Faculty Bibliography

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.

Excessive alcohol consumption is associated with spontaneous burning pain, hyperalgesia and allodynia. Although acetaldehyde has been implicated in the painful alcoholic neuropathy, the mechanism by which the ethanol metabolite causes pain symptoms is unknown. Acute ethanol ingestion caused delayed mechanical allodynia in mice. Inhibition of alcohol dehydrogenase (ADH) or deletion of transient receptor potential ankyrin 1 (TRPA1), a sensor for oxidative and carbonyl stress, prevented allodynia. Acetaldehyde generated by ADH in both liver and Schwann cells surrounding nociceptors was required for TRPA1-induced mechanical allodynia. Plp1-Cre;Trpa1fl/fl mice with a tamoxifen-inducible specific deletion of TRPA1 in Schwann cells revealed that channel activation by acetaldehyde in these cells initiates a NADPH oxidase-1 (NOX-1)-dependent production of hydrogen peroxide (H2O2) and 4-hydroxynonenal (4-HNE), which sustains allodynia by paracrine targeting of nociceptor TRPA1. Chronic ethanol ingestion caused prolonged mechanical allodynia and loss of intraepidermal small nerve fibers in WT mice. While Trpa1-/- or Plp1-Cre;Trpa1fl/fl mice did not develop mechanical allodynia, they did not show any protection from the small fiber neuropathy. Human Schwann cells express ADH/TRPA1/NOX1 and recapitulate the proalgesic functions of mouse Schwann cells. TRPA1 antagonists might attenuate some symptoms of alcohol-related pain.

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.

Neutrophil elastase is a serine protease that has been implicated in the pathogenesis of inflammatory bowel disease. Due to post-translational control of its activation and high expression of its inhibitors in the gut, measurements of total expression poorly reflect the pool of active, functional neutrophil elastase. Fluorogenic substrate probes have been used to measure neutrophil elastase activity, though these tools lack specificity and traceability. PK105 is a recently described fluorescent activity-based probe, which binds to neutrophil elastase in an activity-dependent manner. The irreversible nature of this probe allows for accurate identification of its targets in complex protein mixtures. We describe the reactivity profile of PK105b, a new analogue of PK105, against recombinant serine proteases and in tissue extracts from healthy mice and from models of inflammation induced by oral cancer and Legionella pneumophila infection. We apply PK105b to measure neutrophil elastase activation in an acute model of experimental colitis. Neutrophil elastase activity is detected in inflamed, but not healthy, colons. We corroborate this finding in mucosal biopsies from patients with ulcerative colitis. Thus, PK105b facilitates detection of neutrophil elastase activity in tissue lysates, and we have applied it to demonstrate that this protease is unequivocally activated during colitis.

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.

Proteases sustain hyperexcitability and pain by cleaving protease-activated receptor-2 (PAR₂) on nociceptors through distinct mechanisms. Whereas trypsin induces PAR₂ coupling to Gα_q, Gα_s, and β-arrestins, cathepsin-S (CS) and neutrophil elastase (NE) cleave PAR₂ at distinct sites and activate it by biased mechanisms that induce coupling to Gα_s, but not to Gα_q or β-arrestins. Because proteases activate PAR₂ by irreversible cleavage, and activated PAR₂ is degraded in lysosomes, sustained extracellular protease-mediated signaling requires mobilization of intact PAR₂ from the Golgi apparatus or de novo synthesis of new receptors by incompletely understood mechanisms. We found here that trypsin, CS, and NE stimulate PAR₂-dependent activation of protein kinase D (PKD) in the Golgi of HEK293 cells, in which PKD regulates protein trafficking. The proteases stimulated translocation of the PKD activator Gβγ to the Golgi, coinciding with PAR₂ mobilization from the Golgi. Proteases also induced translocation of a photoconverted PAR₂-Kaede fusion protein from the Golgi to the plasma membrane of KNRK cells. After incubation of HEK293 cells and dorsal root ganglia neurons with CS, NE, or trypsin, PAR₂ responsiveness initially declined, consistent with PAR₂ cleavage and desensitization, and then gradually recovered. Inhibitors of PKD, Gβγ, and protein translation inhibited recovery of PAR₂ responsiveness. PKD and Gβγ inhibitors also attenuated protease-evoked mechanical allodynia in mice. We conclude that proteases that activate PAR₂ by canonical and biased mechanisms stimulate PKD in the Golgi; PAR₂ mobilization and de novo synthesis repopulate the cell surface with intact receptors and sustain nociceptive signaling by extracellular proteases.

G protein-coupled receptors (GPCRs) are the largest family of transmembrane signaling proteins. Within the gastrointestinal tract, GPCRs expressed by epithelial cells sense contents of the lumen, and GPCRs expressed by epithelial cells, myocytes, neurons, and immune cells participate in communication amongst cells. GPCRs control digestion, mediate digestive diseases, and coordinate repair and growth. GPCRs are the target of over one third of therapeutic drugs, including many drugs used to treat digestive diseases. Recent advances in structural, chemical, and cell biology research have revealed that GPCRs are not static binary switches that operate from the plasma membrane to control a defined set of intracellular signals. Rather, GPCRs are dynamic signaling proteins that adopt distinct conformations and subcellular distributions when associated with different ligands and intracellular effectors. An understanding of the dynamic nature of GPCRs has provided insights into the mechanism of activation and signaling of GPCRs, and has revealed opportunities for drug discovery. We review the allosteric modulation, biased agonism, oligomerization, and compartmentalized signaling of GPCRs that control digestion and digestive diseases. We highlight the implications of these concepts for the development of selective and effective drugs to treat diseases of the gastrointestinal tract.

Introduction/UNASSIGNED:knockout mice show attenuated pain behaviours after peripheral nerve injury, compared with control mice. The type I transmembrane isoform of Sez6 is cleaved by the β-amyloid precursor protein cleavage enzyme 1 (BACE1), resulting in Sez6 extracellular domain shedding from the neuron surface. Objectives/UNASSIGNED:To determine whether this BACE1-shed form of Sez6 can be detected in the cerebrospinal fluid (CSF) and whether Sez6 levels in the CSF are altered in neuropathic pain or chronic inflammatory pain (IP). Methods/UNASSIGNED:We analysed the CSF samples collected during surgery from patients with chronic neuropathic pain (n = 8) or IP (n = 33), comparing them to the CSF samples from patients with suspected subarachnoid haemorrhage that was subsequently excluded (nonsurgical group, n = 5). Western blots were used to determine the relative Sez6 levels in the CSF from the different patient and nonsurgical comparison groups. Results/UNASSIGNED:The results show that BACE1-shed Sez6 can be readily detected in the CSF by Western blot and that the levels of Sez6 are significantly higher in the IP group than in the nonsurgical comparison group. Conclusion/UNASSIGNED:The association between elevated Sez6 levels in the CSF and IP is further evidence for persistent alterations in central nervous system activity in chronic IP conditions.