Ian Spreadbury

Transient receptor potential ankyrin-1 has a major role in mediating visceral pain in mice

The excitatory ion channel transient receptor potential ankyrin-1 (TRPA1) is prominently expressed by primary afferent neurons and is a mediator of inflammatory pain. Inflammatory agents can directly activate [e.g., hydroxynonenal (HNE), prostaglandin metabolites] or indirectly sensitize [e.g., agonists of protease-activated receptor (PAR(2))] TRPA1 to induce somatic pain and hyperalgesia. However, the contribution of TRPA1 to visceral pain is unknown. We investigated the role of TRPA1 in visceral hyperalgesia by measuring abdominal visceromotor responses (VMR) to colorectal distention (CRD) after intracolonic administration of TRPA1 agonists [mustard oil (MO), HNE], sensitizing agents [PAR(2) activating peptide (PAR(2)-AP)], and the inflammatory agent trinitrobenzene sulfonic acid (TNBS) in trpa1(+/+) and trpa1(-/-) mice. Sensory neurons innervating the colon, identified by retrograde tracing, coexpressed immunoreactive TRPA1, calcitonin gene-related peptide, and substance P, expressed TRPA1 mRNA and responded to MO with depolarizing currents. Intracolonic MO and HNE increased VMR to CRD and induced immunoreactive c-fos in spinal neurons in trpa1+/+ but not in trpa1(-/-) mice. Intracolonic PAR(2)-AP induced mechanical hyperalgesia in trpa1+/+ but not in trpa1(-/-) mice. TNBS-induced colitis increased in VMR to CRD and induced c-fos in spinal neurons in trpa1(+/+) but not in trpa1(-/-) mice. Thus TRPA1 is expressed by colonic primary afferent neurons. Direct activation of TRPA1 causes visceral hyperalgesia, and TRPA1 mediates PAR(2)-induced hyperalgesia. TRPA1 deletion markedly reduces colitis-induced mechanical hyperalgesia in the colon. Our results suggest that TRPA1 has a major role in visceral nociception and may be a therapeutic target for colonic inflammatory pain.

BACTERIAL CELL PRODUCTS SIGNAL TO MOUSE COLONIC NOCICEPTIVE DORSAL ROOT GANGLIA NEURONS

This study examined whether bacterial cell products that might gain access to the intestinal interstitium could activate mouse colonic nociceptive dorsal root ganglion (DRG) neurons using molecular and electrophysiological recording techniques. Colonic projecting neurons were identified by using the retrograde tracer fast blue and Toll-like receptor (TLR) 1, 2, 3, 4, 5, 6, 9, adapter proteins Md-1 and Md-2, and MYD88 mRNA expression was observed in laser-captured fast blue-labeled neurons. Ultrapure LPS 1 microg/ml phosphorylated p65 NF-kappaB subunits increased transcript for TNF-alpha and IL-1beta and stimulated secretion of TNF-alpha from acutely dissociated DRG neurons. In current-clamp recordings from colonic DRG neurons, chronic incubation (24 h) of ultrapure LPS significantly increased neuronal excitability. In acute studies, 3-min superfusion of standard-grade LPS (3-30 microg/ml) reduced the rheobase by up to 40% and doubled action potential discharge rate. The LPS effects were not significantly different in TLR4 knockout mice compared with wild-type mice. In contrast to standard-grade LPS, acute application of ultrapure LPS did not increase neuronal excitability in whole cell recordings or afferent nerve recordings from colonic mesenteric nerves. However, acute application of bacterial lysate (Escherichia coli NLM28) increased action potential discharge over 60% compared with control medium. Moreover, lysate also activated afferent discharge from colonic mesenteric nerves, and this was significantly increased in chronic dextran sulfate sodium salt mice. These data demonstrate that bacterial cell products can directly activate colonic DRG neurons leading to production of inflammatory cytokines by neurons and increased excitability. Standard-grade LPS may also have actions independent of TLR signaling.

Sensitization of peripheral sensory nerves by mediators from colonic biopsies of diarrhea-predominant irritable bowel syndrome patients: a role for PAR2.

OBJECTIVES:This study examined whether mediators from biopsies of human irritable bowel syndrome (IBS) colons alter intrinsic excitability of colonic nociceptive dorsal root ganglion (DRG) neurons by a protease activated receptor 2 (PAR2)-mediated mechanism.METHODS:Colonic mucosal biopsies from IBS patients with constipation (IBS-C) or diarrhea (IBS-D) and from healthy controls were incubated in medium, and supernatants were collected. Small-diameter mouse colonic DRG neurons were incubated in supernatants overnight and perforated patch current-clamp recordings obtained. Measurements of rheobase and action potential discharge at twice rheobase were compared between IBS and controls to assess differences in intrinsic excitability.RESULTS:Supernatants from IBS-D patients elicited a marked increase in neuronal excitability compared with controls. These changes were consistent among individual patients but the relative contribution of rheobase and action potential discharge varied. In contrast, no differences in neuronal excitability were seen with IBS-C patient supernatants. The increased excitability seen with IBS-D supernatant was not observed in PAR2 knockout mice. A cysteine protease inhibitor, which had no effect on the pronociceptive actions of a serine protease, inhibited the proexcitatory actions of IBS-D supernatant.CONCLUSIONS:Soluble mediators from colonic biopsies from IBS-D but not IBS-C patients sensitized colonic nociceptive DRG neurons, suggesting differences between these two groups. PAR2 signaling plays a role in this action and this protease signaling pathway could provide novel biomarkers and therapeutic targets for treatment.

Mediators of Chronic Stress and Tissue Proteases Interact to Potentiate the Excitability of Colonic Nociceptive Dorsal Root Ganglia Neurons in a Model of Post-Infectious IBS

To examine mechanisms underlying the role of chronic stress in post-infectious irritable bowel syndrome (PI-IBS), we previously studied the effect of chronic stress and prior infectious colitis in the C. rodentium infected mouse (day 30 post infection), a model of human E. coli self-limiting colitis (NGM A:257,2010). Water avoidance stress (WAS; 1 hr on days 21-30 following infection) increased stress hormones (corticosterone and epinephrine), excitability of colonic nociceptive DRG neurons and colonic multi-unit afferent firing, compared to post-infected animals alone The current study examined pathways underlying this stress-post-infection effect using patch clamp recordings from Fast Blue labeled colonic DRG neurons to measure changes in excitability (rheobase (Rh) and/or increases in action potential discharge (APD)). At day 30, infection had resolved and histopathology was normal. However, tissue trypsin-like activity (>10 fold; p<0.01) and serine and cysteine proteases (~ 0.5-1 fold; p < 0.05) were elevated in unstressed post-infected animals. In patch clamp studies, excitability of neurons incubated in colonic tissue supernatants from unstressed post-infected mice was significantly inhibited by a global protease inhibitor (post-infected Rh = 61.5 +/7.5 pA vs. post-infected + inhibitor = 96.3 +/11.5 pA; p = 0.0162 and post-infected APD = 4.4 +/0.7 vs. post-infected + inhibitor 1.8 +/0.4; p = 0.007). In contrast, neuronal excitability with supernatants from uninfected WAS animals was not altered by the protease inhibitor and excitability did not differ between neurons exposed toWAS or control supernatants. Colonic histopathological scoring andWestern blots of tight junction proteins (occludin) were also not different between WAS and control groups. We therefore tested whether stress hormones could signal directly to DRG neurons. Labeled neurons were isolated using laser captured microdissection and corticosterone and β2 receptor mRNA identified by PCR. In patch clamp studies, colonic DRG neurons incubated in epinephrine (5 nM) and corticosterone (1 μM) were hyperexcitable compared to controls (epinephrine /corticosterone Rh = 48.6 +/10.9 pA vs control = 85.8 +/12.3 pA, p = 0.03 and epinephrine/corticosterone APD = 2.6 +/0.4 vs. control = 1.6 +/0.2; p = 0.04). Compared to controls, incubating neurons with protease activating receptor2 activating peptide (PAR2-AP; 30 μM) or WAS alone had no effect on Rh. However, WAS and PAR2-AP (30 μM) combined markedly decreased Rh (control + PAR2-AP = 85.8 +/12.3 pA vs. WAS + PAR2-AP 19.2 +/3.5 pA, p = 0.002). These data suggest tissue proteases and circulating stress hormones converge on DRG nociceptive neurons to increase sensory signaling from the colon. Moreover, this interaction enables sub-threshold levels of proteases to enhance peripheral sensory signaling.