The objective of the present study was to examine the effect of 5-HT4 receptor ligands on the peristaltic reflex in the mouse colon. 5-hydroxytryptamine, a neurotransmitter found mainly within the gastrointestinal tract, has been implicated in the contraction and relaxation of smooth muscle within this region. The actions of 5-HT are mediated by at least one or more of seven subtype receptors. The receptor subtype that will be the focus of attention in this study is the 5-HT4 receptor.
Segments of the proximal colon obtained from MF1 mice, were cannulated at the anal and aboral ends, and secured horizontally in a water jacketed bath containing oxygenated Krebs solution. The intraluminal distension pressure was controlled by adjusting the elevation of the reservoir, and the volume ejected to the aboral side was recorded and measured via a a pressure transducer and Power Lab system using Chart v4.1.2 software for Windows.. All drugs were administered serosally. Cumulative concentration response curves of 5-HT and tegaserod (agonists) were obtained by adding increasing concentrations of drugs at an interval of 5-15 minutes. The antagonists GR113808 and SB204070 were added to the tissues after regular peristalsis was obtained and allowed to equilibrate for 15 minutes, after which either 5-HT or tegaserod were added cumulatively. All results are expressed as mean±SEM from number of animals indicated by n.
A regular peristalsis was established before the addition of 5-HT (average rate of peristalsis was 77±7, n=7. 5-HT facilitated peristalsis at 10-7M and further cumulative addition of 5-HT caused a slow decrease in peristalsis until at 10-4M, rate of peristalsis was inhibited. In all tissues in which peristalsis was inhibited, it recovered once tissue was washed. In the presence of SB204070 and GR113808, there was no significant change in the rate of peristalsis. The addition of tegaserod produced only a slow decrease in peristalsis until peristalsis was abolished in all tissues at 10-4M. The peristalsis abolished by tegaserod could not be re-established in any tissue by washing.
In all tissues, it was possible to obtain peristalsis so the effects of drug testing could be established. The addition of the 5-HT4 selective receptor antagonists, SB204070 and GR113808, showed no significant change in the concentration-response curves. The partial 5-HT4 agonist, tegaserod, also did not facilitate peristalsis in the current study. Both these findings suggest that the 5-HT4 receptor is not implicated in the mouse proximal colon
INTRODUCTION1.1 The gastrointestinal tract
The digestive system is a vital component of the human body; the overall function being to provide nourishment for over a trillion cells within the body. To be able to do this, the digestive system is specialised to ingest food, propel it through the digestive tract, digest it, and absorb water, electrolytes and other nutrients from the lumen of the gastrointestinal tract (Seeley et al, 2006). The absorbed substances are transported to the cells, via the circulatory system, whilst the undigested substances are eliminated from the anus.
The digestive system consists of the main digestive tract, a tube extending from the mouth to the anus, as well as its associated component organs and accessory organs, which are primarily glands located outside the digestive tract that secrete fluids into the digestive tract (Seeley et al, 2006). The component organs include the oral cavity (mouth), pharynx (throat), oesophagus, stomach, small intestine, large intestine and anus. The accessory organs include three pairs of salivary glands, the exocrine pancreas and the exocrine liver. To enable the homoeostatic environment within the body to be maintained, it is vital that the digestive system is functioning efficiently. The large intestine, which consists of the caecum, colon and rectum, has sufficient homeostatic functions, and contributes to the overall stability of the homoeostatic environment within the body. The colon is the central part of the large intestine, and constitutes the last 150cm of the gastrointestinal tract. It is approximately a 6cm tube, which extends from the ileum to the anus. Its main function is to store faecal material and regulate its release into the external environment (Smith et al, 2006). It also produces a thick mucous secretion, which lubricates the passage of faecal material during defecation.
The gastrointestinal tract of a mouse consists of the oesophagus, stomach, small intestine and large intestine. The mouse colon is similar to the human colon, consisting of the ascending, transverse and descending parts but lacking the sigmoid part (Cook, 1965).
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/G/GITract.htmlThe main physiological processes of the digestive system are digestion, absorption, motility, secretion, and excretion. Digestion involves the breakdown of larger molecules to smaller ones (i.e. glucose and amino acids) to allow efficient utilisation and absorption of these molecules. The ingested material and secretions are transported across the epithelial cell membrane, mainly within the small intestine. Subsequently, the transported molecules enter the circulation; a central physiological process of the digestive system.
The gastrointestinal tract is approximately a 15 feet long tube, and food must be moved along it to reach the correct sites for digestion, mixing and absorption (Smith et al, 2001). This process, known as peristalsis, is aided by the smooth muscle lining the tract, which contracts and relaxes mixing the ingested material, whilst at the same time propelling it through the tract. Propulsion of the intestinal contents is a crucial part of digestion that depends on the coordinated activity of circular and longitudinal smooth muscles brought about by the peristaltic reflex (Shiinaa et al, 2005). The peristaltic reflex is initiated by either stimulation of the gastrointestinal mucosa or by stretching of the intestinal wall, resulting in a circular contraction behind the stimulus and an area of relaxation in front of it (Shiinaa et al, 2005). This wave of contraction moves in the oral-anal direction, and subsequently propels the contents within the lumen forward.
The reflex is co-ordinated by the intramural nerve plexuses within the intestine and so, can be obtained even in the isolated tissues. Many studies investigating the mechanisms which mediate intestinal motility have predominantly focused on peristalsis. Trendelengburg (1917) carried out the first in vitro study investigating the peristaltic reflex in guinea pig ileum. Within the ileum, the reflex was found to consist of contractions of both the longitudinal and circular muscles that were both regular as well as coordinated. The increase in intraluminal pressure, which causes the ileum to distend, is followed by an increase in longitudinal muscle contraction, and subsequently, by an increase in circular muscle contraction, which propels the contents towards the anal section (Trendelengburg, 1917).
Gastrointestinal disorders are a common problem in today’s society, and many lead to long term diseases and even morbidity, as well as having a negative impact on healthcare costs (Crowell et al, 2004) However, due to the complexity and the differing functions of the various organs of the GI tract, the treatment of disorders within the tract is a very complex task and has not, as of yet, been fully understood. Diseases of the colon can lead to a whole host of illnesses, including diarrhoea, constipation, Crohn’s disease, Inflammatory Bowel disease, Irritable Bowel Syndrome (IBS), and many more. Symptoms occurring outside of the GI tract, in particular symptoms associated with ibs, including anxiety, depression and schizophrenia, have been related to the morbidity of such disorders. It has been suggested by research, that altered levels of the neurotransmitter, 5-hydroxytryptamine (serotonin), may lead to both intestinal and extra intestinal symptoms in IBS, as well as being implicated in other functional bowel diseases
It is therefore important that further studies are carried investigating the link between 5-hydroxytryptamine and disorders of the gastrointestinal tract, and to further understand the pathogenesis of these disorders, so that new, more effective treatments can be formulated.
1.25-hydroxytryptamine
5-hydroxytryptamine, also more commonly referred to as serotonin, is a monoamine neurotransmitter, and is predominantly synthesised, stored and released in the enterchromaffin cells of the intestinal mucosa (Costedio et al, 2007). According to Gershon et al (1965), 5-HT is synthesised through the action of two tryptophan hydroxylases, TpH1 and TpH2, which are found within the enterochromaffin cells and neurons. Approximately 95% of all mammalian 5-hydroxytryptamine is found within the gastrointestinal tract (Sanger, G.J, 2008)
5-HT initiates the peristaltic and secretory reflex, and transmits information to the central nervous system, by activating both the intrinsic and extrinsic primary afferent neurones (Sikandar et al 2009). It can also modulate a wide range of biological processes such as mood, cognition, perception, feeding behaviour, smooth muscle contractility, and platelet aggregation (Setola et al, 2003). Within the guinea pig ileum, 5-HT has been found to cause both facilitation and inhibition of peristalsis (Tuladhar et al), and has been found to facilitate peristalsis, when added serosally in the marmoset ileum (Tuladhar et al, 1996).
The actions of 5-HT, particularly contraction or relaxation responses, are mediated by at least one or more of seven subtype receptors (Setola et al, 2003), ranging from 5-HT1 to 5-HT7. 5-HT1 and 5-HT2 receptors have been further subdivided, as can be seen in figure 2.With the exception of the 5-HT3 receptor, the other receptors are, at molecular level, G protein couple metabotropic receptors which span the membrane. The 5-HT3 receptor is a ligand-gated ion channel (Barnes et al, 1999). Many 5-HT receptors can now be associated with various physiological responses, ranging from modulation of neuronal activity and transmitter release to behavioural change (Barnes et al, 1999).
Receptor Subtype Transduction Mechanism Localization Function Specific Agonists Specific Antagonists 5-HT1AvAC (Gi/o)Limbic system (hippocampus, lateral septum, cortical areas), mesencephalic raphe nucleiHyperpolarization, modulation of neurotransmitter release, anxiolysis, hypothermia, hyperphagiaXaliprofen (2491) S 14506 (1771) Ipsapirone (1869) BP 554 (0556) U 92016A (2739) Tandospirone (2854)*MM 77 (0933) NAN-190 (0553) Spiroxatrine (0631) (S)-WAY 100135 (1253) 5-HT1BvAC (Gi/o)Basal ganglia, striatum, amygdala, trigeminal ganglion, vascular smooth muscleAutoreceptor, locomotion, hypophagia, hypothermia, modulation of neurotransmitter release, vasoconstrictionCGS 12066B (0638) CP 93129 (1032) CP 94253 (1317) 5-Nonyloxytryptamine (0901)GR 55562 (1054) Isamoltane (0992) SB 224289 (1221) NAS-181 (1413) 5-HT1DvAC (Gi/o)Basal ganglia, hippocampus, cortex, spinal cord, vascular smooth muscleAutoreceptor, modulation of neurotransmitter releaseL-694,247 (0781) GR 46611 (0864) PNU 109291 (2556) PNU 142633 (1985)BRL 15572 (1207) Cyanopindolol (0993) LY 310762 (3078) 5-ht1EvAC (Gi/o)Cortex, caudate putamen, claustrum, hippocampus, amygdalaUnknownBRL 54443 (1129)– 5-HT1FvAC (Gi/o)Hippocampus, cortex, dorsal raphe nucleus, uterusSpeculative role in visual and cognitive functionBRL 5443 (1129) LY 344864 (2451) LY 334370 (3079)– 5-HT2A^ PLCForebrain, caudate nucleus, nucleus accumbens, hippocampus, olfactory tubercle, vascular smooth muscle, blood plateletsNeuronal depolarization, head twitch, hyperthermia, modulation of neurotransmitter release smooth muscle contraction, platelet activationTBC-2 (2592)R-96544(1742) Spiperone (0995) MDL 11,939 (0870) 4F 4PP (0523) Risperidone (2865) 5-HT2B^ PLCBrain, stomach fundus (rat), gut, heart, kidney, lungContraction of the stomach fundus, anxietyBW 723C86 (1059)SB 204741 (1372) LY 272015 (3077) 5-HT2C^ PLCChoroid plexus, cortex, limbic system, basal gangliaHypolocomotion, hypophagia, penile erection, hyperthermia, anxiety, v noradrenalin and dopamine releaseMK 212 (0941) Ro 60-0175 (1854) WAY 161503 (1801) CP 809101 (3041) 1-Methylpsilocin (3017)N-Desmethylclozapine (1007) RS 102221 (1050) SB 242084 (2901) 5-HT3Ion channel (Na+, K+, Ca2+)Dorsal vagal complex, hippocampus, amygdala, caudate, cerebral cortex, heart, intestinesAnxiety, cognition, pain , reward/withdrawal, vomiting reflex, vasodilation, intestinal tone and secretionSR 57227 (1205) Quipazine (0629) 1-phenylbiguanide (0969) m-chlorophenylbiguanide (0440)MDL 72222 (0640) Tropisetron (2459) Y-25130 (0380) Ondansetron (2891) Granisetron (2903) 5-HT4^ AC (Gs)Cerebral cortex, limbic areas, hippocampus, colliculus, intestinesLearning and memory, visual perception, anxiety, motor coordination, arousal, smooth muscle relaxation, modulation of neurotransmitter releaseCisapride (1695) RS 67333 (0989)* RS 67506 (0990)* CJ 033466 (3089)*GR 113808 (1322) GR 125487 (1658) RS 39604 (0991) RS 23597-190 (0728) 5-ht5Av AC (Gi/o)Amygdala, hippocampus, caudate nucleus, cerebellum, hypothalamus, thalamus, substantia nigra, spinal cordModulation of exploratory behavior and locomotion–SB 699551 (3188) 5-HT6^ AC (Gs)Striatum, olfactory tubercles, nucleus accumbens, hippocampus, stomach, adrenal glandsMemory and learning, modulation of neurotransmitter release5-Methyl-5-hydroxytryptamine (0558) EMD 386088 (2382)SB 258585 (1961) Ro 47-1816/001 (2911) SB 399885 (3189) NPS ALX Compound 4a (3285) 5-HT7^ AC (Gs)Thalamus, hypothalamus, hippocampus, cerebral cortex, amygdala, GI and vascular smooth muscle, heartCircadian rhythms, smooth muscle relaxation, nociception, hypotension, modulation of REM sleep, learning and memory, LH releaseAS 19 (1968) LP 44 (2534) LP 12 (2925)Pimozide (0937) SB 269970 (1612) SB 259719 (2726)Figure 2: A table summarising the properties of 5-HT receptors and subtypes
(http://www.tocris.com/pharmacologicalBrowser.php?ItemId=5101)
1.3 5-HT4 receptor subtype
The receptor subtype that will be the focus of attention in this study is the 5-HT4 receptor subtype. These receptors are located primarily in the nigrostriatal and mesolimbic systems and smooth muscle in the gastrointestinal tract, and play a role in gastrointestinal motility (Craig & Clark, 1989), as well as in anxiety, visual perception, memory and learning.
The 5-HT4 receptors on intrinsic primary and afferent neurones, are activated by endogenous serotonin released from enterchromaffin cells, in response to mechanical or chemical stimuli. These neurons release transmitters such as calcitonin gene-related peptide (CGRP), activating interneurons which in turn stimulate excitatory neurons on the orad side of the mucosal stimulus and stimulate inhibitory neurons on the caudad side (Ji et al, 2004). Subsequently, this results in peristaltic reflexes occurring at the site of the originating stimuli. The effect of 5-HT4 receptor modulated peristalsis has been found in guinea pig ileum (Tuladhar, 1994; Tuladhar et al, 1995). Also, stimulation of 5-HT4 receptors have been reported to enhance the peristaltic reflex in the rat distal colon (Kadowaki et al, 2002).
5-HT4 receptor agonists, such as tegaserod and 5-HT, stimulate gastrointestinal motility and secretion through release of acetylcholine from excitatory neurones. It is important to note that 5-HT4 agonists strengthen, rather than directly activate the peristaltic reflexes
1.45-HT4 receptor agonists and antagonists
The 5-HT4 receptor agonists that will be focus of this study will be tegaserod and 5-hydroxytryptamine (as mentioned above), and antagonists will be GR113808 and SB204070.
Tegaserod [3-(5-methoxy-1H-indol-3-ylmethylene)- N-pentyl-carbazimidamide] hydrogen maleate, is a partial 5-HT4 agonist that has been implicated in gastro-intestinal motility. In the guinea pig ileum, tegaserod was found to stimulate peristalsis by increasing the number of circular muscle contractions (Ji et al, 2004). It has been used in the treatment of symptoms of irritable bowel syndrome, including abdominal pain, bloating and constipation (Muller-Lissner et al, 2001).
The responses mediated by 5-HT4 receptors have been greatly facilitated by a number of highly selective antagonists e.g. GR113808, SB204070
SB204070 (1-Butyl-4-piperidinyl)methyl-8-amino-7-chloro-1,4-benzodioxane-5-carboxylate hydrochloride) is a selective 5-HT4 serotonin receptor antagonist. In the guinea pig distal colon, SB204070 was found to antagonize 5-HT4 receptor mediated-contractions Although the nature of the antagonism is quite complex, it has been suggested that SB204070 acts has a pseudo-irreversible antagonist (Wardle et al, 1994).
GR113808 (1-methyl-1H-indole-3-carboxylic acid, [1-[2-[(methylsulfonyl)amino]ethyl]
-4-piperidinyl]methyl ester) is a potent, selective 5-HT4 receptor antagonist. In the guinea-pig ascending colon, GR113808 behaved as an antagonist of 5-hydroxytryptamine -induced contraction, with a high affinity for the 5-HT4 receptor (Gale et al, 1994).
1.3Aims of the study
The aims of our investigation were to investigate whether 5-HT4 receptor ligands were able to modulate the peristaltic reflex within the mouse proximal colon. This study also allowed us to investigate the effect of pharmacological manipulations that have been designed to study the role of 5-hydroxytryptamine on the peristaltic reflex within this region of the intestine. This study was undertaken using a range of 5-HT4 receptor agonists and antagonists.
METHOD2.1 Krebs solution
The Krebs-Heinslet solution was prepared at the start of the experiment. To prepare one litre of the solution, 2.1g of sodium hydrogen carbonate (NaHCO3) and 2g of glucose were dissolved in 300ml of dissolved water. 40ml of the Krebs-Heinslet concentration was added to the solution, and the preparation was made up to 1 litre using distilled water. It was found that 5 litres was an adequate volume for the experiment, therefore this was prepared by multiplying each quantity by 5. The marriotte bottle containing the Krebs-Heinslet solution was attached to the apparatus, and used to wash out each organ bath three times, and then added to the required level.
2.2 Preparation of the tissue
The experiment was carried out using MF1 mice. The animals were killed by cervical dislocation, and the GI tract was removed. Segments of the proximal colon (approx. 2-3cm) were carefully dissected on a polystyrene board, taking care not to puncture the colon and to disturb it as little as possible. This section was then quickly transferred to the water jacketed glass bath, which contained Krebs-Heinslet solution aerated with 95% oxygen and 5% CO2, and maintained at 37OC. This was to prevent hypoxia of the tissue and abnormal temperature. All tissues were equilibrated for at least 20 minutes prior to the start of the experiment.
The oral end of the proximal colon was cannulated to the inflow glass tube, which was connected to the reservoir containing saline solution, and secured with thread. The intraluminal contents of the colon were allowed to expel naturally via peristalsis, brought about by the raising of the height of the reservoir by 4cm. After the contents had been expelled, the reservoir was lowered and the aboral end was then cannulated to the opposing outflow glass tube. The tissue was then left to equilibrate for at least 20 minutes prior to the start of the experiment. Finally, to induce peristalsis, the intraluminal pressure was raised, by raising the reservoir by 4cm for at least 15 minutes, until peristalsis became regular and the drugs could be administered; the height of the reservoir needed to achieve steady peristalsis was determined in preliminary experiments.
2.3 Experimental preparation
The outflow tube was connected, via a plastic tube, to a T glass tube, which was open to the atmosphere. Changes to the volume of fluid driven into this vertical tube during peristalsis were measured as a pressure changes, and recorded using pressure transducers connected to a quad bridge amplifier and Power Lab system using Chart v4.1.2 software for Windows. Before the proximal colon was cannulated, the computer software was calibrated to zero, and set to commence recording.
Figure 3: A schematic diagram representing apparatus used to study peristalsis in mouse proximal colon.
The peristalsis trace on the power lab software was recorded as a series of peaks and troughs. During peristalsis the peaks were formed when the tissue contracted, and the troughs formed when the tissue relaxed. This cycle was repeated with each peristaltic stroke.
2.4 Administration of drugs
After regular peristalsis had been established, the drugs could be administered serosally. A cumulative response curve for the agonist tegaserod was obtained by adding increasing concentrations of tegaserod (0.01µM– 10µM). In the preliminary experiments, it was found that tegaserod 10-2M did not allow the tissue to exhibit peristalsis sufficiently, and was too potent, therefore the highest concentration used was 10-3M. Each concentration had a 15 minute contact time with the tissue before the next concentration was administered. The volume ejected and the rate of peristalsis was measured and recorded. Changes to the rate of peristalsis were then compared to the control values obtained 15 minutes prior to administering the first drug. In the preliminary experiments, it was found that washing the tissue between each drug administration had a negative effect on peristalsis, and subsequently the tissue didn’t recover. Therefore drugs were administered continuously without washout. This process was repeated with the agonist, 5-HT, with concentrations ranging from 0.1µM-100µM.
To examine the effects of GR113808 and SB204070 (antagonists) on 5-HT and tegaserod responses, either antagonist was added to the tissues after regular peristalsis was obtained and allowed to equilibrate for 15 minutes, after which either 5-HT or tegaserod were added cumulatively and their effects on peristalsis were measured as described above
2.5 Statistical analysis
All results are expressed as mean±SEM from number of animals indicated by n. The difference between the values was determined by using the unpaired t test when two groups were compared and using the one way ANOVA followed by Fisher’s PLSD when more than two groups were compared.
2.6Consideration of safety issues
The chemicals used within this investigation were obtained from Tocris bioscience. To ensure the safety of all members of the group throughout the duration of the investigation, a chemical risk assessment form (COSHH) was formulated and signed by all members. This form highlighted all the chemicals that were to be used throughout the experiment, and the risks and precautions associated with each of them. It was ensured that the precautions were adhered to at all stages of the experiment, and general laboratory regulations were also put into place i.e. no eating or drinking in labs, wearing a lab coat etc.
RESULTS
The peristaltic reflex was investigated within the mouse proximal colon. Segments of the proximal colon (approximately 3cm in length) were cannulated in vitro, and regular peristalsis was achieved by raising of the intraluminal pressure. Peristalsis was distinguished as circular muscle contractions arising from the oral side and travelling to the anal side. The proximal part of the colon was distinguished from the distal part by striations across the surface, and also by it containing softer faecal pellets
3.1 The effects of 5-HT on the peristaltic reflex in the mouse proximal colon
A regular peristalsis was established before the addition of 5-HT (average rate of peristalsis was 77±7, n=7). The fluid was ejected from the oral to anal direction. The addition of 5-HT 10-7M caused a significant increase in the rate of peristalsis. At this concentration the rate of peristalsis was 101 ± 8, n=7, which was a substantial increase from the control value. Further cumulative addition of 5-HT caused a slow decrease in peristalsis until at 10-4M, rate of peristalsis, at 56 ± 19, n=7 , was lower than the control at the beginning. In all tissues in which peristalsis was inhibited, it recovered once tissue was washed.
3.2 The effects of the 5-HT4 receptor antagonist, SB204070, on the cumulative addition of 5-HT on the peristaltic reflex in the mouse proximal colon
3.3 The effects of tegaserod on the peristaltic reflex in the mouse proximal colon
3.4The effects of the 5-HT4 receptor antagonist, GR113808, on the cumulative addition Tegaserod on the peristaltic reflex in the mouse proximal colon
DISCUSSION
Gastrointestinal disorders within humans and animals have become much more common, and as such more effective treatments need to be formulated. Research has implicated 5-hydroxytryptamine within these disorders, and thus the 5-HT receptors, which are involved in gastrointestinal motility, are potential targets for treating such disorders.
The present investigation was designed to study the peristaltic reflex within the mouse proximal colon, and further, to investigate the 5-HT4 receptor, which has been found to modulate peristalsis in the guinea pig ileum (Tuladhar et al., 1995). Peristalsis is the principle mechanism controlling the movement of chyme within the intestine, and takes place without the conscious control. The nervous pathway of the peristaltic reflex is entirely intrinsic (Bulbring et al,1958), and therefore we can obtain this reflex even in isolated tissues. The method used to study peristalsis was similar to the one used by Trendelengburg (1917), in which the peristaltic reflex was triggered by raising of the intraluminal pressure, allowing the measurement of different parameters of peristalsis, including the rate of peristaltic stokes and the volume of intraluminal fluid ejected to the anal side with each peristaltic stroke.
Within the present study, regular peristalsis was obtained so the effects of the 5-HT4 receptor ligands could be established. Craig and Clarke (1991) suggested that the 5-HT4 receptors had a facilitatory effect on 5-HT in the guinea-pig ileum and this was further confirmed by Tuladhar et al (1993). However, this finding was not reciprocated within the mouse proximal colon.
Cumulative addition of 5-HT produced facilitation of peristalsis within the mouse proximal colon, characterised by an increase in the number of peristaltic strokes per hour and thus the rate of peristalsis at 10-7M.. This facilitation was observed at concentrations up until10 -4M, where inhibition of peristalsis was observed. At this concentration, 5-HT desensitised the tissues so no further peristalsis could be established. However, peristalsis was re-established in 6 out of the 7 tissues after washing of the tissues. This facilitatory and inhibitory effect of 5-HT was also observed in various studies carried out by Tuladhar et al, Bulbring & Crema (1958) and others. Therefore, this study has shown that the addition of 5-HT in vitro can modulate peristalsis within the mouse proximal colon. At lower concentrations, 5-HT facilitates peristalsis, whereas at higher concentrations 5-HT can both facilitate and inhibit peristalsis.
In the present study both GR113808 and SB204070 failed to affect the facilitatory effect of 5-HT in the mouse proximal colon. GR113808 and SB204070 are both highly potent 5-HT4 receptor antagonists (Gale et al., 1994; Wardle et al., 1994). This suggests that the 5-HT4 receptor is not implicated within the mouse proximal; had it been implicated both SB204070 and GR113808 would have antagonised the 5-HT4 receptor mediated contractions, and there would have been a significant decrease in the rate of peristalsis. This is in contrast to the findings by Costall et al (1993), where the 5-HT4 receptor was implicated in the guinea pig ileum, in which it exhibited a facilitatory effect on 5-HT.
In the guinea-pig ileum tegaserod has also been shown to facilitate peristalsis ( Ji et al, 2004 ), which was not observed in the current study. This further suggests that the 5-HT4 receptor is not implicated within the mouse proximal colon; as tegaserod is a partial 5-HT4 agonist, had 5-HT4 receptor been implicated, facilitation of peristalsis would have been observed.
The inhibitory effect of 5-HT on peristalsis has been reported to involve the 5-HT7 receptor (Tuladhar et al, 2003). Further studies are required to examine the receptor involved in the inhibitory effect of 5-HT in the mouse colon and to examine whether 5-HT7 receptors are involved. It is interesting to note that the inhibitory effect of tegaserod could involve a completely different mechanism as peristalsis could not be recovered by washing in any tissue, in contrast with 5-HT.
In conclusion, the current study has shown that 5-HT can both facilitate and inhibit peristalsis. However, the 5-HT receptors mediating these effects are likely to be different from the ones involved in the modulation of peristalsis in the guinea-pig ileum. Further studies are required to establish the receptors involved.
REFERENCESARBAB SIKANDERA, S. V. R., A, AND KAUSHAL KISHOR PRASADA (2009).
Role of serotonin in gastrointestinal motility and irritable bowel syndrome. Clinica Chimica Acta, 403, 47-55
BARNES, N. M. & SHARP, T. (1999). A review of central 5-HT receptors and their function. Neuropharmacology, 38, 1083-1152.
BULBRING, E., LIN RC (1958)The effect of intraluminal application of 5-hydroxytryptamine and 5-hydroxytryptophan on peristalsis; the local production of 5-HT and its release in relation to intraluminal pressure and propulsive activity. J Physiol 2006 Aug 15;575(Pt 1):1-2.
COOK, M. J. (1965) The anatomy of the laboratory mouse. London (&New York):
Academic Press.
COSTEDIO, M., HYMAN, N. & MAWE, G. (2007). Serotonin and Its Role in Colonic Function and in Gastrointestinal Disorders. Diseases of the Colon & Rectum, 50, 376-388.
CRAIG, D. A. & CLARKE, D. E. 1990. Pharmacological characterization of a neuronal receptor for 5-hydroxytryptamine in guinea pig ileum with properties similar to the 5-hydroxytryptamine receptor. Journal of Pharmacology and Experimental Therapeutics, 252, 1378-1386.
CROWELL, M. D. 2004. Role of serotonin in the pathophysiology of the irritable bowel syndrome. British journal of pharmacology, 141, 1285-93.
GALE, J. D., GROSSMAN, C. J., WHITEHEAD, J. W., OXFORD, A. W., BUNCE, K. T. & HUMPHREY, P. P. (1994). GR113808: a novel, selective antagonist with high affinity at the 5-HT4 receptor. British journal of pharmacology, 111, 332-8.
GERSHON, M. D., DRAKONTIDES, A. B. & ROSS, L. L. 1965. Serotonin: Synthesis and Release from the Myenteric Plexus of the Mouse Intestine. Science, 149, 197-9.
JI, S. W., PARK, H., CHUNG, J. P., LEE, S. I. & LEE, Y. H. 2004. Effects of tegaserod on ileal peristalsis of guinea pig in vitro. Journal of pharmacological sciences, 94, 144-52.
Kadowaki, M., Wang, X.B., Shimatani, H., Yoneda, S. & Takaki, M. (2002). 5-HT4 receptor enhances the propulsive power of the peristaltic reflex in the rat distal colon. Naunyn-Schmied. Arch. Pharmacol, 99, 62-65.
MARGARET E. SMITH, D. G. M. (2001). The digestive system- Basic science and clinical conditions, Elsevier ltd.
MULLER-LISSNER, S. A. (2001) Tegaserod, a 5-HT(4) receptor partial agonist, relieves
symptoms in irritable bowel syndrome patients with abdominal pain, bloating and
constipation Aliment Pharmacol Ther
ROD R. SEELEY, T. D. S., PHILIPSTATE(2006). Anatomy and physiology. McGraw
Hill Higher Education
SANGER, G. J. (2008). 5-hydroxytryptamine and the gastrointestinal tract: where nextTrends in pharmacological sciences, 29, 465-71.
SETOLA, V. & ROTH, B. L. (2003). Why Mice Are Neither Miniature Humans nor Small Rats: A Cautionary Tale Involving 5-Hydroxytryptamine-6 Serotonin Receptor Species Variants. Molecular Pharmacology, 64, 1277-1278.
SHIINA, T., SHIMIZU, Y., SUZUKI, Y., NIKAMI, H. & TAKEWAKI, T. (2005). Measurement of the propelled liquid by isolated hamster ileum as a parameter to evaluate peristalsis. European Journal of Pharmacology, 517, 120-126.
SIKANDER, A., RANA, S. V. & PRASAD, K. K. (2009). Role of serotonin in gastrointestinal motility and irritable bowel syndrome. Clinica chimica acta; international journal of clinical chemistry, 403, 47-55.
TULADHAR, B.R., COSTALL, B. & NAYLOR, R.J. (1995). Evidence of a 5-Ht3 Receptor-Mediated Facilitation of the Emptying Phase of the Peristaltic Reflex in the Isolated Guinea-Pig Ileum. Br J Pharmacol, 114, P374-P374.
TULADHAR, B.R., COSTALL, B. & NAYLOR, R.J. (2002). Modulation of 5-HT4 receptor function in the rat isolated ileum by fluoxetine: the involvement of endogenous 5-hydroxytryptamine. Br J Pharmacol, 136, 150-6.
TULADHAR B.R (2003) 5-HT7 receptors mediate the inhibitory effect of 5-HT on
peristalsis in the isolated guinea-pig ileum. Br J Pharmacol
WARDLE, K. A., ELLIS, E. S., BAXTER, G. S., KENNETT, G. A., GASTER, L. M. & SANGER, G. J. (1994). The effects of SB 204070, a highly potent and selective 5-HT4
receptor antagonist, on guinea-pig distal colon. British journal of pharmacology,
112, 789-94.
HTTP://WWW.TOCRIS.COM/PHARMACOLOGICALBROWSER.PHP?ITEMID=5115.
APPENDIXTable 1: 5-HT alone
S.No -log C (M) RATE OF PERISTALSIS 1 2 3 4 5 6 7 AVG SEM N 1 Before58.7 56.9 71.6 114.9 73.9 77.1 82.2 77 7 7 2 7.00 126.8 77.6 95.1 115.3 119.4 76.8 95.6 101 8 7 3 6.00 120.4 58.1 77.3 102.7 127.7 92.1 70.2 93 10 7 4 5.00 42.9 26.1 61.9 104.5 93.0 67.9 93.7 70 11 7 5 4.00 0.0 0.0 37.9 105.7 113.7 29.8 105.0 56 19 7Table 1 shows the rate of peristalsis after adding the agonist,5-HT 10-8 – 10-4 M, to mouse proximal colon. The values are expressed as mean±SEM.
Table 2: 5-HT in the presence of SB204070
S.No -log C (M) RATE OF PERISTALSIS 1 2 3 4 5 6 7 AVG SEM N 1 SB-204070 7.00 79.4 129.3 44.1 31.2 101.6 79.2 82.8 78.2 12.4 7 2 5-HT 5.00 149.9 124.6 71.3 30.2 102.7 58.8 95.1 90.4 15.3 7 3 5HT 4.00 113.8 132.2 19.7 27.9 96.3 51.2 130.3 81.6 18.1 7 4 5HT 3.00 90.8 114.9 23.0 25.9 99.2 77.2 0.0 61.6 16.8 7 5 5HT 2.00 0.0 122.1 0.0 0.0 0.0 55.8 0.0 25.4 17.9 7Table 2 shows the rate of peristalsis after adding 5-HT 10-5 – 10-2 in the presence of the 5-HT4 antagonist, SB204070 10-7M, to the mouse proximal colon. The values are expressed as mean±SEM.
Table 3: Tegaserod alone
S.No -logC (M) RATE OF PERISTALSIS 1 2 3 4 5 AVG SEM N 1 Before 42.6 45.6 151.3 45.6 116.6 80.322.5 5 2 8.00 40.9 40.9 127.3 41.3 123.0 74.7 20.6 5 3 7.00 38.7 35.2 107.4 32.6 0.0 42.8 17.5 5 4 6.00 31.1 38.1 97.9 27.9 123.3 63.7 19.6 5 5 5.00 0.0 0.00 0.00 0.0 45.4 9.0 9.0 5
Table 3 shows the rate of peristalsis after adding the agonist, tegaserod 10-8 – 10-5 M, to mouse proximal colon. The values are expressed as mean±SEM.
Table 4: Tegaserod in the presence of SB204070
S.No -logC (M) RATE OF PERISTALSIS 1 2 3 4 5 AVG SEM N 1 BEFORE 99.4 107.5 121.2 124.3 105.4 111.5 4.7 5 2 SB-204070 7.00 64.5 98.1 65.1 59.8 53.6 68.2 7.7 5 3 Tegaserod 8.00 100.1 105.8 91.3 110.8 54.3 92.5 10.0 5 4 Tegaserod 7.00 107.3 122.2 74.5 72.5 59.7 87.3 11.7 5 5 Tegaserod 6.00 111.4 113.4 99.2 90.5 83.799.6 5.7 5 6 Tegaserod 5.00 0.00.0 104.9 213.4 75.7 78.8 39.5 5Table 4 shows the rate of peristalsis after adding tegaserod 10-8 – 10-5 in the presence of the 5-HT4 antagonist, SB204070 10-7M, to the mouse proximal colon. The values are expressed as mean±SEM.
Table 5: Tegaserod in the presence of GR113808
S.No -logC (M) RATE OF PERISTALSIS 1 2 3 4 5 6 7 AVG SEM N 1 BEFORE 33.0 108.2 129.9 50.7 31.9 34.1 35.0 64.8 17.6 7 2 GR113808 6.00 15.6 82.6 121.6 47.6 25.2 18.4 19.6 52.0 17.2 7 3 Tegaserod 8.00 37.8 267.2 105.9 48.0 22.2 26.12 21.5 75.5 33.8 7 4 Tegaserod 7.00 71.3 339.7 112.2 49.7 17.4 24.00 9.2 89.0 43.9 7 5 Tegaserod 6.00 74.4 277.4 49.0 37.2 19.9 14.53 0.0 67.4 36.2 7 6 Tegaserod 5.00 20.1 0.0 0.0 30.7 -1.5 10.02 0.0 8.4 4.7 7
Table 5 shows the rate of peristalsis after adding tegaserod 10-8 – 10-5 in the presence of the 5-HT4 antagonist, GR11380810-6M, to the mouse proximal colon. The values are expressed as mean±SEM.
