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Human platelet α2-adrenergic receptors: Labeling with 3H-yohimbine, a selective antagonist ligand
Life Sciences, Vol. 28, pp. 2705-2717 Printed in the U.S.A.
Pergamon Press
HUMAN PLATELET a2-ADRENERGIC RECEPTORS: LABELING WITH 3H-YOHIMBINE, A SELECTIVE ANTAGONIST LIGAND Makoto Daiguji, Herbert Y. Meltzer and David C. U'Prichard Department of Pharmacology, Northwestern University School of Medicine, Chicago, Illinois 60611, and Department of Psychiatry, Chicago University Pritzker School of Medicine, Chicago, Illinois 60637 (Received in final form April 2, 1981) Summary 3H-Yohimbine, a potent and selective pharmacological antagonist of a2-adrenergic receptors, labeled human platelet membrane a2-receptors with high affinity. Binding was rapid Both saturation and kinetic experiand reversible at 25oC. ments indicated a single order of binding sites, with an equilibrium KO value of 1.0-1.5 nM. Low Mg2+ concentrations increased the KD for 3H-yohimbine without altering the Bmax. The 3H-yohimbine site exhibited a2-receptor specificity: (-)-norepinephrine and (-)-isoproterenol were 4.8 and 330 times less potent than (-)-epinephrine; (-)-catecholamines were 17-35 times more potent than corresponding (t)-catecholamines; the selective al-antagonist prazosin was 340 times less potent than yohimbine. Catecholamine agonists exhibited shallow curves in inhibiting 3H-yohimbine binding, with pseudo-Hill coefficients (nH) of less than 1.0, whereas the nH of antagonists was 1.0. No specific binding of 3H-prazosin to platelet membranes was observed, indicating the absence of a -receptors. 3H-Yohimbine labeled fewer olatelet sites than did 3H-a. ihydroergocryptine under identical conditions (80 vs 130 receptors/ cell), and may be a more s ecific and useful antagonist probe of platelet a2-receptors than !_ H dihydroergocryptine. a2-Adrenergic receptors have been extensively studied in brain and some peripheral tissues using agonist or partial agonist radioligands such as 3Hcatecholamines and 3H-imidazolines, which have selectively high affinity for this receptor subtype (l-5). Another approach to labeling the a2-receptor has been to examine the binding of the non-selective a-antagonist 3H-dihydroergocryptine (3H-DHEC), and to distinguish the a2-receptor component of 3H-DHEC binding, which has high affinity for the a2-selective antagonist yohimbine and low affinity for the al-selective antagonist prazosin (6-9). Human platelets contain an a-adrenergic receptor that mediates inhibition of basal and prostaglandin-stimulated adenylate cyclase activity (lo), and platelet aggregation (11). These responses appear to be “2, and not
0024-3205/81/242705-13$02.00/O Copyright (c) 1981 Pergamon Press Ltd.
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3H-yohimbine
Binding
Vol.
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1981
(12,13), and platelet radioligand binding studies, which have "13 in nature been mainlv oerformed usina 3H-DHEC (14-161. have indicated that olatelets contain a2-receptors (7,8).J From extensive' studies of the interaction of 3H-DHEC with platelet a2-receptors, Lefkowitz and co-workers have concluded like the erythrocyte B-receptor, can exist in two conthat this receptor, formations differentiated by high and low affinity respectively for agonists, and designated a2(H) and a2(L) (17-18). According to this model, 3H-agonist ligands label with high affinity only the a2(H) state, and not the total a2receptor population (19).
the selectivity of 3H-DHEC as a platelet a2-receptor probe Recently, has been questioned, as has the concept that human platelet membranes contain 3H-DHEC, an ergot alkaloid, only a2-receptors, and no al-receptors (20,21). has as high affinity for al-receptors (17,22) and for serotonin receptors (23) as for a2-receptors, and serotonin receptors have been suggested to occur on another platelet a-receptor probe platelet membranes (24). 3H-Phentolamine, (25) also has, like 3H-DHEC, equal affinity for al- and u2-receptors (26).
We therefore decided to characterize a2-receptors on human platelet Yohimbine is a potent and membranes using recently available 3H-yohimbine. selective a2-receptor antagonist in pharmacological studies (27), and exhibits 3H-DHEC sites and at the a2-receptor high affinity (Ki 1-2 nM) at platelet 3H-DHEC binding in other tissues containing both a-receptor component of The results obtained show that 3H-yohimbine selectively subtypes (7,8). labels human platelet a2-receptors, and appears to label fewer platelet membrane sites than 3H-DHEC under the same conditions. In addition, an examination of platelet 3H-prazosin binding indicates that human platelet membranes contain no al-receptors.
Materials --___
and Methods
The majority of experiments to characterize Platelet membrane preparation: 3H-vohimbine bindinq were performed usinq platelet-rich plasma (PRP) obtained from local blood banks. Membranes were prepared from PRP which had been stored Platelets at 250C for no more than three days from the time blood was drawn. were lysed and membranes prepared by the method of Newman et al. (15), with PRP was centrifuged at 300 x g for lOTinto eliminate some modifications. and the supernatant centrifuged at 16,000 x g for 10 min. residual erythrocytes, The resulting pellet was resuspended in ice-cold 50 mM Tris-HCl buffer (pH 8.0 at OoC) containing O.llM NaCl and 0.02M Na2EDTA (final pH 6.3j and centriAfter a further resuspension and centrifugafuged at 16,000 x g for 10 min. tion in the same buffer, washed intact platelets were resuspended in ice-cold 5.CmM Tris-HCl buffer (pH 8.0 at OoC) containing 5.0 mM Na2EDTA, and disrupted using a Brinkmann Polytron PT-10 (setting 5, 30 set) before cenAll the above procedures were pertrifugation at 39,000 x g for 10 min. Membrane pellets were rapidly frozen in an acetone-CO2 formed at 4oC. bath and stored at -700C until assay.
To examine binding to freshly prepared platelet membranes, blood samples were obtained from healthy human volunteers and centrifuged in citratecentrifuged at 16,000 dextrose at 600 x g for 2.5 min, and the supernatant All subsequent steps were carried out as above, except that x g for 10 min. 3H-yohimbine binding assays were performed on platelet membranes on the with no intervening -7OOC storage. day of membrane preparation,
Vol. 28, No. 24, 1981
Platelet 3H-yohimbine Binding
2707
Thawed or freshly prepared membrane pellets were Ligand binding assays: resuspended in 50 mM Tris-HCl (pH 7.0 at 25OC), centrifuged at 50,000 x g Incubaand resuspended in the same buffer for binding assays. for 10 min, tion tubes kept on ice received lo-60 ul diluted 3H-yohimbine (New England Nuclear, 80-90 Ci/mmole), 20 pl of various concentrations of drugs dissolved in 0.1% ascorbic acid, 1.0 ml of membrane suspension (0.4 mg membrane protein) Samples were pH 7.0, for a final volume of 2.0 ml. and 50 mM Tris-HCl, incubated with shaking for 60 min at 25oC, and the assay was terminated by The rapid filtration under reduced pressure over Whatman GF/B filters. filters were rinsed with 3 x 5 ml of ice-cold 50 mM Tris-HCl buffer (pH 8.0 at OoC), transferred to vials containing 8.0 ml Formula 947 (New England Nuclear) and subsequently counted by liquid scintillation spectrometry at 35% efficiency.
Non-specific binding was determined in parallel samples containing 100 pM (-)-norepinephrine (NE), and specific binding was obtained by subtracting nonIn typical experiments employing 0.2 nM 3Hspecific from total binding. yohimbine and 0.4 mg platelet membrane protein, total binding was 1500 c.p.m. and non-specific binding was 120 c.p.m. A variety of structurally dissimilar unlabeled yohimbine, at maximally effective agents, including a-receptor concentrations, inhibited 3H-yohimbine binding to the same extent as 100 pM Inhibition by two or more of these agents was not additive, indicat(-)-NE. ing that all a-receptor agents inhibited the same component of binding. The bound was always less than 10% of the added radioamount of 3H-yohimbine 3H-yohimbine (0.2 nM) binding to the filter paper represented activity. and was not inhibited by co-incubation only 2-3% of the total c.p.m. bound, with 100 pM (-)-NE.
3H-DHEC and 3H-WB-4101 binding to platelet membranes were performed as previously described in brain experiments (28,29), except that a 1.0 ml incubation volume was used for 3H-DHEC assays. 3H-prazosin binding to platelet membranes was assayed according to the methods of Greengrass and Bremner (30). Specific binding for these three ligands was defined as binding inhibited by 100 pM (-)-NE. Preliminary experiments showed that the same component of 3H-DHEC binding was inhibited by 100 pM (-)-NE or 10 uM phentolamine, a common indicator of specific binding in this assay (14-16). All assays were performed in triplicate or duplicate. Protein concentrations were determined by the method of Lowry (31), using bovine albumin as a standard, and specific binding is expressed as fmoles of 3H-yohimbine bound per mg platelet membrane protein. In several experiments, the numbers of platelets in 300 x g or 600 x g supernatants were determined using a Coulter counter, and binding constants were subsequently expressed as number of 3H-yohimbine sites per platelet.
In preliminary experiments, storage of platelet membranes at -7oOC for up to 15 days had no effect on specific binding of 3H-yohimbine. The effect of PRP storage time at 250C was assessed, and specific binding of 0.2 nM 3Hyohimbine declined from 22-24 fmol/mg protein (2 or 3 days old) to 14 and 9 fmol/mg protein after storage for 5 and 6 days respectively. Differences 3H-yohimbine binding to membranes from freshly-prepared platelets between and 3 day old platelets are discussed below. Dru s: Drugs were kindly donated by the following isomers Sterling-Winthrop); oxymetazoline (Schering), line and phentolamine (CIBA-Geigy); tramazoline and
--T-
sources: catecholamine tolazoline, naphazoclonidine (Boehringer
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3H-yohimbine
Binding
Vol.
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1981
Ingel heim); amidephrine (Mead Johnson); o-ergocryptine and dihydroergocryptine (Sandoz); yohimbine (Regis); WB-4101 (Ward Blenkisop); prazosin (Pfizer). p-kinoclonidine was obtained from Dr. Bruno Rouot, and a-yohimbine from Drs. Carl Roth and David J. Jones. Other drugs and reagents were obtained from commercial sources. Results
Kinetic characteristics of _?H-yohimbine binding: In preliminary experiments specific bindinq of 3H-vohimbine at equilibrium did not vary appreciably 'between pH 6.3- and 7.3; but decreased' sharply at pH 7.7-8.0: Binding was optimal at pH 7.0, which was chosen for further experiments. Specific binding of 0.2 nM 3H-yohimbine was linear at tissue concentrations between 0.25 and 2.0 mg protein/ml (data not shown).
Association of 3H-yohimbine binding to platelet membranes at 250C was rapid, with a t1/2 of 7 min (Fig. lA), reaching equilibrium levels by 4060 min. Specific binding was observed to decrease somewhat after 90 min An inincubation, possibly reflecting some degradation of the radioligand. cubation time of 60 min was chosen to represent equilibrium binding in subsequent experiments. 3H-Yohimbine associated to platelet sites in a hyperbolic manner (Fig. lA, Inset), and the mean observed initial rate constant (k,b) from three experiments was 0.076 + 0.021 min-l for 0.2 nM 3H-yohimbine. Dissociation of bound 3H-yohimbine at 250C was determined by incubating platelet membranes to equilibrium, and then adding 100 uM (-)-NE at time zero and measuring residual specific binding at subsequent time intervals (Fig. 1B). When plotted on a semilogarithmic scale, dissociation was linear, indicating a first-order process with a t1/2 of 12 min. The rate constant for dissociation (k-1) was 0,061 min-1. The second order rate constant for association (k of 3H-yohimbine binding was determined from the equation kl = (k,b - kl)/ [t3H-yohimbine] to be 7.5 x 107 ~-1 min-1, and the equilibrium dissociation constant (KD), determined from the ratio k_l/kl, was 0.81 nM.
Specific binding of 3H-yohimbine to platelet membranes was saturable with increasing concentrations of ligand (Fig. 2). Non-specific binding increased linearly between 0.1 and 8 nM, whereas specific binding plateaued between 3 and 8 nM. A Scatchard (32) plot of the data indicated a single population of sites with an apparent KD of 1.23 nM, similar to the value obtained from kinetic experiments, and a maximum number of sites (Bmax) on membranes prepared from 2-3 day old platelets of 200 fmoles/mg protein, or about 80 binding sites per cell. From 5 similar experiments, the mean values (+ S.E.M.) were 1.25 + 0.10 nM (KD) and 182 + 29 fmoles/mg protein (Bmax). fie mean Hill coefficient (nH) of binding of-H-yohimbine was 0.93 + 0.05, In these experimenTs, the indicating the absence of cooperative interactions. binding site concentration was 30-40 pM (0.4 mg protein per assay), approximately 5% of the ligand KD value. Experiments were performed to determine whether the apparent KD value of 3H-yohimbine would decrease with decreasing binding site concentration (33). The KD was not however si nificantly different at membrane protein concentrations of 0.1 mg/ml (1.02 nM 3 , 0.5 mg/ml (1.03 nM) and 1.0 mg/ml (1.07 nM). Low concentrations of magnesium ion in the assay medium decreased the specific binding of 3H-yohimbine (Fig. 3). Tsai and Lefkowitz have prethe affinity of catecholamine agonists viously observed that Mg2+ increased
Platelet 3H-yohirnbineBinding
vol. 28, No. 24,1981
800
[
700
protein]
A
nM
[YOH]=OZ
2709
=0.4 mg /assay
20
200-e I
0
IO
60
20
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/
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60
80
(rnln)
0
1603 FIG.
40
20 TIME
60
(mln)
1
Kinetics at 25°C of 3H-yohimbine binding to human platelet membranes (A) Association: 0.2 nM 3H-yohimbine was incubated with membranes for various times before filtration in the absence (total) or presence (non-specific) of 100 uM (-)-NE. Insert: semi-logarithmic plot of increase in specific binding with time. (B) Dissociation: 0.2 nM 3H-yohimbine was incubated to equilibrium (60 min) with or without 100 uM (-)-NE, before addition of 100 pM(-)-NE to all Samples were filtered at various times thereafter. Insert: samples. semi-logarithmic plot of decrease in specific binding with time. Values are from a representative experiment performed in triplicate.
sites labeled by 3H-DHEC (34). Thus the decrease H yohimbine binding could be due to enhanced interactions of residual circulating catecholamines with "2 (H) sites on platelet membranes. However the fact that the platelet membranes were repeatedly washed, and that the decrease in binding was due to a dose-dependent increase in the apparent KB of 3H-yohimbine with increasing Mg2+ (Fig. 3, Inset), rather than a reduction in the number of sites, suggested that this was not the explanation.
p;
latelet T_ . .-c1 receptor
Pharmacological properties of ?H-yohimbine binding: Catecholamines and other drugs inhibited JH-yohimbineTnding to membranes obtained from 2-3 day old platelets with a potency order suggesting an o-adrenergic receptor
Platelet
2710
3H-yohimbine
vol.
Binding
28, No.
24,
1981
Thus (-)-epinephrine was 4.8 times more potent than interaction (Table 1). (-)-NE and 330 times more potent that the B-receptor agonist (-)-isoproterenol. since (-)-epinephrine and (-)-NE were Marked stereoselectivity was observed, Imidazoline 20 and 16 times more potent than their respective (+)-isomers. binding, with apparent derivatives were very potent inhibitors of 3H-yohimbine Among Ki values ranging from 6.0 nM (oxymetazoline) to 52 nM (clonidine). inhibitor was dihydroergocryptine a-receptor antagonists, the most potent (Ki 600 PM), followed by the a-stereoisomer of yohimbine (rauwolscine), aergocryptine and yohimbine itself (Kis 670, 760 and 820 PM). That 3H-yohimbine receptor on platelet membranes was specifically labeled an a2-adrenergic indicated by the 450-fold greater affinity of yohimbine compared to prazosin, the greater affinity of rauwolscine compared to yohimbine (35), and the much greater affinity of clonidine compared to the selective al-receptor agonists Thus the affinity of yohimbine (-)-phenylephrine (36) and amidephrine (37). at platelet a -receptors is substantially greater than at brain a2-receptors labeled with 32H-clonidine (29, 38) or 3H-p-aminoclonidine (2).
KO=I 23nM
54
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1
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I
6
7
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3H-Yohimbine binding to human platelet membranes (0.4 mg protein) Nonas a function of increasing concentrations of 3H-yohimbine. specific binding was determined by incubating samples with 100 uM Values are (-)-NE. Insert: Scatchard plot of specific binding. from a representative experiment performed in triplicate.
Lefkowitz and colleagues have demonstrated that at platelet 3H-DHEC binding sites, full agonists such as catecholamines exhibit shallow inhibition with pseudo-Hill coefficients (nH) less than 1.0, which are made curves, steeper and shifted in the direction of decreased affinity by guanine nucleoantagonists on the other hand have steep curves (nH equals tides or sodium; and partial agonists show intermediate 1.0) and no nucleotide-or sodium-shift, These data have been modeled to two steepness of slope and shifts (34,39). states
of
the
platelet
a2-receptor
differing
in
affinity
for
agonists
(18).
Vol. 28,
No.
24,
Platelet 3H-yohimbine Binding
1981
//
I
I
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lwg2+
300
(mM)
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mM
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I 5
3
Effects of magnesium on 3H-yohimbine binding. 0.2 nM 3H-yohimbine was incubated with platelet membranes (0.4 mg protein) as described in Materials and Methods in the absence and presence of various Scatchard plots of Insert: concentrations of magnesium chloride. 3H-yohimbine saturation isotherms determined in the presence of 0,l or 5 mM magnesium. Values are from a representative experiment In three such experiments, mean K values performed in duplicate. for 3H-yohimbine (k S.E.M.) were 1.14 k 0.03 nM (no Mg 2P2, 1.38 f 0.09 nM (1.0 mM Mg2+) and 1.93 f 0.03 nM (5.0 mM Mg2 ). No curvilinearity of the significant changes in B,,,, or significant Scatchard plots were detected at any magnesium concentration.
2711
2712
Platelet
3H-yohimbine
TABLE
INHIBITION OF PLATELET a-ADRENERGIC
Drugs
Vol.
Binding
,,1,L;;;o;;iterenol OxymetazoTine Naphazoline Tramazoline p-Aminoclonidine Clonidine
3H-YOHIMBINE BINDING BY RECEPTOR AGENTS
wgocryptine Rauwolscine a-Ergocryptine Yohimbine WB-4101 Phentolamine Piperoxan Tolazoline Prazosin
"H
75 360 620 1500 5900 25000
+ T T T T z
20 50 160 400 600 4000
(4) (4) (4) (3) (3) (3)
0.72 0.71 0.70 0.69 0.80 0.66
+ T 7 T T 5
0.04 0.04 0.06 0.03 0.04 0.06
6.0 15 20 29
+ T T +
1.4 1.3 2.8 5.5
(3) (3) (3) (4)
11
(4)
1.06 + 0.83 T 1.00 T 0.94 T l.OlZ
0.11 0.01 0.09 0.05 0.04
920 + 19 6700 z 640
(3) (3)
0.73 + 0.02 1.03 z 0.05
0.60 + 0.67 + 0.76 T 0.82 + 2.4 T 3.77 13.6> 210 ? 370 5
(4) (4) (3) (6) (3) (5) (3) (3) (3)
0.88 +
0.04
1.04 0.95 0.98 0.98 0.96 0.92 0.93
0.03 0.03 0.05 0.04 0.09 0.06 0.09
52+-
Amidephrine
24, 1981
1
Ki(nM)
Catecholamines (-)-Epinephrine (-)-Norepinephrine (-)-a-Methylnorepinephrine (+)-Epinephrine (+)-Norepinephrine
28, No.
0.22 0.14 0.05 0.24 0.6 0.9 2.3 50 120
+ T T + + T 5
Platelet membranes were incubated with 0.2 nM 3H-yohimbine and 6-18 concenin Materials and Methods. trations of unlabeled drugs, as described IC50 values (concentration inhibiting 50% of specific binding) and pseudo-Hill Coefficients (nH) were determined from Hill plots of the data, and apparent Ki values were derived using the formula of Cheng and Prusoff (41): Ki = IC5O/(l + [3H-YOH]/KD), where Kg of 3H-yohimbine is 1.25 nM. Values are mean -+ S.E.M. from number of experiments shown in parentheses.
binding revealed Analysis of the nH values for competitors of 3H-yohimbine similar properties for agonist and antagonist interactions at this site All catecholamine competitors had nH values of about 0.7, whereas (Table 1). Imidazoall antagonists had nH values not significantly different from 1.0. lines are generally believed to be basically antagonists with little or no intrinsic activity at platelet a2-receptors (40), and had nH values of about 1.0.
The examined
inhibition of in more detail
platelet (Fig. 4).
3H-yohimbine Over a 10-8
binding by a few drugs was - lo-5 M concentration range,
28, No.
Vol.
24,
1981
Platelet
3H-yohimbine
271:
Binding
x Phentolanxne \
,
x
40 .(-)-Norephinephrine \,
t
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FIG. 4 Upper: Inhibition of 3H- ohimbine specific binding by agonists and antagonists. 0.2 nM Y H-yohimbine was incubated with platelet membranes (0.4 mg protein) and various concentrations of unlabeled drugs as described in Materials and Methods. Points shown are the means of two experiments, each performed in duplicate. Corresponding values from each experiment differed by less than 5% from each other. Lower: logit-log slopes of inhibition curves. Dotted lines represent a slope of 1.0.
inhibition of binding by (-)-epinephrine and (-)-norepinephrine was shallow (nH 0.61 and 0.67), suggesting a two-site interaction. In some competition experiments both catecholamines at low concentrations (lo-10 - 10-g M) appeared to cause a slight increase in 3H-yohimbine binding (Fig. 4). This effect was not however consistently seen in every experiment, and its significance is unclear at present. Clonidine and p-aminoclonidine inhibited 90% of 3H-yohimbine binding with nH close to 1.0, but appeared to inhibit the initial 10% of binding with greater affinity, while phentolamine inhibited binding with nH of about 1.0 (Fig. 4).
3H-Prazosin and AH-WB-4101 binding to platelet membranes: 3HPrazosin labels al-adrenergic receptors in brain and peripheral tissues with high selectivity and affinity (KD 0.1 - 1.0 nM) (26,30). In human platelet membranes, we observed no specific binding of 3H-prazosin at 0.3 - 2.6 nM radioligand concentrations, confirming previous findings that platelets contain only the ap-type of adrenergic receptor (8, 18).
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Platelet 3H-yohimbine Binding
Vol. 28, No. 24, 1981
In brain membranes, 3H-WB-4101 is also a highly selective label for "IHowever it has been reported to have decreased al-receptor receptors (1,29). selectivity in some peripheral tissues (42), and recently both tritiated and unlabeled WB-4101 were found to label rabbit uterine oI- and a2-receptors with the same high affinity (43). The inhibitory potency of unlabeled WB-4101 at platelet 3H-yohimbine sites (Table 1) was 80-100 times greater than its Since potency at rat brain a2-receptor sites labeled by 3H-clonidine (29). binding are those of an o2the pharmacological properties of 3H-yohimbine and kinetic, saturation and antagonist inhibition studies show that receptor, 3H-yohimbine labels one receptor type, it appears that platelet and brain o2receptors have structural differences reflected in the disparity of WB-4101 potencies. Specific NE-displaceable 3H-WB-4101 binding to human platelet membranes was observed and in three saturation studies, over a concentration range of 0.1 - 6.0 nM jH-WB-4101 the apparent KD of 3H-WB-4101 was 2.0 + 0.5 nM, Thus, contrary similar to the Ki of WB-4101, inhibiting 3H-yohimbine binding. to previous suggestions (l), but in support of recent findings in the rabbit results indicate that 3H-WB-4101 in some tissues uterus (43), the present 3H-WB-4101, however, labeled many fewer sites on may label o2-receptors. platelet membranes (Bmax 42+4 fmoles/mg protein) than did 3H-yohimbine, for reasons which are uncleardt present. Compari_son of platelet ?H-yohimbine and ?H-DHEC binding: Specific binding of JH-DHEC to membranes obtained from 2-3 day old platelets was 50-70X of total binding when 0.5 nM 3H-DHEC and 1.0 mg protein/assay were used. In two saturation experiments, 3H-DHEC bound to a single order of sites with an apparent KD value of 4.9 + 1.2 nM, and a Bmax value of 427 + 5 fmoles/mg protein, or 134 + 10 receptors per cell. 3H-Yohimbine binding to the same batch of membranes was also monophasic (KD 1.1 + 0.2 nM), but the number of sites labeled (256 + 32 fmoles/mg protein; 82 + 5 receptors per cell) was only 60% of the number of 3H-DHEC sites. ConGivably, 3H-DHEC could label another population of o2-receptors, which, like rat brain, have lower affinity for yohimbine and WB-4101. To test this hypothesis, we analyzed the inhibition of platelet 3H-DHEC binding by yohimbine, WB-4101 and phentolamine (Fig. 5). While the IC50 for phentolamine inhibiting 3H-DHEC was similar to its apparent Ki in inhibiting 3H-yohimbine (Table 1) , Thimbine and WB-4101 were 6-7 times less potent inhibitors of 3H-DHEC than of H-yohimbine binding. In addition, the curve for inhibition of 3H-DHEC binding by WB-4101 was shallow with a definite tail at higher concentrations (nH = 0.75). However in these experiments the curve for yohimbine inhibition of 3H-DHEC binding Thus the present results, while indicating some was steep (nH = 0.96). anomalies in antagonist competition at 3H-DHEC sites, do not provide a conclusive explanation for the discrepancy in our studies between the B,,, of 3H-yohimbine and 3H-DHEC.
Alexander et al. (16) 3H-Yohimbine binding to fresh platelet membranes: have reported that the number of JH-DHEC u2-receptor sites is about 60% greater in membranes prepared from freshly-obtained platelets compared to stored plateWe examined the saturation characteristics of 3H-yohimbine binding in lets. platelet membranes prepared on the same day from blood from seven normal volunteers. The values (mean + S.E.M.) for apparent KD (1.16 + 0.08nM) and Bmax (168 + 16 fmoles/mg protein; 93 + 12 receptors per cell) of3H-yohimbine were not sTgnificantly different from-those obtained using membranes from 2-3 day old platelets.
Vol. 28, No. 24, 1981
Platelet 3H-yohimbine Binding
IC,&nM ~
I
2715
“H -
.
150
0
56
0 75 096
A
3.9
0 80
-loo IINHIBITOR~CM)
FIG.
5
Inhibition of 3H-DHEC specific binding by antagonists. 0.5 nM 3H-DHEC was incubated with platelet membranes (1.0 mg protein) and various concentrations of unlabeled drugs as described in Materials and Methods. Points shown are the means of three experiments, each performed in triplicate. IC50 and nH values were obtained from logit-log slopes of inhibition curves shown.
Discussion
The major finding of this study is that the antagonist radioligand 3Hyohimbine labels a single class of sites on human platelet membranes as judged and competition by unlabeled antagonfrom kinetic and saturation experiments, ists. The relative potencies of a series of agonist and antagonist competitors The of binding indicate that 3H-yohimbine binds to an a2-adrenergic receptor. indicates that human absence of specific high-affinity binding of 3H-prazosin platelets possess no al-receptor.
Catecholamine agonists, but not imidazoline drugs or antagonists, exhibited shallow competition curves in inhibiting 3H-yohimbine binding, with nH values of about 0.7. Similar shallow curves in the absence of guanine nucleotides and sodium ion have been observed for agonist interactions at platelet 3H-DHEC sites (34,39), and these data have been fitted to a model where the platelet a2-receptor exists in two affinity states for agonists, CY~(H) and az(L)(lB). The apparent Ki values for agonists inhibiting platelet 3H-yohimbine binding are IO-50 times greater than Ki values for the same drugs inhibiting 3Hcatecholamine and 3H-imidazoline binding to a2-receptors in brain membranes
2716
Platelet 3H-yohimbine Binding
Vol. 28, No. 24, 1981
[$i384i;14), and at these brain sites agonist competitors exhibi;H_steep curves . Therefore it seems likely that 3H-catecholamines and imiiazolines selectively label the a2(H) form of the receptor, while 3Hyohimbine labels both a2(H) and a2(L). Recently we have shown that (-)-3Hepinephrine and 3H-p-aminoclonidine a2-receptor binding to human platelet membranes also shows high-affinity interactions for agonist competitors, indicating that these ligands selectively label the o2(H) form in platelets (45). In addition, agonist inhibition of platelet 3H-yohimbine binding is influenced by guanine nucleotides and metal ions in a manner analogous to 3H-DHEC sites (Daiguji et al., in preparation).
Brain and platelet a2-receptors are not identical with respect to antagonist affinities, since WB-4101 and yohimbine itself were much more potent competitors of platelet 3H-yohimbine binding than of rat brain 3H-catecholamine and 3H-imidazoline binding (38,44). Tissue, and possibly species, heterogeneity of a2-receptors has also been suggested on the basis of different potencies of WB-4101 in inhibiting the a2-receptor mediated suppression of NE release (42).
A comparison of platelet 3H-DHEC and 3H-yohimbine binding suggests that the latter radioligand may be more useful for biochemical and clinical studies. Firstly, in similar experimental conditions, specific binding is a much larger component of total ligand binding for 3H-yohimbine. Secondly, the observations that 3H-DHEC labels more platelet sites than 3H-yohimbine, and that some antagonist drugs inhibit 3H-DHEC in a manner indicating interactions at more than site, would suggest that NE- or phentolamine-displaceable platelet 3HDHEC binding incorporates some non-a2-receptor interactions.
Acknowledgements
This research was supported by USPHS grants NS-15591, MH-30059 and MH-30938, H.Y.M is recipient of and a grant-in-aid from the American Heart Association. We thank Joan Mitrius for excellent technical assistance. USPHS RCSA MH-47808.
References
1 2. 3. 4. 5. 6. 7. 8. 9. ::: 12.
D.C. U'PRICHARD and S.H. SNYDER, Life Sci. 24 79-88 (1979). B.R. ROUOT and S.H. SNYDER, Life Sci. 25 76F774 (1979). D.C. U'PRICHARD and S.H. SNYDER, J. NeFochem. 34 385-394 (1980). B. JARROTT, W.J. LOUIS and R.J. SUMMERS, Brit. x Pharmacol. -65 663-670 (1979). T. TANAKA and K. STARKE, Naunyn-Schmied. Arch. Pharmacol. 309 207-215 i1:7i)IkCH J P DAUSSE and P MEYER Nature (Lond ) 274 492-494 (1978) L'ife B:B: HOFFMAN; A. DELEAN, C.L:WOOD, b.D. SCHOCKEN and R.J. LEFKOWITZ, Sci. 24 1739-1746 (1979). C.L. WOO, C.D. ARNETT, W.R. CLARKE, B.S. TSAI and R.J. LEFKOWITZ, Biochem. Pharmacol. 28 1277-1282 (1979). B.B. HOFFMAN and R.JTLEFKOWITZ, Biochem. Pharmacol. 29 452-454 (1979). E.W. SALZMAN and L.L. NERI, Nature (Lond.) 224 609-617(1969). W. BARTHEL and F. MARKWARDT, Biochem. Pharmzl. 24 37-46 (1974). K.H. JAKOBS, W. SAUR and G. SCHULTZ, Mol. Pharmacg. (1978). -14 1073-1078
vol. 28, No. 24, 1981
13. ::: 16.
:i: 19. 20. 21. 22. 23.
Platelet 3H-yohimbine Binding
2717
GRANT and M.C. SCRUTTON, Nature (Land.) 277 659-661 (1979). M.S. KAFKA, J.F. TALLMAN and C.C. SMITH, Life-i. 21 1429-1438 (1977). K.D. NEWMAN, L.T. WILLIAMS, N.H. BISHOPRIC and R.J.TEFKOWITZ, J. Clin. Invest. 61 395-402 (1978).. R.W. ALEmNDER, B. COOPER and R.I.HANDIN, J. Clin. Invest. -61 11361144 (1978). B.B. HOFFMAN, and R.J. LEFKOWITZ, Ann. Rev. Pharmacol. 20 581-608 (1980). B.B. HOFFMAN, D. MULLIKIN-KILPATRICK and R.J. LEFKOWITZTJ. Biol. Chem. -255 4645-4652 (1980). B.B. HOFFMAN, T. MICHEL, D.M. KILPATRICK, R.J. LEFKOWITZ, M.E.M. TOLBERT, H. GILMAN and J.N. FAIN, Proc. Natl. Acad. Sci. USA 77 4569-4573 (1980). M.C. SCRUTTON and J.A. GRANT, Nature (Lond.) 280 70071979). J.M. ELLIOTT and D.G. GRAHAME-SMITH, Proc. Brit. Pharmacol. Sot., April 1980, p.19. S.J. PEROUTKA, D.A. GREENBERG, D.C. U'PRICHARD and S.H. SNYDER, Mol. Pharmacol. 14 403-412 (1978). D.C. U'PRICTiARD and S.H. SNYDER, in Animal Models in Psychiatry and ;;;~?;;o~;~~;~.HANIN and E. USDIN, Eds., pp. 477-495, Pergamon Press,
J.A.
A.H. DRUMMOND and J.L. GORDON, Biochem. J. 150 129-132 (1975). M.L. STEER, J. KHORANA and B. GALGOCI, Mol.Tarmacol. 16 719-728 (1979). D.C. U'PRICHARD, M.E. CHARNESS, D. ROBERTSON and S.H. SNYDER, Eur. J. Pharmacol. 50 87-89 (1978). 27. K. STARKE, E. mROWSK1 and T. ENDO, Eur. J. Pharmacol. 34 385-388 (1975). 28. D.A. GREENBERG and S.H. SNYDER, Mol. Pharmacol. 14 38-49(1978). Pharmacol. -13 29. D.C. U'PRICHARD, D.A. GREENBERG and S.H. SNYDER,%l. 454-473 (1977). P. GREENGRASS and R. BREMNER, Eur. J. Pharmacol. 55 323-326 (1979). 33:: O.H. LOWRY, N.J. ROSEBROUGH, A.L. FARR and R.J. RANDALL, 3. Biol. Chem. 193 265-275 (1951). __ 32. G. SCATCHARD, Ann. N.Y. Acad. Sci. 51 660-672 (1949). 33. J.Z. FIELDS, W.R. ROESKE, E. MORKINTnd H.I. YAMAMURA, J. Biol. Chem. 253 3251-3258 (1978). 34. B.S. TSAI and R.J. LEFKOWITZ, Mol. Pharmacol. 14 540-548 (1978). T. TANAKA and K. STARKE, Eur. J. Pharmacol. 63791-194 (1980). %: K. STARKE, T. END0 and H.D. TAUBE, Naunyn-Scfiied. Arch. Pharmacol. 291 55-78 (1975). 37. D.H. JENKINSON, D.G. HAYLETT, K. KOLLER and G. BURGESS, in Recent Advances in the Pharmacology of Adrenoceptors, E. SZABADI, -ADSHAW and P. BEVAN, Eds., pp. 23-33, Elsevier-North Holland, Amsterdam (1978). 38. D.C. U'PRICHARD, W.D. BECHTEL, B. ROUOT and S.H. SNYDER, Mol. Pharmacol. 16 47-60 (1979). 39. ES. TSAI and R.J. LEFKOWITZ, Mol. Pharmacol. 16 61-68 (1979). 40. P. LASCH and K.H. JAKOBS, Naunyn-Schmied. ArchTPharmacol. -306 119-125 (1979). 41. Y. CHENG and W.H. PRUSOFF, Biochem. Pharmacol. 22 3099-3108 (1973). 42. S.Z. LANGER, R. MASSINGHAM and N.B. SHEPPERSON,%oc. Brit. Pharmacol. sot., April 1980, p. 20. 43. B.B. HOFFMAN and R.J. LEFKOWITZ, Biochem. Pharmacol. 29 1537-1541 (1980). 44. D.C. U'PRICHARD and S.H. SNYDER, J. Biol. Chem. 52 64?%6463 (1977). 45. D.C. U'PRICHARD, J. MITRIUS, D.J. KAHN and M. DAXUJI, in Psychopharmacology and Biochemistry of Neurotransmitter Receptors, R.W. OLSEN and H.I. YAMAMURA, Eds., Elsevier North-Holland, Amsterdam, pp.247-259, 1980.
24. 25. 26.
Pergamon Press
HUMAN PLATELET a2-ADRENERGIC RECEPTORS: LABELING WITH 3H-YOHIMBINE, A SELECTIVE ANTAGONIST LIGAND Makoto Daiguji, Herbert Y. Meltzer and David C. U'Prichard Department of Pharmacology, Northwestern University School of Medicine, Chicago, Illinois 60611, and Department of Psychiatry, Chicago University Pritzker School of Medicine, Chicago, Illinois 60637 (Received in final form April 2, 1981) Summary 3H-Yohimbine, a potent and selective pharmacological antagonist of a2-adrenergic receptors, labeled human platelet membrane a2-receptors with high affinity. Binding was rapid Both saturation and kinetic experiand reversible at 25oC. ments indicated a single order of binding sites, with an equilibrium KO value of 1.0-1.5 nM. Low Mg2+ concentrations increased the KD for 3H-yohimbine without altering the Bmax. The 3H-yohimbine site exhibited a2-receptor specificity: (-)-norepinephrine and (-)-isoproterenol were 4.8 and 330 times less potent than (-)-epinephrine; (-)-catecholamines were 17-35 times more potent than corresponding (t)-catecholamines; the selective al-antagonist prazosin was 340 times less potent than yohimbine. Catecholamine agonists exhibited shallow curves in inhibiting 3H-yohimbine binding, with pseudo-Hill coefficients (nH) of less than 1.0, whereas the nH of antagonists was 1.0. No specific binding of 3H-prazosin to platelet membranes was observed, indicating the absence of a -receptors. 3H-Yohimbine labeled fewer olatelet sites than did 3H-a. ihydroergocryptine under identical conditions (80 vs 130 receptors/ cell), and may be a more s ecific and useful antagonist probe of platelet a2-receptors than !_ H dihydroergocryptine. a2-Adrenergic receptors have been extensively studied in brain and some peripheral tissues using agonist or partial agonist radioligands such as 3Hcatecholamines and 3H-imidazolines, which have selectively high affinity for this receptor subtype (l-5). Another approach to labeling the a2-receptor has been to examine the binding of the non-selective a-antagonist 3H-dihydroergocryptine (3H-DHEC), and to distinguish the a2-receptor component of 3H-DHEC binding, which has high affinity for the a2-selective antagonist yohimbine and low affinity for the al-selective antagonist prazosin (6-9). Human platelets contain an a-adrenergic receptor that mediates inhibition of basal and prostaglandin-stimulated adenylate cyclase activity (lo), and platelet aggregation (11). These responses appear to be “2, and not
0024-3205/81/242705-13$02.00/O Copyright (c) 1981 Pergamon Press Ltd.
2706
Platelet
3H-yohimbine
Binding
Vol.
28, No.
24,
1981
(12,13), and platelet radioligand binding studies, which have "13 in nature been mainlv oerformed usina 3H-DHEC (14-161. have indicated that olatelets contain a2-receptors (7,8).J From extensive' studies of the interaction of 3H-DHEC with platelet a2-receptors, Lefkowitz and co-workers have concluded like the erythrocyte B-receptor, can exist in two conthat this receptor, formations differentiated by high and low affinity respectively for agonists, and designated a2(H) and a2(L) (17-18). According to this model, 3H-agonist ligands label with high affinity only the a2(H) state, and not the total a2receptor population (19).
the selectivity of 3H-DHEC as a platelet a2-receptor probe Recently, has been questioned, as has the concept that human platelet membranes contain 3H-DHEC, an ergot alkaloid, only a2-receptors, and no al-receptors (20,21). has as high affinity for al-receptors (17,22) and for serotonin receptors (23) as for a2-receptors, and serotonin receptors have been suggested to occur on another platelet a-receptor probe platelet membranes (24). 3H-Phentolamine, (25) also has, like 3H-DHEC, equal affinity for al- and u2-receptors (26).
We therefore decided to characterize a2-receptors on human platelet Yohimbine is a potent and membranes using recently available 3H-yohimbine. selective a2-receptor antagonist in pharmacological studies (27), and exhibits 3H-DHEC sites and at the a2-receptor high affinity (Ki 1-2 nM) at platelet 3H-DHEC binding in other tissues containing both a-receptor component of The results obtained show that 3H-yohimbine selectively subtypes (7,8). labels human platelet a2-receptors, and appears to label fewer platelet membrane sites than 3H-DHEC under the same conditions. In addition, an examination of platelet 3H-prazosin binding indicates that human platelet membranes contain no al-receptors.
Materials --___
and Methods
The majority of experiments to characterize Platelet membrane preparation: 3H-vohimbine bindinq were performed usinq platelet-rich plasma (PRP) obtained from local blood banks. Membranes were prepared from PRP which had been stored Platelets at 250C for no more than three days from the time blood was drawn. were lysed and membranes prepared by the method of Newman et al. (15), with PRP was centrifuged at 300 x g for lOTinto eliminate some modifications. and the supernatant centrifuged at 16,000 x g for 10 min. residual erythrocytes, The resulting pellet was resuspended in ice-cold 50 mM Tris-HCl buffer (pH 8.0 at OoC) containing O.llM NaCl and 0.02M Na2EDTA (final pH 6.3j and centriAfter a further resuspension and centrifugafuged at 16,000 x g for 10 min. tion in the same buffer, washed intact platelets were resuspended in ice-cold 5.CmM Tris-HCl buffer (pH 8.0 at OoC) containing 5.0 mM Na2EDTA, and disrupted using a Brinkmann Polytron PT-10 (setting 5, 30 set) before cenAll the above procedures were pertrifugation at 39,000 x g for 10 min. Membrane pellets were rapidly frozen in an acetone-CO2 formed at 4oC. bath and stored at -700C until assay.
To examine binding to freshly prepared platelet membranes, blood samples were obtained from healthy human volunteers and centrifuged in citratecentrifuged at 16,000 dextrose at 600 x g for 2.5 min, and the supernatant All subsequent steps were carried out as above, except that x g for 10 min. 3H-yohimbine binding assays were performed on platelet membranes on the with no intervening -7OOC storage. day of membrane preparation,
Vol. 28, No. 24, 1981
Platelet 3H-yohimbine Binding
2707
Thawed or freshly prepared membrane pellets were Ligand binding assays: resuspended in 50 mM Tris-HCl (pH 7.0 at 25OC), centrifuged at 50,000 x g Incubaand resuspended in the same buffer for binding assays. for 10 min, tion tubes kept on ice received lo-60 ul diluted 3H-yohimbine (New England Nuclear, 80-90 Ci/mmole), 20 pl of various concentrations of drugs dissolved in 0.1% ascorbic acid, 1.0 ml of membrane suspension (0.4 mg membrane protein) Samples were pH 7.0, for a final volume of 2.0 ml. and 50 mM Tris-HCl, incubated with shaking for 60 min at 25oC, and the assay was terminated by The rapid filtration under reduced pressure over Whatman GF/B filters. filters were rinsed with 3 x 5 ml of ice-cold 50 mM Tris-HCl buffer (pH 8.0 at OoC), transferred to vials containing 8.0 ml Formula 947 (New England Nuclear) and subsequently counted by liquid scintillation spectrometry at 35% efficiency.
Non-specific binding was determined in parallel samples containing 100 pM (-)-norepinephrine (NE), and specific binding was obtained by subtracting nonIn typical experiments employing 0.2 nM 3Hspecific from total binding. yohimbine and 0.4 mg platelet membrane protein, total binding was 1500 c.p.m. and non-specific binding was 120 c.p.m. A variety of structurally dissimilar unlabeled yohimbine, at maximally effective agents, including a-receptor concentrations, inhibited 3H-yohimbine binding to the same extent as 100 pM Inhibition by two or more of these agents was not additive, indicat(-)-NE. ing that all a-receptor agents inhibited the same component of binding. The bound was always less than 10% of the added radioamount of 3H-yohimbine 3H-yohimbine (0.2 nM) binding to the filter paper represented activity. and was not inhibited by co-incubation only 2-3% of the total c.p.m. bound, with 100 pM (-)-NE.
3H-DHEC and 3H-WB-4101 binding to platelet membranes were performed as previously described in brain experiments (28,29), except that a 1.0 ml incubation volume was used for 3H-DHEC assays. 3H-prazosin binding to platelet membranes was assayed according to the methods of Greengrass and Bremner (30). Specific binding for these three ligands was defined as binding inhibited by 100 pM (-)-NE. Preliminary experiments showed that the same component of 3H-DHEC binding was inhibited by 100 pM (-)-NE or 10 uM phentolamine, a common indicator of specific binding in this assay (14-16). All assays were performed in triplicate or duplicate. Protein concentrations were determined by the method of Lowry (31), using bovine albumin as a standard, and specific binding is expressed as fmoles of 3H-yohimbine bound per mg platelet membrane protein. In several experiments, the numbers of platelets in 300 x g or 600 x g supernatants were determined using a Coulter counter, and binding constants were subsequently expressed as number of 3H-yohimbine sites per platelet.
In preliminary experiments, storage of platelet membranes at -7oOC for up to 15 days had no effect on specific binding of 3H-yohimbine. The effect of PRP storage time at 250C was assessed, and specific binding of 0.2 nM 3Hyohimbine declined from 22-24 fmol/mg protein (2 or 3 days old) to 14 and 9 fmol/mg protein after storage for 5 and 6 days respectively. Differences 3H-yohimbine binding to membranes from freshly-prepared platelets between and 3 day old platelets are discussed below. Dru s: Drugs were kindly donated by the following isomers Sterling-Winthrop); oxymetazoline (Schering), line and phentolamine (CIBA-Geigy); tramazoline and
--T-
sources: catecholamine tolazoline, naphazoclonidine (Boehringer
2708
Platelet
3H-yohimbine
Binding
Vol.
28, No.
24,
1981
Ingel heim); amidephrine (Mead Johnson); o-ergocryptine and dihydroergocryptine (Sandoz); yohimbine (Regis); WB-4101 (Ward Blenkisop); prazosin (Pfizer). p-kinoclonidine was obtained from Dr. Bruno Rouot, and a-yohimbine from Drs. Carl Roth and David J. Jones. Other drugs and reagents were obtained from commercial sources. Results
Kinetic characteristics of _?H-yohimbine binding: In preliminary experiments specific bindinq of 3H-vohimbine at equilibrium did not vary appreciably 'between pH 6.3- and 7.3; but decreased' sharply at pH 7.7-8.0: Binding was optimal at pH 7.0, which was chosen for further experiments. Specific binding of 0.2 nM 3H-yohimbine was linear at tissue concentrations between 0.25 and 2.0 mg protein/ml (data not shown).
Association of 3H-yohimbine binding to platelet membranes at 250C was rapid, with a t1/2 of 7 min (Fig. lA), reaching equilibrium levels by 4060 min. Specific binding was observed to decrease somewhat after 90 min An inincubation, possibly reflecting some degradation of the radioligand. cubation time of 60 min was chosen to represent equilibrium binding in subsequent experiments. 3H-Yohimbine associated to platelet sites in a hyperbolic manner (Fig. lA, Inset), and the mean observed initial rate constant (k,b) from three experiments was 0.076 + 0.021 min-l for 0.2 nM 3H-yohimbine. Dissociation of bound 3H-yohimbine at 250C was determined by incubating platelet membranes to equilibrium, and then adding 100 uM (-)-NE at time zero and measuring residual specific binding at subsequent time intervals (Fig. 1B). When plotted on a semilogarithmic scale, dissociation was linear, indicating a first-order process with a t1/2 of 12 min. The rate constant for dissociation (k-1) was 0,061 min-1. The second order rate constant for association (k of 3H-yohimbine binding was determined from the equation kl = (k,b - kl)/ [t3H-yohimbine] to be 7.5 x 107 ~-1 min-1, and the equilibrium dissociation constant (KD), determined from the ratio k_l/kl, was 0.81 nM.
Specific binding of 3H-yohimbine to platelet membranes was saturable with increasing concentrations of ligand (Fig. 2). Non-specific binding increased linearly between 0.1 and 8 nM, whereas specific binding plateaued between 3 and 8 nM. A Scatchard (32) plot of the data indicated a single population of sites with an apparent KD of 1.23 nM, similar to the value obtained from kinetic experiments, and a maximum number of sites (Bmax) on membranes prepared from 2-3 day old platelets of 200 fmoles/mg protein, or about 80 binding sites per cell. From 5 similar experiments, the mean values (+ S.E.M.) were 1.25 + 0.10 nM (KD) and 182 + 29 fmoles/mg protein (Bmax). fie mean Hill coefficient (nH) of binding of-H-yohimbine was 0.93 + 0.05, In these experimenTs, the indicating the absence of cooperative interactions. binding site concentration was 30-40 pM (0.4 mg protein per assay), approximately 5% of the ligand KD value. Experiments were performed to determine whether the apparent KD value of 3H-yohimbine would decrease with decreasing binding site concentration (33). The KD was not however si nificantly different at membrane protein concentrations of 0.1 mg/ml (1.02 nM 3 , 0.5 mg/ml (1.03 nM) and 1.0 mg/ml (1.07 nM). Low concentrations of magnesium ion in the assay medium decreased the specific binding of 3H-yohimbine (Fig. 3). Tsai and Lefkowitz have prethe affinity of catecholamine agonists viously observed that Mg2+ increased
Platelet 3H-yohirnbineBinding
vol. 28, No. 24,1981
800
[
700
protein]
A
nM
[YOH]=OZ
2709
=0.4 mg /assay
20
200-e I
0
IO
60
20
mln
,~~_~~__~_~~~~~~~_“~__~~~” 3
1 0
40
non-speclfk
1
/
20
I
40 TIME
60
80
(rnln)
0
1603 FIG.
40
20 TIME
60
(mln)
1
Kinetics at 25°C of 3H-yohimbine binding to human platelet membranes (A) Association: 0.2 nM 3H-yohimbine was incubated with membranes for various times before filtration in the absence (total) or presence (non-specific) of 100 uM (-)-NE. Insert: semi-logarithmic plot of increase in specific binding with time. (B) Dissociation: 0.2 nM 3H-yohimbine was incubated to equilibrium (60 min) with or without 100 uM (-)-NE, before addition of 100 pM(-)-NE to all Samples were filtered at various times thereafter. Insert: samples. semi-logarithmic plot of decrease in specific binding with time. Values are from a representative experiment performed in triplicate.
sites labeled by 3H-DHEC (34). Thus the decrease H yohimbine binding could be due to enhanced interactions of residual circulating catecholamines with "2 (H) sites on platelet membranes. However the fact that the platelet membranes were repeatedly washed, and that the decrease in binding was due to a dose-dependent increase in the apparent KB of 3H-yohimbine with increasing Mg2+ (Fig. 3, Inset), rather than a reduction in the number of sites, suggested that this was not the explanation.
p;
latelet T_ . .-c1 receptor
Pharmacological properties of ?H-yohimbine binding: Catecholamines and other drugs inhibited JH-yohimbineTnding to membranes obtained from 2-3 day old platelets with a potency order suggesting an o-adrenergic receptor
Platelet
2710
3H-yohimbine
vol.
Binding
28, No.
24,
1981
Thus (-)-epinephrine was 4.8 times more potent than interaction (Table 1). (-)-NE and 330 times more potent that the B-receptor agonist (-)-isoproterenol. since (-)-epinephrine and (-)-NE were Marked stereoselectivity was observed, Imidazoline 20 and 16 times more potent than their respective (+)-isomers. binding, with apparent derivatives were very potent inhibitors of 3H-yohimbine Among Ki values ranging from 6.0 nM (oxymetazoline) to 52 nM (clonidine). inhibitor was dihydroergocryptine a-receptor antagonists, the most potent (Ki 600 PM), followed by the a-stereoisomer of yohimbine (rauwolscine), aergocryptine and yohimbine itself (Kis 670, 760 and 820 PM). That 3H-yohimbine receptor on platelet membranes was specifically labeled an a2-adrenergic indicated by the 450-fold greater affinity of yohimbine compared to prazosin, the greater affinity of rauwolscine compared to yohimbine (35), and the much greater affinity of clonidine compared to the selective al-receptor agonists Thus the affinity of yohimbine (-)-phenylephrine (36) and amidephrine (37). at platelet a -receptors is substantially greater than at brain a2-receptors labeled with 32H-clonidine (29, 38) or 3H-p-aminoclonidine (2).
KO=I 23nM
54
k I
/,I
I.7
100
d&specific I
2
j50=
, , ;l. B(fmol/mg ,
1
3
4
I
5
FIG.
200
prot) I
I
6
7
I
I
I
8
2
3H-Yohimbine binding to human platelet membranes (0.4 mg protein) Nonas a function of increasing concentrations of 3H-yohimbine. specific binding was determined by incubating samples with 100 uM Values are (-)-NE. Insert: Scatchard plot of specific binding. from a representative experiment performed in triplicate.
Lefkowitz and colleagues have demonstrated that at platelet 3H-DHEC binding sites, full agonists such as catecholamines exhibit shallow inhibition with pseudo-Hill coefficients (nH) less than 1.0, which are made curves, steeper and shifted in the direction of decreased affinity by guanine nucleoantagonists on the other hand have steep curves (nH equals tides or sodium; and partial agonists show intermediate 1.0) and no nucleotide-or sodium-shift, These data have been modeled to two steepness of slope and shifts (34,39). states
of
the
platelet
a2-receptor
differing
in
affinity
for
agonists
(18).
Vol. 28,
No.
24,
Platelet 3H-yohimbine Binding
1981
//
I
I
I
lwg2+
300
(mM)
D
0 1
A
5
l
.
200
\
100
‘lo
(ii,
I 14 I. 39 IQ0
200
B (fmol/mg I
I
1
I
I 2
300 prot)
I 3
1 4
[MAGNESIUM],
mM
I
I
FIG.
I 5
3
Effects of magnesium on 3H-yohimbine binding. 0.2 nM 3H-yohimbine was incubated with platelet membranes (0.4 mg protein) as described in Materials and Methods in the absence and presence of various Scatchard plots of Insert: concentrations of magnesium chloride. 3H-yohimbine saturation isotherms determined in the presence of 0,l or 5 mM magnesium. Values are from a representative experiment In three such experiments, mean K values performed in duplicate. for 3H-yohimbine (k S.E.M.) were 1.14 k 0.03 nM (no Mg 2P2, 1.38 f 0.09 nM (1.0 mM Mg2+) and 1.93 f 0.03 nM (5.0 mM Mg2 ). No curvilinearity of the significant changes in B,,,, or significant Scatchard plots were detected at any magnesium concentration.
2711
2712
Platelet
3H-yohimbine
TABLE
INHIBITION OF PLATELET a-ADRENERGIC
Drugs
Vol.
Binding
,,1,L;;;o;;iterenol OxymetazoTine Naphazoline Tramazoline p-Aminoclonidine Clonidine
3H-YOHIMBINE BINDING BY RECEPTOR AGENTS
wgocryptine Rauwolscine a-Ergocryptine Yohimbine WB-4101 Phentolamine Piperoxan Tolazoline Prazosin
"H
75 360 620 1500 5900 25000
+ T T T T z
20 50 160 400 600 4000
(4) (4) (4) (3) (3) (3)
0.72 0.71 0.70 0.69 0.80 0.66
+ T 7 T T 5
0.04 0.04 0.06 0.03 0.04 0.06
6.0 15 20 29
+ T T +
1.4 1.3 2.8 5.5
(3) (3) (3) (4)
11
(4)
1.06 + 0.83 T 1.00 T 0.94 T l.OlZ
0.11 0.01 0.09 0.05 0.04
920 + 19 6700 z 640
(3) (3)
0.73 + 0.02 1.03 z 0.05
0.60 + 0.67 + 0.76 T 0.82 + 2.4 T 3.77 13.6> 210 ? 370 5
(4) (4) (3) (6) (3) (5) (3) (3) (3)
0.88 +
0.04
1.04 0.95 0.98 0.98 0.96 0.92 0.93
0.03 0.03 0.05 0.04 0.09 0.06 0.09
52+-
Amidephrine
24, 1981
1
Ki(nM)
Catecholamines (-)-Epinephrine (-)-Norepinephrine (-)-a-Methylnorepinephrine (+)-Epinephrine (+)-Norepinephrine
28, No.
0.22 0.14 0.05 0.24 0.6 0.9 2.3 50 120
+ T T + + T 5
Platelet membranes were incubated with 0.2 nM 3H-yohimbine and 6-18 concenin Materials and Methods. trations of unlabeled drugs, as described IC50 values (concentration inhibiting 50% of specific binding) and pseudo-Hill Coefficients (nH) were determined from Hill plots of the data, and apparent Ki values were derived using the formula of Cheng and Prusoff (41): Ki = IC5O/(l + [3H-YOH]/KD), where Kg of 3H-yohimbine is 1.25 nM. Values are mean -+ S.E.M. from number of experiments shown in parentheses.
binding revealed Analysis of the nH values for competitors of 3H-yohimbine similar properties for agonist and antagonist interactions at this site All catecholamine competitors had nH values of about 0.7, whereas (Table 1). Imidazoall antagonists had nH values not significantly different from 1.0. lines are generally believed to be basically antagonists with little or no intrinsic activity at platelet a2-receptors (40), and had nH values of about 1.0.
The examined
inhibition of in more detail
platelet (Fig. 4).
3H-yohimbine Over a 10-8
binding by a few drugs was - lo-5 M concentration range,
28, No.
Vol.
24,
1981
Platelet
3H-yohimbine
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Binding
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FIG. 4 Upper: Inhibition of 3H- ohimbine specific binding by agonists and antagonists. 0.2 nM Y H-yohimbine was incubated with platelet membranes (0.4 mg protein) and various concentrations of unlabeled drugs as described in Materials and Methods. Points shown are the means of two experiments, each performed in duplicate. Corresponding values from each experiment differed by less than 5% from each other. Lower: logit-log slopes of inhibition curves. Dotted lines represent a slope of 1.0.
inhibition of binding by (-)-epinephrine and (-)-norepinephrine was shallow (nH 0.61 and 0.67), suggesting a two-site interaction. In some competition experiments both catecholamines at low concentrations (lo-10 - 10-g M) appeared to cause a slight increase in 3H-yohimbine binding (Fig. 4). This effect was not however consistently seen in every experiment, and its significance is unclear at present. Clonidine and p-aminoclonidine inhibited 90% of 3H-yohimbine binding with nH close to 1.0, but appeared to inhibit the initial 10% of binding with greater affinity, while phentolamine inhibited binding with nH of about 1.0 (Fig. 4).
3H-Prazosin and AH-WB-4101 binding to platelet membranes: 3HPrazosin labels al-adrenergic receptors in brain and peripheral tissues with high selectivity and affinity (KD 0.1 - 1.0 nM) (26,30). In human platelet membranes, we observed no specific binding of 3H-prazosin at 0.3 - 2.6 nM radioligand concentrations, confirming previous findings that platelets contain only the ap-type of adrenergic receptor (8, 18).
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Platelet 3H-yohimbine Binding
Vol. 28, No. 24, 1981
In brain membranes, 3H-WB-4101 is also a highly selective label for "IHowever it has been reported to have decreased al-receptor receptors (1,29). selectivity in some peripheral tissues (42), and recently both tritiated and unlabeled WB-4101 were found to label rabbit uterine oI- and a2-receptors with the same high affinity (43). The inhibitory potency of unlabeled WB-4101 at platelet 3H-yohimbine sites (Table 1) was 80-100 times greater than its Since potency at rat brain a2-receptor sites labeled by 3H-clonidine (29). binding are those of an o2the pharmacological properties of 3H-yohimbine and kinetic, saturation and antagonist inhibition studies show that receptor, 3H-yohimbine labels one receptor type, it appears that platelet and brain o2receptors have structural differences reflected in the disparity of WB-4101 potencies. Specific NE-displaceable 3H-WB-4101 binding to human platelet membranes was observed and in three saturation studies, over a concentration range of 0.1 - 6.0 nM jH-WB-4101 the apparent KD of 3H-WB-4101 was 2.0 + 0.5 nM, Thus, contrary similar to the Ki of WB-4101, inhibiting 3H-yohimbine binding. to previous suggestions (l), but in support of recent findings in the rabbit results indicate that 3H-WB-4101 in some tissues uterus (43), the present 3H-WB-4101, however, labeled many fewer sites on may label o2-receptors. platelet membranes (Bmax 42+4 fmoles/mg protein) than did 3H-yohimbine, for reasons which are uncleardt present. Compari_son of platelet ?H-yohimbine and ?H-DHEC binding: Specific binding of JH-DHEC to membranes obtained from 2-3 day old platelets was 50-70X of total binding when 0.5 nM 3H-DHEC and 1.0 mg protein/assay were used. In two saturation experiments, 3H-DHEC bound to a single order of sites with an apparent KD value of 4.9 + 1.2 nM, and a Bmax value of 427 + 5 fmoles/mg protein, or 134 + 10 receptors per cell. 3H-Yohimbine binding to the same batch of membranes was also monophasic (KD 1.1 + 0.2 nM), but the number of sites labeled (256 + 32 fmoles/mg protein; 82 + 5 receptors per cell) was only 60% of the number of 3H-DHEC sites. ConGivably, 3H-DHEC could label another population of o2-receptors, which, like rat brain, have lower affinity for yohimbine and WB-4101. To test this hypothesis, we analyzed the inhibition of platelet 3H-DHEC binding by yohimbine, WB-4101 and phentolamine (Fig. 5). While the IC50 for phentolamine inhibiting 3H-DHEC was similar to its apparent Ki in inhibiting 3H-yohimbine (Table 1) , Thimbine and WB-4101 were 6-7 times less potent inhibitors of 3H-DHEC than of H-yohimbine binding. In addition, the curve for inhibition of 3H-DHEC binding by WB-4101 was shallow with a definite tail at higher concentrations (nH = 0.75). However in these experiments the curve for yohimbine inhibition of 3H-DHEC binding Thus the present results, while indicating some was steep (nH = 0.96). anomalies in antagonist competition at 3H-DHEC sites, do not provide a conclusive explanation for the discrepancy in our studies between the B,,, of 3H-yohimbine and 3H-DHEC.
Alexander et al. (16) 3H-Yohimbine binding to fresh platelet membranes: have reported that the number of JH-DHEC u2-receptor sites is about 60% greater in membranes prepared from freshly-obtained platelets compared to stored plateWe examined the saturation characteristics of 3H-yohimbine binding in lets. platelet membranes prepared on the same day from blood from seven normal volunteers. The values (mean + S.E.M.) for apparent KD (1.16 + 0.08nM) and Bmax (168 + 16 fmoles/mg protein; 93 + 12 receptors per cell) of3H-yohimbine were not sTgnificantly different from-those obtained using membranes from 2-3 day old platelets.
Vol. 28, No. 24, 1981
Platelet 3H-yohimbine Binding
IC,&nM ~
I
2715
“H -
.
150
0
56
0 75 096
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5
Inhibition of 3H-DHEC specific binding by antagonists. 0.5 nM 3H-DHEC was incubated with platelet membranes (1.0 mg protein) and various concentrations of unlabeled drugs as described in Materials and Methods. Points shown are the means of three experiments, each performed in triplicate. IC50 and nH values were obtained from logit-log slopes of inhibition curves shown.
Discussion
The major finding of this study is that the antagonist radioligand 3Hyohimbine labels a single class of sites on human platelet membranes as judged and competition by unlabeled antagonfrom kinetic and saturation experiments, ists. The relative potencies of a series of agonist and antagonist competitors The of binding indicate that 3H-yohimbine binds to an a2-adrenergic receptor. indicates that human absence of specific high-affinity binding of 3H-prazosin platelets possess no al-receptor.
Catecholamine agonists, but not imidazoline drugs or antagonists, exhibited shallow competition curves in inhibiting 3H-yohimbine binding, with nH values of about 0.7. Similar shallow curves in the absence of guanine nucleotides and sodium ion have been observed for agonist interactions at platelet 3H-DHEC sites (34,39), and these data have been fitted to a model where the platelet a2-receptor exists in two affinity states for agonists, CY~(H) and az(L)(lB). The apparent Ki values for agonists inhibiting platelet 3H-yohimbine binding are IO-50 times greater than Ki values for the same drugs inhibiting 3Hcatecholamine and 3H-imidazoline binding to a2-receptors in brain membranes
2716
Platelet 3H-yohimbine Binding
Vol. 28, No. 24, 1981
[$i384i;14), and at these brain sites agonist competitors exhibi;H_steep curves . Therefore it seems likely that 3H-catecholamines and imiiazolines selectively label the a2(H) form of the receptor, while 3Hyohimbine labels both a2(H) and a2(L). Recently we have shown that (-)-3Hepinephrine and 3H-p-aminoclonidine a2-receptor binding to human platelet membranes also shows high-affinity interactions for agonist competitors, indicating that these ligands selectively label the o2(H) form in platelets (45). In addition, agonist inhibition of platelet 3H-yohimbine binding is influenced by guanine nucleotides and metal ions in a manner analogous to 3H-DHEC sites (Daiguji et al., in preparation).
Brain and platelet a2-receptors are not identical with respect to antagonist affinities, since WB-4101 and yohimbine itself were much more potent competitors of platelet 3H-yohimbine binding than of rat brain 3H-catecholamine and 3H-imidazoline binding (38,44). Tissue, and possibly species, heterogeneity of a2-receptors has also been suggested on the basis of different potencies of WB-4101 in inhibiting the a2-receptor mediated suppression of NE release (42).
A comparison of platelet 3H-DHEC and 3H-yohimbine binding suggests that the latter radioligand may be more useful for biochemical and clinical studies. Firstly, in similar experimental conditions, specific binding is a much larger component of total ligand binding for 3H-yohimbine. Secondly, the observations that 3H-DHEC labels more platelet sites than 3H-yohimbine, and that some antagonist drugs inhibit 3H-DHEC in a manner indicating interactions at more than site, would suggest that NE- or phentolamine-displaceable platelet 3HDHEC binding incorporates some non-a2-receptor interactions.
Acknowledgements
This research was supported by USPHS grants NS-15591, MH-30059 and MH-30938, H.Y.M is recipient of and a grant-in-aid from the American Heart Association. We thank Joan Mitrius for excellent technical assistance. USPHS RCSA MH-47808.
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