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Keywords:

  • Endothelin;
  • mouse proximal and distal colon;
  • endothelin receptors

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References
  • The study investigated the role of endothelin (ET) and the ET receptor subtypes ETA and ETB in mediating longitudinal contraction in the mouse proximal and distal colon.

  • Cumulative concentration–response curves to a range of ET agonists (ET-1, ET-2, ET-3, (Ala1,3,11,13) ET and IRL 1620) were established by administering concentrations ranging from 0.01 nM to 0.3 μM. Concentration–response curves to ET-1, which exhibits a high affinity for both ETA and ETB receptor subtypes, were also established in the presence of the ETA antagonist BMS 182874 and the ETB antagonist IRL1038.

  • The addition of the selective ETA receptor antagonist BMS 182874 caused a rightward shift of the concentration–response curve to ET-1 in both sections of the colon. The ETB receptor antagonist IRL1038 (0.3–1 μM) did not significantly effect the response to ET-1 in the proximal colon but caused a significant decrease in response towards higher concentrations ranges (geqslant R: gt-or-equal, slanted3 nM) in the distal colon.

  • A comparison of the concentration–response curves to ET-1, ET-2 and ET-3 showed a rank order of potency ET-1geqslant R: gt-or-equal, slantedET-2≫ET-3 in the proximal colon and ET-1geqslant R: gt-or-equal, slantedET-2geqslant R: gt-or-equal, slantedET-3 in the distal colon. The selective ETB receptor agonists, (Ala1,3,11,13) ET and IRL 1620 did not produce any response in the proximal sections of the colon but produced a smaller contraction in the distal segments.

  • The data indicate that ET can contract the proximal tissues of the mouse colon predominantly via ETA receptors and in the distal tissues via ETA and ETB receptors.

British Journal of Pharmacology (2006) 147, 607–611. doi:10.1038/sj.bjp.0706657


Abbreviations:
EDCF

endothelium-derived contracting factor

ET-1

endothelin-1

ET-2

endothelin-2

ET-3

endothelin-3

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

The initial discovery of the peptidergic endothelium-derived contracting factor (EDCF) began with the pioneering work of Hickey et al. (1985) and was confirmed in subsequent studies (Gillespie et al., 1986; O'Brian et al., 1987). In 1987, Masaki and co-workers isolated, purified, sequenced and cloned this peptide and coined the name ‘endothelin’ (ET) (Yanagisawa et al., 1988; Masaki et al., 1994). Subsequent studies have identified three different ET genes in the human genome, which are differentially regulated (Rubanyi & Polokoff, 1994) and express three different isopeptides-ET-1, ET-2 and ET-3 (Inoue et al., 1989). Moreover these isopeptides are differentially distributed among several tissues (Kasuya et al., 1991) in varied proportions and exert their effects through a number of ET receptor subtypes. Among these the ETA and ETB receptors have been molecularly characterized (Sakurai et al., 1992), but the existence of a third subtype, ETC (Karne et al., 1993), as a species variant or a new subtype is yet to be ascertained.

ET-binding proteins are widely distributed throughout the gastrointestinal tract, brain, kidneys, lungs, trachea and vascular tissue (Takahashi et al., 1990). The gastrointestinal tract of the rat has a specific binding area for ET in the fundus of the stomach, jejenum, ileum and colon, where relatively low concentrations of ET have been shown to elicit a contractile response (Wallace et al., 1989; Takahashi et al., 1990; Peskar et al., 1992).

The mounting interest in ET research, coupled with the discovery of potent agonists and antagonists, may lead to a better understanding of the pathogenic mechanisms in the gastrointestinal tract and the development of novel therapies for gastrointestinal disorders. Moreover, the discovery of potent ET antagonists and the recent demonstration of their effectiveness in animal models of pathology such as Hirschsprung's disease (Zhu et al., 2004), has provided preliminary evidence that endogenous ETs may be of importance in the pathogenesis of human diseases (Rubanyi & Polokoff, 1994; Tekin et al., 1999; Davenport & Maguire, 2002). The present study investigated the role of ET and its receptors ETA and ETB in mediating longitudinal muscle contraction responses in the mouse proximal and distal colon.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

Animals and housing conditions

The experiments were carried out on adult Bantin Kingman White (BKW) mice of either sex weighing between 25 and 35 g. The mice were housed in single sex groups of 10 per cage and allowed food and water ad libitum. The polypropylene cages were floored with sawdust and cleaned on a regular basis. The animals were maintained at a humidity of 40–50% at 21°C on a 14/10-h light/dark cycle. Animals were allowed to acclimatize in holding rooms for at least 3 weeks before commencing the experiments.

Tissue preparation

Animals were killed by cervical dislocation and the entire length of the colon, approximately 6–7 cm in length, was removed. The section was kept in Krebs–Heinseleit (KH) (composition mM: NaCl 118, KCl 4.5, KH2PO4 1.2, MgSO4 1.2, CaCl2 2.5, NaHCO3 25.0, glucose 10.0) solution oxygenated with 95% O2 and 5% CO2. The mesentery and fatty tissue were removed and the luminal contents were carefully washed out with KH solution. The proximal and distal segment of the colon (approximately 2 cm in length and taken 1–2 cm and 4–5 cm, respectively, from the ileo-caecal junction), was dissected and mounted vertically in 10 ml, water-jacketed organ baths containing KH solution kept at 37°C and oxygenated with 95% O2 and 5% CO2. Longitudinal contractions were measured isometrically using Grass Force Displacement transducers (FT03C, Grass Instrument Co., MA, U.S.A.) and recorded on a multichannel Powerlab recorder (Powerlab/8SP Data recording system SP8088). The tissues were placed under a resting tension of 1 g and allowed to equilibrate for 30 min with two washouts every 15 min.

Drug administration

Concentration–response curves to ET receptor agonists (0.01 nM–0.3 μM) were established, once a stable baseline tension was obtained, by cumulative administration of the agonist with contact time of 1–2 min for each concentration. In experiments examining the effects of tetrodotoxin (TTX), ETA and ETB receptor antagonists on ET-1 responses, the tissues were equilibrated with TTX or the antagonist for at least 30 min before the cumulative concentration–response curves to ET-1 were established.

Drugs

(Ala1,3,11,13) ET, BMS 182874 (5-(dimethylamino)-N-(3,4-dimethyl-5-isoxazolyl)-1-naphthalene sulfonamide), ET-1, ET-2, ET-3, IRL 1038((Cys11, Cys15) ET-1), IRL 1620 (Suc-(Glu9, Ala11, 15) ET-1 (8–21)) (Tocris) and TTX citrate (Tocris, Bristol, U.K.). All drugs, except TTX which was dissolved in distilled water, were dissolved and diluted in bovine serum albumin (0.01%). Drugs were added directly into the organ baths in volumes not exceeding 1% of the bath volume.

Analysis of results

A difference between two groups was compared using the Student's t-test (unpaired, two-tailed). When a multiple comparison was involved, one-way ANOVA was used to check the difference between the treatments groups. Wherever necessary, a difference between any of the test groups with the control was assessed using the t-test with Dunnett's correction. A probability of P<0.05 was accepted as showing a significant difference between treatments and the control values. All results are expressed as the means±s.e.m. The potencies of the ET agonists were expressed as pEC50 values relative to individual maxima.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

The cumulative addition of ET (0.01 nM–0.3 μM) to the proximal and distal sections of the mouse colon produced a concentration-dependent contractile response, with pEC50 value of 9.20±0.09 and Emax of 1.24±0.08 g at 100 nM in the proximal colon and pEC50 value of 9.16±0.09 and Emax of 1.36±0.14 at 100 nM in distal sections, respectively (n=8) (Figure 1). The contractile response to ET was unaffected in the presence of TTX. The concentration–response curves to ET in the presence and absence of TTX were superimposable in both the proximal and distal segments of the colon (n=8, data not shown).

image

Figure 1. Representative tracing showing the contractile response induced by the cumulative addition of increasing concentrations of endothelin-1 (ET-1) (0.01 nM–0.3 μM) on the mouse (a) proximal and (b) distal colon. (c) Concentration–response curves to the cumulative addition of ET-1 (0.01 nM–0.3 μM) in the mouse proximal and distal colon (n=8). The graph represents the mean±s.e.m. as taken from eight animals.

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The addition of the ETA receptor-selective antagonist, BMS 182874 (0.3, 3 and 30 μM) caused a rightward shift of the concentration–response curve to ET-1 in both sections of the colon, without the suppression of the maximum response. The rightward shift of the curve to ET-1 increased as the concentration of BMS 182874 increased (Figure 2a and b).

image

Figure 2. Antagonism of the contractile response to ET-1 in the mouse colon by BMS 182874. Concentration–response curves to endothelin-1 in the absence (ET-1 alone, n=8) and presence of 0.3 μM (+0.3 μM BMS 182874, n=8), 3 μM (+3 μM BMS 182874, n=8) and 30 μM (+30 μM BMS 182874, n=8) of BMS 182874 in the (a) proximal and (b) distal mouse colon. Each point represents the means with the s.e.m. shown by vertical bars.

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The addition of the ETB receptor antagonist, IRL 1038 (0.3 and 1 μM), did not significantly effect the response to ET-1 in the proximal sections of the colon but caused a significant decrease towards higher concentration ranges (>3 nM, P<0.05) in the distal colon (Figure 3a and b).

image

Figure 3. Antagonism of the contractile response to endothelin-1 (ET-1) in the mouse colon by IRL 1038. Concentration–response curves to endothelin-1 in the absence (ET-1 alone, n=8) and presence of 0.3 μM (+0.3 μM IRL 1038, n=8) and 1 μM (+1 μM IRL 1038, n=8) of IRL 1038 in the (a) proximal and (b) distal mouse colon. Each point represents the means with the s.e.m. shown by vertical bars.

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The cumulative addition of ET-1, ET-2, and ET-3 in concentrations ranging from 0.01 nM to 0.3 μM produced concentration-dependent contractions in both the proximal and distal sections of the colon (Figure 4a and b). The pEC50 values and Emax values were: 9.20±0.09 and 1.24±0.08 g at 100 nM for ET-1; 8.8±0.09 and 1.15±0.07 g at 100 nM for ET-2; 7.33±0.19 and 0.92±0.2 g at 300 nM for ET-3, (n=4–8) in the proximal colon and 9.16±0.09 and 1.36±0.14 g at 100 nM for ET-1; 9.08±0.07 and 1.18±0.05 g at 100 nM for ET-2; 7.9±0.25 and 1.91±0.26 g at 300 nM for ET-3 (n=4–8) in the distal colon. The addition of (Ala1,3,11,13) ET and IRL1620 (0.01 nM–0.3 μM) did not produce any response in the proximal section of the colon but produced a smaller contraction in the distal sections (pEC50 values and Emax values were: 0±0 and 0±0 for (Ala1,3,11,13) ET; 0±0 and 0±0 for IRL1620, n=4, in the proximal section of the colon and pEC50 values and Emax values were: 8.1±0.22 and 0.57±0.12 g at 10 nM for (Ala1,3,11,13) ET; 7.15±0.12 and 0.62±0.10 g for IRL1620 at 10 nM, n=4, in distal sections of the colon).

image

Figure 4. Graphical representation of the effect of the cumulative addition of ET-1 (n=8), ET-2 (n=8), ET-3 (n=4), (Ala1,3,11,13) endothelin (n=4) and IRL 1620 (n=4) in concentrations ranging from 0.01 nM to 0.3 μM in the mouse (a) proximal and (b) distal colon. Each value represents the mean±s.e.m. as taken from eight animals.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

In the mouse, the role of ET and its receptors has previously only been demonstrated in sections taken from the mouse ileum where ET was capable of causing a dose-dependent contraction (Ishida et al., 1989). Moreover, it was shown that a new peptide family, ET consisting of three members in mammals, appeared to be present in mice (Saida et al., 1989). Of these, two ET-related genes were identified by cloning and sequence analysis of a mouse genome; one encoded a peptide identical to the porcine and human vasoconstrictor peptide ET while the other encoded a novel peptide differing from ET in three amino-acid residues. This peptide, so named ‘vasoactive intestinal contractor,’ was expressed in the intestine and in no other tissues or endothelial cells (Saida et al., 1989). To date however, ET receptor(s) have not been fully studied in murine colonic tissues.

The results from the present study showed that ET could contract both the proximal and distal sections of the mouse colon showing the existence of ET receptors in the mouse lower bowel. The receptors mediating ET contraction in both proximal and distal colon are likely to be located on the smooth muscles as the TTX did not alter response to ET. The addition of the nonpeptide ETA receptor, selective antagonist BMS 182874 (Webb et al., 1995) caused a rightward shift of the concentration–response curve to ET-1 in both sections of the colon, without suppression of the maximum response, indicating an involvement of the ETA receptor in the contractile response to ET-1 in both proximal and distal sections of the mouse colon.

The ETB receptor antagonist IRL1038 (0.3–1 μM) (Masaki et al., 1994) did not significantly affect the response to ET-1 in the proximal colon. However, IRL 1038 caused a significant decrease in the response to ET-1 in higher concentrations (>3 nM, P<0.05) in the distal colon, indicting the involvement of the ETB receptors in this section of the mouse colon. This was further supported by the ability of the two ETB receptor agonists, (Ala1,3,11,13) ET and IRL1620 to produce contractile responses in the distal (but not proximal sections) of the colon. Furthermore, the rank order of the potency of agonists was, ET-1geqslant R: gt-or-equal, slantedET-2≫ET-3 in the proximal colon and ET-1geqslant R: gt-or-equal, slantedET-2geqslant R: gt-or-equal, slantedET-3 in the distal colon, indicative of the presence of ETA receptors in both the proximal and distal colon (Masaki et al., 1994).

Therefore, the results indicate the presence of ET receptors in the mouse colon to mediate a contraction response. It seems that ET mediates its effects predominantly via ETA receptors in the proximal colon and through both ETA and ETB receptors in the distal section of the mouse colon. Moreover, the present study also illustrates the potential role of the various ET receptor ligands in moderating contractility in the lower bowel. With the advent of knockout mice models, the importance of the ET receptors in colonic contraction and measures of motility can now be studied (Davenport & Maguire, 2002).

The present study demonstrated that ET is highly potent in the mouse colon as it initiates the contraction response at concentrations as low as 10−11 M. Similar values have been obtained from studies carried out in rat stomach, jejenum, ileum and colon where concentrations of 10−11–10−7 M ET could contract the stomach and colon of rats and also the ileum of the guinea pig (Takahashi et al., 1990). The potency of ET-1 observed in the present study is also comparable to the potency of ET-1 reported for contraction of the guinea-pig ileum (Ishida et al., 1989).

ET-1 and ET-3 peptides have been shown to be present in the gastrointestinal tract; they have been found in the rat gastric mucosa by immunoassay (Matsumoto et al., 1989), by in situ hybridization (MacCumber et al., 1989), and by ET binding (Takahashi et al., 1990). ET-3 has also been shown to play an important role in the development of the mouse enteric nervous system (Gershon, 1995). Both ETB and ET-3 receptors are necessary to prevent the premature differentiation of crest-derived cells, which leads to agangliosis (Wu et al., 1999). A number of studies have shown that the pharmacological effects of exogenous ET-1 in the intestine are revealing of a potent intestinal secretagogue and an effect to increase colon contraction by direct stimulation of the smooth muscle (Brown and Smith, 1991; Moummi et al., 1992; Kiyohara et al., 1993). A study by Egidy et al. (2000) showed the presence of the ETB receptors in neurons and glial cells, indicating that they may be involved in the formation of enteric neurons as has been shown in the mouse (Wu et al., 1999). Moreover, the various cellular localizations of the ET components suggested that they might be implicated in the modulation of intestinal motility and secretion.

There have also been reports on the distribution of ET in the human colon. Inagaki et al. (1994) reported ET-like immunoreactivity and binding sites for ET-1 in the human colon and competition studies have shown that there are two populations of ET receptors (Escrig et al., 1992). Mutations of ETB receptors have been found in some families with Hirschsprung's disease or agangliosis (Puffenberger et al., 1994; Zhu et al., 2004).

The findings from this study therefore provide evidence for the involvement of ET and ETA and ETB receptors in mediating a contraction response in the mouse colon. This may suggest a role for ET in the modulation of colonic and intestinal function and pathophysiology. It would be of interest to further investigate the possibility that a pharmacological manipulation of the ET receptor subtypes might reveal a therapeutic potential.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References
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