Andrew D. Grant

5,6-EET Is Released upon Neuronal Activity and Induces Mechanical Pain Hypersensitivity via TRPA1 on Central Afferent Terminals

Epoxyeicosatrienoic acids (EETs) are cytochrome P450-epoxygenase-derived metabolites of arachidonic acid that act as endogenous signaling molecules in multiple biological systems. Here we have investigated the specific contribution of 5,6-EET to transient receptor potential (TRP) channel activation in nociceptor neurons and its consequence for nociceptive processing. We found that, during capsaicin-induced nociception, 5,6-EET levels increased in dorsal root ganglia (DRGs) and the dorsal spinal cord, and 5,6-EET is released from activated sensory neurons in vitro. 5,6-EET potently induced a calcium flux (100 nm) in cultured DRG neurons that was completely abolished when TRPA1 was deleted or inhibited. In spinal cord slices, 5,6-EET dose dependently enhanced the frequency, but not the amplitude, of spontaneous EPSCs (sEPSCs) in lamina II neurons that also responded to mustard oil (allyl isothiocyanate), indicating a presynaptic action. Furthermore, 5,6-EET-induced enhancement of sEPSC frequency was abolished in TRPA1-null mice, suggesting that 5,6-EET presynaptically facilitated spinal cord synaptic transmission by TRPA1. Finally, in vivo intrathecal injection of 5,6-EET caused mechanical allodynia in wild-type but not TRPA1-null mice. We conclude that 5,6-EET is synthesized on the acute activation of nociceptors and can produce mechanical hypersensitivity via TRPA1 at central afferent terminals in the spinal cord.

Differing effects of exogenous and endogenous hydrogen sulphide in carrageenan-induced knee joint synovitis in the rat.

Background and purpose:  Recent findings suggest that the noxious gas H2S is produced endogenously, and that physiological concentrations of H2S are able to modulate pain and inflammation in rodents. This study was undertaken to evaluate the ability of endogenous and exogenous H2S to modulate carrageenan‐induced synovitis in the rat knee.

Evidence of a role for NK1 and CGRP receptors in mediating neurogenic vasodilatation in the mouse ear

The aims of this study were to develop a technique to measure blood flow in the mouse ear and to investigate the nature of the vasodilator mediator(s) involved in the response to capsaicin. The response to capsaicin, applied topically, was investigated in anaesthetized CD1 or Sv129+C57BL/6 wild‐type (+/+) or NK1 receptor knockout mice (−/−). Blood flow was assessed by laser Doppler flowmetry and oedema formation by 125I‐albumin accumulation. Capsaicin induced significant increases in blood flow (0.2–200 μg in 20 μl) and oedema (2–200 μg in 20 μl). The oedema response was absent in NK1−/− mice and NK1+/+mice treated with the selective NK1 receptor antagonist SR140333 (480 nmol kg−1) as expected. Furthermore, the capsaicin‐evoked increase in blood flow was significantly potentiated in the knockout mice (203% of wild‐type response, P<0.05) and wild‐type mice treated with SR140333 (201%, P<0.05). The CGRP receptor antagonist CGRP8–37 (400 nmol kg−1) had no effect on capsaicin‐induced blood flow in NK1+/+mice but abolished the increased blood flow to capsaicin in NK1−/−, and NK1+/+wild‐type mice pre‐treated with SR140333. The results indicate that neurogenic vasodilatation can be measured in the mouse ear. The capsaicin‐induced increased blood flow involves activation of, and possible interactions between, both NK1 and CGRP1 receptors.

Role of PKC and CaV1.2 in Detrusor Overactivity in a Model of Obesity Associated with Insulin Resistance in Mice

Obesity/metabolic syndrome are common risk factors for overactive bladder. This study aimed to investigate the functional and molecular changes of detrusor smooth muscle (DSM) in high-fat insulin resistant obese mice, focusing on the role of protein kinase C (PKC) and Cav1.2 in causing bladder dysfunction. Male C57BL/6 mice were fed with high-fat diet for 10 weeks. In vitro functional responses and cystometry, as well as PKC and Cav1.2 expression in bladder were evaluated. Obese mice exhibited higher body weight, epididymal fat mass, fasting glucose and insulin resistance. Carbachol (0.001–100 µM), α,β-methylene ATP (1–10 µM), KCl (1–300 mM), extracellular Ca2+ (0.01–100 mM) and phorbol-12,13-dibutyrate (PDBu; 0.001–3 µM) all produced greater DSM contractions in obese mice, which were fully reversed by the Cav1.2 blocker amlodipine. Cystometry evidenced augmented frequency, non-void contractions and post-void pressure in obese mice that were also prevented by amlodipine. Metformin treatment improved the insulin sensitivity, and normalized the in vitro bladder hypercontractility and cystometric dysfunction in obese mice. The PKC inhibitor GF109203X (1 µM) also reduced the carbachol induced contractions. PKC protein expression was markedly higher in bladder tissues from obese mice, which was normalized by metformin treatment. The Cav1.2 channel protein expression was not modified in any experimental group. Our findings show that Cav1.2 blockade and improvement of insulin sensitization restores the enhanced PKC protein expression in bladder tissues and normalizes the overactive detrusor. It is likely that insulin resistance importantly contributes for the pathophysiology of this urological disorder in obese mice.

A reactive oxygen species-mediated component in neurogenic vasodilatation

AIMS Activation of the transient receptor potential vanilloid receptor 1 (TRPV1) leads to release of potent microvascular vasodilator neuropeptides. This study was designed to investigate in vivo mechanisms involved in TRPV1-mediated peripheral vasodilatation. METHODS AND RESULTS Wildtype (WT) and TRPV1 knockout (KO) mice were investigated in a model of peripheral vasodilatation. Blood flow was measured by laser Doppler flowmetry under anaesthesia and following local application of the TRPV1 agonist capsaicin. A sustained (60 min) increase in blood flow was observed in WT but not TRPV1 KO mouse ears. This response was resistant to blockers of classic vasodilators but inhibited in pharmacogenetic experiments that targeted blockade of the substance P (SP) and calcitonin gene-related peptide (CGRP) pathways. The TRPV1-mediated vasodilatation was also attenuated by treatment with superoxide dismutase and the hydrogen peroxide scavenger catalase, but not by deactivated enzymes, supporting a novel role for reactive oxygen species (ROS) generation. Furthermore, neurogenic vasodilatation was observed neither in the presence of the selective NADPH inhibitor apocynin, nor in gp91 phox KO mice, under conditions where prostaglandin E1-induced vasodilatation occurred. Finally, a role of neuropeptides in initiating a ROS-dependent component was verified as superoxide dismutase, catalase, and apocynin inhibited SP and CGRP vasodilatation. CONCLUSION These studies provide in vivo evidence that ROS are involved in mediating TRPV1- and neuropeptide-dependent neurogenic vasodilatation. An essential role of NADPH oxidase-dependent ROS is revealed that may be of fundamental importance to the neurogenic vasodilator component involved in circulatory homeostasis and the pathophysiology of certain cardiovascular diseases.

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.

Enhanced Vascular Responses to Adrenomedullin in Mice Overexpressing Receptor-Activity-Modifying Protein 2

Adrenomedullin (AM) levels are elevated in cardiovascular disease, but little is known of the role of specific receptor components. AM acts via the calcitonin receptor-like receptor (CLR) interacting with a receptor-activity–modifying protein (RAMP). The AM1 receptor is composed of CLR and RAMP2, and the calcitonin gene–related peptide (CGRP) receptor of CLR and RAMP1, as determined by molecular and cell-based analysis. This study examines the relevance of RAMP2 in vivo. Transgenic (TG) mice that overexpress RAMP2 in smooth muscle were generated. The role of RAMP2 in the regulation of blood pressure and in vascular function was investigated. Basal blood pressure, acute angiotensin II–raised blood pressure, and cardiovascular properties were similar in wild-type (WT) and TG mice. However, the hypotensive effect of IV AM, unlike CGRP, was enhanced in TG mice (P<0.05), whereas a negative inotropic action was excluded by left-ventricular pressure–volume analysis. In aorta relaxation studies, TG vessels responded in a more sensitive manner to AM (EC50, 8.0±1.5 nmol/L) than WT (EC50, 17.9±3.6 nmol/L). These responses were attenuated by the AM receptor antagonist, AM22-52, such that residual responses were identical in all mice. Remaining relaxations were further inhibited by CGRP receptor antagonists, although neither affected AM responses when given alone. Mesenteric and cutaneous resistance vessels were also more sensitive to AM in TG than WT mice. Thus RAMP2 plays a key role in the sensitivity and potency of AM-induced hypotensive responses via the AM1 receptor, providing evidence that this receptor is a selective target for novel therapeutic approaches.

Neurokinin B induces oedema formation in mouse lung via tachykinin receptor-independent mechanisms

The tachykinin neurokinin B (NKB) has been implicated in the hypertension that characterises pre‐eclampsia, a condition where tissue oedema is also observed. The ability of NKB, administered intradermally or intravenously, to induce oedema formation (assessed as plasma extravasation) was examined by extravascular accumulation of intravenously injected 125I‐albumin in wild‐type and tachykinin NK1 receptor knockout mice. Intradermal NKB (30‐300 pmol) caused dose‐dependent plasma extravasation in wild‐type (P < 0.05) but not NK1 knockout mice, indicating an essential role for the NK1 receptor in mediating NKB‐induced skin oedema. Intravenous administration of NKB to wild‐type mice produced plasma extravasation in skin, uterus, liver (P < 0.05) and particularly in the lung (P < 0.01). Surprisingly, the same doses of NKB led to plasma extravasation in the lung and liver of NK1 knockout mice. By comparison, the tachykinin substance P induced only minimal plasma extravasation in the lungs of wild‐type mice. The plasma extravasation produced by NKB in the lungs of NK1 receptor knockout mice was unaffected by treatment with the NK2 receptor antagonist SR48968 (3 mg kg−1), by the NK3 receptor antagonists SR142801 (3 mg kg−1) and SB‐222200 (5 mg kg−1) or by the cyclo‐oxygenase (COX) inhibitor indomethacin (20 mg kg−1). L‐Nitro‐arginine methyl ester (15 mg kg−1), an inhibitor of endothelial nitric oxide synthase (eNOS), produced only a partial inhibition. We conclude that NKB is a potent stimulator of plasma extravasation through two distinct pathways: via activation of NK1 receptors, and via a novel neurokinin receptor‐independent pathway specific to NKB that operates in the mouse lung. These findings are in keeping with a role for NKB in mediating plasma extravasation in diseases such as pre‐eclampsia.

TRPA1 and TRPV4 Activation in Human Odontoblasts Stimulates ATP Release

The mechanism of pain in dentine hypersensitivity is poorly understood but proposed to result from the activation of dental sensory neurons in response to dentinal fluid movements. Odontoblasts have been suggested to contribute to thermal and mechanosensation in the tooth via expression of transient receptor potential (TRP) channels. However, a mechanism by which odontoblasts could modulate neuronal activity has not been demonstrated. In this study, we investigated functional TRP channel expression in human odontoblast-like cells and measured ATP release in response to TRP channel activation. Human immortalized dental pulp cells were driven toward an odontoblast phenotype by culture in conditioned media. Functional expression of TRP channels was determined with reverse transcription polymerase chain reaction and ratiometric calcium imaging with Fura-2. ATP release was measured using a luciferin-luciferase assay. Expression of mRNA for TRPA1, TRPV1, and TRPV4 but not TRPM8 was detected in odontoblasts by reverse transcription polymerase chain reaction. Expression of TRPV4 protein was detected by Western blotting and immunocytochemistry. The TRPA1 agonists allyl isothiocyanate and cinnamaldehyde and the TRPV4 agonist GSK1016790A caused a concentration-dependent increase in intracellular Ca2+ concentration that was inhibited by the selective antagonists HC030031, AP18, and HC067047, respectively. In contrast, exposure to the TRPV1 agonist capsaicin or the TRPM8 agonist icilin had no effect on intracellular Ca2+ concentration. Treatment with allyl isothiocyanate, cinnamaldehyde, or GSK1016790A caused an increase in ATP concentration in culture medium that was abolished by preincubation with TRP channel antagonists. These data demonstrate that activation of TRPA1 and TRPV4 channels in human odontoblast-like cells can stimulate ATP release. We were unable to confirm the presence of thermosensitive TRPV1 and TRPM8 that has previously been reported in odontoblasts.

Protein kinase D isoforms are expressed in rat and mouse primary sensory neurons and are activated by agonists of protease-activated receptor 2

Serine proteases generated during injury and inflammation cleave protease‐activated receptor 2 (PAR2) on primary sensory neurons to induce neurogenic inflammation and hyperalgesia. Hyperalgesia requires sensitization of transient receptor potential vanilloid (TRPV) ion channels by mechanisms involving phospholipase C and protein kinase C (PKC). The protein kinase D (PKD) serine/threonine kinases are activated by diacylglycerol and PKCs and can phosphorylate TRPV1. Thus, PKDs may participate in novel signal transduction pathways triggered by serine proteases during inflammation and pain. However, it is not known whether PAR2 activates PKD, and the expression of PKD isoforms by nociceptive neurons is poorly characterized. By using HEK293 cells transfected with PKDs, we found that PAR2 stimulation promoted plasma membrane translocation and phosphorylation of PKD1, PKD2, and PKD3, indicating activation. This effect was partially dependent on PKCϵ. By immunofluorescence and confocal microscopy, with antibodies against PKD1/PKD2 and PKD3 and neuronal markers, we found that PKDs were expressed in rat and mouse dorsal root ganglia (DRG) neurons, including nociceptive neurons that expressed TRPV1, PAR2, and neuropeptides. PAR2 agonist induced phosphorylation of PKD in cultured DRG neurons, indicating PKD activation. Intraplantar injection of PAR2 agonist also caused phosphorylation of PKD in neurons of lumbar DRG, confirming activation in vivo. Thus, PKD1, PKD2, and PKD3 are expressed in primary sensory neurons that mediate neurogenic inflammation and pain transmission, and PAR2 agonists activate PKDs in HEK293 cells and DRG neurons in culture and in intact animals. PKD may be a novel component of a signal transduction pathway for protease‐induced activation of nociceptive neurons and an important new target for antiinflammatory and analgesic therapies. J. Comp. Neurol. 516:141–156, 2009.