Faculty Bibliography

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.

Oral squamous cell carcinoma (OSCC) is one of the most painful cancers, which interferes with orofacial function including talking and eating. We report that legumain (Lgmn) cleaves protease-activated receptor-2 (PAR₂) in the acidic OSCC microenvironment to cause pain. Lgmn is a cysteine protease of late endosomes and lysosomes that can be secreted; it exhibits maximal activity in acidic environments. The role of Lgmn in PAR₂-dependent cancer pain is unknown. We studied Lgmn activation in human oral cancers and oral cancer mouse models. Lgmn was activated in OSCC patient tumors, compared to matched normal oral tissue. After intraplantar, facial or lingual injection, Lgmn evoked nociception in wild-type (WT) female mice but not in female mice lacking PAR₂ in Na_V1.8-positive neurons (Par₂Na_v1.8), nor in female mice treated with a Lgmn inhibitor, LI-1. Inoculation of an OSCC cell line caused mechanical and thermal hyperalgesia that was reversed by LI-1. Par₂Na_v1.8 and Lgmn deletion attenuated mechanical allodynia in female mice with carcinogen-induced OSCC. Lgmn caused PAR₂-dependent hyperexcitability of trigeminal neurons from WT female mice. Par₂ deletion, LI-1 and inhibitors of adenylyl cyclase or protein kinase A prevented the effects of Lgmn. Under acidified conditions, Lgmn cleaved within the extracellular N-terminus of PAR₂ at Asn³⁰↓Arg³¹, proximal to the canonical trypsin activation site. Lgmn activated PAR₂ by biased mechanisms in HEK293 cells to induce Ca²⁺ mobilization, cAMP formation and protein kinase A/D activation, but not β-arrestin recruitment or PAR₂ endocytosis. Thus, in the acidified OSCC microenvironment Lgmn activates PAR₂ by biased mechanisms that evoke cancer pain.SIGNIFICANCE STATEMENTOral squamous cell carcinoma (OSCC) is one of the most painful cancers. We report that legumain (Lgmn), which exhibits maximal activity in acidic environments, cleaves protease-activated receptor-2 (PAR₂) on neurons to produce OSCC pain. Active Lgmn was elevated in OSCC patient tumors, compared to matched normal oral tissue. Lgmn evokes pain-like behavior through PAR₂ Exposure of pain-sensing neurons to Lgmn decreased the current required to generate an action potential through PAR₂ Inhibitors of adenylyl cyclase and protein kinase A prevented the effects of Lgmn. Lgmn activated PAR₂ to induce calcium mobilization, cAMP formation and activation of protein kinase D and A, but not β-arrestin recruitment or PAR₂ endocytosis. Thus, Lgmn is a biased agonist of PAR₂ that evokes cancer pain.

Whether G protein-coupled receptors signal from endosomes to control important pathophysiological processes and are therapeutic targets is uncertain. We report that opioids from the inflamed colon activate δ-opioid receptors (DOPr) in endosomes of nociceptors. Biopsy samples of inflamed colonic mucosa from patients and mice with colitis released opioids that activated DOPr on nociceptors to cause a sustained decrease in excitability. DOPr agonists inhibited mechanically sensitive colonic nociceptors. DOPr endocytosis and endosomal signaling by protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) pathways mediated the sustained inhibitory actions of endogenous opioids and DOPr agonists. DOPr agonists stimulated the recruitment of Gα_i/o and β-arrestin1/2 to endosomes. Analysis of compartmentalized signaling revealed a requirement of DOPr endocytosis for activation of PKC at the plasma membrane and in the cytosol and ERK in the nucleus. We explored a nanoparticle delivery strategy to evaluate whether endosomal DOPr might be a therapeutic target for pain. The DOPr agonist DADLE was coupled to a liposome shell for targeting DOPr-positive nociceptors and incorporated into a mesoporous silica core for release in the acidic and reducing endosomal environment. Nanoparticles activated DOPr at the plasma membrane, were preferentially endocytosed by DOPr-expressing cells, and were delivered to DOPr-positive early endosomes. Nanoparticles caused a long-lasting activation of DOPr in endosomes, which provided sustained inhibition of nociceptor excitability and relief from inflammatory pain. Conversely, nanoparticles containing a DOPr antagonist abolished the sustained inhibitory effects of DADLE. Thus, DOPr in endosomes is an endogenous mechanism and a therapeutic target for relief from chronic inflammatory pain.

Endothelial barrier disruption is a hallmark of tissue injury, edema, and inflammation. Vascular endothelial cells express the G protein-coupled receptor (GPCR) protease acctivated receptor 1 (PAR1) and the ion channel transient receptor potential vanilloid 4 (TRPV4), and these signaling proteins are known to respond to inflammatory conditions and promote edema through remodeling of cell-cell junctions and modulation of endothelial barriers. It has previously been established that signaling initiated by the related protease activated receptor 2 (PAR2) is enhanced by TRPV4 in sensory neurons and that this functional interaction plays a critical role in the development of neurogenic inflammation and nociception. Here, we investigated the PAR1-TRPV4 axis, to determine if TRPV4 plays a similar role in the control of edema mediated by thrombin-induced signaling. Using Evans Blue permeation and retention as an indication of increased vascular permeability in vivo, we showed that TRPV4 contributes to PAR1-induced vascular hyperpermeability in the airways and upper gastrointestinal tract of mice. TRPV4 contributes to sustained PAR1-induced Ca²⁺ signaling in recombinant cell systems and to PAR1-dependent endothelial junction remodeling in vitro. This study supports the role of GPCR-TRP channel functional interactions in inflammatory-associated changes to vascular function and indicates that TRPV4 is a signaling effector for multiple PAR family members.

Cathepsin X/Z/P is cysteine cathepsin with unique carboxypeptidase activity. Its expression is associated with cancer and neurodegenerative diseases, although its roles during normal physiology are still poorly understood. Advances in our understanding of its function have been hindered by a lack of available tools that can specifically measure the proteolytic activity of cathepsin X. We present a series of activity-based probes that incorporate a sulfoxonium ylide warhead, which exhibit improved specificity for cathepsin X compared to previously reported probes. We apply these probes to detect cathepsin X activity in cell and tissue lysates, in live cells and in vivo and to localize active cathepsin X in mouse tissues by microscopy. Finally, we utilize an improved method to generate chloromethylketones, necessary intermediates for synthesis of acyloxymethylketones probes, by way of sulfoxonium ylide intermediates. In conclusion, the probes presented in this study will be valuable for investigating cathepsin X pathophysiology.

Duodenogastroesophageal reflux (DGER) is associated with chronic lung disease. Bile acids (BAs) are established markers of DGER aspiration and are important risk factors for reduced post-transplant lung allograft survival by disrupting the organ-specific innate immunity facilitating airway infection and allograft failure. However, it is unknown whether BAs also affect airway reactivity. We investigated the acute effects of 13 BAs detected in post-lung transplant surveillance bronchial washings (BW) on airway contraction. We exposed precision-cut slices from human and mouse lungs to BAs and monitored dynamic changes in cross-sectional luminal area of peripheral airways using video phase-contrast microscopy. We also used guinea-pig tracheal rings in organ baths to study BA effects in proximal airway contraction induced by electrical field stimulation. We found that most secondary BAs at low micromolar concentrations strongly and reversibly relaxed smooth muscle and inhibited peripheral airway constriction induced by acetylcholine but not by non-cholinergic bronchoconstrictors. Similarly, secondary BAs strongly inhibited cholinergic constrictions in tracheal rings. In contrast, TC-G 1005, a specific agonist of the BA receptor TGR5, did not cause airway relaxation, and Tgr5 deletion in knockout mice did not affect BA-induced relaxation, suggesting that this receptor is not involved. BAs inhibited acetylcholine-induced inositol phosphate synthesis in human airway smooth muscle cells overexpressing the muscarinic M3 receptor. Our results demonstrate that select BAs that are found in BW of patients with lung transplantation can affect airway reactivity by inhibiting the cholinergic contractile responses of the proximal and peripheral airways, possibly by acting as antagonists of M3 muscarinic receptors.

Nanoparticle-mediated drug delivery is especially useful for targets within endosomes because of the endosomal transport mechanisms of many nanomedicines within cells. Here, we report the design of a pH-responsive, soft polymeric nanoparticle for the targeting of acidified endosomes to precisely inhibit endosomal signalling events leading to chronic pain. In chronic pain, the substance P (SP) neurokinin 1 receptor (NK₁R) redistributes from the plasma membrane to acidified endosomes, where it signals to maintain pain. Therefore, the NK₁R in endosomes provides an important target for pain relief. The pH-responsive nanoparticles enter cells by clathrin- and dynamin-dependent endocytosis and accumulate in NK₁R-containing endosomes. Following intrathecal injection into rodents, the nanoparticles, containing the FDA-approved NK₁R antagonist aprepitant, inhibit SP-induced activation of spinal neurons and thus prevent pain transmission. Treatment with the nanoparticles leads to complete and persistent relief from nociceptive, inflammatory and neuropathic nociception and offers a much-needed non-opioid treatment option for chronic pain.

Itch induces scratching that removes irritants from the skin, whereas pain initiates withdrawal or avoidance of tissue damage. While pain arises from both the skin and viscera, we investigated whether pruritogenic irritant mechanisms also function within visceral pathways. We show that subsets of colon-innervating sensory neurons in mice express, either individually or in combination, the pruritogenic receptors Tgr5 and the Mas-gene-related GPCRs Mrgpra3 and Mrgprc11. Agonists of these receptors activated subsets of colonic sensory neurons and evoked colonic afferent mechanical hypersensitivity via a TRPA1-dependent mechanism. In vivo intracolonic administration of individual TGR5, MrgprA3, or MrgprC11 agonists induced pronounced visceral hypersensitivity to colorectal distension. Coadministration of these agonists as an "itch cocktail" augmented hypersensitivity to colorectal distension and changed mouse behavior. These irritant mechanisms were maintained and enhanced in a model of chronic visceral hypersensitivity relevant to irritable bowel syndrome. Neurons from human dorsal root ganglia also expressed TGR5, as well as the human ortholog MrgprX1, and showed increased responsiveness to pruritogenic agonists in pathological states. These data support the existence of an irritant-sensing system in the colon that is a visceral representation of the itch pathways found in skin, thereby contributing to sensory disturbances accompanying common intestinal disorders.

BACKGROUND & AIMS:Mood disorders and constipation are often comorbid, yet their shared etiologies have rarely been explored. The neurotransmitter serotonin (5-HT) regulates central nervous system and enteric nervous system (ENS) development and long-term functions, including gastrointestinal (GI) motility and mood. Therefore, defects in neuron production of 5-HT might result in brain and intestinal dysfunction. Tryptophan hydroxylase 2 (TPH2) is the rate-limiting enzyme in 5-HT biosynthesis. A variant of TPH2 that encodes the R441H substitution (TPH2-R441H) was identified in individuals with severe depression. We studied mice with an analogous mutation (TPH2-R439H), which results in a 60%-80% decrease in levels of 5-HT in the central nervous system and behaviors associated with depression in humans. Feeding chow that contains 5-HTP slow release (5-HTP SR) to TPH2-R439H mice restores levels of 5-HT in the central nervous system and reduces depressive-like behaviors. METHODS:We compared the effects of feeding chow, with or without 5-HTP SR, to mice with the TPH2-R439H mutation and without this mutation (control mice). Myenteric and submucosal plexuses were isolated from all 4 groups of mice, and immunocytochemistry was used to quantify total enteric neurons, serotonergic neurons, and 5-HT-dependent subsets of neurons. We performed calcium imaging experiments to evaluate responses of enteric neurons to tryptamine-evoked release of endogenous 5-HT. In live mice, we measured total GI transit, gastric emptying, small intestinal transit, and propulsive colorectal motility. To measure colonic migrating motor complexes (CMMCs), we isolated colons and constructed spatiotemporal maps along the proximodistal length to quantify the frequency, velocity, and length of CMMCs. We measured villus height, crypt perimeter, and relative densities of enterochromaffin and enteroendocrine cells in small intestinal tissue. RESULTS:Levels of 5-HT were significantly lower in enteric neurons from TPH2-R439H mice than from control mice. TPH2-R439H mice had abnormalities in ENS development and ENS-mediated GI functions, including reduced motility and intestinal epithelial growth. Total GI transit and propulsive colorectal motility were slower in TPH2-R439H mice than controls, and CMMCs were slower and less frequent. Villus height and crypt perimeter were significantly decreased in colon tissues from TPH2-R439H mice compared with controls. Administration of 5-HTP SR to adult TPH2-R439H mice restored 5-HT to enteric neurons and reversed these abnormalities. Adult TPH2-R439H mice given oral 5-HTP SR had normalized numbers of enteric neurons, total GI transit, and colonic motility. Intestinal tissue from these mice had normal measures of CMMCs and enteric epithelial growth CONCLUSIONS: In studies of TPH2-R439H mice, we found evidence for reduced release of 5-HT from enteric neurons that results in defects in ENS development and GI motility. Our findings indicate that neuron production of 5-HT links constipation with mood dysfunction. Administration of 5-HTP SR to mice restored 5-HT to the ENS and normalized GI motility and growth of the enteric epithelium. 5-HTP SR might be used to treat patients with intestinal dysfunction associated with low levels of 5-HT.