Klin. Wochenschr. 56 (Suppl. I), 13%145 (1978)

Klinische Wochenschrift

© Springer-Verlag 1978

[3H]Dihydroergonine Binding to e-Adrenergic Receptors in Human Platelets K.H. Jakobs and R. Rauschek Department of Pharmacology, Universityof Heidelberg

Bindung von [3H]Dihydroergonin an ~-adrenerge Rezeptoren in menschlichen Thrombozyten Zusammenfassung. Die Bindung von [3H]Dihydroergonin, einem potenten ct-adrenergen Blocker in menschlichen Thrombozyten, wurde in intakten menschlichen Thrombozyten und ThrombozytenMembranen untersucht. Die Bindung erreichte ihr )kquilibrium innerhalb von 10 rain bei 25 ° C und war reversibel nach Zugabe eines 12berschusses yon Phentolamin. Die Geschwindigkeitskonstanten betrugen 0,31 min- 1 ffir die Vorwfirtsreaktion und 0,027 rain 1 fur die Rfickw~irtsreaktion, woraus eine Assoziationsgeschwindigkeitskonstante von 2,2 x 1 0 7 M - l m i n -1 berechnet wurde. Die [3H]Dihydroergonin-Bindung erreichte Sfittigung mit 220 fmol [3H]Dihydroergonin gebunden pro mg MembranProtein und etwa 200 Bindungsstellen pro Thrombozyt. Aquilibriums-Untersuchungen ergaben eine einheitliche Population yon Bindungsstellen ohne Anzeichen einer Kooperativitfit und eine DissoziationsKonstante von 6 bis 7 nM. Die Bindung yon [3H]Dihydroergonin zeigte alle typischen Charakteristika der Bindung an einen c~-adrenergen Rezeptor and Stereoselektivitfit: Adrenerge Agonisten bzw. Antagonisten konkurrierten um die Bindungsstellen in der Potenzrcihe: (1)-Adrenalin > (1)-Noradrenalin > (d)-Noradrenalin >>(1)-Isoprenalin bzw. Yohimbin > Dihydroergotamin > Phentolamin >> Tolazolin > Azapetin ~> (1)-Propranolol > (1)-Pindolol. Es wnrde versucht, die Bindungsdaten verschiedener e-adrenerger Agonisten mit der ThrombozytenAggregation und der Hemmung der Adenylat-Cyclase zu korrelieren. Unter verschiedenen getesteten ~-adrenergen Agonisten waren nur Adrenalin und Noradrenalin ffihig, eine primfire Thrombozyten-Aggregation auszul6sen und die Adenylat-Cyclase zu hemOffprint requests to: Dr. K.H. Jakobs (address see page 145)

men. Verschiedene andere Substanzen, die in anderen Systemen c~-adrenerge Agonisten sind, verdrfingten [3H)Dihydroergonin yon seinen Bindungsstetlen mit etwas geringeren Affinitfiten als Adrenalin, so Phenylephrin und Methoxamin, oder mit weit h6heren Affinitfiten, so die Imidazoline Xylometazolin, Oxymetazolin, Naphazolin und Tetryzolin. Im Gegensatz zu Adrenalin und Noradrenalin waren diese Substanzen aber nicht f/ihig, eine prim/ire Thrombozyten-Aggregation (bis 1 mM) und eine Hemmung der AdenylatCyclase (bis 100 ~tM) zu induzieren. Die Ergebnisse zeigen, dab der thrombozyt/ire ~-adrenerge Rezeptor sich nicht wesentlich yon ~-adrenergen Rezeptoren in anderen Systemen unterscheidet, was die Bindung yon Substanzen an den Rezeptor angeht, dab aber nur ein limitiertes Spektrum von Substanzen f/ihig ist, fiber diesen Rezeptor biologische Antworten hervorzurufen.

Schliisselwiirter: c~-adrenerge Rezeptoren -

Thrombozyten-Aggregation - Thrombozyt~ire AdenylatCyclase - ct-adrenerge Wirkungen.

Summary. Binding of [3H]dihydroergonine, a potent e-adrenergic blocking agent, was studied in intact human platelets and platelet membranes. The binding process reached equilibrium within 10 rain at 25 ° C and was reversible upon addition of excess phentolamine with forward and reverse rate constants of 0.31 and 0.027 rain- 1, respectively, and with a second ol"der association rate constant of 2.2 x 107 M 1 min- 1. The [3H]dihydroergonine binding reached saturation with 220 fmol of [3H]dihydroergonine bound per mg of platelet membrane protein and about 200 binding sites per platelet. Equilibrium studies indicated a single population of binding sites with no apparent cooperativity and a dissociation constant of 6 to 7 nM. Binding of [3H]dihydroergonine showed all the

140

K.H. Jakobs and R. Rauschek: ~-AdrenergicReceptors in Human Platelets

typical characteristics of binding to an c~-adrenergic receptor and stereoselectivity: Adrenergic agonists and antagonists competed for the binding sites with the following order of potency: (1)-adrenaline> (1)- noradrenaline > (d)- noradrenaline >> (1)- isoprenaline and yohimbine > dihydroergotamine > phentolamine >> tolazoline > azapetine >> (1)-propranolol > ( 1)-pindol ol, respectively. It was tried to correlate the binding data of various e-adrenergic agonists with platelet aggregation and inhibition of adenylate cyclase. Out of many c+adrenergic agonists tested, only adrenaline and noradrenaline were able to induce primary platelet aggregation and inhibition of adenylate cyclase. Various other compounds that are c~-adrenergic agonists in other systems displaced [3H]dihydroergonine from its binding sites with somewhat lower affinities than adrenaline, e.g., phenylephrine and methoxamine, or with much higher affinities, e.g., the imidazolines, xylometazoline, oxymetazoline, naphazoline and tetryzolin. In contrast to adrenaline and noradrenaline, these compounds were unable to induce primary platelet aggregation (up to 1 mM) and inhibition of adenylate cyclase (up to 100 gM). These data indicate that the platelet ~-adrenergic receptor does not greatly differ from ~-adrenergic receptors found in other tissues with respect to binding of drugs to the receptor but that the platelet receptor is unique with regard to the limited spectrum of compounds that are capable of inducing biological responses. Key words: c~-adrenergic receptors - platelet aggregation - platelet adenylate cyclase - e-adrenergic responses.

Adrenaline and noradrenaline induce aggregation of human platelets [4, 18, 20, 21], cause a rapid decrease in prostaglandin El-enhanced cyclic A M P levels in intact platelets [6, 9, 10, 17, 22, 23] and induce immediate inhibition of platelet adenylate cyclase in cellfree preparations [12, 13, 14]. The findings that these responses are blocked by c+adrenergic blocking agents such as dihydroergotamine and phentolamine, but not by /~-adrenergic blocking agents, indicate that these adrenergic effects are mediated by c+adrenergic receptors. Recently, [3H]dihydroergocryptine, an c~-adrenergic antagonist, has been shown to bind specifically to ~-adrenergic receptors in different tissues of various species [7, 24, 25, 26] including human platelets [1, 15, 19]. We have recently shown that out of many c~-adrenergic agonists tested only adrenaline and nor-

adrenaline are able to induce primary platelet aggregation and inhibition of adenylate cyclase (14). This narrow spectrum of agonists, which is up to now found only in platelets, stimulated us to study the binding characteristics of the cz-adrenergic receptor in human platelets, with special interest on e-adrenergic agonists. In the present study, we report on the identification and characterization of e-adrenergic receptors in intact human platelets and platelet membranes with [3H]dihydroergonine as labelled ligand and on the effects of various adrenergic agonists and antagonists on [3H]dihydroergonine binding. This study indicates that various agents, which act as c+adrenergic agonists in other systems and which are without any apparent intrinsic activity in platelets with respect to aggregation and inhibition of adenylate cyclase, compete for [3H]dihydroergonine binding sites with similar or even higher affinities than adrenaline and noradrenaline. Materials and Methods a) Reagents

[3H]Dihydroergoninehydrochloride(26.8 Ci/mmol, tritiated at position 13 of the dihydrolysergicacid residue, Fig. 1), tmlabelled dihydroergoninehydrochloride, dihydroergotaminemethansulfonate and (1)-pindolol hydrochloride were obtained from Sandoz AG, Basel. The bitartrates of (1)-adrenaline and (1)-noradrenaline and the hydrochlorides of (1)-phenylephrineand yohimbine were purchased from Sigma, Mtinchen. (1)-isoprenaline bitartrate was donated by Boehringer Ingelheim, (1)-propranolol hydrochloride by ICI-Pharma, Plankstadt, phentolamine methansulfonate, naphazoline nitrate and the hydrochlorides of xylometazolineand tolazoline were obtained from CIBA-Geigy, Basel, (d)-noradrenalinebitartrate from Adams Chemicals, Chicago, (d,1)-methoxamine hydrochloridefrom Wellcome, Grol3burgwedel,oxymetazolinehydrochloride from E. Merck, Darmstadt, and tetryzolin hydrochloride from Pfizer, Karlsruhe. Aqueous solutions of adrenergic agonists and antagonistswereprepared shortly beforeexperiments.Ethanol, which was necessary for dissolving the ergot alkaloids, did not affect the binding at the highest concentration used (0.5%).

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K.H. Jakobs and R. Rauschek: e-Adrenergic Receptors in Human Platelets

b) Preparationof Intact Platetetsand Ptatelet Membranes Platelets from blood of healthy volunteers and ptatelet membranes were prepared as described previously [14]. Platelet-rich plasma, anticoagulated with sodium citrate (0.38 %, w/v) was used as source of intact platelets. Platelet membranes were obtained by freeze thawing of isolated platelets and centrifugation of the lysates at 30,000 x g for 20 min and resuspension of the pellets in 50 mM Tris-HC1 buffer, pH 7.5.

E 200 kob s = 031 min 1 k l = 22 107 ~4-1 m 1

d) Other Methods Protein was determined as described by Lowry et al. [16], using bovine serum albumin as standard. Platetet aggregation was monitored in platelet-rich plasma anticoagulated by 0.38% (w/v) sodium citrate at 37° C [3]. Platelet counting was performed through phase contrast microscopy. The dissociation constants (Kd) were calculated from the concentrations required to cause 50% inhibition of [3H]dihydroergonine binding (ICs0) by the method of Cheng and Prusoff [5] according to the relationship: K~=ICso/[l+[ligand]/K~ ligand],

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c) [3H]Dihydroergonine Binding Assay [3H]Dihydroergonine binding in platelet membranes (40 to 80 gg of protein) was assayed with 10 nM [3H]dihydroergonine (about 20,000 cpm) or at the indicated concentration and 10 mM MgC12 in 50 mM Tris-HC1, pH 7.5. Incubation was performed at 25° C in a total volume of 100 gl for 10 min or as indicated. In intact platelets, [3H]dihydroergonine binding was assayed with 10 nM [3H]dihydroergonine (about 120,000 cpm) for 10 rain at 25° C in a total volume of 600 gI with about 1 to 3 x l0 s platelets per sample. Assays were run in triplicate. Similar results to those described were obtained in at least two separate experiments. Separation of bound and free [3H]dihydroergonine was achieved by rapid filtration of the incubation mixture through glass fiber filters (Schleicher and Schfill). The filters were washed 4 times with 5 ml of 50 mM Tris-HC1 buffer, pH 7.5, and the radioactivity in the filters was determined in a Triton-toluene scintillation mixture. Specific binding of [3H]dihydroergonine was defined as the difference between total binding and binding measured in the presence of 100 gM phentolamine and was about 80% and 50% of the total binding in platelet membranes and intact platelets, respectively.

141

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Fig. 2. Binding of [3H]dihydroergonine to platelet membranes as function of time. [3H]Dihydroergonine (12.8 nM) was incubated with platelet membranes for the indicated times at 25° C. Indicated on the left panel is the specific binding as a function of time. Right panel: Pseudo-first order kinetic plot of [3H]dihydroergonine binding: beq, amount of [3H]dihydroergonine bound at equilibrium, b, amount of [3Hldihydroergouine bound at time t. The line has a slope, kobs, equal to the observed forward rate constant. The second order association rate constant, kt, is calculated from k~ = (kob~-kz)/[DHE] where kz is the reverse rate constant and [DHE] is the concentration of [3H]dihydroergonine in the assay

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Fig. 3. Reversibility of [3H]dihydroergonine binding to platelet membranes. Platelet membranes were incubated with [3H]dihydroergonine (12.8 nM) for 10 rain at 25 ° C after which time an excess of phentolamine (100 gM) was added. The time point of phentolamine addition is defined as t=0. Left panel: At the indicated times, specific [aH]dihydroergonine binding was determined. Right panel: First order rate plot of the dissociation of the receptorligand complex; beq amount of [3H]dihydroergonine bound immediately prior to the addition of phentolamine. The line has a slope, - k z , equal to the reverse rate constant

T h e t i m e - c o u r s e o f specific b i n d i n g o f [ 3 H ] d i h y d r o e r g o n i n e to p l a t e l e t m e m b r a n e s is s h o w n in Fig. 2. B i n d i n g was r a p i d a n d r e a c h e d e q u i l i b r i u m w i t h i n 6 to 10 m i n . T h e o b s e r v e d f o r w a r d r a t e c o n s t a n t (kobs) was 0.31 m i n - 1. T h e b i n d i n g was a l s o r e v e r s i b l e u p o n a d d i t i o n o f excess p h e n t o l a m i n e , a n d t h e r e v e r s e r a t e c o n s t a n t (kz) was 0.027 m i n 1 (Fig. 3). T h e s e c o n d order association rate constant (kl) derived from t h e s e d a t a was 2.2 x 107 M - 1 m i n - 1. T h e Ka c a l c u l a t e d f r o m t h e s e d a t a was 1.2 n M w h i c h is a b o u t o n e f i f t h o f t h e Ka d e r i v e d f r o m t h e e q u i l i b r i u m data.

T h e b i n d i n g o f [ 3 H ] d i h y d r o e r g o n i n e to p l a t e l e t m e m b r a n e s was a s a t u r a b l e p r o c e s s w i t h h i g h a f f i n i t y (Fig. 4). S a t u r a t i o n w a s a c h i e v e d w i t h 220 f m o l o f [3H]dihydroergonine bound per mg of protein. Halfm a x i m a l s a t u r a t i o n o c c u r r e d at 6.27 n M , as a n estim a t e o f t h e e q u i l i b r i u m d i s s o c i a t i o n c o n s t a n t (Ka). T h e S c a t c h a r d - p l o t a n a l y s i s r e v e a l e d a single s t r a i g h t l i n e ; this f i n d i n g i n d i c a t e s a single p o p u l a t i o n o f b i n d i n g sites w i t h n o o b v i o u s t e n d e n c y o f c o o p e r a t i v i t y .

Results

142

K.H. Jakobs and R. Rauschek: ~-Adrenergic Receptors in Human Platelets 80 ,

Kd ~ 6 27 nM n ,, 220 fmot/mg protein

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Fig 6. Specific [3H]dihydroergonine binding to intact platelets as a function of [3H]dihydroergonine concentration. Ptatelets (as platelet-rich plasma, 2.4 x 108 platelets per tube) were incubated with the indicated concentrations of [3H]dihydroergonine for 10 min at 25 ° C. Left panel: Specific binding as a function of [aH]dihydroergonine concentration, Right panel: Scatchard plot of [3H]dihydroergonine binding to intact platelets

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5 10 15 2'0 incubation time (rain) Fig. 5. Binding of [3H]dihydroergonine to intact platelets as a function of time. [3H]Dihydroergonine (10 nM) was incubated with platelet-rich plasma (PRP, 1.1 x 108 platelets per tube) and plateletpoor plasma (PPP) for the indicated times at 25 ° C in the absence and presence of 100 gM phentolamine. Indicated on the ordinate is the total [SH]dihydroergonine bound per tube

Specific binding of [3H]dihydroergonine to intact platelets was also a rapid process which reached equilibrium within about 10 min (Fig. 5). Binding was also a saturable process with 186 binding sites per platelet (Fig. 6). Half-maximal saturation occurred at 7.45 nM [3tt]dihydroergonine, compared to about 6 nM in platelet membranes.

Characterization of the [ H]Dihydroergonine Binding Sites as ~-Adrenergic Receptors Adrenergic agonists competed for the [3H]dihydroergonine binding sites with the order of potency

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ctdrenergic agonist (tog M) Fig. 7. Inhibition of [3tt]dihydroergonine binding to platelet membranes by various adrenergic agonists. Platelet membranes were incubated with [3H]dihydroergonine (10 nM) in the absence and presence of the indicated adrenergic agonists, and specific binding was determined. Indicated is the inhibition of [3H]dihydroergonine specific binding at the indicated concentrations of adrenergic agonists compared to the binding in their absence

(t)-adrenaline > (1)-noradrenatine >> (1)-isoprenaline (Fig. 7) with Kd values of 0.216, 0.642 and more than 100 gM, respectively. The (d)-stereoisomer of noradrenaline competed for the binding sites with a Ke value of 6.02 gM. Thus, the [3H]dihydroergonine binding sites showed the typical pattern of an czadrenergic receptor with respect to the affinity of adrenergic agonists and to stereoselectivity. c~-Adrenergic antagonists competed for the [3H]dihydroergonine binding sites with high affinities. The order of potency was yohimbine>dihydroergotamine > phentolamine >> tolazoline > azapetine with Ka values of 0.004, 0.011, 0.036, 0.853 and 1.26 gM, respectively (Fig. 8). These values are

K.H. Jakobs and R. Rauschek: c~-AdrenergicReceptors in Human Platelets

143

1. Effectsof various ~-adrenergicagonists on [3H]dihydroergonine binding, ptatelet aggregation and adenylate cyclaseactivity

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(1)-adrenaline (1)-noradrenaline (1)-phenylephrine (d,1)-methoxamine xylometazoline oxymetazoline naphazoline tetryzolin

0.216% 0.269b 0.642~ 0.751" 4.99a 0.018b 0.016b 0.031b 0.058b

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adrenergic antagonist (log M) Fig. 8. Inhibition of [3H]dihydroergoninebinding to platelet membranes by various adrenergicantagonists. Conditions were identical to those describedin Figure 7

in good agreement with the order of potency of these c~-adrenergic antagonists to inhibit adrenaline-induced aggregation and reduction of adenylate cyclase activity [14]. In contrast, fl-adrenergic antagonist such as (1)-propranolol and (1)-pindolot competed with [3H]dihydroergonine for the binding sites only at high concentrations with Ka values of 23.5 and 32.5 gM, respectively. Thus, [3H]dihydroergonine binding sites also showed the typical pattern of an ~-adrenergic receptor with respect to adrenergic antagonists.

Comparison of [ 3H] Dihy&oergonine Binding and Biological Response with Respect to e-Adrenergic Agonists Indicated in Table 1 are the effects of various agents, which are potent c~-adrenergic agonists in various biological systems, on [3H]dihydroergonine binding and their efficacies to induce primary platelet aggregation and inhibition of adenylate cyclase. (1)-Adrenaline displaced [3H]dihydroergonine from its binding sites with high affinity and was able to induce primary platelet aggregation and inhibition of adenylate cyclase. (1)-Noradrenaline, which was several times less potent than adrenaline in binding displacement, was also weaker in inducing platelet aggregation and inhibition of adenylate cyclase. Other phenylethylamine derivatives, such as phenylephrine and methoxamine, which compared to adrenaline had about 5- and 20fold less affinity for the binding sites, respectively, were unable to induce primary platelet aggregation (tested up to 1 mM, not shown) and to inhibit adenylate cyclase (tested up to 100 gM) [14].

12 4--5 n.d. n.d. n.d. n.d. n.d. n,d.

* Data from Reference[14] Kd=values derived from experimentswith (a) platelet membranes, (b) intact platelets N.D. =no detectable primary aggregation at concentrations up to 1 mM n.d. =no detectable adenylate cyclase inhibition at concentrations up to 100 gM

Various imidazoline derivatives, which act as c~adrenergic agonists in several systems, were also studied with regard to their effects on [3H]dihydroergonine binding (Table 1), All imidazoline derivatives tested displaced [3H]dihydroergonine from its binding sites with much higher affinities than adrenaline. Xylometazoline, oxymetazoline, naphazoline and tetryzolin had Ka values of 0.018, 0.016, 0.031 and 0.058 ]aM, respectively.

Discussion

The binding sites for [3H]dihydroergonine identified in intact human platelets and platelet membranes appear to be e-adrenergic receptors. The binding exhibited high affinity, saturabitity, reversibility and stereospecificity. The catecholamine potency series, adrenaline > noradrenaline >>isoprenaline, for [3H]dihydroergonine displacement was consistent with findings at various c~-adrenergic receptors, e-Adrenergic antagonists such as yohimbine and phentolamine were much more potent than fi-adrenergie antagonists such as propranolol and pindolol. The binding characteristics found with [3H]dihydroergonine agree closely with those of [3H]dihydroergocryptine recently described in platelets, including the number of receptors per platelet (about 200) found in studies with intact platelets [1, 15, 19]. The relative potency of the catecholamines, adrenaline, noradrenaline and isoprenaline, in displacing [3H]dihydroergonine binding was welt correlated with their effects to induce primary platelet aggregation

144

K.H. Jakobs and R. Rauschek: e-Adrenergic Receptors in Human Platelets

and inhibition of adenylate cyclase, although somewhat higher concentrations were required to induce effects on adenylate cyclase than on [3H]dihydroergonine binding and aggregation. The reasons for this finding were discussed elsewhere [19]. Similarly, eadrenergic antagonists such as yohimbine, dihydroergotamine and phentolamine, which were potent in displacing [3H]dihydroergonine binding, were also potent inhibitors of adrenaline-induced platelet aggregation and inhibition of adenylate cyclase, whereas tolazotine and azapetine, which were weak inhibitors of the above adrenaline effects, were also less potent in binding displacement. The fl-adrenergic antagonists, propranolol and pindolol, neither blocked the biological responses induced by adrenaline nor competed for [3H]dihydroergonine binding with high affinities. The most striking results obtained in the [3H]dihydroergonine binding studies are the effects of various synthetic adrenergic agents that are full agonists at various peripheral or central e-adrenergic receptors. Especially, agonists of the imidazoline type such as xylometazoline, oxymetazoline, naphazoline and tetryzolin competed for the [3H]dihydroergonine binding sites with very high affinities. The Ka values obt a i n e d with these agents were about two orders of magnitude less than those obtained with adrenaline and noradrenaline and were of the same order as those of the most potent typical c~-adrenergic antagonists. Similar observations were made with another imidazoline derivative, clonidine, using [3H]dihydroergocryptine as radioligand [1, 19]. In contrast with the findings in these binding studies, we have recently shown that none of tested synthetic eadrenergic agonists of the phenylethylamine and the imidazoline type is capable of inducing primary platelet aggregation and inhibition of adenylate cyclase [14], even when added at concentrations up to 1 mM and 100 gM, respectively. Thus, these synthetic e-adrenergic agonists appear to be devoid of any intrinsic activity in the platelet system. From these diverging results we have concluded that the e-adrenergic agonists, which undergo the primary reaction with high affinities, but are unable to induce biological responses, should behave as c~adrenergic antagonists in the platelet system. We actually found that these synthetic ~-adrenergic agonists of the phenylethylamine and imidazoline type are potent ~-adrenergic antagonists in human platelets (11, P. Lasch and K.H. Jakobs, in preparation). The platelet c~-adrenergic receptor has many similarities with the presynaptic type of c~-adrenergic receptors with respect to the binding specificities in as much as imidazoline derivatives including clonidine bind with high affinities whereas the typical postsyn-

aptic c~-adrenergic agonist, phenylephrine, has much lower affinity [2].With regard to triggering biological responses, however, the platelet e-adrenergic receptor completely differs from that found in other tissues. Acknowledgements. This work was supported by the Deutsche Forschungsgemeinschaft. We are indepted to Miss Gabriele Gabel for excellent technical assistance and to Dr. R. Markstein, Sandoz AG, Basel, for donating [3H]dihydroergonine.

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K.H. Jakobs and R. Rauschek: c~-Adrenergic Receptors in Human Platelets 16. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J.: Protein measurement with the Foiin phenol reagent. J. Biol. Chem. 193, 265-275 (1951) 17. Marquis, N.R., Becker, J.A., Vigdahl, R.L. : Platelet aggregation. III. An epinephrine induced decrease in cyclic AMP synthesis. Biochem Biophys. Res. Commun. 39, 783-~789 (1970) 18. Mills, D.C.B., Roberts, G,C.K. : Effects of adrenaline on human blood platelets. J. Physiol. 193, 443-453 (1967) 19. Newman, K.D., Williams, L,T., Bishopric, N.H., Lefkowitz, R.J. : Identification of c~-adrenergic receptors in human platelets by [3H]dihydroergocryptine binding. J. Clin. Invest. 61,395M02 (1978) 20. O'Brien, J,R. : Some effects of adrenaline and anti-adrenaline compounds on platelets in vitro and in vivo. Nature 200, 763-764 (1963) 21. O'Brien, J.R. : A comparison of platelet aggregation produced by seven compounds and a comparison of their inhibitors. J. Clin. Pathol. 17, 275-281 (1964) 22. Robison, G.A., Arnold, A., Hartmann, R.C. : Divergent effects of epinephrine and prostaglandin E1 on the level of cyclic AMP in human blood platelets. Pharmacol. Res. Commun. 1, 325-332 (1969)

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23. Salzman, E.W., Neri, L.L. : Cyclic 3',5'-adenosine monophosphate in human blood pletelets. Nature 224, 609-610 (1969) 24. Strittmatter, W.J., Davis, J.N., Lefkowitz, R.J. : c~-Adrenergic receptors in rat parotid cells. I. Correlation of [3H]dihydroergocryptine binding and catecholamine-stimulated potassium efflux. J. Biol. Chem. 252, 5472-5477 (1977) 25. Tsai, S., Lefkowitz, R.J.: [3H]Dihydroergocryptine binding to alpha-adrenergic receptors in canine aortic membranes. J. Pharmacol. Exp. Ther. 204, 606-614 (1978) 26. Williams, L.T., Mullikin, D., Lefkowitz, R.J. : Identification of ~-adrenergic receptors in uterine smooth muscle membranes by [3H]dihydroergocryptine binding. J. Biol. Chem. 251, 69t5-6923 (1976) Received August 16, 1978 Dr. Karl H. Jakobs Pharmakalogisches Institut der Universit/it Im Neuenheimer Feld 366 D-6900 Heidelberg Federal Republic of Germany