ACETYLCHOLINE

I. Overview classes of neurochemical messengers

II. Structure of ACh

A. Could be classified as an amine; a substituted ammonia. It is an aliphatic amine
B. Is an ester of choline and acetyl CoA

III. History

A. First identified transmitter substance--'vagusstuff'
B. One of the hardest to understand its role in the CNS
Much early work done on neuromuscular junction or the electric eel or torpedo preparations; CNS neurons seem to operate differently.

IV. Distribution

A. PNS
1. Somatic Motor nerves: cranial and spinal
2. Autonomic (sympathetic and parasympathetic) preganglionic nerves
3. Parasympathetic postganglionic nerves B. CNS
1. Only recently that precise picture of cholinergic neurons has emerged. Based on new methods, but particularly on availability of antibodies to synthesizing enzyme, CAT. CAT is a weak immunogen and it was difficult to produce antibodies against it.
2. Local Circuit neurons
Caudate-putamen, nuc. accumbens, possibly cerebral ctx & hippocampus
3. Projection Neurons
a. Basal forebrain projection system
medial septal nucleus, magnocellular preoptic area, substanstia innominata, nucleus basalis. Project to entire nonstriatal telencephalon.
b. Pontine tegmental projection system
pedunculopontine tegmental area & dorsolateral tegmental nucleus. Project to thalamus, globus pallidus, subthalamic nuc., lateral hypothalamus, basal forebrain; descending projection to brainstem nuclei.

V. Biochemistry of ACh

A. Synthesis 1. Enzyme of synthesis is choline acetyltransferase (CAT) a. Unique marker for cholinergic neurons. while present in non-neural tissue (placenta, sperm cells), it is not present in glia cells.
b. Enzyme synthesized in cell body but transported to terminal; highest amount in terminal.
c. Probably a cytoplasmic enzyme. Early indication of high proportion being membrane bound due to enzyme trapped in synaptosomes. Part of enzyme does behave as if it were firmly bound to nerve terminal membranes.
d. Substrates
1. Acetyl CoA and Choline
Km for choline is 0.4-1mM and for ACoA is 7-46 µM.
Cytoplasmic concentrations (estimated): choline-0.05 mM and ACoA-5 µM.Thus, CAT is not saturated and could operate at ~<1/20 of maximal velocity.
2. Sources
ACoA: Derived from glucose via pyruvate produced in glycolysis. Acetate is poor source of ACoA for ACh synthesis. Produced in mitochondria; must be transported across inner mitochondrial membrane because membrane is not permeable to ACoA.
Choline: Source of choline for ACh synthesis
1. Under most conditions, choline used for synthesis of ACh in cholinergic neurons originates from extracellular fluid surrounding terminals, 50-60% of which comes from choline cleaved by action of ACh esterase on released ACh. Hi aff. carrier responsible for transport at nerve endings. Saturable, Na+ dependent, Kt+0.1 to 10 µM. Lo aff. carrier mediates transport elsewhere, Kt~30 to 200 µM. There appears to be little stored choline in nerve cells, can replace all of ACh in terminal with labelled choline in incubation medium.
2. Circulating Choline (or phosphatidyl choline). Physiologically choline can be produced by a series of methylation reactions of phosphatidylethanolamine to phosphatidylcholine (PC). Choline is released from PC. PC is synthesized mainly in liver and is main store & source of free choline in body. Brain is not thought to produce choline de nova--but see below.
However, chloine transport across brain capillaries is bidirectional; only after ingesting a meal is plasma conc. of choline higher than brain extracellular fluid conc.
3. Evidence that Ach synthesis does not require circulating choline:
Resting synthesis of ACh in brain slices does not require addition of choline to medium. (not so for symp ganglia or diaphragm preparations). If Na+ is absent from medium, choline uptake is blocked & brain slices lose most of ACh, but they recover ACh stores after addition of Na+ even in medium to which no choline is added. But if only 50-60% of released choline is recaptured by nerve terminal, how do terminals maintain ACh synthesis constant.
4. An enzymatic system that forms PC has been characterized in brain. The enzyme, PEMT(phosphatidyl enthanolamine-N-methyltransferase) may be regulated by various neurotransmitters. Remains to be proven whether this system is extensive enough to provide choline necessary for synthesis of ACh. 2. Regulation of synthesis a. ACh levels are stable in mammalian CNS over wide range of neural activity.
Rate of ACh synthesis adapts to rate of ACh release so as to keep ACh constant
Availability of choline is limiting factor
According to law of mass action when reaction catalyzed by ChAT at equillibrium: [ACh]=Keq[choline][AcCoA]/[CoA] b. Two important regulatory factors 1. Uptake of choline; Increased synaptic activity leads to increased rate of uptake. Release of ACh appears to be main factor for increased choline uptake. If release is prevented, uptake is inhibited.
2. [Ach] in compartment of synthesis; if ACh decreases during synaptic activity, there will be a shift in the equilibrium of reaction catalyzed by ChAT leading to increased production of ACh and there will be disinhibition of the hi. aff. choline carriers which will increase supply of choline in nerve endings. c. Turnover Rate: Time it takes to replinish available pool (amount synthesized per unit time to replace tha utilized. No good method to measure ACh turnover. No good synthesis inhibitors. But all estimates indicate that brain contains only a few min. supply of ACh, thus turnover is probably very rapid. Study by Potter et al most reliable.

B. Inactivation of ACh

1. By cholinesterases; ACh-E is only one such enzyme. Hydrolyze ACh to choline & acetic acid. ACh-E is very stable enzyme, accounts for postmortem breakdown of ACh.
2. Unusual for a transmitter to be enzymatically degraded after release; action must have biologic advantage.
3. Enzyme action serves two functions; reduces time of exposure of ACh to receptors and maintains supply of choline.
4. Esterases are located both presynaptically in cholinergic neurons & on postsynaptic membrane; hence enzyme is not a marker of cholinergic neurons. Enzyme is also present in neurons like nigrastriatal DA & NE projections from locus coeruleus that do not contain ACh or receive ACh input. Enzyme may have physiologic actions unrelated to the hydrolysis of ACh.
5. Enzyme is sometimes released from neurons, dendrites as well as axons. Also released into CSF, release does not parallel hydrolysis of ACh.

C. Release of ACh

1. Ca++ dependent
2. Newly synthesized seems more readily releasable; special transporter for Ach on vesicle membranes.
3. Is vesicle necessary for release?
a. ACh in terminal seems compartmentalized: 50% is vesicle bound and 50% is cytoplasmic. Cytoplasmic may be artifactual.
b. In PNS, a surplus ACh is defined as that ACh which increases after ACh-E inhibition.
c. In PNS, good evidence that vesicles play a role in ACh release.

Source: The Cholinergic Synapse, V.P. Whittaker, Ed. Handbook of Experimental Pharmacol., vol 86, 1988.
Cooper, J. R., Unsolved problems in the cholinergic nervous system. J. Neruochem., 63:395-399, 1994.

Last modified: 9/8/97