• 
  

  The common designation of a group of neurotransmitters as purines is a misnomer; what are called purinergic signaling molecules are the nucleoside and nucleotide derivatives of purine and perhaps pyrimidine bases.

  
  
• 
  

  The two principal purinergic signaling molecules are adenosine and adenosine triphosphate (ATP). ATP is stored in small synaptic vesicles and released in a Ca²⁺-dependent fashion, whereas adenosine is released from nonvesicular cytoplasmic stores, likely via bidirectional nucleoside transporters.

  
  
• 
  

  Purine receptors form a relatively large and diverse group and have been categorized as P1 and P2 receptors.

  
  
• 
  

  P1 receptors, also called adenosine receptors, bind adenosine and its analogs and are G protein–coupled. Stimulant drugs of the methylxanthine family, including caffeine, are antagonists of adenosine receptors. P2 receptors consist of both ligand-gated ion channels termed P2X receptors and G protein–coupled receptors termed P2Y receptors. P2X receptors play an important role in pain processing.

  
  
• 
  

  Cannabinoids, the principal active ingredients of marijuana, primarily act in the brain on the CB₁ receptor, a G protein–coupled receptor found on presynaptic terminals in the central nervous system (CNS).

  
  
• 
  

  Anandamide and 2-arachidonylglycerol are endogenous cannabinoids (endocannabinoids), which are released from postsynaptic cells and activate presynaptic CB₁ receptors.

  
  
• 
  

  Nitric oxide (NO) is generated from arginine by NO synthase, which is stimulated by activation of postsynaptic NMDA receptors and increases in cellular Ca²⁺ levels. It diffuses out of cells and activates soluble guanylyl cyclase leading to the production of cGMP in adjacent cells and nerve terminals.

  
  
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  Carbon monoxide (CO) is produced by the breakdown of heme by heme oxygenase-2 and also may function as an atypical, diffusible messenger. Hydrogen sulfide (H₂S) is another “gas transmitter”: it is generated from cyst(e)ine by the enzyme cystathionine β-synthase.

  
  
• 
  

  Neurotrophic factors are polypeptides or small proteins that support the growth, differentiation, and survival of neurons. They produce their effects by activation of tyrosine kinases.

  
  
• 
  

  The neurotrophins, which comprise nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4), act by binding to a family of tropomyosin receptor kinase (Trk) receptors, TrkA, TrkB, and TrkC, with intrinsic tyrosine kinase activity.

  
  
• 
  

  Numerous other growth factors, such as glial cell line–derived neurotrophic factor (GDNF), vascular endothelial growth factor (VEGF), and neuregulin, are important in regulating the nervous system.

  
  
• 
  

  Several cytokine-like factors, including ciliary neurotrophic factor (CNTF) and interleukin-6 (IL-6), are characterized by binding to receptors that activate a family of protein tyrosine kinases called Janus kinases (JAKs), which in turn activate transcription factors called signal transducers and activators of transcription (STATs).

  
  
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  Many additional cytokines, best understood for their role in the immune system and inflammatory responses, also are important in the regulation of CNS function. Prominent examples include interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), and transforming growth factor-β (TGF-β).

  
  
• 
  

  Chemokines are small proteins involved in immune responses; in the brain, chemokines are expressed predominately by microglia.

Atypical neurotransmitters include a host of intercellular signaling molecules that have unusual properties or more recent dates of discovery compared with better known small-molecule and neuropeptide neurotransmitters that were reviewed in Chapters 5 to 7. The atypical neurotransmitters include the purinergic neurotransmitters adenosine and adenosine triphosphate (ATP), endogenous cannabinoids (endocannabinoids), the gases nitric oxide (NO) and carbon monoxide (CO), and families of neurotrophic factors and cytokines. Each of these neurotransmitters is found in specific subsets of neurons (and in some cases glia), has specific biochemic machinery for its synthesis, and exerts its biologic effects via activation of specific receptors or enzymes. Because of their discrete anatomic distributions and, in many cases, modulatory functions, these atypical neurotransmitters, their receptors, and the proteins involved in their production and degradation are potentially significant targets for drug development. Not discussed in this chapter are transient receptor potential (TRP) channels, which are covered in Chapters 2 and 11. Many of these channels respond to a host of endogenous molecules (eg, lipids, nucleotides, inorganic ions) or environmental signals (eg, temperature, mechanical stress) and thus function analogously to other signaling mechanisms presented in this chapter.

The purinergic neurotransmitters, adenosine and ATP, were long considered improbable mediators of neurotransmission because of their roles in intermediary metabolism and as building blocks of RNA and DNA. However, we now know that they are concentrated at certain synapses, they are released in response to synaptic stimulation, and they activate specific receptors to produce significant responses in target neurons. It is also established that purinergic receptors mediate the actions of several pharmacologic agents, most notably caffeine and related stimulants.

Biochemistry

The practice of referring to these signaling molecules as purines is to some degree a misnomer. Purines are ring-structured basic compounds that include adenine and guanine. These molecules do not function as neurotransmitters, nor do the pyrimidines, which include uracil, thymidine, and cytosine. So-called purinergic signaling molecules are in fact the nucleoside and nucleotide derivatives of purine bases 8–1A. The two principal neurotransmitters in this family are adenosine and ATP. Related to these are the adenine dinucleotides, which consist of two adenosine molecules covalently linked by a chain of two to six phosphate groups. Adenine dinucleotides are represented by the abbreviation ApnA, wherein n equals the number of phosphates between the two adenosines 8–1B. ApnA molecules are released by neurons and thus may be considered part of the purinergic signaling family. There is also some evidence that nucleoside and nucleotide derivatives of pyrimidine bases may serve as neurotransmitters, although this possibility remains poorly characterized.

Storage and Release

Despite their similarities, adenosine and ATP have distinct properties. ATP and ApnA are stored in small synaptic vesicles and are released in response to action potentials in a Ca²⁺-dependent process similar to the release process for classic neurotransmitters (Chapter 3). Indeed, ATP and classic neurotransmitters often can be detected in the same synapses and even in the same synaptic vesicles indicating that they can be coreleased. Release of ATP from sympathetic nerve terminals as well as vascular endothelial cells and tumor cells may play a particularly important role in various pain states. In contrast, adenosine is released from nonvesicular cytoplasmic stores and most likely reaches the extracellular space by one of two means. First, any of several bidirectional nucleoside transporters can secrete adenosine into the extracellular space, including the synapse. Second, ATP is very rapidly metabolized into adenosine once it is released from a cell. A membrane-bound ectodiphosphohydrolase converts ATP into ADP and AMP, and subsequently a membrane-associated or soluble ecto-5´-nucleotidase converts AMP into adenosine. Because the conversion of ATP to adenosine takes place in less than a second, the synaptic release of ATP should be regarded as an important source of extracellular adenosine.

The schematic representation of the purinergic synapse in 8–2 shows the release of ATP leading to the production of other purinergic compounds and to the subsequent activation of several types of purinergic receptors. It should be noted that ApnA is hydrolyzed much more slowly than ATP and can reside in the synaptic cleft for longer periods of time.

Transporters

Nucleoside transporters are membrane-bound proteins that shuttle purine and pyrimidine nucleosides into and out of many cells, including neurons. The transporters are purine-selective or pyrimidine-selective. Some act to concentrate the nucleosides in a cell in a Na⁺-dependent fashion. Others transport nucleosides down their concentration gradients. Pharmacologic and genetic studies indicate that there are at least seven nucleoside transporters, which belong to two gene families. The so-called equilibrative nucleoside transporter gene family (Slc29A1–A4 or ENT1–4), which consists of four subtypes (Slc29A1 or ENT1–4), mediates both efflux and influx of nucleosides; the concentrative nucleoside transporter family, which has three members (Slc28A1–A3 or CNT1–3), mediates Na⁺-dependent influx of nucleosides. Although much remains to be learned about the structure and function of these proteins, they have been exploited in the development of powerful therapeutic agents. A number of cancer chemotherapeutic drugs, such as gemcitabine, and several potent antiviral compounds, such as zidovudine (AZT), which are used in the treatment of AIDS, are nucleoside analogs that enter target cells by means of nucleoside transporters. Given the powerful effects that adenosine exerts on brain function, it is possible that drugs that regulate nucleoside transporters might eventually prove useful in the treatment of neuropsychiatric disorders.

Receptors

Purine receptors form a relatively large and diverse group of proteins that comprise two main subgroups, referred to as P1 and P2 receptors. Characteristics of both of these receptor families are discussed here and are summarized in 8–1. P1 receptors, also known as adenosine receptors (A₁ and A₂), bind adenosine and its analogs and are coupled to G proteins. Four subtypes have been identified (A₁, A_2A, A_2B, A₃), and each displays the canonical seven-transmembrane domains found in other members of the G protein–coupled receptor superfamily. The A₁ receptor subtype is the most widely expressed in the brain and spinal cord and has the highest affinity for adenosine. Its activation has been implicated in the putative anxiolytic, anticonvulsant, analgesic, and sedative properties of adenosine. Conversely, antagonism of A₁ receptors by methylxanthines such as caffeine and similar drugs results in stimulatory effects, including increased alertness at low doses and anxiety and irritability at much higher doses (8–1; see also Chapter 13).

The two subtypes of the A₂ receptor, A_2A and A_2B, have a somewhat lower affinity for adenosine than do A₁ receptors. The A_2B receptor is ubiquitous in the human body yet is expressed only at very low levels in the brain and spinal cord. In contrast, the A_2A receptor is highly concentrated in the dorsal striatum, nucleus accumbens, and olfactory tubercle—three brain regions that receive rich dopaminergic innervation (Chapters 6, 16–18). Indeed, the actions of adenosine and dopamine are interrelated in these regions. In the striatum, A_2A receptor agonists inhibit dopamine D₂ receptor–mediated behaviors, and A_2A antagonists mimic D₂ receptor agonists. The latter action likely contributes to the locomotor-activating properties of methylxanthines. The inverse relationship between adenosine and dopamine has led to speculation that selective A_2A antagonists might be useful in the treatment of Parkinson disease, as discussed in the next section of this chapter.

The A₃ receptor is expressed at low levels in the brain, and its function is not yet known. It is distinguished from the other subtypes in that it has a much lower affinity for adenosine. Whereas A₁ and A₂ receptors bind adenosine with nanomolar affinity, micromolar concentrations are necessary to activate A₃ receptors.

The P2 receptor family is intriguing because it comprises both G protein–coupled receptors (the P2Y family) and ligand-gated ion channels (the P2X family). Eight human P2Y receptor subtypes have been cloned and characterized. These receptors display unique pharmacologic profiles, and bind purine and pyrimidine nucleotide diphosphates and triphosphates, as well as ApnA molecules, with varying affinities 8–1. The P2Y₁ receptor, for example, binds ATP and ADP but not UTP or UDP. In contrast, the P2Y₂ receptor is activated by ATP and UTP with equal affinity. The regional distribution and functional significance of P2Y receptors in the central nervous system (CNS) are still being characterized.

Seven P2X receptor subtypes are known. Each is an ATP- or ApnA-gated cation channel composed of multiple subunits. Although the exact stoichiometry of P2X receptors is unknown, functional homomeric receptors have been constituted in vitro, and there is evidence for heteromeric P2X_2/3 receptors. Because these receptors have multiple subunits, each of the cloned proteins in 8–1 should be regarded as a subunit rather than as a complete receptor. P2X receptor activation leads to rapid Na⁺, K⁺, and Ca²⁺ flux and subsequent membrane depolarization. These fast-acting channels have been detected in the peripheral nervous system, neuromuscular junction, spinal cord, and many brain regions. When on postsynaptic cells, their activation leads to membrane depolarization. They are also found on many presynaptic nerve terminals throughout the CNS, and their activation routinely leads to enhancement of transmitter release. Via their actions in both the periphery and the spinal cord, they appear to play important roles in mediating a variety of different forms of pain and thus are important targets for the development of novel analgesic medications (Chapter 11).

Purine Functions

The cloning of the P1 and P2 receptor families opened up numerous avenues for investigating purine function in the nervous system. Receptor-selective agonists and antagonists are being developed, and transgenic animals that underexpress or overexpress individual receptor subtypes have been generated. Although many putative roles for nucleosides and nucleotides have been hypothesized, only a handful have been clearly established in the CNS.

Adenosine has both anxiolytic and hypnotic properties, and the administration of adenosine receptor antagonists confirms these observations. As described in 8–1, caffeine and other methylxanthine compounds, by blocking endogenous adenosine action, increase alertness and improve cognitive performance. The cognition-enhancing properties of these drugs are particularly interesting, partly because of the wide use of caffeine, and partly because adenosine receptor antagonists may prove useful as symptomatic treatments for the cognitive deficits associated with Alzheimer disease and other forms of dementia (Chapter 18).

Adenosine also is being investigated for its neuroprotective effects; it is hoped that these properties might be exploited in the treatment of stroke. Stroke is characterized, in part, by an ischemia-induced increase in glutamate levels followed by an overstimulation of glutamate receptors and massive influx of Ca²⁺ into neurons, which unleashes a cascade of cytotoxic events (Chapter 20). Thus, an ideal neuroprotective agent would inhibit glutamate release presynaptically and prevent postsynaptic membrane depolarization and subsequent Ca²⁺ influx. Adenosine can accomplish both feats. Adenosine activation of presynaptic A₁ receptors inhibits the release of glutamate and other neurotransmitters at many synapses throughout the brain. Activation of postsynaptic A₁ receptors opens K⁺ channels, and thereby hyperpolarizes neurons and counters the excitatory effects of glutamate. The net result is believed to be decreased Ca²⁺ influx and decreased neuronal death. Experiments in animal models support this hypothesis. The administration of selective A₁ agonists just before or during an ischemic event in animals significantly reduces neuronal loss. Conversely, the administration of A₁ antagonists augments ischemic brain damage. It is important to note that ischemia dramatically increases adenosine levels in the brain that will likely influence the net effects of drugs that target adenosine receptors.

Although activation of A₁ receptors may provide some measure of neuroprotection, the actions of adenosine at other receptor subtypes may exert the opposite effect. Activation of A₂ receptors, for example, has had deleterious effects on animals in models of stroke. Conversely, A₂-selective antagonists can produce neuroprotective effects. Acute activation of A₃ receptors with selective agonists also profoundly increases brain damage and mortality in animal models. In contrast, chronic administration of these adenosine agonists improves survival. Adenosine’s actions on vascular tone and platelet function likely contribute to these various observations. The interplay that occurs among adenosine receptor subtypes during ischemia clearly requires further investigation.

Adenosine systems also may prove to be promising targets for drugs designed to treat Parkinson disease. As discussed in Chapter 18, this disease is caused by a loss of dopaminergic innervation to the striatum, which leads to rigidity, tremor, and slowness of movement. The striatum, as previously mentioned, expresses high levels of adenosine A_2A receptors. A_2A receptor agonists produce biochemical and behavioral effects (decreased motor activity) that mimic those associated with Parkinson disease because they decrease dopamine neurotransmission in the striatum. Accordingly, blocking A_2A receptors might have the opposite effect; that is, dopamine function in the striatum and motor activity might be expected to increase.

Several orally active compounds that selectively block A_2A receptors have been described. When used in animal models of Parkinson disease, these drugs significantly reverse motor deficits while inducing little, if any, dyskinesia, a side effect associated with L-dopa therapy (Chapter 18). Moreover, when coadministered with L-dopa, A_2A antagonists diminish L-dopa-induced dyskinesia. Currently, A_2A receptor antagonists are in development for the treatment of Parkinson disease.

Adenosine also has been reported to exhibit anticonvulsant, anxiolytic, and antidepressant activity, as well as both analgesic and pain-enhancing properties. Although these purported functions require further investigation, they point to the therapeutic potential of selective adenosine receptor agonists and antagonists.

Over the last decade, a role for ATP and P2X receptors in pain has become apparent. Application of ATP to human skin elicits acute pain. Several different subtypes of P2X receptors are found in sensory neurons in dorsal root ganglia and other sensory ganglia with P2X₃ and P2X_2/3 receptors generally having the highest levels of expression. These receptors are also found at nociceptive nerve terminals in the periphery where they can respond to the release of ATP from various sources including tumors and vascular endothelium 8–3.

Molecular and pharmacologic studies of P2X receptors, in particular P2X₃ and P2X_2/3 receptors, suggest that while they may not be critically involved in acute pain sensation, changes in their levels and functions importantly contribute to the development and maintenance of various forms of neuropathic and inflammatory pain. As these pain states are common, and among the most resistant to treatment, P2X receptors appear to be important targets for potential novel analgesic medications (Chapter 11).

In 1964, identification of the psychoactive ingredient in marijuana as Δ⁹-tetrahydrocannabinol (THC) led to the synthesis of related compounds that displayed saturable binding and pharmacology consistent with the existence of a so-called cannabinoid receptor. Some of these agents 8–4 were found to cause a GTP-dependent inhibition of adenylyl cyclase, which suggested that the receptor for cannabinoids is a member of the large superfamily of G protein–coupled receptors. These observations led to the eventual cloning of two cannabinoid receptors known as the CB₁ and CB₂ receptors. CB₁ receptors are found at very high concentrations throughout the brain including the cerebellum, hippocampus, basal ganglia, cortex, brainstem, thalamus, and hypothalamus. This broad anatomic distribution of the CB₁ receptor helps to explain the diverse effects elicited by marijuana 8–2. The CB₂ receptor has low overall homology with CB₁ and is found primarily in immune cells with some limited expression in the brain.

CB₁ and CB₂ receptors couple to effectors by means of G_i/o proteins. Like other receptors that couple with these G proteins, CB₁ receptors inhibit adenylyl cyclase and, depending on the cell type, inhibit voltage-gated Ca²⁺ channels or stimulate inwardly rectifying K⁺ channels. As they are primarily found on presynaptic terminals of both excitatory and inhibitory neurons, activation of CB₁ receptors inhibits transmitter release and thereby synaptic transmission in a wide range of brain regions.

The identification of cannabinoid receptors led to the speculation that an endogenous cannabinoid-like substance might exist; such speculation was partly based on the discovery of endogenous opioid peptides soon after the detection of opioid receptors (Chapter 7). Because cannabinoids are highly lipophilic, the search for an endogenous substance focused on membrane extracts from the brain. This led to the identification of two substances, anandamide and 2-arachidonylglycerol (2-AG), which function as endogenous cannabinoids or endocannabinoids: they are released in response to neural activity and activate CB₁ receptors. As shown in 8–5A, both anandamide and 2-AG are thought to be synthesized from membrane-derived lipid precursors. Abundant evidence suggests that their release from postsynaptic neurons is coupled to their synthesis. Specifically, in a variety of cell types, strong depolarization leads to Ca²⁺ influx via voltage-dependent Ca²⁺ channels and this in turn causes activation of the enzymes responsible for generating anandamide or 2-AG 8–6. In certain cell types, the generation of these endocannabinoids can also be stimulated by activation of postsynaptic G protein–coupled receptors, specifically G_q-linked metabotropic glutamate receptors or muscarinic receptors 8–6.