Inflammatory proteases (mast cell tryptase and trypsins) cleave protease-activated receptor 2 (PAR2) on spinal afferent neurons and cause persistent inflammation and hyperalgesia by unknown ...mechanisms. We determined whether transient receptor potential vanilloid receptor 1 (TRPV1), a cation channel activated by capsaicin, protons, and noxious heat, mediates PAR2-induced hyperalgesia. PAR2 was coexpressed with TRPV1 in small- to medium-diameter neurons of the dorsal root ganglia (DRG), as determined by immunofluorescence. PAR2 agonists increased intracellular Ca2+ (Ca2+i) in these neurons in culture, and PAR2-responsive neurons also responded to the TRPV1 agonist capsaicin, confirming coexpression of PAR2 and TRPV1. PAR2 agonists potentiated capsaicin-induced increases in Ca2+i in TRPV1-transfected human embryonic kidney (HEK) cells and DRG neurons and potentiated capsaicin-induced currents in DRG neurons. Inhibitors of phospholipase C and protein kinase C (PKC) suppressed PAR2-induced sensitization of TRPV1-mediated changes in Ca2+i and TRPV1 currents. Activation of PAR2 or PKC induced phosphorylation of TRPV1 in HEK cells, suggesting a direct regulation of the channel. Intraplantar injection of a PAR2 agonist caused persistent thermal hyperalgesia that was prevented by antagonism or deletion of TRPV1. Coinjection of nonhyperalgesic doses of PAR2 agonist and capsaicin induced hyperalgesia that was inhibited by deletion of TRPV1 or antagonism of PKC. PAR2 activation also potentiated capsaicin-induced release of substance P and calcitonin gene-related peptide from superfused segments of the dorsal horn of the spinal cord, where they mediate hyperalgesia. We have identified a novel mechanism by which proteases that activate PAR2 sensitize TRPV1 through PKC. Antagonism of PAR2, TRPV1, or PKC may abrogate protease-induced thermal hyperalgesia.
Background & Aims
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The mechanisms underlying abdominal pain perception in irritable bowel syndrome (IBS) are poorly understood. Intestinal mast cell infiltration may perturb nerve function leading ...to symptom perception. We assessed colonic mast cell infiltration, mediator release, and spatial interactions with mucosal innervation and their correlation with abdominal pain in IBS patients.
Methods
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IBS patients were diagnosed according to Rome II criteria and abdominal pain quantified according to a validated questionnaire. Colonic mucosal mast cells were identified immunohistochemically and quantified with a computer-assisted counting method. Mast cell tryptase and histamine release were analyzed immunoenzymatically. Intestinal nerve to mast cell distance was assessed with electron microscopy.
Results
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Thirty-four out of 44 IBS patients (77%) showed an increased area of mucosa occupied by mast cells as compared with controls (9.2% ± 2.5% vs. 3.3 ± 0.8%, respectively;
P < 0.001). There was a 150% increase in the number of degranulating mast cells (4.76 ± 3.18/field vs. 2.42 ± 2.26/field, respectively;
P = 0.026). Mucosal content of tryptase was increased in IBS and mast cells spontaneously released more tryptase (3.22 ± 3.48 pmol/min/mg vs. 0.87 ± 0.65 pmol/min/mg, respectively;
P = 0.015) and histamine (339.7 ± 59.0 ng/g vs. 169.3 ± 130.6 ng/g, respectively;
P = 0.015). Mast cells located within 5 μm of nerve fibers were 7.14 ± 3.87/field vs. 2.27 ± 1.63/field in IBS vs. controls (
P < 0.001). Only mast cells in close proximity to nerves were significantly correlated with severity and frequency of abdominal pain/discomfort (
P < 0.001 and
P = 0.003, respectively).
Conclusions
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Colonic mast cell infiltration and mediator release in proximity to mucosal innervation may contribute to abdominal pain perception in IBS patients.
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.
Background & Aims Transient receptor potential ankyrin (TRPA) 1, an excitatory ion channel expressed by sensory neurons, mediates somatic and visceral pain in response to direct activation or noxious ...mechanical stimulation. Although the intestine is routinely exposed to irritant alimentary compounds and inflammatory mediators that activate TRPA1, there is no direct evidence for functional TRPA1 receptors on enteric neurons, and the effects of TRPA1 activation on intestinal function have not been determined. We characterized expression of TRPA1 by enteric neurons and determined its involvement in the control of intestinal contractility and transit. Methods TRPA1 expression was characterized by reverse-transcription polymerase chain reaction and immunofluorescence analyses. TRPA1 function was examined by Ca2+ imaging and by assays of contractile activity and transit. Results We detected TRPA1 messenger RNA in the mouse intestine and TRPA1 immunoreactivity in enteric neurons. The cecum and colon had immunoreactivity for neuronal TRPA1, but the duodenum did not. TRPA1 immunoreactivity was also detected in inhibitory motoneurons and descending interneurons, cholinergic neurons, and intrinsic primary afferent neurons. TRPA1 activators, including cinnamaldehyde, allyl isothiocyanate (AITC), and 4-hydroxynonenal, increased Ca2+ i in myenteric neurons. These were reduced by a TRPA1 antagonist (HC-030031) or deletion of Trpa1 . TRPA1 activation inhibited contractility of the segments of colon but not stomach or small intestine of Trpa1+/+ but not Trpa1−/− mice; this effect was reduced by tetrodotoxin or N G -nitro- l -arginine methyl ester. Administration of AITC by gavage did not alter gastric emptying or small intestinal transit, but luminal AITC inhibited colonic transit via TRPA1. Conclusions Functional TRPA1 is expressed by enteric neurons, and activation of neuronal TRPA1 inhibits spontaneous neurogenic contractions and transit of the colon.
IL‐6 contributes to the delayed resolution of inflammation and promotes a tumorgenic microenvironment in the pancreas of obese mice.
Obesity increases severity of acute pancreatitis and risk of ...pancreatic cancer. Pancreatitis and obesity are associated with elevated IL‐6, a cytokine involved in inflammation and tumorigenesis. We studied the role of IL‐6 in the response of lean and obese mice to pancreatitis induced by IL‐12 + IL‐18. Lean and diet‐induced obese (DIO) WT and IL‐6 KO mice and ob/ob mice pretreated with anti‐IL‐6 antibodies were evaluated at Days 1, 7, and 15 after induction of pancreatitis. Prolonged elevation of IL‐6 in serum and visceral adipose tissue was observed in DIO versus lean WT mice, whereas circulating sIL‐6R declined in DIO but not lean mice with pancreatitis. The severe inflammation and lethality of DIO mice were also observed in IL‐6 KO mice. However, the delayed resolution of neutrophil infiltration; sustained production of CXCL1, CXCL2, and CCL2; prolonged activation of STAT‐3; and induction of MMP‐7 in the pancreas, as well as heightened induction of serum amylase A of DIO mice, were blunted significantly in DIO IL‐6 KO mice. In DIO mice, production of OPN and TIMP‐1 was increased for a prolonged period, and this was mediated by IL‐6 in the liver but not the pancreas. Results obtained in IL‐6 KO mice were confirmed in ob/ob mice pretreated with anti‐IL‐6 antibodies. In conclusion, IL‐6 does not contribute to the increased severity of pancreatitis of obese mice but participates in delayed recovery from acute inflammation and may favor development of a protumorigenic environment through prolonged activation of STAT‐3, induction of MMP‐7, and sustained production of chemokines.
Certain serine proteases signal to cells by cleaving protease-activated receptors (PARs) and thereby regulate hemostasis,
inflammation, pain and healing. However, in many tissues the proteases that ...activate PARs are unknown. Although pancreatic
trypsin may be a physiological agonist of PAR 2 and PAR 4 in the small intestine and pancreas, these receptors are expressed by cells not normally exposed pancreatic trypsin. We investigated
whether extrapancreatic forms of trypsin are PAR agonists. Epithelial cells lines from prostate, colon, and airway and human
colonic mucosa expressed mRNA encoding PAR 2 , trypsinogen IV, and enteropeptidase, which activates the zymogen. Immunoreactive trypsinogen IV was detected in vesicles
in these cells. Trypsinogen IV was cloned from PC-3 cells and expressed in CHO cells, where it was also localized to cytoplasmic
vesicles. We expressed trypsinogen IV with an N-terminal Igκ signal peptide to direct constitutive secretion and allow enzymatic
characterization. Treatment of conditioned medium with enteropeptidase reduced the apparent molecular mass of trypsinogen
IV from 36 to 30 kDa and generated enzymatic activity, consistent with formation of trypsin IV. In contrast to pancreatic
trypsin, trypsin IV was completely resistant to inhibition by polypeptide inhibitors. Exposure of cell lines expressing PAR 2 and PAR 4 to trypsin IV increased Ca 2+ i and strongly desensitized cells to PAR agonists, whereas there were no responses in cells lacking these receptors. Thus,
trypsin IV is a potential agonist of PAR 2 and PAR 4 in epithelial tissues where its resistance to endogenous trypsin inhibitors may permit prolonged signaling.
Mast cells that are in close proximity to autonomic and enteric nerves release several mediators that cause neuronal hyperexcitability.
This study examined whether mast cell tryptase evokes acute and ...long-term hyperexcitability in submucosal neurons from the
guinea-pig ileum by activating proteinase-activated receptor 2 (PAR2) on these neurons. We detected the expression of PAR2
in the submucosal plexus using RT-PCR. Most submucosal neurons displayed PAR2 immunoreactivity, including those colocalizing
VIP. Brief (minutes) application of selective PAR2 agonists, including trypsin, the activating peptide SL-NH 2 and mast cell tryptase, evoked depolarizations of the submucosal neurons, as measured with intracellular recording techniques.
The membrane potential returned to resting values following washout of agonists, but most neurons were hyperexcitable for
the duration of recordings (> 30 minâhours) and exhibited an increased input resistance and amplitude of fast EPSPs. Trypsin,
in the presence of soybean trypsin inhibitor, and the reverse sequence of the activating peptide (LR-NH 2 ) had no effect on neuronal membrane potential or long-term excitability. Degranulation of mast cells in the presence of antagonists
of established excitatory mast cell mediators (histamine, 5-HT, prostaglandins) also caused depolarization, and following
washout of antigen, long-term excitation was observed. Mast cell degranulation resulted in the release of proteases, which
desensitized neurons to other agonists of PAR2. Our results suggest that proteases from degranulated mast cells cleave PAR2
on submucosal neurons to cause acute and long-term hyperexcitability. This signalling pathway between immune cells and neurons
is a previously unrecognized mechanism that could contribute to chronic alterations in visceral function.
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.
Mechanisms that arrest G-protein-coupled receptor (GPCR) signaling prevent uncontrolled stimulation that could cause disease. Although uncoupling from heterotrimeric G-proteins, which transiently ...arrests signaling, is well described, little is known about the mechanisms that permanently arrest signaling. Here we reported on the mechanisms that terminate signaling by protease-activated receptor 2 (PAR2), which mediated the proinflammatory and nociceptive actions of proteases. Given its irreversible mechanism of proteolytic activation, PAR2 is a model to study the permanent arrest of GPCR signaling. By immunoprecipitation and immunoblotting, we observed that activated PAR2 was mono-ubiquitinated. Immunofluorescence indicated that activated PAR2 translocated from the plasma membrane to early endosomes and lysosomes where it was degraded, as determined by immunoblotting. Mutant PAR2 lacking intracellular lysine residues (PAR2Δ14K/R) was expressed at the plasma membrane and signaled normally but was not ubiquitinated. Activated PAR2 Δ14K/R internalized but was retained in early endosomes and avoided lysosomal degradation. Activation of wild type PAR2 stimulated tyrosine phosphorylation of the ubiquitin-protein isopeptide ligase c-Cbl and promoted its interaction with PAR2 at the plasma membrane and in endosomes in an Src-dependent manner. Dominant negative c-Cbl lacking the ring finger domain inhibited PAR2 ubiquitination and induced retention in early endosomes, thereby impeding lysosomal degradation. Although wild type PAR2 was degraded, and recovery of agonist responses required synthesis of new receptors, lysine mutation and dominant negative c-Cbl impeded receptor ubiquitination and degradation and allowed PAR2 to recycle and continue to signal. Thus, c-Cbl mediated ubiquitination and lysosomal degradation of PAR2 to irrevocably terminate signaling by this and perhaps other GPCRs.
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