[48]
GINKGOLIDESAS PAF ANTAGONISTS
433
nists. It has been used to study the membrane receptor regulation and desensitization. 12 Most recently, it has also been used, either in conjunction with, or in replacement of, the radioimmunoassay for the detection of sulfidopeptide leukotrienes in biological fluids (Mong et al., results not shown).
[48] G i n k g o l i d e s a n d P l a t e l e t - A c t i v a t i n g F a c t o r B i n d i n g Sites By D. J. HOSFORD, M. T. DOMINGO, P. E. CHABRIER, and
P. BRAQUET Introduction
Specific membrane receptors are assumed to mediate the various biological responses to platelet-activating factor (PAF), 1 the inflammatory autacoid produced by neutrophils, 2 eosinophils, 3 monocytes, 4 macrophages, 5 platelets, 6 and endothelial cells. 7 However, the failure to isolate and characterize the putative receptor leaves open the question of the nature and heterogenity of the binding site(s) in various cells and tissues. Some progress has been made in the formulation of a model of the PAF receptor by studies with various compounds that can specifically antagonize PAF-induced biological effects both in oitro and in oivo. 8"9 Among these compounds, the unique 20-carbon cage molecules named ginkgolides 1°'11 (Fig. 1) have been shown to specifically inhibit PAF-induced actions, such as platelet aggregation, hypotension, bronchoconstriction, i p. Braquet, L. Touqui, T. S. Shen, and B. B. Vargaftig, Pharmacol. Reo. 39, 97 (1987). 2 j. M. Lynch, G. Z. Lotner, S. J. Betz, and P. M. Henson, J. lmmunol. 123, 1219 (1979). 3 T. C. Lee, D. J. Lenihan, B. Malone, L. L. Roddy, and S. I. Wasserman, J. Biol. Chem. 259, 5530 (1984). 4 G. Camussi, M. Aglietta, R. Coda, F. Bussolino, W. Piacibeilo, and C. Tetta, Immunology 42, 191 (1981). 5 j. M. Mencia-Huerta and J. Benveniste, Eur. J. lmmunol. 9, 409 (1979). 6 M. Chignard, J. P. Le Couedic, M. Tenc6, B. B. Vargaftig, and J. Benveniste, Nature (London) 275, 799 (1979). 7 S. M. Prescott, G. A. Zimmerman, and T. M. Mclntyre, Med. Sci. 81, 3534 (1984). 8 p. Braquet, P. E. Chabrier, and J. M. Mencia-Huerta, Adv. Inflammation Res. 12, 135 (1987). 9 D. Hosford, J. M. Mencia-Huerta, C. Page, and P. Braquet, Phytother. Res. 2, 1 (1988). to p. Braquet, B. Spinnewyn, M. Braquet, R. H. Bourgain, J. E. Taylor, A. Etienne, and K. Drieu, Blood Vessels 16, 559 (1985). 11 p. Braquet, Drug Future 12, 643 (1987).
METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
434
PHARMACOLOGY: ANTAGONIST/SYNTHESIS INHIBITORS
GinkgolidIHB e nomenclature R1 A BN 52020 OH B BN 52021 OH C J
M
BN 52022 BN 52024 BN 52023
OH OH H
R2 H OH OH H OH
R3 H H OH OH OH
[48]
0 o HO--q .H
/
r'Ht.u 19+ /
18
~
20
~,s~~I-H
R,
0 +` ' ~ ' 0 FIo. 1. Structure of ginkgolides A, B, C, J, and M.
and to exert a protective effect in experimentally induced diseases such as brain ischemia ~2,13and eye inflammation. 14At the present time, five ginkgolides (A, B, C, M, and J) have been identified. Coded BN 52020, BN 52021, BN 52022, BN 52023, and BN 52024, respectively, by the Institut Henri Beaufour, studies with ginkgolides and other PAF antagonists have implicated PAF in a wide range of pathologies including asthma, shock, ischemia, and graft rejection. 1~,]5 The development of these compounds into potential therapeutic agents is currently being assessed in clinical trials, t6 The interaction of ginkgolides with PAF binding sites has been investigated in various tissues by binding studies using tritiated PAF ([3H]PAF), 12 B. Spinnewyn, N. Blavet, F. Clostre, N. G. Bazan, and P. Braquet, Prostaglandins 34, 333 (1987). 13 p. Braquet, M. Paubert-Braquet, M. Koltai, R. Bourgain, F. Bussolino, and D. Hosford, Trends Pharmacol. Sci. 10, 23 (1989). 14 N. L. J. Verbeij and N. J. van Haerigen, in "Ginkgolides: Chemistry, Biology, Pharmacology and Clinical Perspectives" (P. Braquet, ed.), Vol. 1, pp. 749-758. Prous Science, Barcelona, 1988. 15 p. Braquet (ed.), "The Ginkgolides: Chemistry, Biology, Pharmacology and Clinical Perspectives." Vol. 1. Prous Science, Barcelona, 1988. 16 D. Hosford and P. Braquet, Prog. Med. Chem. 27, 325-379 (1990).
[48]
GINKGOLIDES AS P A F ANTAGONISTS
435
since high specific activity radiolabeled ginkgolides are not currently available. Ginkgolide binding has been measured on membranes from platelet or tissue homogenates, as a function of time and concentration, in order to further elucidate the nature, location, and physiological relevance of PAF receptor sites in these systems. This chapter examines ginkgolide interactions with PAF binding sites in three different preparations: rabbit platelet membranes, gerbil brain tissue, and rabbit iris and ciliary body. Methods and Materials Biologically relevant studies on [3H]PAF binding require investigation of the concentration range 10-11 to 10 -6 M, in the presence of 0.025% bovine serum albumin (BSA), the latter agent acting as a carrier for the PAF. In our investigations the bound ligand is separated from the free ligand by filtration procedures and characteristics of PAF binding such as dissociation constant (KD) and number of sites (Bmax) are obtained from concentration-dependent experiments, subjected to Scatchard analysis, and plotted as bound/free versus bound (B/F vs B). The equilibrium dissociation constant (Ki) of ginkgolides on PAF binding sites in the various tissues is derived from displacement studies and calculated according to the Cheng-Prusoff equation:
g~--
IC50 1 + [PAF]/KD
where [PAF] is the molar concentration of the labeled PAF and KA is the PAF dissociation constant. Rabbit Platelet Membranes In this study, the effect of the ginkgolides on [3H]PAF binding is evaluated using ginkgolide B (BN 52021), ginkgolide A (BN 52020), ginkgolide C (BN 52022), and a mixture (BN 52063) of the three ginkgolides B, A, and C in the molar ratio 2 : 2 : 1. Chemicals. Synthetic [3H]PAF (specific activity: 59.5 Ci/mmol) in ethanol solution is purchased from New England Nuclear (Boston, MA). Unlabeled PAF and lyso-PAF (Calbiochem, Switzerland), are solubilized in ethanol solution and stored at - 8 0 °. BN 52021, BN 52020, BN 52022, and BN 52063 are solubilized in dimethyl sulfoxide (DMSO). Platelet Membrane Preparation. Rabbit whole blood (6 volumes) is drawn from the central ear artery into I volume of ACD solution (citric acid, 1.4 g, sodium citrate, 2.5 g, and dextrose, 2 g per 100 ml of water) and centrifuged at 150 g for 15 min at 4°. The platelet-rich plasma (PRP) is
436
PHARMACOLOGY: ANTAGONIST/SYNTHESIS INHIBITORS
[48]
carefully removed and centrifuged for an additional 15 min at 1000 g at 4°. The platelet pellet is then washed three times, twice in 10 mM Tris-HC1 buffer, pH 7.4, containing NaCI 150 mM, MgCI2 5 mM, and EDTA 2 mM and, finally, in the same buffer but without sodium. The platelet pellet is resuspended in this latter buffer, quickly frozen in liquid nitrogen, and slowly thawed at room temperature. The freeze/thaw cycle is repeated at least three times as described by Shen et al. 17The lysed platelets are then centrifuged at 100,000 g for 30 min at 4° in a Beckman L8.55 ultracentrifuge (rotor 50.2 Ti). The platelet membrane homogenate is stored at - 8 0 ° and used within 2 weeks. Protein content is determined by the Lowry method TMusing BSA as standard. Binding Assay. Sixty to 100/.~gof membrane protein is added to plastic tubes containing 1 nM [3H]PAF and 1 ml of 10 mM Tris-HC1 buffer, pH 7.0, containing 0.025% BSA and incubated with or without unlabeled PAF or ginkgolides. The incubation is carried out for 90 min at 0°. The bound [3H]PAF is separated from the free [3H]PAF by immediate filtration through Whatman GF/C glass fiber filters under a Brandel vacuum system (Brandel Biomedical Research, Gaithersburg, MD). Filters are washed three times with 5 ml of ice-cold buffer. The filters are then placed in polyethylene vials containing 10 ml of liquid scintillation fluid (Instagel, Packard, Paris) and the radioactivity measured by an LKB/3-counter with 45% efficiency. The nonspecific binding is determined in the presence of 10 -6 M of unlabeled PAF. The specific binding is calculated by subtracting nonspecific from total binding. The inhibition by ginkgolides of the specific [3H]PAF binding is expressed as percentage inhibition (% I) using the equation: [3H]PAF (tb) - [3H]PAF bound in presence of ginkgolides %1= x 100 [3H]PAF specifically bound where [3H]PAF (tb) is [aH]PAF totally bound. Gerbil Brain Tissue
The involvement of PAF in cerebral ischemia has been suggested by studies showing that PAF antagonists including BN 52021, kadsurenone, and brotizolam improve cerebral metabolism and protect the brain in the postischemic phase induced by bilateral carotid ligature in the gerbil.12,13 Thus, we investigated the existence of specific [3H]PAF binding sites in gerbil brain membrane homogenate. 17 T. Y. Shen, S. B. Hwang, M. N. Chang, T. W. Doebber, M. H. T. Lain, M. S. Wu, X. Wang, G. Q. Han, and R. Z. Li, Proc. Natl. Acad. Sci. U.S.A. 82, 672 (1985). la O. H. Lowry, N. J. Rosebrough, A. L. Fan-, and J. Randall, J. Biol. Chem. 193, 265 (1951).
[48]
GINKGOLIDES AS P A F ANTAGONISTS
437
Brain Tissue Preparation. Male gerbils Meriones unguiculatus (6070 g) are purchased from Tumblebrook farm (West-Brookfield, UK). Animals are allowed free access to food and water prior to experimentation. Gerbils are killed by decapitation and the brain quickly dissected out and placed into ice-cold 50 mM Tris-HCl buffer, pH 7.4, containing MgCI2 10 mM, EDTA 2 mM, Trasylol (aprotinin) 50 IU/ml, and PMSF 1 × 10 - 4 M. In some experiments the brain is perfused using the homogenizing buffer, through the left ventricle and under anesthesia. Brain tissue is then washed and homogenized in the same buffer using a Dounce homogenizer at 4°. The homogenates are centrifuged at 1000 g for 15 min at 4°. The microsome, mitochondria, and synaptosome-rich supernatant is harvested and recentrifuged at 10,000 g for 30 min. The P2 pellet is then resuspended in the same buffer, assayed for protein content, and used for the binding assay. Protein content is determined by the Bradford method 19 using Bio-Rad protein assay reagent and BSA as the standard. The brain tissue preparation is used within the same day as isolation. Binding Assay. Protein, 150-350/xg, from the P2 preparation is added to plastic tubes containing [3H]PAF (3 nM for kinetic and displacement studies) and 1 ml 10 mM Tris-HCl buffer, pH 7.0, containing MgCI2 5 mM, Trasylol (50 IU/ml), PMSF 1 x 10-4 M, and 0.025% BSA without or with 1000-fold unlabeled PAF (for nonspecific binding). The reaction is carried out at 25° for 30 min. The unbound PAF is separated from the bound by immediate filtration of the reaction mixture through Whatman GF/B glass fiber filters presoaked in the binding buffer. Tubes and filters are washed three times with 5 ml of precooled binding buffer. The filters are then placed into polyethylene vials containing 10 ml of liquid scintillation fluid (Instagel, Packard) and the radioactivity measured in a LKB/3-counter with 60% efficiency. The PAF specific binding is calculated by subtracting the nonspecific binding (binding in presence of 10 - 6 M unlabeled PAF) from the total binding. Binding data are analyzed by the Scatchard method using the computerized program of Vindimian et al. 2° The specificity of binding is investigated by competition experiments using lyso-PAF, unlabeled PAF, and BN 52021 to displace 1 nM [3H]PAF. Rabbit Iris and Ciliary Body The demonstration that BN 52021 exerts significant protective effects in inflammatory eye diseases, 14 suggests the involvement of PAF in such conditions. In order to investigate the possible presence of PAF receptor 19 M. Bradford, Anal. Biochem. 72, 248 (1976). 20 E. Vindimian, C. Robaut, and G. Fillion, Appl. Biochem. 5, 261 (1983).
438
PHARMACOLOGY; ANTAGONIST/SYNTHESIS INHIBITORS
[48]
sites in eye tissue, we carried out binding studies on rabbit iris and ciliary body. Tissue Preparation. Pigmented rabbits (either sex) are killed by an overdose of pentobarbital 5 min after injection of heparin (1000 IU per rabbit). The eyes are enucleated and placed into precooled 50 mM Tris buffer, pH 7.4, containing 10 mM MgCI2,2 mM EDTA, 50 IU Trasylol per ml, and 1 × 10-4 M PMSF. The eyes are perfused arterially in order to remove the blood from the anterior segment. The iris and ciliary body are carefully dissected out, homogenized in a Dounce homogenizer, the homogenates filtered through two layers of gauze, and centrifugated twice at 40,000 g for 20 min at 4 °. The pellets are finally resuspended in the homogenization buffer and analyzed for protein content by the Bradford method 19 using Bio-Rad protein assay reagent and BSA as standard. Binding Assay. Inhibition of [3H]PAF binding to iris and ciliary homogenates is performed in a final volume of 1 ml buffer, using plastic tubes that contain [3H]PAF (10 -9 M), unlabeled PAF (1/zM, for nonspecific binding), lyso PAF or BN 52021, and 40 to 70/~g of protein diluted in the binding buffer. The binding buffer (pH 7.0) contains Tris I0 mM, MgCI2 5 mM, Trasylol 50 IU per ml, PMSF 0.1 mM, and BSA 0.025%. After 40-min incubation at 25 °, the unbound PAF is separated from the bound by filtration through Whatman GF/B glass fiber filters presoaked for 24 hr in the binding buffer and using a Brandel vacuum system. The assay tubes and filters are washed three times with 3 ml of precooled binding buffer and the filters placed in polyethylene vials containing 10 ml of liquid scintillation fluid (Instagel, Packard). The radioactivity is measured in a LKB/3 counter with 45% efficiency. The percentage inhibition is determined as previously described. In some experiments, stability of [3H]PAF is analyzed after a 40-min incubation by extraction from the reaction mixture, using the solvent system, chloroform: methanol (2 : 1, v/v) and separation on TLC silica plates using a chloroform : methanol : ammonia (70 : 35 : 7, v/v/v) system. In our binding conditions, intact [3H]PAF represents 93% of the total radioactivity recovered from the plates. Results
Rabbit Platelet Membranes The binding of [3H]PAF to rabbit platelet membrane preparations is found to be specific, saturable, time-dependent, reversible, and of high affinity. The specific binding is a linear function of membrane protein concentration from 60 to 120/.~g protein. Experiments using increasing concentrations of [3H]PAF from 1 to 8 nM demonstrate a specific and
[48]
GINKGOLIDESASPAF ANTAGONISTS 0 •
~
•
439
•
\. -~
o
40-
o 6070-
90100 10-6
,
,
,
10-7
10-8
10-9
Antagonist concentration (M) FIG. 2. Inhibition of [3H]PAF (1 nM) specific binding to rabbit platelet membrane by BN 52020 (O), BN 52021 (11), BN 52022 (&), and BN 52063 (D).
saturable binding, specific [3H]PAF binding accounting for about 45% of the total. Scatchard analysis of the binding data reveals a single class of binding sites with a KD of 3 nM and a Bm~, of 2.25 ± 0.2 pmol/mg protein. The displacement of [3H]PAF (1 nM) binding in rabbit platelet membranes by BN 52020, BN 52021, BN 52022, and BN 52063 is shown in Fig. 2. All these compounds inhibit [3H]PAF binding in a dose-dependent manner. Maximum inhibition of [3H]PAF binding by the ginkgolides is observed with BN 52021 with an IC50 value of 2.5 × 10 - 7 M. BN 52063, BN 52020, and BN 52022 exhibit ICs0 values of 7 × 10-7 M, 8.3 × 10 - 7 M , and 6 × 10 - 6 M , respectively. In comparison, the concentration required to inhibit specific [3H]PAF binding by 50% is 2~× 10 - 9 M for unlabeled PAF, whereas lyso-PAF is unable to displace [3H]PAF binding below concentrations of 10-5 M. The specificity of the ginkgolide compounds for PAF binding sites is also assessed in experiments using other radioligands. BN 52021 and the other ginkgolides (10-8-I 0-5 M ) are found to be totally inactive in inhibiting the binding of the appropriate 3H-labeled ligands to al-, a2-, fix-, and fl2-adrenergic receptors, H1 and H2 histamine receptors, 5HTI and 5HT2 serotoninergic receptors, /x, 8, and X opiate receptors, benzodiazepine, and muscarinic and imipraminic receptors.
440
PHARMACOLOGY: ANTAGONIST/SYNTHESIS INHIBITORS
[48]
As reported previously, ~1 the binding of [3H]-PAF is modulated by the presence of mono- and divalent cations. A decrease of the binding is observed with the monovalent cations Li + and Na ÷, whereas K + and divalent cations such as Mg 2+ and Ca 2÷ enhanced the specific [3H]PAF binding (data not shown). In this respect, the inhibition of [3H]PAF binding by BN 52021, the most effective ginkgolide, is investigated in the presence of high concentrations of NaCI or MgC12. In the presence of 150 mM NaCI, the ICs0 of the displacement curve is shifted to 4.8 x 10-8 M, while with 5 mM MgCI2 the displacement curve is shifted to the right with an increase of the IC5o value to 5 × 10 - 7 M.
Gerbil Brain Tissue The specific binding is saturable, representing 20-25% of the total binding and constituting about 5% of the total radioactivity added to the incubation medium. Scatchard plot of the binding data from saturation and competition experiments is curvilinear and could be described by a twocomponent binding model on computerized analysis, indicating the existence of two apparent classes of binding sites (Fig. 3). The first population of PAF recognition sites exhibits a Ko, of 3.66 -+ 0.56 nM and a Bma,,, of 0.83 --- 0.23 pmol/mg of membrane protein, while the second population has a KD2 of 20.4 - 0.56 nM and a Bm~2 of 1.1 --- 0.32 pmol/mg of membrane protein (n = 5). The association of [3H]PAF to gerbil brain homogenate at 25 ° reaches equilibrium within 15 min and remains stable for at least 60 rain. The specificity of binding is established by displacing 1 nM [3H]PAF by lyso-PAF, unlabeled PAF, and by BN 52021 in competition experiments. Lyso-PAF is ineffective up to 10-5 M in competition with [3H]PAF for the binding sites. Unlabeled PAF ( 1 0 - 1 ° - 1 0 - 6 M ) and BN 52021 (10 -1°10-5 M) dose-dependently inhibits the specific binding of [3H]PAF to brain membrane preparations with IC50 values of 2.5 × 10 - 9 M and 3.5 x I0 -s M, respectively (Fig. 4). Interestingly, in this tissue, BN 52021 fails to totally inhibit the [3H]PAF binding. We further evaluated whether the binding of [3H]PAF is associated to discrete brain areas by studying the distribution of [3H]PAF binding in various regions of the gerbil brain. The maximum amount of specific binding is found in the midbrain and the hippocampus, with less binding being apparent in the olfactory bulb, frontal cortex, and cerebellum.
Rabbit Iris and Ciliary Body Specific [3H]PAF binding is found in cornea, iris, and ciliary body and is characterized in the latter two. The specific binding represents 20% of 2~ S. B. Hwang, M. H. Lam, and S. S. Pong, J. Biol. Chem. 261, 13720 (1986).
[48]
GINKGOLIDES AS P A F ANTAGONISTS
441
40-
36-~
Kdl
32" , . , ~ 28
\
= 3.66+ 0.92 IO~M
Bmax 1 = 0.83 + 0.30 pmol / mg Kd =20,4 + 0.5610 ~ M
e~
2
;
,1
+_
4 0
i
0
,
.
=
=
•
•
=
,
•
=
i
|
•
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4
Bound (nM) FIG. 3. Scatchard analysis of [3H]PAF binding in gerbil brain homogenate. The experiments are performed at 25° and each point is the average of triplicate determinations ( n = 5).
the total binding and is linear over the concentration range assayed, up to 90/zg of protein per tube. For kinetic and competition studies, [3H]PAF is used at a concentration of 3 nM. At 25° the association reaches equilibrium within the first 30 min of incubation and remains stable up to 60 min with this latter concentration. An excess of unlabeled PAF added to the reaction mixture at 25 min demonstrates a reversibility of 50% within 15 min. No reversibility is observed at 45 min, even after addition of an excess of unlabeled PAF. Increasing concentrations of labeled PAF over the range of 1 to 14 nM exhibit saturable binding and show a two-step saturation isotherm. The first maximum is reached at 4-5 nM and Scatchard analysis derived from both saturation and competition studies demonstrates a curvilinear repartition of the points suggesting two subtype components (Fig. 5). For the iris, a high-affinity component with a KD, of 4.9 - 0.47 nM and Bmaxt of 3.17 + 0.5 pmol/mg protein and a low-affinity component with a K,t2 of 11.6 -+ 0.33 nM and Bmax2 of 12.46 +- 2.3 pmol/mg is observed. Similar values are found for ciliary body: Knt =" 5.7 +-- 0.09 nM; Bronx, = 3.41 -+ 1 pmol/mg and KD2 = 24.4 +- 0.91 nM; Bmax~--- 16.6 -+ 0.51 pmol/mg. In our study, the specificity of PAF binding to iris and ciliary body is confirmed by inhibition of the [3H]PAF binding in the presence of un-
442
PHARMACOLOGY: ANTAGONIST/SYNTHESIS INHIBITORS
[48]
0"
10.
20. = 30. .o ..¢ r-
40-
•
50-
•
¢1 o 60.
•
70-
80-
~
90. 100 10-10
,
i
.
•
|
10-9
lO-S
10-7
10-6
10-5
(MI FIG. 4. Inhibition of [3H]PAF (1 nM) binding to gerbil brain homogenate by unlabeled PAF ( 0 ; 10-1°-10 -6 M, C5o = 5 x 10 -8 M ) and BN 52021 ( 1 ; 10-1°-10 -5 M). Each point is
the mean of triplicate determinations(n = 3).
labeled PAF, lyso-PAF or BN 52021. Displacement of the binding by unlabeled PAF demonstrates a biphasic inhibition curve. Lyso-PAF at a concentration below 10 -6 M fails to inhibit the binding. The inhibition curve obtained with BN 52021 differs from that observed with unlabeled PAF and is only partial (Fig. 6). The maximum inhibition obtained is about 60% and appears to be associated with only one component of PAF displacement curve. Discussion Ginkgolides are specific PAF antagonists isolated from the roots and leaves of the fossil tree Ginkgo biloba. Extracts of this tree have been used by the Chinese for some 5000 years to alleviate asthma and other inflammatory conditions, the beneficial effects of the extract being largely attributable to the PAF antagonizing effects of its constituents. The chemistry, biology, and pharmacology of these compounds have been extensively
[48]
GINKGOUDZS AS PAF ANTAGONISTS
443
80-
70-
60-
'7
s0.
o × LL
40-
m
30-
20-
10"
O" 0
0.2
0.4
0.6
0.8
1
[3H]PAF bound (nM) FIG. 5. Scatchard analysis of [3H]PAFbinding to iris homogenate in 10 mM Tris buffer, pH 7, containing 5 mM MgC12,PMSF 1 x 10.4 M, Trasylo150IU/m|, and BSA 0.025%at 25°. Each point is the average of triplicate determinations ( n = 4). reviewed in various recent publications. ~l '15 The results presented here demonstrate the presence o f specific binding sites for [3H]PAF in rabbit platelet membranes, gerbil brain tissue, and rabbit iris and ciliary body, the binding of the mediator in these systems being specifically antagonized by B N 52021 and other ginkgolides. In rabbit platelet membranes, the ginkgolides dose-dependently antagonize specific P A F binding, with ICso values of 2.5 × 10 -7 M, 8.3 x 10 -7 M, and 6 × 10 -6 M for B N 52021, B N 52020, and B N 52022, respectively. The inhibition o f P A F recognition sites is highly specific since the ginkgolides do not interact with any other known receptors (see section on Results). Characterized by the presence of two hydroxyl groups on car-
444
[48]
PHARMACOLOGY" ANTAGONIST/SYNTHESIS INHIBITORS
O"
20 r-
.o ,.~_ 40 e'e-
~
6o
80
100 I 10-10
, 10-9
10-8
10-7
10-6
10-5
Antagonist concentration (M) FIG. 6. Inhibition of [3H]PAF (1-3 riM) binding to iris homogenate by unlabeled PAF ( O - - O ) , BN 52021 (11--11), and lyso-PAF ( • determinations (n = 4).
• ) . Each point is the average of triplicate
bons 1 and 3, BN 52021 is the most powerful antagonist. The loss of the hydroxyl group on carbon 1 in BN 52020 and the presence of a hydroxyl group on carbon 7, in the a position of the lipophilic tert-butyl moiety in BN 52022 render these compounds less active. Studies reported elsewhere ~~have shown BN 52024 to possess an IC50 value of 5.4 x 10-5 M in this assay, the loss of the hydroxyl group on carbon 1 further decreasing the activity in comparison to BN 52022. These data corroborate physicochemical findings showing that as the ginkgolide becomes less polar, its PAF antagonistic activity increases. 1~ These binding studies corroborate results obtained for the inhibition of PAF-induced platelet aggregation, where BN 52021 is the most effective antagonist followed by BN 52020 and 52022.tl Moreover, the affinity of BN 52021 for PAF receptor sites is altered by Na + and Mg 2÷. Indeed, the state of affinity of PAF receptor appears to be low in the presence of Na ÷ and high in the presence of Mg 2÷.21 Accordingly the ability of BN 52021 to displace specific [3H]PAF binding is dependent on the ionic conditions. This phenomenon has also been observed with other PAF antagonists, such as CV-3988, ONO 6240, and L-652,569, but, not with kadsurenone which binds equally well to the PAF receptor at both the high- and lowaffinity state. 21
[48]
~IrqKGOLXI)ESAS PAF ANTAGONISTS
445
The finding that BN 52021 inhibits specific [3H]PAF in gerbil brain tissue may account for the protective effect of the compound in various models of cerebral ischemia. 12'13 The direct radioligand binding studies reported here indicate that the PAF recognition sites are most likely located on brain cells and not on blood elements since PAF binding characteristics were similar whether or not the brain was perfused and did not correlate with the level of cerebral vasculature. Scatchard analysis of the binding data indicates the existence of two populations of PAF binding sites in gerbil brain while other tissues such as platelets (see section on Methods and Materials) and lung 22 exhibit only one class of site. Furthermore, the [3H]PAF specific binding to whole brain homogenate is totally displaced by unlabeled PAF but only partially inhibited by BN 52021. Thus, it is possible that BN 52021 and other PAF antagonists interact only with one site, although the present data do not indicate which receptor population is involved. Similar to the case with brain tissue and that recently reported for rat retina, 23 two populations of PAF binding sites were also detected in iris and ciliary body. The specific binding in these latter tissues (20% of the total binding) is low as compared to that obtained, in the same buffer conditions, for platelets (70-80%), but is similar to the 20-25% found in brain tissue or to the 30-40% in lung tissue. In rabbit iris and ciliary body, specific PAF binding exhibited a twostep saturation isotherm, suggesting heterogenous binding characteristics. The displacement by unlabeled PAF exhibited a biphasic curve, which could be described by a two-component computerized analysis. Inhibition of [3H]PAF binding by BN 52021 was monophasic, partial (60%), and only involved the first step of the PAF inhibition curve. Despite this lack of total inhibition of PAF binding, BN 52021 significantly inhibits PAF-induced plasma leakage and edema formation in the rat retina 24 and the acute rise in intraocular pressure (IOP) caused by topical administration of the mediator in the rabbit eye. 25 In addition, the ginkgolide also abolishes the deleterious effects of PAF on the electroretinogram of rat eyes in oitro, 26 prevents cell infiltration and edema formation provoked by immune kerati-
22 S. B. Hwang, M. H. Lam, and T. Y. Shen, Biochem. Biophys. Res. Comrnun. 128, 972 (1985). z3 M. Doly, B. Bonhomme, P. Braquet, P. E. Chabrier, and G. Meyniel, lmmunopharmacology 13, 189 (1987). 24 M. Doly, P. Braquet, K. Drieu, B. Bonhomme, and M. T. Droy, in "Colour Vision Deficiencies" (G. Verrist, ed.), Vol. 8, p. 515. W. Junk, Dordrecht, 1987. 25 p. Braquet, R. F. Vidal, M. Paubert-Braquet, H. Hamard, and B. B. Vargaftig, Agents Actions 15, 82 (1984). 26 M. Doly, P. Braquet, B. Bonhomme, and G. Meyniel, Int. J. Tissue React. 9, 33 (1987).
446
PHARMACOLOGY: ANTAGONIST/SYNTHESIS 1NHIBITORS
[49]
tis in the cornea of the rabbit eye,14 and inhibits the increased IOP and breakdown of the blood-aqueous barrier after laser irradiation of the rabbit iris. 27 In conclusion, the identification of specific PAF binding sites in various cells and tissues and their blockade by ginkgolides explains the beneficial effects of ginkgolides in numerous experimental diseases and highlights their potential as valuable therapeutic agents for man. 27 N. L. Verbeij, J. L. van Delft, N. J. van Haeringen, and P. Braquet, in "The Ginkgolides: Chemistry, Biology, Pharmacologyand ClinicalPerspectives" (P. Braquet, ed.), Vol. 2. Prous Science, Barcelona, 1990, in press.
[49] K a d s u r e n o n e a n d O t h e r R e l a t e d L i g n a n s as A n t a g o n i s t s of Platelet-Activating Factor Receptor B y T. Y. SrIEN and I. M. HUSSAINI
Introduction Platelet-activating factor (PAF) is a highly potent ether-linked phospholipid (1-O-alkyl-2-acetyl-sn-glycerol-3-phosphorylcholine) which activates platelets as well as modulates the function of leukocytes and other target cells. Interaction of P A F with a specific membrane recognition site coupled to phosphatidylinositol metabolism produces the biological actions of the phospholipid. 1-3 A number of specific reversible and irreversible P A F receptor antagonists have been developed using both in vitro (platelet aggregation; binding studies) and in vivo (hypotension; bronchoconstriction) screening methods. Some of these antagonists include PAF analogs, e.g., CV-3989, 4 ONO-6248, 5 SR-63441, 6 natural products, l F. Snyder, Med. Res. Rev. 5, 107 (1985). 2 D. J. Hanahan, Annu. Rev. Biochem. 55, 483 (1986). 3 p. Braquet, T. Y. Shen, L. Touqui, and B. B. Vargaftig, Pharmacol. Rev. 39, 97 (1987).
4 Z. Terrashita, S. Tsuchima, Y. Yoshiota, J. Normura, Y. Inada, and S. Nishikoda, Life Sci. 32, 1975 (1983). 5 C. M. Winslow, H. U. Gubler, A. K. Delillo, F. J. Davies, G. E. Frisch, J. C. Tomesch, and R. N. Saunders, Proc. 2nd Intern. Conf. Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Gatlinburg, Tennessee, p. 33 (1986). 6 T. Mitamoto, H. Ohno, T. Tano, T. Okada, N. Hamanaka, and A. Kawasaki, Proc. 3rd Intern. Conf. Inflammation, Paris, France, p. 513 (1984). METHODSIN ENZYMOLOGY,VOL. 187
Copyright @1990by AcademicPress, Inc. All fightsof reproductionin any formreserved.
GINKGOLIDESAS PAF ANTAGONISTS
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nists. It has been used to study the membrane receptor regulation and desensitization. 12 Most recently, it has also been used, either in conjunction with, or in replacement of, the radioimmunoassay for the detection of sulfidopeptide leukotrienes in biological fluids (Mong et al., results not shown).
[48] G i n k g o l i d e s a n d P l a t e l e t - A c t i v a t i n g F a c t o r B i n d i n g Sites By D. J. HOSFORD, M. T. DOMINGO, P. E. CHABRIER, and
P. BRAQUET Introduction
Specific membrane receptors are assumed to mediate the various biological responses to platelet-activating factor (PAF), 1 the inflammatory autacoid produced by neutrophils, 2 eosinophils, 3 monocytes, 4 macrophages, 5 platelets, 6 and endothelial cells. 7 However, the failure to isolate and characterize the putative receptor leaves open the question of the nature and heterogenity of the binding site(s) in various cells and tissues. Some progress has been made in the formulation of a model of the PAF receptor by studies with various compounds that can specifically antagonize PAF-induced biological effects both in oitro and in oivo. 8"9 Among these compounds, the unique 20-carbon cage molecules named ginkgolides 1°'11 (Fig. 1) have been shown to specifically inhibit PAF-induced actions, such as platelet aggregation, hypotension, bronchoconstriction, i p. Braquet, L. Touqui, T. S. Shen, and B. B. Vargaftig, Pharmacol. Reo. 39, 97 (1987). 2 j. M. Lynch, G. Z. Lotner, S. J. Betz, and P. M. Henson, J. lmmunol. 123, 1219 (1979). 3 T. C. Lee, D. J. Lenihan, B. Malone, L. L. Roddy, and S. I. Wasserman, J. Biol. Chem. 259, 5530 (1984). 4 G. Camussi, M. Aglietta, R. Coda, F. Bussolino, W. Piacibeilo, and C. Tetta, Immunology 42, 191 (1981). 5 j. M. Mencia-Huerta and J. Benveniste, Eur. J. lmmunol. 9, 409 (1979). 6 M. Chignard, J. P. Le Couedic, M. Tenc6, B. B. Vargaftig, and J. Benveniste, Nature (London) 275, 799 (1979). 7 S. M. Prescott, G. A. Zimmerman, and T. M. Mclntyre, Med. Sci. 81, 3534 (1984). 8 p. Braquet, P. E. Chabrier, and J. M. Mencia-Huerta, Adv. Inflammation Res. 12, 135 (1987). 9 D. Hosford, J. M. Mencia-Huerta, C. Page, and P. Braquet, Phytother. Res. 2, 1 (1988). to p. Braquet, B. Spinnewyn, M. Braquet, R. H. Bourgain, J. E. Taylor, A. Etienne, and K. Drieu, Blood Vessels 16, 559 (1985). 11 p. Braquet, Drug Future 12, 643 (1987).
METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
434
PHARMACOLOGY: ANTAGONIST/SYNTHESIS INHIBITORS
GinkgolidIHB e nomenclature R1 A BN 52020 OH B BN 52021 OH C J
M
BN 52022 BN 52024 BN 52023
OH OH H
R2 H OH OH H OH
R3 H H OH OH OH
[48]
0 o HO--q .H
/
r'Ht.u 19+ /
18
~
20
~,s~~I-H
R,
0 +` ' ~ ' 0 FIo. 1. Structure of ginkgolides A, B, C, J, and M.
and to exert a protective effect in experimentally induced diseases such as brain ischemia ~2,13and eye inflammation. 14At the present time, five ginkgolides (A, B, C, M, and J) have been identified. Coded BN 52020, BN 52021, BN 52022, BN 52023, and BN 52024, respectively, by the Institut Henri Beaufour, studies with ginkgolides and other PAF antagonists have implicated PAF in a wide range of pathologies including asthma, shock, ischemia, and graft rejection. 1~,]5 The development of these compounds into potential therapeutic agents is currently being assessed in clinical trials, t6 The interaction of ginkgolides with PAF binding sites has been investigated in various tissues by binding studies using tritiated PAF ([3H]PAF), 12 B. Spinnewyn, N. Blavet, F. Clostre, N. G. Bazan, and P. Braquet, Prostaglandins 34, 333 (1987). 13 p. Braquet, M. Paubert-Braquet, M. Koltai, R. Bourgain, F. Bussolino, and D. Hosford, Trends Pharmacol. Sci. 10, 23 (1989). 14 N. L. J. Verbeij and N. J. van Haerigen, in "Ginkgolides: Chemistry, Biology, Pharmacology and Clinical Perspectives" (P. Braquet, ed.), Vol. 1, pp. 749-758. Prous Science, Barcelona, 1988. 15 p. Braquet (ed.), "The Ginkgolides: Chemistry, Biology, Pharmacology and Clinical Perspectives." Vol. 1. Prous Science, Barcelona, 1988. 16 D. Hosford and P. Braquet, Prog. Med. Chem. 27, 325-379 (1990).
[48]
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435
since high specific activity radiolabeled ginkgolides are not currently available. Ginkgolide binding has been measured on membranes from platelet or tissue homogenates, as a function of time and concentration, in order to further elucidate the nature, location, and physiological relevance of PAF receptor sites in these systems. This chapter examines ginkgolide interactions with PAF binding sites in three different preparations: rabbit platelet membranes, gerbil brain tissue, and rabbit iris and ciliary body. Methods and Materials Biologically relevant studies on [3H]PAF binding require investigation of the concentration range 10-11 to 10 -6 M, in the presence of 0.025% bovine serum albumin (BSA), the latter agent acting as a carrier for the PAF. In our investigations the bound ligand is separated from the free ligand by filtration procedures and characteristics of PAF binding such as dissociation constant (KD) and number of sites (Bmax) are obtained from concentration-dependent experiments, subjected to Scatchard analysis, and plotted as bound/free versus bound (B/F vs B). The equilibrium dissociation constant (Ki) of ginkgolides on PAF binding sites in the various tissues is derived from displacement studies and calculated according to the Cheng-Prusoff equation:
g~--
IC50 1 + [PAF]/KD
where [PAF] is the molar concentration of the labeled PAF and KA is the PAF dissociation constant. Rabbit Platelet Membranes In this study, the effect of the ginkgolides on [3H]PAF binding is evaluated using ginkgolide B (BN 52021), ginkgolide A (BN 52020), ginkgolide C (BN 52022), and a mixture (BN 52063) of the three ginkgolides B, A, and C in the molar ratio 2 : 2 : 1. Chemicals. Synthetic [3H]PAF (specific activity: 59.5 Ci/mmol) in ethanol solution is purchased from New England Nuclear (Boston, MA). Unlabeled PAF and lyso-PAF (Calbiochem, Switzerland), are solubilized in ethanol solution and stored at - 8 0 °. BN 52021, BN 52020, BN 52022, and BN 52063 are solubilized in dimethyl sulfoxide (DMSO). Platelet Membrane Preparation. Rabbit whole blood (6 volumes) is drawn from the central ear artery into I volume of ACD solution (citric acid, 1.4 g, sodium citrate, 2.5 g, and dextrose, 2 g per 100 ml of water) and centrifuged at 150 g for 15 min at 4°. The platelet-rich plasma (PRP) is
436
PHARMACOLOGY: ANTAGONIST/SYNTHESIS INHIBITORS
[48]
carefully removed and centrifuged for an additional 15 min at 1000 g at 4°. The platelet pellet is then washed three times, twice in 10 mM Tris-HC1 buffer, pH 7.4, containing NaCI 150 mM, MgCI2 5 mM, and EDTA 2 mM and, finally, in the same buffer but without sodium. The platelet pellet is resuspended in this latter buffer, quickly frozen in liquid nitrogen, and slowly thawed at room temperature. The freeze/thaw cycle is repeated at least three times as described by Shen et al. 17The lysed platelets are then centrifuged at 100,000 g for 30 min at 4° in a Beckman L8.55 ultracentrifuge (rotor 50.2 Ti). The platelet membrane homogenate is stored at - 8 0 ° and used within 2 weeks. Protein content is determined by the Lowry method TMusing BSA as standard. Binding Assay. Sixty to 100/.~gof membrane protein is added to plastic tubes containing 1 nM [3H]PAF and 1 ml of 10 mM Tris-HC1 buffer, pH 7.0, containing 0.025% BSA and incubated with or without unlabeled PAF or ginkgolides. The incubation is carried out for 90 min at 0°. The bound [3H]PAF is separated from the free [3H]PAF by immediate filtration through Whatman GF/C glass fiber filters under a Brandel vacuum system (Brandel Biomedical Research, Gaithersburg, MD). Filters are washed three times with 5 ml of ice-cold buffer. The filters are then placed in polyethylene vials containing 10 ml of liquid scintillation fluid (Instagel, Packard, Paris) and the radioactivity measured by an LKB/3-counter with 45% efficiency. The nonspecific binding is determined in the presence of 10 -6 M of unlabeled PAF. The specific binding is calculated by subtracting nonspecific from total binding. The inhibition by ginkgolides of the specific [3H]PAF binding is expressed as percentage inhibition (% I) using the equation: [3H]PAF (tb) - [3H]PAF bound in presence of ginkgolides %1= x 100 [3H]PAF specifically bound where [3H]PAF (tb) is [aH]PAF totally bound. Gerbil Brain Tissue
The involvement of PAF in cerebral ischemia has been suggested by studies showing that PAF antagonists including BN 52021, kadsurenone, and brotizolam improve cerebral metabolism and protect the brain in the postischemic phase induced by bilateral carotid ligature in the gerbil.12,13 Thus, we investigated the existence of specific [3H]PAF binding sites in gerbil brain membrane homogenate. 17 T. Y. Shen, S. B. Hwang, M. N. Chang, T. W. Doebber, M. H. T. Lain, M. S. Wu, X. Wang, G. Q. Han, and R. Z. Li, Proc. Natl. Acad. Sci. U.S.A. 82, 672 (1985). la O. H. Lowry, N. J. Rosebrough, A. L. Fan-, and J. Randall, J. Biol. Chem. 193, 265 (1951).
[48]
GINKGOLIDES AS P A F ANTAGONISTS
437
Brain Tissue Preparation. Male gerbils Meriones unguiculatus (6070 g) are purchased from Tumblebrook farm (West-Brookfield, UK). Animals are allowed free access to food and water prior to experimentation. Gerbils are killed by decapitation and the brain quickly dissected out and placed into ice-cold 50 mM Tris-HCl buffer, pH 7.4, containing MgCI2 10 mM, EDTA 2 mM, Trasylol (aprotinin) 50 IU/ml, and PMSF 1 × 10 - 4 M. In some experiments the brain is perfused using the homogenizing buffer, through the left ventricle and under anesthesia. Brain tissue is then washed and homogenized in the same buffer using a Dounce homogenizer at 4°. The homogenates are centrifuged at 1000 g for 15 min at 4°. The microsome, mitochondria, and synaptosome-rich supernatant is harvested and recentrifuged at 10,000 g for 30 min. The P2 pellet is then resuspended in the same buffer, assayed for protein content, and used for the binding assay. Protein content is determined by the Bradford method 19 using Bio-Rad protein assay reagent and BSA as the standard. The brain tissue preparation is used within the same day as isolation. Binding Assay. Protein, 150-350/xg, from the P2 preparation is added to plastic tubes containing [3H]PAF (3 nM for kinetic and displacement studies) and 1 ml 10 mM Tris-HCl buffer, pH 7.0, containing MgCI2 5 mM, Trasylol (50 IU/ml), PMSF 1 x 10-4 M, and 0.025% BSA without or with 1000-fold unlabeled PAF (for nonspecific binding). The reaction is carried out at 25° for 30 min. The unbound PAF is separated from the bound by immediate filtration of the reaction mixture through Whatman GF/B glass fiber filters presoaked in the binding buffer. Tubes and filters are washed three times with 5 ml of precooled binding buffer. The filters are then placed into polyethylene vials containing 10 ml of liquid scintillation fluid (Instagel, Packard) and the radioactivity measured in a LKB/3-counter with 60% efficiency. The PAF specific binding is calculated by subtracting the nonspecific binding (binding in presence of 10 - 6 M unlabeled PAF) from the total binding. Binding data are analyzed by the Scatchard method using the computerized program of Vindimian et al. 2° The specificity of binding is investigated by competition experiments using lyso-PAF, unlabeled PAF, and BN 52021 to displace 1 nM [3H]PAF. Rabbit Iris and Ciliary Body The demonstration that BN 52021 exerts significant protective effects in inflammatory eye diseases, 14 suggests the involvement of PAF in such conditions. In order to investigate the possible presence of PAF receptor 19 M. Bradford, Anal. Biochem. 72, 248 (1976). 20 E. Vindimian, C. Robaut, and G. Fillion, Appl. Biochem. 5, 261 (1983).
438
PHARMACOLOGY; ANTAGONIST/SYNTHESIS INHIBITORS
[48]
sites in eye tissue, we carried out binding studies on rabbit iris and ciliary body. Tissue Preparation. Pigmented rabbits (either sex) are killed by an overdose of pentobarbital 5 min after injection of heparin (1000 IU per rabbit). The eyes are enucleated and placed into precooled 50 mM Tris buffer, pH 7.4, containing 10 mM MgCI2,2 mM EDTA, 50 IU Trasylol per ml, and 1 × 10-4 M PMSF. The eyes are perfused arterially in order to remove the blood from the anterior segment. The iris and ciliary body are carefully dissected out, homogenized in a Dounce homogenizer, the homogenates filtered through two layers of gauze, and centrifugated twice at 40,000 g for 20 min at 4 °. The pellets are finally resuspended in the homogenization buffer and analyzed for protein content by the Bradford method 19 using Bio-Rad protein assay reagent and BSA as standard. Binding Assay. Inhibition of [3H]PAF binding to iris and ciliary homogenates is performed in a final volume of 1 ml buffer, using plastic tubes that contain [3H]PAF (10 -9 M), unlabeled PAF (1/zM, for nonspecific binding), lyso PAF or BN 52021, and 40 to 70/~g of protein diluted in the binding buffer. The binding buffer (pH 7.0) contains Tris I0 mM, MgCI2 5 mM, Trasylol 50 IU per ml, PMSF 0.1 mM, and BSA 0.025%. After 40-min incubation at 25 °, the unbound PAF is separated from the bound by filtration through Whatman GF/B glass fiber filters presoaked for 24 hr in the binding buffer and using a Brandel vacuum system. The assay tubes and filters are washed three times with 3 ml of precooled binding buffer and the filters placed in polyethylene vials containing 10 ml of liquid scintillation fluid (Instagel, Packard). The radioactivity is measured in a LKB/3 counter with 45% efficiency. The percentage inhibition is determined as previously described. In some experiments, stability of [3H]PAF is analyzed after a 40-min incubation by extraction from the reaction mixture, using the solvent system, chloroform: methanol (2 : 1, v/v) and separation on TLC silica plates using a chloroform : methanol : ammonia (70 : 35 : 7, v/v/v) system. In our binding conditions, intact [3H]PAF represents 93% of the total radioactivity recovered from the plates. Results
Rabbit Platelet Membranes The binding of [3H]PAF to rabbit platelet membrane preparations is found to be specific, saturable, time-dependent, reversible, and of high affinity. The specific binding is a linear function of membrane protein concentration from 60 to 120/.~g protein. Experiments using increasing concentrations of [3H]PAF from 1 to 8 nM demonstrate a specific and
[48]
GINKGOLIDESASPAF ANTAGONISTS 0 •
~
•
439
•
\. -~
o
40-
o 6070-
90100 10-6
,
,
,
10-7
10-8
10-9
Antagonist concentration (M) FIG. 2. Inhibition of [3H]PAF (1 nM) specific binding to rabbit platelet membrane by BN 52020 (O), BN 52021 (11), BN 52022 (&), and BN 52063 (D).
saturable binding, specific [3H]PAF binding accounting for about 45% of the total. Scatchard analysis of the binding data reveals a single class of binding sites with a KD of 3 nM and a Bm~, of 2.25 ± 0.2 pmol/mg protein. The displacement of [3H]PAF (1 nM) binding in rabbit platelet membranes by BN 52020, BN 52021, BN 52022, and BN 52063 is shown in Fig. 2. All these compounds inhibit [3H]PAF binding in a dose-dependent manner. Maximum inhibition of [3H]PAF binding by the ginkgolides is observed with BN 52021 with an IC50 value of 2.5 × 10 - 7 M. BN 52063, BN 52020, and BN 52022 exhibit ICs0 values of 7 × 10-7 M, 8.3 × 10 - 7 M , and 6 × 10 - 6 M , respectively. In comparison, the concentration required to inhibit specific [3H]PAF binding by 50% is 2~× 10 - 9 M for unlabeled PAF, whereas lyso-PAF is unable to displace [3H]PAF binding below concentrations of 10-5 M. The specificity of the ginkgolide compounds for PAF binding sites is also assessed in experiments using other radioligands. BN 52021 and the other ginkgolides (10-8-I 0-5 M ) are found to be totally inactive in inhibiting the binding of the appropriate 3H-labeled ligands to al-, a2-, fix-, and fl2-adrenergic receptors, H1 and H2 histamine receptors, 5HTI and 5HT2 serotoninergic receptors, /x, 8, and X opiate receptors, benzodiazepine, and muscarinic and imipraminic receptors.
440
PHARMACOLOGY: ANTAGONIST/SYNTHESIS INHIBITORS
[48]
As reported previously, ~1 the binding of [3H]-PAF is modulated by the presence of mono- and divalent cations. A decrease of the binding is observed with the monovalent cations Li + and Na ÷, whereas K + and divalent cations such as Mg 2+ and Ca 2÷ enhanced the specific [3H]PAF binding (data not shown). In this respect, the inhibition of [3H]PAF binding by BN 52021, the most effective ginkgolide, is investigated in the presence of high concentrations of NaCI or MgC12. In the presence of 150 mM NaCI, the ICs0 of the displacement curve is shifted to 4.8 x 10-8 M, while with 5 mM MgCI2 the displacement curve is shifted to the right with an increase of the IC5o value to 5 × 10 - 7 M.
Gerbil Brain Tissue The specific binding is saturable, representing 20-25% of the total binding and constituting about 5% of the total radioactivity added to the incubation medium. Scatchard plot of the binding data from saturation and competition experiments is curvilinear and could be described by a twocomponent binding model on computerized analysis, indicating the existence of two apparent classes of binding sites (Fig. 3). The first population of PAF recognition sites exhibits a Ko, of 3.66 -+ 0.56 nM and a Bma,,, of 0.83 --- 0.23 pmol/mg of membrane protein, while the second population has a KD2 of 20.4 - 0.56 nM and a Bm~2 of 1.1 --- 0.32 pmol/mg of membrane protein (n = 5). The association of [3H]PAF to gerbil brain homogenate at 25 ° reaches equilibrium within 15 min and remains stable for at least 60 rain. The specificity of binding is established by displacing 1 nM [3H]PAF by lyso-PAF, unlabeled PAF, and by BN 52021 in competition experiments. Lyso-PAF is ineffective up to 10-5 M in competition with [3H]PAF for the binding sites. Unlabeled PAF ( 1 0 - 1 ° - 1 0 - 6 M ) and BN 52021 (10 -1°10-5 M) dose-dependently inhibits the specific binding of [3H]PAF to brain membrane preparations with IC50 values of 2.5 × 10 - 9 M and 3.5 x I0 -s M, respectively (Fig. 4). Interestingly, in this tissue, BN 52021 fails to totally inhibit the [3H]PAF binding. We further evaluated whether the binding of [3H]PAF is associated to discrete brain areas by studying the distribution of [3H]PAF binding in various regions of the gerbil brain. The maximum amount of specific binding is found in the midbrain and the hippocampus, with less binding being apparent in the olfactory bulb, frontal cortex, and cerebellum.
Rabbit Iris and Ciliary Body Specific [3H]PAF binding is found in cornea, iris, and ciliary body and is characterized in the latter two. The specific binding represents 20% of 2~ S. B. Hwang, M. H. Lam, and S. S. Pong, J. Biol. Chem. 261, 13720 (1986).
[48]
GINKGOLIDES AS P A F ANTAGONISTS
441
40-
36-~
Kdl
32" , . , ~ 28
\
= 3.66+ 0.92 IO~M
Bmax 1 = 0.83 + 0.30 pmol / mg Kd =20,4 + 0.5610 ~ M
e~
2
;
,1
+_
4 0
i
0
,
.
=
=
•
•
=
,
•
=
i
|
•
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4
Bound (nM) FIG. 3. Scatchard analysis of [3H]PAF binding in gerbil brain homogenate. The experiments are performed at 25° and each point is the average of triplicate determinations ( n = 5).
the total binding and is linear over the concentration range assayed, up to 90/zg of protein per tube. For kinetic and competition studies, [3H]PAF is used at a concentration of 3 nM. At 25° the association reaches equilibrium within the first 30 min of incubation and remains stable up to 60 min with this latter concentration. An excess of unlabeled PAF added to the reaction mixture at 25 min demonstrates a reversibility of 50% within 15 min. No reversibility is observed at 45 min, even after addition of an excess of unlabeled PAF. Increasing concentrations of labeled PAF over the range of 1 to 14 nM exhibit saturable binding and show a two-step saturation isotherm. The first maximum is reached at 4-5 nM and Scatchard analysis derived from both saturation and competition studies demonstrates a curvilinear repartition of the points suggesting two subtype components (Fig. 5). For the iris, a high-affinity component with a KD, of 4.9 - 0.47 nM and Bmaxt of 3.17 + 0.5 pmol/mg protein and a low-affinity component with a K,t2 of 11.6 -+ 0.33 nM and Bmax2 of 12.46 +- 2.3 pmol/mg is observed. Similar values are found for ciliary body: Knt =" 5.7 +-- 0.09 nM; Bronx, = 3.41 -+ 1 pmol/mg and KD2 = 24.4 +- 0.91 nM; Bmax~--- 16.6 -+ 0.51 pmol/mg. In our study, the specificity of PAF binding to iris and ciliary body is confirmed by inhibition of the [3H]PAF binding in the presence of un-
442
PHARMACOLOGY: ANTAGONIST/SYNTHESIS INHIBITORS
[48]
0"
10.
20. = 30. .o ..¢ r-
40-
•
50-
•
¢1 o 60.
•
70-
80-
~
90. 100 10-10
,
i
.
•
|
10-9
lO-S
10-7
10-6
10-5
(MI FIG. 4. Inhibition of [3H]PAF (1 nM) binding to gerbil brain homogenate by unlabeled PAF ( 0 ; 10-1°-10 -6 M, C5o = 5 x 10 -8 M ) and BN 52021 ( 1 ; 10-1°-10 -5 M). Each point is
the mean of triplicate determinations(n = 3).
labeled PAF, lyso-PAF or BN 52021. Displacement of the binding by unlabeled PAF demonstrates a biphasic inhibition curve. Lyso-PAF at a concentration below 10 -6 M fails to inhibit the binding. The inhibition curve obtained with BN 52021 differs from that observed with unlabeled PAF and is only partial (Fig. 6). The maximum inhibition obtained is about 60% and appears to be associated with only one component of PAF displacement curve. Discussion Ginkgolides are specific PAF antagonists isolated from the roots and leaves of the fossil tree Ginkgo biloba. Extracts of this tree have been used by the Chinese for some 5000 years to alleviate asthma and other inflammatory conditions, the beneficial effects of the extract being largely attributable to the PAF antagonizing effects of its constituents. The chemistry, biology, and pharmacology of these compounds have been extensively
[48]
GINKGOUDZS AS PAF ANTAGONISTS
443
80-
70-
60-
'7
s0.
o × LL
40-
m
30-
20-
10"
O" 0
0.2
0.4
0.6
0.8
1
[3H]PAF bound (nM) FIG. 5. Scatchard analysis of [3H]PAFbinding to iris homogenate in 10 mM Tris buffer, pH 7, containing 5 mM MgC12,PMSF 1 x 10.4 M, Trasylo150IU/m|, and BSA 0.025%at 25°. Each point is the average of triplicate determinations ( n = 4). reviewed in various recent publications. ~l '15 The results presented here demonstrate the presence o f specific binding sites for [3H]PAF in rabbit platelet membranes, gerbil brain tissue, and rabbit iris and ciliary body, the binding of the mediator in these systems being specifically antagonized by B N 52021 and other ginkgolides. In rabbit platelet membranes, the ginkgolides dose-dependently antagonize specific P A F binding, with ICso values of 2.5 × 10 -7 M, 8.3 x 10 -7 M, and 6 × 10 -6 M for B N 52021, B N 52020, and B N 52022, respectively. The inhibition o f P A F recognition sites is highly specific since the ginkgolides do not interact with any other known receptors (see section on Results). Characterized by the presence of two hydroxyl groups on car-
444
[48]
PHARMACOLOGY" ANTAGONIST/SYNTHESIS INHIBITORS
O"
20 r-
.o ,.~_ 40 e'e-
~
6o
80
100 I 10-10
, 10-9
10-8
10-7
10-6
10-5
Antagonist concentration (M) FIG. 6. Inhibition of [3H]PAF (1-3 riM) binding to iris homogenate by unlabeled PAF ( O - - O ) , BN 52021 (11--11), and lyso-PAF ( • determinations (n = 4).
• ) . Each point is the average of triplicate
bons 1 and 3, BN 52021 is the most powerful antagonist. The loss of the hydroxyl group on carbon 1 in BN 52020 and the presence of a hydroxyl group on carbon 7, in the a position of the lipophilic tert-butyl moiety in BN 52022 render these compounds less active. Studies reported elsewhere ~~have shown BN 52024 to possess an IC50 value of 5.4 x 10-5 M in this assay, the loss of the hydroxyl group on carbon 1 further decreasing the activity in comparison to BN 52022. These data corroborate physicochemical findings showing that as the ginkgolide becomes less polar, its PAF antagonistic activity increases. 1~ These binding studies corroborate results obtained for the inhibition of PAF-induced platelet aggregation, where BN 52021 is the most effective antagonist followed by BN 52020 and 52022.tl Moreover, the affinity of BN 52021 for PAF receptor sites is altered by Na + and Mg 2÷. Indeed, the state of affinity of PAF receptor appears to be low in the presence of Na ÷ and high in the presence of Mg 2÷.21 Accordingly the ability of BN 52021 to displace specific [3H]PAF binding is dependent on the ionic conditions. This phenomenon has also been observed with other PAF antagonists, such as CV-3988, ONO 6240, and L-652,569, but, not with kadsurenone which binds equally well to the PAF receptor at both the high- and lowaffinity state. 21
[48]
~IrqKGOLXI)ESAS PAF ANTAGONISTS
445
The finding that BN 52021 inhibits specific [3H]PAF in gerbil brain tissue may account for the protective effect of the compound in various models of cerebral ischemia. 12'13 The direct radioligand binding studies reported here indicate that the PAF recognition sites are most likely located on brain cells and not on blood elements since PAF binding characteristics were similar whether or not the brain was perfused and did not correlate with the level of cerebral vasculature. Scatchard analysis of the binding data indicates the existence of two populations of PAF binding sites in gerbil brain while other tissues such as platelets (see section on Methods and Materials) and lung 22 exhibit only one class of site. Furthermore, the [3H]PAF specific binding to whole brain homogenate is totally displaced by unlabeled PAF but only partially inhibited by BN 52021. Thus, it is possible that BN 52021 and other PAF antagonists interact only with one site, although the present data do not indicate which receptor population is involved. Similar to the case with brain tissue and that recently reported for rat retina, 23 two populations of PAF binding sites were also detected in iris and ciliary body. The specific binding in these latter tissues (20% of the total binding) is low as compared to that obtained, in the same buffer conditions, for platelets (70-80%), but is similar to the 20-25% found in brain tissue or to the 30-40% in lung tissue. In rabbit iris and ciliary body, specific PAF binding exhibited a twostep saturation isotherm, suggesting heterogenous binding characteristics. The displacement by unlabeled PAF exhibited a biphasic curve, which could be described by a two-component computerized analysis. Inhibition of [3H]PAF binding by BN 52021 was monophasic, partial (60%), and only involved the first step of the PAF inhibition curve. Despite this lack of total inhibition of PAF binding, BN 52021 significantly inhibits PAF-induced plasma leakage and edema formation in the rat retina 24 and the acute rise in intraocular pressure (IOP) caused by topical administration of the mediator in the rabbit eye. 25 In addition, the ginkgolide also abolishes the deleterious effects of PAF on the electroretinogram of rat eyes in oitro, 26 prevents cell infiltration and edema formation provoked by immune kerati-
22 S. B. Hwang, M. H. Lam, and T. Y. Shen, Biochem. Biophys. Res. Comrnun. 128, 972 (1985). z3 M. Doly, B. Bonhomme, P. Braquet, P. E. Chabrier, and G. Meyniel, lmmunopharmacology 13, 189 (1987). 24 M. Doly, P. Braquet, K. Drieu, B. Bonhomme, and M. T. Droy, in "Colour Vision Deficiencies" (G. Verrist, ed.), Vol. 8, p. 515. W. Junk, Dordrecht, 1987. 25 p. Braquet, R. F. Vidal, M. Paubert-Braquet, H. Hamard, and B. B. Vargaftig, Agents Actions 15, 82 (1984). 26 M. Doly, P. Braquet, B. Bonhomme, and G. Meyniel, Int. J. Tissue React. 9, 33 (1987).
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tis in the cornea of the rabbit eye,14 and inhibits the increased IOP and breakdown of the blood-aqueous barrier after laser irradiation of the rabbit iris. 27 In conclusion, the identification of specific PAF binding sites in various cells and tissues and their blockade by ginkgolides explains the beneficial effects of ginkgolides in numerous experimental diseases and highlights their potential as valuable therapeutic agents for man. 27 N. L. Verbeij, J. L. van Delft, N. J. van Haeringen, and P. Braquet, in "The Ginkgolides: Chemistry, Biology, Pharmacologyand ClinicalPerspectives" (P. Braquet, ed.), Vol. 2. Prous Science, Barcelona, 1990, in press.
[49] K a d s u r e n o n e a n d O t h e r R e l a t e d L i g n a n s as A n t a g o n i s t s of Platelet-Activating Factor Receptor B y T. Y. SrIEN and I. M. HUSSAINI
Introduction Platelet-activating factor (PAF) is a highly potent ether-linked phospholipid (1-O-alkyl-2-acetyl-sn-glycerol-3-phosphorylcholine) which activates platelets as well as modulates the function of leukocytes and other target cells. Interaction of P A F with a specific membrane recognition site coupled to phosphatidylinositol metabolism produces the biological actions of the phospholipid. 1-3 A number of specific reversible and irreversible P A F receptor antagonists have been developed using both in vitro (platelet aggregation; binding studies) and in vivo (hypotension; bronchoconstriction) screening methods. Some of these antagonists include PAF analogs, e.g., CV-3989, 4 ONO-6248, 5 SR-63441, 6 natural products, l F. Snyder, Med. Res. Rev. 5, 107 (1985). 2 D. J. Hanahan, Annu. Rev. Biochem. 55, 483 (1986). 3 p. Braquet, T. Y. Shen, L. Touqui, and B. B. Vargaftig, Pharmacol. Rev. 39, 97 (1987).
4 Z. Terrashita, S. Tsuchima, Y. Yoshiota, J. Normura, Y. Inada, and S. Nishikoda, Life Sci. 32, 1975 (1983). 5 C. M. Winslow, H. U. Gubler, A. K. Delillo, F. J. Davies, G. E. Frisch, J. C. Tomesch, and R. N. Saunders, Proc. 2nd Intern. Conf. Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Gatlinburg, Tennessee, p. 33 (1986). 6 T. Mitamoto, H. Ohno, T. Tano, T. Okada, N. Hamanaka, and A. Kawasaki, Proc. 3rd Intern. Conf. Inflammation, Paris, France, p. 513 (1984). METHODSIN ENZYMOLOGY,VOL. 187
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