1 chapter 11 Acetylcholine Palmer Taylor Joan Heller Brown CHEMITY F ACETYCHINE 186 GANIZATIN F THE CHINEGIC NEVU YTEM 186 Acetylcholine receptors have been classified into subtypes based on early studies of pharmacologic selectivity 186 The intrinsic complexity and the multiplicity of cholinergic receptors became evident upon elucidation of their primary structures 189 FUNCTINA APECT F CHINEGIC NEUTANMIIN 189 Both muscarinic and nicotinic responses are elicited in brain and spinal cord 189 Neurotransmission in autonomic ganglia is more complex than depolarization mediated by a single transmitter 190 Muscarinic receptors are widely distributed at postsynaptic parasympathetic effector sites 190 timulation of the motoneuron releases acetylcholine onto the muscle endplate and results in contraction of the muscle fiber 191 Competitive blocking agents cause muscle paralysis by preventing access of acetylcholine to its binding site on the receptor 191 YNTHEI, TAGE AND EEAE F ACETYCHINE 192 Acetylcholine formation is limited by the intracellular concentration of choline, which is determined by active transport of choline into the nerve ending 192 A second transport system concentrates acetylcholine in the synaptic vesicle 193 Choline is supplied to the neuron either from plasma or by metabolism of choline-containing compounds 193 A slow release of acetylcholine from neurons at rest probably occurs at all cholinergic synapses 194 The relationship between acetylcholine content in a vesicle and the quanta of acetylcholine released can only be estimated 194 Depolarization of the nerve terminal by an action potential increases the number of quanta released per unit time 194 All the acetylcholine contained within the cholinergic neuron does not behave as if in a single compartment 194 ACETYCHINETEAE AND THE TEMINATIN F ACETYCHINE ACTIN 195 Cholinesterases are widely distributed throughout the body in both neuronal and non-neuronal tissues 195 The primary and tertiary structures of the cholinesterases are known 195 Acetylcholinesterases exist in several molecular forms governed by alternative splicing 195 Cholinesterase catalysis and inhibition mechanisms involve formation of reversible complexes and covalent conjugates 197 Consequences of acetylcholinesterase inhibition differ with effector site 197 NICTINIC ECEPT 197 The nicotinic acetylcholine receptor (nach) was the first characterized neurotransmitter receptor 197 The nach consists of five subunits arranged around a pseudoaxis of symmetry 198 Analysis of the opening and closing events of individual channels has provided information about ligand binding and activation of the receptor 201 Continued exposure of nicotinic receptors to agonist leads to desensitization of the receptors 201 Nicotinic receptor subunits are part of a large superfamily of ligand-gated channels 202 Both nicotinic receptors and acetylcholinesterase are regulated tightly during differentiation and synapse formation 202 MUCAINIC ECEPT 203 Muscarinic receptor activation causes inhibition of adenylyl cyclase, stimulation of phospholipase C and regulation of ion channels 203 adioligand-binding studies have been used to characterize muscarinic receptors 205 The binding properties of the antagonist pirenzepine led to the initial classification of muscarinic receptors 205 Transgenic mice are being generated to assess the functions of receptor subtypes in vivo 207 Acetylcholine (ACh) and its targets of interaction have played a long-standing and critical role in the basic concepts of neurochemistry. At the turn of the last century, ACh emerged as a pivotal mediator in chemical neurotransmission and as a ligand for defining receptors. The natural alkaloids physostigmine (by inhibiting acetylcholinesterase) and nicotine and muscarine (for distinguishing Basic Neurochemistry: Molecular, Cellular and Medical Aspects X , American ociety for Neurochemistry. All rights reserved. Published by Elsevier, Inc.
2 186 PAT II Intercellular ignaling receptor subtype) were invaluable tools for identifying ACh as a transmitter and delineating its sites of action. The unusual spatial localization of nicotinic acetylcholine receptors (nach) at the neuromuscular junction endplates enabled the discoveries of quantal release of neurotransmitter, receptor desensitization and channel gating properties of individual receptor molecules through single channel and noise analysis. ACh is widely distributed in the nervous system. It subserves all motor transmission in vertebrates, is the primary transmitter for peripheral ganglia, mediates parasympathetic actions of the autonomic nervous system and is a dominant transmitter in the central nervous system. Toxins from various predatory species have evolved to block motor activity of prey; others from coral and plants use paralysis induced by cholinergic agents for protection from predation. The high affinity and selectivity of these toxins enabled the nach to emerge as the first chemically characterized neurotransmitter receptor. ther toxins from snakes and snails have proved useful to identify receptor subtypes in various tissues. In Alzheimer s dementias, ACh-containing nerve terminals are preferentially affected and therapy is directed to extending the surface area and duration of action of released ACh (Ch. 47). Cholinergic nerve terminals are also the site of action in the treatment of certain myasthenias and disorders involving compromised or excessive smooth muscle activity and exocrine secretion in the periphery. Carbamate and organophosphate pesticides act to delay the termination of action of acetylcholine by inhibiting acetylcholinesterase, and volatile organophosphates that have been used in terrorism incidents also act in this manner. ACh arrived within the evolutionary scheme long before the design of the nervous system and functional synapses. Bacteria, fungi, protozoa and plants store ACh and possess biosynthetic and degradative capacities for turnover of the molecule. Even in higher organisms, ACh distribution is far wider than the nervous system. For example, ACh is found in the cornea, certain ciliated epithelia, the spleen of ungulates and the human placenta . Although definitive evidence is lacking, ACh has been proposed to play a role in development and tissue differentiation. ACh was first proposed as a mediator of cellular function by Hunt in 1907, and in 1914 Dale  pointed out that its action closely mimicked the response of parasympathetic nerve stimulation (see Ch. 10). oewi, in 1921, provided clear evidence for ACh release by nerve stimulation. eparate receptors that explained the variety of actions of ACh became apparent in Dale s early experiments . The nicotinic ACh receptor was the first transmitter receptor to be purified and to have its primary structure determined [3, 4]. The primary structures of most subtypes of both nicotinic and muscarinic receptors, the cholin esterases (ChE), choline acetyltransferase (ChAT), the choline and ACh transporters have been ascertained. Threedimensional structures for several of these proteins or surrogates within the same protein family are also known. B A C 3 H H H + C CH 2 CH 2 N C 3 H N + N + C H H H H CHEMITY F ACETYCHINE Torsional rotation in the ACh molecule can occur around bonds τ 1, τ 2 and τ 3 (Fig. 11-1). ince the methyl groups are disposed symmetrically around τ 3 and constraints are placed on τ 1 by the planar acetoxy group, the most important torsion angle determining ACh conformation in solution is τ 2. A view from the β-methylene carbon of the molecule (Fig. 11-1B,C) shows the energy configurations around τ 2. Nuclear magnetic resonance (NM) studies indicate that the gauche conformation is predominant in solution [5, 6]. tudies of the activities of rigid analogs of ACh suggest that the trans conformation may be the active conformation at muscarinic receptors , while results of NM and X-ray crystallography studies suggest a change in configuration when ACh is bound to the nicotinic receptor [6, 8]. Hence the bound conformations of this flexible molecule appear to differ substantially with receptor subtype. This finding should not be a great surprise, since structural modifications of ACh that enhance or diminish activity on muscarinic receptors are very different from those modifications that influence activity on nicotinic receptors. GANIZATIN F THE CHINEGIC NEVU YTEM Acetylcholine receptors have been classified into subtypes based on early studies of pharmacologic selectivity. ong before structures were known, two crude alkaloid fractions, containing nicotine and muscarine (Fig. 11-2), were used to subclassify receptors in the cholinergic nervous system (Fig. 11-3). The greatly different C H H C FIGUE 11-1 tructure of acetylcholine. (A) The three torsion angles, τ 1, τ 2 and τ 3. (B) Newman projection of the gauche conformation. (C) Newman projection of the trans conformation. The molecule is viewed in the plane of the paper from the left side, and the bond angles around τ 2 are compared.
3 CHAPTE 11 Acetylcholine 187 Cholinergic tage 3 tage 2 tage 1 Nicotinic muscle Agonist: Phenyltrimethylammonium Antagonist: Elapid α-toxins, d-tubocurarine Nicotinic Junctional [(α1) 2 βεδ] Nicotinic Embryonic [(α1) 2 βγδ] Nicotinic Agonist: Nicotine Antagonist: d-tubocurarine Nicotinic neuronal β2 Nicotinic neuronal β3 Nicotinic neuronal β4 Nicotinic neuronal β5 Agonist: Acetylcholine Nicotinic neuronal (α+β) Agonist: Dimethylphenylpiperazinium, Cytisine Antagonist: Trimethaphan, Neuronal bungarotoxin Nicotinic neuronal α2 (α-toxin insensitive) Nicotinic neuronal α3 (α-toxin insensitive) Nicotinic neuronal α4 (α-toxin insensitive) Nicotinic neuronal α5 (non-functional without other α subunits) Nicotinic neuronal α6 (α-toxin insensitive) Nicotinic neuronal α7 (α-toxin sensitive) Muscarinic Agonist: Muscarine, Pilocarpine Antagonist: Atropine Agonist: xotremorine Antagonist: Propylbenzilylcholine mustard, Quinuclidinyl benzilate Muscarinic m 1 M 1 Antagonist: Pirenzepine Muscarinic m 2 M 2 Antagonist: Methoctramine Muscarinic m 3 M 3 Antagonist: Hexahydrosiladifenidol Muscarinic m 4 M 4 Antagonist: Himbacine Muscarinic m 5 M 5 Nicotinic neuronal α8 (α-toxin sensitive) Nicotinic neuronal α9 Nicotinic neuronal α10 FIGUE 11-2 Classification of cholinergic receptors. The diagram shows a historical classification of receptors analyzed on the basis of distinct responses with crude alkaloids (stage 1), the partial resolution of receptor subtypes with chemically synthesized agonists and antagonists (stage 2) and the distinction of primary structures of the receptors principally through cloning by recombinant DNA techniques (stage 3). activities of the antagonists atropine on muscarinic receptors and d-tubocurarine on nicotinic receptors further supported the argument that multiple classes of receptors existed for ACh. ubsequently, it was found that all nicotinic receptors are not identical. Nicotinic receptors in the neuromuscular junction, sometimes denoted as N 1 receptors, show selectivity for phenyltrimethylammonium as an agonist; elicit membrane depolarization in response to bisquaternary amines, with decamethonium being the most potent; are preferentially blocked by the competitive antagonist d-tubocurarine and are blocked irreversibly by the snake α-toxins. Nicotinic receptors in ganglia, N 2 receptors, are stimulated preferentially by 1,1-dimethyl-4-phenylpiperazinium, blocked competitively by trimethaphan, blocked noncompetitively by bisquaternary amines, with hexamethonium being the
4 188 PAT II Intercellular ignaling Nicotinic and muscarinic agonists CH H + 3 CH + 3 C CH 2 CH 2 N N CH H 3 3 C Acetylcholine (N + M) Phenyltrimethylammonium (N 1 ) CH 2 N Muscarine (M) + + N N 1,1-Dimethyl-4- phenylpiperazinium (N 2 ) C + N CH 2 C C CH 2 N xotremorine-m (M) N N Nicotine (N 1 + N 2 ) Nicotinic antagonists C CH 2 N N CH 2 ( ) 2 N + CH 2 H HC CH H 2 C HC CH + 2 H 2 C H 2 C Trimethaphan (N 2 ) H 3 C CH 2 + H N H d-tubocurarine (N 1 ) H 3 C + N (CH2)6 + N H 3 C + N (CH2)10 + N Hexamethonium (N 2 ) Decamethonium (N 1 ) Muscarinic antagonists H N C H i CH 2 CH 2 CH 2 N N N C N CH 2 N N Hexahydrosiladifenidol (M 3 ) Pirenzepine (M 1 ) Himbacine (M 4 ) N C Atropine (M) CH 2 H CH NH (CH 2 ) 6 NH (CH 2 ) 8 NH (CH 2 ) 6 NH Methoctramine (M 2 ) FIGUE 11-3 tructure of compounds important to the classification of receptor subtypes at cholinergic synapses. Compounds are subdivided as nicotinic (N) and muscarinic (M). The compounds interacting with nicotinic receptors are subdivided further according to whether they are neuromuscular (N 1 ) or ganglionic (N 2 ). Compounds with muscarinic subtype selectivity (M 1, M 2, M 3, M 4 ) are also noted.
5 CHAPTE 11 Acetylcholine 189 most potent, and show resistance to the snake α-toxins . A large number of distinct neuronal nicotinic receptors are found in the CN; they are closer relatives of the nicotinic receptors in ganglia than of those in muscle. Muscarinic receptors also exhibit distinct subtypes. The antagonist pirenzepine (PZ) has the highest affinity for one subtype, M 1, which is found mainly in neuronal tissues. Another antagonist, methoctramine, has a higher affinity for M 2 receptors, which are the predominant muscarinic receptor subtype in mammalian heart. Hexahydrosiladifenidol is relatively selective for the M 3 receptors present in smooth muscle and glands, whereas himbacine exhibits high affinity for M 4 receptors. With this level of multiplicity of receptor subtypes, limitations on specificity preclude a single antagonist defining a distinct subtype. The intrinsic complexity and the multiplicity of cholinergic receptors became evident upon elucidation of their primary structures. In the CN, at least nine different sequences of α subunits and three different sequences of β subunits of the nicotinic receptor have been identified [10, 11]. Expression of the cloned genes encoding certain subunit combinations yields functional receptors with different sensitivities toward various toxins and agonists. At least five distinct muscarinic receptor genes have been cloned and sequenced. The genes are called m 1 to m 5. They correlate with the M 1 to M 5 receptors identified pharmacologically. The subtypes differ in their ability to couple to different G proteins and, hence, to elicit cellular signaling events. Thus, cholinergic receptor classification can be considered in terms of three stages of development. Initially, Dale  distinguished nicotinic and muscarinic receptor subtypes with crude alkaloids. Then, chemical synthesis and structure-activity relationships clearly revealed that nicotinic and muscarinic receptors were heterogeneous, but chemical selectivity could not come close to uncovering the true diversity of receptor subtypes. astly, analysis of subtypes came from molecular cloning, making possible the classification of receptors on the basis of primary structure (Fig. 11-2). FUNCTINA APECT F CHINEGIC NEUTANMIIN The individual subtypes of receptors often show discrete anatomical locations in the peripheral nervous system, and this has facilitated their classification. Nicotinic receptors are found in peripheral ganglia and skeletal muscle. Upon innervation of skeletal muscle, receptors congregate in the junctional or postsynaptic endplate area. Upon denervation or in noninnervated embryonic muscle, the receptors are distributed across the surface of the muscle, and these extrajunctional receptors are synthesized and degraded rapidly. Junctional receptors exhibit far slower rates of turnover and are distinguished by an ε subunit replacing a γ subunit in the assembled pentameric receptor. Ganglionic nicotinic receptors are found on postsynaptic neurons in both parasympathetic and sympathetic ganglia and in the adrenal gland. Ganglionic nicotinic receptors appear in tissues of neural crest embryonic origin and exhibit identical properties in sympathetic and parasympathetic ganglia. Muscarinic receptors are responsible for postganglionic parasympathetic neurotransmission and thus for control of a wide range of smooth muscle, cardiac muscle and secretory responses. ome responses originating in the sympathetic division of the autonomic nervous system, such as sweating and piloerection, also are mediated through muscarinic receptors. Both muscarinic and nicotinic responses are elicited in brain and spinal cord. A few specific central cholinergic pathways have been characterized. In higher brain centers, ACh is concentrated in interneurons and long projection neurons. In the striatum, cholinergic interneurons provide critical intercircuitry for extrapyramidal motor control and implicit memory. Four predominant nuclei in the basal forebrain send projecton neurons to the hippocampus, olfactory bulb and cerebral cortex. These include the nucleus basalis of Meynert, a region of primary degeneration in Alzheimer s disease. Four areas of the brain stem send cholinergic projections to the thalamus, interpeduncular nucleus and the superior colliculus. enshaw cells in the spinal cord play a role in modulating motoneuron activity by a feedback mechanism. timulation of enshaw cells occurs through branches of the motoneuron, and the transmitter is ACh acting on nicotinic receptors. Both nicotinic and muscarinic receptors are widespread in the CN. Muscarinic receptors with a high affinity for pirenzepine (PZ), M 1 receptors, predominate in the hippocampus and cerebral cortex, whereas M 2 receptors predominate in the cerebellum and brainstem, and M 4 receptors are most abundant in the striatum. Central muscarinic and nicotinic receptors are targets of intense pharmacological interest for their potential roles in regulating abnormal neurological signaling in Alzheimer s disease, Parkinson s disease and certain seizure disorders. Nicotinic receptors are largely localized at prejunctional sites and control the release of neuro transmitters [10, 11]. Histochemical studies using antibodies selective for receptor subtype, ChAT and presynaptic transport proteins, along with receptor autoradiography with labeled ligands, have produced detailed maps of the CN. In addition, the nerve cell bodies containing the mna encoding these proteins have been defined through in situ hybridization with a cdna or antisense mna. tudies involving iontophoretic application of transmitter, local stimulation and intracellular or cellsurface measurements of responses establish appropriate
6 190 PAT II Intercellular ignaling functional correlates. ecently, knockout mice strains, harboring deletions of M1 M5 machs and various nach subunits, have been generated, providing a direct means of determining the localization of function of these receptors [12, 13]. Neurotransmission in autonomic ganglia is more complex than depolarization mediated by a single transmitter. In autonomic ganglia, the primary electrophysiological event following preganglionic nerve stimulation is the rapid depolarization of postsynaptic sites by released ACh acting on nicotinic receptors. This activation gives rise to an initial excitatory postsynaptic potential (EPP), which is due to an inward current through a cation channel (see Chs 6 and 10). This mechanism is virtually identical to that in the neuromuscular junction, with an immediate onset of the depolarization and decay within a few milliseconds. Nicotinic antagonists such as trimethaphan competitively block ganglionic transmission, whereas agents such as hexamethonium produce blockade by occluding the channel. An action potential is generated in the postganglionic nerve when the initial EPP attains a threshold amplitude. everal secondary events amplify or suppress this signal. These include the slow EPP, the late, slow EPP and an inhibitory postsynaptic potential (IPP). The slow EPP is generated by ACh acting on muscarinic receptors and is blocked by atropine. It has a latency of approximately 1 sec and a duration of seconds. The late, slow EPP can last for several minutes and is mediated by peptides found in ganglia, including substance P, angiotensin, luteinizing-hormone-releasing hormone (HH) and the enkephalins. The slow EPP and late, slow EPP result from decreased K + conductance and are believed to regulate the sensitivity of the postsynaptic neuron to repetitive depolarization . The IPP seems to be mediated by the catecholamines dopamine and/or norepinephrine and is blocked by α-adrenergic antagonists as well as by atropine. ACh released from presynaptic terminals may act on a catecholamine-containing interneuron to release dopamine and perhaps norepinephrine. imilar to the slow EPP, the IPP has a prolonged latency and duration of action. These secondary events vary with the individual ganglia and are believed to modulate the sensitivity to the primary event. Hence, drugs that selectively block the slow EPP, such as atropine, will diminish the efficiency of ganglionic transmission rather than eliminate it. imilarly, agonists such as muscarine and the ganglion-selective muscarinic agent, McN-A-343, are not thought of as primary ganglionic stimulants. ather, they enhance the initial EPP under conditions of repetitive stimulation. ince parasympathetic and sympathetic ganglia exhibit comparable sensitivities to nicotinic agonists such as nicotine and ACh in producing the initial EPP, the pharmacological effect of ganglionic stimulation depends on the profile of innervation to particular organs or tissues (Table 11-1). For example, blood vessels are innervated only by the sympathetic nervous system; thus, ganglionic stimulation should produce only vasoconstriction. imilarly, the pharmacological effects of ganglionic blockade will depend on which component of the autonomic nervous system is exerting the predominant tone at the effector organ. Muscarinic receptors are widely distributed at postsynaptic parasympathetic effector sites. The response to systemically administered ACh is characteristic of stimulation of postganglionic effector sites rather than of ganglia. This is a consequence of the greater abundance of muscarinic receptors at effector sites in innervated tissues and the limited blood flow to ganglia. Muscarinic receptors are found in visceral smooth muscle, cardiac muscle, secretory glands and endothelial cells of the vasculature. Except for endothelial cells, each of these sites receives cholinergic innervation. esponses can be excitatory or inhibitory, depending on the tissue. Even within a single tissue the responses may vary. For example, muscarinic stimulation causes gastrointestinal smooth muscle to depolarize and contract, except at sphincters, where hyperpolarization and relaxation are seen (Table 11-2). In most tissues innervated by the cholinergic nervous system, smooth muscle exhibits intrinsic electrical and/or mechanical activity. This activity is modulated rather than initiated by cholinergic nerve stimulation. Cardiac muscle and smooth muscle exhibit spikes of electrical activity that are propagated between cells. These spikes are initiated by rhythmic fluctuations in resting membrane potential. In intestinal smooth muscle, cholinergic stimulation TABE 11-1 Predominance of sympathetic or parasympathetic tone at effector sites: effects of autonomic ganglionic blockade ite Predominant tone Primary effects of ganglionic blockade Arterioles ympathetic (adrenergic) Vasodilation, increased peripheral blood flow, hypotension Veins ympathetic (adrenergic) Dilation, pooling of blood, decreased venous return, decreased cardiac output Heart Parasympathetic (cholinergic) Tachycardia Iris Parasympathetic (cholinergic) Mydriasis Ciliary muscle Parasympathetic (cholinergic) Cycloplegia (focus to far vision) Gastrointestinal tract Parasympathetic (cholinergic) educed tone and motility of smooth muscle, constipation, decreased gastric and pancreatic secretions Urinary bladder Parasympathetic (cholinergic) Urinary retention alivary glands Parasympathetic (cholinergic) Xerostomia weat glands ympathetic (cholinergic) Anhidrosis
7 CHAPTE 11 Acetylcholine 191 TABE 11-2 Effects of acetylcholine (ACh) stimulation on peripheral tissues Tissue Effects of ACh Vasculature (endothelial cells) elease of endothelium-derived relaxing factor (nitric oxide) and vasodilation Eye iris (pupillae sphincter muscle) Contraction and miosis Ciliary muscle Contraction and accommodation of lens to near vision alivary glands and lacrimal glands ecretion thin and watery Bronchi Constriction, increased secretions Heart Bradycardia, decreased conduction (atrioventricular block at high doses), small negative inotropic action Gastrointestinal tract Increased tone, increased gastrointestinal secretions, relaxation at sphincters Urinary bladder Contraction of detrusor muscle, relaxation of the sphincter weat glands Diaphoresis eproductive tract, male Erection Uterus Variable, dependent on hormonal influence will cause a partial depolarization and increase the frequency of spike production. In contrast, cholinergic stimulation of atria will decrease the generation of spikes through hyperpolarization of the membrane. Membrane depolarization typically results from an increase in Na + conductance. In addition, mobilization of intracellular Ca 2+ from the endoplasmic or sarcoplasmic reticulum and the influx of extracellular Ca 2+ appear to be elicited by ACh acting on muscarinic receptors (see Ch. 22). The resulting increase in intracellular free Ca 2+ is involved in activation of contractile, metabolic and secretory events. timulation of muscarinic receptors has been linked to changes in cyclic nucleotide concentrations. eductions in camp concentrations and increases in cgmp concentrations are typical responses (see Ch. 21). These cyclic nucleotides may facilitate contraction or relaxation, depending on the particular tissue. Inhibitory responses also are associated with membrane hyperpolarization, and this is a consequence of an increased K + conductance. Increases in K + conductance may be mediated by a direct receptor linkage to a K + channel or by increases in intracellular Ca 2+, which in turn activate K + channels. Mechanisms by which muscarinic receptors couple to multiple cellular responses are considered later. timulation of the motoneuron releases acetylcholine onto the muscle endplate and results in contraction of the muscle fiber. Contraction and associated electrical events can be produced by intra-arterial injection of ACh close to the muscle. ince skeletal muscle does not possess inherent myogenic tone, the tone of apparently resting muscle is maintained by spontaneous and intermittent release of ACh. The consequences of spontaneous release at the motor endplate of skeletal muscle are small depolarizations from the quantized release of ACh, termed miniature endplate potentials (MEPPs)  (see Ch. 10). Decay times for the MEPPs range between 1 and 2 ms, a duration similar to the mean channel open time seen with ACh stimulation of individual receptor molecules. timulation of the motoneuron results in the release of several hundred quanta of ACh. The summation of MEPPs gives rise to a postsynaptic excitatory potential (PEP), also termed motor endplate potential. A sufficiently large and abrupt potential change at the endplate will elicit an action potential by activating voltage-sensitive Na + channels. The action potential propagates in two-dimensional space across the surface of the muscle to release Ca 2+ and elicit contraction. Therefore, the PEP may be thought of as a generator potential, found only in junctional regions and arising from the opening of multiple receptor channels. Normal resting potentials in endplates are about 70 mv. The PEP causes the endplate to depolarize partially to about 55 mv. It is the rapid and transient changes from 70 to 55 mv in localized areas of the endplate that trigger action potential generation [9, 15]. Competitive blocking agents cause muscle paralysis by preventing access of acetylcholine to its binding site on the receptor. Competitive blockade with agents such as d-tubocurarine result in maintenance of the endplate potential at 70 mv. Without frequent PEPs, action potentials are not triggered and flaccid paralysis of the muscle results. The actions of competitive blocking agents can be surmounted by excess ACh. Depolarizing neuromuscular blocking agents, such as decamethonium and succinylcholine, produce depolarization of the endplate such that the endplate potential is 55 mv. The high concentrations of depolarizing agent that are maintained in this synapse do not allow regions of the endplate to repolarize, as would occur with a labile transmitter such as ACh. ince it is the transition in membrane potential between 70 and 55 mv that triggers the action potential, flaccid paralysis also will occur with a depolarizing block . Excess ACh will not reverse the paralysis by depolarizing blocking agents. As might be expected if depolarization occurs in a non-uniform manner in microscopic areas within individual endplates and in individual motor units, the onset of depolarization blockade is characterized by muscle twitching and fasciculations that are not evident in competitive block. nce paralysis occurs, the overall pharmacological actions of competitive and depolarizing blocking agents are similar, yet intracellular measurements of endplate potential can distinguish these two classes of agent.
8 192 PAT II Intercellular ignaling YNTHEI, TAGE AND EEAE F ACETYCHINE The biosynthesis and storage of ACh can be divided into three processes that allow for recovery of hydrolyzed transmitter by choline transport back into the nerve ending, conversion by acetylation to active transmitter and then storage in a vesicle for subsequent release [16 21] (Fig. 11-4). The synthesis reaction is a single step catalyzed by the enzyme choline acetyl transferase (ChAT): Choline + Acetyl coenzyme A a Acetylcholine + Coenzyme A. ChAT, first assayed in a cell-free preparation in 1943, has subsequently been purified and cloned from several sources . The purification of ChAT has allowed ACh ACh ACh AcCoA ChAT Choline = VAChT = ChT AChE Choline + Ac ACh FIGUE 11-4 Transport, synthesis and degradative processes in a cholinergic presynaptic nerve terminal and synapse. The choline transport protein (ChT) functions at the nerve ending membrane to transport choline into the cytoplasm, where its acetylation by acetyl CoA is catalyzed by choline acetyltransferase (ChAT) to generate acetylcholine (ACh) in the vicinity of the synaptic vesicle. The vesicular acetylcholine transporter (VAChT) concentrates acetylcholine in the vesicle. ChT is also found on the vesicle but in a functionally inactive state. Upon nerve stimulation, depolarization and Ca 2+ entry, ACh-containing vesicles fuse with the membrane and release their contents. The fusion of the membrane results in more ChT being exposed to the synaptic gap, where it becomes active. ACh is hydrolyzed to acetate and choline catalyzed by acetylcholinesterase (AChE), allowing for recapture of much of the choline by ChT. Because of the differing ionic compositions in the extracellular milieu and within the cell, ChT is thought to be active only when situated on the nerve cell membrane. imilarly, the VAChT may only be active when encapsulated in the synaptic vesicle . production of specific antibodies. Whereas acetylcholinesterase (AChE), the enzyme responsible for degradation of ACh, is produced by cells containing cholinoreceptive sites as well as in cholinergic neurons, ChAT is found in the nervous system specifically at sites where ACh synthesis takes place. Within cholinergic neurons, ChAT is concentrated in nerve terminals, although it is also present in axons, where it is transported from its site of synthesis in the soma. When subcellular fractionation studies are carried out, ChAT is recovered in the synaptosomal fraction, and within synaptosomes it is primarily cytoplasmic. It has been suggested that ChAT also binds to the outside of the storage vesicle under physiological conditions and that ACh synthesized in that location may be situated favorably to enter the vesicle. Brain ChAT has a K D for choline of approximately 1 mmol/l and for acetyl coenzyme A (CoA) of approximately 10 µmol/l. The activity of the isolated enzyme, assayed in the presence of optimal concentrations of cofactors and substrates, appears far greater than the rate at which choline is converted to ACh in vivo. This suggests that the activity of ChAT is repressed in vivo. urprisingly, inhibitors of ChAT do not decrease ACh synthesis when used in vivo; this may reflect a failure to achieve a sufficient local concentration of inhibitor, but also suggests that this step is not rate-limiting in the synthesis of ACh [18 20]. The acetyl CoA used for ACh synthesis in mammalian brain comes from pyruvate formed from glucose. It is unclear how the acetyl CoA, generally thought to be formed at the inner membrane of the mitochondria, accesses the cytoplasmic ChAT. This may also be a regulated, rate-limiting step. Acetylcholine formation is limited by the intracellular concentration of choline, which is determined by active transport of choline into the nerve ending. Choline is present in the plasma at a concentration of about 10 µmol/l. A low-affinity choline uptake system with a K m of µmol/l is present in all tissues, but cholinergic neurons also have a Na + -dependent high-affinity choline uptake system, with a K m for choline of 1 5 µmol/l [17 20]. The high-affinity uptake mechanisms should be saturated at 10 µmol/l choline, so the plasma choline concentration is probably adequate for sustained ACh synthesis even under conditions of high demand, as observed in ganglia. ince the plasma concentration of choline is above the K m of the high-affinity choline-transport system, it is not expected that choline concentrations in the nerve ending would be increased by increasing the plasma concentration of choline or by changing the K m of the uptake system. However, neuronal choline content can be influenced by altering the capacity of the high-affinity choline-uptake mechanism, i.e. by changing the maximum velocity (V max ) for transport. The cloning of the high affinity choline transporter has provided considerable insight into the mechanisms by which events associated with neuronal
9 CHAPTE 11 Acetylcholine N H N H Vesamicol H H 3 C H 3 C Hemicholinium (HC-3) FIGUE 11-5 tructures of hemicholinium (HC-3) and vesamicol. activity enhance choline entry into neurons [17, 19]. If the K m of ChAT for choline in vivo is as high as that seen with the purified enzyme, one would expect ACh synthesis to increase in proportion to the greater availability of choline. Conversely, ACh synthesis should be diminished when high-affinity choline uptake is blocked. Hemicholinium-3 is a potent inhibitor of the highaffinity choline-uptake system, with a K i in the submicromolar range (Fig. 11-5). Indeed, treatment with this drug decreases ACh synthesis and leads to a reduction in ACh release during prolonged stimulation; these findings lend support to the notion that choline uptake is the ratelimiting factor in the biosynthesis of ACh . Characterization and regulation of the high affinity choline transporter. The successful cloning of the high affinity choline transporter , now referred to as the CHT, was spurred by the discovery of a Caenorhabditis elegans transporter exhibiting cholinergic neuron specificity. The homologous rat CHT cdna conferred high affinity Na + and Cl dependent choline transport when expressed in Xenopus oocytes, and subsequently homologous cdnas were cloned from numerous species including mice and humans . The gene structure is highly conserved amongst the species, and the encoded protein is a member of a family of Na + -dependent transporters. The structure reveals 13 transmembrane spans, glycosylation sites on the extracellular surface and several potential sites for phosphorylation on intracellular loops and at the C- terminus. The development of antibodies to CHT revealed the anticipated localization of CHT at presynaptic sites and exclusively in cholinergic neurons. urprisingly, however, much of the CHT immunoreactivity was found associated with intracellular vesicles rather than at the presynaptic plasma membrane. These vesicles appear to be those involved in ACh synthesis (and its subsequent + N storage and release) as CHT is co-localized with the vesicular ACh transporter (VAChT) described below. Interestingly, blocking endocytosis leads to CHT accumulation at the plasma membrane and an enhanced capacity for choline uptake . Thus, it appears that the transporter is normally removed from the plasma membrane and recycled. Conversely, stimuli that trigger ACh release have been shown to increase CHT density at the plasma membrane, providing a mechanism by which uptake can be tightly coupled to the rate of ACh release. It is likely that post-translational modification, e.g. phosphorylation, also serves to regulate the transporter in response to altered synaptic activity. Whether this occurs via activation of a resident plasma membrane pool of CHT or alterations in activity or recycling of the vesicular CHT pool remains to be determined. The transporter precursor progresses down the axons by axoplasmic transport and is localized to the vesicular environment by poorly understood mechanisms. A second transport system concentrates acetylcholine in the synaptic vesicle. The vesicular ACh transporter (VAChT) also has been cloned and expressed (see also Ch. 5). Its sequence places it in the 12-membranespanning family characteristic of the other biogenic amine transporters found in adrenergic nerve endings [18, 20, 21]. Interestingly, the gene encoding the transporter is located within an intron of the ChAT gene, suggesting a mechanism for coregulation of gene expression for ChAT and VAChT . ACh uptake into the vesicle is driven by a proton-pumping ATPase. Coupled countertransport of H + and ACh allows the vesicle to remain isoosmotic and electroneutral . A selective inhibitor of ACh transport, vesamicol (Fig. 11-5), inhibits vesicular ACh uptake with an IC 50 of 40 nnol/l [16 18]. Inhibition appears noncompetitive, suggesting that this inhibitor acts on a site other than the ACh-binding site on the transporter. Vesamicol blocks the evoked release of newly synthesized ACh without significantly affecting high-affinity choline uptake, ACh synthesis or Ca 2+ influx. The fact that ACh release is lost secondary to the blockade of uptake by the vesicle supports the concept that the vesicle is the site of ACh release. The activity of the recombinant transporter also is inhibited by vesamicol [19, 20]. Choline is supplied to the neuron either from plasma or by metabolism of choline-containing compounds. The majority of the choline used in ACh synthesis is thought to come directly from recycling of released ACh, hydrolyzed to choline by acetylcholinesterase (Fig. 11-4). Presumably, uptake of this neurotransmitter-derived choline occurs rapidly, before the choline diffuses away from the synaptic cleft. Another source of choline is the breakdown of phosphatidylcholine, which may be stimulated by locally released ACh. Choline derived from these two sources is then subject to high-affinity uptake into the
10 194 PAT II Intercellular ignaling nerve ending. In the CN, these metabolic sources of choline appear to be particularly important because choline in the plasma cannot readily pass the blood brain barrier. Thus, the high-affinity uptake of choline into cholinergic neurons in the CN might not always be saturated, and ACh synthesis could be limited by the supply of choline, at least during sustained activity. This would be consistent with the finding that ACh stores in the brain are subject to variation, whereas ACh stores in ganglia and muscles remain relatively constant. A slow release of acetylcholine from neurons at rest probably occurs at all cholinergic synapses. Quantized ACh release was described first by Fatt and Katz, who recorded small, spontaneous depolarizations at frog neuromuscular junctions that were subthreshold for triggering action potentials. These MEPPs were shown to be due to the release of ACh. When the nerve was stimulated and endplate potentials recorded and analyzed, the magnitude of these potentials always was found to be some multiple of the magnitude of the individual MEPPs. It was suggested that each MEPP resulted from a finite quantity or quantum of released ACh and that the endplate potentials resulted from release of greater numbers of quanta during nerve stimulation (see Ch. 10). A possible structural basis for these discrete units of transmitter was discovered shortly thereafter when independent electron microscopic and subcellular fractionation studies by de obertis and Whittaker revealed the presence of vesicles in cholinergic nerve endings. These investigators defined procedures for subcellular fractionation of mammalian brain and Torpedo electric organs that yield resealed nerve endings, or synaptosomes, that can be lysed to release a fraction enriched in vesicles. More than half of the ACh in the synaptosome is associated with particles that look like the vesicles seen under an electron microscope. Therefore, it is clear that ACh is associated with a vesicle fraction, and it is likely that it is contained within the vesicle. The origin of the free ACh within the synaptosome is less clear. It may be ACh that is normally in the cytosol of the nerve ending, or it may be an artifact of release from the vesicles during their preparation (see Ch. 10). The relationship between acetylcholine content in a vesicle and the quanta of acetylcholine released can only be estimated. Estimates of the amount of ACh contained within cholinergic vesicles vary considerably . Whittaker estimated that there are about 2,000 molecules of ACh in a cholinergic vesicle from the CN. A similar estimate of about 1,600 molecules of ACh per vesicle was made using sympathetic ganglia. The most abundant source of cholinergic synaptic vesicles is the electric organ of Torpedo. Vesicles from Torpedo are far larger than those from mammalian species and are estimated to contain up to 100 times more ACh per vesicle. The Torpedo vesicle also contains ATP and, in its core, a proteoglycan of the heparin sulfate type. Both of these constituents may serve as counter-ions for ACh, which otherwise would be at a hyperosmotic concentration. The amount of ACh in a quantum has been estimated by comparing the potential changes associated with MEPPs to those obtained by iontophoresis of known quantities of ACh. Based on such analysis, the amount of ACh per quantum at the snake neuromuscular junction was estimated to be something less than 10,000 molecules . ince this compares with estimates of vesicle content, quanta are likely defined by the amount of releasable ACh in the vesicle. An alternative favored by some investigators is that ACh is released directly from the cytoplasm. In this model, definable quanta are evident because channels in the membrane are open for finite periods of time when Ca 2+ is elevated. A presynaptic membrane protein suggested to mediate Ca 2+ -dependent translocation of ACh has been isolated . Although some arguments support this model, most investigators favor the notion that the vesicle serves not only as a unit of storage but also as a unit of release. The vesicle hypothesis and release of neurotransmitters are discussed also in Chapter 10. Depolarization of the nerve terminal by an action potential increases the number of quanta released per unit time. elease of ACh requires the presence of extracellular Ca 2+, which enters the neuron when it is depolarized. Most investigators are of the opinion that a voltage-dependent Ca 2+ current is the initial event responsible for transmitter release, which occurs about 200 µs later. The mechanism through which elevated Ca 2+ increases the probability of ACh release is not yet known; presumably activation of proteins in the synaptic vesicle membrane fusion complex causes the vesicle to fuse with the neuronal membrane. Dependence on Ca 2+ is a common feature of all exocytotic release mechanisms and it is likely that exocytosis is a conserved mechanism for transmitter release. There is good evidence that adrenergic vesicles empty their contents into the synaptic cleft because norepinephrine and epinephrine are released along with other contents of the storage vesicle. Although less rigorous data are available for cholinergic systems, cholinergic vesicles contain ATP, and release of ATP has been shown to accompany ACh secretion from these vesicles. Furthermore, Heuser and eese demonstrated, in electron microscopic studies at frog nerve terminals, that vesicles fuse with the nerve membrane and that vesicular contents appear to be released by exocytosis; it has been difficult to ascertain, however, whether the fusions are sufficiently frequent to account for release on stimulation. The nerve ending also appears to endocytose the outer vesicle membrane to form vesicles that subsequently are refilled with ACh [19, 23]. All the acetylcholine contained within the cholinergic neuron does not behave as if in a single compartment. esults of a variety of neurophysiological and biochemical experiments suggest the presence of two distinguishable
11 CHAPTE 11 Acetylcholine 195 pools of ACh, only one of which is readily available for release. These have been referred to as the readily available or depot pool and the reserve or stationary pool. The reserve pool refills the readily available pool as it is utilized. Unless the rate of mobilization of ACh into the readily available pool is adequate, the amount of ACh that can be released may be limited. It is also likely that newly synthesized ACh is used to fill the readily available pool of ACh because it is the newly synthesized ACh that is released preferentially during nerve stimulation. The precise relationship between these functionally defined pools and ACh storage vesicles is not known. It is possible that the readily available pool resides in vesicles poised for release near the nerve ending membrane, whereas the reserve pool is in more distant vesicles. ACETYCHINETEAE AND THE TEMINATIN F ACETYCHINE ACTIN Cholinesterases are widely distributed throughout the body in both neuronal and non-neuronal tissues. Based largely on substrate specificity, the cholinesterases are subdivided into the acetylcholinesterases (AChEs) (EC ) and the butyrylcholinesterase (BuChE) (EC ) [24 26]. Choline esters with acyl groups of the size of butyric acid or larger are hydrolyzed very slowly by AChE; selective inhibitors for each enzyme have been identified. AChE and BuChE are encoded by single but distinct genes. BuChE is synthesized primarily in the liver and appears in plasma; however, it is highly unlikely that appreciable concentrations of ACh diffuse from the locality of the synapse and elicit a systemic response. That the population distribution of BuChE mutations correlates with resistance of the enzyme to naturally occurring inhibitors suggests that this enzyme hydrolyzes dietary esters of potential toxicity. Although BuChE is localized in the nervous system during development, the existence of nonexpressing mutations in the BuChE gene within the human population also demonstrates that this enzyme is not essential for nervous system function. In general, AChE distribution correlates with innervation and development in the nervous system. The AChEs also exhibit synaptic localization upon synapse formation. Total knock out of the AChE gene in mice gives rise to an animal with continuous tremors, stunted growth and deficits in neurologic function . The primary and tertiary structures of the cholinesterases are known. The primary structures of the cholinesterases initially defined a large and functionally eclectic superfamily of proteins, the α,β hydrolase fold family, that function not only catalytically as hydrolases but also as surface adhesion molecules forming heterologous cell contacts, as seen in the structurally related proteins neuroligin and the tactins. A sequence homologous to the cholinesterases and a presumed common structural matrix are found in thyroglobulin, in which tyrosine residues become iodinated and conjugated to form thyroid hormone [24, 25]. The initial solution of the crystal structure of the Torpedo enzyme , followed by the mammalian AChE structure , revealed that the active center serine lies at the base of a rather narrow gorge that is lined heavily with aromatic residues (Fig. 11-6). The enzyme carries a net negative charge, and an electrostatic dipole is oriented on the enzyme to facilitate diffusional entry of cationic ligands. Crystal structures of several inhibitors in a complex with AChE also have been elucidated . Acetylcholinesterases exist in several molecular forms governed by alternative splicing. The open reading frame in mammalian AChE genes is encoded by three invariant exons (exons 2, 3 and 4) followed by three splicing alternatives. Continuation through exon 4 gives rise to a monomeric species. plicing to exon 5 gives rise to the carboxyl-terminal sequence signal for addition of glycophospholipid, while splicing to exon 6 encodes a sequence containing a cysteine that links to other catalytic or structural subunits. These species of AChE differ only in the last 40 residues in their C-termini. FIGUE 11-6 View of the active center gorge of mammalian acetylcholinesterase looking into the gorge cavity. The gorge is Å in depth in a molecule of 40 Å diameter and is heavily lined with aromatic amino acid side chains. ide chains from several sets of critical residues are shown emanating from the α carbon of the α carbon-amide backbone: (a) A catalytic triad between Glu 334, His 447 and er 203 is shown by dotted lines to denote the hydrogen-bonding pattern. This renders er 203 more nucleophilic to attack the carbon of acetylcholine (shown in white with the van der Waals surface). This leads to formation of an acetyl enzyme, which is deacetylated rapidly. (b) The acyl pocket outline by Phe 295 and 297 is of restricted size in acetylcholinesterase. In butyrylcholinesterase, these side chains are aliphatic, increasing the size and flexibility in the acyl pocket. (c) The choline subsite lined by the aromatic residues Trp 86, Tyr 337 and Tyr 449 and the anionic residue Glu 202. (d) A peripheral site which resides at the rim of the gorge encompasses Trp 286, Tyr 72, Tyr 124 and Asp 74. This site modulates catalysis by binding inhibitors or, at high concentrations, a second substrate molecule.