An important traditional classification of autonomic nerves is based on the primary transmitter molecules—acetylcholine or norepinephrine—released from their terminals 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 some sympathetic postganglionic fibers are cholinergic. A significant number of parasympathetic postganglionic neurons use nitric oxide or peptides as the primary transmitter or as cotransmitters.
Most postganglionic sympathetic fibers (Figure 6–1) release norepinephrine (also known as noradrenaline); they are noradrenergic (often called simply “adrenergic”) fibers; that is, they work by releasing norepinephrine (noradrenaline). As noted, some sympathetic fibers release acetylcholine. Dopamine is a very important transmitter in the CNS, and it may be released by some peripheral sympathetic fibers under certain circumstances. 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 following text), in addition to the primary transmitters just described.
Five key features of neurotransmitter function provide potential targets for pharmacologic therapy: synthesis, storage, release, 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 portion of the cell membrane facing the synapse (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 may be synthesized in the neuron cell body and carried to the terminal by axonal transport. They may also be recycled several times within the terminal after each exocytotic release of transmitter. Ultra-fast neuronal firing appears to be supported by rapid recycling of clathrin-coated vesicles from endosomes in the nerve 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. VAMPs and SNAPs are collectively called fusion proteins.
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 (ACh) is then transported into the storage vesicle by a 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 transmitters 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, synaptosomal nerve-associated proteins; VAMPs, vesicle-associated membrane proteins.
TABLE 6–1Some of the transmitter substances found in autonomic nervous system, enteric nervous system, and nonadrenergic, noncholinergic neurons.1 ||Download (.pdf) TABLE 6–1 Some of the transmitter substances found in autonomic nervous system, enteric nervous system, and nonadrenergic, noncholinergic neurons.1
|Substance ||Functions |
|Acetylcholine (ACh) ||The primary transmitter at ANS ganglia, at the somatic neuromuscular junction, and at parasympathetic postganglionic nerve endings. A primary excitatory transmitter to smooth muscle and secretory cells in the ENS. Probably also the major neuron-to-neuron (“ganglionic”) transmitter in the ENS. |
|Adenosine triphosphate (ATP) ||Acts as a transmitter or cotransmitter at many ANS-effector synapses. |
|Calcitonin gene-related peptide (CGRP) ||Found with substance P in cardiovascular sensory nerve fibers. Present in some secretomotor ENS neurons and interneurons. A cardiac stimulant. |
|Cholecystokinin (CCK) ||May act as a cotransmitter in some excitatory neuromuscular ENS neurons. |
|Dopamine ||A modulatory transmitter in some ganglia and the ENS. Possibly a postganglionic sympathetic transmitter in renal blood vessels. |
|Enkephalin and related opioid peptides ||Present in some secretomotor and interneurons in the ENS. Appear to inhibit ACh release and thereby inhibit peristalsis. May stimulate secretion. |
|Galanin ||Present in secretomotor neurons; may play a role in appetite-satiety mechanisms. |
|GABA (γ-aminobutyric acid) ||May have presynaptic effects on excitatory ENS nerve terminals. Has some relaxant effect on the gut. Probably not a major transmitter in the ENS. |
|Gastrin-releasing peptide (GRP) ||Extremely potent excitatory transmitter to gastrin cells. Also known as mammalian bombesin. |
|Neuropeptide Y (NPY) ||Found in many noradrenergic neurons. Present in some secretomotor neurons in the ENS and may inhibit secretion of water and electrolytes by the gut. Causes long-lasting vasoconstriction. It is also a cotransmitter in some parasympathetic postganglionic neurons. |
|Nitric oxide (NO) ||A cotransmitter at inhibitory ENS and other neuromuscular junctions; may be especially important at sphincters. Cholinergic nerves innervating blood vessels appear to activate the synthesis of NO by vascular endothelium. NO is not stored, it is synthesized on demand by nitric oxide synthase, NOS; see Chapter 19. |
|Norepinephrine (NE) ||The primary transmitter at most sympathetic postganglionic nerve endings. |
|Serotonin (5-HT) ||An important transmitter or cotransmitter at excitatory neuron-to-neuron junctions in the ENS. |
|Substance P, related tachykinins ||Substance P is an important sensory neurotransmitter in the ENS and elsewhere. Tachykinins appear to be excitatory cotransmitters with ACh at ENS neuromuscular junctions. Found with CGRP in cardiovascular sensory neurons. Substance P is a vasodilator (probably via release of nitric oxide). |
|Vasoactive intestinal peptide (VIP) ||Excitatory secretomotor transmitter in the ENS; may also be an inhibitory ENS neuromuscular cotransmitter. A probable cotransmitter in many cholinergic neurons. A vasodilator (found in many perivascular neurons) and cardiac stimulant. |
Acetylcholine (ACh) 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 1000–50,000 molecules in each vesicle). Most of the vesicular acetylcholine (a positively charged quaternary amine) 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 cleavage 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 the precursor amino acid tyrosine into the nerve ending, convert it to dopa, and then synthesize a catecholamine transmitter (dopamine, norepinephrine, or epinephrine; Figure 6–5), and 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 by tyrosine hydroxylase, 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 and related drugs (tetrabenazine, deutetrabenazine) cause 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 certain antidepressant drugs, resulting in an increase of transmitter activity in the synaptic cleft (see Box: Neurotransmitter Uptake Carriers).
Schematic diagram of a generalized noradrenergic junction (not to scale). Tyrosine is transported into the noradrenergic nerve 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 and tetrabenazine. The same carrier transports norepinephrine (NE) and several related amines into these vesicles. Dopamine is converted to NE in the vesicle by dopamine-β-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-β-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 certain antidepressants, or into postjunctional or perijunctional cells. Regulatory receptors are present on the presynaptic terminal. SNAPs, synaptosome-associated proteins; VAMPs, vesicle-associated membrane proteins.
Biosynthesis of catecholamines. The rate-limiting step, conversion of tyrosine to dopa, can be inhibited by metyrosine (α-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 Gardner DG, Shoback D [editors]: Greenspan’s Basic & Clinical Endocrinology, 9th ed. McGraw-Hill, 2011. Copyright © The McGraw-Hill Companies, Inc.)
Neurotransmitter Uptake Carriers
As noted in Chapters 1, 4, and 5, 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 use 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 using 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, γ-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-β-hydroxylase, and peptide cotransmitters are simultaneously released from the same vesicles. Indirectly acting and mixed-action 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 measurement 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.
Metabolism of catecholamines by catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO). (Reproduced, with permission, from Gardner DG, Shoback D [editors]: Greenspan’s Basic & Clinical Endocrinology, 9th ed. McGraw-Hill, 2011. Copyright © The McGraw-Hill Companies, Inc.)
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.
Growth of neurons and transmitter expression in specific neurons is a dynamic process. For example, neurotrophic factors released from target tissues influence growth and synapse formation by neurons. In addition, the transmitters released from a specific population of neurons can change in response to environmental factors such as the light-dark cycle.