Ursodeoxycholic acid action on the transport function of the small intestine in normal and cystic fibrosis mice.

Ursodeoxycholic acid possesses choleretic and cytoprotective properties and in cystic fibrosis (CF) it is used to treat the hepatobiliary symptoms of the disease. This study investigated the effects of this bile acid on the transport function of the small intestine in normal and CF mice. The effects of ursodeoxycholic acid were monitored as changes in short-circuit current (SCC) in stripped sheets of small intestine from normal (Swiss MF1) and transgenic CF (Cftr(tm2Cam)) mice. In ileal sheets from Swiss MF1 mice, mucosal ursodeoxycholic acid caused a biphasic increase in SCC. The first phase was reduced by lowering the mucosal Na+ concentration, while the second phase was inhibited by (Cl-)-free conditions, serosal furosemide or mucosal diphenylamine-2-carboxylic acid (DPC), suggesting an initial Na+-dependent bile acid absorption followed by a stimulation of electrogenic Cl- secretion. Serosal application of ursodeoxycholic acid to the ileum and mucosal or serosal application to the mid-intestine and jejunum elicited a secretory response only. Secretion was Ca2+-dependent, but did not involve neural mechanisms. Mucosal mast cells, histamine and serotonin (5-HT) were implicated in the secretory response. Responses in tissues from transgenic wild-type mice were similar to those obtained with Swiss MF1 mice, but the secretory response to mucosal or serosal application of the bile acid was impaired in CF tissues. In ilea from CF mice the initial absorptive phase of the response to mucosal ursodeoxycholic acid was still observed. It is concluded that ursodeoxycholic acid induces secretion throughout the murine small intestine by a mechanism that involves degranulation of mucosal mast cells. In the ileum Na+-dependent absorption can also be detected. The secretory response is defective in CF intestine, but the absorptive effect is still present.

Taurocholic acid-induced secretion in normal and cystic fibrosis mouse ileum.

Bile acids cause secretion throughout the intestinal tract and this process contributes to maintaining the fluidity of intestinal contents. In cystic fibrosis (CF) defective intestinal secretion can lead to excessive dehydration of the luminal contents and the development of clinical symptoms. This study was designed to investigate bile acid-induced secretion in mouse ileum and to determine whether this process was defective in CF. Taurocholic acid-induced secretion was monitored as a rise in short-circuit current (SCC) in ileal sheets from normal (Swiss MF1) and transgenic CF mice. Taurocholic acid increased the SCC in both intact and stripped ileal sheets from Swiss MF1 mice. This effect was due to a stimulation of electrogenic Cl- secretion as it was inhibited by Cl(-)-free conditions, serosal furosemide (frusemide), mucosal diphenylamine-2-carboxylic acid (DPC) and increased serosal K+ concentration, without being affected by reduced mucosal Na+ concentration. Taurocholic acid-induced secretion was inhibited by tetrodotoxin, indicating the involvement of a neural pathway, but this did not include capsaicin-sensitive afferent neurons or muscarinic cholinoreceptors. Mucosal mast cells also contributed to the response. Responses in tissues from transgenic wild-type mice were similar to those obtained with Swiss MF1 animals, but ilea from CF mice exhibited a lower basal SCC with significantly reduced secretory responses to acetylcholine and taurocholic acid. We concluded that taurocholic acid induces ileal secretion by a mechanism that entails activation of enteric nerves and degranulation of mucosal mast cells. Impaired bile acid-induced secretion in CF may contribute to luminal dehydration.

Acetylcholine induces cytosolic Ca2+ mobilization in isolated distal colonic crypts from normal and cystic fibrosis mice.

In intestinal biopsies from cystic fibrosis (CF) patients acetylcholine fails to elicit a chloride secretion response, and this observation can be explained by a defect in the Ca2+ signalling pathway in CF secretory cells. We tested the hypothesis that in CF intestine, the generation of an intracellular Ca2+ signal upon cholinergic stimulation is absent. A transgenic CF mouse model was used. Electrical measurements on intact jejunum and unstripped colon were performed in Ussing chambers. Intact distal colonic crypts were isolated, and the intracellular Ca2+ concentration was monitored using the Ca2+-sensitive dye fura-2. Acetylcholine increased the short-circuit current generated by wild-type jejunum and colon, but failed to induce a response in CF tissues. Acetylcholine caused a transient elevation in the intracellular Ca2+ concentration in colonic crypts from both wild-type and CF mice; the amplitude and timing of the response in CF crypts was indistinguishable from that in wild-type crypts. The response to acetylcholine was also observed in the absence of extracellular calcium, indicating intracellular stores as the source from which the cytosolic Ca2+ concentration increased. We conclude that the absence of a cholinergically-induced secretory response in CF intestine is not due to a defect in the generation of a Ca2+ signal in intestinal cells upon cholinergic stimulation.

Effect of loperamide on Na+/D-glucose cotransporter activity in mouse small intestine.

The mu-opioid agonist loperamide is an antidiarrhoeal drug which inhibits intestinal motility and secretion. Its anti-absorptive effects are less well investigated, but may be mediated through calmodulin. We have investigated further the effect of loperamide on the intestinal Na+-dependent D-glucose transporter (SGLT1). Brush-border membrane vesicles were prepared from mouse small intestine, and uptake of [3H]glucose was measured. Na+-dependent glucose uptake displayed the typical overshoot at 34 s; the peak value was 1.6 nmol mg(-1). The overshoot disappeared in the presence of phlorizin or when Na+ was replaced by K+. Extravesicular loperamide dose-dependently inhibited SGLT1 activity with an IC50 value of 450 micromol L(-1). Loperamide displayed a mixed inhibition type: the apparent Vmax decreased from 0.9 to 0.5 nmol mg(-1)/15 s, the apparent Km increased from 0.23 to 1.13 mmol L(-1) glucose. Na+ kinetics were more complex, but loperamide inhibited net glucose uptake by 90% at 100 mmol L(-1) Na+. Glucose uptake was unchanged by agents affecting calmodulin activity. Loperamide inhibited intestinal Na+, K+-ATPase activity, whilst sucrase activity was unaffected. SGLT1 activity was inhibited by loperamide, but this effect was not mediated through calmodulin. As this action is only evident at high concentrations of loperamide a nonspecific mechanism may be involved.

The action of 5-hydroxytryptamine on normal and cystic fibrosis mouse colon: effects on secretion and intracellular calcium.

The ability of mouse colon to generate a secretory response to stimulation by 5-hydroxytryptamine (5-HT) was investigated in intact colonic sheets mounted in Ussing chambers. A preparation of intact isolated crypts was used to determine whether 5-HT action was associated with an elevation of cytosolic calcium levels, measured using the calcium-sensitive fluorescent dye, fura-2. 5-HT increased the short-circuit current, an effect that was inhibited by 55% in the absence of chloride and by 83% in the presence of serosal frusemide, consistent with the stimulation of electrogenic chloride secretion. This was confirmed by the observation that colonic tissue from transgenic cystic fibrosis mice (n = 4) failed to respond to 5-HT, although wild-type tissues generated an increased short-circuit current of 52.4+/-1.1 microAcm(-2) (n = 9). The electrical response to 5-HT was calcium-dependent. 5-HT action was unaffected by tetrodotoxin and was not mimicked by the 5-HT3 agonist 1-phenylbiguanide, indicating that neural mechanisms are not involved. The cyclooxygenase inhibitor indomethacin, however, reduced the 5-HT-induced rise in short-circuit current by 73%, suggesting that prostaglandin production contributes to the response. Stimulation of crypts with acetylcholine elicited an increase in cytosolic calcium levels, but no such response was detected on application of 5-HT (10(-6) to 10(-4) M), suggesting that 5-HT does not directly modulate intracellular calcium in colonic crypt cells. It is concluded that mouse colon responds to 5-HT challenge with a stimulation of electrogenic chloride secretion and that this effect is mediated by indirect mechanisms that might involve immune elements within the colonic wall.

Effects of 5-hydroxytryptamine on the transintestinal electrical activity and cardiovascular function of fawn-hooded rats in-vivo.

Fawn-hooded rats, which have abnormal serotonergic function, were used to investigate the receptors involved in 5-hydroxytryptamine (5-HT)-induced intestinal secretion. The effects of 5-HT on secretion by the small intestine and proximal colon, monitored as increased transintestinal electrical activity, and on cardiovascular function, measured as changes in heart rate and blood pressure, were compared in fawn-hooded and Wistar rats. The maximum fall in heart rate induced by 5-HT (mediated by 5-HT3 receptors) was greater in fawn-hooded than in Wistar rats. ED50 values (the doses resulting in 50% of the maximum effect) for the 5-HT2-mediated increases in systolic pressure were lower for both 5-HT and 5-methoxytryptamine in the fawn-hooded group. The prolonged fall in diastolic pressure mediated by 5-HT1-like receptors was significantly attenuated in fawn-hooded rats, with the maximum responses to 5-HT, 5-methoxytryptamine and 6-hydroxyindalpine reduced to 21%, 42% and 28%, respectively, of the values obtained for Wistar rats. In fawn-hooded rats the small intestine was less sensitive to the effects of 5-HT (ED50 = 47 nmol kg(-1); ED50 for Wistar rats = 23 nmol kg(-1)) and the maximum colonic response to 5-methoxytryptamine was greater (7.0 mV compared with 4.3 mV in Wistar rats), but other indices did not differ for the two strains. The responses to 6-hydroxyindalpine were similar in fawn-hooded and Wistar rats. It is concluded that although the cardiovascular response of fawn-hooded rats to 5-HT challenge is very different from that of Wistar rats, this difference is not reflected in marked alterations in 5-HT-induced intestinal secretion. This is consistent with 5-HT stimulating secretion via the activation of several different receptor subtypes so that any changes in the receptor profile in fawn-hooded rats results in little alteration in the overall intestinal response.

5-Hydroxytryptamine-induced secretion by rat jejunum in-vitro involves several 5-hydroxytryptamine receptor subtypes.

The receptors contributing to 5-hydroxytryptamine (5-HT)-induced anion secretion by rat jejunum have been investigated by testing the effects of selective agonists and antagonists in-vitro using both intact and stripped intestinal sheets. In both intact and stripped jejunum 5-HT and 5-methoxytryptamine, an agonist that lacks affinity for 5-HT3 receptors, induced concentration-dependent increases in the short-circuit current (SCC), although 5-methoxytryptamine induced a smaller maximum response. In intact sheets 1-phenylbiguanide, a selective 5-HT3 agonist, induced a response that was similar in magnitude to that of 5-methoxytryptamine, but in stripped preparations it had little effect. Tetrodotoxin inhibited the response of intact jejunum to 5-HT (by 86%) and 5-methoxytryptamine (by 85%) and abolished the response to 1-phenylbiguanide. In stripped sheets inhibition of the 5-HT response by tetrodotoxin was reduced to 27%. Desensitization to 1-phenylbiguanide reduced the response to 5-HT in intact but not stripped sheets whereas, in contrast, desensitization to 5-methoxytryptamine inhibited the 5-HT response in stripped sheets but was without effect in intact sheets. Mianserin, a 5-HT1, 5-HT2 and 5-HT3 antagonist, and renzapride, a 5-HT1 and 5-HT3 antagonist, both reduced the maximum response to 5-HT, but 5-HTP-DP, a 5-HT1 antagonist, was without effect. The 5-HT3 antagonist granisetron reduced the response to 5-HT in intact, but not in stripped sheets. Tropisetron, a 5-HT3 and 5-HT4 antagonist, inhibited the response to 5-methoxytryptamine in both preparations, but did not alter the response to 5-HT. It is concluded that 5-HT-induced jejunal secretion involves more than one 5-HT receptor subtype, with both neural and non-neural mechanisms contributing to the response.

Several receptor subtypes contribute to 5-hydroxytryptamine-induced secretion by rat ileum in-vitro.

The receptors contributing to 5-hydroxytryptamine (5-HT)-induced secretion by rat ileum were investigated in-vitro using selective agonists and antagonists. 5-HT induced a dose-dependent increase in the short-circuit current (SCC) generated by both intact and stripped sheets of rat ileum. 1-Phenylbiguanide, a selective 5-HT3 agonist, and 5-methoxytryptamine, an agonist that lacks affinity for 5-HT3 receptors, also increased the SCC. In intact sheets 5-HT was more effective than either 1-phenylbiguanide or 5-methoxytryptamine, whereas in stripped sheets 5-HT and 5-methoxytryptamine were equipotent, with 1-phenylbiguanide having little effect. Tetrodotoxin abolished the response of intact sheets to 1-phenylbiguanide but only reduced the responses to 5-HT and 5-methoxytryptamine by 57% and 54%, respectively. This inhibition was reduced to 25% in stripped sheets. The 5-HT3 antagonist granisetron abolished the response to 1-phenylbiguanide, but did not alter the effects of 5-HT. Ketanserin, a 5-HT2 antagonist, had a small effect on the actions of 5-methoxytryptamine in intact, but not stripped, sheets and no effect on the response to 5-HT in either preparation. Tropisetron, a 5-HT3 and 5-HT4 antagonist, inhibited the response to 5-methoxytryptamine, but had less effect on the response to 5-HT. Desensitization to 1-phenylbiguanide inhibited the response to 5-HT in intact, but not stripped sheets, whereas desensitization to 5-methoxytryptamine abolished the 5-HT response in stripped sheets, but induced only 42% inhibition in intact sheets. Previous exposure to a combination of both 1-phenylbiguanide and 5-methoxytryptamine abolished the 5-HT-induced increase in SCC in both preparations. The findings suggest that 5-HT-induced ileal secretion involves more than one 5-HT receptor subtype and that both neural and non-neural mechanisms contribute to the response.

Comparison of the intestinal secretory response to 5-hydroxytryptamine in the rat jejunum and ileum in-vitro.

A secretory response to 5-hydroxytryptamine (5-HT) is observed throughout the intestinal tract; this investigation has compared the nature of this response in the jejunum and ileum of the rat in-vitro. Different basal electrical activity was observed for jejunal and ileal sheets of rat small intestine. In both intact and stripped preparations the basal short-circuit current (SCC) was greater and the tissue resistance lower in the jejunum than in the ileum. 5-HT caused concentration-dependent increases in SCC in intact and stripped preparations of both regions. EC50 values were similar in the jejunum and ileum, stripped sheets from both regions showing greater sensitivity. In the ileum the maximum increase in SCC induced by 5-HT was similar in intact and stripped sheets, but in the jejunum the response was greater in intact preparations. The jejunal response to 5-HT was reduced in the absence of bicarbonate but unaffected by lack of chloride, whereas the ileal response was inhibited by removal of chloride but unaltered in bicarbonate-free conditions. In intact sheets the tetrodotoxin-sensitive neural component was greater in the jejunum. In stripped sheets a neural component could still be detected in the ileum, but not in the jejunum. There are, therefore, fundamental differences in the way in which the jejunum and ileum respond to 5-HT stimulation--the jejunal response is primarily a result of stimulation of bicarbonate secretion whereas chloride secretion predominates in the ileum. The myenteric plexus appears to play a more prominent role in the jejunum; in the ileum other neural elements also contribute to the response.

Chloride conductance and sodium-dependent glucose transport in rat and human enterocytes.

BACKGROUND & AIMS: In cystic fibrosis intestine, there is an increase in the rate of Na+-dependent glucose absorption. This may result from enterocyte hyperpolarization after defective Cl- channel function, but only if Cl- secretion and Na+/glucose cotransport occur in the same membrane. This study examined the effects of Cl- gradients on Na+/glucose uptake in brush border membrane vesicles from rat and human small intestine. METHODS: Vesicles were prepared by Mg2+-precipitation, and the active uptake of tritiated glucose was measured using a filtration-stop protocol. RESULTS: An outwardly directed Cl- gradient inhibited active glucose uptake in rat vesicles, whereas an inward Cl- gradient stimulated uptake. These effects were sensitive to blockers of the cystic fibrosis transmembrane regulator but not to inhibitors of other Cl- channels. Active glucose uptake into vesicles prepared from normal human intestine was also inhibited by an outward Cl- gradient, whereas uptake into vesicles prepared from a single sample of human cystic fibrosis intestine was not. CONCLUSIONS: A Cl- conductance resembling the cystic fibrosis transmembrane regulator is colocalized with Na+/glucose cotransport in rat and human small intestine, supporting the possibility that abnormalities in glucose absorption in cystic fibrosis may be a secondary effect of defects in Cl- channel function.

Influence of peppermint oil on absorptive and secretory processes in rat small intestine.

BACKGROUND: Peppermint oil is used to relieve the symptoms of irritable bowel syndrome, relaxing intestinal smooth muscle by reducing the availability of calcium, but its effects on intestinal transport are unknown. AIMS: To determine the effect of peppermint oil on intestinal transport processes. METHODS: The influence of peppermint oil on intestinal transport was investigated in rat jejunum using both intestinal sheets mounted in Ussing chambers and brush border membrane vesicles. RESULTS: Mucosal peppermint oil (1 and 5 mg/ml) had no significant effect on basal short circuit current, but inhibited the increase associated with sodium dependent glucose absorption. The increased short circuit current induced by serosal acetylcholine, a reflection of calcium mediated electrogenic chloride secretion, was unaffected by mucosal peppermint oil (5 mg/ml). In contrast, serosal peppermint oil (1 mg/ml) inhibited the response to acetylcholine without reducing the effect of mucosal glucose. In brush border membrane vesicles active glucose uptake was inhibited by extravesicular peppermint oil at concentrations of 0.5 and 1 mg/ml. CONCLUSIONS: Peppermint oil in the intestinal lumen inhibits enterocyte glucose uptake via a direct action at the brush border membrane. Inhibition of secretion by serosal peppermint oil is consistent with a reduced availability of calcium.

Neural involvement in 5-hydroxytryptamine-induced net electrogenic ion secretion in the rat intestine in-vivo.

5-Hydroxytryptamine (5-HT) induces active electrogenic anion secretion by both the small intestine and the colon, responses that can be detected from measurements of transmural electrical activity. This approach was adopted to examine the involvement of neural mechanisms in 5-HT-induced secretion in rat proximal jejunum, distal ileum and proximal colon in-vivo. Under control conditions, 5-HT caused maximum rises in transintestinal potential difference of 4.7 +/- 0.3, 3.8 +/- 0.4 and 7.6 +/- 0.3 mV, respectively, with corresponding ED50 values of 28 +/- 3, 38 +/- 4 and 41 +/- 4 nmol kg-1 (n = 12). In each region examined a neural component in the secretory response to 5-HT was identified. Hexamethonium (22 mumol kg-1) reduced the 5-HT response in each region: in the jejunum and colon, it also attenuated the responses to the 5-HT3 agonist, phenylbiguanide and to 5-methoxytryptamine (5-MeOT), an agonist at all 5-HT receptors except 5-HT3, indicating that in these regions the nicotinic pathway can be activated by more than one 5-HT receptor subtype. Atropine (0.27 and 2.7 mumol kg-1) was found to have regional effects on the intestinal responses to 5-HT receptor agonists. In the jejunum, evidence for a pro-secretory muscarinic pathway which could be activated by more than one 5-HT receptor subtype was found. In the ileum and colon no muscarinic pro-secretory pathway was identified, indeed in the colon, an anti-secretory pathway may be present. This muscarinic anti-secretory pathway was observed with phenylbiguanide and 5-MeOT, but not 5-HT. Substance P release does not appear to be involved in mediating the intestinal secretory response to 5-HT. 5-HT-induced intestinal anion secretion may involve a direct secretory action on the enterocyte which can be modified by neurally-mediated pro-secretory and anti-secretory pathways, the balance between these processes varying down the length of the gut.

Evidence that the secretory response of rat intestine to 5-hydroxytryptamine in-vivo involves more than one 5-hydroxytryptamine-receptor subtype.

The transintestinal potential difference (PD) across rat mid-small intestine and proximal colon was measured in-vivo. The 5-hydroxytryptamine (5-HT)-induced increase in PD, which reflects a stimulation of electrogenic C1 secretion, was mimicked by both 2-methyl-5-hydroxytryptamine (2-CH3-5-HT), an agonist at 5-HT3 receptors, and 5-methoxytryptamine (5-MT), an agonist that lacks affinity for 5-HT3 receptors. The 5-HT3 antagonist granisetron caused a marked inhibition of the response to 2-CH3-5-HT in both regions, but only produced a small inhibition of the small intestinal response to 5-HT, with a more pronounced effect in the colon. The failure of granisetron to produce a marked antagonism of the 5-HT-induced rise in the transintestinal PD, coupled with the ability of 5-MT to induce a secretory response, indicates that 5-HT3 receptors are not the only ones involved in the stimulation of C1 secretion. The 5-HT2 antagonist ketanserin failed to influence the response to 5-HT in either the small intestine or the colon, but it did inhibit the action of 5-MT, having a much greater effect in the small intestine. In the presence of granisetron however, ketanserin also inhibited the small intestinal response to 5-HT, having only a minimal effect in the colon. This suggests that 5-HT2 receptors can also play a role in the activation of C1 secretion. These observations suggest that both 5-HT2 and 5-HT3 receptors contribute to the stimulation of electrogenic C1 secretion by 5-HT, with 5-HT2 receptors playing a more prominent role in the small intestine and 5-HT3 receptors being more important in the colon.

Do 5-HT4 receptors mediate the intestinal secretory response to 5-HT in rat in-vivo?

The involvement of the recently characterized 5-HT4 receptor in the actions of 5-hydroxytryptamine (5-HT) on jejunal, ileal and colonic electrogenic ion secretion was investigated in the rat in-vivo. 5-HT and the 5-HT1-, 5-HT2- and 5-HT4-receptor agonist 5-methoxytryptamine (5-MeOT), induced a rise in transintestinal PD in all regions of the gut. However, the 5-HT4-receptor agonists renzapride and cisapride had no effect. Furthermore, the 5-HT4-receptor antagonists SDZ 205-557 (2-diethylaminoethyl-[2-methoxy-4-amino-5-chloro] benzoate), tropisetron and SB 204070 ([1-butyl-4-piperidinylmethyl]-8-amino-7-chloro-1,4- benzodioxan-5-carboxylate hydrochloride) did not affect the secretory response to either 5-HT or 5-MeOT in the jejunum, but did cause a small inhibition in the ileum and colon. It is concluded that 5-HT4 receptors do not make a contribution to the electrically monitored 5-HT intestinal secretory response in the rat jejunum in-vivo, but may play a small role in the ileum and colon.

Comparison of a computer simulation program and a traditional laboratory practical class for teaching the principles of intestinal absorption.

Here we describe an evaluation of the effectiveness, compared with a traditional laboratory, of an interactive computer-assisted learning (CAL) program, which simulates a series of experiments performed using isolated, everted sacs of rat small intestine. The program is aimed at undergraduate students of physiology and is designed to offer an alternative student-centered learning approach to the traditional laboratory-based practical class. The evaluative study compared two groups of second-year undergraduate students studying a module on epithelial transport: one group worked independently using the CAL program and associated learning materials, and the other group followed a conventional practical class approach, working in the laboratory under supervision. Knowledge gain of each group was measured by means of a test consisting of a range of question types (e.g., short-answer factual, calculation, interpretation) given to students before and after the module. Student attitude to both approaches was assessed by questionnaire, and the resource requirements were also compared. It was found that the knowledge gain of both groups of students was the same, that students had a positive attitude toward using CAL programs of this type, and that the cost of the conventional laboratory-based approach was five times greater. The potential for integrating CAL programs into the undergraduate curriculum is discussed.

Is desensitization of intestinal 5-hydroxytryptamine receptors an in-vitro phenomenon?

The responses of proximal jejunum and distal ileum to successive applications of 5-hydroxytryptamine (5-HT) were examined in-vitro and in-vivo by measuring the electrical changes that reflect the stimulation of Cl- secretion. In stripped intestinal sheets the second application of a maximal concentration of 5-HT failed to elicit any response, indicating that complete desensitization had occurred. If submaximal concentrations were used, a second response was observed, although it was smaller than the first, indicating partial desensitization. Replacing the bathing solutions following application of a maximal 5-HT concentration also reduced, but did not abolish, the degree of desensitization observed with a second application of 5-HT. In an in-vivo preparation, however, there was no diminution of the responses to four successive maximal doses of 5-HT. This lack of desensitization was also evident in the cardiovascular responses to 5-HT. It is concluded that desensitization to 5-HT is a phenomenon that is readily observed only in-vitro and which is probably related to the inability of a small amount of isolated tissue to eliminate 5-HT.