Following the cloning of the classical opioid receptors, the G-protein coupled opiate receptor-like protein (ORL1 or NOP) was cloned based on its structural homology (48-49% identity) to other members of the opioid receptor family; it has an endogenous ligand, nociceptin/orphanin (FQ: N/OFQ) (Stevens, 2009). See Figure 18–2. Since this system does not display an opioid pharmacology (Fioravanti et al., 2008), it will not be discussed in detail in this chapter.
N/OFQ binding sites have been found to be largely in the CNS and to be densely distributed in cortical regions, ventral forebrain, hippocampus, brainstem, and spinal cord, as well as in a number of peripheral cells including basophils, endothelial cells, and macrophages.
Opiate Receptor Subtypes
The existence of three classes of opiate receptors (MOR, DOR, and KOR) is widely accepted. The opioid receptors appear early in vertebrate evolution, being already present with the appearance of jawed vertebrates. The human opiate receptors have been mapped to chromosome 1p355-33 (DOR), chromosome 8q11.23-21 (KOR), chromosome 6q25-26 (MOR), and chromosome 20q13.33 (NOR) (Dreborg et al., 2008). Low-stringency hybridization procedures have identified no opioid receptor types other than the cloned opioid receptors. Nevertheless, pharmacological studies have suggested the possible existence of at least two subtypes of each receptor. While cloning studies have not supported the existence of these subtypes as distinct classes, the modified specificity for opioid ligands may result from several underlying events.
Heterodimerization. In the membrane, opiate receptors can form both homo- and heterodimers. Dimerization can alter the pharmacological properties of the respective receptors. For example, DORs form heterodimers with both MORs and KORs. Thus, MOR-DOR and DOR-KOR heterodimers show less affinity for highly selective agonists, reduced agonist-induced receptor trafficking, and mutual synergy between receptor-selective agonists in binding to the respective agonists and also in the agonist-induced intracellular signaling (Gupta et al., 2006).
Alternative Splicing of Receptor RNA
Alternative splicing of receptor heteronuclear RNA (e.g., exon skipping and intron retention) is thought to play an important role in producing in vivo diversity within many members of the GPCR superfamily (Chapter 3). Splice variants exist within each of the three opioid receptor families, and this alternative splicing of receptor transcripts may be crucial for the diversity of opioid receptors. As an example, the human Oprm gene has at least two promoters, multiple exons, with many exons generating at least 11 splice variants that encode multiple morphine-binding isoforms, varying largely at their carboxy (intracellular) terminus. Given the functional importance of the intracellular components of the GPCRs, it is not surprising that significant differences exist for the receptor isoforms in terms of agonist-induced G protein activation and receptor internalization. Importantly, these splice variants have been found expressed in vivo (Pan, 2005).
Receptor Subtype Agonists/Antagonists
Studies on the presence and function of these receptors have depended upon the convergence between agonist/antagonist structure-activity studies and a variety of functional assays in biological and cloned receptor systems. Highly selective agonists have been developed that show specific affinity for the respective binding site and G-protein coupled reporter activation in cloned expression systems (e.g., DAMGO for MOR, DPDPE for DOR, and U-50,488, and U-69,593 for KOR) (Table 18–1). The study of the biological functions of opioid receptors in vivo has been aided by synthesis of selective antagonists. Among the most commonly used antagonists are cyclic analogs of somatostatin such as CTOP as a MOR antagonist, a derivative of naloxone called naltrindole as a DOR-receptor antagonist, and a bivalent derivative of naltrexone called nor-binaltorphimine (nor-BNI) as a KOR antagonist. These tools have made possible the characterization of the distribution of binding which, in conjunction with immunohistochemistry using antibodies derived from cloned receptors, has served to define the anatomical distribution of the receptors and the roles of the respective receptors in biological functions. Positron emission tomography (PET) has been used to characterize in vivo binding in brain with selective isotopically labeled ligands (Henriksen and Willoch, 2008). In vivo delivery of selective antagonists and agonists into specific brain regions has established the receptor types and anatomical distribution involved in mediating various opioid effects (see below).
Each receptor consists of an extracellular N-terminus, seven transmembrane helices, three extra- and intracellular loops, and an intracellular C-terminus characteristic of the GPCRs. The opioid receptors also possess two conserved cysteine residues in the first and second extracellular loops, which form a disulfide bridge.
Structural Correlates of Binding/Coupling Requirements for Opiate Ligands
Opiate Receptor Structures. Studies of chimeric receptors and site-directed mutagenesis of cloned receptors have provided definitive insights into structural determinants of opioid ligand–receptor interaction. Though there is significant complexity (Kane et al., 2006), several general principles define binding and selectivity. First, all opioid receptors display a binding pocket formed by TM3-TM7. Second, the pocket in the respective receptor is partially covered by the extracellular loops, which together with the extracellular termini of the TM segments, provide a gate conferring selectivity, allowing ligands, particularly peptides, to be differentially accessible to the different receptor types. Thus, alkaloids (e.g., morphine) bind in the core of the transmembrane portion of the receptor, whereas large peptidyl ligands bind at the extracellular loops. As noted, it is the extracellular loops that show the greatest structural diversity across receptors. Third, selectivity has been attributed to extracellular loops: first and third for the MOR, second for the KOR, and third for the DOR (Waldhoer et al., 2004). Alkaloid antagonists are thought to bind deeper into the pocket sterically hindering conformational changes leading to a functional antagonism.
Structure-Activity Relationships. Receptor selectivity by the various opiate agonists is commonly explained in terms of the "message-address" concept (Takemori and Portoghese, 1992). Thus, elements shared by all structures (reflecting agents that bind at all sites, such as naltrexone) represent the "message," while elements associated with a ligand binding at a specific receptor represent the structural "address." The common structural features constituting the message are:
To this "message" are added a variable "linker" region and the "address" that specifies opiate receptor selectivity (Figure 18–3). Refinements of this message-linker-address model have led to the synthesis of new compounds with the predicted specificity. For the KOR and DOR, elements constituting the address have been defined. Thus, for the KOR, a second basic hydrophobic group is implicated in forming a specific salt bridge; for the DOR, a hydrophobic group such as an indole forms the address. MOR ligands such as morphine lack a common chemical moiety and thus other elements are thought to contribute to ligand specificity at that receptor (Kane et al., 2006).
Opiate Receptor Coupling to Membrane Function
Agonist binding results in conformational changes in the GPCR, initiating the G protein activation/inactivation cycle (Chapter 3). The μ, δ, and κ receptors largely couple through pertussis toxin-sensitive, Gi/Go proteins (but occasionally to Gs or Gz). Upon receptor activation, the Gi/Go coupling results in a large number of intracellular events, including:
Inhibition of adenylyl cyclase activity
Reduced opening of voltage-gated Ca2+ channels
Stimulation of K+ current though several channels including G protein-activated inwardly rectifying K+ channels (GIRKs)
Activation of PKC and PLCβ
As with other GPCRs, the second intracellular loop is involved in the efficacy of G-protein activation while the third loop defines the α subunit that is activated (Gether, 2000).
Regulation of Opiate Receptor Disposition
Like other GPCRs, MOR and DORs can undergo rapid agonist-mediated internalization via a classic endocytic, β-arrestin-mediated pathway, whereas KORs do not internalize after prolonged agonist exposure (Chu et al., 1997). Internalization of the MOR and DORs apparently occurs via partially distinct endocytic pathways, suggesting receptor-specific interactions with different mediators of intracellular trafficking. These processes may be induced differentially as a function of the structure of the ligand. For example, certain agonists, such as etorphine and enkephalins, cause rapid internalization of the receptor, whereas morphine does not cause MOR internalization, even though it decreases adenylyl cyclase activity equally well. In addition, a truncated receptor with normal G-protein coupling recycles constitutively from the membrane to the cytosol (Segredo et al., 1997), suggesting that activation of signal transduction and internalization are controlled by distinct molecular mechanisms. These studies also support the hypothesis that different ligands induce different conformational changes in the receptor that result in divergent intracellular events, and they may provide an explanation for differences in the spectrum of effects of various opioids.
Functional Consequences of Acute and Chronic Opiate Receptor Activation
The loss of effect with exposure to opiates occurs over short- and long-term intervals.
Desensitization. Acute agonist occupancy of the opiate receptors results in activation of the intracellular signaling outlined previously. In the face of a transient activation (minutes to hours), a phenomenon called acute tolerance or desensitization can be observed that is specific for that receptor and disappears with a time course parallel to the clearance of the agonist. Short-term desensitization probably involves phosphorylation of the receptors resulting in an uncoupling of the receptor from its G-protein and/or internalization of the receptor.
Tolerance. Sustained administration of an opiate agonist (days to weeks) leads to progressive loss of drug effect. Here tolerance refers to a decrease in the apparent effectiveness of a drug with continuous or repeated agonist administration, and with the removal of the agonist disappears over several weeks. This tolerance is reflected by a reduction in the maximum achievable effect or a right shift in the dose-effect curve. This phenomenon can be manifested at the level of the intracellular cascade (e.g., reduced inhibition of adenylyl cyclase) and at the organ system level (e.g., loss of sedative and analgesic effects).
This loss of effect with persistent exposure to an opiate agonist has several key properties:
Changes in response are time-dependent, with changes occurring over short-term (minutes to hours, as with desensitization) and long-term intervals (weeks to months).
Tolerance to drug effect is surmountable with higher doses of the opioid.
Tolerance is reversible over time after termination of the drug.
Different physiological responses develop tolerance at different rates. Thus, at the organ system level, some end points show little or no tolerance development (pupillary miosis), some show moderate tolerance (constipation, emesis, analgesia, sedation), and some show rapid tolerance (euphorogenic).
In general, opiate agonists of a given class will typically show a reduced response in a system rendered tolerant to another agent of that class (e.g., cross-tolerance between the μ agonists, such as morphine and fentanyl).
The completeness of this cross-tolerance is not consistent and forms the basis for the switching between opioid drugs used in clinical therapy. This incomplete cross-tolerance has been hypothesized to reflect small but important differences in the receptors with which the different opiates of the same class bind (Pasternak, 2005).
Dependence. During the state of tolerance, the phenomenon of dependence is observed. Dependence represents a state of adaptation manifested by receptor/drug class-specific withdrawal syndrome produced by cessation of drug exposure (e.g., by drug abstinence) or administration of an antagonist (e.g., naloxone). The withdrawal is manifested by the exaggerated appearance of enhanced signs of cellular activation. In the CNS, increased adenylyl cyclase, release of excitatory amino acids and cytokines, activation of microglia and astrocytes, and the initiation of apoptotic processes have been reported. Such indices of hyperexcitability are also noted in peripheral plexi such as are present in the GI tract and in autonomic ganglia (discussed later). At the organ system level, withdrawal is manifested by significant somatomotor and autonomic outflow (reflected by agitation, hyperalgesia, hyperthermia, hypertension, diarrhea, pupillary dilation, and release of virtually all pituitary and adrenomedullary hormones) and by affective symptoms (dysphoria, anxiety, and depression) (Kreek et al., 1998). These phenomena are considered to be highly aversive and motivate the drug recipient to make robust efforts to avoid the withdrawal state. Consistent with the receptor selectivity of the effects, the withdrawal signs observed in an animal tolerant to a given opiate can be suppressed by application of another drug from the same class.
Addiction. Addiction is a behavioral pattern characterized by compulsive use of a drug and overwhelming involvement with its procurement and use. The positive, rewarding effects of opiates are considered to be the driving component for initiating the recreational use of opiates. This positive reward property in humans and animals is subject to the development of tolerance. Given the aversive nature of withdrawal symptoms, in the dependent organism, it is not surprising that avoidance and alleviation of withdrawal symptoms may become a primary motivation for compulsive drug taking (Kreek and Koob, 1998). When the drive to acquire the drug leads to drug-seeking behaviors that occur in spite of the physical, emotional, or societal damage suffered by the drug seeker, then the obsession or compulsion to acquire and use the drug is considered to reflect an addicted state. In animals, this may be manifest by willingness to tolerate very stressful conditions to acquire drug delivery. In humans, aberrant behaviors considered to be signs of addiction include prescription forgery, stealing drugs from others, and obtaining prescription drugs from nonmedical sources; these behaviors are considered indicators of an addiction disorder. Note that drug dependence is not synonymous with drug addiction. Any individual who is exposed for some period to opiates will display some degree of tolerance and should the drug be removed abruptly, there will be an expression of withdrawal signs, the severity of which will depend upon the dose and duration of drug exposure. Such a condition does not itself indicate an addicted state. Thus, tolerance and dependence are physiological responses seen in all patients but are not predictors of addiction (Chapter 24). For example, cancer pain often requires prolonged treatment with high doses of opioids, leading to tolerance and dependence. Yet abuse in this setting is considered to be unusual (Foley, 1993).
Mechanisms of Tolerance/Dependence-Withdrawal
The mechanisms underlying chronic tolerance and dependence/ withdrawal are controversial. Several types of events are considered to contribute.
Receptor Disposition. With chronic opiate exposure, the general consensus is that the loss of effect is not related to the density of membrane receptors. As noted, acute desensitization or receptor internalization may play a role in the initiation of chronic tolerance but is not sufficient to explain persistent changes observed with chronic exposure. Thus, morphine, unlike other μ agonists, does not promote μ receptor internalization or receptor phosphorylation and desensitization (Koch et al., 2005; von Zastrow et al., 2003). These studies suggest that receptor desensitization and down-regulation are agonist specific. Other studies of GPCRs indicate that endocytosis and sequestration of receptors do not invariably lead to receptor degradation but can also result in receptor dephosphorylation and recycling to the surface of the cell (Krupnick and Benovic, 1998). Accordingly, opioid tolerance may not be related to receptor desensitization but rather to a lack of desensitization. Agonists that rapidly internalize opioid receptors also would rapidly desensitize signaling, but this desensitization would be at least partially reset by recycling of "reactivated" opioid receptors. The lack of desensitization caused by morphine may result in prolonged receptor signaling, which even though less efficient than that observed with other agonists, would lead to further downstream cellular adaptations that increase tolerance development. The measurement of relative agonist signaling versus endocytosis (RAVE) for opioid agonists could be predictive of the potential for tolerance development (Waldhoer et al., 2004).
Adaptation of Intracellular Signaling Mechanisms in the Opioid Receptor-Bearing Neurons. Coupling of MOR to cellular effectors, such as inhibition of adenylyl cyclase, activation of inwardly rectifying K+ channels, inhibition of Ca2+ currents, and inhibition of terminal release of transmitters demonstrates functional uncoupling of receptor occupancy from effector function (Williams et al., 2001). Importantly, the chronic opioid effect initiates adaptive counterregulatory change. The best example of such cellular counterregulatory processes is the rebound increase in cellular cyclic AMP levels produced by "superactivation" of adenylyl cyclase and up-regulation of the amount of enzyme. Such superactivation leads to an induction of an excitatory state mediated by an increased cation current through activation of PKA. Terminal transmitter release is thus commonly enhanced during opioid withdrawal (Bailey and Connor, 2005).
System Level Counteradaptation. In the face of chronic opiate exposure, an evident loss of drug effect is noted. An important line of speculation is that the apparent loss of inhibitory effect may reflect an enhanced excitability of the regulated link. Thus, tolerance to the analgesic action of chronically administered μ opiates may result in an activation of bulbospinal pathways that increases the excitability of spinal dorsal horn pain transmission linkages. Similarly, in the face of chronic opiate exposure, opiate receptor occupancy will lead to the activation of PKC, which can phosphorylate and, accordingly, enhance the activation of local glutamate receptors of the N-methyl-D-aspartate (NMDA) type (Chapter 14). These receptors are known to mediate a facilitated state leading to enhanced spinal pain processing. Blocking of these receptors can at least partially attenuate the loss of analgesic efficacy with continued opiate exposure (Trujillo and Akil, 1991). These system level counteradaptation hypotheses may represent mechanisms that apply to specific systems (e.g., pain modulation) but not necessarily to others (e.g., sedation or miosis) (Christie, 2008).
Differential Tolerance Development and Fractional Occupancy Requirements. An interesting problem posed in explaining tolerance relates to the differential rates of tolerance development noted earlier. Why responses such as miosis show no tolerance over extended exposure (indeed, it is considered symptomatic in drug overdose of highly tolerant patients) while analgesia and sedation are more likely to show a reduction is not clear. One possibility is that tolerance represents a functional uncoupling of some fraction of the receptor population and that different physiological end points may require activation of different fractions of their coupled receptors to produce a given physiological effect. Accordingly, it would be consistent that receptors in the miosis pathways need to activate a small fraction of their receptors, relative to those systems mediating pain control to produce a significant physiological action (e.g., the systems mediating miosis have a greater functional receptor reserve).