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Targeting G protein-coupled receptors for the treatment of chronic pain in the digestive system
  1. Lena Gottesman-Katz1,2,
  2. Rocco Latorre1,
  3. Stephen Vanner3,
  4. Brian L Schmidt4,
  5. Nigel W Bunnett1
  1. 1 Molecular Pathobiology, New York University, New York, New York, USA
  2. 2 Division of Pediatric Gastroenterology, Columbia University Medical Center/New York Presbyterian, New York, New York, USA
  3. 3 Gastrointestinal Diseases Research Unit, Division of Gastroenterology, Queens University, Kingston, Ontario, Canada
  4. 4 Bluestone Center, New York University, New York, New York, USA
  1. Correspondence to Professor Nigel W Bunnett, Molecular Pathobiology, New York University, New York, NY 10010, USA; nwb2{at}


Chronic pain is a hallmark of functional disorders, inflammatory diseases and cancer of the digestive system. The mechanisms that initiate and sustain chronic pain are incompletely understood, and available therapies are inadequate. This review highlights recent advances in the structure and function of pronociceptive and antinociceptive G protein-coupled receptors (GPCRs) that provide insights into the mechanisms and treatment of chronic pain. This knowledge, derived from studies of somatic pain, can guide research into visceral pain. Mediators from injured tissues transiently activate GPCRs at the plasma membrane of neurons, leading to sensitisation of ion channels and acute hyperexcitability and nociception. Sustained agonist release evokes GPCR redistribution to endosomes, where persistent signalling regulates activity of channels and genes that control chronic hyperexcitability and nociception. Endosomally targeted GPCR antagonists provide superior pain relief in preclinical models. Biased agonists stabilise GPCR conformations that favour signalling of beneficial actions at the expense of detrimental side effects. Biased agonists of µ-opioid receptors (MOPrs) can provide analgesia without addiction, respiratory depression and constipation. Opioids that preferentially bind to MOPrs in the acidic microenvironment of diseased tissues produce analgesia without side effects. Allosteric modulators of GPCRs fine-tune actions of endogenous ligands, offering the prospect of refined pain control. GPCR dimers might function as distinct therapeutic targets for nociception. The discovery that GPCRs that control itch also mediate irritant sensation in the colon has revealed new targets. A deeper understanding of GPCR structure and function in different microenvironments offers the potential of developing superior treatments for GI pain.

  • abdominal pain
  • cell signalling
  • neurobiology
  • neurogastroenterology
  • receptor characterisation

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Key messages

Chronification of pain in the digestive system

  • Chronic pain in the digestive system accompanies functional disorders, inflammatory diseases and cancer. It afflicts one quarter of the US population.

  • The transition from acute (physiological) to chronic (pathological) pain in the digestive system is a poorly understood and complex process. Chronic hypersensitivity of pain-sensing nerves innervating the digestive system manifests as allodynia and hyperalgesia, hallmarks of chronic abdominal pain.

  • There is a dearth of long-term, effective treatment options for chronic pain in the digestive system. The opioid crisis, a leading cause of mortality, highlights the need to understand the aetiology of chronic pain and to develop more effective treatments.

G protein-coupled receptors (GPCRs) are complex and dynamic signalling proteins that control chronic pain

  • GPCRs are the largest family of receptors. They regulate most physiological and pathological processes, including pain.

  • GPCRs expressed by primary sensory neurons and second-order spinal neurons can stimulate and inhibit pain transmission.

  • New knowledge about GPCR structure and function has revealed that GPCRs are complex and dynamic signalling proteins. Once activated, GPCRs translocate to subcellular microdomains and adopt distinct conformations. An understanding of this dynamic behaviour provides insights into how GPCRs control pain and has revealed new opportunities for therapy.

Key messages

Insights into the dynamic properties of GPCRs reveals new therapeutic options for chronic pain

  • GPCRs are the largest class of drug targets. One-third of Federal Drug Administration-approved drugs target GPCRs.

  • The discovery that activated GPCRs internalise and that GPCRs in endosomes can generate persistent signals that contribute to neuronal excitation and pain transmission raises the possibility that GPCRs in endosomes are an appropriate target for treating chronic pain.

  • Antagonists of neuropeptide and protease receptors conjugated to transmembrane lipids or encapsulated into nanoparticles target GPCRs in endosomes and provide superior pain relief in preclinical models.

  • Biased agonists of GPCRs might stabilise receptor conformations that favour signalling of beneficial pathways. Biased agonists of µ-opioid receptors (MOPrs) have the potential to provide analgesia without addiction, respiratory depression, nausea and constipation.

  • Molecular modelling has facilitated the development of opioids that preferentially activate MOPrs in the acid extracellular fluid of diseased tissues (eg, cancer and inflammation). These agonists provide analgesia in preclinical models of pain without side effects.

  • By altering the conformation of GPCRs, allosteric modulators can fine-tune the effects of endogenous ligands. Positive allosteric modulators offer the prospect of enhancing the analgesic properties of endogenous opioids for pain control.

  • The discovery that GPCR dimers with distinct pharmacological properties may control pain transmission has revealed new therapeutic targets. Drugs that target dimers of MOPrs and δ-opioid receptors have been advanced to treat pain.

  • GPCRs expressed by sensory nerves can transmit distinct sensations in different tissues. The discovery that GPCRs that mediate itch in the skin also represent a mechanism of irritant sensation in the colon has led to the identification of new targets for chronic pain in the digestive system.

  • Knowledge of the mechanisms and treatment of chronic pain largely derives form studies of somatic pain in preclinical models. There are opportunities to apply this information to chronic pain in the digestive system. Challenges for future research into chronic pain in the digestive system lie in identification of the fundamental processes that control sensitivity of neurons that sense and transmit painful signals in humans.


Acute pain is a physiological mechanism of protection that allows the awareness and avoidance of injury. Its importance is illustrated by the severe injuries sustained by individuals with a congenital insensitivity to detect pain.1 Chronic pain, defined as lasting greater than 3 months, can persist after healing, is often debilitating and afflicts a significant portion of the world’s population.2 Chronic GI pain is a common type of visceral pain that can arise due to multiple underlying aetiologies and, like most chronic pain, is often difficult to treat.3 The mechanisms that underlie the transition from acute to chronic pain are complex, multifactorial and not fully elucidated. Consequently, there are a dearth of effective treatments for chronic pain. Existing and widely used therapies, including non-steroidal anti-inflammatory drugs and opioids, are often ineffective and have severe and sometimes life-threatening side effects.4 The large number of deaths attributable to opioid overdoses highlights the need for new treatments.5 The neuronal pathway of pain transmission from the digestive tract is well established.6 Primary sensory neurons, with cell bodies within dorsal root ganglia (DRGs), project fibres to the digestive system and dorsal horn of the spinal cord. A subset of these neurons, called nociceptors, detect painful stimuli that are transmitted centrally via second-order neurons in the dorsal horn. Gut afferent nerves synapse at multiple levels in the spinal cord, which accounts for the poorly localised nature of visceral pain.7

Multiple mechanisms drive chronic visceral pain, many of which have garnered significant research attention. This review concerns one such mechanism, the role of G protein-coupled receptors (GPCRs) in chronic visceral pain. With almost 850 members in the human genome, GPCRs are the largest and most functionally diverse family of receptors. GPCRs are characterised by seven transmembrane domains, an extracellular amino-terminus and an intracellular carboxyl-terminus. GPCRs can both stimulate and inhibit pain transmission. Pronociceptive GPCRs (eg, substance P (SP) neurokinin-1 receptor (NK1R)) stimulate signalling molecules (eg, Ca2+ and cyclic AMP (cAMP)) that excite neurons of the pain pathway and evoke pain. In contrast, antinociceptive GPCRs (eg, opioid receptors (OPrs)) inhibit these pathways and thereby depress neuronal excitability to provide an endogenous mechanism of pain control. In light of the ubiquitous and vital roles of GPCRs in human physiology and pathology, it is not surprising that they are prime therapeutic targets; one-third of Federal Drug Administration-approved drugs target GPCRs.8 GPCRs regulate pain transmission at multiple levels and are established and emerging targets for the treatment of chronic pain.9 10 Table 1 summarises GPCRs that can promote or inhibit chronic pain in the digestive system.

Table 1

GPCRs implicated in painful digestive diseases

Recent advances in understanding the structure and function of GPCRs have provided new insights into the mechanisms that signal chronic pain and reveal novel opportunities for therapy.9 10 This review highlights the implications of these advances for understanding the mechanisms that signal chronic pain in the digestive system and discusses how this information might inform the development of more effective pharmacological treatments. Rather than discuss every GPCRs that control pain, the review focuses on select GPCRs and peripheral signalling pathways that illustrate unifying concepts. While some of these concepts have not been examined in the context of GI pain, we discuss how studies of other types of pain can guide future investigations of pain in the digestive system.

Signalling of chronic pain in the digestive system

Functional disorders, inflammatory diseases and cancer are associated with chronic pain in the digestive system. These conditions can be inter-related. Although patients with IBD usually experience acute pain during active disease, a significant subset of patients experience chronic pain during remission, often meeting criteria for IBS.11 IBS, previously considered a functional chronic pain syndrome without an overt cause, has been linked to sensitisation of colonic afferent neurons after acute inflammation or infection, as in the case of postinflammatory or postinfective IBS.12 The severe pain of pancreatitis and pancreatic cancer has inflammatory and neuropathic components.13 Mechanical stress due to obstruction of ducts, mediators from immune cells and damaged acinar cells, and injury of splanchnic nerves contribute to pancreatic pain. Pancreatic cancer cells invade the perineural space, releasing factors that sensitise nerve endings and lead to pain.14

Chronic visceral hypersensitivity (CVH) is a hallmark of chronic pain in the digestive system. CVH is caused by sensitisation of nociceptors, lowering the threshold for stimulus-evoked excitation.6 7 These changes lead to increased pain perception, which manifests as allodynia or hyperalgesia. The pathogenesis underlying the transition from acute pain to CVH is complex and incompletely understood. The sustained release of nociceptive mediators from injured tissues, recurrent inflammation and immune dysregulation, microbial dysbiosis, enteric glial cell influences, decreased antinociceptive mechanisms and epigenetic influences can all lead to sensitisation of ion channels and neuroplastic changes that contribute to CVH (figure 1).15 16 GPCRs play a major role in many of these processes.

Figure 1

Peripheral and central mechanisms of neuronal sensitisation. Many factors contribute to neuronal sensitisation and chronic visceral pain. Mediators that influence signalling pathways and induce neuronal hypersensitivity in peripheral nociceptors arise from sustained release of nociceptive mediators due to recurrent inflammation, enteric glial cells, immune cells, enteroendocrine cells and microbial dysbiosis. Endogenous opioids suppress neuronal activity. Epigenetic changes within the soma of primary afferent neurons in the dorsal root ganglia contribute to long-term sensitisation. Central mechanisms involve release of excitatory neurotransmitters and decreased inhibitory pathways.

GPCRs and pain in the digestive system

The concept that GPCRs are simple binary switches that couple to a defined set of signalling partners and regulate a single cellular process has been superseded by the realisation that GPCRs are complex and dynamic signalling proteins.9 10 Thus, GPCRs can adopt different conformations when associated with different agonists, antagonists or signalling partners, and can redistribute to distinct subcellular microdomains. This conformation and positional dynamism underlies some of the key properties of GPCRs, which depend on subcellular microdomains (eg, compartmentalised signalling and pH dependence), the nature of the ligand (eg, biased agonism and allosteric modulation), the relationship with other receptors (eg, dimerisation) and the specific tissue of action. An understanding of these complexities, which are discussed further, can provide new information about how GPCRs signal pain and can offer the prospect of developing more selective and effective agonists or antagonists for the treatment of pain.

Compartmentalised signalling of GPCRs


How can GPCRs that often activate a common set of second messengers and enzymes selectively regulate cellular responses?

The answer may lie in compartmentalised signalling, the capacity of GPCRs to interact with particular signalling partners in defined subcellular microdomains. GPCRs were once considered to function primarily at the plasma membrane, where ligands in the extracellular fluid bind to extracellular and transmembrane domains of receptors and intracellular domains couple to heterotrimeric G proteins. G protein-mediated signalling from the plasma membrane leads to activation of secondary messengers (eg, cAMP, inositol trisphosphate, diacyl glycerol and Ca2+) and subsequent activation of kinases (eg, protein kinase C (PKC), protein kinase A (PKA), phosphorylated extracellular regulated kinase) that phosphorylate and sensitise ligand-gated and voltage-gated ion channels. Ion fluxes depolarise nociceptors and trigger action potentials leading to acute nociception. However, it is now known that GPCR signalling at the plasma membrane can be transient. GPCR kinases phosphorylate activated GPCRs, which serves to increase receptor affinity for β-arrestins (β-ARRs).17 β-ARRs interdict GPCR association with G proteins and desensitise signalling at the plasma membrane.

Given the fleeting nature of plasma membrane signalling, how can GPCRs generate sustained signals that might underlie long-lasting nociception and neuronal sensitisation?

GPCR signalling in endosomes might provide an answer.18 19 In addition to desensitisation, β-ARRs mediate endocytosis and intracellular signalling. β-ARRs couple GPCRs to clathrin and adaptor protein-2, which mediate endocytosis of the receptor. Although once considered as a conduit for receptor recycling to the plasma membrane or degradation in lysosomes, endosomes are now considered important sites of GPCR signalling. Endosomal signalling is mediated by both β-ARR-dependent and G protein-dependent processes, which generate sustained intracellular signals via secondary messengers (eg, cAMP) and kinases (eg, PKC and ERK1/2). These pathways can regulate the activity of ion channels and the transcription of genes that underlie persistent neuronal sensitisation (figure 2). Endosomal signalling, as well as sensitisation of ion channels, such as transient receptor potential ion channels, has been shown to play a significant role in driving chronic visceral pain.20 21 Several GPCRs, some of which have been studied in GI pain, are known to signal from endosomes, including protease-activated receptor-2 (PAR2),22 the SP, NK1R,23 24 the µ-opioid receptor (MOPr) and δ-opioid receptor (DOPr).25 26

Figure 2

Compartmentalised signalling of GPCRs. Several GPCRs signal nociception from endosomes. (1) Noxious stimuli activate sensory afferents leading to nociceptive transmission; (2) membrane-bound GPCRs couple to heterotrimeric G proteins, which initiate intracellular signalling pathways that contribute to transient nociception; (3) GRKs phosphorylate activated GPCRs and promote the binding of βARRs, which terminate G protein-dependent signalling at the plasma membrane. βARRs facilitate clathrin-mediated receptor endocytosis by linking the GPCR to internalisation machinery. Endocytic proteins include the AP2 complex, which allows clathrin to coat and internalise cargo, and dynamin, which ligates the budding vesicle. (4) GPCRs continue to signal pain from endosomes through both β-ARRs and G-protein-dependent processes, which generate secondary messengers such as cAMP, pERK and PKC. Endosomally derived pathways contribute to sustained nociception and chronic visceral pain via regulation of transcription as well as transcriptionally independent ion channel sensitisation. AP2, adaptor protein-2; βARR, β-arrestin; cAMP, cyclic AMP; GPCR, G protein-coupled receptor; GRK, GPCR kinase; pERK, phosphorylated extracellular regulated kinase; PKC, protein kinase C.

Endosomal signalling of PAR2

PAR2 is a GPCR for serine and cysteine proteases, which are activated in disease.27 PAR2 is highly expressed in colonocytes, selectively expressed in a subset of primary sensory neurons and has been implicated in the development of visceral hypersensitivity in IBS. In humans, trypsin isoforms from the exocrine pancreas and intestinal epithelial cells, mast cell-derived tryptase and proteases produced by luminal bacteria can cleave within the extracellular amino-terminus of PAR2, exposing a tethered ligand domain that binds to and activates the cleaved receptor.28 Trypsin-3, tryptase and PAR2 are upregulated in biopsies of colonic mucosa from patients with IBS.29 30 Supernatants from patients with IBS release increased proteases, and injections of these supernatants into colons of awake mice lead to increased visceral hyperalgesia.31 PAR2-dependent mechanisms have been suggested to play a significant role in sensitisation of colonic nociceptors and colonic hyperalgesia.22 27 30 Similarly, in a model of postinfectious IBS in mice induced by the nematode parasite Trichinella spiralis elevated protease activity and PAR2 expression in the colon accompany PAR2-dependent visceral hypersensitivity.32

A recent study suggests that endosomal signalling of PAR2 underlies sustained hyperexcitability of nociceptors in colonic afferent nerves.22 Trypsin-activated PAR2 traffics to endosomes and continues to signal by β-ARR and Gαq-mediated mechanisms, leading to activation of ERK in the nucleus and cytosol and activation of PKC in the cytosol. Disruption of clathrin-mediated endocytosis of PAR2 abrogates the ability of IBS supernatants and trypsin to cause sustained hyperexcitability of nociceptors in neurons. A PAR2 antagonist conjugated to the transmembrane lipid cholestanol, which preferentially delivers the antagonist to endosomes, also curtails the sustained hyperexcitability of nociceptors.

Compartmentalised signalling of NK1R

SP belongs to the tachykinin family of neuropeptides that are prominently expressed by primary sensory and enteric neurons.33 SP interacts with the NK1R, which is localised to second order spinal neurons, as well as neurons of the myenteric and submucosal nerve plexuses of the intestine.33 34 NK1R antagonism or deletion attenuates intestinal and pancreatic nociception and inflammation in preclinical mouse models.35 36 Nociceptive and proinflammatory stimuli in the intestine, pancreas and skin induce NK1R endocytosis in spinal and enteric neurons, which is attributable to activation of nociceptors and release of SP.23 24 34 37 38 The NK1R continues to signal from endosomes by G-protein-mediated and β-ARR-mediated mechanisms that lead to activation of nuclear ERK as well as cytosolic cAMP and PKC.24 39 These signals mediate sustained SP-induced excitation of spinal neurons, gene transcription and somatic nociception. Inhibitors of clathrin-mediated and dynamin-mediated endocytosis and endosomally targeted NK1R antagonists suppress NK1R endosomal signalling, neuronal excitation and somatic nociception.24 39 This provides evidence for a major role of endosomal NK1R signalling in nociceptive transmission in the spinal cord. Whether NK1R signalling in endosomes mediates GI pain is unknown but worthy of further investigation in light of the marked redistribution of the NK1R to endosomes of spinal and enteric neurons in preclinical models of colonic and pancreatic inflammatory pain.33 34 37 38

Compartmentalised signalling of MOPr and DOPr

In addition to pronociceptive PAR2 and NK1R, antinociceptive GPCRs, exemplified by MOPr and DOPr, can also signal from endosomes to regulate the excitability of neurons in the pain pathway. MOPr and DOPr are expressed by primary sensory neurons and spinal neurons, where agonists depress excitability and thereby dampen nociceptive transmission. Despite their severe detrimental on-target side effects of respiratory depression, constipation and addiction, MOPr agonists, such as morphine, are widely used to treat pain. However, recent studies suggest that peptides and non-peptide opiate drugs can activate OPrs in distinctly different ways. A genetically encoded biosensor derived from a conformation-specific nanobody has been used to detect real-time MOPr and DOPr signalling in subcellular compartments of living neurons.26 These studies reveal that membrane impermeant opioid peptides initially activate OPrs at the plasma membrane, and that sustained signals then propagate to endosomes coincident with receptor endocytosis. Non-peptide drugs, including morphine, which are membrane permeant distort this normal pattern of plasma membrane and endosomal signalling by rapidly activating internal pools of OPrs in the Golgi apparatus. The functional relevance of this Golgi-directed signalling by opiate drugs for pain control remains to be determined.

Endosomal signalling of DOPr might represent an endogenous mechanism for controlling inflammatory pain in the colon.25 Chronic colitis is associated with an upregulation of opioids, which derive from infiltrating immune cells and activate DOPr on nociceptors to suppress excitability and nociception.40 Colonic biopsies from patients with IBD and mice with chronic colitis release opioids that activate the DOPr and suppress the excitability of mouse nociceptors.25 DOPr agonists that evoke DOPr endocytosis cause long-lasting antinociception, whereas non-internalising DOPr agonists exert only transient inhibitory effects. Inhibitors of clathrin-dependent and dynamin-dependent endocytosis block the sustained actions of opioids from colonic biopsies, supporting a role for endosomal DOPr signalling in sustained suppression of inflammatory pain in humans. Opioids also evoke DOPr endocytosis in enteric neurons, where the role of endosomal signalling remains to be defined.41

Therapeutic implications

Efforts to discover drugs that are antagonists or agonists of GPCRs have largely focused on the identification of ligands that interact with receptors at the cell surface. The discovery that GPCRs in subcellular compartments, including endosomes and the Golgi apparatus, can generate sustained signals suggests that intracellular rather than cell surface GPCRs are a more appropriate therapeutic target.19 Drugs that target GPCRs in endosomes must traverse the plasma and endosomal membranes as well as the cytosol, and be capable of engaging GPCRs in the acidic endosomal microenvironment, where receptors are likely assembled in signalling complexes comprising ligands, GPCRs, G proteins, β-ARRs and kinases. Although GPCR drugs can fail in clinical trials for many reasons, it is possible that the failure of certain drugs for pain control relates to their inability to effectively engage with GPCRs in endosomes. Several strategies have been devised to target GPCRs in endosomes for the treatment of pain (figure 3).

Figure 3

Therapeutic targeting of GPCRs in endosomes. (A) Inhibitors of clathrin, dynamin and βARRs prevent receptor endocytosis and thus inhibit endosomal nociception transmission. (B) Tripartite lipidated probes can deliver GPCR antagonists to endosomes. Tripartite probes are composed of cholestanol, which enables membrane insertion, a polyethylene glycol (PEG) linker and a GPCR antagonist cargo. Tripartite probes insert into the plasma membrane and undergo endocytosis, which delivers the cargo to GPCRs in endosomes. (C.) Nanoparticle drug delivery systems deliver and release therapies to endosomes. pH-dependent nanoparticles are composed of a hydrophobic core and a hydrophilic shell. They self-assemble in an aqueous environment, encapsulating a hydrophobic drug cargo. Nanoparticles undergo endocytosis. In the acidic endosomal environment, protonation of the hydrophobic monomer results in like–like charge repulsion, nanoparticle disassembly and cargo release. βARR, β-arrestin; GPCR, G protein-coupled receptor; GRK, GPCR kinase.

Inhibitors of clathrin, dynamin and β-ARRs block endocytosis of PAR2 and NK1R, attenuate endosomal signalling, and prevent sustained activation of primary sensory and spinal neurons (figure 3A). When injected intrathecally, clathrin and dynamin inhibitors and dynamin-1 siRNA suppress NK1R endocytosis and attenuate nociception in mice and rats.23 After intraplantar injection, clathrin and dynamin inhibitors attenuate trypsin-evoked and PAR2-dependent nociception. Endocytosis inhibitors also block PAR2-dependent sensitisation of colonic nociceptors.22 Whether these drugs attenuate visceral pain in the GI tract is unknown. Given the widespread roles of clathrin and dynamin in protein trafficking and vesicle formation, side effects may preclude clinical use.

Other approaches to target endosomal GPCRs have focused on drug delivery. Tripartite lipidated probes are composed of three components: cholestanol, which enables membrane insertion; a polyethylene glycol linker; and a GPCR antagonist cargo (figure 3B).22 39 Tripartite probes insert into the plasma membrane and then accumulate in early endosomes. Tripartite NK1R antagonists cause long-lasting inhibition of endosomal signalling and attenuate persistent SP-evoked excitation of spinal neurons. A tripartite PAR2 antagonist accumulates in endosomes of DRG neurons and blocks endosomal signalling of PAR2. A tripartite PAR2 antagonist blocks sustained hypersensitivity in DRG nociceptors treated with supernatant of biopsies from patients with IBS.22 Given these findings, the effects of tripartite antagonists in colonic visceral pain is worthy of further investigation. Despite encouraging preclinical findings, lipid-conjugated antagonists incorporate indiscriminately into membrane of all cells, which may complicate clinical development.

Nanoparticle drug delivery offers the tantalising prospect of selectively delivering antagonists or agonists of GPCRs to endosomes of neurons that sense and transmit pain, which may limit drug dosing, minimise on-target side effects in other cell types and mitigate systemic toxicity. There are multiple methods to target nanoparticles to an area of interest, including based on size, material and charge, and molecular recognition.42 Stimulus-responsive nanoparticles are designed to release their cargo in response to certain triggers, such as acidity, redox potential and protease activity. Although nanoparticles have primarily been developed for the treatment of cancer,42 their use has been extended for the treatment of pain.24 25

Aprepitant is an NK1R antagonist that is approved for the treatment of chemotherapy-induced nausea and vomiting but has failed in clinical trials for the treatment of pain.33 Aprepitant has been incorporated into pH-responsive soft-polymer nanoparticles designed to enter endosomes and disassemble in acidic endosomes, where they release drug cargo (figure 3C).24 Nanoparticle-encapsulated aprepitant causes sustained inhibition of endosomal NK1R signalling and attenuates SP-evoked excitation of spinal neurons. Nanoparticle-encapsulated aprepitant provides more efficacious and sustained reversal of somatic inflammatory and neuropathic pain than free aprepitant. Nanoparticle delivery can be further refined by coating nanoparticles with a ligand that promotes targeting to endosomes of nociceptors or spinal neurons. Nanoparticles with both a liposome shell conjugated to the DOPr agonist (DADLE) and a pH-sensitive mesoporous silica core containing DADLE are preferentially endocytosed by DOPr-expressing cells and gradually release cargo in the acidic and reducing endosomal environment.25 These nanoparticles have sustained antinociceptive effects on primary sensory neurons and colonic afferent neurons from mice. Nanoparticles also offer the capability of incorporating antagonists of several pronociceptive GPCRs into a single particle, which might overcome the redundancy that is inherent in pain transmission pathways.

pH-dependent activation of GPCRS


Is it possible to devise strategies that selectively activate GPCRs in diseased tissue and thereby obviate side effects in healthy tissues?

Ligands that activate GPCRs in endosomes must be capable of binding to receptors in an acidic endosomal environment of pH<6.0. The extracellular fluid of diseased tissues (eg, tumours, sites of inflammation and infection) can also become acidified, which might alter ligand–receptor interactions and signalling.43 The concept of pH-dependent ligand/GPCR interaction and signalling has been exploited for the development of drugs that selectively engage GPCRs in acidified diseased tissues. The MOPr exemplifies the potential usefulness of such an approach. MOPr agonists such as morphine and fentanyl inhibit pain by dampening the excitability of primary sensory and spinal neurons. In a mouse model of IBD, endogenous opioids from infiltrating immune cells activate MOPr on peripheral gut nociceptors to evoke anti-inflammatory and antinociceptive signalling pathways.40 44 However, the usefulness of MOPr agonists for the treatment of pain is limited by on-target side effects that are mediated by MOPrs in other cell types. These include MOPrs in the enteric nervous system, where activation leads to constipation, and the central nervous system, where MOPr agonists cause respiratory depression, nausea and addiction.

Therapeutic implications

Information about the atomic structure of the MOPr has been used to selectively activate it in diseased tissue.45 46 Molecular modelling of the effects of extracellular pH on docking of fentanyl to MOPr enabled the design of a fluorinated fentanyl analogue; fluorination enablesthe drug to be preferentially protonated in acidic microenvironments of diseased tissues, which facilitates MOPr binding (figure 4A). This analogue preferentially binds to and activates MOPr in model cells in acidified extracellular fluid and attenuates inflammatory pain without causing addiction, respiratory depression or constipation in preclinical models. These results have far-reaching implications for the development of new opioid treatments that successfully alleviate pain while mitigating unwanted side effects, including abuse potential. Whether such analogues of fentanyl suppress inflammatory pain in the digestive system without causing constipation remains to be determined. Similar approaches might be used to enhance the on-target selectivity of agonists and antagonists of other GPCRs while minimising on-target detrimental actions in healthy tissues.

Figure 4

pH-dependent agonism, biased agonism and allosteric modulation of GPCRs. (A) pH-dependent agonism. A fluorinated opioid molecule is modified to selectively bind to GPCRs in acidic extracellular fluid such as at sites of cancer, inflammation and infection. (B) Biased agonism. The process by which different ligands bind to the same GPCR orthosteric site but activate different signalling pathways via stabilisation of specific receptor conformations. As opposed to balanced agonism, whereby the ligand triggers both therapeutic and deleterious pathways, biased agonists are designed to stabilise a specific conformation to favour therapeutic pathways over deleterious pathways. (C) Allosteric modulation. The process by which an effector molecule binds to a region of a GPCR (an allosteric site) that is distinct from that occupied by an endogenous (orthosteric) ligand. Allosteric binding modifies the ability of the endogenous ligand to activate the receptor. Positive allosteric modulators increase the potency of endogenous ligands. Negative allosteric modulators decrease the potency of endogenous ligands. GPCR, G protein-coupled receptor.

Biased agonism of GPCRs


Can a particular GPCR signalling pathway be preferentially targeted?

Biased agonism refers to the concept that different agonists of the same GPCR can stabilise distinct conformations that might favour signalling by one pathway over another (figure 4B).47 Recent advances in our understanding of how different proteases activate PAR2 illustrate the relevance of biased agonism to signalling of pain. In the intestine, proteases that activate PAR2 arise from multiple sources, including immune cells, microbial cells and pancreatic tissue (figure 5A).28 As discussed in the aforementioned section on compartmentalised signalling, proteases such as trypsin activate PAR2 and signal via canonical mechanisms. Cleavage by trypsin induces coupling to Gαq and β-ARRs, which mediate endocytosis and endosomal signalling that are necessary for the sustained sensitisation of colonic sensory nerves during IBS (figure 5B).22 48 However, endosomal signalling of PAR2 is not always the mediator of long-term neuronal sensitisation. Other proteases activated in the digestive system during inflammation and cancer, including cathepsin S from macrophages, elastase from neutrophils,and legumain from tumour cells, cleave PAR2 at unique sites and activate biased signalling pathways that do not lead to receptor endocytosis (figure 5C).16 22 49 50 Once activated by these proteases, PAR2 remains at the plasma membrane where it continues to generate pronociceptive signals by mechanisms that entail activation of adenylyl cyclase and PKA, which may phosphorylate and sensitise ion channels. These findings indicate that both endocytosis-dependent as well as endocytosis-independent pathways can contribute to ion channel sensitisation by PAR2 (figure 5).16

Figure 5

Canonical and biased signalling of PAR2. (A) Proteases are released from multiple sources including immune cells (ie, T from mast cells, CS and matrix metalloproteinase from granulocytes and NE), exocrine pancreas tissue (ie, trypsin-1/2) and microbial breakdown products. (1) Proteases activate PAR2 on peripheral nerve endings. (B) Tryptase and trypsin activate canonical signalling, which include (2) G-protein mediated signalling from the plasma membrane, leading to activation of secondary messengers (ie, PKC). (3) Canonical signalling also entails β-ARR-mediated endocytosis of PAR2 and endosomal signalling by G protein-mediated and β-ARR-mediated processes that activate pERK and mediate persistent hypersensitivity and chronic nociception. (C) Cathepsin S and neutrophil elastase activate biased pathways. They cleave PAR2 at different sites from tryptase and trypsin, stabilising different receptor conformations and activating biased pathways (4). These include G-protein-mediated signalling from the plasma membrane via cAMP and PKA. They do not induce β-ARR-mediated endocytosis of PAR2. Secondary messengers activated by both canonical and biased signalling can contribute to sensitisation of ion channels, another mechanism underlying chronic visceral pain (5). Ion movement leads to membrane depolarisation and triggers action potentials leading to nociception. β-ARR, β-arrestin; cAMP, cyclic AMP; CS, cathepsin S; NE, neutrophil elastase; PAR2, protease-activated receptor-2; pERK, phosphorylated extracellular regulated kinase; PKA, protein kinase A; PKC, protein kinase C; T, tryptase; TRPV, transient receptor potential vanilloid.

Therapeutic implications

The concept of biased agonism is of particular interest for drug development because it may allow the identification of GPCR agonists that activate therapeutically beneficial pathways at the expense of those that invoke deleterious side effects.9 10 51 There is intense interest in biased agonism of OPrs to mitigate unwanted side effects. The concept of developing biased agonists of MOPr arose from studies of β-ARR2-deficient mice, which implicated β-ARR2 signalling pathways in morphine-evoked respiratory depression and constipation, whereas analgesia was signalled by G protein pathways.52 These findings spurred efforts to identify biased agonists that evoke MOPr signalling by G proteins but not β-ARR2, some of which have been evaluated in clinical trials.53 54

TRV130 (oliceridine) is a biased MOPr agonist that activates G protein pathways with minimal β-ARR2 activation. A phase III double-blinded randomised control trial comparing oliceridine, morphine and placebo for treatment of acute postoperative abdominoplasty pain showed that oliceridine provided comparable analgesia to morphine with a favourable side-effect profile, although abuse potential with repeated doses was similar to morphine.55 56 The Federal Drug Administration declined approval, citing that benefits of analgesia did not outweigh risks compared with morphine. Multiple other biased agonists of MOPr and DOPr have been developed.55 57–59

Despite the theoretical benefits of biased agonism, clinical success has been elusive. The sine qua non of biased agonism is that the signalling pathways underlying the therapeutically beneficial versus detrimental actions of GPCR agonists are known and different. However, controversy remains as to whether analgesia and constipation are truly signalled by distinct pathways in human tissues. Indeed, recent reports question the role of β-ARR2-mediated pathways in constipation and respiratory depression. Mice expressing a phosphorylation-defective MOPr mutant that is unable to recruit β-ARR2 show augmented analgesia and decreased tolerance compared with wild-type mice when administered opioids, confirming a role for β-ARR2 in MOPr desensitisation.60 However, opioid-induced constipation and respiratory depression were maintained. Recent studies have found that opioids induce respiratory depression and constipation to a similar extent in β-ARR2 knockout and wild-type mice.61 These findings cast doubt on β-ARR2 as a mediator of the side effects of opioid, which may explain the poor efficacy of biased opioids. A pharmacological assessment of MOR-biased ligands such as oliceridine suggests that low intrinsic efficacy (ability for an agonist to trigger a receptor response) compared with morphine, and not biased agonism, may account for the lack of β-ARR2-mediated actions.62 Thus, although opioids have been developed that preferentially activate certain signalling pathways in model cell lines, controversy about the underlying mechanisms and signalling pathways in human tissues has cast doubt on the importance of biased agonism for drug development. Despite these setbacks, there remains sustained interest in developing biased agonists of GPCRs. Atomic-level molecular modelling has identified GPCR conformations that favour signalling of one pathway over another.63 64 Such studies might enable the rational design of GPCR-biased agonists.

Allosteric modulation of GPCRs


Allosteric modulators are ligands that bind to an allosteric site of a GPCR that is different from that occupied by endogenous ligands, termed the orthosteric site.65 Allosteric modulators can increase (positive allosteric modulators (PAMs)) or decrease (negative allosteric modulators) the affinity or efficacy of endogenous ligands (figure 4C). Allosteric modulation has been exploited in efforts to develop drugs to treat pain.10

Therapeutic implications

By fine-tuning the effects of endogenous analgesics such as opioids, PAMs of OPrs might provide refined control of pain. Allosteric modulators also hold the potential for increased selectivity for GPCR subtypes. Orthosteric sites are evolutionarily conserved so that the same ligand can bind to different GPCR subtypes. In contrast, allosteric sites are typically specific to each GPCR subtype. Thus, allosteric modulators have been attractive drug targets to alter ligand binding on a specific GPCR and enhance receptor subtype specificity.

Multiple allosteric modulators have been developed for the treatment of central nervous system diseases, including Alzheimer’s disease and schizophrenia, with limited success.9 These modulators target muscarinic receptors and increase the affinity of receptor subtypes to acetylcholine. However, despite selectivity for CNS located M1 muscarinic receptor, PAM MK-7622 caused significant cholinergic side effects, specifically diarrhoea, in peripheral tissues.66

There has been considerable interesting in developing allosteric modulators of DOPr and MOPr for the improved treatment of pain.67 68 While most allosteric modulators have not been tested in preclinical models of GI pain, the focus remains on increasing pain relief while mitigating side effects, including those affecting the GI tract.67 68 For example, morphine and fentanyl interact with the orthosteric site of MOPr, and allosteric modulation, theoretically, could decrease the dosage needed to achieve analgesia or enhance the effects of endogenous opioids. While MOPr and DOPr PAMs have been developed, clinical testing has not progressed. Nevertheless, the ability to modulate the effects of opioids for the treatment of pain and diarrhoea remains of considerable interest.

GPCR dimerisation


GPCR dimerisation adds to the complexity of GPCRs while offering new avenues for therapeutic design.69 Despite initial excitement in the concept that different GPCRs might associate to create a receptor with unique physiological roles and pharmacological properties, the concept of GPCR dimerisation remains controversial.10 It has been challenging to convincingly demonstrate the existence of GPCR dimers in native tissues. The dimerisation of OPrs is a controversial area that has garnered much attention. Early studies provided evidence that MOPr and DOPr signalling is inter-related. Thus, DOPr antagonism or deletion attenuated morphine-evoked tolerance and dependence in mice.70 Binding low-dose DOPr antagonists increased the potency of MOPr agonists, inviting new therapeutic strategies with bifunctional ligands.71 Later studies confirmed that they colocalise in certain neuronal populations in both the peripheral and central nervous system and function as heterodimers.72 However, other studies have disputed these conclusions. Mice expressing fluorescently tagged DOPrs show little coexpression of DOPr and MOPr in primary neurons.73 Furthermore, where they are coexpressed in interneurons and secondary neurons, there is no evidence for heterodimer formation. Electrophysiological and molecular studies support DOPr and MOPr coexpression by sensory DRG neurons originating in the mouse colon, although heterodimer formation of these receptors remains unclear.31

Therapeutic implications

Despite controversy, drugs composed of bifunctional ligands to target both DOPr and MOPr remain an active area of therapeutic research. Eluxadoline is a compound with MOPr agonist and DOPr antagonist activity that demonstrated relief of abdominal pain in patients with IBS-induced diarrhoea without evidence for abuse potential in phase II and III trials.74 Further investigation regarding the coexpression and interaction of GPCRs in the GI system is needed to understand therapeutic potential.

Tissue-specific outcomes of GPCR signalling


The realisation that GPCRs can convey distinct sensory information in different tissues has led to the identification of new mediators of visceral pain. Studies of GPCRs that mediate itch in the skin, such as mas-related G-protein receptors (Mrgprs) and Takeda GPCR 5 (TGR5), have revealed new roles for irritant sensation and visceral hypersensitivity in the colon.15 The Mrgpr family of GPCRs are expressed by cutaneous afferent neurons, where they mediate the sensation of itch.75 Chloroquine, an antimalarial drug, evokes pruritus by activating MrgprA3/MrgprX1 in cutaneous afferent nerves. TGR5, a receptor for secondary bile acids, is also expressed by cutaneous afferents and has been implicated in bile acid-evoked scratching in mice and cholestatic itch.76 77

Accumulating evidence suggests that certain Mrgprs and TGR5 are expressed by colonic afferent neurons where they might mediate hypersensitivity and pain, rather than itch. TGR5 is expressed by enteric neurons, where activation by luminal bile acids evokes the peristaltic reflex.76 Mrgprc11 is expressed on splanchnic and pelvic DRG neurons in mice.78 Activation with the endogenous ligand, bovine adrenal medulla 8–22, causes visceral hypersensitivity and increased visceromotor responses to colorectal distension. Colonic biopsies from patients with IBS show elevated levels of certain metabolites of polyunsaturated fatty acids compared with healthy controls.79 80 One metabolite, 5-oxoeicosatetraenoic acid, evokes visceral hypersensitivity and nociceptor activation through the Mrgprd receptor pathway in mice.79

Mrgpra3, Mrgprc11 and TGR5 are expressed in DRG neurons innervating the mouse colon and have been implicated in afferent nerve sensitisation associated with IBS pain.15 Agonists of these receptors sensitise nociceptors innervating isolated segments of mouse colon, which is not seen in receptor knockout mice. Intraluminal administration of these agonists triggers visceral hypersensitivity to colorectal distension. These receptors induce mechanical hypersensitivity through the transient receptor potential ankyrin-1 ion channel, a major mediator of itch.

Therapeutic implications

This irritant-sensing pathway in the colon might represent a visceral representation of the cutaneous itch pathway and could contribute to sensory disturbances accompanying IBS. Thus, antagonists of Mrgprs, TGR5 or downstream channels such as TRPA1, might be considered for the treatment of chronic pain in the digestive system.

Conclusions and future directions

Chronic pain within the digestive tract remains a major unmet medical problem. It accompanies common digestive diseases, is poorly understood and is difficult to treat. Analgesic drugs such as opioids have major GI side effects. Given the importance and complexity of GPCRs in controlling the pathways of CVH, it is not surprising that they have been a focus of drug discovery and development. GPCR-targeted therapies are usually based on observations in preclinical disease models. Few drugs progress to clinic; of those that do progress, many fail in clinical trials of pain. Reasons for failure might include the inability to generate preclinical models of visceral pain that faithfully replicate human diseases; the challenge of studying visceral pain in animals, the inherent redundancy of pain signalling pathways where multiple receptors and channels participate in important protective pain mechanisms; and the lack of understanding of how receptors and channels regulate pain signalling in relevant human cells.

Despite these challenges, new information about how GPCRs signal nociception holds the potential to develop improved therapies. These include antagonists that selectively target endosomal GPCRs; opioids that preferentially activate OPrs in the acidified extracellular milieu of disease tissues; biased agonists that stabilise OPr conformations that favour signalling of therapeutically desirable pathways but not those that mediate the detrimental actions of opioids; and PAMs that subtly amplify the analgesic properties of endogenous opioids. Many of these approaches have been evaluated for the treatment of somatic pain. Further studies are necessary to test their effectiveness against visceral pain in the digestive system. The opioid crisis, which has highlighted the problem of chronic pain and its treatment, will continue to spur efforts to discover new information about the mechanisms and treatment of chronic pain in the digestive system.



  • Contributors LGK, RL, SV, BS and NB researched field and wrote the manuscript.

  • Funding Supported by grants from National Institutes of Health (NS102722, DE026806, DE029951, DK118971; NWB and BLS) and Department of Defense (W81XWH1810431, NWB and BLS).

  • Competing interests NWB is a founding scientist of Endosome Therapeutics Inc.

  • Patient and public involvement Patients and/or the public were not involved in the design, conduct, reporting or dissemination plans of this research.

  • Provenance and peer review Not commissioned; externally peer reviewed.