Background
Endothelin is one of many local mediators that are important in pain generation and the modulation of nociceptor responsiveness to painful stimuli. The endothelins, ET-1, ET-2 and ET-3, are vasoactive peptides, originally cloned from endothelial cells [
1], but also produced by other cell types, including some tumor cells [
2‐
5]. Endothelins act on ET
A and ET
B receptors (ET
ARs and ET
BRs) [
6,
7], both G protein-coupled receptors that can activate multiple G protein types and influence various signaling pathways [
8].
ET-1 injection excites nociceptors [
9,
10] and induces nocifensive behaviour in animals [
11‐
13], and severe pain and tactile allodynia in humans [
14]. ET receptor antagonists have been reported to reduce neuropathic and inflammatory pain, and pain in patients with metastatic prostate cancer (see [
15,
16] for reviews). Given the number of reports on the involvement of ET-1 in nociception, relatively little is known about the signaling cascade and effectors that lead to the nociceptive responses to ET-1 in primary sensory neurons.
Activation of the ET
AR, which is expressed in sensory neurons [
17], results in small increases in [Ca
2+]
i in a sensory neuron-derived cell line [
18] and DRG neurons [
19], and in a protein kinase C(PKC)-ε-mediated potentiation of Ca
2+ responses to capsaicin [
19]. The increased responsiveness of sensory neurons may result from an ET
AR-mediated lowering of the threshold for activation of tetrodotoxin (TTX)-insensitive Na
+ channels [
20], but may involve other effectors. One possibility is that ET-1 affects other channels like the nonselective cation channel TRPV1, an integrator of a number of noxious stimuli, including heat (> 42°C), capsaicin, endocannabinoids and H
+ [
21], which is essential for thermal hyperalgesia in inflammation [
22,
23]. TRPV1 activation results in depolarization and excitation of sensory neurons. In a preliminary conference report we showed that activation of the ET
AR potentiated TRPV1 responses to capsaicin in HEK 293 cells [
24]. A number of modulators sensitize nociceptors by potentiating TRPV1 responses [
25‐
30]. Possible mechanisms involved in potentiation are phosphorylation
via PKC-ε [
31] and protein kinase A (PKA) [
32,
33], disinhibition of TRPV1 by hydrolysis of phosphatidylinositol bisphosphate (PIP
2) [
28], or modulation via phophatidylinositol-3-kinase and extracellular signal-related kinases 1/2 [
34].
In this study, we investigated ET receptor expression in DRG and, using the patch clamp technique, the effects of ET-1 on responses to capsaicin in DRG neurons. A subpopulation of neurons responded to ET-1 with a potentiation of the capsaicin-mediated responses. To investigate the signaling pathways involved in potentiation, we studied the effects of ET-1 in HEK293 cells coexpressing the ETAR and TRPV1.
Discussion
We show here that DRG neurons mainly express ETARs and that their expression partially overlaps with the expression of TRPV1. We also show that ET-1 potently modulates the functional activity of TRPV1 in a subpopulation of sensory neurons and in HEK293 cells co-expressing the ETAR and TRPV1. A significant modulation of TRPV1 activity was also found for HEK293 cells co-expressing TRPV1 and ETBRs.
Even though the ET
AR can stimulate pathways leading to both PKA and PKC activation, receptor-mediated potentiation of TRPV1 in HEK293 cells was predominantly mediated by PKC. Evidence for this is that ET-1 effects were completely inhibited by the PKC inhibitor BIM X, and prevented in the PKC phosphorylation site mutant TRPV1-S800A. There is also a strong similarity between the extent of potentiation by ET-1 and that elicited by the PKC-activating phorbol ester PMA. In contrast to the effects of PKC activation and inhibition, the effects of PKA activation by forskolin were comparatively weak, and dbcAMP had no significant effect. Potentiation by ET-1 also persisted in the presence of H-89, an inhibitor of PKA. In addition, some potentiation was observed with the ET
BR which did not increase cAMP. These results indicate that, under the conditions used, i.e. in the absence of extracellular Ca
2+, ET-1-mediated potentiation is unlikely to occur via G
s and AC, nor to a great extent
via PKC-mediated activation of AC. Because the cAMP/PKA pathway acts, at least partly, by decreasing Ca
2+-dependent desensitization [
32,
43], we cannot rule out that this pathway provides an additional component of potentiation of TRPV1 by ET-1 at physiological Ca
2+ concentrations. Our work lends support to a recent study showing that PKC-ε is involved in the ET-1-mediated enhancement of capsaicin-induced Ca
2+ increases in sensory neurons, but which did not show that TRPV1 is the target for PKC-ε-mediated phosphorylation [
19]. Our observation is also in line with the finding that S800 is crucially involved in PMA-mediated sensitization of TRPV1 via PKC-ε [
44]. From our data, we cannot rule out that different pathways may be involved in the responses to ET-1 in sensory neurons, but there is a striking similarity between the effects in DRGs and in HEK293 cells. The evidence for an involvement of PKC in potentiation by ET-1 supports studies showing an involvement of PKC in potentiation of TRPV1 by bradykinin
via B
2 receptors [
26,
27,
31], ATP
via G
q-coupled P
2Y
1 receptors [
41,
45], the chemokine CCL
3 via CCR
1 [
46], 5-HT
via 5-HT
2 receptors [
47], and in some of the effects of prostaglandins
via EP
1 or IP receptors [
48], but contrasts with that showing that the effects of bradykinin and NGF result from PLC-mediated release of TRPV1 from inhibition by PIP
2 [
28,
49]. The increase of TRPV1 currents in response to PKC activation in sensory neurons most likely results from a phosphorylation-induced increase in the activity of channels at a given agonist concentration [
50], but has also been attributed to an increased recruitment of intracellularly-stored vesicles carrying TRPV1 to the plasma membrane [
51].
In DRG neurons, we observed two effects of ET-1; potentiation of responses to capsaicin and, in a smaller population of neurons, the activation of an inward current. These effects are very similar to those of bradykinin, which also activates an inward current, most likely a cation current, in some sensory neurons [
26,
52,
53], and potentiates currents through TRPV1 [
26,
27,
31]. It remains unclear whether the channel activated by bradykinin is TRPV1 because not all heat-sensitive neurons with temperature thresholds of 42°C, characteristic for TRPV1, show a current response to bradykinin [
26]. On the other hand, high concentrations of bradykinin can shift the temperature threshold of TRPV1 sufficiently to produce a current at room temperature [
27]. The inward current activated by ET-1 was not characterized directly, but it differed from TRPV1 currents in the absence of current noise for currents of comparable amplitudes, and there was no link between the amplitude of the ET-1-activated and capsaicin-activated currents. Other possible candidates for the ET-1-activated channel include other cation channels, like e.g. TRPA1, which has been shown to be activated by bradykinin [
54]. It is also notable that in HEK293 cells co-transfected with TRPV1 and ET
ARs or ET
BRs, ET-1 did not induce a rapidly activating and inactivating inward current like that in DRG neurons, but did result in a small slow increase in an outwardly-rectifying current that resembled TRPV1. Thus, the molecular basis and nature of the fast inward current seen in DRG neurons in response to ET-1 remains to be clarified.
Our data at the cellular level support a role of potentiation of currents through TRPV1 in ET-1-induced excitation of nociceptors by increasing their sensitivity to algogenic stimuli. Furthermore, ET-1 could produce an initial transient excitation of some neurons by the activation of an inward current. Previous studies have postulated that part of the effects of ET-1 on nociception occur
via modulation of TTX-resistant Na
+ channels [
20] shifting their potential dependence of activation to more negative membrane potentials leading to enhanced excitability of the sensory neurons. Effects on Na
+ channels have been observed with the hyperalgesic modulators PGE
2, 5-HT, epinephrine and adenosine (for reviews see [
55,
56]), and may be mediated by PKA or PKC activation [
57]. Thus, the actions of ET-1 in sensory neurons involve contributions of Na
+ channels and TRPV1, and may be mediated by both PKA and PKC. By its concerted actions on TRPV1 and Na
+ channels, ET-1 could increase the depolarization of sensory endings in response to noxious stimuli and concomitantly reduce the threshold for activation of a population of Na
+ channels.
The effects of ET-1 on nociception are complex. Different experimental models have been used to study the role of ET-1 in nociception, and receptor subtype-specific agonists and antagonists to identify the ET receptor subtype mediating the effects. The pronociceptive actions of ET-1 have been reported to involve either ET
ARs [
9,
10,
58‐
62] or ET
BRs [
63,
64], or both ET
ARs and ET
BRs [
12,
65,
66]. Our results indicate that the ET
AR is expressed in sensory neurons and could contribute to the algogenic effects of ET-1 by sensitizing TRPV1. This suggestion is supported by a recent study on mouse DRGs which showed that the Ca
2+ release in response to ET-1, and the potentiatory effect of ET-1 on capsaicin-induced Ca
2+ responses are mediated exclusively by ET
ARs [
19]. Even though the ET
BR is able to potentiate TRPV1 in HEK293 cells, the low level of ET
BR expression in DRG neurons indicates that the algogenic effects of ET
BR agonists [
12,
64] and the analgesic effects of ET
BR antagonists [
63‐
66] are more likely to result from indirect effects on the ET
BR in Schwann and other glial cells where receptor expression is high [
17]. It is difficult to extrapolate from data obtained on single isolated neurons to the situation
in vivo, but our results could explain the excitatory effects of ET-1 injection on nociceptors and some of the amplification of responses to thermal and mechanical stimuli. The rapid inward current, if occurring in the periphery, could rapidly excite some ET receptor-expressing nociceptive neurons. Thereafter, the potentiation of TRPV1 could be responsible for the excitation, and for the hyperalgesia and allodynia seen after ET-1 application. Potentiation of TRPV1 with high ET-1 concentrations occurs rapidly and, although it decreases with time, by extrapolating our data (e.g. those in Fig.
2A) can probably persist for several tens of minutes. It is more difficult to explain the TRPV1-mediated prolongation of tactile allodynia in response to low ET-1 concentrations where a role of TRPV1 is only significant at times longer than 30 minutes after ET-1 application [
67]. While the role of the ET
AR in the action of ET-1 on nociception is relatively clear, the role of the ET
BR is less well understood. The ET
BR in glial cells and possibly in neurons is likely to have pronociceptive effects, whereas the ET
BR in keratinocytes has been reported to mediate the analgesic effects of ET-1 and ET
B agonists by inducing the release of β-endorphin [
68].
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
TP conceived and designed the study, performed the electrophysiological experiments, analyzed the data and drafted the manuscript. CZ isolated the DRGs for immunohistochemical work and binding experiments, established primary cultures and perfomed part of the cAMP and inositol phosphate experiments. FK performed electrophysiological experiments and analyzed the data. SSM performed the immunohistochemistry. JE conducted binding experiments with membrane preparations of DRGs. MS provided rTRPV1-YFP and generated the TRPV1-S800A mutant. JF conducted part of the cAMP and inositol phosphate experiments, and performed analysis of cAMP and inositol phosphates from cell extracts. CS supervised the part of the study on DRGs. AO conceived and designed the study, performed laser scanning microscopy, supervised binding experiments and respective analysis, and drafted the manuscript.
All authors read and approved the manuscript.