Corticotropin-releasing factor receptor 2 mediates sex-specific cellular stress responses.

Although females suffer twice as much as males from stress-related disorders, sex-specific participating and pathogenic cellular stress mechanisms remain uncharacterized. Using corticotropin-releasing factor receptor 2-deficient (Crhr2-/-) and wild-type (WT) mice, we show that CRF receptor type 2 (CRF2) and its high-affinity ligand, urocortin 1 (Ucn1), are key mediators of the endoplasmic reticulum (ER) stress response in a murine model of acute pancreatic inflammation. Ucn1 was expressed de novo in acinar cells of male, but not female WT mice during acute inflammation. Upon insult, acinar Ucn1 induction was markedly attenuated in male but not female Crhr2-/- mice. Crhr2-/- mice of both sexes show exacerbated acinar cell inflammation and necrosis. Electron microscopy showed mild ER damage in WT male mice and markedly distorted ER structure in Crhr2-/- male mice during pancreatitis. WT and Crhr2-/- female mice showed similarly distorted ER ultrastructure that was less severe than distortion seen in Crhr2-/- male mice. Damage in ER structure was accompanied by increased ubiquitination, peIF2, and mistargeted localization of vimentin in WT mice that was further exacerbated in Crhr2-/- mice of both sexes during pancreatitis. Exogenous Ucn1 rescued many aspects of histological damage and cellular stress response, including restoration of ER structure in male WT and Crhr2-/- mice, but not in females. Instead, females often showed increased damage. Thus, specific cellular pathways involved in coping and resolution seem to be distinct to each sex. Our results demonstrate the importance of identifying sex-specific pathogenic mechanisms and their value in designing effective therapeutics.

Local injection of dsRNA targeting calcitonin receptor-like receptor (CLR) ameliorates Clostridium difficile toxin A-induced ileitis.

Enteritis caused by Clostridium difficile toxin (Tx) is a nosocomial disease of increasing clinical concern, but the local mediators of C. difficile TxA inflammation are unknown. The potent vasodilator calcitonin gene-related peptide mediates neurogenic inflammation via the calcitonin receptor-like receptor (CLR). Here we examined the ileum-specific effects of reducing CLR on TxA ileitis by local preinjection of double-stranded RNAs. Treatment with CLR dsRNA for 7 d decreased CLR immunoreactivity, whereas treatment with non-CLR dsRNA did not. Subsequent injection of TxA in the same location increased CLR in rats treated with non-CLR dsRNA but not in rats treated with CLR dsRNA, documenting that local injection of dsRNA is effective in preventing the increase in CLR immunoreactivity in response to local TxA. After non-CLR dsRNA pretreatment, TxA induced robust intestinal secretion, myeloperoxidase activity, and histopathologic indications of inflammation including epithelial damage, congestion, neutrophil infiltration, loss of mucin from goblet cells, and increase in mast cell numbers. After CLR dsRNA pretreatment, TxA-induced changes in intestinal secretion and histopathologic inflammation were improved, including normal mucin staining and fewer resident mast cells. Loss of CLR prevented TxA-mediated activation of NF-κB and concomitant increases in pERK1/2 and TNF-α mRNA. Locally produced CLR plays a proinflammatory role in TxA ileitis via MAPK signaling and TNF-α. The results reported here strongly suggest that a local injection of dsRNA targeting CLR could be an effective local therapeutic approach at the inflammation site in the treatment of a growing, clinically relevant hospital-acquired disease, C. difficile infection.

Active cathepsins B, L, and S in murine and human pancreatitis.

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.

Rosiglitazone improves survival and hastens recovery from pancreatic inflammation in obese mice.

Obesity increases severity of acute pancreatitis (AP) by unclear mechanisms. We investigated the effect of the PPAR-gamma agonist rosiglitazone (RGZ, 0.01% in the diet) on severity of AP induced by administration of IL-12+ IL-18 in male C57BL6 mice fed a low fat (LFD) or high fat diet (HFD), under the hypothesis that RGZ would reduce disease severity in HFD-fed obese animals. In both LFD and HFD mice without AP, RGZ significantly increased body weight and % fat mass, with significant upregulation of adiponectin and suppression of erythropoiesis. In HFD mice with AP, RGZ significantly increased survival and hastened recovery from pancreatic inflammation, as evaluated by significantly improved pancreatic histology, reduced saponification of visceral adipose tissue and less severe suppression of erythropoiesis at Day 7 post-AP. This was associated with significantly lower circulating and pancreas-associated levels of IL-6, Galectin-3, osteopontin and TIMP-1 in HFD + RGZ mice, particularly at Day 7 post-AP. In LFD mice with AP, RGZ significantly worsened the degree of intrapancreatic acinar and fat necrosis as well as visceral fat saponification, without affecting other parameters of disease severity or inflammation. Induction of AP lead to major suppression of adiponectin levels at Day 7 in both HFD and HFD + RGZ mice. In conclusion, RGZ prevents development of severe AP in obese mice even though it significantly increases adiposity, indicating that obesity can be dissociated from AP severity by improving the metabolic and inflammatory milieu. However, RGZ worsens selective parameters of AP severity in LFD mice.

Localization of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) in human gastrointestinal tract.

Calcitonin gene-related peptide (CGRP) exerts its diverse effects on vasodilation, nociception, secretion, and motor function through a heterodimeric receptor comprising of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). Despite the importance of CLR·RAMP1 in human disease, little is known about its distribution in the human gastrointestinal (GI) tract, where it participates in inflammation and pain. In this study, we determined that CLR and RAMP1 mRNAs are expressed in normal human stomach, ileum and colon by RT-PCR. We next characterized antibodies that we generated to rat CLR and RAMP1 in transfected HEK cells. Having characterized these antibodies in vitro, we then localized CLR-, RAMP1-, CGRP- and intermedin-immunoreactivity (IMD-IR) in various human GI segments. In the stomach, nerve bundles in the myenteric plexus and nerve fibers throughout the circular and longitudinal muscle had prominent CLR-IR. In the proximal colon and ileum, CLR was found in nerve varicosities of the myenteric plexus and surrounding submucosal neurons. Interestingly, CGRP expressing fibers did not co-localize, but were in close proximity to CLR. However, CLR and RAMP1, the two subunits of a functional CGRP receptor were clearly localized in myenteric plexus, where they may form functional cell-surface receptors. IMD, another member of calcitonin peptide family was also found in close proximity to CLR, and like CGRP, did not co-localize with either CLR or RAMP1 receptors. Thus, CGRP and IMD appear to be released locally, where they can mediate their effect on their receptors regulating diverse functions such as inflammation, pain and motility.

Role of IL-6 in the resolution of pancreatitis in 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.

Transient receptor potential ankyrin 1 is expressed by inhibitory motoneurons of the mouse intestine.

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 Ca(2+) 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 [Ca(2+)](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.

Serine proteases mediate inflammatory pain in acute pancreatitis.

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.

Transient receptor potential ion channels V4 and A1 contribute to pancreatitis pain in mice.

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.

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.

Transient receptor potential vanilloid 4 mediates protease activated receptor 2-induced sensitization of colonic afferent nerves and visceral hyperalgesia.

Protease-activated receptor (PAR(2)) is expressed by nociceptive neurons and activated during inflammation by proteases from mast cells, the intestinal lumen, and the circulation. Agonists of PAR(2) cause hyperexcitability of intestinal sensory neurons and hyperalgesia to distensive stimuli by unknown mechanisms. We evaluated the role of the transient receptor potential vanilloid 4 (TRPV4) in PAR(2)-induced mechanical hyperalgesia of the mouse colon. Colonic sensory neurons, identified by retrograde tracing, expressed immunoreactive TRPV4, PAR(2), and calcitonin gene-related peptide and are thus implicated in nociception. To assess nociception, visceromotor responses (VMR) to colorectal distension (CRD) were measured by electromyography of abdominal muscles. In TRPV4(+/+) mice, intraluminal PAR(2) activating peptide (PAR(2)-AP) exacerbated VMR to graded CRD from 6-24 h, indicative of mechanical hyperalgesia. PAR(2)-induced hyperalgesia was not observed in TRPV4(-/-) mice. PAR(2)-AP evoked discharge of action potentials from colonic afferent neurons in TRPV4(+/+) mice, but not from TRPV4(-/-) mice. The TRPV4 agonists 5',6'-epoxyeicosatrienoic acid and 4alpha-phorbol 12,13-didecanoate stimulated discharge of action potentials in colonic afferent fibers and enhanced current responses recorded from retrogradely labeled colonic dorsal root ganglia neurons, confirming expression of functional TRPV4. PAR(2)-AP enhanced these responses, indicating sensitization of TRPV4. Thus TRPV4 is expressed by primary spinal afferent neurons innervating the colon. Activation of PAR(2) increases currents in these neurons, evokes discharge of action potentials from colonic afferent fibers, and induces mechanical hyperalgesia. These responses require the presence of functional TRPV4. Therefore, TRPV4 is required for PAR(2)-induced mechanical hyperalgesia and excitation of colonic afferent neurons.

Calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), and calcitonin gene-related peptide (CGRP) immunoreactivity in the rat trigeminovascular system: differences between peripheral and central CGRP receptor distribution.

Calcitonin gene-related peptide (CGRP) is a key mediator in primary headaches including migraine. Animal models of meningeal nociception demonstrate both peripheral and central CGRP effects; however, the target structures remain unclear. To study the distribution of CGRP receptors in the rat trigeminovascular system we used antibodies recognizing two components of the CGRP receptor, the calcitonin receptor-like receptor (CLR) and the receptor activity-modifying protein 1 (RAMP1). In the cranial dura mater, CLR and RAMP1 immunoreactivity (-ir) was found within arterial blood vessels, mononuclear cells, and Schwann cells, but not sensory axons. In the trigeminal ganglion, besides Schwann and satellite cells, CLR- and RAMP1-ir was found in subpopulations of CGRP-ir neurons where colocalization of CGRP- and RAMP1-ir was very rare ( approximately 0.6%). CLR- and RAMP1-ir was present on central, but not peripheral, axons. In the spinal trigeminal nucleus, CLR- and RAMP1-ir was localized to "glomerular structures," partly colocalized with CGRP-ir. However, CLR- and RAMP1-ir was lacking in central glia and neuronal cell bodies. We conclude that CGRP receptors are associated with structural targets of known CGRP effects (vasodilation, mast cell degranulation) and targets of unknown function (Schwann cells). In the spinal trigeminal nucleus, CGRP receptors are probably located on neuronal processes, including primary afferent endings, suggesting involvement in presynaptic regulation of nociceptive transmission. Thus, in the trigeminovascular system CGRP receptor localization suggests multiple targets for CGRP in the pathogenesis of primary headaches.

Substance P released by TRPV1-expressing neurons produces reactive oxygen species that mediate ethanol-induced gastric injury.

Although neurokinin 1 receptor antagonists prevent ethanol (EtOH)-induced gastric lesions, the mechanisms by which EtOH releases substance P (SP) and SP damages the mucosa are unknown. We hypothesized that EtOH activates transient receptor potential vanilloid 1 (TRPV1) on sensory nerves to release SP, which stimulates epithelial neurokinin 1 receptors to generate damaging reactive oxygen species (ROS). SP release was assayed in the mouse stomach, ROS were detected using dichlorofluorescein diacetate, and neurokinin 1 receptors were localized by immunofluorescence. EtOH-induced SP release was prevented by TRPV1 antagonism. High dose EtOH caused lesions, and TRPV1 or neurokinin 1 receptor antagonism and neurokinin 1 receptor deletion inhibited lesion formation. Coadministration of low, innocuous doses of EtOH and SP caused lesions by a TRPV1-independent but neurokinin 1 receptor-dependent process. EtOH, capsaicin, and SP stimulated generation of ROS by superficial gastric epithelial cells expressing neurokinin 1 receptors by a neurokinin 1 receptor-dependent mechanism. ROS scavengers prevented lesions induced by a high EtOH dose or a low EtOH dose plus SP. Gastric lesions are caused by an initial detrimental effect of EtOH, which is damaging only if associated with TRPV1 activation, SP release from sensory nerves, stimulation of neurokinin 1 receptors on epithelial cells, and ROS generation.

Protease-activated receptor 2, dipeptidyl peptidase I, and proteases mediate Clostridium difficile toxin A enteritis.

BACKGROUND & AIMS: We studied the role of protease-activated receptor 2 (PAR(2)) and its activating enzymes, trypsins and tryptase, in Clostridium difficile toxin A (TxA)-induced enteritis. METHODS: We injected TxA into ileal loops in PAR(2) or dipeptidyl peptidase I (DPPI) knockout mice or in wild-type mice pretreated with tryptase inhibitors (FUT-175 or MPI-0442352) or soybean trypsin inhibitor. We examined the effect of TxA on expression and activity of PAR(2) and trypsin IV messenger RNA in the ileum and cultured colonocytes. We injected activating peptide (AP), trypsins, tryptase, and p23 in wild-type mice, some pretreated with the neurokinin 1 receptor antagonist SR140333. RESULTS: TxA increased fluid secretion, myeloperoxidase activity in fluid and tissue, and histologic damage. PAR(2) deletion decreased TxA-induced ileitis, reduced luminal fluid secretion by 20%, decreased tissue and fluid myeloperoxidase by 50%, and diminished epithelial damage, edema, and neutrophil infiltration. DPPI deletion reduced secretion by 20% and fluid myeloperoxidase by 55%. In wild-type mice, FUT-175 or MPI-0442352 inhibited secretion by 24%-28% and tissue and fluid myeloperoxidase by 31%-71%. Soybean trypsin inhibitor reduced secretion to background levels and tissue myeloperoxidase by up to 50%. TxA increased expression of PAR(2) and trypsin IV in enterocytes and colonocytes and caused a 2-fold increase in Ca(2+) responses to PAR(2) AP. AP, tryptase, and trypsin isozymes (trypsin I/II, trypsin IV, p23) caused ileitis. SR140333 prevented AP-induced ileitis. CONCLUSIONS: PAR(2) and its activators are proinflammatory in TxA-induced enteritis. TxA stimulates existing PAR(2) and up-regulates PAR(2) and activating proteases, and PAR(2) causes inflammation by neurogenic mechanisms.

Role of calcitonin receptor-like receptor in colonic motility and inflammation.

Calcitonin gene-related peptide (CGRP) mediates neurogenic inflammation and modulates intestinal motility. The CGRP receptor is a heterodimer of calcitonin receptor-like receptor (CLR) and receptor-associated modifying protein 1. We used RNA interference to elucidate the specific role of CLR in colonic motility and inflammation. Intramural injection of double-stranded RNA (dsRNA) against CLR (dsCLR) into the colonic wall at two sites caused the spatial and temporal downregulation of CLR in the colon within 1 day of dsRNA injection. Knockdown of CLR persisted for 7-9 days, and the effect of knockdown spread to approximately 2 cm proximal and distal to the injection sites, whereas control dsRNA injection did not affect CLR expression. Measurement of isometric contractions of isolated colonic muscle segments revealed that in control dsRNA-injected rats, CGRP abrogated contractions entirely and decreased resting muscular tone, whereas in dsCLR-injected rats, CGRP decreased muscle tone but slow-wave contractions of varying amplitude persisted. In trinitrobenzene sulfonic acid-induced colitis, rats with knockdown of CLR displayed a significantly greater degree of edema and necrosis than saline- or control dsRNA-injected rats. Levels of the proinflammatory cytokines TNF-alpha and IL-6 were markedly upregulated by trinitrobenzene sulfonic acid treatment. TNF-alpha mRNA levels were further increased in CLR knockdown rats, whereas levels of IL-6 were unaltered. Thus this study demonstrates that CLR is a functional receptor for CGRP.

Post-endocytic sorting of calcitonin receptor-like receptor and receptor activity-modifying protein 1.

Calcitonin receptor-like receptor (CLR) and the receptor activity-modifying protein 1 (RAMP1) comprise a receptor for calcitonin gene-related peptide (CGRP). Although CGRP induces endocytosis of CLR/RAMP1, little is known about post-endocytic sorting of these proteins. We observed that the duration of stimulation with CGRP markedly affected post-endocytic sorting of CLR/RAMP1. In HEK and SK-N-MC cells, transient stimulation (10(-7) M CGRP, 1 h), induced CLR/RAMP1 recycling with similar kinetics (2-6 h), demonstrated by labeling receptors in living cells with antibodies to extracellular epitopes. Recycling of CLR/RAMP1 correlated with resensitization of CGRP-induced increases in [Ca(2+)](i). Cycloheximide did not affect resensitization, but bafilomycin A(1), an inhibitor of vacuolar H(+)-ATPases, abolished resensitization. Recycling CLR and RAMP1 were detected in endosomes containing Rab4a and Rab11a, and expression of GTPase-defective Rab4aS22N and Rab11aS25N inhibited resensitization. After sustained stimulation (10(-7) M CGRP, >2 h), CLR/RAMP1 trafficked to lysosomes. RAMP1 was degraded approximately 4-fold more rapidly than CLR (RAMP1, 45% degradation, 5 h; CLR, 54% degradation, 16 h), determined by Western blotting. Inhibitors of lysosomal, but not proteasomal, proteases prevented degradation. Sustained stimulation did not induce detectable mono- or polyubiquitination of CLR or RAMP1, determined by immunoprecipitation and Western blotting. Moreover, a RAMP1 mutant lacking the only intracellular lysine (RAMP1K142R) internalized and was degraded normally. Thus, after transient stimulation with CGRP, CLR and RAMP1 traffic from endosomes to the plasma membrane, which mediates resensitization. After sustained stimulation, CLR and RAMP1 traffic from endosomes to lysosomes by ubiquitin-independent mechanisms, where they are degraded at different rates.

Ubiquitin-dependent down-regulation of the neurokinin-1 receptor.

Transient stimulation with substance P (SP) induces endocytosis and recycling of the neurokinin-1 receptor (NK(1)R). The effects of sustained stimulation by high concentrations of SP on NK(1)R trafficking and Ca(2+) signaling, as may occur during chronic inflammation and pain, are unknown. Chronic exposure to SP (100 nm, 3 h) completely desensitized Ca(2+) signaling by wild-type NK(1)R (NK(1)Rwt). Resensitization occurred after 16 h, and cycloheximide prevented resensitization, implicating new receptor synthesis. Lysine ubiquitination of G-protein-coupled receptors is a signal for their trafficking and degradation. Lysine-deficient mutant receptors (NK(1)RDelta5K/R, C-terminal tail lysines; and NK(1)RDelta10K/R, all intracellular lysines) were expressed at the plasma membrane and were functional because they responded to SP by endocytosis and by mobilization of Ca(2+) ions. SP desensitized NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. However, NK(1)RDelta5K/R and NK(1)RDelta10K/R resensitized 4-8-fold faster than NK(1)Rwt by cycloheximide-independent mechanisms. NK(1)RDelta325 (a naturally occurring truncated variant) showed incomplete desensitization, followed by a marked sensitization of signaling. Upon labeling receptors in living cells using antibodies to extracellular epitopes, we observed that SP induced endocytosis of NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. After 4 h in SP-free medium, NK(1)RDelta5K/R and NK(1)RDelta10K/R recycled to the plasma membrane, whereas NK(1)Rwt remained internalized. SP induced ubiquitination of NK(1)Rwt and NK(1)RDelta5K/R as determined by immunoprecipitation under nondenaturing and denaturing conditions and detected with antibodies for mono- and polyubiquitin. NK(1)RDelta10K/R was not ubiquitinated. Whereas SP induced degradation of NK(1)Rwt, NK(1)RDelta5K/R and NK(1)RDelta10K/R showed approximately 50% diminished degradation. Thus, chronic stimulation with SP induces ubiquitination of the NK(1)R, which mediates its degradation and down-regulation.

Protease-activated receptor 2 sensitizes TRPV1 by protein kinase Cepsilon- and A-dependent mechanisms in rats and mice.

Proteases that are released during inflammation and injury cleave protease-activated receptor 2 (PAR2) on primary afferent neurons to cause neurogenic inflammation and hyperalgesia. PAR2-induced thermal hyperalgesia depends on sensitization of transient receptor potential vanilloid receptor 1 (TRPV1), which is gated by capsaicin, protons and noxious heat. However, the signalling mechanisms by which PAR2 sensitizes TRPV1 are not fully characterized. Using immunofluorescence and confocal microscopy, we observed that PAR2 was colocalized with protein kinase (PK) Cepsilon and PKA in a subset of dorsal root ganglia neurons in rats, and that PAR2 agonists promoted translocation of PKCepsilon and PKA catalytic subunits from the cytosol to the plasma membrane of cultured neurons and HEK 293 cells. Subcellular fractionation and Western blotting confirmed this redistribution of kinases, which is indicative of activation. Although PAR2 couples to phospholipase Cbeta, leading to stimulation of PKC, we also observed that PAR2 agonists increased cAMP generation in neurons and HEK 293 cells, which would activate PKA. PAR2 agonists enhanced capsaicin-stimulated increases in [Ca2+]i and whole-cell currents in HEK 293 cells, indicating TRPV1 sensitization. The combined intraplantar injection of non-algesic doses of PAR2 agonist and capsaicin decreased the latency of paw withdrawal to radiant heat in mice, indicative of thermal hyperalgesia. Antagonists of PKCepsilon and PKA prevented sensitization of TRPV1 Ca2+ signals and currents in HEK 293 cells, and suppressed thermal hyperalgesia in mice. Thus, PAR2 activates PKCepsilon and PKA in sensory neurons, and thereby sensitizes TRPV1 to cause thermal hyperalgesia. These mechanisms may underlie inflammatory pain, where multiple proteases are generated and released.

Calcitonin gene-related peptide partially mediates nociception in acute experimental pancreatitis.

BACKGROUND: The mechanism by which pancreatitis causes pain is unknown. The neuropeptide calcitonin gene-related peptide (CGRP) is released after sensory nerve activation and promotes nociceptive signaling in models of visceral pain. We hypothesized that acute pancreatitis leads to the activation of pancreatic sensory neurons that release CGRP in the dorsal horn of the spinal cord. This signal is ultimately transmitted to the brain, and pain is sensed. METHODS: To induce pancreatitis, rats were injected with l-arginine (500 mg/kg) intraperitoneally or saline (control). Pancreatitis was confirmed by measuring serum amylase and evaluating pancreatic histology. Activation of nociceptive pathways was evaluated by counting Fos-like immunoreactive nuclei (FLI) in the dorsal horn of the spinal cord at T3-L1. Some animals received the CGRP antagonist CGRP(8-37) (50 microg intrathecally) 2 hours before perfusion. Animals were compared using a 2-tailed t test. RESULTS: l-Arginine treatment induced acute necrotizing pancreatitis in the rat at 24 hours. l-Arginine (24 hours) increased FLI in the dorsal horn of the spinal cord, with a peak effect at L1. Intrathecal administration of CGRP(8-37) significantly decreased the number of FLI nuclei in the dorsal horn of the spinal cord in T11-L1. CONCLUSIONS: Nociception in the l-arginine model of acute pancreatitis is partially mediated by the release of CGRP in the dorsal horn of the spinal cord. Antagonism of CGRP or its receptors may be useful in treating pain from acute pancreatitis.