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Keywords:

  • endothelin-1;
  • ETA receptors;
  • ETB receptors;
  • BQ123;
  • BQ788;
  • pruritus;
  • itch;
  • IRL1620;
  • BALB/c mice;
  • pruriceptors

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Conflict of interest
  9. References

Background and purpose:

Endothelin-1 (ET-1) is present in murine and human skin and causes itch (pruritus) when injected in humans. This behavioural study examined the scratch reflex evoked by ET-1 in mice.

Experimental approach:

An automated detector was used to determine whether ET-1 causes reflex scratching, the behavioural correlate of itching, in BALB/c mice. Selective agonists and antagonists were used to probe the ET receptor(s) involved.

Key results:

ET-1 evoked dose-related reflex scratching lasting up to 20 min following intradermal injection (0.1-100 ng; 0.04-40 pmol). The ED50 for ET-1 induced scratching was 2.1 ng and desensitization occurred with cumulative dosing. High doses of the ETB receptor agonist IRL1620 (10 μg; 5.5 nmol), also caused scratching (ED50 1.3 μg, 0.7 nmol). The ETA receptor antagonist BQ123 significantly reduced scratching evoked by ET-1 and IRL 1620, suggesting that both agonists caused scratching via an ETA receptor-dependent mechanism. The ETB receptor antagonist BQ788 significantly reduced scratching evoked by IRL1620 but had no effect on scratching evoked by ET-1. This indicated that activation of ETB receptors by high doses of ETB agonist, but not ET-1, can trigger scratching.

Conclusion and implications:

ET-1 is a potent endogenous activator of reflex scratching (itch). Mechanisms for ET-induced scratching are considered, including direct action of ET-1 on pruriceptive nerve endings and indirect actions via release of endogenous mediators such as histamine from mast cells. ET-1 and ETA receptors, possibly also ETB receptors, are potential targets for developing specific anti-pruritic drugs to treat pruritic skin disorders such as atopic dermatitis.

British Journal of Pharmacology (2007) 151, 278–284. doi:10.1038/sj.bjp.0707216


Abbreviations:
AD

atopic dermatitis

CGRP

calcitonin Gene-related peptide

CI

confidence interval

ET-1

endothelin-1

ETA

endothelin A receptor

ETB

endothelin B receptor

H1

histamine H1 receptor

PBS

phosphate-buffered saline

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Conflict of interest
  9. References

Endothelin-1 (ET-1) is a 21 amino-acid peptide which is an endogenous agonist at Endothelin A (ETA) and Endothelin B (ETB) receptors (Masaki et al., 1994). ET-1 has been reported to cause itch (pruritus) when injected intradermally in man (Ferreira et al., 1989), but detailed study of this pruritogenic action has not been undertaken. The peptide is present in human skin (Bull and Dowd, 1993) and its synthesis and release has been demonstrated in cultures of human endothelial cells and keratinocytes (Terenghi et al., 1991; Yohn et al., 1993). ET-1 is upregulated in response to hypoxia, sheer stress and a range of inflammatory cytokines (Kurihara et al., 1989; Yoshizumi et al., 1990; Horio et al., 1991; Elton et al., 1992). It has potent vasoconstrictor actions (Yanagisawa and Masaki, 1989), and can evoke the triple response in skin (Brain et al., 1992). ET-1 is also involved in cell signalling (Simonson and Dunn, 1990) and inflammatory processes (Xu et al., 1998; Kuryliszyn-Moskal et al., 2005), which are important in chronic itchy skin disorders such as atopic dermatitis (Goligorsky et al., 1999; Stander and Steinhoff, 2002).

The present investigation was performed to establish whether ET-1, which is present in murine skin (Ahn et al., 1998), causes pruritus when injected into the outer layers of the skin in BALB/c mice. Selective endothelin receptor agonists and antagonists (Davenport, 2002) were used to characterize the pharmacological receptors involved in the response obtained. Pruritus was measured non-invasively using an automated scratch detector to record reflex scratching (Brash et al., 2005), which is the behavioural correlate of itch in animals. There is considerable clinical need for specific anti-itch drugs (Rees and Murray, 2005; Summey and Yosipovitch, 2005), and the information obtained from this functional study should be valuable for the development of new drugs.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Conflict of interest
  9. References

Animals

All experiments were performed in accordance with Local Ethics Committee approval and UK Home Office regulations. Female BALB/c mice (n=84; Charles River, Margate, Kent, UK), 10-week-old adults, were used in the experiments. Mice weighed 16–21 g and were housed under controlled light (0700–1900 hours) and temperature (22°C) with food and water available ad libitum.

Procedure

The itch-inducing properties of ET-1 and the ETB agonist IRL1620 (Davenport, 2002) were studied by injecting the drugs intra-dermally (i.d.; into the outer layer of skin; could also be termed subdermal or intra-cutaneous) in the nape of the unshaven neck via a 26 G needle and measuring the subsequent reflex scratching of the area by the hind paw during the 20 min post-injection period. Doses of ET-1 or IRL1620 were administered at 90 min intervals on a cumulative basis. Control values were taken from the results of phosphate-buffered saline (PBS) injections given during this study and in previous experiments using the same protocol (Bell et al., 2004). After dose–response experiments involving ET-1, 5-HT (10 μg, i.d.) was administered 60 min after the last ET-1 dose to check that mice still retained the ability to scratch in response to a standard pruritogen.

In experiments involving the antagonist, scratching was induced by a mid-range dose of ET-1 (3 ng; 1.2 pmol) or IRL1620 (10 μg; 5.5 nmol), with a repeat injection 90 min after the initial dose. These doses were chosen on the basis of results from the dose–response experiments (see Results). The ETA antagonist, BQ123 (Davenport, 2002), was administered at a dose of 500 μg kg−1 intra-peritoneally (i.p.), 30 min before the second injection of agonist. This dose was chosen on the basis of its effectiveness in reducing scratching in earlier protocols involving 10, 50 and 500 μg kg−1 (i.p.) BQ123. The effect of the same dose of BQ123 on IRL1620-induced scratching was also assessed. The ETB antagonist, BQ788 (Davenport, 2002), was administered cumulatively in doses of 5, 50 and 500 μg kg−1 (i.p.), following the same protocol as used for studying BQ123.

To minimize any behavioural changes owing to the novel environment of the recording boxes, all mice were acclimatized to the boxes for 20–30 min before the experiment, with another 30 min in the holding cage in advance of any recording.

Measurement of scratching

Scratching was measured using the repetitive movement detector system designed, built and validated in our laboratory (Brash et al., 2005). Briefly, four mice were placed in individual lightweight Styrofoam boxes, which were placed on separate force platforms, positioned over sensitive strain gauges. Scratching evoked by i.d. injection in this strain of mice was mainly in the 11–15 Hz range, and the detector was set to record all repetitive motor movements of a frequency of 11–20 Hz; grooming behaviour occurs below this frequency. A ‘bout’ of scratching was recorded when three or more repetitive scratch movements or ‘beats’ made by the hindlimb were recorded and ended when the interval between successive movements was greater than 0.2 s. The total number of scratch movements in each bout was recorded, and on average there were five scratch movements per bout, a value that was independent of the pruritogen and the dose injected.

Data were recorded and processed using a MacLab Recorder. To verify that only scratching events were being automatically processed, the mice were filmed using a video camera (Vista, NCD 132) positioned above the recording boxes, and the image was recorded on a VCR (Panasonic, NV-HD640). This arrangement enabled the observer remotely to ensure the animals' welfare, observe behaviour, and note any ‘non-scratch’ events for checking against the automated data record. The scratch score from the automated system correlated very well with the very time-consuming operator-derived manual scoring of activity recorded on video tape, as reported previously (Brash et al., 2005).

Scratching was recorded over a 20 min period immediately following the i.d. injection, on the basis of previous studies using histamine as a pruritogen in man and animals (Keele and Armstrong, 1964; Laidlaw et al., 2002; Bell et al., 2004), and during pilot experiments in this study.

Statistical analysis

Data are shown as mean±s.e.m. for n values or mean with 95% confidence intervals (CI), unless otherwise stated. Log dose–response curves were plotted using the nonlinear regression (sigmoidal log dose–response) function in GraphPad Prism v4.0. The mean ‘apparent’ ED50, the estimated dose to elicit half the apparent maximal response under non-steady conditions in vivo, was calculated from the pooled data. Non-parametric statistical tests used are given in the text, and the null hypothesis was rejected at P<0.05.

Materials

ET-1 (MW 2492; Tocris, Avonmouth, UK), BQ123 (ETA antagonist, MW 611; Tocris), BQ788 (ETB antagonist MW 664; Tocris), histamine diphosphate (MW 307; Sigma, Poole, UK) and 5-HT (serotonin creatinine sulphate, MW 387; Sigma) were all dissolved in PBS, (pH=7.4). IRL1620 (ETB agonist, MW 1820; Tocris), was dissolved in PBS (pH=7.4; stock solution: saline with 4% hydrochloric acid). ET-1 and IRL1620 were injected in a volume of 100 μl. The antagonists BQ123 and BQ788 were injected i.p. in a volume of 100 μl.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Conflict of interest
  9. References

Scratching induced by ET-1

ET-1 caused dose-dependent scratching when injected i.d. in doses ranging from 0.1 to 1000 ng (0.04–400 pmol), as summarized in Figure 1. The scratch response tended to diminish following doses of ET-1 greater than 10 ng, and the overall log dose–response plot for cumulative dosing was bell-shaped. Statistical analysis showed that ET-1 induced scratching which, apart from the lowest (0.1 ng) and highest (1000 ng) cumulative doses of ET-1 was significantly greater than that caused by a control injection of the same volume of PBS (P<0.05; Kruskal–Wallis test with Dunn's post hoc test versus PBS).

image

Figure 1. Pooled data for ET-1-induced scratching in female BALB/c mice during the 20-min post-injection period (n=8–40). ET-1 induced dose-dependent scratching. Dashed line represents the mean level of scratching induced by injection of 100 μl PBS i.d., which served as a control (31±8, scratch movements in 20 min, n=18). The solid line shows a sigmoidal curve fitted to scratching evoked by ET-1 over the range 10−10–10−7 g.

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The mean apparent ED50 for scratching induced by ET-1 i.d. was calculated as 2.5 ng (1.0 pmol; 95% CI 0.6–9.5 ng) from a sigmoidal plot fitted to the pooled data for doses in the range 10−10–10−7 g (Figure 1).

Desensitization

Injection of ET-1 (100 ng) as the first, rather than the last dose of an increasing sequence, evoked 662±66 scratches (n=5), which was significantly more than observed during cumulative dose–response experiments (Figure 1; P<0.05; Mann–Whitney test), and was not significantly different from the response to 10 ng ET-1 during cumulative dosing (Figure 1; P>0.05, Mann–Whitney test).

The time to onset of scratching following ET-1 or IRL1620 was somewhat variable and not dose-dependent. Most of the scratching occurred during the first 15 min following injection, and had normally ended by 20 min. Scratching induced by a mid-range dose of ET-1 (3 ng) started significantly later than that evoked by a standard dose of histamine (data by Bell et al., 2004), as can be seen from the summary in Figure 2.

image

Figure 2. Box and whisker plot showing the range, median, upper and lower quartiles for the delay to onset of scratching and the time at which the maximal response occurred following i.d. injection of PBS (100 μl; n=4), ET-1 (3 ng; n=29), IRL1620 (10 μg; n=23), 5-HT (10 μg; n=15) and histamine (1000 μg; n=10). Scratching evoked by ET-1 or IRL1620 began significantly later than that caused by 5-HT or histamine (P<0.05; Kruskal–Wallis, with Dunn's post hoc test) and this test also showed that the onset for scratching following injection of ET-1 was not significantly different from that for IRL1620. There was no significant difference between 5-HT and histamine in the delay to onset of scratching. The lower part of the figure shows the time at which scratching was most intense following an i.d. injection, which varied considerably between individual animals within treatment groups, and between groups. There was no significant difference between the group median values, apart from for the peak response to IRL1620 which occurred significantly later than that to 5-HT (P<0.05; Kruskal–Wallis, with Dunn's post hoc test; the other combinations did not differ significantly; P>0.05).

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A single injection of 5-HT 10 μg (25 nmol) was made 60 min after the last dose of ET-1, to determine whether mice retained the ability to respond to a pruritogenic compound despite the apparent decline in responsiveness following cumulative high doses of ET-1. The mean response to 5-HT was 325±49 scratches (n=16), which was not significantly different from control responses to 5-HT 10 μg given before any other substance (369±45 scratch movements, n=7; P>0.05 Mann–Whitney test versus 5-HT after preceding doses of ET-1).

Subjectively, no abnormal behaviour was observed in the mice after administration of ET-1. However, following the highest doses (100 and 1000 ng) the mice generally were less active and spent more time in the corner of the recording box.

Role of ETA receptors in scratching induced by ET-1

The ETA antagonist, BQ123, reduced scratching induced by the 3 ng (1.2 pmol) mid-range dose of ET-1, as shown in Figure 3. Lower doses of BQ123 (10 and 50 μg kg−1 i.p.) reduced scratching, but with some variability such that group mean values before and after the antagonist were not significantly different. A higher dose of antagonist (500 μg kg−1) was required to achieve a statistically significant reduction in ET-1-induced scratching. The scratch response to ET-1 returned to normal approximately 3 h after BQ123 500 μg kg−1. Subjectively, no alteration in behaviour was observed in mice given BQ123.

image

Figure 3. The ETA receptor antagonist, BQ123, (500 μg kg−1 i.p.), significantly reduced ET-1-induced scratching in BALB/c mice (P<0.05; Wilcoxon signed rank test). Open symbols show response before, filled symbols after the antagonist, and lines connect responses from individual animals (n=7). Horizontal bar shows the median value.

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Effect of the ETB receptor agonist IRL1620 on scratching behaviour

IRL1620 evoked dose-dependent scratching, as summarized in Figure 4a. The mean apparent ED50 for the agonist determined from the pooled data was 1.3 μg (0.7 nmol) with 95% CI 0.08–21 μg. On average, the scratching induced by IRL1620 started 266±48 s (n=12) after i.d. injection, which was not significantly different from the mean onset of response to ET-1 (P>0.05; Mann–Whitney test). Scratching evoked by IRL1620 was only significantly different from that caused by PBS at the highest dose (10 μg; 5.5 nmol; Kruskal–Wallis test with Dunn's post hoc test versus PBS). The number of scratch movements caused by 10 μg IRL1620 was not significantly different from that evoked by ET-1, 3 ng, (P>0.05; Mann–Whitney test, Figures 1 and 4a).

image

Figure 4. (a) Pooled data for scratching evoked by the ETB receptor agonist, IRL-1620 during the 20 min post-injection period (n=4–8). Sigmoidal curve fitted to data points. Dashed line represents the mean level of scratching induced by PBS, which served as a control for the injection. (b) The effect of ETA receptor antagonist, BQ123 (500 μg kg−1 i.p.), on scratching induced by the 10 μg dose of IRL1620 (n=8). Responses before (open symbols) were significantly reduced after (closed symbols) the antagonist (P<0.05; Wilcoxon paired rank test). The lines join paired responses from individual animals, and the horizontal bars represent the median values.

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Effect of the ETA receptor antagonist BQ123 on scratching induced by IRL1620

The ETA antagonist, BQ123 (500 μg kg−1 i.p.), significantly reduced scratching induced by the 10 μg dose of IRL1620 (Figure 4b; P<0.05; Wilcoxon paired rank test). The antagonism lasted for several hours before responses to the agonist recovered. Subjectively, no change in behaviour was observed after the ETA antagonist.

Effect of the ETB receptor antagonist BQ788 on scratching induced by ET-1 and IRL 1620

The ETB antagonist, BQ788 (5–500 μg kg−1 i.p.), had no significant effect on scratching induced by the 3 ng mid-range dose of ET-1 (P>0.05; Kruskal–Wallis test with Dunn's post hoc test versus control), as shown in Figure 5a. However, scratching evoked by the 10 μg dose of IRL1620 was significantly reduced by the 50 and 500 μg kg−1 doses of BQ788 (Figure 5b; P<0.01; Kruskal–Wallis test with Dunn's post hoc test versus control).

image

Figure 5. (a) The effect of the ETB receptor antagonist BQ788 (5–500 μg kg−1 i.p.) on scratching induced by ET-1 (3 ng; n=7–11). Control responses to ET-1 (open column) did not differ significantly from those observed following increasing doses of antagonist (shaded columns; P>0.05; Kruskal–Wallis test with Dunn's post hoc test versus control). (b) The ETB receptor antagonist, BQ788 (5–500 μg kg−1 i.p.), antagonized scratching induced by the ETB agonist IRL1620 10 μg (n=8). Responses were significantly reduced by the two highest doses of the antagonist (shaded columns), in comparison with scratching in the pre-antagonist control (open column; *P<0.01; Kruskal–Wallis test with Dunn's post hoc test versus control).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Conflict of interest
  9. References

The results of this study show that ET-1 is a very potent pruritogen in mice, causing dose-dependent scratching, the behavioural correlate of itch, with a mean apparent ED50 of 1 pmol (2.5 ng). ET-1 is therefore about six million times more potent as a pruritogen than histamine, which has an apparent ED50 of 5.8 μmol in this strain of mice (Bell et al., 2004). The log dose–response plot for ET-1-induced scratching was bell-shaped and cumulative dosing with the peptide led to desensitization of the scratch reflex.

Two previous studies found that ET-1 causes pruritus when administered to human skin. Both Ferreira et al. (1989) and Katugampola et al. (2000) described the development of a burning pruritus in human subjects following intradermal injection of ET-1. The present experiments in mice used an automated repetitive movement detector (Brash et al., 2005) to measure accurately the scratching induced by injecting ET-1, and we thought this was the first report of the pruritogenic action of ET-1 in BALB/c mice. However, after the work was submitted for publication, a paper was published which demonstrated that ET-1 causes pruritus in mice via similarly potent actions involving ETA receptors in murine skin (Trentin et al., 2006).

Scratching in animals as a behavioural correlate of itch

It is impossible to determine what sensation any animal experiences, so behavioural correlates have to be used. Animals may scratch in response to painful stimuli as well as to those that are associated with different human sensations, such as itch and tickle (McMahon and Koltzenburg, 1992). However, it has been shown that whereas the pruritogenic agents compound 48/80 and substance P cause scratching when injected subcutaneously in mice, the algogenic (pain producing) agents capsaicin and formalin do not, but instead usually cause vocalization, biting and withdrawal (Kuraishi et al., 1995). We found that histamine or 5-HT caused dose-related scratching in mice, whereas injection of algogens evoked slight scratching which did not differ in intensity from that induced by PBS (Bell et al., 2004). Injection of histamine into human skin is invariably associated with the sensation of itch and reflex scratching (Keele and Armstrong, 1964), and even very high doses of ET-1 (up to 50 μg i.d.) only caused itch (Ferreira et al., 1989). We therefore consider it probable, by analogy with the results from man, that mice in the present study were scratching in response to ‘itch’ rather than ‘pain’ evoked by low doses of ET-1. It is the reflex scratching which causes damage to the skin in response to pruritogens both in man and mice, and that behaviour can be studied objectively in animals.

Involvement of ETA and ETB receptors in ET-1-induced scratching

The selective ETA antagonist, BQ123 (Ihara et al., 1992), significantly reduced scratching evoked by a mid-range dose of ET-1 (Figure 3), which strongly suggests the involvement of ETA receptors in ET-1-induced itch. The selective ETB agonist, IRL-1620 (Takai et al., 1992), also induced scratching, which could be interpreted as evidence for involvement of ETB receptors in the scratch reflex response to ET-1. However, the ED50 for the scratch response to IRL-1620 was 875 times greater than that of ET-1 (Figure 4a), and only scratching evoked by the highest dose of IRL-1620 was significantly greater than the vehicle-induced response. It therefore seemed probable that the high dose of IRL-1620 evoked scratching via weak agonist action at ETA receptors (Takai et al., 1992). This was confirmed by showing that the ETA antagonist, BQ123, significantly reduced scratching in response to IRL-1620 (Figure 4b).

The possibility existed that BQ123 might exert its antipruritic action through sedation, as occurs with sedating histamine H1 receptor antagonists (Nicholson, 1983; Shuster, 1988). However, this was rejected because there is no evidence that ETA antagonists such as BQ123 cause sedation, and we did not see any change in spontaneous exploration or grooming behaviour following BQ123 administration.

Taken together, this functional integrative behavioural evidence from intact animals suggested that ET-1 acts via ETA receptors to evoke reflex scratching. Further studies were undertaken using the ETB antagonist, BQ788, in the expectation that these would provide additional evidence that IRL 1620 evokes scratching via an ETA receptor-dependent mechanism, that is, they would serve as a negative control. In fact, we found that scratching evoked by IRL1620 was antagonized by BQ788, whereas that caused by ET-1 was unaffected. This evidence therefore may mean that ETB receptors are involved in scratching evoked by the ETB agonist, IRL1620, but not in scratching caused by ET-1, which is an endogenous non-selective ETA and ETB receptor agonist.

Trentin et al. (2006) used male Swiss mice and measured bouts of scratching evoked by ET-1 (1–20 pmol). Scratching was antagonized by co-injecting the ETA antagonist BQ123 into the skin, or by i.p. administration of a different ETA receptor antagonist, atrasentan. Our results with ET-1 and BQ123 in female BALB/c mice confirm some of the findings of Trentin et al. (2006) and provide additional evidence that ET-1 is a potent pruritogen in mice, apparently acting via ETA receptors in the skin. These authors also reported that IRL1620 (10 pmol) was not pruritic, and that accords with our findings using this dose (Figure 4a). However, we observed that higher doses of the ETB agonist are pruritic, whereas they found that, when co-injected, it was antipruritic, inhibiting scratching evoked by ET-1 or histamine, an effect we did not investigate.

Our results showed that systemic administration of the ETB antagonist, BQ788, did not affect the scratch response to the non-selective ETA/B receptor agonist ET-1 (Figure 5a), in contrast to the finding of Trentin et al. (2006) that co-injecting BQ788 enhanced scratching evoked by ET-1, suggesting that ETB receptors inhibit scratching. Our results are thus discrepant, but Trentin et al. (2006) did comment on their unexpected finding that systemic administration of the ETB antagonist A-1292621 diminished scratching induced by ET-1.

Attempting to characterize receptors on the basis of behavioural studies involving high doses of agonists and antagonists is complicated by the issue of the selectivity of drugs for receptors in particular species. Thus, we found that only high doses of the ETB agonist IRL1620 cause scratching, which was antagonized by the ETA receptor antagonist BQ123 and also by the ETB receptor antagonist BQ788. This suggests that ETB receptors, as well as ETA receptors are involved in evoking scratching, but that could be a misinterpretation if high doses of agonist and/or antagonist are acting non-selectively in mice to affect ETA receptors. We did not co-inject drugs locally, and the situation is complicated by the possibility that systemic and local effects of a drug will differ in vivo. Also, as suggested by the delay to the start of scratching, ET-1 may be acting indirectly on pruriceptors via release of pruritogenic mediators (e.g. histamine from mast cells; Yamamura et al., 1994b). It seems probable that ETA and ETB receptors interact via receptor ‘crosstalk’ (Mickley et al., 1997), which makes it difficult to establish what contribution is made by the individual receptor subtypes in response to the endogenous ligand, ET-1. Further studies using in vitro techniques to establish the location and the type(s) of ET receptor involved in evoking the scratching reflex are clearly needed.

Mechanisms of ET-1-induced itch: possible indirect action via histamine release

The relatively long delay to onset of scratching following ET-1 injection into the skin is suggestive of an indirect action of the peptide. Several lines of evidence support the involvement of histamine in ET-1-induced pruritus. When injected into human skin ET-1 evokes pallor at the injection site, with flare in the surrounding tissue of similar character to that evoked by histamine (Brain et al., 1992). This flare response is markedly reduced by H1 receptor antagonists (Crossman et al., 1991; Brain et al., 1992). ET-1-induced release of substance P from primary afferent neurons in human skin has been proposed (Bunker et al., 1992), and substance P induces itch via histamine-dependent and independent mechanisms in both humans (Hagermark et al., 1978; Hagermark, 1992) and mice (Andoh et al., 1998).

Research on murine foetal skin-derived mast cells shows the presence of ETA receptors on their surface and ET-1 degranulates these cells in a dose- and ETA receptor-dependent manner (Matsushima et al., 2004). Histamine release caused by ET-1 has also been demonstrated in both peritoneal and bone marrow-derived mast cells harvested from BALB/c mice (Yamamura et al., 1994a, 1994b). It is thus possible that ET-1 degranulates mast cells in BALB/c mice and the delayed onset of itch is caused by liberated histamine. However, against this possibility, Brain et al. (1992) described the inability of ET-1 to activate human skin mast cells in vitro, and Katugampola et al. (2000) found only a minimal rise in histamine in the skin microvasculature after infusing ET-1, suggesting that histamine release is not important for the development of ET-1-induced pruritus in humans. A reciprocal interaction between ET-1 and mast cells has been reported (Hultner and Ehrenreich, 2005) and it will be of considerable interest to perform more detailed studies in normal and genetically modified mice, for example. mast cell deficient (Wershil and Galli, 1994), to establish whether or not ET-1 acts via ETA and possibly ETB receptors to trigger scratching indirectly, through release of histamine or other mediator(s) from neurones, glia, immune cells and blood vessels in the skin (Pomonis et al., 2001).

Direct activation of pruriceptive C-fibres

Although the overall onset of scratching following injection of ET-1 or IRL1620 was significantly slower in comparison with the pruritogens histamine and 5-HT, several mice began scratching quite rapidly after injection, and were close to the median delay for histamine or 5-HT (Figure 2). The high variance in time to onset of response may reflect the time taken for injected substances such as ET-1 and histamine to reach a threshold concentration for activating pruriceptive C-fibres in a particular receptive field, which may result from slight differences in the positioning of the needle in the skin. The fact that scratching started fairly soon after injection in some animals could be interpreted as direct action of ET-1 on ETA receptors located on the small-diameter pruriceptive C-fibres described by Schmelz et al. (1997). ET-1 binding sites have been identified within the dorsal root ganglia (DRG) of the rat, rabbit and monkey, with ETA receptors localized to small diameter DRG neuronal cell bodies (Kar et al., 1991; Pomonis et al., 2001), which may be involved in nociception or pruriception. Moreover, colocalization of ETA receptors and the C-fibre marker, CGRP, has been demonstrated on these cell bodies. This evidence points to the presence of ETA receptors on neurones in DRG which might be pruriceptive, but this is speculative. Detailed neuropharmacological investigation is required to identify mechanically insensitive pruriceptive C-fibres in murine skin which respond to low doses of ET-1 in an ETA and possibly ETB receptor-dependent manner.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Conflict of interest
  9. References

Our finding that ET-1 induces dose-dependent scratching in BALB/c mice via an ETA receptor in skin confirms the work of Trentin et al. (2006) and complements the preliminary reports from studies in man in showing that this endogenous peptide is a potent activator of the scratch reflex (itch). The low ED50 for ET-1-induced scratching in mice implies that a small increase in ET-1 concentration within the skin could trigger pruritus. This may be very relevant in chronic skin disorders such as atopic dermatitis which are associated with distressing and damaging scratching. Our study of the acute effects of ET-1 needs to be extended to include animal models of chronic scratching, and a combination of selective drugs and suitable genetically modified strains of mice, a particular advantage of using this species, could be used to help establish how ET-1 acts to induce reflex scratching. ET-1 is clearly a putative mediator of itch, making the peptide and its receptors potential targets for developing much needed specific anti-itch drugs.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Conflict of interest
  9. References