Patients with cholestatic disease exhibit pruritus and analgesia, but the mechanisms underlying these symptoms are unknown. We report that bile acids, which are elevated in the circulation and tissues during cholestasis, cause itch and analgesia by activating the GPCR TGR5. TGR5 was detected in peptidergic neurons of mouse dorsal root ganglia and spinal cord that transmit itch and pain, and in dermal macrophages that contain opioids. Bile acids and a TGR5-selective agonist induced hyperexcitability of dorsal root ganglia neurons and stimulated the release of the itch and analgesia transmitters gastrin-releasing peptide and leucineenkephalin. Intradermal injection of bile acids and a TGR5-selective agonist stimulated scratching behavior by gastrin-releasing peptide-and opioid-dependent mechanisms in mice. Scratching was attenuated in Tgr5-KO mice but exacerbated in Tgr5-Tg mice (overexpressing mouse TGR5), which exhibited spontaneous pruritus. Intraplantar and intrathecal injection of bile acids caused analgesia to mechanical stimulation of the paw by an opioid-dependent mechanism. Both peripheral and central mechanisms of analgesia were absent from Tgr5-KO mice. Thus, bile acids activate TGR5 on sensory nerves, stimulating the release of neuropeptides in the spinal cord that transmit itch and analgesia. These mechanisms could contribute to pruritus and painless jaundice that occur during cholestatic liver diseases.

Background & Aims Although proteases control inflammation and pain, the identity, cellular origin, mechanism of action, and causative role of proteases that are activated during disease are not defined. We investigated the activation and function of cysteine cathepsins (Cat) in colitis. Methods Since protease activity, rather than expression, is regulated, we treated mice with fluorescent activity-based probes that covalently modify activated cathepsins. Activated proteases were localized by tomographic imaging of intact mice and confocal imaging of tissues, and were identified by electrophoresis and immunoprecipitation. We examined the effects of activated cathepsins on excitability of colonic nociceptors and on colonic pain, and determined their role in colonic inflammatory pain by gene deletion. Results Tomography and magnetic resonance imaging localized activated cathepsins to the inflamed colon of piroxicam-treated il10−/− mice. Confocal imaging detected activated cathepsins in colonic macrophages and spinal neurons and microglial cells of mice with colitis. Gel electrophoresis and immunoprecipitation identified activated Cat-B, Cat-L and Cat-S in colon and spinal cord, and Cat-S was preferentially secreted into the colonic lumen. Intraluminal Cat-S amplified visceromotor responses to colorectal distension and induced hyperexcitability of colonic nociceptors, which required expression of protease-activated receptor-2. Cat-S deletion attenuated colonic inflammatory pain induced with trinitrobenzene sulfonic acid. Conclusions Activity-based probes enable non-invasive detection, cellular localization and proteomic identification of proteases activated during colitis and are potential diagnostic tools for detection of predictive disease biomarkers. Macrophage cathepsins are activated during colitis, and Cat-S activates nociceptors to induce visceral pain via protease-activated receptor-2. Cat-S mediates colitis pain and is a potential therapeutic target.

The mechanisms of pancreatic pain, a cardinal symptom of pancreatitis, are unknown. Proinflammatory agents that activate transient receptor potential (TRP) channels in nociceptive neurons can cause neurogenic inflammation and pain. We report a major role for TRPV4, which detects osmotic pressure and arachidonic acid metabolites, and TRPA1, which responds to 4-hydroxynonenal and cyclopentenone prostaglandins, in pancreatic inflammation and pain in mice. Immunoreactive TRPV4 and TRPA1 were detected in pancreatic nerve fibers and in dorsal root ganglia neurons innervating the pancreas, which were identified by retrograde tracing. Agonists of TRPV4 and TRPA1 increased intracellular Ca(2+) concentration ([Ca(2+)](i)) in these neurons in culture, and neurons also responded to the TRPV1 agonist capsaicin and are thus nociceptors. Intraductal injection of TRPV4 and TRPA1 agonists increased c-Fos expression in spinal neurons, indicative of nociceptor activation, and intraductal TRPA1 agonists also caused pancreatic inflammation. The effects of TRPV4 and TRPA1 agonists on [Ca(2+)](i), pain and inflammation were markedly diminished or abolished in trpv4 and trpa1 knockout mice. The secretagogue cerulein induced pancreatitis, c-Fos expression in spinal neurons, and pain behavior in wild-type mice. Deletion of trpv4 or trpa1 suppressed c-Fos expression and pain behavior, and deletion of trpa1 attenuated pancreatitis. Thus TRPV4 and TRPA1 contribute to pancreatic pain, and TRPA1 also mediates pancreatic inflammation. Our results provide new information about the contributions of TRPV4 and TRPA1 to inflammatory pain and suggest that channel antagonists are an effective therapy for pancreatitis, when multiple proinflammatory agents are generated that can activate and sensitize these channels.

Cathepsins regulate premature trypsinogen activation within acinar cells, a key initial step in pancreatitis. The identity, origin, and causative roles of activated cathepsins in pancreatic inflammation and pain are not defined. By using a near infrared-labeled activity-based probe (GB123) that covalently modifies active cathepsins, we localized and identified activated cathepsins in mice with cerulein-induced pancreatitis and in pancreatic juice from patients with chronic pancreatitis. We used inhibitors of activated cathepsins to define their causative role in pancreatic inflammation and pain. After GB123 administration to mice with pancreatitis, reflectance and confocal imaging showed significant accumulation of the probe in inflamed pancreas compared with controls, particularly in acinar cells and macrophages, and in spinal cord microglia and neurons. Biochemical analysis of pancreatic extracts identified them as cathepsins B, L, and S (Cat-B, Cat-L, and Cat-S, respectively). These active cathepsins were also identified in pancreatic juice from patients with chronic pancreatitis undergoing an endoscopic procedure for the treatment of pain, indicating cathepsin secretion. The cathepsin inhibitor K11777 suppressed cerulein-induced activation of Cat-B, Cat-L, and Cat-S in the pancreas and ameliorated pancreatic inflammation, nocifensive behavior, and activation of spinal nociceptive neurons. Thus pancreatitis is associated with an increase in the active forms of the proteases Cat-B, Cat-L, and Cat-S in pancreatic acinar cells and macrophages, and in spinal neurons and microglial cells. Inhibition of cathepsin activation ameliorated pancreatic inflammation and pain. Activity-based probes permit identification of proteases that are predictive biomarkers of disease progression and response to therapy and may be useful noninvasive tools for the detection of pancreatic inflammation.

Acute pancreatitis is a life-threatening inflammatory disease characterized by abdominal pain of unknown etiology. Trypsin, a key mediator of pancreatitis, causes inflammation and pain by activating protease-activated receptor 2 (PAR(2)), but the isoforms of trypsin that cause pancreatitis and pancreatic pain are unknown. We hypothesized that human trypsin IV and rat P23, which activate PAR(2) and are resistant to pancreatic trypsin inhibitors, contribute to pancreatic inflammation and pain. Injections of a subinflammatory dose of exogenous trypsin increased c-Fos immunoreactivity, indicative of spinal nociceptive activation, but did not cause inflammation, as assessed by measuring serum amylase and myeloperoxidase activity and by histology. The same dose of trypsin IV and P23 increased some inflammatory end points and caused a more robust effect on nociception, which was blocked by melagatran, a trypsin inhibitor that also inhibits polypeptide-resistant trypsin isoforms. To determine the contribution of endogenous activation of trypsin and its minor isoforms, recombinant enterokinase (ENK), which activates trypsins in the duodenum, was administered into the pancreas. Intraductal ENK caused nociception and inflammation that were diminished by polypeptide inhibitors, including soybean trypsin inhibitor and a specific trypsin inhibitor (type I-P), and by melagatran. Finally, the secretagogue cerulein induced pancreatic nociceptive activation and nocifensive behavior that were reversed by melagatran. Thus trypsin and its minor isoforms mediate pancreatic pain and inflammation. In particular, the inhibitor-resistant isoforms trypsin IV and P23 may be important in mediating prolonged pancreatic inflammatory pain in pancreatitis. Our results suggest that inhibitors of these isoforms could be novel therapies for pancreatitis pain.