Background
The leukotrienes (LTs) are a family of biologically active lipid mediators. They are synthesized from arachidonic acid (AA)
via the 5-lipoxygenase pathway. AA is enzymatically converted to LTB4, LTC4, LTD4 and LTE4 that are known as bioactive LTs. LTC4, LTD4 and LTE4 are collectively termed the cysteinyl leukotrienes (CysLTs). LTs are peripherally produced by activated leukocytes in response to peripheral inflammation, such as asthma and atopic dermatitis [
1,
2]. Four different types (BLT1, BLT2, CysLT1 and CysLT2) of G-protein-coupled receptor for LT have been cloned [
3‐
6]. LTB4 activates BLT1 and BLT2, and CysLTs activate CysLT1 and CysLT2.
Peripheral inflammation often elicits mechanical and thermal hyperalgesia. The most studied of these lipid mediators are the prostaglandins (PGs) of the cyclooxygenase pathway of AA metabolism [
7,
8]. Expression of G-protein-coupled receptors of EP for E-type PG is localized in C-fibers, unmyelinated nociceptive fibers, in the dorsal root ganglion (DRG) [
8]. Activation of EP signaling plays a role in neuronal sensitization mediating modulation of the transient receptor potential vanilloid subfamily 1 (TRPV1) receptor and P2X3 receptor [
9,
10].
Intradermal injection of LTB4 has been shown to produce both thermal and mechanical hyperalgesia [
11,
12]. Jain et al. have reported that LTs are involved in inflammatory pain induced by carrageenan [
13]. Furthermore, we demonstrated that an increase in LT synthesis in microglia in the spinal cord induced by peripheral nerve injury contributes to neuropathic pain [
14]. However, in the periphery, the mechanism of the nociception induced by LTs is unknown and the precise expression pattern of LT receptors in the DRG has not been clarified. The purpose of this study is to examine the expression of LT receptor mRNAs in the DRG to assess whether LT receptors are expressed in nociceptive neurons. Furthermore, we attempted to determine the nociceptive role of LT receptors in DRG by behavioral analyses.
Discussion
LTs are lipid mediators with a proinflammatory profile and have been implicated in the pathogenesis of several types of inflammation [
1]. For example, the blood and synovial fluids of patients with rheumatoid arthritis contain higher levels of LTB4 than people without rheumatoid arthritis [
18]. LTB4 is known as a potent neutrophil chemotactic agent. It is considered that the neutrophils that are infiltrated by rheumatoid arthritis produce LTB4 in synovial fluids and induce the inflammatory condition. Several studies have demonstrated that LTs are involved in inflammatory pain [
11‐
13]. It is well known that nerve growth factor (NGF) is up-regulated in inflammatory tissue and sensitizes nociceptors [
19] leading to thermal hyperalgesia [
20]. It has reported that NGF increased LTB4 in the rat paw skin and these results suggested the participation of LTB4 in NGF-induced local thermal hyperalgesia [
21]. Furthermore, Trang et al. have reported that intrathecal administration of LTB4 leads to thermal hyperalgesia, and a BLT1 receptor antagonist suppresses this hyperalgesia [
22]. These previous reports indicate that LTs in peripheral tissues may have an effect on primary afferents.
In the present study, we demonstrated the expression of LT receptors, BLT1, BLT2, CysLT1, and CysLT2, in the adult rat DRG. We could not detect BLT2 and CysLT1 mRNAs in the DRG. We found the BLT1 mRNA expression in non-neuronal cells, but Andoh et al. reported expression of BLT1 in mouse DRG neurons [
23]. This discrepancy may be due to the difference of the species (rat versus mouse) or the methods (ISHH versus IHC). In contrast to the expression of BLT1 mRNA, CysLT2 mRNA was expressed in DRG neurons. CysLT2 was cloned in 2000 [
5], however, there has been limited information of its tissue distribution in nervous system, such as in the astrocyte in brain [
24]. CysLT2 is involved in apoptosis induced by oxygen-glucose deprivation
in vitro [
24], but its functional role remains largely unknown. We precisely quantified CysLT2 mRNA in the adult rat DRG showing that about 40% of DRG neurons expressed CysLT2 mRNA (S/N > 20) and small sized DRG neurons preferentially expressed CysLT2. Double-labeling analysis with NF-200 and CysLT2 showed that most CysLT2-labeled cells did not express NF-200. Moreover, a lot of CysLT2-positive profiles exclusively co-localized with IB4-binding, a quarter of CGRP-positive neurons expressed CysLT2 mRNA. These results indicate that CysLT2 was mainly expressed in unmyelinated and non-peptidergic neurons.
Interestingly, CysLT2 mRNA expressing neurons were heavily co-localized with TRPV1- or P2X3-positive neurons. TRPV1, one of the TRPV family, has been cloned and is a thermosensitive channel with a threshold of 42 degrees Celsius [
25]. TRPV1 is expressed in small sized neurons [
26] and is modulated by various G-protein coupled receptors, such as EP4 [
8], protease-activated receptor 2 [
27] and neurokinin-1 receptor [
28]
via the protein kinase C (PKC) pathway. 12-(S)-HPETE, a product of 12-lipoxygenase, potentiates the TRPV1 current in HEK cells [
29]. Thompson et al. have reported that the signaling pathway for CysLT2 is involved in the activation of PKC pathway
via Gq-proteins [
30]. Because it is possible that CysLT2 can sensitize TRPV1 in primary sensory neurons, we examined whether intraplantar injection of LTC4 leads to thermal hyperalgesia. All doses of LTC4 (8 fmol, 0.8 pmol and 80 pmol) did not affect on heat sensitivity at 10, 30 and 60 min after the injection in normal rats. The data indicate LTC4 does not have a role on thermal hyperalgesia in a normal condition. However, a further study is required to know the role of LTC4 on thermal sensitivity in tissue inflammation.
P2X3 is a ligand-gated ion channel for ATP, and belongs to P2X family. P2X3 is of particular interest in the context of pain pathways, because it is selectively expressed at high levels by nociceptors [
31], and electrophysiological studies suggest that the P2X receptors in sensory neurons may play an important role in the generation and/or modulation of the pain signaling from the periphery to the spinal cord [
32]. Furthermore, we previously reported that P2X3 in peripheral afferents plays a role in the induction of the hypersensitivity to mechanical stimulation observed during peripheral inflammation [
33] and many P2X3s are co-expressed with protease-activated receptor 2 in the rat dorsal root ganglion neurons. Nocifensive behaviors induced by αβ-me-ATP injection to the hind paw were significantly augmented after the application of protease-activated receptor 2 agonists [
17]. Fos expression induced by the αβ-me-ATP injection in dorsal horn neurons was also increased after the pre-application of protease-activated receptor 2 agonists [
34]. These previous studies led us to behavioral experiments to study whether the LTC4 have a role in potentiation of pain sensation induced by αβ-me-ATP. Intraplantar injection of LTC4 before the αβ-me-ATP injection induced a significant increase of paw-lifting behaviors and Fos expression in the spinal dorsal horn. Based on the finding described in the present study, we concluded that CysLT2, the receptor of LTC4, located in the primary afferent, might modulate the activation of P2X3 by the injection of αβ-me-ATP.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MO with KK and HY designed and performed all of experiments, analyzed data and drafted the paper. HY, KK, TF, YD and KN supervised the project and edited the manuscript. All authors contributed to data interpretation, have read and approved the final manuscript.