Neurochemistry

The basic biochemistry of the synthesis and destruction of ACh is outlined in Fig. 6.1 and put into the context of the cholinergic synapse in Fig. 6.2.

Presynaptic terminal

ACh metabolism

+ acetylcholinesterase - anticholinesterase

Muscarinic rcccptors G prot:eins

-Pirenzapine M | messengers

Direct Nicotinic receptors opening of Na+ channels

-DHflE

ACh metabolism

+ acetylcholinesterase - anticholinesterase

Postsynaptic cell

Muscarinic rcccptors G prot:eins

-Pirenzapine M | messengers

Direct Nicotinic receptors opening of Na+ channels

-DHflE

^ Receptors)

Figure 6.2 Diagrammatic representation of a cholinergic synapse. Some 80% of neuronal acetylcholine (ACh) is found in the nerve terminal or synaptosome and the remainder in the cell body or axon. Within the synaptosome it is almost equally divided between two pools, as shown. ACh is synthesised from choline, which has been taken up into the nerve terminal, and to which it is broken down again, after release, by acetylcholinesterase. Postsynaptically the nicotinic receptor is directly linked to the opening of Na+ channels and can be blocked by compounds like dihydro-/i-erythroidine (DH/?E). Muscarinic receptors appear to inhibit K+ efflux to increase cell activity. For full details see text

SYNTHESIS

Acetylcholine is synthesised in nerve terminals from its precursor choline, which is not formed in the CNS but transported there in free form in the blood. It is found in many foods such as egg yolk, liver and vegetables although it is also produced in the liver and its brain concentration rises after meals. Choline is taken up into the cytoplasm by a high-affinity (Km = 1-5 ^M), saturable, uptake which is Na+ and ATP dependent and while it does not appear to occur during the depolarisation produced by high concentrations of potassium it is increased by neuronal activity and is specific to cholinergic nerves. A separate low-affinity uptake, or diffusion (Km = 50 ^M), which is linearly related to choline concentration and not saturable, is of less interest since it is not specific to cholinergic neurons.

The reaction of choline with mitochondrial bound acetylcoenzyme A is catalysed by the cytoplasmic enzyme choline acetyltransferase (ChAT) (see Fig. 6.1). ChAT itelf is synthesised in the rough endoplasmic reticulum of the cell body and transported to the axon terminal. Although the precise location of the synthesis of ACh is uncertain most of that formed is stored in vesicles. It appears that while ChAT is not saturated with either acetyl-CoA or choline its synthesising activity is limited by the actual availability of choline, i.e. its uptake into the nerve terminal. No inhibitors of ChAT itself have been developed but the rate of synthesis of ACh can, however, be inhibited by drugs like hemicholinium or triethylcholine, which compete for choline uptake into the nerve.

STORAGE AND RELEASE

ACh is not distributed evenly within the neuron. If brain tissue is homogenised in isotonic salt solution containing an anticholinesterase, about 20% of the total ACh is released into solution, presumably from cell bodies, and it is found in the supernatant fraction on centrifugation. The remaining 80% settles within the sedimenting pellet and if this is resuspended and spun through a sucrose gradient it is all found in the synaptosome (nerve ending) fraction. After analysis of the synaptosomes and further centrifugation about half of this ACh, i.e. 40% of the original total still remains in the spun-down pellet. This is referred to as the firmly bound or stable 'pool' of ACh since it is not subject to hydrolysis by cholinesterase during the separation procedure. The other half (again 40% or the original) is found in the supernatant and undergoes hydrolysis unless protected by anticholinesterases. While it is generally assumed that some of this latter ACh was always in the synaptosomal cytoplasm probably half of it (20% of the original) comes from disrupted vesicles. This mixture of vesicular and cytoplasmic ACh is called the labile pool and is probably the most important source of releasable ACh, and also where newly synthesised ACh is found. Thus in studies in which tissue has been incubated with labelled precursor choline not only is this pool (fraction) heavily labelled but since most of the released ACh is also labelled it is assumed to come from this pool. With the passage of time there is interchange of ACh between the labile and the so-called fixed pool and in the absence of adequate resynthesis, i.e. blockage of choline uptake, it is likely that ACh will be released from the latter source as well.

Morphological evidence has also been obtained for two distinct vesicles with one designated VP1 (see Whittaker 1987; Zinnerman et al. 1993) being larger but less dense than the other (VP2). It is the latter which are thought to be incompletely filled with

ACh and considered to be the vesicles in the labile releasable pool. The evidence for and the actual mechanism of the vesicular release of ACh, mostly gained from studies at peripheral synapses, has been covered in Chapter 4.

Apart from inhibiting the uptake of choline and hence its availability for ACh synthesis, with hemicholinium (see above), there are no drugs that directly affect the actual storage or release of ACh. Some experimental tools have, however, been used such as vesamicol, which appears to block the packaging of ACh into its vesicles and thus initiates the slow rundown of releasable vesicular ACh. Some toxins also inhibit ACh release.

Botulinum toxin produced by the anaerobic bacillus Clostridium botulinum is unbelievably toxic with a minimum lethal mouse dose of 10~12g. Its occurrence in certain, generally preserved, foods leads to an extremely serious form of poisoning (botulism) resulting in progressive parasympathetic, motor and eventually respiratory paralysis and death. There are no antidotes and mortality is high. Despite this frightening profile, the toxin is finding increasing therapeutic use in relieving some forms of localised muscle spasm such as those of the eyelids (blepharospasm). Obviously it has to be injected directly into the muscle in carefully calculated small amounts. Provided this is achieved its firm binding and slow dissociation ensures a local effect that can last a number of weeks.

Beta-bungarotoxin, a protein in cobra snake venom, also binds to cholinergic nerves to stop ACh release while a-bungarotoxin (from the same source) binds firmly to peripheral postsynaptic nicotinic receptors. The combined effect ensures the paralysis of the snake's victim.

While there is no active neuronal uptake of ACh itself, cholinergic nerve terminals do possess autoreceptors. Since these are stimulated by ACh rather than by the choline, to which ACh is normally rapidly broken down, it is unlikely that they would be activated unless the synaptic release of ACh was so great that it had not been adequately hydrolysed by cholinesterase.

ACh is widely distributed throughout the brain and parts of the spinal cord (ventral horn and dorsal columns). Whole brain concentrations of 10nmolg_1 tissue have been reported with highest concentrations in the interpeduncular, caudate and dorsal raphe nuclei. Turnover figures of 0.15-2.0 nmolg^min-1 vary with the area studied and the method of measurement, e.g. synthesis of labelled ACh from [14C]-choline uptake or rundown of ACh after inhibition of choline uptake by hemicholinium. They are all sufficiently high, however, to suggest that in the absence of synthesis depletion could occur within minutes.

METABOLISM

Released ACh is broken down by membrane-bound acetylcholinesterase, often called the true or specific cholinesterase to distinguish it from butyrylcholinesterase, a pseudo-or non-specific plasma cholinesterase. It is an extremely efficient enzyme with one molecule capable of dealing with something like 10 000 molecules of ACh each second, which means a short life and rapid turnover (100 ps) for each molecule of ACh. It seems that about 50% of the choline freed by the hydrolysis of ACh is taken back into the nerve. There is a wide range of anticholinesterases which can be used to prolong and potentiate the action of ACh. Some of these, such as physostigmine, which can cross the blood-brain barrier to produce central effects and neostigmine, which does not readily do so, combine reversibly with the enzyme. Others such as the pesticide, disopropylpho-sphofluorate (DYFLOS), form an irreversible complex requiring the synthesis of new enzyme before recovery. Recently longer acting but reversible inhibitors such as tetrahydro aminoacridine have found some use in the therapy of Alzheimer's disease (Chapter 8). The manner in which acetylcholinesterase is thought to bind to and react with ACh and how drugs may inhibit it are shown in Fig. 6.3.

In addition to its vital role in the metabolism of ACh, acetylcholinesterase has been shown somewhat surprisingly to be released in the substantia nigra, along with DA,

The Function The Brain Drugs

Figure 6.3 Modes of action of the anticholinesterase drugs. Cholinesterase, which has both an anionic and an ester site (Fig. 6.1), can be inhibited by drugs acting reversibly and irreversibly. Edrophonium is a short-acting inhibitor that binds reversibly with the anionic site (1) while DYFLOS reacts almost irreversibly with the esteratic site (2). Since hydrolysis of the enzyme is negligible new enzyme must be synthesised to overcome the effect of this very toxic compound. Clinically useful anticholinesterase like neostigmine have a medium duration of action (21 = 1 h). It binds to both sites on the enzyme (3) with the result that neostigmine itself is hydrolysed but the transfer of its carbamyl group to the enzyme's esteratic site produces a carbamylated enzyme which recovers by hydrolysis much more slowly (min) than after its acetylation by ACh (ms)

Figure 6.3 Modes of action of the anticholinesterase drugs. Cholinesterase, which has both an anionic and an ester site (Fig. 6.1), can be inhibited by drugs acting reversibly and irreversibly. Edrophonium is a short-acting inhibitor that binds reversibly with the anionic site (1) while DYFLOS reacts almost irreversibly with the esteratic site (2). Since hydrolysis of the enzyme is negligible new enzyme must be synthesised to overcome the effect of this very toxic compound. Clinically useful anticholinesterase like neostigmine have a medium duration of action (21 = 1 h). It binds to both sites on the enzyme (3) with the result that neostigmine itself is hydrolysed but the transfer of its carbamyl group to the enzyme's esteratic site produces a carbamylated enzyme which recovers by hydrolysis much more slowly (min) than after its acetylation by ACh (ms)

presumably from the soma and dendrites of DA neurons. Its function there is uncertain but purified preparation of the enzyme infused into the substantia nigra cause not only hyperpolarisation of the neurons, due to the opening of K+ channels, but also a variety of motor effects in rats that are not related to its enzymatic activity and the turnover of ACh (see Greenfield 1991).

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