Primarily because of their potent analgesic properties, but also because of their antitussive and antidiarrheal effects, opiates have long ranked among the most important drugs in the pharmacopoeia. Indeed, of the drugs commonly used today, morphine is one of a small number that were available in the 19th century. The medical importance of opiate analgesics (Chapter 11), combined with their addictive liabilities (Chapter 16), has produced much research in an unsuccessful attempt to develop potent opiate analgesics that are also nonaddictive. Products of this research include the discovery of lipophilic, small-molecule opioid receptor antagonists, such as naloxone and naltrexone, which have been critical tools for investigating the physiology and behavioral actions of opiates. In addition, naloxone is effective in the treatment of opiate overdoses, and naltrexone, which is longer-acting, is used in the long-term treatment of opiate addiction (by blocking binding of opiates to their receptors) and of alcoholism (presumably by blocking endogenous opioid peptides that contribute to rewarding responses to alcohol). More significantly, this research motivated the discoveries of opioid receptors and endogenous opioid peptides in the nervous system 7–1.
7–1 The Body’s Own Opiates
Historically, three types of observations suggested the presence of specific opioid receptors in the human body. (The term opioid refers to endogenous peptides with opiate-like pharmacology, whereas opiate refers to morphine and related nonpeptide analogs.) (1) Opiate analgesics such as morphine produce effects at extremely low concentrations; for example, a few milligrams of morphine can produce clinically significant analgesia. This suggested action at a small number of high-affinity receptors rather than “mass action.” (2) Opiate drugs exhibit stereoselectivity, suggesting a receptor that could only bind a drug with a specific conformation. (3) A competitive antagonist of opiate action (naloxone) had been identified in early studies. In the 1970s, several independent laboratories used radiolabeled ligand binding methods in preparations of synaptic membranes to produce convincing evidence of opioid receptors in neural tissues.
The discovery of opioid receptors raised the question of whether the body contained endogenous opioids. A more convincing indicator of endogenous opiate-like activity came from physiologic experiments. Under certain conditions of stress, animals can exhibit markedly elevated pain thresholds (stress-induced analgesia). The injection of naloxone can prevent the development of this stress effect, which suggested the involvement of an endogenous substance that bound to opioid receptors. Using a sensitive bioassay contraction of guinea pig ileum (opiates are clinically constipating), and extracts of porcine brain, it was possible to identify opiate-like activity in brain tissue. These early studies discovered two pentapeptides with opioid activity, which were named enkephalins (Greek for in the head). Subsequently, it has been determined that three separate genes encode at least 18 endogenous peptides with opiate-like activity.
Opioid peptides are encoded by three distinct genes 7–6. These precursors include POMC, from which the opioid peptide β-endorphin and several nonopioid peptides are derived, as discussed earlier; proenkephalin, from which met-enkephalin and leu-enkephalin are derived; and prodynorphin, which is the precursor of dynorphin and related peptides. Although they come from different precursors, opioid peptides share significant amino acid sequence identity. Specifically, all of the well-validated endogenous opioids contain the same four N-terminal amino acids (Tyr–Gly–Gly–Phe), followed by either Met or Leu (see 7–6).
Opioid peptides. A. Structures of the three opioid precursors. Proopiomelanocortin (POMC) gives rise to the opioid β-endorphin and other nonopioid peptides, including melanocyte-stimulating hormones (MSH), adrenocorticotropin (ACTH), and corticotropin-like intermediate lobe peptide (CLIP). Proenkephalin (Pro-enk) gives rise to multiple copies of the pentapeptide met-enkephalin (ME), one copy of the pentapeptide leu-enkephalin (LE), and several extended enkephalin-containing peptides, including two extended versions of met-enkephalin, ME-Arg–Gly–Leu (ME-RGL) and ME-Arg–Phe (ME-RF). Other large enkephalin fragments are designated peptides E, F, and B. Prodynorphin (Pro-dyn) gives rise to dynorphin and α-neo-endorphin. B. Shared opioid peptide sequences. Although they vary in length from as few as 5 amino acids (enkephalins) to as many as 31 (β-endorphin), the endogenous opioid peptides shown here contain a shared N-terminal sequence followed by either Met or Leu.
There are three opioid receptors, μ, κ, and δ 7–4, and a fourth related receptor (ORL-1 that binds nociceptin; see below). However, there is growing evidence that heterodimers form (eg, μ–δ dimers), yielding a receptor with distinct properties. As well, several isoforms of the μ receptor, generated by alternative splicing, have been shown to mediate distinct functions. The σ receptor, a single transmembrane-spanning protein that binds phencyclidine,1 was once considered to be a possible fourth opioid receptor because it binds benzomorphan opiate drugs such as pentazocine. However, it is not blocked by opioid receptor antagonists, and instead binds diverse drugs that are unrelated to opioids. The functional significance of the σ receptor remains poorly understood.
The μ, κ, and δ opioid receptors are all linked to the Gi/o family of G proteins (Chapter 4). Among endogenous opioid peptides, β-endorphin binds preferentially to μ receptors. Two other brain peptides, endomorphin-1 and -2, which lack the signature opioid peptide sequence shown in 7–6 (Tyr–Gly–Gly–Phe), bind selectively and with high affinity to μ receptors as well. Enkephalins bind with high affinity to δ receptors and dynorphin peptides to κ receptors. However, opioid peptides do not bind exclusively to the receptors for which they have highest affinity; in vivo binding is also likely to be influenced by relative locations of released peptides and opioid receptors. Moreover, the complex pharmacology of opiate drugs suggests that posttranslational modifications and the aforementioned heterodimerization of μ and δ receptors create a far richer possibility for opiate drug and peptide binding than the three cloned receptors would initially suggest.
Morphine-like opiate drugs preferentially bind to μ receptors, which are concentrated in regions associated with descending analgesic pathways, such as the periaqueductal gray matter, rostroventral medulla, medial thalamus, and dorsal horn of the spinal cord (Chapter 11). Significantly, these receptors also reside in reward-related regions including the ventral tegmental area (VTA) of the midbrain and the nucleus accumbens, where they are responsible for the addictive effects of opiates (Chapter 16) and may play a more general role in the hedonic effects of natural rewards, such as food. In addition, μ receptors are expressed in the dorsal striatum and in the locus ceruleus (LC). In the LC, μ receptors mediate important aspects of opiate physical dependence and withdrawal (Chapter 16).
Enkephalins, rather than any clinically available drugs, are the molecules with the greatest affinity for δ opioid receptors. δ receptors are expressed not only in the dorsal horn of the spinal cord where they play a role in analgesia but also in many brain regions.
Some κ receptor agonists such as nalbuphine and butorphanol exert clinically useful analgesic effects acting via κ receptors (Chapter 11), but are also potent μ receptor antagonists. Pentazocine, also used as an analgesic, is a κ receptor agonist and a partial μ receptor agonist or weak antagonist. As such, these drugs may precipitate withdrawal if used as analgesics in individuals who are dependent on morphine or heroin. κ receptors are found in the dorsal horn of the spinal cord, in the dorsal striatum and nucleus accumbens, in deep cortical layers, and in many other brain regions. Even though they are analgesic, κ receptor agonists and dynorphin peptides may produce dysphoria rather than euphoria because they are expressed on the presynaptic terminals of dopamine neurons that project from the VTA to the nucleus accumbens and other forebrain regions. Because κ receptors are coupled via Gi/o to a K+ conductance that hyperpolarizes dopamine terminals, κ agonists decrease dopamine release. In contrast, μ receptors are expressed on inhibitory interneurons in the VTA that suppress firing of dopamine neurons. Morphine-like opiates acting via μ receptors thereby disinhibit dopamine neurons and stimulate dopamine release, an effect opposite to κ agonists.
Another peptide, alternatively termed nociceptin or orphanin FQ, binds to a G protein–coupled receptor termed the nociceptin receptor, also known as ORL1. The terminology derives from ORL1 having been an orphan receptor because it did not have a known ligand (hence the term orphanin). Nociceptin/orphanin F/Q is a hectadecapeptide closely related to dynorphin A. It is derived from the pronociceptin/orphanin FQ gene. Nociceptin/orphanin F/Q has been reported to have antiopioid effects, and thus pronociceptive functions at least in some experimental paradigms, raising the possibility that ORL1 antagonists may be analgesic. However, possible antinociceptive effects of nociceptin/orphanin F/Q are also under investigation.
CRF (also called CRH) is a 41-amino-acid peptide that was first isolated in the search for a hypothalamic releasing factor that causes ACTH secretion from the anterior lobe of the pituitary gland. CRF shares this capability with vasopressin in many species, including humans (see below). It is synthesized by a subset of neurons in the paraventricular nucleus (PVN) of the hypothalamus. A subset of these neurons project to the median eminence from which CRF is released into the portal hypophyseal circulation, from which it acts on pituitary corticotrophs (these neuroendocrine functions are described in Chapter 10). CRF is not only delivered into the portal circulation, however; PVN neurons and other neurons, located elsewhere in brain, synthesize CRF and release it synaptically in the brainstem, cerebral cortex, central nucleus of the amygdala, and the bed nucleus of the stria terminalis (BNST), among other regions. CRF localized within the amygdala and BNST appears to play a role in anxiety and fear-related behavior (Chapter 15). Amygdala CRF is also important in mediating the negative emotional symptoms of withdrawal from most, and possibly all, addictive drugs (Chapter 16).
Two CRF receptors have been identified and cloned. CRF1 receptors are expressed widely in the brain, while CRF2 receptors exhibit a much narrower distribution. They are concentrated in the lateral septal nuclei of the forebrain. The endogenous ligand for CRF2 is not CRF, but a related 40-amino-acid peptide, urocortin. Urocortin can exert potent hypotensive and anorexigenic effects. Other members of the CRF family are urocortin II and III, urotensin I (isolated from certain fish), and sauvagine (isolated from frog skin).
CRF1 receptor antagonists have been studied extensively for use as potential anxiolytic and antidepressant drugs (Chapter 15). Despite a great deal of research, no large clinical trial has yet demonstrated clear efficacy. One of the challenges is the difficulty in generating brain-penetrant CRF1 antagonists, as noted earlier in this chapter. However, it is also possible that CRF, released in several brain areas in response to stress, actually serves an adaptive function such that antagonism of CRF might not be beneficial. The case may be stronger for CRF1 antagonists being useful in the treatment of drug addiction withdrawal states (Chapter 16), but convincing clinical trials have yet to be performed. One might be concerned about the neuroendocrine side effects of CRF1 antagonists—in producing a dangerous syndrome of cortisol deficiency (Chapter 10); however, administration of these drugs appears safe perhaps because vasopressin has an independent ability to release ACTH. Interest remains in the exploration of CRF2 antagonists, although little clinical validation is currently available.
These closely related nonapeptides 7–7 have both neuroendocrine and more purely neural functions. Oxytocin and vasopressin are synthesized in the PVN and supraoptic nucleus of the hypothalamus and transported within the long axons of these neurons for storage and ultimately release into the blood as neurohormones. Axons of the magnocellular (large) neurons of these two hypothalamic nuclei project to the posterior pituitary (neurohypophysis) where the two peptides are stored in presynaptic terminals and released into the systemic circulation. Vasopressin acts in the distal tubules of the kidney to facilitate water reabsorption (it is also called antidiuretic hormone [ADH]), and oxytocin stimulates uterine contraction at parturition and milk letdown during nursing. Axons from the parvocellular (small) neurons of the PVN and supraoptic nucleus also project to the portal hypophyseal vessels. Vasopressin can act in the anterior pituitary, like CRF, to stimulate synthesis and release of ACTH, as stated above. The neuroendocrine functions of these peptides are described in Chapter 10; however, they also act within the brain.
Vasopressin and oxytocin. A. Sequence comparison of vasopressin and oxytocin. These are closely related nonapeptides (ie, composed of nine amino acids) that differ by only two amino acids. Shared amino acids are underlined. B. Both peptides also share the feature of an internal disulfide bond between the two cysteine residues. The structure of oxytocin is shown.
Vasopressin has three receptors 7–4, V1a and V1b (once called V3), and V2. All are G protein–linked, V1a and V1b to Gq/11 and thus to the phosphatidylinositol second messenger system, and V2 to Gs and to adenylyl cyclase (Chapter 4). A large body of research has implicated vasopressin and its V1a receptor in the regulation of affiliative behavior 7–2. The V1b receptor is located in the anterior pituitary where it stimulates ACTH secretion in concert with CRF. It is also expressed in the brain. V1b knockout mice exhibit reduced aggression. Oxytocin has a single receptor that is linked to Gq/11.
7–2 Vasopressin and Affiliative Behavior
Species differences in the promoter of the V1a receptor gene result in markedly different patterns of receptor expression in the brain, which in turn lead to variations in behavioral responses to vasopressin. V1a receptor expression patterns influence male reproductive and social behavior in several rodent species, most notably in two types of voles. The male montane vole, which has a V1a receptor pattern similar to that found in mice, tends to be asocial and promiscuous. In contrast, the male prairie vole, which exhibits more extensive V1a receptor expression in the brain, is highly social and monogamous. Studies of mice have provided direct evidence that such differences in V1a receptor expression affect affiliative behavior. Transgenic mice that express the prairie vole V1a receptor gene show prairie vole-like patterns of V1a receptor distribution superimposed on their normal, endogenous expression of V1a receptors. Such mice respond to vasopressin administration with an increase in affiliative behavior, whereas control mice do not exhibit this response. These experiments not only show significant effects of vasopressin on social behavior but also underscore the importance of patterns of receptor expression, and thus neural circuit activation, on the actions of any neurotransmitter.
Overall vasopressin and oxytocin play important roles in the ability of rodents to recognize previously encountered individuals of the same species and regulate social behavior through actions in diverse regions of the brain. Oxytocin appears to increase male–female bonding after mating and mother–infant bonding. In the amygdala, vasopressin acting via V1a receptors may increase anxiety. Acting in a different region of the amygdala, oxytocin may decrease fear and anxiety and may promote prosocial behaviors by inhibiting avoidance and decreasing aggression. Is there any relevance for humans? Oxytocin can be delivered to humans via nasal spray, but resulting levels in CSF have not been adequately established. In a functional MRI experiment, oxytocin decreased amygdala activation in human volunteers in response to fearful faces or scenes 7–8. In several human experiments, oxytocin spray increased trusting behavior compared with a placebo spray. These results in humans are quite early, but suggest a role for oxytocin in regulating social interactions. Indeed, clinical trials are under way to evaluate the utility of oxytocin in treating autism (Chapter 14).
Effect of oxytocin on human amygdala activation. A. Coronal sections at the level of the anterior commissure. The response to fearful or angry faces is on the left and to fearful or threatening scenes is on the right. The effect of oxytocin is greater for faces, a social stimulus. B. Compared with placebo, oxytocin suppresses amygdala activation, predominantly on the left. C. This result is shown quantitatively as a function of BOLD functional MRI signal. (Reproduced with permission from Kirsch P, Esslinger C, Chen Q, et al. Oxytocin modulates neural circuitry for social cognition and fear in humans. J Neurosci. 2005;25(49):11489–11493.)
Substance P, NKA (previously known as substance K), NKB, and neuropeptide K are members of the tachykinin family, all of which share the C-terminal sequence Phe–X–Gly–Leu–Met–NH2. Substance P, NKA, and neuropeptide K are encoded by the PPT-A gene, and are produced by alternative splicing 7–3. The PPT-B gene encodes NKB. The three known tachykinin receptors, all of which are Gq/11-coupled, are designated NK1, NK2, and NK3 7–4. Substance P binds with highest affinity to the NK1 receptor, NKA preferentially binds to the NK2 receptor, and NKB binds somewhat selectively to the NK3 receptor.
Of the tachykinins, substance P has gained the most attention. It was first discovered in 1931 in extracts of brain and gut. It is expressed in the dorsal horn of the spinal cord, amygdala, medulla, hypothalamus, substantia nigra, cerebral cortex, and striatum. In the latter region, it is colocalized with GABA and dynorphin in the striatonigral neurons that make up the “direct” striatothalamocortical pathway of great interest in Parkinson disease (Chapters 14 and 18).
Substance P was long thought to be a critically important neurotransmitter carrying pain signals. Primary afferent nociceptors that synapse in the dorsal horn of the spinal cord (Chapter 11) express a complex array of neuropeptides, of which the most abundant include substance P, NKA, and CGRP. Substance P is colocalized with glutamate in the synaptic terminals of a class of nociceptors called C fibers. NK1 receptors are found in abundance in the dorsal horn. Accordingly, NK1 receptors were considered an important target for the development of nonopioid analgesics. The first clues that this strategy might not succeed came from mice in which the PPT-A or NK1 receptor gene had been knocked out; the phenotypes did not show substantial alterations in pain threshold. More definitively, potent and selective NK1 antagonist drugs did not have significant effects in clinical tests on humans. Based on NK1 receptor expression in the amygdala and on animal models of distress, NK1 antagonists have also been studied as possible antidepressants. To date, most clinical trials have shown no efficacy. The main clinical use of NK1 antagonists has been in blocking the nausea and vomiting caused by cancer chemotherapy. These drugs, such as aprepitant, are likely exerting their actions in the medullary vomiting centers.
Substance P is released not only in the dorsal horn but also in retrograde fashion from the free nerve endings of nociceptive neurons. This retrograde release contributes to the phenomenon of neurogenic inflammation; other peptides such as bradykinin also contribute (Chapter 11).
Neurotensin is a 13-amino-acid peptide derived from a larger precursor that also contains neuromedin N, a related 6-amino-acid peptide. Neurotensin is expressed in the brain, adrenal gland, and gut in slightly different forms that illustrate tissue-specific posttranslational processing. Its C terminus contains one of three different Lys–Arg sequences, which are differentially cleaved depending on the tissue in which neurotensin is processed. In the brain, the precursor gives rise to both neurotensin and neuromedin N, whereas in the adrenal gland, neurotensin, a larger form of neuromedin N, and a larger form of neurotensin sequence are produced. In the gut, the most common products are neurotensin and large neuromedin N. Central administration of neurotensin produces hypothermia and analgesia.
There are three neurotensin receptors 7–4. Two of these, NTS1 and NTS2, are G protein–coupled receptors 7–4. NTS3 is a single membrane-spanning receptor analogous to type I amino acid receptors, exceptional for neuropeptide signaling. Neurotensin mRNA is induced in striatopallidal neurons of the striatum by D2 receptor antagonists, including antipsychotic drugs, and in striatonigral neurons by psychostimulant drugs such as cocaine and amphetamine. Therefore, it has been hypothesized that neurotensin influences dopamine signaling and perhaps contributes to the plasticity induced by drugs that act on dopamine systems in the brain. These observations, however, have not yet led to new treatments for psychotic disorders or drug addiction.
Orexin A and B (also referred to as hypocretin 1 and 2), products of a single gene, were discovered by convergent approaches in rat brain. One group of investigators found a peptide that stimulated feeding (thus the name “orexin”), and the other group was searching for ligands for “orphan” hypothalamic G protein–linked receptors (thus the alternative name, hypocretin). The two known orexin receptors, OX1 and OX2, are coupled via Gq and Gi/o, respectively. There are reports of OX2 coupling to Gq as well. Soon after the peptides were discovered, a loss-of-function mutation in the OX2 receptor was found to be the cause of canine narcolepsy (Chapter 13), and orexin peptide knockout mice were shown to exhibit a narcolepsy-like syndrome. Subsequently, humans with narcolepsy were found to have presumed autoimmune destruction of orexin neurons and depletion of orexin from cerebrospinal fluid. These discoveries have stimulated great interest in the development of orexin receptor ligands to modulate sleep and alertness. The diverse functions of orexin peptides, which in addition to sleep and arousal include feeding and reward, result from an anatomic organization that is reminiscent of monoamine neurotransmitters (Chapter 6). Orexin peptides are synthesized solely by neurons in the lateral and posterior hypothalamus and project to and activate monoaminergic and cholinergic neurons, among many other neuronal types 6–25. Orexins are discussed further in Chapters 6 and 13.
NPY is the best known of a related peptide family that also includes peptide YY (PYY) and pancreatic polypeptide (PP). NPY gains its name for its N- and C-terminal tyrosines (“Y” is the single letter symbol for tyrosine). NPY is the most abundant neuropeptide in cerebral cortex. It also is concentrated in the dorsal horn of the spinal cord and arcute nucleus of hypothalamus. It is colocalized with norepinephrine in both the LC and sympathetic nervous system 7–3. NPY is a potent stimulator of feeding. Leptin, which inhibits feeding, acts partly by inhibiting the synthesis and release of hypothalamic NPY. Nonetheless, mice lacking NPY continue to feed normally, underscoring the complex interactions of hypothalamic systems that control feeding and energy balance (Chapter 10). In the periphery, NPY sensitizes smooth muscle to the effects of norepinephrine, resulting in a potent vasoconstrictor effect.
NPY, PYY, and PP bind to a group of six G protein–linked receptors, designated Y1 to Y6 7–4 with varying selectivity; all couple to Gi/o. These receptors display marked region-specific distributions in brain and are located on both postsynaptic and presynaptic sites. Activation of the Y1 receptor is thought to cause a decrease in anxiety-like behavior, acting, perhaps, in the amygdala. This hypothesis has generated interest in exploring the use of Y1 agonists for the treatment of anxiety disorders. In contrast, activation of the Y5 receptor has been proven to stimulate feeding, acting presumably within the hypothalamus; accordingly, Y5 antagonists may be useful medications for the treatment of obesity. Applications of NPY pharmacology have not yet borne fruit in the clinic.