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Keywords:

  • 5-hydroxytryptamine-4;
  • cholinergic;
  • colon;
  • human;
  • nitrergic;
  • nitric oxide

Abstract

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

Abstract  Previous studies have demonstrated mixed inhibitory and facilitatory effects of 5-hydroxytryptamine-4 (5-HT4) receptor agonists on electrical field stimulation (EFS)-induced responses in human isolated colon. Here we report three types of responses to EFS in human isolated colon circular muscle: monophasic cholinergic contraction during EFS, biphasic response (nitrergic relaxation during EFS followed by cholinergic contraction after termination of EFS) and triphasic response (cholinergic contraction followed by nitrergic relaxation during EFS and a tachykininergic contraction after EFS). The effects of two 5-HT4 receptor agonists, prucalopride and tegaserod were then investigated on monophasic responses only. Each compound inhibited contractions during EFS in a concentration-dependent manner. In the presence of Nω-nitro-l-arginine methyl ester (l-NAME) however, prucalopride and tegaserod enhanced the contractions in a concentration-dependent manner. In strips where the tone was elevated with substance-P and treated with scopolamine, EFS-induced relaxations were enhanced by the two agonists. The above observed effects by the two agonists were abolished by 5-HT4 receptor antagonist SB-204070. The two agonists did not alter the tone raised by substance-P in the presence of scopolamine and l-NAME and did not affect carbachol-induced contractions in the presence of tetrodotoxin. These results suggest that in the circular muscle of human colon, 5-HT4 receptor agonists simultaneously facilitate the activity of neurones which release the inhibitory and excitatory neurotransmitters, nitric oxide and acetylcholine respectively.


Introduction

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

5-Hydroxytryptamine-4 (5-HT4) receptor agonists such as prucalopride and tegaserod have been utilized in the clinic to enhance peristalsis and speed colonic transit therefore relieving constipation and symptoms experienced by patients with constipation-predominant irritable bowel syndrome.1 In terms of mechanism of action, 5-HT4 receptor agonists have been shown to facilitate cholinergic contractions in longitudinal preparations of human isolated colon.2 In the circular muscle of the human colon, 5-HT4 receptor agonists have been shown to cause inhibition of spontaneous activity3,4 and to elicit relaxant activity directly on the smooth muscle.4–6 Therefore, it has been proposed that these compounds facilitate colonic propulsion via a coordinated combination of circular muscle relaxation and longitudinal muscle contraction.2 Although this model has been widely acknowledged as the mechanism of action of 5-HT4 receptor agonists in treatment of idiopathic chronic constipation and constipation-dominant irritable bowel syndrome,1 it is not clear how these simple actions stimulate propulsive activity, given that the circular muscle of the colon is generally thought of as providing the major mechanical force behind intestinal propulsion. A recent report has demonstrated mixed inhibitory and facilitatory effects of prucalopride on electrical field stimulation (EFS)-induced contractions in human colon circular muscle.6 We have therefore investigated the effects of prucalopride and tegaserod on nerve-evoked responses elicited by EFS in human isolated colon circular muscle. Our results suggest that modulation of neuronal transmission in human colon circular muscle is the major mechanism by which 5-HT4 receptor agonists facilitate propulsive activity.

Methods

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

Collection of human tissues

Segments of colon (transverse or sigmoid) were obtained from patients undergoing surgery for colorectal cancer. The study was approved by the local ethics committee and written informed consent was obtained from patients. The segments were transferred from the hospital to the research laboratories within 2 h after resection in ice-cold Kreb's solution (containing in mmol L−1: NaCl 121.5, CaCl2 2.5, KH2PO4 1.2, KCl 4.7, MgSO4 1.2, NaHCO3 25, glucose 5.6) equilibrated with 5% CO2 and 95% O2. The segment was cut open longitudinally and the three teania coli were discarded. The remaining three intertaenial parts of the segment were rinsed and stored overnight at 8 °C in fresh, oxygenated Kreb's solution. The next day the mucosa was removed and strips (3–4 × 15 mm) parallel to the circular muscle fibres were cut. The strips (n = 4–16 from each patient) were mounted in tissue baths (10 mL) containing Kreb's solution at 37 °C and gassed with 5% CO2 in O2.

Experimental protocol

The change in the tension was recorded using isometric force transducers (AD Instruments, Chalgrove, UK) on a data acquisition system (Biopac Systems Inc., Goleta, CA, USA). The strips were given 2 g tension and allowed to equilibrate for 45 min during which time the bath solutions were changed every 15 min. At the end of the equilibration period the strips were stimulated via two parallel platinum ring electrodes connected to a stimulator (STG2008, Scientifica, Uckfield, UK). The stimulation parameters were 50 V (c. 200 mA), 5 Hz, 0.5 ms bipolar pulse duration, for 10 s, every 1 min. EFS parameters were chosen according to our preliminary experiments where tetrodotoxin (TTX)-sensitive optimum contractile responses were achieved. The effects of compounds were investigated either by adding in cumulative fashion to construct a concentration–response curve or at a single concentration. They were expressed as a percentage of the mean of at least three predrug EFS-induced responses, which was expressed as 100%. The effect of the compounds in a single strip was determined from at least five EFS-induced responses. Minimum contact time for Nω-nitro-l-arginine methyl ester (l-NAME), scopolamine, TTX and SB-204070 was 20 min.

In the experiments with carbachol-induced tone, carbachol (1 μmol L−1) was applied to the bath and was washed once the contraction reached a plateau. Carbachol application was repeated twice more with 15–20 min intervals. Fifteen minutes before the last application, prucalopride or tegaserod (3 μmol L−1) was added to the bath. During this particular experiment 1 μmol L−1 TTX was present throughout.

Statistical analysis

Data are expressed as mean ± standard error of the mean; n values are the numbers of patients. Differences between the means were determined using Student's t-test for unpaired observations; P < 0.05 is considered as statistically significant. The correlations between the type of tissues, gender of patients and the type of response were tested using a Mann–Whitney test. Correlations between the patients’ age and the type of response were assessed using Pearson correlation coefficients.

Compounds

Carbachol, scopolamine, l-NAME, L-732138 were obtained from Sigma, Poole, UK. TTX and MDL-29913 were obtained from Tocris, Bristol, UK. Substance-P was obtained from Calbiochem, Nottingham, UK. SB-204070, SB-235375, prucalopride and tegaserod were synthesized inhouse. SB-235375, MDL-29913 and L-732138 were dissolved in ethanol. Tegaserod was dissolved in DMSO. All other compounds were dissolved in Kreb's solution. Maximum final concentrations of ethanol and DMSO in the tissue baths were 0.01% which did not alter EFS-induced responses.

Results

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

Patient details

The age, gender, site of the cancer and region of the tissue used in this study are summarized in Table 1. Mean age of the patients was 69.1 ± 2.8 years (range 44–86; n = 17); male to female ratio was 6 : 11. Transverse to sigmoid ratio was 6 : 11.

Table 1.   The age and gender of patients, the sites of cancer and tissue and the percentage of strips showing monophasic, biphasic and triphasic responses to electrical field stimulation
Patient no.Age (years)GenderCancer siteTissue siteMonophasicBiphasicTriphasic
 172FColonTransverse10000
 274FCaecumTransverse80020
 366FColonTransverse57.1042.9
 459MColonSigmoid67033
 552MColonSigmoid37.52537.5
 665MRectumSigmoid43.817.837.4
 744MColonTransverse12.5087.5
 886FColonTransverse42.828.628.6
 972MRectumSigmoid10000
1071FColonSigmoid037.562.5
1182FColonTransverse10000
1278FColonSigmoid37.537.525
1368FColonSigmoid37.52537.5
1452FColonSigmoid62.52512.5
1582FRectumSigmoid37.537.525
1677MRectumSigmoid502525
1774FRectumSigmoid10000
Mean ± SEM69.1 ± 2.8   56.8 ± 7.515.3 ± 3.827.9 ± 5.7

Responses to EFS

The responses elicited by EFS are divided into three main groups:

  • 1
    Monophasic response. A contraction which began at the initiation of the EFS (Fig. 1). This response was the most dominant one, observed in 56.8 ± 7.5% of the strips (Table 1).
  • 2
    Biphasic response. A relaxation which began at the initiation of the EFS and followed by a contraction which began at the termination of EFS (Fig. 2). This type of response was observed in 15.3 ± 3.8% of the strips (Table 1).
  • 3
    Triphasic response. An initial contraction followed by a relaxation during the EFS was observed. At the termination of EFS a second contraction was observed (Fig. 3). This triphasic response was evident in 27.9 ± 5.7% of the strips (Table 1).
image

Figure 1.  Effect of Nω-nitro-l-arginine methyl ester (l-NAME) and scopolamine on electrical field stimulation (EFS)-induced monophasic contractions. (A) A representative tracing depicting a monophasic contraction evoked by EFS (shown with black rectangle; 50 V, 5 Hz, 0.5 ms, 10 s). Treatment with l-NAME (300 μmol L−1; 30 min incubation) enhanced EFS-induced contraction and with scopolamine (B) (scop; 10 μmol L−1, 30 min incubation) blocked EFS-induced contraction.

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image

Figure 2.  Effect of Nω-nitro-l-arginine methyl ester (l-NAME) and scopolamine on electrical field stimulation (EFS)-induced biphasic responses. (A) A representative tracing depicting a biphasic response evoked by EFS (shown with black rectangle; 50 V, 5 Hz, 0.5 ms, 10 s). Note the relaxation phase during EFS. In the presence of l-NAME (300 μmol L−1; 30 min incubation), the relaxation phase is inhibited and a monophasic contraction was observed during and after EFS. (B) Scopolamine (scop; 10 μmol L−1; 30 min incubation) inhibited the contraction after EFS which led to an enhanced initial relaxation phase during EFS.

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image

Figure 3.  Effect of Nω-nitro-l-arginine methyl ester (l-NAME), scopolamine and NK receptor antagonists on electrical field stimulation (EFS)-induced triphasic responses. (A) A representative tracing depicting a triphasic response evoked by EFS (shown with black rectangle; 50 V, 5 Hz, 0.5 ms, 10 s). Note the initial contraction followed by relaxation during EFS. In the presence of l-NAME (300 μmol L−1; 30 min incubation), the relaxation phase is inhibited and the contraction during EFS was enhanced while the post-EFS contraction was decreased. (B) Scopolamine (scop; 10 μmol L−1; 30 min incubation) inhibited the contraction during EFS which led to an enhanced post-EFS contraction. (C) Further addition of NK receptor antagonists (1 μmol L−1 L-732138 + 1 μmol L−1 MDL-29913 + 0.1 μmol L−1 SB-235375) completely inhibited the after-contraction.

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All the responses were abolished in the presence of TTX (1 μmol L−1; not shown; n = 17). No correlation could be found between the type of tissues, the age or gender of patients and the type of response (Table 1).

Effect of l-NAME

In strips with a monophasic response to EFS, an inhibitor of nitric oxide (NO) synthase l-NAME (300 μmol L−1), enhanced the contractions during EFS by 25.6 ± 4.8% (n = 7; Fig. 1A). In the biphasic response group, l-NAME completely inhibited the relaxation response during EFS; instead, a contraction during EFS was observed which was 32.8 ± 9.3% greater than the control (n = 5; Fig. 2A). In the triphasic response group, l-NAME enhanced the initial contraction (31.2 ± 9.3%), completely inhibited the relaxation during EFS and reduced the contraction after EFS by 43.6 ± 4.9% (n = 5; Fig. 3A).

Effect of scopolamine and neurokinin receptor antagonists

The addition of scopolamine (10 μmol L−1) prevented the contractions evoked by EFS in the monophasic response group (97.7 ± 1.5 % inhibition; n = 4; Fig. 1B). In the strips with a biphasic response, scopolamine inhibited the contraction after EFS (89.7 ± 5.6% inhibition; n = 4; Fig. 2B). In the triphasic group, scopolamine inhibited the initial contraction observed during EFS by 84.7 ± 9.1% and enhanced the contraction after EFS by 43.7 ± 12.8% (n = 5; Fig. 3B,C). Further addition of a combination of antagonists of NK-R1 (L-732138; 1 μmol L−1), NK-R2 (MDL-29913; 1 μmol L−1) and NK-R3 (SB-235375; 0.1 μmol L−1) resulted in complete inhibition of the after-contraction (n = 5; Fig. 3C).

Effect of 5-HT4 receptor agonists on monophasic contractions to EFS

The effects of prucalopride and tegaserod were investigated only on the strips with a monophasic response to EFS. All strips showing monophasic response were included. Under control conditions, both compounds decreased the magnitude of EFS-induced contractions in a concentration-dependent manner without affecting the basal tone (Figs 4 and 5). Maximum inhibitions observed with prucalopride and tegaserod were 38.1 ± 1.3% and 23.5 ± 2.5% respectively (P < 0.05; n = 5). In the presence of the 5-HT4 receptor antagonist, SB-204070 (1 μmol L−1) the effects of the two agonists were abolished (n = 5; Fig. 5). SB-204070 on its own had no effect on the tone or on the EFS-induced contractions (data not shown).

image

Figure 4.  Effect of prucalopride and SB-204070 on electrical field stimulation (EFS)-induced monophasic contractions. Prucalopride inhibited EFS-induced contractions (shown here at 3 μmol L−1); an effect reversed by SB-204070 (1 μmol L−1).

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image

Figure 5.  Effect of prucalopride (squares) and tegaserod (circles) (each from 1 nM to 10 μmol L−1) on electrical field stimulation (EFS)-induced monophasic contractions in the absence (closed symbols) or presence (open symbols) of SB-204070 (1 μmol L−1). Both compounds inhibited EFS-induced contractions which was prevented when the tissues were pretreated with SB-204070 (n = 5).

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In the presence of the NO synthase inhibitor, l-NAME (300 μmol L−1), EFS-induced contractions were further enhanced by prucalopride and tegaserod with maximum potentiation of 122.5 ± 14.5% and 47.3 ± 5.1% respectively (P < 0.05; n = 4; Figs 6 and 7). SB-204070 (1 μmol L−1) abolished the effects of the compounds on EFS-induced contractions in the presence of l-NAME (n = 4; Figs 6 and 7).

image

Figure 6.  Effect of prucalopride on electrical field stimulation (EFS)-induced monophasic contractions after pretreatment with Nω-nitro-l-arginine methyl ester (l-NAME) (300 μmol L−1). Treatment with l-NAME enhanced the contractions which were further potentiated by prucalopride (shown here at 10 μmol L−1). SB-204070 (1 μmol L−1) reversed the effect of prucalopride (right panel) while prucalopride effect was stable in a parallel experiment (left panel).

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image

Figure 7.  Effect of prucalopride (squares) and tegaserod (circles) (each from 1 nM to 30 μmol L−1) on electrical field stimulation (EFS)-induced monophasic contractions after pretreatment with Nω-nitro-l-arginine methyl ester (l-NAME) (300 μmol L−1) in the absence (closed symbols) or presence (open symbols) of SB-204070 (1 μmol L−1). Both compounds enhanced EFS-induced contractions in the presence of l-NAME which was prevented when the tissues were pretreated with SB-204070 (n = 4).

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Effect of 5-HT4 receptor agonists on non-adrenergic non-cholinergic relaxations to EFS

In the presence of scopolamine (10 μmol L−1), the addition of substance-P (1 μmol L−1) evoked a muscle contraction which was maintained for at least 30 min. During this time, EFS elicited relaxations which were abolished with l-NAME (300 μmol L−1) or TTX (1 μmol L−1) (data not shown; n = 4). These relaxation responses were potentiated with 1 μmol L−1 prucalopride or tegaserod by 49.2 ± 7.8% and 26.4 ± 5.3% respectively (n = 4; Fig. 8); an effect which was reversed by 1 μmol L−1 SB-204070 (96.1 ± 5.8% and 87.8 ± 5.4% of control respectively; n = 4). l-NAME on its own did not affect substance-P induced tone (data not shown).

image

Figure 8.  Effect of prucalopride (A) and tegaserod (B) (each 1 μmol L−1) on electrical field stimulation (EFS)-induced non-adrenergic non-cholinergic (NANC) relaxations. Representative tracings depicting that both compounds enhance NANC relaxations in the presence of scopolamine (10 μmol L−1) and after the elevation of the tone with substance-P (1 μmol L−1). EFS (50 V, 5 Hz, 0.5 ms, 10 s) is shown by black rectangle.

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Effect of 5-HT4 receptor agonists on elevated muscle tone and carbachol-induced contractions

To assess whether or not the two agonists have any direct effect on the smooth muscle, the compounds were tested on the raised tone with substance-P (1 μmol L−1) in the presence of l-NAME (300 μmol L−1) and scopolamine (10 μmol L−1). Neither compound when tested at 10 μmol L−1 affected the muscle tone (n = 3; data not shown).

To assess whether or not the two agonists have any direct effect on smooth muscle contractility, the compounds were tested against submaximally effective, carbachol-induced contractions in the presence of TTX (1 μmol L−1). Neither compound had any effect on the carbachol-induced responses (Fig. 9). In the presence of prucalopride or tegaserod, the contractions to carbachol were 101.6 ± 4.6% and 107.9 ± 5.3% of control contractions respectively (P > 0.05; n = 3 each).

image

Figure 9.  Prucalopride or tegaserod do not affect carbachol-induced contraction in the presence of tetrodotoxin (TTX) (1 μmol L−1). A representative tracing depicting that treatment with prucalopride (upper panel) or tegaserod (lower panel) had no effect on carbachol-induced responses in the presence of TTX. Carbachol (1 μmol L−1) was added to the bath (closed triangles) and then washed (open triangles).

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Discussion

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

We have demonstrated in this study that the responses to EFS in the human isolated colon circular muscle can be classified into three groups: monophasic contraction, which was the most abundant type therefore was used in experiments with 5-HT4 receptor agonists, was purely cholinergic and modulated by nitrergic inhibitory transmission. Biphasic responses were composed of a nitrergic relaxation during EFS followed by a cholinergic contraction after EFS. In the presence of l-NAME, the relaxation was abolished and the cholinergic contraction was enhanced and started during and not after EFS. This type of response is suggestive of a strong nitrergic component, most probably stronger than that present in the strips which exhibited monophasic contractions to EFS. As the contractions during both monophasic and biphasic responses were abolished by scopolamine, non-cholinergic contractile neurotransmitters can be excluded in these response types. Previously similar responses were observed in human sigmoid colon preparations where the authors observed a ‘latent period’ during EFS and an ‘off-contraction’ after EFS.7 They suggested that the ‘latent period’ was mediated by NO and ATP (through P2Y1 receptor) and the ‘off-contraction’ was mediated by acetylcholine. In our experiments, we obtained complete inhibition by l-NAME of the initial relaxation during EFS, suggesting little or no involvement of a purinergic pathway. However, we would not exclude the possibility that under different stimulation parameters such activity could have been evident.

Triphasic responses to EFS were more complicated. Initial contraction during EFS was cholinergic as it was inhibited by scopolamine. The relaxation observed during EFS was nitrergic as it was blocked by l-NAME. The contraction after EFS were non-cholinergic and were mediated by tachykinins as suggested previously8–10 as scopolamine did not block and actually enhanced these contractions and they were blocked by a combination of antagonists of the three NK receptors. Interestingly, a similar potentiation of EFS-evoked after-contractions by atropine has previously been observed in an opossum oesophageal–stomach preparation.11 These observations were suggested by the authors as being consistent with an ability of acetylcholine to inhibit the release of substance-P.

To our knowledge this is the first account of such diverse neurogenic responses in human colon circular muscle. It was interesting to note that different parts of the colon even from the same patient could give such different responses to EFS. This could be due to different density of intrinsic nerve fibres in the strips and/or the orientation of muscle fibres within each strip. In particular, the muscle strips used in our experiments have only a limited width which may include only a small part of the motor and interneurones which project to this piece of muscle; therefore it is possible that only the pharmacology of a limited set of neurones with short projection lengths are studied in each preparation. Nevertheless, by careful attention to the nature and pharmacology of the different responses evoked by EFS, it becomes possible to investigate the action of drugs or substances on the functions of different nerve phenotypes within the human colon.

In this study, we have shown that EFS-induced, neuronally mediated monophasic contractions are reduced by the 5-HT4 receptor agonists tegaserod and prucalopride. This effect was reversed by a selective 5-HT4 receptor antagonist, confirming that the effect observed by the two compounds is through this receptor. We have further demonstrated that the inhibitory action of the two agonists is not due to a direct effect on the smooth muscle as they did not affect the tone elevated by substance-P or carbachol. Once the tissues were treated with an inhibitor of NO synthase, instead of inhibiting, 5-HT4 receptor agonists enhanced the EFS-induced contractions. These results suggest that 5-HT4 receptor activation potentiates nitrergic modulation of cholinergic responses and that once this modulation is lifted by l-NAME, cholinergic activity is further enhanced by the 5-HT4 receptor agonists. Enhancement of nitrergic responses by the two 5-HT4 receptor agonists was further demonstrated in the experiments where non-adrenergic non-cholinergic (NANC) relaxation responses to EFS were obtained in the presence of scopolamine and substance-P. These NANC relaxation responses were nitrergic in nature and neuronal in origin as they were inhibited by l-NAME or TTX. 5-HT4 receptor agonists enhanced the magnitude and the duration of these nitrergic relaxations. To our knowledge this is the first demonstration of a nitrergic component to the actions of 5-HT4 receptor agonists.

The 5-HT4 receptor is known to be positively linked to adenylate cyclase (AC), activation of which leads to an increase in intracellular cyclic AMP (cAMP) levels in the human colon.12,13 An increase in cAMP levels and consequent activation of protein kinase A (PKA) have been shown to mediate the direct smooth muscle relaxant effect of 5-HT4 receptor agonists.12,13 A similar activation of PKA in the excitatory cholinergic nerves by the 5-HT4 receptor agonists might account for the increased acetylcholine release akin to that in the frog neuromuscular junction.14 Activation of cAMP-PKA pathway has also been shown to increase NO release from perivascular nitrergic nerves via activation of neuronal NO synthase (nNOS). Therefore, we speculate that facilitation of inhibitory responses in the human colon by 5-HT4 receptor agonists in our experiments might be due to activation of nNOS in the nitrergic nerves by the cAMP-PKA pathway. Further research is needed to elucidate the exact molecular mechanisms by which 5-HT4 receptor agonism facilitates both excitatory cholinergic and inhibitory nitrergic transmission.

An inhibitory effect of 5-HT4 receptor agonists has previously been shown on human isolated colon circular muscle tone which has been attributed to a direct effect on the smooth muscle.5 In this study, the authors used high concentrations of potassium to elevate muscle tension to reproducibly detect drug-induced changes in the tension. However, we were unable to reproduce this technique, i.e. the elevated tone to high potassium could not be maintained in our hands (our unpublished observations). Furthermore, the two 5-HT4 receptor agonists did not affect the raised muscle tone in the presence of l-NAME and scopolamine or the contractions evoked by carbachol in the presence of TTX in our study suggesting that at least in our conditions 5-HT4 receptor agonism does not cause direct smooth muscle relaxation.

Our results showing that in the presence of l-NAME, EFS-induced cholinergic contractions are further potentiated by the 5-HT4 receptor agonists are in accordance with a previous study6 where 5-HT4 receptor agonists have been shown to inhibit or enhance EFS-induced cholinergic contractions in the presence of an inhibitor of NO synthase and to enhance EFS-induced acetylcholine release in the human isolated colon circular strips. In that study,6 the authors suggested that the heterogenous action of 5-HT4 receptor agonists on cholinergic contractions could be explained by the presence of direct inhibitory effect of these agonists on the smooth muscle as discussed above. In our experiments, however, we did not observe any such direct effect, the two agonists showed facilitatory effect in all strips studied in the presence of l-NAME. This discrepancy could partly be due to the heterogeneity of responses to EFS observed in human colon preparations. We have in this study investigated the effects of 5-HT4 receptor agonists only on those strips showing monophasic contractions. It is possible that the heterogeneity of responses to agonists observed by Leclere et al. (2005) could be a result of including different types of responders to EFS.

Previously 5-HT4 receptor agonists have been shown to relax or inhibit spontaneous contractions in human isolated colon preparations.4,15 This has been attributed to the direct relaxant effect of the compounds. Our results suggest another plausible scenario where the compounds might be exerting their relaxant effect via release of NO.

In summary, we have confirmed the ability of 5-HT4 receptor agonists to enhance cholinergic excitatory action and added a new paradigm of activation of nitrergic inhibitory action. Although 5-HT4 receptors have been localized in both smooth muscle and myenteric plexus in the human colon16 and in spite of the reports of an ability of 5-HT4 receptors agonists to directly relax human colonic muscle,6 we were unable to confirm these findings. The reason for this difference remains unknown and is likely to involve several factors, one of which may be the involvement of nitrergic pathways activated via 5-HT4 receptors.

Acknowledgments

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

The authors wish to thank Simon T. Bate for his help in the statistical analysis. Preliminary results of this work were presented as an abstract in the 20th International Symposium on Neurogastroenterology and Motility, Toulouse, France, July 3–6, 2005.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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    Serio R, Mule F, Bonvissuto F, Postorino A. Tachykinins mediate noncholinergic excitatory neural responses in the circular muscle of rat proximal colon. Can J Physiol Pharmacol 1998; 76: 6849.
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    Domoto T, Jury J, Berezin I, Fox JE, Daniel EE. Does substance P comediate with acetylcholine in nerves of opossum esophageal muscularis mucosa? Am J Physiol 1983; 245: G1928.
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    McLean PG, Coupar IM. Stimulation of cyclic AMP formation in the circular smooth muscle of human colon by activation of 5-HT4-like receptors. Br J Pharmacol 1996; 117: 2389.
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