SEARCH

SEARCH BY CITATION

Keywords:

  • acetylcholine;
  • cholinergic nerves;
  • 5-hydroxytryptamine 4 receptor;
  • human colon

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Tissue preparation
  6. Dissection and preparation of muscle strips
  7. Contractility study
  8. Experimental protocol of contractility study
  9. Release study
  10. Experimental protocol of release study
  11. Measurement of radioactivity and separation by HPLC of radioactive compounds
  12. Statistics
  13. Compounds
  14. Results
  15. Contractility study
  16. Release study
  17. Discussion
  18. Acknowledgements
  19. References

Abstract  5-Hydroxytryptamine 4 (5-HT4) receptor agonists promote colonic propulsion. The alteration of circular muscle (CM) motility underlying this involves inhibition of contractility via smooth muscle 5-HT4 receptors and proximal colonic motility stimulation, the mechanism of the latter not having been characterized. Our aim was to identify and characterize a 5-HT4 receptor-mediated stimulation of human colon CM contractile activity. 5-HT4 receptor ligands were tested on electrical field stimulation (EFS)-induced contractions of human colonic muscle strips cut in the circular direction (called ‘whole tissue’ strips). Additionally, after incubation of tissues with [3H]-choline these compounds were tested on EFS-induced release of tritium in whole tissue strips and in ‘isolated’ CM strips, obtained by superficial cutting in the CM layer. Tetrodotoxin and atropine blocked EFS-induced contractions of whole tissue CM strips. Prucalopride (0.3 μmol L−1) evoked a heterogenous response on EFS-induced contraction, ranging from inhibition (most frequently observed) to enhancement. In the release experiments, EFS-induced tritium efflux was blocked by tetrodotoxin. Prucalopride increased EFS-induced tritium and [3H]-acetylcholine efflux in whole tissue and in isolated CM strips. All effects of prucalopride were antagonized by the selective 5-HT4 receptor antagonist GR113808. The results obtained indicate the presence of excitatory 5-HT4 receptors on cholinergic nerves within the CM of human colon.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Tissue preparation
  6. Dissection and preparation of muscle strips
  7. Contractility study
  8. Experimental protocol of contractility study
  9. Release study
  10. Experimental protocol of release study
  11. Measurement of radioactivity and separation by HPLC of radioactive compounds
  12. Statistics
  13. Compounds
  14. Results
  15. Contractility study
  16. Release study
  17. Discussion
  18. Acknowledgements
  19. References

5-Hydroxytryptamine 4 (5-HT4) receptors have been described to be abundantly expressed in the gastrointestinal tract, mediating various aspects of gut motor function.1 Although most pharmacological studies were performed with intact rodents or rodent isolated tissues, dogs and humans are endowed with these receptors as well. This was demonstrated in recent in vivo and in vitro studies. In healthy humans, 5-HT4 receptor agonists have been demonstrated to stimulate whole gut transit, and colonic transit as well.2, 3 In patients with constipation, prucalopride increased stool frequency and decreased stool consistency.4, 5 Colonic contractility patterns were influenced by the 5-HT4 receptor agonist prucalopride in conscious dogs, equipped with circularly orientated force transducers on the various regions of large intestine6 and the change of colonic motility was often associated with increased stool frequency. Prucalopride stimulated proximal, and inhibited distal colonic motility, and reduced the time to the first giant migrating contraction (GMC). GMCs are held responsible for mass movements over longer distances of large intestine.7 As assessed by ambulant colonic manometry in healthy volunteers, prucalopride increased the frequency of high-amplitude propagated contractions (the human equivalent of GMCs).8 We have attempted to establish correlates of these in vivo actions by means of isolated tissue studies.

In the longitudinal muscle layer of both canine large intestine (from ascending to descending colon) and human large intestine (from ascending colon to rectum), 5-HT4 receptors on cholinergic nerves mediate facilitation of cholinergic neurotransmission resulting in enhanced longitudinal muscle contractility.9 In longitudinal muscle–myenteric plexus preparations of guinea-pig ileum, it was shown that 5-HT4 receptors facilitate cholinergic neuro-neuronal transmission.10 In canine large intestine circular muscle (CM), smooth muscle 5-HT4 receptors mediate relaxation, a phenomenon that gradually increases in efficiency going from the ascending colon to the rectum.11 In human large intestine CM, 5-HT4 receptors located on smooth muscle mediate relaxation, irrespective of the region observed.12 This inhibitory action on CM tone demonstrated in vitro may explain the distally increasing CM inhibition observed with prucalopride in dog large intestine in vivo.6 It can be anticipated from the inhibitory 5-HT4 receptors on colon CM that also in humans, activation of 5-HT4 receptors will be associated with reduction of large intestine luminal resistance. However, the stimulation of human colonic transit by 5-HT4 receptor agonists cannot be triggered by inhibition of CM activity alone. It remains thus to be investigated what mechanism underlies the 5-HT4 receptor-mediated stimulation of colonic motility in the CM. We hypothesized that in human colon CM, in addition to the presence of inhibitory smooth muscle 5-HT4 receptors, excitatory 5-HT4 receptors are present on cholinergic neurones.

We set out to investigate this via two experimental approaches. First, in classical organ bath experiments we tested the effect of a 5-HT4 receptor agonist and antagonist on electrical field stimulation (EFS)-induced contractions of strips from human large intestinal CM. Second, we assessed the effect of these ligands on EFS-triggered release of tritiated acetylcholine from those muscle strips.

Dissection and preparation of muscle strips

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Tissue preparation
  6. Dissection and preparation of muscle strips
  7. Contractility study
  8. Experimental protocol of contractility study
  9. Release study
  10. Experimental protocol of release study
  11. Measurement of radioactivity and separation by HPLC of radioactive compounds
  12. Statistics
  13. Compounds
  14. Results
  15. Contractility study
  16. Release study
  17. Discussion
  18. Acknowledgements
  19. References

The segments were cut open along the longitudinal axis. Luminal contents were rinsed out with Krebs–Henseleit solution (composition in mmol L−1: glucose 11.1, CaCl2 2.51, NaHCO3 25, MgSO4 1.18, KH2PO4 1.18, KCl 4.69 and NaCl 118) and the mucosa and adhering mesentery were removed. The tissue was stored in fresh solution at 4 °C to be used the next day.

There were two ways of dissection of CM strips. The first one was obtained by cutting through the entire wall in the circular direction. This conventional dissection method yielded CM strips that contained CM, some longitudinal muscle, and the intermediate myenteric plexus. These strips were called ‘whole tissue’ CM strips and were used for both organ bath experiments and release experiments. Using a light microscope, the other type of CM strips was obtained by superficial cutting in the CM layer with a pair of curved scissors. This provided CM strips that did not contain longitudinal muscle and they were called for future reference ‘isolated’ CM strips. Isolated CM strips were used for release experiments only. This dissection procedure was performed to evaluate the possible contribution of excitatory 5-HT4 receptors on cholinergic neurones in the longitudinal muscle layer.9 We were aided in this dissection procedure by the use of intertaenial tissue of human specimens, in which the longitudinal muscle layer is clearly less pronounced.

Contractility study

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Tissue preparation
  6. Dissection and preparation of muscle strips
  7. Contractility study
  8. Experimental protocol of contractility study
  9. Release study
  10. Experimental protocol of release study
  11. Measurement of radioactivity and separation by HPLC of radioactive compounds
  12. Statistics
  13. Compounds
  14. Results
  15. Contractility study
  16. Release study
  17. Discussion
  18. Acknowledgements
  19. References

Only ‘whole tissue’ muscle strips (measuring approximately 2–3 cm in length and 2–3 mm in width) were used to study contractility as isolated CM strips only showed very weak contractions to electrical stimulation. The strips were anchored to organ bath hooks between platinum wire electrodes (40 mm length, 0.5 mm width) and suspended in a classical organ bath set-up for isotonic measurement (2 g load; Harvard Apparatus Research Grade Isotonic Transducer, Model 72–4487, Holiston, MA, USA). Responses were registered on Kipp en Zonen recorders (Model BD112, Delft, The Netherlands). The 20 mL organ baths were filled with Krebs–Henseleit solution, kept at 37 °C and gassed with carbogen (95% O2, 5% CO2). EFS was applied using stimulation equipment made at the Janssen Research Foundation (Beerse, Belgium).

Experimental protocol of contractility study

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Tissue preparation
  6. Dissection and preparation of muscle strips
  7. Contractility study
  8. Experimental protocol of contractility study
  9. Release study
  10. Experimental protocol of release study
  11. Measurement of radioactivity and separation by HPLC of radioactive compounds
  12. Statistics
  13. Compounds
  14. Results
  15. Contractility study
  16. Release study
  17. Discussion
  18. Acknowledgements
  19. References

After a 30-min period of stabilization, the strips were contracted with carbachol (10 μmol L−1) to test their viability and responsiveness. After washout, NG-nitro-l-arginine (L-NNA; 0.1 mmol L−1) was added to the organ bath solution in order to prevent relaxation due to EFS-induced release of nitric oxide. After 30 min of incubation, the muscle strips were electrically stimulated (initial parameters: 1 ms pulses in trains of 10 s, at an interval of 3 min, 20 V at 12 Hz). Each pulse train resulted in a contraction, and after four to five consecutive pulse trains, contractions were reproducible. The voltage was then reduced until reproducible contractions with an amplitude approximating 30–50% of the contraction observed at 20 V was obtained. This submaximal contraction permits that both inhibition and enhancement of the response can be observed when studying agents interfering with cholinergic neurotransmission. Stimulation at this voltage was continued for at least 15 min, followed by addition of the selective 5-HT4 receptor antagonist, GR113808 or solvent, which, in turn, was left to incubate for 15 min, while EFS was continued. Following this, the selective 5-HT4 receptor agonist, prucalopride was added to the organ bath solution under continued EFS and the response was followed for another 15 min.

Release study

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Tissue preparation
  6. Dissection and preparation of muscle strips
  7. Contractility study
  8. Experimental protocol of contractility study
  9. Release study
  10. Experimental protocol of release study
  11. Measurement of radioactivity and separation by HPLC of radioactive compounds
  12. Statistics
  13. Compounds
  14. Results
  15. Contractility study
  16. Release study
  17. Discussion
  18. Acknowledgements
  19. References

The muscle strips (whole tissue or isolated CM strips) were transported in ice-chilled Krebs–Henseleit solution to the laboratory where the release study was conducted. Upon receipt, the Krebs–Henseleit solution was replaced by physiological salt solution (PSS; composition in mmol L−1: glucose 11.5, CaCl2 2.5, NaHCO3 25, MgCl2 1.2, KH2PO4 1.2, KCl 4.7, NaCl 112, choline 0.0015 and ascorbic acid 0.057). The human colon tissue preparations weighed 34.28 ± 2.57 (whole tissue CM; n = 58) and 7.32 ± 0.80 (isolated CM; n = 100) mg, respectively. Whole tissue preparations used for release experiments were 1 cm in length and 3 mm in width, while measures of isolated CM preparations were more variable: 3–6 mm in length and 2–3 mm in width. A first series of experiments with single isolated CM strips revealed that the amount of total radioactivity (TR) released was too small to allow separation of the different components by HPLC. Therefore, a second series of experiments was performed whereby four isolated CM strips were mounted in one organ bath. As no differences between the different regions of the human colon were observed, the results were pooled. All human strips were used within 24 h after surgery. Strips were mounted vertically without load between two platinum wire electrodes (40 × 0.5 mm) in 2 mL organ baths containing PSS, maintained at 37 °C and gassed with carbogen (95% O2/5% CO2). Guanethidine (4 μmol L−1) was present in the medium throughout all experiments to avoid noradrenergic influences. EFS was applied by means of a Grass stimulator (S88, Quincy, MA, USA).

Experimental protocol of release study

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Tissue preparation
  6. Dissection and preparation of muscle strips
  7. Contractility study
  8. Experimental protocol of contractility study
  9. Release study
  10. Experimental protocol of release study
  11. Measurement of radioactivity and separation by HPLC of radioactive compounds
  12. Statistics
  13. Compounds
  14. Results
  15. Contractility study
  16. Release study
  17. Discussion
  18. Acknowledgements
  19. References

Basically, the same method was used as described for the labelling of acetylcholine pools in pig gastric fundus.13 Briefly, using a peristaltic pump (Gilson Minipuls, Villiers-le Bel France), the tissues were superfused at a rate of 2 mL min−1 during 60 min, and continuous EFS (40 V, 1 ms, 0.5 Hz) was applied the last 20 min. After this equilibration period, superfusion was stopped and the preparations were incubated for 30 min with [3H]-choline (5 μCi mL−1) during which the tissues were stimulated electrically (40 V, 1 ms, 2 Hz) in order to label their cholinergic transmitter stores.

After the labelling procedure, the strips were superfused (2 mL min−1) for 60 min with PSS to remove loosely bound radioactivity. From now on the PSS contained hemicholinium-3 (10 μmol L−1) to prevent the re-uptake of choline, physostigmine (10 μmol L−1) to prevent the hydrolysis of acetylcholine and atropine (1 μmol L−1) to prevent the auto-inhibition of acetylcholine release.

After the washout period, the strips were no longer superfused but the content of the organ bath, filled with 1 mL, was collected at 3 min intervals. A total of 35 samples were collected. A 0.5 mL of the samples was mixed with 2 mL of the scintillator containing solution Ultima Gold (Packard Bioscience, Groningen, The Netherlands). The strips were stimulated twice for 2 min (S1 and S2; 15 V, 1 ms, 4 Hz), at 13 min (S1, fifth sample), and 73 min (S2, 25th sample) after the end of the washout period. Tetrodotoxin, calcium-free medium and ω-conotoxin-GVIA were added 30 min (15th sample) before S2, GR113808 was added 36 min (13th sample) before S2 and prucalopride was added 15 min (20th sample) before S2, and they remained present until the end of the experiment. At the end of the experiment, tissues were blotted and weighed.

Measurement of radioactivity and separation by HPLC of radioactive compounds

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Tissue preparation
  6. Dissection and preparation of muscle strips
  7. Contractility study
  8. Experimental protocol of contractility study
  9. Release study
  10. Experimental protocol of release study
  11. Measurement of radioactivity and separation by HPLC of radioactive compounds
  12. Statistics
  13. Compounds
  14. Results
  15. Contractility study
  16. Release study
  17. Discussion
  18. Acknowledgements
  19. References

Radioactivity of all samples was measured by liquid scintillation counting (Packard Tri-Carb 2100 TR; Canberra Packard, USA). External standardization was used to correct for counting efficiency. Electrical stimulation induced an increase in tritium overflow, not only in samples 5 (S1) and 25 (S2) but also in the two till four following samples. The stimulation-induced increase in tritium overflow was calculated by subtracting basal tritium overflow. Basal tritium overflow during the period of enhanced tritium overflow was calculated by fitting a regression line through the values of the three samples just before stimulation and the values of the three samples starting from where overflow had returned to basal values after stimulation.

The amount of [3H]-acetylcholine, [3H]-choline and [3H]-phosphorylcholine in the samples was analysed by reverse phase HPLC (Bischoff Chromatography, Leonberg Germany; Hyperchrome-HPLC column, 250 × 4.6 mm, prepacked with Hypersil – ODS 5.0 μm). A 0.1 mol L−1 phosphate buffer (pH 4.7) was used, containing methanol (8 vol.%) and tetramethylammonium (0.2 mmol L−1). The flow was 0.5 mL min−1 and the effluent was collected in 1 min fractions. This is a suitable method to separate the different components as demonstrated previously.13

HPLC was performed on one sample before S1 and S2 (sample 3 and sample 23, respectively), and on the sample during stimulation (sample 5 and 25). A 100 μL of the sample was injected into the HPLC; 27 fractions were collected, and each fraction was mixed with 2 mL of Ultima Gold. Fractions 7–12 contained the peaks of [3H]-phosphorylcholine and [3H]-choline and were taken together to calculate the amount of [3H]-phosphorylcholine and [3H]-choline. Fractions 14–25 were summed to calculate the amount of [3H]-acetylcholine (see Fig. 2B,C). The absolute amount of [3H]-phosphorylcholine plus [3H]-choline and of [3H]-acetylcholine was calculated by subtracting the background counting. Background counting was calculated by fitting a regression line through the values of the first five fractions and fractions 26 and 27. Finally, the percentage of [3H]-acetylcholine in each sample was calculated.

image

Figure 2. Influence of electrical field stimulation on the release of total radioactivity (TR) and [3H]-acetylcholine from control whole tissue strips of human colon. (A) TR outflow from human colon whole tissue preparations pre-incubated with [3H]-choline. The abscissa starts at the end of the washout period. Tissues were stimulated twice (S1 and S2: 15 V, 1 ms, 4 Hz, 2 min), and the superfusate (1 mL) was collected in 3 min fractions. (B) HPLC-separation of the radioactive outflow before (sample 3) and during (sample 5) S1. Arrows indicate the peaks for [3H]-phosphorylcholine, [3H]-choline and [3H]-acetylcholine. (C) HPLC-separation of the radioactive outflow before (sample 23) and during (sample 25) S2. A 100 μL of the samples were injected into the HPLC, and every min 500 μL was collected. Results are given as mean ± SEM (panel A, n = 8; panels B and C, n = 7).

Download figure to PowerPoint

Contractility study  For each individual (whole) muscle strip, the average contraction to five EFS pulse trains before addition of treatment or solvent was taken as 100% (called the initial value) and all contractions of this strip were related to this initial value. Repeated measures over time were analysed using PROC MIXED (sas PC version 6.12, SAS, Cary, NY, USA) for data with an unbalanced covariance structure.

Release study  The ratios S2/S1 for TR and for tritiated acetylcholine were calculated. Results were compared by a paired or unpaired t-test, where appropriate. In the case where more than two responses were assessed, anova was performed, followed by a post hoct-test corrected for multiple comparisons (Bonferroni procedure).

For both types of study, experimental data are expressed as means ± SEM, n referring to the number of tissues obtained from different human specimens. P values of less than 0.05 were considered significant.

Compounds

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Tissue preparation
  6. Dissection and preparation of muscle strips
  7. Contractility study
  8. Experimental protocol of contractility study
  9. Release study
  10. Experimental protocol of release study
  11. Measurement of radioactivity and separation by HPLC of radioactive compounds
  12. Statistics
  13. Compounds
  14. Results
  15. Contractility study
  16. Release study
  17. Discussion
  18. Acknowledgements
  19. References

The following compounds were used in the contractility study (with their pharmaceutical names and respective suppliers given in parentheses): [1-[2-[(methylsulphonyl)amino]ethyl]-4-piperidinyl] methyl 1-methyl-1H-indole-3-carboxylate (GR113808), 4-amino-5-chloro-2,3-dihydro-N-(1-[3-methoxypropyl]-4-piperidinyl)-7-benzofurancarboxamide HCl (prucalopride; R093877) (Janssen Research Foundation), atropine sulphate, carbachol, NG-nitro-l-arginine (Janssen Chimica, Beerse, Belgium), tetrodotoxin (Serva, Germany). All compounds were dissolved in 0.9% NaCl solution, except for GR113808, which was dissolved in 0.9% NaCl acidified with tartaric acid in the stock solution. Solutions were prepared freshly on the day of the experiment and all dilutions were made using 0.9% NaCl solution. The solvents did not affect EFS-induced contractions.

In the release study, l-ascorbic acid, atropine sulphate, choline chloride and guanethidine sulphate were obtained from Sigma (St Louis, MO, USA), hemicholinium-3-bromide from RBI (Natick, MA, USA), methanol from Lab-Scan (Dublin, Ireland), [methyl-3H]-choline chloride (2775 GBq mmol−1) from NEN (Boston, MA, USA), physostigmine salicylate from Federa (Brussels, Belgium), prucalopride, and GR113808 from Janssen Research Foundation, tetramethylammonium chloride from Merck-Schuchardt (Hohenbrunn, Germany), and tetrodotoxin and ω-conotoxin-GVIA from Alomone Labs (Jerusalem, Israel). The calcium-free medium was prepared by replacing 2.5 mmol L−1 CaCl2 by 2.5 mmol L−1 MgCl2 in the PSS. Drugs were dissolved and diluted with deionized water. Stock solutions of tetrodotoxin (1 mmol L−1), prucalopride (10 mmol L−1) and GR113808 (10 mmol L−1) were kept frozen at −20 °C. Dilutions were made on the day of the experiment.

Contractility study

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Tissue preparation
  6. Dissection and preparation of muscle strips
  7. Contractility study
  8. Experimental protocol of contractility study
  9. Release study
  10. Experimental protocol of release study
  11. Measurement of radioactivity and separation by HPLC of radioactive compounds
  12. Statistics
  13. Compounds
  14. Results
  15. Contractility study
  16. Release study
  17. Discussion
  18. Acknowledgements
  19. References

The human large intestinal (whole tissue) CM strips were spontaneously active after they had been mounted in the organ baths. After washout of the contraction to carbachol (10 μmol L−1), that induced a stable contraction in all preparations, the strips returned to baseline, and spontaneous activity was reduced. Spontaneous contractility was further reduced during EFS (to constitute roughly 5% of maximal contraction to EFS).

Tetrodotoxin (0.3 μmol L−1) and atropine (1 μmol L−1) blocked the contractions elicited by EFS in every preparation. The effect of prucalopride (0.3 μmol L−1) on EFS-evoked contractions of human large intestinal preparations was variable. Prucalopride stimulated EFS-induced contractions in one specimen (by 26%), inhibited them in four out of eight preparations (inhibition ranging from 22 to 100%), and was without effect in three preparations (Fig. 1A). On average, prucalopride (0.3 μmol L−1) tended to inhibit EFS-induced contractions, albeit this effect was not significant (71 ± 15%vs initial value, n = 8, P > 0.05). In all human muscle strips, GR113808 (0.1 μmol L−1) prevented any effect of prucalopride (111 ± 8%vs initial value, n = 8, P > 0.05, Fig. 1B).

image

Figure 1. Effect of prucalopride on electrical field stimulation (EFS)-evoked contractions of human large intestine circular muscle. All experiments were carried out in the presence of NG-nitro-l-arginine (0.1 mmol L−1). The left panel shows individual data points of the effect of prucalopride, that are connected by a within-specimen solid line. The origin of the tissues in the large intestine is indicated: A, ascending colon, T, transverse colon, S, sigmoid colon. The right panel shows mean contraction ± SEM (n = 8), calculated as percentage of the mean contraction to five EFS pulse trains immediately prior to any addition of compound (initial value). The response to prucalopride after addition of solvent or GR113808 is shown.

Download figure to PowerPoint

Whole tissue of human colon  In human tissues, field stimulation caused a clear-cut increase in TR and 9–12 min were required after stimulation to re-establish the basal release of tritium (Fig. 2A). The mean amount of TR in samples 3 and 23 (prestimulation), and in the samples with increased release because of stimulation (S1 and S2), is given in Table 1. This yielded a S2/S1 ratio for TR of 0.67 ± 0.02 (n = 8). [3H]-Acetylcholine could be detected when HPLC was used to separate the different components present in the samples. In basal conditions, TR contained more [3H]-phosphorylcholine plus [3H]-choline than [3H]-acetylcholine. Stimulation induced a pronounced increase in the release of [3H]-acetylcholine, although there was also a two- to threefold increase in the release of [3H]-phosphorylcholine and [3H]-choline during stimulation (Fig. 2B,C). The mean amounts of [3H]-choline plus [3H]-phosphorylcholine, and [3H]-acetylcholine before and during stimulation, and the percentage of [3H]-acetylcholine released are given in Table 1. The S2/S1 ratio for [3H]-acetylcholine was 0.71 ± 0.06 (n = 7).

Table 1.  Outflow of total radioactivity, [3H]-phosphorylcholine plus [3H]-choline and [3H]-acetylcholine for control whole tissue (WT) and isolated (ISO) circular muscle strips of human colon
 TRPh.-Ch. + Ch.ACh% ACh
  1. ACh, [3H]-acetylcholine; Ph.-Ch. + Ch., [3H]-phosphorylcholine plus [3H]-choline; TR, total radioactivity. Four isolated circular muscle strips were mounted in one organ bath for each experiment. S1 and S2 for TR are the sum of respectively sample 5 and sample 25 and the following samples (see text), while S1 and S2 for Ph.-Ch. + Ch. and ACh are, respectively, samples 5 and 25. Radioactivity is expressed as dpm g−1 tissue. *P < 0.05, **P < 0.01, ***P < 0.001: Significantly different from values before S1 (sample 3). #P < 0.05, ##P < 0.01, ###P < 0.001: Significantly different from values before S2 (sample 23).

WT
 Sample 358 280 ± 983037 440 ± 41906180 ± 44009 ± 5
 S1698 840 ± 196 510100 180 ± 14 400***178 360 ± 33 250***63 ± 4***
 Sample 2335 760 ± 714019 910 ± 46003690 ± 30207 ± 4
 S2466 090 ± 132 53041 180 ± 4050##125 370 ± 23 260###72 ± 4###
 S2/S10.67 ± 0.02 0.71 ± 0.06 
 n8777
ISO
 Sample 375 200 ± 764069 060 ± 12 4802920 ± 15905 ± 2
 S1850 950 ± 181 690232 520 ± 69 680*320 860 ± 57 690**59 ± 5***
 Sample 2348 040 ± 693041 480 ± 62803850 ± 20607 ± 3
 S2509 340 ± 95 46089 330 ± 21 460#202 760 ± 38 710##68 ± 5###
 S2/S10.63 ± 0.03 0.63 ± 0.03 
 n6666

Tetrodotoxin (3 μmol L−1; n = 5), ω-conotoxin-GVIA (1 μmol L−1; n = 1) or removal of extracellular calcium (n = 2) did not influence basal release of TR. Tetrodotoxin nearly abolished the electrically evoked tritium release when compared with control. In control tissues (n = 5), the S2/S1 ratio for release of TR was 0.70 ± 0.02. The S2/S1 ratio for TR after superfusion with tetrodotoxin was 0.08 ± 0.01 (P < 0.001). The S2/S1 ratio for TR in the presence of ω-conotoxin was 0.16 (n = 1), while the ratio was 0.67 for control tissue. In the absence of calcium (n = 2), the S2/S1 ratio for TR was 0.05 and 0.13, while this was 0.65 and 0.67 in control tissues.

Prucalopride (0.3 μmol L−1) did not alter the basal efflux of TR. However, prucalopride increased the electrically evoked release of TR and [3H]-acetylcholine; the mean increase in electrically evoked release of [3H]-acetylcholine by prucalopride was significant (Fig. 3A). The 5-HT4 receptor antagonist GR113808 (0.1 μmol L−1) did not alter the basal release of TR (n = 8), nor did it influence the S2/S1 ratio for TR (S2/S1 ratio for control: 0.69 ± 0.05, n = 4, and for GR113808: 0.66 ± 0.03, n = 4, P > 0.05) and for [3H]-acetylcholine (S2/S1 ratio for control: 0.75 ± 0.01, n = 3, and for GR113808: 0.78 ± 0.05, n = 3, P > 0.05). In the presence of GR113808 (0.1 μmol L−1), prucalopride had no influence on the electrically evoked release of TR (n = 4; Fig. 3A); the influence of prucalopride on the electrically evoked release of [3H]-acetylcholine was reduced by GR113808 (0.1 μmol L−1) and the S2/S1 ratio for [3H]-acetylcholine was no longer significantly different from that in control conditions (Fig. 3A). In a separate series of experiments (Table 2), the influence of lower concentrations of GR113808 (0.01 and 0.03 μmol L−1) was tested vs prucalopride (0.3 μmol L−1). In this series, the mean increases in electrically evoked release of TR and [3H]-acetylcholine by prucalopride were both significant. As well 0.01 as 0.03 μmol L−1 GR113808 was able to antagonize the effect of prucalopride. This series further showed that also the antagonism by GR113808 of the effect of prucalopride on electrically induced release of TR was not always complete (see result with 0.03 μmol L−1 GR113808).

image

Figure 3. Effects of prucalopride (pruca) and prucalopride in the presence of GR113808 (GR) on the electrically evoked release of total radioactivity (TR) and [3H]-acetylcholine from preparations of (A) human colon whole tissue preparations and (B) human colon isolated circular muscle preparations pre-incubated with [3H]-choline (in B, four isolated circular muscle strips were mounted together in one organ bath for each experiment). Tissues were stimulated twice (S1 and S2: 15 V, 1 ms, 4 Hz, 2 min); GR113808 (0.1 μmol L−1) was added 36 min and prucalopride (0.3 μmol L−1) 15 min before S2. The electrically evoked efflux by S2 is expressed as a ratio of that by S1. Each column represents the mean ± SEM. *P < 0.05 and **P < 0.01: Significantly different from control. Numbers above the columns refer to the number of experiments.

Download figure to PowerPoint

Table 2.  Influence of prucalopride (Pruca) and prucalopride in the presence of GR113808 (GR; 0.01 and 0.03 μmol L−1) on the S2/S1 ratio for TR and ACh in whole tissue circular muscle strips of human colon
 ControlPruca0.01 μmol L−1 of GR + pruca0.03 μmol L−1 of GR + pruca
  1. ACh, [3H]-acetylcholine; TR, total radioactivity. Tissues were stimulated twice (S1 and S2: 15 V, 1 ms, 4 Hz, 2 min); GR (0.01 or 0.03 μmol L−1) was added 36 min and prucalopride (0.3 μmol L−1) 15 min before S2. The electrically evoked efflux by S2 is expressed as a ratio of that by S1. Mean ± SEM of n = 5–6 is given. *P < 0.05: Significantly different from control.

TR0.71 ± 0.040.89 ± 0.04*0.73 ± 0.040.77 ± 0.05
ACh0.76 ± 0.031.02 ± 0.08*0.80 ± 0.070.82 ± 0.06

Isolated circular smooth muscle of human colon  Electrical stimulation induced an increase in tritium overflow in isolated human colon CM, and 6–12 min were required after stimulation to re-establish the basal release of tritium. The S2/S1 ratio for TR was 0.53 ± 0.03 (n = 11). However, the amount of TR released by the very small isolated CM strips was very low, and reached the detection limit of the HPLC; a reliable separation by HPLC of the different components present in the samples was therefore not feasible.

Tetrodotoxin (3 μmol L−1; n = 4), ω-conotoxin-GVIA (1 μmol L−1; n = 1) or removal of extracellular calcium (n = 2) did not influence basal release of TR. Tetrodotoxin nearly abolished the electrically evoked tritium release when compared with control in three out of four experiments (S2/S1 ratios for release of TR: control, 0.55 ± 0.03 vs tetrodotoxin, 0.13 ± 0.03; n = 3; P < 0.001). In one experiment, the electrically evoked tritium release was not abolished by tetrodotoxin (S2/S1 ratios for release of TR: control, 0.53 vs tetrodotoxin, 0.51), but in the whole tissue preparation of the same patient, tetrodotoxin abolished the electrically evoked tritium release (S2/S1 ratios for release of TR: control, 0.65 vs tetrodotoxin, 0.09). The S2/S1 ratio for TR in the presence of ω-conotoxin was 0.15 (n = 1), while the ratio was 0.49 for control tissue. After superfusion with calcium-free medium (n = 2), the S2/S1 ratio for TR was 0.24 and 0.10, while this was 0.59 and 0.49 in control tissues.

Prucalopride (0.3 μmol L−1) did not alter the basal efflux of TR. However, prucalopride significantly enhanced the amount of TR (S2/S1 ratio for TR: control, 0.54 ± 0.03 vs prucalopride, 0.77 ± 0.05; P < 0.001; n = 9). GR113808 (0.1 μmol L−1) did not alter the basal release of TR (n = 7). When added before prucalopride, GR113808 antagonized the increase of the electrically evoked release of TR by prucalopride (S2/S1 ratio for TR: 0.62 ± 0.02; n = 7).

In order to be able to measure [3H]-acetylcholine by HPLC, an additional series of experiments was performed where four isolated CM strips were mounted together in one organ bath. This allowed to separate the different components by HPLC, as enough radioactivity was released.

In basal conditions, released radioactivity contained only a small fraction of [3H]-acetylcholine. Stimulation induced a significant increase in the release of [3H]-acetylcholine, although there was some increase in the release of [3H]-phosphorylcholine and [3H]-choline during stimulation. The mean amounts of TR, [3H]-choline plus [3H]-phosphorylcholine and [3H]-acetylcholine before and during stimulation, and the percentage of [3H]-acetylcholine released are given in Table 1. The S2/S1 ratios were 0.63 ± 0.03 for both TR and [3H]-acetylcholine (n = 6).

Tetrodotoxin (3 μmol L−1; n = 5) did not influence basal release of TR. However, tetrodotoxin nearly abolished the electrically evoked tritium release when compared with control (the S2/S1 ratios for release of TR and [3H]-acetylcholine for control tissues were respectively 0.58 ± 0.03 and 0.69 ± 0.06, while the ratios for tetrodotoxin were respectively 0.15 ± 0.03 and 0.03 ± 0.01; P < 0.001; n = 5).

Prucalopride (0.3 μmol L−1) did not alter the basal efflux of TR. However, prucalopride significantly enhanced the amount of TR and [3H]-acetylcholine (P < 0.01; n = 5; Fig. 3B). GR113808 (0.1 μmol L−1; n = 7), added before prucalopride, had no effect on basal release, however, it antagonized the increase by prucalopride of the electrically evoked release of TR and [3H]-acetylcholine (Fig. 3B).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Tissue preparation
  6. Dissection and preparation of muscle strips
  7. Contractility study
  8. Experimental protocol of contractility study
  9. Release study
  10. Experimental protocol of release study
  11. Measurement of radioactivity and separation by HPLC of radioactive compounds
  12. Statistics
  13. Compounds
  14. Results
  15. Contractility study
  16. Release study
  17. Discussion
  18. Acknowledgements
  19. References

The data presented here suggest that the human colon CM is endowed with 5-HT4 receptors located on cholinergic nerves, mediating facilitation of cholinergic neurotransmission resulting in enhanced contraction. Colonic CM may be the first single muscle layer expressing both an inhibitory smooth muscle and an excitatory neuronal 5-HT4 receptor component.

Stimulation-induced contractions and release of TR and [3H]-acetylcholine in colon tissues were reduced by tetrodotoxin and calcium-free medium (the latter only tested in the release study). This indicates a neuronal release dependent upon the opening of sodium channels, and the presence of calcium in the external medium, respectively. The blockade of EFS-induced contractions by atropine in all whole tissue muscle strips, suggested that these nerves are cholinergic in origin. Calcium enters the cholinergic nerves via N-type calcium channels as ω-conotoxin-GVIA abolished the electrically evoked release of TR. [3H]-Choline and [3H]-phosphorylcholine may be released by leakage from the nerve terminals in basal conditions; they indeed formed the major part of basally released radioactivity, and this was not influenced in the presence of tetrodotoxin and ω-conotoxin. Tetrodotoxin abolished stimulation-induced increase of [3H]-acetylcholine, while stimulation-induced increase of TR was clearly reduced but not abolished. This supports the full neuronal release of acetylcholine, but suggests that stimulation induces some non-neuronal release of [3H]-choline and [3H]-phosphorylcholine.

Prucalopride has been shown to be a selective 5-HT4 receptor agonist in isolated tissues of rodents,14 dogs11 as well as humans.12 At 0.3 μmol L−1, prucalopride stimulates 5-HT4 receptors on cholinergic nerves in canine ascending and descending colon and human large intestine longitudinal muscle.9 Here, this concentration of prucalopride had a variable influence on contractions due to EFS in human colonic muscle strips, ranging from stimulation to inhibition; all effects were absent in the presence of the selective 5-HT4 receptor antagonist GR113808 (0.1 μmol L−1;15). The competitive nature of the 5-HT4 receptor antagonism produced by GR113808 has been confirmed in studies with canine and human gastrointestinal preparations (pKB 9.1;11 pKB 9.412), showing that 0.1 μmol L−1 would induce over a two log unit rightward shift of the curve to a 5-HT4 receptor agonist. Prucalopride at 0.3 μmol L−1 induces an approximate EC50-EC90 response via 5-HT4 receptors as determined in various bioassays of rodent,14 dog11 and human gastrointestinal tissue.12 Hence, GR113808 (0.1 μmol L−1) is expected to block a 5-HT4 receptor-mediated response to prucalopride (0.3 μmol L−1). In this manner, it is conceivable that the effects of prucalopride on EFS-induced CM contraction are related to activation of 5-HT4 receptors.

The release study showed that, when interpreting the effect of prucalopride in the functional study, the presence of excitatory 5-HT4 receptors on cholinergic nerves in CM of human colon must be taken in account. Indeed, prucalopride evoked a systematic increase in TR and [3H]-acetylcholine efflux from whole tissue CM, an effect that was antagonized by GR113808 (0.1 μmol L−1). Still, the whole tissue CM strips contain a small amount of longitudinally directed muscle, where 5-HT4 receptors are present on the cholinergic nerves9 and that might contribute to the acetylcholine release detected in the whole tissue preparations. However, in isolated CM strips, only containing CM and nerve endings between the muscle fibres, prucalopride stimulated TR and [3H]-acetylcholine release and its effect was antagonized by GR113808. Both in whole tissue CM strips and in isolated CM strips, the antagonizing effect of GR113808 (0.1 μmol L−1) vs prucalopride seemed less pronounced in case of [3H]-acetylcholine. It can be excluded that this is due to interaction of prucalopride with another 5-HT receptor subtype, as lower concentrations of GR113808 (0.03 and even 0.01 μmol L−1) were still able to antagonize the effect of prucalopride on the electrically induced release of TR and [3H]-acetylcholine. In view of the potency of GR113808, a rightward shift of the concentration-response curve of prucalopride of at least one log unit can still be expected with 0.01 μmol L−1 GR113808. This would mean that the EC50–EC90 response of 0.3 μmol L−1 prucalopride in this study will be reduced to 10–20% by 0.01 μmol L−1 GR113808, which is as observed. Some variability from series-to-series might thus explain the apparently less pronounced influence of GR113808 vs the effect of prucalopride on electrically induced release of [3H]-acetylcholine in some tissues. Hence, in human colon CM, excitatory 5-HT4 receptors are located on cholinergic nerves within the CM wall. As GR113808 did not influence the S2/S1 ratio for TR and [3H]-acetylcholine, it is unlikely that electrical stimulation causes the release of endogenous 5-HT which might act on 5-HT4 receptors to enhance [3H]-acetylcholine release. Our study thus shows that in human colon, excitatory 5-HT4 receptors are present on cholinergic nerves within the CM layer, similar to those shown before on the cholinergic nerves within the longitudinal muscle layer.9 The non-effect of cisapride on electrically induced acetylcholine release in human colonic taenial longitudinal muscle reported earlier16 is probably due to the partial agonist effect of cisapride on 5-HT4 receptors being insufficient to enhance acetylcholine release from cholinergic nerves. The presence of excitatory 5-HT4 receptors on cholinergic nerve endings in human colon CM, and longitudinal muscle9 contrasts with findings in longitudinally mounted guinea-pig colon segments, suggesting that 5-HT4 receptors are primarily located on the soma of intrinsic motor neurones.17 This illustrates the importance of species differences.

The question can then be raised why the corresponding functional response, i.e. increased contraction after cholinergic nerve stimulation in the presence of the 5-HT4 receptor agonist, is not systematically observed in the contractility study as prucalopride even clearly reduced the EFS-induced contractions in some strips. This is probably explained by the presence of inhibitory smooth muscle 5-HT4 receptors in human colonic CM, for which a number of reports have provided evidence.12, 18–21 Inducing contractions by EFS does not prevent prucalopride from activating these smooth muscle receptors, irrespective of prucalopride stimulating neuronal receptors as well. Hence, functional antagonism of the EFS-induced contraction is expected due to activation of the inhibitory smooth muscle 5-HT4 receptors. In view of the above, across-specimen variation in the expression of both functional responses (inhibition vs stimulation of contractility) may explain the heterogenous nature of the prucalopride-induced response in the contractility study. 5-HT4 receptors might also be located at the terminals of inhibitory motor neurones. Facilitation of the release of a relaxant neurotransmitter will also counteract the cholinergic nerve facilitation. This seems less likely as the principal inhibitory neurotransmitter in human colon CM is NO;22, 23 NO cannot be involved in our contractility study as it was performed under inhibition of NO synthesis by L-NNA. The outcome of this study is novel, as the human colon CM may represent the first tissue entity in which both locations (muscular vs neuronal) and functions (relaxation vs facilitation of contraction) of 5-HT4 receptors have been detected. Our functional data illustrating the presence of both excitatory neuronal 5-HT4 receptors, in addition to muscular inhibitory 5-HT4 receptors12 in human colon CM corresponds with autoradiographic data, showing 5-HT4 binding sites in human colon muscle and myenteric plexus.24 The net effect of a 5-HT4 receptor agonist on human colon CM will thus depend on a fine balance between its inhibitory and excitatory effects. Stimulation of the 5-HT4 receptors on the cholinergic nerves towards the CM in human colon probably contributes to the stimulatory action of 5-HT4 receptor agonists on human colonic transit.2, 3

In conclusion, this study illustrates the presence of 5-HT4 receptors on cholinergic nerves within the CM of the human colon.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Tissue preparation
  6. Dissection and preparation of muscle strips
  7. Contractility study
  8. Experimental protocol of contractility study
  9. Release study
  10. Experimental protocol of release study
  11. Measurement of radioactivity and separation by HPLC of radioactive compounds
  12. Statistics
  13. Compounds
  14. Results
  15. Contractility study
  16. Release study
  17. Discussion
  18. Acknowledgements
  19. References

This study was financially supported by grant no. 3G0053.02 from the Fund for Scientific Research Flanders and by Interuniversity Attraction Poles Programme P5/20 (Federal Public Planning Service Science Policy). The authors thank Mrs. I. Van Colen for technical assistance in part of the experiments, Prof. Dr P. Pattyn from the Department of Surgery of the Ghent University Hospital for his help in obtaining the human specimens and Mr. W. De Ridder for his help with the statistical analysis.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Tissue preparation
  6. Dissection and preparation of muscle strips
  7. Contractility study
  8. Experimental protocol of contractility study
  9. Release study
  10. Experimental protocol of release study
  11. Measurement of radioactivity and separation by HPLC of radioactive compounds
  12. Statistics
  13. Compounds
  14. Results
  15. Contractility study
  16. Release study
  17. Discussion
  18. Acknowledgements
  19. References
  • 1
    Hegde SS, Eglen RM. Peripheral 5-HT4 receptors. FASEB 1996; 10: 1398407.
  • 2
    Emmanuel AV, Kamm MA, Roy AJ et al. Effect of a novel prokinetic drug, R093877, on gastrointestinal transit in healthy volunteers. Gut 1998; 42: 5116.
  • 3
    Bouras EP, Camilleri M, Burton DD et al. Selective stimulation of colonic transit by the benzofuran 5-HT(4) agonist, prucalopride, in healthy humans. Gut 1999; 44: 6826.
  • 4
    Sloots CEJ, Poen AC, Kerstens R et al. Effects of prucalopride on colonic transit, anorectal function and bowel habits in patients with chronic constipation. Aliment Pharmacol Ther 2002; 16: 75967.
  • 5
    Emmanuel AV, Roy AJ, Nicholls TJ et al. Prucalopride, a systemic enterokinetic, for the treatment of constipation. Aliment Pharmacol Ther 2002; 16: 134756.
  • 6
    Briejer MR, Prins NH, Schuurkes JAJ. Effects of the enterokinetic prucalopride (R093877) on colonic motility in fasted dogs. Neurogastroenterol Mot 2001; 13: 46572.
  • 7
    Karaus M, Sarna SK. Giant migrating contractions during defecation in the dog colon. Gastroenterology 1987; 92: 92533.
  • 8
    De Schryver AMP, Andriesse GI, Samsom M et al. The effects of the specific 5-HT4 receptor agonist, prucalopride, on colonic motility in healthy volunteers. Aliment Pharmacol Ther 2002; 16: 60312.
  • 9
    Prins NH, Akkermans LMA, Lefebvre RA et al. 5-HT4 receptors on cholinergic nerves involved in contractility of canine and human large intestine longitudinal muscle. Br J Pharmacol 2000; 131: 92732.
  • 10
    Lepard KJ, Ren J, Galligan JJ. Presynaptic modulation of cholinergic and non-cholinergic fast synaptic transmission in the myenteric plexus of guinea pig ileum. Neurogastroenterol Mot 2004; 16: 35564.
  • 11
    Prins NH, Van Haselen JFWR, Lefebvre RA et al. Pharmacological characterization of 5-HT4 receptors mediating relaxation of canine isolated rectum circular smooth muscle. Br J Pharmacol 1999; 127: 14317.
  • 12
    Prins NH, Shankley NP, Welsh NJ et al. An improved in vitro bioassay for the study of 5-HT4 receptors in the human isolated large intestinal circular muscle. Br J Pharmacol 2000; 129: 16018.
  • 13
    Leclere PG, Lefebvre RA. Influence of nitric oxide donors and of the α2-agonist UK-14,304 on acetylcholine release in the pig gastric fundus. Neuropharmacology 2001; 40: 2708.
  • 14
    Briejer MR, Bosmans J-P, Van Daele P et al. The in vitro pharmacological profile of prucalopride, a novel enterokinetic compound. Eur J Pharmacol 2001; 423: 7183.
  • 15
    Gale JD, Grossman CJ, Whitehead JW et al. GR113808: a novel, selective antagonist with high affinity at the 5-HT4 receptor. Br J Pharmacol 1994; 111: 3328.
  • 16
    Burleigh DE, Trout SJ. Evidence against an acetylcholine releasing action of cisapride in the human colon. Br J Clin Pharmacol 1985; 20: 4758.
  • 17
    Briejer MR, Schuurkes JAJ. 5-HT3 and 5-HT4 receptors and cholinergic and tachykininergic neurotransmission in the guinea-pig proximal colon. Eur J Pharmacol 1996; 308: 17380.
  • 18
    Meulemans AL, Ghoos E, Cheyns P et al. 5-HT-induced relaxations of human sigmoid colon are mediated via 5-HT4 receptors. Pflüger's Arch Eur J Physiol 1995; 429: R9.
  • 19
    Tam FS, Bunce KT, Hillier K et al. Characterization of the 5-hydroxytryptamine receptor type involved in inhibition of spontaneous activity of human isolated colonic circular muscle. Br J Pharmacol 1994; 113: 14350.
  • 20
    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.
  • 21
    McLean PG, Coupar IM. Further investigation into the signal transduction mechanism of the 5-HT4-like receptor in the circular smooth muscle of human colon. Br J Pharmacol 1996; 118: 105864.
  • 22
    Boeckxstaens GE, Pelckmans PA, Herman AG, Van Maercke YM. Involvement of nitric oxide in the inhibitory innervation of the human isolated colon. Gastroenterology 1993; 104: 6907.
  • 23
    Keef KD, Du C, Ward SM, McGregor B, Sanders KM. Enteric inhibitory neural regulation of human colonic circular muscle : role of nitric oxide. Gastroenterology 1993; 105: 100916.
  • 24
    Sakurai-Yamashita Y, Yamashita K, Kanematsu T, Taniyama K. Localization of the 5-HT4 receptor in the human and the guineapig colon. Eur J Pharmacol 1999; 383: 2815.