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Hypotensive actions of ketanserin in dogs: Involvement of a centrally mediated inhibition of sympathetic vascular tone
European Journal of Pharmacology, 111 (1985) 319-327
319
Elsevier
H Y P O T E N S I V E A C T I O N S O F K E T A N S E R I N I N D O G S : I N V O L V E M E N T O F A CENTRALLY M E D I A T E D I N H I B I T I O N O F S Y M P A T H E T I C VASCULAR T O N E CHRISTINE A. PHILLIPS, EWAN J. MYLECHARANE *, JENNIFER K. MARKUS and JOHN SHAW
Department of Pharmacology, University of Sydney, N.S.W. 2006, Australia Received 24 October 1984, revised MS received 5 February 1985, accepted 19 February 1985
C.A. PHILLIPS, E.J. MYLECHARANE, J.K. MARKUS and J. SHAW, Hypotensive actions of ketanserin in dogs: involvement of a centrally mediated inhibition of sympathetic vascular tone, European J. Pharmaeol. 111 (1985) 319-327. The intravenous (i.v.) administration of ketanserin (0.1-0.4 mg/kg) produced immediate and sustained decreases in systemic blood pressure and heart rate in pentobarbitone-anaesthetized dogs. These doses of ketanserin did not inhibit common carotid vasoconstrictor responses to intraarterial (i.a.) noradrenaline, pre-ganglionic stimulation of the sectioned cervical sympathetic nerve, or i.v. nicotine, thus the effects of ketanserin are not due to blockade of vascular a-adrenoceptors, adrenergic neurone blockade, or ganglionic blockade. Systemic pressor responses to i.v. nicotine, which produces sympathetic activation by both central and ganglionic stimulating actions, and to common carotid artery occlusion, were inhibited by 0.1-0.4 mg/kg of ketanserin i.v. These results suggest that in the anaesthetized dog, the hypotensive action of ketanserin involves a centrally mediated inhibition of sympathetic tone. Peripheral vascular 5-HT2 receptor blockade does not appear to be responsible for the hypotensive effect of ketanserin in this model, although this does not preclude the involvement of such a mechanism in its clinical antihypertensive action. Ketanserin
Central sympathetic inhibition
Hypotensive action
1. Introduction The recently developed 5-hydroxytryptamine (serotonin; 5-HT) blocking drug ketanserin, which has been termed a 5-HT 2 receptor antagonist on the basis of binding studies (Leysen et al., 1981), has also been reported to have hypotensive activity in rats (Van Nueten et al., 1981) and in humans (De Cree et al., 1981). It has been claimed that these effects are mediated by vascular 5-HT 2 receptor blockade, thereby inhibiting 5-HT-induced vasoconstriction and amplification of other vasoconstrictors (Van Nueten et al., 1981; Janssen, 1983). As there is little evidence to link 5-HT with hypertension, other investigators have attributed
* To whom all correspondence should be addressed: Department of Pharmacology, University of Sydney, N,S.W. 2006, Australia. 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V.
Dogs
this action of ketanserin to its a-adrenoceptor blocking activity (Fozard, 1982, 1983; Kalkman et al., 1982; Persson et al., 1982). During the course of other investigations into the dilator effects of 5-HT in the common carotid and femoral arterial circulations of the dog, we have noted the hypotensive properties of ketanserin (Markus et al., 1984; Phillips et al., 1985). Administration of ketanserin (0.1-0.4 m g / k g i.v.) produced immediate and sustained decreases in systemic blood pressure and in vascular resistance in each circulation, but these doses of ketanserin had no effect on the vasoconstrictor responses to intraarterial (i.a) noradrenaline (NA) in these circulations. These observations suggested the possibility that ketanserin is able to decrease blood pressure in some manner other than vascular a-adrenoceptor blockade. The purpose of the present series of experiments was to investigate further the mechanisms
320 underlying the hypotensive action of ketanserin in dogs. Preliminary reports of these studies have been presented at recent scientific meetings (Mylecharane et al., 1985; Phillips et al., 1984).
2. Materials and methods
2.1. Preparations Experiments were carried out in a total of 14 dogs (12-32 kg) not selected for breed or sex. The dogs were anaesthetized with pentobarbitone sodium (30 m g / k g i.v.) and artificially ventilated with room air via an endotracheal tube. Expiratory CO 2 levels were monitored with a Datex CD-102 capnograph to ensure adequate ventilation. Systemic arterial blood pressure (BP) was measured by means of a polythene catheter passed into the aorta via a femoral artery and connected to a Statham P23a pressure transducer. Mean systemic BP was used in all calculations, and was obtained by adding one-third of the difference between systolic and diastolic pressures to the diastolic pressure. Heart rate (HR) was measured using a Grass Model 7P4 tachograph triggered by the systemic BP signals. A polythene catheter was inserted into a femoral vein for i.v. administration of drugs and fluids. Anaesthesia was maintained at a stable level by the continuous i.v. infusion of pentobarbitone sodium (6 m g / k g per h) in a solution of 0.9% w / v NaC1 (60 ml/h). Body temperature was measured using a rectal thermometer, and was maintained at 37-38°C. The fight common carotid artery was exposed and cleared of connective tissue. An electromagnetic flow probe (IVM, Model A) with a lumen diameter of 3 or 4 mm was positioned around the exposed vessel. Mean common carotid blood flow (CCBF) was measured using an EMI Type 28 electromagnetic flowmeter. Zero flow references were obtained periodically by briefly occluding the vessel distal to the probe. A fine vinyl catheter with a small aperture cut into its wall was passed into and out of the common carotid artery distal to the flow probe, so that the aperture lay within the lumen of the vessel. One end of this catheter was used for the i.a. infusion of NA by a motor-
driven syringe (Braun Perfusor). The other end of the catheter was connected to a Statham P23a pressure transducer, so that local perfusion pressure in the common carotid artery (CCBP) could be measured. The catheter used for i.a. infusion was kept patent by a slow continuous infusion of 0.9% w / v NaC1 solution containing 10 units/ml of heparin (0.06 ml/min). The thyro-lingual branch of the common carotid artery was tied off. The right cervical vagosympathetic nerve trunk (between the inferior and superior cervical ganglia) was carefully dissected free and sectioned proximally, so that the distal part could be electrically stimulated using stainless steel hook electrodes connected to a Grass stimulator (Model SD9). The left cervical vagosympathetic trunk was left undisturbed, but the left common carotid artery was exposed and a snare was placed around the vessel so that a reflex pressor response to unilateral common carotid artery occlusion (CCO) could be produced. Expiratory CO 2 levels, systemic BP, HR, CCBF and CCBP were recorded continuously on a Grass Model 7 polygraph. Common carotid vascular resistance (CCVR) was calculated using the equation: CCVR = C C B P / C C B F ; the inverse of this value was common carotid vascular conductance (CCVC). The CCVR and CCVC values following the various experimental procedures were expressed as % changes from the respective resistance and conductance levels which preceded each of the procedures. Common carotid vasodilator effects were always assessed in terms of the % decreases in CCVR, while vasoconstrictor effects were always assessed as % decreases in CCVC, thereby ensuring that all effects were measured on a comparable scale, i.e. from 0 to -100%.
2.2. Experimental procedures The dogs were atropinized for the duration of the experiments (0.1 m g / k g i.v. initially and supplemented when necessary); this inhibited any systemic responses to afferent vagal fibre activation when the distal cervical vagosympathetic nerve trunk was electrically stimulated. In each experiment, initial cumulative dose-response relationships were established for the common carotid
321 vasoconstrictor effects of i.a. infusions of NA during the first 120 min of the experimental recording period. The doses of NA were increased in a stepwise fashion when each dose had produced stable effects, by increasing the infusion rate of the NA solution through the range 0.03-3 ml/min, until maximal effects were obtained. Common carotid vasoconstrictor responses to electrical stimulation of the cervical sympathetic nerves were also established during this initial recording period. The nerves were stimulated for 15 s periods using 2 ms pulses at a supramaximal voltage (10-25 V) and at frequencies of 0.25, 0.5, 1 and 2.5 Hz. Stimulations at the various frequencies were separated by 3 min intervals. Initial common carotid vasoconstrictor and systemic pressor responses to nicotine (0.2 mg/kg i.v.), and initial systemic pressor responses to unilateral CCO of 20 s duration, were also elicited during this period. The effects of ketanserin were studied in 7 dogs. After establishing the initial responses to the various test procedures in the first 120 rain, three doses of ketanserin were administered i.v. at 60 min intervals, so as to produce cumulatively increasing total doses of 0.1, 0.2 and 0.4 mg/kg. The test procedures were repeated after each dose of ketanserin; a period of 15 min was allowed after each administration of ketanserin before commencement of the test procedures. The effect of subsequent ganglionic blockade (hexamethonium or mecamylamine, 5 mg/kg i.v.) on the various procedures (except for NA) was then tested. As a control measure, the effects of prazosin (0.01, 0.02 and 0.04 mg/kg i.v.) were tested similarly in one dog. The remaining 6 dogs were used as a control series of experiments; after establishing the initial responses to the test procedures in the first 120 rain, they were repeated at corresponding times but in the absence of ketanserin, prazosin or ganglion blocking drug. Data from replicated experimental procedures in any one experiment during the initial 120 min or during any of the succeeding 60 min test periods were always pooled before further analysis. In each experiment, log dose-response curves for the common carotid vasoconstrictor effects of NA were plotted, and the dose of NA producing 50% of the maximal response (EDso) was obtained from each
curve. The effect of ketanserin on the NA response was assessed in terms of agonist dose ratios (i.e. ratio of dose of agonist producing an equivalent response in the presence of antagonist to that before the antagonist). The effects of ketanserin on the other responses (to cervical sympathetic nerve stimulation, nicotine and unilateral CCO) were all assessed in terms of the % changes from the initial responses in each experiment. (Note: these % changes should be distinguished from the units of measurement of some of these responses which happen to be % decreases in CCVC.) Ketanserininduced effects on systemic BP, HR and CCVR were assessed as the changes in these parameters from the pre-ketanserin levels. In the prazosin experiment, and in the control series of experiments, data were analysed in an analogous manner.
2.3. Statistical analysis The EDso and dose ratio values for NA obtained from each experiment were pooled to give geometric means and 95% confidence limits. Other data from each experiment were pooled to give means and S.E.M. values. Statistical evaluation of mean results was performed using Student's t-test (2-tailed) for correlated or non-correlated data as appropriate (Snedecor and Cochran, 1967). Resuits were considered significant when P < 0.05.
2. 4. Drugs The following drugs were used: (-)-noradrenaline hydrogen tartrate (Sigma); nicotine hydrogen tartrate (BDH); ketanserin tartrate (Janssen); hexamethonium bromide (Sigma); mecamylamine hydrochloride (Merck, Sharpe and Dohme); prazosin hydrochloride (Pfizer); atropine sulphate (Boehringer Ingelheim); and pentobarbitone sodium (May and Baker). Solutions of all drugs were prepared by dissolution in 0.9% w / v NaC1 solution, except for prazosin which was first suspended in glycerine (1 in 50) and then dissolved in 5% (+)-glucose solution. Fresh solutions were made at the commencement of each experiment, and were kept on ice during the experiment. Ascorbic acid (20 ~g/ml) was included in the NA
322 solutions to minimize oxidation. Doses of all drugs are expressed in terms of the above salts.
3. Results 3.1. Control experiments
The results obtained in the control series of experiments (n = 6) showed that there were no significant spontaneous changes in systemic BP, H R or CCVR during the experimental recording period. In the common carotid circulation, vasoconstrictor responses (i.e. decreases in CCVC) were produced by the cumulative i.a. infusion of NA (0.01-1 /~g/kg per rain), by cervical sympathetic nerve stimulation (0.25, 0.5, 1 and 2.5 Hz), and by the i.v. administration of nicotine (0.2 mg/kg). The common carotid vasoconstrictor responses to N A were dose-dependent, and those to cervical sympathetic nerve stimulation were frequency-dependent. Systemic pressor responses were produced by nicotine (0.2 m g / k g i.v.) and unilateral CCO for 20 s. There were no significant spontaneous changes in any of these vasoconstrictor or pressor responses during the experimental recording period. 3.2. Effects on systemic BP, H R and C C V R
In the ketanserin-treated dogs, average resting values during the initial 120 min experimental recording period prior to administration of ketanserin were as follows: systemic BP, 131 + 5 mmHg; HR, 160 + 8 beats/rain; CCBF, 190 + 32 m l / m i n ; and CCBP, 123 + 4 mmHg (means + S.E.M., n = 7). Administration of ketanserin (0.1, 0.2 and 0.4 m g / k g i.v.) consistently produced significant decreases in systemic BP. These falls in systemic BP occurred immediately following ketanserin administration and usually reached a stable level within 10 rain; the hypotensive effects were maintained for the 60 min periods between successive doses of ketanserin. H R also fell following each dose of ketanserin. The decreases in H R were significant after the 0.2 and 0.4 m g / k g doses. CCVR was not significantly reduced by ketanserin, and expiratory
CO 2 levels were unaffected. The mean ketanserininduced changes in systemic BP, H R and CCVR are listed in table 1. The subsequent administration of hexamethonium or mecamylamine (5 m g / k g i.v.) produced further decreases in systemic BP and HR, but did not alter CCVR or expiratory CO 2 levels. In the control dog treated with prazosin (0.01, 0.02 and 0.04 m g / k g i.v.), there were falls in systemic BP comparable to those produced by ketanserin, but H R was slightly elevated. The CCVR was unaffected. 3.3. Effects on common carotid vasoconstrictor responses
During the initial 120 min period in the ketanserin-treated dogs, before administration of ketanserin, the EDs0 for NA was 0.04 (0.03-0.06) # g / k g per rain (geometric mean and 95% confidence limits, n = 7). The maximal response to NA was a change in CCVC of - 9 0 + 2% from the pre-infusion level (mean +__S.E.M., n = 7). The common carotid vasoconstrictor responses to cervical sympathetic nerve stimulation at frequencies of 0.25, 0.5, 1 and 2.5 Hz during the preketanserin period were changes in CCVC of - 5 3 +4%, -63___4%, - 7 2 + 4 % and - 7 7 + 3 % , respectively, from the pre-stimulation levels (means ___S.E.M., n = 7). The common carotid vasoconstrictor response to nicotine (0.2 m g / k g i.v.) during this pre-ketanserin period was a change in CCVC of - 5 6 + 7% from the pre-injection level (mean + S.E.M., n -- 6). Ketanserin (0.1-0.4 m g / k g i.v.) failed to produce any significant inhibition of these vasoconstrictor responses to NA, cervical sympathetic nerve stimulation or nicotine. The CCBF and CCBP responses to NA and to cervical sympathetic nerve stimulation in typical experiments before and after 0.4 m g / k g of ketanserin are illustrated in figs. 1 and 2, respectively. The mean values of the NA dose ratios and the % changes in the responses to cervical sympathetic nerve stimulation and nicotine following ketanserin are given in table 1. Although there were slight apparent shifts to the right of the NA log dose-response curves, the NA dose ratios were not significantly
323 TABLE 1 Ketanserin-induced changes in: systemic blood pressure (BP); heart rate (HR); common carotid vascular resistance (CCVR); common carotid (CC) vasoconstrictor responses to noradrenaline (NA), cervical sympathetic nerve stimulation (CSNS) and nicotine; and systemic pressor responses to nicotine and unilateral common carotid occlusion (CCO). The changes in systemic BP, HR and CCVR are means -+S.E.M. of the changes from the pre-ketanserin levels. The NA dose ratios are geometric means (and 95% confidence limits) of the NA dose ratios in the presence of kctanserin. The changes in the responses to CSNS, nicotine and CCO are means + S.E.M. of the % changes in the pre-ketanserin responses. Parameter
n
Systemic BP (mmHg) HR(beats/min) CCVR (% change in pre-ketanserin CCVR level) CC vasoconstrictor response to NA (dose ratio) CC vasoconstrictor response to CSNS (% change in pre-ketanserin response): 0.25Hz 0.5Hz 1Hz 2.5Hz CC vasoconstrictor response to nicotine (% change in pre-ketanserin response) Systemic pressor response to nicotine (% change in pre-ketanserin response) Systemic pressor response to CCO (% change in pre-ketanserin response)
7 7 7
Ketanserin (mg/kg) 0.1
0.2
0.4
- 18 + 3 a -6-+ 3 - 7 -+ 9
- 25 -+ 3 " -15± 5 ~ + 1 -t- 7
-29+__ 3 a -27+ 6 a +3_+ 6
7
1.4 (1.2-1.8)
1.6 (1.2-2.1)
1.7 (1.2-2.4)
7 7 7 7 6
+14-+ +9+ +2-+ +5-+ - 3+
6 3~ 2 2~ 18
+9-+ 5 +12+ 4 ~ +5-+ 2 a +2_+ 3 - 17 -+ 19
+5_+ 8 +14+ 4 a +7-+ 2 a +5-+ 3 -33-+18
6
- 35 -+ 10 ~
- 43 -+ 6 a
-52_+ 4 a
5
- 34 -+ 11 a
- 37 _+13 a
-43_+13 a
Significant changes (P < 0.05).
Control
Ketanserin 0.4 mglkg
200]
different from those obtained
at c o r r e s p o n d i n g
t i m e s i n t h e c o n t r o l series o f e x p e r i m e n t s , w h i c h
CCBF 10o1 ml/min oa
w e r e 1.1 (0.9-1.3), 1.1 (0.5-2.5) a n d 1.0 (0.6-1.6), r e s p e c t i v e l y ( g e o m e t r i c m e a n s a n d 95% c o n f i d e n c e l i m i t s , n = 6). I n s o m e i n s t a n c e s , t h e r e w e r e v e r y small but significant e n h a n c e m e n t s of the vaso-
CCBP 200] mmHg 1001 0 a
c o n s t r i c t o r r e s p o n s e s to cervical s y m p a t h e t i c nerve stimulation.
~ !
! !
o0,o05 0.02
i
I
!
02 0. 1
i
,
o;,
0.5
0o;o;; 0.02
0.1
methonium,
0.5 I'
NA IJg/kg per min
Following ganglionic blockade with either m e c a m y l a m i n e (as i l l u s t r a t e d i n fig. 2) o r h e x a -
•
2 min
Fig. 1. Traces from a typical experiment showing common carotid blood flow (CCBF) and common carotid blood pressure (CCBP) responses to cumulative i.a. infusion of noradrenaline (NA) at the dose levels indicated, initially (Control), and after administration of ketanserin (0.4 mg/kg i.v.).
the common
carotid vasoconstrictor
r e s p o n s e s to cervical s y m p a t h e t i c n e r v e stimulat i o n w e r e c o m p l e t e l y a b o l i s h e d , as w e r e t h o s e t o nicotine. In the prazosin-treated control dog, common carotid vasoconstrictor responses to NA, cervical sympathetic nerve stimulation and nicotine were
324 Control
Me¢~mylsmlne 5 mglkg
Ketanserln 0.4 mg/kg
200] CCBF ml/min 1001 0,=
CCBP mmHg
2oo 1
1°°1 OJ
t
t
0.25 0.5
t 1.0 Hz
t
t
t
t
t
t
t
t
t
2.5
0.25
0.5
1.0
2.5
0.25
0.5
1.0 Hz
2.5
Hz
2 mln
Fig. 2. Traces from a typical experiment showing common carotid blood flow (CCBF) and common carotid blood pressure (CCBP) responses to cervical sympathetic nerve stimulation at the frequencies indicated, initially (Control), after administration of ketanserin (0.4 mg/kg i.v.), and after the subsequent administration of mecamylamine (5 mg/kg i.v.).
all substantially inhibited following each dose of prazosin.
period resulted in a pressor response of 16 + 2 mmHg above the pre-occlusion level of systemic BP. These systemic pressor responses were significantly reduced after each dose of ketanserin. The effect of the 0.4 mg/kg dose is illustrated in fig. 3. Table 1 contains the mean % decreases in the systemic pressor responses produced by ketanserin. Subsequent ganglionic blockade with either hexamethonium (as illustrated in fig. 3) or mecamylamine abolished the systemic pressor re-
3. 4. Effects on systemic pressor responses
In the group of dogs treated with ketanserin, the i.v. administration of 0.2 mg/kg of nicotine during the initial pre-ketanserin period produced an increase in systemic BP of 68 + 13 mmHg from the pre-injection level (mean +S.E.M., n = 6). Unilateral CCO for 20 s in the pre-ketanserin Control
Ketanserln 0.4 mglkg
,0o 1
,oo 1 1001
mmHg 04
Hexarnethonlum 5 mg/kg
•
•
04
•
Nicotine 0.2 mglkg
200] mmHgB,.
1oo.,
1
OJ
-
c c o 20 s
; .~
Fig. 3. Traces from a typical experiment showing systemic blood pressure (aortic BP) responses to nicotine (0.2 mg/kg i.v.) and unilateral common carotid occlusion (CCO) for 20 s, initially (Control), after administration of ketanserin (0.4 mg/kg i.v.), and after the subsequent administration of hexamethonium (5 mg/kg i.v.)
325 sponses. Prazosin markedly inhibited the systemic pressor responses to nicotine and unilateral CCO in the prazosin control dog.
4. Discussion Ketanserin, at doses of 0.1-0.4 mg/kg i.v., produced significant decreases in systemic BP in the present experiments in pentobarbitone-anaesthetized dogs. These falls were comparable with those produced in our other experiments in anaesthetized dogs (Markus et al., 1984; Phillips et al., 1985). Ketanserin has been shown to have aadrenoceptor blocking activity at the doses required to produce hypotension in rats in vivo (Fozard, 1982; Kalkman et al., 1982; Persson et al., 1982). However, the hypotensive effects which we have observed in the present experiments cannot have been due to a-adrenoceptor blockade, because these doses of ketanserin had no inhibitory effects on the common carotid constrictor responses to NA, cervical sympathetic nerve stimulation or nicotine. The observations in the prazosin-treated control dog confirm that a-adrenoceptor blocking activity is able to be demonstrated in our experimental model. It has been well established that prazosin produces selective a-adrenoceptor blockade of responses to both exogenous and neuronally released NA, in parallel with its hypotensive effects, in numerous in vivo experimental models, including pentobarbitone-anaesthetized normotensive dogs (see Cavero et al., 1978; Cavero and Roach, 1980; Massingham et al., 1981, for references). It has been reported that in the dog hind-limb in vivo, prazosin inhibits the vasoconstrictor response to exogenous N A less effectively than it inhibits those to neuronally released NA or the oq-adrenoceptor selective agonist phenylephrine (Langer et al., 1980). The explanation proposed for these observations is that neuronally released NA acts mainly on post-synaptic ax-adrenoceptors, whereas exogenous NA acts on both al- and az-adrenoceptors located postsynaptically on the vascular smooth muscle; because prazosin is selective for al-adrenoceptors, the a2-adrenoceptormediated component of the response to exogenous
NA is not affected. Ketanserin is considered to be selective for ax-adrenoceptors, on the basis of binding studies (Leysen et al., 1981), and because in rats in vivo it antagonizes pressor responses to sympathetic nerve stimulation and the selective al-adrenoceptor agonists phenylephrine and methoxamine more effectively than it blocks pressor responses to N A and the selective a2-adrenoceptor agonist B-HT920 (Fozard, 1982; Kalkman et al., 1982). If this were the situation in dogs, it is conceivable that ketanserin could produce hypotension by antagonizing neuronally released NA without affecting responses to exogenous N A . However, this is not the case in our experiments, because ketanserin at doses of 0.1-0.4 mg/kg produced hypotension but did not inhibit responses to exogenous or neuronally released NA. At higher dose levels in dogs (1-4 mg/kg i.v.), ketanserin does inhibit the vasoconstrictor effects of NA (Markus et al., 1984; Phillips et al., 1985), thus a-adrenoceptor blockade could account for the hypotensive activity of these higher doses. Clearly, however, some other mechanism is responsible for the hypotensive activity of 0.1-0.4 mg/kg of ketanserin in dogs. Blockade of 5-HT2 receptor-mediated vasoconstriction or amplification of the effects of other endogenous vasoconstrictors is unlikely to have been responsible for the hypotensive effects observed in our experiments, as the resultant vasodilatation and hypotension would be expected to produce at least some reflex increases in HR, as has been well documented for other direct vasodilators and a-adrenoceptor antagonists, including prazosin (Cavero and Roach, 1980). In fact, HR was significantly reduced after the 0.2 and 0.4 mg/kg doses of ketanserin in the present experiments. In our other experiments in anaesthetized dogs, we found that pizotifen, at doses well in excess of those required for so-called 'D' or 5-HT2 receptor antagonism, had no effect on systemic BP or on resting vascular resistance in the common carotid and femoral arterial circulations (Markus et al., 1984; Phillips et al., 1985). Although peripheral vascular 5-HT2 receptor blockade is unlikely to be the mechanism involved in the hypotensive action of ketanserin in our experiments, this does not exclude the possibility that such a mechanism
326 might contribute to its efficacy in hypertension, if 5-HT could be shown to have a pathophysiological role in this disease. In our experiments, the hypotensive actions of ketanserin cannot have been due to increases in vagal outflow, as the right vagal trunk was sectioned, and the dogs were atropinized throughout the experiment. The effects of 0.1-0.4 m g / k g of ketanserin on systemic BP and HR, and the absence of a-adrenoceptor blocking activity at these dose levels, suggest that ketanserin has an inhibitory action in the sympathetic nervous system in some manner other, than a-adrenoceptor antagonism. In our other experiments in dogs (Markus et al., 1984; Phillips et al., 1985), ketanserin also produced significant decreases in resting vascular resistance in the femoral and common carotid arterial circulations. In the present experiments, there were no significant reductions in CCVR, but this is to be expected, because the cervical sympathetic nerve trunk was sectioned at the outset in these experiments, thereby removing a large component of resting tone from the common carotid circulation and hence masking any inhibitory effects on sympathetic vascular tone. The other test procedures used in the present experiments were designed to investigate the level at which ketanserin acts in the sympathetic nervous system. The cervical sympathetic nerves which were stimulated are mostly pre-ganglionic, as confirmed by the abolition of common carotid vasoconstrictor responses to electrical stimulation and nicotine following ganglionic blockade with hexamethonium or mecamylamine. The fact that ketanserin had no effect on these common carotid vasoconstrictor responses shows that its hypotensive actions were not due to adrenergic neurone blockade or ganglionic blockade. The findings that ketanserin inhibited the systemic pressor responses to nicotine and to unilateral common carotid occlusion indicate that its sympathetic inhibitory effects are mediated proximally to the sympathetic ganglia, in the central nervous system. The systemic pressor effect of nicotine is due to sympathetic nerve excitation mediated by both central and ganglionic stimulating components, but its common carotid vasoconstrictor action in the present experiments involves only the ganglionic
stimulating component, because the pre-ganglionic sympathetic nerves to this circulation were sectioned. The failure of ketanserin to block the latter effect is consistent with ketanserin having only a centrally mediated inhibitory effect on sympathetic nerve activity. Our results therefore indicate that ketanserin is acting at a site proximal to the sympathetic ganglia, and that its hypotensive effects may be due to a centrally mediated inhibition of sympathetic nerve activity. Although it has been claimed that the main site of action of ketanserin is peripheral (Janssen, 1983), there is increasing evidence that systemically administered ketanserin readily enters the central nervous system. Laduron et al. (1982) reported that the i.v. administration of 0.005 m g / k g of [3H]ketanserin in rats produced considerable specific 5-HT2 receptor binding in various regions of the brain. When administered i.p. at doses of 0.01-1 mg/kg, ketanserin modified central 5-HT receptor-mediated behavioural changes in rats (Yap and Taylor, 1983). Although direct evidence was not obtained, the possibility of a central site of action for ketanserin has been suggested on the basis of the bradycardia which ~it produces in rats (Fozard, 1982) and in man (Fagard et al., i985). Persson et al. (1982) concluded that the hypotensive effect of ketanserin in rats was not due to a central mechanism, but this was based on the effects of ketanserin administered directly into the lateral cerebral ventricle; it is possible that the doses and the route of administration used did not permit ketanserin to gain access to its putative central site of action. However, a central effect of ketanserin may not be of importance in rats; in other studies, these authors found that chronic oral administration of ketanserin to rats did not modify cardiovascular responses to stress, baroreceptor sensitivity, or central catecholamine and 5-HT synthesis rates, and concluded that a central mechanism was not involved in its hypotensive action (Pettersson et al., 1984). Since the completion of the present studies, findings in anaesthetized cats consistent with our conclusions have been published (Ramage, 1983; McCall and Schuette, 1984). These authors found that the i.v. administration of ketanserin (0.05-0.8 mg/kg) produced dose-dependent decreases in
327
systemic BP and HR, and parallel decreases in activity of pre-ganglionic sympathetic nerve recordings. Interestingly, McCall and Schuette (1984) concluded that the central sympathetic inhibitory effect of ketanserin was mediated by central a 1adrenoceptors. The role of central serotonergic pathways in the control of blood pressure remains controversial, as both pressor and depressor responses to activation of these pathways have been described (see Howe et al., 1983, for references). Thus our experiments indicate that in anaesthetized dogs, the hypotensive action of 0.1-0.4 m g / k g of ketanserin is not due to vascular aadrenoceptor or 5-HT2 receptor blockade, but to a centrally mediated inhibition of sympathetic nerve activity. A central effect of ketanserin may make an important contribution to its hypotensive action, and may be responsible for its efficacy in hypertension. Other compounds which are known to reduce sympathetic nerve activity by a central mechanism, such as clonidine, are useful antihypertensive agents; ketanserin may prove to be another compound of this type.
Acknowledgements These studies were supported in part by the National Health and Medical Research Council of Australia, and Janssen Pharmaceutica (Australia). Prazosin was a gift from Pfizer.
References Cavero, I., S. Fenard, R. Gomeni, F. Lefevre and A.G. Roach, 1978, Studies on the mechanism of the vasodilator effects of prazosin in dogs and rabbits, European J. Pharmacol. 49, 259. Cavero, I. and A.G. Roach, 1980, The pharmacology of prazosin, a novel antihypertensive agent, Life Sci. 27, 1525. De Cree, J., H. Verhaegen and J. Symoens, 1981, Acute bloodpressure-lowering effect of ketanserin, Lancet i, 1161. Fagard, R., P. Lijnen, J. Staessen, R. Fiocchi and A. Amery, 1985, Responses of the systemic and pulmonary circulation to chronic ketanserin treatment in hypertension, J. Cardiovasc. Pharmacol. (in press). Fozard, J.R., 1982, Mechanism of the hypotensive effect of ketanserin, J. Cardiovasc. Pharmacol. 4, 829. Fozard, J.R., 1983, The mode of action of ketanserin, Trends Pharmacol. Sci. 4, 412. Howe, P.R.C., D.M. Kuhn, J.B. Minson, B.H. Stead and J.P. Chalmers, 1983, Evidence for a bulbospinal serotonergic pressor pathway in the rat brain, Brain Res. 270, 29. Janssen, P.A.J., 1983, 5-HT2 receptor blockade to study serotonin-induced pathology, Trends Pharmacol. Sci. 4, 198. Kalkman, H.O., P.B.M.W.M. Timmermans and P.A. Van Zwieten, 1982, Characterization of the antihypertensive
properties of ketanserin (R 41 468) in rats, J. Pharmacol. Exp. Ther. 222, 227. Laduron, P.M., P.F.M. Janssen and J.E. Leysen, 1982, In vivo binding of [3H]ketanserin on serotonin S2-receptors in rat brain, European J. Pharmacol. 81, 43. Langer, S.Z., R. Massingham and N.B. Shepperson, 1980, Presence of postsynaptic a2-adrenoceptors of predominantly extrasynaptic location in the vascular smooth muscle of the dog hind limb, Clin. Sci. 59, Suppl. 6, 225s. Leysen, J.E., F. Awouters, L. Kennis, P.M. Laduron, J. Vandenberk and P.A.J. Janssen, 1981, Receptor binding profile of R 41 468, a novel antagonist at 5-HT2 receptors, Life Sci. 28, 1015. McCall, R.B. and M.R. Schuette, 1984, Evidence for an alpha-1 receptor-mediated central sympathoinhibitory action of ketanserin, J. Pharmacol. Exp. Ther. 228, 704. Markus, J.K., C.A. Phillips, E.J. Mylecharane and J. Shaw, 1984, Antagonism by ketanserin and pizotifen of vasodilator responses to 5-hydroxytryptamine in the dog common carotid arterial circulation, Clin. Exp. Pharmacol. Physiol. Suppl. 8, 42. Massingham, R., M.L. Dubocovich, N.B. Shepp0rson and S.Z. Langer, 1981, In vivo selectivity of prazosin but not of WB4101 for postsynaptic alpha-1 adrenoceptors, J. Pharmacol. Exp. Ther. 217, 467. Mylecharane, E.J., C.A. Phillips, J.K. Markus and J. Shaw, 1985, Evidence for a central component to the hypotensive action of ketanserin in the dog, J. Cardiovasc. Pharmacol. (in press). Persson, B., T. Hedner and M. Henning, 1982, Cardiovascular effects in the rat of ketanserin, a novel 5-hydroxytryptamine receptor blocking agent, J. Pharm. Pharmacol. 34, 442. Pettersson, A., B. Persson, M. Henning and T. Hedner, 1984, Antihypertensive effects of chronic 5-hydroxytryptamine (5-HT2) receptor blockade with ketanserin in the spontaneously hypertensive rat, Naunyn-Schmiedeb. Arch. Pharmacol. 327, 43. Phillips, C.A., E.J. Mylecharane, J.K. Markus and J. Shaw, 1984, Does the hypotensive action of ketanserin involve an inhibition of sympathetic nerve activity?, Clin. Exp. Pharmacol. Physiol. Suppl. 8, 43. Phillips, C.A., E.J. Mylecharane and J. Shaw, 1985, Mechanisms involved in the vasodilator action of 5-hydroxytryptamine in the dog femoral arterial circulation in vivo, European J. Pharmacol. (in press). Ramage, A.G., 1983, Effects of ketanserin and methysergide on the cardiovascular system of the cat, Br. J. Pharmacol. 80, 608P. Snedecor, G.W. and W.G. Cochran, 1967, Statistical Methods, 6th edn. (Iowa State University Press, Ames) p. 91. Van Nueten, J.M., P.A.J. Janssen, J. Van Beek, R. Xhonneux, T.J. Verbeuren and P.M. Vanhoutte, 1981, Vascular effects of ketanserin (R 41 468), a novel antagonist of 5-HT2 serotonergic receptors, J. Pharmacol. Exp. Ther. 218, 217. Yap, C.Y. and D.A. Taylor, 1983, Involvement of 5-HT2 receptors in the wet-dog shake behaviour induced by 5-hydroxytryptophan in the rat, Neuropharmacology 22, 801.
319
Elsevier
H Y P O T E N S I V E A C T I O N S O F K E T A N S E R I N I N D O G S : I N V O L V E M E N T O F A CENTRALLY M E D I A T E D I N H I B I T I O N O F S Y M P A T H E T I C VASCULAR T O N E CHRISTINE A. PHILLIPS, EWAN J. MYLECHARANE *, JENNIFER K. MARKUS and JOHN SHAW
Department of Pharmacology, University of Sydney, N.S.W. 2006, Australia Received 24 October 1984, revised MS received 5 February 1985, accepted 19 February 1985
C.A. PHILLIPS, E.J. MYLECHARANE, J.K. MARKUS and J. SHAW, Hypotensive actions of ketanserin in dogs: involvement of a centrally mediated inhibition of sympathetic vascular tone, European J. Pharmaeol. 111 (1985) 319-327. The intravenous (i.v.) administration of ketanserin (0.1-0.4 mg/kg) produced immediate and sustained decreases in systemic blood pressure and heart rate in pentobarbitone-anaesthetized dogs. These doses of ketanserin did not inhibit common carotid vasoconstrictor responses to intraarterial (i.a.) noradrenaline, pre-ganglionic stimulation of the sectioned cervical sympathetic nerve, or i.v. nicotine, thus the effects of ketanserin are not due to blockade of vascular a-adrenoceptors, adrenergic neurone blockade, or ganglionic blockade. Systemic pressor responses to i.v. nicotine, which produces sympathetic activation by both central and ganglionic stimulating actions, and to common carotid artery occlusion, were inhibited by 0.1-0.4 mg/kg of ketanserin i.v. These results suggest that in the anaesthetized dog, the hypotensive action of ketanserin involves a centrally mediated inhibition of sympathetic tone. Peripheral vascular 5-HT2 receptor blockade does not appear to be responsible for the hypotensive effect of ketanserin in this model, although this does not preclude the involvement of such a mechanism in its clinical antihypertensive action. Ketanserin
Central sympathetic inhibition
Hypotensive action
1. Introduction The recently developed 5-hydroxytryptamine (serotonin; 5-HT) blocking drug ketanserin, which has been termed a 5-HT 2 receptor antagonist on the basis of binding studies (Leysen et al., 1981), has also been reported to have hypotensive activity in rats (Van Nueten et al., 1981) and in humans (De Cree et al., 1981). It has been claimed that these effects are mediated by vascular 5-HT 2 receptor blockade, thereby inhibiting 5-HT-induced vasoconstriction and amplification of other vasoconstrictors (Van Nueten et al., 1981; Janssen, 1983). As there is little evidence to link 5-HT with hypertension, other investigators have attributed
* To whom all correspondence should be addressed: Department of Pharmacology, University of Sydney, N,S.W. 2006, Australia. 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V.
Dogs
this action of ketanserin to its a-adrenoceptor blocking activity (Fozard, 1982, 1983; Kalkman et al., 1982; Persson et al., 1982). During the course of other investigations into the dilator effects of 5-HT in the common carotid and femoral arterial circulations of the dog, we have noted the hypotensive properties of ketanserin (Markus et al., 1984; Phillips et al., 1985). Administration of ketanserin (0.1-0.4 m g / k g i.v.) produced immediate and sustained decreases in systemic blood pressure and in vascular resistance in each circulation, but these doses of ketanserin had no effect on the vasoconstrictor responses to intraarterial (i.a) noradrenaline (NA) in these circulations. These observations suggested the possibility that ketanserin is able to decrease blood pressure in some manner other than vascular a-adrenoceptor blockade. The purpose of the present series of experiments was to investigate further the mechanisms
320 underlying the hypotensive action of ketanserin in dogs. Preliminary reports of these studies have been presented at recent scientific meetings (Mylecharane et al., 1985; Phillips et al., 1984).
2. Materials and methods
2.1. Preparations Experiments were carried out in a total of 14 dogs (12-32 kg) not selected for breed or sex. The dogs were anaesthetized with pentobarbitone sodium (30 m g / k g i.v.) and artificially ventilated with room air via an endotracheal tube. Expiratory CO 2 levels were monitored with a Datex CD-102 capnograph to ensure adequate ventilation. Systemic arterial blood pressure (BP) was measured by means of a polythene catheter passed into the aorta via a femoral artery and connected to a Statham P23a pressure transducer. Mean systemic BP was used in all calculations, and was obtained by adding one-third of the difference between systolic and diastolic pressures to the diastolic pressure. Heart rate (HR) was measured using a Grass Model 7P4 tachograph triggered by the systemic BP signals. A polythene catheter was inserted into a femoral vein for i.v. administration of drugs and fluids. Anaesthesia was maintained at a stable level by the continuous i.v. infusion of pentobarbitone sodium (6 m g / k g per h) in a solution of 0.9% w / v NaC1 (60 ml/h). Body temperature was measured using a rectal thermometer, and was maintained at 37-38°C. The fight common carotid artery was exposed and cleared of connective tissue. An electromagnetic flow probe (IVM, Model A) with a lumen diameter of 3 or 4 mm was positioned around the exposed vessel. Mean common carotid blood flow (CCBF) was measured using an EMI Type 28 electromagnetic flowmeter. Zero flow references were obtained periodically by briefly occluding the vessel distal to the probe. A fine vinyl catheter with a small aperture cut into its wall was passed into and out of the common carotid artery distal to the flow probe, so that the aperture lay within the lumen of the vessel. One end of this catheter was used for the i.a. infusion of NA by a motor-
driven syringe (Braun Perfusor). The other end of the catheter was connected to a Statham P23a pressure transducer, so that local perfusion pressure in the common carotid artery (CCBP) could be measured. The catheter used for i.a. infusion was kept patent by a slow continuous infusion of 0.9% w / v NaC1 solution containing 10 units/ml of heparin (0.06 ml/min). The thyro-lingual branch of the common carotid artery was tied off. The right cervical vagosympathetic nerve trunk (between the inferior and superior cervical ganglia) was carefully dissected free and sectioned proximally, so that the distal part could be electrically stimulated using stainless steel hook electrodes connected to a Grass stimulator (Model SD9). The left cervical vagosympathetic trunk was left undisturbed, but the left common carotid artery was exposed and a snare was placed around the vessel so that a reflex pressor response to unilateral common carotid artery occlusion (CCO) could be produced. Expiratory CO 2 levels, systemic BP, HR, CCBF and CCBP were recorded continuously on a Grass Model 7 polygraph. Common carotid vascular resistance (CCVR) was calculated using the equation: CCVR = C C B P / C C B F ; the inverse of this value was common carotid vascular conductance (CCVC). The CCVR and CCVC values following the various experimental procedures were expressed as % changes from the respective resistance and conductance levels which preceded each of the procedures. Common carotid vasodilator effects were always assessed in terms of the % decreases in CCVR, while vasoconstrictor effects were always assessed as % decreases in CCVC, thereby ensuring that all effects were measured on a comparable scale, i.e. from 0 to -100%.
2.2. Experimental procedures The dogs were atropinized for the duration of the experiments (0.1 m g / k g i.v. initially and supplemented when necessary); this inhibited any systemic responses to afferent vagal fibre activation when the distal cervical vagosympathetic nerve trunk was electrically stimulated. In each experiment, initial cumulative dose-response relationships were established for the common carotid
321 vasoconstrictor effects of i.a. infusions of NA during the first 120 min of the experimental recording period. The doses of NA were increased in a stepwise fashion when each dose had produced stable effects, by increasing the infusion rate of the NA solution through the range 0.03-3 ml/min, until maximal effects were obtained. Common carotid vasoconstrictor responses to electrical stimulation of the cervical sympathetic nerves were also established during this initial recording period. The nerves were stimulated for 15 s periods using 2 ms pulses at a supramaximal voltage (10-25 V) and at frequencies of 0.25, 0.5, 1 and 2.5 Hz. Stimulations at the various frequencies were separated by 3 min intervals. Initial common carotid vasoconstrictor and systemic pressor responses to nicotine (0.2 mg/kg i.v.), and initial systemic pressor responses to unilateral CCO of 20 s duration, were also elicited during this period. The effects of ketanserin were studied in 7 dogs. After establishing the initial responses to the various test procedures in the first 120 rain, three doses of ketanserin were administered i.v. at 60 min intervals, so as to produce cumulatively increasing total doses of 0.1, 0.2 and 0.4 mg/kg. The test procedures were repeated after each dose of ketanserin; a period of 15 min was allowed after each administration of ketanserin before commencement of the test procedures. The effect of subsequent ganglionic blockade (hexamethonium or mecamylamine, 5 mg/kg i.v.) on the various procedures (except for NA) was then tested. As a control measure, the effects of prazosin (0.01, 0.02 and 0.04 mg/kg i.v.) were tested similarly in one dog. The remaining 6 dogs were used as a control series of experiments; after establishing the initial responses to the test procedures in the first 120 rain, they were repeated at corresponding times but in the absence of ketanserin, prazosin or ganglion blocking drug. Data from replicated experimental procedures in any one experiment during the initial 120 min or during any of the succeeding 60 min test periods were always pooled before further analysis. In each experiment, log dose-response curves for the common carotid vasoconstrictor effects of NA were plotted, and the dose of NA producing 50% of the maximal response (EDso) was obtained from each
curve. The effect of ketanserin on the NA response was assessed in terms of agonist dose ratios (i.e. ratio of dose of agonist producing an equivalent response in the presence of antagonist to that before the antagonist). The effects of ketanserin on the other responses (to cervical sympathetic nerve stimulation, nicotine and unilateral CCO) were all assessed in terms of the % changes from the initial responses in each experiment. (Note: these % changes should be distinguished from the units of measurement of some of these responses which happen to be % decreases in CCVC.) Ketanserininduced effects on systemic BP, HR and CCVR were assessed as the changes in these parameters from the pre-ketanserin levels. In the prazosin experiment, and in the control series of experiments, data were analysed in an analogous manner.
2.3. Statistical analysis The EDso and dose ratio values for NA obtained from each experiment were pooled to give geometric means and 95% confidence limits. Other data from each experiment were pooled to give means and S.E.M. values. Statistical evaluation of mean results was performed using Student's t-test (2-tailed) for correlated or non-correlated data as appropriate (Snedecor and Cochran, 1967). Resuits were considered significant when P < 0.05.
2. 4. Drugs The following drugs were used: (-)-noradrenaline hydrogen tartrate (Sigma); nicotine hydrogen tartrate (BDH); ketanserin tartrate (Janssen); hexamethonium bromide (Sigma); mecamylamine hydrochloride (Merck, Sharpe and Dohme); prazosin hydrochloride (Pfizer); atropine sulphate (Boehringer Ingelheim); and pentobarbitone sodium (May and Baker). Solutions of all drugs were prepared by dissolution in 0.9% w / v NaC1 solution, except for prazosin which was first suspended in glycerine (1 in 50) and then dissolved in 5% (+)-glucose solution. Fresh solutions were made at the commencement of each experiment, and were kept on ice during the experiment. Ascorbic acid (20 ~g/ml) was included in the NA
322 solutions to minimize oxidation. Doses of all drugs are expressed in terms of the above salts.
3. Results 3.1. Control experiments
The results obtained in the control series of experiments (n = 6) showed that there were no significant spontaneous changes in systemic BP, H R or CCVR during the experimental recording period. In the common carotid circulation, vasoconstrictor responses (i.e. decreases in CCVC) were produced by the cumulative i.a. infusion of NA (0.01-1 /~g/kg per rain), by cervical sympathetic nerve stimulation (0.25, 0.5, 1 and 2.5 Hz), and by the i.v. administration of nicotine (0.2 mg/kg). The common carotid vasoconstrictor responses to N A were dose-dependent, and those to cervical sympathetic nerve stimulation were frequency-dependent. Systemic pressor responses were produced by nicotine (0.2 m g / k g i.v.) and unilateral CCO for 20 s. There were no significant spontaneous changes in any of these vasoconstrictor or pressor responses during the experimental recording period. 3.2. Effects on systemic BP, H R and C C V R
In the ketanserin-treated dogs, average resting values during the initial 120 min experimental recording period prior to administration of ketanserin were as follows: systemic BP, 131 + 5 mmHg; HR, 160 + 8 beats/rain; CCBF, 190 + 32 m l / m i n ; and CCBP, 123 + 4 mmHg (means + S.E.M., n = 7). Administration of ketanserin (0.1, 0.2 and 0.4 m g / k g i.v.) consistently produced significant decreases in systemic BP. These falls in systemic BP occurred immediately following ketanserin administration and usually reached a stable level within 10 rain; the hypotensive effects were maintained for the 60 min periods between successive doses of ketanserin. H R also fell following each dose of ketanserin. The decreases in H R were significant after the 0.2 and 0.4 m g / k g doses. CCVR was not significantly reduced by ketanserin, and expiratory
CO 2 levels were unaffected. The mean ketanserininduced changes in systemic BP, H R and CCVR are listed in table 1. The subsequent administration of hexamethonium or mecamylamine (5 m g / k g i.v.) produced further decreases in systemic BP and HR, but did not alter CCVR or expiratory CO 2 levels. In the control dog treated with prazosin (0.01, 0.02 and 0.04 m g / k g i.v.), there were falls in systemic BP comparable to those produced by ketanserin, but H R was slightly elevated. The CCVR was unaffected. 3.3. Effects on common carotid vasoconstrictor responses
During the initial 120 min period in the ketanserin-treated dogs, before administration of ketanserin, the EDs0 for NA was 0.04 (0.03-0.06) # g / k g per rain (geometric mean and 95% confidence limits, n = 7). The maximal response to NA was a change in CCVC of - 9 0 + 2% from the pre-infusion level (mean +__S.E.M., n = 7). The common carotid vasoconstrictor responses to cervical sympathetic nerve stimulation at frequencies of 0.25, 0.5, 1 and 2.5 Hz during the preketanserin period were changes in CCVC of - 5 3 +4%, -63___4%, - 7 2 + 4 % and - 7 7 + 3 % , respectively, from the pre-stimulation levels (means ___S.E.M., n = 7). The common carotid vasoconstrictor response to nicotine (0.2 m g / k g i.v.) during this pre-ketanserin period was a change in CCVC of - 5 6 + 7% from the pre-injection level (mean + S.E.M., n -- 6). Ketanserin (0.1-0.4 m g / k g i.v.) failed to produce any significant inhibition of these vasoconstrictor responses to NA, cervical sympathetic nerve stimulation or nicotine. The CCBF and CCBP responses to NA and to cervical sympathetic nerve stimulation in typical experiments before and after 0.4 m g / k g of ketanserin are illustrated in figs. 1 and 2, respectively. The mean values of the NA dose ratios and the % changes in the responses to cervical sympathetic nerve stimulation and nicotine following ketanserin are given in table 1. Although there were slight apparent shifts to the right of the NA log dose-response curves, the NA dose ratios were not significantly
323 TABLE 1 Ketanserin-induced changes in: systemic blood pressure (BP); heart rate (HR); common carotid vascular resistance (CCVR); common carotid (CC) vasoconstrictor responses to noradrenaline (NA), cervical sympathetic nerve stimulation (CSNS) and nicotine; and systemic pressor responses to nicotine and unilateral common carotid occlusion (CCO). The changes in systemic BP, HR and CCVR are means -+S.E.M. of the changes from the pre-ketanserin levels. The NA dose ratios are geometric means (and 95% confidence limits) of the NA dose ratios in the presence of kctanserin. The changes in the responses to CSNS, nicotine and CCO are means + S.E.M. of the % changes in the pre-ketanserin responses. Parameter
n
Systemic BP (mmHg) HR(beats/min) CCVR (% change in pre-ketanserin CCVR level) CC vasoconstrictor response to NA (dose ratio) CC vasoconstrictor response to CSNS (% change in pre-ketanserin response): 0.25Hz 0.5Hz 1Hz 2.5Hz CC vasoconstrictor response to nicotine (% change in pre-ketanserin response) Systemic pressor response to nicotine (% change in pre-ketanserin response) Systemic pressor response to CCO (% change in pre-ketanserin response)
7 7 7
Ketanserin (mg/kg) 0.1
0.2
0.4
- 18 + 3 a -6-+ 3 - 7 -+ 9
- 25 -+ 3 " -15± 5 ~ + 1 -t- 7
-29+__ 3 a -27+ 6 a +3_+ 6
7
1.4 (1.2-1.8)
1.6 (1.2-2.1)
1.7 (1.2-2.4)
7 7 7 7 6
+14-+ +9+ +2-+ +5-+ - 3+
6 3~ 2 2~ 18
+9-+ 5 +12+ 4 ~ +5-+ 2 a +2_+ 3 - 17 -+ 19
+5_+ 8 +14+ 4 a +7-+ 2 a +5-+ 3 -33-+18
6
- 35 -+ 10 ~
- 43 -+ 6 a
-52_+ 4 a
5
- 34 -+ 11 a
- 37 _+13 a
-43_+13 a
Significant changes (P < 0.05).
Control
Ketanserin 0.4 mglkg
200]
different from those obtained
at c o r r e s p o n d i n g
t i m e s i n t h e c o n t r o l series o f e x p e r i m e n t s , w h i c h
CCBF 10o1 ml/min oa
w e r e 1.1 (0.9-1.3), 1.1 (0.5-2.5) a n d 1.0 (0.6-1.6), r e s p e c t i v e l y ( g e o m e t r i c m e a n s a n d 95% c o n f i d e n c e l i m i t s , n = 6). I n s o m e i n s t a n c e s , t h e r e w e r e v e r y small but significant e n h a n c e m e n t s of the vaso-
CCBP 200] mmHg 1001 0 a
c o n s t r i c t o r r e s p o n s e s to cervical s y m p a t h e t i c nerve stimulation.
~ !
! !
o0,o05 0.02
i
I
!
02 0. 1
i
,
o;,
0.5
0o;o;; 0.02
0.1
methonium,
0.5 I'
NA IJg/kg per min
Following ganglionic blockade with either m e c a m y l a m i n e (as i l l u s t r a t e d i n fig. 2) o r h e x a -
•
2 min
Fig. 1. Traces from a typical experiment showing common carotid blood flow (CCBF) and common carotid blood pressure (CCBP) responses to cumulative i.a. infusion of noradrenaline (NA) at the dose levels indicated, initially (Control), and after administration of ketanserin (0.4 mg/kg i.v.).
the common
carotid vasoconstrictor
r e s p o n s e s to cervical s y m p a t h e t i c n e r v e stimulat i o n w e r e c o m p l e t e l y a b o l i s h e d , as w e r e t h o s e t o nicotine. In the prazosin-treated control dog, common carotid vasoconstrictor responses to NA, cervical sympathetic nerve stimulation and nicotine were
324 Control
Me¢~mylsmlne 5 mglkg
Ketanserln 0.4 mg/kg
200] CCBF ml/min 1001 0,=
CCBP mmHg
2oo 1
1°°1 OJ
t
t
0.25 0.5
t 1.0 Hz
t
t
t
t
t
t
t
t
t
2.5
0.25
0.5
1.0
2.5
0.25
0.5
1.0 Hz
2.5
Hz
2 mln
Fig. 2. Traces from a typical experiment showing common carotid blood flow (CCBF) and common carotid blood pressure (CCBP) responses to cervical sympathetic nerve stimulation at the frequencies indicated, initially (Control), after administration of ketanserin (0.4 mg/kg i.v.), and after the subsequent administration of mecamylamine (5 mg/kg i.v.).
all substantially inhibited following each dose of prazosin.
period resulted in a pressor response of 16 + 2 mmHg above the pre-occlusion level of systemic BP. These systemic pressor responses were significantly reduced after each dose of ketanserin. The effect of the 0.4 mg/kg dose is illustrated in fig. 3. Table 1 contains the mean % decreases in the systemic pressor responses produced by ketanserin. Subsequent ganglionic blockade with either hexamethonium (as illustrated in fig. 3) or mecamylamine abolished the systemic pressor re-
3. 4. Effects on systemic pressor responses
In the group of dogs treated with ketanserin, the i.v. administration of 0.2 mg/kg of nicotine during the initial pre-ketanserin period produced an increase in systemic BP of 68 + 13 mmHg from the pre-injection level (mean +S.E.M., n = 6). Unilateral CCO for 20 s in the pre-ketanserin Control
Ketanserln 0.4 mglkg
,0o 1
,oo 1 1001
mmHg 04
Hexarnethonlum 5 mg/kg
•
•
04
•
Nicotine 0.2 mglkg
200] mmHgB,.
1oo.,
1
OJ
-
c c o 20 s
; .~
Fig. 3. Traces from a typical experiment showing systemic blood pressure (aortic BP) responses to nicotine (0.2 mg/kg i.v.) and unilateral common carotid occlusion (CCO) for 20 s, initially (Control), after administration of ketanserin (0.4 mg/kg i.v.), and after the subsequent administration of hexamethonium (5 mg/kg i.v.)
325 sponses. Prazosin markedly inhibited the systemic pressor responses to nicotine and unilateral CCO in the prazosin control dog.
4. Discussion Ketanserin, at doses of 0.1-0.4 mg/kg i.v., produced significant decreases in systemic BP in the present experiments in pentobarbitone-anaesthetized dogs. These falls were comparable with those produced in our other experiments in anaesthetized dogs (Markus et al., 1984; Phillips et al., 1985). Ketanserin has been shown to have aadrenoceptor blocking activity at the doses required to produce hypotension in rats in vivo (Fozard, 1982; Kalkman et al., 1982; Persson et al., 1982). However, the hypotensive effects which we have observed in the present experiments cannot have been due to a-adrenoceptor blockade, because these doses of ketanserin had no inhibitory effects on the common carotid constrictor responses to NA, cervical sympathetic nerve stimulation or nicotine. The observations in the prazosin-treated control dog confirm that a-adrenoceptor blocking activity is able to be demonstrated in our experimental model. It has been well established that prazosin produces selective a-adrenoceptor blockade of responses to both exogenous and neuronally released NA, in parallel with its hypotensive effects, in numerous in vivo experimental models, including pentobarbitone-anaesthetized normotensive dogs (see Cavero et al., 1978; Cavero and Roach, 1980; Massingham et al., 1981, for references). It has been reported that in the dog hind-limb in vivo, prazosin inhibits the vasoconstrictor response to exogenous N A less effectively than it inhibits those to neuronally released NA or the oq-adrenoceptor selective agonist phenylephrine (Langer et al., 1980). The explanation proposed for these observations is that neuronally released NA acts mainly on post-synaptic ax-adrenoceptors, whereas exogenous NA acts on both al- and az-adrenoceptors located postsynaptically on the vascular smooth muscle; because prazosin is selective for al-adrenoceptors, the a2-adrenoceptormediated component of the response to exogenous
NA is not affected. Ketanserin is considered to be selective for ax-adrenoceptors, on the basis of binding studies (Leysen et al., 1981), and because in rats in vivo it antagonizes pressor responses to sympathetic nerve stimulation and the selective al-adrenoceptor agonists phenylephrine and methoxamine more effectively than it blocks pressor responses to N A and the selective a2-adrenoceptor agonist B-HT920 (Fozard, 1982; Kalkman et al., 1982). If this were the situation in dogs, it is conceivable that ketanserin could produce hypotension by antagonizing neuronally released NA without affecting responses to exogenous N A . However, this is not the case in our experiments, because ketanserin at doses of 0.1-0.4 mg/kg produced hypotension but did not inhibit responses to exogenous or neuronally released NA. At higher dose levels in dogs (1-4 mg/kg i.v.), ketanserin does inhibit the vasoconstrictor effects of NA (Markus et al., 1984; Phillips et al., 1985), thus a-adrenoceptor blockade could account for the hypotensive activity of these higher doses. Clearly, however, some other mechanism is responsible for the hypotensive activity of 0.1-0.4 mg/kg of ketanserin in dogs. Blockade of 5-HT2 receptor-mediated vasoconstriction or amplification of the effects of other endogenous vasoconstrictors is unlikely to have been responsible for the hypotensive effects observed in our experiments, as the resultant vasodilatation and hypotension would be expected to produce at least some reflex increases in HR, as has been well documented for other direct vasodilators and a-adrenoceptor antagonists, including prazosin (Cavero and Roach, 1980). In fact, HR was significantly reduced after the 0.2 and 0.4 mg/kg doses of ketanserin in the present experiments. In our other experiments in anaesthetized dogs, we found that pizotifen, at doses well in excess of those required for so-called 'D' or 5-HT2 receptor antagonism, had no effect on systemic BP or on resting vascular resistance in the common carotid and femoral arterial circulations (Markus et al., 1984; Phillips et al., 1985). Although peripheral vascular 5-HT2 receptor blockade is unlikely to be the mechanism involved in the hypotensive action of ketanserin in our experiments, this does not exclude the possibility that such a mechanism
326 might contribute to its efficacy in hypertension, if 5-HT could be shown to have a pathophysiological role in this disease. In our experiments, the hypotensive actions of ketanserin cannot have been due to increases in vagal outflow, as the right vagal trunk was sectioned, and the dogs were atropinized throughout the experiment. The effects of 0.1-0.4 m g / k g of ketanserin on systemic BP and HR, and the absence of a-adrenoceptor blocking activity at these dose levels, suggest that ketanserin has an inhibitory action in the sympathetic nervous system in some manner other, than a-adrenoceptor antagonism. In our other experiments in dogs (Markus et al., 1984; Phillips et al., 1985), ketanserin also produced significant decreases in resting vascular resistance in the femoral and common carotid arterial circulations. In the present experiments, there were no significant reductions in CCVR, but this is to be expected, because the cervical sympathetic nerve trunk was sectioned at the outset in these experiments, thereby removing a large component of resting tone from the common carotid circulation and hence masking any inhibitory effects on sympathetic vascular tone. The other test procedures used in the present experiments were designed to investigate the level at which ketanserin acts in the sympathetic nervous system. The cervical sympathetic nerves which were stimulated are mostly pre-ganglionic, as confirmed by the abolition of common carotid vasoconstrictor responses to electrical stimulation and nicotine following ganglionic blockade with hexamethonium or mecamylamine. The fact that ketanserin had no effect on these common carotid vasoconstrictor responses shows that its hypotensive actions were not due to adrenergic neurone blockade or ganglionic blockade. The findings that ketanserin inhibited the systemic pressor responses to nicotine and to unilateral common carotid occlusion indicate that its sympathetic inhibitory effects are mediated proximally to the sympathetic ganglia, in the central nervous system. The systemic pressor effect of nicotine is due to sympathetic nerve excitation mediated by both central and ganglionic stimulating components, but its common carotid vasoconstrictor action in the present experiments involves only the ganglionic
stimulating component, because the pre-ganglionic sympathetic nerves to this circulation were sectioned. The failure of ketanserin to block the latter effect is consistent with ketanserin having only a centrally mediated inhibitory effect on sympathetic nerve activity. Our results therefore indicate that ketanserin is acting at a site proximal to the sympathetic ganglia, and that its hypotensive effects may be due to a centrally mediated inhibition of sympathetic nerve activity. Although it has been claimed that the main site of action of ketanserin is peripheral (Janssen, 1983), there is increasing evidence that systemically administered ketanserin readily enters the central nervous system. Laduron et al. (1982) reported that the i.v. administration of 0.005 m g / k g of [3H]ketanserin in rats produced considerable specific 5-HT2 receptor binding in various regions of the brain. When administered i.p. at doses of 0.01-1 mg/kg, ketanserin modified central 5-HT receptor-mediated behavioural changes in rats (Yap and Taylor, 1983). Although direct evidence was not obtained, the possibility of a central site of action for ketanserin has been suggested on the basis of the bradycardia which ~it produces in rats (Fozard, 1982) and in man (Fagard et al., i985). Persson et al. (1982) concluded that the hypotensive effect of ketanserin in rats was not due to a central mechanism, but this was based on the effects of ketanserin administered directly into the lateral cerebral ventricle; it is possible that the doses and the route of administration used did not permit ketanserin to gain access to its putative central site of action. However, a central effect of ketanserin may not be of importance in rats; in other studies, these authors found that chronic oral administration of ketanserin to rats did not modify cardiovascular responses to stress, baroreceptor sensitivity, or central catecholamine and 5-HT synthesis rates, and concluded that a central mechanism was not involved in its hypotensive action (Pettersson et al., 1984). Since the completion of the present studies, findings in anaesthetized cats consistent with our conclusions have been published (Ramage, 1983; McCall and Schuette, 1984). These authors found that the i.v. administration of ketanserin (0.05-0.8 mg/kg) produced dose-dependent decreases in
327
systemic BP and HR, and parallel decreases in activity of pre-ganglionic sympathetic nerve recordings. Interestingly, McCall and Schuette (1984) concluded that the central sympathetic inhibitory effect of ketanserin was mediated by central a 1adrenoceptors. The role of central serotonergic pathways in the control of blood pressure remains controversial, as both pressor and depressor responses to activation of these pathways have been described (see Howe et al., 1983, for references). Thus our experiments indicate that in anaesthetized dogs, the hypotensive action of 0.1-0.4 m g / k g of ketanserin is not due to vascular aadrenoceptor or 5-HT2 receptor blockade, but to a centrally mediated inhibition of sympathetic nerve activity. A central effect of ketanserin may make an important contribution to its hypotensive action, and may be responsible for its efficacy in hypertension. Other compounds which are known to reduce sympathetic nerve activity by a central mechanism, such as clonidine, are useful antihypertensive agents; ketanserin may prove to be another compound of this type.
Acknowledgements These studies were supported in part by the National Health and Medical Research Council of Australia, and Janssen Pharmaceutica (Australia). Prazosin was a gift from Pfizer.
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