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Siegel GJ, Agranoff BW, Albers RW, et al., editors. Basic Neurochemistry: Molecular, Cellular and Medical Aspects. 6th edition. Philadelphia: Lippincott-Raven; 1999.

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Basic Neurochemistry: Molecular, Cellular and Medical Aspects. 6th edition.

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Synthesis, Storage and Release of Acetylcholine

and .

Correspondence to Palmer Taylor and Joan Heller Brown, Department of Pharmacology, 0636, University of California, San Diego, La Jolla, California 92093.

Acetylcholine is synthesized from its two immediate precursors, choline and acetyl coenzyme A

The synthesis reaction is a single step catalyzed by the enzyme ChAT (EC 2.3.1.6):

Choline+Acetyl coenzyme AAcetylcoline+Coenzyme A

ChAT, first assayed in a cell-free preparation in 1943, subsequently has been purified and cloned from several sources [15]. The purification of ChAT has allowed 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 mM and for acetyl coenzyme A (CoA) of approximately 10 μM. 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. 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.

The acetyl CoA used for ACh synthesis in mammalian brain comes from pyruvate formed from glucose. It is uncertain how the acetyl CoA, generally thought to be formed at the inner membrane of the mitochondria, accesses the cytoplasmic ChAT, and it is possible that this is a rate-limiting step.

Acetylcholine formation is limited by the intracellular concentration of choline, which is determined by uptake of choline into the nerve ending

Choline is present in the plasma at a concentration of about 10 μM. A “low-affinity” choline uptake system with a K m of 10 to 100 μM is present in all tissues, but cholinergic neurons also have an Na+-dependent “high-affinity” choline uptake system, with a K m for choline of 1 to 5 μM [16]. The high-affinity uptake mechanisms should be saturated at 10 μM choline, so the plasma choline concentration is probably adequate for sustained ACh synthesis even under conditions of high demand, as observed in ganglia. Since 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 might be changed by altering the capacity of the high-affinity choline-uptake mechanism, such as changing the maximum velocity (V max) for transport, and this has been reported to occur in some brain regions in response to increased or decreased neuronal activity. There is some dispute about whether the capacity of the uptake system is increased or whether choline influx is regulated by changes in the intraterminal concentration of choline; it is agreed, however, that some event associated with neuronal activity enhances choline entry into neurons [16]. 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 high-affinity choline-uptake system, with a K i in the submicromolar range (Fig. 11-4). 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 rate-limiting factor in the biosynthesis of ACh. To date, the high-affinity choline-uptake system has not been cloned successfully.

Figure 11-4. Structures of hemicholinium (HC-3) and vesamicol.

Figure 11-4

Structures of hemicholinium (HC-3) and vesamicol.

A second transport system concentrates acetylcholine in the synaptic vesicle

ACh is transported into storage vesicles following its synthesis by ChAT in the nerve ending [16]. The vesicular ACh transporter (VAChT) has been cloned and expressed. Its sequence places it in the 12-membrane-spanning family characteristic of other biogenic amine transporters found in adrenergic nerve endings [17,18]. 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 in the vesicle is driven by a proton-pumping ATPase. Coupled countertransport of H+ and ACh allows the vesicle to remain isoosmotic and electroneutral [16].

A selective inhibitor of ACh transport, vesamicol (Fig. 11-4), inhibits vesicular ACh uptake with an IC50 of 40 nM [1618]. Inhibition appears noncompetitive, suggesting that it acts on some 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 Ca2+ influx. The fact that ACh release is lost secondary to the blockade of uptake by the vesicle strongly suggests that the vesicle is the site of ACh release. The expressed transporter from the cloned cDNA also is inhibited by vesamicol [17,18].

Choline is supplied to the neuron either from plasma or by metabolism of choline-containing compounds

At least half of the choline used in ACh synthesis is thought to come directly from recycling of released ACh, hydrolyzed to choline by cholinesterase. Presumably, uptake of this metabolically 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 becomes available in the extracellular space and is then subject to high-affinity uptake into the nerve ending. In the CNS, these metabolic sources of choline appear to be particularly important because choline in the plasma cannot pass the blood—brain barrier. Thus, in the CNS, the high-affinity uptake of choline into cholinergic neurons might not 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

This 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 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 Chap. 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 Robertis and Whittaker revealed the presence of vesicles in cholinergic nerve endings. Subcellular fractionation of mammalian brain and Torpedo electric organs yields 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 Chap. 10).

The relationship between the amount of acetylcholine in a vesicle and the quanta of acetylcholine released can only be estimated

Estimates of the amount of ACh contained within cholinergic vesicles vary, and there is obviously some subjectivity in correcting the values obtained, such as the percent of vesicles that are cholinergic or how much ACh may be lost during their preparation [19]. Whittaker estimated that there are about 2,000 molecules of ACh in a cholinergic vesicle from the CNS. 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, that is, 200,000 molecules 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 [19]. Given the possible error in these calculations, this would be within the range of that estimated to be contained in a vesicle. Therefore, it is likely that quanta are 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 Ca2+ is elevated. A presynaptic membrane protein suggested to mediate Ca2+-dependent translocation of ACh has been isolated [18]. Although there are some compelling arguments in support of 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

Release of ACh requires the presence of extracellular Ca2+, which enters the neuron when it is depolarized. Most investigators are of the opinion that a voltage-dependent Ca2+ current is the initial event responsible for transmitter release, which occurs about 200 μsec later. The mechanism through which elevated Ca2+ increases the probability of ACh release is not yet known; phosphorylation or activation of proteins that causes the vesicle to fuse with the neuronal membrane, are among the possibilities. Dependence on Ca2+ 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 Reese 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].

All of the acetylcholine contained within the cholinergic neuron does not behave as if in a single compartment

Results of a variety of neurophysiological and biochemical experiments suggest that there are at least two distinguishable 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. Although cholinergic vesicles appear to be homogeneous, there may be subpopulations of vesicles that differ in size and density.

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Copyright © 1999, American Society for Neurochemistry.
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