- Email: [email protected]
Properties of [3H]diazepam binding sites on rat blood platelets
Life Sciences, Vol. 27, pp. 1881-1888 Printed in the U.S.A.
Pergamon Press
PROPERTIES OF [3H]DIAZEPAM BINDING SITES ON RAT BLOOD PLATELETS James K. T. Wang, Takashi Tanlguchl I and Sydney Spector I Department of Pharmacology, Columbia University, New York, New York 10032 and IDepartment of Physiological Chemistry and Pharmacology Roche Institute of Molecular Biology Nutley, New Jersey 07110 (Received in final form September 9, 1980) S,,mm~ry Intact rat blood platelets are shown to possess benzodlazepine binding sites of the peripheral type, binding of [3H]diazepam being strongly inhibited by Ro5-4864 (Ki = 3.6 ± 0.5 nM) but only weakly inhibited by clonazepam (Ki - 35.1 + 18.2 ~M). Binding of [3H]dlazepam is specific and saturable, Seatchard analysis reveals a single class of bindlng sites with K D = 14.7 ± 1.0 nM and Bma x = 564 ± 75 fmoles/108 platelets. The Hill coefficient is 0.94, indicating a lack of binding site heterogeneity or negative cooperativity. Binding reaches equilibrium at 6 mln, with k+l = 2.9 x 107 M-I min -I, and is rapidly reversible (tl/2 = 2.2 min) with k_i=0.315 mln -I. K D derived from the rate constants agrees with that estimated by Scatchard analysis. K D of the crude membrane fraction of platelets is also close to that of intact platelets. Binding of [3H]dlazepam is linear with platelet number (between 0.25-2 x 108 platelets), is temperature sensitive with maxlmumbinding at 0°C, and has a broad optimal pH range between pH 5-9. The benzodiazeplnes are widely used drugs that have been extensively studied. Their major clinical use has been to treat the symptoms of anxiety, but they also produce muscle relaxant, anticonvulsant and sedative-hypnotic effects (1,2,3). Specific benzodiazepine binding sites with high affinity for [3H]diazepam have been identified in the rat brain (4,5,6) and are thought to be of physiologic significance. Binding sites for [3H]diazepam also exist in peripheral tissues such as lung, liver and kidney (7). These binding sites are fundamentally different from the brain sites, as their affinities for dlazepam are 5-30 times lower and they exhibit a different specificity profile (7, 8). The peripheral binding sites have not been as intensively investigated as the brain sites, probably because all clinically used benzodiazepine compounds are presumed to act centrally. We have initiated a series of investigations into the peripheral benzodlazeplne binding sites. Recently, we demonstrated the presence of the peripheral sites on rat peritoneal mast cells and reported the first detailed characterization of these sites (8). The present study characterizes the binding of [3H]dlazepam to rat platelets.
0024-3205/80/461881-08502.00/0 Copyright (c) 1980 Pergamon Press Ltd
1882
[3H]Diazepam Binding to Rat Platelets
Vol. 27, No. 20, 1980
Methods Preparation of intact platelets and platelet crude membrane fraction. Male Sprague-Dawley rats (Camm Research Inc., Wayne, NJ) weighing 200 to 300 g each were anesthesized with ether and their abdominal cavities opened. Blood (10-15 ml from each rat) was withdrawn via the portal vein into a plastic syringe containing the anticoagulant described by Rossi (9). The platelets were isolated and washed once according to Rossi (9), the only modification being the addition of 25 mM Tris HCI to the medium. The platelets were finally resuspended in the modified medium at a concentration of 1-2 x 109 platelets/ml. To prepare the crude membrane fraction platelets, isolated as described, were lysed in 5 mM Tris HCI, pH 7.5 and subjected to 20 sec of homogenization by a Brinkmann (Westbury, NY) Polytron at maximum speed. The homogenate was then centrifuged at 46,000 x g for i0 min at 4°C. The total period of exposure to 5 mM Tris HCI before centrifugation was 3 min. The supernatant was discarded and the pellet resuspended in 50 mM Tris HCI, pH 7.5. The final suspension was examined under phase-contrast microscope and showed complete lysis of platelets. [3H]Diazepam bindin$ assay. Binding of [3H]diazepamwas assayed by incubating 0.5-1 x i0 ~ platelets (preincubated at 0°C for at least i0 min) in 250 ~I total volume of the modified medium containing 2.9 rum [~]diazepam, with or without i0 ~ M unlabeled diazepam. Incubation was carried out at 0°C for ii min. Reaction was terminated by the addition of 3 ml of ice-cold Dulbecco's phosphate-buffered s~line and rapid filtration through Whatman GF/B glass fiber filters under reduced pressure. The incubation tube and the filter were then washed twice with 3 ml of ice-cold phosphate-buffered saline. The filters were dried and placed in mini-scintillation vials. Four ml of Aquasol (New England Nuclear, Boston, MA) was added to each vial before liquid scintillation counting. For the platelet crude membrane preparation the incubation conditions were similar, except that the buffer was 50 mM Tris HCI, pH 7.5 and 0.2 mg protein of the crude membrane fraction was used for each assay. All assays were done in duplicate. Nonspecific binding was defined as binding which was not displaced ~M of unlabeled diazepam. Total binding was defined as binding in the of unlabeled ligand. Specific binding was calculated as the difference total and nonspecific binding and was usually at least 90% of the total
by i0 absence between binding.
To determine whether [3H]diazepam was degraded after binding to platelets, binding assays were carried out at 0°C and 370C as described. The platelets were collected by centrifugation. The radioactivity in the pellet was extracted with 100% ethanol. This and aliquots of the supernatant were ehromatographed on thin layer plates in the following solvent systems: chloroform, acetone (9:1) and chloroform, heptane, acetic acid (5:5:1). Both the bound and free [3H]diazepam had Rf values identical to that of authentic [3H]dlazepam. Scatchard analysis, Hill plot and determination of rate constants were performed according to Bennett (i0). Protein concentrations were determined according to Lowry et al (ii) using bovine serum albumin as standard. Chemicals and Reasents. [3H]diazepam (83.5 Ci/nlnol) was obtained from New England Nuclear, Boston, MA., diazepam, clonazepam and Ro5-4864 were provided by Hoffmann-LaRoche Inc., Nutley, NJ. All other chemicals used were of reagent grade and were commercially available.
Vol. 27, No. 20, 1980
[3H]Diazepam Binding to Rat Platelets
1883
Results Saturability of [3H]diazepam binding. In intact platelets specific binding of increasing concentration of [~H]diazepum (1.5-58.0 ruM) was saturable while nonspecific binding increased linearly (Fig. i). Scatchard analysis indicated a single class of binding sites with an apparent equilibrium dissociation binding constant (Kn) of 14.7 -+ 1.0 rum (N=6). Maximum binding capacity (Bma x) was 564 + 75 fmoles[108 platelets (N=6), or about 3,400 binding sites per platelet. Hill plot of mean data from six saturation experiments resulted in a single straight line (Fig. 2) with a Hill coefficient of 0.94,which is indicative of binding site homogeneity and noncooperativity. The K D estimated from the Hill plot was 15.2 nM, almost identical to that derived from Scatchard analysis.
500
._e o 400 E Q Z
D O m
300
W N
200
5
-
i"I\
7
i
I00 I~9 1
0
0
20
30
40
50
60
[3HI DIAZEPAM (nM) Figure 1
Saturation of [3H]dlazepam binding. Intact platelets (i x 10S/assay) were incubated as described in Methods with increasing concentration of [3H]dlazepam (1.5-58.0 nM). A representative experiment out of a total of six is shown. Dark circles represent specific binding, clear circles represent nonspecific binding. A Scatchard plot of the data is shown in the inset. Bound = fmoles specifically bound [3H]diazepam per 1 x 108 platelets. Free = concentration of [3H]dlazepam (nM) present in the incubation medium. The regression llne (r = 0.97), determined by least-square fit, indicates a ~ of 14.3 nM and a Bma x of 564 fmoles/108 platelets.
1884
[3H]Diazepam Binding to Rat Platelets
I
I
1 0.5
I.O
Vol. 27, No. 20, 1980
I
0.5
-~
-O,5
o, -I.O
O
LOGIo
1.5
2.0
([3HIDIAZEPAM-nM)
Fisure 2. Hill plot of mean data from six saturation experiments. [3H]Diazepam-nM= concentration of [3H]diazepam (1.5-58.0 nM). B = fmoles specifically bound [3H]diazepam/10 8 platelets for each concentration of [3H]diazepam. Bma x = 564 fmoles/108 platelets. The line is determined by least-square fit (r = 0.99), with a slope of 0.94. K D is estimated as the abscissa value where the ordinate = O, which is 15.2 nM. Scatchard analysis of [3H]diazepam binding in the platelet crude membrane fraction (data not shown) yielded a K D of 19.2 ± 1.8 nM (N=5), similar to that found in intact platelets. The Bma x in the crude membrane preparation was 602 ± 71 fmoles/mg protein (N=5). Since there is i mg protein/7.1 x 108 platelets, this Bma x corresponds to only 85 fmoles/108 platelets, or 6.6 times lower than the Bma x of intact platelets. Rate constants of [3H]diazepam bindin$. The binding of [3H]diazepam was tlme dependent and equilibrium was achieved after 6 min (Fig. 3). The bimolecular rate constant of association (k+l) was 2.9 x 10 7 M-I min-l. Dissociation of [3H]diazepam binding was rapid and followed first order kinetics, with tl/2 = 2.2 min (Fig. 4) and a rate constant of dissociation (k_ 1 = 0.693/ tl/2) of 0.315 mln -I. The binding constant calculated from the equation K D = k_i/k+l was 10.9 nM, in good agreement with the Scatchard estimate of K D. Specificity of [3Hldiazepam bindin$. Binding of [3H]diazepam was specific and was not influenced by i0 p M of each of the following: norepinephrine, epinephrine, propranolol, phentolamine, serotonin, histamine, diphenhydramine, acetylcholine, atropine, carbachol, hexamethonium, morphine, levallorphan and y-aminobutyric acid (GABA).
Vol. 27, No. 20, 1980
[3H]Diazepam Binding to Rat Platelets
1885
40 O
E
"l-v
50
c~ Z D O El
20 LLI N
// C
10 ~ I
~
Jl
J~"
TIME (rnin)
I
I
0
I
I
,
I
i
I
I
I
5
I
l0
,
,
,
,
I
15
TIME (rain) Figure 3. Time course of [3H]diazepam binding in intact platelets. Platelets (0.5 x 108 ) were incubated as described in Methods for various periods of time. Each point is the mean ± S.E. of data from six experiments. Dark circles represent specific binding, clear circles represent nonspecific binding. The inset plots a regression line that was determined by least-square fit (r = 0.95). Ben = fmoles specifically bound [3H]diazepam at equilibrium, and B = ~moles specifically bound [3H]diazepam at time t. kob s is the t slope of this line and is 0.4. k+i can be calculated from the equation k+l = (kob s - k_ 1 )/[3H]diazepam, where k_ 1 is the rate constant of dissociation (Fig. 4) and [3H]diazepam is the concentration of labeled ligand (2.9 ruM). Unlabeled diazepam inhibited the binding of [3H]diazepamwith an IC50 of 18.6 ± 2.4 rum (N=8). Using the equation Ki = KD.IC50 /(L + KD) , where L = concentration of [3H]diazepam (2.9 nM), Ki was calculated to be 15.5 nM. This is very close to the KD values previously described. Ro5-4864, a 4-chloro derivative of dlazepam that is selective for the peripheral benzodiazeplne sites, was m o r e potent than diazepam and displayed a K i of 3.6 ± 0.5 nM (N=7). Clonazepam, which is selective for the brain sites, was very weak in displacing the binding of [3H]diazepam in intact platelets (K i = 35.1 ± 18.2 ~ M , N=4). Effects of platelet number t temperature and pH on [3H]dlazepam binding. Figure 5 shows that the binding of [JH]dlazepam increased linearly with increasing platelet number in the range of 0.25-2 x 10 s platelets while the nonspecific binding remained constant. [3H]Diazepam binding was also temperature sensitive. Binding was maximal at 0°C and decreased substantially as the temperature was raised to 37°C (Fig. 6). At 37°C, the amount of binding was only 38.6% of that occurring at O°C. The nonspecific binding, however, was not influenced by temperature changes. Figure 7 shows that the binding of [3H]diazepam had a broad pH optima (pH 5-9) with maximum binding at pH 7.4.
1886
[3H]Diazepam Binding to Rat Platelets
0
I00
I
I
I
Vol. 27, No. 20, 1980
I
1
Z
o m .J
W
50
X
o..~
30
-,
20
N
,
IC
I
2
0
,
I
,
3 TIME (rain)
I
,
4
I
5
Figure 4. Dissociation of [3H]diazepam binding. Platelets (i x 10 8) were incubated as described in Methods and after ii min at 0°C, unlabeled diazepam was added to a final concentration of i0 p M (time 0). Specifically bound counts were determined at the indicated times. Results are expressed as percent of counts present at i0 sec after a d d i t i o n of unlabeled diazepam.
I
"6
200
o z
o nn =E w N
IOO
'
I
'
I
J
//z l
I
2
3
P L A T E L E T N U M B E R (x IO8) Figure 5. [3H]Diazepam binding with increasing concentration of platelets. Incubations were performed as described in Methods. Dark circles represent specific binding, clear circles represent nonspecific binding. Each point is the mean ± S.E. of data from six experiments.
Vol. 27, No. 20, 1980
I
I
[3H]Diazepam Binding to Rat Platelets
I
I
'
I
E
60
a
60
z
(:3 Z
O t,n
0 m 40
40
,,¢ Q.
=E
N
1887
zo
_. 1~
1 0
r-
,
I I0
"2_
,
I 20
-i
,
I 30
,
2O
r-~ I 40
TEMPERATURE-°C
4
6
8
I0
pH
Figure 6. Effect of different incubation temperatures on [3H]diazepam binding. Platelets (i x 10 8 ) were incubated as described in Methods at the indicated temperatures. Dark circles represent specific binding, clear circles represent nonspecific binding. Each point is the mean ± S.E. of data from four experiments. Figure 7. Effect of different pH on [ ~]diazepam binding. Platelets (i x 10 8) were incubated as described in Methods at different pH. Dark circles represent specific binding, clear circles represent nonspecific binding. Each point is the mean ± S.E. of data from four experiments.
Discussion The benzodiazepine binding sites in peripheral tissues are distinct from those in brain, their specificity for clonazepam and Ro5-4864 being completely opposite to that in the brain (7,8). Clonazepam is very potent in the brain in displacing [3H]diazepam binding while Ro5-4864 is not effective. In contrast, in peripheral tissues Ro5-4864 is much more potent than clonazepam. Our data therefore indicate that the binding sites on platelets are of the peripheral type, the K i of Ro5-4864 (3.6 nM) being i0 ~ times lower than that of clonazepam (35.1 p M ) . These values are consistent with previous findings that in all peripheral tissues examined (lung, liver, kidney and mast cells) the IC50 of Ro5-4864 is always 3-4 orders of magnitude lower than that of clonazepam (7,8). The benzodiazepine binding sites on platelets exhibit high affinity, specificity and saturability, with excellent agreement between K D values derived from several approaches. Involvement of active uptake is unlikely since binding is maximal at 0°C, a temperature at which energy dependent uptake is probably not optimal. In addition, the crude membrane fraction of platelets shows the same K D for [ 3H]diazepam as the intact platelets. Although this evidence is also consistent with [3H]diazepam binding sites being located on plasma membrane, binding to platelet granules has not been ruled out. The far lower Bma x in the crude membrane fraction, as compared to the Bma x in intact platelets, may be due to loss or inactivation of binding sites during the severe treatment of the platelets.
1888
[3H]Diazepam Binding to Rat Platelets
Vol. 27, No. 20, 1980
It has been reported that GABA increases the affinity of the benzodiazepine receptors in the brain (12-15), and a model has been proposed linking the GABA system with the benzodiazepines (16). However, we were unable to detect any GABA binding sites on platelets (data not shown), nor did GABA affect the binding of [3H]diazepam to platelets. We have previously observed the lack of interaction between GABA and diazepam in rat mast cells (8), and this lack may be common to other peripheral benzodiazepine sites. The finding of specific and high affinity [3H]diazepam receptors in the brain has stimulated a search for an endogenous ligand for these receptors (17). Analogously we may ask if there exist any endogenous ligand(s) for the specific and high affinity [3H]diazepam binding sites on platelets. Platelets constitute a specialized component of circulating blood and play a critical role in the clotting mechanism. When stimulated they undergo a series of reactions that include shape change, adhesion, aggregation and release of granular content, all of which form the initial stages of a complex chain of events that could culminate in the formation of a blood clot. Neumann et al (18) have reported that in hyperlipidemic rats diazepam inhibits the enhancement of blood coagulation by Triton WR-1339. This suggests that diazepam might influence one or more of the platelet reactions. Studies are now in progress to ascertain the effects of benzodiazepine compounds on platelet functions. Acknowledgment The authors wish to thank Drs. A. J. Blume and F. L. Margolis for critical reading of the manuscript, and Ms. M. Radigan for her expert secretarial assistance. J. K. T. Wang was supported by NIH Training Grant No. GM-07182. References i. 2.
3. 4. 5. 6. 7.
G. ZBINDEN and L. O. RANDALL, Adv. Pharmacol. 5 : 2 1 3 - 2 9 1 (1967). L. O. RANDALL, W. SCHALLEK, L. H. STERNBACH and R. Y. NING, in Psychopharmacologlcal Agents, ed. by M. GORDON, Vol. 3: pp. 175-281, Academic Press, New York (1974). H. C. B. DENBER, in Psychopharmacological Treatment: Theory and Practice, ed. by H. C. B. DENBER, pp. 157-173, Marcel Dekker, Inc., New York (197D). R. F. SQUIRES and C. BRAESTRUP, Nature (London) 266:732-734 (1977). H. MOHLER and T. OKADA, Science 198:849-851 (1977). C. R. MACHERER, R. L. KOCHMAN, B. A. BIERSCHENK and S. S. BREMNER, J. Pharmacol. Exp. Ther. 206:405-413 (1978). C. BRAESTRUP and R. F. SQUIRES, Proc. Natl. Acad. Sci. USA 74:3805-3809
(1977). 8. 9. i0. ii. 12. 13. 14. 15. 16. 17. 18.
T. TANIGUCHI, J. K. T. WANG and S. SPECTOR, Life Sci. 2 7 : 1 7 1 - 1 7 8 (1980). E. C. ROSSl, J. Lab. Clin. Med. 78:483-498 (1971). J. P. BENNETT, In Neurotransmitte--~ Receptor Binding~ ed. by H. I. YAMAMURA, S. J. ENNA and M. J. KUIIAR, pp. 57-90, Raven Press, New York (1978). O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR and R. J. RANDALL, J. Biol. Chem. 193:265-275 (1951). J. F. TALLMAN, J. W. THOMAS and D. W. GALLAGER, Nature (London) 274: 383385 (1978). M. S. BRILEY and S. Z. LANGER, Eur. J. Pharmacol. __52:129-132 (1978). G. J. WASTEK, R. C. SPETH, T. D. REISINE and H. I. YAMAMURA, Eur. J. Pharmacol. 50:445-447 (1978). I. L. MARTIN---and J. M. CANDY, Neuropharmacol. 1 7 : 9 9 3 - 9 9 8 (1978). E. COSTA and A. GUIDOTTI, Ann. Key. Pharmacol. Toxicol. 19:531-545 (1979). J. F. TALLMAN, S. M. PAUL, P. SKOLNICK, D. W. GALLAGER, Science 207: 274281 (1980). E. NEUMANN, J. HORAK, B. CUPARENCU, M. CUCIANU and E. SEUSAN, Agressologie, 19 (3): 201-209 (i978).
Pergamon Press
PROPERTIES OF [3H]DIAZEPAM BINDING SITES ON RAT BLOOD PLATELETS James K. T. Wang, Takashi Tanlguchl I and Sydney Spector I Department of Pharmacology, Columbia University, New York, New York 10032 and IDepartment of Physiological Chemistry and Pharmacology Roche Institute of Molecular Biology Nutley, New Jersey 07110 (Received in final form September 9, 1980) S,,mm~ry Intact rat blood platelets are shown to possess benzodlazepine binding sites of the peripheral type, binding of [3H]diazepam being strongly inhibited by Ro5-4864 (Ki = 3.6 ± 0.5 nM) but only weakly inhibited by clonazepam (Ki - 35.1 + 18.2 ~M). Binding of [3H]dlazepam is specific and saturable, Seatchard analysis reveals a single class of bindlng sites with K D = 14.7 ± 1.0 nM and Bma x = 564 ± 75 fmoles/108 platelets. The Hill coefficient is 0.94, indicating a lack of binding site heterogeneity or negative cooperativity. Binding reaches equilibrium at 6 mln, with k+l = 2.9 x 107 M-I min -I, and is rapidly reversible (tl/2 = 2.2 min) with k_i=0.315 mln -I. K D derived from the rate constants agrees with that estimated by Scatchard analysis. K D of the crude membrane fraction of platelets is also close to that of intact platelets. Binding of [3H]dlazepam is linear with platelet number (between 0.25-2 x 108 platelets), is temperature sensitive with maxlmumbinding at 0°C, and has a broad optimal pH range between pH 5-9. The benzodiazeplnes are widely used drugs that have been extensively studied. Their major clinical use has been to treat the symptoms of anxiety, but they also produce muscle relaxant, anticonvulsant and sedative-hypnotic effects (1,2,3). Specific benzodiazepine binding sites with high affinity for [3H]diazepam have been identified in the rat brain (4,5,6) and are thought to be of physiologic significance. Binding sites for [3H]diazepam also exist in peripheral tissues such as lung, liver and kidney (7). These binding sites are fundamentally different from the brain sites, as their affinities for dlazepam are 5-30 times lower and they exhibit a different specificity profile (7, 8). The peripheral binding sites have not been as intensively investigated as the brain sites, probably because all clinically used benzodiazepine compounds are presumed to act centrally. We have initiated a series of investigations into the peripheral benzodlazeplne binding sites. Recently, we demonstrated the presence of the peripheral sites on rat peritoneal mast cells and reported the first detailed characterization of these sites (8). The present study characterizes the binding of [3H]dlazepam to rat platelets.
0024-3205/80/461881-08502.00/0 Copyright (c) 1980 Pergamon Press Ltd
1882
[3H]Diazepam Binding to Rat Platelets
Vol. 27, No. 20, 1980
Methods Preparation of intact platelets and platelet crude membrane fraction. Male Sprague-Dawley rats (Camm Research Inc., Wayne, NJ) weighing 200 to 300 g each were anesthesized with ether and their abdominal cavities opened. Blood (10-15 ml from each rat) was withdrawn via the portal vein into a plastic syringe containing the anticoagulant described by Rossi (9). The platelets were isolated and washed once according to Rossi (9), the only modification being the addition of 25 mM Tris HCI to the medium. The platelets were finally resuspended in the modified medium at a concentration of 1-2 x 109 platelets/ml. To prepare the crude membrane fraction platelets, isolated as described, were lysed in 5 mM Tris HCI, pH 7.5 and subjected to 20 sec of homogenization by a Brinkmann (Westbury, NY) Polytron at maximum speed. The homogenate was then centrifuged at 46,000 x g for i0 min at 4°C. The total period of exposure to 5 mM Tris HCI before centrifugation was 3 min. The supernatant was discarded and the pellet resuspended in 50 mM Tris HCI, pH 7.5. The final suspension was examined under phase-contrast microscope and showed complete lysis of platelets. [3H]Diazepam bindin$ assay. Binding of [3H]diazepamwas assayed by incubating 0.5-1 x i0 ~ platelets (preincubated at 0°C for at least i0 min) in 250 ~I total volume of the modified medium containing 2.9 rum [~]diazepam, with or without i0 ~ M unlabeled diazepam. Incubation was carried out at 0°C for ii min. Reaction was terminated by the addition of 3 ml of ice-cold Dulbecco's phosphate-buffered s~line and rapid filtration through Whatman GF/B glass fiber filters under reduced pressure. The incubation tube and the filter were then washed twice with 3 ml of ice-cold phosphate-buffered saline. The filters were dried and placed in mini-scintillation vials. Four ml of Aquasol (New England Nuclear, Boston, MA) was added to each vial before liquid scintillation counting. For the platelet crude membrane preparation the incubation conditions were similar, except that the buffer was 50 mM Tris HCI, pH 7.5 and 0.2 mg protein of the crude membrane fraction was used for each assay. All assays were done in duplicate. Nonspecific binding was defined as binding which was not displaced ~M of unlabeled diazepam. Total binding was defined as binding in the of unlabeled ligand. Specific binding was calculated as the difference total and nonspecific binding and was usually at least 90% of the total
by i0 absence between binding.
To determine whether [3H]diazepam was degraded after binding to platelets, binding assays were carried out at 0°C and 370C as described. The platelets were collected by centrifugation. The radioactivity in the pellet was extracted with 100% ethanol. This and aliquots of the supernatant were ehromatographed on thin layer plates in the following solvent systems: chloroform, acetone (9:1) and chloroform, heptane, acetic acid (5:5:1). Both the bound and free [3H]diazepam had Rf values identical to that of authentic [3H]dlazepam. Scatchard analysis, Hill plot and determination of rate constants were performed according to Bennett (i0). Protein concentrations were determined according to Lowry et al (ii) using bovine serum albumin as standard. Chemicals and Reasents. [3H]diazepam (83.5 Ci/nlnol) was obtained from New England Nuclear, Boston, MA., diazepam, clonazepam and Ro5-4864 were provided by Hoffmann-LaRoche Inc., Nutley, NJ. All other chemicals used were of reagent grade and were commercially available.
Vol. 27, No. 20, 1980
[3H]Diazepam Binding to Rat Platelets
1883
Results Saturability of [3H]diazepam binding. In intact platelets specific binding of increasing concentration of [~H]diazepum (1.5-58.0 ruM) was saturable while nonspecific binding increased linearly (Fig. i). Scatchard analysis indicated a single class of binding sites with an apparent equilibrium dissociation binding constant (Kn) of 14.7 -+ 1.0 rum (N=6). Maximum binding capacity (Bma x) was 564 + 75 fmoles[108 platelets (N=6), or about 3,400 binding sites per platelet. Hill plot of mean data from six saturation experiments resulted in a single straight line (Fig. 2) with a Hill coefficient of 0.94,which is indicative of binding site homogeneity and noncooperativity. The K D estimated from the Hill plot was 15.2 nM, almost identical to that derived from Scatchard analysis.
500
._e o 400 E Q Z
D O m
300
W N
200
5
-
i"I\
7
i
I00 I~9 1
0
0
20
30
40
50
60
[3HI DIAZEPAM (nM) Figure 1
Saturation of [3H]dlazepam binding. Intact platelets (i x 10S/assay) were incubated as described in Methods with increasing concentration of [3H]dlazepam (1.5-58.0 nM). A representative experiment out of a total of six is shown. Dark circles represent specific binding, clear circles represent nonspecific binding. A Scatchard plot of the data is shown in the inset. Bound = fmoles specifically bound [3H]diazepam per 1 x 108 platelets. Free = concentration of [3H]dlazepam (nM) present in the incubation medium. The regression llne (r = 0.97), determined by least-square fit, indicates a ~ of 14.3 nM and a Bma x of 564 fmoles/108 platelets.
1884
[3H]Diazepam Binding to Rat Platelets
I
I
1 0.5
I.O
Vol. 27, No. 20, 1980
I
0.5
-~
-O,5
o, -I.O
O
LOGIo
1.5
2.0
([3HIDIAZEPAM-nM)
Fisure 2. Hill plot of mean data from six saturation experiments. [3H]Diazepam-nM= concentration of [3H]diazepam (1.5-58.0 nM). B = fmoles specifically bound [3H]diazepam/10 8 platelets for each concentration of [3H]diazepam. Bma x = 564 fmoles/108 platelets. The line is determined by least-square fit (r = 0.99), with a slope of 0.94. K D is estimated as the abscissa value where the ordinate = O, which is 15.2 nM. Scatchard analysis of [3H]diazepam binding in the platelet crude membrane fraction (data not shown) yielded a K D of 19.2 ± 1.8 nM (N=5), similar to that found in intact platelets. The Bma x in the crude membrane preparation was 602 ± 71 fmoles/mg protein (N=5). Since there is i mg protein/7.1 x 108 platelets, this Bma x corresponds to only 85 fmoles/108 platelets, or 6.6 times lower than the Bma x of intact platelets. Rate constants of [3H]diazepam bindin$. The binding of [3H]diazepam was tlme dependent and equilibrium was achieved after 6 min (Fig. 3). The bimolecular rate constant of association (k+l) was 2.9 x 10 7 M-I min-l. Dissociation of [3H]diazepam binding was rapid and followed first order kinetics, with tl/2 = 2.2 min (Fig. 4) and a rate constant of dissociation (k_ 1 = 0.693/ tl/2) of 0.315 mln -I. The binding constant calculated from the equation K D = k_i/k+l was 10.9 nM, in good agreement with the Scatchard estimate of K D. Specificity of [3Hldiazepam bindin$. Binding of [3H]diazepam was specific and was not influenced by i0 p M of each of the following: norepinephrine, epinephrine, propranolol, phentolamine, serotonin, histamine, diphenhydramine, acetylcholine, atropine, carbachol, hexamethonium, morphine, levallorphan and y-aminobutyric acid (GABA).
Vol. 27, No. 20, 1980
[3H]Diazepam Binding to Rat Platelets
1885
40 O
E
"l-v
50
c~ Z D O El
20 LLI N
// C
10 ~ I
~
Jl
J~"
TIME (rnin)
I
I
0
I
I
,
I
i
I
I
I
5
I
l0
,
,
,
,
I
15
TIME (rain) Figure 3. Time course of [3H]diazepam binding in intact platelets. Platelets (0.5 x 108 ) were incubated as described in Methods for various periods of time. Each point is the mean ± S.E. of data from six experiments. Dark circles represent specific binding, clear circles represent nonspecific binding. The inset plots a regression line that was determined by least-square fit (r = 0.95). Ben = fmoles specifically bound [3H]diazepam at equilibrium, and B = ~moles specifically bound [3H]diazepam at time t. kob s is the t slope of this line and is 0.4. k+i can be calculated from the equation k+l = (kob s - k_ 1 )/[3H]diazepam, where k_ 1 is the rate constant of dissociation (Fig. 4) and [3H]diazepam is the concentration of labeled ligand (2.9 ruM). Unlabeled diazepam inhibited the binding of [3H]diazepamwith an IC50 of 18.6 ± 2.4 rum (N=8). Using the equation Ki = KD.IC50 /(L + KD) , where L = concentration of [3H]diazepam (2.9 nM), Ki was calculated to be 15.5 nM. This is very close to the KD values previously described. Ro5-4864, a 4-chloro derivative of dlazepam that is selective for the peripheral benzodiazeplne sites, was m o r e potent than diazepam and displayed a K i of 3.6 ± 0.5 nM (N=7). Clonazepam, which is selective for the brain sites, was very weak in displacing the binding of [3H]diazepam in intact platelets (K i = 35.1 ± 18.2 ~ M , N=4). Effects of platelet number t temperature and pH on [3H]dlazepam binding. Figure 5 shows that the binding of [JH]dlazepam increased linearly with increasing platelet number in the range of 0.25-2 x 10 s platelets while the nonspecific binding remained constant. [3H]Diazepam binding was also temperature sensitive. Binding was maximal at 0°C and decreased substantially as the temperature was raised to 37°C (Fig. 6). At 37°C, the amount of binding was only 38.6% of that occurring at O°C. The nonspecific binding, however, was not influenced by temperature changes. Figure 7 shows that the binding of [3H]diazepam had a broad pH optima (pH 5-9) with maximum binding at pH 7.4.
1886
[3H]Diazepam Binding to Rat Platelets
0
I00
I
I
I
Vol. 27, No. 20, 1980
I
1
Z
o m .J
W
50
X
o..~
30
-,
20
N
,
IC
I
2
0
,
I
,
3 TIME (rain)
I
,
4
I
5
Figure 4. Dissociation of [3H]diazepam binding. Platelets (i x 10 8) were incubated as described in Methods and after ii min at 0°C, unlabeled diazepam was added to a final concentration of i0 p M (time 0). Specifically bound counts were determined at the indicated times. Results are expressed as percent of counts present at i0 sec after a d d i t i o n of unlabeled diazepam.
I
"6
200
o z
o nn =E w N
IOO
'
I
'
I
J
//z l
I
2
3
P L A T E L E T N U M B E R (x IO8) Figure 5. [3H]Diazepam binding with increasing concentration of platelets. Incubations were performed as described in Methods. Dark circles represent specific binding, clear circles represent nonspecific binding. Each point is the mean ± S.E. of data from six experiments.
Vol. 27, No. 20, 1980
I
I
[3H]Diazepam Binding to Rat Platelets
I
I
'
I
E
60
a
60
z
(:3 Z
O t,n
0 m 40
40
,,¢ Q.
=E
N
1887
zo
_. 1~
1 0
r-
,
I I0
"2_
,
I 20
-i
,
I 30
,
2O
r-~ I 40
TEMPERATURE-°C
4
6
8
I0
pH
Figure 6. Effect of different incubation temperatures on [3H]diazepam binding. Platelets (i x 10 8 ) were incubated as described in Methods at the indicated temperatures. Dark circles represent specific binding, clear circles represent nonspecific binding. Each point is the mean ± S.E. of data from four experiments. Figure 7. Effect of different pH on [ ~]diazepam binding. Platelets (i x 10 8) were incubated as described in Methods at different pH. Dark circles represent specific binding, clear circles represent nonspecific binding. Each point is the mean ± S.E. of data from four experiments.
Discussion The benzodiazepine binding sites in peripheral tissues are distinct from those in brain, their specificity for clonazepam and Ro5-4864 being completely opposite to that in the brain (7,8). Clonazepam is very potent in the brain in displacing [3H]diazepam binding while Ro5-4864 is not effective. In contrast, in peripheral tissues Ro5-4864 is much more potent than clonazepam. Our data therefore indicate that the binding sites on platelets are of the peripheral type, the K i of Ro5-4864 (3.6 nM) being i0 ~ times lower than that of clonazepam (35.1 p M ) . These values are consistent with previous findings that in all peripheral tissues examined (lung, liver, kidney and mast cells) the IC50 of Ro5-4864 is always 3-4 orders of magnitude lower than that of clonazepam (7,8). The benzodiazepine binding sites on platelets exhibit high affinity, specificity and saturability, with excellent agreement between K D values derived from several approaches. Involvement of active uptake is unlikely since binding is maximal at 0°C, a temperature at which energy dependent uptake is probably not optimal. In addition, the crude membrane fraction of platelets shows the same K D for [ 3H]diazepam as the intact platelets. Although this evidence is also consistent with [3H]diazepam binding sites being located on plasma membrane, binding to platelet granules has not been ruled out. The far lower Bma x in the crude membrane fraction, as compared to the Bma x in intact platelets, may be due to loss or inactivation of binding sites during the severe treatment of the platelets.
1888
[3H]Diazepam Binding to Rat Platelets
Vol. 27, No. 20, 1980
It has been reported that GABA increases the affinity of the benzodiazepine receptors in the brain (12-15), and a model has been proposed linking the GABA system with the benzodiazepines (16). However, we were unable to detect any GABA binding sites on platelets (data not shown), nor did GABA affect the binding of [3H]diazepam to platelets. We have previously observed the lack of interaction between GABA and diazepam in rat mast cells (8), and this lack may be common to other peripheral benzodiazepine sites. The finding of specific and high affinity [3H]diazepam receptors in the brain has stimulated a search for an endogenous ligand for these receptors (17). Analogously we may ask if there exist any endogenous ligand(s) for the specific and high affinity [3H]diazepam binding sites on platelets. Platelets constitute a specialized component of circulating blood and play a critical role in the clotting mechanism. When stimulated they undergo a series of reactions that include shape change, adhesion, aggregation and release of granular content, all of which form the initial stages of a complex chain of events that could culminate in the formation of a blood clot. Neumann et al (18) have reported that in hyperlipidemic rats diazepam inhibits the enhancement of blood coagulation by Triton WR-1339. This suggests that diazepam might influence one or more of the platelet reactions. Studies are now in progress to ascertain the effects of benzodiazepine compounds on platelet functions. Acknowledgment The authors wish to thank Drs. A. J. Blume and F. L. Margolis for critical reading of the manuscript, and Ms. M. Radigan for her expert secretarial assistance. J. K. T. Wang was supported by NIH Training Grant No. GM-07182. References i. 2.
3. 4. 5. 6. 7.
G. ZBINDEN and L. O. RANDALL, Adv. Pharmacol. 5 : 2 1 3 - 2 9 1 (1967). L. O. RANDALL, W. SCHALLEK, L. H. STERNBACH and R. Y. NING, in Psychopharmacologlcal Agents, ed. by M. GORDON, Vol. 3: pp. 175-281, Academic Press, New York (1974). H. C. B. DENBER, in Psychopharmacological Treatment: Theory and Practice, ed. by H. C. B. DENBER, pp. 157-173, Marcel Dekker, Inc., New York (197D). R. F. SQUIRES and C. BRAESTRUP, Nature (London) 266:732-734 (1977). H. MOHLER and T. OKADA, Science 198:849-851 (1977). C. R. MACHERER, R. L. KOCHMAN, B. A. BIERSCHENK and S. S. BREMNER, J. Pharmacol. Exp. Ther. 206:405-413 (1978). C. BRAESTRUP and R. F. SQUIRES, Proc. Natl. Acad. Sci. USA 74:3805-3809
(1977). 8. 9. i0. ii. 12. 13. 14. 15. 16. 17. 18.
T. TANIGUCHI, J. K. T. WANG and S. SPECTOR, Life Sci. 2 7 : 1 7 1 - 1 7 8 (1980). E. C. ROSSl, J. Lab. Clin. Med. 78:483-498 (1971). J. P. BENNETT, In Neurotransmitte--~ Receptor Binding~ ed. by H. I. YAMAMURA, S. J. ENNA and M. J. KUIIAR, pp. 57-90, Raven Press, New York (1978). O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR and R. J. RANDALL, J. Biol. Chem. 193:265-275 (1951). J. F. TALLMAN, J. W. THOMAS and D. W. GALLAGER, Nature (London) 274: 383385 (1978). M. S. BRILEY and S. Z. LANGER, Eur. J. Pharmacol. __52:129-132 (1978). G. J. WASTEK, R. C. SPETH, T. D. REISINE and H. I. YAMAMURA, Eur. J. Pharmacol. 50:445-447 (1978). I. L. MARTIN---and J. M. CANDY, Neuropharmacol. 1 7 : 9 9 3 - 9 9 8 (1978). E. COSTA and A. GUIDOTTI, Ann. Key. Pharmacol. Toxicol. 19:531-545 (1979). J. F. TALLMAN, S. M. PAUL, P. SKOLNICK, D. W. GALLAGER, Science 207: 274281 (1980). E. NEUMANN, J. HORAK, B. CUPARENCU, M. CUCIANU and E. SEUSAN, Agressologie, 19 (3): 201-209 (i978).