THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The Amerrcan Socrety of Brolopcal Chemists, Inc.

Vol. 262, No. 9, Issue of March 25, pp. 4034-4041,1987 Printed in U.S.A.

Identification and Characterization of Leubotriene D4 Receptors and Signal Transduction Processes in Rat Basophilic Leukemia Cells*

(Received for publication, August 6, 1986)

Henry M. Saraug, Seymour Mong, James J. Foley, Hsiao-Ling Wu, and Stanley T. Crooke From the Department of Molecular Pharmacology, Smith Kline & French Laboratories, Swedeland, Pennsylvania 19479

The leukotriene D, (LTD,) receptor on rat basophilic leukemia (RBL- 1) cell membranes was characterized using a radioligand binding assay. t3H)LTD4 binding to RBL-1 membrane receptors was stereoselective, specific, and saturable. The binding affinity and max- imum binding density of [’HJLTD, to RBL-1 membrane receptors were 0.9 f 0.2 nM and 800 2 126 fmol/mg protein, respectively. Binding of {“HJLTD, to the re- ceptors was enhanced by divalent cations (Cas+, Mg2+, and Mna+) and inhibited by guanine nucleotides and sodium ions, specifically, indicating that a guanine nucleotide-binding protein may regulate the agonist- receptor interaction. LTD4, LTE, agonist and antago- nist analogs competed with the radioligand in binding to the RBL-1 LTD, receptors. The binding affinities of these analogs correlated with (a) those determined from the guinea pig lung LTDa receptors and (b) the pharmacological activities in smooth muscle contrac- tion. LTD, and related agonists also induced time- and concentration-dependent phosphatidylinositol hydrol- ysis in RBL-1 cells. The LTD, induction of inositol 1- phosphate was potent, stereoselective, specific, and was blocked by LTD4 receptor antagonists. The rank order potency of agonist-induced inositol 1-phosphate formation in RBL-1 cells was equivalent to the recep- tor binding affinity determined using either RBL-1 cell or guinea pig lung membranes. These studies have demonstrated the G protein coupled LTD, receptors on RBL-1 cell membranes. Binding of agonists to the re- ceptor may activate the G protein-regulated phospho- lipase C to induce hydrolysis of phosphatidylinositol. The hydrolytic products of phosphatidylinositol, pos- sibly inositol trisphosphate and diacylglycerol, may be the intracellular messengers for LTD, receptors in RBL-1 cells.

Significant advances in the identification and characteri- zation of peptidoleukotriene receptors and signal transduction processes have been reported (1-18). [3H]LTD41 specific bind-

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “Ddvertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

___- I

$ To whom correspondence should be addressed. I The abbreviations used are: LTD4, (5S)-hydroxy-(GR)-S-l-cys-

teinylglycyl-(7E,9E,11Z,142)-eicosatetraenoic acid; LTC+, (SS)-hy- droxy-(6R)-S-glutathionyl-(7E,9E,11Z,142)-eicosatetraenoic acid; LTE., (5S)-hydroxy-(6R)-S-l-cysteinyl-(7E,9E,11Z,14Z)-eicosate- traenoic acid; LTB, (5S,12R)-dihy&oxy-6,8,10,14-eicosatetraenoic acid; (5R,6S) - LTD,, (5R) - hydroxy - (6s) - S - 1 - cysteinylglycyl- (7E,9E,11Z,14Z)-eicosatetraenoic acid; LTDI, (5S)-hydroxy-(6R)-l- cysteinylglycyl-7-cis-eicosenoic acid (5R,GS)-LTD1, (5R)-hydroxy- (6S)-l-cysteinylgIycyl-7-cis-eicosenoic acid; homo-LTDl, (6S)-hy- droxy-(7R)-l-cysteinylglycyl-8-trans-heneicosenoic acid; (6R,7S)-

ing sites have been shown and characterized in human lung membranes, in guinea pig tracheal, lung, and myocardium membranes. Binding of [3H]LTD4 to the lung membranes was specifically regulated by Na*, CaZ+, M P , and guanine nu- cleotides (1, 8, 10-13). These specific binding sites in human and guinea pig lung membranes were shown to be receptors by comparing the rank order potency of agonists and antag- onists in radioligand competition assays to that observed in guinea pig lung, tracheal smooth muscle, and human lung smooth muscle contraction assay systems (5,13,19-23). LTE, and LTD, pharmacologically interact at the same receptor and they share many of the characteristics in G protein and cation regulation (6, 12). Also, [’HH]LTD, bound receptor and G protein ternary complexes have been solubilized from guinea pig lung membranes and partially purified (11). These studies have firmly established that I3H]LTD4 receptors in guinea pig and human lung are biochemically and physiolog- ically important and that the pharmacological activities of LTD, and LTE, are mediated through this class of receptors.

Although significant advances in understanding LTD, re- ceptors and LTD, receptor subtypes have been reported (13, 14), little is known about the signal transduction processes coupled to these receptors. The guanine nucleotide triphos- phate and Na’ sensitivity of binding is generally associated with Gi (or Go)-linked receptors. This suggests that LTD, receptor activation may result in inhibition of adenylate cy- clase and a lowering of intracellular cAMP levels. Anderson et al. (41) have reported such an effect for LTC, and possibly LTD, in guinea pig trachea. However, in our laboratory we have been unable to repeat this observation (9). Also, other laboratories have reported negative results, e.g. cAMP levels measured by radioimmunoassay were unchanged in tracheal smooth muscle induced to contract with LTD, (42). Other

homo-LTD,, (GR)-hydroxy-(7S)-l-cysteinylglycyl-Il-tra~-heneico- senoic acid; 2-nor-LTD1, (4R)-hydroxy-(5S)-S-l-cysteinylglycyl- (62)-nonadecenoic acid; desamino-LTE,, 5-hydzoxy-6-carboxylethyl- thio-(Z)-eicosenoic acid; FPL 55712,7-[3-(4-acetyl-3-hydroxy-2-pro- pyl-phenoxy)-2-hydroxypropoxy]-4-oxo-8-pro~yl-4H-l-benzopyran- 2-carboxylic acid; SKF 103944,2-hydroxy-3-(2-carboxylethylthio)-3- [2-(8-phenyloctyl)phenyl]propanoic acid; SKF 104353, the (2S,3R) isomer of SKF 103944; SKF 104373, the (2R,3S) isomer of SKF 103944; SKF 102922, 4,6-dlth1a-S-[2-(8-phenyloctyl)phenyl]nonane- dioic acid; SKF 102081, 4,6-dithia-S-(2-dodecyIphenyl]nonanedioic acid, Gpp(NH)p, guanyl-5’-yl imidodiphosphate; Gpp(CHa)p, guanyl- 5”yl (P,a-methylene)diphosphonate; App(NH)p, adenyl-5’-yl imido- diphosphate; App(CH2)p, adenyl-5’-yl(P-a-methylene)diphosphonate; DTNB, 5,5‘-dithiobis-(2-nitrobenzoic acid); NEM, N-ethylmaleim- ide; pHMB, p-hydroxymercuribenzoate; L-DTT, (-)-1,4-dithio-~- threitol; DTE, dithioerythritol; HEPES, 4-(2-hydroxyethyl)-l-piper- azineethanesulfonic acid; PI, phosphatidylinositol 4,5-bisphosphate; IP,, inositol monophosphate; IP2, inositol bisphosphate; IPS, inositol trisphosphate; DAG, 1,2-diacylglycerol; IAP, islet-activating protein from B. pertussis; G;, inhibitory guanine nucleotide-binding protein; Go, extracellular guanine nucleotide-binding protein; RBL, rat baso- philic leukemia.

4034

LTD, Receptor and Phosphatidylinositol Turnover 4035

questions about the LTD, receptor remain to be answered. For example, are leukotriene receptors subject to homologous or heterologous desensitization or, receptor down regulation? What are the intracellular messengers? How is the membrane receptor-activated signal transduced to other intracellular biochemical processes, resulting in smooth muscle contrac- tion, arachidonic acid release (17, 19, 20), and synthesis of cyclooxygenase metabolites of arachidonic acid (16-18)?

To answer such questions, clonally derived cells in culture are necessary. In the past, many attempts in our laboratory and others to answer such questions about the LTD, receptors (4, 5, 16, 17) were thwarted because none of the smooth muscle cells or endothelial cells investigated contained signif- icant numbers of [3H]LTD4 specific binding sites. We report here for the first time, that [3H]LTD, specific binding sites in rat basophilic leukemia (RBL-1) cells can be identified and characterized. RBL-1 cells have been used extensively for studies on leukotriene biosynthesis (24-26). Thus the dem- onstration of LTD, receptors on RBL-1 cells will also allow characterization of any possible links between LTD, receptor occupancy and leukotriene biosynthesis. The [3H]LTD4 spe- cific binding sites on RBL-1 cell membranes are bona fide LTD, receptors with similar characteristics to the receptors found in lung. Presented here are the first conclusive data that LTD, receptors are coupled in a guanine nucleotide- sensitive manner to the hydrolysis of PI lipids, forming IPS and DAG, which may be the intracellular messengers for the LTD, receptors.

EXPERIMENTAL PROCEDURES

Materials

[14,15-3H]LTD4 (32.0 Ci/mmol) and ~-myo-[1,2-~H)-inositol (40- 60 Ci/mmol) were obtained from New England Nuclear. LTB,, LTC,, LTD,, LTE,, (5R,6S)-LTD4, LTD1, (5R,6S)-LTD1, homo-LTD1, (6R,7S)-homo-LTD1, 2-nor-LTD1, desamino-LTE1, SKF 103944, SKF 104353, SKF 104373, and SKF 102922 were prepared by total synthesis and supplied by Dr. J. G. Gleason in the Dept. of Medicinal Chemistry, Smith Kline & French Laboratories. Gpp(NH)p,

pHMB, HEPES, L-DTT, and DTE were purchased from Sigma. FPL 55712 was generously supplied by Fison Ltd. Eagle's minimum essen- tial medium was obtained from GIBCO. Islet-activating protein was purchased from List Laboratories.

Gpp(CHz)p, GTP, App(NH)p, App(CHz)p, ATP, DTNB, NEM,

Methods

Cell Culture Conditions-RBL-1 cells were obtained from American Type Tissue Collection (CRL-1378) and grown in Eagle's minimum essential medium supplemented with 10% (v/v) heat-inactivated fetal calf serum, in spinner culture in a humidified environment of 5% COz, 95% air at 37 'C. Cells were grown to a density of 0.7-1.1 X lo6 cells/ml and harvested for the experiments.

Preparation of Membrane-enriched Fraction-RBL-1 cells were harvested by centrifugation at 400 X g for 10 min. Cells were washed twice with 50 mM sodium phosphate buffer (pH 7.0) containing 1 mM EDTA and 0.1% gelatin (buffer A). Cells were resuspended in buffer A at 5 X lo7 cells/ml and disrupted by nitrogen cavitationwith a Parr bomb at 750 p.s.i. for 10 min at 0 "C. The broken cell preparation was centrifuged at 1,000 X g for 10 min. The supernatant was centrifuged at 100,000 X g for 60 min. The high speed pellet was resuspended in 10 mM Tris-HC1 (pH 7.5 at 25 'C; buffer B) containing 10% (w/v) sucrose. The suspension (15 ml) was carefully layered onto 15 ml of buffer B containing 40% (w/v) sucrose in nitrocellulose centrifuge tubes. The tubes were centrifuged at 100,OOO X g for 60 min in a SW-27 swinging bucket rotor. Membranes that settled at the boundary layer were collected and diluted with 5 volumes of 50 mM Tris-HC1 (pH 7.5 at 25 "C; buffer C) and centrifuged at 50,000 X g for 30 min. Pellets were washed by resuspension and centrifugation

pended at a protein concentration of 1 mg/ml in buffer C using the ih an equal volume of buffer C. The membrane pellets were resus-

Bradford protein assay (28) and bovine serum albumin as the stand- ard. The membrane suspension was rapidly frozen in liquid nitrogen

and stored at -80 "C. No loss in LTD,-receptor binding activity was obtained with storage for up to 5 weeks.

Binding of pH]LTD4 to Membrane~-[~H]LTD, binding assays were performed in a total volume of 200 p1 in Nunc minisorb tubes. In addition to membrane protein (500 pg/ml), samples contained, unless otherwise indicated, buffer C containing 10 mM, MgC12,lO mM CaC12, and 13H]LTD4 (1 nM) in the presence or absence of 2 p M unlabeled LTD4. Equilibrium of [3H]LTD, binding was reached with incubation for 40 min at 25 "C. The free and bound ligand were separated after diluting samples with 5 ml of ice-cold buffer D (25 mM Tris-HC1, pH 7.5, at 25 "C) and rapid filtration through Whatman GF/C filters. The filters were rapidly washed with 15 ml of buffer D. Filters were placed in scintillation vials with 10 ml of Aquasol-2 (New England Nuclear) and the radioactivity was determined by scintilla- tion spectrometry. All assays were carried out in triplicate assay tubes. Binding data are presented as specific 13H]LTD, binding which is defined as the difference between the binding in the absence and presence of 2 p~ unlabeled LTD,. At 1-2 nM [3H]LTD, the specific binding accounted for 60-70% of the total binding.

The binding affinity of agonists and antagonists for the LTD, receptor was determined in radioligand competitive binding assays and expressed as K,. The assay tubes contained RBL-1 membrane protein (250-500 pg/ml) and [3H]LTD, (1 nM) in buffer C under standard conditions. The K, was calculated from the equations for competitive inhibition described by Cheng and Prusoff (29). Where applicable, experimental values are expressed as the mean & standard error (S.E.) determined from at least three experiments.

Equilibrium Saturatwn Binding of Radioligand to Receptors-The saturation binding experiments were performed with 25-50 pg of protein and varying concentrations of [3H]LTD4 (from 0.2 to 5 nM) under standard assay conditions. Nonspecific binding for these sam- ples was defined with 2 p~ or a 1000-fold excess of unlabeled LTD,. The saturation data were subjected to computer assisted nonlinear least square curve fitting analysis (30) and further evaluated by the method of Scatchard (31).

Labeling RBL-I Cells with myo-~H]Imsitol-RBL-l cells were cultured and harvested as described above. The cells were washed twice and resuspended in buffer E (Krebs-Ringer-Henseleit buffer, in mM; NaC1, 118; KCl, 4.6; MgSO,.7H,O, 1.1; NaHC03, 24.9; KHzP04, 1.0; and glucose, 11.1) containing 5 mM HEPES (pH 7.4) and 0.1% bovine serum albumin at 5 x lo7 cells/ml. Cells were labeled with my~-[~H]inositoI by addition of 10 pCi/ml to the cell suspension and incubation at 37 "C for 90 min in plastic tubes in an oxygen- enriched (95% 02, 5% CO,) atmosphere. LiCl(2 M) was added to the tubes for a final concentration of 10 mM and incubated for another 10 min at 37 "C. Tubes were centrifuged and the cells were washed 2 times with 50 ml of buffer E containing 10 mM LiCl to remove unincorporated my~-[~H]inositol. The labeled cells were resuspended in buffer E containing 10 mM LiCl at a concentration of 1-2 X lo7 cells/ml for PI metabolism studies.

Formation of pH]Znositol Phosphates in RBL-1 Cells-The myo- [3H]inositol-labeled cell suspension, described above, was equilibrated at 37 "C for 5 min. For agonist analog studies, aliquots (300 pl) of the cell suspension were added to tubes containing 3 pl of varying con- centrations of LTD, or other agonist analogs and incubation contin- ued for 20 min at 37 "C. For antagonist studies, SKF 104353 (5 p ~ ) and SKF 104373 (5 p ~ ) were added to the labeled cell suspension and incubated at 37 "C for 10 min. Aliquots (300 p l ) of the suspension were added to 3 pl of varying LTDl concentrations and incubated for an additional 30 min at 37 "C. All assays were performed in triplicate assay tubes.

Measurement of ~H]Imsitol Phosphate Formation-After incu- bation, the reaction was stopped and the lipids extracted by the addition of 0.93 ml of ch1oroform:methanol (1:2). Tubes were left at room temperature for 15 min and then 0.31 ml of chloroform and 0.31 ml of water were added. Samples were vortexed vigorously for 10 s and centrifuged at 2000 X g for 5 min to separate the phases. [3H]IPl, [3H]IP,, and [3H]IP3 present in the aqueous phase were fractionated and determined by anion exchange column chromatog- raphy as described (32). Briefly, Dowex (Bio-Rad AG l-X8,100-200 mesh, formate form) was resuspended in water at a ratio of 1:l (w/ v). Aliquots (0.75 ml) of the aqueous phase were transferred to disposable polystyrene columns (0.5 x 6 cm) containing 0.5 ml of Dowex suspension and 5 ml of water. The resin was washed 4 times with 5 ml each of water. [3H]IPI was eluted into scintillation vials with 1.5 ml of 0.2 M ammonium formate, 0.1 M formic acid (solution F) twice. The columns were washed 4 times with 5 ml each of solution F. [3H]IP2 was eluted into scintillation vials with 1.5 ml of 0.4 M

4036 LTD, Receptor and Phosphatidylinositol Turnover

ammonium formate, 0.1 M formic acid (solution G ) twice. The column was washed 4 times with 5 ml each of solution G. Finally, [3H]IP3 was eluted into vials with 1.5 ml of 1.0 M ammonium formate, 0.1 M formic acid twice. Aquasol-2 (15 ml) was added to each vial and the radioactivity determined by scintillation spectrometry with efficiency of 30%. Recent reports have demonstrated that the IPB fraction as described above may contain the cyclic form of IP3 (33), IP3 isomers, i.e. inositol 1,3,4-trisphosphate and inositol 1,4,5-trisphosphate, and possibly inositol 1,3,4,5-tetrakisphosphate (34). The [3H]IP3 fraction reported here probably contains all of these inositol phosphates.

RESULTS

Effects of Cations on fH]LTD, Binding-When 1 nM of [3H]LTD4 was incubated with 60 pg of RBL-1 membrane protein in 50 mM Tris-HC1 (pH 7.5) at 25 "C, [3H]LTD4 binding to the membranes was dependent on the time of incubation. After 15 min, [3H]LTD4 binding to the RBL-1 membranes reached a stable, steady-state level. The nonspe- cific binding (binding in the presence of 2 p~ LTD,) and the specific binding (total binding minus the nonspecific binding) of [3H]LTD4 were 30% and 70% of the RBL-1 membrane bound [3H]LTD4, respectively (data not shown). The effects of divalent cations on LTD, specific binding to RBL-1 mem- branes were determined and shown in Fig. lA. In the concen- tration range of 10-20 mM, Ca2+, Mg+, and Mn2+ enhanced ['HILTD, specific binding to RBL-1 membranes by 3- to 4- fold. The specific binding of ['HILTD, was inhibited by monovalent cations (Fig. 1B). The [3H]LTD4 specific binding was selectively inhibited by Na+ with an IC, of 8 k 4 mM. Other monovalent cations were less active than Na'. The rank order potency of the monovalent cations in regulating [3H]LTD4 specific binding was Na+ >> Li+ > K+ > Cs' = Rb'. These results demonstrate that [3H]LTD4 specific binding to RBL-1 membranes was enhanced by divalent cations and specifically inhibited by Na". These characteristics were equivalent to the ['HILTD, binding to guinea pig lung (but not heart) membrane receptors reported previously (1, 11- 13).

Previous [3H]LTD4 binding studies from our laboratory incorporated cysteine and glycine to inhibit the metabolism of LTD, to LTE,. Preliminary studies with the RBL-1 cell membranes showed that cysteine and glycine alone or in combination had no effect in the ['H]LTD4 saturation-bind- ing studies or the LTD, competition studies. In addition, high performance liquid chromatography analysis of the leuko- trienes present before and after incubation showed less than 5% conversion of the ['H]LTD4 to [3H]LTE4 (results not shown). Based on these results, all the following ['HILTD, binding studies were performed under the following standard conditions: 50 mM Tris-HC1 (pH 7.5), 10 mM MgC12, 10 mM CaC12, [3H]LTD4 (0.1-5 nM), 25-50 pg (250-500 pg/ml) of RBL-1 cell membrane protein, and incubation at 25 "C for 40 min. The tissue preparation used was prepared on a sucrose cushion and contained mostly plasma membranes but mito- chondria and microsomes were present. A preliminary study using sucrose gradient centrifugation showed an enrichment of [3H]LTD4 binding to the plasma membrane fraction (data not shown) suggesting that the LTD, receptors are present primarily in the plasma membrane of RBL-1 cells. Addition- ally, [3H]LTD4 binding was detected on the membranes of intact RBL-1 cells? Thus, our data suggests that LTD, recep- tors are located primarily in the plasma membrane of RBL-1 cells.

Equilibrium Saturation Binding of PHILTD, Specific Bind- ing-Under the standard conditions, specific binding of [3H]

H. M. Sarau, S. Mong, J. J. Foley, H.-L. Wu, and S. T. Crooke, unpublished observations.

I I

u I .o IO I( Divalent Cation Concentration (mM)

0

I 1 I J I .o io 100 1000

Monovalent Cation Concentration (mMi

FIG. 1. Effects of cations on specific binding of ['HILTD, to RBL-1 cell membranes. A, concentration-dependent enhancement of [3H]LTD4 specific binding by Ca" (01, M$+ (O), and Mn2+ (X). RBL-1 cell membrane protein (36 pg) was incubated with 1 nM [3H] LTD, at 25 "C for 40 min in the presence of varying concentrations of cations. In the control, where no exogenous cations were added, [3H]LTD4 specific binding was 115 f 12 fmol/mg protein. B, concen- tration-dependent inhibition of ['HILTD, specific binding by mon- ovalent cations. Binding of 13H]LTD4 to RBL-1 membranes was performed under standard conditions, in the presence of varying concentrations of Na+ (O), K+ (O), Li' (X), Cs+ (m), and Rb+ (0). The counter ion was chloride for all divalent and monovalent cations. Mean k S.E. from the average of three separate experiments was calculated and is shown.

LTD, to RBL-1 cell membranes was linearly dependent on the time of incubation for the first 10 min and then reached a steady-state level from 20 to 60 min (results not shown). At the steady-state level, the specific binding of 1 nM [3H]LTD4 was linearly dependent on the concentration of RBL-1 cell membrane protein from 100 to 750 pg/ml (results not shown). Saturation binding of [3H]LTD4 to RBL-1 membranes was studied under standard conditions (Fig. 2 4 ) . Specific binding of [3H]LTD4 to RBL-1 cell membranes increased linearly, dependent on the concentration of [3H]LTD4, from 0.1 to 1.0 nM and then gradually reached a plateau level at higher concentrations of [3H]LTD,. The nonspecific binding of [3H] LTD4 to RBL-1 membranes was linearly related to the con- centration of [3H]LTD4. These results demonstrated that [3H] LTD, could bind to high affinity, low capacity, and saturable specific sites on RBL-1 cell membranes. Using the computer assisted nonlinear least square best fit analysis program (30), we found that the saturation binding data were best described by the model for a single class of specific binding sites. The binding affinity (Kdr dissociation constant) and the maximum number of binding sites (Bmax) were 0.9 f 0.2 nM and 800 -t

LTD, Receptor and Phosphatidylinositol Turnover 4037

I .o 2.0 3.0 4.0 C'HI-LTO,(nM)

7 \ \

200 400 600 800 1000 Bound (frnol/mg protein)

FIG. 2. Saturation binding of ['H]LTD4 to RBL-1 mem- branes. Binding of [3H]LTD, was performed under standard condi- tions with 150 pg/ml RBL-1 cell membranes and 0.1-4 nM of [3H] LTD,. A , nonspecific binding (0), specific binding (O), and total binding (X) of ['HILTD,; B, Scatchard plot of the specific binding data from A. Data shown was a representative (of five) experiments.

125 fmol/mg protein, respectively (the average of five exper- iments). Transformation of the saturation binding data by the Scatchard method (31) yielded a linear plot (Fig. 2B), confirming the single class, high affinity specific binding model. The Kd and B,,, by this method were the same as that obtained by saturation analysis.

Effects of Nucleotides on pH/LTD4 Specific Bindir~g"[~H] LTD, binding to guinea pig lung membrane receptors was regulated by guanine nucleotides (1,13,44). The results shown in Fig. 3 demonstrate that the [3H]LTD4 specific binding to RBL-1 membranes was also specifically regulated by guanine nucleotides. Gpp(NH)p, Gpp(CH,)p, and GTP inhibited [3H] LTD, specific binding in a concentration-dependent manner. The ICso values were 13 k 5, 95 +- 10, and 190 +- 25 KM, respectively. GDP and the adenine nucleotides, e.g. ATP, App(NH)p, and App(CH.Jp, showed little or no significant inhibitory effects on [3H]LTD4 specific binding at concentra- tions lower than 100 phi. These results demonstrate that the triphosphate forms of guanine nucleotides modulate [3H] LTD, specific binding, suggesting that a guanine nucleotide- binding protein (G protein, Ref. 35) may regulate [3H]LTD4

0 LP I 1 I I

0.1 I .o 10 100 1000 Nucleotide Concentration (I."

FIG. 3. Effects of nucleotides on ['H]LTD4 specific binding. RBL-1 cell membranes (35 pg) were incubated with 1 nM [3H]LTD4 and varying concentrations of GTP (O), Gpp(NH)p (x), Gpp(CHAp (O), ATP (a), App(NH)p (W), and App(CH2)p (A) under standard experimental conditions. The [3H]LTD4 specific binding, in the ab- sence of added nucleotide, was taken as 100%. Results from three separate experiments were averaged and shown. S.E., when not shown, was less than 5% of the mean.

120

- - t" I00

u" -

2 8 0 -

1 5 6 0 - d 5

c

0

'-9 I 40 - u " - 2 2 0 - 0

v)

1 I U 1 I O ;:; 6 5 4 3 2

-LOG CONCENTRATION (M)

FIG. 4. Effects of sulfhydryl reagents on ['HILTD, specific binding. RBL-1 cell membranes were incubated with sulfhydryl reagents a t 25 "C for 30 min. Binding of [3H]LTD4 to the modified membranes was initiated by addition of membranes to [3H]LTD, (1 nM) under standard conditions. The reagents studied include pHMB (0), DTNB (m), NEM (O), DTT (X), and DTE (0). Pretreatment of membranes with sulfhydryl reagents did not significantly affect the nonspecific binding of [3H]LTD4 to membranes. Control value was the specific binding of [3H]LTD, determined from membranes that were treated identically in the absence of sulfhydryl reagents.

binding to specific sites on RBL-1 cell membranes. The G protein regulation of [3H]LTD4 binding to guinea pig lung membrane receptors was quite similar to that in RBL-1 cell membranes.

Effects of Sulfhydryl Reagents on PHILTD, Specific Bind- ing-The effects of sulfhydryl directed reagents on [3H]LTD4 specific binding to RBL-1 membranes were evaluated and shown in Fig. 4. Binding of ['HILTD, was determined after the membranes were preincubated with these sulfhydryl re- agents for 30 min at 25 "C. The preincubation procedure had no significant effect on total or nonspecific binding of 13H] LTD,. pHMB induced a concentration-dependent inhibition of [3H]LTD4 specific binding with an ICso of 9.5 & 3 p ~ . Another alkylating reagent, NEM, also induced a concentra- tion-dependent inhibition of [3H]LTD4 specific binding with an ICso of 82 f 25 p ~ . The slope of the inhibition curve induced by NEM was shallow in comparison to that induced by pHMB. DTNB, a disulfide oxidizing reagent, inhibited [3H]LTD4 specific binding with an IC, of 10 p ~ . Reducing agents, such as DTT and DTE, were relatively ineffective in

4038 LTD, Receptor and Phosphatidylinositol Turnover

the inhibition of [3H]LTD, specific binding to RBL-1 cell membranes. The results from these studies suggested that free sulfhydryl groups on RBL-1 cell membranes were essen- tial for [3H]LTD4 specific binding, disruption of certain di- sulfide bonds by DTNB, but not by DTT or DTE, was also inhibitory to [3H]LTD4 specific binding.

Pharmacological Specificity of PHILTD, Binding to RBL-1 Cell Membranes-The binding affinities of several leuko- trienes, a series of agonist analogs, and several newly de- scribed receptor antagonists (36) were determined using a radioligand (['HILTD,) competition method. The results (Fig. 5A) showed that the agonists competed with [3H]LTD4 in binding to the specific sites in a concentration-dependent fashion. The Ki values for LTD,, LTC,, LTE,, (5R,6S)-LTD4, LTD,, and desamino-LTEl were 1.2, 4.2, 6.2, 83, 33, and 17 nM, respectively. LTB, was a weak competitor with a Ki of 10.5 FM. These results demonstrate that the LTD and LTE type agonists compete with [3H]LTD4 for binding to RBL-1 cell membrane-specific sites with stereoselectivity and speci- ficity. The binding affinity of LTC, under standard competi- tion binding conditions was 4.2 -t- 0.8 nM. However, it is possible that under these conditions the LTC, might have been metabolized to LTD, and LTE,. Thus the true Ki for LTC, has not been accurately determined (13, 37). Recent

120 A 1

5 4 -LOG CONCENTRATION (MI

120

-LOG CONCENTRATION (M)

FIG. 5. Competition of ['HILTD., specific binding by leuko- triene analogs. A, varying concentrations of LTD, (01, LTC4 (01, LTEl (A), desamino-LTE, (O), (5R,6S)-LTD4 (m), and LTB4 (X) were incubated with [3H]LTD4 and RBL-1 cell membranes, under standard conditions as described under "Methods," to determine the competition binding of these leukotriene agonist analogs to the [3H] LTD4 specific sites. B, the competitive binding activity of leukotriene antagonists, SKF 104353 (O), SKF 103944 (O), SKF 104373 (A), 2- nor-LTD1 (0), SKF 102922 (m), and FPL 55712 (X) was determined under identical conditions. The control value (100%) was defined as the specific binding of [3H]LTD, in the absence of these analogs. Values are the mean * S.E. taken from the average of three experi- ments.

TABLE I Relative effects of bukotrienes, agonists, and antagonist analogs on

radioligand competition activity and phosphatidylinositol metabolism X , values were determined by radioligand competition studies as

described in the legend to Fig. 5 and expressed as mean f S.E. from three experiments. ECM values are the concentration of agonist required to obtain 50% of its maximal stimulation. Values are the mean & S.E. of three experiments.

(3H]LTD, binding, K, PI, ECm

Agonists

LTC,

desamino-LTE,

homo-LTD, LTD,

(6R,7S)-homo-LTD1 LTB,

SKF 103944 SKF 104353 SKF 104373

SKF 102922 SKF 101132 (2-nor-LTDJ

FPL 55712 SKF 102081

LTD,

LTEs

(6R,GS)-LTD,

(5R,6S)-LTD,

Antagonists

nM

1.2 k 0.3 4.2 f 0.8 6.2 f 1.1 17 f 2 83 f 15 48 f 3 33 f 5 95 f 21

225 f 31 10,500 & 725

1 1 k 3 7.5 f 1.5

295 f 21 570 f 36

1,800 +- 85 3,250 +- 240 1.090 +- 105

nM

ND" 5 + - 4

12 f 8 150 f 30 280 f 30 620 f 50 380 & 45 850 & 180

1,200 f 200 ND

a ND, not determined.

studies utilizing 20 mM serine borate to inhibit y-glutamyl transpeptidase and therefore the conversion of LTC, to LTD, showed LTC, to be considerably weaker as a competitor for [3H]LTD, binding. The Ki in the presence of serine borate was 45 nM. The rank order potency of these agonists in binding to RBL-1 cell membrane-specific sites was equivalent to that determined from the guinea pig lung LTD4-receptor binding assay (1, 13) or from the guinea pig tracheal (or lung) smooth muscle contraction assay (13, 19-23).

Recently reported LTD, receptor antagonists3 competed with [3H]LTD4 for binding to RBL-1 membrane receptors (Fig. 5B; Ref. 36,46). SKF 103944 is a racemic mixture of an active enantiomer SKF 104353 and a less active enantiomer SKF 104373. The Ki values were 11 & 3,7.5 -+ 1.5, and 295 k 21 nM, respectively (Fig. 5B, Table I). FPL 55712 is a SRS-A functional antagonist that has a KL of 3.25 @M. Other antag- onists showed varying degrees of competition in binding to [3H]LTD4 specific sites. These results showed that the recep- tor antagonists bind to the RBL-1 LTD,-specific sites with correct stereoselectivity and specificity. With the exception of FPL 55712, the antagonist activities of these compounds (against LTD,-induced guinea pig tracheal smooth muscle contraction) correlated directly with the binding affinities of these analogs (13) in the RBL-1 membrane [3H]LTD4 specific binding assay (Table I).

LTD,-induced Phosphatidylinositol Hydrolysis in RBL-1 Cells-When my~-[~H]inositol-labeled RBL-1 cells were treated with LTD,, rapid hydrolysis of PI lipids was observed. The results (Fig. 6) demonstrate that LTD4-induced [3H]IP3 formation in RBL-1 cells was significantly higher than control release at 1-min postaddition. LTD, also induced formation of [3H]IP2 and [3H]IP1. The kinetic responses of the LTD,-

Gleason, J. G., Hall, R. F., Perchonock, C. D., Erhard, K. F., Quezee, J. S., Ku, T. W., Kondrad, K., McCarthy, M. E., Mong, S., Crooke, S. T., Chi Rosso, G., Wasserman, M. A., Torphy, T. J., Maccitelli, R. M., Tucker, S. S., and Vickery-Clark, L. (1987) manu- script submitted to J. Med. Chem.

LTDl Receptor and Phosphatidylinositol Turnover 4039

3, ,3

TIME ( M I N )

FIG. 6. Kinetic responses of LTD4-induced [SH]inositol phosphate formation in RBL-A cells. RBL-1 cells were labeled with 10 pCi/ml of my~-[~H]inositol in the presence of 10 mM LiCl as described. These cells were incubated with 1 p~ of LTD, from 0 to 30 min. The 3H-labeled inositol phosphates were extracted and quan- titated by the anion exchange method as described under "Methods." Formation of [3H]IP1 (A), [3H]IP2 (O), and [3H]IP3 (0) was deter- mined from triplicate samples of a representative experiment (of three). The difference of the LTD4-stimulated and the control level of inositol phosphate, a t each point of time, is defined as the net accumulation of [3H]inositol phosphates and is shown.

induced PI hydrolysis and formation of the inositol phos- phates were consistent with the currently accepted mecha- nism of receptor-mediated PI hydrolysis. Thus, agonists bound the membrane receptors and activated a G protein- coupled phospholipase C. The phospholipase C hydrolyzed PI to give rise to the intracellular messengers DAG and IP3 (27). We have measured the agonist induced accumulation of [3H] IP, in the presence of 10 mM LiC1, as an indication of LTD4- induced PI hydrolysis in RBL-1 cells.

Effects of Agonists on IPl Synthesis in RBL-1 Celk-LTD4, LTE,, and the other agonist analogs studied induced a con- centration-dependent synthesis of [3H]IPI in RBL-1 cells. The ECso of LTD, was 5 f 4 nM and the maximal stimulation occurred at 1 p~ (Fig. 7). The stereoisomer (5R,6S)-LTD, was 60-fold less active (EC, = 280 k 30 nM) yet achieved the maximum level of activity equivalent to that of LTD4 (a full agonist). LTE, was a potent agonist with an ECS0 of 12 +- 8 nM. Desami,no-LTEl, LTDI, and homo-LTDl also induced dose-dependent synthesis of 13H]IP1 in RBL-1 cells with ECsO values of 150 k 30, 380 45, and 620 ? 50 nM, respectively. The intrinsic activities (or the maximal efficacies) of these agonists, however, were less than those of LTD, or LTE,. Therefore, LTDI, desamino-LTEl, and homo-LTD4 are "par- tial agonists." The stereospecificity of the agonist-induced response was shown by the fact that (5R,6S)-LTD,, (5R,6S)- LTD1, and (6R,7S)-homo-LTD, were 60-, 3-, and 3.5-fold, respectively, less active than the corresponding natural form agonists in promoting [3H]IP1 formation. The rank order potency for the agonist-induced [3H]IPl synthesis was equiv- alent to that for the RBL-1 cell membrane [3H]LTD4 specific binding. A direct correlation of the membrane-specific site binding affinity to the [3H]IPl synthesis activity for LTD, and its agonist analogs was established (Fig. 7B) with a coefficient of correlation y = 0.95. Thus, these results clearly show that PI hydrolysis in RBL-1 cells induced by LTD, and related agonists was mediated via the membrane-specific re- ceptors for LTD,.

Effects of Antagonists on LTD4-induced IP1 Formation- Additional evidence that the LTD,-induced [3H]IPI formation was mediated via the membrane LTD, receptors was provided

-LOG CONCENTRATION (M)

- 1 0 9 8 7 6 1

RADIOLIGAND COMPETITION, -lop CKII

FIG. 7. Concentration-dependent ['H]IP1 formation in- duced by LTDl and analogs. A , RBL-1 cells were labeled with my~-[~H]inositol and then treated with increasing concentrations of LTD, (O), (5R,6S)-LTD4 (O), LTE, (m), LTD, (A), desamino-LTEi (A), and homo-LTD1 (0) for 20 min at 37 "C. The [3H]IP1 fraction was extracted and quantitated. The maximal net accumulation of [3H]IPl induced by 1 p~ LTD, was defined as 100% maxima1 response in each experiment. It was usually 125-150% of the basal (control) level of [3H]IPl. The extent of [3H]IP1 induced by each of the agonists was determined as net accumulation of ['HH]IP1 and expressed as the fraction to the maximal response, defined by 1 PM LTD, in each experiment. The results from three experiments were averaged and are shown. B , the ECS0 values of maximal stimulation for each of the leukotriene agonist analogs were obtained from the dose-response curves in A. The binding affinities of the leukotriene agonist analogs were taken from Fig. 5A and Table I. The symbols used are the same as in A, plus (5R,6S)-LTD1 (X) and (6R,7S)-homo-LTD1 (7).

,

- log CLT41 (M)

FIG. 8. Inhibition of LTD4-induced ['H]IPI formation by receptor antagonists. RBL-1 cells were labeled with rn~o-[~H] inositol as described under "Methods." 5 pM SKF 104353 (O), 5 pM SKF 104373 (A), or buffer (0, as controls) was added and preincu- bated at 37 "C for 10 min. These samples were then stimulated with increasing concentrations of LTD, in the presence of the antagonists for an additional 20 min. The net accumulation of [3H]IPl in each sample was quantitated. The receptor antagonists SKF 104353 and SKF 104373 did not affect the basal level of [3HJIP1 formation.

4040 LTD, Receptor and Phosphatidylinositol Turnover TABLE I1

Comparative properties of the 13HlLTD4 receptors on RBL-1, guinea pig lung, and h a r t membranes Cation modulation* Sensitivity to "SH reagents

Affinity it^ B-b K d b Divalent (Ca2+,

Guanine nucleotide Alkylating Reducing M2+,Mn2+) modulation (transi- (NEM, (DTT, (Na+) tion of affinity state) ~ H M B Y DTE)'

nM pnwl/ng protein RBL-1 0.9 k 0.2 0.80 +- 0.12 Stimulation Lung 0.2 2 0.05 1-2 Stimulation

Inhibition ICm = 16 f 3 p~ Sensitive Resistant

Heart 3.4 k 2.1 Inhibition ICW = 1.2 & 0.5 pM Sensitive Resistant

0.85 k 0.09 None None None Insensitive Sensitive 'Monovalent, divalent, guanine nucleotide, and sulfhydryl reagent data for lung and heart [3H]LTD4 specific

bAffnity and density of ['HILTD, binding were determined by computer-based, best fit analysis (30, 31) as binding were previously reported by this laboratory and others (1,9, 44).

described in Fig. 2, A and B. Data for lung and heart membranes as previously published (13).

from antagonist studies. The results, shown in Fig. 8, dem- onstrate that preincubation with SKF 104353 and SKF 104373 attenuated the dose-dependent I3H]IP1 synthesis in- duced by LTD,. At a concentration of 5 PM, SKF 104353 and SKF 104373 shifted the LTD, dose-response curves by 4.5 and 2.8 orders of magnitude, respectively, indicating that receptor antagonists stereospecifically inhibited LTD,-in- duced PI hydrolysis. Preliminary data (not shown) suggest that these receptor antagonists were competitive and revers- ible. Preliminary studies suggest that LTD, receptor-mediated PI hydrolysis in RBL-1 cells was not affected by preincuba- tion of the cells with islet activating protein (IAP) isolated from Bordetellapertussis (data not shown).

DISCUSSION

Since the initial reports identifying and characterizing LTD, receptors in guinea pig lung and heart membranes (1, 13, 14, 44), we have sought tissue culture cells that display membrane-bound receptors and receptor-mediated biochem- ical responses (16, 17). This work led to the recent discovery of the LTD, receptor-mediated transcriptional and transla- tional activation of a phosphatidylcholine-specific phospho- lipase A2 and phospholipase A,-activating protein (17, 18). Another major objective, to demonstrate that G protein- regulated [3H]LTD4-specific receptors were coupled to an intracellular second messenger system, in cultured cells, was unsuccessful until this report, primarily because of the diffi- culty in identifying LTD, receptors.

The current study has demonstrated, for the first time, high affinity, stereoselective, and specific membrane-bound LTD, receptors from clonally derived cells in tissue culture. Al- though RBL-1 cells are a homogeneous cell population of transformed cells and therefore, it is possible that the recep- tors in these cells may not be regulated in the same way as in normal cells, we believe that the cells provide a very useful model.

The LTD, and LTE, type agonists and antagonists com- peted with [3H]LTD4 for binding to the RBL-1 cell membrane receptors. The rank order potency of binding affinities of these agonist and antagonist analogs was equivalent to that defined in guinea pig lung membrane LTD, receptor radioli- gand studies and to that defined in the guinea pig tracheal smooth muscle contraction assay systems (19-23) and to that in human lung (8). Thus, it is clear from these studies that RBL-1 cell membrane LTD, receptors accurately reflect the biochemical and pharmacological specificity of the LTD, re- ceptors in guinea pig and human lung and guinea pig tracheal smooth muscle.

Using radioligand binding techniques, two types of LTD, receptors, lung and heart, have been reported and character- ized (13,14). The pharmacological characteristi!.s of the LTD,

receptors on RBL-1 cells resembled those found on guinea pig lung, but not heart membranes (Table 11). Lung and RBL- 1 cell membranes shared the common characteristics of so- dium ion and guanine nucleotide regulation of agonist bind- ing, enhancement of agonist binding by divalent cations, and sensitivity to sulfhydryl alkylating reagents. Thus, these re- sults suggest that the LTD, receptors on RBL-1 cell mem- branes are of the lung type LTD, receptor.

Binding of [3H]LTD4 to the membrane receptors was spe- cifically regulated by guanine nucleotides and Na+, suggesting that a guanine nucleotide-binding protein (possibly Gi or Go) was coupled to the LTD, receptors. These observations sug- gest that LTD, receptors are coupled via Gi protein to mem- brane-bound adenylate cyclase, therefore, activation of LTD, receptors may result in an inhibition of adenylate cyclase (35, 41). However, support for this G protein-regulated second messenger system has not been forthcoming and our labora- tory was unable to demonstrate inhibition of adenylate cyclase in broken cell preparations of guinea pig lung (9). Further- more, a recent study showed that intracellular CAMP levels were unchanged in trachea induced to contract with LTD, (42). Interestingly, intracellular cGMP levels were elevated with the LTD4-induced contraction. These negative results encouraged us to look for alternative signal transduction mechanisms for the LTD, receptor. Recently G proteins have been clearly shown to regulate membrane receptors that are linked to PI metabolism and the formation of IP3 and DAG (43).

In the current study, the LTD4-induced PI hydrolysis and formation of inositol phosphate was demonstrated. The for- mation of IP, (and possibly its precursor IP3) induced by LTD, and analogs was stereoselective, specific, and dependent upon the concentration of the agonists. In addition, the LTD,- induced formation of IP1 was stereoselectively inhibited by high affinity LTD, receptor antagonists. Furthermore, the receptor binding affinities of LTD, and agonist analogs cor- related directly with the agonist potencies in promoting the formation of IPl in RBL-1 cells. Thus, results from these studies clearly demonstrate that the increase in PI hydrolysis in RBL-1 cells induced by LTD, and its analogs was appar- ently mediated through LTD, receptors. The phospholipase C hydrolytic products of PI, i.e. IP3 and DAG, may serve as the intracellular messengers of LTD, receptors in RBL-1 cells.

Pretreatment of the RBL-1 cells with 250 ng/ml IAP for 2.5 h at 37 "C prevented the [32P]ADP-ribosylation of Gi (or Go) in a subsequent treatment with [32P]NAD and IAP. Cells untreated with IAP readily [32P]ADP ribosylated a 41,000-Da protein (Gi or Go). The results suggest that during the pre- treatment IAP enters the cell and Gi (or Go) is ADP-ribosyl- ated utilizing endogenous substrates. Under these conditions, the LTD, receptor-induced PI hydrolysis was not affected by

LTDl Receptor and Phosphatidylinositol Turnover 4041

IAP, suggesting that Gi- or Go-like proteins were not involved in the receptor-mediated signal transduction process in RBL- 1 cells? These results differ from results we have recently reported in bovine endothelial, CPAE, cells (45). Conse- quently some heterogeneity exists in the G proteins to which LTD, receptors are coupled.

The physiological and pharmacological effects of LTD, in smooth muscle, smooth muscle cells, endothelial cells, or other tissues vary. In general, they can be divided into cyclooxygen- ase dependent (such as phospholipase Az-activating protein, prostacyclin, and thromboxane B2 synthesis) or cyclooxygen- ase independent (such as smooth muscle contraction) pro- cesses. The concept of LTDl receptor-mediated PI hydrolysis and thus the formation of IPS (possibly inositol 1,4,5-tris- phosphate) and DAG, explains many specific biochemical activities in tissues and cells. This is because IP3 has been shown to mobilize Caz+ and DAG activates protein kinase C. Intracellular Ca2+ and the activated protein kinase C, either independently or synergistically, may activate a variety of biochemical processes ranging from: synthesis of phospholi- pase Az-activating protein with subsequent activation of phos- pholipase A2 (18), release of arachidonic acid, synthesis of thromboxane B2 and prostacyclin (16, 17, 19), mucous hy- persecretion (38), plasma extravasation, to smooth muscle contraction (19-24). The transcriptional and translational activation of phospholipase Az that we observed previously in bovine endothelial cells and murine smooth muscle cells oc- curred 3-5 min after LTD4 stimulation (18), thus suggesting that these effects are secondary effects of receptor activation and PI hydrolysis. Whether similar events occur in the RBL- 1 cell needs to be investigated.

Preliminary studies demonstrate that in RBL-1 cells, LTD, induces calcium mobilization detected by fura-2, the fluores- cent Ca2+ probe, in a dose-dependent manner: thus confirm- ing this scheme. The present study on PI metabolism leads us to propose that IP3 and DAG should be considered the intracellular messengers for LTD4 receptors in RBL-1 cells and possibly in other cells and tissues. Detailed characteri- zation of the kinetics and specificity of the LTD4-induced PI hydrolysis and Ca2+ mobilization responses in smooth muscle cells, endothelial cells, and tissues are in progress to further understand the biochemical and molecular pharmacology of

It is of interest to note that RBL-1 cells used in this study can mobilize large amounts of arachidonic acid and convert it into either the cyclooxygenase or the lipoxygenase metab- olites (25, 26, 39). RBL cells were used as a source for the purification of LTC4, LTD,, LTE, (24), and 5-lipoxygenase (39). RBL-1 cells when stimulated with calcium ionophore or immunological stimuli, release substantial amounts of LTC,, LTD,, LTB,, PGDz, 5-HETE, and 5,12-diHETES (40). Thus these cells can actively express the enzymes for the biosyn- thesis of prostaglandins and leukotrienes, the LTD, mem- brane receptors, and the signal transduction proteins and factors (current study). This observation suggests that LTD, biosynthesis, receptor regulation, and signal transduction pro- cesses may be regulated coordinately. Studies are in progress to better define these relationships.

LTD,.

S. Mong, H. Wu, and S. T. Crooke, unpublished data.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15. 16.

17.

18.

19.

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