Opioids Receptors and Classification
The mu receptors are a class of receptors that neuromodulate different physiological functions, but above all, nociception but also stress, temperature, respiration, endocrine activity, gastrointestinal activity, memory, mood, and motivation. Because these receptors bind opioids, they are also commonly referred to as mu-opioid receptors (MORs). However, opioid receptors are a very large family of receptors that includes in addition to MORs also delta-opioid receptors (DORs), kappa opioid receptors (KORs), and nociceptin receptor (NOR) also referred to as opioid-receptor-like receptor 1 (ORL1) which seems to have a critical role in the development of tolerance to mu-opioid agonists used as analgesics. Also, there are some other opioid receptors such as the zeta, the epsilon, the lambda, and the iota opioid receptors. Sigma receptors are no longer considered to be opioid receptors as the opioid antagonist naloxone does not reverse their activation. Although according to the International Union of Basic and Clinical Pharmacology (IUPHAR) recommendation the appropriate terminology for the three classical opioid receptors and the nociceptin receptor should be MOP ("Mu OPioid receptor"), DOP, KOP and NOP respectively, in this chapter we will refer to the acronym MOR for indicating mu-opioid receptors as it is the most used in the scientific literature.
Opioid receptors are part of the G protein-coupled receptors (GPCRs) family. Crystallographic studies have allowed researchers to characterize this important superfamily of receptors that control different aspects of cellular function and are implicated in a vast number of neurotransmitter processes. Their basic structure consists of a single polypeptide chain that crosses the cell membrane seven times (seven-transmembrane domain receptors), has an N-terminal extracellular domain of variable length, and a C-terminal intracellular domain, and interacts with heterotrimeric G proteins. GPCRs divide into three distinct families (type A, B, and C) that share the same heptahelical structure but differ in various aspects, mainly due to the length of the N-terminal sequence and the location of the binding site for the agonist. The connection between the receptor and the first stage of signal transduction becomes established through the heterotrimeric (alpha, beta, and gamma subunits) G proteins. The main targets of G proteins through which GPCRs work are the adenyl cyclase that is the enzyme responsible for the formation of the second messenger (intracellular signal transduction) cyclic adenosine monophosphate (cAMP); the phospholipase C that is the enzyme responsible for the formation of inositol triphosphate and diacylglycerol; and several ion channels such as the calcium and potassium channels. According to this last mechanism, GPCRs can directly control the activity of ion channels through mechanisms that do not involve the second messengers (e.g., cAMP). Opioids, for example, reduce neuronal excitability through the opening of the G protein-dependent inward rectifying potassium (irk) channels (GIRK) and subsequent cell membrane hyperpolarization. The opening of the channel occurs by the direct interaction between the subunits (beta-gamma complex from the inactive heterotrimeric G protein complexes G-alpha-beta-gamma) of the G protein and the potassium ion channel. Several GIRK subtypes have been isolated, such as the GIRK1 to GIRK3 types (distributed broadly in the brain), and GIRK4 found primarily in the heart. Interestingly, this type of channel is highly studied as it could be a target for new drugs.
Endogenous and exogenous opioids operate through both inhibitory and excitatory action at the presynaptic and postsynaptic sites. In particular, the MORs interact with a G protein of the inhibitory type, the G-alpha-iota/o class of adenylate cyclase inhibitory G-alpha proteins. Based on the structure of the alpha subunit, there are the G-alpha-iota forms (G-alpha-iota1, 2, and 3), G-alpha-omicron types (A and B), and G-alpha-zeta type. On the other hand, the beta-gamma heterodimer forms from one of the five different betas and one of the twelve different gamma subtypes. In the resting state, there is a G-alpha-beta-gamma complex, and the subunit α binds guanosine diphosphate (GDP). The binding of the opioid agonist (endogenous or exogenous) to the extracellular N-terminus domain of the MOR induces dissociation of GDP from the G-alpha subunit, which is replaced by guanosine triphosphate (GTP), and subsequent dissociation of the G-alpha-GTP from the beta-gamma heterodimer. The now active G-alpha-GTP and beta-gamma subunit complex interact with different intracellular signaling pathways such as the phospholipase C and the mitogen-activated protein kinase (MAPK) pathway, as well as irk-mediated hyperpolarization mechanisms and calcium channels processes. The intracellular signal ends with the action of the GTPase, which hydrolyzes the G-alpha bound GTP to GDP. G-alpha-GDP is not able to activate effector proteins and re-associates with the beta-gamma heterodimer to restore the inactive GDP-bound heterotrimer. Because the enzymatic GTP turnover lasts for approximately 2 to 5 minutes, a new signal may find the receptor still not ready to respond. However, the regulator of G-protein signaling (RGS) protein speed up the GTP hydrolysis up to 100-fold. This protein binds the G-alpha subunit and removes the active G-alpha-GTP and beta-gamma species. In turn, RGS works as a negative regulator of GPCR signaling. RGSs are a family of proteins and represent another interesting perspective for targeted therapy as their specific pharmacological inhibitors could potentiate opioids effects.
MOR subtypes and tissue expression
There are several subtypes of MOR, which are splice variant forms. These variant forms were designated MOR-1A through MOR-1X; some of the variants express truncated forms of the receptor. The B, C, and D variants differ in the amino acid composition at the C-terminus. All variants get transcribed from a single gene (OPRM1 gene, chromosomal location 6q24-q25). Because different variants have undergone isolation in both human and invertebrate tissues, these subtypes get conserved during evolution. Research has identified several single nucleotide polymorphisms in the human receptor. For instance, the variant receptor Ser268 -> Pro possesses an important reduction in coupling efficiency and is less desensitized upon agonist exposure.
MORs are present in the central nervous system (CNS) and represent the most highly expressed of all the opioid receptors. These receptors get expressed in neurons throughout the dorsal horn of the spinal cord and in different brain regions (mainly somatosensorial cerebral cortex) involved in processing nociceptive information. In particular, in the spinal cord, MORs are localized (presynaptic and postsynaptic) into the substantia gelatinosa of Rolando (laminae I and II), which receives sensory information from primary afferent nerve fibers innervating the skin and deeper tissues of the body. Presynaptic MORs activation inhibits the release of excitatory neurotransmitters (e.g., substance P and glutamate), whereas the postsynaptic binding to MORs involves direct hyperpolarization of postsynaptic neurons and, in turn, inhibition of the afferent neural transmission of the painful information, and other types of information. Apart from the somatosensory system, MORs are localized in the extrapyramidal system and the limbic system including the limbic lobe, orbitofrontal cortex (involved in the process of decision-making), piriform cortex, entorhinal cortex (memory and associative functions), hippocampus (opioid-induced consolidation of new memories by increasing LTP in CA3 neurons), fornix, septal nuclei, amygdala (emotional processes), nucleus accumbens involved in reward, pleasure, and addiction, diencephalic structures such as hypothalamus that regulates many autonomic processes, mammillary bodies. Immunohistochemistry, in situ hybridization, and radioligand binding, also demonstrated that MORs get distributed in the mesencephalon (ventral tegmental area, interpeduncular nucleus, pars reticulata of the substantia nigra, superior colliculus), pons (locus coeruleus), thalamus, and caudate-putamen. MORs are also localized in gastrointestinal tract where are responsible for the opioid-induced constipation effect; pupil (miosis); and in the immune cells (e.g., CEM x174 T/B lymphocytes, Raji B cells, CD4+, monocytes/macrophages, neutrophils) where, for instance, regulate interleukin-4 activity in T lymphocytes and modulate macrophage phagocytosis and macrophage secretion of TNF-alpha. Numerous preclinic studies have taken place investigating the effects of opioids on cancer growth and progression.
Endogenous opioids are the natural ligands of opioid receptors that play a role in neurotransmission, pain modulation, and other homeostatic and functional pathways of the brain and peripherally. Beta-endorphin serves as an agonist for MORs and less for DORs. This peptide is derived from a larger precursor peptide, proopiomelanocortin (POMC), and is secreted by the arcuate nucleus of the hypothalamus (via the anterior lobe of the pituitary gland) during stress and exercise, inducing euphoria, inhibition of postexercise pain, and muscular fatigue, and stimulating glucose uptake. Moreover, because beta-endorphin exerts a tonic inhibitory influence upon the gonadotropin-releasing hormone (GnRH) secretion, it is involved in the regulation of the reproductive function. Other endogenous opioids are the enkephalins that bind mainly the DORs and less the MORs, whereas the dynorphins bind mainly the KORs. Enkephalins are short (5-amino-acid) polypeptides, including met-enkephalin (YGGFM), and Leu-enkephalin (YGGFL). These pentapeptides are generated from a precursor protein called proenkephalin and are found primarily in the amygdala, brainstem, dorsal horn of the spinal cord, adrenal medulla, and other peripheral tissues. Again, dynorphins include dynorphin A (17 amino-acids of which the first five are Leu-enkephalin), dynorphin B (rimorphin), and dynorphin 1-8. They get secreted in the hippocampus, amygdala, hypothalamus, striatum, and spinal cord and are involved in numerous functions related to learning and memory, emotional control, stress response, and pain. The effects of both endogenous and exogenous opioids are characteristically reversed by naloxone.
Drugs that activate MORs are useful for their pharmacological benefit in providing pain relief. These agents (i.e., opioid drugs) include the so-called weak opioids codeine and tramadol, and the strong opioids oxycodone, morphine, hydromorphone, meperidine, tapentadol, methadone, fentanyl, sufentanil, remifentanil. Experiments conducted on MORs knockout mice proved that in addition to the pain-relieving effects, the binding of opioids to MORs could also induce various effects in multiple organ systems. These effects can assume the shape of side effects and are associated with acute and chronic opioids use. Acute effects include but are not limited to depressed respiration, slowing of the gastrointestinal motility, nausea, vomiting, constipation, dizziness, itch, cough suppression, miosis, hallucinations, dysphoria, and sedation. Furthermore, the chronic use of opioids induces continued activation of the MORs-related signaling pathways (G protein signaling) and can lead to homeostatic changes, e.g., tolerance, hyperalgesia, and physical dependence. Again, MORs mediate opioid rewarding and euphoric effects. Therefore, the misuse and/or abuse of prescribed opioid drugs after an initial therapeutic use or in patients that self-medicate led to the opioid crisis that broke out in North America in the early years of the 2000s. In 2016, more than 20,000 deaths in the United States (US) resulted from an overdose of prescription opioids, and another 13,000 deaths resulted from heroin overdose. As a consequence, epidemiological data indicate that drug overdoses are the leading cause of death in US adults under age 50, and opioids account for more than half of all drug overdose deaths.