Neurotransmitter Chemistry of the Autonomic Nervous System
An important traditional classification of autonomic nerves is based on the primary transmitter molecules—acetylcholine or norepinephrine—released from their terminal boutons and varicosities. A large number of peripheral ANS fibers synthesize and release acetylcholine; they are cholinergic fibers; that is, they work by releasing acetylcholine. As shown in Figure 6–1, these include all preganglionic efferent autonomic fibers and the somatic (nonautonomic) motor fibers to skeletal muscle as well. Thus, almost all efferent fibers leaving the CNS are cholinergic. In addition, most parasympathetic postganglionic and a few sympathetic postganglionic fibers are cholinergic. A significant number of parasympathetic postganglionic neurons utilize nitric oxide or peptides as the primary transmitter or cotransmitters.
Most postganglionic sympathetic fibers release norepinephrine (also known as noradrenaline); they are noradrenergic (often called simply "adrenergic") fibers; that is, they work by releasing norepinephrine (noradrenaline). These transmitter characteristics are presented schematically in Figure 6–1. As noted, some sympathetic fibers release acetylcholine. Dopamine is a very important transmitter in the CNS, and there is evidence that it may be released by some peripheral sympathetic fibers. Adrenal medullary cells, which are embryologically analogous to postganglionic sympathetic neurons, release a mixture of epinephrine and norepinephrine. Finally, most autonomic nerves also release several cotransmitter substances (described in the text that follows), in addition to the primary transmitters just described.
Five key features of neurotransmitter function provide potential targets for pharmacologic therapy: synthesis, storage, release, and termination of action of the transmitter, and receptor effects. These processes are discussed next.
The terminals and varicosities of cholinergic neurons contain large numbers of small membrane-bound vesicles concentrated near the synaptic portion of the cell membrane (Figure 6–3) as well as a smaller number of large dense-cored vesicles located farther from the synaptic membrane. The large vesicles contain a high concentration of peptide cotransmitters (Table 6–1), whereas the smaller clear vesicles contain most of the acetylcholine. Vesicles are initially synthesized in the neuron cell body and carried to the terminal by axonal transport. They may also be recycled several times within the terminal. Vesicles are provided with vesicle-associated membrane proteins (VAMPs), which serve to align them with release sites on the inner neuronal cell membrane and participate in triggering the release of transmitter. The release site on the inner surface of the nerve terminal membrane contains synaptosomal nerve-associated proteins (SNAPs), which interact with VAMPs.
Figure 6–3 Add to 'My Saved Images'
Schematic illustration of a generalized cholinergic junction (not to scale). Choline is transported into the presynaptic nerve terminal by a sodium-dependent choline transporter (CHT). This transporter can be inhibited by hemicholinium drugs. In the cytoplasm, acetylcholine is synthesized from choline and acetyl-CoA (AcCoA) by the enzyme choline acetyltransferase (ChAT). Acetylcholine is then transported into the storage vesicle by a second carrier, the vesicle-associated transporter (VAT), which can be inhibited by vesamicol. Peptides (P), adenosine triphosphate (ATP), and proteoglycan are also stored in the vesicle. Release of transmitter occurs when voltage-sensitive calcium channels in the terminal membrane are opened, allowing an influx of calcium. The resulting increase in intracellular calcium causes fusion of vesicles with the surface membrane and exocytotic expulsion of acetylcholine and cotransmitters into the junctional cleft (see text). This step can be blocked by botulinum toxin. Acetylcholine's action is terminated by metabolism by the enzyme acetylcholinesterase. Receptors on the presynaptic nerve ending modulate transmitter release. SNAPs, synaptosome-associated proteins; VAMPs, vesicle-associated membrane proteins.
Table 6–1 Some of the Transmitter Substances Found in Autonomic Nervous System, Enteric Nervous System, and Nonadrenergic, Noncholinergic Neurons.1
Acetylcholine is synthesized in the cytoplasm from acetyl-CoA and choline through the catalytic action of the enzyme choline acetyltransferase (ChAT). Acetyl-CoA is synthesized in mitochondria, which are present in large numbers in the nerve ending. Choline is transported from the extracellular fluid into the neuron terminal by a sodium-dependent membrane choline transporter (CHT; Figure 6–3). This symporter can be blocked by a group of research drugs called hemicholiniums. Once synthesized, acetylcholine is transported from the cytoplasm into the vesicles by a vesicle-associated transporter (VAT) that is driven by proton efflux (Figure 6–3). This antiporter can be blocked by the research drug vesamicol. Acetylcholine synthesis is a rapid process capable of supporting a very high rate of transmitter release. Storage of acetylcholine is accomplished by the packaging of "quanta" of acetylcholine molecules (usually 1000 to 50,000 molecules in each vesicle). Most of the vesicular acetylcholine (ACh) is bound to negatively charged vesicular proteoglycan (VPG).
Vesicles are concentrated on the inner surface of the nerve terminal facing the synapse through the interaction of so-called SNARE proteins on the vesicle (a subgroup of VAMPs called v-SNAREs, especially synaptobrevin) and on the inside of the terminal cell membrane (SNAPs called t-SNAREs, especially syntaxin and SNAP-25). Physiologic release of transmitter from the vesicles is dependent on extracellular calcium and occurs when an action potential reaches the terminal and triggers sufficient influx of calcium ions via N-type calcium channels. Calcium interacts with the VAMP synaptotagmin on the vesicle membrane and triggers fusion of the vesicle membrane with the terminal membrane and opening of a pore into the synapse. The opening of the pore and inrush of cations results in release of the acetylcholine from the proteoglycan and exocytotic expulsion into the synaptic cleft. One depolarization of a somatic motor nerve may release several hundred quanta into the synaptic cleft. One depolarization of an autonomic postganglionic nerve varicosity or terminal probably releases less and releases it over a larger area. In addition to acetylcholine, several cotransmitters are released at the same time (Table 6–1). The acetylcholine vesicle release process is blocked by botulinum toxin through the enzymatic removal of two amino acids from one or more of the fusion proteins.
After release from the presynaptic terminal, acetylcholine molecules may bind to and activate an acetylcholine receptor (cholinoceptor). Eventually (and usually very rapidly), all of the acetylcholine released diffuses within range of an acetylcholinesterase (AChE) molecule. AChE very efficiently splits acetylcholine into choline and acetate, neither of which has significant transmitter effect, and thereby terminates the action of the transmitter (Figure 6–3). Most cholinergic synapses are richly supplied with acetylcholinesterase; the half-life of acetylcholine molecules in the synapse is therefore very short (a fraction of a second). Acetylcholinesterase is also found in other tissues, eg, red blood cells. (Other cholinesterases with a lower specificity for acetylcholine, including butyrylcholinesterase [pseudocholinesterase], are found in blood plasma, liver, glia, and many other tissues.)
Adrenergic neurons (Figure 6–4) transport a precursor amino acid (tyrosine) into the nerve ending, then synthesize the catecholamine transmitter (Figure 6–5), and finally store it in membrane-bound vesicles. In most sympathetic postganglionic neurons, norepinephrine is the final product. In the adrenal medulla and certain areas of the brain, some norepinephrine is further converted to epinephrine. In dopaminergic neurons, synthesis terminates with dopamine. Several processes in these nerve terminals are potential sites of drug action. One of these, the conversion of tyrosine to dopa, is the rate-limiting step in catecholamine transmitter synthesis. It can be inhibited by the tyrosine analog metyrosine. A high-affinity antiporter for catecholamines located in the wall of the storage vesicle (vesicular monoamine transporter, VMAT) can be inhibited by the reserpine alkaloids. Reserpine causes depletion of transmitter stores. Another transporter (norepinephrine transporter, NET) carries norepinephrine and similar molecules back into the cell cytoplasm from the synaptic cleft (Figure 6–4; NET). NET is also commonly called uptake 1 or reuptake 1 and is partially responsible for the termination of synaptic activity. NET can be inhibited by cocaine and tricyclic antidepressant drugs, resulting in an increase of transmitter activity in the synaptic cleft (see Neurotransmitter Uptake Carriers).
Figure 6–4 Add to 'My Saved Images'
Schematic diagram of a generalized noradrenergic junction (not to scale). Tyrosine is transported into the noradrenergic ending or varicosity by a sodium-dependent carrier (A). Tyrosine is converted to dopamine (see Figure 6–5 for details), and transported into the vesicle by the vesicular monoamine transporter (VMAT), which can be blocked by reserpine. The same carrier transports norepinephrine (NE) and several other amines into these granules. Dopamine is converted to NE in the vesicle by dopamine--hydroxylase. Physiologic release of transmitter occurs when an action potential opens voltage-sensitive calcium channels and increases intracellular calcium. Fusion of vesicles with the surface membrane results in expulsion of norepinephrine, cotransmitters, and dopamine--hydroxylase. Release can be blocked by drugs such as guanethidine and bretylium. After release, norepinephrine diffuses out of the cleft or is transported into the cytoplasm of the terminal by the norepinephrine transporter (NET), which can be blocked by cocaine and tricyclic antidepressants, or into postjunctional or perijunctional cells. Regulatory receptors are present on the presynaptic terminal. SNAPs, synaptosome-associated proteins; VAMPs, vesicle-associated membrane proteins.
Figure 6–5 Add to 'My Saved Images'
Biosynthesis of catecholamines. The rate-limiting step, conversion of tyrosine to dopa, can be inhibited by metyrosine (-methyltyrosine). The alternative pathway shown by the dashed arrows has not been found to be of physiologic significance in humans. However, tyramine and octopamine may accumulate in patients treated with monoamine oxidase inhibitors. (Reproduced, with permission, from Greenspan FS, Gardner DG [editors]: Basic and Clinical Endocrinology, 7th ed. McGraw-Hill, 2003.)
Neurotransmitter Uptake Carriers
As noted in Chapter 1, several large families of transport proteins have been identified. The most important of these are the ABC (ATP-Binding Cassette) and SLC (SoLute Carrier) transporter families. As indicated by the name, the ABC carriers utilize ATP for transport. The SLC proteins are cotransporters and in most cases, use the movement of sodium down its concentration gradient as the energy source. Under some circumstances, they also transport transmitters in the reverse direction in a sodium-independent fashion.
NET, SLC6A2, the norepinephrine transporter, is a member of the SLC family, as are similar transporters responsible for the reuptake of dopamine (DAT, SLC6A3) and 5-HT (serotonin, SERT, SLC6A4) into the neurons that release these transmitters. These transport proteins are found in peripheral tissues and in the CNS wherever neurons utilizing these transmitters are located.
NET is important in the peripheral actions of cocaine and the amphetamines. In the CNS, NET and SERT are important targets of several antidepressant drug classes (see Chapter 30). The most important inhibitory transmitter in the CNS, -aminobutyric acid (GABA), is the substrate for at least three SLC transporters: GAT1, GAT2, and GAT3. GAT1 is the target of an antiseizure medication (see Chapter 24). Other SLC proteins transport glutamate, the major excitatory CNS transmitter.
Release of the vesicular transmitter store from noradrenergic nerve endings is similar to the calcium-dependent process previously described for cholinergic terminals. In addition to the primary transmitter (norepinephrine), adenosine triphosphate (ATP), dopamine--hydroxylase, and peptide cotransmitters are also released into the synaptic cleft. Indirectly acting and mixed sympathomimetics, eg, tyramine, amphetamines, and ephedrine, are capable of releasing stored transmitter from noradrenergic nerve endings by a calcium-independent process. These drugs are poor agonists (some are inactive) at adrenoceptors, but they are excellent substrates for monoamine transporters. As a result, they are avidly taken up into noradrenergic nerve endings by NET. In the nerve ending, they are then transported by VMAT into the vesicles, displacing norepinephrine, which is subsequently expelled into the synaptic space by reverse transport via NET. Amphetamines also inhibit monoamine oxidase and have other effects that result in increased norepinephrine activity in the synapse. Their action does not require vesicle exocytosis.
Norepinephrine and epinephrine can be metabolized by several enzymes, as shown in Figure 6–6. Because of the high activity of monoamine oxidase in the mitochondria of the nerve terminal, there is significant turnover of norepinephrine even in the resting terminal. Since the metabolic products are excreted in the urine, an estimate of catecholamine turnover can be obtained from laboratory analysis of total metabolites (sometimes referred to as "VMA and metanephrines") in a 24-hour urine sample. However, metabolism is not the primary mechanism for termination of action of norepinephrine physiologically released from noradrenergic nerves. Termination of noradrenergic transmission results from two processes: simple diffusion away from the receptor site (with eventual metabolism in the plasma or liver) and reuptake into the nerve terminal by NET (Figure 6–4) or into perisynaptic glia or other cells.
Figure 6–6 Add to 'My Saved Images'
Metabolism of catecholamines by catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO). (Modified and reproduced, with permission, from Greenspan FS, Gardner DG [editors]: Basic and Clinical Endocrinology, 7th ed. McGraw-Hill, 2003.)
Cotransmitters in Cholinergic & Adrenergic Nerves
As previously noted, the vesicles of both cholinergic and adrenergic nerves contain other substances in addition to the primary transmitter, sometimes in the same vesicles and sometimes in a separate vesicle population. Some of the substances identified to date are listed in Table 6–1. Many of these substances are also primary transmitters in the nonadrenergic, noncholinergic nerves described in the text that follows. They appear to play several roles in the function of nerves that release acetylcholine or norepinephrine. In some cases, they provide a faster or slower action to supplement or modulate the effects of the primary transmitter. They also participate in feedback inhibition of the same and nearby nerve terminals.