Muscarinic acetylcholine receptor

P ro g ress in N e u r o b i o l o g y , 1978. Vol. 11. pp. 171-188. Pergamon Press Ltd. Printed in Great Britain

MUSCARINIC

ACETYLCHOLINE

RECEPTOR

EDITH HEILBRONN Unit of Biochemistry, National Defence Research Institute, Department 4, S-172 04 Sundbyber9 4, Sweden and TAMAS BARTFAI Department of Biochemistry, Arrhenius Laboratory, University of Stockholm, S-106 91 Stockholm, Sweden (Receiced 21 February 1978)

Contents 1. Introduction 2. Methods for measurement of muscarinic receptors 2.1. Physiologicalresponses 2.2. Biochemical responses 2.2.1. Indirect measurements 2.2.2. Direct measurements 3. Localization of the muscarinic receptor 3.1. Muscarinic innervation in the periphery 3.2. Muscarinic receptors in the CNS 3.3. Post- and pre-synaptic localization of the muscarinic receptor 4. Characterization of the receptor 4.1. Ligand structure 4.2. Binding of antagonists and agonists to the muscarinic receptor 4.3. Solubilization and purification attempts 5. Muscarinic binding and phosphatidyl inositol turnover 6. Muscarinic receptor mediated increases in cGMP levels 7. Model of muscarinic cholinergic synapse 8. Pharmacological significance 9. Current research on muscarinic receptors References

171 172 172 173 173 173 174 174 175 176 177 177 178 178 179 180 183 183 184 184

1. Introduction Acetylcholine, a putative neurotransmitter in the peripheral and in the central nervous system mediates two kinds of responses dependent on the identity of the neurotransmitter receptor (cf. for review Michaelson and Zeimal, 1973). The fast responses (latency < msec, durations 30-100 msec) to ACh such as contraction of skeletal muscle are mediated through nicotinic acetylcholine receptors. The slow responses (latency ~ 100 msec and duration ~ 0.5 sec) such as smooth muscle contraction are mediated through muscarinic acetylcholine receptors. The nicotinic receptors are stimulated by nicotine, carbachol and inhibited by o-tubocurarine and snake ~-toxins. The muscarinic receptors are stimulated by muscarine, acetylcholine, acetyl-fl-methylcholine, carbachol, bethanechol and inhibited by atropine, scopolamine, and 3-quinuclidinyl benzylate. The large kinetic differences between the two types of acetylcholine receptors are based on differences in their mechanism of action and are of physiological significance (Purves, 1976). The last decade represented a breakthrough in studies on neurotransmitter receptors. The most intensely studied receptors are the nicotinic acetylcholine receptors from the electric organs of eels and rays which were solubilized and purified by biospecific affinity chromatography on snake ~-toxin-Sepharose and other cholinergic ligands (Cohen et al., 1972; Karlsson et al., 1972; Klett et al., 1973). It was established that these receptors represented the ACh recognition site and it was suggested that they may also act as monovalent cation channel. Namely outflow of Na + from loaded "microsacs" was stimulated by nicotinic agonists and this action could be blocked by the nicotinic antagonist Dtubocurarine (Kasai and Changeux, 1971). Recently it was discovered that these receptors JPN I 1:3 4 h 171

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are substrates for protein phosphorylation (Gordon et al., 1977; Teichberg et al., 1977). Studies on the muscarinic receptor have been hampered by difficulties to solubilize this membrane protein. Additional problems were the lack of a receptor-rich tissue comparable to the electric organ and the lack of specific, high affinity ligands of high specific radioactivity. This latter obstacle has been removed in 1974 with the introduction of [3H]-propylbenzilylcholine mustard ([3H]PrBCM), an irreversible muscarinic antagonist (Burgen et al., 1974a, b) developed from benzilylcholine mustard (Gill and Rang, 1966) and with the introduction of [3H]-3-quinuclidinyl benzylate (3[H]-3-QNB) (Yamamura et al., 1974a-c), which is the radioactively labeled form of the compound studied by Albanus (1970) and MeyerhOffer (1972) and originally described by Abood and Biel (1962). These compounds have high affinity (Kd < 10.9 M) and are now available in high enough specific activity (2-20 Ci/mM). The advent of these probes has accelerated biochemical work on the in situ muscarinic receptor. Important progress in the understanding of muscarinic action in the peripheral and the central nervous system was made by the observation that ACh acting at muscarinic receptor sites raised tissue levels of cGMP. George et aL (1970) observed that cGMP levels were increased when rat hearts were perfused with ACh containing medium. This observation has soon been extended to the central nervous system (Lee et al., 1972) and subsequently to all tissues in the periphery in the CNS which possess muscarinic innervation (cf. for reviews Goldberg and Haddox, 1977; Bartfai et al., 1977). The physiological significance of the muscarinic innervation in parasympathetic responses has been well documented (cf. Koelle, 1976). In the CNS most of the cholinergic innervation is of the muscarinic type (Brimblecombe, 1973; Curtis and Crawford, 1969; Krnjevi6, 1974). Cholinergic mechanisms play an important role in higher nervous functions such as sleep, avoidance behavior and learning (cf. van Woert, 1976; De Feudis, 1974; Weiss et al., 1976). As mechanisms of vital importance cholinergic mechanisms are targets for drug and poison action and susceptible to pathological malfunctioning. In the last years several observations were made which point to the role of malfunction of muscarinicdopaminergic balance in the etiology of Parkinsonism, Huntington's chorea, tardive dyskinesia and hyperactivity (cf. van Woert, 1976; Weiss et al., 1976). This review cannot attempt to cover all papers appearing on the physiology, electrophysiology, pharmacology and biochemistry of muscarinic innervation in the periphery and in the CNS. Our aim was to discuss some of the developments in the work on the biochemistry of the receptor. We also tried to refer to the pharmacological and physiological implications of the findings discussed.

2. Methods for Measurement of Muscurinic Receptors

The methodology of measurement of muscarinic action or of receptors is based on Dale's classical definition (Dale, 1914) of the muscarinic receptor, which is stimulated by muscarine or acetylcholine and blocked by atropine. These characteristics have to be fulfilled by response or binding before it can be termed muscarinic, independent of the methodology used. 2.1. PHYSIOLOGICALRESPONSES The parasympathetic actions of ACh are mediated via muscarinic receptors. Pupiilary contraction, contraction of smooth muscle, stimulation of sweat, gastric and salivary glands, and complex cardiovascular events are among the most marked effects of muscarinic agonists (Koelle, 1975). Pupillary contraction-dilation upon local application of a drug offers a fast qualitative test of its muscarinic potency. Contraction of smooth muscle: increase in tone and motility can be used as a quantitative tool of measurement of muscarinic effects. Most of the classical pharmacological studies on muscarinic actions were based on measurements of smooth muscle contraction (of. Stephenson, 1956; Rang, 1966; Parker, 1972). Another method which was often used in estimating muscarinic activity of

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drugs is based on drop of arterial blood pressure upon application of muscarinic agonists (e.g. Bebbington et al., 1966). Measurements of changes in membrane potential and membrane resistance of neurons upon iontophoresis of muscarinic agonists (Krnjevi6 and Phillis, 1963a-c) and iontophoretic studies on smooth muscle cells (Purves, 1974; Bolton, 1971, 1972) are the electrophysiological techniques which have provided much of the information on localization of muscarinic receptors. Measurement of a physiological response several reaction steps away from the muscarinic receptor has the advantage of high sensitivity--since most of the chemical reactions occurring between receptor occupancy and e.g. smooth muscle contraction represent a cascade type amplification of the synaptic signal. This amplification, however, is not linear. Among the disadvantages are the indirect nature of the effect measured (e.g. the observation that less than all receptors are occupied when a full response is elicited: "spare receptors" are present). Finally, there are numerous methodological problems involved in this type of bioassay such as poor selectivity (i.e. other substances can evoke the same response) calibration problems, specific and nonspecific desensitization of the preparation, few samples can be measured on the same preparation and the methods are time consuming. 2.2. BIOCHEMICAL RESPONSES 2.2.1. Indirect measurements Among the muscarinic responses which require the presence of whole cells and can be studied with standard in vitro biochemical techniques are changes in ion fluxes, stimulation of phosphatidylinositol (PI) turnover, and stimulation of guanosine 3',5'-cyclic monophosphate (cGMP) synthesis. Efflux of K + from preloaded smooth muscle fragments has been measured by Burgen and Spero (1968). Gardner and his colleagues (1975) used the measurement of Ca 2+ effiux from pancreatic cells to study muscarinic action. The correlation between bi.nding of muscarinic agonists and enzyme secretion from the pancreas has also been studied (Hokin, 1968). Measurements of increased turnover of phosphatidylinositol have been used to characterize muscarinic responses in tissue slices and smooth muscle and have been reviewed by Michell (1975). Recently measurements of increased cGMP levels as a consequence of muscarinic stimulation in nervous tissue have been used by several groups (for review, cf. Goldberg and Haddox, 1977; Bartfai et al., 1977). All of the above mentioned muscarinic responses are dependent on the presence of extracellular Ca 2+ and abolished when Ca 2+ is removed by EGTA. Whereas the effiux of K + from smooth muscle (Burgen and Spero, 1970) and phosphatidylinositol turnover (Jones and Mitchell, 1975) are responses that can be studied in Ca 2+ free medium. 2.2.2. Direct measurements Direct "assessment of the number of muscarinic binding sites in subcellular fractions or on whole cells became feasible with the advent of high specific radioactivity, high affinity ligands such as [3H]-quinuclidinyl benzylate ([3H]-3-QNB) (Yamamura and Snyder, 1974), [3H]-benzetimide (Beld and .Ariens, 1973), [3H]-atropine (O'Brien et al., 1970; Farrow and O'Brien, 1973; Bartfai et al., 1974) and [3H]-propylbenzilylcholine mustard ([3H]-PrBCM) (Burgen et al., 1974). These ligands are all muscarinic antagonists with high affinity for the receptor (Kd = 10-~°-10 -9 M) (cf. Table 1). The first three ligands bind reversibly whereas PrBCM is an alkylating agent. The muscarinic receptor has been labeled by other irreversibly bound ligands such as dibenamine (Takagi et al., 1965) and benzilylcholine mustard (Gill and Rang, 1966; Gupta et al., 1976) or acetylcholine mustard (Robinson et al., 1975). Binding sludies directly determine the total number of receptor sites (i.e. even those receptors will be measured which are not required for eliciting the physiological response). Thus measurements of physiological response and ligand binding studies provide complementary information. During the work on characterization of several neurotransmitter and hormone receptors by binding studies several criteria have been defined for examining specific receptor-related

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E. HEILBRONN AND T. BARTFAI TABLE 1. LIGANDS GENERALLY USED IN STUDIES ON MUSCARINIC RECEPTORS

Agonist

ICsot (M)

Carbachol Acetylcholine Acetyl-/3-methylcholine Pilocarpine Oxotremorine

Antagonist

Reversible 1.5 x 10-' (2) Atropine 2-4 x 10-6 (1) Scopolamine 3-5 x 10-6 (1) 3-Quinuclidinyl benzylate 7 x 10-6 (1) (_~) Benzetimide 5-8 × 10-r (1) N-methyl-4-piperidylbenzylate

ICsot (M)

1.5 x 10-9 (1) 7 ~ 10-~° (1) 4.5 x 10-t° (1) 8.9 x 10-~° (4) 4 × 10-~° (5)

Muscarine Acetylcholine mustard

Irreversible 8 × 10 6 (3) Propylbenzilylcholine mustard Dibenamine

5 x 10-9 (2)

(1) Snyder et al. (1975). (2) Birdsall and Hulme (1976). (3) Robinson et al. (1975). (4) Beld and A.riens (1974). (5) Kloog and Sokolovsky (1977). t To compile this table we choose data from pharmacological experiments on smooth muscle contraction as well as we selected values derived by ligand binding studies. IC,o is the concentration of the compound at which either half maximal response was elicited or blocked or a 50~ inhibition of binding of another specific ligand was attained. binding (cf. Birdsall and Hulme, t976): (i) Saturable specific binding: the specific binding is defined as the difference in the number of bound (radioactive) ligands in the absence and presence of great excess of another specific ligand; (ii) Pharmacological specificity: pharmacologically known agonists and antagonists when applied in excess to their affinity constant should be able to block binding of the ligand (stereospecificity of the binding should be the same as of the pharmacologically active drug) and (iii) in s i t u localization and quantitation: localization of the receptor anatomically and subcellularly should follow the pattern of innervation established by lesion and autoradiographic or immunofluorescence studies. Binding of reversible ligands is measured either by equilibrium techniques (equilibrium dialysis or get filtration) or by direct measurement of the ligand-receptor complex after centrifugation or filtration. The equilibrium techniques yield true binding constants and are not subject to errors due to dissociation of the ligand-receptor complex during separation of bound and free ligand. Among the disadvantages are slowness (24-36 hr) of the method which often allows proteolytic degradation of the sample. This process can be effectively slowed down by use of protease inhibitors such as phenylmethylsulfonylfluoride (PMSF), trasylol and EDTA. Presently the direct methods are the most popular. Filtration of antagonist-receptor complexes on glass-fiber filters (most often used is Whatman GF/B) can be done rapidly as compared to the off-rate of the very tightly bound ligand ([3H]-3-QNB) from the receptor so that dissociation of the complex becomes negligible (Yamamura and Snyder, 1974b). The irreversible affinity label PrBCM is a strong alkylating agent which binds to muscarinic sites with high affinity. Use of this ligand for a limited time circumvents the problems of possible dissociation of the receptor-ligand complex during filtration or centrifugation and subsequent washing. Long exposure of the tissue will, however, lead to labeling of a number of nonspecific sites, also loss of label from the tissue occurs when lhe tissue is stored.

3. Localization of the Muscarinic Receptor 3.1. MUSCARINIC INNERVATION IN THE PERIPHERY Muscarinic receptors mediate the parasympathetic actions of acetylcholine on the periphery. Small doses of antimuscarinic agents such as atropine depress sweat and salivary

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secretion. Larger doses of atropine dilate the pupils, decrease the tone and motility of the gut and increase the heart rate through vagal blockade. The parasympathetic effects of muscarinic agonist have often been studied in the guinea pig ileum where ACh evokes dose-dependent contraction which can be blocked by atropine. The density of muscarinic receptors in the periphery varies widely; human erythrocyte membranes have a receptor density of23.10 -15 moles mg -1 (Aronstam et al., 1977), while smooth muscle has a receptor concentration of 2-5.10 i2 moles mg protein -1. Muscarinic receptors appear on a great variety of locations in the periphery; it seems that their binding properties are quite similar and that these properties are also very close to those found with central muscarinic receptors (Beld et al., 1975). 3.2. MUSCARINIC RECEPTORS IN THE C N S

Most of the cholinergic innervation of the brain is of the muscarinic type except for the spinal cord where nicotinic innervation dominates. The main cholinergic pathways in the CNS are (i) the septal-hippocampal pathway where the cholinergic cell bodies from the medial septal nucleus project to the hippocampus and (ii) the habenulo-interpeduncular tract in which the interpeduncular nucleus receives cholinergic afferens from the habenula through the fasciculus retroflexus. These tracts were mapped by lesioning techniques (for review: Kuhar, 1976). Cholinergic neurons with muscarinic receptors are also found in great number on the interneurons in the caudate nucleus (McGeer et al., 1975) and on the amocrine cells of the retina. In the cerebral cortex most of the Renshaw cells have muscarinic receptors according to iontophoretic studies (Krnjevi6 and Phillis, 1963a-c). In the cerebellum only Purkinje cells seem to possess muscarinic receptors (Crawford et al., 1966). The receptor density of different brain areas has been determined by dissecting the different areas and measuring [3H]-propyl benzilylcholine mustard binding (Hiley and Burgen, 1974) and [3H]-3-quinuclidinyl benzylate binding (Yamamura and Snyder, 1974b). The results show reasonably good agreement. Most of the binding sites (480 pmoles g protein -1) are found in the caudate nucleus, the lowest binding capacity is shown by the cerebellar cortex (3-40 pmoles g protein-I). The different cortical and subcortical areas vary in receptor density much more than in the specific activity of two other cholinergic marker proteins, choline acetyltransferase and acetylcholinesterase (cf. Table 2). This difference in distribution might be explained by assuming that most of the muscarinic receptors are located on the soma and dendrites of cholinoreceptive cells. Choline acetyltransferase on the other hand is localized outside the soma of the cholinelgic neuron in the nerve ending which releases ACh and self is juxtapositioned to the cholinoreceptive neuron. Acetylcholinesterase is present on both sides of the cholinergic synapse and along significant portions of neuronal plasma membrane as indicated by histochemical methods. Therefore TABLE 2. REGIONAL DISTRIBUTION OF PROTEINS IN CHOLINERGIC TRANSMISSION

Muscarinict receptor Cerebral cortex Cerebellum Caudate nucleus Hippocampus Brain stem

100' 9 122 62 13

Nicotinic++ Choline receptor acetyltransferaset Relative activities 100' 23 128 81 157

100' 27 420 126 282

Acetylcholinesteraset 100' 86 546 87 134

1 Data taken from Snyder et al. (1975). ~ Data taken from Salvaterra et al. (1975). * 100 % muscarinic binding corresponds to 390 pmoles [aH]-3-QNB bound g protein -~. 100% of nicotinic binding corresponds to 0.021 pmoles [t25I]-labeled bungarotoxin-binding sites mg protein -a. 100% choline acetyl transferase activity corresponds to 8.6 mMol mg-t x hr. 100% acetylcholinesteraseactivity corresponds to 28.2 nMol mg-1 x min.

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it serves as more diffuse marker for the cholinergic system than 3-QNB binding or choline acetyltransferase. The nicotinic receptor also occurs in brain. Its distribution follows a different pattern from that of the muscarinic receptor and it is present in far less quantities (cf. Table 2). Autoradiographic studies on the light microscopic level using [3H]-3-QNB have indicated the uneven distribution of receptors within brain areas and suggested that many more receptors may be localized on dendrites than on cell bodies (Kuhar and Yamamura, 1976). The tight binding and low off-rate of [3H]-3-QNB made it possible to study binding of this ligand after i.v. injection of the compound followed decapitation and dissection (Yamamura et al., 1974). The distribution of in vivo accumulated ligand followed that of the in vitro binding. The subcellular localization of the receptor has been determined using [3H]-3-QNB (Yamamura and Snyder, 1974b) and [3H]-atropine (Alberts and Bartfai, 1976) as specific ligands. It was found that practically all muscarinic receptors are membrane bound. Fractionation of the membrane pellet (10,000 g x 30 rain) on a discontinuous sucrose density gradient showed that most of the receptors, as measured by [3H]-PrBCM binding (Burgen et al., 1974b), are found in the synaptosomal membrane fraction. When synaptosomal membranes are further fractionated subsequent to phospholipase A2 treatment, rigid membrane fragments of possible post-synaptic origin are obtained. The receptor density of these membranes is manyfold higher than that of the synaptic membrane fraction indicating that the receptors are concentrated to the synaptic site and are not evenly distributed in a synaptosomal membrane preparation which also contains large quantities of plasma membrane (Bartfai et al., 1976). 3.3. POST- AND PRE-SYNAPTIC LOCALIZATIONOF THE MUSCARINIC RECEPTOR Classical studies on the physiology of smooth muscle contraction were aimed at the postsynaptic muscarinic receptor. Mapping of musearinic cholinoreceptive cells in the CNS by microiontophoresis was also based on detection of post-synaptic muscarinic receptors. These post-synaptic receptors seem to be involved in mediation of most of the muscarinic actions of ACh in the periphery and in the CNS. The muscarinic agonists and antagonists were tested, new ones developed and their relative potencies determined using smooth muscle contraction which is a response mediated through post-synaptic muscarinic receptors. Discussions on the effect of muscarinic ligands have until recently assumed that these compounds act exclusively at post-synaptic sites. In the last 10 years we got several indications that also pre-synaptic muscarinic receptor (autoreceptors) which regulate release of ACh exist in the CNS (Table 3). Muscarinic agonists inhibited and atropine stimulated ACh release from cerebral cortex and cortical slices (Polak and Bertel-Meeuws, 1966; Bertel-Meeuws and Polak, 1968; Polak, 1971; Szerb and Somogyi, 1973) and from hippocampal slices (Hadhazy and Szerb, 1977). It appears that the pre-synaptic receptor TABLE3. PRE-SYNAPTICMUSCARINICRECEPTORS Condition Rat cerebral cortex (1) Control (K+: 4.7 mM) High K + (25 raM) High K + (25 mM)+atropine (3.4 ~M) Guinea pig ileum (2) Control Control+atropine (3.10-7 M) Control+ oxotremorine (10-4 M) Control+ oxotremorin¢(10-4 M) +atropine (3.10-7 M)

ACh release

100 520 1700 100 100 67 96

(1) Polak and Meeuws (1966). (2) Kilbinger and Wagner (1975).

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might have somewhat lower affinity for 3-QNB than the post-synaptic receptor (Szerb et al., 1977) and therefore might not have been included in the measurement of receptor content of CNS with low 3-QNB (1 nM) concentrations used when collecting data for Table 2. Pre-synaptic muscarinic receptors have also been demonstrated in smooth muscle (Kilbinger and Wagner, 1975). The physiological significance of these pre-synaptic receptors is difficult to evaluate before good estimates of their affinity for ACh are available. It is assumed that pre-synaptic receptors become important at repetitive nerve stimulation when ACh is accumulated in the synaptic gap. The data from two systems, guinea pig ileum and cerebral cortical slices, indicate, however, that too strong stimulation (e.g. depolarization with K + concentrations higher than 25 mM or high frequency (>3 Hz stimulation) overcomes the inhibitory effect on ACh release mediated by the autoreceptor. Thus the receptor may play an inhibitory role in ACh release at medium strong repeated stimuli. The mechanism of action of muscarinic autoreceptor seems to be similar to that of well studied ~-adrenergic autoreceptor (cf. for review Stj/~rne, 1976).

4. Characteristics of the Receptor 4.1. LIGAND STRUCTURE

Early exploration of mAChR was done by variations of the structure of cholinergic compounds. Synthetic drugs serving as muscarinic ligands may be derived from ACh by the introduction of large lipophilic groups into the acidic part of the molecule. The presence of a quaternary or tertiary amine group or a nitrogen-containing heterocycle is important. Tertiary amines may cause psychotomimetic effects in addition to peripheral anticholinergic symptoms. Blocking cholinergic agents are found among a variety of compounds such as: amines, amino alcohols, derivatives of amino alcohols (esters, ethers, carbamates), amides etc.; but possess usually a cationic head, an alcoholic hydroxyl, an ester group, and a cyclic substituent, which is generally aromatic. X-ray structure determinations of a number of potent agonists, e.g. ACh, muscarine, 2-methyl-4-trimethylammonium-methyl-l,3-dioxolane and oxotremorine (Chotia and Pauling, 1968; Chotia, 1970), and antagonists atropine 3-QNB (Meyerh6ffer and Carlstr/3m, 1969), NMR studies (Culvenor and Ham, 1966) and MO-calculations (Kier, 1971) have reasonably well established the preferred conformation of muscarinic agents. Beckett et al. (1971) and Meyerh~3ffer(1972) have suggested a three point attachment of cholinergic compounds to the receptor. The three important points of receptor attachment are probably: (1) a cationic head, usually a tertiary or a quaternary amino group, to form an ionic bond; (2) a hydroxylic group for a hydrogen bond and (3) at least one aromatic ring, which binds by van der Waal's forces. In addition, there are dipole-dipole interactions or hydrogen bonds involving the carbamyl group and the ether oxygen. The protonated form rather than the full base of tertiary amines has muscarinic effects. This agrees well with the finding by many (see e.g. Waser, 1961) that replacement of O by S in the ether group lowers activity. Interaction of the keto group in ACh and muscarone, the OH-group in muscarine and the second O in the dioxolane I with the receptor is more difficult to explain. Bebbington and Brimblecombe (1969) and earlier Waser (1961) suggested a hydrogen bond between this part of the receptor and the compound. This, however, is not in agreement with the high muscarinic activity of e.g. oxathiolane Ill, a compound with SH group instead of OH (Elferink and Salemink, 1975). Oxathiolane should be expected to have lower activity on account of the small ability of sulfur to form hydrogen bonds. This compound is, however, very active. The importance of hydrophobic binding in the potency of various psychotomimetic glycolates was examined by Baumgold et al. (1977) who found that hydrophobic bonding accounts largely for the tight binding and that addition of phosphatidylserine enhanced binding of [3H]-3-QNB with 20 %. This increase is relatively low to allow any conclusions on the effect or mode of action of phospholipids in ligand binding. Enantiomers of muscarinic ligands often show large differences in pharmacological potency. The (+)benzetimide

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binds two orders of magnitude tighter to muscarinic receptor than does the ( - ) f o r m (Beld and Ariens, 1974). This difference was utilized to assay stereoselectivity of the receptor. Detergents have been found to abolish this stereoselectivity. Other agonists e.g. methacholine (Beld and )~riens, 1974) and antagonists e.g. 3-QNB (Baumgold et al., 1977) also possess large differences in their affinity towards the receptor. 4.2. BINDING OF ANTAGONISTS TO THE MUSCARINIC RECEPTOR AS indicated in Table l, the receptor shows a much higher affinity for antagonists than for agonists. Binding of antagonists--atropine, 3-QNB, PrBCM, benzetimide--has been studied by use of the radiolabeled compounds. The specific binding of reversible and irreversible antagonists is saturable, and obeys the classical binding isotherm suggesting that a homogenous population of receptor sites binds the antagonists. The binding curve has not indicated any sign of cooperativity between binding sites; thus it is not possible to estimate from antagonist binding data how many antagonist binding subunits are in: cluded in a receptor molecule. The dissociation constants found in the binding studies are in close agreement with the values derived from studies on pharmacologically effective doses of the corresponding ligand. Because of the lower affinity of the receptor towards agonists and because of the lack of availability of high specific radioactivity agonists binding of these (acetylcholine, oxotremorine, carbachol) was often studied by measuring their inhibitory effect on [3H]antagonist binding (Birdsall and Hulme, 1976). Studies of agonist binding showed a strong deviation from classical saturation curves (Birdsall et al., 1976) and the Hill coefficient of agonist binding was significantly lower than one (Burgen et al., 1974a). The appearance of intermediary plateau regions was apparent when protection of receptor by ACh against alkylation by PrBCM was measured. Birdsall and Hulme (1976) explained their results by assuming that two receptor populations, different in agonist binding affinity, exist. These populations have the same affinity towards antagonist, thus they were not revealed before binding of agonist was studied. The authors also suggest that the population of lower affinity conformer of the receptor is that which might be involved in the physiological response. Studies on desensitization of agonist binding in smooth muscle (Young, 1974) would tend to support this idea. In these experiments it was observed that the protection by carbachol against alkylation of the receptor by [3H]-PrBCM had a Hill coefficient of 0.44+_0.08. If the smooth muscle strips were first exposed to the agonist, which was rapidly removed before labeling with [3H]-PrBCM and protection by newly added carbachol was measured the Hill coefficient increased to 0.90_+0.2. In preparations from rat brain no desensitization of agonist binding was observed yet. Recent studies by Aronstam et al. (1977) also suggest the presence of two populations of musearinic receptors in synaptosomal membranes. In these studies treatment of the membranes with the alkylating agent N-ethyl maleimide (NEM) increased the affinity of receptors for agonists without affecting the binding of antagonists. Hill coefficient of agonist binding increases from n = 0.68 to 0.91 indicating that the treatment either abolishes negative cooperativity between binding sites or converts most of the receptor into one homogenous population of binding sites. The effect of NEM indicates the presence of essential --SH or --NH2 groups in the receptor's active site. The notion of important SH groups is further strengthened by the finding that Cd ~+ in low concentration (5/tM) inhibits 3-QNB binding to the receptor (Hedlund and Bartfai, unpublished). Recent studies by Gupta et al. (1976) on recovery from benzilylcholine mustard inhibition suggest that this alkylating affinity label binds at least to two sites on the receptor. Further, more detailed studies on the effect of alkylating agents without specific active site directed groups are required before the mechanism of changes in agonist binding properties can be delineated. 4.3. SOLUBILIZATIONAND PURIFICATIONATTEMPTS Solubilization of the receptor for a long time was completely unsuccessful. Several workers have concluded that charged and nonionic detergents denature the receptor (Beld

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179

and ~riens, 1974; Bartfai et al., 1974). The first successful solubilization was reported by Beld and ,~riens (1974) who used 1% digitonin. An 80% solubilization of receptors from bovine tracheal muscle and 20 % solubilization from bovine caudate was achieved. If the freeze-dried caudate was first extracted with hexane prior to digitonin treatment a complete solubilization was found even there. The molecular properties of the solubilized material have not been reported and there is some question whether digitonin really solubilized the receptor or only broke up the membrane into smaller pieces which did not sediment at 100,000 g × 1 hr centrifugation. Digitonin is known to complex with cholesterol and might interrupt the membrane structure at cholesterols. Bartfai et al. (1974) have reported a systematic study of the solubilizing effect of charged, nonionic detergents and of chaotropic agents. They found that 2 M NaCI released an atropine binding protein from membranes prepared from cerebral cortex. This protein had an isoelectric point of 4.8-4.9. Determination of the molecular weight of the solubilized material indicated that three protein components were able to bind atropine or could be labeled with [3H]-PrBCM (Alberts and Bartfai, 1976). The molecular weight of these proteins were 30,000, 70,000 and 260,000. The latter had some acetylcholine esterase activity and it is known that this enzyme can also bind atropine. However, the Ka value for binding to the esterase is about 10-5 M (Kato et al., 1972) whereas that for the solubilized receptor ( ~ 10 8 M) which makes it unlikely that the atropine binding component is ACHE. Alberts and Bartfai (1976), and later Carson et al. (1977), carried out comparison of the binding properties of the membrane-bound and NaCl-solubilized receptor. It was found that the solubilized protein has lower affinity for both antagonists and agonists than the membrane-bound protein. ACh for example has two binding sites with 3.10 -8 and 3.10 -6 M dissociation constants in the membrane while the solubilized receptor shows only one class of binding site with lower affinity than any of the membrane bound sites (Carson et al., 1977). Similar changes take place in antagonist binding. This change may result from partial denaturation of the solubilized receptor. Another possibility is that selective solubilization of a receptor population with lower affinity was achieved. Purification of the atropine binding protein which was solubilized by 2 M NaC1 from synaptic membranes from rat brain by Alberts and Bartfai (1976) has been attempted. By the use of conventional gel filtration and ion exchange chromatography a 200-fold purification was achieved. The yield was 4 %. The instability of the solubilized material and lack of a good affinity chromatographic technique hampered the purification efforts. Preparation of a matrix for affinity chromatography is difficult despite the fact that several reversibly binding, high affinity ligands (atropine, 3-QNB) are known. Substitution of these structures which would enable us to attach them to the matrix, diminishes their affinity for the receptor substantially. The partially purified receptor represents approximately 1-5 % of the total protein in the purified sample if we assume that one antagonist is bound per 30,000 molecular weight and that the density of receptors is 0.1-0.2 nmoles mg protein -1 in the starting material. Binding of atropine to the solubilized receptor shows a pH optimum at pH = 7.2. The binding capacity varies with Ca 2 ~-concentration of the Krebs-Ringer's medium. Optimal binding is found at 0.5 mM Ca 2+.

5. Muscarinic Binding and Phosphatidylinositol Turnover Binding studies gave information about the amount and distribution of the ligand binding sites. The mode of action by which receptor occupancy by agonist is transduced into signal for the receiving cell is not known. One possibility is that a second messenger role is played by Ca z+ which is a necessary requirement for all muscarinic action. The other phenomena elicited by muscarinic stimulus are increased turnover of phosphatidylinositol (PI) and stimulation of cGMP synthesis. Increased turnover of phosphatidylinositol (PI) is observed when muscarinic cholinergic, ~-adrenergic or H~-histaminergic receptors are occupied by agonists (cf. for review Michell, 1975; Jafferji and Michell, 1976a). The concentration of PI, which represents 2-10 ~ of the

180

E. HEmBRONNAND T. B~TEAI

total phospholipids, does not increase during the period of stimulation but an increased synthesis and degradation can be followed by labeling the phosphate (Heilbronn and Widlund, 1970) or the inositol part of the phospholipid. Receptor occupancy vs agonist concentration and increased turnover of PI vs agonist concentration curves show good agreement (Michell e t al., 1976). Atropine blocks the effect of muscarinic agonists. Recently it has been shown that blockers of Ca 2+ movement such as D 600 do not block the muscarinic agonist mediated increase in turnover of PI while they block the physiological response (in this case contraction of smooth muscle) (Jafferji and Michell, 1976b). This suggests that though Ca 2÷ is required for the physiological response, turnover of PI is either preceding entry of Ca 2+ or is completely independent from it (Jones and Michell, 1975). Interestingly, the alkylating c~-adrenergic antagonist phenoxybenzamine can block PI turnover stimulated not only by ~-adrenergic agonists but also by muscarinic or histaminergic (H1) agonists (Jafferji and Michell, 1976b). On the basis of these experiments it is suggested that PI might participate in mediation of muscarinic (~-adrenergic or H1histaminergic) stimulation according to the following scheme: mAChR ~ PI

~

C a 2+

entry ~ physiol, response

or

mAChR ~ Ca 2+ entry ---) physiol, response PI Increased turnover of PI leads to transient appearance of fat-soluble diacylglycerate in the membranes and of water-soluble inositolphosphate in the cytosol. Any of these or both may affect enzymes such as protein kinases or guanylate cyclases (which both occur in membrane-bound and soluble forms) and thereby serve as a link between receptor occupancy and physiological response.

6. Muscarinic Receptor Mediated Increases in cGMP Levels

In several peripheral tissues and in tissue slices from sympathetic ganglion and brain increases in c G M P levels upon muscarinic stimulation have been observed (Table 4). The largest increase in c G M P levels (200-fold) with muscarinic stimulation was found in studies on N 1E 115 neuroblastoma cells (Matsuzawa and Nirenberg, 1975). The muscarinic receptor mediated stimulation of c G M P synthesis in each instance studied is dependent TABLE 4. MUSCARINIC RECEPTOR-MEDIATED INCREASES IN TISSUE LEVELS OF CYCLIC G M P

Tissue

Species

Fold increase

Reference

Heart (perfuscd) Heart slices Ductus deferens Uterus Lung Ileum Umbilical artery Cerebral cortex Cerebellum Cerebral cortex (slices) Neuroblastoma (N1E 115) Sympathetic ganglion (slices) Submaxillary gland Thyroid (slices) Renal cortex (slices) Exocrin¢ pancreas Ncutrophils Lymphocytes

rat rat rat rat rabbit guinea pig human rabbit rabbit rat mouse bovine rat rat rat guinea pig human human

2.5 5 2-3 2 2 2 3 2-3 1.5-2 1.5 200 5 2-3 5 5 5-20 2 3

George et al., 1970 Lee et al., 1972 Schultz et al., 1973 Goldbcrg et al., 1973 Goldberg et al., 1973 Lee et aL, 1972 Clyman et al., 1975 Lee et al., 1972 Kuo et al., 1972 Palmer and Duszynski, 1975 Matsuzawaand Nirenberg, 1975 Kebabian et aL, 1975 Schultz et al., 1973 van Sande et al., 1975 De Rubertis and Craven, 1976 Haymovits and Seheele, 1976 Smith and Igrmrro, 1975 Goldberg et al., 1973

MUSCARINIC ACETYLCHOLINE RECEPTOR

181

on the presence of extracellular Ca 2+. The muscariniC stimulus evoked--increase in intracellular cGMP levels--was additive to the PGE mediated increase in cGMP in these cells (Matsuzawa and Nirenberg, 1975) or to the histamine mediated increase in cGMP in slices of bovine sympathetic ganglion (Bartfai et al., 1977) indicating that either each type of receptor is coupled to a guanylate cyclase or, if a common pool of guanylate cyclase exists, then this pool is sufficiently large not to be a limiting factor when several receptor types are stimulated. The neurotransmitter evoked increases in cGMP levels are maximal within 0.5-1 rain and rapidly fall towards the basal value. This decrease in cGMP values is due mostly to hydrolysis of cGMP by Y,5'-cyclic nucleotide phosphodiesterase and to some transport of the cyclic nucleotide out from the cells. The muscarinic receptors show high and low affinity binding of agonists (Birdsall and Hulme, 1976). It appears that increase in cellular cGMP levels is correlated with occupancy of the low affinity population of receptors in N1E 115, the system where dose-response relationship was best studied (Matsuzawa and Nirenberg, 1975; Strange et al., 1977; Bartfai and Breakefield, 1977). Several attempts to demonstrate an acetylcholine-sensitive guanylate cyclase in membrane preparations similar to the hormone sensitive adenylate cyclases which can be studied in broken cell preparations have failed (Limbird and Lefkowitz, 1975; Bartfai et al., unpublished). It appears that there can only be an indirect coupling between the fully membrane bound receptor and the mostly soluble guanylate cyclase. It was postulated that Ca 2+ may play this mediator role (Schultz et al., 1973). However, rather high (raM concentrations of Ca 2+) are required for stimulation of the guanylate cyclase in vitro whereas the intracellular C a 2 ~ concentration is in the range of 10-6-10 -8 M. It is possible that Ca 2~ after being channeled in by means of some intermediary component is capable of stimulating the guanylate cyclase. Recent experiments show that the Ca 2+ binding protein (CDR) which regulates both adenylate cyclase and the cyclic nucleotide phosphodiesterase does not affect the Ca2+-guanylate cyclase interaction (Olson et al., 1976) thus is not likely to be the intermediate component of Ca2+-activation of guanylate cyclases. Nevertheless the requirement for extracellular Ca 2+ is puzzling and suggests that influx of Ca z+ directly or indirectly may be controlled by muscarinic receptor. Depolarizing agents can also raise cGMP levels in whole cells or slices prepared from brain if Ca 2+ is present in the medium (Ferrendelli et al., 1976). Recently it was observed (Breakefield et al., in preparation) that the increases in cGMP levels in N1E 115 neuroblastoma cells mediated by the receptor and depolarizing agents are additive even though saturating concentrations of the muscarinic agonist are used. This suggests that depolarizing agents and the muscarinic receptor may regulate separate Ca 2+ channels. It is postulated that increased cGMP levels exert their effect by activation of cGMP dependent protein kinases present in smooth muscle and nervous tissue (Kuo, 1974). Phosphorylation of several endogenous proteins by the cGMP dependent protein kinases might be the route via which muscarinic stimulus elicits a physiological response. Such cGMP dependent phosphorylation of membrane protein in smooth muscle has indeed been observed (Casnellie and Greengard, 1976; De Jonge, 1976). Though in smooth muscle increased cGMP levels upon muscarinic stimulation are well documented, there is evidence dissociating this phenomenon from that of the contraction of smooth muscle. Several agents which raise cGMP levels (NO2, NAN3) relax smooth muscle but their potencies in stimulating cGMP synthesis and muscle relaxation do not agree well (Diamond and Hartle, 1974). In the case of neurons in the CNS it seems more likely that cGMP is involved in mediation of the muscarinic response. Iontophoretic studies showed that cGMP can mimic the muscarinic action of ACh on neurons in the cortex and on Purkinje cells in the cerebellum (Stone et al., 1975; Bloom, 1975). In recent studies Woody (1977) applied cGMP either onto the surface of cortical neurons or into the nerve cell--in both cases cGMP in low concentrations mimics the effect of ACh in changing membrane resistance. (In the case of cat motor neurons, cGMP could not mimic the actions of ACh (Krnjevi6, 1977).) In this context it is interesting to point out the "Ying-Yang" concept of regulation via cyclic nucleotides in which Goldberg et al. proposed (1973) that regulation of certain cell

E. HEILBRONNAND T. BARTFAI

182

',, f

) /-h ~

Chotnergi i cneuron

~o

o

o

\ ~

FIG. la.

/

~~Unergic reuron

8~

7

8

\ /5J ( A T P 1 1 . % _ ~ . ~

/

~"'ACh C&2÷

~

~

g

C~,2"

/ GTP

cO#P ~

.r-.. / ADP

5'GMP

FIO. lb.

Chotinoreceptineuron ve

N~,+~K* perme~bd~ty

MUSCARINIC ACETYLCHOLINE RECEPTOR

183

functions can be based on the opposing actions of cAMP and cGMP. It is well documented that other putative transmitters such as dopamine, noradrenaline acting at fl-receptor sites, histamine acting at H2-receptor sites raise c A M P levels in neurons whereas the c G M P levels are regulated by ACh acting at muscarinic receptors by histamine acting at Hi-receptor sites and by noradrenaline acting at c~-adrenergic receptor sites (cf. for review Nathanson, 1977). It is also known that most neurons have more than one type of neurotransmitter receptor. Thus cAMP and c G M P levels can be regulated by several neurotransmitters independently within the same neuron.

7. Model of Muscarinic Cholinergic Synapse It appears that muscarinic receptors are present both pre- and post-synaptically. There is evidence that both phosphatidylinositol turnover and c G M P synthesis are stimulated by occupancy of the receptors with agonists. Increased phosphatidylinositol turnover is independent whereas c G M P synthesis is dependent on the presence of extracellular Ca 2+. The formed c G M P activates c G M P dependent protein kinases which phosphorylate endogenous membrane and cytosolic proteins. It is postulated that changes in the phosphorylation state of these proteins regulate permeability changes at the synaptic membrane. Whether c G M P is involved in pre-synaptic processes is not known. Figure 1 summarizes the above model of muscarinic synapse.

8. Pharmacological Significance The parasympathetic effects of ACh are mediated via muscarinic cholinergic receptors. Regulation of smooth muscle contraction, enzyme secretion from lymphocytes and pancreatic acinar cells by ACh involves muscarinic receptors. In the CNS muscarinic receptors are distributed unevenly. Highest receptor density is found in the striatum and cerebral cortex. Cholinergic innervation of striatum gained attention with the studies on dopaminergiccholinergic balances in this structure (cf. for reviews Weiss et al., 1976; van Woert, 1976). Several disorders may be described as loss of balance of dopaminergic and cholinergic activities. Parkinsonism can be described as hypoactivity of the dopaminergic input into the limbic system as compared to that of cholinergic activity there (van Woert, 1976). Huntington's chorea is characterized by an extensive loss of muscarinic receptors (Enna et al., 1976, 1977). Tardive dyskinesia which appears as a result of long lasting treatment with phenothiazines is another example of a disorder with hypoactivity of the cholinergic system (cf. van Woert, 1976). The disorder hyperactivity is characterized by loss of activity of the cholinergic system which usually has an inhibitory effect on motor activity (Silbergeld and Goldberg, 1976). Cross reactivity of drugs of different classes with the muscarinic receptor is a common problem in the therapy of neurological disorders. Antidepressants such as imipramine and amitriptyline bind to the muscarinic receptor with IC5o values of 10 and 78 nM (Snyder and Yamamura, 1977). Binding of antischizophrenic drugs such as chlorpromazine (Haigh and Young, 1975; Y a m a m u r a et al., 1976) and pimozide (Yamamura et al., 1976) to the receptor was also FIG. 1. The components of muscarinic synapse are: (1) pre-synaptic muscarinic receptor (autoreceptor); (2) post-synaptic muscarinic receptor; (3) Ca 2÷ channel; (4) synaptic vesicle; (5) high affinity choline uptake system (a specific marker of cholinergic nerve endings); (6) choline acetyl transferase; (7) mitochondrion; (8) receptors for other neurotransmitters which influence ACh release; (9) acetylcholinesterase; (10) Ca2+-dependent protein kinase; (1 I) membrane-bound guanylate cyclase (which might be coupled to the receptor); (12) soluble guanylate cyclase; (13) cGMP-dependent protein kinase; (14) endogenous membrane protein which might be involved in changes of membrane potential (a substrate for the protein kinase); (15)ion channels; (16) protein phosphatase and (17) 3', 5'-cyclicnucleotide phosphodiesterase. Most of the data on generation of cGMP through muscarinic stimulus refer to a post-synaptic site, however, the existence of pre-synaptic guanylate cyclases is also likely. PI turnover has similarly been described mostly as a post-synaptic event.

184

E. HEILBRONNAND T. BARTFAI

observed. A close inverse correlation was found between extrapyramidal side effects of several antipsychotic drugs (phenothiazines and butyrophenones), and their affinity for the muscarinic receptor: drugs such as clozapine and thioradizine which bind with high affinity to muscarinic receptors have the lowest incidence of extrapyramidal side effects (Snyder et al., 1974). Richelson (1977) has extended these studies by showing that psychoactive drugs not only block binding of antagonists (3-QNB) to the receptor but also block the increase in c G M P levels mediated by carbachol in NI E 115 cells. The pharmacology and significance of the pre-synaptic muscarinic receptor is not understood. The present status of our knowledge can be summarized as it appears that ACh via these pre-synaptic receptors inhibits its own release preventing overflow of ACh in the synaptic cleft. The rate of synthesis or choline uptake might also be regulated via these receptors.

9. Current Research on Muscarinic Receptors Studies on the muscarinic receptors are presently concentrated along several lines. The biochemical studies are aimed at finding better methods to solubilize large quantities of active receptor from smooth muscle and brain. Such solubilization is followed by purification and incorporation of the purified protein into phospholipid vesicles to study its relation to Ca 2+ channeling mechanisms and to stimulation of c G M P synthesis. It is hoped that studies on receptor-cGMP coupling and on receptor mediated increase in phosphatidylinositol turnover may be facilitated by the recognition of the fact that ~-adrenergic and Hl-histaminergic receptors mediate the same type of changes. Experiments on the additivity of these three types of stimuli in stimulating PI turnover and c G M P synthesis are in progress. The importance of the heterogenous population of receptors towards agonist binding and its possible relation to desensitization mechanisms is presently at trial in numerous laboratories. The electrophysiology of the receptors is being studied by techniques of single unit recording. The second messenger role of c G M P in muscarinic synaptic transmission is in the focus of investigation in several laboratories. The changes in the distribution, affinity and number of muscarinic receptors in the pathology of several diseases of the nervous system are under study by means of binding studies utilizing [3H]-3-QNB binding.

References

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