magnitude (Fig. 5B). Serine-borate complex did not affect specific and nonspecific binding of [3H]LTD4, and it did not shift the competition curve for LTD4. DISCUSSION

Scatchard analysis of equilibrium binding indicated a single class of LTD4 binding sites in guinea pig lung with a Kd in good agreement with EC50 values from studies of LTD4-induced contractions of guinea pig lung parenchymal strips (4, 8, 9, 14). The slopes of Hill plots suggested no cooperativity among binding sites. The conformation of this binding site may undergo significant changes at various temperatures because the affinity was 5-fold higher at 200C than at 0°C. A Kd value can also be obtained independently from the kinetic data obtained at 20°C by using the equation, Kd = k 1/k+1, where k+I is the rate constant of association (3.71 x 10 M-'-min-') and L1 is the rate constant of dissociation (0.0144 min-1 for the fast phase of dissociation). The Kd determined in this way was 3.89 X 10-11 M, which was close to that obtained by Scatchard analysis at equilibrium at 200C (5.46 x 10-11 M). However, the contribution of the slow phase of dissociation in the overall equilibrium is unclear. The Kd value determined at 0°C by saturation experiments should be considered as tentative because the value is not confirmed independently by kinetic analysis. Dissociation of the [3H]LTD4-receptor complex proceeded relatively slowly at 20°C and even slower at 0°C. The very slow dissociation at the lower temperature does not seem to be due to the formation of a covalent bond between the ligand and the binding site or to a physical sequestration of the ligand because in the presence of NaCl and GTP dissociation was rapid and nearly complete. In addition, bound [3H]LTD4 could be extracted with methanol and recovered unchanged. The slow association and dissociation of LTD4 binding is compatible with the slow onset and reversal of contractions induced by leukotrienes as compared with histamine in guinea pig lung parenchymal strips (1, 15, 16). (5R,6S)-LTD4, (5R,6R)-LTD4, and (5S,6S)-LTD4 possess 100 mM). A similar effect of divalent cations but not of Na+ has been observed for [3H]LTC4 binding (10). Similar ionic interactions have also been found for the histamine, a- and 3-adrenergic, angiotensin II, and opiate receptors and are generally attributed to an association with adenylate cyclase (22-26). Inhibition of receptor binding by Na+ is confined to those receptors that inhibit adenylate cyclase (27, 28). The observation that slow-reacting substance of anaphylaxis reduces cAMP levels (29) together with our findings of the inhibition of LTD4 binding by Na+ and the acceleration of dissociation by GTP provide evidence that the LTD4 receptor may be linked to adenylate cyclase via the inhibitory guanine nucleotide regulatory unit. In conclusion, we have demonstrated a LTD4 binding site that may mediate the effects of LTD4 and LTE4 in guinea pig for lung. This observation, together with our previous evidenceand a specific LTC4 receptor in rat lung, suggests that LTD4 LTE4 may interact with a receptor different from that for LTC4. These data raise crucial questions about the distribution of these two receptors and the metabolic enzymes that regulate the levels of leukotrienes in various tissues. Such distributions may indicate uniquely different roles for LTC4 and LTD4 in some

Biochemistry: Pong and DeHaven instances, including various physiological or pathological con-

ditions. We thank Frederick A. Kuehl, Jr., and R. W. Egan for encouragement, advice, and critical evaluation of this manuscript and R. L. Vandlen for his help in computer analysis of data. We also thank Y. Girard of Merck-Frosst for leukotrienes and D. Ahern of New England Nu-

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