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[3H] diazepam binding to human granulocytes
Life Sciences, Vol. 37, pp. 11-16 Printed in the U.S.A.
Pergamon Press
[3H] DIAZEPAM BINDING TO HUMANGRANULOCYTES Peter A Bond, Richard L Cundall and Brenda Rolfe. St. John's Hospital, Stone, Aylesbury, Bucks, England. (Received in final form April 23, 1985)
Summary [ 3H]-diazepam binds to sites on human granulocyte membranes, with l i t t l e or no binding to platelets or lymphocytes. These [3H]-diazepam binding sites are of the peripheral type, being strongly inhibited by R05-4864 (Ki=6.23nM) but only weakly by clonazepam (Ki=14pM). Binding of [3H] diazepam at 0° is saturable, specific and stereoselective. Scatchard analysis indicates a single class of sites with Bmax of 109±17f moles per mg of protein and KD of 3.07±0.53nM. Hill plots of saturation experiments gave straight lines with a mean Hill coefficient of 1.03±0.014. Binding is time dependent and reversible and i t varies linearly with granulocyte protein concentration over the range 0.025-0.300 mg of protein. Binding sites for benzodiazepines have been reported in the brains of several n~mmalian species (1,2). [3H] diazepam or [3H] flunitrazepam were used for binding studies. These receptors were believed to be a single homogeneous population but later work has suggested a heterogeneous population of benzodiazepine binding sites (3,4,5). Benzodiazepine binding sites have also been described in the periphery (6), for example in renal tissue (7), in rat blood platelets (8) and rat peritoneal mast cells (9). These peripheral receptors differ from those in brain with respect to their a f f i n i t y for certain benzodiazepines. We have looked for binding sites in platelets, from human blood.
lymphocytes and granulocytes
Methods Separation of Blood Cells Blood was obtained from the Blood Transfusion Service as 500ml packs with acid citrate dextrose as anticoagulant. The separation of blood cells was accomplished by a method based on Boyum (10) and described by Bond and Cundall (11), which was briefly as follows. Blood was centrifuged at 190g for 15 minutes to separate platelet rich plasma which was removed and centrifuged at 1900g for 10 mins to give a platelet pellet. The separated plasma was added back to the blood and this was layered on to a mixture of 2 parts of 6% Dextran T500 (Pharmacia Fine Chemicals A-B, Uppsala, Sweden) in 0.9% NaCl and 1 part Triosil 440 (Nyegaard ~ Co. A.S.,Oslo) (diluted 2 parts Triosil to 3 parts water) and allowed to stand at room temperature for 1/2 to 2 hrs until the erythrocytes had passed into the lower layer, leaving plasma which contained leucocytes. Leucocyte rich plasma was diluted (1:1) with phosphate buffered saline pH 7.4 and layered onto a mixture of 8% Ficoll (Pharmacia, Uppsala, Sweden) and Triosil 440 (29.5 ml T r i o s i l , diluted 2 parts Triosil to 3 parts water, made up to 100 0024-3205/8S $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
12
Diazepam Binding to Human Granulocytes
Vol. 37, No. i, 1985
ml with 8% Ficoll) and centrifuged at 400g for 35 mins. The cells removed from the interphase were lymphocytes while those at the bottom of the tube were granulocytes. Platelets, lymphocytes, and granulocytes were washed with phosphate-buffered saline and then homogenised in lysing buffer (5mM Tris-HCl, 5mM EDTA), wasEed again in phosphate buffered saline and resuspended in incubation buffer (O.05M Tris HCI with lOmM MgCl2). The Binding Assay The binding of [3H] diazepam to washed cell membranes was carried out for 25 mins at O°C. The reaction mixture contained [3H] diazepam 0.1 - 1OhM (specific activity 94 Ci per mmol ) (Amersham International P.L.C. U.K.), cell membranes (0.1 - 0.3 mg protein) in 100 ~l of incubation buffer added to start the reaction. Incubation buffer made up the total volume to 500 ~l. The reaction was terminated by addition of 5 ml of cold incubation buffer and f i l t r a t i o n through Whatman GF/F glassfibre f i l t e r s . The f i l t e r s were washed with 2 x 5 ml aliquots of cold incubation buffer and the f i l t e r s were counted in a Triton X-lOO-toluene s c i n t i l l a t i o n Cocktail in a LKB Rackbeta liquid s c i n t i l l a t i o n spectrometer. "Non-specific" binding as determined by carrying out duplicate assays in the presence of 5 x 10-4M unlabelled diazepam. Specific binding is the total binding minus non-specific binding. Values were calculated from Scatchard Plots. Mean values are quoted with standard error (S.E.). Results [ 3H] diazepam bound specifically to human granulocytes but not consistently to platelets or lymphocytes. Lysed and whole cells gave similar results, but the lysed cells proved easier to handle. The specific binding of [3H] diazepam to granulocyte membranes was saturable while non-specific binding increased linearly with concentration (Fig 1). Scatchard plots of the specific binding of [3H] diazepam to granulocyte membranes were linear over the substrate concentration range 0.1 - 6.0nM and indicated a single class of binding site with an apparent equilibrium dissociation binding constant (KD) of 3.07±0.53 nM (n=12). Maximumbinding was 109±17 f moles per mg protein. Binding was also measured using a centrifugation method to check whether equilibration during washing of the cell membranes could lead to loss of bound radioactivity. No difference was found between centrifugation and f i l t r a t i o n . However, membrane concentration had to be such that f i l t r a t i o n rate did not slow down too much i.e. not above 0.3 mg. Binding of [3H] diazepam reached equilibrium within 15 mins with t l / 2 of 2.2 mins and was reversible ( t l / 2 = 0.7 mins) (Fig 2). From the rate constant of association (KI=4.6±0.6 xlO7M-Imin-1,N=6) and the rate constant of dissociation (K2 =O.131±O.034min-1 ,N=6) the dissociation constant (KD =K2 /K I) was calculated as 2.86±0.63nM (N=6). Hill plots of 12 saturation experiments gave straight lines with a mean Hill coefficient of 1.03±0.014 (Fig 3). Binding was proportional to cell concentration o v e r the range 0.025 to 0.3 mg of protein.
Vol.
37, No.
I, 1985
Diazepam Binding to Human Granulocytes
13
80
200 60
E
in
0 (3. nn o~
EE
~
w 40 100
x~ c o
no
on
20
~E
i
I
2 4 [3HI Diazepam (nM)
I
!
I
6
100
200
Bound
Figure 1 The effect of concentration on the binding of [3H] diazepam, with Scatchard plot of the same data. In the f i r s t plot open circles represent total binding while solid circles show non-specific binding. The Scatchard plot is of specific binding.
c 14
~ - Diazcpam
10-4M
ol
E ~6 I::3 .m
4'
e--i
3:
2 f:} o m
i
10
I
I
20 30 Time at 0" (rains)
I
I
I
10
20
30
Figure 2 The time course of [ 3H] diazepam (1.29nM) binding displacement by unl abel led di azepam.
and
its
14
Diazepam Binding to Human Granulocytes
Vol. 37, No. i, 1985
The a b i l i t y of various benzodiazepines to displace [ 3H] diazepam from granulocyte receptors is shown in Table I. The most potent compound in displacing [ 3H] diazepam binding was R05-4864, a 4-chloro derivative of diazepam, selective for peripheral sites, while clonazepam, which is selective for brain sites, is much less potent. The pharmacological activity of benzodiazepines containing an asymmetric carbon atom (C3 ) is markedly stereospecific, the S-enantiomer being more active than the R. The S-enantiomer of a benzodiazepine (R011-6896) was almost 500 times more potent in displacing [ 3H] diazepam than the R-enantiomer (R011-6893). Another pair of enantiomers (R011-3128 and R011-3624) were less active but there was s t i l l a f i f t y - f o l d difference between them.
0 -1 -2 -3
I=, 0 0
•-=
-6 l
i
-2
-1
I
0
I
I,
.1
.2
l
,,3
Loglo ((3H) Diazepam - nM)
Figure
3
Hill plots of five separate saturation experiments. diazepam specifically bound per mg of protein.
B = moles of
[ 3H]
As alcohol was used to make up solutions of benzodiazepines (10% to give a final 1% in the reaction mixture) the effect of alcohol on binding was investigated. Up to 4% in the incubation medium had no effect, while 10% caused a 26% reduction.
Vol. 37, No. i, 1985
Diazepam Binding to Human Granulocytes
15
TABLE I A f f i n i t y of various benzodiazepines for the diazepam binding site. Ki values (M) (N) 6.23±4.4x10-9 (4) R05-4864 R011-6896
4.2±2.15x10-8
(3)
Medazepam
8.5±5.4xi0-8
(4)
Diazepam
4.3±1.9x10-7
(3)
Chlordiazepoxide
1.6±1.3x10-6
(4)
R011-3128
2 . 2 ± 1 . 5 x 1 0 - 6 (3)
Flurazepam
4.1±3.1x10-6 (3)
Flunitrazepam
5.5±1.5x10-6 (3)
Clonazepam
1.4±0.4x10-5 (3)
R011-6893
2.4±1.8xi0 -5
(3)
R011-3624
>10-4
(4)
Ki calculated from the formula : - Ki =
IC.50 1 + C
Rb
where IC.50 is the concentration producing half maximal inhibition. C is the concentration of [3H] diazepam. K~ is the dissociation constant for t3H] diazepam. [3H]-diazepam was used at a concentration of lnM. Discussion The binding site for benzodiazepines which we have found in granulocyte membranes has characteristics which are typical of receptors. Binding is saturable and Scatchard analysis is consistent with a single site with a KD of 3.07~0.53 nM and maximum binding 109±17 f moles per mg protein. Hill plots of saturation experiments gave straight lines with a mean Hill coefficient of 1.03 ±0.014 indicating homogeneity of the binding site and that binding was non cooperative. Binding was reversible and time dependent and the association rate constant calculated from such timed experiments of 2.86nM, is close to the KD of 3.07 obtained from Scatchard analysis. Binding is stereoselective, two pairs of benzodiazepine enantiomorphs showing a marked difference in their a f f i n i t i e s for the site. Two distinct types of benzodiazepine binding site have been described in mammalian tissue, that in brain being distinct from those found in peripheral tissues (6). The main distinguishing feature lies in the a f f i n i t y of the receptor for clonazepam and R05-4864. In the brain clonazepam is a potent agent for displacing [ 3H] diazepam binding, while ROS-4864 is relatively weak. Conversely in peripheral tissues R05-4864 is more potent than clonazepam. The binding of [3H] diazepam to human granulocytes is of a peripheral type, the Ki for displacement of [3H] diazepam binding by R05-4864 is over 1000 times less than that for clonazepam. Being readily distinguishable from the receptor in
16
Diazepam Binding to Human Granulocytes
Vol. 37, No. 1, 1985
brain, the high a f f i n i t y site binding [3H] diazepam to granulocyte membranes has the properties of a receptor, in being reversible, saturable and stereospecific. Wang and Coworkers (8) have described [3H] diazepam binding sites on rat blood platelets, while Taniguchi and co-workers (9) found [3H]-diazepam sites on rat peritoneal mast cells. These have characteristics of peripheral binding sites with respect for their a f f i n i t y for R05-4864 and clonazepam. The a f f i n i t y of [ 3H]-diazepam for the sites is relatively low (KD=9OnM), though in other respects they resemble the granulocyte sites. Our search for diazepam receptors on platelets and lymphocytes has led to inconsistent results, and results in granulocytes are more variable than those in the rat platelet. However, this is to be expected when comparing a genetically homogeneous population of rats with humans. Acknowledgments We thank Miss J. Kenworthyof the Oxford Regional Blood Transfusion Service for supplying blood, Mrs. G. Jackson for secretarial assistance, the Oxford Regional Health Authority and the Cooper Charitable Trust for financial support, and the Medical Research Council for a grant to purchase a liquid s c i n t i l l a t i o n spectrometer. References 1. 2. 3.
R.F.SQUIRESand C.BRAESTRUP, Nature 266, 732-734 (1977) H.MOHLERand T.OKADA, Science 198, 8~F~T-851(1977) R.F.SQUIRES, D.I.BENSON, C.BR)~I~STRUP, J.COUPET, C.A.KLEPNER, V.MYERSand B.BEER, Pharmacol. Biochem. Behav. 10, 825-830 (1979) 4. W.SIEGHARTand M.KAROBATH, Nature, ~ 6 , 285-287 (1980) 5. I.YOKOI, S.E.ROSE and T.YANAGIHARA,~fe Sci. 28, 1591-1595 (1981) 6. C.BRAESTRUPand R.F.SQUIRES, Proc. Natl. Acad.~ci. 74, 3805-3809 (1977) 7. J.W.REGAN, H.I.YAMAMURA, S.YAMADAand W.R.ROESKE, ~ i f e Sci. 28, 991-998 (1981) 8. J.K.T.WANG, T.TANIGUCHI and S.SPECTOR, Life Sci. 27, 1881-1888 (1980) 9. T.TANIGUCHI,J.K.T.WANG and S.SPECTOR, Life Sci. ~__, 171-178 (1980) 10. A.BOYUM, Tissue Antigens 4, 269-274 (1974) 11. P.A.BOND and R.L.CUNDALL,-CIin. Chim. Acta 80, 317-326 (1977)
Pergamon Press
[3H] DIAZEPAM BINDING TO HUMANGRANULOCYTES Peter A Bond, Richard L Cundall and Brenda Rolfe. St. John's Hospital, Stone, Aylesbury, Bucks, England. (Received in final form April 23, 1985)
Summary [ 3H]-diazepam binds to sites on human granulocyte membranes, with l i t t l e or no binding to platelets or lymphocytes. These [3H]-diazepam binding sites are of the peripheral type, being strongly inhibited by R05-4864 (Ki=6.23nM) but only weakly by clonazepam (Ki=14pM). Binding of [3H] diazepam at 0° is saturable, specific and stereoselective. Scatchard analysis indicates a single class of sites with Bmax of 109±17f moles per mg of protein and KD of 3.07±0.53nM. Hill plots of saturation experiments gave straight lines with a mean Hill coefficient of 1.03±0.014. Binding is time dependent and reversible and i t varies linearly with granulocyte protein concentration over the range 0.025-0.300 mg of protein. Binding sites for benzodiazepines have been reported in the brains of several n~mmalian species (1,2). [3H] diazepam or [3H] flunitrazepam were used for binding studies. These receptors were believed to be a single homogeneous population but later work has suggested a heterogeneous population of benzodiazepine binding sites (3,4,5). Benzodiazepine binding sites have also been described in the periphery (6), for example in renal tissue (7), in rat blood platelets (8) and rat peritoneal mast cells (9). These peripheral receptors differ from those in brain with respect to their a f f i n i t y for certain benzodiazepines. We have looked for binding sites in platelets, from human blood.
lymphocytes and granulocytes
Methods Separation of Blood Cells Blood was obtained from the Blood Transfusion Service as 500ml packs with acid citrate dextrose as anticoagulant. The separation of blood cells was accomplished by a method based on Boyum (10) and described by Bond and Cundall (11), which was briefly as follows. Blood was centrifuged at 190g for 15 minutes to separate platelet rich plasma which was removed and centrifuged at 1900g for 10 mins to give a platelet pellet. The separated plasma was added back to the blood and this was layered on to a mixture of 2 parts of 6% Dextran T500 (Pharmacia Fine Chemicals A-B, Uppsala, Sweden) in 0.9% NaCl and 1 part Triosil 440 (Nyegaard ~ Co. A.S.,Oslo) (diluted 2 parts Triosil to 3 parts water) and allowed to stand at room temperature for 1/2 to 2 hrs until the erythrocytes had passed into the lower layer, leaving plasma which contained leucocytes. Leucocyte rich plasma was diluted (1:1) with phosphate buffered saline pH 7.4 and layered onto a mixture of 8% Ficoll (Pharmacia, Uppsala, Sweden) and Triosil 440 (29.5 ml T r i o s i l , diluted 2 parts Triosil to 3 parts water, made up to 100 0024-3205/8S $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
12
Diazepam Binding to Human Granulocytes
Vol. 37, No. i, 1985
ml with 8% Ficoll) and centrifuged at 400g for 35 mins. The cells removed from the interphase were lymphocytes while those at the bottom of the tube were granulocytes. Platelets, lymphocytes, and granulocytes were washed with phosphate-buffered saline and then homogenised in lysing buffer (5mM Tris-HCl, 5mM EDTA), wasEed again in phosphate buffered saline and resuspended in incubation buffer (O.05M Tris HCI with lOmM MgCl2). The Binding Assay The binding of [3H] diazepam to washed cell membranes was carried out for 25 mins at O°C. The reaction mixture contained [3H] diazepam 0.1 - 1OhM (specific activity 94 Ci per mmol ) (Amersham International P.L.C. U.K.), cell membranes (0.1 - 0.3 mg protein) in 100 ~l of incubation buffer added to start the reaction. Incubation buffer made up the total volume to 500 ~l. The reaction was terminated by addition of 5 ml of cold incubation buffer and f i l t r a t i o n through Whatman GF/F glassfibre f i l t e r s . The f i l t e r s were washed with 2 x 5 ml aliquots of cold incubation buffer and the f i l t e r s were counted in a Triton X-lOO-toluene s c i n t i l l a t i o n Cocktail in a LKB Rackbeta liquid s c i n t i l l a t i o n spectrometer. "Non-specific" binding as determined by carrying out duplicate assays in the presence of 5 x 10-4M unlabelled diazepam. Specific binding is the total binding minus non-specific binding. Values were calculated from Scatchard Plots. Mean values are quoted with standard error (S.E.). Results [ 3H] diazepam bound specifically to human granulocytes but not consistently to platelets or lymphocytes. Lysed and whole cells gave similar results, but the lysed cells proved easier to handle. The specific binding of [3H] diazepam to granulocyte membranes was saturable while non-specific binding increased linearly with concentration (Fig 1). Scatchard plots of the specific binding of [3H] diazepam to granulocyte membranes were linear over the substrate concentration range 0.1 - 6.0nM and indicated a single class of binding site with an apparent equilibrium dissociation binding constant (KD) of 3.07±0.53 nM (n=12). Maximumbinding was 109±17 f moles per mg protein. Binding was also measured using a centrifugation method to check whether equilibration during washing of the cell membranes could lead to loss of bound radioactivity. No difference was found between centrifugation and f i l t r a t i o n . However, membrane concentration had to be such that f i l t r a t i o n rate did not slow down too much i.e. not above 0.3 mg. Binding of [3H] diazepam reached equilibrium within 15 mins with t l / 2 of 2.2 mins and was reversible ( t l / 2 = 0.7 mins) (Fig 2). From the rate constant of association (KI=4.6±0.6 xlO7M-Imin-1,N=6) and the rate constant of dissociation (K2 =O.131±O.034min-1 ,N=6) the dissociation constant (KD =K2 /K I) was calculated as 2.86±0.63nM (N=6). Hill plots of 12 saturation experiments gave straight lines with a mean Hill coefficient of 1.03±0.014 (Fig 3). Binding was proportional to cell concentration o v e r the range 0.025 to 0.3 mg of protein.
Vol.
37, No.
I, 1985
Diazepam Binding to Human Granulocytes
13
80
200 60
E
in
0 (3. nn o~
EE
~
w 40 100
x~ c o
no
on
20
~E
i
I
2 4 [3HI Diazepam (nM)
I
!
I
6
100
200
Bound
Figure 1 The effect of concentration on the binding of [3H] diazepam, with Scatchard plot of the same data. In the f i r s t plot open circles represent total binding while solid circles show non-specific binding. The Scatchard plot is of specific binding.
c 14
~ - Diazcpam
10-4M
ol
E ~6 I::3 .m
4'
e--i
3:
2 f:} o m
i
10
I
I
20 30 Time at 0" (rains)
I
I
I
10
20
30
Figure 2 The time course of [ 3H] diazepam (1.29nM) binding displacement by unl abel led di azepam.
and
its
14
Diazepam Binding to Human Granulocytes
Vol. 37, No. i, 1985
The a b i l i t y of various benzodiazepines to displace [ 3H] diazepam from granulocyte receptors is shown in Table I. The most potent compound in displacing [ 3H] diazepam binding was R05-4864, a 4-chloro derivative of diazepam, selective for peripheral sites, while clonazepam, which is selective for brain sites, is much less potent. The pharmacological activity of benzodiazepines containing an asymmetric carbon atom (C3 ) is markedly stereospecific, the S-enantiomer being more active than the R. The S-enantiomer of a benzodiazepine (R011-6896) was almost 500 times more potent in displacing [ 3H] diazepam than the R-enantiomer (R011-6893). Another pair of enantiomers (R011-3128 and R011-3624) were less active but there was s t i l l a f i f t y - f o l d difference between them.
0 -1 -2 -3
I=, 0 0
•-=
-6 l
i
-2
-1
I
0
I
I,
.1
.2
l
,,3
Loglo ((3H) Diazepam - nM)
Figure
3
Hill plots of five separate saturation experiments. diazepam specifically bound per mg of protein.
B = moles of
[ 3H]
As alcohol was used to make up solutions of benzodiazepines (10% to give a final 1% in the reaction mixture) the effect of alcohol on binding was investigated. Up to 4% in the incubation medium had no effect, while 10% caused a 26% reduction.
Vol. 37, No. i, 1985
Diazepam Binding to Human Granulocytes
15
TABLE I A f f i n i t y of various benzodiazepines for the diazepam binding site. Ki values (M) (N) 6.23±4.4x10-9 (4) R05-4864 R011-6896
4.2±2.15x10-8
(3)
Medazepam
8.5±5.4xi0-8
(4)
Diazepam
4.3±1.9x10-7
(3)
Chlordiazepoxide
1.6±1.3x10-6
(4)
R011-3128
2 . 2 ± 1 . 5 x 1 0 - 6 (3)
Flurazepam
4.1±3.1x10-6 (3)
Flunitrazepam
5.5±1.5x10-6 (3)
Clonazepam
1.4±0.4x10-5 (3)
R011-6893
2.4±1.8xi0 -5
(3)
R011-3624
>10-4
(4)
Ki calculated from the formula : - Ki =
IC.50 1 + C
Rb
where IC.50 is the concentration producing half maximal inhibition. C is the concentration of [3H] diazepam. K~ is the dissociation constant for t3H] diazepam. [3H]-diazepam was used at a concentration of lnM. Discussion The binding site for benzodiazepines which we have found in granulocyte membranes has characteristics which are typical of receptors. Binding is saturable and Scatchard analysis is consistent with a single site with a KD of 3.07~0.53 nM and maximum binding 109±17 f moles per mg protein. Hill plots of saturation experiments gave straight lines with a mean Hill coefficient of 1.03 ±0.014 indicating homogeneity of the binding site and that binding was non cooperative. Binding was reversible and time dependent and the association rate constant calculated from such timed experiments of 2.86nM, is close to the KD of 3.07 obtained from Scatchard analysis. Binding is stereoselective, two pairs of benzodiazepine enantiomorphs showing a marked difference in their a f f i n i t i e s for the site. Two distinct types of benzodiazepine binding site have been described in mammalian tissue, that in brain being distinct from those found in peripheral tissues (6). The main distinguishing feature lies in the a f f i n i t y of the receptor for clonazepam and R05-4864. In the brain clonazepam is a potent agent for displacing [ 3H] diazepam binding, while ROS-4864 is relatively weak. Conversely in peripheral tissues R05-4864 is more potent than clonazepam. The binding of [3H] diazepam to human granulocytes is of a peripheral type, the Ki for displacement of [3H] diazepam binding by R05-4864 is over 1000 times less than that for clonazepam. Being readily distinguishable from the receptor in
16
Diazepam Binding to Human Granulocytes
Vol. 37, No. 1, 1985
brain, the high a f f i n i t y site binding [3H] diazepam to granulocyte membranes has the properties of a receptor, in being reversible, saturable and stereospecific. Wang and Coworkers (8) have described [3H] diazepam binding sites on rat blood platelets, while Taniguchi and co-workers (9) found [3H]-diazepam sites on rat peritoneal mast cells. These have characteristics of peripheral binding sites with respect for their a f f i n i t y for R05-4864 and clonazepam. The a f f i n i t y of [ 3H]-diazepam for the sites is relatively low (KD=9OnM), though in other respects they resemble the granulocyte sites. Our search for diazepam receptors on platelets and lymphocytes has led to inconsistent results, and results in granulocytes are more variable than those in the rat platelet. However, this is to be expected when comparing a genetically homogeneous population of rats with humans. Acknowledgments We thank Miss J. Kenworthyof the Oxford Regional Blood Transfusion Service for supplying blood, Mrs. G. Jackson for secretarial assistance, the Oxford Regional Health Authority and the Cooper Charitable Trust for financial support, and the Medical Research Council for a grant to purchase a liquid s c i n t i l l a t i o n spectrometer. References 1. 2. 3.
R.F.SQUIRESand C.BRAESTRUP, Nature 266, 732-734 (1977) H.MOHLERand T.OKADA, Science 198, 8~F~T-851(1977) R.F.SQUIRES, D.I.BENSON, C.BR)~I~STRUP, J.COUPET, C.A.KLEPNER, V.MYERSand B.BEER, Pharmacol. Biochem. Behav. 10, 825-830 (1979) 4. W.SIEGHARTand M.KAROBATH, Nature, ~ 6 , 285-287 (1980) 5. I.YOKOI, S.E.ROSE and T.YANAGIHARA,~fe Sci. 28, 1591-1595 (1981) 6. C.BRAESTRUPand R.F.SQUIRES, Proc. Natl. Acad.~ci. 74, 3805-3809 (1977) 7. J.W.REGAN, H.I.YAMAMURA, S.YAMADAand W.R.ROESKE, ~ i f e Sci. 28, 991-998 (1981) 8. J.K.T.WANG, T.TANIGUCHI and S.SPECTOR, Life Sci. 27, 1881-1888 (1980) 9. T.TANIGUCHI,J.K.T.WANG and S.SPECTOR, Life Sci. ~__, 171-178 (1980) 10. A.BOYUM, Tissue Antigens 4, 269-274 (1974) 11. P.A.BOND and R.L.CUNDALL,-CIin. Chim. Acta 80, 317-326 (1977)