Chapter 7: Neurotransmitters & Neuromodulators

After studying this chapter, youshould be able to:

• List the major types of neurotransmitters.

• Summarize the steps involved in the biosynthesis, release, action, and removal from the synaptic cleft of the major neurotransmitters.

• Describe the various types of receptors for amino acids, acetylcholine, monoamines, ATP, opioids, nitric oxide, and cannabinoids.

• Identify the endogenous opioid peptides, their receptors, and their functions.

Nerve endings have been called biologic transducers that convert electrical energy into chemical energy. An observation made by Otto Loewi, a German pharmacologist, serves as the foundation for the concept of chemical neurotransmission and his receipt of the Nobel Prize in Physiology and Medicine in 1936. He provided the first decisive evidence that a chemical messenger was released by the vagus nerve supplying the heart to reduce heart rate. The experimental design came to him in a dream on Easter Sunday of 1920. He awoke from the dream, jotted down notes, but the next morning they were indecipherable. The next night, the dream recurred and he went to his laboratory at 3:00 AM to conduct a simple experiment on a frog heart. He isolated the hearts from two frogs, one with and one without its innervation. Both hearts were attached to cannulas filled with a saline solution. The vagus nerve of the first heart was stimulated, and then the saline solution from that heart was transferred to the noninnervated heart. The rate of its contractions slowed as if its vagus nerve had been stimulated. Loewi called the chemical released by the vagus nerve vagusstoff. Not long after, it was identified chemically to be acetylcholine. Loewi also showed that when the sympathetic nerve of the first heart was stimulated and its effluent was passed to the second heart, the rate of contraction of the “donor” heart increased as if its sympathetic fibers had been stimulated. These results proved that nerve terminals release chemicals that cause the modifications of cardiac function that occur in response to stimulation of its nerve supply.

Regardless of the type of chemical mediator involved, several common steps comprise the process of transmission at a chemical synapse. The first steps are the synthesis of the neurotransmitter usually within the nerve terminal and its storage within synaptic vesicles. This is followed by release of the chemical into the synaptic cleft in response to nerve impulses. The secreted neurotransmitter can then act on receptors on the membrane of the postsynaptic neuron, effector organ (eg, muscle or gland), or even on the presynaptic nerveterminal. Thefinal steps in the process lead to termination of the actions of the neurotransmitter and include diffusion away from the synaptic cleft, reuptake into the nerve terminal, and enzymatic degradation. All of these processes, plus the events in the postsynaptic neuron, are regulated by many physiologic factors and can be altered by drugs. Therefore, pharmacologists (in theory) should be able to develop drugs that regulate not only somatic and visceral motor activity but also emotions, behavior, and all the other complex functions of the brain. Some chemicals released by neurons have little or no direct effects on their own but can modify the effects of neurotransmitters. These chemicals are called neuromodulators.

CHEMISTRY OF TRANSMITTERS

Many neurotransmitters and the enzymes involved in their synthesis and catabolism have been localized in nerve endings by immunohistochemistry, a technique in which antibodies to a given substance are labeled and applied to the brain and other tissues. The antibodies bind to the substance, and the location of the substance is then determined by locating the label with the light or electron microscope. In situ hybridization histochemistry, which permits localization of the mRNAs for particular synthesizing enzymes or receptors, has also been a valuable tool.

There are two main classes of chemical substances that serve as neurotransmitters and neuromodulators: small-molecule transmitters and large-molecule transmitters. Small-molecule transmitters include amino acids (eg, glutamate, γ-aminobutyric acid [GABA], and glycine), acetylcholine, monoamines (eg, norepinephrine, epinephrine, dopamine, and serotonin), and adenosine triphosphate (ATP). Large-molecule transmitters include neuropeptides such as substance P, enkephalin, vasopressin, and a host of others. In general, neuropeptides are co-localized with one of the small-molecule neurotransmitters (Table 7–1).

Figure 7–1 shows the biosynthesis of some common small-molecule transmitters released by neurons in the central nervous system (CNS) or peripheral nervous system. Figure 7–2 shows the location of major groups of neurons that contain norepinephrine, epinephrine, dopamine, and acetylcholine. These are some of the major central neurotransmitter and neuromodulatory systems.

RECEPTORS

The action of a chemical mediator on its target structure is more dependent on the type of receptor on which it acts than on the properties of the mediator per se. Cloning and other molecular biology techniques have permitted spectacular advances in knowledge about the structure and function of receptors for neurotransmitters and other chemical messengers. The individual receptors, along with their ligands (the molecules that bind to them), are discussed in the following parts of this chapter. However, five themes have emerged that should be mentioned in this introductory discussion.

First, in every instance studied in detail to date, each chemical mediator has the potential to act on many subtypes of receptors. Thus, for example, norepinephrine acts on α₁-, α₂-, β₁-, β₂-, and β₃-adrenergic receptors. Obviously, this multiplies the possible effects of a given ligand and makes its effects in a given cell more selective.

Second, there are receptors on the presynaptic as well as the postsynaptic elements for many secreted transmitters. One type of presynaptic receptor called an autoreceptor often inhibits further secretion of the transmitter, providing feedback control. For example, norepinephrine acts on α₂-presynaptic receptors to inhibit additional norepinephrine secretion. A second type of presynaptic receptor is called a heteroreceptor whose ligand is a chemical other than the transmitter released by the nerve ending on which the receptor is located. For example, norepinephrine acts on a heteroreceptor on a cholinergic nerve terminal to inhibit the release of acetylcholine. In some cases, presynaptic receptors facilitate the release of neurotransmitters.

Third, although there are many neurotransmitters and many subtypes of receptors for each ligand, the receptors tend to group in two large families in terms of structure and function: ligand-gated channels (also known as ionotropic receptors) and metabotropic receptors. In the case of ionotropic receptors, a membrane channel is opened when a ligand binds to the receptor; and activation of the channel usually elicits a brief (few to tens of milliseconds) increase in ionic conductance. Thus, these receptors are important for fast synaptic transmission. Metabotropic receptors are 7-transmembrane G-protein–coupled receptors (GPCR), and binding of a neurotransmitter to these receptors initiates the production of a second messenger that modulates the voltage-gated channels on neuronal membranes. The receptors for some neurotransmitters and neuromodulators are listed in Table 7–2, along with their principal second messengers and, where established, their net effect on ion channels. It should be noted that this table is an over-simplification. For example, activation of α₂-adrenergic receptors decreases intracellular cyclic adenosine monophosphate (cAMP) concentrations, but there is evidence that the G-protein activated by α₂-adrenergic presynaptic receptors also acts directly on Ca²⁺ channels to inhibit norepinephrine release.

Fourth, receptors are concentrated in clusters on the postsynaptic membrane close to the endings of neurons that secrete the neurotransmitters specific for them. This is generally due to the presence of specific binding proteins for them.

Fifth, in response to prolonged exposure to their ligands, most receptors become unresponsive; that is, they undergo desensitization. This can be of two types: homologous desensitization, with loss of responsiveness only to the particular ligand and maintained responsiveness of the cell to other ligands; and heterologous desensitization, in which the cell becomes unresponsive to other ligands as well.

REUPTAKE

Neurotransmitters are rapidly transported from the synaptic cleft back into the cytoplasm of the neurons that secreted them via a process called reuptake, which involves a high-affinity, Na⁺-dependent membrane transporter. Figure 7–3 illustrates the principle of reuptake of norepinephrine released from a sympathetic postganglionic nerve. After release of norepinephrine into the synaptic cleft, it is rapidly routed back into the sympathetic nerve terminal by a norepinephrine transporter (NET). A portion of the norepinephrine that re-enters the neuron is sequestered into the synaptic vesicles through the vesicular monoamine transporter (VMAT). There are analogous membrane and vesicular transporters for other small-molecule neurotransmitters released at other synapses in the CNS and peripheral nervous system.

Reuptake is a major factor in terminating the action of transmitters, and when it is inhibited, the effects of transmitter release are increased and prolonged. This has clinical consequences. For example, several effective antidepressant drugs are inhibitors of the reuptake of amine transmitters, and cocaine may inhibit dopamine reuptake. Glutamate uptake into neurons and glia is important because glutamate is an excitotoxin that can kill cells by overstimulating them (see Clinical Box 7–1). There is evidence that during ischemia and anoxia, loss of neurons is increased because glutamate reuptake is inhibited.

Synaptic physiology is a rapidly expanding, complex field that cannot be covered in detail in this book. However, it is appropriate to summarize information about the principal neurotransmitters and their receptors.

EXCITATORY & INHIBITORY AMINO ACIDS

Glutamate

Glutamate is the main excitatory transmitter in the brain and spinal cord and has been calculated to be responsible for 75% of the excitatory transmission in the CNS. There are two distinct pathways involved in the synthesis of glutamate (Figure 7–1). In one pathway, α-ketoglutarate produced by the Krebs cycle is converted to glutamate by the enzyme GABA transaminase (GABA-T). In the second pathway, glutamate is released from the nerve terminal into the synaptic cleft by Ca²⁺-dependent exocytosis and transported via a glutamate reuptake transporter into glia, where it is converted to glutamine by the enzyme glutamine synthetase (Figure 7–4). Glutamine then diffuses back into the nerve terminal where it is hydrolyzed back to glutamate by the enzyme glutaminase. In addition to uptake of released glutamate into glia, the membrane transporters also return glutamate directly into the nerve terminal. Within glutamatergic neurons, glutamate is highly concentrated in synaptic vesicles by a vesicular glutamate transporter.

Glutamate Receptors

Glutamate acts on both ionotropic and metabotropic receptors in the CNS (Figure 7–4). There are three subtypes of ionotropic glutamate receptors, each named for its relatively specific agonist. These are the AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionate), kainate (kainate is an acid isolated from seaweed), and NMDA (N-methyl-D-aspartate) receptors. Table 7–2 summarizes some of the major properties of these receptors. Ionotropic glutamate receptors are tetramers composed of different subunits whose helical domains span the membranes three times and a short sequence that forms the channel pore. Four AMPA (GluR1–GluR4), five kainate (GluR5–GluR7, KA1, KA2), and six NMDA (NR1, NR2A–NR2D) subunits have been identified, and each is coded by a different gene.

The release of glutamate and its binding to AMPA or kainate receptors primarily permits the influx of Na⁺ and the efflux of K⁺, accounting for fast excitatory postsynaptic potential (EPSP). Most AMPA receptors have low Ca²⁺ permeability, but the absence of certain subunits in the receptor complex at some sites allows for the influx of Ca²⁺, which may contribute to the excitotoxic effect of glutamate (see Clinical Box 7–1).

Activation of the NMDA receptor permits the influx of relatively large amounts of Ca²⁺ along with Na⁺. When glutamate is in excess in the synaptic cleft, the NMDA receptor-induced influx of Ca²⁺ into neurons is the major basis for the excitotoxic actions of glutamate. The NMDA receptor is unique in several ways (Figure 7–5). First, glycine facilitates its function by binding to the receptor. In fact, glycine binding is essential for the receptor to respond to glutamate. Second, when glutamate binds to the NMDA receptor, it opens, but at normal membrane potentials, the channel is blocked by extracellular Mg²⁺. This block is removed only when the neuron containing the receptor is partially depolarized by the activation of adjacent AMPA and kainate receptors. Third, the excitatory postsynaptic potential induced by activation of NMDA receptors is slower than that elicited by activation of the AMPA and kainate receptors.

Essentially all neurons in the CNS have both AMPA and NMDA receptors. Kainate receptors are located presynaptically on GABA-secreting nerve endings and postsynaptically at various sites, most notably in the hippocampus, cerebellum, and spinal cord. Kainate and AMPA receptors are found in glia as well as neurons, but NMDA receptors occur only in neurons. The concentration of NMDA receptors in the hippocampus is high, and blockade of these receptors prevents long-term potentiation, a long-lasting facilitation of transmission in neural pathways following a brief period of high-frequency stimulation. Thus, these receptors may well be involved in memory and learning (see Chapter 15).

Activation of the metabotropic glutamate receptors (mGluR) leads to either an increase in intracellular inositol 1,4,5-triphosphate (IP₃) and diacylglycerol (DAG) levels or a decrease in intracellular cAMP levels (Table 7–2). There are eight known subtypes of mGluR. These receptors are located at both presynaptic (mGluR_{2-4, 6-8}) and postsynaptic sites (mGluR_{1, 5}), and they are widely distributed in the brain. They appear to be involved in the production of synaptic plasticity, particularly in the hippocampus and the cerebellum. Activation of presynaptic mGluR autoreceptors on neurons in the hippocampus limits the release of glutamate from these neurons. Knockout of the gene mGluR1 causes severe motor incoordination and deficits in spatial learning.

A characteristic of an excitatory synapse is the presence of a thickened area called a postsynaptic density (PSD) on the membrane of the postsynaptic neuron. This is a complex structure containing ionotropic glutamate receptors and signaling, scaffolding, and cytoskeletal proteins. The mGluR are located adjacent to the PSD.

Pharmacology of Glutamate Synapses

Table 7–2 shows some of the pharmacologic properties of various types of glutamate receptors, examples of the agonists that bind to these receptors, and some of the antagonists that prevent activation of the receptors. The clinical applications of drugs that modulate glutamatergic transmission are still in their infancy. This is because the role of glutamate as a neurotransmitter was discovered much later than that of most other small-molecule transmitters. It was not identified as a neurotransmitter until the 1970s, more than 50 years after the discovery of chemical neurotransmission. One area of drug development is intraspinal or extradural administration of NMDA receptor antagonists for the treatment of chronic pain.

GABA

GABA is the major inhibitory mediator in the brain and mediates both presynaptic and postsynaptic inhibition. GABA, which exists as β-aminobutyrate in the body fluids, is formed by decarboxylation of glutamate (Figure 7–1) by the enzyme glutamate decarboxylase (GAD), which is present in nerve endings in many parts of the brain. GABA is metabolized primarily by transamination to succinic semialdehyde and then to succinate in the citric acid cycle. GABA-T is the enzyme that catalyzes the transamination. In addition, there is an active reuptake of GABA via the GABA transporter. A vesicular GABA transporter (VGAT) transports GABA and glycine into secretory vesicles.

GABA Receptors

Three subtypes of GABA receptors have been identified: GABA_A, GABA_B, and GABA_C (Table 7–2). The GABA_A and GABA_B receptors (Figure 7–6) are widely distributed in the CNS, whereas in adult vertebrates the GABA_C receptors are found almost exclusively in the retina. The GABA_A and GABA_C receptors are examples of ionotropic receptors, activation of which allows the entry of Cl^– into neurons to mediate fast inhibitory postsynaptic potentials (IPSP). The GABA_B receptors are examples of GPCR that are linked via G-proteins to alter the influx of K⁺ and Ca²⁺; G_i inhibits adenylyl cyclase to open a K⁺ channel, and G_o inhibits or delays Ca²⁺ influx. Activation of GABA_B receptors mediates both presynaptic and slow postsynaptic inhibition.

The GABA_A receptors are pentamers made up of various combinations of six α subunits, four β, four γ, one δ, and one ε. This endows them with considerably different properties from one location to another. However, most synaptic GABA_A receptors have two α, two β, and one γ subunit (Figure 7–6). GABA_A receptors on dendrites, axons, or somas often contain δ and ε subunits in place of the γ subunit. The GABA_C receptors are relatively simple in that they are pentamers of three ρ subunits in various combinations.

There is a chronic low-level stimulation of GABA_A receptors in the CNS that is aided by GABA in the interstitial fluid. This background stimulation cuts down on the “noise” caused by incidental discharge of the billions of neural units and greatly improves the signal-to-noise ratio in the brain.

Pharmacology of GABA Synapses

Table 7–2 shows some of the pharmacologic properties of GABA receptors, including examples of agonists that bind to receptors and some of the antagonists that prevent activation of these receptors. The increase in Cl^– conductance produced by GABA_A receptors is potentiated by benzodiazepines (eg, diazepam). Thus, these are examples of neuromodulators. These drugs have marked antianxiety activity and are also effective muscle relaxants, anticonvulsants, and sedatives. Benzodiazepines bind to α subunits of GABA_A receptors. Barbiturates such as phenobarbital are effective anticonvulsants because they enhance GABA_A receptor—mediated inhibition as well as suppress AMPA receptor—mediated excitation. The anesthetic actions of barbiturates (thiopental, pentobarbital, and methoxital) result from their actions as agonists at GABA_A receptors as well as by acting as neuromodulators of GABA transmission. Regional variation in anesthetic actions in the brain seems to parallel the variation in subtypes of GABA_A receptors. Other inhaled anesthetics do not act by increasing GABA receptor activity; rather, they act by inhibiting NMDA and AMPA receptors.

Glycine

Glycine has both excitatory and inhibitory effects in the CNS. When it binds to NMDA receptors, it makes them more sensitive to the actions of glutamate. Glycine may spill over from synaptic junctions into the interstitial fluid and in the spinal cord; for example, it may facilitate pain transmission by NMDA receptors in the dorsal horn. However, glycine is also responsible in part for direct inhibition, primarily in the brainstem and spinal cord. Like GABA, it acts by increasing Cl^– conductance. Its action is antagonized by strychnine. The clinical picture of convulsions and muscular hyperactivity produced by strychnine emphasizes the importance of postsynaptic inhibition in normal neural function. The glycine receptor responsible for inhibition is a Cl^– channel. It is a pentamer made up of two subunits: the ligand-binding α subunit and the structural β subunit. There are three kinds of neurons that are responsible for direct inhibition in the spinal cord: neurons that secrete glycine, neurons that secrete GABA, and neurons that secrete both. Neurons that secrete only glycine have the glycine transporter GLYT2, those that secrete only GABA have GAD, and those that secrete glycine and GABA have both. This third type of neuron is of special interest because the neurons seem to have glycine and GABA in the same vesicles.

Acetylcholine

Acetylcholine is the transmitter at the neuromuscular junction, in autonomic ganglia, and in postganglionic parasympathetic nerve-target organ junctions and some postganglionic sympathetic nerve-target junctions (see Chapter 13). In fact, acetylcholine is the transmitter released by all neurons that exit the CNS (cranial nerves, motor neurons, and preganglionic neurons). Acetylcholine is also found in the basal forebrain complex (septal nuclei and nucleus basalis), which projects to the hippocampus and neocortex, and the pontomesencephalic cholinergic complex, which projects to the dorsal thalamus and forebrain (Figure 7–2). These systems may be involved in regulation of sleep-wake states, learning, and memory (see Chapters 14 and 15).

Acetylcholine is largely enclosed in small, clear synaptic vesicles in high concentration in the terminals of cholinergic neurons. It is synthesized in the nerve terminal from choline and acetyl-CoA by the enzyme choline acetyltransferase (ChAT) (Figure 7–1 and Figure 7–7). Choline used in the synthesis of acetylcholine is transported from the extracellular space into the nerve terminal via a Na⁺-dependent choline transporter (CHT). Following its synthesis, acetylcholine is transported from the cytoplasm into vesicles by a vesicle-associated transporter (VAT). Acetylcholine is released when a nerve impulse triggers the influx of Ca²⁺ into the nerve terminal.

Acetylcholine must be rapidly removed from the synapse if repolarization is to occur. The removal occurs by way of hydrolysis of acetylcholine to choline and acetate, a reaction catalyzed by the enzyme acetylcholinesterase in the synaptic cleft. This enzyme is also called true or specific cholinesterase. Its greatest affinity is for acetylcholine, but it also hydrolyzes other choline esters. Acetylcholinesterase molecules are clustered in the postsynaptic membrane of cholinergic synapses. Hydrolysis of acetylcholine by this enzyme is rapid enough to explain the observed changes in Na⁺ conductance and electrical activity during synaptic transmission. There are a variety of cholinesterases in the body that are not specific for acetylcholine. One found in plasma is capable of hydrolyzing acetylcholine but has different properties from acetylcholinesterase. It is called pseudocholinesterase. The plasma moiety is partly under endocrine control and is affected by variations in liver function.

Acetylcholine Receptors

Acetylcholine receptors are divided into two main types on the basis of their pharmacologic properties. Muscarine, the alkaloid responsible for the toxicity of toadstools, mimics the stimulatory action of acetylcholine on smooth muscle and glands. These actions of acetylcholine are called muscarinic actions, and the receptors involved are muscarinic cholinergic receptors. In sympathetic ganglia and skeletal muscle, nicotine mimics the stimulatory actions of acetylcholine. These actions of acetylcholine are called nicotinic actions, and the receptors involved are nicotinic cholinergic receptors. Nicotinic receptors are subdivided into those found in muscle at the neuromuscular junction (N_M) and those found in the CNS and autonomic ganglia (N_N). Both muscarinic and nicotinic acetylcholine receptors are also found within the brain.

The nicotinic acetylcholine receptors are members of a superfamily of ligand-gated ion channels (ionotropic receptors) that also includes the GABA_A and glycine receptors and some of the glutamate receptors. Each nicotinic cholinergic receptor is made up of five subunits that form a central channel which, when the receptor is activated, permits the passage of Na⁺ and other cations. The five subunits come from several types designated as α, β, γ, δ, and ε that are each coded by different genes. The N_M receptor is comprised of two α, one β, one δ, and either one γ or one ε subunit (Figure 7–8). The N_N receptors are comprised of only α and β subunits. Each α subunit has a binding site for acetylcholine, and binding of an acetylcholine molecule to each of them induces a conformational change in the protein so that the channel opens. This increases the conductance of Na⁺, and the resulting influx of Na⁺ produces a depolarizing potential. A prominent feature of neuronal nicotinic cholinergic receptors is their high permeability to Ca²⁺. Many of the nicotinic cholinergic receptors in the brain are located presynaptically on glutamate-secreting axon terminals, and they facilitate the release of this transmitter.

There are five types of muscarinic cholinergic receptors (M₁–M₅), which are encoded by five separate genes. These are metabotropic receptors that are coupled via G-proteins to adenylyl cyclase, K⁺ channels, and/or phospholipase C (Table 7–2). M₁, M₄, and M₅ receptors are located in the CNS; M₂ receptors are in the heart, M₃ are on glands and smooth muscle. M₁ receptors are also located on autonomic ganglia where they can modulate neurotransmission.

Pharmacology of Cholinergic Synapses

Table 7–2 shows some of the major agonists that bind to cholinergic receptors as well as some of the cholinergic receptor antagonists. Figure 7–7 also shows the site of action of various drugs that alter cholinergic transmission. For example, hemicholinium blocks the choline transporter that moves choline into the nerve terminal, and vesamicol blocks the VAT that moves acetylcholine into the synaptic vesicle. Also, botulinum toxin prevents the release of acetylcholine from the nerve terminal.

MONOAMINES

Norepinephrine & Epinephrine

The chemical transmitter present at most sympathetic postganglionic endings is norepinephrine. It is stored in the synaptic knobs of the neurons that secrete it in characteristic small vesicles that have a dense core (granulated vesicles). Norepinephrine and its methyl derivative, epinephrine, are also secreted by the adrenal medulla (see Chapter 20), but epinephrine is not a mediator at postganglionic sympathetic endings. As discussed in Chapter 6, each sympathetic postganglionic neuron has multiple varicosities along its course, and each of these varicosities appears to be a site at which norepinephrine is secreted.

There are also norepinephrine-secreting and epinephrine-secreting neurons in the brain. Norepinephrine-secreting neurons are properly called noradrenergic neurons, although the term adrenergic neurons is also applied. However, the latter term should be reserved for epinephrine-secreting neurons. The cell bodies of the norepinephrine-containing neurons are located in the locus coeruleus and other medullary and pontine nuclei (Figure 7–2). From the locus coeruleus, the axons of the noradrenergic neurons descend into the spinal cord, enter the cerebellum, and ascend to innervate the paraventricular, supraoptic, and periventricular nuclei of the hypothalamus, the thalamus, the basal telencephalon, and the entire neocortex. The action of norepinephrine in these regions is primarily as a neuromodulator.

Biosynthesis & Release of Catecholamines

The principal catecholamines found in the body (norepinephrine, epinephrine, and dopamine) are formed by hydroxylation and decarboxylation of the amino acid tyrosine (Figure 7–1 and Figure 7–9). Some of the tyrosine is formed from phenylalanine, but most is of dietary origin. Phenylalanine hydroxylase is found primarily in the liver (see Clinical Box 7–2). Tyrosine is transported into catecholamine-secreting neurons via a Na⁺-dependent carrier. It is converted to dihydroxy-phenylalanine (dopa) and then to dopamine in the cytoplasm of the cells by tyrosine hydroxylase and dopa decarboxylase, respectively. The decarboxylase is also called amino acid decarboxylase. The rate-limiting step in synthesis of catecholamines is the conversion of tyrosine to dopa. Tyrosine hydroxylase is subject to feedback inhibition by dopamine and norepinephrine, thus providing internal control of the synthetic process.

Once dopamine is synthesized, it is transported into the vesicle by the VMAT. Here the dopamine is converted to norepinephrine by dopamine β-hydroxylase. Norepinephrine is the only small-molecule transmitter that is synthesized in synaptic vesicles instead of being transported into the vesicle after its synthesis.

Some neurons in the CNS and adrenal medullary cells also contain the cytoplasmic enzyme phenylethanolamine-N-methyltransferase, which catalyzes the conversion of norepinephrine to epinephrine. In these cells, norepinephrine leaves the vesicles, is converted to epinephrine in the cytoplasm, and then enters other vesicles for storage until it is released by exocytosis.

Catabolism of Catecholamines

Norepinephrine, like other amine and amino acid transmitters, is removed from the synaptic cleft by binding to postsynaptic receptors, binding to presynaptic receptors, reuptake into the presynaptic neurons, or catabolism. Reuptake via a NET is a major mechanism to terminate the actions of norepinephrine (Figure 7–3), and the hypersensitivity of sympathetically denervated structures is explained in part on this basis. After the noradrenergic neurons are cut, their endings degenerate with loss of NET to remove norepinephrine from the synaptic cleft. Consequently, more norepinephrine from other sources is available to stimulate the receptors on the autonomic effectors.

Epinephrine and norepinephrine are metabolized to biologically inactive products by oxidation and methylation. The former reaction is catalyzed by monoamine oxidase (MAO) and the latter by catechol-O-methyltransferase (COMT). MAO is located on the outer surface of the mitochondria. MAO is widely distributed, being particularly plentiful in the nerve endings at which catecholamines are secreted. COMT is also widely distributed, particularly in the liver, kidneys, and smooth muscles. In the brain, it is present in glial cells, and small amounts are found in postsynaptic neurons, but none is found in presynaptic noradrenergic neurons. Consequently, catecholamine metabolism has two different patterns.

Extracellular epinephrine and norepinephrine are for the most part O-methylated, and measurement of the concentrations of the O-methylated derivatives normetanephrine and metanephrine in the urine is a good index of the rate of secretion of norepinephrine and epinephrine. The O-methylated derivatives that are not excreted are largely oxidized, and vanillylmandelic acid is the most plentiful catecholamine metabolite in the urine.

In the noradrenergic nerve terminals, some of the norepinephrine is constantly being converted by intracellular MAO to the physiologically inactive deaminated derivatives, 3,4-dihydroxymandelic acid and its corresponding glycol. These are subsequently converted to their corresponding O-methyl derivatives, vanillylmandelic acid, and 3-methoxy-4-hydroxyphenylglycol.

α- & β-Adrenoceptors

Epinephrine and norepinephrine both act on α- and β-adrenergic receptors (adrenoceptors), with norepinephrine having a greater affinity for α-adrenoceptors and epinephrine for β-adrenoceptors. These receptors are metabotropic GPCR, and each has multiple subtypes (α_1A, α_1B, α_1D, α_2A, α_2B, α_2C, and β₁β₃). Most α₁-adrenoceptors are coupled via G_q proteins to phospholipase C, leading to the formation of IP₃ and DAG, which mobilizes intracellular Ca²⁺ stores and activates protein kinase C, respectively. Thus, at many synapses, activation of α₁-adrenoceptors is excitatory to the postsynaptic target. In contrast, α₂-adrenoceptors activate G_i inhibitory proteins to inhibit adenylyl cyclase and decrease cAMP. Other actions of α₂-adrenoceptors are to activate G-protein–coupled inward rectifier K⁺ channels to cause membrane hyperpolarization and to inhibit neuronal Ca²⁺ channels. Thus, at many synapses, activation of α₂-adrenoceptors inhibits the postsynaptic target. Presynaptic α₂-adrenoceptors are autoreceptors which, when activated, inhibit further release of norepinephrine from postganglionic sympathetic nerve terminals. β-Adrenoceptors activate a stimulatory G_S protein to activate adenylyl cyclase to increase cAMP.

α₁-Adrenoceptors are located on smooth muscle and the heart, and α₂-adrenoceptors are located in the CNS and on pancreatic islets cells and nerve terminals. β₁-Adrenoceptors are located in the heart and renal juxtaglomerular cells. β₂-Adrenoceptors are located in bronchial smooth muscle and skeletal muscle. β₃-Adrenoceptors are located in adipose tissue.

Pharmacology of Noradrenergic Synapses

Table 7–2 shows some of the common agonists that bind to adrenoceptors as well as some of the common adrenoceptor antagonists. Figure 7–9 also shows the site of action of various drugs that alter noradrenergic transmission. For example, metyrosine blocks the action of tyrosine hydroxylase, the rate-limiting step in the synthetic pathway for catecholamine production in the nerve terminal. Reserpine blocks the VMAT that moves dopamine into the synaptic vesicle. Also, bretylium and guanethidine prevent the release of norepinephrine from the nerve terminal. Cocaine and tricyclic antidepressants block the NET. In addition to the agonists listed in Table 7–2, some drugs mimic the actions of norepinephrine by releasing stored transmitter from the noradrenergic endings. These are called sympathomimetics and include amphetamines and ephedrine.

Dopamine

In some parts of the brain, catecholamine synthesis stops at dopamine (Figure 7–1), which can then be secreted into the synaptic cleft. Active reuptake of dopamine occurs via a Na⁺- and Cl^–-dependent dopamine transporter. Dopamine is metabolized to inactive compounds by MAO and COMT in a manner analogous to the inactivation of norepinephrine. 3,4-Dihydroxyphenylacetic acid and homovanillic acid are conjugated, primarily to sulfate.

Dopaminergic neurons are located in several brain regions (Figure 7–2). One region is the nigrostriatal system, which projects from the midbrain substantia nigra to the striatum in the basal ganglia and is involved in motor control. Another dopaminergic system is the mesocortical system; it arises primarily in the ventral tegmental area, which projects to the nucleus accumbens and limbic subcortical areas. The mesocortical system is involved in reward behavior and addiction and in psychiatric disorders such as schizophrenia (see Clinical Box 7–3). Studies using positive emission tomography (PET) scanning in healthy humans show that a steady loss of dopamine receptors occurs in the basal ganglia with age. The loss is greater in men than in women.

Dopamine Receptors

Five dopamine receptors have been cloned, but they fall into two major categories: D₁-like (D₁ and D₅) and D₂-like (D₂, D₃, and D₄). All dopamine receptors are metabotropic GPCR. Activation of D₁-type receptors leads to an increase in cAMP, whereas activation of D₂-like receptors reduces cAMP levels. Overstimulation of D₂ receptors may contribute to the pathophysiology of schizophrenia (Clinical Box 7–3). D₃ receptors are highly localized, especially to the nucleus accumbens (Figure 7–2). D₄ receptors have a greater affinity than the other dopamine receptors for the “atypical” antipsychotic drug clozapine, which is used primarily to treat schizophrenia in individuals who do not respond to other therapies.

Serotonin

Serotonin (5-hydroxytryptamine; 5-HT) is present in highest concentration in blood platelets and in the gastrointestinal tract, where it is found in the enterochromaffin cells and the myenteric plexus. It is also found within the brainstem in the midline raphe nuclei, which project to a wide portion of the CNS including the hypothalamus, limbic system, neocortex, cerebellum, and spinal cord (Figure 7–2).

Serotonin is synthesized from the essential amino acid tryptophan (Figure 7–1 and Figure 7–10). The rate-limiting step is the conversion of the amino acid to 5-hydroxytryptophan by tryptophan hydroxylase. This is then converted to serotonin by the aromatic L-amino acid decarboxylase. Serotonin is transported into the vesicles by the VMAT. After release from serotonergic neurons, much of the released serotonin is recaptured by the relatively selective serotonin transporter (SERT). Once serotonin is returned to the nerve terminal, it is either taken back into the vesicles or is inactivated by MAO to form 5-hydroxyindoleacetic acid (5-HIAA). This substance is the principal urinary metabolite of serotonin, and urinary output of 5-HIAA is used as an index of the rate of serotonin metabolism in the body.

Tryptophan hydroxylase in the CNS is slightly different from the tryptophan hydroxylase in peripheral tissues and is coded by a different gene. This is presumably why knockout of the TPH1 gene, which codes for tryptophan hydroxylase in peripheral tissues, has much less effect on brain serotonin production than on peripheral serotonin production.

Serotonergic Receptors

There are seven classes of 5-HT receptors (from 5-HT₁ through 5-HT₇ receptors), and all except one (5-HT₃) are GPCR and affect adenylyl cyclase or phospholipase C (Table 7–2). Within the 5-HT₁ group are the 5-HT_1A, 5-HT_1B, 5-HT_1D, 5-HT_1E, and 5-HT_1F subtypes. Within the 5-HT₂ group there are 5-HT_2A, 5-HT_2B, and 5-HT_2C subtypes. There are two 5-HT₅ subtypes: 5-HT_5A and 5-HT_5B. Some of the serotonin receptors are presynaptic and others are postsynaptic.

5-HT_2A receptors mediate platelet aggregation and smooth muscle contraction. Mice in which the gene for 5-HT_2C receptors has been knocked out are obese as a result of increased food intake despite normal responses to leptin (see Chapter 26), and they are prone to fatal seizures. 5-HT₃ receptors are present in the gastrointestinal tract and the area postrema and are related to vomiting. 5-HT₄ receptors are also present in the gastrointestinal tract, where they facilitate secretion and peristalsis, and in the brain. 5-HT₆ and 5-HT₇ receptors in the brain are distributed throughout the limbic system, and the 5-HT₆ receptors have a high affinity for antidepressant drugs.

Pharmacology of Serotonergic Synapses

Table 7–2 shows some of the common agonists that bind to 5-HT receptors as well as some of the common 5-HT receptor antagonists. In addition, tricyclic antidepressants inhibit the uptake of serotonin by block of SERT, similar to what was described for their actions at noradrenergic synapses. Selective serotonin uptake inhibitors (SSRIs) such as fluoxetine are widely used in the treatment of depression (see Clinical Box 7–4).

Histamine

Histaminergic neurons have their cell bodies in the tuberomammillary nucleus of the posterior hypothalamus, and their axons project to all parts of the brain, including the cerebral cortex and the spinal cord. Histamine is also found in cells in the gastric mucosa and in heparin-containing cells called mast cells that are plentiful in the anterior and posterior lobes of the pituitary gland as well as at body surfaces. Histamine is formed by decarboxylation of the amino acid histidine. The three well-characterized types of histamine receptors (H₁, H₂, and H₃) are all found in both peripheral tissues and the brain. Most, if not all, of the H₃ receptors are presynaptic, and they mediate inhibition of the release of histamine and other transmitters via a G-protein. H₁ receptors activate phospholipase C, and H₂ receptors increase intracellular cAMP. The function of this diffuse histaminergic system is unknown, but evidence links brain histamine to arousal, sexual behavior, blood pressure, drinking, pain thresholds, and regulation of the secretion of several anterior pituitary hormones. In addition, a recently described histamine H₄ receptor appears to play a role in regulating cells of the immune system.

ATP

ATP is an example of a small-molecule that is often co-localized and co-released from synaptic vesicles such as those in noradrenergic postganglionic sympathetic neurons (Figure 7–7), and it has recently been identified as a neurotransmitter. ATP has been shown to mediate rapid synaptic responses in the autonomic nervous system and a fast response in the habenula. ATP binds to P_2X receptors, which are ligand-gated ion channel receptors. P_2X receptors have widespread distributions throughout the body, including the dorsal horn, which implicates a role for ATP in sensory transmission. Antagonists of P_2X receptors are under development for the treatment of chronic pain. ATP also binds to P_2Y and P_2U receptors, which are GPCR.

LARGE-MOLECULE TRANSMITTERS: NEUROPEPTIDES

Substance P

Substance P is a polypeptide containing 11 amino acid residues that is found in the intestine, various peripheral nerves, and many parts of the CNS. It is one of a family of polypeptides called tachykinins that differ at the amino terminal end but have in common the carboxyl terminal sequence of Phe-X-Gly-LeuMet-NH₂, where X is Val, His, Lys, or Phe. Other members of the family include neurokinin A and neurokinin B.

There are three neurokinin receptors (NK₁–NK₃), which are metabotropic GPCR. Substance P is the preferred ligand for NK₁ receptors in the CNS, and activation of this receptor leads to increased formation of IP₃ and DAG.

Substance P is found in high concentrations in the endings of primary afferent neurons in the spinal cord, and it is probably the mediator at the first synapse in the pathways for pain transmission in the dorsal horn. It is also found in high concentrations in the nigrostriatal system, where its concentration is proportional to that of dopamine, and in the hypothalamus, where it may play a role in neuroendocrine regulation. Upon injection into the skin, it causes redness and swelling, and it is probably the mediator released by nerve fibers that is responsible for the axon reflex. In the intestine, it is involved in peristalsis. Several recently developed centrally active NK-1 receptor antagonists have been shown to have antidepressant activity. They have also been used as antiemetics in patients undergoing chemotherapy.

Opioid Peptides

The brain and the gastrointestinal tract contain receptors that bind morphine. The search for endogenous ligands for these receptors led to the discovery of two closely related pentapeptides (enkephalins) that bind to these opioid receptors: met-enkephalin and leu-enkephalin. These and other peptides that bind to opioid receptors are called opioid peptides. The enkephalins are found in nerve endings in the gastrointestinal tract and many different parts of the brain, and they appear to function as synaptic transmitters. They are found in the substantia gelatinosa and have analgesic activity when injected into the brainstem. They also decrease intestinal motility. Enkephalins are metabolized primarily by two peptidases: enkephalinase A, which splits the Gly-Phe bond, and enkephalinase B, which splits the Gly-Gly bond. Aminopeptidase, which splits the Tyr-Gly bond, also contributes to their metabolism.

Like other small peptides, the endogenous opioid peptides are synthesized as part of larger precursor molecules. More than 20 active opioid peptides have been identified. Unlike other peptides, however, the opioid peptides have a number of different precursors. Each has a prepro form and a pro form from which the signal peptide has been cleaved. Proenkephalin was first identified in the adrenal medulla, but it is also the precursor for met-enkephalin and leu-enkephalin in the brain. Each proenkephalin molecule contains four met-enkephalins, one leuenkephalin, one octapeptide, and one heptapeptide. Proopiomelanocortin, a large precursor molecule found in the anterior and intermediate lobes of the pituitary gland and the brain, contains β-endorphin, a polypeptide of 31 amino acid residues that has metenkephalin at its amino terminal. There are separate enkephalin-secreting and β-endorphin–secreting systems of neurons in the brain. β-Endorphin is also secreted into the bloodstream by the pituitary gland. A third precursor molecule is prodynorphin, a protein that contains three leuenkephalin residues associated with dynorphin and neoendorphin. Different types of dynorphins are found in the duodenum and the posterior pituitary and hypothalamus; β-neoendorphins are also found in the hypothalamus.

There are three classes of opioid receptors: μ, κ, and δ with various subtypes of each of these, but genes encoding only one subtype for each has been identified and characterized. As shown in Table 7–3, they differ in physiologic effects and affinity for various opioid peptides. All three are GPCR, and all inhibit adenylyl cyclase. Activation of μ receptors increases K⁺ conductance, hyperpolarizing central neurons and primary afferents. Activation of κ receptors and δ receptors closes Ca²⁺ channels.

Other Polypeptides

Numerous other polypeptides are found in the brain. For example, somatostatin is found in various parts of the brain, where it may function as a neurotransmitter with effects on sensory input, locomotor activity, and cognitive function. In the hypothalamus, this growth hormone—inhibiting hormone is secreted into the portal hypophysial vessels; in the endocrine pancreas, it inhibits insulin secretion and the secretion of other pancreatic hormones; and in the gastrointestinal tract, it is an important inhibitory gastrointestinal regulator. A family of five different somatostatin receptors have been identified (SSTR1 through SSTR5). All are GPCR that inhibit adenylyl cyclase and exert various other effects on intracellular messenger systems. It appears that SSTR2 mediates cognitive effects and inhibition of growth hormone secretion, whereas SSTR5 mediates the inhibition of insulin secretion.

Vasopressin and oxytocin are not only secreted as hormones but also are present in neurons that project to the brainstem and spinal cord. The brain contains bradykinin, angiotensin II, and endothelin. The gastrointestinal hormones, including vasoactive intestinal polypeptide (VIP), cholecystokinin (CCK-4 and CCK-8), are also found in the brain. There are two kinds of CCK receptors in the brain, CCK-A and CCK-B. CCK-8 acts at both binding sites, whereas CCK-4 acts at the CCK-B sites. Gastrin, neurotensin, galanin, and gastrin-releasing peptide are also found in the gastrointestinal tract and brain. Neurotensin, VIP, and CCK receptors have been cloned and shown to be GPCR. The hypothalamus contains both gastrin 17 and gastrin 34. VIP produces vasodilation and is found in vasomotor nerve fibers. The functions of these peptides in the nervous system are unknown, although some of the peptides also expressed in the gastrointestinal system have been implicated in satiety (see Chapter 26).

Calcitonin gene-related peptide (CGRP) is present in the CNS and peripheral nervous system, gastrointestinal tract, cardiovascular system, and urogenital system. CGRP is co-localized with either substance P or acetylcholine. CGRP-like immunoreactivity is present in the circulation, and injection of CGRP causes vasodilation. CGRP and the calcium-lowering hormone calcitonin are both products of the calcitonin gene. In the thyroid gland, splicing produces the mRNA that codes for calcitonin, whereas in the brain, alternative splicing produces the mRNA that codes for CGRP. CGRP has little effect on Ca²⁺ metabolism, and calcitonin is only a weak vasodilator. Release of CGRP from trigeminal afferent fibers may contribute to the pathophysiology of migraine. Actions of this peptide are mediated by two types of metabotropic CGRP receptors.

Neuropeptide Y is a polypeptide that is very abundant throughout the brain and the autonomic nervous system. Neuropeptide Y acts on eight identified receptors: Y₁–Y₈; except for Y₃, these are GPCR. Activation of these receptors mobilizes Ca²⁺ and inhibits adenylyl cyclase. It acts within the CNS to increase food intake, and Y₁ and Y₅ receptor antagonists may be used to treat obesity. It also acts in the periphery to cause vasoconstriction. It acts on heteroreceptors on postganglionic sympathetic nerve terminals to reduce release of norepinephrine.

OTHER CHEMICAL TRANSMITTERS

Nitric oxide (NO), a compound released by the endothelium of blood vessels as endothelium-derived relaxing factor, is also produced in the brain. It is synthesized from arginine, a reaction catalyzed in the brain by one of the three forms of NO synthase. It activates guanylyl cyclase and, unlike other transmitters, it is a gas, which crosses cell membranes with ease and binds directly to guanylyl cyclase. NO is not stored in vesicles like other classic transmitters; it is synthesized on demand at postsynaptic sites and diffuses to adjacent sites on the neuron. Synthesis may be triggered by activation of NMDA receptors, which leads to an influx of Ca²⁺ and activation of neuronal nitric oxide synthase. It may be the signal by which postsynaptic neurons communicate with presynaptic endings to enhance release of glutamate. It may also play a role in synaptic plasticity and thus in memory and learning.

Two types of endogenous cannabinoids have been identified as neurotransmitters: 2-arachidonyl glycerol and anandamide. These are also not stored in vesicles; they are rapidly synthesized in response to Ca²⁺ influx after a neuron is depolarized. Both act on a cannabinoid receptor (CB₁) with a high affinity for Δ⁹-tetrahydrocannabinol, the psychoactive ingredient in marijuana. These receptors are primarily located on presynaptic nerve terminals. The CB₁ receptor triggers a G-protein–mediated decrease in intracellular cAMP levels and is common in central pain pathways as well as in parts of the cerebellum, hippocampus, and cerebral cortex. In addition to inducing euphoria, CB₁ receptor agonists have an anti-nociceptive effect, and CB₁ receptor antagonists enhance nociception. Endogenous cannabinoids also act as retrograde synaptic messengers; they travel back across a synapse after release and bind to presynaptic CB₁ receptors to inhibit further transmitter release. A CB₂ receptor, which also couples to G-proteins, has also been cloned; it is located primarily in the periphery. Agonists of this class of receptor do not induce the euphoric effects of activation of CB₁ receptors and they may have potential for use in the treatment of chronic pain.

• Neurotransmitters and neuromodulators are divided into two major categories: small-molecule transmitters and large-molecule transmitters (neuropeptides). Usually neuropeptides are co-localized with one of the small-molecule neurotransmitters.

• Rapid removal of the chemical transmitter from the synaptic cleft occurs by diffusion, metabolism, and, in many instances, reuptake into the presynaptic neuron.

• Major neurotransmitters include glutamate, GABA, and glycine, acetylcholine, norepinephrine, serotonin, and opioids. ATP, NO, and cannabinoids can also act as neurotransmitters or neuromodulators.

• The amino acid glutamate is the main excitatory transmitter in the CNS. There are two major types of glutamate receptors: metabotropic (GPCR) and ionotropic (ligand-gated ion channels receptors, including kainate, AMPA, and NMDA).

• GABA is the major inhibitory mediator in the brain. Three subtypes of GABA receptors have been identified: GABA_A and GABA_C (ligand-gated ion channel) and GABA_B (G-protein–coupled). The GABA_A and GABA_B receptors are widely distributed in the CNS.

• Acetylcholine is found at the neuromuscular junction, in autonomic ganglia, and in postganglionic parasympathetic nerve-target organ junctions and a few postganglionic sympathetic nerve-target junctions. It is also found in the basal forebrain complex and pontomesencephalic cholinergic complex. There are two major types of cholinergic receptors: muscarinic (GPCR) and nicotinic (ligand-gated ion channel receptors).

• Norepinephrine-containing neurons are in the locus coeruleus and other medullary and pontine nuclei. Some neurons also contain phenylethanolamine-N-methyltransferase, which catalyzes the conversion of norepinephrine to epinephrine. Epinephrine and norepinephrine act on α- and β-adrenoceptors, with norepinephrine having a greater affinity for α-adrenoceptors and epinephrine for β-adrenoceptors. They are GPCR, and each has multiple forms.

• Serotonin (5-HT) is found within the brainstem in the midline raphe nuclei, which project to portions of the hypothalamus, the limbic system, the neocortex, the cerebellum, and the spinal cord. There are at least seven types of 5-HT receptors, and many of these contain subtypes. Most are GPCR.

• The three types of opioid receptors are GPCR that differ in physiologic effects, distribution in the brain and elsewhere, and affinity for various opioid peptides.