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Serotonin induced vasodilatation in the human forearm is antagonized by the selective 5-HT3 receptor antagonist ICS 205–930
Life Sciences, Vol. 43, pp. 1441-1449 Printed in the U.S.A.
Pergamon Press
SEROTONIN INDUCED VASODILATATION IN THE HUMAN FOREARM IS A N T A G O N I Z E D BY T H E S E L E C T I V E 5-HT 3 RECEPTOR ANTAGONIST ICS 205-930 Gerard J. Blauw, Peter van Brummelen* and Pieter A. van Zwieten**.
Department of Nephrology, University Hospital Leiden, The Netherlands. *Present address: Department of Clinical Research, Hoffman-la Roche & Co. AG, Basel, Switzerland. **Department of Pharmacology, Academic Medical Centre, Amsterdam, The Netherlands. (Received in final form September 7, 1988)
The role of 5-HT~ receptors in the biphasic vasodilatator response to serotonin (5-nydroxytryptamine; 5-HT) was investigated in the forearm of 7 young healthy volunteers (aged 22-32 years). Single dose infusions of 5-HT (i ng/kg/min) and of acetylcholine (ACh, 500 ng/kg/min) were administered into the brachial artery. Subsequently combined infusions of 5-HT together with the selective 5-HT receptor antagonist ICS 205-930 (350 and 700 ng/kg/min), an~ ACh together with ICS 205-930 (700 ng/kg/min) were given. After a pause of at least 1 hour the single infusions of 5-HT and ACh were repeated. Subsequently, 5 - H T a n d A C h w e r e infused together with atropine (i00 ng/kg/min). Forearm blood flow (FBF) was m e a s u r e d b y R-wave triggered venous occlusion plethysmography. Heart rate (HR)' and i.a. blood pressure (BP) were recorded semi-continuously. None of the drugs in the doses used did induce systemic hemodynamic effects. After an initial rapid transient increase in FBF of 316 ± 55%, 5-HT elicited a persistent increase in FBF of 90 ± 22% (mean ± SEM, i><0.05 for both). ACh induced a monophasic vasodilatatlon of 475 ± 123% (p<0.05). Both the initial transient and the persistent dilatator response to 5-HT were attenuated by ICS 205-930 350 ng/kg/min (p~0.057, n=5) and 700 ng/kg/min (p<0.05, n=7). The highest dose of ICS 205-930 did not significantly influence the dilatator response to ACh. Atropine abolished the ACh induced vasodilatation (I)<0.05), but did not influence the biphasic dilatator response to 5-HT. Thus the 5-HT induced biphasic vasodilatation was antagonized by ICS 205-930, indicating that this response was mediated by 5-HT 3 receptor activation. The fact that atropine did not influence the vascular response to 5-HT suggests that 5-HT did not induce vascular relaxation indirectly by the release of ACh from cholinergic nerve endings. The serotonin (5-hydroxytryptamine; 5-HT) receptors involved in the complex cardiovascular effects of 5-HT are currently classified into three different receptorsSubtypes' appearedViZ" 5-HT_,~o 5-HT2 and 5-HT3 receptors (1-3). Since the 5-HT 1 be a heterogenous entity, they are tentatively classified as "5-HTl-like" receptors. Both "5-HT.-like" and 5-HT_ receptors are assumed to be located in the vascular wall (~or reviews see ~ef. 3-5). Depending on the species and type of blood vessels investigated, stimulation of "5-HT_-like" receptors may induce either vasodilatation or vasoconstriction, whereas activation of 5-HT 2 receptors elicits vasoconstriction in all species investigated so far, including man (I-9). In prior studies we have found evidence that intra-arterially (i.a.) infused 0024-3205/88 $3.00 + .00 Copyright (c) 1988 Pergamon Press plc
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5-HT induced a biphasic vascular response in the forearm consisting of an initial rapid transient vascular relaxation, followed by a persistent vasodilatation for low doses, and a vasoconstriction only for high pharmacological doses of 5-HT (8,9). It was demonstrated that the 5-HT induced vasoconstriction was mediated by 5-HT 2 receptor stimulation, since it could be inhibited by the selective 5-HT~ receptor antagonists ketanserin and ritanserin (8,9). The mechanism underlyifig the biphasic vasodilatator response could not be established in those experiments, but it was speculated that a "5-HTl-like" receptor could be involved (8). In contrast to the "5-HT_-like" and 5-HT_ receptors, there is little evidence 1 ~ . . that the 5-HT~ receptor subtype plays a role in the peripheral vascular effects of 5-RT (3-5). So far 5-HT 3 receptors have been found on autonomic and sensory neurons, causing depolarization upon stimulation (for reviews see ref. 10-12). However, recent evidence from studies in the porcine carotid vascular bed suggests, that 5-HT~ receptor stimulation is involved in the vascular relaxation induced by 5:HT (7). Since 5-HT~ receptor stimulation evokes neuronal depolarization, the mechanism underlying vascular relaxation could well be the release of acetylcholine (ACh) from cholinergic nerve terminals (11-13). Alternatively, this vasodilatation could be induced by excitation of sensory neurons, via a so called "axon-reflex" (10,11,14). In the present study the highly selective 5-HT 3 receptor antagonist ICS 205-930 was used to investigate the 5-HT induced biphasic vasodilatation (15). The involvement of cholinergic neurons in the vasodilatator response to 5-HT was also investigated (16,17). Methods
Subjects Seven non-patient volunteers (males; mean age 28 years, range of 22-32 years) participated in this study. Their medical history, physical examination and routine laboratory tests did not show evidence of any relevant disease. None of the subjects was receiving any medication at the time of the study or in the previous two weeks. On the day of the study and 12 hours before, all subjects had refrained from smoking and caffeine containing beverages. Informed consent was obtained from all subjects and the protocol was approved by the Ethics Committee of the Leiden University Hospital. Procedures The studies were performed in a quiet room with a temperature between 21°C and 23°C. During the experiments the subjects were in the supine position with the non-dominant arm stabilized slightly above the level of the heart. After local anaesthesia of the skin, the brachial artery of the non-dominant arm was cannulated in the cubital fossa. The cannula (Autocath 1453.13, Saint Leu-La-Foret, France) thus applied was used for infusion of drugs with a Sage constant rate infusion pump (model 351, Sage Instruments Inc., Cambridge, MA, USA) and for intra-arteriel (i.a.) blood pressure (BP) recording with a Statham P23 Id pressure transducer (Gould Inc., Oxnard, CA, USA). Heart rate (HR) was derived from a continuously recorded one-lead electrocardiogram (ECG). Forearm blood flow (FBF) was measured 5 times per minute by venous occlusion plethysmography (Hokanson EC-2 plethysmograph, Hokanson Inc., Issaquah, WA, USA), using mercury-in-silastic strain gauges and a rapid cuff inflator (Hokanson E-10). Tracings of ECG, BP and FBF were directly recorded on a polygraph (Mingograph 803, Siemens-Elema, Stockholm, Sweden). A personal computer (IBM AT III) extended with an analog digital convertor (model DT 2801, Data Translation Inc., Malborough, MA, USA) was used for R-wave triggered control of the rapid cuff inflator, and also for on-line analysis of FBF, BP and HR recordings (18-19). During the measurements of FBF, the
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blood flow in the hand was excluded from the circulation using a small wrist cuff, inflated to 40 mmHg above the systolic blood pressure (SBP). The experiments started at least 45 minutes after cannulation of the brachial artery. Between the various infusions the wrist cuff was deflated and sufficient time (20 minutes) was allowed for FBF to return to basal levels. Dru~s and Solutions The following drugs were infused into the brachial artery: 5-hydroxytryptamine HCl (Janssen Chimica), [1H~-indol-3-carbonic-acid-tropine -ester HCl (ICS 205-930, Sandoz AG), acethylcboline HCl (tested according to United States Pharmacopoeia XXI) and atropine-sulphate (tested according to European Pharmacopoeia). All drugs were dissolved in saline. The solutions were prepared from sterile stock solutions and ampoules on the day of the
study. Study protocol Infusions of 5-HT, acetylcholine and ICS 205-930 In the 7 subjects 5-HT and ACh were infused in a random order in single doses of 1 and 500 ng/kg/min, respectively, together with a continuous infusion of saline (0.4 ml/min). Each infusion lasted for 8 minutes. Subsequently, 5-HT (i ng/kg/min) was infused together with ICS 205-930 350 ng/kg/min (n=5) and 700 ng/kg/min (n=7). Finally, ACh (500 ng/kg/min) was infused in the presence of the highest dose of ICS 205-930. The infusions of ICS 205-930 started 6 minutes before the 5-HT and ACh infusions. Infusions of 5-HT, acet~lcholine and atropine At least one hour after the last infusion of ICS 205-930, the infusions of 5-HT (I ng/kg/min) and ACh (500 ng/kg/min) were repeated in the presence of a continuous infusion of saline (0.4 ml/min). Subsequently, 5-HT and ACh were administered together with atropine 100 ng/kg/min, respectively. The infusion of atropine started 6 minutes before the 5-HT and ACh infusions. The hemodynamic variables FBF, i.a. BP and HR were recorded semi-continuously during the various infusions. Basal values were recorded during the 2 minutes preceding each infusion. The hemodynamic Values measured during the first 2 minutes, and during the last 2 minutes of each infusion were used for analysis. Statistical analysis Results are given as means ± SEM. FBF values are expressed as percent changes from baseline. Wilcoxon's signed rank test for matched pairs was used to evaluate the statistical significance of the data. P-values lower than 0.05 were regarded as significant. Results In all experiments only small changes in i.a. BP and HR were observed (table I). During some infusions in which 5-HT and ACh increased FBF, i.a. BP was slightly but significantly reduced (table I). Since HR was uninfluenced by these infusions of 5-HT and ACh, the minimal reductions in i.a. BP are more likely explained by local vasodilatation in the forearm, rather than by systemic hemodynamic effects. Therefore changes in FBF during the various infusions can be interpreted as local vascular effects of the drugs. Baseline levels of FBF observed in the various experiments are listed in table I. Single infusions of serotonin and acet~lcholine In all subjects the infusion of 5-HT i ng/kg/min together with saline induced a biphasic dilatator response, in which the following two phases could be distinguished:
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1) a rapid transient increase in FBF within the first 2 minutes after the infusion was started, followed by 2) a persistent increase in FBF, reaching a steady state 5 minutes after the start of the infusion. The maximum increase in FBF during the initial phase (AFBF ) was 316 ± 55% max (p<0.05, fig. i, table II). During the persistent dilatator response FBF was increased by 90 ± 22% (p<0.05, fig. 1, table II). The infusion of ACh 500 ng/kg/min together with saline elicited a significant monophasic increase in FBF of 475 ± 123% (p
4.00
SEROTONIN (M+-SEM)
300
n
/I
-I00
-
,
0
2
~
SALINE 0.4 ml.min"i
o--o
'ICS 205-930
4
350 ng.kg".min"
6
(minutes)
FIG. 1 Mean percent changes In forearm blood flow (+ SEN) induced by simultaneous infusions of serotonln wlth saline, and serotonin with two doses of ICS 205-930 (for values and statistics see text and table II).
Combined infusions of serotonin with ICS 205-930 and acet~lcholine with ICS 205-930 During the infusion of ICS 205-930 350 ng/kg/min both the initial transient vasodilatation and the persistent dilatator phase induced by 5-HT 1 ng/kg/min were reduced (p=0.057 for both, fig. i, table II). The highest dose of ICS 205-930 (700 ng/kg/min) further attenuated the initial transient vasodilatation (p<0.05), and reduced the persistent vasodilatation elicited by 5-HT to the same extent as the dose of 350 ng/kg/min (p<0.05, fig. 1, table II). The highest dose of ICS 205-930 did not significantly influence the vasodilatation induced by Ach (fig. 2).
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V a s c u l a r E f f e c t s of 5-HT a n d ICS 2 0 5 - 9 3 0
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TABLE I Mean baseline levels (e SEM) of Foreana Blood Flow before each infusion, and Mean Arterial Pressure and Heart Rate before and after the various infusions. INFUSION (ng/kg/min)
n
BASAL FBF (ml/dl/min)
M A P (mmHg) before after
5-HT 1 A C h 500 ICS 350 ICS 700 ATR 100
7 7 5 7 7
2.3 2.2 2.5 2.3 2.7
80 80 78 80 80
5 - H T + ICS 350 5 - H T + ICS 700 Ach + ICS 700
5 7 7
2.8 ± 0 . 4 2.7 ± 0 . 9 3.3 + 1.3
77 ± 2 78 + 2 78 ± 1
76 ± 2 78 ± 1 77 ± 1
57 ± 2 54 ± 4 55 ± 4
54 + 3 57 ± 4 59 + 4
5 - H T (after ICS:) i ACh (after ICS ) 5 - H T + A T R i00 ACh + A T R i00
7 7 7 7
2.0 2.2 2.8 3.0
80 77 79 79
77 74 77 79
60 60 58 59
60 59 57 58
+ ± ± ¢ +
± ± ± _+
0.4 0.3 0.3 0.5 0.5
0.3 0.4 0.5 0.5
+ ¢ * ± -+
± ± ± +
2 1 1 2 2
2 1 2 2
77 75 77 78 79
+ ± ± ¢ ±
± ± -+ +
H R (beats/mln) before after 2* 2* 2 2 2
57 58 58 54 61
2 2* 1 2
+ ± ± ± ±
± ± ± ±
3 2 3 3 4
3 1 4 3
56 58 57 54 60
± ± ± ± +
~ ± ± ±
2 2 2 4 4
3 3 4 3
FBF = forearm blood flow. MAP = nman arterial pressure, ER = heart rate, 5-HT = serotonln, ACh = acetylchollne, ICS = ICS 205-930, ATR -- atropine, t indicates infusions of serotonln and acetylchollne given one hour after the last infusion of ICS 205-930 {see text). * indicates slgnlflcant difference at the level of I)<0.05 coapared with baseline.
ACETYLCHOLINE (M+SEM)
~ 600 h 400 SALINE
O-
m
:
-
ICS 2 0 5 - 9 3 0
A
A
ATROPINE
.
z
~
FIG. 2 Mean percent changes in forearm blood flow (± SEM) induced by simultaneous infusions of acetylcholine with saline, acetylchollne wlth ICS 205-930 and acetylcholine with atropine.
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T A B L E II
The influence of two doses of ICS 205-930 on the blphaslc vascular response to Serotonln. Infusion (ng/kg/min)
First p h a s e A F B F m a x (%)
5-HT + saline 5-HT + ICS 350 5-HT + ICS 700
316 ± 55 50 ± 18 t I0 ± 7*
Second phase AFBF (%)
n
90 ± 22 18 ± 12 t 16 ± 22*
7 5 7
FBF = maximal increase in forearm blood flow measured during the first two minutes after the ~nfums~on was started, FBF = mean percent change in forearm blood flow measured during the last two minutes of the infusion, 5-HT = serotonln, ICS = ICS 205-930. Asterisk end dagger indicates difference compared wlth the Infusion of serotonin with saline (T I)=0.057, * p
400
SEROTONIN (M_+SEM)
'~
SALINE 0.4 ml.min"
A
A T ~
300A
I00
ng.kg'Imin"l
200¢:3
IO0-
-I00
-- ,
FIG. 3 Mea~ percent changes in forearm blood flow (±S~) induced by the simultaneous infusions of serotonln wlth saline and serotonln with atropine, which were given at least one hour after the last infusion of ICS 205-930 (see study protocol).
Single i n f u s i o n s of s e r o t o n i n a n d a c e t ~ l c h o l i n e a d m i n i s t e r e d 1 h o u r a f t e r the last i n f u s i o n of ICS 2 0 5 - 9 3 0 One h o u r a f t e r the last i n f u s i o n s of ICS 205-930, the single infusion of 5-HT again i n d u c e d a b i p h a s i c d i l a t a t o r response (fig. 3). C o m p a r e d w i t h the first single i n f u s i o n of 5-HT, the i n i t i a l t r a n s i e n t v a s o d i l a t a t i o n was p a r t i a l l y r e c o v e r e d (AFBF = 316 ± 55% and 167 ± 50%, r e s p e c t i v e l y , p < O . 0 5 for both), max
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whereas the persistent vasodilatation was completely recovered (~FBF = 90 ± 22% and 128 ± 29%, respectively, p<0.05 for both, fig. 1,3). ACh elicited the same vasodilatator response as during the first single infusion of ACh (AFBF = 541 ± 123% and 476 ± 123%, respectively). Combined infusions of eerotonin with atropine and of acet~icholine with atropine The infusion of atropine 100 ng/kg/min did neither influence the initial transient phase, nor the persistent vasodilatation induced by 5-HT significantly, when compared with the single infusion of 5-HT given one hour after the last infusion of ICS 205-930 (fig. 3). The increase in FBF induced by acetylcholine was abolished by the simultaneous infusion of atropine (fig. 2). Single infusions of ItS 205-930 and atropine The single infusions of both ICS 205-930 and atropine did not significantly influence FBF in the doses used. Discussion The main finding in the present study was that 5-HT 3 receptors were involved in the complex vascular response to 5-HT. The 5-HT induced initial transient vasodilatation as well as the second persistent dilatator phase were antagonized by ICS 205-930. Since ICS 205-930 is a highly selective 5-HT 3 receptor antagonist, with negligible affinity for other receptor subtypes (15), the present results strongly suggests that in the human forearm the 5-HT induced vasodilatation is mediated by 5-HTR receptor stimulation. These results corroborate those of a recent study of Saxena et al. (7), who have found evidence in the porcine carotid vascular bed that the 5-HT induced vasodilatation is mediated by 5-HT 3 receptor activation. Bradley et al. (1,2) have proposed that a 5-HT~ receptor mediated response should be characterized by the following three-criteria: i) it is susceptible to antagonism by a 5-HT 3 receptor antagonist like MDL 72222, ICS 205-930 and BRL 43694, 2) it is resistant to 5-HT 2 receptor blockade by ketanserin, and 3) the response can be mimlced by the selective 5-HT 3 agonist 2-methyl-5-HT. In a prior study we have demonstrated that the 5-HT induced biphasic dilatator response was not antagonized by the selective 5-HT~ antagonists ketanserin and ritanserin (8,9). In fact these 5-HT 2 recepto~ antagonists enhanced the dilatator response to 5-HT. However, the third requirement, whether the dilatator response to 5-HT could be mimiced by 2-methyl-5-HT could not be established, since this 5-HT analogue has not been tested for i.a. human use. These findings together with the present results favour the presence and functional relevance of a 5-HT 3 receptor subtype in the vascular response to 5-HT in the human forearm. It has been shown that 5-HT 3 receptors are primarily present on autonomic and sensory neurons, where they were shown to elicit depolarization upon stimulation (i0,ii). It seems unlikely that the 5-HT induced vasodilatation is mediated via stimulation of 5-HT_ receptors present on adrenergic neurons, 3 since excitation of these neurons would induce the release of norepinephrine (ii), resulting in vasoconstriction via activation of postsynaptic ~i- and ~2-adrenoceptors (20). A role for cholinergic neurons in the dilatat6r response to 5-HT was considered to be more likely, since depolarization of these nerve fibers can induce vasodilatation via the release of ACh (ii). However, the present findings that the dilatator response to i.a. infused 5-HT was not attenuated by the anticholinergic drug atropine, whereas the vasodilatation induced by i.a. infused ACh was completely blocked by the same
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Vascular Effects of 5-HT and ICS 205-930
dose of atropine
Vol. 43, No. 18, 1988
(fig. 2,3), excludes this possible mechanism.
The finding that the 5-HT induced vasodilatation could be attributed neither to adrenergic nor to cholinergic 5-HT_ receptor stimulation, the possibility 3 that this dilatator response was mediated via excitation of sensory nerves should be considered. It is well-established that 5-HT can induce orthodromic as well as antidromic stimulation of afferent neurons (10-14). 5-HT induced orthodromic impulse conduction occurs for instance in afferent neurons involved in pain perception, and in the afferent limb of the Bezold-Jarisch reflex (11). Antidromic impulse conduction in afferent neurons elicited by 5-HT, also called the "axon-reflex", evokes cutaneous vasodilatation, probably via the release of vasoactive substances like bradykinin and substance P (14,21). It has been shown that both the 5-HT induced Bezold-Jarisch reflex and 5-HT generated pain on a blister base can be attenuated by ICS 205-930, and also that the 5-HT induced axon-reflex can be inhibited by the 5-HT 3 receptor antagonists MDL 72222 and BRL 43694. These findings support the view that the 5-HT evoked depolarization of sensory neurons is mediated via 5-HT_ receptor stimulation (13-15,22). On the basis of the evidence available, i~ is likely that the 5-HT induced biphasic vasodilatation is mediated via activation of 5-HT~ receptors present on sensory neurons. For this type of receptors the t~rm 5-HT3s receptors has been proposed (12). The finding that the single infusions of both doses of ICS 205-930 did not influence FBF provides evidence that in the human forearm the 5-HTi receptors 3 are not activated by endogenous 5-HT under basal conditions. These results are in accordance with those obtained from studies performed with the 5-HT_ antagonist MDL 72222 in humans (14) and in pigs (7), where MDL 72222 did nat influence the hemodynamic variables measured. The fact that atropine did not influence FBF is in accordance with the literature, and confirms that under basal conditions endogenous ACh does not influence basal vascular tone in the forearm (17). Conclusions In these experiments it was shown that in the human forearm the biphasic vasodilatator response to i.a. infused 5-HT was antagonized by the selective 5-HT 3 antagonist ICS 205-930, which provides evidence that this response was mediated by neuronal 5-HT~ receptor stimulation. Furthermore, it was demonstrated that 5-HT do~s not induce vascular relaxation indirectly via the release of ACh from cholinergic nerve terminals. It was reasoned that the 5-HT 3 receptors involved in this response are located on sensory neurons. The fact that ICS 205-930 did not influence FBF suggests that in the forearm 5-HT 3 receptors are not tonically active under basal conditions. Acknowledgements We appreciate the assistance of dr. P. Vermeij (Ph.D.) for preparing the various solutions of the drugs, and the secretarial help of Mrs. H.C.N. Kappelle-de Vries. ICS 205-930 was kindly supplied by Sandoz AG, Basel, Switzerland. References I.
2.
P.B. BRADLEY, G. ENGEL, W. FENIUK, J.R. FOZARD, P.P.A. HUMPHREY, D.N. MIDDLEMISS, E.J. MYLECHARANE, B.P. RIDCHARDSON, P.R. SAXENA, Neuropharmacol. 25 563-576 (1986). P.R. SAXENA, B.P. RIDCHARDSON, E.J. MYLECHARANE, et al., Trends Pharmacol. Sci. 7 3-4 (1986).
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3. 4.
5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
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M. C~STHERT, Progr. Pharmacol. 6/2 155-172 (1986). J.M. VAN NUETEN, W.J. JANSSENS, P.M. VANHOUTTE, Serotonin and the Cardiovascular System (ed. P.M. Vanhoutte), p. 95-103, Raven Press, New York (1985). H.S. HOUSTON, P.M. VANHOUTTE, Drugs 31 149-163 (1986). H.E. CONNOR, W. FENIUK, P.P.A. HUMPHREY, P.J. PERREN, Br. J. Pharmac. 87 417-426 (1986). P.R. SAXENA, D.J. DUNCKER, A.N. BOM, J. HEILIGERS, P.D. VERDOUW, Naunyn-Schmiedeberg ts Arch. Pharmacol. 333 198-204 (1986). G.J. BLAUW, P. VAN BRUMMELEN, P.C. CHANG, P. VERMEIJ, P.A. VAN ZWIETEN, Hypertension 11 256-263 (1988). G.J. BLAUW, P. VAN BRUMMELEN, P. VERMEIJ, P.A. VAN ZWIETEN, Br. J. clin. Pharmac. 25 629P-630P (1988). D. WALLIS, Life Sci. 29 2345-2355 (1981). J.R. FOZARD, Neuropharmacol. 23 1473-1486 (1984). M. C~STHERT, E. SCHLICKER, J. Cardiovasc. Pharmacol. i0 (suppl 3) $3-$7 (1987). H.O. KALKMAN, G. ENGEL, D. HOYER, J. Hypertension 2 (suppl 3) 143-145 (1984). J.M. ORWIN, J.R. FOZARD, Eur. J. Clin. Pharmacol. 30 209-212 (1986). B.P. RIDCHARDSON, G. ENGEL, P. DONATSCH, P.A. STADLER, Nature 316 126-131 (1985). F. DUFF, A.D.M. GREENFIELD, J.T. SHEPHERD, I.D. THOMPSON, J. Physiol. 120 160-170 (1953). P. GASKELL, J. Physiol. 131 639-646 (1956). P.C. CHANG, P. VAN BRUMMELEN, J. Cardiovasc. Pharmacol. i0 (suppl 5) S123-S125 (1987). P.C. CHANG, R. VERLINDE, T. BRUNING, P. VAN BRUMMELEN, Comput. Biol. Med. 18 157-163 (1988). K. JIE, P. VAN BRUMMELEN, P. VERMEIJ, P.B.M.W.M. TIMMERMANS, P.A. VAN ZWIETEN, Eur. J. Clin. Invest. 17 174-181 (1987). F. LEMBECK, P. HOLZER, Naunyn-Schmiedeberg's Arch. Pharmacol. 310 175-183 (1979). S.M. COOPER, B.D.C. ARNOLD, W.G. RAPEPORT, Br. J. clin. Pharmac. 25 I06P-107P (1988).
Pergamon Press
SEROTONIN INDUCED VASODILATATION IN THE HUMAN FOREARM IS A N T A G O N I Z E D BY T H E S E L E C T I V E 5-HT 3 RECEPTOR ANTAGONIST ICS 205-930 Gerard J. Blauw, Peter van Brummelen* and Pieter A. van Zwieten**.
Department of Nephrology, University Hospital Leiden, The Netherlands. *Present address: Department of Clinical Research, Hoffman-la Roche & Co. AG, Basel, Switzerland. **Department of Pharmacology, Academic Medical Centre, Amsterdam, The Netherlands. (Received in final form September 7, 1988)
The role of 5-HT~ receptors in the biphasic vasodilatator response to serotonin (5-nydroxytryptamine; 5-HT) was investigated in the forearm of 7 young healthy volunteers (aged 22-32 years). Single dose infusions of 5-HT (i ng/kg/min) and of acetylcholine (ACh, 500 ng/kg/min) were administered into the brachial artery. Subsequently combined infusions of 5-HT together with the selective 5-HT receptor antagonist ICS 205-930 (350 and 700 ng/kg/min), an~ ACh together with ICS 205-930 (700 ng/kg/min) were given. After a pause of at least 1 hour the single infusions of 5-HT and ACh were repeated. Subsequently, 5 - H T a n d A C h w e r e infused together with atropine (i00 ng/kg/min). Forearm blood flow (FBF) was m e a s u r e d b y R-wave triggered venous occlusion plethysmography. Heart rate (HR)' and i.a. blood pressure (BP) were recorded semi-continuously. None of the drugs in the doses used did induce systemic hemodynamic effects. After an initial rapid transient increase in FBF of 316 ± 55%, 5-HT elicited a persistent increase in FBF of 90 ± 22% (mean ± SEM, i><0.05 for both). ACh induced a monophasic vasodilatatlon of 475 ± 123% (p<0.05). Both the initial transient and the persistent dilatator response to 5-HT were attenuated by ICS 205-930 350 ng/kg/min (p~0.057, n=5) and 700 ng/kg/min (p<0.05, n=7). The highest dose of ICS 205-930 did not significantly influence the dilatator response to ACh. Atropine abolished the ACh induced vasodilatation (I)<0.05), but did not influence the biphasic dilatator response to 5-HT. Thus the 5-HT induced biphasic vasodilatation was antagonized by ICS 205-930, indicating that this response was mediated by 5-HT 3 receptor activation. The fact that atropine did not influence the vascular response to 5-HT suggests that 5-HT did not induce vascular relaxation indirectly by the release of ACh from cholinergic nerve endings. The serotonin (5-hydroxytryptamine; 5-HT) receptors involved in the complex cardiovascular effects of 5-HT are currently classified into three different receptorsSubtypes' appearedViZ" 5-HT_,~o 5-HT2 and 5-HT3 receptors (1-3). Since the 5-HT 1 be a heterogenous entity, they are tentatively classified as "5-HTl-like" receptors. Both "5-HT.-like" and 5-HT_ receptors are assumed to be located in the vascular wall (~or reviews see ~ef. 3-5). Depending on the species and type of blood vessels investigated, stimulation of "5-HT_-like" receptors may induce either vasodilatation or vasoconstriction, whereas activation of 5-HT 2 receptors elicits vasoconstriction in all species investigated so far, including man (I-9). In prior studies we have found evidence that intra-arterially (i.a.) infused 0024-3205/88 $3.00 + .00 Copyright (c) 1988 Pergamon Press plc
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5-HT induced a biphasic vascular response in the forearm consisting of an initial rapid transient vascular relaxation, followed by a persistent vasodilatation for low doses, and a vasoconstriction only for high pharmacological doses of 5-HT (8,9). It was demonstrated that the 5-HT induced vasoconstriction was mediated by 5-HT 2 receptor stimulation, since it could be inhibited by the selective 5-HT~ receptor antagonists ketanserin and ritanserin (8,9). The mechanism underlyifig the biphasic vasodilatator response could not be established in those experiments, but it was speculated that a "5-HTl-like" receptor could be involved (8). In contrast to the "5-HT_-like" and 5-HT_ receptors, there is little evidence 1 ~ . . that the 5-HT~ receptor subtype plays a role in the peripheral vascular effects of 5-RT (3-5). So far 5-HT 3 receptors have been found on autonomic and sensory neurons, causing depolarization upon stimulation (for reviews see ref. 10-12). However, recent evidence from studies in the porcine carotid vascular bed suggests, that 5-HT~ receptor stimulation is involved in the vascular relaxation induced by 5:HT (7). Since 5-HT~ receptor stimulation evokes neuronal depolarization, the mechanism underlying vascular relaxation could well be the release of acetylcholine (ACh) from cholinergic nerve terminals (11-13). Alternatively, this vasodilatation could be induced by excitation of sensory neurons, via a so called "axon-reflex" (10,11,14). In the present study the highly selective 5-HT 3 receptor antagonist ICS 205-930 was used to investigate the 5-HT induced biphasic vasodilatation (15). The involvement of cholinergic neurons in the vasodilatator response to 5-HT was also investigated (16,17). Methods
Subjects Seven non-patient volunteers (males; mean age 28 years, range of 22-32 years) participated in this study. Their medical history, physical examination and routine laboratory tests did not show evidence of any relevant disease. None of the subjects was receiving any medication at the time of the study or in the previous two weeks. On the day of the study and 12 hours before, all subjects had refrained from smoking and caffeine containing beverages. Informed consent was obtained from all subjects and the protocol was approved by the Ethics Committee of the Leiden University Hospital. Procedures The studies were performed in a quiet room with a temperature between 21°C and 23°C. During the experiments the subjects were in the supine position with the non-dominant arm stabilized slightly above the level of the heart. After local anaesthesia of the skin, the brachial artery of the non-dominant arm was cannulated in the cubital fossa. The cannula (Autocath 1453.13, Saint Leu-La-Foret, France) thus applied was used for infusion of drugs with a Sage constant rate infusion pump (model 351, Sage Instruments Inc., Cambridge, MA, USA) and for intra-arteriel (i.a.) blood pressure (BP) recording with a Statham P23 Id pressure transducer (Gould Inc., Oxnard, CA, USA). Heart rate (HR) was derived from a continuously recorded one-lead electrocardiogram (ECG). Forearm blood flow (FBF) was measured 5 times per minute by venous occlusion plethysmography (Hokanson EC-2 plethysmograph, Hokanson Inc., Issaquah, WA, USA), using mercury-in-silastic strain gauges and a rapid cuff inflator (Hokanson E-10). Tracings of ECG, BP and FBF were directly recorded on a polygraph (Mingograph 803, Siemens-Elema, Stockholm, Sweden). A personal computer (IBM AT III) extended with an analog digital convertor (model DT 2801, Data Translation Inc., Malborough, MA, USA) was used for R-wave triggered control of the rapid cuff inflator, and also for on-line analysis of FBF, BP and HR recordings (18-19). During the measurements of FBF, the
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Vascular Effects of 5-HT and ICS 205-930
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blood flow in the hand was excluded from the circulation using a small wrist cuff, inflated to 40 mmHg above the systolic blood pressure (SBP). The experiments started at least 45 minutes after cannulation of the brachial artery. Between the various infusions the wrist cuff was deflated and sufficient time (20 minutes) was allowed for FBF to return to basal levels. Dru~s and Solutions The following drugs were infused into the brachial artery: 5-hydroxytryptamine HCl (Janssen Chimica), [1H~-indol-3-carbonic-acid-tropine -ester HCl (ICS 205-930, Sandoz AG), acethylcboline HCl (tested according to United States Pharmacopoeia XXI) and atropine-sulphate (tested according to European Pharmacopoeia). All drugs were dissolved in saline. The solutions were prepared from sterile stock solutions and ampoules on the day of the
study. Study protocol Infusions of 5-HT, acetylcholine and ICS 205-930 In the 7 subjects 5-HT and ACh were infused in a random order in single doses of 1 and 500 ng/kg/min, respectively, together with a continuous infusion of saline (0.4 ml/min). Each infusion lasted for 8 minutes. Subsequently, 5-HT (i ng/kg/min) was infused together with ICS 205-930 350 ng/kg/min (n=5) and 700 ng/kg/min (n=7). Finally, ACh (500 ng/kg/min) was infused in the presence of the highest dose of ICS 205-930. The infusions of ICS 205-930 started 6 minutes before the 5-HT and ACh infusions. Infusions of 5-HT, acet~lcholine and atropine At least one hour after the last infusion of ICS 205-930, the infusions of 5-HT (I ng/kg/min) and ACh (500 ng/kg/min) were repeated in the presence of a continuous infusion of saline (0.4 ml/min). Subsequently, 5-HT and ACh were administered together with atropine 100 ng/kg/min, respectively. The infusion of atropine started 6 minutes before the 5-HT and ACh infusions. The hemodynamic variables FBF, i.a. BP and HR were recorded semi-continuously during the various infusions. Basal values were recorded during the 2 minutes preceding each infusion. The hemodynamic Values measured during the first 2 minutes, and during the last 2 minutes of each infusion were used for analysis. Statistical analysis Results are given as means ± SEM. FBF values are expressed as percent changes from baseline. Wilcoxon's signed rank test for matched pairs was used to evaluate the statistical significance of the data. P-values lower than 0.05 were regarded as significant. Results In all experiments only small changes in i.a. BP and HR were observed (table I). During some infusions in which 5-HT and ACh increased FBF, i.a. BP was slightly but significantly reduced (table I). Since HR was uninfluenced by these infusions of 5-HT and ACh, the minimal reductions in i.a. BP are more likely explained by local vasodilatation in the forearm, rather than by systemic hemodynamic effects. Therefore changes in FBF during the various infusions can be interpreted as local vascular effects of the drugs. Baseline levels of FBF observed in the various experiments are listed in table I. Single infusions of serotonin and acet~lcholine In all subjects the infusion of 5-HT i ng/kg/min together with saline induced a biphasic dilatator response, in which the following two phases could be distinguished:
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Vascular Effects of 5-HT and ItS 205-930
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1) a rapid transient increase in FBF within the first 2 minutes after the infusion was started, followed by 2) a persistent increase in FBF, reaching a steady state 5 minutes after the start of the infusion. The maximum increase in FBF during the initial phase (AFBF ) was 316 ± 55% max (p<0.05, fig. i, table II). During the persistent dilatator response FBF was increased by 90 ± 22% (p<0.05, fig. 1, table II). The infusion of ACh 500 ng/kg/min together with saline elicited a significant monophasic increase in FBF of 475 ± 123% (p
4.00
SEROTONIN (M+-SEM)
300
n
/I
-I00
-
,
0
2
~
SALINE 0.4 ml.min"i
o--o
'ICS 205-930
4
350 ng.kg".min"
6
(minutes)
FIG. 1 Mean percent changes In forearm blood flow (+ SEN) induced by simultaneous infusions of serotonln wlth saline, and serotonin with two doses of ICS 205-930 (for values and statistics see text and table II).
Combined infusions of serotonin with ICS 205-930 and acet~lcholine with ICS 205-930 During the infusion of ICS 205-930 350 ng/kg/min both the initial transient vasodilatation and the persistent dilatator phase induced by 5-HT 1 ng/kg/min were reduced (p=0.057 for both, fig. i, table II). The highest dose of ICS 205-930 (700 ng/kg/min) further attenuated the initial transient vasodilatation (p<0.05), and reduced the persistent vasodilatation elicited by 5-HT to the same extent as the dose of 350 ng/kg/min (p<0.05, fig. 1, table II). The highest dose of ICS 205-930 did not significantly influence the vasodilatation induced by Ach (fig. 2).
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18, 1 9 8 8
V a s c u l a r E f f e c t s of 5-HT a n d ICS 2 0 5 - 9 3 0
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TABLE I Mean baseline levels (e SEM) of Foreana Blood Flow before each infusion, and Mean Arterial Pressure and Heart Rate before and after the various infusions. INFUSION (ng/kg/min)
n
BASAL FBF (ml/dl/min)
M A P (mmHg) before after
5-HT 1 A C h 500 ICS 350 ICS 700 ATR 100
7 7 5 7 7
2.3 2.2 2.5 2.3 2.7
80 80 78 80 80
5 - H T + ICS 350 5 - H T + ICS 700 Ach + ICS 700
5 7 7
2.8 ± 0 . 4 2.7 ± 0 . 9 3.3 + 1.3
77 ± 2 78 + 2 78 ± 1
76 ± 2 78 ± 1 77 ± 1
57 ± 2 54 ± 4 55 ± 4
54 + 3 57 ± 4 59 + 4
5 - H T (after ICS:) i ACh (after ICS ) 5 - H T + A T R i00 ACh + A T R i00
7 7 7 7
2.0 2.2 2.8 3.0
80 77 79 79
77 74 77 79
60 60 58 59
60 59 57 58
+ ± ± ¢ +
± ± ± _+
0.4 0.3 0.3 0.5 0.5
0.3 0.4 0.5 0.5
+ ¢ * ± -+
± ± ± +
2 1 1 2 2
2 1 2 2
77 75 77 78 79
+ ± ± ¢ ±
± ± -+ +
H R (beats/mln) before after 2* 2* 2 2 2
57 58 58 54 61
2 2* 1 2
+ ± ± ± ±
± ± ± ±
3 2 3 3 4
3 1 4 3
56 58 57 54 60
± ± ± ± +
~ ± ± ±
2 2 2 4 4
3 3 4 3
FBF = forearm blood flow. MAP = nman arterial pressure, ER = heart rate, 5-HT = serotonln, ACh = acetylchollne, ICS = ICS 205-930, ATR -- atropine, t indicates infusions of serotonln and acetylchollne given one hour after the last infusion of ICS 205-930 {see text). * indicates slgnlflcant difference at the level of I)<0.05 coapared with baseline.
ACETYLCHOLINE (M+SEM)
~ 600 h 400 SALINE
O-
m
:
-
ICS 2 0 5 - 9 3 0
A
A
ATROPINE
.
z
~
FIG. 2 Mean percent changes in forearm blood flow (± SEM) induced by simultaneous infusions of acetylcholine with saline, acetylchollne wlth ICS 205-930 and acetylcholine with atropine.
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V a s c u l a r E f f e c t s of 5-HT and !CS 2 0 5 - 9 3 0
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43, No.
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T A B L E II
The influence of two doses of ICS 205-930 on the blphaslc vascular response to Serotonln. Infusion (ng/kg/min)
First p h a s e A F B F m a x (%)
5-HT + saline 5-HT + ICS 350 5-HT + ICS 700
316 ± 55 50 ± 18 t I0 ± 7*
Second phase AFBF (%)
n
90 ± 22 18 ± 12 t 16 ± 22*
7 5 7
FBF = maximal increase in forearm blood flow measured during the first two minutes after the ~nfums~on was started, FBF = mean percent change in forearm blood flow measured during the last two minutes of the infusion, 5-HT = serotonln, ICS = ICS 205-930. Asterisk end dagger indicates difference compared wlth the Infusion of serotonin with saline (T I)=0.057, * p
400
SEROTONIN (M_+SEM)
'~
SALINE 0.4 ml.min"
A
A T ~
300A
I00
ng.kg'Imin"l
200¢:3
IO0-
-I00
-- ,
FIG. 3 Mea~ percent changes in forearm blood flow (±S~) induced by the simultaneous infusions of serotonln wlth saline and serotonln with atropine, which were given at least one hour after the last infusion of ICS 205-930 (see study protocol).
Single i n f u s i o n s of s e r o t o n i n a n d a c e t ~ l c h o l i n e a d m i n i s t e r e d 1 h o u r a f t e r the last i n f u s i o n of ICS 2 0 5 - 9 3 0 One h o u r a f t e r the last i n f u s i o n s of ICS 205-930, the single infusion of 5-HT again i n d u c e d a b i p h a s i c d i l a t a t o r response (fig. 3). C o m p a r e d w i t h the first single i n f u s i o n of 5-HT, the i n i t i a l t r a n s i e n t v a s o d i l a t a t i o n was p a r t i a l l y r e c o v e r e d (AFBF = 316 ± 55% and 167 ± 50%, r e s p e c t i v e l y , p < O . 0 5 for both), max
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Vascular Effects of 5-HT and ICS 205-930
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whereas the persistent vasodilatation was completely recovered (~FBF = 90 ± 22% and 128 ± 29%, respectively, p<0.05 for both, fig. 1,3). ACh elicited the same vasodilatator response as during the first single infusion of ACh (AFBF = 541 ± 123% and 476 ± 123%, respectively). Combined infusions of eerotonin with atropine and of acet~icholine with atropine The infusion of atropine 100 ng/kg/min did neither influence the initial transient phase, nor the persistent vasodilatation induced by 5-HT significantly, when compared with the single infusion of 5-HT given one hour after the last infusion of ICS 205-930 (fig. 3). The increase in FBF induced by acetylcholine was abolished by the simultaneous infusion of atropine (fig. 2). Single infusions of ItS 205-930 and atropine The single infusions of both ICS 205-930 and atropine did not significantly influence FBF in the doses used. Discussion The main finding in the present study was that 5-HT 3 receptors were involved in the complex vascular response to 5-HT. The 5-HT induced initial transient vasodilatation as well as the second persistent dilatator phase were antagonized by ICS 205-930. Since ICS 205-930 is a highly selective 5-HT 3 receptor antagonist, with negligible affinity for other receptor subtypes (15), the present results strongly suggests that in the human forearm the 5-HT induced vasodilatation is mediated by 5-HTR receptor stimulation. These results corroborate those of a recent study of Saxena et al. (7), who have found evidence in the porcine carotid vascular bed that the 5-HT induced vasodilatation is mediated by 5-HT 3 receptor activation. Bradley et al. (1,2) have proposed that a 5-HT~ receptor mediated response should be characterized by the following three-criteria: i) it is susceptible to antagonism by a 5-HT 3 receptor antagonist like MDL 72222, ICS 205-930 and BRL 43694, 2) it is resistant to 5-HT 2 receptor blockade by ketanserin, and 3) the response can be mimlced by the selective 5-HT 3 agonist 2-methyl-5-HT. In a prior study we have demonstrated that the 5-HT induced biphasic dilatator response was not antagonized by the selective 5-HT~ antagonists ketanserin and ritanserin (8,9). In fact these 5-HT 2 recepto~ antagonists enhanced the dilatator response to 5-HT. However, the third requirement, whether the dilatator response to 5-HT could be mimiced by 2-methyl-5-HT could not be established, since this 5-HT analogue has not been tested for i.a. human use. These findings together with the present results favour the presence and functional relevance of a 5-HT 3 receptor subtype in the vascular response to 5-HT in the human forearm. It has been shown that 5-HT 3 receptors are primarily present on autonomic and sensory neurons, where they were shown to elicit depolarization upon stimulation (i0,ii). It seems unlikely that the 5-HT induced vasodilatation is mediated via stimulation of 5-HT_ receptors present on adrenergic neurons, 3 since excitation of these neurons would induce the release of norepinephrine (ii), resulting in vasoconstriction via activation of postsynaptic ~i- and ~2-adrenoceptors (20). A role for cholinergic neurons in the dilatat6r response to 5-HT was considered to be more likely, since depolarization of these nerve fibers can induce vasodilatation via the release of ACh (ii). However, the present findings that the dilatator response to i.a. infused 5-HT was not attenuated by the anticholinergic drug atropine, whereas the vasodilatation induced by i.a. infused ACh was completely blocked by the same
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Vascular Effects of 5-HT and ICS 205-930
dose of atropine
Vol. 43, No. 18, 1988
(fig. 2,3), excludes this possible mechanism.
The finding that the 5-HT induced vasodilatation could be attributed neither to adrenergic nor to cholinergic 5-HT_ receptor stimulation, the possibility 3 that this dilatator response was mediated via excitation of sensory nerves should be considered. It is well-established that 5-HT can induce orthodromic as well as antidromic stimulation of afferent neurons (10-14). 5-HT induced orthodromic impulse conduction occurs for instance in afferent neurons involved in pain perception, and in the afferent limb of the Bezold-Jarisch reflex (11). Antidromic impulse conduction in afferent neurons elicited by 5-HT, also called the "axon-reflex", evokes cutaneous vasodilatation, probably via the release of vasoactive substances like bradykinin and substance P (14,21). It has been shown that both the 5-HT induced Bezold-Jarisch reflex and 5-HT generated pain on a blister base can be attenuated by ICS 205-930, and also that the 5-HT induced axon-reflex can be inhibited by the 5-HT 3 receptor antagonists MDL 72222 and BRL 43694. These findings support the view that the 5-HT evoked depolarization of sensory neurons is mediated via 5-HT_ receptor stimulation (13-15,22). On the basis of the evidence available, i~ is likely that the 5-HT induced biphasic vasodilatation is mediated via activation of 5-HT~ receptors present on sensory neurons. For this type of receptors the t~rm 5-HT3s receptors has been proposed (12). The finding that the single infusions of both doses of ICS 205-930 did not influence FBF provides evidence that in the human forearm the 5-HTi receptors 3 are not activated by endogenous 5-HT under basal conditions. These results are in accordance with those obtained from studies performed with the 5-HT_ antagonist MDL 72222 in humans (14) and in pigs (7), where MDL 72222 did nat influence the hemodynamic variables measured. The fact that atropine did not influence FBF is in accordance with the literature, and confirms that under basal conditions endogenous ACh does not influence basal vascular tone in the forearm (17). Conclusions In these experiments it was shown that in the human forearm the biphasic vasodilatator response to i.a. infused 5-HT was antagonized by the selective 5-HT 3 antagonist ICS 205-930, which provides evidence that this response was mediated by neuronal 5-HT~ receptor stimulation. Furthermore, it was demonstrated that 5-HT do~s not induce vascular relaxation indirectly via the release of ACh from cholinergic nerve terminals. It was reasoned that the 5-HT 3 receptors involved in this response are located on sensory neurons. The fact that ICS 205-930 did not influence FBF suggests that in the forearm 5-HT 3 receptors are not tonically active under basal conditions. Acknowledgements We appreciate the assistance of dr. P. Vermeij (Ph.D.) for preparing the various solutions of the drugs, and the secretarial help of Mrs. H.C.N. Kappelle-de Vries. ICS 205-930 was kindly supplied by Sandoz AG, Basel, Switzerland. References I.
2.
P.B. BRADLEY, G. ENGEL, W. FENIUK, J.R. FOZARD, P.P.A. HUMPHREY, D.N. MIDDLEMISS, E.J. MYLECHARANE, B.P. RIDCHARDSON, P.R. SAXENA, Neuropharmacol. 25 563-576 (1986). P.R. SAXENA, B.P. RIDCHARDSON, E.J. MYLECHARANE, et al., Trends Pharmacol. Sci. 7 3-4 (1986).
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5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
Vascular Effects of 5-HT and ICS 205-930
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