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Comparison of the actions of serotoninergic agents on human saphenous veins and platelets
European Journal of Pharmacology, 124 (1986) 107-111 Elsevier
107
C O M P A R I S O N OF T H E A C T I O N S OF S E R O T O N I N E R G I C AGENTS O N H U M A N S A P H E N O U S V E I N S AND P L A T E L E T S MIKAEL VICTORZON,CARLO TAPPARELLIand ELSE MI]LLER-SCHWEINITZER* Preclinical Research, Sandoz Ltd., CH-4002 Basel, Switzerland
Received 14 November 1985, revised MS received 23 January 1986, accepted 11 February 1986
M. VICTORZON, C. TAPPARELLI and E. MOLLER-SCHWEINITZER, Comparison of the actions ofserotoninergic agents on human saphenous veins and platelets, European J. Pharmacol. 124 (1986) 107-111. Changes in tension of helical strips from human saphenous veins and reversible aggregation of human platelets were recorded in vitro. Comparison of the activities of 12 serotonin receptor agonists revealed that only 4 of the investigated tryptamine derivatives acted as full agonists on both tissues. 5-Carboxamidotryptamine, a drug with selective affinity for both 5-HTIA and 5-HT~B binding sites, though the most potent agonist on veins, failed to produce platelet aggregation but acted as a weak antagonist of the 5-HT-induced reversible aggregation. The tetralin derivative 8-OH-DPAT, a drug with selective affinity for 5-HT~A binding sites, was a weak partial agonist on veins and completely devoid of any activity on the platelets. The antagonism of 5-HT by spiperone was monophasic on platelets but biphasic on veins. These data are in line with the contention that the 5-HT-induced reversible aggregation of human platelets is initiated by 5-HT2-1ike recognition sites while the evidence suggests that the contractile response of human saphenous vein to 5-HT reflects activation of both 5-HT2- and 5-HTl-like recognition sites. Human saphenous vein
Tryptamine derivatives
Platelet aggregation
1. Introduction More than 100 years ago, Virchow emphasised that besides alterations in the vessel wall, changes of both blood flow and blood composition predispose to thrombosis. While the development of one of these alterations may only facilitate thrombogenesis, the concomitant appearance of two of these changes will lead to the formation of a thrombus. It has been shown that 5-hydroxytryptamine (5-HT) receptors are involved in both venoconstriction and platelet aggregation. Thus 5-HT is a potent venoconstrictor of human hand veins in situ (Aellig, 1982) and of human femoral and saphenous veins in vitro (Glusa, 1984; Glusa and Markwardt, 1984; Miiller-Schweinitzer, 1984). Furthermore, 5-HT induces shape change and reversible aggregation of human platelets and may, in addition, potentiate aggregation brought about * To whom all correspondenceshould be addressed. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
5-HT receptors
In vitro
by various other stimuli such as ADP, collagen, adrenaline and noradrenaline (Ball et al., 1977; Baumgartner and Born, 1968; DeClerck et al., 1982; Michal and Motamed, 1976). The present study was performed to investigate whether there exist differences between the 5-HT receptors mediating venoconstriction and those initiating reversible platelets aggregation.
2. Materials and methods 2.1. Vein strips
Human saphenous veins were obtained from patients undergoing surgical removal of varicose veins. The patients had usually been premedicated with droperidol (2.5-5 mg) and atropine (0.5 mg) and the anaesthetic used was a barbiturate and a halothane-nitrous oxide mixture. The veins were placed in Krebs-Henseleit solution (mmol/1):
108 NaC1 118, KC1 4.7, MgSO 4 1.2, CaCI 2 1.2, K H z P O 4 1.2, N a H C O 3 25, glucose 11, EDTA 0.03, gassed with 5% CO 2 in oxygen and transported to the laboratory within 2-4 h. The veins were cleaned of loose connective tissue and stored overnight in Krebs-Henseleit solution at 4°C. Thereafter the veins were cut into helical strips (15 × 2 mm) with a ring at one end and suspended in 10 ml organ baths containing Krebs-Henseleit solution at 37°C, gassed continuously with 5% CO 2 in 02. Changes in the tone of the preparations were monitored isometrically. At the beginning of the experiments the strips were stretched to an initial tension of 7.5 mN and allowed to equilibrate for at least 4 h in the bathing medium which was changed every 15 rain. During this time the preparations were stimulated twice with 5-HT (1 /~mol/1) before the baseline tension was readjusted to about 3 mN. Thereafter a cumulative concentration-response curve for 5-HT was determined the maximum of which was taken as a point of comparison for subsequent responses. Repeated washouts were followed by the determination of cumulative concentration-response curves for 5-HT and various tryptamine derivatives. The curves were obtained by increasing the concentration of the agonist in the organ bath by a factor 4 when the maximum effect had been produced by the preceding concentration. These curves, consisting of 6-7 concentrations, were determined in the presence of 30 /tmol/l pargyline added 20 min before the agonist in order to prevent inactivation of the indole derivatives by monoamine oxidase (Vane, 1959). When the antagonism of 5-HT by spiperone was investigated, an incubation period of 15 min was allowed. Responses to the agonists are expressed as percentages of the maximal response to 5-HT. In each experiment 6 strips of the same vein were investigated at the same time, one of the strips being stimulated only with 5-HT and thus serving as a control preparation to correct for any sensitivity changes during the experiment. 2.2. Aggregation
Human platelet-rich plasma (PRP) was prepared from blood obtained from healthy volun-
teers. The blood was mixed with 3.8% (w/v) trisodium citrate (9:1) and platelet-rich plasma (PRP) was separated by centrifugation of 25 ml blood samples in 50 ml plastic tubes (Falcon) for 10 min at 1100 rpm (RT 6000; Sorvall) and kept at room temperature (max. 4-6 h) until used. Drugs were dissolved daily before use and diluted with RCD buffer. The RCD buffer consisted of Ringer solution (mmol/l: NaC1 150, KCI 5.63, CaC12 2.25) mixed with 3.8% (w/v) trisodium citrate and 1% (w/v) dextrose ( 6 : 2 : 1) at pH 7.4. Platelet aggregation was studied in a double-channel Payton aggregometer (Model 300 BD-5) connected to a Rikadenki recorder. PRP (0.5 ml) was placed in a cuvette containing a steel stirring bar and stirred (1100 rpm) at 37°C for 2 min prior to the addition of 20 #1 of the agonist. The aggregation induced by the agonist was expressed as a percentage of the maximum reversible aggregation induced by 5-HT alone. When the antagonism of 5-HT was investigated, an incubation period of 2 min was allowed before 5-HT (20/~1) was added. 2.3 Drugs used and their sources
The following pharmacological agents were used: 5-hydroxytryptamine creatinine sulfate (5HT), 5-methoxytryptamine hydrochloride (5OCH3-T, Fluka, Buchs, Switzerland), 5-methyltryptamine (5-CH3-T), tryptamine hydrochloride (T), pargyline hydrochloride (Sigma, Munich, FRG), a-methyl-5-hydroxytryptamine creatinine sulfate (a-CH3-5-HT, Upjohn, Kalamazoo, MI, USA), spiperone (Janssen, Beerse, Belgium), /3methyl-5-hydroxytryptamine hydrogen oxalate (/3C H 3-5-HT), ~o-N-methyl-5-hydroxytryptamine oxalate (~o-N-CH3-5-HT), N,N-dimethyl-5-hydroxytryptamine binoxalate (N,N-(CH3)2-5-HT), 5-aminotryptamine oxalate (5-NH2-T), 5-carboxamidotryptamine hydrogen maleinate (5-CONHz-T ), 4-hydroxytryptamine creatinine sulfate (4-HT) and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) were synthesized at Sandoz Ltd., Basel, Switzerland. All compounds were dissolved just before use. Drug concentrations are given as molar concentrations throughout. Statistical analysis of data was performed using Student's t-test.
109 TABLE 1 Parameters calculated for various serotonin receptor agonists on isolated human saphenous veins and platelets. ECso values for stimulation and ICs0 values for antagonism of 5-HT-induced reversible platelets aggregation are given as the negative logarithms, maximal effects (Era,x) of the agonists are expressed in relation to the the maximum effect of 5-HT ( = 1.0). Data are presented as means_+ S.E.M. Number of determinations for 5-HT n = 15, for the other compounds n = 5 on veins, n = 4 on platelets. Veins
(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12)
5-HT 5-CH3-T 5-OCH3-T a-CH3-5-HT fl-CH3-5-HT to-CHa-5-HT NN(CH3)2-5-HT 5-NH2-T 5-CONH2-T 4-HT Tryptamine 8-OH-DPAT
Platelets
ECso
_+S.E.M.
7.33 7.38 7.15 6.24 6.09 7.16 6.81 6.02 7.56 6.53 6.12 5.55
0.11 0.10 0.04 0.06 0.11 0.25 0.12 0.06 0.13 0.11 0.04 0.24
ECs0
< < < < < < < <
6.29 6.13 6.05 5.93 4.0 4.0 4.0 5.0 4.0 5.0 5.0 4.0
Platelets _+S.E.M.
3.1. Serotonin receptor agonists As can be seen from table 1, besides 5-HT only 5-CH3-T, 5-OCH3-T and ot-CH3-5-HT proved to be full agonists on both saphenous veins and
PLATELETS
6.34 7.53 7.24 6.78 6.43 6.90 6.81 < 5.00
3 x 104°mo1/I
÷1
10"9mo1/I I-"r-
3x 10"9 mol/I
,fi ,/
Emax
_+S.E.M.
1.00 1.13 1.00 0.89 0.86 0.97 0.79 0.90 0.87 0.79 0.82 0.67
0.06 0.01 0.05 0.07 0.09 0.06 0.04 0.02 0.06 0.03 0.12
Emax
_+S.E.M.
1.00 1.08 1.12 0.94
0.07 0.16 0.09
i
~___7=
SAPHENOUS VEIN
100] ==so
50-
0.21 0.12 0.11 0.39 0.10 0.17 0.23
Platelets
platelets. By contrast, the other tryptamine derivatives, though acting as full agonists on veins, were devoid of stimulant activity on platelets but produced antagonism of the 5-HT-induced reversible aggregation. The most potent venoconstrictor agent, 5-CONH2-T, acted as pure but weak antagonist on platelets and the weakest veno-
HUMAN
100
_+S.E.M.
0.01 0.12 0.05 0.13
3. Results
HUMAN
ICso
Veins
I "/
/ /. ~~ "
'~/
xo/-.'< .v
o\~..-"r
,~"..-~ ~ ' " p'%"" %.'T ~'¢" ,,"
,It,'--'~"J~----~--t"
10"8mo1/I 0-
concentration
of
5-HT
concentration
of
5-HT
Fig. 1. Left: concentration-response curve for the reversible aggregation of human platelets induced by 5-HT in the absence ( ) and presence ( . . . . ) of spiperone. The bars represent means_+ S.E.M., for each point n = 3. Right: cumulative concentration-response curve for the contractile response of human saphenous vein strips (treated with 30 p.mol/l cocaine plus 30 # m o l / l pargyline) to 5-HT in the absence ( ) and presence ( - - - - - - ) of spiperone. The bars represent means_+ S.E.M., for each point n = 3.
110 constrictor agent, 8-OH-DPAT, possessed no affinity at all for the 5-HT receptor on platelets, either as agonist or as antagonist.
3.2. Antagonism of 5-HT by spiperone The antagonism of 5-HT by spiperone was investigated in a second series of experiments. Spiperone blocked the 5-HT-induced reversible aggregation of human platelets in a dose-dependent non-competitive way producing a - l o g ICs0 value of 8.75 + 0.06 (mean + S.E.M., n = 9, fig. 1, left). By contrast, the spiperone-induced shift of the 5-HT curve on saphenous vein strips was biphasic. While the antagonism of the upper part of the 5-HT curve consisted of an apparent shift to the right, the lower part of the curve was largely resistant to blockade by spiperone (fig. 1, right). Only about 70% of the venoconstrictor response to to 5-HT seemed to be susceptible to blockade by spiperone. A - log ICs0 value of 7.4 was estimated for this proportion of the venoconstrictor response to 5-HT. The shift of the curve by 10-300 nmol/1 spiperone at the 70% effect level of the control curve yielded a pA 2 value of 8.41 + 0.15 (mean _ S.E.M., n = 12).
4. Discussion
A comparison of the effects of various serotonin receptor agonists on human saphenous veins to their actions on human blood platelets indicates considerable differences between the 5-HT receptor types in the two tissues. In accordance with observations reported by Born et al. (1972) and Boullin et al. (1975a) only 4 out of 12 compounds, namely 5-HT, 5-CH3-T, 5-OCH3-T and a-CHa-5HT, proved to be full agonists on both tissue preparations. The introduction of methyl groups at the amine or at the fl-carbon of the side-chain, the introduction of a hydroxy group at position 4 of the indole, or the replacement of the hydroxy group at position 5 of the indole by a hydrogen or by an amino group eliminated the stimulant activity on the platelets, thereby leading to compounds with antagonistic activity at the 5-HT receptor mediating reversible aggregation. The tetralin
derivative, 8-OH-DPAT, a drug with high and selective affinity for 5-HT1A binding sites in rat brain cortex (Engel et al., 1986; Hoyer et al., 1985; Middlemiss and Fozard, 1983), proved to be a very weak agonist on saphenous veins and was completely inactive on the platelets. This suggests that neither saphenous veins nor platelets from man possess a considerable proportion of 5-HT1A-like recognition sites. In the same radioligand binding studies the compound 5-CONH2-T has been characterized as an agent with high and selective affinity for both 5-HTIA and 5-HT1a binding sites (Engel et al., 1986). The weak antagonism of the 5-HT-induced aggregation by 5-CONH2-T when used at about 10 times higher concentrations than required for venoconstriction might suggest some affinity of 5-CONH2-T for the 5-HT receptor on platelets. However, the observation that neither 5-CONH2-T nor 8-OH-DPAT induced platelet aggregation supports the contention that the 5-HT receptors mediating aggregation of human platelets do not involve 5-HT l-like recognition sites but belong to the 5-HT2 subtype (Born et al., 1972; Boullin et al., 1975b; DeClerk et al., 1982; Glusa and Markwardt, 1984; Markwardt, 1984; Michal, 1969; Michal and Motamed, 1976). Further evidence for this was provided by the potent and monophasic antagonism by spiperone of the 5-HT-induced reversible aggregation of human platelets. Spiperone, a drug with high and selective affinity for 5-HT2 recognition sites (Engel et al., 1986; Hoyer et al., 1985) inhibited the 5-HT-induced aggregation of human platelets with a - l o g ICs0 value of 8.75 which is identical with the pK d value (8.76) of spiperone at the 5-HT2 binding sites (Engel et al., 1986; Hoyer et al., 1985) in rat brain membranes, thus indicating that the reversible 5-HT-induced aggregation of human platelets is mediated through 5-HT: recognition sites. In contrast, spiperone displaced the 5-HT curve for human saphenous vein strips in a biphasic manner, suggesting the involvement of more than one serotoninergic recognition site in the venoconstrictor response to 5-HT. Only about 70% of the venoconstrictor response to 5-HT proved to be susceptible to spiperone. The pA 2 value of 8.41 calculated from the shift to the right of this proportion of the 5-HT curve by spiperone
111
again suggests stimulation of 5-HT2 receptors while the spiperone-resistant proportion of the 5-HT curve might reflect stimulation of additional 5-HT recognition site(s). The potent stimulant activity of 5-CONH2-T suggests that a considerable proportion of the contractile response to 5-HT of human saphenous vein might be mediated through 5-HTl-like recognition sites. However, further studies using selective 5-HT antagonists are required to identify and characterise in detail the 5-HT receptor subtype(s) mediating constrictor responses of human saphenous vein to 5-HT. In conclusion, the present data support the contention that the 5-HT-induced reversible aggregation of human platelets is mediated through 5-HT2-1ike receptors whereas the evidence suggests that the contractile response to 5-HT of human saphenous vein reflects activation of 5-HT2 receptors and in addition stimulation of 5-HTl-like recognition sites.
Acknowledgement The authors gratefully acknowledge the valuable technical assistance of Mr P. Gfeller.
References Aellig, W.H., 1972, Agonists and antagonists of 5-hydroxytryptamine on venomotor receptors, Adv. Neurol. 33, 321. Ball, S.E., D.J. Boullin and P.A.M. Glenton, 1977, Interactions between noradrenaline and 5-hydroxytryptamine involving platelet aggregation, J. Physiol. 272, 98P. Baumgartner, H.R. and G.V.R. Born, 1968, Effects of 5-hydroxytryptamine on platelet aggregation, Nature 218, 137. Born, G.V.R., K. Jueng~aroen and F. Michal, 1972, Relative activities on and uptake by human blood platelets of 5-hydroxytryptamine and several analogues, Br. J. Pharmacol. 44, 117.
Boullin, D.J., A.R. Green and R.P.J. Grimes, 1975a, Human blood platelet aggregation induced by dopamine, 5-hydroxytryptamine and analogues, J. Physiol. (London) 252, 46P. Boullin, D.J., R.P.J. Grimes and M.W. Orr, 1975b, The actions of fluphenthixol upon 5-hydroxytryptamine-induced aggregation and the uptake of 5-hydroxytryptamine and dopamine by human blood platelets, Br. J. Pharmacol. 55, 555. DeClerck, F., J.-L. David and P.A.J. Janssen, 1982, Inhibition of 5-hydroxytryptamine-induced and -amplified human platelet aggregation by ketanserin (R 41 468), a selective 5-HT2-receptor antagonist, Agents Actions 12, 388. Engel, G., M. Gt~thert, D. Hoyer, E. Schlicker and K. Hillenbrand, 1986, Identity of inhibitory presynaptic 5-hydroxytryptamine (5-HT) autoreceptors in the rat brain cortex with 5-HT 1B-binding sites, Naunyn-Schmiedeb. Arch. Pharmacol. 332, 1. Glusa, E., 1984, Experimentell pharmakologische Untersuchungen zur venentonisierenden Wirkung von Dihydroergotamin, Z. Ges. Inn. Med. 39, 414. Glusa, E. and F. Markwardt, 1984, Inhibition of 5-hydroxytryptamine-potentiated aggregation of human blood platelets by 5-hydroxytryptamine receptor blocking agents, Biochem. Biochim. Acta 43, 217. Hoyer, D., G. Engel and H.O. Kalkman, 1985, Molecular pharmacology of 5-HT 1 and 5-HT2 recognition sites in rat and pig brain membranes: Radioligand binding studies with [3H]5-HT, [3H]8-OH-DPAT, (-)[125I]iodocyanopindolol, [3H]mesulergine and [3H]ketanserin, European J. Pharmacol. 118, 13. Michal, F., 1969, D-Receptor for serotonin on blood platelets, Nature (London) 221, 1253. Michal, F. and M. Motamed, 1976, Shape change and aggregation of blood platelets: interaction between the effects of adenosine diphosphate, 5-hydroxytryptamine and adrenaline, Br. J. Pharmacol. 56, 209. Middlemiss, D.N. and J.R. Fozard, 1983, 8-Hydroxy-2-(di-npropylamino)-tetralin discriminates between subtypes of the 5-HT~ recognition site, European J. Pharmacol. 90, 151. Mialler-Schweinitzer, E., 1984, Alpha-adrenoceptors, 5-hydroxytryptamine receptors and the action of dihydroergotamine in human venous preparations, NaunynSchmiedeb. Arch. Pharmacol. 237, 299. Vane, J.R., 1959, The relative activities of some tryptamine analogues on the isolated rat stomach strip preparation, Br. J. Pharmacol. 14, 87.
107
C O M P A R I S O N OF T H E A C T I O N S OF S E R O T O N I N E R G I C AGENTS O N H U M A N S A P H E N O U S V E I N S AND P L A T E L E T S MIKAEL VICTORZON,CARLO TAPPARELLIand ELSE MI]LLER-SCHWEINITZER* Preclinical Research, Sandoz Ltd., CH-4002 Basel, Switzerland
Received 14 November 1985, revised MS received 23 January 1986, accepted 11 February 1986
M. VICTORZON, C. TAPPARELLI and E. MOLLER-SCHWEINITZER, Comparison of the actions ofserotoninergic agents on human saphenous veins and platelets, European J. Pharmacol. 124 (1986) 107-111. Changes in tension of helical strips from human saphenous veins and reversible aggregation of human platelets were recorded in vitro. Comparison of the activities of 12 serotonin receptor agonists revealed that only 4 of the investigated tryptamine derivatives acted as full agonists on both tissues. 5-Carboxamidotryptamine, a drug with selective affinity for both 5-HTIA and 5-HT~B binding sites, though the most potent agonist on veins, failed to produce platelet aggregation but acted as a weak antagonist of the 5-HT-induced reversible aggregation. The tetralin derivative 8-OH-DPAT, a drug with selective affinity for 5-HT~A binding sites, was a weak partial agonist on veins and completely devoid of any activity on the platelets. The antagonism of 5-HT by spiperone was monophasic on platelets but biphasic on veins. These data are in line with the contention that the 5-HT-induced reversible aggregation of human platelets is initiated by 5-HT2-1ike recognition sites while the evidence suggests that the contractile response of human saphenous vein to 5-HT reflects activation of both 5-HT2- and 5-HTl-like recognition sites. Human saphenous vein
Tryptamine derivatives
Platelet aggregation
1. Introduction More than 100 years ago, Virchow emphasised that besides alterations in the vessel wall, changes of both blood flow and blood composition predispose to thrombosis. While the development of one of these alterations may only facilitate thrombogenesis, the concomitant appearance of two of these changes will lead to the formation of a thrombus. It has been shown that 5-hydroxytryptamine (5-HT) receptors are involved in both venoconstriction and platelet aggregation. Thus 5-HT is a potent venoconstrictor of human hand veins in situ (Aellig, 1982) and of human femoral and saphenous veins in vitro (Glusa, 1984; Glusa and Markwardt, 1984; Miiller-Schweinitzer, 1984). Furthermore, 5-HT induces shape change and reversible aggregation of human platelets and may, in addition, potentiate aggregation brought about * To whom all correspondenceshould be addressed. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
5-HT receptors
In vitro
by various other stimuli such as ADP, collagen, adrenaline and noradrenaline (Ball et al., 1977; Baumgartner and Born, 1968; DeClerck et al., 1982; Michal and Motamed, 1976). The present study was performed to investigate whether there exist differences between the 5-HT receptors mediating venoconstriction and those initiating reversible platelets aggregation.
2. Materials and methods 2.1. Vein strips
Human saphenous veins were obtained from patients undergoing surgical removal of varicose veins. The patients had usually been premedicated with droperidol (2.5-5 mg) and atropine (0.5 mg) and the anaesthetic used was a barbiturate and a halothane-nitrous oxide mixture. The veins were placed in Krebs-Henseleit solution (mmol/1):
108 NaC1 118, KC1 4.7, MgSO 4 1.2, CaCI 2 1.2, K H z P O 4 1.2, N a H C O 3 25, glucose 11, EDTA 0.03, gassed with 5% CO 2 in oxygen and transported to the laboratory within 2-4 h. The veins were cleaned of loose connective tissue and stored overnight in Krebs-Henseleit solution at 4°C. Thereafter the veins were cut into helical strips (15 × 2 mm) with a ring at one end and suspended in 10 ml organ baths containing Krebs-Henseleit solution at 37°C, gassed continuously with 5% CO 2 in 02. Changes in the tone of the preparations were monitored isometrically. At the beginning of the experiments the strips were stretched to an initial tension of 7.5 mN and allowed to equilibrate for at least 4 h in the bathing medium which was changed every 15 rain. During this time the preparations were stimulated twice with 5-HT (1 /~mol/1) before the baseline tension was readjusted to about 3 mN. Thereafter a cumulative concentration-response curve for 5-HT was determined the maximum of which was taken as a point of comparison for subsequent responses. Repeated washouts were followed by the determination of cumulative concentration-response curves for 5-HT and various tryptamine derivatives. The curves were obtained by increasing the concentration of the agonist in the organ bath by a factor 4 when the maximum effect had been produced by the preceding concentration. These curves, consisting of 6-7 concentrations, were determined in the presence of 30 /tmol/l pargyline added 20 min before the agonist in order to prevent inactivation of the indole derivatives by monoamine oxidase (Vane, 1959). When the antagonism of 5-HT by spiperone was investigated, an incubation period of 15 min was allowed. Responses to the agonists are expressed as percentages of the maximal response to 5-HT. In each experiment 6 strips of the same vein were investigated at the same time, one of the strips being stimulated only with 5-HT and thus serving as a control preparation to correct for any sensitivity changes during the experiment. 2.2. Aggregation
Human platelet-rich plasma (PRP) was prepared from blood obtained from healthy volun-
teers. The blood was mixed with 3.8% (w/v) trisodium citrate (9:1) and platelet-rich plasma (PRP) was separated by centrifugation of 25 ml blood samples in 50 ml plastic tubes (Falcon) for 10 min at 1100 rpm (RT 6000; Sorvall) and kept at room temperature (max. 4-6 h) until used. Drugs were dissolved daily before use and diluted with RCD buffer. The RCD buffer consisted of Ringer solution (mmol/l: NaC1 150, KCI 5.63, CaC12 2.25) mixed with 3.8% (w/v) trisodium citrate and 1% (w/v) dextrose ( 6 : 2 : 1) at pH 7.4. Platelet aggregation was studied in a double-channel Payton aggregometer (Model 300 BD-5) connected to a Rikadenki recorder. PRP (0.5 ml) was placed in a cuvette containing a steel stirring bar and stirred (1100 rpm) at 37°C for 2 min prior to the addition of 20 #1 of the agonist. The aggregation induced by the agonist was expressed as a percentage of the maximum reversible aggregation induced by 5-HT alone. When the antagonism of 5-HT was investigated, an incubation period of 2 min was allowed before 5-HT (20/~1) was added. 2.3 Drugs used and their sources
The following pharmacological agents were used: 5-hydroxytryptamine creatinine sulfate (5HT), 5-methoxytryptamine hydrochloride (5OCH3-T, Fluka, Buchs, Switzerland), 5-methyltryptamine (5-CH3-T), tryptamine hydrochloride (T), pargyline hydrochloride (Sigma, Munich, FRG), a-methyl-5-hydroxytryptamine creatinine sulfate (a-CH3-5-HT, Upjohn, Kalamazoo, MI, USA), spiperone (Janssen, Beerse, Belgium), /3methyl-5-hydroxytryptamine hydrogen oxalate (/3C H 3-5-HT), ~o-N-methyl-5-hydroxytryptamine oxalate (~o-N-CH3-5-HT), N,N-dimethyl-5-hydroxytryptamine binoxalate (N,N-(CH3)2-5-HT), 5-aminotryptamine oxalate (5-NH2-T), 5-carboxamidotryptamine hydrogen maleinate (5-CONHz-T ), 4-hydroxytryptamine creatinine sulfate (4-HT) and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) were synthesized at Sandoz Ltd., Basel, Switzerland. All compounds were dissolved just before use. Drug concentrations are given as molar concentrations throughout. Statistical analysis of data was performed using Student's t-test.
109 TABLE 1 Parameters calculated for various serotonin receptor agonists on isolated human saphenous veins and platelets. ECso values for stimulation and ICs0 values for antagonism of 5-HT-induced reversible platelets aggregation are given as the negative logarithms, maximal effects (Era,x) of the agonists are expressed in relation to the the maximum effect of 5-HT ( = 1.0). Data are presented as means_+ S.E.M. Number of determinations for 5-HT n = 15, for the other compounds n = 5 on veins, n = 4 on platelets. Veins
(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12)
5-HT 5-CH3-T 5-OCH3-T a-CH3-5-HT fl-CH3-5-HT to-CHa-5-HT NN(CH3)2-5-HT 5-NH2-T 5-CONH2-T 4-HT Tryptamine 8-OH-DPAT
Platelets
ECso
_+S.E.M.
7.33 7.38 7.15 6.24 6.09 7.16 6.81 6.02 7.56 6.53 6.12 5.55
0.11 0.10 0.04 0.06 0.11 0.25 0.12 0.06 0.13 0.11 0.04 0.24
ECs0
< < < < < < < <
6.29 6.13 6.05 5.93 4.0 4.0 4.0 5.0 4.0 5.0 5.0 4.0
Platelets _+S.E.M.
3.1. Serotonin receptor agonists As can be seen from table 1, besides 5-HT only 5-CH3-T, 5-OCH3-T and ot-CH3-5-HT proved to be full agonists on both saphenous veins and
PLATELETS
6.34 7.53 7.24 6.78 6.43 6.90 6.81 < 5.00
3 x 104°mo1/I
÷1
10"9mo1/I I-"r-
3x 10"9 mol/I
,fi ,/
Emax
_+S.E.M.
1.00 1.13 1.00 0.89 0.86 0.97 0.79 0.90 0.87 0.79 0.82 0.67
0.06 0.01 0.05 0.07 0.09 0.06 0.04 0.02 0.06 0.03 0.12
Emax
_+S.E.M.
1.00 1.08 1.12 0.94
0.07 0.16 0.09
i
~___7=
SAPHENOUS VEIN
100] ==so
50-
0.21 0.12 0.11 0.39 0.10 0.17 0.23
Platelets
platelets. By contrast, the other tryptamine derivatives, though acting as full agonists on veins, were devoid of stimulant activity on platelets but produced antagonism of the 5-HT-induced reversible aggregation. The most potent venoconstrictor agent, 5-CONH2-T, acted as pure but weak antagonist on platelets and the weakest veno-
HUMAN
100
_+S.E.M.
0.01 0.12 0.05 0.13
3. Results
HUMAN
ICso
Veins
I "/
/ /. ~~ "
'~/
xo/-.'< .v
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,~"..-~ ~ ' " p'%"" %.'T ~'¢" ,,"
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concentration
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concentration
of
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Fig. 1. Left: concentration-response curve for the reversible aggregation of human platelets induced by 5-HT in the absence ( ) and presence ( . . . . ) of spiperone. The bars represent means_+ S.E.M., for each point n = 3. Right: cumulative concentration-response curve for the contractile response of human saphenous vein strips (treated with 30 p.mol/l cocaine plus 30 # m o l / l pargyline) to 5-HT in the absence ( ) and presence ( - - - - - - ) of spiperone. The bars represent means_+ S.E.M., for each point n = 3.
110 constrictor agent, 8-OH-DPAT, possessed no affinity at all for the 5-HT receptor on platelets, either as agonist or as antagonist.
3.2. Antagonism of 5-HT by spiperone The antagonism of 5-HT by spiperone was investigated in a second series of experiments. Spiperone blocked the 5-HT-induced reversible aggregation of human platelets in a dose-dependent non-competitive way producing a - l o g ICs0 value of 8.75 + 0.06 (mean + S.E.M., n = 9, fig. 1, left). By contrast, the spiperone-induced shift of the 5-HT curve on saphenous vein strips was biphasic. While the antagonism of the upper part of the 5-HT curve consisted of an apparent shift to the right, the lower part of the curve was largely resistant to blockade by spiperone (fig. 1, right). Only about 70% of the venoconstrictor response to to 5-HT seemed to be susceptible to blockade by spiperone. A - log ICs0 value of 7.4 was estimated for this proportion of the venoconstrictor response to 5-HT. The shift of the curve by 10-300 nmol/1 spiperone at the 70% effect level of the control curve yielded a pA 2 value of 8.41 + 0.15 (mean _ S.E.M., n = 12).
4. Discussion
A comparison of the effects of various serotonin receptor agonists on human saphenous veins to their actions on human blood platelets indicates considerable differences between the 5-HT receptor types in the two tissues. In accordance with observations reported by Born et al. (1972) and Boullin et al. (1975a) only 4 out of 12 compounds, namely 5-HT, 5-CH3-T, 5-OCH3-T and a-CHa-5HT, proved to be full agonists on both tissue preparations. The introduction of methyl groups at the amine or at the fl-carbon of the side-chain, the introduction of a hydroxy group at position 4 of the indole, or the replacement of the hydroxy group at position 5 of the indole by a hydrogen or by an amino group eliminated the stimulant activity on the platelets, thereby leading to compounds with antagonistic activity at the 5-HT receptor mediating reversible aggregation. The tetralin
derivative, 8-OH-DPAT, a drug with high and selective affinity for 5-HT1A binding sites in rat brain cortex (Engel et al., 1986; Hoyer et al., 1985; Middlemiss and Fozard, 1983), proved to be a very weak agonist on saphenous veins and was completely inactive on the platelets. This suggests that neither saphenous veins nor platelets from man possess a considerable proportion of 5-HT1A-like recognition sites. In the same radioligand binding studies the compound 5-CONH2-T has been characterized as an agent with high and selective affinity for both 5-HTIA and 5-HT1a binding sites (Engel et al., 1986). The weak antagonism of the 5-HT-induced aggregation by 5-CONH2-T when used at about 10 times higher concentrations than required for venoconstriction might suggest some affinity of 5-CONH2-T for the 5-HT receptor on platelets. However, the observation that neither 5-CONH2-T nor 8-OH-DPAT induced platelet aggregation supports the contention that the 5-HT receptors mediating aggregation of human platelets do not involve 5-HT l-like recognition sites but belong to the 5-HT2 subtype (Born et al., 1972; Boullin et al., 1975b; DeClerk et al., 1982; Glusa and Markwardt, 1984; Markwardt, 1984; Michal, 1969; Michal and Motamed, 1976). Further evidence for this was provided by the potent and monophasic antagonism by spiperone of the 5-HT-induced reversible aggregation of human platelets. Spiperone, a drug with high and selective affinity for 5-HT2 recognition sites (Engel et al., 1986; Hoyer et al., 1985) inhibited the 5-HT-induced aggregation of human platelets with a - l o g ICs0 value of 8.75 which is identical with the pK d value (8.76) of spiperone at the 5-HT2 binding sites (Engel et al., 1986; Hoyer et al., 1985) in rat brain membranes, thus indicating that the reversible 5-HT-induced aggregation of human platelets is mediated through 5-HT: recognition sites. In contrast, spiperone displaced the 5-HT curve for human saphenous vein strips in a biphasic manner, suggesting the involvement of more than one serotoninergic recognition site in the venoconstrictor response to 5-HT. Only about 70% of the venoconstrictor response to 5-HT proved to be susceptible to spiperone. The pA 2 value of 8.41 calculated from the shift to the right of this proportion of the 5-HT curve by spiperone
111
again suggests stimulation of 5-HT2 receptors while the spiperone-resistant proportion of the 5-HT curve might reflect stimulation of additional 5-HT recognition site(s). The potent stimulant activity of 5-CONH2-T suggests that a considerable proportion of the contractile response to 5-HT of human saphenous vein might be mediated through 5-HTl-like recognition sites. However, further studies using selective 5-HT antagonists are required to identify and characterise in detail the 5-HT receptor subtype(s) mediating constrictor responses of human saphenous vein to 5-HT. In conclusion, the present data support the contention that the 5-HT-induced reversible aggregation of human platelets is mediated through 5-HT2-1ike receptors whereas the evidence suggests that the contractile response to 5-HT of human saphenous vein reflects activation of 5-HT2 receptors and in addition stimulation of 5-HTl-like recognition sites.
Acknowledgement The authors gratefully acknowledge the valuable technical assistance of Mr P. Gfeller.
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