- Email: [email protected]
Dicyclomine- and pirenzepine-sensitive muscarinic receptors mediate inhibition of [3H]serotonin release in different rat brain areas
European Journal of Pharmacology, 129 (1986) 353-357
353
Elsevier EJP 442SC Short communication
Dicyclomine- and pirenzepine-sensitive muscarinic receptors mediate inhibition of [3H]serotonin release in different rat brain areas M a r i o Marchi, P a o l o Paudice, M a r i a g r a z i a Bella a n d M a u r i z i o Raiteri * lstituto di Farmacologiae Farmacognosia, Universit~di Genova, Viale Cembrano 4, 16148 Genova, Italy
Received 30 July 1986, accepted 12 August 1986
The effects of acetylcholine (ACh) on the release of [3H]5-hydroxytryptamine ([3H]5-HT) were investigated in synaptosomes prepared from rat cerebral cortex, hypothalamus and hippocampus and depolarized with 15 mM KCI under superfusion conditions. ACh inhibited the release of [3H]5-HT in all three brain ares. This effect was not modified by hexamethonium but was antagonized by atropine and by the non-classical antagonists pirenzepine and dicyclomine. Serotonin release; Muscarinic receptor subtypes; Dicyclomine; Pirenzepine; (Superfused synaptosomes)
1. Introduction The cholinergic modulation of serotonin (5-HT) release has been little investigated. The basal release of [3H]5-HT newly synthesized from [3H] tryptophan in rat hypothalamic slices (H6ry et al., 1977), as well as the basal release of previously accumulated [3H]5-HT or of endogenous 5-HT in rat striatal slices (Westfall et al., 1983) were found to be increased following activation of nicotinic receptors. Inhibition of the basal release of newly formed [3H]5-HT from rat hypothalamic slices was observed instead when muscarinic receptors were activated (H~ry et al., 1977). Atropine-sensitive muscarinic receptors were found also to mediate the inhibition of the K +-evoked release of [3H]5-HT from hypothalamic slices (Ennis and Cox, 1982). The present study of the effects of muscarinic receptor activation on the release of 5-HT originated from the following considerations: (1) since cholinergic and serotoninergic innervations of dif-
* To whom all correspondence should be addressed. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
ferent brain areas have different origins, it was important to investigate different cerebral regions. (2) In experiments with brain slices the use of acetylcholine (ACh) is not easy due to its rapid inactivation. Moreover, the anatomical localization of the receptors involved in release regulation cannot be determined directly with brain slices. We have utilized superfused synaptosomes, an experimental condition in which ACh can be used without acetylcholinesterase inhibitors and which allows to ascertain directly whether a receptor is located on nerve endings. (3) Since the existence of muscarinic receptor subtypes is well accepted (see for reviews: Hirschowitz et al., 1984), we have characterized the muscarinic receptors involved in the modulation of 5-HT release in terms of receptor subtypes.
2. Materials and methods 2.1. Preparation of synaptosomes
Nerve endings were prepared from rat cerebral cortex, hypothalamus and hippocampus of adult
354 S p r a g u e - D a w l e y rats ( 2 0 0 - 2 5 0 g) essentially acc o r d i n g to G r a y a n d W h i t t a k e r (1962). The pellet (P2 fraction) was r e s u s p e n d e d (at a protein conc e n t r a t i o n of 0.5-0.6 m g / m l ) in 5 ml of m e d i u m having the following c o m p o s i t i o n ( m M ) : N a C I 125, KC1 3, M g S O 4 1.2, CaCI 2 1.2, N a H z P O 4 1.2, N a H C O 3 20 a n d glucose 10 (aeration with 95% 02 a n d 5% C O 2 at 3 7 ° C ) p H 7.2-7.4.
2.4. Drugs
2.2. Release experiments"
SJS,
T h e release of [3H]5-HT was studied using a superfusion a p p a r a t u s (see Raiteri et al., 1974; Raiteri a n d Levi, 1978). Briefly, the s y n a p t o s o m e suspension was p r e i n c u b a t e d for 10 min at 3 7 ° C ; [ 3 H ] 5 - H T (specific activity 29.6 C i / m m o l ; N E N ) was a d d e d to give a final c o n c e n t r a t i o n of 0.03 /~M. U p t a k e was s t o p p e d after 15 rain of i n c u b a tion b y a d d i n g 100 ml of m e d i u m a n d the labelled nerved endings were d i s t r i b u t e d in the superfusion c h a m b e r s . The s y n a p t o s o m e s were then superfused at 3 7 ° C with c o n t i n u o u s l y o x y g e n a t e d m e d i u m at the rate of 0.6 m l / m i n . The nerve endings were s t i m u l a t e d for two p e r i o d s of 45 s each (S 1, $2), starting after 38 a n d 66 min of superfusion, b y raising the K + c o n c e n t r a t i o n of the m e d i u m to 15 m m o l / l . M u s c a r i n i c agonists or a n t a g o n i s t s were a d d e d c o n c o m i t a n t l y with KC1 at min 66 of superfusion ($2). The s t i m u l a t i o n e v o k e d release of tritium was c a l c u l a t e d b y subtraction of the b a s a l outflow. The latter was the average of the tritium r a d i o a c t i v i t y present in the two 3 min fractions collected before a n d after K + stimulation. The release evoked was expressed as a p e r c e n t a g e of the tritium c o n t e n t of the s y n a p t o somes at the onset of the respective s t i m u l a t i o n p e r i o d s (S 1 a n d $2). D r u g effects were e v a l u a t e d b y calculating the ratio (S2/Sx) of the 3H release e v o k e d d u r i n g the two s t i m u l a t i o n periods. T h e statistical significance of differences was d e t e r m i n e d b y analysis of variance (one-way).
2.3. Separation of [ 3H]5-HT from ~H-metabolites T h e [3H]5-HT in the fractions a n d in the superfused s y n a p t o s o m e s was d e t e r m i n e d after sepa r a t i o n from 3 H - m e t a b o l i t e s (Raiteri et al., 1984). T h e a m o u n t of [3H]5-HT f o u n d in s y n a p t o s o m e s a c c o u n t e d for 45-55% of total 3H radioactivity.
A C h chloride, a t r o p i n e sulfate, h e x a m e t h o n i u m c h l o r i d e a n d o x o t r e m o r i n e were p u r c h a s e d from Sigma Chemical Co. (St. Louis, MO). The following drugs were gifts: p i r e n z e p i n e d i h y d r o c h l o r i d e ( l s t . D e Angeli, Milano, Italy) a n d d i c y c l o m i n e ( C a m i i l o Corvi, Piacenza, Italy).
®
16 14
T
12
T
I0
T
08 08 04 02
+
+
Q
Q
§ [27)
(21)
(21}
(201
(81
SdS,
®
10 .I_
08 06 +
04 02
[80]
[151
[211
t21]
(80]
t15]
(22]
Fig. 1. Effects of acetylcholine receptor agonists and antagonists on the K+-induced release of [3H]5-HT from rat hypothalamic (A), and cortical (B) synaptosomes. Nerve endings prelabeled with the radioactive indoleamine were superfused and stimulated for two periods of 45 s each (S], S2) by increasing the K + concentration of the medium to 15 mmol/1. Data are expressed as the ratio S2/SI of the K+-induced [3H]5-HT or 3H release evoked by the two stimulation periods. The basal [3H]5-HT release from hypothalamic synaptosomes amounted to 0.57_+ 0.04% of the total [3H]5-HT. The K ~-evoked release (S~) in the controls was 2.01 + 0.12%. The basal tritium efflux from cortical synaptosomes amounted to 2.11 _+0.10% and the K+-evoked release (S l) in the controls was 3.21 + 0.39%. The data represent the means_+S.E.M, and the number of separate determinations is reported in parentheses. *P < 0.05: **P < 0.01 compared to the control (ANOVA test). HEXA = hexamethonium; OXO = oxotremorine; A = atropine.
355 3. Results
Figure 1A shows that ACh (10 /tM) inhibited the release of [3H]5-HT from rat hypothalamic synaptosomes exposed in superfusion to 15 mM KC1. The inhibitory action of ACh was not counteracted by hexamethonium (10/~M). However, 10 /~M ACh was totally antagonized by 1 #M atropine. The release of [3H]5-HT was inhibited by oxotremorine. The results obtained by measuring tritium radioactivity were qualitatively superimposable to those obtained when [3H]5-HT was separated from metabolites. The results of experiments carried out with cerebral cortex synaptosomes are illustrated in fig. lB. Acetylcholine dose dependently inhibited the SdSi 14 12
®
T
T
T L
K+-induced [3H]5-HT release. Oxotremorine mimicked ACh. Atropine, but not hexamethonium, antagonized ACh. The effect of ACh on [3H]5-HT release was also studied in hippocampus synaptosomes. While significant, the inhibitory action of ACh was less pronounced in this area than in cortex and hypothalamus (not shown). Atropine or hexamethonium, when tested alone, did not cause changes of [3H]5-HT release in the areas considered (not shown). The data obtained with the muscarinic antagonists dicyclomine and pirenzepine are shown in fig. 2. Both in hypothalamus (fig. 2A) and in the cerebral cortex (fig. 2B) the inhibitory action of ACh (10/tM) on the K+-evoked release of [3H]5H T was concentration dependently antagonized by dicyclomine or pirenzepine. Dicycl0mine or pirenzepine themselves did not affect [3H]5-HT release (not shown).
10
4. Discussion 0.8 E.
06 04
._= +
+
c~
c~
(8}
(TJ
02
(12}
(12)
(12)
S2/Sl
®
r2 10
T
T
08 86 04 82
116}
[16)
(101
[8}
(9}
{101
Fig. 2. Effectsof dicyclomineand pirenzepineon the inhibition of the K +-induced releaseof [3H]5-HT from hypothalamic(A) and cortical (B) synaptosomes by ACh. The procedure was similar to that described in the legend to fig. 1. The data represent the means+S.E.M, and the number of separate determinations is reported in parentheses. *P < 0.05; **P < 0.01 compared to the control (ANOVA test). DICY= dicyclomine; PIRE = pirenzepine.
Studying the spontaneous release of newly synthesized [3H]5-HT from rat hypothalamic slices Hdry et al. (1977) found that nicotinic agonists, including ACh, potentiated whereas the muscarinic agonists oxotremorine inhibited the release of the indoleamine. The authors hypothesized the presence of cholinoceptors on serotoninergic terminals but also considered alternative explanations implying indirect mechanisms. We have examined the effects of ACh on the depolarization-evoked release of [3H]5-HT previously taken up into synaptosomes prepared from various areas of the rat brain, including hypothalamus. At the concentration (10 /zM) used by Hdry et al. (1977) ACh did not enhance the 15 mM K+-evoked release of [3H]5-HT. Even ACh 100 /~M did not enhance [3H]5-HT release but decreased it instead. However, the presence of the nicotinic antagonist hexamethonium did not increase the inhibitory action of ACh as one would have expected if ACh was activating inhibitory (muscarinic) and excitatory (nicotinic) receptors concomitantly. It is possible that nicotinic receptor activation potentiates only the spontaneous
356 release of 5-HT without affecting the release evoked by depolarization. Another possibility is that newly synthesized and taken up [3H]5-HT are differentially sensitive to nicotinic receptor activation. A third possibility is that nicotinic receptors are not located on serotoninergic terminals. Muscarinic receptor activation (by ACh or oxotremorine) inhibited the K÷-evoked release of previously accumulated [3H]5-HT in synaptosomes obtained from rat hypothalamus, cerebral cortex and hippocampus. These data are consistent with those obtained when hypothalamic slices were used to study either the basal release of newly synthesized [3H]5-HT (H6ry et al., 1977) or the K÷-evoked release of preaccumulated [3H]5H T (Ennis and Cox, 1982). Our results with isolated nerve endings in superfusion, a condition in which indirect effects between transmitters are minimized, provide direct demonstration that 5-HT nerve terminals in rat cortex, hypothalamus and hippocampus are endowed with muscarinic receptors whose activation by ACh or muscarinic agonists brings about inhibition of 5-HT release. The existence of subtypes of muscarinic binding sites in the CNS is well documented (for reviews see: Hirschowitz et al., 1984). These different binding sites appear to reflect different receptors. It was found in a study comparing the pharmacological characteristics of the muscarinic autoreceptors regulating ACh release and the muscarinic heteroreceptors located on dopamine terminals and mediating the potentiation of dopamine release that the action of ACh was blocked by dicyclomine or by pirenzepine only at the heteroreceptors (Marchi and Raiteri, 1985). The autoreceptors were relatively insensitive to these muscarinic antagonists. On the other hand, atropine, scopolamine or quinuclydinylbenzylate blocked equally well both the auto- and the heteroreceptors (Raiteri et al., 1984; Marchi and Raiteri, 1985). The results obtained with dicyclomine and pirenzepine (fig. 2A,B) suggest that the muscarinic presynaptic heteroreceptors regulating the depolarization-evoked release of [3H]5H T from cortical and hypothalamic nerve endings differ pharmacologically from the muscarinic autoreceptors, where these compounds have been shown to be poorly effective (Marchi and Raiteri,
1985). It seems premature however to use the M~ nomenclature for the presynaptic receptors characterized in this study. The following main conclusions can be drawn from the present investigation: (1) the muscarinic receptors mediating the inhibition of 5-HT release previously identified by H6ry et al. (1977) and by Ennis and Cox (1982) in slices of rat hypothalamus are localized on serotoninergic nerve endings. (2) Three muscarinic receptors in the cerebral cortex of the rat have now been characterized in terms of localization, function and pharmacology: (a) an autoreceptor located on cholinergic terminals, mediating inhibition of ACh release, insensitive to dicyclomine or pirenzepine (Marchi and Raiteri, 1985); (b) a heteroreceptor, located on dopaminergic nerve endings, mediating potentiation of dopamine release, sensitive to dicyclomine or pirenzepine (Marchi and Raiteri, 1985) and (c) a heteroreceptor, located on serotoninergic terminals, mediating inhibition of 5-HT release, dicyclomine- and pirenzepine-sensitive.
Acknowledgements This work was supported by grants from the Italian Ministry of Education and from the Italian National Research Council. The authors wish to thank Mrs. Maura Agate for her expert secretarial assistance.
References Ennis, C. and B. Cox, 1982, Inhibitory muscarinic receptors modulate the potassium-evoked release of [3H]serotonin from rat hypothalamic slices, European J. Pharmacol. 81, 159. Gray, E.G. and V.P. Whittaker, 1962, The isolation of nerve endings from brain: an electron-microscopicstudy of cell fragments derived by homogenization and centrifugation, J. Anat. (London) 96, 79. H6ry, F., S. Bourgoin, M. Hamon, J.P. Ternaux and J. Glowinski, 1977, Control of the release of newly synthesized 3H-5-hydroxytryptamine by nicotinic and muscarinic receptors in rat hypothalamic slices, Naunyn-Schmiedeb, Arch. Pharmacol. 296, 91. Hirschowitz, B.I., R. Hammer, A. Giacheni, J.J. Keirns and R.R. Levine(eds.), 1984, Subtypes of Muscarinic Receptors (Elsevier North Holland). Marchi, M. and M. Raiteri, 1985, On the presence in the cerebral cortex of muscarinic receptor subtypes which dif-
357 fer in neuronal localization, function and pharmacological properties, J. Pharmacol. Exp. Ther. 235, 230. Raiteri, M., F. Angelini and G. Levi, 1974, A simple apparatus for studying the release of neurotransmitters from synaptosomes, European J. Pharmacol. 25, 411. Raiteri, M., R. Leardi and M. Marchi, 1984, Heterogeneity of presynaptic muscarinic receptors regulating neurotransmitter release in the rat brain, J. Pharmacol. Exp. Ther. 228, 209.
Raiteri, M. and G. Levi, 1978, Release mechanisms for catecholamines and serotonin in synaptosomes, in: Reviews of Neuroscience, Vol. 3, eds. S. Ehrenpreis and I. Kopin (Raven Press, New York) p. 77. Westfall, T.C., H. Grant and H. Perry, 1983, Release of dopamine and 5-hydroxytryptamine from rat striatal slices following activation of nicotinic cholinergic receptors, Gen. Pharmacol. 14, 321.
353
Elsevier EJP 442SC Short communication
Dicyclomine- and pirenzepine-sensitive muscarinic receptors mediate inhibition of [3H]serotonin release in different rat brain areas M a r i o Marchi, P a o l o Paudice, M a r i a g r a z i a Bella a n d M a u r i z i o Raiteri * lstituto di Farmacologiae Farmacognosia, Universit~di Genova, Viale Cembrano 4, 16148 Genova, Italy
Received 30 July 1986, accepted 12 August 1986
The effects of acetylcholine (ACh) on the release of [3H]5-hydroxytryptamine ([3H]5-HT) were investigated in synaptosomes prepared from rat cerebral cortex, hypothalamus and hippocampus and depolarized with 15 mM KCI under superfusion conditions. ACh inhibited the release of [3H]5-HT in all three brain ares. This effect was not modified by hexamethonium but was antagonized by atropine and by the non-classical antagonists pirenzepine and dicyclomine. Serotonin release; Muscarinic receptor subtypes; Dicyclomine; Pirenzepine; (Superfused synaptosomes)
1. Introduction The cholinergic modulation of serotonin (5-HT) release has been little investigated. The basal release of [3H]5-HT newly synthesized from [3H] tryptophan in rat hypothalamic slices (H6ry et al., 1977), as well as the basal release of previously accumulated [3H]5-HT or of endogenous 5-HT in rat striatal slices (Westfall et al., 1983) were found to be increased following activation of nicotinic receptors. Inhibition of the basal release of newly formed [3H]5-HT from rat hypothalamic slices was observed instead when muscarinic receptors were activated (H~ry et al., 1977). Atropine-sensitive muscarinic receptors were found also to mediate the inhibition of the K +-evoked release of [3H]5-HT from hypothalamic slices (Ennis and Cox, 1982). The present study of the effects of muscarinic receptor activation on the release of 5-HT originated from the following considerations: (1) since cholinergic and serotoninergic innervations of dif-
* To whom all correspondence should be addressed. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
ferent brain areas have different origins, it was important to investigate different cerebral regions. (2) In experiments with brain slices the use of acetylcholine (ACh) is not easy due to its rapid inactivation. Moreover, the anatomical localization of the receptors involved in release regulation cannot be determined directly with brain slices. We have utilized superfused synaptosomes, an experimental condition in which ACh can be used without acetylcholinesterase inhibitors and which allows to ascertain directly whether a receptor is located on nerve endings. (3) Since the existence of muscarinic receptor subtypes is well accepted (see for reviews: Hirschowitz et al., 1984), we have characterized the muscarinic receptors involved in the modulation of 5-HT release in terms of receptor subtypes.
2. Materials and methods 2.1. Preparation of synaptosomes
Nerve endings were prepared from rat cerebral cortex, hypothalamus and hippocampus of adult
354 S p r a g u e - D a w l e y rats ( 2 0 0 - 2 5 0 g) essentially acc o r d i n g to G r a y a n d W h i t t a k e r (1962). The pellet (P2 fraction) was r e s u s p e n d e d (at a protein conc e n t r a t i o n of 0.5-0.6 m g / m l ) in 5 ml of m e d i u m having the following c o m p o s i t i o n ( m M ) : N a C I 125, KC1 3, M g S O 4 1.2, CaCI 2 1.2, N a H z P O 4 1.2, N a H C O 3 20 a n d glucose 10 (aeration with 95% 02 a n d 5% C O 2 at 3 7 ° C ) p H 7.2-7.4.
2.4. Drugs
2.2. Release experiments"
SJS,
T h e release of [3H]5-HT was studied using a superfusion a p p a r a t u s (see Raiteri et al., 1974; Raiteri a n d Levi, 1978). Briefly, the s y n a p t o s o m e suspension was p r e i n c u b a t e d for 10 min at 3 7 ° C ; [ 3 H ] 5 - H T (specific activity 29.6 C i / m m o l ; N E N ) was a d d e d to give a final c o n c e n t r a t i o n of 0.03 /~M. U p t a k e was s t o p p e d after 15 rain of i n c u b a tion b y a d d i n g 100 ml of m e d i u m a n d the labelled nerved endings were d i s t r i b u t e d in the superfusion c h a m b e r s . The s y n a p t o s o m e s were then superfused at 3 7 ° C with c o n t i n u o u s l y o x y g e n a t e d m e d i u m at the rate of 0.6 m l / m i n . The nerve endings were s t i m u l a t e d for two p e r i o d s of 45 s each (S 1, $2), starting after 38 a n d 66 min of superfusion, b y raising the K + c o n c e n t r a t i o n of the m e d i u m to 15 m m o l / l . M u s c a r i n i c agonists or a n t a g o n i s t s were a d d e d c o n c o m i t a n t l y with KC1 at min 66 of superfusion ($2). The s t i m u l a t i o n e v o k e d release of tritium was c a l c u l a t e d b y subtraction of the b a s a l outflow. The latter was the average of the tritium r a d i o a c t i v i t y present in the two 3 min fractions collected before a n d after K + stimulation. The release evoked was expressed as a p e r c e n t a g e of the tritium c o n t e n t of the s y n a p t o somes at the onset of the respective s t i m u l a t i o n p e r i o d s (S 1 a n d $2). D r u g effects were e v a l u a t e d b y calculating the ratio (S2/Sx) of the 3H release e v o k e d d u r i n g the two s t i m u l a t i o n periods. T h e statistical significance of differences was d e t e r m i n e d b y analysis of variance (one-way).
2.3. Separation of [ 3H]5-HT from ~H-metabolites T h e [3H]5-HT in the fractions a n d in the superfused s y n a p t o s o m e s was d e t e r m i n e d after sepa r a t i o n from 3 H - m e t a b o l i t e s (Raiteri et al., 1984). T h e a m o u n t of [3H]5-HT f o u n d in s y n a p t o s o m e s a c c o u n t e d for 45-55% of total 3H radioactivity.
A C h chloride, a t r o p i n e sulfate, h e x a m e t h o n i u m c h l o r i d e a n d o x o t r e m o r i n e were p u r c h a s e d from Sigma Chemical Co. (St. Louis, MO). The following drugs were gifts: p i r e n z e p i n e d i h y d r o c h l o r i d e ( l s t . D e Angeli, Milano, Italy) a n d d i c y c l o m i n e ( C a m i i l o Corvi, Piacenza, Italy).
®
16 14
T
12
T
I0
T
08 08 04 02
+
+
Q
Q
§ [27)
(21)
(21}
(201
(81
SdS,
®
10 .I_
08 06 +
04 02
[80]
[151
[211
t21]
(80]
t15]
(22]
Fig. 1. Effects of acetylcholine receptor agonists and antagonists on the K+-induced release of [3H]5-HT from rat hypothalamic (A), and cortical (B) synaptosomes. Nerve endings prelabeled with the radioactive indoleamine were superfused and stimulated for two periods of 45 s each (S], S2) by increasing the K + concentration of the medium to 15 mmol/1. Data are expressed as the ratio S2/SI of the K+-induced [3H]5-HT or 3H release evoked by the two stimulation periods. The basal [3H]5-HT release from hypothalamic synaptosomes amounted to 0.57_+ 0.04% of the total [3H]5-HT. The K ~-evoked release (S~) in the controls was 2.01 + 0.12%. The basal tritium efflux from cortical synaptosomes amounted to 2.11 _+0.10% and the K+-evoked release (S l) in the controls was 3.21 + 0.39%. The data represent the means_+S.E.M, and the number of separate determinations is reported in parentheses. *P < 0.05: **P < 0.01 compared to the control (ANOVA test). HEXA = hexamethonium; OXO = oxotremorine; A = atropine.
355 3. Results
Figure 1A shows that ACh (10 /tM) inhibited the release of [3H]5-HT from rat hypothalamic synaptosomes exposed in superfusion to 15 mM KC1. The inhibitory action of ACh was not counteracted by hexamethonium (10/~M). However, 10 /~M ACh was totally antagonized by 1 #M atropine. The release of [3H]5-HT was inhibited by oxotremorine. The results obtained by measuring tritium radioactivity were qualitatively superimposable to those obtained when [3H]5-HT was separated from metabolites. The results of experiments carried out with cerebral cortex synaptosomes are illustrated in fig. lB. Acetylcholine dose dependently inhibited the SdSi 14 12
®
T
T
T L
K+-induced [3H]5-HT release. Oxotremorine mimicked ACh. Atropine, but not hexamethonium, antagonized ACh. The effect of ACh on [3H]5-HT release was also studied in hippocampus synaptosomes. While significant, the inhibitory action of ACh was less pronounced in this area than in cortex and hypothalamus (not shown). Atropine or hexamethonium, when tested alone, did not cause changes of [3H]5-HT release in the areas considered (not shown). The data obtained with the muscarinic antagonists dicyclomine and pirenzepine are shown in fig. 2. Both in hypothalamus (fig. 2A) and in the cerebral cortex (fig. 2B) the inhibitory action of ACh (10/tM) on the K+-evoked release of [3H]5H T was concentration dependently antagonized by dicyclomine or pirenzepine. Dicycl0mine or pirenzepine themselves did not affect [3H]5-HT release (not shown).
10
4. Discussion 0.8 E.
06 04
._= +
+
c~
c~
(8}
(TJ
02
(12}
(12)
(12)
S2/Sl
®
r2 10
T
T
08 86 04 82
116}
[16)
(101
[8}
(9}
{101
Fig. 2. Effectsof dicyclomineand pirenzepineon the inhibition of the K +-induced releaseof [3H]5-HT from hypothalamic(A) and cortical (B) synaptosomes by ACh. The procedure was similar to that described in the legend to fig. 1. The data represent the means+S.E.M, and the number of separate determinations is reported in parentheses. *P < 0.05; **P < 0.01 compared to the control (ANOVA test). DICY= dicyclomine; PIRE = pirenzepine.
Studying the spontaneous release of newly synthesized [3H]5-HT from rat hypothalamic slices Hdry et al. (1977) found that nicotinic agonists, including ACh, potentiated whereas the muscarinic agonists oxotremorine inhibited the release of the indoleamine. The authors hypothesized the presence of cholinoceptors on serotoninergic terminals but also considered alternative explanations implying indirect mechanisms. We have examined the effects of ACh on the depolarization-evoked release of [3H]5-HT previously taken up into synaptosomes prepared from various areas of the rat brain, including hypothalamus. At the concentration (10 /zM) used by Hdry et al. (1977) ACh did not enhance the 15 mM K+-evoked release of [3H]5-HT. Even ACh 100 /~M did not enhance [3H]5-HT release but decreased it instead. However, the presence of the nicotinic antagonist hexamethonium did not increase the inhibitory action of ACh as one would have expected if ACh was activating inhibitory (muscarinic) and excitatory (nicotinic) receptors concomitantly. It is possible that nicotinic receptor activation potentiates only the spontaneous
356 release of 5-HT without affecting the release evoked by depolarization. Another possibility is that newly synthesized and taken up [3H]5-HT are differentially sensitive to nicotinic receptor activation. A third possibility is that nicotinic receptors are not located on serotoninergic terminals. Muscarinic receptor activation (by ACh or oxotremorine) inhibited the K÷-evoked release of previously accumulated [3H]5-HT in synaptosomes obtained from rat hypothalamus, cerebral cortex and hippocampus. These data are consistent with those obtained when hypothalamic slices were used to study either the basal release of newly synthesized [3H]5-HT (H6ry et al., 1977) or the K÷-evoked release of preaccumulated [3H]5H T (Ennis and Cox, 1982). Our results with isolated nerve endings in superfusion, a condition in which indirect effects between transmitters are minimized, provide direct demonstration that 5-HT nerve terminals in rat cortex, hypothalamus and hippocampus are endowed with muscarinic receptors whose activation by ACh or muscarinic agonists brings about inhibition of 5-HT release. The existence of subtypes of muscarinic binding sites in the CNS is well documented (for reviews see: Hirschowitz et al., 1984). These different binding sites appear to reflect different receptors. It was found in a study comparing the pharmacological characteristics of the muscarinic autoreceptors regulating ACh release and the muscarinic heteroreceptors located on dopamine terminals and mediating the potentiation of dopamine release that the action of ACh was blocked by dicyclomine or by pirenzepine only at the heteroreceptors (Marchi and Raiteri, 1985). The autoreceptors were relatively insensitive to these muscarinic antagonists. On the other hand, atropine, scopolamine or quinuclydinylbenzylate blocked equally well both the auto- and the heteroreceptors (Raiteri et al., 1984; Marchi and Raiteri, 1985). The results obtained with dicyclomine and pirenzepine (fig. 2A,B) suggest that the muscarinic presynaptic heteroreceptors regulating the depolarization-evoked release of [3H]5H T from cortical and hypothalamic nerve endings differ pharmacologically from the muscarinic autoreceptors, where these compounds have been shown to be poorly effective (Marchi and Raiteri,
1985). It seems premature however to use the M~ nomenclature for the presynaptic receptors characterized in this study. The following main conclusions can be drawn from the present investigation: (1) the muscarinic receptors mediating the inhibition of 5-HT release previously identified by H6ry et al. (1977) and by Ennis and Cox (1982) in slices of rat hypothalamus are localized on serotoninergic nerve endings. (2) Three muscarinic receptors in the cerebral cortex of the rat have now been characterized in terms of localization, function and pharmacology: (a) an autoreceptor located on cholinergic terminals, mediating inhibition of ACh release, insensitive to dicyclomine or pirenzepine (Marchi and Raiteri, 1985); (b) a heteroreceptor, located on dopaminergic nerve endings, mediating potentiation of dopamine release, sensitive to dicyclomine or pirenzepine (Marchi and Raiteri, 1985) and (c) a heteroreceptor, located on serotoninergic terminals, mediating inhibition of 5-HT release, dicyclomine- and pirenzepine-sensitive.
Acknowledgements This work was supported by grants from the Italian Ministry of Education and from the Italian National Research Council. The authors wish to thank Mrs. Maura Agate for her expert secretarial assistance.
References Ennis, C. and B. Cox, 1982, Inhibitory muscarinic receptors modulate the potassium-evoked release of [3H]serotonin from rat hypothalamic slices, European J. Pharmacol. 81, 159. Gray, E.G. and V.P. Whittaker, 1962, The isolation of nerve endings from brain: an electron-microscopicstudy of cell fragments derived by homogenization and centrifugation, J. Anat. (London) 96, 79. H6ry, F., S. Bourgoin, M. Hamon, J.P. Ternaux and J. Glowinski, 1977, Control of the release of newly synthesized 3H-5-hydroxytryptamine by nicotinic and muscarinic receptors in rat hypothalamic slices, Naunyn-Schmiedeb, Arch. Pharmacol. 296, 91. Hirschowitz, B.I., R. Hammer, A. Giacheni, J.J. Keirns and R.R. Levine(eds.), 1984, Subtypes of Muscarinic Receptors (Elsevier North Holland). Marchi, M. and M. Raiteri, 1985, On the presence in the cerebral cortex of muscarinic receptor subtypes which dif-
357 fer in neuronal localization, function and pharmacological properties, J. Pharmacol. Exp. Ther. 235, 230. Raiteri, M., F. Angelini and G. Levi, 1974, A simple apparatus for studying the release of neurotransmitters from synaptosomes, European J. Pharmacol. 25, 411. Raiteri, M., R. Leardi and M. Marchi, 1984, Heterogeneity of presynaptic muscarinic receptors regulating neurotransmitter release in the rat brain, J. Pharmacol. Exp. Ther. 228, 209.
Raiteri, M. and G. Levi, 1978, Release mechanisms for catecholamines and serotonin in synaptosomes, in: Reviews of Neuroscience, Vol. 3, eds. S. Ehrenpreis and I. Kopin (Raven Press, New York) p. 77. Westfall, T.C., H. Grant and H. Perry, 1983, Release of dopamine and 5-hydroxytryptamine from rat striatal slices following activation of nicotinic cholinergic receptors, Gen. Pharmacol. 14, 321.