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Mianserin — An analysis of its peripheral autonomic actions
European Journal o f Pharmacology, 51 (1978) 1--10 © Elsevier/North-Holland Biomedical Press
1
MIANSERIN -- AN ANALYSIS OF ITS P E R I P H E R A L AUTONOMIC ACTIONS JOHN C. DOXEY, JOHN EVERITT and GEOFFREY METCALF
R e c k i t t and Colman, Pharmaceutical Division, Dansom Lane, Hull, HU8 7DS, England Received 10 November 1977, revised MS received 12 April 1978, accepted 16 May 1978
J.C. DOXEY, J. EVERITT and G. METCALF, Mianserin -- an analysis o f its peripheral autonomic actions, European J. Pharmacol. 51 (1978) 1--10. The autonomic profile of mianserin has been compared with that of yohimbine, phentolamine, phenoxybenzamine and desmethylimipramine. The effects of mianserin on tyramine and noradrenaline pressor responses in the pithed rat were consistent with c~-adrenoceptor antagonist and uptake blocking properties. In isolated tissue experiments, the selectivity of mianserin for pre- and postsynaptic ~-adrenoceptors was similar to that of phentolamine. In pithed rats mianserin antagonised the pressor response produced by clonidine and reversed the inhibitory actions of clonidine on cardiac nerve stimulation. In contrast mianserin only caused slight reversal of the inhibitory effects of clonidine on hypogastric nerve stimulation. Uptake blockade itself inhibits hypogastric nerve stimulation and this could counteract any antagonism of the effects of clonidine at the presynaptic ~-adrenoceptor. The results demonstrate the uptake blocking properties of mianserin and its antagonism at both pre- and postsynaptic ~-adrenoceptors. Mianserin
Presynaptic ~-adrenoceptors
Uptake blockade
1. Introduction Mianserin hydrochloride (ORG. GB 94) was recently introduced as an antidepressant agent (Van der Burg et al., 1970) and was found to have an efficacy similarto that of amitriptyline (Ghose et al., 1976). It has been reported (Goodlet and Sugrue, 1974) that mianserin, like desmethylimipramine (DMI), blocks central catecholaminergic uptake systems. These workers also reported that mianserin and DMI antagonise tyramine and potentiate noradrenaline pressor responses in the pithed rat. In contrast mianserin had no significant effect on tyramine pressor responses in man (Ghose et al., 1976). Despite their similarities on monoamine uptake processes, mianserin, unlike DMI, was essentially devoid of effect in conventional antidepressant screening tests (Goodlet and Sugrue, 1974}. Van Zwieten (1975) reported that mianserin was a p o t e n t a-adrenoceptor antagonist in the
Postsynaptic ~-adrenoceptors
periphery and Baumann and Maitre (1977) concluded that a presynaptic a-adrenoceptor antagonist c o m p o n e n t was included in the action of mianserin. In this study the profile of mianserin has been compared with those of yohimbine, phentolamine, phenoxybenzamine and DMI.
2. Materials and methods 2.1. P i t h e d rats
Male rats in the b o d y weight range 200-300 g were pithed during a brief period of halothane anaesthesia. The animals were subsequently artificially respired with r o o m air (100 strokes/min, 1 ml/100 g}. Blood pressure was recorded with a Hewlett Packard 1280c pressure transducer from the right c o m m o n carotid artery. Heart rate was recorded using the arterial pressure wave to trigger a ratemeter.
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J.C. DOXEY ET AL.
In experiments where contractions of the vas deferens were monitored an isometric transducer (Statham Gold Cell, model uC3) was employed. All parameters were displayed on a Hewlett Packard 7700 recorder. The right femoral vein was cannulated to facilitate administration of drugs. In experiments where sympathetic outflow was stimulated, tubocurarine (1 mg/kg, i.v.) was injected before stimulation commenced.
and 300 pg/kg, i.v.) or noradrenaline (0.1, 0.3 and 1.0 pg/kg, i.v.) was constructed in pithed rats prior to injection of the drug under examination. The test c o m p o u n d was then injected i.v. and the dose response to the pressor agent re-examined in its presence i.e. each animal acted as its own control. Saline (0.9% w/v NaC1 solution) was injected into further animals for control studies.
2.2. Assessment o f presynap tic a-adrenoceptor antagonist activity in pithed rats
2.4. Assessment o f pre- and postsynaptic aadrenoceptor antagonist activity in isolated tissue experiments
Selective stimulation of cardiac or hypogastric nerves in the pithed rat (Gillespie et al., 1970) leads to corresponding increases in heart rate and vas deferens tension with little effect on sympathetic discharge to other organs. The presynaptic a-adrenoceptor agonist actions of clonidine inhibit the effects of electrical stimulation on both cardiac nerves (Armstrong and Boura, 1973; Drew, 1976) and hypogastric nerves (Doxey and Everitt, 1977). Clonidine also produces a pressor response in pithed rats; an action attributable to its postsynaptic a-adrenoceptor agonist action. The ability of mianserin to reverse the presynaptic actions of clonidine (30 pg/kg, i.v.) was compared with that of yohimbine, phentolamine, phenoxybenzamine and DMI following i.v. injection Ca minimum of 3 rats was used for each drug studied and the figures represent typical experimental results. The stimulus parameters for stimulation of cardiac nerves and hypogastric nerves were 10 V, 0.5 msec, 1 Hz for 10 sec every 2 min and 20 V, 50 psec, 6 Hz for 3 sec every 30 sec respectively. 2.3. Tyramine and noradrenaline responses in the pithed rat
pressor
The i.v. effects of mianserin on noradrenaline and tyramine pressor responses were studied in pithed rats. Each pressor agent was studied in a different group of 5 rats. A dose response curve to either tyramine (30, 100
The pre- and postsynpatic a-adrenoceptor antagonist activity of mianserin was studied using an identical procedure to that used by Doxey et al. (1977). Presynaptic a-adrenoceptor antagonist activity was assessed by studying the effects of increasing concentrations of mianserin on cumulative clonidine dose--response curves on the rat vas deferens stimulated at 0.1 Hz. Postsynaptic a-adrenoceptor antagonist activity was assessed by comparison of control cumulative noradrenaline dose--response curves with those in the presence of increasing concentrations of mianserin using the rat anococcygeus muscle. Corticosterone 40 pM, DMI 10 nM and propranolol 0.1 #M were present in the Krebs solution throughout the experiments to block extraneuronal and neuronal uptake and fl-adrenoceptors. 2.5. Drugs used The following drugs were used in this study: clonidine hydrochloride (Boehringer Ingelheim); corticosterone (Sigma); desmethylimipramine hydrochloride (Geigy); mianserin hydrochloride (Organon); noradrenaline bitartrate (Koch Light Laboratories Ltd); phenoxybenzamine hydrochloride (SKF); phentolamine mesylate (CIBA); (+)-propranolol hydrochloride (ICI); tubocurarine chloride (Burroughs Wellcome);tyramine hydrochloride (Koch Light Laboratories Ltd), and yohimbine hydrochloride (Sigma). All drugs, with the
M I A N S E R I N -- A N A U T O N O M I C P R O F I L E
3
exception of phenoxybenzamine which was made up at pH 3, were dissolved in saline and doses mentioned in the text are in terms of their respective salts.
3. Results 3.1. Effect o f mianserin on noradrenaline and tyramine pressor responses in the pithed rat
The antagonism of noradrenaline pressor responses produced by phentolamine (0.3 and 1.0 mg/kg, i.v.), yohimbine (0.3 and 1.0 mg/kg, i.v.) and phenoxybenzamine (10 mg/kg, i.v.), fig. 1, was quantitatively similar to their antagonism of tyramine pressor responses, fig. 2. Whilst DMI (0.1 and 0.3 mg/kg, i.v.) decreased tyramine responses, noradrenaline responses were potentiated (fig. 1 and 2). Mianserin (1
PHEN TOLAMINE
YOHIMBINE
and 3 mg/kg, i.v.) has a similar action to DMI on tyramine pressor responses, fig. 2, but its effects on noradrenaline pressor responses were qualitatively different. At 1 mg/kg i.v., mianserin had little effect on noradrenaline pressor responses, at 3 mg/kg, i.v. the slope of the noradrenaline dose--response curve was reduced by mianserin, fig. 1. In control experiments noradrenaline and tyramine responses were not significantly changed (P < 0.05). 3.2. Determination of the in vitro antagonist properties o f mianserin at pre- and postsynaptic a-adrenoceptors
Presynaptic a-adrenoceptor antagonist activity was assessed by studying the effects of increasing concentrations of mianserin on cumulative clonidine dose--response curves on
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Fig. 1. Dose--response curves for the increase in diastolic blood pressure p r o d u c e d by noradrenaline in the pithed rat. Controls (e); p h e n t o l a m i n e 0.3 (m) and 1.0 (A); y o h i m b i n e 0.3 ( I ) and 1.0 (A); p h e n o x y b e n z a m i n e 10 (m); DMI, 0.1 ( I ) and 0.3 (A) and mianserin 1.0 (B) and 3.0 (A); doses are all mg/kg, i.v. The results are the m e a n o f 5 animals -+ S.E.M. Asterisks indicate a m e a n response differing significantly (P < 0.05) f r o m initial values, using a t-test for paired data.
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J.C. D O X E Y ET AL. PHENTOLAMINE
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Fig. 2. Dose--response curves for the increase in diastolic b l o o d pressure p r o d u c e d by t y r a m i n e in the p i t h e d rat, controls (e); p h e n t o l a m i n e 0.3 (m) and 1.0 (A); y o h i m b i n e 0.3 (m) and 1.0 (A); p h e n o x y b e n z a m i n e 10 (m); DMI, 0.1 i.v. (m) and 0.3 (4) and mianserin 1.0 (m) and 3.0 (4); doses are all mg/kg, i.v. The results are the m e a n o f 5 animals _+S.E.M. Asterisks indicate a m e a n response differing significantly (P < 0.05) from initial values, using t-test for paired data.
the rat vas deferens, stimulated at 0.1 Hz. At the concentrations used mianserin competitively displaced the clonidine dose--response curve. The pA2 value of mianserin against clonidine was 7.3 + 0.1, table 1. Postsynaptic ~-adrenoceptor antagonist activity was assessed by comparing control cumulative noradrenaline dose--response curves with those in the presence of increasing
concentrations of mianserin on the rat anococcygeus muscle. Mianserin competitively displaced the noradrenaline dose--response curve, the pA2 value of mianserin against noradrenaline was 6.6 + 0.1, table 2. The pA2 values of mianserin against clonidine and noradrenaline were significantly different (P < 0.05). These results suggest that mianserin acts preferentially at presynaptic c~-adrenoceptors.
TABLE 1
TABLE 2
Drug antagonism at the presynaptic a - a d r e n o c e p t o r of the rat vas deferens.
Drug antagonism at the postsynaptic 0~-adrenoceptor o f the rat anococcygeus muscle.
The Krebs solution contained c o r t i c o s t e r o n e 40/~M, DMI 10 nM and propranolol 0.1 pM. The results are expressed as the m e a n + S.E.M.
The Krebs solution c o n t a i n e d corticosterone 40 pM, DMI 10 nM and propranolol 0.1 pM. The results are expressed as the m e a n -+ S.E.M.
Agonist
Antagonist
Drug parameter
n
Agonist
pD2 = 8.8 -+ 0.1 pA2 = 7.3 -+ 0.1
3 6
Noradrenaline
Mianserin
Clonidine
Antagonist
Drug parameter
n
Mianserin
PD2 = 6.7 + 0.2 PA2 = 6.6 -+ 0.1
4 8
MIANSERIN -- AN AUTONOMIC PROFILE
5
3.3. The effect o f mianserin on clonidine induced inhibition o f hypogastric nerve stimulation in the pithed rat
dine, fig. 3a. Phenoxybenzamine (10 mg/kg, i.v.) antagonised the pressor response produced by clonidine but had no effect on the inhibitory actions of clonidine on hypogastric nerve stimulation, fig. 3b. Neither the pressor effect of clonidine nor its action on hypogastric nerves was antagonised by DMI (0.1 and 0.3 mg/kg, i.v.), fig. 3c. Mianserin (1 mg/kg, i.v.) antagonised the pressor response produced by clonidine but even at 3 mg/kg, i.v., mianserin only partially reversed the inhibitory actions of clonidine on hypogastric nerves, fig. 3d. In a separate experiment both DMI (0.3 mg/ kg, i.v.) and mianserin (3 mg/kg, i.v.) inhibited
Selective stimulation of the hypogastric nerve of the pithed rat produced regular contractions of the vas deferens. Clonidine, 30 pg/kg, i.v., produced a long lasting (> 30 min) inhibition of hypogastric nerve stimulation and an associated, but shorter acting, pressor response. Phentolamine (0.3 mg/kg, i.v.) like yohimbine (0.3 mg/kg, i.v.) reversed both the pressor effect and the inhibition of hypogastric nerve stimulation produced by cloni-
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Fig. 3. T h e effects o f c l o n i d i n e ( 3 0 p g / k g , i.v.) o n b l o o d pressure a n d c o n t r a c t i o n s o f t h e vas d e f e r e n s a n d its m o d i f i c a t i o n b y (a) y o h i m b i n e , 0.3 a n d 1.0 m g / k g , i.v., (b) p h e n o x y b e n z a m i n e , 10 m g / k g , i.v., (e) DMI, 0.1 a n d 0.3 m g / k g , i.v. a n d (d) m i a n s e r i n 1 a n d 3 mg/kg, i.v. T h e h y p o g a s t r i c nerves o f p i t h e d rats w e r e s t i m u l a t e d at 20 V, 50/zsec, 6 Hz for 3 sec every 30 sec. U p p e r traces, b l o o d pressure; l o w e r traces, vas deferens.
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J.C. D O X E Y ET AL.
hypogastric nerve stimulation in the pithed rat. These effects were antagonised by yohimbine (0.3--1.0 mg/kg, i.v.), figs. 4a and 4b. 3.4. The effect o f mianserin on clonidine induced inhibition o f cardiac nerve stimulation in the pithed rat
Cardiac nerves can be stimulated selectively in pithed rats to produce constant positive chronotropic responses. Clonidine (30 pg/kg, i.v.) inhibited the cardiac acceleration produced by low frequency selective stimulation of cardiac nerves in the pithed rat. Phentolamine (0.3 mg/kg, i.v.) like yohimbine (0.3 mg/kg, i.v.) antagonised the inhibitory effects
of clonidine on cardiac nerve stimulation, fig. 5a. The inhibitory actions of clonidine on cardiac nerves were unaffected by phenoxybenzamine (10 mg/kg, i.v.), fig. 5b and DMI (0.1 mg/kg, i.v.), fig. 5c. In contrast to its actions on hypogastric nerves, mianserin (1 mg/kg, i.v.} potentiated the tachycardia produced by cardiac nerve stimulation, fig. 6. The inhibitory effects of clonidine on cardiac nerves were reversed by mianserin (1 mg/kg, i.v.), fig. 5d.
4. Discussion
The lack of activity of mianserin in conventional antidepressant screening tests (Van Riezen, 1972) prompted an investigation o f its peripheral autonomic actions in pithed rats. It has been suggested that mianserin possesses activity at several sites on the sympathetic neurone (Baumann and Maitre, 1977} and it was considered that these activities could act in opposition to one another and thus mask properties associated with antidepressant activity in animal tests. Assessment of uptake blocking properties in compounds which also possess ~-adrenoceptor antagonist properties was carried out in pithed rats. The effects of the compounds on pressor responses produced by i.v. injections of noradrenaline and tyramine were studied. Noradrenaline pressor responses are potentiated by the uptake blocking properties of a c o m p o u n d (Goodlet and Sugrue, 1974}, whereas ~-adrenoceptor antagonist properties would inhibit noradrenaline pressor responses. In contrast uptake blocking properties and ~-adrenoceptor antagonist properties would be, on the whole, additive in antagonising tyramine pressor responses. The pressor responses produced by noradrenaline and tyramine were equally susceptible to inhibition by the ~-adrenoceptor antagonists, phentolamine, yohimbine and phenoxybenzamine. DMI displayed a typical uptake blocking profile with little or no postsynaptic ~-adrenoceptor antagonist properties
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Fig. 4. T h e effects o f (a) m i a n s e r i n (3 mg/kg, i.v.) a n d (b) DMI (0.3 mg/kg, i.v.) o n b l o o d pressure a n d cont r a c t i o n s o f t h e vas d e f e r e n s a n d t h e i r reversal b y y o h i m b i n e (0.3 a n d 1.0 m g / k g , i.v.). T h e h y p o g a s t r i c n e r v e s o f p i t h e d rats were s t i m u l a t e d at 20 V, 50 psec, 6 Hz f o r 3 sec every 30 sec. U p p e r traces, b l o o d pressure; l o w e r traces, vas d e f e r e n s .
MIANSERIN -- AN AUTONOMIC PROFILE
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Fig. 5. The effects of clonidine (30 pg/kg, i.v.) on blood pressure and cardiac acceleration and its modification by (a) yohimbine, 0.3 mg/kg, i.v., (b) phenoxybenzamine, 10 mg/kg, i.v., (c) DMI, 0.1 mg/kg, i.v. and (d) mianserin 1 mg/kg, i.v. The car diac, i.v. and (d) mianserin 1 mg/kg, i.v. The cardiac nerves of pithed rats were stimulated at 10 V, 0.5 msec, 1 Hz for 10 sec every 2 min (A). Upper traces, blood pressure; lower traces, heart rate. in t h a t n o r a d r e n a l i n e pressor responses were p o t e n t i a t e d and t y r a m i n e pressor responses antagonised. At l o w doses (1 mg/kg, i.v.), mianserin m a r k e d l y antagonised t y r a m i n e pressor responses b u t had little or n o e f f e c t o n n o r a d r e n a l i n e pressor responses. At 3 mg/ kg, i.v. mianserin r e d u c e d t h e slope o f t h e n o r a d r e n a l i n e r e s p o n s e line and t y r a m i n e pressor responses were f u r t h e r r e d u c e d . It w o u l d a p p e a r t h e r e f o r e t h a t mianserin possessed b o t h u p t a k e b l o c k i n g p r o p e r t i e s and p o s t s y n a p t i c a - a d r e n o c e p t o r antagonist properties at t h e doses studied.
T h e r e is considerable evidence t o s u p p o r t the concept that sympathetic presynaptic nerve terminals possess a - a d r e n o c e p t o r s . Activation o f these p r e s y n a p t i c a - a d r e n o c e p t o r s inhibits, and their b l o c k a d e p o t e n t i a t e s , the release o f n o r a d r e n a l i n e b y nerve impulses (Langer, 1 9 7 4 ) . T h e r e is also evidence t h a t p r e s y n a p t i c and p o s t s y n a p t i c a - a d r e n o c e p t o r s d i f f e r in t h e i r sensitivity t o b o t h agonists (Starke, 1 9 7 2 ; Starke e t al., 1 9 7 4 ; Starke et al., 1 9 7 5 b ) , and antagonists ( T h o e n e n et al., 1964; D u b o c o v i c h and Langer, 1 9 7 4 ; S t a r k e et al., 1 9 7 5 a ; D o x e y e t al., 1977).
8
J.C. D O X E Y ET AL.
i mmHg 200 0 j 500 ~
beats/min {h
o
AAianserin 1mg/kgj[.v. Fig. 6. The effect of mianserin(1 mg/kg i.v.)
on
cardiac acceleration in the pithed rat. Cardiac nerves were s t i m u l a t e d a t 10 V, 0.5 msec, 1 Hz for 10 sec every 2 m i n (A). U p p e r trace, b l o o d pressure; l o w e r trace, heart rate.
In isolated tissue experiments where the relative potency of mianserin at presynaptic a-adrenoceptors was studied, mianserin competitively antagonised both the presynaptic actions of clonidine and the postsynaptic actions of noradrenaline. The pA2 value for mianserin against clonidine (7.3 + 0.1) at the presynaptic a-adrenoceptor was significantly (P < 0.05) greater than its value against noradrenaline (6.6 + 0.1) at the postsynaptic aadrenoceptor. Under the same experimental conditions, phentolamine (pA2 vs. clonidine 8.38 + 0.1 and pA2 vs. noradrenaline 7.7. + 0.17) had a pre- to postsynaptic ratio very similar to that of mianserin, yohimbine (pA2 vs. clonidine 8.18+ 0.1 and pA2 vs. noradrenaline 6.4-+ 0.2) was more selective for presynaptic a-adrenoceptors than either phentolamine or mianserin and phenoxybenzamine (pA2 vs. c l o n i d i n e < 7.54, pA2 vs. noradrenaline 8.3 + 0.1) acted preferentially at postsynaptic a-adrenoceptors (Doxey et al., 1977). The activity of DMI at pre- and postsynaptic a-adrenoceptors was not studied since this compound, together with propranolol and corticosterone, was already present
in the physiological solution. The a-adrenoceptor antagonist properties of mianserin were investigated further in pithed rats. Sympathetic outflow to either the heart or the vas deferens was stimulated via the cardiac and hypogastric nerves respectively. The increase in heart rate or vas deferens tension produced by electrical stimulation was inhibited by the presynaptic agonist actions of clonidine. Clonidine also produced a pressor response in pithed rats, an action attributable to its postsynaptic a-adrenoceptor agonist action. Previous studies (Doxey and Everitt, 1977) have shown that phentolamine antagonised the presynaptic action of clonidine on cardiac and hypogastric nerves, and its postsynaptic action on blood vessels. In these studies both yohimbine and phentolamine antagonised the pressor response produced by clonidine and reversed the inhibitory effects of clonidine on cardiac and hypogastric nerves. It was clear therefore that both these compounds possessed pre- and postsynaptic a-adrenoceptor antagonist properties. Phenoxybenzamine reduced the pressor response produced by clonidine but had no effect on the presynaptic actions of clonidine on either cardiac or hypogastric nerves. In this experimental model therefore phenoxybenzamine was devoid of presynaptic activity. Desmethylimipramine did not antagonise either the pre- or postsynaptic actions of clonidine. At low doses mianserin reduced the pressor response produced by clonidine and completely reversed the inhibitory actions of clonidine on cardiac nerves. In contrast the inhibitory effect of clonidine on the hypogastric nerves was only slightly antagonised by mianserin. It would have been predicted from in vitro studies that mianserin and phentolamine would have had similar properties and that mianserin would have readily reversed the presynaptic effects of clonidine on hypogastric nerves. As discussed previously mianserin also possesses uptake blocking properties and it was considered that these could have been involved in the actions of mianserin on the hypogastric nerves of the pithed rat.
MIANSERIN -- AN AUTONOMIC PROFILE
In experiments where the effects of either mianserin or DMI were studied on hypogastric nerve stimulation both caused inhibition of the vas deferens twitch although the inhibition was less with mianserin. The inhibition produced was reversed by yohimbine. It has been shown previously (Marshall et al., 1977) that blockade of uptake in the vas deferens inhibits the effect of sympathetic stimulation. This action has been attributed to increases in endogenous noradrenaline which activate presynaptic ~-adrenoceptors and this inhibits further transmitter release. Thus in hypogastric nerves the effects of mianserin on uptake and presynaptic ~-adrenoceptors act in opposition to one another and as a consequence the presynaptic actions of clonidine were not antagonised. In view of the results with mianserin, yohimbine and phentolamine it appears that selective presynaptic ~-adrenoceptor antagonists will only reverse the presynaptic actions of clonidine on the hypogastric nerves of pithed rats if they lack strong uptake blocking properties. There is conflicting evidence relating to the role of noradrenaline in m o t o r transmission in the vas deferens and this has led to the formulation of several hypotheses to explain pharmacologicai observations (Swedin, 1971; Ambache and A b o o Zar, 1971; Furness, 1974; Jones and Spriggs, 1975; Jenkins et al., 1976). The atypical transmission was exemplified by the inhibition of hypogastric nerve stimulation produced b y DMI in the pithed rat in these studies and b y cocaine in earlier in vitro studies using the mouse vas deferens (Marshall et al., 1977). In the in vitro experiments carried out in these and previous studies (Doxey et al., 1977) the Krebs solution was renewed continuously thus preventing the accumulation of noradrenaline in the bathing fluid. Under these conditions very high concentrations of DMI (10 -s M) were required to inhibit the twitch response of the rat vas deferens (Doxey and Easingwood, unpublished observations). In contrast to their effects on hypogastric nerves, mianserin and DMI (Doxey, 1977) potentiate the effects of cardiac nerve
9
stimulation in the pithed rat. The failure of DMI to reverse either the preor postsynaptic agonist actions of clonidine is in contrast to other studies where similar doses of DMI injected into the vertebral artery of anaesthetised cats antagonised the central hypotensive effects of clonidine (Van Spanning and Van Zwieten, 1973; Van Zwieten, 1976}. These workers concluded however that this was the result of a competitive interaction at an ~-adrenoceptor within the central nervous system and that it was unlikely that the uptake blocking properties of DMI were responsible, since cocaine did not diminish the effect of clonidine. In conclusion, the present experiments have shown that, in the periphery, doses of mianserin which inhibit the re-uptake of noradrenaline into nerve endings also exert antagonist activity at both pre- and postsynaptic ~-adrenoceptors. Although mianserin was less potent than phentolamine the pre- to postsynaptic ratio of these two c o m p o u n d s was very similar. Although it is difficult to extrapolate peripheral observations to the central nervous system such profiling could prove tiseful in investigating new antidepressant molecules which are not detected by conventional animal tests. References Ambache, N. and M. Aboo Zar, 1971, Evidence against adrenergic motor transmission in the guinea-pig vas deferens, J. Physiol. London 216, 359. Armstrong, J.M. and A.L.A. Boura, 1973, Effects of clonidine and guanethidine on peripheral sympathetic nerve function in the pithed rat, Brit. J. Pharmacol. 4 7 , 8 5 0 . Baumann, P.A. and L. Maitre, 1977, Blockade of the presynaptic s-receptor a n d of amine uptake in the rat brain by the antidepressant mianserine, Naunyn-Schmiedeb. Arch. Pharmacol. 300, 31. Doxey, J.C., 1977, Effect of clonidine on cardiac accerelation in pithed rats, J. Pharm. Pharmacol. 29, 173. Doxey, J.C. and J. Everitt, 1977, Inhibitory effects of clonidine on responses to s y m p a t h e t i c nerve stimulation in the pithed rat, Brit. J. Pharmacol. 61,559.
10 Doxey, J.C., C.F.C. Smith and J.M. Walker, 1977, Selectivity of blocking agents for pre- and postsynaptic a-adrenoceptors, Brit. J. Pharmacol. 60, 91. Drew, G.M., 1976, Effects of a-adrenoceptor agonists a a d antagonists on pre- and postsynaptically located 0~-adrenoceptors, European J. Pharmacol. 36, 313. Dubocovich, M.L. and S.Z. Langer, 1974, Negative feedback regulation of noradrenaline release by nerve stimulation in the perfused cats spleen: differences in potency of phenoxybenzamine in blocking the pre- and postsynaptic adrenergic receptors, J. Physiol. (London) 237, 505. Furness, J.B., 1974, Transmission on the longitudinal muscle of the guinea pig vas deferens: the effect of pretreatment with guanethidine, Brit. J. Pharmacol. 50, 63. Ghose, K., A. Coppen and P. Turner, 1976, Autonomic actions and interactions of mianserin hydrochloride (Org. GB 94) and amitriptyline in patients with depressive illness, Psychopharmacol. 49, 201. Gillespie, J.S., A. Maclaren and D. Pollock, 1970, A method of stimulating different segments of the autonomic outflow from the spinal column to various organs in the pithed cat and rat, Brit. J. Pharmacol. 40, 257. Goodlet, L. and M.F. Sugrue, 1974, The effect of a new antidepressant, Org. GB 94 on amine uptake mechanisms, Brit. J. Pharmacol. 52, 431. Jenkins, D.A., I. Marshall and P.A. Nasmyth, 1976, Is noradrenaline the m o t o r transmitter in the mouse vas deferens?, J. Physiol. (London) 254, 49P. Jones, M.E.L. and T.L.B. Springs, 1975, Noradrenaline and m o t o r transmission in the vas deferens of the mouse, Brit. J. Pharmacol. 53,323. Langer, S.Z., 1974, Presynaptic regulation of catecholamine release, Biochem. Pharmacol. 23, 1793. Marshall, I., P.A. Nasmyth and N.B. Shepperson, 1977, Pre-synaptic ~-adrenoceptors and the
J.C. DOXEY ET AL. inhibition by uptake blocking agents of the twitch response of the mouse vas deferens, Brit. J. Pharmacol. 59, 511P. Starke, K., 1972, 0~-Sympathomimetic inhibition of adrenergic and cholinergic traaaraission in the rabbit heart, Naunyn-Schmiedeb. Arch. Pharmacol. 274, 18. Starke, K., E. Borowski and T. Endo, 1975a, Preferential blockade of presynaptic ~-adrenoceptors by yohimbine, European J. Pharlnacol. 34,385. Starke, K., T. Endo and H.D. Taube, 1975b, Pre- and postsynaptic components of effect of drugs with aadrenoceptor affinity, Nature, London 254,440. Starke, K., H. Montel, N. Gayk and R. Merker, 1974, Comparison of the effects of clonidine on pre- and postsynaptic adrenoceptors in the rabbit pulmonary artery, Arch. Pharmacol. 285, 133. Swedin, G., 1971, Studies on neurotransmission mechanisms in the rat and guinea pig vas deferens, Acta Physiol. Scand. 83, Suppl. 369, 1. Thoenen, H., A. Hurlimann and W. Haefely, 1964, Dual site of action of phenoxybenzamine in the cats spleen, Experientia 20, 272. Van der Burg, B.J., I.L. Bonta, J. Debobelle, C. Ramon and B. Vargaftig, 1970, A novel type of substituted piperazine with high antiserotonin potency, J. Med. Chem. 1, 35. Van Riezen, H., 1972, Different central effects of the 5HT antagonists mianserine and cyproheptadine, Arch. Intern. Pharmacodyn. Therap. 198, 256. Van Spanning, H.W. and P.A. Van Zwieten, 1973, The interference of tricyclic antidepressants with the central hypotensive effect of clonidine, European J. Pharmacol. 24,402. Van Zwieten, P.A. 1975, Interaction between centrally acting hypotensive drugs and tricylcic antidepressants, Arch. Intern. Pharmacodyn. 214, 12. Van Zwieten, P.A., 1976, The reversal of clonidineinduced hypotension by protriptyline and desipramine, Pharmacol. 14,227.
1
MIANSERIN -- AN ANALYSIS OF ITS P E R I P H E R A L AUTONOMIC ACTIONS JOHN C. DOXEY, JOHN EVERITT and GEOFFREY METCALF
R e c k i t t and Colman, Pharmaceutical Division, Dansom Lane, Hull, HU8 7DS, England Received 10 November 1977, revised MS received 12 April 1978, accepted 16 May 1978
J.C. DOXEY, J. EVERITT and G. METCALF, Mianserin -- an analysis o f its peripheral autonomic actions, European J. Pharmacol. 51 (1978) 1--10. The autonomic profile of mianserin has been compared with that of yohimbine, phentolamine, phenoxybenzamine and desmethylimipramine. The effects of mianserin on tyramine and noradrenaline pressor responses in the pithed rat were consistent with c~-adrenoceptor antagonist and uptake blocking properties. In isolated tissue experiments, the selectivity of mianserin for pre- and postsynaptic ~-adrenoceptors was similar to that of phentolamine. In pithed rats mianserin antagonised the pressor response produced by clonidine and reversed the inhibitory actions of clonidine on cardiac nerve stimulation. In contrast mianserin only caused slight reversal of the inhibitory effects of clonidine on hypogastric nerve stimulation. Uptake blockade itself inhibits hypogastric nerve stimulation and this could counteract any antagonism of the effects of clonidine at the presynaptic ~-adrenoceptor. The results demonstrate the uptake blocking properties of mianserin and its antagonism at both pre- and postsynaptic ~-adrenoceptors. Mianserin
Presynaptic ~-adrenoceptors
Uptake blockade
1. Introduction Mianserin hydrochloride (ORG. GB 94) was recently introduced as an antidepressant agent (Van der Burg et al., 1970) and was found to have an efficacy similarto that of amitriptyline (Ghose et al., 1976). It has been reported (Goodlet and Sugrue, 1974) that mianserin, like desmethylimipramine (DMI), blocks central catecholaminergic uptake systems. These workers also reported that mianserin and DMI antagonise tyramine and potentiate noradrenaline pressor responses in the pithed rat. In contrast mianserin had no significant effect on tyramine pressor responses in man (Ghose et al., 1976). Despite their similarities on monoamine uptake processes, mianserin, unlike DMI, was essentially devoid of effect in conventional antidepressant screening tests (Goodlet and Sugrue, 1974}. Van Zwieten (1975) reported that mianserin was a p o t e n t a-adrenoceptor antagonist in the
Postsynaptic ~-adrenoceptors
periphery and Baumann and Maitre (1977) concluded that a presynaptic a-adrenoceptor antagonist c o m p o n e n t was included in the action of mianserin. In this study the profile of mianserin has been compared with those of yohimbine, phentolamine, phenoxybenzamine and DMI.
2. Materials and methods 2.1. P i t h e d rats
Male rats in the b o d y weight range 200-300 g were pithed during a brief period of halothane anaesthesia. The animals were subsequently artificially respired with r o o m air (100 strokes/min, 1 ml/100 g}. Blood pressure was recorded with a Hewlett Packard 1280c pressure transducer from the right c o m m o n carotid artery. Heart rate was recorded using the arterial pressure wave to trigger a ratemeter.
2
J.C. DOXEY ET AL.
In experiments where contractions of the vas deferens were monitored an isometric transducer (Statham Gold Cell, model uC3) was employed. All parameters were displayed on a Hewlett Packard 7700 recorder. The right femoral vein was cannulated to facilitate administration of drugs. In experiments where sympathetic outflow was stimulated, tubocurarine (1 mg/kg, i.v.) was injected before stimulation commenced.
and 300 pg/kg, i.v.) or noradrenaline (0.1, 0.3 and 1.0 pg/kg, i.v.) was constructed in pithed rats prior to injection of the drug under examination. The test c o m p o u n d was then injected i.v. and the dose response to the pressor agent re-examined in its presence i.e. each animal acted as its own control. Saline (0.9% w/v NaC1 solution) was injected into further animals for control studies.
2.2. Assessment o f presynap tic a-adrenoceptor antagonist activity in pithed rats
2.4. Assessment o f pre- and postsynaptic aadrenoceptor antagonist activity in isolated tissue experiments
Selective stimulation of cardiac or hypogastric nerves in the pithed rat (Gillespie et al., 1970) leads to corresponding increases in heart rate and vas deferens tension with little effect on sympathetic discharge to other organs. The presynaptic a-adrenoceptor agonist actions of clonidine inhibit the effects of electrical stimulation on both cardiac nerves (Armstrong and Boura, 1973; Drew, 1976) and hypogastric nerves (Doxey and Everitt, 1977). Clonidine also produces a pressor response in pithed rats; an action attributable to its postsynaptic a-adrenoceptor agonist action. The ability of mianserin to reverse the presynaptic actions of clonidine (30 pg/kg, i.v.) was compared with that of yohimbine, phentolamine, phenoxybenzamine and DMI following i.v. injection Ca minimum of 3 rats was used for each drug studied and the figures represent typical experimental results. The stimulus parameters for stimulation of cardiac nerves and hypogastric nerves were 10 V, 0.5 msec, 1 Hz for 10 sec every 2 min and 20 V, 50 psec, 6 Hz for 3 sec every 30 sec respectively. 2.3. Tyramine and noradrenaline responses in the pithed rat
pressor
The i.v. effects of mianserin on noradrenaline and tyramine pressor responses were studied in pithed rats. Each pressor agent was studied in a different group of 5 rats. A dose response curve to either tyramine (30, 100
The pre- and postsynpatic a-adrenoceptor antagonist activity of mianserin was studied using an identical procedure to that used by Doxey et al. (1977). Presynaptic a-adrenoceptor antagonist activity was assessed by studying the effects of increasing concentrations of mianserin on cumulative clonidine dose--response curves on the rat vas deferens stimulated at 0.1 Hz. Postsynaptic a-adrenoceptor antagonist activity was assessed by comparison of control cumulative noradrenaline dose--response curves with those in the presence of increasing concentrations of mianserin using the rat anococcygeus muscle. Corticosterone 40 pM, DMI 10 nM and propranolol 0.1 #M were present in the Krebs solution throughout the experiments to block extraneuronal and neuronal uptake and fl-adrenoceptors. 2.5. Drugs used The following drugs were used in this study: clonidine hydrochloride (Boehringer Ingelheim); corticosterone (Sigma); desmethylimipramine hydrochloride (Geigy); mianserin hydrochloride (Organon); noradrenaline bitartrate (Koch Light Laboratories Ltd); phenoxybenzamine hydrochloride (SKF); phentolamine mesylate (CIBA); (+)-propranolol hydrochloride (ICI); tubocurarine chloride (Burroughs Wellcome);tyramine hydrochloride (Koch Light Laboratories Ltd), and yohimbine hydrochloride (Sigma). All drugs, with the
M I A N S E R I N -- A N A U T O N O M I C P R O F I L E
3
exception of phenoxybenzamine which was made up at pH 3, were dissolved in saline and doses mentioned in the text are in terms of their respective salts.
3. Results 3.1. Effect o f mianserin on noradrenaline and tyramine pressor responses in the pithed rat
The antagonism of noradrenaline pressor responses produced by phentolamine (0.3 and 1.0 mg/kg, i.v.), yohimbine (0.3 and 1.0 mg/kg, i.v.) and phenoxybenzamine (10 mg/kg, i.v.), fig. 1, was quantitatively similar to their antagonism of tyramine pressor responses, fig. 2. Whilst DMI (0.1 and 0.3 mg/kg, i.v.) decreased tyramine responses, noradrenaline responses were potentiated (fig. 1 and 2). Mianserin (1
PHEN TOLAMINE
YOHIMBINE
and 3 mg/kg, i.v.) has a similar action to DMI on tyramine pressor responses, fig. 2, but its effects on noradrenaline pressor responses were qualitatively different. At 1 mg/kg i.v., mianserin had little effect on noradrenaline pressor responses, at 3 mg/kg, i.v. the slope of the noradrenaline dose--response curve was reduced by mianserin, fig. 1. In control experiments noradrenaline and tyramine responses were not significantly changed (P < 0.05). 3.2. Determination of the in vitro antagonist properties o f mianserin at pre- and postsynaptic a-adrenoceptors
Presynaptic a-adrenoceptor antagonist activity was assessed by studying the effects of increasing concentrations of mianserin on cumulative clonidine dose--response curves on
PHENOXYBE NZAMINE
DMI
MIANSERIN
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Fig. 1. Dose--response curves for the increase in diastolic blood pressure p r o d u c e d by noradrenaline in the pithed rat. Controls (e); p h e n t o l a m i n e 0.3 (m) and 1.0 (A); y o h i m b i n e 0.3 ( I ) and 1.0 (A); p h e n o x y b e n z a m i n e 10 (m); DMI, 0.1 ( I ) and 0.3 (A) and mianserin 1.0 (B) and 3.0 (A); doses are all mg/kg, i.v. The results are the m e a n o f 5 animals -+ S.E.M. Asterisks indicate a m e a n response differing significantly (P < 0.05) f r o m initial values, using a t-test for paired data.
4
J.C. D O X E Y ET AL. PHENTOLAMINE
Y©HIMBINE
PHE NOXYBENZAMINE
DMI
MIAN~ERIN
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Fig. 2. Dose--response curves for the increase in diastolic b l o o d pressure p r o d u c e d by t y r a m i n e in the p i t h e d rat, controls (e); p h e n t o l a m i n e 0.3 (m) and 1.0 (A); y o h i m b i n e 0.3 (m) and 1.0 (A); p h e n o x y b e n z a m i n e 10 (m); DMI, 0.1 i.v. (m) and 0.3 (4) and mianserin 1.0 (m) and 3.0 (4); doses are all mg/kg, i.v. The results are the m e a n o f 5 animals _+S.E.M. Asterisks indicate a m e a n response differing significantly (P < 0.05) from initial values, using t-test for paired data.
the rat vas deferens, stimulated at 0.1 Hz. At the concentrations used mianserin competitively displaced the clonidine dose--response curve. The pA2 value of mianserin against clonidine was 7.3 + 0.1, table 1. Postsynaptic ~-adrenoceptor antagonist activity was assessed by comparing control cumulative noradrenaline dose--response curves with those in the presence of increasing
concentrations of mianserin on the rat anococcygeus muscle. Mianserin competitively displaced the noradrenaline dose--response curve, the pA2 value of mianserin against noradrenaline was 6.6 + 0.1, table 2. The pA2 values of mianserin against clonidine and noradrenaline were significantly different (P < 0.05). These results suggest that mianserin acts preferentially at presynaptic c~-adrenoceptors.
TABLE 1
TABLE 2
Drug antagonism at the presynaptic a - a d r e n o c e p t o r of the rat vas deferens.
Drug antagonism at the postsynaptic 0~-adrenoceptor o f the rat anococcygeus muscle.
The Krebs solution contained c o r t i c o s t e r o n e 40/~M, DMI 10 nM and propranolol 0.1 pM. The results are expressed as the m e a n + S.E.M.
The Krebs solution c o n t a i n e d corticosterone 40 pM, DMI 10 nM and propranolol 0.1 pM. The results are expressed as the m e a n -+ S.E.M.
Agonist
Antagonist
Drug parameter
n
Agonist
pD2 = 8.8 -+ 0.1 pA2 = 7.3 -+ 0.1
3 6
Noradrenaline
Mianserin
Clonidine
Antagonist
Drug parameter
n
Mianserin
PD2 = 6.7 + 0.2 PA2 = 6.6 -+ 0.1
4 8
MIANSERIN -- AN AUTONOMIC PROFILE
5
3.3. The effect o f mianserin on clonidine induced inhibition o f hypogastric nerve stimulation in the pithed rat
dine, fig. 3a. Phenoxybenzamine (10 mg/kg, i.v.) antagonised the pressor response produced by clonidine but had no effect on the inhibitory actions of clonidine on hypogastric nerve stimulation, fig. 3b. Neither the pressor effect of clonidine nor its action on hypogastric nerves was antagonised by DMI (0.1 and 0.3 mg/kg, i.v.), fig. 3c. Mianserin (1 mg/kg, i.v.) antagonised the pressor response produced by clonidine but even at 3 mg/kg, i.v., mianserin only partially reversed the inhibitory actions of clonidine on hypogastric nerves, fig. 3d. In a separate experiment both DMI (0.3 mg/ kg, i.v.) and mianserin (3 mg/kg, i.v.) inhibited
Selective stimulation of the hypogastric nerve of the pithed rat produced regular contractions of the vas deferens. Clonidine, 30 pg/kg, i.v., produced a long lasting (> 30 min) inhibition of hypogastric nerve stimulation and an associated, but shorter acting, pressor response. Phentolamine (0.3 mg/kg, i.v.) like yohimbine (0.3 mg/kg, i.v.) reversed both the pressor effect and the inhibition of hypogastric nerve stimulation produced by cloni-
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Fig. 3. T h e effects o f c l o n i d i n e ( 3 0 p g / k g , i.v.) o n b l o o d pressure a n d c o n t r a c t i o n s o f t h e vas d e f e r e n s a n d its m o d i f i c a t i o n b y (a) y o h i m b i n e , 0.3 a n d 1.0 m g / k g , i.v., (b) p h e n o x y b e n z a m i n e , 10 m g / k g , i.v., (e) DMI, 0.1 a n d 0.3 m g / k g , i.v. a n d (d) m i a n s e r i n 1 a n d 3 mg/kg, i.v. T h e h y p o g a s t r i c nerves o f p i t h e d rats w e r e s t i m u l a t e d at 20 V, 50/zsec, 6 Hz for 3 sec every 30 sec. U p p e r traces, b l o o d pressure; l o w e r traces, vas deferens.
6
J.C. D O X E Y ET AL.
hypogastric nerve stimulation in the pithed rat. These effects were antagonised by yohimbine (0.3--1.0 mg/kg, i.v.), figs. 4a and 4b. 3.4. The effect o f mianserin on clonidine induced inhibition o f cardiac nerve stimulation in the pithed rat
Cardiac nerves can be stimulated selectively in pithed rats to produce constant positive chronotropic responses. Clonidine (30 pg/kg, i.v.) inhibited the cardiac acceleration produced by low frequency selective stimulation of cardiac nerves in the pithed rat. Phentolamine (0.3 mg/kg, i.v.) like yohimbine (0.3 mg/kg, i.v.) antagonised the inhibitory effects
of clonidine on cardiac nerve stimulation, fig. 5a. The inhibitory actions of clonidine on cardiac nerves were unaffected by phenoxybenzamine (10 mg/kg, i.v.), fig. 5b and DMI (0.1 mg/kg, i.v.), fig. 5c. In contrast to its actions on hypogastric nerves, mianserin (1 mg/kg, i.v.} potentiated the tachycardia produced by cardiac nerve stimulation, fig. 6. The inhibitory effects of clonidine on cardiac nerves were reversed by mianserin (1 mg/kg, i.v.), fig. 5d.
4. Discussion
The lack of activity of mianserin in conventional antidepressant screening tests (Van Riezen, 1972) prompted an investigation o f its peripheral autonomic actions in pithed rats. It has been suggested that mianserin possesses activity at several sites on the sympathetic neurone (Baumann and Maitre, 1977} and it was considered that these activities could act in opposition to one another and thus mask properties associated with antidepressant activity in animal tests. Assessment of uptake blocking properties in compounds which also possess ~-adrenoceptor antagonist properties was carried out in pithed rats. The effects of the compounds on pressor responses produced by i.v. injections of noradrenaline and tyramine were studied. Noradrenaline pressor responses are potentiated by the uptake blocking properties of a c o m p o u n d (Goodlet and Sugrue, 1974}, whereas ~-adrenoceptor antagonist properties would inhibit noradrenaline pressor responses. In contrast uptake blocking properties and ~-adrenoceptor antagonist properties would be, on the whole, additive in antagonising tyramine pressor responses. The pressor responses produced by noradrenaline and tyramine were equally susceptible to inhibition by the ~-adrenoceptor antagonists, phentolamine, yohimbine and phenoxybenzamine. DMI displayed a typical uptake blocking profile with little or no postsynaptic ~-adrenoceptor antagonist properties
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Yohimbine 0.3 mg/kg, i.v,
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Fig. 4. T h e effects o f (a) m i a n s e r i n (3 mg/kg, i.v.) a n d (b) DMI (0.3 mg/kg, i.v.) o n b l o o d pressure a n d cont r a c t i o n s o f t h e vas d e f e r e n s a n d t h e i r reversal b y y o h i m b i n e (0.3 a n d 1.0 m g / k g , i.v.). T h e h y p o g a s t r i c n e r v e s o f p i t h e d rats were s t i m u l a t e d at 20 V, 50 psec, 6 Hz f o r 3 sec every 30 sec. U p p e r traces, b l o o d pressure; l o w e r traces, vas d e f e r e n s .
MIANSERIN -- AN AUTONOMIC PROFILE
7 200
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mmHg
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• Jo
Fig. 5. The effects of clonidine (30 pg/kg, i.v.) on blood pressure and cardiac acceleration and its modification by (a) yohimbine, 0.3 mg/kg, i.v., (b) phenoxybenzamine, 10 mg/kg, i.v., (c) DMI, 0.1 mg/kg, i.v. and (d) mianserin 1 mg/kg, i.v. The car diac, i.v. and (d) mianserin 1 mg/kg, i.v. The cardiac nerves of pithed rats were stimulated at 10 V, 0.5 msec, 1 Hz for 10 sec every 2 min (A). Upper traces, blood pressure; lower traces, heart rate. in t h a t n o r a d r e n a l i n e pressor responses were p o t e n t i a t e d and t y r a m i n e pressor responses antagonised. At l o w doses (1 mg/kg, i.v.), mianserin m a r k e d l y antagonised t y r a m i n e pressor responses b u t had little or n o e f f e c t o n n o r a d r e n a l i n e pressor responses. At 3 mg/ kg, i.v. mianserin r e d u c e d t h e slope o f t h e n o r a d r e n a l i n e r e s p o n s e line and t y r a m i n e pressor responses were f u r t h e r r e d u c e d . It w o u l d a p p e a r t h e r e f o r e t h a t mianserin possessed b o t h u p t a k e b l o c k i n g p r o p e r t i e s and p o s t s y n a p t i c a - a d r e n o c e p t o r antagonist properties at t h e doses studied.
T h e r e is considerable evidence t o s u p p o r t the concept that sympathetic presynaptic nerve terminals possess a - a d r e n o c e p t o r s . Activation o f these p r e s y n a p t i c a - a d r e n o c e p t o r s inhibits, and their b l o c k a d e p o t e n t i a t e s , the release o f n o r a d r e n a l i n e b y nerve impulses (Langer, 1 9 7 4 ) . T h e r e is also evidence t h a t p r e s y n a p t i c and p o s t s y n a p t i c a - a d r e n o c e p t o r s d i f f e r in t h e i r sensitivity t o b o t h agonists (Starke, 1 9 7 2 ; Starke e t al., 1 9 7 4 ; Starke et al., 1 9 7 5 b ) , and antagonists ( T h o e n e n et al., 1964; D u b o c o v i c h and Langer, 1 9 7 4 ; S t a r k e et al., 1 9 7 5 a ; D o x e y e t al., 1977).
8
J.C. D O X E Y ET AL.
i mmHg 200 0 j 500 ~
beats/min {h
o
AAianserin 1mg/kgj[.v. Fig. 6. The effect of mianserin(1 mg/kg i.v.)
on
cardiac acceleration in the pithed rat. Cardiac nerves were s t i m u l a t e d a t 10 V, 0.5 msec, 1 Hz for 10 sec every 2 m i n (A). U p p e r trace, b l o o d pressure; l o w e r trace, heart rate.
In isolated tissue experiments where the relative potency of mianserin at presynaptic a-adrenoceptors was studied, mianserin competitively antagonised both the presynaptic actions of clonidine and the postsynaptic actions of noradrenaline. The pA2 value for mianserin against clonidine (7.3 + 0.1) at the presynaptic a-adrenoceptor was significantly (P < 0.05) greater than its value against noradrenaline (6.6 + 0.1) at the postsynaptic aadrenoceptor. Under the same experimental conditions, phentolamine (pA2 vs. clonidine 8.38 + 0.1 and pA2 vs. noradrenaline 7.7. + 0.17) had a pre- to postsynaptic ratio very similar to that of mianserin, yohimbine (pA2 vs. clonidine 8.18+ 0.1 and pA2 vs. noradrenaline 6.4-+ 0.2) was more selective for presynaptic a-adrenoceptors than either phentolamine or mianserin and phenoxybenzamine (pA2 vs. c l o n i d i n e < 7.54, pA2 vs. noradrenaline 8.3 + 0.1) acted preferentially at postsynaptic a-adrenoceptors (Doxey et al., 1977). The activity of DMI at pre- and postsynaptic a-adrenoceptors was not studied since this compound, together with propranolol and corticosterone, was already present
in the physiological solution. The a-adrenoceptor antagonist properties of mianserin were investigated further in pithed rats. Sympathetic outflow to either the heart or the vas deferens was stimulated via the cardiac and hypogastric nerves respectively. The increase in heart rate or vas deferens tension produced by electrical stimulation was inhibited by the presynaptic agonist actions of clonidine. Clonidine also produced a pressor response in pithed rats, an action attributable to its postsynaptic a-adrenoceptor agonist action. Previous studies (Doxey and Everitt, 1977) have shown that phentolamine antagonised the presynaptic action of clonidine on cardiac and hypogastric nerves, and its postsynaptic action on blood vessels. In these studies both yohimbine and phentolamine antagonised the pressor response produced by clonidine and reversed the inhibitory effects of clonidine on cardiac and hypogastric nerves. It was clear therefore that both these compounds possessed pre- and postsynaptic a-adrenoceptor antagonist properties. Phenoxybenzamine reduced the pressor response produced by clonidine but had no effect on the presynaptic actions of clonidine on either cardiac or hypogastric nerves. In this experimental model therefore phenoxybenzamine was devoid of presynaptic activity. Desmethylimipramine did not antagonise either the pre- or postsynaptic actions of clonidine. At low doses mianserin reduced the pressor response produced by clonidine and completely reversed the inhibitory actions of clonidine on cardiac nerves. In contrast the inhibitory effect of clonidine on the hypogastric nerves was only slightly antagonised by mianserin. It would have been predicted from in vitro studies that mianserin and phentolamine would have had similar properties and that mianserin would have readily reversed the presynaptic effects of clonidine on hypogastric nerves. As discussed previously mianserin also possesses uptake blocking properties and it was considered that these could have been involved in the actions of mianserin on the hypogastric nerves of the pithed rat.
MIANSERIN -- AN AUTONOMIC PROFILE
In experiments where the effects of either mianserin or DMI were studied on hypogastric nerve stimulation both caused inhibition of the vas deferens twitch although the inhibition was less with mianserin. The inhibition produced was reversed by yohimbine. It has been shown previously (Marshall et al., 1977) that blockade of uptake in the vas deferens inhibits the effect of sympathetic stimulation. This action has been attributed to increases in endogenous noradrenaline which activate presynaptic ~-adrenoceptors and this inhibits further transmitter release. Thus in hypogastric nerves the effects of mianserin on uptake and presynaptic ~-adrenoceptors act in opposition to one another and as a consequence the presynaptic actions of clonidine were not antagonised. In view of the results with mianserin, yohimbine and phentolamine it appears that selective presynaptic ~-adrenoceptor antagonists will only reverse the presynaptic actions of clonidine on the hypogastric nerves of pithed rats if they lack strong uptake blocking properties. There is conflicting evidence relating to the role of noradrenaline in m o t o r transmission in the vas deferens and this has led to the formulation of several hypotheses to explain pharmacologicai observations (Swedin, 1971; Ambache and A b o o Zar, 1971; Furness, 1974; Jones and Spriggs, 1975; Jenkins et al., 1976). The atypical transmission was exemplified by the inhibition of hypogastric nerve stimulation produced b y DMI in the pithed rat in these studies and b y cocaine in earlier in vitro studies using the mouse vas deferens (Marshall et al., 1977). In the in vitro experiments carried out in these and previous studies (Doxey et al., 1977) the Krebs solution was renewed continuously thus preventing the accumulation of noradrenaline in the bathing fluid. Under these conditions very high concentrations of DMI (10 -s M) were required to inhibit the twitch response of the rat vas deferens (Doxey and Easingwood, unpublished observations). In contrast to their effects on hypogastric nerves, mianserin and DMI (Doxey, 1977) potentiate the effects of cardiac nerve
9
stimulation in the pithed rat. The failure of DMI to reverse either the preor postsynaptic agonist actions of clonidine is in contrast to other studies where similar doses of DMI injected into the vertebral artery of anaesthetised cats antagonised the central hypotensive effects of clonidine (Van Spanning and Van Zwieten, 1973; Van Zwieten, 1976}. These workers concluded however that this was the result of a competitive interaction at an ~-adrenoceptor within the central nervous system and that it was unlikely that the uptake blocking properties of DMI were responsible, since cocaine did not diminish the effect of clonidine. In conclusion, the present experiments have shown that, in the periphery, doses of mianserin which inhibit the re-uptake of noradrenaline into nerve endings also exert antagonist activity at both pre- and postsynaptic ~-adrenoceptors. Although mianserin was less potent than phentolamine the pre- to postsynaptic ratio of these two c o m p o u n d s was very similar. Although it is difficult to extrapolate peripheral observations to the central nervous system such profiling could prove tiseful in investigating new antidepressant molecules which are not detected by conventional animal tests. References Ambache, N. and M. Aboo Zar, 1971, Evidence against adrenergic motor transmission in the guinea-pig vas deferens, J. Physiol. London 216, 359. Armstrong, J.M. and A.L.A. Boura, 1973, Effects of clonidine and guanethidine on peripheral sympathetic nerve function in the pithed rat, Brit. J. Pharmacol. 4 7 , 8 5 0 . Baumann, P.A. and L. Maitre, 1977, Blockade of the presynaptic s-receptor a n d of amine uptake in the rat brain by the antidepressant mianserine, Naunyn-Schmiedeb. Arch. Pharmacol. 300, 31. Doxey, J.C., 1977, Effect of clonidine on cardiac accerelation in pithed rats, J. Pharm. Pharmacol. 29, 173. Doxey, J.C. and J. Everitt, 1977, Inhibitory effects of clonidine on responses to s y m p a t h e t i c nerve stimulation in the pithed rat, Brit. J. Pharmacol. 61,559.
10 Doxey, J.C., C.F.C. Smith and J.M. Walker, 1977, Selectivity of blocking agents for pre- and postsynaptic a-adrenoceptors, Brit. J. Pharmacol. 60, 91. Drew, G.M., 1976, Effects of a-adrenoceptor agonists a a d antagonists on pre- and postsynaptically located 0~-adrenoceptors, European J. Pharmacol. 36, 313. Dubocovich, M.L. and S.Z. Langer, 1974, Negative feedback regulation of noradrenaline release by nerve stimulation in the perfused cats spleen: differences in potency of phenoxybenzamine in blocking the pre- and postsynaptic adrenergic receptors, J. Physiol. (London) 237, 505. Furness, J.B., 1974, Transmission on the longitudinal muscle of the guinea pig vas deferens: the effect of pretreatment with guanethidine, Brit. J. Pharmacol. 50, 63. Ghose, K., A. Coppen and P. Turner, 1976, Autonomic actions and interactions of mianserin hydrochloride (Org. GB 94) and amitriptyline in patients with depressive illness, Psychopharmacol. 49, 201. Gillespie, J.S., A. Maclaren and D. Pollock, 1970, A method of stimulating different segments of the autonomic outflow from the spinal column to various organs in the pithed cat and rat, Brit. J. Pharmacol. 40, 257. Goodlet, L. and M.F. Sugrue, 1974, The effect of a new antidepressant, Org. GB 94 on amine uptake mechanisms, Brit. J. Pharmacol. 52, 431. Jenkins, D.A., I. Marshall and P.A. Nasmyth, 1976, Is noradrenaline the m o t o r transmitter in the mouse vas deferens?, J. Physiol. (London) 254, 49P. Jones, M.E.L. and T.L.B. Springs, 1975, Noradrenaline and m o t o r transmission in the vas deferens of the mouse, Brit. J. Pharmacol. 53,323. Langer, S.Z., 1974, Presynaptic regulation of catecholamine release, Biochem. Pharmacol. 23, 1793. Marshall, I., P.A. Nasmyth and N.B. Shepperson, 1977, Pre-synaptic ~-adrenoceptors and the
J.C. DOXEY ET AL. inhibition by uptake blocking agents of the twitch response of the mouse vas deferens, Brit. J. Pharmacol. 59, 511P. Starke, K., 1972, 0~-Sympathomimetic inhibition of adrenergic and cholinergic traaaraission in the rabbit heart, Naunyn-Schmiedeb. Arch. Pharmacol. 274, 18. Starke, K., E. Borowski and T. Endo, 1975a, Preferential blockade of presynaptic ~-adrenoceptors by yohimbine, European J. Pharlnacol. 34,385. Starke, K., T. Endo and H.D. Taube, 1975b, Pre- and postsynaptic components of effect of drugs with aadrenoceptor affinity, Nature, London 254,440. Starke, K., H. Montel, N. Gayk and R. Merker, 1974, Comparison of the effects of clonidine on pre- and postsynaptic adrenoceptors in the rabbit pulmonary artery, Arch. Pharmacol. 285, 133. Swedin, G., 1971, Studies on neurotransmission mechanisms in the rat and guinea pig vas deferens, Acta Physiol. Scand. 83, Suppl. 369, 1. Thoenen, H., A. Hurlimann and W. Haefely, 1964, Dual site of action of phenoxybenzamine in the cats spleen, Experientia 20, 272. Van der Burg, B.J., I.L. Bonta, J. Debobelle, C. Ramon and B. Vargaftig, 1970, A novel type of substituted piperazine with high antiserotonin potency, J. Med. Chem. 1, 35. Van Riezen, H., 1972, Different central effects of the 5HT antagonists mianserine and cyproheptadine, Arch. Intern. Pharmacodyn. Therap. 198, 256. Van Spanning, H.W. and P.A. Van Zwieten, 1973, The interference of tricyclic antidepressants with the central hypotensive effect of clonidine, European J. Pharmacol. 24,402. Van Zwieten, P.A. 1975, Interaction between centrally acting hypotensive drugs and tricylcic antidepressants, Arch. Intern. Pharmacodyn. 214, 12. Van Zwieten, P.A., 1976, The reversal of clonidineinduced hypotension by protriptyline and desipramine, Pharmacol. 14,227.