Effects of neo-adjuvant chemotherapy for oesophago-gastric cancer on neuro-muscular gastric function.

Delayed gastric emptying symptoms are often reported after chemotherapy. This study aims to characterise the effects of chemotherapy on gastric neuro-muscular function. Patients undergoing elective surgery for oesophago-gastric cancer were recruited. Acetylcholinesterase, nNOS, ghrelin receptor and motilin expressions were studied in gastric sections from patients receiving no chemotherapy (n = 3) or oesophageal (n = 2) or gastric (n = 2) chemotherapy. A scoring system quantified staining intensity (0-3; no staining to strong). Stomach sections were separately suspended in tissue baths for electrical field stimulation (EFS) and exposure to erythromycin or carbachol; three patients had no chemotherapy; four completed cisplatin-based chemotherapy within 6 weeks prior to surgery. AChE expression was markedly decreased after chemotherapy (scores 2.3 ± 0.7, 0.5 ± 0.2 and 0 ± 0 in non-chemotherapy, oesophageal- and gastric-chemotherapy groups (p < 0.03 each) respectively. Ghrelin receptor and motilin expression tended to increase (ghrelin: 0.7 ± 0.4 vs 2.0 ± 0.4 and 1.2 ± 0.2 respectively; p = 0.04 and p = 0.2; motilin: 0.7 ± 0.5 vs 2.2 ± 0.5 and 2.0 ± 0.7; p = 0.06 and p = 0.16). Maximal contraction to carbachol was 3.7 ± 0.7 g and 1.9 ± 0.8 g (longitudinal muscle) and 3.4 ± 0.4 g and 1.6 ± 0.6 (circular) in non-chemotherapy and chemotherapy tissues respectively (p < 0.05 each). There were loss of AChE and reduction in contractility to carbachol. The tendency for ghrelin receptors to increase suggests an attempt to upregulate compensating systems. Our study offers a mechanism by which chemotherapy markedly alters neuro-muscular gastric function.

Activation of prostaglandin EP receptors by lubiprostone in rat and human stomach and colon.

BACKGROUND AND PURPOSE: Lubiprostone (Amitiza), a possible ClC-2 channel opener derived from prostaglandin E(1) and indicated for the treatment of constipation, increases chloride ion transport and fluid secretion into the intestinal lumen. As lubiprostone may also directly modulate gastrointestinal motility, we investigated its actions and the possible involvement of prostaglandin EP receptor activation on rat and human isolated gastrointestinal preparations. EXPERIMENTAL APPROACH: Rat and human isolated preparations were mounted in tissue baths for isometric recording. The effects of lubiprostone on muscle tension and on electrically stimulated, neuronal contractions were investigated in the absence and presence of EP receptor antagonists. KEY RESULTS: In rat and human stomach longitudinal muscle, lubiprostone induced a contraction (pEC(50) of 7.0+/-0.0, n=4 and 6.4+/-0.2, n=3, respectively), which was inhibited by pretreatment with the EP(1) receptor antagonist, EP(1)A 300 nM (pEC(50) reduced to 6.2+/-0.2, n=6), but not by the EP(3) or EP(4) receptor antagonists (L-798106 and GW627368X, respectively, 1 microM, P>0.05). Lubiprostone also reduced electrically stimulated, neuronal contractions in rat and human colon circular muscle preparations (pIC(50) of 8.9+/-0.4, n=7 and 8.7+/-0.9, n=6, respectively), an effect mediated pre-junctionally. This effect was reduced by the EP(4) receptor antagonist (pIC(50) of 6.7+/-1.1, n=7 and 7.7+/-0.4, n=6, respectively) but not by EP(1) or EP(3) receptor antagonists. CONCLUSIONS AND IMPLICATIONS: In rats and humans, lubiprostone contracts stomach longitudinal muscle and inhibits neuronally mediated contractions of colon circular muscle. Experiments are now needed to determine if this additional activity of lubiprostone contributes to its clinical efficacy and/or side-effect profile.

Evidence for the involvement of a 5-HT4 receptor in the secretory response of human small intestine to 5-HT.

5-Hydroxytryptamine increases transmucosal short-circuit current across human isolated small intestinal mucosa. The competitive 5-HT4 antagonist, DAU 6285 evoked a concentration-dependent, dextral and parallel shift of the concentration-response curve to 5-HT, with no alteration of the maximum response. Schild analysis of this antagonism produced a Schild regression with a slope of 1.00 and an apparent pA2 estimate of 6.17. It appears that a 5-HT4 receptor may mediate the short-circuit current response of human small intestinal mucosa to 5-HT.

Functional evidence for a 5-HT2B receptor mediating contraction of longitudinal muscle in human small intestine.

Application of 5-hydroxytryptamine induces contraction of longitudinal muscle strips from human terminal ileum. The response was resistant to antagonism by ketanserin, ondansetron or DAU6285, but was non-surmountably antagonized by methysergide. The selective 5-HT2B/2C receptor antagonist, SB 200646A evoked a concentration-dependent, parallel and dextral displacement of the concentration-response curve to 5-HT, yielding a pA2 estimate of 7.17. Application of yohimbine, a 5-HT1 and 5-HT2B receptor antagonist, also induced a rightward shift of the response curve to 5-HT, yielding a pA2 estimate of 8.10. In conclusion, it appears that a 5-HT2B receptor mediates the contractile response of the longitudinal muscle of human small intestine to 5-HT.

Investigation of the effects of platelet-activating factor (PAF) on ion transport and prostaglandin synthesis in human colonic mucosa in vitro.

1 We have investigated the effects of platelet-activating factor (PAF), an endogenous mediator of inflammation, on ion transport and prostaglandin synthesis in the human isolated colon. 2 Application of PAF to the serosal surface of human colonic mucosa induced a marked, concentration-dependent increase in ion transport. Mucosal application was without effect. 3 The secretory response to PAF was significantly inhibited by prior application of a specific PAF receptor antagonist WEB 2170, indicating that the response is dependent on PAF receptor activation. 4 The response to PAF was attenuated by prior application of indomethacin or piroxicam, implicating products of the cyclo-oxygenase pathway in the response. 5 The response to PAF was attenuated by the loop diuretic bumetanide, indicating an involvement of chloride ion secretion in the response. 6 Addition of PAF to the serosal surface induced a significant increase in serosal prostaglandin E2 (PGE2), but not 6-oxo-PGF1alpha release. There was no effect on mucosal application of PAF. 7 In summary, we have shown that PAF is a potent secretagogue in isolated preparations of human colon and that the response is dependent on a specific PAF receptor, cyclo-oxygenase products and bumetanide-sensitive chloride ion transport.

5-HT(2B) receptors play a key role in mediating the excitatory effects of 5-HT in human colon in vitro.

1. 5-Hydroxytryptamine (5-HT) is known to produce a number of different effects in the gastrointestinal tract of various species, and has been proposed to play a key role in a number of intestinal disorders in man, including irritable bowel syndrome (IBS), although the receptors involved have yet to be established. The aim of the present study was to investigate the distribution and function of 5-HT(2B) receptors in human colon, and to establish their possible role in the aetiology of IBS. 2. The distribution of 5-HT(2B) receptor mRNA and protein were investigated by quantitative RT - PCR, Western analysis and immunocytochemistry. High levels of both mRNA and protein for 5-HT(2B) receptors were found throughout the human gastrointestinal tract, and in particular in colon, where 5-HT(2B) receptors were found predominantly in the longitudinal and circular smooth muscle layers within the muscularis externa, and in the myenteric nerve plexus lying between these two layers. 3. Electrical field stimulation of longitudinal muscle preparations of human colon mounted in organ baths resulted in neuronally-mediated contractile responses, that were significantly potentiated by application of 5-HT (up to 10(-7) M), with a pEC(50) of 8.2 +/- 0.1 (n=49 donors). The response to 5-HT was inhibited by a number of selective 5-HT(2B) receptor antagonists. 4. This study has shown for the first time that, in contrast to animal studies, the excitatory effects of 5-HT in human colon are mediated by 5-HT(2B) receptors. It is proposed that these receptors contribute to the putative 5-HT-induced colonic smooth muscle hypersensitivity associated with IBS.

5-HT1D and 5-HT2B receptors mediate contraction of smooth muscle in human small intestine.

In conclusion, 5-HT has been shown to contract both circular and longitudinal muscle layers of human terminal ileum. By the use of selective antagonists, the receptors mediating these actions have been identified. In the circular muscle of human small intestine, 5-HT-induced contraction is mediated via a receptor of the 5-HT1D sub-type, whereas a receptor of the 5-HT2B sub-type mediates the contractile response to 5-HT of longitudinal muscle layers.

Heterogeneity of 5-HT receptors mediating secretion in the human intestine.

In conclusion, application of 5-HT has been shown to induce fluid secretion in both the small and large intestine of man. By the use of selective antagonists, the receptors mediating these effects have been identified and characterized. 5-HT induces secretion across human ileal mucosa via a receptor of the 5-HT4 sub-type, whereas a receptor of the 5-HT2A sub-type appears to mediate the effect in human sigmoid colon. The response in human ascending colonic mucosa cannot be definitively classified at this time, but may reflect the presence of a heterogenous receptor population.

The effect of atrial natriuretic peptides on ion transport by muscle-stripped and full-thickness preparations of rat colon.

The effects of three atrial natriuretic peptides (ANPs) have been tested for their ability to increase ion transport in muscle-stripped and intact preparations of rat colon. Anaritide (fragment 4-28 of human ANP), human ANP and rat atriopeptin III had no effect on short-circuit current in muscle-stripped preparations. Such insensitivity could not be explained by desensitisation, enzymic destruction or non-specific absorption. Short-circuit current was increased by 8-Br-cGMP and NaNP. The pharmacological activity of anaritide was demonstrated by abolition of phenylephrine-induced tone in rat aortic smooth muscle. In contrast to muscle-stripped colon, intact preparations responded with a tetrodotoxin-sensitive increase in short-circuit current to anaritide even though such preparations were less sensitive to acetylcholine and substance P. These results imply that selective neural damage or the lack of certain nerve pathways, caused by complete muscle stripping, will prevent the pro-secretory effects of anaritide in rat colon.

Short-circuit current responses to 5-hydroxytryptamine in human ileal mucosa are mediated by a 5-HT4 receptor.

5-Hydroxytryptamine (5-HT) increases short-circuit current when added to the serosal side of human isolated ileal mucosa; mucosally applied 5-HT was ineffective. Tetrodotoxin reduced both basal short-circuit current and increases in short-circuit current due to electrical field stimulation of mucosal nerves. However, neither tetrodotoxin, ondansetron nor methysergide plus ketanserin affected 5-HT induced increases in short-circuit current. Application of SDZ 205-557 (2-diethylaminoethyl-(2-methoxy-4-amino-5-chloro) benzoate) to the tissue caused a significant increase in the concentration ratio between two successive 5-HT response curves. It is concluded that the effect of 5-HT on short-circuit current of human ileal mucosa appears to be due to stimulation of a 5-HT4 receptor.

Human colonic mucosa possesses a mixed population of 5-HT receptors.

The aim of this study was to characterise the 5-HT receptor(s) mediating secretory responses of isolated human colonic mucosa to 5-HT. Sheets of muscle-stripped mucosa from proximal (ascending) and distal (sigmoid) human colon were set up in Ussing chambers for measurement of short-circuit current (Isc). Serosal application of 5-HT led to non-neural, concentration-dependent increases in Isc. Desensitisation to 5-HT was observed with ascending and sigmoid colonic mucosa. Using selective 5-HT antagonists we have shown that 5-HT induces secretion in sigmoid colon via a 5-HT2A receptor. In ascending colon a combination of 5-HT2A and 5-HT4 receptors appears to be involved.

Characterization of the EP receptor types that mediate longitudinal smooth muscle contraction of human colon, mouse colon and mouse ileum.

BACKGROUND: Prostaglandin E(2) (PGE(2) ) is an inflammatory mediator implicated in several gastrointestinal pathologies that affect normal intestinal transit. The aim was to establish the contribution of the four EP receptor types (EP(1-4) ), in human colon, that mediate PGE(2) -induced longitudinal smooth muscle contraction. METHODS: Changes in isometric muscle tension of human colon, mouse colon and mouse ileum were measured in organ baths in response to receptor-specific agonists and antagonists. In addition, lidocaine was used to block neurogenic activity to investigate whether EP receptors were pre- or post-junctional. KEY RESULTS: PGE(2) contracted longitudinal muscle from human and mouse colon and mouse ileum. These contractions were inhibited by the EP(1) receptor antagonist, EP(1) A in human colon, whereas a combination of EP(1) A and the EP(3) antagonist, L798106 inhibited agonist responses in both mouse preparations. The EP(3) agonist, sulprostone also increased muscle tension in both mouse tissues, and these responses were inhibited by lidocaine in the colon but not in the ileum. Although PGE(2) consistently contracted all three muscle preparations, butaprost decreased tension by activating smooth muscle EP(2) receptors in both colonic tissues. Alternatively, in mouse ileum, butaprost responses were lidocaine-sensitive, suggesting that it was activating prejunctional EP(2) receptors on inhibitory motor neurons. Conversely, EP(4) receptors were not functional in all the intestinal muscle preparations tested. CONCLUSIONS & INFERENCES: PGE(2) -induced contraction of longitudinal smooth muscle is mediated by EP(1) receptors in human colon and by a combination of EP(1) and EP(3) receptors in mouse intestine, whereas EP(2) receptors modulate relaxation in all three preparations.

GSK962040: a small molecule, selective motilin receptor agonist, effective as a stimulant of human and rabbit gastrointestinal motility.

There is an urgent clinical need for a safe, efficacious stimulant of gastric emptying; current therapies include erythromycin (an antibiotic with additional properties which preclude chronic use) and metoclopramide (a 5-hydroxytryptamine type 4 receptor agonist and an antagonist at brain D2 receptors, associated with movement disorders). To move away from the complex motilide structure of erythromycin, a small molecule motilin receptor agonist, GSK962040, was identified and characterized. The compound was evaluated using recombinant human receptors, rabbit and human isolated stomach preparations known to respond to motilin and in vivo, by measuring its ability to increase defecation in conscious rabbits. At the human motilin receptor, the pEC50 (the negative logarithm to base 10 of the EC50 value, the concentration of agonist that produces 50% of the maximal response) values for GSK962040 and erythromycin as agonists were, respectively, 7.9 and 7.3; GSK962040 had no significant activity at a range of other receptors (including ghrelin), ion channels and enzymes. In rabbit gastric antrum, GSK962040 300 nmol L(-1)-10 micromol L(-1) caused a prolonged facilitation of the amplitude of cholinergically mediated contractions, to a maximum of 248 +/- 47% at 3 micromol L(-1). In human-isolated stomach, GSK962040 10 micromol L(-1), erythromycin 10 micromol L(-1) and [Nle13]-motilin 100 nmol L(-1), each caused muscle contraction of similar amplitude. In conscious rabbits, intravenous doses of 5 mg kg(-1) GSK962040 or 10 mg kg(-1) erythromycin significantly increased faecal output over a 2-h period. Together, these data show that GSK962040, a non-motilide structure, selectively activates the motilin receptor. Simplification of the structural requirements to activate this receptor greatly facilitates the design of potentially new medicines for gastroparesis.

Evidence for a nonneural electrogenic effect of cholera toxin on human isolated ileal mucosa.

Cholera toxin-induced intestinal secretion in intact rats requires a functioning myenteric plexus. The aim of this investigation was to determine whether neural elements were essential for cholera toxin to produce a secretory effect in human isolated ileum. Mucosal preparations were mounted in Ussing chambers. Cholera toxin was applied apically and short-circuit current monitored for 3 hr, at which point forskolin was given. Cholera toxin (10 microg/ml) induced a tetrodotoxin-insensitive increase in short-circuit current in muscle-stripped preparations of human ileum. The increase was not additive with the action of forskolin (25 microM). Cholera toxin exerts a marked nonneural secretory effect in human ileal mucosa in vitro, probably by the same mechanism as forskolin, namely elevation of cyclic AMP.