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Chapter 15 Presynaptic modulation of transmitter release by nicotinic receptors
A. Nordberg. K. Fuxe. B. Holmstedt and A. Sundwall (Eds.) Progress in Brain Research, Vol. 79 0 1989 Elsevier Science Publishers B.V. (Biomedical Division)
157
CHAPTER 15
Presynaptic modulation of transmitter release by nicotinic receptors Susan Wonnacott, Jane Irons, Catherine Rapier, Beverley Thorne and George G. Lunt Department of Biochemistry, University of Bath, Bath BA2 7A Y, U.K.
Introduction Nicotine acts on many transmitter systems in different parts of the brain to promote transmitter release, and these modulatory actions may underlie some of the psychopharmacological and behavioural effects of nicotine (reviewed by Balfour, 1982). Although many of these early studies were ascribed to a direct presynaptic action of nicotine, mediated by nicotinic acetylcholine receptors on the nerve terminals, high concentrations of nicotine were commonly used and the nicotinic pharmacology of the effect was often poorly established. With increasing evidence in favour of multiple classes of putative nicotinic receptors in the brain from ligand binding studies (Wonnacott, 1987), protein chemistry (Whiting and Lindstrom, 1987) and molecular biology (Wada et al., 1988), we have re-examined the presynaptic actions of nicotine. Using isolated nerve terminals (synaptosomes) we can be confident that nicotine is acting presynaptically, and we have developed a superfusion technique permitting the repetitive stimulation of the preparation (Rapier et al., 1988). Perhaps the best characterised presynaptic action of nicotine concerns the enhancement of dopamine release in the striatum (reviewed by Chesselet, 1984), so our initial studies focussed on this system. Subsequently we have extended this research to the hippocampus. The phar-
macological profile of the presynaptic nicotinic receptor suggests a correlation with high affinity binding sites for [3H]nicotine in the brain, and leads us to propose a model for the presynaptic modulation of transmitter release by nicotinic receptors. Nicotinic modulation of dopamine release from striatal synaptosomes Nicotine provoked the release of [3H]dopamine from synaptosomes isolated from rat striata and preincubated with radiolabelled transmitter (Rapier et al., 1985, 1987, 1988). Nicotine-evoked release was concentration-dependent over the range lo-* to M, and the half maximal response was observed with 3.8 pM nicotine (Rapier et al., 1988). Although a dose-response relationship has not previously been reported for nicotine-evoked dopamine release in the striatum, Giorguieff-Chesselet et al. (1979a) demonstrated that 1 pM nicotine caused the release of [3H]dopamine (newly synthesised from [3H]tyrosine) from striatal slices. Nicotine-evoked dopamine release from nucleus accumbens (minced tissue) had an EC,, of 0.5 pM (Rowel1 et al., 1987). Moreover, low micromolar concentrations are likely to be in the range of smoking doses of nicotine. Clearly, studies using high ( > M) concentrations of nicotine are of dubious
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physiological significance, and such concentrations may promote transmitter release by other mechanisms (Arqueros et al., 1978). In Fig. 1, nicotine is compared with other agonists for their ability to stimulate [3H]dopamine release from striatal synaptosomes. At 1 pM (Fig. la), cytisine is marginally more potent while dimethylphenylpiperazinium (DMPP) is slightly weaker than (-)nicotine. The action of nicotine is markedly stereoselective. Indeed, dose-response curves for ( - ) and ( + )nicotine indicate that a 100200-
100-
8 O O r b. 10pM
c. 100pM
Fig. 1. Release of [3H]dopamine from striatal synaptosomes by nicotinic agonists. Agonists were compared at a single concentration of (a) 1 pM (b) 10 pM or (c) 100 pM for their ability to release [3H]dopamine from perfused synaptosomes (Rapier et al., 1988). Basal release was subtracted and stimulated release converted to fmol/mg protein by reference to the specific activity of the [3H]dopamine.
fold higher concentration of ( + )nicotine is necessary to elicit the half maximal response (Rapier et al., 1988). The lack of stereoselectivity reported by Connelly and Littleton (1983) for the nicotine-induced release of [3H]dopamine from whole brain synaptosomes may again reflect the high concentrations M) employed; taken together with the slight Ca2+ dependence and meagre inhibition by pempidine seen in this study it seems probable that non-specific mechanisms were contributing to the observed effects. Nicotine and acetylcholine were equipotent when compared at both 10 pM (Fig. lb) and 100 pM (Fig. lc) concentrations, and choline proved to be effective in releasing [3H]dopamine. The efficacy of nicotinic agonists favours a nicotinic receptor mechanism, and this is supported by the sensitivity of agonist-evoked [3H]dopamine release to nicotinic antagonists (Fig. 2). Thus release elicited by nicotine and DMPP could be inhibited by the ganglionic blocking drugs mecamylamine and pempidine, by dihydro-P-erythroidine which is effective at both ganglionic and neuromuscular synapses, and by the novel marine toxin neosurugatoxin (Rapier et al., 1985). The specificity of these agents was exemplified by their inability to inhibit transmitter release stimulated by a depolarising concentration of potassium (Fig. 2). Neosurugatoxin is a very potent ganglionic nicotinic antagonist (Hayashi et al., 1984) devoid of activity at the neurornuscular junction. In contrast, a-bungarotoxin failed to attenuate agonistevoked [3H]dopamine release (Fig. 2; Rapier et al., 1985). De Belleroche and Bradford (1978) previously reported a small inhibition by abungarotoxin (0.19 pM) of acetylcholine (0.3 mM) evoked release of [3H]dopamine from striatal synaptosomes, but this effect was not significant and possible contamination of the toxin by neuronal bungarotoxin was not excluded. Intrinsic nicotinic excitation demonstrated electrophysiologically in rat striatal slices (Misgeld et al., 1980) was suppressed by mecamylamine and dtubocurarine, but not by a-bungarotoxin. In
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agreement with the data from synaptosomes (Fig. 2), nicotine- or acetylcholine-induced release of [3H]dopaminefrom rat striatal slices was partially blocked by mecamylamine and pempidine (Giorguieff et al., 1976, 1977; Giorguieff-Chesselet et al., 1979a), consistent with a presynaptic nicotinic receptor of the ganglionic (C6) type. The stereoselectivity for ( - )nicotine displayed by this presynaptic receptor and its sensitivity to neosurugatoxin but not a-bungarotoxin are also characteristic of the high affinity binding sites for [3H]nicotine in rat brain (Rapier et al., 1985; Wonnacott, 1986) and distinguish this ligand bin1 p M nicotine
1
2
3
4
5
4
5
1 pM DMPP
r
1
2
3
2OmM K C I
Fig. 2. Effect of nicotinic antagonists on evoked [3H]dopamine release. Synaptosomes loaded with [3H]dopamine were perfused in the presence or absence of antagonist for 20 min before stimulation with nicotinic agonists (1 pM) or KCI (20 mM). Evoked release in the presence of antagonist is presented as a percentage of the control response in the absence of antagonist. 1: Mecamylamine (5 pM); 2: pempidine (5 pM); 3: neosurugatoxin (0.05 pM); 4:dihydro-P-erythroidine (0.5 pM); 5: a-bungarotoxin (0.25 pM).
ding site from that for a-[1251]bungarotoxin. On this evidence we can propose that the presynaptic nicotinic receptor on dopaminergic nerve terminals and [3H]nicotine binding sites may be equivalent. There are moderate numbers of [3H]nicotine binding sites in the rat striatum compared with low levels of a-[1251]bungarotoxin binding sites (Clarke et al., 1985; Marks et al., 1986). Lesion experiments with 6-hydroxydopamine support the presence of both types of binding sites on dopaminergic nerve terminals in the striatum (de Belleroche et al., 1979; McGeer et al., 1979; Schwartz et al., 1984; Clarke and Pert, 1985). However, the functional significance of abungarotoxin binding sites in mammalian brain is presently unclear, whereas there is very good evidence for the nicotinic receptor status of [3H]nicotine binding sites. Immunoaffinity purification of the [3H]nicotine binding protein (Whiting and Lindstrom, 1987) followed by Nterminal amino acid sequencing of the agonist binding subunit (Whiting et al., 1987) demonstrates that this subunit is identical to the protein product of the a4-gene (coding for a nicotinic receptor asubunit) cloned from rat brain (Goldman et al., 1987). Functional expression in Xenopus oocytes of a4 in combination with the &-gene product (Boulter et al., 1987) results in depolarising responses to acetylcholine or nicotine (1 pM) that are blocked by neuronal bungarotoxin but not by abungarotoxin. These results are consistent with the agonist sensitivity of the presynaptic nicotinic receptor modulating dopamine release (Fig. 1) and its insensitivity to a-bungarotoxin (Fig. 2). Moreover, neuronal bungarotoxin inhibits nicotine-evoked dopamine release from striatal slices (Zigmond et al., this volume). The characteristic nicotinic depolarisations observed in the oocyte expression system imply that the receptor protein includes a n integral ion transduction mechanism. Nicotine-evoked [3H]dopamine release is inhibited by histrionicotoxin (Rapier et al., 1987), a non-competitive antagonist that acts at the ion channel of the muscle nicotinic receptor (Albuquerque et al., 1973). The
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concentration of histrionicotoxin producing 50% blockade of striatal [3H]dopamine release was 5 pM, in close agreement with the sensitivity of muscle responses (Spivak et al., 1982). These data suggest that the presynaptic receptor operates via a cation channel that may closely resemble that of the muscle receptor. This is also supported by their mutual sensitivity to ketamine (Rapier et al., 1987). A disparity between the presynaptic nicotinic receptor mediating dopamine release and high affinity binding sites for [3H]nicotine is the sensitivity of the former to antagonists such as mecamylamine and pempidine (Fig. 2) that fail to inhibit ligand binding (MacAllan et al., 1988). It is increasingly recognised however that such blocking drugs are likely to be non-competitive antagonists that act at the level of the ion channel (Varanda et al., 1985). The in vitro demonstration of nicotine-evoked dopamine release from striatal nerve terminals does not reveal the physiological significance of such a mechanism. There are cholinergic interneurones in the striatum (McGeer et al., 1975) in intimate association with dopaminergic terminals that could provide an endogenous source of nicotinic agonist. Acetylcholine-evoked release of [3H]dopamine from cat caudate nucleus in vivo a. 3H-ACh
has been demonstrated (Giorguieff et al., 1976) and was partially blocked by hexamethonium and mecamylamine. These researchers subsequently reported that the presynaptic enhancement of release is seen only in the absence of nigral activation (Giorguieff-Chesselet et al., 1979b). However, in this study substance P was employed to stimulate nigro-striatal neurones; substance P can itself inhibit nicotinic receptor activation (Eardley and McGee, 1985) and this may be an alternative explanation of the absence of response to acetylcholine under these conditions. The effects of nicotine in the presence of depolarising stimuli have not been assessed in striatal preparations in vitro. Nicotinic modulation of transmitter release from hippocampal synaptosomes
To address the question of how widespread presynaptic nicotinic receptors are in the mammalian brain, we have commenced a study of neurotransmitter release in the hippocampus. Micromolar concentrations of nicotine stimulate the release of both [3H]acetylcholine and [3H]GABA (Fig. 3) and the dose-response data for [3H]GABA reIease indicate that 5 pM nicotine
b. 3H-GABA 3H-ACh
'*,Iroot
-
20 40 60 Fraction number
T
1
3H-GABA
0.5p M DHpE
n O . l p L M QBGT
4L2-
60 Fraction number
Fig. 3. Nicotine-evoked transmitter release from hippocampal synaptosomes. Hippocampal synaptosomes preloaded with (a) [3H]choline or (b) [3H]GABA were stimulated with successive pulses (50 pl) of 10 pM (-)nicotine.' Transmitter release was monitored in successive fractions (350 pI) collected from the perfused preparation.
Fig. 4. Inhibition of nicotine-evoked transmitter release from hippocampal synaptosomes. Hippocampal synaptosomes preloaded with ['Hlcholine or [3H]GABA were perfused in the presence or absence of antagonist as described in the legend to Fig. 2.
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produces the half maximal response. The nicotinic stimulation of both transmitters is insensitive to abungarotoxin but inhibited by dihydro-p-erythroidine (Fig. 4). Histrionicotoxin also inhibits nicotine-evoked [3H]acetylcholine release (Rapier et al., 1987) and [3H]GABA release (Fig. 5 ) from hippocampal synaptosomes. Thus far the presynaptic nicotinic receptors on cholinergic and GABAergic terminals in the hippocampus appear identical to those responsible for the modulation of striatal dopamine release. Nicotinic autoreceptors on cholinergic terminals may subserve a physiological role in the feedback regulation of acetylcholine release. Hexamethonium sensitive nicotine- and DMPP-evoked release of acetylcholine from cortical synaptosomes has been reported (Rowell and Winkler, 1984), and a similar phenomenon has been demonstrated in synaptosomes of the myenteric plexus (Briggs and Cooper, 1982). The specific nicotinic agonist methylcarbamylcholine elicits [3H]acetylcholine release from hippocampal and cortical slices but not from striatal slices (Araujo et al., 1988) suggesting a regional specificity in the distribution of nicotinic autoreceptors. The presence of nicotinic receptors corresponding to [3H]nicotine binding
sites on cholinergic terminals in cortical and limbic regions may explain the deficits in these ligand binding sites in the brains of Alzheimer patients (Nordberg and Winblad, 1986; Whitehouse et al., 1986).
A model for the mechanism of nicotine-evoked transmitter release
From the data available concerning the presynaptic modulation of transmitter release by nicotine and other agonists, we can propose a model to account for this phenomenon (Fig. 6). Thus the receptor is shown schematically as a pentameric transmembrane protein as in the case of the muscle nicotinic receptor, except that the neuronal protein is likely to consist of only two types of subunits (Whiting and Lindstrom, 1987; Wada et al., 1988). The recognition site on the a-subunits will bind nicotine and other agonists, and the competitive antagonist dihydro-0-erythroidine, but unlike the corresponding subunit in muscle, the neuronal site has lost the ability to bind a-bungarotoxin. Instead, neosurugatoxin and neuronal bungarotoxin are likely to act in the vicinity of this site in neuronal but not muscle receptors.
Neosurugatox in
(abungorotox in insensitive)
T+
Local depolarisation
20
I
I
40
60
Fraction number
Fig. 5 . Inhibition of nicotine-evoked [3H]GABA release from hippocampal synaptosomes by perhydrohistrionicotoxin. Hippocampal synaptosomes preloaded with [3H]GABA were stimulated with successive pulses of (-)nicotine (10 pM). Perhydrohistrionicotoxin (H,,HTX; 10 pM) was introduced into the perfusion buffer for the period indicated. The response to nicotine is seen to recover after removal of the toxin.
4
opens voltage-dependent Ca++ channels
1
e n t r y of Ca+ +
t
Triggers transmitter release
Fig. 6 . Schematic model for the presynaptic modulation of transmitter release by nicotinic receptors.
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By analogy with the muscle nicotinic receptor, agonist binding is presumed to promote an allosteric change in the protein that opens an integral ion channel, permitting the influx of cations into the nerve terminal. This is supported by the histrionicotoxin sensitivity, and it is plausible that the non-competitive antagonists mecamylamine and pempidine also act at the channel. Nicotineevoked dopamine release in the striatum (Giorguieff-Chesselet et al., 1979a; Rapier et al., 1988) and methylcarbamylcholine-evoked acetylcholine release in the hippocampus (Araujo et al., 1988) is not inhibited by tetrodotoxin which indicates that the voltage-dependent Na+ channel is not involved in the response. The local depolarisation arising from cation flux through' the nicotinic channel results in Ca2+-dependent transmitter release. Omission of Ca2+ from the perfusion buffer resulted in a 60% inhibition of nicotineevoked release of [3H]dopamine (Rapier et al., 1988), [3H]GABA and [3H]acetylcholine. Similar Ca2+-dependence has been reported by other groups (e.g. Giorguieff-Chesselet et al., 1979a; Rowel1 et al., 1984, 1987) and accords with the Ca2 -dependence exhibited by K + - and veratridine-stimulated transmitter release (Rapier et al., 1988). Thus it is envisaged that neurotransmitter release triggered by nicotine occurs by the same Ca2+-dependent process as that induced by nerve stimulation. It remains to be established what contribution presynaptic nicotinic stimulation may make in the presence of cell firing and in concert with other presynaptic receptors, both inhibitory and stimulatory, that may be present on the same nerve terminal. Nevertheless, the widespread distribution of presynaptic nicotinic receptors in the brain makes these likely targets for nicotine, whereby it may subtly influence the synaptic output of many neurotransmitters. +
Acknowledgements This research was supported by grants from The Tobacco Advisory Council, R J Reynolds Tobacco Co., and The Mental Health Foundation, and
postgraduate studentships (to C.R. and B.T.) from The Science and Engineering Research Council.
References Albuquerque, E.X., Barnard, E.A., Chiu, T.H., Lapa, A.J., Dolly, J.O., Jansson, S.E., Daly, J.W. and Witkop, B. (1973) Acetylcholine receptor and ion conductance modulator sites at the murine neuromuscular junction: evidence from specific toxin reactions. Proc. Natl. Acad. Sci. U.S.A., 70: 949-953. Araujo, D.M., Lapchak, P.A., Collier, B. and Quirion, R. (1988) Characterisation of [3H]N-methylcarbamylcholine binding sites and effect of N-methylcarbamylcholine on acetylcholine release in rat brain. J. Neurochem., 51: 292 - 299. Arqueros, L., Naquira, D. and Zunino, E. (1978) Nicotine induced release of catecholamines from rat hippocampus and striatum. Biochem. Pharmacol., 27: 2667 - 2674. Balfour, D.J.K. (1982) The effects of nicotine on brain neurotransmitter sysrems. Pharmacol. Ther., 16: 269- 282. Boulter, J . , Connolly, J., Denens, E., Goldman, D., Heinemann, S. and Patrick, J . (1987) Functional expression of two neuronal nicotinic acetylcholine receptors from cDNA clones identifies a gene family. Proc. Nut/. Acad. Sci. U.S.A., 84: 7763-7769. Briggs. C.A. and Cooper, J.R. (1982) Cholinergic modulation of the release of ['H]acetylcholine from synaptosornes of the myenteric plexus. J . Neurochem., 38: 501 - 508. Chesselet, M.F. (1984) Presynaptic regulation of neurotransmitter release in the brain. Neuroscience, 12: 347 - 375. Clarke, P.B.S. and Pert, A. (1985) Autoradiographic evidence for nicotine receptors on nigrostriatal and mesolimbic dopaminergic neurons. Bruin Res., 348: 355 - 358. Clarke, P.B.S., Schwartz, R.D., Paul, S.M., Pert, C.B. and Pert, A. (1985) Nicotinic binding in rat brain: autoradiographic comparison of [3H]acetylcholine, [3H]nicotine and ['251]olbungarotoxin. J . Neurosci., 5: 1307- 1315. Connelly, M.S. and Littleton, J.M. (1983) Lack of stereoselectivity in ability of nicotine to release dopamine from rat synaptosomal preparations. J. Neurochem., 41 : 1297 - 1302. De Belleroche, J . and Bradford, H.F. (1978) Biochemical evidence for the presence of presynaptic receptors on dopaminergic nerve terminals. Brain Res., 142: 53 - 68. De Belleroche, J . , Lugmani, Y. and Bradford, H.F. (1979) Evidence for presynaptic cholinergic receptors on dopaminergic terminals: degeneration studies with 6hydroxydopamine. Neurosci. Lett., 11: 209-213. Eardley, D. and McGee, R. (1985) Both substance P agonists and antagonists inhibit ion conductance through nicotinic acetylcholine receptors on PC12 cells. Eur. J. Pharmacol., 114: 101-104.
163 Giorguieff, M.F., le Floc’h, M.L., Westfall, T.C., Glowinski, J. and Besson, M.J. (1976) Nicotinic effect of acetylcholine on the release of newly synthesised [3H]dopamine in rat striatal slices and cat caudate nucleus. Brain Rex, 106: 117- 131. Giorguieff, M.F., le Floc’h, M.L., Glowinski, J . and Besson, M.J. (1977) Involvement of cholinergic presynaptic receptors of nicotinic and muscarinic types in the control of the spontaneous release of dopamine from striatal dopaminergic terminals in the rat. J . Pharmacol. Exp. Ther., 200: 535 - 544. Giorguieff-Chesselet, M.F., Kernel, M.L., Wandscheer, D. and Glowinski. J. (1979a) Regulation of dopamine release by presynaptic nicotinic receptors in rat striatal slices: effect of nicotine in a low concentration. Life Sci., 25: 1257 - 1262. Giorguieff-Chesselet, M.F., Kernel, M.F. and Glowinski, J . (1979b) The presynaptic stimulating effect of acetylcholine on dopamine release is suppressed during activation of nigrostriatal dopaminergic neurons in the cat. Neurosci. Lett., 14: 177- 182. Goldman, D., Deneris, E., Luyten, W., Kochhar, A., Patrick, J. and Heinemann, S. (1987) Members of a nicotinic acetylcholine receptor gene family are expressed in different regions of the mammalian central nervous system. Cell, 48: 965 - 973. Hayashi, E., Isogai, M., Kagawa, Y., Takayanagi, N. and Yamada, S. (1984) Neosurugatoxin, a specific antagonist of nicotinic acetylcholine receptors. J. Neurochem., 42: 1491 - 1494. MacAllan, D., Lunt, G.G., Wonnacott, S., Swanson, K., Rapoport, J. and Albuquerque, E.X. (1988) Methyllycaconitine and anatoxin-a differentiate between nicotinic receptors in vertebrate and invertebrate nervous systems. FEBS Lett., 226: 357 - 363. Marks, M.J., Slitzel, J.A., Romm, E., Wehner, J.M. and Collins, A.C. (1986) Nicotinic binding sites in rat and mouse brain: comparison of acetylcholine, nicotine and abungarotoxin. Mol. Pharmacol., 30: 427 -436. McGeer, E.G., McGeer, P.L., Grewaal, D.S. and Singh, V.K. (1975) Striatal cholinergic interneurones and their relation t o dopaminergic nerve endings. J. Pharmacol. (Paris), 6: 143 - 152. McGeer, P.L., McGeer, E.G. and Innanen, V.T. (1979) Dendro axonic transmission. I . Evidence from receptor binding of dopaminergic and cholinergic agents. Brain Res., 169: 433-441. Misgeld, V., Weiler, M.H. and Bak, I.J. (1980) Intrinsic cholinergic excitation in the rat neostriatum: nicotinic and muscarinic receptors. Exp. Brain Res., 39: 401 - 409. Nordberg, A. and Winblad, B. (1986) Reduced number of [3H]nicotine and [3H]acetylcholine binding sites in the frontal cortex of Alzheimer brains. Neurosci. Lett., 72: 115-121.
Rapier, C., Harrison, R., Lunt, G.G. and Wonnacott, S. (1985) Neosurugatoxin blocks nicotinic acetylcholine receptors in the brain. Neurochem. Int., 7: 389 - 396. Rapier, C., Wonnacott, S., Lunt, G.G. and Albuquerque, E.X. (1987) The neurotoxin histrionicotoxin interacts with the putative ion channel of the nicotinic acetylcholine receptors in the central nervous system. FEBS Lett., 212: 292 - 296. Rapier, C., Lunt, G.G. and Wonnacott, S. (1988) Stereoselective nicotine-induced release of doparnine from striatal synaptosomes: concentration dependence and repetitive stimulation. J . Neurochem., 50: 1123 - 1130. Rowell, P.P. and Winkler, D.L. (1984) Nicotinic stimulation of [3H]acetylcholine release from mouse cerebral cortical synaptosomes. J. Neurochem., 43: 1593 - 1598. Rowell, P.P., Carr, L.A. and Garner, A.C. (1987) Stimulation of [3H]dopamine release by nicotine in rat nucleus accumbens. J. Neurochem., 49: 1449 - 1454. Schwartz, R.D., Lehmann, J. and Kellar, K.J. (1984) Presynaptic nicotinic cholinergic receptors labelled by [3H]acetylcholine on catecholamine and serotonin axons in brain. J. Neurochem., 42: 1495- 1498. Spivak, C.E., Maleque, M.A., Oliveira, A.C., Masukawa, L.M., Tokuyama, T., Daly, J.W. and Albuquerque, E.X. (1982) Actions of the histrionicotoxins at the ion channel of the nicotinic acetylcholine receptor and at the voltagesensitive ion channels of muscle membranes. Mot. Pharmacol., 21: 351-361. Varanda, W.A., Aracava, Y., Sherby, S.M., VanMeter, W.G., Eldefrawi, M.E. and Albuquerque, E.X. (1985) The acetylcholine receptor of the neuromuscular junction recognises mecamylamine as a noncompetitive antagonist. Mot. Pharrnacol., 28: 128- 137. Wada, K., Ballivet, M., Boulter, J., Connolly, J., Wada, E., Deneris, E.S., Swanson, L.W., Heinemann, S. and Patrick, J. (1988) Functional expression of a new pharmacological subtype of brain nicotinic acetylcholine receptor. Science, 240: 330 - 332. Whitehouse, P.J., Martino, A.M., Antuono, P.G., Lowenstein, P.R., Coyle, J.T., Price, D.L. and Kellar, K. J. (1986) Nicotinic acetylcholine binding in Alzheimer’s disease. Brain Rex, 371: 146-151. Whiting, P. and Lindstrom, J . (1987) Purification and characterisation of a nicotinic acetylcholine receptor from rat brain. Proc. Natl. Acad. Sci. U.S.A., 84: 595-599. Whiting, P., Esch, F., Schimasaki, S. and Lindstrom, J . (1987) Neuronal nicotinic acetylcholine receptor 6-subunit is coded for by the cDNA clone a4.FEBS Lett., 219: 459-463. Wonnacott, S. (1986) aBungarotoxin binds to low affinity nicotine binding sites in rat brain. J. Neurochem., 47: 1706- 1712. Wonnacott, S. (1987) Brain nicotine binding sites. Human Toxicol.. 6: 343 - 353.
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CHAPTER 15
Presynaptic modulation of transmitter release by nicotinic receptors Susan Wonnacott, Jane Irons, Catherine Rapier, Beverley Thorne and George G. Lunt Department of Biochemistry, University of Bath, Bath BA2 7A Y, U.K.
Introduction Nicotine acts on many transmitter systems in different parts of the brain to promote transmitter release, and these modulatory actions may underlie some of the psychopharmacological and behavioural effects of nicotine (reviewed by Balfour, 1982). Although many of these early studies were ascribed to a direct presynaptic action of nicotine, mediated by nicotinic acetylcholine receptors on the nerve terminals, high concentrations of nicotine were commonly used and the nicotinic pharmacology of the effect was often poorly established. With increasing evidence in favour of multiple classes of putative nicotinic receptors in the brain from ligand binding studies (Wonnacott, 1987), protein chemistry (Whiting and Lindstrom, 1987) and molecular biology (Wada et al., 1988), we have re-examined the presynaptic actions of nicotine. Using isolated nerve terminals (synaptosomes) we can be confident that nicotine is acting presynaptically, and we have developed a superfusion technique permitting the repetitive stimulation of the preparation (Rapier et al., 1988). Perhaps the best characterised presynaptic action of nicotine concerns the enhancement of dopamine release in the striatum (reviewed by Chesselet, 1984), so our initial studies focussed on this system. Subsequently we have extended this research to the hippocampus. The phar-
macological profile of the presynaptic nicotinic receptor suggests a correlation with high affinity binding sites for [3H]nicotine in the brain, and leads us to propose a model for the presynaptic modulation of transmitter release by nicotinic receptors. Nicotinic modulation of dopamine release from striatal synaptosomes Nicotine provoked the release of [3H]dopamine from synaptosomes isolated from rat striata and preincubated with radiolabelled transmitter (Rapier et al., 1985, 1987, 1988). Nicotine-evoked release was concentration-dependent over the range lo-* to M, and the half maximal response was observed with 3.8 pM nicotine (Rapier et al., 1988). Although a dose-response relationship has not previously been reported for nicotine-evoked dopamine release in the striatum, Giorguieff-Chesselet et al. (1979a) demonstrated that 1 pM nicotine caused the release of [3H]dopamine (newly synthesised from [3H]tyrosine) from striatal slices. Nicotine-evoked dopamine release from nucleus accumbens (minced tissue) had an EC,, of 0.5 pM (Rowel1 et al., 1987). Moreover, low micromolar concentrations are likely to be in the range of smoking doses of nicotine. Clearly, studies using high ( > M) concentrations of nicotine are of dubious
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physiological significance, and such concentrations may promote transmitter release by other mechanisms (Arqueros et al., 1978). In Fig. 1, nicotine is compared with other agonists for their ability to stimulate [3H]dopamine release from striatal synaptosomes. At 1 pM (Fig. la), cytisine is marginally more potent while dimethylphenylpiperazinium (DMPP) is slightly weaker than (-)nicotine. The action of nicotine is markedly stereoselective. Indeed, dose-response curves for ( - ) and ( + )nicotine indicate that a 100200-
100-
8 O O r b. 10pM
c. 100pM
Fig. 1. Release of [3H]dopamine from striatal synaptosomes by nicotinic agonists. Agonists were compared at a single concentration of (a) 1 pM (b) 10 pM or (c) 100 pM for their ability to release [3H]dopamine from perfused synaptosomes (Rapier et al., 1988). Basal release was subtracted and stimulated release converted to fmol/mg protein by reference to the specific activity of the [3H]dopamine.
fold higher concentration of ( + )nicotine is necessary to elicit the half maximal response (Rapier et al., 1988). The lack of stereoselectivity reported by Connelly and Littleton (1983) for the nicotine-induced release of [3H]dopamine from whole brain synaptosomes may again reflect the high concentrations M) employed; taken together with the slight Ca2+ dependence and meagre inhibition by pempidine seen in this study it seems probable that non-specific mechanisms were contributing to the observed effects. Nicotine and acetylcholine were equipotent when compared at both 10 pM (Fig. lb) and 100 pM (Fig. lc) concentrations, and choline proved to be effective in releasing [3H]dopamine. The efficacy of nicotinic agonists favours a nicotinic receptor mechanism, and this is supported by the sensitivity of agonist-evoked [3H]dopamine release to nicotinic antagonists (Fig. 2). Thus release elicited by nicotine and DMPP could be inhibited by the ganglionic blocking drugs mecamylamine and pempidine, by dihydro-P-erythroidine which is effective at both ganglionic and neuromuscular synapses, and by the novel marine toxin neosurugatoxin (Rapier et al., 1985). The specificity of these agents was exemplified by their inability to inhibit transmitter release stimulated by a depolarising concentration of potassium (Fig. 2). Neosurugatoxin is a very potent ganglionic nicotinic antagonist (Hayashi et al., 1984) devoid of activity at the neurornuscular junction. In contrast, a-bungarotoxin failed to attenuate agonistevoked [3H]dopamine release (Fig. 2; Rapier et al., 1985). De Belleroche and Bradford (1978) previously reported a small inhibition by abungarotoxin (0.19 pM) of acetylcholine (0.3 mM) evoked release of [3H]dopamine from striatal synaptosomes, but this effect was not significant and possible contamination of the toxin by neuronal bungarotoxin was not excluded. Intrinsic nicotinic excitation demonstrated electrophysiologically in rat striatal slices (Misgeld et al., 1980) was suppressed by mecamylamine and dtubocurarine, but not by a-bungarotoxin. In
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agreement with the data from synaptosomes (Fig. 2), nicotine- or acetylcholine-induced release of [3H]dopaminefrom rat striatal slices was partially blocked by mecamylamine and pempidine (Giorguieff et al., 1976, 1977; Giorguieff-Chesselet et al., 1979a), consistent with a presynaptic nicotinic receptor of the ganglionic (C6) type. The stereoselectivity for ( - )nicotine displayed by this presynaptic receptor and its sensitivity to neosurugatoxin but not a-bungarotoxin are also characteristic of the high affinity binding sites for [3H]nicotine in rat brain (Rapier et al., 1985; Wonnacott, 1986) and distinguish this ligand bin1 p M nicotine
1
2
3
4
5
4
5
1 pM DMPP
r
1
2
3
2OmM K C I
Fig. 2. Effect of nicotinic antagonists on evoked [3H]dopamine release. Synaptosomes loaded with [3H]dopamine were perfused in the presence or absence of antagonist for 20 min before stimulation with nicotinic agonists (1 pM) or KCI (20 mM). Evoked release in the presence of antagonist is presented as a percentage of the control response in the absence of antagonist. 1: Mecamylamine (5 pM); 2: pempidine (5 pM); 3: neosurugatoxin (0.05 pM); 4:dihydro-P-erythroidine (0.5 pM); 5: a-bungarotoxin (0.25 pM).
ding site from that for a-[1251]bungarotoxin. On this evidence we can propose that the presynaptic nicotinic receptor on dopaminergic nerve terminals and [3H]nicotine binding sites may be equivalent. There are moderate numbers of [3H]nicotine binding sites in the rat striatum compared with low levels of a-[1251]bungarotoxin binding sites (Clarke et al., 1985; Marks et al., 1986). Lesion experiments with 6-hydroxydopamine support the presence of both types of binding sites on dopaminergic nerve terminals in the striatum (de Belleroche et al., 1979; McGeer et al., 1979; Schwartz et al., 1984; Clarke and Pert, 1985). However, the functional significance of abungarotoxin binding sites in mammalian brain is presently unclear, whereas there is very good evidence for the nicotinic receptor status of [3H]nicotine binding sites. Immunoaffinity purification of the [3H]nicotine binding protein (Whiting and Lindstrom, 1987) followed by Nterminal amino acid sequencing of the agonist binding subunit (Whiting et al., 1987) demonstrates that this subunit is identical to the protein product of the a4-gene (coding for a nicotinic receptor asubunit) cloned from rat brain (Goldman et al., 1987). Functional expression in Xenopus oocytes of a4 in combination with the &-gene product (Boulter et al., 1987) results in depolarising responses to acetylcholine or nicotine (1 pM) that are blocked by neuronal bungarotoxin but not by abungarotoxin. These results are consistent with the agonist sensitivity of the presynaptic nicotinic receptor modulating dopamine release (Fig. 1) and its insensitivity to a-bungarotoxin (Fig. 2). Moreover, neuronal bungarotoxin inhibits nicotine-evoked dopamine release from striatal slices (Zigmond et al., this volume). The characteristic nicotinic depolarisations observed in the oocyte expression system imply that the receptor protein includes a n integral ion transduction mechanism. Nicotine-evoked [3H]dopamine release is inhibited by histrionicotoxin (Rapier et al., 1987), a non-competitive antagonist that acts at the ion channel of the muscle nicotinic receptor (Albuquerque et al., 1973). The
160
concentration of histrionicotoxin producing 50% blockade of striatal [3H]dopamine release was 5 pM, in close agreement with the sensitivity of muscle responses (Spivak et al., 1982). These data suggest that the presynaptic receptor operates via a cation channel that may closely resemble that of the muscle receptor. This is also supported by their mutual sensitivity to ketamine (Rapier et al., 1987). A disparity between the presynaptic nicotinic receptor mediating dopamine release and high affinity binding sites for [3H]nicotine is the sensitivity of the former to antagonists such as mecamylamine and pempidine (Fig. 2) that fail to inhibit ligand binding (MacAllan et al., 1988). It is increasingly recognised however that such blocking drugs are likely to be non-competitive antagonists that act at the level of the ion channel (Varanda et al., 1985). The in vitro demonstration of nicotine-evoked dopamine release from striatal nerve terminals does not reveal the physiological significance of such a mechanism. There are cholinergic interneurones in the striatum (McGeer et al., 1975) in intimate association with dopaminergic terminals that could provide an endogenous source of nicotinic agonist. Acetylcholine-evoked release of [3H]dopamine from cat caudate nucleus in vivo a. 3H-ACh
has been demonstrated (Giorguieff et al., 1976) and was partially blocked by hexamethonium and mecamylamine. These researchers subsequently reported that the presynaptic enhancement of release is seen only in the absence of nigral activation (Giorguieff-Chesselet et al., 1979b). However, in this study substance P was employed to stimulate nigro-striatal neurones; substance P can itself inhibit nicotinic receptor activation (Eardley and McGee, 1985) and this may be an alternative explanation of the absence of response to acetylcholine under these conditions. The effects of nicotine in the presence of depolarising stimuli have not been assessed in striatal preparations in vitro. Nicotinic modulation of transmitter release from hippocampal synaptosomes
To address the question of how widespread presynaptic nicotinic receptors are in the mammalian brain, we have commenced a study of neurotransmitter release in the hippocampus. Micromolar concentrations of nicotine stimulate the release of both [3H]acetylcholine and [3H]GABA (Fig. 3) and the dose-response data for [3H]GABA reIease indicate that 5 pM nicotine
b. 3H-GABA 3H-ACh
'*,Iroot
-
20 40 60 Fraction number
T
1
3H-GABA
0.5p M DHpE
n O . l p L M QBGT
4L2-
60 Fraction number
Fig. 3. Nicotine-evoked transmitter release from hippocampal synaptosomes. Hippocampal synaptosomes preloaded with (a) [3H]choline or (b) [3H]GABA were stimulated with successive pulses (50 pl) of 10 pM (-)nicotine.' Transmitter release was monitored in successive fractions (350 pI) collected from the perfused preparation.
Fig. 4. Inhibition of nicotine-evoked transmitter release from hippocampal synaptosomes. Hippocampal synaptosomes preloaded with ['Hlcholine or [3H]GABA were perfused in the presence or absence of antagonist as described in the legend to Fig. 2.
161
produces the half maximal response. The nicotinic stimulation of both transmitters is insensitive to abungarotoxin but inhibited by dihydro-p-erythroidine (Fig. 4). Histrionicotoxin also inhibits nicotine-evoked [3H]acetylcholine release (Rapier et al., 1987) and [3H]GABA release (Fig. 5 ) from hippocampal synaptosomes. Thus far the presynaptic nicotinic receptors on cholinergic and GABAergic terminals in the hippocampus appear identical to those responsible for the modulation of striatal dopamine release. Nicotinic autoreceptors on cholinergic terminals may subserve a physiological role in the feedback regulation of acetylcholine release. Hexamethonium sensitive nicotine- and DMPP-evoked release of acetylcholine from cortical synaptosomes has been reported (Rowell and Winkler, 1984), and a similar phenomenon has been demonstrated in synaptosomes of the myenteric plexus (Briggs and Cooper, 1982). The specific nicotinic agonist methylcarbamylcholine elicits [3H]acetylcholine release from hippocampal and cortical slices but not from striatal slices (Araujo et al., 1988) suggesting a regional specificity in the distribution of nicotinic autoreceptors. The presence of nicotinic receptors corresponding to [3H]nicotine binding
sites on cholinergic terminals in cortical and limbic regions may explain the deficits in these ligand binding sites in the brains of Alzheimer patients (Nordberg and Winblad, 1986; Whitehouse et al., 1986).
A model for the mechanism of nicotine-evoked transmitter release
From the data available concerning the presynaptic modulation of transmitter release by nicotine and other agonists, we can propose a model to account for this phenomenon (Fig. 6). Thus the receptor is shown schematically as a pentameric transmembrane protein as in the case of the muscle nicotinic receptor, except that the neuronal protein is likely to consist of only two types of subunits (Whiting and Lindstrom, 1987; Wada et al., 1988). The recognition site on the a-subunits will bind nicotine and other agonists, and the competitive antagonist dihydro-0-erythroidine, but unlike the corresponding subunit in muscle, the neuronal site has lost the ability to bind a-bungarotoxin. Instead, neosurugatoxin and neuronal bungarotoxin are likely to act in the vicinity of this site in neuronal but not muscle receptors.
Neosurugatox in
(abungorotox in insensitive)
T+
Local depolarisation
20
I
I
40
60
Fraction number
Fig. 5 . Inhibition of nicotine-evoked [3H]GABA release from hippocampal synaptosomes by perhydrohistrionicotoxin. Hippocampal synaptosomes preloaded with [3H]GABA were stimulated with successive pulses of (-)nicotine (10 pM). Perhydrohistrionicotoxin (H,,HTX; 10 pM) was introduced into the perfusion buffer for the period indicated. The response to nicotine is seen to recover after removal of the toxin.
4
opens voltage-dependent Ca++ channels
1
e n t r y of Ca+ +
t
Triggers transmitter release
Fig. 6 . Schematic model for the presynaptic modulation of transmitter release by nicotinic receptors.
162
By analogy with the muscle nicotinic receptor, agonist binding is presumed to promote an allosteric change in the protein that opens an integral ion channel, permitting the influx of cations into the nerve terminal. This is supported by the histrionicotoxin sensitivity, and it is plausible that the non-competitive antagonists mecamylamine and pempidine also act at the channel. Nicotineevoked dopamine release in the striatum (Giorguieff-Chesselet et al., 1979a; Rapier et al., 1988) and methylcarbamylcholine-evoked acetylcholine release in the hippocampus (Araujo et al., 1988) is not inhibited by tetrodotoxin which indicates that the voltage-dependent Na+ channel is not involved in the response. The local depolarisation arising from cation flux through' the nicotinic channel results in Ca2+-dependent transmitter release. Omission of Ca2+ from the perfusion buffer resulted in a 60% inhibition of nicotineevoked release of [3H]dopamine (Rapier et al., 1988), [3H]GABA and [3H]acetylcholine. Similar Ca2+-dependence has been reported by other groups (e.g. Giorguieff-Chesselet et al., 1979a; Rowel1 et al., 1984, 1987) and accords with the Ca2 -dependence exhibited by K + - and veratridine-stimulated transmitter release (Rapier et al., 1988). Thus it is envisaged that neurotransmitter release triggered by nicotine occurs by the same Ca2+-dependent process as that induced by nerve stimulation. It remains to be established what contribution presynaptic nicotinic stimulation may make in the presence of cell firing and in concert with other presynaptic receptors, both inhibitory and stimulatory, that may be present on the same nerve terminal. Nevertheless, the widespread distribution of presynaptic nicotinic receptors in the brain makes these likely targets for nicotine, whereby it may subtly influence the synaptic output of many neurotransmitters. +
Acknowledgements This research was supported by grants from The Tobacco Advisory Council, R J Reynolds Tobacco Co., and The Mental Health Foundation, and
postgraduate studentships (to C.R. and B.T.) from The Science and Engineering Research Council.
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