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Influence of alpha- and beta-adrenoceptors on thrombin-induced serotonin release in rat platelets
THROMBOSIS RESEARCH 40; 147-159, l-985 0049-3848/85 $3.00 + .OO Printed in the USA. Copyright (c) 1985 Pergamon Press Ltd. All rights reserved.
INFLUENCE OF ALPHA- AND BETA-AORENOCEPTORS ON THROMBIN-INDUCED SEROTONIN RELEASE IN RAT PLATELETS
S. Koutouzov, L. Cothenet-Vernoux, P. Marche and J.P.
Dausse
U7 INSERM, Department of Pharmacology, Hdpital Necker, 161 rue de Sevres 75015 Paris, France
(Received 22.5.1985; Accepted in original form 25.6.1985 by Editor B. Vargaftig) ABSTRACT This work was designed to investigate the influence of rat platelet adrenoceptors on the early thrombin-induced serotonin release. In washed platelets prelabeled with [3H]-serotonin, adrenaline and isoproterenol both inhibited, in a dose-dependent manner, the early thrombin-induced secretion of serotonin. Inhibitory responses of both adrenaline and isoproterenol were blocked in the presence of beta-adrenoceptor antagonists, suggesting that the catecholamine acted solely through beta-adrenoceptors. However, isoproterenol inhibited the thrombin-induced serotonin release to a much greater extent than the catecholamine, suggesting that the alpha2-component of adrenaline might account for the difference observed between the two compounds. Our observation that selective alpha2-adrenoceptor antagonists as yohimbine and rauwolscine potentiated the inhibitory effect of adrenaline to a level close to that observed with isoproterenol, lends support to the above hypothesis. This latter result suggested that, conversely, alpha2-adrenergic compounds might exert a counteracting effect on a full beta-adrenoceptor mediated inhibition. Although synthetic alpha2-adrenergic agents failed to influence isoproterenol inhibitory effect, our study shows that prestimulation of beta-adrenoceptors by isoproterenol, followed by addition of adrenaline or noradrenaline markedly diminished the inhibitory effect of isoproterenol to a level close to that which characterized the inhibition observed with catecholamines, when tested alone. Our work favours the hypothesis that, in rat platelets, early after platelet stimulation, catecholamines might counteract a beta-adrenoceptor- mediated inhibition, through alpha2-adrenoceptor sites. Key words: Platelets, catecholamines, rat, serotonin release, adrenoceptors Abbreviations: 5HT: 5-hydroxytryptamine (serotonin); Hepes: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; EDTA: [ethylene dinitrilol-tetraacetic acid; ADP: adenosine diphosphate. 147
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INTRODUCTION The synergistic effect of adrenaline on thrombin, collagen and ADPinduced aggregation of human platelets (1, 2, 3) and the aggregatory effect of the catecholamine in platelet-rich plasma appear to be a consequence of alpha-adrenoceptor stimulation (4, 5, 6). The high absolute density of alpha2-adrenoceptor sites as well as the alpha2/beta density ratio of 3-4 (71 might explain the alpha2 excitatory effect of adrenaline in human platelets. In contrast, in rat platelets, adrenaline has been shown to inhibit the thrombin-induced aggregation, thus suggesting a beta-adrenergic-mediated effect of catecholamine (81. Since approximately equal numbers of alpha2- and beta-adrenoceptors have been found in rat platelets (71, the question arises as to the role played by the alpha2-adrenoceptors in this species. There is now ample evidence that platelet activation by physiological agents induces rapid modifications of membrane components which may participate to the platelet shape-change, the release reaction and aggregation (9). To our knowledge, the effects of adrenaline on the thrombin-induced platelet responses have been studied so far only in terms of aggregation and at long times after stimulation. This prompted us to further characterize the mechanism whereby adrenaline influences the thrombin-induced responses in rat platelets. For this purpose we investigated the effect of adrenaline - and alpha- and beta-adrenergic agents - on the thrombin-induced serotonin release, at early times after platelet stimulation. This physiological response allows accurate measurements of the early changes promoted by platelet stimulation_and may represent a sensitive index of the initial platelet/agonist interaction. Our results describe in a detailed functional study, the role of alphaand beta-adrenoceptors in rat platelets, measured as variation of thrombininduced serotonin release. MATERIALS AND METHODS 5-hydroytryptamine-creatine sulfate, L-epinephrine-bitartrate, DLisoproterenol-HCl, L-alprenolol-D-tartrate,yohimbine-HCl were obtained from Sigma (St. Louis, Mo; USA). (-l-norepinephrine was obtained from SterlingWinthrop Research Institute (Rensselaer, NY; USA). Clonidine-HCl was from Boehringer-Ingelheim (FRG). Prazosin was supplied by Pfizer (Brussels, Belgium). Guanfacine was from Sandoz Ltd (Basel, Switzerland), rauwolscine was from Carl Roth KG (Karlsruhe, FRG) and methoxamine, from Wellcome Research Laboratories (USA). Timolol-maleate was provided by Merck (FRG). BHT 920 was kindly provided by Pr. Schmitt (France). C3Hl-5_hydroxytryptamine (14.9 Ci/mmole) was obtained from Amersham (The Radiochemical Center, UK). Bovin thrombin (63 U/mg) was from Hoffmann-La Roche (Basel, Switzerland). METHODS Preparation of [3H]-serotonin loaded washed platelets. Under light ether anaesthesia, blood was withdrawn from the abdominal aorta of male Wistar rats (~250 g, If fa- Credo, France) on 0.1 volume of acid-citrate- dextrose (ACD/C) (170 mM trisodium citrate, 130 mM citric acid, 101 mM dextrose) as anticoagulant. Platelet-rich plasma was obtained by centrifugation at 180 g for 20 min at room temperature. Platelet-rich plasma (PRP) from 6-8 rats was pooled for each experiment. PRP was incubated with lo-6M Ei-hydroxytryp-
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tamine containing 5yCi [3H]-serotonin (14.9 Ci/mnol) for 5 min at 37°C. All subsequent experiments for preparing platelets were carried out at room temperature. PRP was then centrifuged at 400 g for 15 min and platelets were washed twice with a calcium-free Tyrode's buffer (RaCl 137 mM; KC1 2.6 mM; NaHC03 12 mM; MgC12 0.9 mM; glucose 5.5 mM; gelatin 0.25 %, pti 6.5) by centrifugation at 400 g for 15 min. The washed platelets were finally resuspended in a calcium-free Tyrode's HEPES buffer (NaCl 137 mM; KC1 2.6 mM; HEPES 5 mM; MgC12 0.9 mM; glucose 5.5 mM; gelatin 0.25 %, pH 7.4) (10). Measurement of [3H]-serotonin platelet release. Washed-platelet suspensions were adJusted to 2-4 x 100 platelets/ml and calcium (1.3 mM final concentration) was added 20 min before starting the experiments. Platelet samples (0.4 ml)-were placed in aggregometer tubes and stirred for 3 min at 37°C in a Chronolog aggregometer before the addition of 4 ~1 thrombin to initiate the release reaction. The adrenergic agents and catecholamines to be tested were added in a volume of 4~1 at 2 and 1 min, respectively prior to the addition of thrombin. In some experiments, 1 min before addition of catecholamines, either 1 (iM imipramine or 2 mM creatin phosphate/20 U/ml creatin phosphokinase were added to the platelet suspension to determine respectively the ability of serotonin uptake or trace concentrations of ADP to interfere with the effects of catecholamines upon the thrombin-induced serotonin release. Light transmission was recorded throughout the experiment and displayed on a chart recorder. Decreased light transmission represents the shape change of platelets from a disc to a sphere, while increased light transmission reflects platelet aggregation. Platelet serotonin release was determined in every experiment by assaying the appearence of C3H1-serotonin in the platelet-free suspension. Following thrombin addition and incubation for the designated times, the samples were transfered to tubes containing 80 ~1 ice-cold EDTA (0.1 Ml for terminating the release reaction. The samples were immediately centrifuged for 30 set in an Eppendorf centrifuge and, on an aliquot of the supernatant, [3H]-serotonin was determined by scintillation counting. The percent of [3H]-serotonin released was calculated by comparing the radioactivity present in the supernatants of stimulated platelets (Rsl to the total amount of releasable radioactivity (Rt). The latter was obtained by stimulating the platelet suspensions with thrombin (5 U/ml) for 5 min at 37°C. The amount of radioactivity in the supernatants of unstimulated platelet suspensions (Ru) (i.e. spontaneous release) was subtracted from that observed following stimulation and the % C3HI-serotonin release was expressed as the ratio: Rs-Ru/Rt-Ru. "Spontaneous" serotonin release (Ru) averaged 3-5 % of the total releasable serotonin and was not affected by the various drugs used throught this study.
RESULTS Kinetics of the thrombin-induced serotonin release and effects of adrenergic Klnetlcs of serotonln release were first studied as a fumclon of agents. -in concentration (Fig. 1). The results revealed that the percentage of serotonin release incremith the dose of thrombin up to 30 set and plateaued between 30 and 60 set of stimulation. The addition of 50~ M adrenaline 1 min prior to thrombin stimulation induced a decrease in serotonin release (Fig. 1). Other adrenergic agents such as isoproterenol and noradrenaline actedsimilarly (not shown); the relative order of potency of these agonists for inhibiting the thrombin-induced serotonin release was adrenaline isoproterenol noradrenaline. >/ >
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0
10
20
30
TIME
60
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FIG. 1 Kinetics of thrombin-induced secretion of [3Hl-serotonin in presence or absence of adrenal1ne. Suspensions of washed rat platelets prelabeled with [3H]-serotonin were placed into aggregometer tubes and preincubated for 3 min at 37°C before exposure to thrombin (closed symbols) for the indicated times of stirmlation (A, 2 U/ml; 0, 0.5 U/ml; 4, 0.25 U/ml). Adrenaline (50 MM) was added 1 minute before exposure to thrombin (open symbols). Data were expressed as % of the total releasable serotonin. (O,U, A,& Values are mean of two independent experiments performed in duplicate. (O,O) Values are mean + S.E.M. from 6-8 independent experiments performed in duplicate.
In order to study the effect of adrenergic agents on the serotonin release early after platelet activation by thrombin and to obtain a measurable release as well as a consistent inhibition of the release, without using high thrombin concentration, 0.5 U/ml thrombin and 20 set of thrombin stimulation appeared to be as the most appropriate conditions for our purpose. Thus, all subsequent experiments were performed using these latter conditions. Dose-response of the inhibitory effect of isoproterenol and adrenaline upon thrombin-induced platelet responses. Isoproterenol and adrenaline both inhibit d th thrombin-induced serotonin release in a dose-dependant manner (Fig. 27. Th"emaximum inhibitory effect observed with saturating concentratmlO))M) of isoproterenol and adrenaline reached 81.5 + 2.5 % and 44.7 + 2.2 X, respectively. Such inhibitions were not affected bythe presence of ADP scavengers and of imipramine in the incubation medium. The molar cdncentrations required to produce half-maxinum inhibitory effect were 0.082 + 0.009pM and 0.19 + 0.02 PM, respectively for isoproterenol and adrenaline-
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FIG.2 Dose-response curves of the effect of adrenoceptor agonists on the thrombininduced serotonin release. Suspensions of washed rat platelets prelabeled with [3H1-serotonin were placed into aggregometer tubes and preincubated for 3 min before exposure to 0.5 U/ml thrombin. After 20 set of stimulation by thrombin, the platelet samples were analysed for serotonin release as described in "Methods". When present, the adrenoceptor agonists were added 1 min before thrombin. (HI, adrenaline. The drug-induced inhibition of serotonin isoproterenol; (-1, release is expressed as the percentage of serotonin release in the presence of drugs compared to the release observed with thrombin alone. Values are mean + S.E.M. of 3-4 independent experiments using duplicate determinations. The aggregation studies performed in parallel show that the pretreatment of-platelets with either isoproterenol or adrenaline diminished the extent of thrombin-induced aggregation (Fig. 3) without any significant change in aggregation velocity (not shown).Thenhibitory effect of the adrenergic agents increased in a dose-dependent manner and isoproterenol appeared to be more potent than adrenaline for inhibiting thrombin-induced aggregation. In addition, it should be noted that the lag-phase of the thrombin-induced aggregation, provoked by increasing concentrations of these drugs, increased with a concomitant unmasking of platelet shape-change. Characterization of the adrenergic components responsible for inhibiting the Prior addition of beta-adrenergic tirombin-Induced serotonin release. blockers, alprenolol or tlmolol (It MI abolished the inhibitory effect of he thrombin-induced serotonin release adrenaline and isoproterenol on (Table 1). Moreover, in the presence of these antagonists, adrenaline exhibited a slight potentiating effect on thrombin-induced serotonin release.
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(a) A
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(M)
FIG. 3 Effect of adrenoceptor agonists on the thrombin-induced aggregation. Experimental conditions were as in Fig. 2. Aggregation was recorded for 20 set and when present, the adrenoceptor agonists were added I min before thrombin. (Hatched column), isoproterenol; (white column), adrenaline. Data were expressed as % aggregation observed with drugs compared with aggregation observed with thrombin alone (taken as 100 %) (black column). Each point is a single determination and this experiment is representative of two others that yielded similar results. Example traces of thrombin-induced aggregation (a), in presence of 1OrM isoproterenol (bl or in presence of 10 PM adrenaline (cl. TABLE 1 Effect of beta-adrenoceptor antagonists on isoproterenol- and adrenalineinduced inhibition of serotonin release Additions
- None Alprenolol (IpM) 'imolol (1pM)
Adrenaline (10t~M)
Isoproterenol (1OpM)
- 35.4
- 74.3
+
5.5
-
5.9
+
3.0
-
3.0
Experimental conditions were as in Fig. 2. When present, adrenergic antagonists were added 2 min before 0.5 U/ml thrombin, followed, 1 min later by 10 \'M adrenaline or 10~ M isoproterenol. Results are expressed as percent inhibition (-1 or potentiation (+I of 5HT release versus controls, thrombin
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alone or thrombin + beta-antagonists (the beta-blockers (lr M) did not af feet the thrombin-induced 5HT release). Results are the mean of two experiments performed in duplicate. In order to elucidate the difference observed in the inhibitory potencies between adrenaline and isoproterenol, experiments were carried out in which alpha-adrenergic blocking agents were added 1 min before the addition of a saturating concentration of adrenaline (10~ M) (i.e. 2 min prior to thrombin stimulation). Results (Fig. 4) revealed that razosin, an alphaladrenergic antagonist, in the range of IO-8-5.10~& did not affect the potency of adrenaline for inhibiting thrombin-induced'serotoninrelease. Under the same experimental conditions, rauwolscine and yohimbine (lo-9-5.10-5M), both alpha2-adrenergic blockers, increased in a dose-dependent manner the inhibitory effect of adrenaline. The maximum values of increase (expressed as the difference between the percentages of adrenaline-induced inhibition with or without alpha2-adrenergic blockers) were 29.2 + 2.3 % and 26.7 + 2.2 %, for rauwolscine and yohimbine, respectively. Under these condition?, the inhibitory extent then reached by adrenaline in the presence of the alpha2-antagonists was close to that obtained by isoproterenol, when tested alone. The molar concentrations required to produce half-maximum effect in the extent of inhibition were 0.08 + 0.01~ M and 0.32 + 0.14~M, respectively for rauwolscine and yohimbine. fi addition, it shouTd be noted that yohimbine and rauwolscine did not influence the effectiveness of isoproterenol to inhibit the thrombin-induced serotonin release (not shown). In this/study, recordings of aggregation (not shown) also indicated that alpha2-adrenergic blockers increased adrenaline inhibitory effect upon thrombin-induced aggregation to an extent close to that observed with isoproterenol.
-Log
[DRUG
1(Ml
FIG.4 Effect of alpha-adrenoceptor antagonists on adrenaline inhibitory effect of thrombin-induced serotonin release. Experimental conditions were as in Fig. 2. Adrenaline (1OpM)
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min before thrombin and when present, alpha-adrenoceptor antagonists were added 1 min before adrenaline. ( II 1, rauwolscine; ( A-A 1, yohimbine; (WI,, prazosin. Data were expressed as the variation observed in extent of inhibition: [% inhibition of serotonin release with adrenaline in the presence of alpha-adrenoceptor antagonists - % inhibition of serotonin release with adrenaline alone (taken as reference point 0 1. (M) Each point represents mean + SEM of 3 experiments using duplicates; (+l is a single determination usiiig duplicate; (A) is representative of two other experiments performed in duplicate that yielded similar results.
Conversely, the results obtained with the alpha2-adrenergic antagonists led us to test the effect of different alpha-adrenergic agonists upon isoproterenol effectiveness in inhibiting thrombin-induced serotonin release. 10-g-5. Methoxamine, an alphal-adrenergic the agonist, in range lO-!jM,had no effect upon the ability of isoproterenol to inhibit thrombin-induced serotonin release (not shown). Surprisingly, under the same experimental conditions, alphaZ-adrenergic agonists such as clonidine, guanfacine or BHT 920, tested in the range of 10-8-5.10'5M, were completely devoid of action upon isoproterenol effectiveness to inhibit thrombin-induced secretion (not shown). Since among these alpha2-adrenergic agonists, clonidine and guanfacine have been described as partial agonists or even antagonists on human platelets, these drugs were tested on the ability of adrenaline to inhibit thrombin-induced serotonin release. Results, reported in Table 2, show that both clonidine and guanfacine, when added 1 min before anoptimal concentration of adrenaline (lOpM), increased the potency of the catecholamine for inhibiting thrombin-induced serotonin release. Furthermore, the maximum extent of increase as well as the molar concentrations required to produce half-maximum effect were close to those obtained with the alpha2- adrenergic antagonists, under the same experimental conditions. TABLE 2 Antagonistic effect of alpha2-adrenoceptor agonists on the adrenalineinduced inhibitory effect of the serotonin release
Maximal Variation in Extent of Inhibition (A) %
EC50 (PM)
Drugs
Guanfacine
0.21 + 0.05 (n = 3)
+ 32.7 + 3.1 (n = 3)
Clonidine
0.75 _t 0.10 (n = 3)
+ 25.8 + 4.8 (n = 3)
BHT 920
>
10
(n = 2)
Experimental conditions were as in Fig. 2. Adrenaline (1OpM) was added 1 min before thrombin and when present, alpha2-adrenoceptor drugs were added 1 min before adrenaline. Data were expressed as the variation observed in extent of inhibition: [% inhibition of serotonin release with adrenaline in the presence of drugs - % inhibition of serotonin release with adrenaline alone]. EC50 values represent the molar concentrations of drugs required to produce the half- maximum effect in the extent of inhibition. The values given are mean 2 S.E.M. (where indicated) with the number of experiments in
1.
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parentheses. Since the presence of alphaZ-adrenoceptor blockers rendered adrenaline as inhibitory potent as isoproterenol, this suggested that the alpha2adrenergic component of the catecholamine might be involved in the difference observed in the inhibitory potencies between the two compounds. This led us to test whether the alpha2-component of catecholamines might be able to counteract the beta-adrenoceptor-mediated inhibition of isoproterenol. When platelqts were first challenged for 1 min with a beta-adrenergic agonist, isoproterenol (10 Ml, the further addition of catecholamines (adrenaline or noradrenaline) prgvoked - as a function of catecholamine concentration - a decrease in the effectiveness of isoproterenol for inhibiting the thrombininduced serotonin release (Fig. 5). This suggested that the alpha-component of catecholamines might be involved in counteracting the isoproterenol inhibiting effect upon thrombin-induced serotonin release. The maximum effect of both drugs was reached at 1OpM and, at this concentration, the variation in extent of inhibition (expressed as the difference between the percentages of isoproterenol-induced inhibition with or without catecholamine) reached 23.6 + 2.3 % and 35.8 + 0.9 %, respectively for noradrenaline and adrenaline. It iJ noteworthy that; under these conditions, the inhibitory effect observed reached an extent close to that which characterized the effect of catecholamines upon thrombin-induced serotonin release, when tested alone. The molar concentrations required to produce half- maximum effect of variation in the extent of inhibition were 0.72 + 0.05pM and 0.39 + 0.06 $4, respectively for noradrenaline and adrenaline. In addition, in this study, recordings of aggregation also indicated that catecholamines decreased the inhibitory effect of isoproterenol on thrombin-induced aggregation to an extent close to that obtained with catecholamines when tested alone (not shown).
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FIG. 5 Effect of catecholamines on isoproterenol inhibitory potency thrombln-induced serotonin release.
upon
the
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Experimental conditions were as in Fig. 2. Isoproterenol (1OpM) was added 2 min before thrombin and, when present, catecholamines were added 1 min after isoproterenol. (0-O) noradrenaline; (O-O), adrenaline. Data were expressed as the variation observed in extent of inhibition of serotonin release: 1% inhibition of serotonin release with isoproterenol in the presence of catecholamines - % inhibition of serotonin release with isoproterenol alone (taken as reference point 0 1. Each point is a single determination using duplicates and is representative of 3 others that yielded similar results.
DISCUSSION The present experiments were designed to study the influence of platelet adrenoceptors on the early thrombin-induced serotonin release. This study shows that, on washed rat platelets, adrenaline and isoproterenol inhibited, in a dose-dependent manner, the early thrombin-induced serotonin secretion and aggregation. Since beta-adrenoceptor antagonists abolished the inhibition of the thrombin-induced serotonin release, this' suggested that the inhibitory effects of both compounds might be mediated through betaadrenoceptor sites. Our results also show that, in the presence of betaadrenoceptor blockers, adrenaline exhibited only a slight potentiating effect of the thrombin-induced serotonin release. It seems therefore that under these experimental conditions, the alpha2-adrenergic component of adrenaline could only act weakly in potentiating the thrombin-induced response. Nevertheless, the present study shows that when rat platelets were challenged with isoproterenol, the inhibition was far more pronounced than with adrenaline. Furthermore, this difference in the inhibitory potency between the two compounds remained whatever their concentrations or the radioligand binding studies length of thrombin stimulation. Recently, demonstrated the presence of both beta-adrenoceptor sites (11) and alpha2adrenoceptor sites (7) on rat platelets. One may therefore envisage that the alpha2-component of adrenaline may account for the difference in inhibitory potency between isoproterenol and the catecholamine. Our observation that the highly selective alpha2-adrenoceptor antagonists rauwolscine and yohimbine (12, 13) potentiated the inhibitory effect of adrenaline to an extent close to that which characterized isoproterenol, lends support to the above hypothesis. Furthermore, this result suggests that, conversely, alpha2adrenoceptor agonists might exert a counteracting effect on the beta-adrenoceptor-mediated inhibition of thrombin-induced serotonin release. Nevertheless, the well known alpha2-adrenoceptor agonists clonidine, guanfacine and BtiT 920 (see 14 for review) failed to influence isoproterenol inhibitory effect upon thrombin-induced serotonin release. Inversely, clonidine and guanfacine potentiated the adrenaline inhibitory effect, as did rauwolscine and yohimbine, therefore suggesting an antagonistic activity. These results were not completely unexpected since several authors have reported that, in human platelets, clonidine and related compounds might act as partial agonists or even antagonists with respect to adrenaline-induced aggregation and adenylate cyclase inhibition (15-19). These latter results suggested that the alpha2-adrenergic component of the natural catecholamines might be able to counteract a beta-adrenoceptor-mediated inhibition. Interestingly, our data show that, in the presence of optimal concentration of isoproterenol, the further addition of adrenaline or noradrenaline provoked a marked decrease in the beta-adrenoceptor agonist inhibitory effect. Under these conditions, inhibition of thrombin-induced serotonin release reached an extent close to that which characterized this observed with catecholamines when tested alone. Finally, our study shows that methoxamine, an alphal-
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adrenergic agonist (201 is unable to mimic the ability of catecholamine to counteract the beta-adrenoceptor-mediated inhibition of the thrombin-induced serotonin release. Prazosin, an alphal-adrenergic antagonist (21) also failed to potentiate the inhibitory effect of adrenaline. Taken together, these results are consistent with an alphaZ-adrenergic counteracting effect of catecholamines on the beta-adrenoceptor-mediated inhibition of thrombininduced serotonin release. Thus, such an effect might account for the difference we observed in the inhibitory potency between isoproterenol and adrenaline. There is compelling evidence that the platelet release reaction triggered by thrombin is associated with a rapid mobilization of Ca2+ ions from intracellular binding sites into the cytosol (22-24). On the other hand, it is known that beta-adrenoceptor stimulation causes an increase in intracellular CAMP via stimulation of adenylate cyclase (251 whereas, in contrast, alpha2-adrenoceptors decrease CAMP concentration, via inhibition of adenylate cyclase (26-301. Although an action of thrombin on the intracellular CAMP level - which may or may not be mediated by adenylate cyclase - cannot be ruled out (31, 321, our results suggest that, on rat platelets, catecholamines, by increasin CAMP concentration, may exert an inhibitory early after platelet activaeffect on intracellular Ca 29 mobilization, tion. Furthermore, our finding that catecholamines may counteract a betainhibition of thrombin-induced serotonin release adrenoceptor-mediated suggests the ability of these compounds to antagonize stimulators of adenylto promote the mobilization of ate cyclase and thereby, indirectly, [Ca2+]i and secretion by thrombin.
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21. CAMBRIDGE, D., DAVEY, M.J. and MASSINGHAM, R. Prazosin, a selective antagonist of postsynaptic alpha-adrenoceptors. Br. J. Pharmacol. -59, 514P, 1977. 22. CHARO, I.F., FEINMAN, secretion by an antagonist Comm. 72, 1462-1467, 1976. --
R.D. and DETWILER, T.C. Inhibition of platelet of intracellular calcium. Biochem. Biophys. Res.
23. FEINMAN, R.D. and DETWILER, T.C. Platelet cation inophores. ~Nature, 249, 172-173, 1974.
secretion
induced by divalent
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24. MASSIMI, P. and LUSCHER, E.F. On the significance of influx of Ca2+ ions into stimulated human blood platelets. Biochim. Biophys. Acta, -' 436 652-663. 1976. 25. ABDULLA, Y.H. Beta-adrenergic receptors in human platelets. J. Atheroscler. Res. 2, 171-177, 1969. 26. ZIEVE, P.D. and GREENOUGH, W.B. Adenylcyclase in human platelets: acti462-466, 1969. vity and responsiveness. Biochem. Biophys. Res. Comm. 2, 27. MAR@IS, M.R., BECKER, J.A. and VIGDHAL, R.L. Platelet aggregation III. An epinephrine-induced decrease in cyclic AMP synthesis. Biochem. Biophys. Res. Comn. -39. 783-789. 1970. 28. HASLAM, R.J. and TAYLOR, A. Effects of catecholamines on the formation of adenosine 3', 5'-cyclic monophosphate in human blood platelets. Biochem. 125 377-379, 1971. -J. -’ 29. HARWOOD, J.P., MOSKOWITZ, J. and KRISHNA, G. Dynamic interaction of prostaglandin and norepinephrine in the formation of adenosine 3', 5'-monophosphate in human and rabbit platelets. Biochim. Biophys. Acta, 261, 444456, 1972. 30. JAKOBS, K.H., SAUR, W. and SCHULTZ, G. Reduction of adenylate cyclase activity in lysates of human platelets by the alpha-adrenergic component of epinephrine. J. Cycl. Nucleotide Res. 2, 381-392, 1976. 31. BRODIE, G.N., BAEZIGER, M.L., CHASE, L.R. and MAJERUS, P.W. The effects of thrombin on adenyl cyclase activity and a membrane protein from human platelets. J. Clin. Invest. -51, 81-88, 1972. 32. SALZMAN, E.W. and NERI, L.L. Cyclic 3',5'-adenosine monophosphate in human blood platelets. Nature, 224, 609-610, 1969.
159
INFLUENCE OF ALPHA- AND BETA-AORENOCEPTORS ON THROMBIN-INDUCED SEROTONIN RELEASE IN RAT PLATELETS
S. Koutouzov, L. Cothenet-Vernoux, P. Marche and J.P.
Dausse
U7 INSERM, Department of Pharmacology, Hdpital Necker, 161 rue de Sevres 75015 Paris, France
(Received 22.5.1985; Accepted in original form 25.6.1985 by Editor B. Vargaftig) ABSTRACT This work was designed to investigate the influence of rat platelet adrenoceptors on the early thrombin-induced serotonin release. In washed platelets prelabeled with [3H]-serotonin, adrenaline and isoproterenol both inhibited, in a dose-dependent manner, the early thrombin-induced secretion of serotonin. Inhibitory responses of both adrenaline and isoproterenol were blocked in the presence of beta-adrenoceptor antagonists, suggesting that the catecholamine acted solely through beta-adrenoceptors. However, isoproterenol inhibited the thrombin-induced serotonin release to a much greater extent than the catecholamine, suggesting that the alpha2-component of adrenaline might account for the difference observed between the two compounds. Our observation that selective alpha2-adrenoceptor antagonists as yohimbine and rauwolscine potentiated the inhibitory effect of adrenaline to a level close to that observed with isoproterenol, lends support to the above hypothesis. This latter result suggested that, conversely, alpha2-adrenergic compounds might exert a counteracting effect on a full beta-adrenoceptor mediated inhibition. Although synthetic alpha2-adrenergic agents failed to influence isoproterenol inhibitory effect, our study shows that prestimulation of beta-adrenoceptors by isoproterenol, followed by addition of adrenaline or noradrenaline markedly diminished the inhibitory effect of isoproterenol to a level close to that which characterized the inhibition observed with catecholamines, when tested alone. Our work favours the hypothesis that, in rat platelets, early after platelet stimulation, catecholamines might counteract a beta-adrenoceptor- mediated inhibition, through alpha2-adrenoceptor sites. Key words: Platelets, catecholamines, rat, serotonin release, adrenoceptors Abbreviations: 5HT: 5-hydroxytryptamine (serotonin); Hepes: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; EDTA: [ethylene dinitrilol-tetraacetic acid; ADP: adenosine diphosphate. 147
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INTRODUCTION The synergistic effect of adrenaline on thrombin, collagen and ADPinduced aggregation of human platelets (1, 2, 3) and the aggregatory effect of the catecholamine in platelet-rich plasma appear to be a consequence of alpha-adrenoceptor stimulation (4, 5, 6). The high absolute density of alpha2-adrenoceptor sites as well as the alpha2/beta density ratio of 3-4 (71 might explain the alpha2 excitatory effect of adrenaline in human platelets. In contrast, in rat platelets, adrenaline has been shown to inhibit the thrombin-induced aggregation, thus suggesting a beta-adrenergic-mediated effect of catecholamine (81. Since approximately equal numbers of alpha2- and beta-adrenoceptors have been found in rat platelets (71, the question arises as to the role played by the alpha2-adrenoceptors in this species. There is now ample evidence that platelet activation by physiological agents induces rapid modifications of membrane components which may participate to the platelet shape-change, the release reaction and aggregation (9). To our knowledge, the effects of adrenaline on the thrombin-induced platelet responses have been studied so far only in terms of aggregation and at long times after stimulation. This prompted us to further characterize the mechanism whereby adrenaline influences the thrombin-induced responses in rat platelets. For this purpose we investigated the effect of adrenaline - and alpha- and beta-adrenergic agents - on the thrombin-induced serotonin release, at early times after platelet stimulation. This physiological response allows accurate measurements of the early changes promoted by platelet stimulation_and may represent a sensitive index of the initial platelet/agonist interaction. Our results describe in a detailed functional study, the role of alphaand beta-adrenoceptors in rat platelets, measured as variation of thrombininduced serotonin release. MATERIALS AND METHODS 5-hydroytryptamine-creatine sulfate, L-epinephrine-bitartrate, DLisoproterenol-HCl, L-alprenolol-D-tartrate,yohimbine-HCl were obtained from Sigma (St. Louis, Mo; USA). (-l-norepinephrine was obtained from SterlingWinthrop Research Institute (Rensselaer, NY; USA). Clonidine-HCl was from Boehringer-Ingelheim (FRG). Prazosin was supplied by Pfizer (Brussels, Belgium). Guanfacine was from Sandoz Ltd (Basel, Switzerland), rauwolscine was from Carl Roth KG (Karlsruhe, FRG) and methoxamine, from Wellcome Research Laboratories (USA). Timolol-maleate was provided by Merck (FRG). BHT 920 was kindly provided by Pr. Schmitt (France). C3Hl-5_hydroxytryptamine (14.9 Ci/mmole) was obtained from Amersham (The Radiochemical Center, UK). Bovin thrombin (63 U/mg) was from Hoffmann-La Roche (Basel, Switzerland). METHODS Preparation of [3H]-serotonin loaded washed platelets. Under light ether anaesthesia, blood was withdrawn from the abdominal aorta of male Wistar rats (~250 g, If fa- Credo, France) on 0.1 volume of acid-citrate- dextrose (ACD/C) (170 mM trisodium citrate, 130 mM citric acid, 101 mM dextrose) as anticoagulant. Platelet-rich plasma was obtained by centrifugation at 180 g for 20 min at room temperature. Platelet-rich plasma (PRP) from 6-8 rats was pooled for each experiment. PRP was incubated with lo-6M Ei-hydroxytryp-
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tamine containing 5yCi [3H]-serotonin (14.9 Ci/mnol) for 5 min at 37°C. All subsequent experiments for preparing platelets were carried out at room temperature. PRP was then centrifuged at 400 g for 15 min and platelets were washed twice with a calcium-free Tyrode's buffer (RaCl 137 mM; KC1 2.6 mM; NaHC03 12 mM; MgC12 0.9 mM; glucose 5.5 mM; gelatin 0.25 %, pti 6.5) by centrifugation at 400 g for 15 min. The washed platelets were finally resuspended in a calcium-free Tyrode's HEPES buffer (NaCl 137 mM; KC1 2.6 mM; HEPES 5 mM; MgC12 0.9 mM; glucose 5.5 mM; gelatin 0.25 %, pH 7.4) (10). Measurement of [3H]-serotonin platelet release. Washed-platelet suspensions were adJusted to 2-4 x 100 platelets/ml and calcium (1.3 mM final concentration) was added 20 min before starting the experiments. Platelet samples (0.4 ml)-were placed in aggregometer tubes and stirred for 3 min at 37°C in a Chronolog aggregometer before the addition of 4 ~1 thrombin to initiate the release reaction. The adrenergic agents and catecholamines to be tested were added in a volume of 4~1 at 2 and 1 min, respectively prior to the addition of thrombin. In some experiments, 1 min before addition of catecholamines, either 1 (iM imipramine or 2 mM creatin phosphate/20 U/ml creatin phosphokinase were added to the platelet suspension to determine respectively the ability of serotonin uptake or trace concentrations of ADP to interfere with the effects of catecholamines upon the thrombin-induced serotonin release. Light transmission was recorded throughout the experiment and displayed on a chart recorder. Decreased light transmission represents the shape change of platelets from a disc to a sphere, while increased light transmission reflects platelet aggregation. Platelet serotonin release was determined in every experiment by assaying the appearence of C3H1-serotonin in the platelet-free suspension. Following thrombin addition and incubation for the designated times, the samples were transfered to tubes containing 80 ~1 ice-cold EDTA (0.1 Ml for terminating the release reaction. The samples were immediately centrifuged for 30 set in an Eppendorf centrifuge and, on an aliquot of the supernatant, [3H]-serotonin was determined by scintillation counting. The percent of [3H]-serotonin released was calculated by comparing the radioactivity present in the supernatants of stimulated platelets (Rsl to the total amount of releasable radioactivity (Rt). The latter was obtained by stimulating the platelet suspensions with thrombin (5 U/ml) for 5 min at 37°C. The amount of radioactivity in the supernatants of unstimulated platelet suspensions (Ru) (i.e. spontaneous release) was subtracted from that observed following stimulation and the % C3HI-serotonin release was expressed as the ratio: Rs-Ru/Rt-Ru. "Spontaneous" serotonin release (Ru) averaged 3-5 % of the total releasable serotonin and was not affected by the various drugs used throught this study.
RESULTS Kinetics of the thrombin-induced serotonin release and effects of adrenergic Klnetlcs of serotonln release were first studied as a fumclon of agents. -in concentration (Fig. 1). The results revealed that the percentage of serotonin release incremith the dose of thrombin up to 30 set and plateaued between 30 and 60 set of stimulation. The addition of 50~ M adrenaline 1 min prior to thrombin stimulation induced a decrease in serotonin release (Fig. 1). Other adrenergic agents such as isoproterenol and noradrenaline actedsimilarly (not shown); the relative order of potency of these agonists for inhibiting the thrombin-induced serotonin release was adrenaline isoproterenol noradrenaline. >/ >
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I
0
10
20
30
TIME
60
(seconds)
FIG. 1 Kinetics of thrombin-induced secretion of [3Hl-serotonin in presence or absence of adrenal1ne. Suspensions of washed rat platelets prelabeled with [3H]-serotonin were placed into aggregometer tubes and preincubated for 3 min at 37°C before exposure to thrombin (closed symbols) for the indicated times of stirmlation (A, 2 U/ml; 0, 0.5 U/ml; 4, 0.25 U/ml). Adrenaline (50 MM) was added 1 minute before exposure to thrombin (open symbols). Data were expressed as % of the total releasable serotonin. (O,U, A,& Values are mean of two independent experiments performed in duplicate. (O,O) Values are mean + S.E.M. from 6-8 independent experiments performed in duplicate.
In order to study the effect of adrenergic agents on the serotonin release early after platelet activation by thrombin and to obtain a measurable release as well as a consistent inhibition of the release, without using high thrombin concentration, 0.5 U/ml thrombin and 20 set of thrombin stimulation appeared to be as the most appropriate conditions for our purpose. Thus, all subsequent experiments were performed using these latter conditions. Dose-response of the inhibitory effect of isoproterenol and adrenaline upon thrombin-induced platelet responses. Isoproterenol and adrenaline both inhibit d th thrombin-induced serotonin release in a dose-dependant manner (Fig. 27. Th"emaximum inhibitory effect observed with saturating concentratmlO))M) of isoproterenol and adrenaline reached 81.5 + 2.5 % and 44.7 + 2.2 X, respectively. Such inhibitions were not affected bythe presence of ADP scavengers and of imipramine in the incubation medium. The molar cdncentrations required to produce half-maxinum inhibitory effect were 0.082 + 0.009pM and 0.19 + 0.02 PM, respectively for isoproterenol and adrenaline-
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2’ J 0
I
I
I
I
I
I
9
8
7
6
5
4
-Log
[MIIIENEMIC
ux~usT](~)
FIG.2 Dose-response curves of the effect of adrenoceptor agonists on the thrombininduced serotonin release. Suspensions of washed rat platelets prelabeled with [3H1-serotonin were placed into aggregometer tubes and preincubated for 3 min before exposure to 0.5 U/ml thrombin. After 20 set of stimulation by thrombin, the platelet samples were analysed for serotonin release as described in "Methods". When present, the adrenoceptor agonists were added 1 min before thrombin. (HI, adrenaline. The drug-induced inhibition of serotonin isoproterenol; (-1, release is expressed as the percentage of serotonin release in the presence of drugs compared to the release observed with thrombin alone. Values are mean + S.E.M. of 3-4 independent experiments using duplicate determinations. The aggregation studies performed in parallel show that the pretreatment of-platelets with either isoproterenol or adrenaline diminished the extent of thrombin-induced aggregation (Fig. 3) without any significant change in aggregation velocity (not shown).Thenhibitory effect of the adrenergic agents increased in a dose-dependent manner and isoproterenol appeared to be more potent than adrenaline for inhibiting thrombin-induced aggregation. In addition, it should be noted that the lag-phase of the thrombin-induced aggregation, provoked by increasing concentrations of these drugs, increased with a concomitant unmasking of platelet shape-change. Characterization of the adrenergic components responsible for inhibiting the Prior addition of beta-adrenergic tirombin-Induced serotonin release. blockers, alprenolol or tlmolol (It MI abolished the inhibitory effect of he thrombin-induced serotonin release adrenaline and isoproterenol on (Table 1). Moreover, in the presence of these antagonists, adrenaline exhibited a slight potentiating effect on thrombin-induced serotonin release.
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’i
(a) A
100
I
9
a -Log
I
I
I
.7
6
5
[DRUG]
(M)
FIG. 3 Effect of adrenoceptor agonists on the thrombin-induced aggregation. Experimental conditions were as in Fig. 2. Aggregation was recorded for 20 set and when present, the adrenoceptor agonists were added I min before thrombin. (Hatched column), isoproterenol; (white column), adrenaline. Data were expressed as % aggregation observed with drugs compared with aggregation observed with thrombin alone (taken as 100 %) (black column). Each point is a single determination and this experiment is representative of two others that yielded similar results. Example traces of thrombin-induced aggregation (a), in presence of 1OrM isoproterenol (bl or in presence of 10 PM adrenaline (cl. TABLE 1 Effect of beta-adrenoceptor antagonists on isoproterenol- and adrenalineinduced inhibition of serotonin release Additions
- None Alprenolol (IpM) 'imolol (1pM)
Adrenaline (10t~M)
Isoproterenol (1OpM)
- 35.4
- 74.3
+
5.5
-
5.9
+
3.0
-
3.0
Experimental conditions were as in Fig. 2. When present, adrenergic antagonists were added 2 min before 0.5 U/ml thrombin, followed, 1 min later by 10 \'M adrenaline or 10~ M isoproterenol. Results are expressed as percent inhibition (-1 or potentiation (+I of 5HT release versus controls, thrombin
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alone or thrombin + beta-antagonists (the beta-blockers (lr M) did not af feet the thrombin-induced 5HT release). Results are the mean of two experiments performed in duplicate. In order to elucidate the difference observed in the inhibitory potencies between adrenaline and isoproterenol, experiments were carried out in which alpha-adrenergic blocking agents were added 1 min before the addition of a saturating concentration of adrenaline (10~ M) (i.e. 2 min prior to thrombin stimulation). Results (Fig. 4) revealed that razosin, an alphaladrenergic antagonist, in the range of IO-8-5.10~& did not affect the potency of adrenaline for inhibiting thrombin-induced'serotoninrelease. Under the same experimental conditions, rauwolscine and yohimbine (lo-9-5.10-5M), both alpha2-adrenergic blockers, increased in a dose-dependent manner the inhibitory effect of adrenaline. The maximum values of increase (expressed as the difference between the percentages of adrenaline-induced inhibition with or without alpha2-adrenergic blockers) were 29.2 + 2.3 % and 26.7 + 2.2 %, for rauwolscine and yohimbine, respectively. Under these condition?, the inhibitory extent then reached by adrenaline in the presence of the alpha2-antagonists was close to that obtained by isoproterenol, when tested alone. The molar concentrations required to produce half-maximum effect in the extent of inhibition were 0.08 + 0.01~ M and 0.32 + 0.14~M, respectively for rauwolscine and yohimbine. fi addition, it shouTd be noted that yohimbine and rauwolscine did not influence the effectiveness of isoproterenol to inhibit the thrombin-induced serotonin release (not shown). In this/study, recordings of aggregation (not shown) also indicated that alpha2-adrenergic blockers increased adrenaline inhibitory effect upon thrombin-induced aggregation to an extent close to that observed with isoproterenol.
-Log
[DRUG
1(Ml
FIG.4 Effect of alpha-adrenoceptor antagonists on adrenaline inhibitory effect of thrombin-induced serotonin release. Experimental conditions were as in Fig. 2. Adrenaline (1OpM)
was added 1
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min before thrombin and when present, alpha-adrenoceptor antagonists were added 1 min before adrenaline. ( II 1, rauwolscine; ( A-A 1, yohimbine; (WI,, prazosin. Data were expressed as the variation observed in extent of inhibition: [% inhibition of serotonin release with adrenaline in the presence of alpha-adrenoceptor antagonists - % inhibition of serotonin release with adrenaline alone (taken as reference point 0 1. (M) Each point represents mean + SEM of 3 experiments using duplicates; (+l is a single determination usiiig duplicate; (A) is representative of two other experiments performed in duplicate that yielded similar results.
Conversely, the results obtained with the alpha2-adrenergic antagonists led us to test the effect of different alpha-adrenergic agonists upon isoproterenol effectiveness in inhibiting thrombin-induced serotonin release. 10-g-5. Methoxamine, an alphal-adrenergic the agonist, in range lO-!jM,had no effect upon the ability of isoproterenol to inhibit thrombin-induced serotonin release (not shown). Surprisingly, under the same experimental conditions, alphaZ-adrenergic agonists such as clonidine, guanfacine or BHT 920, tested in the range of 10-8-5.10'5M, were completely devoid of action upon isoproterenol effectiveness to inhibit thrombin-induced secretion (not shown). Since among these alpha2-adrenergic agonists, clonidine and guanfacine have been described as partial agonists or even antagonists on human platelets, these drugs were tested on the ability of adrenaline to inhibit thrombin-induced serotonin release. Results, reported in Table 2, show that both clonidine and guanfacine, when added 1 min before anoptimal concentration of adrenaline (lOpM), increased the potency of the catecholamine for inhibiting thrombin-induced serotonin release. Furthermore, the maximum extent of increase as well as the molar concentrations required to produce half-maximum effect were close to those obtained with the alpha2- adrenergic antagonists, under the same experimental conditions. TABLE 2 Antagonistic effect of alpha2-adrenoceptor agonists on the adrenalineinduced inhibitory effect of the serotonin release
Maximal Variation in Extent of Inhibition (A) %
EC50 (PM)
Drugs
Guanfacine
0.21 + 0.05 (n = 3)
+ 32.7 + 3.1 (n = 3)
Clonidine
0.75 _t 0.10 (n = 3)
+ 25.8 + 4.8 (n = 3)
BHT 920
>
10
(n = 2)
Experimental conditions were as in Fig. 2. Adrenaline (1OpM) was added 1 min before thrombin and when present, alpha2-adrenoceptor drugs were added 1 min before adrenaline. Data were expressed as the variation observed in extent of inhibition: [% inhibition of serotonin release with adrenaline in the presence of drugs - % inhibition of serotonin release with adrenaline alone]. EC50 values represent the molar concentrations of drugs required to produce the half- maximum effect in the extent of inhibition. The values given are mean 2 S.E.M. (where indicated) with the number of experiments in
1.
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parentheses. Since the presence of alphaZ-adrenoceptor blockers rendered adrenaline as inhibitory potent as isoproterenol, this suggested that the alpha2adrenergic component of the catecholamine might be involved in the difference observed in the inhibitory potencies between the two compounds. This led us to test whether the alpha2-component of catecholamines might be able to counteract the beta-adrenoceptor-mediated inhibition of isoproterenol. When platelqts were first challenged for 1 min with a beta-adrenergic agonist, isoproterenol (10 Ml, the further addition of catecholamines (adrenaline or noradrenaline) prgvoked - as a function of catecholamine concentration - a decrease in the effectiveness of isoproterenol for inhibiting the thrombininduced serotonin release (Fig. 5). This suggested that the alpha-component of catecholamines might be involved in counteracting the isoproterenol inhibiting effect upon thrombin-induced serotonin release. The maximum effect of both drugs was reached at 1OpM and, at this concentration, the variation in extent of inhibition (expressed as the difference between the percentages of isoproterenol-induced inhibition with or without catecholamine) reached 23.6 + 2.3 % and 35.8 + 0.9 %, respectively for noradrenaline and adrenaline. It iJ noteworthy that; under these conditions, the inhibitory effect observed reached an extent close to that which characterized the effect of catecholamines upon thrombin-induced serotonin release, when tested alone. The molar concentrations required to produce half- maximum effect of variation in the extent of inhibition were 0.72 + 0.05pM and 0.39 + 0.06 $4, respectively for noradrenaline and adrenaline. In addition, in this study, recordings of aggregation also indicated that catecholamines decreased the inhibitory effect of isoproterenol on thrombin-induced aggregation to an extent close to that obtained with catecholamines when tested alone (not shown).
0
-a-
0
0-0
\
0 \
0
0.
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0
0 \
0
0
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O-I-
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7
6
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FIG. 5 Effect of catecholamines on isoproterenol inhibitory potency thrombln-induced serotonin release.
upon
the
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Experimental conditions were as in Fig. 2. Isoproterenol (1OpM) was added 2 min before thrombin and, when present, catecholamines were added 1 min after isoproterenol. (0-O) noradrenaline; (O-O), adrenaline. Data were expressed as the variation observed in extent of inhibition of serotonin release: 1% inhibition of serotonin release with isoproterenol in the presence of catecholamines - % inhibition of serotonin release with isoproterenol alone (taken as reference point 0 1. Each point is a single determination using duplicates and is representative of 3 others that yielded similar results.
DISCUSSION The present experiments were designed to study the influence of platelet adrenoceptors on the early thrombin-induced serotonin release. This study shows that, on washed rat platelets, adrenaline and isoproterenol inhibited, in a dose-dependent manner, the early thrombin-induced serotonin secretion and aggregation. Since beta-adrenoceptor antagonists abolished the inhibition of the thrombin-induced serotonin release, this' suggested that the inhibitory effects of both compounds might be mediated through betaadrenoceptor sites. Our results also show that, in the presence of betaadrenoceptor blockers, adrenaline exhibited only a slight potentiating effect of the thrombin-induced serotonin release. It seems therefore that under these experimental conditions, the alpha2-adrenergic component of adrenaline could only act weakly in potentiating the thrombin-induced response. Nevertheless, the present study shows that when rat platelets were challenged with isoproterenol, the inhibition was far more pronounced than with adrenaline. Furthermore, this difference in the inhibitory potency between the two compounds remained whatever their concentrations or the radioligand binding studies length of thrombin stimulation. Recently, demonstrated the presence of both beta-adrenoceptor sites (11) and alpha2adrenoceptor sites (7) on rat platelets. One may therefore envisage that the alpha2-component of adrenaline may account for the difference in inhibitory potency between isoproterenol and the catecholamine. Our observation that the highly selective alpha2-adrenoceptor antagonists rauwolscine and yohimbine (12, 13) potentiated the inhibitory effect of adrenaline to an extent close to that which characterized isoproterenol, lends support to the above hypothesis. Furthermore, this result suggests that, conversely, alpha2adrenoceptor agonists might exert a counteracting effect on the beta-adrenoceptor-mediated inhibition of thrombin-induced serotonin release. Nevertheless, the well known alpha2-adrenoceptor agonists clonidine, guanfacine and BtiT 920 (see 14 for review) failed to influence isoproterenol inhibitory effect upon thrombin-induced serotonin release. Inversely, clonidine and guanfacine potentiated the adrenaline inhibitory effect, as did rauwolscine and yohimbine, therefore suggesting an antagonistic activity. These results were not completely unexpected since several authors have reported that, in human platelets, clonidine and related compounds might act as partial agonists or even antagonists with respect to adrenaline-induced aggregation and adenylate cyclase inhibition (15-19). These latter results suggested that the alpha2-adrenergic component of the natural catecholamines might be able to counteract a beta-adrenoceptor-mediated inhibition. Interestingly, our data show that, in the presence of optimal concentration of isoproterenol, the further addition of adrenaline or noradrenaline provoked a marked decrease in the beta-adrenoceptor agonist inhibitory effect. Under these conditions, inhibition of thrombin-induced serotonin release reached an extent close to that which characterized this observed with catecholamines when tested alone. Finally, our study shows that methoxamine, an alphal-
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adrenergic agonist (201 is unable to mimic the ability of catecholamine to counteract the beta-adrenoceptor-mediated inhibition of the thrombin-induced serotonin release. Prazosin, an alphal-adrenergic antagonist (21) also failed to potentiate the inhibitory effect of adrenaline. Taken together, these results are consistent with an alphaZ-adrenergic counteracting effect of catecholamines on the beta-adrenoceptor-mediated inhibition of thrombininduced serotonin release. Thus, such an effect might account for the difference we observed in the inhibitory potency between isoproterenol and adrenaline. There is compelling evidence that the platelet release reaction triggered by thrombin is associated with a rapid mobilization of Ca2+ ions from intracellular binding sites into the cytosol (22-24). On the other hand, it is known that beta-adrenoceptor stimulation causes an increase in intracellular CAMP via stimulation of adenylate cyclase (251 whereas, in contrast, alpha2-adrenoceptors decrease CAMP concentration, via inhibition of adenylate cyclase (26-301. Although an action of thrombin on the intracellular CAMP level - which may or may not be mediated by adenylate cyclase - cannot be ruled out (31, 321, our results suggest that, on rat platelets, catecholamines, by increasin CAMP concentration, may exert an inhibitory early after platelet activaeffect on intracellular Ca 29 mobilization, tion. Furthermore, our finding that catecholamines may counteract a betainhibition of thrombin-induced serotonin release adrenoceptor-mediated suggests the ability of these compounds to antagonize stimulators of adenylto promote the mobilization of ate cyclase and thereby, indirectly, [Ca2+]i and secretion by thrombin.
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on
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4. O'BRIEN, J.R. Some effect of adrenaline and antiadrenaline platelets in vitro and in vivo. -Nature, 200, 763-764, 1963.
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on
5. BYGDEMAN, S. and JOHMSEN, 0. Studies on the effect of adrenergic blocking drugs on catecholamine-induced platelet aggregation and uptake of noradrenaline and 5-hydroxytryptamine. Acta Physiol. Stand. -75, 129-138, 1969. SHARMA, H.M., PANGANAMALA, R-V., GEER, J.C. and CORNWELL, D.G. Inhibi6. tion of platelet aggregation induced by either epinephrine or arachidonic acid. Thromb. Res. 7, 879-884, 1975. 7. KEKRY, R., SCRUTTON, M.C. and WALLIS, R.B. Mammalian platelet adrenoceptors. Br. J. Pharmacol. -’ 81 91-102, 1984. 8. YU, S.K. and LATOUR, J.G. Potentiation by alpha-and inhibition by betaadrenergic stimulations of rat platelet aggregation. Thrombos. Haemostas. 37, 413-422, 1977.
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9. LAPETINA, E.G. and CUATRECASAS, P. Stimulation of phosphatidic acid in platelets precede the formation of arachidonate and parallels the release of serotonin. Biochem. Biophys. Acta, 573, 394-402, 1979. 10. RANDON, J., LEFORT, J. and VARGAFTIG, B. Comparison between gregating effect of ticlopidine in whole blood, platelet-rich masked platelets of the rat. Thromb. Res. -26, 13-20, 1982.
11. KEKRY, K. and SCRUTTON, M.C. Platelet p-adrenoceptors. 79 _'
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