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Characterization of α2-adrenergic receptors on human platelets using [3H]yohimbine
Vol. 97, No. 4, 1980 December 31, 1980
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 1562-1570
CHARACTERIZATION OF e2-ADRENERGIC RECEPTORS ON HUMANPLATELETS USING [3H]YOHIMBINE Harvey d. Motulsky*, Sanford J. Shattil f ,
Paul A. Insel*
*Division of Pharmacology, M-013, Department of Medicine, U~iversity of California San Diego, La dolla, California 92093; and the ~Division of Hematology-Oncology, Department of Medicine, VA Medical Center, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Received November 14, 1980 SUMMARY: In order to investigate properties of a~pha-adrenergic receptors of human platelets, we have examined the binding of [JH]yohimbine, a potent ap-adrenergicqantagonist, to intact platelets and platelet membranes. There w~re 207±41 [~H]yohimbine sites per p]atelet. These had an equilibrium dissociation constant (Kn) of 2.7 ± 0.7nM, a Hill coefficient of 1.02±0.11, and were competed for b9 adrenergic compounds stemeoselective]~ and with a rank order expected at ep-adrenergic receptors. Addition of Na and G~P synergistically decreased the affinity of epinephrine in competing for [~H]yohimbine sites in platelet membranes and the resulting affinity of epinephrine in membranes was similar to that obtained with intact platelets. Substitution of s~crose for NaCI increased the affinity of epinephrine for intact platelets. [ H]yohimbine thus appears to be a useful ]igand for characterization of p]atelet ~9-adrenergic recep~grs. Our results suggest that extracellular Na + and intmac~l]ular GTP and Mg-- may be physiological determinants of epinephrine binding to these receptors. INTRODUCTION:
Epinephrine, acting via an a-adrenergic receptor, stimulates
human plate]ets to aggregate and secrete, potentiates the aggregation and
secretion induced by unrelated agonists such as ADP, and inhibits adenylate cyclase (1-3).
Therefore, platelets may be useful for studying changes in
~-adrenergic receptor number or function in human diseases and for defining molecular events involved in a-adrenergic action. The radioligands [3H]dihydroergocryptine ([3H]DHE), [3H]phentolamine, and [3H]clonidine have been used to identify and characterize these receptors on intact platelets and membrane preparations (4-8).
Two features of studies using these ligands with intact
platelets are high non-specific binding and the unexplained low a f f i n i t y of ~-adrenergic agonists in competing for radioligand binding sites (5,6,9). Recent studies suggest that ~-adrenergic receptors on human platelets may be 0006-291X/80/241562-09501.00/0 Copyrtght© 1980byAcademtc Press, ~c. AHrigh~ofreproduction m anyform reserved.
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exclusively of the ~2 subclass (8,10,11).
In t h i s report we show that the
~2-adrenergic antagonist [3H]yohimbine binds s p e c i f i c a l l y and with high a f f i n i t y to a2-adrenergic receptors on intact platelets and platelet membranes, and we present an explanation for the low a f f i n i t y of a-adrenergic agonists in binding studies with intact p l a t e l e t s .
MATERIALS AND METHODS: Blood was drawn from healthy men, aged 22-45, who had taken no medication for at least one week. P l a t e l e t - r i c h plasma (PRP) was prepared, aggregation studies were performed, and platelets were washed as described (9,12). The washed platelets were then suspended at 25°C in a freshly-prepared incubation buffer (50mM Tris-HCl, lOOmM NaCI, 5mM EDTA, and O.8mM ascorbic acid, pH=7.5). In some experiments the platelets were homogenized as described (8) and subsequently maintained at 4°C. The homogenate was centrifuged at 500 g for i0 min, the pellet discarded, and the remaining supernatant spun at 30,000 g for 10 min. The pellet was washed 3 times and resuspended in an incubation buffer containing 50mM Tris HCI, 0.5mM EDTA, pH 7.5. This particulate "membrane" preparation contained less than 0.5% of the a c t i v i t y of a cytoplasmic marker enzyme, lactate dehydrogenase, contained in the platelets from which the membranes were derived. Membrane protein concentration was assayed as described (8). Platelets or platelet membranes were incubated with [3H]yohimbine (82 Ci/mmole, New England Nuclear, Boston, Mass.) and various competing compounds in a total volume of 0.25mi at 25 ° . At the appropriate time (30 min in equilibrium binding experiments), lOml of incubation buffer (25°C) was added to each tube and the contents were f i l t e r e d over glass f i b e r f i l t e r s (Whatman GF/C). The test tube and f i l t e r were washed with an additional lOml of buffer and the binding retained on the f i l t e r determined. Specific binding, defined as total binding minus binding a c t i v i t y that could not be competed for by IO~M phentolamine, was linear with platelet number from 3.5 to 22x10" p l a t e l e t s / sample. Purity of [3H]yohimbine was determined by thin layer chromatography on s i l i c a gel G plates with methanol:acetic acid (19:1) as the mobile phase and r ~ d i o a c t i v i t y bound to platelets for 60 min co-migrated with native [ H]yohimbine. Results are shown as mean ± 1 standard deviation and data points are average of duplicate determinations unless otherwise noted. RESULTS: Platelet aggregation studies were performed in PRP and demonstrate that yohimbine is a pure ~-adrenergic antagonist. failed to aggregate p l a t e l e t s , but as l i t t l e
As much as 6~M yohimbine
as O.03~M inhibited the i n i t i a l
rate and the extent of platelet aggregation induced by epinephrine (IO~M). The i n h i b i t i o n was half-maximal at ~O.2uM and was maximal at I~M.
We also
confirmed that yohimbine i n h i b i t s the a b i l i t y of epinephrine (O.I~M) to potentiate ADP-induced aggregation (2,13).
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[3H-Yohlmbine] (nM) Equilibrium binding of [3H]yohimbin~ to platelet membranes and atelets. Varying concentrations of [ H]yohimbine were incubated with platelet membranes (bottom) and intact platelets (top) for 30 min at 25% Total binding (F'I), nonspecific binding (X) and specific binding (a) are indicated. The results shown are typical of 8 experiments with intact platelets and 4 experiments with platelet membranes.
Specific binding of [3H]yohimbine equilibrated in 20 min at concentrations as low as O.5nM and was stable for at least I hr.
When IO~M phentola-
mine was subsequently added to the incubation, more than 90% of s p e c i f i c a l l y bound [3H]yohimbine dissociated in a mono-exponential manner from either platelet membranes or intact p l a t e l e t s .
For intact platelets the dissociation
rate constant (k2) was 0.037±0.004 min -1 (n = 3) and the association rate constant ( k l ) was 0.10±0.04 min-lnM -1 (n = 5).
The ratio of k 2 to k I (O.4nM), a
k i n e t i c a l l y derived estimate of the dissociation constant of [3H]yohimbine is similar to the value derived from equilibrium studies presented below. [3H]yohimbine bound saturably to both platelet membranes and intact platelets (Fig. 1).
Nonspecific [3H]yohimbine binding was less than 35% of
total binding at all concentrations tested.
Figure 2 shows the pooled results
from 8 experiments in which various concentrations of [3H]yohimbine were incubated with intact platelets and the Scatchard plot (inset) derived from these data.
The equilibrium dissociation constant (KD) in these experiments
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3ooI ¢'N
m
\ @ @
@ "100 c~ 7 0 BOUNI)Csltls/©e113 i
S FREE
i
i
i
I0 IS 20 2S ~ H - Y O H I H B I N E (nH)
Figure 2. Equilibrium binding of [3H]yohimbine to intact plate~ets. Platelets were incubated for 30 min with varying concentrations of [ H]yohimbine a~d the specific binding determined. Eight experiments, each with 5 or 6 [ H]yohimbine concentrations are pooled to yield the results shown. The error bars are standard deviations; the small digits are the number of experiments run at each concentration. The inset is a Scatchard plot derived from the pooled data yielding a Kn of 3.0nM and a maximum binding of 225 receptors/ platelet. Values in the-text are the averages from the 8 individual Scatchard plots.
was 2.7±0.7nM (range 1.6-3.8), a value that corresponds well with the KD for unlabelled yohimbine determined by competition with [3H]yohimbine (Table I ) . There were 207±41 (range 140-288) [3H]yohimbine binding sites per p l a t e l e t and the H i l l
c o e f f i c i e n t for these sites was 1.02±0.11.
Four experiments were
done with p l a t e l e t membranes. The KD was 2.8±0.9nM and the maximum binding was 334±161 fmol. per mg. membrane protein. The potency of nonradioactive compounds in competition with [3H]yohimbine binding to i n t a c t p l a t e l e t s was stereoselective and as expected for ~2-adrenergic receptors:
clonidine > phenylephrine > isoproterenol;
(-)-epinephrine >
(-)-norepinephrine > (+)-epinephrine > (+)-norepinephrine; yohimbine > DHE > phentolamine > prazosin > propranolol
(Table I ) .
The KD'S of dopamine and
serotonin (~lmM) were several orders of magnitude greater than those reported 4 in s i m i l a r experiments with [3H]DHE and [3H]phentolamine binding to p l a t e l e t membranes (4-7). Imipramine competed for [3H]yohimbine sites about lO00-fold
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Table 1
Competition for [3H]yohimbine Binding Sites on Intact Platelets by Adrenergic and Non-adrenergic Compounds Dissociation Constant (KD)
(~M) Agonists clonidine phenylephrine (-)-epinephrine (-)-norepinephrine (+)-epinephrine (+)-norepinephrine (-)-isoproterenol
0.13 1.7 1.7 5.2 15 58 360
Antagonlsts yohimbine dihydroergocryptine phentolamine prazosin (-)-propranolol Other dopamine serotonin imipramine dihydroxyphenylalanine pyrocatechol
0.0020 0.0051 0.020 2.0 13 >i00 >i00 7.5 N.I. N.I.
Platelets ~#ere ~ncbbated with [3H]yohimbine and varying concentrations of the competing compound, and the concentration of the competitor competing for 50% of the specific [ H]yohimbine binding was determined (ICon). The KD of each drug was Galculated from the ICon using the:formula, Kn~ICKn (Diss. constant for [~H]yohimbine/~Diss. constant for [~H]yohimbin~ + 5]) where the dissociation constant for [~H]yohimbine was 2.7nM and S was the concentration of [ H]yohimbine used in a particular experiment. Results shown are means of 2-4 experiments in which 6-10 concentrations of competitor were used. N.I. indicates no inhibition at concentrations greater than 300uM.
less well than [3H]imiprimine
is reported to bind to p l a t e l e t membranes (14).
In competition with [3H]DHE b i n d i n g ,
( - ) - e p i n e p h r i n e has been reported to
have a lower a f f i n i t y
in i n t a c t p l a t e l e t s
(KD=I.0-2.4~M, r e f . 5,6,9) than in
membrane preparations
(KD:O.O5-O.4~M , r e f . 4 - 6 , 8 ) .
We f i n d a s i m i l a r d i f f e r -
ence with epinephrine competition f o r [3H]yohimbine binding s i t e s : 0.8-2.7~M and H i l l
coefficient
of .62-.87 in i n t a c t
1566
platelets
a KD of
(n=7) and a KD
Vol. 97, No. 4, 1980
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
°L ssE
Iog[ EPINEPHRINE]
F~gure 3. Effects of NaCl and GTP on epinephrine competition for specific ~-~H']yo-~mbine binding. Variable concentrations of (-)epinephrine were incubated with platelets and platelet membranes plus 3.3nM [ H]yohimbine. The (-)-epinephrine competition curve is shown for intact cells ( [ ] ) , and membranes (o), and membranes with added lOOm/~NaCI (X), O.In~M GTP (A), or both (0). Binding to both membranes and intact platelets was in 50mMTris-HCl, O.5mM EDTAand 8n~4MgClp. NaCl (lOOn@i) was added to the buffer used with the intact cells. The expeFiment is typical of three performed.
0.02-0.17~M and H i l l coefficients of .60-.74 with platelet membranes (n=3). The a f f i n i t y of epinephrine for the platelet e2-adrenergic receptor in platelet membranes is known to be influenced by Na+, Mg++ and GTP (15,16).
Addi-
tion of MgCI2 (8mM) to the incubation buffer used with platelet membranes decreased the KD of (-)-epinephrine from 0.17pM to O.07~M. Addition of either NaCl (lOOmM) or GTP (O.lmM) (plus 8mM MgCI2) increased the KD of epinephrine in platelet membranes to O.3pM, and increased the H i l l coefficient from ~0.6 to ~0.8.
When added together GTP and N a C I
increased the KD of ( - ) - e p i -
nephrine to 2~M, a value that was greater than that obtained in intact platelets in the same experiment (Fig. 3), and increased the H i l l coefficiemt to 1.0.
Blocking epinephrine uptake and metabolism with IO~M pargyline (a
monoamine oxidase i n h i b i t o r ) , inhibitor),
300~M catechol (a catechol-o-methyl transferase-
IpM imipramine (an amine uptake i n h i b i t o r ) ,
and lOpM normetaneph-
rine (a catecholamine uptake i n h i b i t o r ) did not change the a b i l i t y of epinephrine to compete with [3H]yohimbine for binding to intact platelets (Fig. 4). Substituting sucrose (200mN to maintain i s o t o n i c i t y )
for the NaCl decreased
the epinephrine KD of intact platelets from 1.8±0.8 to .32±.11~M (n = 4, p<.05 by paired t - t e s t ) .
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mlOIB
x
c~ ~
8~
-7
-6 -~ I osEEPINEPHRTNE3
-4
Figure 4o Epinephrine competition for specific [3H]yohi~bine binding to intact platelets. Platelets were incubated with 2.7nM [ H]yohimbine and varying concentrations of (-)-epinephrine in incubation buffer alone (o), with added 10~M pargyline, 300pM catechol, lpM imipramine, and 10~M normetanephrine (A) and with 200 mM sucrose substituting for the NaCl ( n ) . Platelets were incubated wit~ the metabolic and uptake inhibitors for 30 min prior to the addition of [ H]yohimbine and epinephrine.
DISCUSSION These results indicate that yohimbine is a pure antagonist at the platelet a-adrenergic receptor and that [3H]yohimbine binding is rapid, reversible, saturable, and competed for by adrenergic compounds in a rank order of potency expected for a2-adrenergic receptors.
The a b i l i t y of dopamine, serotonin, and
imipramine to compete for [3H]yohimbine binding sites is low, indicating that [3H]yohimbine recognizes true m-adrenergic receptors and not catecholamine uptake sites.
[3H]yohimbine also does not appear to bind to monoamine oxidase
or catechol-o-methyltransferase. Previous work from Lefkowitz's laboratory has shown that Na+ decreases affinities
of agonists in competing for [3H]DHE sites on rabbit platelet mem-
branes (15) and that GTP and Mg++ together decrease agonist a f f i n i t i e s these sites on human platelet membranes (10,16).
for
Our results using [3H]yohim-
bine confirm and extend those observations by showing that the combined effect of GTP, Mg++ and Na+ are able to account for the lower a f f i n i t y of epinephrine in binding to m2-adrenergic receptors of intact platelets compared to platelet membranes. Moreover, replacing NaCI with sucrose in the buffer used with intact platelets shifts the competition curve for (-)-epinephrine to a value ++ similar to that observed with membranes to which Mg and GTP are added. These results are consistent with the interpretation that i n t r a c e l l u l a r GTP
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and Mg++ and e x t r a c e l l u l a r Na+ are physiological regulators of the p l a t e l e t ~2-adrenergic receptor.
Such results are also consistent with the role that
i n t r a c e l l u l a r GTP appears to exert in producing lower a f f i n i t y of agonist binding to B-adrenergic receptors of intact cells compared to receptors in washed membranes of other c e l l s types (eq. 17). Work from a number of laboratories has emphasized the importance of c l a s s i f i c a t i o n of a-adrenergic receptors into ~1 and a2 subtypes (18).
As an
~ - s e l e c t i v e antagonist, r3H]yohimbine appears to have a number of advantages compared to results obtained with previously employed radioligands, including lower nonspecific binding, higher specific a c t i v i t y , and smaller requirements of blood needed for studies with intact p l a t e l e t s .
Platelets obtainable from
lml of blood are s u f f i c i e n t for each determination, and thus 30-50mi of blood are adequate to ascertain the number of platelet ~2-adrenergic receptors and t h e i r a f f i n i t i e s for [3H]yohimbine and ~-adrenergic agonists.
Those advan-
tages, plus i t s high a f f i n i t y and s e l e c t i v i t y , lead us to conclude that [3H]yohimbine w i l l be useful for further characterization of p l a t e l e t ~2-adrenergic receptors and perhaps for studies of other tissues containing ~2-adrenergic receptors.
ACKNOWLEDGEMENTS: This work was supported in part by grants from the American Heart Association (76-680 and 80-955), National I n s t i t u t e s of Health (HL-18827 and HL-25457) and California Heart Association (80-SI15), and a postdoctoral fellowship of the National I n s t i t u t e s of Health to HJM (IF32 HL06148-01). Dr. Insel is an Established Investigator, American Heart Association.
REFERENCES 1. 2. 3. 4. 5. 6. 7.
M i l l s , D.C.B. and Roberts, G.C.K. (1967) J. Physiol. (Lond.) 193,443-453. Grant, J.A. and Scrutton, M.C. (1979) Nature 277,659-661. Jakobs, K.H. (1978) Nature 274,819-820. Kafka, M.S., Tallman, J.F. and Smith, C.C. (1977) Life Sciences 21,1429-1438. New,nan,K.D., Williams, L.T., Bishopric, N.H. and Lefkowitz, R.J. (1978) J. Clin. Invest. 61,719-728. Alexander, R.W., Cooper, B. and Handin, R.I. (1978) J. Clin. Invest. 61,1136-1144. Steer, M.L., Khorana, J. and Galgoci, B. (1979) Mol. Pharmacol. 16,719-728.
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8. 9. 10. ii. 12. 13. 14. 15. 16. 17. 18.
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
S h a t t i l , S.J., McDonough, M., Turnbull, d. and Insel, P.A. (1981) Mol. Pharmacol., in press. Insel, P.A., Nirenberg, P., Turnbull, J. and S h a t t i l , S.J. (1978) Biochemistry 17,5269-5274. Hoffman, B.B., Mullikin-Kilpatrick, D. and Lefkowitz, R.J. (1980) J. Biol. Chem. 255,4645-4652. Hoffman, B.B., DeLean, A., Wood, C.L., Schocken, D.D. and Lefkowitz, R.J. (1979) Life Sci. 24,1739-1746. S h a t t i l , S.J., Anaya-Galindo, R., Bennett, d., Colman, R.W. and Cooper, R.A. (1975) J. Clin. Invest. 55,636-643. Glusa, E., Markwardt, F. and Barthel, W. (1979) Phamacology 19,196-201. Paul, S.M., Rehavi, M., Skolnick, P. and Goodwin, F . K . . (1980) Life Sci. 26,953-59. Tsai, B.S. and Lefkowitz, R.J. (1978) Mol. Pharmacol. 14,540-548. Tsai, B.S. and Lefkowitz, R.J. (1979) Mol. Phamacol. 16,61-68. Insel, PA and Stoolman, L.M. (1978) Mol. Pharmacol. 16, 549-561. Hoffman, B.B. and Lefkowitz, R.J. (1980) New Engl. d. Med. 302,1390-1396.
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Pages 1562-1570
CHARACTERIZATION OF e2-ADRENERGIC RECEPTORS ON HUMANPLATELETS USING [3H]YOHIMBINE Harvey d. Motulsky*, Sanford J. Shattil f ,
Paul A. Insel*
*Division of Pharmacology, M-013, Department of Medicine, U~iversity of California San Diego, La dolla, California 92093; and the ~Division of Hematology-Oncology, Department of Medicine, VA Medical Center, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Received November 14, 1980 SUMMARY: In order to investigate properties of a~pha-adrenergic receptors of human platelets, we have examined the binding of [JH]yohimbine, a potent ap-adrenergicqantagonist, to intact platelets and platelet membranes. There w~re 207±41 [~H]yohimbine sites per p]atelet. These had an equilibrium dissociation constant (Kn) of 2.7 ± 0.7nM, a Hill coefficient of 1.02±0.11, and were competed for b9 adrenergic compounds stemeoselective]~ and with a rank order expected at ep-adrenergic receptors. Addition of Na and G~P synergistically decreased the affinity of epinephrine in competing for [~H]yohimbine sites in platelet membranes and the resulting affinity of epinephrine in membranes was similar to that obtained with intact platelets. Substitution of s~crose for NaCI increased the affinity of epinephrine for intact platelets. [ H]yohimbine thus appears to be a useful ]igand for characterization of p]atelet ~9-adrenergic recep~grs. Our results suggest that extracellular Na + and intmac~l]ular GTP and Mg-- may be physiological determinants of epinephrine binding to these receptors. INTRODUCTION:
Epinephrine, acting via an a-adrenergic receptor, stimulates
human plate]ets to aggregate and secrete, potentiates the aggregation and
secretion induced by unrelated agonists such as ADP, and inhibits adenylate cyclase (1-3).
Therefore, platelets may be useful for studying changes in
~-adrenergic receptor number or function in human diseases and for defining molecular events involved in a-adrenergic action. The radioligands [3H]dihydroergocryptine ([3H]DHE), [3H]phentolamine, and [3H]clonidine have been used to identify and characterize these receptors on intact platelets and membrane preparations (4-8).
Two features of studies using these ligands with intact
platelets are high non-specific binding and the unexplained low a f f i n i t y of ~-adrenergic agonists in competing for radioligand binding sites (5,6,9). Recent studies suggest that ~-adrenergic receptors on human platelets may be 0006-291X/80/241562-09501.00/0 Copyrtght© 1980byAcademtc Press, ~c. AHrigh~ofreproduction m anyform reserved.
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exclusively of the ~2 subclass (8,10,11).
In t h i s report we show that the
~2-adrenergic antagonist [3H]yohimbine binds s p e c i f i c a l l y and with high a f f i n i t y to a2-adrenergic receptors on intact platelets and platelet membranes, and we present an explanation for the low a f f i n i t y of a-adrenergic agonists in binding studies with intact p l a t e l e t s .
MATERIALS AND METHODS: Blood was drawn from healthy men, aged 22-45, who had taken no medication for at least one week. P l a t e l e t - r i c h plasma (PRP) was prepared, aggregation studies were performed, and platelets were washed as described (9,12). The washed platelets were then suspended at 25°C in a freshly-prepared incubation buffer (50mM Tris-HCl, lOOmM NaCI, 5mM EDTA, and O.8mM ascorbic acid, pH=7.5). In some experiments the platelets were homogenized as described (8) and subsequently maintained at 4°C. The homogenate was centrifuged at 500 g for i0 min, the pellet discarded, and the remaining supernatant spun at 30,000 g for 10 min. The pellet was washed 3 times and resuspended in an incubation buffer containing 50mM Tris HCI, 0.5mM EDTA, pH 7.5. This particulate "membrane" preparation contained less than 0.5% of the a c t i v i t y of a cytoplasmic marker enzyme, lactate dehydrogenase, contained in the platelets from which the membranes were derived. Membrane protein concentration was assayed as described (8). Platelets or platelet membranes were incubated with [3H]yohimbine (82 Ci/mmole, New England Nuclear, Boston, Mass.) and various competing compounds in a total volume of 0.25mi at 25 ° . At the appropriate time (30 min in equilibrium binding experiments), lOml of incubation buffer (25°C) was added to each tube and the contents were f i l t e r e d over glass f i b e r f i l t e r s (Whatman GF/C). The test tube and f i l t e r were washed with an additional lOml of buffer and the binding retained on the f i l t e r determined. Specific binding, defined as total binding minus binding a c t i v i t y that could not be competed for by IO~M phentolamine, was linear with platelet number from 3.5 to 22x10" p l a t e l e t s / sample. Purity of [3H]yohimbine was determined by thin layer chromatography on s i l i c a gel G plates with methanol:acetic acid (19:1) as the mobile phase and r ~ d i o a c t i v i t y bound to platelets for 60 min co-migrated with native [ H]yohimbine. Results are shown as mean ± 1 standard deviation and data points are average of duplicate determinations unless otherwise noted. RESULTS: Platelet aggregation studies were performed in PRP and demonstrate that yohimbine is a pure ~-adrenergic antagonist. failed to aggregate p l a t e l e t s , but as l i t t l e
As much as 6~M yohimbine
as O.03~M inhibited the i n i t i a l
rate and the extent of platelet aggregation induced by epinephrine (IO~M). The i n h i b i t i o n was half-maximal at ~O.2uM and was maximal at I~M.
We also
confirmed that yohimbine i n h i b i t s the a b i l i t y of epinephrine (O.I~M) to potentiate ADP-induced aggregation (2,13).
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400
Jnfac-~Plafelefs
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~ o
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c ©
400
o L CL
300
Pla{elef M e m b r a n ~
200 E h_
100
5
10
15
20
25
[3H-Yohlmbine] (nM) Equilibrium binding of [3H]yohimbin~ to platelet membranes and atelets. Varying concentrations of [ H]yohimbine were incubated with platelet membranes (bottom) and intact platelets (top) for 30 min at 25% Total binding (F'I), nonspecific binding (X) and specific binding (a) are indicated. The results shown are typical of 8 experiments with intact platelets and 4 experiments with platelet membranes.
Specific binding of [3H]yohimbine equilibrated in 20 min at concentrations as low as O.5nM and was stable for at least I hr.
When IO~M phentola-
mine was subsequently added to the incubation, more than 90% of s p e c i f i c a l l y bound [3H]yohimbine dissociated in a mono-exponential manner from either platelet membranes or intact p l a t e l e t s .
For intact platelets the dissociation
rate constant (k2) was 0.037±0.004 min -1 (n = 3) and the association rate constant ( k l ) was 0.10±0.04 min-lnM -1 (n = 5).
The ratio of k 2 to k I (O.4nM), a
k i n e t i c a l l y derived estimate of the dissociation constant of [3H]yohimbine is similar to the value derived from equilibrium studies presented below. [3H]yohimbine bound saturably to both platelet membranes and intact platelets (Fig. 1).
Nonspecific [3H]yohimbine binding was less than 35% of
total binding at all concentrations tested.
Figure 2 shows the pooled results
from 8 experiments in which various concentrations of [3H]yohimbine were incubated with intact platelets and the Scatchard plot (inset) derived from these data.
The equilibrium dissociation constant (KD) in these experiments
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3ooI ¢'N
m
\ @ @
@ "100 c~ 7 0 BOUNI)Csltls/©e113 i
S FREE
i
i
i
I0 IS 20 2S ~ H - Y O H I H B I N E (nH)
Figure 2. Equilibrium binding of [3H]yohimbine to intact plate~ets. Platelets were incubated for 30 min with varying concentrations of [ H]yohimbine a~d the specific binding determined. Eight experiments, each with 5 or 6 [ H]yohimbine concentrations are pooled to yield the results shown. The error bars are standard deviations; the small digits are the number of experiments run at each concentration. The inset is a Scatchard plot derived from the pooled data yielding a Kn of 3.0nM and a maximum binding of 225 receptors/ platelet. Values in the-text are the averages from the 8 individual Scatchard plots.
was 2.7±0.7nM (range 1.6-3.8), a value that corresponds well with the KD for unlabelled yohimbine determined by competition with [3H]yohimbine (Table I ) . There were 207±41 (range 140-288) [3H]yohimbine binding sites per p l a t e l e t and the H i l l
c o e f f i c i e n t for these sites was 1.02±0.11.
Four experiments were
done with p l a t e l e t membranes. The KD was 2.8±0.9nM and the maximum binding was 334±161 fmol. per mg. membrane protein. The potency of nonradioactive compounds in competition with [3H]yohimbine binding to i n t a c t p l a t e l e t s was stereoselective and as expected for ~2-adrenergic receptors:
clonidine > phenylephrine > isoproterenol;
(-)-epinephrine >
(-)-norepinephrine > (+)-epinephrine > (+)-norepinephrine; yohimbine > DHE > phentolamine > prazosin > propranolol
(Table I ) .
The KD'S of dopamine and
serotonin (~lmM) were several orders of magnitude greater than those reported 4 in s i m i l a r experiments with [3H]DHE and [3H]phentolamine binding to p l a t e l e t membranes (4-7). Imipramine competed for [3H]yohimbine sites about lO00-fold
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Table 1
Competition for [3H]yohimbine Binding Sites on Intact Platelets by Adrenergic and Non-adrenergic Compounds Dissociation Constant (KD)
(~M) Agonists clonidine phenylephrine (-)-epinephrine (-)-norepinephrine (+)-epinephrine (+)-norepinephrine (-)-isoproterenol
0.13 1.7 1.7 5.2 15 58 360
Antagonlsts yohimbine dihydroergocryptine phentolamine prazosin (-)-propranolol Other dopamine serotonin imipramine dihydroxyphenylalanine pyrocatechol
0.0020 0.0051 0.020 2.0 13 >i00 >i00 7.5 N.I. N.I.
Platelets ~#ere ~ncbbated with [3H]yohimbine and varying concentrations of the competing compound, and the concentration of the competitor competing for 50% of the specific [ H]yohimbine binding was determined (ICon). The KD of each drug was Galculated from the ICon using the:formula, Kn~ICKn (Diss. constant for [~H]yohimbine/~Diss. constant for [~H]yohimbin~ + 5]) where the dissociation constant for [~H]yohimbine was 2.7nM and S was the concentration of [ H]yohimbine used in a particular experiment. Results shown are means of 2-4 experiments in which 6-10 concentrations of competitor were used. N.I. indicates no inhibition at concentrations greater than 300uM.
less well than [3H]imiprimine
is reported to bind to p l a t e l e t membranes (14).
In competition with [3H]DHE b i n d i n g ,
( - ) - e p i n e p h r i n e has been reported to
have a lower a f f i n i t y
in i n t a c t p l a t e l e t s
(KD=I.0-2.4~M, r e f . 5,6,9) than in
membrane preparations
(KD:O.O5-O.4~M , r e f . 4 - 6 , 8 ) .
We f i n d a s i m i l a r d i f f e r -
ence with epinephrine competition f o r [3H]yohimbine binding s i t e s : 0.8-2.7~M and H i l l
coefficient
of .62-.87 in i n t a c t
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a KD of
(n=7) and a KD
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°L ssE
Iog[ EPINEPHRINE]
F~gure 3. Effects of NaCl and GTP on epinephrine competition for specific ~-~H']yo-~mbine binding. Variable concentrations of (-)epinephrine were incubated with platelets and platelet membranes plus 3.3nM [ H]yohimbine. The (-)-epinephrine competition curve is shown for intact cells ( [ ] ) , and membranes (o), and membranes with added lOOm/~NaCI (X), O.In~M GTP (A), or both (0). Binding to both membranes and intact platelets was in 50mMTris-HCl, O.5mM EDTAand 8n~4MgClp. NaCl (lOOn@i) was added to the buffer used with the intact cells. The expeFiment is typical of three performed.
0.02-0.17~M and H i l l coefficients of .60-.74 with platelet membranes (n=3). The a f f i n i t y of epinephrine for the platelet e2-adrenergic receptor in platelet membranes is known to be influenced by Na+, Mg++ and GTP (15,16).
Addi-
tion of MgCI2 (8mM) to the incubation buffer used with platelet membranes decreased the KD of (-)-epinephrine from 0.17pM to O.07~M. Addition of either NaCl (lOOmM) or GTP (O.lmM) (plus 8mM MgCI2) increased the KD of epinephrine in platelet membranes to O.3pM, and increased the H i l l coefficient from ~0.6 to ~0.8.
When added together GTP and N a C I
increased the KD of ( - ) - e p i -
nephrine to 2~M, a value that was greater than that obtained in intact platelets in the same experiment (Fig. 3), and increased the H i l l coefficiemt to 1.0.
Blocking epinephrine uptake and metabolism with IO~M pargyline (a
monoamine oxidase i n h i b i t o r ) , inhibitor),
300~M catechol (a catechol-o-methyl transferase-
IpM imipramine (an amine uptake i n h i b i t o r ) ,
and lOpM normetaneph-
rine (a catecholamine uptake i n h i b i t o r ) did not change the a b i l i t y of epinephrine to compete with [3H]yohimbine for binding to intact platelets (Fig. 4). Substituting sucrose (200mN to maintain i s o t o n i c i t y )
for the NaCl decreased
the epinephrine KD of intact platelets from 1.8±0.8 to .32±.11~M (n = 4, p<.05 by paired t - t e s t ) .
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mlOIB
x
c~ ~
8~
-7
-6 -~ I osEEPINEPHRTNE3
-4
Figure 4o Epinephrine competition for specific [3H]yohi~bine binding to intact platelets. Platelets were incubated with 2.7nM [ H]yohimbine and varying concentrations of (-)-epinephrine in incubation buffer alone (o), with added 10~M pargyline, 300pM catechol, lpM imipramine, and 10~M normetanephrine (A) and with 200 mM sucrose substituting for the NaCl ( n ) . Platelets were incubated wit~ the metabolic and uptake inhibitors for 30 min prior to the addition of [ H]yohimbine and epinephrine.
DISCUSSION These results indicate that yohimbine is a pure antagonist at the platelet a-adrenergic receptor and that [3H]yohimbine binding is rapid, reversible, saturable, and competed for by adrenergic compounds in a rank order of potency expected for a2-adrenergic receptors.
The a b i l i t y of dopamine, serotonin, and
imipramine to compete for [3H]yohimbine binding sites is low, indicating that [3H]yohimbine recognizes true m-adrenergic receptors and not catecholamine uptake sites.
[3H]yohimbine also does not appear to bind to monoamine oxidase
or catechol-o-methyltransferase. Previous work from Lefkowitz's laboratory has shown that Na+ decreases affinities
of agonists in competing for [3H]DHE sites on rabbit platelet mem-
branes (15) and that GTP and Mg++ together decrease agonist a f f i n i t i e s these sites on human platelet membranes (10,16).
for
Our results using [3H]yohim-
bine confirm and extend those observations by showing that the combined effect of GTP, Mg++ and Na+ are able to account for the lower a f f i n i t y of epinephrine in binding to m2-adrenergic receptors of intact platelets compared to platelet membranes. Moreover, replacing NaCI with sucrose in the buffer used with intact platelets shifts the competition curve for (-)-epinephrine to a value ++ similar to that observed with membranes to which Mg and GTP are added. These results are consistent with the interpretation that i n t r a c e l l u l a r GTP
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and Mg++ and e x t r a c e l l u l a r Na+ are physiological regulators of the p l a t e l e t ~2-adrenergic receptor.
Such results are also consistent with the role that
i n t r a c e l l u l a r GTP appears to exert in producing lower a f f i n i t y of agonist binding to B-adrenergic receptors of intact cells compared to receptors in washed membranes of other c e l l s types (eq. 17). Work from a number of laboratories has emphasized the importance of c l a s s i f i c a t i o n of a-adrenergic receptors into ~1 and a2 subtypes (18).
As an
~ - s e l e c t i v e antagonist, r3H]yohimbine appears to have a number of advantages compared to results obtained with previously employed radioligands, including lower nonspecific binding, higher specific a c t i v i t y , and smaller requirements of blood needed for studies with intact p l a t e l e t s .
Platelets obtainable from
lml of blood are s u f f i c i e n t for each determination, and thus 30-50mi of blood are adequate to ascertain the number of platelet ~2-adrenergic receptors and t h e i r a f f i n i t i e s for [3H]yohimbine and ~-adrenergic agonists.
Those advan-
tages, plus i t s high a f f i n i t y and s e l e c t i v i t y , lead us to conclude that [3H]yohimbine w i l l be useful for further characterization of p l a t e l e t ~2-adrenergic receptors and perhaps for studies of other tissues containing ~2-adrenergic receptors.
ACKNOWLEDGEMENTS: This work was supported in part by grants from the American Heart Association (76-680 and 80-955), National I n s t i t u t e s of Health (HL-18827 and HL-25457) and California Heart Association (80-SI15), and a postdoctoral fellowship of the National I n s t i t u t e s of Health to HJM (IF32 HL06148-01). Dr. Insel is an Established Investigator, American Heart Association.
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M i l l s , D.C.B. and Roberts, G.C.K. (1967) J. Physiol. (Lond.) 193,443-453. Grant, J.A. and Scrutton, M.C. (1979) Nature 277,659-661. Jakobs, K.H. (1978) Nature 274,819-820. Kafka, M.S., Tallman, J.F. and Smith, C.C. (1977) Life Sciences 21,1429-1438. New,nan,K.D., Williams, L.T., Bishopric, N.H. and Lefkowitz, R.J. (1978) J. Clin. Invest. 61,719-728. Alexander, R.W., Cooper, B. and Handin, R.I. (1978) J. Clin. Invest. 61,1136-1144. Steer, M.L., Khorana, J. and Galgoci, B. (1979) Mol. Pharmacol. 16,719-728.
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8. 9. 10. ii. 12. 13. 14. 15. 16. 17. 18.
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S h a t t i l , S.J., McDonough, M., Turnbull, d. and Insel, P.A. (1981) Mol. Pharmacol., in press. Insel, P.A., Nirenberg, P., Turnbull, J. and S h a t t i l , S.J. (1978) Biochemistry 17,5269-5274. Hoffman, B.B., Mullikin-Kilpatrick, D. and Lefkowitz, R.J. (1980) J. Biol. Chem. 255,4645-4652. Hoffman, B.B., DeLean, A., Wood, C.L., Schocken, D.D. and Lefkowitz, R.J. (1979) Life Sci. 24,1739-1746. S h a t t i l , S.J., Anaya-Galindo, R., Bennett, d., Colman, R.W. and Cooper, R.A. (1975) J. Clin. Invest. 55,636-643. Glusa, E., Markwardt, F. and Barthel, W. (1979) Phamacology 19,196-201. Paul, S.M., Rehavi, M., Skolnick, P. and Goodwin, F . K . . (1980) Life Sci. 26,953-59. Tsai, B.S. and Lefkowitz, R.J. (1978) Mol. Pharmacol. 14,540-548. Tsai, B.S. and Lefkowitz, R.J. (1979) Mol. Phamacol. 16,61-68. Insel, PA and Stoolman, L.M. (1978) Mol. Pharmacol. 16, 549-561. Hoffman, B.B. and Lefkowitz, R.J. (1980) New Engl. d. Med. 302,1390-1396.
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