Basic Mechanisms

Molecular Components of the Opioid System: Receptors, Peptides, and Their Genes

From the very early days of opioid research it seemed obvious that opiate alkaloids act on the nervous system. The existence of specific receptors was demonstrated by the presence of high-affinity and saturable binding sites in brain membrane preparations (Pert and Snyder 1973, Simon et al 1973, Terenius 1973). Naloxone, a synthetic morphine derivative, was found to block morphine activity and was considered the prototypical opioid antagonist. From there, any biological activity that was reversed by naloxone was considered opioid in nature. Synthetic chemistry provided novel alkaloid compounds with opioid activity that revealed several classes of opioid receptors (Martin et al 1976). Ultimately, three major opioid receptor subtypes emerged from pharmacological studies, referred to as mu, delta, and kappa receptors. Their molecular characterization, however, waited almost another 20 years because of the paucity and strongly hydrophobic properties of these membrane receptor proteins.

The first opioid receptor to be characterized at the molecular level was a mouse delta receptor. Molecular cloning of this receptor was achieved by expression cloning (Evans et al 1992, Kieffer et al 1992). Isolation of this cDNA represented a milestone in opioid research (Barnard 1993, Brownstein 1993) and opened the way to functional exploration of the opioid system by mutagenesis approaches in vitro and in vivo. A first step was molecular identification of an opioid receptor gene family, including mu, delta, and kappa, as well as the closely related ORL1 receptors (Taylor and Dickenson 1998). Their genes have now been cloned in many species, including humans, rodents, amphibians, and zebra fish. In humans and in mice the four genes are highly homologous in their intron/exon organization, possibly deriving from a common ancestor (for review, see Kieffer 1995, 1997; see also Table 30-1).

In the early 1970s, demonstration of opioid binding sites launched the search for endogenous ligands. Met- and leu-enkephalins, two closely related pentapeptides, were first purified from brain and sequenced (Hughes et al 1975). Many more peptides have since been isolated from nervous tissue, the pituitary gland, and the adrenals (Akil et al 1984). These peptides share a common N-terminal sequence—YGGFL/M—considered the opioid pharmacophore and partially overlapping the morphine structure (Barnard 1993). Three genes encoding those peptides were cloned in the early 1980s (Nakanishi et al 1979, Comb et al 1982, Kakidani et al 1982). The genes encode large precursor proteins, proopiomelanocortin, preproenkephalin, and preprodynorphin. Proopiomelanocortin produces the largest opioid peptide, β-endorphin, as well as peptides with non-opioid activities. The preproenkephalin and preprodynorphin precursors are processed to generate several copies of enkephalin and dynorphin peptides, respectively. All members of this large family of opioid peptides act as agonists at mu, delta, and kappa receptors with nanomolar affinity and limited receptor selectivity (Akil et al 1998). The discovery of endogenous opioid peptides further broadened the panel of available opioid ligands, and synthesis of a vast plethora of enkephalin derivatives completed the large repertoire of alkaloid-type opioids (Corbett et al 1993) to study mu, delta, and kappa receptor function.

Opioid Receptors: Structure–Activity

Opioid receptors belong to the superfamily of G protein–coupled receptors (GPCRs). This receptor family comprises several hundred members in the human genome (Lagerstrom and Schioth 2008). GPCRs contain seven hydrophobic transmembrane domains interconnected by short loops and display an extracellular N-terminal domain and an intracellular C-terminal tail (Fig. 30-1A). A classic serpentine representation the delta receptor is shown in Figure 30-1B.

Mu, delta, and kappa receptors are highly homologous, with transmembrane domains and intracellular loops best conserved (86%–100%). The extracellular loops, however, as well as N- or C-terminal tails, differ largely. Sequence comparisons, combined with mutagenesis experiments, have led to the identification of receptor domains with specific functions (Fig. 30-1C). Structure–activity relationships in opioid receptors have been evaluated in great detail in several reviews (see Befort and Kieffer 1997, Law et al 1999a, Chaturvedi et al 2000, Décaillot and Kieffer 2003).

Receptor Structure

The first structure of a GPCR, the bovine rhodopsin, was solved by x-ray cristallography (Palczewski et al 2000) and was long used as a template for structure-function predictions at GPCRs. Another 7 years were required until first GPCRs bound to hormones or neurotransmitters were crystallized and their structure solved at atomic level (Audet and Bouvier 2012). In 2012, 20 years after opioid receptor cloning, a structure was reported for each member of the opioid receptor gene family. The receptors were crystallized under an inactive form, were bound to an antagonist, and structures of the mu receptor-beta-funaltrexamine (Manglik et al 2012), the delta receptor-naltrindole (Granier et al 2012), the kappa receptor-JDTic (Wu et al 2012), and the ORL-1 receptor-C-24 (Thompson et al 2012) complexes are now available (PBD accession numbers 4DKL, 4EJ4, ADJH and 4EA3, respectively). The four receptors share a conserved wide-open binding pocket contrasting with buried pockets of other GPCRs crystallized so far. Mu receptors form intimate oligomeric pairs within the crystal (Manglik et al 2012), supporting the view that opioid receptors may function as homo- or heterodimers, or even larger oligomers (Jordan and Devi 1999). This breakthrough in opioid research reveals ligand-binding modes and enables the development of structure-based approaches to design better drugs. A next challenge will be to elucidate receptor activation mechanisms and understand receptor signaling at structural level.

Opioid Receptor Diversity: The Molecular Basis for Pharmacological Subtypes

Opioid receptor pharmacology is complex, and the existence of multiple mu (Pasternak 1993), delta (Traynor and Elliott 1993, Zaki et al 1996), and kappa (Traynor 1989) receptor types has been proposed from a wide plethora of in vitro and in vivo experiments. Gene cloning has provided three receptor genes only, and the molecular basis for pharmacological diversity remains a matter of debate (Zaki et al 1996, Befort and Kieffer 1997). Homology cloning, as well as bioinformatic search in mammalian genomes, shows no evidence of additional closely related opioid receptor genes. Alternative splicing could potentially generate receptor protein variants from the three known opioid receptor genes. Reverse transcriptase polymerase chain reaction experiments have allowed the detection of alternative transcripts in several cell lines or tissues for all three receptors, with their abundance definitely being lower than that of the three known transcripts. Delta and kappa receptor mRNA variants encode truncated—and thus probably non-functional—receptors (for review, see Gavériaux-Ruff and Kieffer 1999). Alternative mu receptor mRNA molecules encode receptors with variable C termini, and some alterations in the binding or signaling properties of the putative encoded receptors have been reported in transfected systems (Bolan et al 2004).

To date, it has been extremely difficult to establish the biological relevance of these alternative transcripts in vivo and to correlate their existence with the multiple opioid receptor subtypes described earlier by the pharmacology. On the other hand, increasing evidence supports the notion that the three known mu, delta, and kappa receptor proteins may adopt multiple active conformations that would contribute to their pharmacological heterogeneity. In an early report, extensive site-directed mutagenesis of the delta receptor opioid binding site showed unique sets of interactions for each of the 12 opioid compounds under study (Befort et al 1996). These data suggested the existence of multiple binding sites, which opened the way to the possible existence of multiple ligand–receptor conformational states. This was later substantiated functionally with the discovery of ligand-dependent receptor responses. Particularly striking was the finding that in contrast to the peptidic mu agonist DAMGO, morphine was unable to induce mu receptor internalization whereas both compounds efficiently signaled within the cell (Arden et al 1995, Keith et al 1996). Further studies in transfected cells confirmed that receptor activation and subsequent regulation are strongly agonist dependent and that agonist efficacy does not necessarily correlate with their ability to induce receptor phosphorylation, internalization, or desensitization (von Zastrow et al 2003). The best possible interpretation of these data is that multiple active conformations of the receptor do exist and are being stabilized by individual ligands, a notion that has been proposed and investigated for other GPCRs (for a recent example, see Beinborn et al 2004). An important implication is that the ligand–receptor complex, rather than the receptor itself, determines the ultimate physiological cellular response. Therefore, a single receptor protein (e.g., the mu receptor) is able to trigger distinct pharmacological responses depending on the bound ligand.

At least as important as the interacting ligand is the cellular environment of the receptor. Many proteins directly interact with the receptor, thereby modulating receptor conformation and activity. Beyond the known Go/Gi alpha proteins calmodulin, kinases, and arrestins, other regulatory proteins, multidomain scaffolding units, or chaperone molecules potentially influence opioid receptor pharmacology, as was demonstrated for several other GPCRs (Brady and Limbird 2002). Proteomic approaches have recently led to the identification of novel interacting partners for opioid receptor C-terminal tails, including signaling (phospholipase A₂), cytoskeletal (filamin and periplakin), and sorting (GASP) proteins (for review, see Contet et al 2004). This research field is expanding rapidly and the importance of these interactions on opioid binding and signaling is being investigated.

Another potential receptor modulator is the receptor itself. The possibility that GPCRs may exist as dimeric or oligomeric complexes has gained much support in recent years. The co-expression of delta and kappa receptors in transfected cells produced delta–kappa heterodimers, as revealed by co-immunoprecipitation experiments, and most importantly, resulted in substantial modifications of ligand binding (Jordan and Devi 1999). In this experimental setting, the decreased binding of selective mu and delta ligands with a concomitant increase in the binding of non-selective opioids was consistent with a kappa-2 pharmacology. Similarly, mu and delta receptor co-expression led to an alteration in opioid binding and signaling that was attributed to receptor dimerization (for review, see Devi 2001, Levac et al 2002). Taken together, the data suggest that the physical association of opioid receptors—either as homodimers or as heterodimers—creates novel receptor entities with unique pharmacological activities that can potentially enlarge opioid receptor heterogeneity. Whether receptor dimerization or interaction with other protein partners truly modulates opioid pharmacology in vivo remains an important question.

Finally, the entire cellular context adds another level of complexity to biological responses to agonists. First, G-alpha subunits, considered the classic signaling effectors, differ across cell types. Second, the number of possible G protein–associated signaling pathways has expanded dramatically beyond the classic second-messenger generating systems (Marinissen and Gutkind 2001). Altogether, the variable combinations of G-alpha proteins and the nature of associated intracellular signaling networks necessarily generate cell-specific responses that represent another source of pharmacological heterogeneity, particularly in the analysis of complex responses such as nociceptive behavior.

In conclusion, molecular approaches have provided a novel prospect of the opioid receptor that should be viewed as a dynamic multicomponent unit rather than as a single protein entity. The specific biological response is elicited and determined by the ligand-bound receptor within its neuronal microenvironment. The possibility of multiple active conformations of the receptor, which are influenced by the chemical nature of the agonist and modulated by interacting membrane or intracellular protein partners, forms the basis for enormous opioid receptor diversity. Hence, there is no doubt that several mu, delta, and kappa pharmacological profiles or “subtypes” can arise from activation of the three known mu, delta, and kappa receptor proteins, depending on the experimental setting.

In fact, as in vivo molecular pharmacology develops, it is likely that the complexity of opioid responses will extend well beyond the previously reported pharmacological subtypes. Particularly intriguing was the recent observation that morphine efficiently internalizes mu receptors in dendrites but not in the cell bodies of nucleus accumbens neurons (Haberstock-Debic et al 2003), a finding that could not be anticipated from previous studies of classic expression systems or from experiments using brain homogenates and whole animals. Cellular compartmentalization—and therefore the precise localization of the receptor—is yet an additional source of pharmacological heterogeneity.

The finding of agonist-driven responses at the mu receptor has important therapeutic implications. Gene knockout experiments have definitively demonstrated that mu receptors mediate all morphine activities (Matthes et al 1996, Kieffer and Gavériaux-Ruff 2002), thereby ruling out the possibility that the beneficial (analgesia) and non-desirable (tolerance, dependence, respiratory depression) activities of the prototypical opiate could be mediated by two separate molecular entities—and hence distinct therapeutic targets. Mu receptor signaling, however, is dissociable from mu receptor phosphorylation, internalization, or down-regulation (see above). Because morphine signaling triggers low levels of receptor regulation, there has been much speculation on the possibility that sustained morphine signaling may in fact induce the cellular and neuronal adaptations responsible for tolerance and dependence (Kieffer and Evans 2002). There are therefore at least two “types” of active mu receptors that could be defined as either highly regulated or poorly regulated receptors and be represented by DAMGO/mu and morphine/mu receptor complexes. Although most presently available mu agonists seem to induce profound tolerance in vivo (Evans et al 2000), the intriguing possibility exists that targeting the highly regulated receptor conformation may lead to a potent non-addictive analgesic drug. Because we are only starting to understand the molecular basis for mu opioid receptor heterogeneity, rational design of such compounds at present represents a far distant goal.

Deleting Opioid Receptor or Peptide Genes in Vivo: Consequences on Pain Pharmacology

Homologous recombination techniques have led to the production of mice with targeted gene deletions, and mouse lines lacking each genetic component of the opioid system have been created, including the knockout of mu, delta, or kappa receptor genes, as well as endorphin/proopiomelanocortin, preproenkephalin, or preprodynorphin genes (for review, see Kieffer and Gavériaux-Ruff 2002). All the mutant mice are viable and fertile, as are triple-mutant mice lacking all the opioid receptor genes, thus indicating that the opioid system is not essential for survival. The spontaneous behavior of these knockout lines has been analyzed by many investigators, as well as the responses of receptor-deficient mice to prototypal opiate or non-opioid drugs (Gavériaux-Ruff and Kieffer 2002, Kieffer and Gavériaux-Ruff 2002, Pintar and Kieffer 2004). Here we will summarize a number of findings that relate to pain research.

One important issue was the clarification of in vivo molecular targets for clinically useful opiates or for opioid compounds that have been classically used in pharmacology to study mu, delta, and kappa receptor function. A significant finding was the abolition of all morphine effects in mice lacking mu receptors, which indicated that a single gene product is essential for the wide spectrum of both the beneficial and adverse activities of morphine. Furthermore, the strong decrease in U50,488H hypolocomotion, analgesia, and aversion in kappa receptor–deficient mice and the maintained U50,488H analgesia in mice lacking either mu or delta receptors confirmed the selective activity of this prototypal kappa agonist in vivo. This also demonstrated that kappa receptor–mediated analgesia is independent from mu- and delta receptor–mediated analgesia, at least in models of acute thermal pain. Finally, examination of the analgesic activities of DPDPE and deltorphin, considered the reference selective delta agonists, has been very informative. Results from studies performed in separate laboratories investigating delta agonist activities in either mu or delta receptor knockout mice have been extremely diverse. Data showed either maintained or decreased antinociceptive properties of the two compounds in both mutant animals (Kieffer and Gavériaux-Ruff 2002). One recent study reported a direct comparison of the two mouse lines in response to DPDPE and deltorphin treatment (Scherrer et al 2004). In the tail immersion test there was no analgesia in the mu null mutant and full analgesia in the delta null mutant, the latter being reversed by the mu antagonist CTOP. In the hot plate test, the two compounds produced full analgesia in the delta knockout mice and lower but significant analgesia in the mu mutants. In this test also, deltorphin elicited weak but measurable analgesia in the mu/kappa double-null mutant, in which only delta receptors were expressed. Together, these data suggest that mu rather than delta receptors are recruited by delta agonists in spinal analgesia and that both mu and delta receptors contribute to supraspinal analgesia. Although the conclusions apply only to behavioral models of acute thermal pain, these data have revealed the need to develop more selective non-peptidic delta agonists to further validate delta receptors as possible therapeutic targets in pain control. Conclusions on opioid pharmacology from knockout mice data are shown in Figure 30-2.

Another important issue is the respective contribution of each component of the opioid system to the regulation of physiological pain. Nociceptive responses to a number of acute noxious stimuli were examined in all the knockout lines and are summarized in Table 30-2. Taken together, the data show mostly enhanced pain sensitivity, concordant with the notion of an antinociceptive opioid tone. Some paradoxical responses have also been described—for example, decreased writhing or inflammatory hyperalgesia in the mu receptor knockout and reduced formalin irritation in the preproenkephalin null mutants (see Table 30-2)—which remain unexplained.

Also of importance are reports from many investigators using mu, delta, or kappa knockout mice suggesting that the three receptors regulate distinct pain modalities. Recently, a direct comparative study of the three mutant lines clarified this issue. Responses of single and combinatorial opioid receptor knockout mice were compared in several models of acute pain, and the distinct patterns of tonic antinociceptive activities were confirmed (Martin et al 2003):

Gender differences were reported in all the phenotypes, and together, the data were consistent with previous pharmacology. Importantly, deletion of all three opioid receptors (triple knockout) strongly enhanced pain sensitivity in all the tests and for both genders, thus suggesting that although the specific contribution of each opioid receptor is subtle, the opioid system as a whole exerts a significant inhibitory tone on physiological pain.

The development of chronic pain in mutant mice has been addressed only recently. Most striking is the fact that delta opioid receptor knockout mice showed no, or only subtle, modification of pain thresholds in models of acute pain but displayed increased pain responses in models of neuropathic (Nadal et al 2006) and inflammatory pain (Gavériaux-Ruff et al 2008). These data clearly establish the existence of an endogenous delta opioid receptor activity that reduces chronic pain. Also, knockout mice were insensitive to nortriptyline, a tricyclic antidepressant drug that fully reverses mechanical allodynia in an animal model of neuropathic pain (Benbouzid et al 2008). The latter data suggest that delta opioid receptors reduce chronic pain downstream of aminergic systems. Finally, a conditional knockout approach was applied to the delta opioid receptor gene. Using Nav1.8-Cre mice, the receptor was mainly deleted in primary nociceptive afferent neurons of dorsal root ganglia and left intact at all other sites of nociceptive pathways. Conditional mutant mice showed significantly enhanced inflammatory pain and strongly reduced antihyperalgesic effects of SNC80. The latter study demonstrates that activity of a highly restricted receptor population of the peripheral nervous system is sufficient to reduce chronic pain and mediate delta opioid analgesia (Gavériaux-Ruff et al 2011).

Finally, mice lacking preprodynorphin have been studied in both inflammatory and neuropathic pain models (Wang et al 2001), and the data indicate a biphasic role of prodynorphin in the control of nociceptive responses that may result from the dual opioid (kappa) and non-opioid (N-methyl-D-aspartate [NMDA]) activities of the peptide.

In conclusion, analysis of the opioid system in knockout mice provides genetic evidence that mu, delta, and kappa receptors, activated by endogenous peptides, all contribute to modulate pain. These mutant mice show altered responses in many other types of behaviors, including addictive (for review, see Kieffer and Gavériaux-Ruff 2002) and emotional behavior (Filliol et al 2000). Extending data from pharmacology, genetic manipulations unambiguously reveal unique contributions of each opioid receptor and peptide in opioid biology. In the future, the development of conditional gene knockout and the sophistication of mouse models of chronic pain should reveal the specific opioid receptor populations that control abnormal pain states, as well as the sites of opioid peptide release.

Opioid Receptor and Peptide Actions: Pathways Relevant to Pain

Coupling of opioid receptors to K⁺ and Ca²⁺ ion channels is believed to be a main mechanism whereby either exogenous or endogenous opioids produce analgesia. Ultimately, activation of all three opioid receptors, as well as the ORL receptor, and subsequent modification of ion channel activities will inhibit neuronal and cellular activity. The location of the receptors on synaptic circuits will have a bearing on what this leads to in terms of function in integrated systems. Thus, the opening of potassium channels—the most well-documented effect of opioid receptor activation—will inhibit release of transmitters if the receptors are on presynaptic terminals and will inhibit neuronal firing if the opioid receptor is found post-synaptically on neuronal cell bodies.

The nature of the neuron is another issue. Opioid receptor activation can cause positive effects whereby inhibition of one neuron allows other neurons downstream to become active. Thus, in integrated systems, opioids do not simply produce inhibition, and this is likely to contribute to low-dose excitations of neurons, supraspinal analgesic mechanisms, emesis, and the rewarding effects of opioids. This disinhibition occurs through opioid inhibition of inhibitory neurons, such as γ-aminobutyric acid (GABA)-containing cells. In this way, reduction of inhibition leads to facilitation in a circuit.

Activation of the opioid system by receptor agonists can provide considerable antinociception as demonstrated by various preclinical studies (Yaksh and Noueihed 1985; Dickenson 1994, 1995; Mao et al 1995; Ossipov et al 1995a, 1995b) and its widespread clinical manipulation (McQuay et al 1992). Endogenous release of the endorphins and enkephalins from intrinsic spinal neurons is stimulated only by high-intensity stimuli (Lucas and Yaksh 1990), and under normal conditions, even though the opioid antagonist naloxone does not produce hyperalgesia (Yaksh 1989), data from knockout studies reveal opioid-mediated tonic control.

The discovery that the endogenous opioid peptides, in particular, the enkephalins, are inactivated by two metallopeptidases—neutral endopeptidase and aminopeptidase N—led to the production of what has been termed “dual inhibitors.” These are single molecules with the ability to block both enzymes and thereby produce protection of a large proportion of the available endogenous opioids of the enkephalin family. These compounds have been used as a way of developing “physiological” analgesics that may lack some of the side effects of morphine by protecting the endogenous opioids that are being released during synaptic activity rather than simply activating a receptor. This approach has led to a number of compounds being produced, and most recently, a series of dual aminophosphinic inhibitors of the two enkephalin-catabolizing enzymes have been designed. These types of compounds are effective in tests such as the hot plate, rat tail flick, writhing, and formalin tests. Under these conditions, extracellular levels of the endogenous enkephalins’ brain areas related to pain are increased. Overall, the compounds produce effects that are about 40% of the maximal analgesia and can be antagonized by prior injection of naloxone. Since the increase in endogenous enkephalin levels parallels the antinociceptive responses, it is clear that the reason for the subtle tone is the rapid metabolism of these peptides (Le Guen et al 2003).

Other than studies on endogenous opioid systems, studies on activation of the opioid receptor system through exogenous agonists have provided data that suggest a substantial amount of plasticity in persistent pain states. Although such changes are beneficial in the presence of inflammation, neuropathic pain caused by peripheral nerve damage more often than not displays reduced sensitivity to opioids. This is evident both preclinically (Mao et al 1995, Ossipov et al 1995a), where the route of application is critical (Suzuki et al 1999), and clinically (Portenoy et al 1990, Jadad et al 1992) and is surrounded by much controversy. Mechanisms by which opioid sensitivity may be reduced after nerve injury include a reduction in spinal opioid receptors (Porreca et al 1998), non-opioid receptor–expressing Aβ fiber–mediated allodynia, increased cholecystokinin (CCK) antagonism of opioid actions (Nichols et al 1995), and NMDA-mediated dorsal horn neuronal hyperexcitability, which probably requires a greater opioid inhibitory counteraction (Dickenson 1997). These issues will be discussed later in this section.

Spinal Analgesia

Autoradiographic and immunohistochemical techniques have demonstrated that within the spinal cord, opioid receptors are located mostly in the superficial dorsal horn (laminae I and II), with a smaller population in deeper layers (Besse et al 1990b, Rahman et al 1998). The contribution of mu, delta, and kappa receptors to the total opiate binding throughout the spinal cord is estimated at 70, 24, and 6%, respectively (Besse et al 1990a, 1990b), at a predominantly (>70%) presynaptic location on the central terminals of only small-diameter nociceptive primary afferents. This is likely to include both C and Aδ fibers but exclude large-diameter A fibers. Opioid receptors are synthesized in small-diameter dorsal root ganglion (DRG) cell bodies and transported centrally and peripherally. This implies that the main mechanism of spinal opioid analgesia, whether it be mediated endogenously or exogenously, is via activation of presynaptic opioid receptors, which act to selectively decrease release of transmitter from nociceptive afferents and thus nociceptive transmission, with innocuous evoked activity being left intact. Indeed, spinally applied morphine can reduce release of substance P and calcitonin gene–related peptide after noxious stimulation (Go and Yaksh 1987), and excitatory but not inhibitory lamina II synaptic transmission is inhibited by a presynaptic opioid mechanism (Kohno et al 1999). Opioid receptors are also found in the periphery since after synthesis they are moved both to the central and peripheral endings of small fibers and their expression is increased in nociceptive primary afferents during inflammation. Endogenously, opiates are released from immune cells, and exogenous agonists developed for peripheral application have been shown to be antinociceptive in inflammatory states (Machelska et al 1999), where their restriction to outside the blood–brain barrier may allow reduced side effects (Janson and Stein 2003).

The remaining 30% of opioid receptors are located post-synaptically on interneurons and the dendrites of projection cells (Besse et al 1990a) visualized functionally as agonist-stimulated receptor internalization (Trafton et al 2000). Any opioid-mediated cell hyperpolarization leading to inhibition of firing of neurons with wide–dynamic range input will not exert nociceptive-specific effects, and from electrophysiological studies a small inhibition of Aβ fiber–evoked responses can be observed (Dickenson 1994, Dickenson and Suzuki 1999). Since the inhibitory effect is much less pronounced than that observed on the C fiber–evoked response, this confirms that the predominant site of action of spinal opioids is via presynaptic opioid receptors on the central terminals of nociceptive afferents (Ossipov et al 1995a, 1995b). This anatomical location of opioid receptors on fine (Aδ and C fiber) afferent terminals means that tactile information, transmitted by Aβ fibers, is mostly unimpaired by opioids since only the post-synaptic receptors will control input from these large fibers that converge onto lamina V wide–dynamic range neurons (Fig. 30-3). Thus, dynamic allodynias thought to be mediated by these fibers may be less well controlled than noxious (Aδ and C) and static allodynias (likely to be Aδ mediated; see Dickenson and Sullivan 1986, Dickenson and Suzuki 1999, Field et al 1999).

The importance of the spinal actions of opioids is evidenced by the rapidity with which the original animal behavioral studies on spinal delivery led to effective human applications. A large number of electrophysiological and behavioral studies have shown that mu, delta, and kappa receptor agonists and nociceptin acting at the ORL1 receptor cause antinociception and inhibition of spinal nociceptive neurons after intrathecal application (Yaksh and Noueihed 1985, Dickenson 1994, Taylor and Dickenson 1998). In normal animals, opioids are selective for noxious-evoked activity, and there are clear rank orders of potency of drugs within a receptor class. The mechanisms by which these effects are produced have been discussed in the preceding sections. In broad terms, the most potent opioids are the mu ligands, presumably reflecting the fact that mu opioid sites are the largest pool in the spinal cord. Delta opioids, nociceptin, and some kappa opioids then follow in order of potency. One important factor is the inverse relationship between lipophilicity and potency for a range of synthetic opioids acting at the mu receptor (McQuay et al 1989, Dickenson et al 1990). Here, the opioid with the highest potency was morphine, which is the least lipophilic. Drugs with high systemic potency as a result of their lipophilicity (e.g., fentanyl) were relatively ineffective after spinal application, probably because of non-specific binding of the lipophilic opioids in the lipid-rich fiber tracts capping the cord or as a consequence of vascular redistribution reducing the dose of opioid reaching opioid receptors in the superficial spinal cord. This is probably one of the principal reasons for the effectiveness of peptides given spinally since they are unlikely to redistribute far from the opioid receptors. Overall, there is a remarkable correlation between animal and human data on effective doses of different opioids given spinally (Yaksh 1997).

High levels of opioid receptors and endogenous opioids are present in the spinal cord. Orphanin FQ–like immunoreactivity and ORL1 receptors are located in the dorsal horn of the spinal cord and in the midbrain and brain stem sites, in some cases alongside the enkephalins, dynorphins, and endomorphins. Nociceptin has been identified in the spinal cord in areas adjacent but mostly separate from the location of the other endogenous opioids (Taylor and Dickenson 1998). The spinal level of all the opioid peptides is not altered by rhizotomy, thus indicating that they are all derived from intrinsic spinal neurons or descending pathways from the brain (Riedl et al 1996). Whereas the enkephalins and endorphins are inhibitory, as are mu and delta synthetic opioids (with the exception of low-dose mu facilitation), dynorphin—the endogenous kappa opioid receptor agonist—has a number of effects that differ from the typical opioid actions. It produces facilitation of some neurons and inhibition of other nociceptive neurons when applied spinally (Knox and Dickenson 1987), and spinal application of a kappa receptor antagonist both increases and decreases individual neuronal activity in normal animals but is considerably more effective in animals with inflammation (Stanfa and Dickenson 1994). What the consequences are for pain transmission is not yet understood.

Recent studies on the mechanisms by which the brain stem controls spinal activity in relation to nerve injury have shown that enhanced spinal levels of dynorphin can contribute to enhanced pain (Lai et al 2001, Wang et al 2001). Whether the effects of dynorphin are mediated by kappa opioid receptors is doubtful in this context, but this is an interesting example of an opioid peptide being pro-nociceptive. The functions of nociceptin in the processing of noxious events are also controversial (Henderson and McKnight 1997, Darland et al 1998, Taylor and Dickenson 1998, Heinricher 2003), although the discrepancies may be resolved on the basis of differential spinal and supraspinal actions of the peptide. In general, when administered into supraspinal sites, nociceptin produces hyperalgesia, whereas spinal administration has clearly been shown to be inhibitory and to produce analgesia (Darland et al 1998, Taylor and Dickenson 1998). These spinal antinociceptive effects of the peptide are observed in behavioral and in vivo and in vitro electrophysiological studies. Thus, nociceptin suppresses spinal reflexes and dorsal horn neuronal activity. Intrathecal orphanin FQ has also been shown to be antinociceptive in the tail flick test at doses that do not induce motor deficits or sedation (Darland et al 1998, Taylor and Dickenson 1998). The recent description of antagonists of the ORL1 receptor should help in clarifying the function of this receptor (Koizumi et al 2004).

Supraspinal Analgesia

Output from the spinal cord is attenuated by opioids. Opioid mechanisms at a number of other supraspinal sites such as the thalamic levels, the amygdala, and the sensory cortex are likely to play key roles in the overall analgesic state. However, from both animal and patient data, it is clear that spinal routes can almost entirely abolish pain responses and therefore prevent supraspinal activation by noxious input. As pain-related activity arrives at higher centers, input from the spinal cord to the parabrachial area, the central gray, and the amygdala may contribute mostly to the emotional aspects of pain, whereas input to the thalamus generates the sensory aspects (Rahman et al 2003). Higher centers play key roles in cognition, memory, attention, punishment, and other processes, and thus research on the ways in which centers involved in the higher processing of pain interact with opioids and sensory transmission may well yield further targets for therapy. One circuit may well be this type of loop whereby areas important in anxiety and attention that will probably be altered in chronic pain patients can facilitate spinal events (Hunt and Mantyh 2001, Rahman et al 2003). The ability not only to image pain but also to examine drug effects in humans will be a powerful tool for understanding the ways in which analgesics alter central nervous system (CNS) function (Rogers et al 2004).

These different projection pathways tend to emanate from different spinal nociceptive neurons such that the affective pathway arises predominantly from lamina I whereas the sensory–discriminative pathway arises more from lamina V. Importantly, in animals both neuronal populations are suppressed by opioids, thus confirming that any dissociation of the emotional and sensory aspects of pain produced by opioids must arise from brain mechanisms.

Important supraspinal sites of opioid action are the midbrain and brain stem structures (i.e., the periaqueductal gray [PAG] and the rostroventral medulla ([RVM]). In fact, the first studies on opioid sites of action pointed to the brain and in particular to sites accessible from the ventricles. Exogenous injection of morphine into either of these sites causes antinociception via increased activity in the inhibitory descending controls terminating in the dorsal horn of the spinal cord (for reviews, see Yaksh 1997, Fields 2000, Heinricher 2003, Heinricher and Neubert 2004). Electrical stimulation or application of glutamate to these sites, which would activate neurons, also causes antinociception, so morphine would appear to act through disinhibition to increase outflow from both sites (see Fig. 30-3). An influential hypothesis to explain this circuitry concerns the electrophysiological identification of two major populations of RVM output neurons: ON cells, whose activity coincides with spinal nociceptive reflexes, and OFF cells, whose activity is related to suppression of these reflexes. Infusion of morphine into the RVM is associated with a pronounced reduction in the activity of ON cells and increased activity of OFF cells, thereby supporting the pro- and antinociceptive labels assigned to ON and OFF cells, respectively:

There are also important interactions between serotonin (5-HT) and nitric oxide (NO) in the PAG. NO appears to be required for the 5-HT–mediated inhibition of PAG output and reversal of antinociception (Hamalainen and Lovick 1997). The circuitry between the PAG and the RVM is complex; when combined with the ascending pathways, a feedback loop in the modulation of nociceptive information becomes apparent.

Fibers descending from the RVM to the dorsal horn of the spinal cord are mostly serotonergic, enkephalinergic, glycinergic, and GABAergic. The nucleus raphe magnus contained within the RVM and the noradrenergic nuclei (locus coeruleus, subcoeruleus, A5 and A7 cell groups) are major PAG relays for noradrenergic and serotonergic descending pathways, respectively, to the dorsal horn (Kwiat and Basbaum 1992). Rather than the RVM being a homogeneous population of serotonergic neurons, GABA- (and glycine)-releasing neurons are now thought to constitute a significant proportion of spinally projecting RVM fibers (Antal et al 1996). The pharmacology of noradrenergic and serotonergic modulation in the dorsal horn is complex, but opioids can also interact with noradrenergic mechanisms, and many studies have shown that the effector mechanism and the location of the major noradrenaline target receptor—the α₂ adrenoceptor—are very similar to those of opioid receptors.

In this context, tramadol, a dual serotonin and noradrenaline uptake inhibitor yet a weak opioid, has been succeeded by the new drug tapentadol, which produces analgesia through a dual mechanism of action within a single molecule. Tapentadol has quite low affinity for the mu opioid receptor, where it acts as an agonist, but the same molecule also acts as a noradrenaline reuptake inhibitor and thus increases levels of the transmitter, which in turn leads to analgesia through activation of the inhibitory α₂ adrenoceptors. This novel dual central mechanism of action been termed MOR-NRI (Tzschentke et al 2007). There are a number of preclinical studies on its mechanisms, as well as phase II/III clinical studies, and the drug is a powerful analgesic, presumably because of these dual actions.

Central Side Effects

The large numbers of opioid receptors in regions such as the solitary tract and adjacent areas are probably related to the respiratory effects of opiates: cough suppression and nausea and vomiting.

Opiates acting in the brain stem reduce the sensitivity of the respiratory centers to PCO₂, and this is the most common cause of death from overdose with the street use of opiates.

Opiates activate the chemoreceptor trigger zone in the medulla to cause nausea and vomiting; cough suppression also occurs because of the inhibitory effects of opiates on brain stem nuclei in the cough reflex pathway. Dextromethorphan is the non-opiate isomer of levorphanol and is an effective cough suppressant.

Actions in the monoamine nuclei (such as the well-demonstrated effects of opioids on noradrenergic transmission in the locus coeruleus and enhancement of dopamine release in the ventral tegmental area) are likely to be associated with reward processes and therefore relate to dependence. Psychological dependence does not seem to occur to any great extent in the presence of pain.

The relative extent of the unwanted effects caused by selective agonists at the different opioid receptors is of great importance in determining whether non-mu opioids will have better spectra of action than morphine. However, there are good indications that the delta and kappa receptor agonists cause less respiratory depression than mu and that prolonged protection of the enkephalins by peptidase inhibitors has no dependence liability (Le Guen et al 2003).

Peripheral Side Effects

A number of side effects of opiates are due to their actions on opiate receptors outside the CNS. Opiates constrict the pupils by acting on the oculomotor nucleus and cause constipation by inducing maintained contraction of the smooth muscle of the gut, which reduces motility. This diminished propulsion, coupled with opiates reducing secretion in the gut, underlies its antidiarrheal effect. Opiates contract sphincters throughout the gastrointestinal tract. Although these effects are predominantly peripheral actions, there are central contributions as well. Morphine can also release histamine from mast cells, which can produce irritation and bronchospasm in extreme cases. Opiates have minimal cardiovascular effects at therapeutic doses.

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