- Email: [email protected]
Blockade of 5-hydroxytryptamine receptors in the central nervous system by β-adrenoceptor antagonists
BLOCKADE OF 5-HYDROXYTRYPTAMINE RECEPTORS IN THE CENTRAL NERVOUS SYSTEM BY P-ADRENOCEPTOR ANTAGONISTS MARTA WEINSTOCK,* CARMELLA WEISS? and Department
of Physiology
and Pharmacology, Ramat (Accepted
Sackler School Aviv, Israel 8 October
S. GITTER$
of Medicine,
Tel Aviv University,
1976)
Summary-( k)-propranolol. oxprenolol and pindolol in doses ranging from 0.5 to 5 mg/kg antagonised the head twitch produced in mice by SHTP. These doses had no effect on the pinna reflex. (+) propranolol was inactive. These P-adrenoceptor antagonists also prevented the induction of sleep in 5-day old chicks by 5HT. Their activities in the two tests were similar to their 5HT receptor blocking potencies in the rat fundus preparation. It is suggested that these P-adrenoceptor antagonists may block some 5HT receptors in the central nervous system.
It was recently reported that propranolol can antagonise, apparently competitively, the stimulant effects of 5-hydroxytryptamine (5HT) on rat stomach and uterine smooth muscle (Schechter and Weinstock, 1974). Propranolol also reduces the stimulant effect of 5HT on sympathetic ganglia (Weinstock and Schechter, 1975). /I-adrenoceptor antagonists, including propranolol (Atsmon, Blum, Wijsenbeek, Maoz, Steiner and Ziegelman, 1970; Yorkston, Malik, Harvard, Zaki and Morrison. 1974), oxprenolol (Volk, Bier, Braun, Griiter and Spiegelberg, 1972) and pindolol (Auriel, Pinar and Bord, 1973) have been found to have a beneficial effect in some acute psychotic states, particularly schizophrenia. Propranolol and pindolol also appear to be useful agents for the treatment of migraine (Anthony, Lance and Somerville, 1972). The involvement of 5HT in psychoses (Puzynski and Jakimow, 1975) and in migraine (Curran, Hinterberger and Lance, 1965; Rydzewski, 1976) has often been suggested. ilt was therefore of interest to see whether fi-adrenoceptor antagonists could block the effects of 5HT in the central nervous system. 5-hydroxytryptophan (SHTP) is a popular tool for increasing 5HT activity in the central nervous system. When administered parenterally either alone, or with a monoamine oxidase inhibitor or peripheral decarboxylase inhibitor, this agent causes an increase in motor activity (Grahame-Smith, 1971; Modigh, 1972). * Present address: Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, MD 20014, U.S.A. i Part of an MSc. Thesis to be submitted by C.W. to Tel Aviv University. 1 Supported by a grant from the Chief Scientist’s Bureau, Ministry of Health, Israel. Key cards: SHTP head twitch, 5HT sleep, chicks, antagonism by propranolol, oxprenolol. pindolol. 213
as well as a characteristic head twitching (Corne, Pickering and Warner, 1963). The hyperactivity has been shown to involve catecholamine release (Maj and Pawlowski, 1975) and so is of doubtful value as a test for specific antiserotonin activity for P-adrenergic blocking drugs. However, the head twitch response may be a more specific indicator of serotonin activity as it is not elicited by catecholamine precursors or blocked by phenoxybenzamine or dopamine antagonists (Corne et al., 1963). A further test for 5HT activity is the induction of sleep in young chicks in which the blood brain barrier to 5HT has not yet developed, by parenteral injection of 5HT (Jalfre, Ruth-Monachon and Haefely, 1974). The present study reports the effects of a number of P-adrenoceptor antagonists and (+)-propranolol in the head twitch test induced by 5HTP in mice, and in sleep produced by 5HT in chicks.
METHODS
Rat isolated stomach fundus preparation The ability of b-adrenoceptor antagonists to block the responses of the isolated rat stomach fundus strip was determined as described previously (Schechter and Weinstock, 1974). The contact time between the potential antagonist and the tissue was however increased from 5 to 30min before 5HT was added. 5HTP
indud
head twitch in mice
Male albino mice, ICR strain, weighing 2G-24 g were injected subcutaneously with 0.2ml saline or with varying doses of a-adrenoceptor antagonist. Fifteen minutes later they were given SHTP, 300mg/kg intraperitoneally. This dose of 5HTP caused persistent head twitches in SO-100% of saline pretreated mice. The number of mice in each group which showed at least five head twitches during a period
274
MARTA WEINSTOCK, CARMELLA WEISS and S. GITTEK
of 2 min, 23 min after injection of SHTP was determined. From this, the dose of each drug needed to reduce the number of mice showing head twitches by 50?<:was computed (ED5,,). Confidence limits were determined by the method of Litchfield and Wilcoxon
(1948). The specificity of 5HT antagonism by these drugs was demonstrated by comparing the ED+ with the doses required to inhibit the pinna reflex as described by Corne, Pickering and Warner (1963). Induction of sleep in young chicks with 5HT A modification of the method of Jalfre et al. (1974) was used. Five-day-old chicks were injected intramliscularly with 5HT, 20 mg/kg. This dose produced sleep in all the chicks within 3-5 min which lasted 15-20 min. The agents under test or saline were injected intramuscularly at several dose levels. 15 min before 5HT. The dose of each drug was determined which prevented the induction of sleep in 50% of the chicks
parallel shift in the dose response curve to 5HT, with no reduction in the maximum response over the concentration range employed. Methysergide caused a non-surmountable blockade which increased in intensity with the time of exposure. The 5HT blocking activity of both isomers of propranolol and oxprenolol was found to be greater by a factor of 2-5 than that reported in our previous study. This difference was probably due to the increased time of exposure of the tissue to the drug used in the present experiments. Pindolol was found to be a slightly more potent antagonist of 5HT than ($)-propranolol. The concentration of each antagonist was determined which caused a shift to the right of the dose response curve for 5HT by a factor of 2,
i.e.
(IC,,).
(ED,,).
concentration 5HT to give response 50’;/, of max. in presence of antagonist concentration 5HT to give response 50% of max. in absence of antagonist
= 2.
These values are shown in Table 1.
Materials The drugs used in this study were dissolved in saline and injected in a volume of 0.1 ml/10 g in mice and 0.1 ml/20 g in chicks. They included 5-hydroxytryptophan and 5-hydroxytryptamine creatinine sulphate (Sigma Ltd.); (k)-oxprenolol hydrochloride (Ciba Ltd.); (f)-propranolol hydrochloride (Sigma Ltd.); (+)-propranolol hydrochloride, (-)-proprano101 hydrochloride (Ayerst Ltd.); pindolol hydrochloride, methysergide maleate (Sandoz). All doses are expressed as mg/kg of the salt. RESULTS
Antagonism qf SHT-induced contraction of the isolated rut stomuch fundus by /I-adrenoceptor antagonists All the drugs tested except methysergide
caused a
Effect qf /I-adrenoceptor head twitch in mice
antagonists on SHTP-induced
(-+)-Propranolol, pindolol and oxprenolol all antagonised SHTP-induced head twitches in mice in doses ranging from 0.5 to 5 mg/kg. These agents were slightly more potent than methyscrgide in this test. (+)-Propranolol was inactive in doses below 50 mg/kg. Higher doses of (+)-propranolol together with SHTP were lethal. None of the above agents had any effect on the pinna reflex at doses which antagonised the SHTPinduced head twitch. Some reduction in the pinna reflex was only achieved when doses in excess of 40mg/kg were given. The ED,, values for SHTP antagonism and doses reducing the pinna reflex are shown in Table 1.
Table 1. Effect of /&adrenoceptor antagonists on 5HT induced
contraction in mice and pinna reflex
of rat stomach,
SHTP-induced
head twitch
Pinna reflex dose to antagonise
Compound (+)
No. of experiments
Antagonism of 5HT induced contraction of rat fundus I& (ng/ml + SE.)
No. of mice
EDSo mg/kg
1.8 (1.3-2.4) (-) >50 0.9 (0.5-1.6) 2.7 (2.c3.6) 4.5 (3.1-5.6)
Head
Propranolol
6
43 * 2
30
(-) Propranolol (+) Propranolol Pindolol
5 6 6
24 +_ 4 520 + 11 24 k 3
(-) 39 21
Oxprenolol
6
41 * 3
18
Methysergide
4
(-)
Not tested.
0.12 * 0.005
20
No. of mice
m&g (“/i) of mice in which reflex abolished
27
40 (25’:,,)
(-) 12 12
(-) 60 (16”;) 50 (167,,)
twitch
(95% limits)
12 10
100(16”/,,) 40
(1o:fJ
5HT antagonism by propranolol Table 2. Effect of /I-adrenoceptor antagonists induced sleep in chicks
on 5HT-
Dose of drug to
Compound
No. of chicks
(*) Propranolol
46
(-)
18
Propranol
(+) Propranolol Pindolol
18 45
Oxprenolol
50
Methysergide
18
Antagonism fi-adrenoceptor
of
Antagonism of 5HT induced sleep E% mg/kg (95”;, limits)
5HT
induced
blocking
drugs
induce
sleep in SOYIof chicks mg,‘kg
9.4 (5.76- 15.32) 6.0 (4.19~8.58) >50 3.x (2.9-4.97) 4.5 (2.9-6.97) 0.45 (0.09- 1.48)
sleep
in
20 20 50 10 20 1.6
chicks
by
(+)-Propranolol, (-)-propranolol, pindolol and oxprenolol prevented the induction of sleep by 5HT in 5-day old chicks. The EDjo for sleep antagonism for each drug is shown in Table 2. (+)-Propranolol was again ineffective as a 5HT antagonist. At lower doses than the ED,,, values these agents did not prevent sleep entirely but shortened its duration considerably. Higher doses, in excess of 10 mg/kg for pindolol and 20mg/kg for the other a-blocking agents caused sleep in chicks when given alone and greatly prolonged the sleep induced by 5HT. Methysergide also antagonised the effect of 5HT in low doses but induced sleep by itself at a dose of 1.6 mg/kg (see Table 2). DISCUSSION
There is considerable evidence that 5HT neurones are involved in the regulation of sleep and also play a part in the central control of movement (Chase and Murphy, 1973). While a large number of different substances can antagonise the effects of 5HT on peripheral receptors in smooth muscle, their effects on central nervous system 5HT receptors are highly variable (Gyermek, 1966; Clineschmidt and Lotti, 1974). Thus many peripheral 5HT antagonists, such as LSD25 may even ‘stimulate 5HT receptors in the brain (Haigler and L\ghajanian, 1974). The finding that a number of ,d-adrenoceptor antagonists could prevent the inducI:ion of head twitches in mice by 5HT in doses well below those which caused general central nervous system depression and blocked the pinna reflex, suggested that these agents may also block some 5HT receptors in the brain. All the drugs were slightly more potent than methysergide as 5HTP antagonists. It is possible that propranolol and the related compounds reduced the behavioural response to SHTP,
275
through an indirect effect of another system. However, this is not likely to result from blockade of catsince catecholamines themecholamine receptors, selves antagonise the head-twitch response to 5HTP (Corne, Pickering and Warner, 1963). In favour of their direct action on serotonin receptors is the similarity between the ability of these agents to antagonise the 5HTP syndrome in mice and their blocking effect of SHT-induced contractions of rat stomach muscle. The slightly reduced activity of oxprenolol as compared with (k)-propranolol. in the mouse head twitch test may be due to its lesser ability to penetrate the blood-brain barrier (Delini-Stula and Meier, 1976). There is also some evidence that b-adrenoceptor antagonists may block the responses of other brain preparations to 5HT. Thus both (f)-propranolol and pindolol have been shown to prevent the increase in cyclic-AMP activity induced by 5HT in rabbit brain (Kocur, Jurkowski, Kedziora and Czernicki, 1975). Sotalol antagonised the excitation of neurones in the somatosensory cortex of the cat caused by 5HT and noradrenaline but not by acetylcholine (Bevan, Bradshaw and Szabadi, 1974). In the present study. it was also found that all of the compounds which antagonised the effect of 5HT on rat stomach muscle, also reduced its ability to cause sleep in chicks. In this test, as in smooth muscle, however, the fl-adrenoceptor antagonists were considerably less potent than methysergide. Their relatively greater efficacy as antagonists of 5HT in mice than in chicks compared with methysergide could be due either to their greater ability to penetrate the blood-brain barrier or to a difference in the type of 5HT receptor involved in the two species. All the P-blocking agents and (+)-propranolol caused sleep by themselves in higher doses, or potentiated the effect of 5HT. In this respect the drugs resembled methysergide and other 5HT antagonists such as LSD25 and BOL (Jalfre, Ruth-Monachon and Haefely, 1974). It is possible that higher doses of these agents alter the balance of 5HT activity between that on the cell bodies in the raphe nuclei and that in the nerve endings. resulting m sleep (Haigler and Aghajanian, 1974). Alternatively sleep may have been due to a general central nervous depressant effect (Singh, Bhandari and Mahawar. 1971; Bainbridge and Greenwood, 1971). It may therefore be concluded that (- )- and (+)-propranolol, oxprenolol and pindolol can block 5HT receptors in the central nervous system as well as those in smooth muscle and sympathetic ganglia. It remains to be determined whether this property is of any importance in their anti-schizophrenic activity and their undoubted value in the prophylactic treatment of migraine. Adderzdum-Since
cation a report
this paper was submitted for publiappeared in which it was shown that
276
MARTAWEJNSTOCK, CARMELLA WEISS
(- )-propranolol in doses of l&40 mg/kg reduced the hyperactivity induced in rats by 5HTP and tranylcypromine (Green and Gra~lame-Smith, 1976). The (+)-isomer did not show any blocking activity at a dose of 20mg/kg. Although the doses used in this test were somewhat higher than those employed by us in mice, and even high enough to cause non-specific depressant effects, the findings of Green and Graham~-S~nith are nevertheless in agreement with those reported in the present study.
REFERENCES Anthony. M., Lance. J. W. and Somerville, B. (1972). A comparative trial of pindolol. clonidine and cdrbamazepine in interval therapy in migraine. &fed. J. Ausl. 59: 134331346. Atsmon. A., Blum, I., Wijsenbeek, H.. Maoz, B., Steiner. M. and Ziegehnan, G. (1970). The short term effects of adrenergic blocking agents in a small group of psychotic patients. Psychiatr. Neural. Nezrrochir. 74: 251-258. Auriel, B., Pinar, N. and Bord, M. (1973). Le pindolol: un nouveau psychotrope. E?l~~p~7~~~ (Pnris) 62: 427441. Bainbridge, J. G. and Greenwood, D. T. (1971). Tranquillizing effects of propranolol demonstrated in rats. Neu~o~~~~~~~u~~~~~~~~ lo: 453-458. Bevan, P., Bradshaw, C. M. and Szabadi, E. (1974). Potentiation and antagonism of neuronal responses to monoamines by methysergide and sotalol. Rr. J. Pharmac. 50: 445P.
Chase, T. N. and Murphy, D. L. (1973). Serotonin and central nervous system function. Aim. Rev. Pharmac. 13: 181~197. Clineschmidt, B. V. and Lotti, V. 1. (1974). Indol~dmine antagonists: Relative potencies as inhibitors of tryptamine and 5-hydroxy tryptophan evoked responses. Br. J. Pharmuc. SO: 311-313.
Come, S. J., Pickering, R. W. and Warner, 3. T. (1963). A method for assessing effects of drugs on central actions of 5-hydroxytryptamine. Br. J. Pharmac. 28: 106-120. Curran, D. A., Hinterberger, H. and Lance, J. W. (1965). Total plasma serotonin, 5-hydroxyindoieaceti~ acid and p-hydroxy-m-methoxymandelic acid excretion in normal and migrainous subjects. Bruin 88: 997.-1010. Delini-Stula, A. and Meier, M. (1976). Inhibitory effects of propranoiol and oxprenolol on excitation induced by a MAO inhibitor and reserpine in the mouse. h’europhurmucology 15: 3X3-389. Grahame-Smith, D. G. (1971). Studies in ci~v on the relationship between brain tryptophall, brain 5HT synthesis and hyperactivity in rats treated with a monoamine oxidase inhibitor and 1-tryptophan. J. Nrurochem. 18: 105331066.
and S. CITTER
Green. A. R. and Grahame-Smith, D. G. (1976). (-)-Propranolol inhibits the behavioural responses of rats to increased ~-hydroxytryptami~e in the central nervous system. Nnrure Nrtv Bioi. 262:. 594-596. Gyermek, L. (1966). Drugs which antagonise: 5-hydroxytryptamine and related indolealkylamines. In: handbook of E~~~~r~~ze~~tffl P~~ari~zucof~~~~: S- Hyd~~.~y~ryp~arn~fze und Related Indolealkylamines. Vol. 19, pp. 471-528.
Springer. Berlin. Haigler, H. J. and Aghajanian, G. K. (1974). Lysergic acid diethylamide and serotonin: A comparison of effects on serotoninergic neurones and neurones receiving a serotoninergic input. 6. Pharmac. esp. T’her. 188: 688-699. Jaifre, h4., Rtlch-Monachon, M.-A. and Haefelv, W. (1974). Methods for assessing the interaction of-drugs with 5-hvdroxvtrvntamine neurones and receutors in the brain. A&. t)iochem. Psychophurmuc. 10: i21-134. Kocur, J., Jurkowski, A., Kedziora, J., Czernicki, J. and Kedziora, H. (1975). ~n~u~nce of 5-hydroxytryptamin~ and some fl-adrenolytic compounds on adenyl cyclase in rabbit brain in aitro. Pal. J. Pharmac. Phurm. 27 Suppl.: 113-117. Litchfield, J. T. Jr. and Wilcoxon, Y. (1948). A simplified method of evaluating dose effect experiments. J. Pharmat. exp. Ther. 149:99-113. Maj, J. and Pawiowski, L. (1975). The effect of I-%hydroxytryptoph~ (5-HTP) on locomotor activity in mice. Poi. J. Phurmuc. Phnrm. 27 Suppl.: 145-149. Modigh, K. (1972). Central and peripheral effects of S-hydroxytryptophan on motor activity in mice. Psych~~h~~~macofogia
23: 48--54.
Puzynski, S. and Jakimow, B. (1975). Disturbances in tryptophan and serotonin metabolism in affective psychoses and schizophrenia. Poi. J. Pharmac. P~a~rn, 27 Suppi.: 47-60. Rydzewski, W. (1976). Serotonin in migraine: Levels in whole blood in and between attacks. Heuduche 16: 16-19. Schechter, Y. and Weinstock, M. (1974). B-adrenoceptor blocking agents and responses to adrenaline and S-hydroxytryptamine in isolated rat stomach and uterus. Br. .I. P~7[~rrn~~c. 52: 283287. Singh. K. P.. Bhandari, D. S. and Mahawar, M. M. (1971). Effects of propranolol (a beta adrenergic blocking agent) on some central nervous system parameters. fnd. J. Med. Rrs. 59: 186-794. Volk, W., Bier, W., Braun, J. P., Gri.iter, W. and Spiegelberg, U. (1972). Behandlungen von erregten Psychosen mit einem Beta-Receptoren Blocker (Oxprenolol*) in hoher Dosierung. Nervenarzt 43: 491492. Weinstock, M. and Schechter, Y. (1975). Antagonism by propranolol of the ganglion stimulant action of 5-hydroxytryptamine. Eur. J. P~l~~rn~c.32: 293301. Yorkston, N. J., Mahk, M. K. IJ., Harvard, C. W. H., Zaki, S. A. and Morrison, R. C. (1974). Propranolol in control of schizophrenic symptoms. 137.Ned. J. 4: 633-63.5.
of Physiology
and Pharmacology, Ramat (Accepted
Sackler School Aviv, Israel 8 October
S. GITTER$
of Medicine,
Tel Aviv University,
1976)
Summary-( k)-propranolol. oxprenolol and pindolol in doses ranging from 0.5 to 5 mg/kg antagonised the head twitch produced in mice by SHTP. These doses had no effect on the pinna reflex. (+) propranolol was inactive. These P-adrenoceptor antagonists also prevented the induction of sleep in 5-day old chicks by 5HT. Their activities in the two tests were similar to their 5HT receptor blocking potencies in the rat fundus preparation. It is suggested that these P-adrenoceptor antagonists may block some 5HT receptors in the central nervous system.
It was recently reported that propranolol can antagonise, apparently competitively, the stimulant effects of 5-hydroxytryptamine (5HT) on rat stomach and uterine smooth muscle (Schechter and Weinstock, 1974). Propranolol also reduces the stimulant effect of 5HT on sympathetic ganglia (Weinstock and Schechter, 1975). /I-adrenoceptor antagonists, including propranolol (Atsmon, Blum, Wijsenbeek, Maoz, Steiner and Ziegelman, 1970; Yorkston, Malik, Harvard, Zaki and Morrison. 1974), oxprenolol (Volk, Bier, Braun, Griiter and Spiegelberg, 1972) and pindolol (Auriel, Pinar and Bord, 1973) have been found to have a beneficial effect in some acute psychotic states, particularly schizophrenia. Propranolol and pindolol also appear to be useful agents for the treatment of migraine (Anthony, Lance and Somerville, 1972). The involvement of 5HT in psychoses (Puzynski and Jakimow, 1975) and in migraine (Curran, Hinterberger and Lance, 1965; Rydzewski, 1976) has often been suggested. ilt was therefore of interest to see whether fi-adrenoceptor antagonists could block the effects of 5HT in the central nervous system. 5-hydroxytryptophan (SHTP) is a popular tool for increasing 5HT activity in the central nervous system. When administered parenterally either alone, or with a monoamine oxidase inhibitor or peripheral decarboxylase inhibitor, this agent causes an increase in motor activity (Grahame-Smith, 1971; Modigh, 1972). * Present address: Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, MD 20014, U.S.A. i Part of an MSc. Thesis to be submitted by C.W. to Tel Aviv University. 1 Supported by a grant from the Chief Scientist’s Bureau, Ministry of Health, Israel. Key cards: SHTP head twitch, 5HT sleep, chicks, antagonism by propranolol, oxprenolol. pindolol. 213
as well as a characteristic head twitching (Corne, Pickering and Warner, 1963). The hyperactivity has been shown to involve catecholamine release (Maj and Pawlowski, 1975) and so is of doubtful value as a test for specific antiserotonin activity for P-adrenergic blocking drugs. However, the head twitch response may be a more specific indicator of serotonin activity as it is not elicited by catecholamine precursors or blocked by phenoxybenzamine or dopamine antagonists (Corne et al., 1963). A further test for 5HT activity is the induction of sleep in young chicks in which the blood brain barrier to 5HT has not yet developed, by parenteral injection of 5HT (Jalfre, Ruth-Monachon and Haefely, 1974). The present study reports the effects of a number of P-adrenoceptor antagonists and (+)-propranolol in the head twitch test induced by 5HTP in mice, and in sleep produced by 5HT in chicks.
METHODS
Rat isolated stomach fundus preparation The ability of b-adrenoceptor antagonists to block the responses of the isolated rat stomach fundus strip was determined as described previously (Schechter and Weinstock, 1974). The contact time between the potential antagonist and the tissue was however increased from 5 to 30min before 5HT was added. 5HTP
indud
head twitch in mice
Male albino mice, ICR strain, weighing 2G-24 g were injected subcutaneously with 0.2ml saline or with varying doses of a-adrenoceptor antagonist. Fifteen minutes later they were given SHTP, 300mg/kg intraperitoneally. This dose of 5HTP caused persistent head twitches in SO-100% of saline pretreated mice. The number of mice in each group which showed at least five head twitches during a period
274
MARTA WEINSTOCK, CARMELLA WEISS and S. GITTEK
of 2 min, 23 min after injection of SHTP was determined. From this, the dose of each drug needed to reduce the number of mice showing head twitches by 50?<:was computed (ED5,,). Confidence limits were determined by the method of Litchfield and Wilcoxon
(1948). The specificity of 5HT antagonism by these drugs was demonstrated by comparing the ED+ with the doses required to inhibit the pinna reflex as described by Corne, Pickering and Warner (1963). Induction of sleep in young chicks with 5HT A modification of the method of Jalfre et al. (1974) was used. Five-day-old chicks were injected intramliscularly with 5HT, 20 mg/kg. This dose produced sleep in all the chicks within 3-5 min which lasted 15-20 min. The agents under test or saline were injected intramuscularly at several dose levels. 15 min before 5HT. The dose of each drug was determined which prevented the induction of sleep in 50% of the chicks
parallel shift in the dose response curve to 5HT, with no reduction in the maximum response over the concentration range employed. Methysergide caused a non-surmountable blockade which increased in intensity with the time of exposure. The 5HT blocking activity of both isomers of propranolol and oxprenolol was found to be greater by a factor of 2-5 than that reported in our previous study. This difference was probably due to the increased time of exposure of the tissue to the drug used in the present experiments. Pindolol was found to be a slightly more potent antagonist of 5HT than ($)-propranolol. The concentration of each antagonist was determined which caused a shift to the right of the dose response curve for 5HT by a factor of 2,
i.e.
(IC,,).
(ED,,).
concentration 5HT to give response 50’;/, of max. in presence of antagonist concentration 5HT to give response 50% of max. in absence of antagonist
= 2.
These values are shown in Table 1.
Materials The drugs used in this study were dissolved in saline and injected in a volume of 0.1 ml/10 g in mice and 0.1 ml/20 g in chicks. They included 5-hydroxytryptophan and 5-hydroxytryptamine creatinine sulphate (Sigma Ltd.); (k)-oxprenolol hydrochloride (Ciba Ltd.); (f)-propranolol hydrochloride (Sigma Ltd.); (+)-propranolol hydrochloride, (-)-proprano101 hydrochloride (Ayerst Ltd.); pindolol hydrochloride, methysergide maleate (Sandoz). All doses are expressed as mg/kg of the salt. RESULTS
Antagonism qf SHT-induced contraction of the isolated rut stomuch fundus by /I-adrenoceptor antagonists All the drugs tested except methysergide
caused a
Effect qf /I-adrenoceptor head twitch in mice
antagonists on SHTP-induced
(-+)-Propranolol, pindolol and oxprenolol all antagonised SHTP-induced head twitches in mice in doses ranging from 0.5 to 5 mg/kg. These agents were slightly more potent than methyscrgide in this test. (+)-Propranolol was inactive in doses below 50 mg/kg. Higher doses of (+)-propranolol together with SHTP were lethal. None of the above agents had any effect on the pinna reflex at doses which antagonised the SHTPinduced head twitch. Some reduction in the pinna reflex was only achieved when doses in excess of 40mg/kg were given. The ED,, values for SHTP antagonism and doses reducing the pinna reflex are shown in Table 1.
Table 1. Effect of /&adrenoceptor antagonists on 5HT induced
contraction in mice and pinna reflex
of rat stomach,
SHTP-induced
head twitch
Pinna reflex dose to antagonise
Compound (+)
No. of experiments
Antagonism of 5HT induced contraction of rat fundus I& (ng/ml + SE.)
No. of mice
EDSo mg/kg
1.8 (1.3-2.4) (-) >50 0.9 (0.5-1.6) 2.7 (2.c3.6) 4.5 (3.1-5.6)
Head
Propranolol
6
43 * 2
30
(-) Propranolol (+) Propranolol Pindolol
5 6 6
24 +_ 4 520 + 11 24 k 3
(-) 39 21
Oxprenolol
6
41 * 3
18
Methysergide
4
(-)
Not tested.
0.12 * 0.005
20
No. of mice
m&g (“/i) of mice in which reflex abolished
27
40 (25’:,,)
(-) 12 12
(-) 60 (16”;) 50 (167,,)
twitch
(95% limits)
12 10
100(16”/,,) 40
(1o:fJ
5HT antagonism by propranolol Table 2. Effect of /I-adrenoceptor antagonists induced sleep in chicks
on 5HT-
Dose of drug to
Compound
No. of chicks
(*) Propranolol
46
(-)
18
Propranol
(+) Propranolol Pindolol
18 45
Oxprenolol
50
Methysergide
18
Antagonism fi-adrenoceptor
of
Antagonism of 5HT induced sleep E% mg/kg (95”;, limits)
5HT
induced
blocking
drugs
induce
sleep in SOYIof chicks mg,‘kg
9.4 (5.76- 15.32) 6.0 (4.19~8.58) >50 3.x (2.9-4.97) 4.5 (2.9-6.97) 0.45 (0.09- 1.48)
sleep
in
20 20 50 10 20 1.6
chicks
by
(+)-Propranolol, (-)-propranolol, pindolol and oxprenolol prevented the induction of sleep by 5HT in 5-day old chicks. The EDjo for sleep antagonism for each drug is shown in Table 2. (+)-Propranolol was again ineffective as a 5HT antagonist. At lower doses than the ED,,, values these agents did not prevent sleep entirely but shortened its duration considerably. Higher doses, in excess of 10 mg/kg for pindolol and 20mg/kg for the other a-blocking agents caused sleep in chicks when given alone and greatly prolonged the sleep induced by 5HT. Methysergide also antagonised the effect of 5HT in low doses but induced sleep by itself at a dose of 1.6 mg/kg (see Table 2). DISCUSSION
There is considerable evidence that 5HT neurones are involved in the regulation of sleep and also play a part in the central control of movement (Chase and Murphy, 1973). While a large number of different substances can antagonise the effects of 5HT on peripheral receptors in smooth muscle, their effects on central nervous system 5HT receptors are highly variable (Gyermek, 1966; Clineschmidt and Lotti, 1974). Thus many peripheral 5HT antagonists, such as LSD25 may even ‘stimulate 5HT receptors in the brain (Haigler and L\ghajanian, 1974). The finding that a number of ,d-adrenoceptor antagonists could prevent the inducI:ion of head twitches in mice by 5HT in doses well below those which caused general central nervous system depression and blocked the pinna reflex, suggested that these agents may also block some 5HT receptors in the brain. All the drugs were slightly more potent than methysergide as 5HTP antagonists. It is possible that propranolol and the related compounds reduced the behavioural response to SHTP,
275
through an indirect effect of another system. However, this is not likely to result from blockade of catsince catecholamines themecholamine receptors, selves antagonise the head-twitch response to 5HTP (Corne, Pickering and Warner, 1963). In favour of their direct action on serotonin receptors is the similarity between the ability of these agents to antagonise the 5HTP syndrome in mice and their blocking effect of SHT-induced contractions of rat stomach muscle. The slightly reduced activity of oxprenolol as compared with (k)-propranolol. in the mouse head twitch test may be due to its lesser ability to penetrate the blood-brain barrier (Delini-Stula and Meier, 1976). There is also some evidence that b-adrenoceptor antagonists may block the responses of other brain preparations to 5HT. Thus both (f)-propranolol and pindolol have been shown to prevent the increase in cyclic-AMP activity induced by 5HT in rabbit brain (Kocur, Jurkowski, Kedziora and Czernicki, 1975). Sotalol antagonised the excitation of neurones in the somatosensory cortex of the cat caused by 5HT and noradrenaline but not by acetylcholine (Bevan, Bradshaw and Szabadi, 1974). In the present study. it was also found that all of the compounds which antagonised the effect of 5HT on rat stomach muscle, also reduced its ability to cause sleep in chicks. In this test, as in smooth muscle, however, the fl-adrenoceptor antagonists were considerably less potent than methysergide. Their relatively greater efficacy as antagonists of 5HT in mice than in chicks compared with methysergide could be due either to their greater ability to penetrate the blood-brain barrier or to a difference in the type of 5HT receptor involved in the two species. All the P-blocking agents and (+)-propranolol caused sleep by themselves in higher doses, or potentiated the effect of 5HT. In this respect the drugs resembled methysergide and other 5HT antagonists such as LSD25 and BOL (Jalfre, Ruth-Monachon and Haefely, 1974). It is possible that higher doses of these agents alter the balance of 5HT activity between that on the cell bodies in the raphe nuclei and that in the nerve endings. resulting m sleep (Haigler and Aghajanian, 1974). Alternatively sleep may have been due to a general central nervous depressant effect (Singh, Bhandari and Mahawar. 1971; Bainbridge and Greenwood, 1971). It may therefore be concluded that (- )- and (+)-propranolol, oxprenolol and pindolol can block 5HT receptors in the central nervous system as well as those in smooth muscle and sympathetic ganglia. It remains to be determined whether this property is of any importance in their anti-schizophrenic activity and their undoubted value in the prophylactic treatment of migraine. Adderzdum-Since
cation a report
this paper was submitted for publiappeared in which it was shown that
276
MARTAWEJNSTOCK, CARMELLA WEISS
(- )-propranolol in doses of l&40 mg/kg reduced the hyperactivity induced in rats by 5HTP and tranylcypromine (Green and Gra~lame-Smith, 1976). The (+)-isomer did not show any blocking activity at a dose of 20mg/kg. Although the doses used in this test were somewhat higher than those employed by us in mice, and even high enough to cause non-specific depressant effects, the findings of Green and Graham~-S~nith are nevertheless in agreement with those reported in the present study.
REFERENCES Anthony. M., Lance. J. W. and Somerville, B. (1972). A comparative trial of pindolol. clonidine and cdrbamazepine in interval therapy in migraine. &fed. J. Ausl. 59: 134331346. Atsmon. A., Blum, I., Wijsenbeek, H.. Maoz, B., Steiner. M. and Ziegehnan, G. (1970). The short term effects of adrenergic blocking agents in a small group of psychotic patients. Psychiatr. Neural. Nezrrochir. 74: 251-258. Auriel, B., Pinar, N. and Bord, M. (1973). Le pindolol: un nouveau psychotrope. E?l~~p~7~~~ (Pnris) 62: 427441. Bainbridge, J. G. and Greenwood, D. T. (1971). Tranquillizing effects of propranolol demonstrated in rats. Neu~o~~~~~~~u~~~~~~~~ lo: 453-458. Bevan, P., Bradshaw, C. M. and Szabadi, E. (1974). Potentiation and antagonism of neuronal responses to monoamines by methysergide and sotalol. Rr. J. Pharmac. 50: 445P.
Chase, T. N. and Murphy, D. L. (1973). Serotonin and central nervous system function. Aim. Rev. Pharmac. 13: 181~197. Clineschmidt, B. V. and Lotti, V. 1. (1974). Indol~dmine antagonists: Relative potencies as inhibitors of tryptamine and 5-hydroxy tryptophan evoked responses. Br. J. Pharmuc. SO: 311-313.
Come, S. J., Pickering, R. W. and Warner, 3. T. (1963). A method for assessing effects of drugs on central actions of 5-hydroxytryptamine. Br. J. Pharmac. 28: 106-120. Curran, D. A., Hinterberger, H. and Lance, J. W. (1965). Total plasma serotonin, 5-hydroxyindoieaceti~ acid and p-hydroxy-m-methoxymandelic acid excretion in normal and migrainous subjects. Bruin 88: 997.-1010. Delini-Stula, A. and Meier, M. (1976). Inhibitory effects of propranoiol and oxprenolol on excitation induced by a MAO inhibitor and reserpine in the mouse. h’europhurmucology 15: 3X3-389. Grahame-Smith, D. G. (1971). Studies in ci~v on the relationship between brain tryptophall, brain 5HT synthesis and hyperactivity in rats treated with a monoamine oxidase inhibitor and 1-tryptophan. J. Nrurochem. 18: 105331066.
and S. CITTER
Green. A. R. and Grahame-Smith, D. G. (1976). (-)-Propranolol inhibits the behavioural responses of rats to increased ~-hydroxytryptami~e in the central nervous system. Nnrure Nrtv Bioi. 262:. 594-596. Gyermek, L. (1966). Drugs which antagonise: 5-hydroxytryptamine and related indolealkylamines. In: handbook of E~~~~r~~ze~~tffl P~~ari~zucof~~~~: S- Hyd~~.~y~ryp~arn~fze und Related Indolealkylamines. Vol. 19, pp. 471-528.
Springer. Berlin. Haigler, H. J. and Aghajanian, G. K. (1974). Lysergic acid diethylamide and serotonin: A comparison of effects on serotoninergic neurones and neurones receiving a serotoninergic input. 6. Pharmac. esp. T’her. 188: 688-699. Jaifre, h4., Rtlch-Monachon, M.-A. and Haefelv, W. (1974). Methods for assessing the interaction of-drugs with 5-hvdroxvtrvntamine neurones and receutors in the brain. A&. t)iochem. Psychophurmuc. 10: i21-134. Kocur, J., Jurkowski, A., Kedziora, J., Czernicki, J. and Kedziora, H. (1975). ~n~u~nce of 5-hydroxytryptamin~ and some fl-adrenolytic compounds on adenyl cyclase in rabbit brain in aitro. Pal. J. Pharmac. Phurm. 27 Suppl.: 113-117. Litchfield, J. T. Jr. and Wilcoxon, Y. (1948). A simplified method of evaluating dose effect experiments. J. Pharmat. exp. Ther. 149:99-113. Maj, J. and Pawiowski, L. (1975). The effect of I-%hydroxytryptoph~ (5-HTP) on locomotor activity in mice. Poi. J. Phurmuc. Phnrm. 27 Suppl.: 145-149. Modigh, K. (1972). Central and peripheral effects of S-hydroxytryptophan on motor activity in mice. Psych~~h~~~macofogia
23: 48--54.
Puzynski, S. and Jakimow, B. (1975). Disturbances in tryptophan and serotonin metabolism in affective psychoses and schizophrenia. Poi. J. Pharmac. P~a~rn, 27 Suppi.: 47-60. Rydzewski, W. (1976). Serotonin in migraine: Levels in whole blood in and between attacks. Heuduche 16: 16-19. Schechter, Y. and Weinstock, M. (1974). B-adrenoceptor blocking agents and responses to adrenaline and S-hydroxytryptamine in isolated rat stomach and uterus. Br. .I. P~7[~rrn~~c. 52: 283287. Singh. K. P.. Bhandari, D. S. and Mahawar, M. M. (1971). Effects of propranolol (a beta adrenergic blocking agent) on some central nervous system parameters. fnd. J. Med. Rrs. 59: 186-794. Volk, W., Bier, W., Braun, J. P., Gri.iter, W. and Spiegelberg, U. (1972). Behandlungen von erregten Psychosen mit einem Beta-Receptoren Blocker (Oxprenolol*) in hoher Dosierung. Nervenarzt 43: 491492. Weinstock, M. and Schechter, Y. (1975). Antagonism by propranolol of the ganglion stimulant action of 5-hydroxytryptamine. Eur. J. P~l~~rn~c.32: 293301. Yorkston, N. J., Mahk, M. K. IJ., Harvard, C. W. H., Zaki, S. A. and Morrison, R. C. (1974). Propranolol in control of schizophrenic symptoms. 137.Ned. J. 4: 633-63.5.