Open Access

Endothelin potentiates TRPV1 via ETA receptor-mediated activation of protein kinase C

  • Tim D Plant1, 2Email author,
  • Christian Zöllner3,
  • Frauke Kepura1,
  • Shaaban S Mousa3,
  • Jenny Eichhorst4,
  • Michael Schaefer2,
  • Jens Furkert4,
  • Christoph Stein3 and
  • Alexander Oksche2
Molecular Pain20073:35

DOI: 10.1186/1744-8069-3-35

Received: 10 May 2007

Accepted: 14 November 2007

Published: 14 November 2007

Abstract

Background

Endothelin-1 (ET-1) both stimulates nociceptors and sensitizes them to noxious stimuli, an effect probably mediated by the ETA receptor (ETAR) expressed in sensory neurons. The cellular mechanisms of this ET-1-mediated effect are only poorly understood. TRPV1, the heat-, pH- and capsaicin-sensitive cation channel already known to be modulated by a number of cellular mediators released in response to noxious stimuli and during inflammation, is a potential target for the action of ET-1.

Results

We studied the effects of ET-1 on TRPV1 in sensory neurons from the dorsal root ganglion (DRG) and in HEK293 cells coexpressing TRPV1 and the ETAR. Specific 125I-ET-1 binding sites (817 ± 92 fmol/mg) were detected in membrane preparations of DRG with an ETAR/ETBR ratio of 60:40. In an immunofluorescence analysis, coexpression of TRPV1 and the ETAR was found in a subpopulation of primary sensory neurons. ET-1 strongly potentiated capsaicin-induced TRPV1 currents in some neurons, and in HEK293 cells co-expressing TRPV1 and the ETAR. Weaker potentiation was observed in HEK293 cells coexpressing TRPV1 and the ETBR. ETAR activation also increased responses to low pH and heat. In HEK293 cells, strong potentiation of TRPV1 like that induced by ET-1 via the ETAR could be induced by PKC activation, but not with activators of the adenylyl cyclase or the PKA pathway. Furthermore, inhibition of PKC with bisindolylmaleimide X (BIM X) or mutation of the PKC phosphorylation site S800 completely prevented ETAR-mediated potentiation.

Conclusion

We conclude that ET-1 potentiates TRPV1 by a PKC-dependent mechanism and that this could play a major role in the algogenic and hyperalgesic effects of ET-1 described in previous studies.

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 [25]. Endothelins act on ETA and ETB receptors (ETARs and ETBRs) [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 [1113], 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 ETAR, which is expressed in sensory neurons [17], results in small increases in [Ca2+]i in a sensory neuron-derived cell line [18] and DRG neurons [19], and in a protein kinase C(PKC)-ε-mediated potentiation of Ca2+ responses to capsaicin [19]. The increased responsiveness of sensory neurons may result from an ETAR-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 ETAR potentiated TRPV1 responses to capsaicin in HEK 293 cells [24]. A number of modulators sensitize nociceptors by potentiating TRPV1 responses [2530]. 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 (PIP2) [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.

Results

Endothelin receptors in dorsal root ganglion neurons

The expression of endothelin receptor subtypes in the rat lumbar DRG was analyzed in binding experiments using 125I-ET-1 as the radioligand. Saturation binding analysis of membranes derived from isolated lumbar DRG (L4 – L5) revealed a maximal binding capacity of 817 ± 92 fmol/mg (mean ± SD of three measurements performed in duplicate). When the ETAR-selective antagonist BQ123 or the ETBR-selective agonist IRL1620 were used as competing ligands (to determine the amount of ETAR or ETBR expression), binding capacities of 503 ± 115 and 313 ± 113 fmol/mg protein were obtained, respectively. Thus, both ETARs and ETBRs are expressed in the rat DRG with an expression ratio of 60:40.

In an immunofluorescence analysis, we further studied the distribution of ETARs and ETBRs in tissue sections of lumbar DRG. To probe whether ETARs and ETBRs are expressed in small sensory neurons that express TRPV1, we performed double staining experiments using affinity-purified antibodies against ETARs or ETBRs in conjunction with antibodies against TRPV1. The data show that ETARs are widely expressed in small and medium-to-large diameter neurons, and, in particular, in TRPV1-expressing small sensory neurons (Fig. 1A). Controls with pre-immune serum showed a weak uniform staining of all neurons (not shown). Coexpression of the ETAR with TRPV1 was found in 31 of 66 TRPV1-positive DRGs (47%). ETBR expression in DRGs was, if present, relatively weak (Fig. 1B), and not easy to distinguish from unspecific staining (not shown). The staining with the ETBR antibody showed a different pattern from that of the ETAR and TRPV1 antibodies and surrounded the perikarya, possibly reflecting expression in satellite cells. More prominent staining with the ETBR antibody was observed when analyzing the axonal extensions of DRG, where the cells stained by the ETB antibody were costained by antibodies directed against the S100 antigen (Fig. 1C), indicating that they are glia. In contrast, for the ETAR no expression was found in glial cells (data not shown). Thus, ETAR are found in sensory neurons, including TRPV1-positive small diameter neurons, whereas ETBRs are mainly found in glial cells. Our data on ETR expression in sensory neurons are consistent with previous reports [17, 35], which also demonstrated ETAR expression in neurons, frequently in small neurons together with calcitonin gene-related peptide [17], a marker of a subpopulation of C fibers. These studies also demonstrated ETBR expression in DRG satellite cells and nonmyelinating Schwann cells, but not in DRGs.
Figure 1

Expression of ET A Rs and ET B Rs in rat dorsal root ganglion. Sections of lumbar rat dorsal root ganglion were analysed for the expression of ETARs (A) or ETBRs (B, C) using affinity-purified antibodies. For the identification of TRPV1-postive neurons co-staining with a TRPV1 antibody (A, B) was performed. Expression of ETBRs in glial cells was demonstrated by co-staining of the S100 antigen (C). Bars: 20 μm.

Effects of ET-1 in isolated sensory neurons

A low concentration of capsaicin (10 nM) evoked currents through TRPV1 in a subpopulation of DRG neurons. In the Ca2+-free extracellular solutions used to minimise the Ca2+-dependent component of desensitization, repetitive applications of capsaicin activated currents which were either stable or which declined slowly. In 9/30 capsaicin-sensitive neurons (30%), application of ET-1 (100 nM) for 30 or 60 s prior to the application of 10 nM capsaicin resulted in a large increase in the capsaicin-induced current (Fig. 2A). On average, currents were increased 14.7 ± 4.1-fold (mean ± SEM, n = 8) when the first capsaicin-elicited response after ET-1 treatment was compared to that before ET-1 addition. The potentiation of the capsaicin responses by a single application of ET-1 was transient; the amplitude of the capsaicin-activated current declined to different degrees in response to repetitive stimulation in the six neurons in which three or more capsaicin responses could be elicited after ET-1 application. However, as shown in Figure 2A, potentiation often persisted for several minutes. In 5/9 neurons that responded to ET-1 with a potentiation of capsaicin-induced TRPV1 activation, ET-1 alone also transiently activated an inward current with a wide range of amplitudes (between -2.0 and -69.3 pA/pF, mean: -25.5 ± 12.3 pA/pF, n = 5) at -60 mV (Fig. 2B). Activation and decay of the ET-1-activated current was rapid. The cells that responded to ET-1 with a potentiation of capsaicin-activated currents could not be distinguished from the non-responsive cells on the basis of their size. Their capacitances, indicative of the cell surface area, were 35.3 ± 5.9 pF (n = 8) and 39.1 ± 3.7 pF (n = 21), respectively. In 13 of the 21 cells that did not respond to ET-1, we subsequently tested for the ability of bradykinin (1 μM) to potentiate capsaicin (10 nM)-activated TRPV1 currents. In 6/13 cells, bradykinin application resulted in a potentiation of capsaicin-activated currents through TRPV1 (16.5 ± 7.6-fold, n = 6), which closely resembled that induced by ET-1 (data not shown). It was notable that responses of DRGs to ET-1 were mainly observed on the day of preparation, or on the day after preparation. Neurons cultured for longer rarely responded to ET-1, but did respond to bradykinin, suggesting that in cultured DRGs the expression of ET receptor subtypes might be down regulated or that there is an impairment in the efficiency of coupling to downstream signaling cascades. Loss of receptors during culture may also explain the lower percentage of cultured capsaicin-sensitive neurons that respond to ET-1, compared to the percentage of DRGs coexpressing TRPV1 and ETAR in sections of ganglia.
Figure 2

Potentiation of capsaicin-activated currents in dorsal root ganglion neurons by ET-1. A, Recording of current at -60 mV showing responses to repetitive applications of capsaicin (10 nM) in a Ca2+-free extracellular solution. Application of ET-1 (100 nM) at the time indicated by the bar resulted in a small increase in current during the ET-1 application and a large potentiation of the capsaicin-activated currents. B, Results of a similar experiment to that in A which shows a larger current response to ET-1 application. C, Bar graph summarising the potentiation of capsaicin responses by ET-1 (100 nM) in ET-1-responsive rat DRG neurons. Control is the normalized capsaicin response before ET-1 application, after ET-1 the first (n = 9, p = 0.0066) and second (n = 8, p = 0.017) normalized responses after ET-1 application.

Effects of ET-1 on HEK293 cells transiently transfected with TRPV1 and the ETA or ETB receptor

Owing to the small fraction of neurons that responded to ET-1, the regulation of TRPV1 by ET-1 was analysed in HEK293 cells transiently co-transfected with plasmids encoding TRPV1-YFP and the ETAR or ETBR. HEK293 cells co-expressing the ETAR and TRPV1 responded to 10 nM capsaicin with currents that displayed the characteristic outwardly-rectifying IV-relation of TRPV1 (Fig. 3A, B). ET-1 application (100 nM) for 30 s or 1 minute resulted in a large potentiation of capsaicin-activated currents through TRPV1 (Fig. 3A – C), like that in sensory neurons. Currents recorded during the first response to capsaicin directly after ET-1 application were increased 8.3 ± 1.5-fold (n = 10), those in response to the second capsaicin application, 2.5 minutes after ET-1 treatment, 10.7 ± 2.0-fold (n = 9). In addition, in most cells, ET-1 application resulted in the slow activation of a current, particularly in the outward direction, with the characteristics of TRPV1 (Fig. 3A, B). However, none of the cells displayed a rapid, transient current like that seen in DRGs, an indication that the ET-1-activated current inn DRGs may be mediated by a different channel.
Figure 3

Potentiation of TRPV1 currents by ET-1 in HEK293 cells cotransfected with TRPV1 and the ET A R. A – C, Potentiation of capsaicin-activated currents by ET-1. A, Recording of current at -60 mV showing the potentiation of responses to capsaicin (10 nM) by the application of ET-1 (100 nM). B, IV-relationships recorded at the times indicated by the numbers 1 – 4 in A. C, Bar graph summarizing the potentiation of TRPV1 responses to capsaicin by ETAR activation in HEK293 cells. The bars show the normalized control response prior to ET-1 application and the normalized first (n = 10, p = 0.0009) and second (n = 9, p = 0.0012) responses after ET-1 application. D – F, Potentiation of H+-activated currents by ET-1. D, ET-1 increased the amplitude of TRPV1 responses to pH 5.1. Currents were recorded at -60 mV and applications of pH 5.1 preceded by a solution of pH 6.8 to prevent the activation of endogenous ASICs. E, IV-relationships recorded at the times indicated by numbers in D. F, Bar graph summarizing the potentiation of the TRPV1 response to pH 5.1 by ET-1, and showing the normalized response before ET-1, and the first (n = 12, p < 0.0001) and second (n = 9, p = 0.0069) responses after ET-1. G – J, Potentiation of heat-activated currents through TRPV1 by ET-1. G, Current recordings at -60 mV (upper traces) showing consecutive responses of a control cell to heating to 44°C (left), and responses from a cell treated with ET-1 after the first response to heat (right). The lower traces show the temperature recorded in the chamber close to the cell. H, I-V relationships recorded at the times indicated by the numbers in G. I, Bar graph summarizing the potentiation of the normalized first (n = 10, p = 0.0252) and second (n = 7, p = 0.0233) responses to heat after ET-1. J, Plot of the current-temperature relationships for the control response (filled squares) and the second response after ET-1 (open squares) from the experiment in G.

In ETAR-expressing cells, application of ET-1 also potentiated currents activated by low pH or by heat (Fig. 3D – J). H+-activated TRPV1 currents were evoked by application of a solution of pH 5.1, a pH which produces a submaximal TRPV1 activation, preceded by the application of a solution of pH 6.8 to desensitize endogenous acid-sensing ion channels (ASICs; Fig. 3D) [36]. Application of ET-1 resulted in a 9.9 ± 1.3-fold (n = 12) potentiation of the first response to pH 5.1 after ET-1 application (Fig. 3D – F), and a significant potentiation of subsequent responses (Fig. 3D, F). Current responses of TRPV1 to heat were recorded upon raising the temperature from room temperature to 44°C (Fig. 3G). In controls, consecutive responses had similar amplitudes (Fig. 3G, left). ET-1 augmented the responses to the increase in temperature (2.5 ± 0.6-fold, n = 10, for the first response after ET-1, Fig. 3I) and, as illustrated for the experiment in Fig.3G, 3H, shifted the threshold for TRPV1 activation to lower temperatures (Fig. 3J).

HEK293 cells transfected with the same amounts of TRPV1 and the ETBR showed a less prominent ET-1-induced potentiation of capsaicin-stimulated TRPV1 currents when compared to that elicited by the ETAR (Fig. 4). The first and second responses after ET-1 application were 2.9 ± 0.8-fold and 2.7 ± 0.5-fold (n = 5) that of the control, respectively.
Figure 4

ET-1 also potentiates capsaicin-activated currents in HEK293 cells cotransfected with TRPV1 and the ET B R. A, Current responses to capsaicin (10 nM) application were potentiated by the application of ET-1 (100 nM). B, Bar graph summarizing the effects of ET-1 on normalized capsaicin responses in cells cotransfected with TRPV1-YFP and the ETBR. Shown are the first (n = 7, p = 0.0102) and second (n = 6, p = 0.0163) capsaicin response after ET-1 normalized to the capsaicin response before ET-1 application.

Role of protein kinases in the ETA receptor-mediated potentiation of TRPV1

Both ET receptor subtypes couple to G proteins from different families and each of the receptor types has its own distinct profile of G proteins that it can activate. The ETAR stimulates G proteins of the Gq, Gs and G12/13 families, the ETBR activates G proteins of the Gqand Gi families e.g. [3740]. To investigate the signalling pathways activated and possibly involved in the modulation of TRPV1 in HEK293 cells, we studied cAMP and inositol phosphate formation in HEK cells stably expressing the ETAR or ETBR. ET-1 induced 22 ± 2.5- and 18 ± 2.0-fold increases in inositol phosphate formation via ETARs and ETBRs, respectively (Table 1). For HEK cells expressing the ETAR, a 34 ± 10.6-fold increase in cAMP levels was noted. Cells expressing the ETBR did not show any ET-1-induced increase in cAMP.
Table 1

Synopsis of ET-1-mediated cAMP and inositol phosphate formation via ETA and ETB receptors

 

HEK ETAR

HEK ETBR

 

-fold of control

EC50 [nM]

-fold of control

EC50 [nM]

formation of inositol phosphates

22.3 ± 2.5

4.5 ± 1.2

18 ± 2.0

7.3 ± 1.7

formation of cAMP

34 ± 10.6

13.3 ± 3.2

n. i.

n.a.

HEK293 cells stably expressing the ETAR or the ETBR were stimulated with increasing concentrations of ET-1 (up to 100 nM) in the presence of 10 mM LiCl (inositol phosphate assays) for 60 min or 1 mM IBMX (cAMP assays) for 30 min. The amount of formed inositol phosphates was determined by anion exchange chromatography and cAMP was determined by cAMP RIA as described in Materials and Methods. Values are means ± SD of at least three independent experiments performed in duplicate. Duplicates differed by less than 10 %. n.i.: no increase, n.a.: not applicable

From the data presented above, stimulation of the Gq-coupled ETAR in HEK293 cells could potentiate TRPV1 by a PLCβ-mediated breakdown of PIP2 or by the production of DAG and activation of PKC. Alternatively, TRPV1 potentiation could be mediated by the activation of PKA. We therefore studied the effects of PKC and adenylyl cyclase (AC)/PKA activation on capsaicin-mediated TRPV1 activation, and compared these with ET-1/ETAR-mediated effects on TRPV1 activity. Application of the phorbol ester, phorbol myristate acetate (1 μM), for 1 minute before the addition of capsaicin (10 nM) resulted in a clear and strong potentiation of the capsaicin responses that closely resembled the potentiation seen with ET-1 (Fig. 5). In contrast to activation of PKC by PMA, the activation of AC with forskolin (50 μM, Fig. 5) or treatment with the membrane-permeable cAMP analog, dibutyryl cAMP (dbcAMP; 5 mM), had comparatively weak effects or no effect on TRPV1 currents, respectively. After 1.5 minutes of treatment, forskolin increased currents 1.5 ± 0.1-fold (n = 4), but the effect after 4 minutes of forskolin treatment was not significant. Similarily, dbcAMP had no significant effect on current responses to capsaicin; they were 0.97 ± 0.11-fold (n = 4, p = 0.802) and 1.5 ± 0.5-fold (n = 4, p = 0.357) of the control, after 1.5 and 4 minutes of dbcAMP application, respectively.
Figure 5

Potentiatory effects of forskolin and PMA onTRPV1. A, Adenylyl cyclase activation with forskolin (50 μM) resulted in a small increase in current responses to capsaicin (caps, 10 nM) whereas application of the PKC activator PMA (1 μM) resulted in a large potentiation of responses to capsaicin. B, Bar graph showing the control and the first two normalized responses to capsaicin after forskolin (forskolin 1: n = 5, p = 0.0271; forskolin 2: n = 4, p = 0.067) and PMA (PMA 1: n = 4, p = 0.0922; PMA 2: n = 4, p = 0.0088) application.

To analyse the role of PKC activation in ET-1-mediated potentiation of TRPV1, we tested the effect of the PKC inhibitor BIM X on the potentiation of capsaicin-induced TRPV1 currents in response to ET-1. Potentiation was completely prevented by a 2 minute incubation with BIM X (500 nM) prior to the ET-1 application (Fig. 6A). Control experiments performed in parallel on the same days with cells transfected at the same time showed strong potentiation (Fig. 6B). In contrast to the results after PKC inhibition, potentiation still occurred after treatment with the protein kinase inhibitor H89 (5 μM, n = 4), which at this concentration most strongly inhibits PKA, and was not significantly different from that without H89 (Fig. 6).
Figure 6

Protein kinase C inhibitors, but not protein kinase A inhibitors prevent the potentiation of TRPV1 by ET-1. A, Current trace from an experiment in which the PKC inhibitor BIM X (500 nM) was applied immediately prior to stimulation with ET-1 (100 nM) illustrating that BIM X treatment prevented potentiation by ET-1. B, Bar graph summarizing the effects of ET-1 on the first response to capsaicin after ET-1 in the absence (n = 6, p = 0.0156) and presence of BIM X (n = 7, p = 0.8964). C, Trace showing ET-1 potentiation of capsaicin-activated currents in the presence of the PKA inhibitor H-89. D, Bar graph summarizing the effects of ET-1 on the first (n = 4, p = 0.0953) and second (n = 4, p = 0.0275) capsaicin responses after treatment with H-89. Cells were cotransfected with TRPV1-YFP and the ETAR.

To confirm the pivotal role of PKC-mediated phosphorylation in the modulation of TRPV1 by ET-1 using an independent approach, we employed a mutant of TRPV1 which does not show PKC-mediated potentiation. The PKC phosphorylation sites involved in PKC-mediated enhancement of capsaicin-activated currents through TRPV1 have been localized to S502 and S800 [41, 42]. Mutation of either site to alanine leads to a very strong reduction in sensitization of currents by PMA [41, 42] and by mediators like ATP that act via the Gq/11-coupled metabotropic P2Y1 receptor leading to an activation of PKC [41]. We therefore tested the effects of ETAR activation on the TRPV1 mutant TRPV1-S800A. The ETAR was coexpressed with YFP-tagged TRPV1-S800A in HEK293 cells. The mutant TRPV1-S800A differed from wild type TRPV1 in that currents tended to increase with consecutive responses, rather than decrease (compare the first and second response to capsaicin in Fig. 7A). In contrast to wild type TRPV1, no significant potentiation of capsaicin-activated currents was observed on application of ET-1 (Fig. 7). It is unclear whether the slight increase after ET-1 in Fig. 7A results from run-up of channel currents or from a very small potentiation that persisted in the mutant. The inability to observe an effect does not result from the briefer capsaicin application than that for the wild type because the application was continued until the current response was close to a plateau (Fig. 7A, lower trace). It is clear, however, that the strong stimulatory effect of ETAR activation is lost following mutation of the PKC phosphorylation site, consistent with sensitization resulting from PKC-mediated phosphorylation.
Figure 7

ET-1 does not potentiate capsaicin responses from the PKC phosphorylation site mutant TRPV1-S800A. A, top, Current trace showing responses to capsaicin (10 nM) before and after ET-1 (100 nM) application to a HEK293 cell cotransfected with TRPV1-S800A and the ETAR. Bottom, Current response to capsaicin after ET-1 in top trace on an expanded time scale. B, Bar graph summarizing the effects of ET-1 on the first (n = 4, p = 0.0735) and second (n = 4, p = 0.0921) capsaicin responses after ET-1 application recorded from TRPV1-S800A.

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 ETAR 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 ETBR which did not increase cAMP. These results indicate that, under the conditions used, i.e. in the absence of extracellular Ca2+, ET-1-mediated potentiation is unlikely to occur via Gs and AC, nor to a great extent via PKC-mediated activation of AC. Because the cAMP/PKA pathway acts, at least partly, by decreasing Ca2+-dependent desensitization [32, 43], we cannot rule out that this pathway provides an additional component of potentiation of TRPV1 by ET-1 at physiological Ca2+ concentrations. Our work lends support to a recent study showing that PKC-ε is involved in the ET-1-mediated enhancement of capsaicin-induced Ca2+ 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 B2 receptors [26, 27, 31], ATP via Gq-coupled P2Y1 receptors [41, 45], the chemokine CCL3 via CCR1 [46], 5-HT via 5-HT2 receptors [47], and in some of the effects of prostaglandins via EP1 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 PIP2 [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 ETARs or ETBRs, 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 PGE2, 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 ETARs [9, 10, 5862] or ETBRs [63, 64], or both ETARs and ETBRs [12, 65, 66]. Our results indicate that the ETAR 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 Ca2+ release in response to ET-1, and the potentiatory effect of ET-1 on capsaicin-induced Ca2+ responses are mediated exclusively by ETARs [19]. Even though the ETBR is able to potentiate TRPV1 in HEK293 cells, the low level of ETBR expression in DRG neurons indicates that the algogenic effects of ETBR agonists [12, 64] and the analgesic effects of ETBR antagonists [6366] are more likely to result from indirect effects on the ETBR 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 ETAR in the action of ET-1 on nociception is relatively clear, the role of the ETBR is less well understood. The ETBR in glial cells and possibly in neurons is likely to have pronociceptive effects, whereas the ETBR in keratinocytes has been reported to mediate the analgesic effects of ET-1 and ETB agonists by inducing the release of β-endorphin [68].

Conclusion

Our results show that ET-1 potentiates TRPV1 via a PKC-mediated effect and we suggest that this could be responsible for a major part of the pain-producing and hyperalgesic effects of ET-1 observed in previous studies.

Methods

Isolation and culture of DRG neurons

Dorsal root ganglia (DRG) were isolated as described recently [69]. In brief, DRG from lumbar segments L4 – L5 of male Wistar rats were isolated and placed in Minimal Essential Medium (MEM, Biochrom AG, Berlin, Germany) at 4°C. Thereafter, they were sequentially digested with collagenase (type II; 3 mg/ml) for 50 min at 37°C, and trypsin (type I; 0.25 mg/ml) for 10 min at 37°C. After careful mechanical dissociation, cells were washed twice and resuspended in fresh medium supplemented with 10% horse serum, 50 ng/ml NGF, 50 U/ml penicillin and 50 μg/ml streptomycin. Neurons were seeded on poly-l-lysine-coated glass coverslips in 6-well culture plates 4 – 36 h before the experiments. Neurons were kept at 37°C in an atmosphere of 5% CO2.

Generation of plasmids encoding mutant TRPV1

To investigate the role of PKC-mediated phosphorylation of TRPV1, a plasmid was generated, which encodes a mutant TRPV1 in which serine 800 is replaced by alanine (TRPV1-S800A). Site-directed mutagenesis was performed with the QuickChange mutagenesis kit (Stratagene, La Jolla, CA), using a plasmid encoding a rat TRPV1-YFP fusion protein [70] and appropriate sense and antisense oligonucleotides spanning the mutated triplet and 15 flanking nucleotides. The construct was verified by DNA sequencing.

HEK culture

HEK293 cells were cultured in MEM-Earle medium (Biochrom), supplemented with 10% (v/v) fetal calf serum (Gibco/Invitrogen, Karlsruhe, Germany) and 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were plated onto glass cover slips 24 – 48 h prior to transfection. The cells were transiently transfected with 1 μg of the plasmid pTRPV1-YFP [70] and 2 μg of the plasmid pETA-myc DNA [71] or the plasmid containing the FLAG-tagged ETB DNA [71] using 6 μl of FuGENE 6 Transfection Reagent (Roche Diagnostics, Mannheim, Germany) in 94 μl of OptiMEM medium (Gibco/Invitrogen) per 85 mm dish. In some experiments, cells were transfected 1 – 2 days after plating into 35 mm dishes using 9 μl TransIT (Mirus, Madison, WI) in serum-free MEM-Earle medium, subsequently trypsinized and plated onto glass coverslips 2 days after transfection. No differences were observed between results obtained with the two methods. Electrophysiological experiments were performed 24 – 72 h after transfection.

Immunofluorescence analysis

Immunofluorescence analysis was performed as described recently [69]. Freshly isolated DRG were embedded in Tissue-Tek compound (OCT, Miles Inc., Elkhart, USA) and frozen. Consecutive sections (9 μm) were prepared with a cryostat and mounted onto gelatin-coated slides. To prevent non-specific binding, the sections were incubated for 60 min in PBS containing 0.3% Triton X-100, 1% BSA, 4% goat serum, and 4% horse serum (block solution). The sections were then incubated overnight at 4°C with a guinea pig polyclonal antibody against TRPV1 (1:1,000; Chemicon, CA, USA) or S100 Antigen (1:200; Abcam, Cambridge, UK) in combination with a peptide-derived rabbit polyclonal antibody directed against the C terminus of the ETAR (1:100) (Plant et al., 2006) or against the N terminus of the ETBR (1:100) [71]. The tissue sections were washed with PBS and then incubated with Texas red-conjugated goat anti-rabbit antibody and FITC-conjugated donkey anti-guinea pig antibody. Thereafter, sections were washed with PBS, mounted in vectashield (Vector Laboratories, Burlingame, CA, USA) and viewed with a Zeiss 510 laser scanning microscope (Zeiss, Oberkochen, Germany).

cAMP and inositol phosphate measurements

Determination of inositol phosphates and of cAMP was performed as described previously [72]. In brief, HEK293 cells stably expressing ETARs or ETBRs were seeded onto 24-well plates (100,000 cells/well). For inositol phosphate determination, cells were incubated the following day with 74 kBq/ml myo- [2-3H]inositol (specific activity 370 – 740 GBq/mmol; Amersham Biosciences) for 20 h at 37°C. Cells were then washed with DMEM, 10 mM HEPES, 0.5% BSA, 10 mM LiCl, and finally stimulated with buffer or increasing concentrations of ET-1 (10 pM to 100 nM) for 60 min at 37°C. Cells were then lysed with 0.1 M NaOH, and inositol phosphates isolated from cleared supernatants by anion exchange chromatography.

For the determination of cAMP, cells were washed with 1 ml of stimulation medium (DMEM without fetal calf serum, supplemented with 10 mM HEPES, 0.5% BSA, 0.25 mM 3-isobutyl-1-methylxanthine) and incubated for 30 min at 37°C with buffer or increasing concentrations of ET-1 (10 pM to 100 nM). Cells were extracted with 750 μl of 0.1% trifluoroacetic acid, 0.005% Triton X-100 for 30 min at 4°C. After acetylation of the samples, the cAMP content was determined using 125I-cAMP-tyrosylmethylester (10,000 cpm, specific activity 81.4 TBq/mM, Biotrend, FRG) and polyclonal rabbit anti-cAMP-antibody (final dilution 1:160,000). After an overnight incubation at 4°C, the antibody-bound fraction was precipitated, and the radioactivity of the precipitate was determined in a β-counter.

125I-ET-1 binding analysis

Binding analysis was performed as described [73, 74]. In brief, membranes (5 μg) of DRGs were incubated in a final volume of 200 μl Tris/BAME buffer with increasing concentrations of 125I-ET-1 (18 to 1000 pM) in the absence or presence of unlabelled ET-1 (1 μM) (to detect ETARs and ETBRs) for 2 hours at 25°C. The ratio of ETA and ETBR expression was determined by 125I-ET-1 saturation binding analysis using ET-1 (1 μM, total binding), IRL1620 (1 μM, ETBR-specific binding) or BQ123 (10 μM, ETAR-specific binding) as competing ligands. The samples were then transferred onto GF/C filters (Whatman International Ltd., Maidstone, UK), and washed twice with PBS using a Brandel cell harvester. Radioactivity was determined in a γ-counter. Data were analyzed with RadLig Software 4.0 (Cambridge, UK).

Patch clamp recordings

Recordings of whole cell currents from single cells were made with an EPC-7 or EPC-10 amplifier using Pulse software (HEKA, Lambrecht, Germany) as described previously [75]. Experiments were performed using the standard whole cell mode of the patch clamp technique. Cells were held at a potential of -60 mV and the current recorded using XChart (HEKA). In HEK293 cells, ramps from -100 to +100 mV with a duration of 400 ms were applied at a frequency of 0.2 Hz. Ramp data were acquired at a frequency of 4 kHz after filtering at 1 kHz.

The standard pipette solution contained 100 mM CH3O3SCs (cesium methane sulfonate), 25 mM CsCl, 3 mM MgCl2, 2 mM Na2ATP, 3.62 mM CaCl2, 10 mM EGTA, 30 mM HEPES (pH 7.2 with CsOH). Pipette tips were filled with the same solution without ATP to avoid the activation of purinergic receptors prior to seal formation. The standard extracellular solution contained: 140 mM NaCl, 5 mM CsCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 10 mM HEPES (pH 7.4 with NaOH). For Ca2+-free solutions, Ca2+ was omitted and 0.5 mM EGTA added. Experiments were performed at room temperature 20 – 25°C. Solutions were applied by bath perfusion. In most cases, with the exception of Fig. 2B, which was not used for quantification of the response, capsaicin was applied until currents approached a steady state value. In experiments on the effects of heat, bath temperature was measured using a thermistor placed close to the cell studied and recorded using XChart. The test solution was preheated to appropriate temperatures using a water jacket connected to a water bath.

Declarations

Acknowledgements

This work was supported by grants from the DFG and Fonds der Chemischen Industrie. We thank Inge Reinsch and Nadine Albrecht for technical assistance.

Authors’ Affiliations

(1)
Institut für Pharmakologie und Toxikologie, FB-Medizin, Philipps-Universität Marburg
(2)
Institut für Pharmakologie, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin
(3)
Klinik für Anästhesiologie und operative Intensivmedizin, Charité-Universitätsmedizin Berlin, Campus Benjamin-Franklin
(4)
Leibniz-Institut für Molekulare Pharmakologie

References

  1. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, Masaki T: A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988, 332: 411–415. 10.1038/332411a0PubMedView ArticleGoogle Scholar
  2. Ehrenreich H, Anderson RW, Fox CH, Rieckmann P, Hoffman GS, Travis WD, Coligan JE, Kehrl JH, Fauci AS: Endothelins, peptides with potent vasoactive properties, are produced by human macrophages. J Exp Med 1990, 172: 1741–1748. 10.1084/jem.172.6.1741PubMedView ArticleGoogle Scholar
  3. Giaid A, Gibson SJ, Ibrahim BN, Legon S, Bloom SR, Yanagisawa M, Masaki T, Varndell IM, Polak JM: Endothelin 1, an endothelium-derived peptide, is expressed in neurons of the human spinal cord and dorsal root ganglia. Proc Natl Acad Sci U S A 1989, 86: 7634–7638. 10.1073/pnas.86.19.7634PubMed CentralPubMedView ArticleGoogle Scholar
  4. MacCumber MW, Ross CA, Snyder SH: Endothelin in brain: receptors, mitogenesis, and biosynthesis in glial cells. Proc Natl Acad Sci U S A 1990, 87: 2359–2363. 10.1073/pnas.87.6.2359PubMed CentralPubMedView ArticleGoogle Scholar
  5. Nelson JB, Hedican SP, George DJ, Reddi AH, Piantadosi S, Eisenberger MA, Simons JW: Identification of endothelin-1 in the pathophysiology of metastatic adenocarcinoma of the prostate. Nat Med 1995, 1: 944–949. 10.1038/nm0995-944PubMedView ArticleGoogle Scholar
  6. Sakurai T, Yanagisawa M, Takuwa Y, Miyazaki H, Kimura S, Goto K, Masaki T: Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor. Nature 1990, 348: 732–735. 10.1038/348732a0PubMedView ArticleGoogle Scholar
  7. Arai H, Hori S, Aramori I, Ohkubo H, Nakanishi S: Cloning and expression of a cDNA encoding an endothelin receptor. Nature 1990, 348: 730–732. 10.1038/348730a0PubMedView ArticleGoogle Scholar
  8. Alexander SP, Mathie A, Peters JA: Endothelin. Br J Pharmacol 2006, 147 Suppl 3: S33–34. 10.1038/sj.bjp.0706612View ArticleGoogle Scholar
  9. Davar G, Hans G, Fareed MU, Sinnott C, Strichartz G: Behavioral signs of acute pain produced by application of endothelin-1 to rat sciatic nerve. Neuroreport 1998, 9: 2279–2283. 10.1097/00001756-199807130-00025PubMedView ArticleGoogle Scholar
  10. Gokin AP, Fareed MU, Pan HL, Hans G, Strichartz GR, Davar G: Local injection of endothelin-1 produces pain-like behavior and excitation of nociceptors in rats. J Neurosci 2001, 21: 5358–5366.PubMedGoogle Scholar
  11. Raffa RB, Schupsky JJ, Lee DK, Jacoby HI: Characterization of endothelin-induced nociception in mice: evidence for a mechanistically distinct analgesic model. J Pharmacol Exp Ther 1996, 278: 1–7.PubMedGoogle Scholar
  12. Raffa RB, Schupsky JJ, Jacoby HI: Endothelin-induced nociception in mice: mediation by ETA and ETB receptors. J Pharmacol Exp Ther 1996, 276: 647–651.PubMedGoogle Scholar
  13. Ferreira SH, Romitelli M, de Nucci G: Endothelin-1 participation in overt and inflammatory pain. J Cardiovasc Pharmacol 1989, 13 Suppl 5: S220–2.PubMedView ArticleGoogle Scholar
  14. Dahlof B, Gustafsson D, Hedner T, Jern S, Hansson L: Regional haemodynamic effects of endothelin-1 in rat and man: unexpected adverse reaction. J Hypertens 1990, 8: 811–817. 10.1097/00004872-199009000-00004PubMedView ArticleGoogle Scholar
  15. Davar G: Endothelin-1 and metastatic cancer pain. Pain Med 2001, 2: 24–27. 10.1046/j.1526-4637.2001.002001024.xPubMedView ArticleGoogle Scholar
  16. Nicol GD: ET--phone the pain clinic. Trends Neurosci 2004, 27: 177–80; discussion 180. 10.1016/j.tins.2004.02.001PubMedView ArticleGoogle Scholar
  17. Pomonis JD, Rogers SD, Peters CM, Ghilardi JR, Mantyh PW: Expression and localization of endothelin receptors: implications for the involvement of peripheral glia in nociception. J Neurosci 2001, 21: 999–1006.PubMedGoogle Scholar
  18. Zhou QL, Strichartz G, Davar G: Endothelin-1 activates ET(A) receptors to increase intracellular calcium in model sensory neurons. Neuroreport 2001, 12: 3853–3857. 10.1097/00001756-200112040-00050PubMedView ArticleGoogle Scholar
  19. Yamamoto H, Kawamata T, Ninomiya T, Omote K, Namiki A: Endothelin-1 enhances capsaicin-evoked intracellular Ca2+ response via activation of endothelin a receptor in a protein kinase Cepsilon-dependent manner in dorsal root ganglion neurons. Neuroscience 2006, 137: 949–960. 10.1016/j.neuroscience.2005.09.036PubMedView ArticleGoogle Scholar
  20. Zhou Z, Davar G, Strichartz G: Endothelin-1 (ET-1) selectively enhances the activation gating of slowly inactivating tetrodotoxin-resistant sodium currents in rat sensory neurons: a mechanism for the pain-inducing actions of ET-1. J Neurosci 2002, 22: 6325–6330.PubMedGoogle Scholar
  21. Julius D, Basbaum AI: Molecular mechanisms of nociception. Nature 2001, 413: 203–210. 10.1038/35093019PubMedView ArticleGoogle Scholar
  22. Caterina MJ, Leffler A, Malmberg AB, Martin WJ, Trafton J, Petersen-Zeitz KR, Koltzenburg M, Basbaum AI, Julius D: Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 2000, 288: 306–313. 10.1126/science.288.5464.306PubMedView ArticleGoogle Scholar
  23. Davis JB, Gray J, Gunthorpe MJ, Hatcher JP, Davey PT, Overend P, Harries MH, Latcham J, Clapham C, Atkinson K, Hughes SA, Rance K, Grau E, Harper AJ, Pugh PL, Rogers DC, Bingham S, Randall A, Sheardown SA: Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia. Nature 2000, 405: 183–187. 10.1038/35012076PubMedView ArticleGoogle Scholar
  24. Plant TD, Zollner C, Mousa SA, Oksche A: Endothelin-1 potentiates capsaicin-induced TRPV1 currents via the endothelin A receptor. Exp Biol Med (Maywood) 2006, 231: 1161–1164.Google Scholar
  25. Lopshire JC, Nicol GD: The cAMP transduction cascade mediates the prostaglandin E2 enhancement of the capsaicin-elicited current in rat sensory neurons: whole-cell and single-channel studies. J Neurosci 1998, 18: 6081–6092.PubMedGoogle Scholar
  26. Cesare P, McNaughton P: A novel heat-activated current in nociceptive neurons and its sensitization by bradykinin. Proc Natl Acad Sci U S A 1996, 93: 15435–15439. 10.1073/pnas.93.26.15435PubMed CentralPubMedView ArticleGoogle Scholar
  27. Sugiura T, Tominaga M, Katsuya H, Mizumura K: Bradykinin lowers the threshold temperature for heat activation of vanilloid receptor 1. J Neurophysiol 2002, 88: 544–548.PubMedGoogle Scholar
  28. Chuang HH, Prescott ED, Kong H, Shields S, Jordt SE, Basbaum AI, Chao MV, Julius D: Bradykinin and nerve growth factor release the capsaicin receptor from PtdIns(4,5)P2-mediated inhibition. Nature 2001, 411: 957–962. 10.1038/35082088PubMedView ArticleGoogle Scholar
  29. Premkumar LS, Ahern GP: Induction of vanilloid receptor channel activity by protein kinase C. Nature 2000, 408: 985–990. 10.1038/35050121PubMedView ArticleGoogle Scholar
  30. Tominaga M, Wada M, Masu M: Potentiation of capsaicin receptor activity by metabotropic ATP receptors as a possible mechanism for ATP-evoked pain and hyperalgesia. Proc Natl Acad Sci U S A 2001, 98: 6951–6956. 10.1073/pnas.111025298PubMed CentralPubMedView ArticleGoogle Scholar
  31. Cesare P, Dekker LV, Sardini A, Parker PJ, McNaughton PA: Specific involvement of PKC-epsilon in sensitization of the neuronal response to painful heat. Neuron 1999, 23: 617–624. 10.1016/S0896-6273(00)80813-2PubMedView ArticleGoogle Scholar
  32. Bhave G, Zhu W, Wang H, Brasier DJ, Oxford GS, Gereau RW: cAMP-dependent protein kinase regulates desensitization of the capsaicin receptor (VR1) by direct phosphorylation. Neuron 2002, 35: 721–731. 10.1016/S0896-6273(02)00802-4PubMedView ArticleGoogle Scholar
  33. Rathee PK, Distler C, Obreja O, Neuhuber W, Wang GK, Wang SY, Nau C, Kress M: PKA/AKAP/VR-1 module: A common link of Gs-mediated signaling to thermal hyperalgesia. J Neurosci 2002, 22: 4740–4745.PubMedGoogle Scholar
  34. Zhuang ZY, Xu H, Clapham DE, Ji RR: Phosphatidylinositol 3-kinase activates ERK in primary sensory neurons and mediates inflammatory heat hyperalgesia through TRPV1 sensitization. J Neurosci 2004, 24: 8300–8309. 10.1523/JNEUROSCI.2893-04.2004PubMedView ArticleGoogle Scholar
  35. Berti-Mattera LN, Gariepy CE, Burke RM, Hall AK: Reduced expression of endothelin B receptors and mechanical hyperalgesia in experimental chronic diabetes. Exp Neurol 2006, 201: 399–406. 10.1016/j.expneurol.2006.04.034PubMedView ArticleGoogle Scholar
  36. Gunthorpe MJ, Smith GD, Davis JB, Randall AD: Characterisation of a human acid-sensing ion channel (hASIC1a) endogenously expressed in HEK293 cells. Pflügers Archiv European Journal of Physiology 2001, 442: 668–674. 10.1007/s004240100584PubMedView ArticleGoogle Scholar
  37. Aramori I, Nakanishi S: Coupling of two endothelin receptor subtypes to differing signal transduction in transfected Chinese hamster ovary cells. J Biol Chem 1992, 267: 12468–12474.PubMedGoogle Scholar
  38. Oda K, Fujitani Y, Watakabe T, Inui T, Okada T, Urade Y, Okuda-Ashitaka E, Ito S: Endothelin stimulates both cAMP formation and phosphatidylinositol hydrolysis in cultured embryonic bovine tracheal cells. FEBS Lett 1992, 299: 187–191. 10.1016/0014-5793(92)80244-BPubMedView ArticleGoogle Scholar
  39. Eguchi S, Hirata Y, Marumo F: Endothelin subtype B receptors are coupled to adenylate cyclase via inhibitory G protein in cultured bovine endothelial cells. J Cardiovasc Pharmacol 1993, 22 Suppl 8: S161–3.PubMedView ArticleGoogle Scholar
  40. Gohla A, Offermanns S, Wilkie TM, Schultz G: Differential involvement of Galpha12 and Galpha13 in receptor-mediated stress fiber formation. J Biol Chem 1999, 274: 17901–17907. 10.1074/jbc.274.25.17901PubMedView ArticleGoogle Scholar
  41. Numazaki M, Tominaga T, Toyooka H, Tominaga M: Direct phosphorylation of capsaicin receptor VR1 by protein kinase Cepsilon and identification of two target serine residues. J Biol Chem 2002, 277: 13375–13378. 10.1074/jbc.C200104200PubMedView ArticleGoogle Scholar
  42. Bhave G, Hu HJ, Glauner KS, Zhu W, Wang H, Brasier DJ, Oxford GS, Gereau RW: Protein kinase C phosphorylation sensitizes but does not activate the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1). Proc Natl Acad Sci U S A 2003, 100: 12480–12485. 10.1073/pnas.2032100100PubMed CentralPubMedView ArticleGoogle Scholar
  43. Mohapatra DP, Nau C: Desensitization of capsaicin-activated currents in the vanilloid receptor TRPV1 is decreased by the cyclic AMP-dependent protein kinase pathway. J Biol Chem 2003, 278: 50080–50090. 10.1074/jbc.M306619200PubMedView ArticleGoogle Scholar
  44. Mandadi S, Tominaga T, Numazaki M, Murayama N, Saito N, Armati PJ, Roufogalis BD, Tominaga M: Increased sensitivity of desensitized TRPV1 by PMA occurs through PKCepsilon-mediated phosphorylation at S800. Pain 2006, 123: 106–116. 10.1016/j.pain.2006.02.016PubMedView ArticleGoogle Scholar
  45. Moriyama T, Iida T, Kobayashi K, Higashi T, Fukuoka T, Tsumura H, Leon C, Suzuki N, Inoue K, Gachet C, Noguchi K, Tominaga M: Possible involvement of P2Y2 metabotropic receptors in ATP-induced transient receptor potential vanilloid receptor 1-mediated thermal hypersensitivity. J Neurosci 2003, 23: 6058–6062.PubMedGoogle Scholar
  46. Zhang N, Inan S, Cowan A, Sun R, Wang JM, Rogers TJ, Caterina M, Oppenheim JJ: A proinflammatory chemokine, CCL3, sensitizes the heat- and capsaicin-gated ion channel TRPV1. Proc Natl Acad Sci U S A 2005, 102: 4536–4541. 10.1073/pnas.0406030102PubMed CentralPubMedView ArticleGoogle Scholar
  47. Sugiura T, Bielefeldt K, Gebhart GF: TRPV1 function in mouse colon sensory neurons is enhanced by metabotropic 5-hydroxytryptamine receptor activation. J Neurosci 2004, 24: 9521–9530. 10.1523/JNEUROSCI.2639-04.2004View ArticleGoogle Scholar
  48. Moriyama T, Higashi T, Togashi K, Iida T, Segi E, Sugimoto Y, Tominaga T, Narumiya S, Tominaga M: Sensitization of TRPV1 by EP1 and IP reveals peripheral nociceptive mechanism of prostaglandins. Mol Pain 2005, 1: 3. 10.1186/1744-8069-1-3PubMed CentralPubMedView ArticleGoogle Scholar
  49. Prescott ED, Julius D: A modular PIP2 binding site as a determinant of capsaicin receptor sensitivity. Science 2003, 300: 1284–1288. 10.1126/science.1083646PubMedView ArticleGoogle Scholar
  50. Vellani V, Mapplebeck S, Moriondo A, Davis JB, McNaughton PA: Protein kinase C activation potentiates gating of the vanilloid receptor VR1 by capsaicin, protons, heat and anandamide. J Physiol 2001, 534: 813–825. 10.1111/j.1469-7793.2001.00813.xPubMed CentralPubMedView ArticleGoogle Scholar
  51. Morenilla-Palao C, Planells-Cases R, Garcia-Sanz N, Ferrer-Montiel A: Regulated exocytosis contributes to protein kinase C potentiation of vanilloid receptor activity. J Biol Chem 2004, 279: 25665–25672. 10.1074/jbc.M311515200PubMedView ArticleGoogle Scholar
  52. Burgess GM, Mullaney I, McNeill M, Dunn PM, Rang HP: Second messengers involved in the mechanism of action of bradykinin in sensory neurons in culture. J Neurosci 1989, 9: 3314–3325.PubMedGoogle Scholar
  53. Nicol GD, Cui M: Enhancement by prostaglandin E2 of bradykinin activation of embryonic rat sensory neurones. J Physiol 1994, 480: 485–492.PubMed CentralPubMedView ArticleGoogle Scholar
  54. Bandell M, Story GM, Hwang SW, Viswanath V, Eid SR, Petrus MJ, Earley TJ, Patapoutian A: Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin. Neuron 2004, 41: 849–857. 10.1016/S0896-6273(04)00150-3PubMedView ArticleGoogle Scholar
  55. McCleskey EW, Gold MS: Ion channels of nociception. Annu Rev Physiol 1999, 61: 835–856. 10.1146/annurev.physiol.61.1.835PubMedView ArticleGoogle Scholar
  56. Lai J, Porreca F, Hunter JC, Gold MS: Voltage-gated sodium channels and hyperalgesia. Annu Rev Pharmacol Toxicol 2004, 44: 371–397. 10.1146/annurev.pharmtox.44.101802.121627PubMedView ArticleGoogle Scholar
  57. Gold MS, Levine JD, Correa AM: Modulation of TTX-R INa by PKC and PKA and their role in PGE2-induced sensitization of rat sensory neurons in vitro. J Neurosci 1998, 18: 10345–10355.PubMedGoogle Scholar
  58. Piovezan AP, D'Orleans-Juste P, Souza GE, Rae GA: Endothelin-1-induced ET(A) receptor-mediated nociception, hyperalgesia and oedema in the mouse hind-paw: modulation by simultaneous ET(B) receptor activation. Br J Pharmacol 2000, 129: 961–968. 10.1038/sj.bjp.0703154PubMed CentralPubMedView ArticleGoogle Scholar
  59. Fareed MU, Hans GH, Atanda A, Strichartz GR, Davar G: Pharmacological characterization of acute pain behavior produced by application of endothelin-1 to rat sciatic nerve. Journal of Pain 2000, 1: 46–53. 10.1016/S1526-5900(00)90087-7View ArticleGoogle Scholar
  60. Jarvis MF, Wessale JL, Zhu CZ, Lynch JJ, Dayton BD, Calzadilla SV, Padley RJ, Opgenorth TJ, Kowaluk EA: ABT-627, an endothelin ET(A) receptor-selective antagonist, attenuates tactile allodynia in a diabetic rat model of neuropathic pain. Eur J Pharmacol 2000, 388: 29–35. 10.1016/S0014-2999(99)00865-1PubMedView ArticleGoogle Scholar
  61. Wacnik PW, Eikmeier LJ, Ruggles TR, Ramnaraine ML, Walcheck BK, Beitz AJ, Wilcox GL: Functional interactions between tumor and peripheral nerve: morphology, algogen identification, and behavioral characterization of a new murine model of cancer pain. J Neurosci 2001, 21: 9355–9366.PubMedGoogle Scholar
  62. Peters CM, Lindsay TH, Pomonis JD, Luger NM, Ghilardi JR, Sevcik MA, Mantyh PW: Endothelin and the tumorigenic component of bone cancer pain. Neuroscience 2004, 126: 1043–1052. 10.1016/j.neuroscience.2004.04.027PubMedView ArticleGoogle Scholar
  63. Griswold DE, Douglas SA, Martin LD, Davis TG, Davis L, Ao Z, Luttmann MA, Pullen M, Nambi P, Hay DW, Ohlstein EH: Endothelin B receptor modulates inflammatory pain and cutaneous inflammation. Mol Pharmacol 1999, 56: 807–812.PubMedGoogle Scholar
  64. da Cunha JM, Rae GA, Ferreira SH, Cunha Fde Q: Endothelins induce ETB receptor-mediated mechanical hypernociception in rat hindpaw: roles of cAMP and protein kinase C. Eur J Pharmacol 2004, 501: 87–94. 10.1016/j.ejphar.2004.08.004PubMedView ArticleGoogle Scholar
  65. De-Melo JD, Tonussi CR, D'Orleans-Juste P, Rae GA: Articular nociception induced by endothelin-1, carrageenan and LPS in naive and previously inflamed knee-joints in the rat: inhibition by endothelin receptor antagonists. Pain 1998, 77: 261–269. 10.1016/S0304-3959(98)00098-0PubMedView ArticleGoogle Scholar
  66. Baamonde A, Lastra A, Villazon M, Bordallo J, Hidalgo A, Menendez L: Involvement of endogenous endothelins in thermal and mechanical inflammatory hyperalgesia in mice. Naunyn Schmiedebergs Arch Pharmacol 2004, 369: 245–251. 10.1007/s00210-003-0841-1PubMedView ArticleGoogle Scholar
  67. Balonov K, Khodorova A, Strichartz GR: Tactile allodynia initiated by local subcutaneous endothelin-1 is prolonged by activation of TRPV-1 receptors. Exp Biol Med (Maywood) 2006, 231: 1165–1170.Google Scholar
  68. Khodorova A, Navarro B, Jouaville LS, Murphy JE, Rice FL, Mazurkiewicz JE, Long-Woodward D, Stoffel M, Strichartz GR, Yukhananov R, Davar G: Endothelin-B receptor activation triggers an endogenous analgesic cascade at sites of peripheral injury. Nat Med 2003, 9: 1055–1061. 10.1038/nm885PubMedView ArticleGoogle Scholar
  69. Zollner C, Shaqura MA, Bopaiah CP, Mousa S, Stein C, Schafer M: Painful inflammation-induced increase in mu-opioid receptor binding and G-protein coupling in primary afferent neurons. Mol Pharmacol 2003, 64: 202–210. 10.1124/mol.64.2.202PubMedView ArticleGoogle Scholar
  70. Hellwig N, Albrecht N, Harteneck C, Schultz G, Schaefer M: Homo- and heteromeric assembly of TRPV channel subunits. J Cell Sci 2005, 118: 917–928. 10.1242/jcs.01675PubMedView ArticleGoogle Scholar
  71. Gregan B, Jurgensen J, Papsdorf G, Furkert J, Schaefer M, Beyermann M, Rosenthal W, Oksche A: Ligand-dependent differences in the internalization of endothelin A and endothelin B receptor heterodimers. J Biol Chem 2004, 279: 27679–27687. 10.1074/jbc.M403601200PubMedView ArticleGoogle Scholar
  72. Grantcharova E, Furkert J, Reusch HP, Krell HW, Papsdorf G, Beyermann M, Schulein R, Rosenthal W, Oksche A: The extracellular N terminus of the endothelin B (ETB) receptor is cleaved by a metalloprotease in an agonist-dependent process. J Biol Chem 2002, 277: 43933–43941. 10.1074/jbc.M208407200PubMedView ArticleGoogle Scholar
  73. Oksche A, Boese G, Horstmeyer A, Furkert J, Beyermann M, Bienert M, Rosenthal W: Late endosomal/lysosomal targeting and lack of recycling of the ligand-occupied endothelin B receptor. Mol Pharmacol 2000, 57: 1104–1113.PubMedGoogle Scholar
  74. Oksche A, Boese G, Horstmeyer A, Papsdorf G, Furkert J, Beyermann M, Bienert M, Rosenthal W: Evidence for downregulation of the endothelin-B-receptor by the use of fluorescent endothelin-1 and a fusion protein consisting of the endothelin-B-receptor and the green fluorescent protein. J Cardiovasc Pharmacol 2000, 36: S44–7.PubMedView ArticleGoogle Scholar
  75. Strotmann R, Harteneck C, Nunnenmacher K, Schultz G, Plant TD: OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarity. Nat Cell Biol 2000, 2: 695–702. 10.1038/35036318PubMedView ArticleGoogle Scholar

Copyright

© Plant et al; licensee BioMed Central Ltd. 2007

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Advertisement