Jennifer V. Bodkin

Evidence for the pathophysiological relevance of TRPA1 receptors in the cardiovascular system in vivo

AIMS The aim of the study is to investigate transient receptor potential ankyrin 1 (TRPA1)-induced responses in the vasculature and on blood pressure and heart rate (HR), in response to TRPA1 agonists using wild-type (WT) and TRPA1 knockout (KO) mice. METHODS AND RESULTS TRPA1 agonists allyl isothiocyanate and cinnamaldehyde (CA) significantly increased blood flow in the skin of anaesthetized WT, but not in TRPA1 KO mice. CA also induced TRPA1-dependent relaxation of mesenteric arteries. Intravenously injected CA induced a transient hypotensive response accompanied by decreased HR that was, depending on genotype and dose, followed by a more sustained dose-dependent pressor response (10-320 micromol/kg). CA (80 micromol/kg) induced a depressor response that was significantly less in TRPA1 KO mice, with minimal pressor effects. The pressor response of a higher CA dose (320 micromol/kg) was observed in WT but not in TRPA1 KO mice, indicating involvement of TRPA1. Experiments using TRP vanilloid 1 (TRPV1) KO and calcitonin gene-related peptide (CGRP) KO mice provided little evidence for the involvement of TRPV1 or CGRP, nor did blocking substance P receptors affect responses. However, the cholinergic antagonist atropine sulphate (5 mg/kg) significantly inhibited the depressor response and slowed HR with CA (80 micromol/kg), but had no effect on pressor responses. The pressor response remained unaffected, even in the presence of the ganglion blocker hexamethonium bromide (1 mg/kg). The alpha-adrenergic blocker prazosin hydrochloride (1 mg/kg) significantly inhibited both components, but not slowed HR. CONCLUSION TRPA1 is involved in mediating vasodilation. TRPA1 can also influence changes in blood pressure of possible relevance to autonomic system reflexes and potentially to vasovagal/neurocardiogenic syncope disorders.

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.

A Role for TRPV1 in Influencing the Onset of Cardiovascular Disease in Obesity

Obesity induced by Western diets is associated with type 2 diabetes mellitus and cardiovascular diseases, although underlying mechanisms are unclear. We investigated a murine model of diet-induced obesity to determine the effect of transient potential receptor vanilloid 1 (TRPV1) deletion on hypertension and metabolic syndrome. Wild-type and TRPV1 knockout mice were fed normal or high-fat diet from 3 to 15 weeks. High-fat diet-fed mice from both genotypes became obese, with similar increases in body and adipose tissue weights. High-fat diet-fed TRPV1 knockout mice showed significantly improved handling of glucose compared with high-fat diet-fed wild-type mice. Hypertension, vascular hypertrophy, and altered nociception were observed in high-fat diet-fed wild-type but not high-fat diet-fed TRPV1 knockout mice. Wild-type, but not high-fat diet-fed TRPV1 knockout, mice demonstrated remodeling in terms of aortic vascular hypertrophy and increased heart and kidney weight, although resistance vessel responses were similar in each. Moreover, the wild-type mice had significantly increased plasma levels of leptin, interleukin 10 and interleukin 1&bgr;, whereas samples from TRPV1 knockout mice did not show significant increases. Our results do not support the concept that TRPV1 plays a major role in influencing weight gain. However, we identified a role of TRPV1 in the deleterious effects observed with high-fat feeding in terms of inducing hypertension, impairing thermal nociception sensitivity, and reducing glucose tolerance. The observation of raised levels of adipokines in wild-type but not TRPV1 knockout mice is in keeping with TRPV1 involvement in stimulating the proinflammatory network that is central to obesity-induced hypertension and sensory neuronal dysfunction.

TRPA1 is essential for the vascular response to environmental cold exposure

The cold-induced vascular response, consisting of vasoconstriction followed by vasodilatation, is critical for protecting the cutaneous tissues against cold injury. Whilst this physiological reflex response is historic knowledge, the mechanisms involved are unclear. Here by using a murine model of local environmental cold exposure, we show that TRPA1 acts as a primary vascular cold sensor, as determined through TRPA1 pharmacological antagonism or gene deletion. The initial cold-induced vasoconstriction is mediated via TRPA1-dependent superoxide production that stimulates α2C-adrenoceptors and Rho-kinase-mediated MLC phosphorylation, downstream of TRPA1 activation. The subsequent restorative blood flow component is also dependent on TRPA1 activation being mediated by sensory nerve-derived dilator neuropeptides CGRP and substance P, and also nNOS-derived NO. The results allow a new understanding of the importance of TRPA1 in cold exposure and provide impetus for further research into developing therapeutic agents aimed at the local protection of the skin in disease and adverse climates.

Superoxide generation and leukocyte accumulation: key elements in the mediation of leukotriene B4-induced itch by transient receptor potential ankyrin 1 and transient receptor potential vanilloid 1

The underlying mechanisms of itch are poorly understood. We have investigated a model involving the chemoattractant leukotriene B4 (LTB4) that is up‐regulated in common skin diseases. Intradermal injection of LTB4 (0.1 nmol/site) into female CD1 mice induced significant scratching movements (used as an itch index) compared with vehicle‐injected (0.1% bovine serum albumin‐saline) mice. Intraperitoneal transient receptor potential (TRP) channel antagonist treatment significantly inhibited itch as follows: TRP vanilloid 1 (TRPV1) antagonist SB366791 (0.5 mg/kg, by 97%) and the TRP ankyrin 1 (TRPA1) antagonists TCS 5861528 (10 mg/kg; 82%) and HC‐030031 (100 mg/kg; 76%). Leukotriene B4 receptor 2 antagonism by LY255283 (5 mg/kg i.p.; 62%) reduced itch. Neither TRPV1‐knockout (TRPV1‐KO) nor TRPA1‐knockout (TRPA1‐KO mice exhibited LTB4‐induced itch compared with their wild‐type counterparts. The reactive oxygen species scavengers N‐acetylcysteine (NAC; 204 mg/kg i.p.; 86%) or superoxide dismutase (SOD; 10 mg/kg i.p.; 83%) also inhibited itch. LTB4‐induced superoxide release was attenuated by TCS 5861528 (56%) and HC‐030031 (66%), NAC (58%), SOD (50%), and LY255283 (59%) but not by the leukotriene B4 receptor 1 antagonist U‐75302 (9 nmol/site) or SB366791. Itch, superoxide, and myeloperoxidase generation were inhibited by the leukocyte migration inhibitor fucoidan (10 mg/kg i.v.) by 80, 61, and 34%, respectively. Myeloperoxidase activity was also reduced by SB366791 (35%) and SOD (28%). TRPV1‐KO mice showed impaired myeloperoxidase release, whereas TRPA1‐KO mice exhibited diminished production of superoxide. This result provides novel evidence that TRPA1 and TRPV1 contribute to itch via distinct mechanisms.—Fernandes, E. S., Vong, C. T., Quek, S., Cheong, J., Awal, S., Gentry, C., Aubdool, A. A., Liang, L., Bodkin, J.V., Bevan, S., Heads, R., Brain, S.D. Superoxide generation and leukocyte accumulation: key elements in the mediation of leukotriene B4‐induced itch by transient receptor potential ankyrin 1 and transient receptor potential vanilloid 1. FASEB J. 27, 1664–1673 (2013). www.fasebj.org

The vasoactive potential of kisspeptin-10 in the peripheral vasculature.

Splice products of the Kiss1 protein (kisspeptins) have been shown to be involved in a diverse range of functions, including puberty, metastasis and vasoconstriction in large human arteries. Circulating Kisspeptin-10 (Kp-10) plasma levels are low in normal individuals but are elevated during various disease states as well as pregnancy. Here, we investigated the potential of Kp-10, the shortest biologically active kisspeptin, to influence microvascular effects, concentrating on the cutaneous vasculature. Kp-10 caused a dose-dependent increase in oedema formation (0.3–10nmol/injection site), assessed by Evans Blue albumin dye extravasation, in the dorsal skin of CD1 mice. Oedema formation was shown to be inhibited by the histamine H1 receptor antagonist mepyramine. The response was characterised by a ring of pallor at the injection site in keeping with vasoconstrictor activity. Therefore, changes in dorsal skin blood flow were assessed by clearance of intradermally injected 99mtechnetium. Kp-10 was found to significantly reduce clearance, in keeping with decreased blood flow and providing further evidence for vasoconstrictor activity. The decreased clearance was partially inhibited by co-treatment with the cyclo-oxygenase inhibitor indomethacin. Finally evidence for the kisspeptin receptor gene (Kiss1R), but not the kisspeptin peptide gene (Kiss1), mRNA expression was observed in heart, aorta and kidney samples from normal and angiotensin II induced hypertensive mice, with similar mRNA levels observed in each. We have evidence for two peripheral vasoactive roles for kisspeptin-10. Firstly, plasma extravasation indicative of ability to induce oedema formation and secondly decreased peripheral blood flow, indicating microvascular constriction. Thus Kp-10 has vasoactive properties in the peripheral microvasculature.

Transient receptor potential ankyrin 1: emerging pharmacology and indications for cardiovascular biology.

Transient receptor potential anykrin 1 (TRPA1) is a member of the TRP superfamily, representing the sole member of the TRPA subfamily. It has many identified endogenous and exogenous agonists, comprising largely of chemical irritants and products of oxidative stress. Classically located on sensory neurone endings, TRPA1 has developed a strong presence in pain and inflammatory studies, where it is now becoming an intriguing clinical drug target. TRPA1 is increasingly recognized in a growing number of neuronal and non‐neuronal locations with expanding expression and activity profiles providing evidence of a role for TRPA1 in other systems. Interest in discovering the pharmacological and functional roles of TRPA1 is increasing and diversifying into many areas. Historically, compounds now known as TRPA1 agonists have demonstrated cardiovascular activity, modulating activities in both the heart and the vasculature. Now TRPA1 has been identified as the receptor via which these compounds can act, these studies are being revisited and expanded on using current techniques. It is therefore timely to review the current knowledge of TRPA1 receptor presence and activities of relevance to the cardiovascular system, summarizing findings to date and identifying potential areas for future investigation.

Design and pharmacological evaluation of PF-4840154, a non-electrophilic reference agonist of the TrpA1 channel.

TrpA1 is an ion channel involved in nociceptive and inflammatory pain. It is implicated in the detection of chemical irritants through covalent binding to a cysteine-rich intracellular region of the protein. While performing an HTS of the Pfizer chemical collection, a class of pyrimidines emerged as a non-reactive, non-covalently binding family of agonists of the rat and human TrpA1 channel. Given the issues identified with the reference agonist Mustard Oil (MO) in screening, a new, non-covalently binding agonist was optimized and proved to be a superior agent to MO for screening purposes. Compound 16a (PF-4840154) is a potent, selective agonist of the rat and human TrpA1 channel and elicited TrpA1-mediated nocifensive behaviour in mouse.

Investigating the potential role of TRPA1 in locomotion and cardiovascular control during hypertension

Radiotelemetry was used to investigate the in vivo cardiovascular and activity phenotype of both TRPA1 (transient receptor potential ankyrin 1) wild‐type (WT) and TRPA1 knockout (KO) mice. After baseline recording, experimental hypertension was induced using angiotensin II infusion (1.1 mg−1 kg−1 a day, for 14 days). TRPA1 WT and KO mice showed similar morphological and functional cardiovascular parameters, including similar basal blood pressure (BP), heart rate, size, and function. Similar hypertension was also displayed in response to angiotensin II (156 ± 7 and 165 ± 11 mmHg, systolic BP ± SEM, n = 5–6). TRPA1 KO mice showed increased hypertensive hypertrophy (heart weight:tibia length: 7.3 ± 1.6 mg mm−1 vs. 8.8 ± 1.7 mg mm−1) and presented with blunted interleukin 6 (IL‐6) production compared with hypertensive WT mice (151 ± 24 vs. 89 ± 16 pg mL−1). TRPA1 expression in dorsal root ganglion (DRG) neurones was upregulated during hypertension (163% of baseline expression). Investigations utilizing the TRPA1 agonist cinnamaldehyde (CA) on mesenteric arterioles isolated from näive mice suggested a lack of TRPA1‐dependent vasoreactivity in this vascular bed; a site with notable ability to alter total peripheral resistance. However, mesenteric arterioles isolated from TRPA1 KO hypertensive mice displayed significantly reduced ability to relax in response to nitric oxide (NO) (P < 0.05). Unexpectedly, naïve TRPA1 KO mice also displayed physical hyperactivity traits at baseline, which was exacerbated during hypertension. In conclusion, our study provides a novel cardiovascular characterization of TRPA1 KO mice in a model of hypertension. Results suggest that TRPA1 has a limited role in global cardiovascular control, but we demonstrate an unexpected capacity for TRPA1 to regulate physical activity.

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.