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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.
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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.