In the present study we used a genetic mouse model to characterise the role of sEH in nociceptive processing. Our findings suggest that the sEH is expressed in a subpopulation of myelinated primary sensory neurons. There, it negatively regulates steady state EET tissue levels as well as release of EETs from neurons. Specifically we show that 8,9-EET activates TRPA1-expressing neurons and sensitizes neuropetide release from TRPA1-expressing fibers. Finally, we find that 8,9-EET can induce mechanical hyperalgesia and that sEH deletion prolongs mechanical hyperalgesia during inflammation.
sEH exhibits lipid phosphatase as well as epoxide hydrolase activity, with the latter resulting in rapid inactivation of EETs and epoxyoctadecenoic acids (EpOMEs) linoleic acid metabolites [11, 30]. Although a putative lipid phosphatase activity may have an impact on nociceptive processing, we focused on the epoxide hydrolase activity of sEH and on EETs/DHETs since they mediate most sEH functions in the cardiovascular system [9, 31].
The EETs are generally attributed with anti-inflammatory effects, largely on the basis of the fact that 11,12-EET can inhibit the IκB kinase and NF-κB signaling . However, although 11,12-EET appears to be the most potent with respect to anti-inflammatory, anti-migratory, and pro-fibrinolytic effects , it has also been reported to increase COX2 expression in endothelial cells, a phenomenon linked to angiogenesis . This finding is of relevance, as one major consequence of immune cell activation after zymosan injection is induction of COX-2 expression at the inflammation site and in the spinal cord to sensitize nociceptive processing [23, 24]. However, we found no obvious change in COX-2 expression or in prostaglandin synthesis in paw tissue from zymosan-treated sEH-/- mice that could be attributed to the higher EET levels in these animals. Our results suggest that sEH deletion, which mainly increases levels of 8-9- and 14,15-EET in the tissue studied, does not alter zymosan-induced inflammatory hyperalgesia by an inhibition of prostaglandin synthesis. These findings contrast with those made by Incoeglu et al. who described that inhibition of sEH reduces PGD2 synthesis and hyperalgesia in a LPS model . While these reports are difficult to reconcile, it is possible that differences in the mechanisms of immune cell activation between the two models may be a determinant factor since LPS and zymosan selectively activate toll like receptor 4 (TLR-4) and TLR-2, respectively  leading to expression and release of a different set of proinflammatory mediators, which may be differentially affected by EETs.
All studies reporting antinociceptive effects of sEH are based on a pharmacological inhibition of the enzyme [10, 36, 37]. The model used in the present investigation was that of Ephx gene deletion resulting in the loss of the N-terminal lipid phosphatase activity and C-terminal soluble epoxide hydrolase activity. Thus, one major difference between our study and those performed previously is the fact that the sEH-associated lipid phosphatase activity was also inhibited. As no endogenous substrates or pharmacological inhibitors have been identified that target sEH lipid phosphatase activity, it is currently not possible to distinguish between the contributions of the lipid phosphatase and the epoxide hydrolase activities to the phenotype of sEH-/- mice . Another possible explanation for the differences between this and previous studies could be related to off-target effects of the urea-based inhibitors used in the pharmacological studies. Although specificity for sEH over other epoxide hydrolases such as mEH is generally good for most inhibitors, some compounds including 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) can also activate PPAR□, which represses COX-2 expression [39, 40].
We did not detect the sEH in invading immune cells within inflamed paw tissue. In contrast, we found prominent expression of sEH in primary afferent neurons in the DRG. It seems that also other components of the EET generating/metabolising pathway are present in sensory neurons. The epoxygenases CYP2J3 and CYP2J4 are expressed together with the sEH in trigeminal ganglia, indicating that the CYP450/sEH pathway is a common, integral component of peripheral sensory neuron signaling .
In the nociceptive models used in this study mechanical or heat stimuli are applied to the plantar surface of the hind paw. Here, nociception is processed by small and medium diameter DRG-neurons with C- and A-δ fibres. We found that the sEH was mainly expressed in NF200 positive large and medium diameter DRG neurons. Most of these cells transmit proprioceptive and low threshold mechanical but not noxious sensations. However, we found that sEH deficient neurons have a higher steady state release of EET into the extracellular medium. Further, we showed that an increase of AA bioavailability predominately affects EET release instead of intracellular accumulation. This implies that the sEH present in myelinated neurons may also affect C-fibre neurons by paracrine signaling. Neuronal activity as well as inflammation activates PLA2 in primary afferents causing increased synthesis of eicosanoids. The restricted expression pattern of sEH in a subset of DRG neurons and its hydrolyzing activity on EET, preventing their release, suggests that the sEH may act to limit EET signaling to nociceptors.
Using cellular imaging experiments we identified 8,9-EET as the sEH substrate most likely to sensitize nociceptor function. 8,9-EET induced a direct calcium influx in ≈5% of sensory neurons. Co-stimulation with different TRP-channel agonists revealed that 8,9-EET only activates a subset of capsaicin responsive nociceptors and not large non nociceptive neurons. Further, we found that within the capsaicin-responsive group most were also AITC-responsive (TRPA1-positive neurons) (92.3% positive cells). Notably, Kwan et al. reported that only AITC-sensitive neurons that also respond to TRPV1 express TRPA1 . Even though we did not further address the specific target of 8,9-EET in this TRPA1-expressing cell population, multiple studies have shown that calcium transients in nociceptive neurons induce plasticity changes through the activation of PKCε, p38 MAPK or ERK which result in reduced activation thresholds and increased firing rates . It could be speculated that 8,9-EET may directly activate TRPA1 at high concentrations although various other possible targets exist. However, due to its electrophilic character, 8,9-EET can potentially sensitize TRPA1 by direct interaction with its intracellular cysteins as previously described for other lipids like 4-HNE or cyclopentons [44, 45]. In addition to a potential direct activation of the TRPA1-positive cell population, we found that lower doses of 8.9-EET potentiate AITC-induced calcium flux. Here, 8,9-EET may modulate TRPA1 indirectly via G-protein coupled receptors as described for bradykinin . Moreover, to increase functionality of certain TRP-channels such as TRPC6, EETs have already been shown to promote membrane translocation . Finally, other TRPA1 independent downstream processes, such as sensitized voltage gated calcium channels or calcium transporters may be involved in the observed increased calcium responses.
We also investigated whether 8,9-EET modulates the activation of TRPA1-expressing neurons. 8,9-EET application to isolated sciatic nerves caused an increased neuropeptide release in response to AITC. Peripheral nerve axons resemble peripheral sensory terminals in their common properties of sensory and signal transduction and CGRP neuropeptides are stored all along axons of small diameter peptidergic neurons . Stimulation of those cells by AITC induces a translocation of TRPA1 to the membrane where it can be activated resulting in calcium influx, subsequent vesicle fusion and neurotransmitter release. CGRP release from sciatic nerves can be sensitized by activation of G-protein-coupled receptors and related protein kinases  and that CGRP can induce mechanical hyperalgesia and central sensitization . TRPA1 expressing neurons appear to mediate mechanotransduction and blockade of TRPA1 attenuates the development of mechanical hyperalgesia [27, 42, 49]. Our finding that 8,9-EET increases CGRP release from TRPA1 expressing neurons strongly suggests that sEH activity in primary afferents may prevents mechanical hypersensitivity.
In keeping with this, 8,9-EET injection into a hind paw lowered the mechanical but not thermal threshold in wild- type mice. Wild- type mice recover from zymosan-induced mechanical hyperalgesia 2 to 4 days after injection, a time during which 8,9-EET levels decrease in the paw tissue. Our finding that 8,9-EET sensitizes primary afferents and reduces mechanical thresholds suggested that hydrolysis of 8,9-EET could potentially contribute to the resolution of mechanical hyperalgesia during inflammation. In accordance with this hypothesis, sEH-deficient mice, which exhibit elevated 8,9-EET levels during inflammation, show a dramatically reduced recovery from mechanical hyperalgesia.