Fiona A. Russell

Calcitonin Gene-Related Peptide: Physiology and Pathophysiology

Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide. Discovered 30 years ago, it is produced as a consequence of alternative RNA processing of the calcitonin gene. CGRP has two major forms (α and β). It belongs to a group of peptides that all act on an unusual receptor family. These receptors consist of calcitonin receptor-like receptor (CLR) linked to an essential receptor activity modifying protein (RAMP) that is necessary for full functionality. CGRP is a highly potent vasodilator and, partly as a consequence, possesses protective mechanisms that are important for physiological and pathological conditions involving the cardiovascular system and wound healing. CGRP is primarily released from sensory nerves and thus is implicated in pain pathways. The proven ability of CGRP antagonists to alleviate migraine has been of most interest in terms of drug development, and knowledge to date concerning this potential therapeutic area is discussed. Other areas covered, where there is less information known on CGRP, include arthritis, skin conditions, diabetes, and obesity. It is concluded that CGRP is an important peptide in mammalian biology, but it is too early at present to know if new medicines for disease treatment will emerge from our knowledge concerning this molecule.

A distinct role for transient receptor potential ankyrin 1, in addition to transient receptor potential vanilloid 1, in tumor necrosis factor α–induced inflammatory hyperalgesia and Freund's complete adjuvant–induced monarthritis

OBJECTIVE To investigate the involvement of transient receptor potential ankyrin 1 (TRPA1) in inflammatory hyperalgesia mediated by tumor necrosis factor α(TNFα) and joint inflammation. METHODS Mechanical hyperalgesia was assessed in CD1 mice, mice lacking functional TRP vanilloid 1 (TRPV1-/-) or TRPA1 (TRPA1-/-), or respective wildtype (WT) mice. An automated von Frey system was used, following unilateral intraplantar injection of TNFα or intraarticular injection of Freunds complete adjuvant (CFA). Knee swelling and histologic changes were determined in mice treated with intraarticular injections of CFA. RESULTS TNFα induced cyclooxygenase-independent bilateral mechanical hyperalgesia in CD1 mice. The selective TRPV1 receptor antagonist SB-366791 had no effect on mechanical hyperalgesia when it was coinjected with TNFα, but intrathecally administered SB- 366791 attenuated bilateral hyperalgesia, indicating the central but not peripheral involvement of TRPV1 receptors. A decrease in pain sensitivity was also observed in TRPV1-/- mice. Intraplantar coadministration of the TRPA1 receptor antagonist AP-18 with TNFα inhibited bilateral hyperalgesia. Intrathecal treatment with AP-18 also reduced TNFα-induced hyperalgesia. CFA-induced mechanical hyperalgesia in CD1 mice was attenuated by AP-18 (administered by intraarticular injection 22 hours after the administration of CFA). Furthermore, intraarticular CFA–induced ipsilateral mechanical hyperalgesia was maintained for 3 weeks in TRPA1 WT mice. In contrast, TRPA1-/- mice exhibited mechanical hyperalgesia for only 24 hours after receiving CFA. CONCLUSION Evidence suggests that endogenous activation of peripheral TRPA1 receptors plays a critical role in the development of TNFα-induced mechanical hyperalgesia and in sustaining the mechanical hyperalgesia observed after intraaarticular injection of CFA. These results suggest that blockade of TRPA1 receptors may be beneficial in reducing the chronic pain associated with arthritis.

Hydrogen peroxide is a novel mediator of inflammatory hyperalgesia, acting via transient receptor potential vanilloid 1-dependent and independent mechanisms

Abstract Inflammatory diseases associated with pain are often difficult to treat in the clinic due to insufficient understanding of the nociceptive pathways involved. Recently, there has been considerable interest in the role of reactive oxygen species (ROS) in inflammatory disease, but little is known of the role of hydrogen peroxide (H2O2) in hyperalgesia. In the present study, intraplantar injection of H2O2‐induced a significant dose‐ and time‐dependent mechanical and thermal hyperalgesia in the mouse hind paw, with increased c‐fos activity observed in the dorsal horn of the spinal cord. H2O2 also induced significant nociceptive behavior such as increased paw licking and decreased body liftings. H2O2 levels were significantly raised in the carrageenan‐induced hind paw inflammation model, showing that this ROS is produced endogenously in a model of inflammation. Moreover, superoxide dismutase and catalase significantly reduced carrageenan‐induced mechanical and thermal hyperalgesia, providing evidence of a functionally significant endogenous role. Thermal, but not mechanical, hyperalgesia in response to H2O2 (i.pl.) was longer lasting in TRPV1 wild type mice compared to TRPV1 knockouts. It is unlikely that downstream lipid peroxidation was increased by H2O2. In conclusion, we demonstrate a notable effect of H2O2 in mediating inflammatory hyperalgesia, thus highlighting H2O2 removal as a novel therapeutic target for anti‐hyperalgesic drugs in the clinic.

Tumour necrosis factor α mediates transient receptor potential vanilloid 1-dependent bilateral thermal hyperalgesia with distinct peripheral roles of interleukin-1β, protein kinase C and cyclooxygenase-2 signalling

ABSTRACT TNFα plays a pivotal role in rheumatoid arthritis (RA) but little is known of the mechanisms that link the inflammatory and nociceptive effects of TNFα. We have established a murine model of TNFα‐induced TRPV1‐dependent bilateral thermal hyperalgesia that then allowed us to identify distinct peripheral mechanisms involved in mediating TNFα‐induced ipsilateral and contralateral hyperalgesia. Thermal hyperalgesia and inflammation were assessed in both hindpaws following unilateral intraplantar (i.pl.) TNFα. The hyperalgesic mechanisms were analysed through pharmacogenetic approaches involving TRPV1−/− mice and TRPV1 antagonists. To study the mediators downstream of TNFα, cyclooxygenase (COX) and PKC inhibitors were utilised and cytokine and prostaglandin levels assessed. The role of neutrophils was determined through use of the selectin inhibitor, fucoidan. We show that TNFα (10 pmol) causes thermal hyperalgesia (1–4 h) in the ipsilateral inflamed and contralateral uninjured hindpaws, which is TRPV1‐dependent. GF109203X, a PKC inhibitor, suppressed the hyperalgesia indicating that PKC is involved in TRPV1 sensitisation. Ipsilateral COX‐2‐derived prostaglandins were also crucial to the development of the bilateral hyperalgesia. The prevention of neutrophil accumulation with fucoidan attenuated hyperalgesia at 4 but not at 1 h, indicating a role in the maintenance but not in the induction of bilateral hyperalgesia. However, TNFα‐induced IL‐1β generation in both paws and the presence of local IL‐1β in the contralateral paw were essential for the development of bilateral hyperalgesia. These results identify a series of peripheral events through which TNFα triggers and maintains bilateral inflammatory pain. This potentially allows a better understanding of mechanisms involved in TNFα‐dependent pain pathways in symmetrical diseases such as arthritis.

Proteinase-Activated Receptor-4 (PAR4) Activation Leads to Sensitization of Rat Joint Primary Afferents Via a Bradykinin B2 Receptor-Dependent Mechanism

The G-protein-linked receptor, proteinase-activated receptor-4 (PAR(4)) is activated by proteinases released into the joint during inflammation. It is unclear whether PAR(4) has a pro- or anti-nociceptive effect and whether it directly affects nerve activity. In this study, we examined the expression of PAR(4) in joints and dorsal root ganglion (DRG) neurons and whether activation of PAR(4) has an effect on nociception in normal rat knee joints. Electrophysiological recordings were made from joint primary afferents in male Wistar rats during both nonnoxious and noxious rotations of the knee. Afferent firing rate was recorded for 15 min post close intra-arterial injection of 10(-9)-10(-5) mol of the PAR(4) activating peptide, AYPGKF-NH(2), or the inactive peptide, YAPGKF-NH(2) (100 mul bolus). Rats were either naive or pretreated with the selective PAR(4) antagonist, pepducin P4pal-10, the transient receptor potential vanilloid-1 (TRPV1) antagonist, SB366791, or the bradykinin B(2) receptor antagonist, HOE140. Immunofluorescence experiments showed extensive PAR(4) expression in the knee joint and in sensory neurons projecting from the joint. AYPGKF-NH(2) significantly increased joint afferent firing during nonnoxious and noxious rotation of the knee. The inactive control peptide, YAPGKF-NH(2) was without effect. Systemic pretreatment with the PAR(4) antagonist, pepducin P4pal-10, inhibited the AYPGKF-NH(2)-induced increase in firing rate. Pretreatment with HOE140, but not SB366791, also blocked this increase in firing rate. These data reveal that in normal rat knee joints, PAR(4) activation increases joint primary afferent activity in response to mechanical stimuli. This PAR(4)-induced sensitization is TRPV1-independent but involves B(2) receptor activation, suggesting a role for kinins in this process.

Transient receptor potential canonical 5 (TRPC5) protects against pain and vascular inflammation in arthritis and joint inflammation

Objective Transient receptor potential canonical 5 (TRPC5) is functionally expressed on a range of cells including fibroblast-like synoviocytes, which play an important role in arthritis. A role for TRPC5 in inflammation has not been previously shown in vivo. We investigated the contribution of TRPC5 in arthritis. Methods Male wild-type and TRPC5 knockout (KO) mice were used in a complete Freunds adjuvant (CFA)-induced unilateral arthritis model, assessed over 14 days. Arthritis was determined by measurement of knee joint diameter, hindlimb weightbearing asymmetry and pain behaviour. Separate studies involved chronic pharmacological antagonism of TRPC5 channels. Synovium from human postmortem control and inflammatory arthritis samples were investigated for TRPC5 gene expression. Results At baseline, no differences were observed. CFA-induced arthritis resulted in increased synovitis in TRPC5 KO mice assessed by histology. Additionally, TRPC5 KO mice demonstrated reduced ispilateral weightbearing and nociceptive thresholds (thermal and mechanical) following CFA-induced arthritis. This was associated with increased mRNA expression of inflammatory mediators in the ipsilateral synovium and increased concentration of cytokines in synovial lavage fluid. Chronic treatment with ML204, a TRPC5 antagonist, augmented weightbearing asymmetry, secondary hyperalgesia and cytokine concentrations in the synovial lavage fluid. Synovia from human inflammatory arthritis demonstrated a reduction in TRPC5 mRNA expression. Conclusions Genetic deletion or pharmacological blockade of TRPC5 results in an enhancement in joint inflammation and hyperalgesia. Our results suggest that activation of TRPC5 may be associated with an endogenous anti-inflammatory/analgesic pathway in inflammatory joint conditions.

Topical diclofenac in the treatment of osteoarthritis of the knee

Osteoarthritis (OA) is a common disease that affects millions of people worldwide. As there is no cure for OA, drug treatment to relieve symptoms is the main form of management. Nonsteroidal anti-inflammatory drugs (NSAIDs) such as diclofenac are the most commonly used drugs to treat knee OA pain. Unfortunately, these agents are associated with gastrointestinal and cardiovascular risks, which limit their chronic use. Topical NSAIDs are emerging as a viable alternative for managing OA pain. Because a pharmacologically effective dose can be restricted to the site of pain, there is superfluous systemic absorption, and the risk of related adverse effects is minimized. This article reviews the currently available preclinical and clinical information on topical diclofenac for the treatment of OA pain, including data from recently published randomized controlled trials regarding the efficacy and safety of topical diclofenac. Most studies confirm topical diclofenac to be as effective as oral diclofenac with significantly reduced side effects; however, the efficacy of NSAIDs is far from optimal, and more research needs to be done to investigate the underlying mechanisms of OA in order to improve treatment options, especially for patients with NSAID-resistant OA pain.

A role for hydrogen peroxide in inflammatory hyperalgesia of the mouse hindpaw

Reactive oxygen species, including hydrogen peroxide (H2O2), are released from activated leucocytes and resident cells in the joint during inflammation. We hypothesize that H2O2 is an important mediator of inflammatory pain and, thus, the aim of our experiments was to investigate the effect of H2O2 on inflammation of the mouse hindpaw. We performed behavioural studies in CD1 mice to determine the effect of H2O2 on both thermal and mechanical hyperalgesia and oedema formation 20 minutes after intraplantar injection into the mouse hindpaw. The Hargreaves technique was used to test thermal hyperalgesia and a dynamic plantar aesthesiometer was used for mechanical hyperalgesia studies. Oedema was determined by measuring paw mass. Our experiments showed intraplantar injection of H2O2 (2200–8800 nmoles) causes significant thermal and mechanical hyperalgesia of the mouse hindpaw. H2O2 also causes significant oedema formation. Thus, we propose that H2O2 is a potential target for the treatment of inflammatory pain.

AB0095 Investigating The Role of Transient Receptor Potential Canonical 5 (TRPC5) in Complete Freund's Adjuvant (CFA)-Induced Arthritis

Background Rheumatoid arthritis (RA) is a chronic autoimmune disease characterised by inflammation of diarthrodial joints. Sensory neurons expressing transient receptor potential (TRP) channels, a wide family of non-selective cation channels, have been shown to play a role in the pathogenesis of RA1. Transient receptor potential 5 (TRPC5) is a member of the canonical family of TRPC channels and is abundantly expressed in the central nervous system and periphery2, including CD55+ fibroblast-like synoviocytes3, but the role of TRPC5 in arthritis is unknown. Objectives To evaluate the for the first time, the contribution of TRPC5 in an in vivo model of arthritis and investigate the role of TRPC5 in the modulation of hyperalgesia and joint inflammation. Methods All experiments were conducted under the guidelines of the United Kingdom Home Office Animals (Scientific Procedures) Act 1986. Age-matched, male 129S1/SvImJ wildtype (WT) and TRPC5 knockout (KO) mice littermates bred from heterozygotic mice provided by Prof. D.E. Clapham, USA2. Induction of arthritis using CFA was performed as previously described1; inflammation was allowed to progress to 14 days. Nociceptive tests were carried out weekly. Synovial blood flow was assessed in anaesthetised WT and TRPC5 KO mice using a full-field laser perfusion imager (FLPI)4 on day 14. Ex vivo analysis included assessment of cellular infiltration in the knee joint, and determination of cytokine concentrations. In separate studies, WT and TRPC5 KO mice received the TRPC4/5 antagonist, ML2045(2mg/kg, i.p., n=6) or vehicle (2% DMSO in saline, n=4) daily following i.art. injection of CFA. Results are presented as mean ± s.e.m. and analysed by 2-way ANOVA and Bonferonni post hoc test. Results WT mice developed primary hyperalgesia with a significant reduction in withdrawal threshold compared to baseline responses (334.7±25.7 vs 452.2±8.2 gram force (gf); p<0.01]). Primary hyperalgesia was exacerbated in TRPC5 KO mice compared to WT mice on day 14 (267.4±34.2 vs 334.7±25.4gf; p<0.05]). This was associated with similar deficits in weight-bearing on the ipsilateral hind-limb which was significantly reduced compared to WT mice. Ipsilateral synovial blood flow was significantly increased in TRPC5 KO mice compared to the contralateral synovium (291.3±50.2 vs 198.2±21.4 flux units (AU); p<0.01), and WT ipsilateral synovium (p<0.001). Pharmacological antagonism of TRPC5 in WT mice corroborated the results observed in TRPC5 KO mice, where hyperalgesia and joint inflammation was significantly exacerbated compared to vehicle treated WT mice. Conclusions Our results reveal a protective ability of TRPC5 in inflammatory joint conditions in vivo. Genetic deletion or pharmacological antagonism of TRPC5 resulted in an exacerbated inflammatory response highlighted by heightened nociceptive responses, increased cellular infiltration and cytokine concentrations in the knee joint. References Fernandes, E.S., et al. 2011. Arthritis Rheum 63, 819–829. Riccio, A., et al. 2009. Cell 137, 761–772. Xu, S.Z., et al. 2008. Nature 451, 69–72 Fernandes, E.S., et al. 2016. Arthritis Res Ther 18(1), 7 Miller, M., et al. 2011. J Biol Chem 286, 33436–46. Acknowledgement Supported by Arthritis Research UK. Disclosure of Interest None declared

Substance P and CGRP do not play an important role in TNFα-induced inflammation

Previously,we have shown a role for TRPV1 in CFA-induced joint inflammation and suggested an involvement ofTNFα [1]. We have now investigated mechanisms involved in TNFα-induced inflammation in the mouse paw. The possible role of neuropeptides was investigated, since TRPV1 activation leads to CGRP and substance P release. NK1−/− and CGRP−/− mice matched with their respective wild-types were used. Mice were given intraplantar injections ofTNF a (10pmol/50μl) and Tyrode (contralateral paw; 50μl). One group ofCG RP-/-) mice were pretreated with the NK1 receptor antagonist, SR140333 (480nmol/kg i.v.). Thermal hyperalgesic thresholds were measured using the Hargreaves technique before and over 4 hours after injection. Paw mass and neutrophil accumulation were measured. All the mice exhibited bilateral hyperalgesia, significant paw oedema and neutrophil accumulation in the TNFα-treated paw. These results suggest that substance P and CGRP are not important in development of hyperalgesia, oedema or neutrophil accumulation in this model.