Faculty Bibliography

Activity-based probes are small molecules that covalently bind to the active site of a protease in an activity-dependent manner. We synthesized and characterized two fluorescent activity-based probes that target serine proteases with trypsin-like or elastase-like activity. We assessed the selectivity and potency of these probes against recombinant enzymes and demonstrated that while they are efficacious at labeling active proteases in complex protein mixtures in vitro, they are less valuable for in vivo studies. We used these probes to evaluate serine protease activity in two mouse models of acute inflammation, including pancreatitis and colitis. As anticipated, the activity of trypsin-like proteases was increased during pancreatitis. Levels of elastase-like proteases were low in pancreatic lysates and colonic luminal fluids, whether healthy or inflamed. Exogenously added recombinant neutrophil elastase was inhibited upon incubation with these samples, an effect that was augmented in inflamed samples compared to controls. These data suggest that endogenous inhibitors and elastase-degrading proteases are upregulated during inflammation.

Pancreatitis is an inflammatory disease of the pancreas characterized by dysregulated activity of digestive enzymes, necrosis, immune infiltration, and pain. Repeated incidence of pancreatitis is an important risk factor for pancreatic cancer. Legumain, a lysosomal cysteine protease, has been linked to inflammatory diseases such as atherosclerosis, stroke, and cancer. Until now, legumain activation has not been studied during pancreatitis. We used a fluorescently quenched activity-based probe to assess legumain activation during caerulein-induced pancreatitis in mice. We detected activated legumain by ex vivo imaging, confocal microscopy, and gel electrophoresis. Compared with healthy controls, legumain activity in the pancreas of caerulein-treated mice was increased in a time-dependent manner. Legumain was localized to CD68(+) macrophages and was not active in pancreatic acinar cells. Using a small-molecule inhibitor of legumain, we found that this protease is not essential for the initiation of pancreatitis. However, it may serve as a biomarker of disease, since patients with chronic pancreatitis show strongly increased legumain expression in macrophages. Moreover, the occurrence of legumain-expressing macrophages in regions of acinar-to-ductal metaplasia suggests that this protease may influence reprogramming events that lead to inflammation-induced pancreatic cancer.

BACKGROUND AND PURPOSE:Diverse proteases cleave protease-activated receptor-2 (PAR2) on primary sensory neurons and epithelial cells to evoke pain and inflammation. Trypsin and tryptase activate PAR2 by a canonical mechanism that entails cleavage within the extracellular N-terminus revealing a tethered ligand that activates the cleaved receptor. Cathepsin-S and elastase are biased agonists that cleave PAR2 at different sites to activate distinct signalling pathways. Although PAR2 is a therapeutic target for inflammatory and painful diseases, the divergent mechanisms of proteolytic activation complicate the development of therapeutically useful antagonists. EXPERIMENTAL APPROACH:We investigated whether the PAR2 antagonist GB88 inhibits protease-evoked activation of nociceptors and protease-stimulated oedema and hyperalgesia in rodents. KEY RESULTS:Intraplantar injection of trypsin, cathespsin-S or elastase stimulated mechanical and thermal hyperalgesia and oedema in mice. Oral GB88 or par2 deletion inhibited the algesic and proinflammatory actions of all three proteases, but did not affect basal responses. GB88 also prevented pronociceptive and proinflammatory effects of the PAR2-selective agonists 2-furoyl-LIGRLO-NH2 and AC264613. GB88 did not affect capsaicin-evoked hyperalgesia or inflammation. Trypsin, cathepsin-S and elastase increased [Ca(2+) ]i in rat nociceptors, which expressed PAR2. GB88 inhibited this activation of nociceptors by all three proteases, but did not affect capsaicin-evoked activation of nociceptors or inhibit the catalytic activity of the three proteases. CONCLUSIONS AND IMPLICATIONS:GB88 inhibits the capacity of canonical and biased protease agonists of PAR2 to cause nociception and inflammation.

The μ-opioid receptor (MOR) is a major regulator of gastrointestinal motility and secretion and mediates opiate-induced bowel dysfunction. Although MOR is of physiological and therapeutic importance to gut function, the cellular and subcellular distribution and regulation of MOR within the enteric nervous system are largely undefined. Herein, we defined the neurochemical coding of MOR-expressing neurons in the guinea pig gut and examined the effects of opioids on MOR trafficking and regulation. MOR expression was restricted to subsets of enteric neurons. In the stomach MOR was mainly localized to nitrergic neurons (∼88%), with some overlap with neuropeptide Y (NPY) and no expression by cholinergic neurons. These neurons are likely to have inhibitory motor and secretomotor functions. MOR was restricted to noncholinergic secretomotor neurons (VIP-positive) of the ileum and distal colon submucosal plexus. MOR was mainly detected in nitrergic neurons of the colon (nitric oxide synthase positive, 87%), with some overlap with choline acetyltransferase (ChAT). No expression of MOR by intrinsic sensory neurons was detected. [d-Ala(2), MePhe(4), Gly(ol)(5)]enkephalin (DAMGO), morphiceptin, and loperamide induced MOR endocytosis in myenteric neurons. After stimulation with DAMGO and morphiceptin, MOR recycled, whereas MOR was retained within endosomes following loperamide treatment. Herkinorin or the δ-opioid receptor agonist [d-Ala(2), d-Leu(5)]enkephalin (DADLE) did not evoke MOR endocytosis. In summary, we have identified the neurochemical coding of MOR-positive enteric neurons and have demonstrated differential trafficking of MOR in these neurons in response to established and putative MOR agonists.