Effects of antisecretory factor-derived peptides on contractions in guinea pig colon.

Two antisecretory factor (AF)-derived peptides have been studied in relation to effects on motility of guinea pig colon. Colon segments were isolated from adult guinea pigs and incubated in Tyrode Ringer. Motility was measured as the force and frequency of contractions upon addition of the derived peptides AF 1 (8 amino acids (aa)) and AF 3 (10 amino acids). At the lowest concentration (5 pM), peptide AF 1 induced a negative effect on the force of contraction in colon segments; an effect that was abolished by the cholinergic agonist carbachol. Peptide AF 3 induced a significant increase in the force of colon contractions at all concentrations (5-180 pM), with carbachol only reducing the effect of peptide AF 3 at a concentration of 15 pM. Both peptides increased contractile frequency, although the overall response was lower for peptide AF 3 than for peptide AF 1. It is concluded that antisecretory factor-derived peptides may play a role in regulating colon motility such that under pathophysiological conditions, they may serve to hasten the evacuation of noxious agents from the large intestine.

Gastrointestinal-tract models and techniques for use in safety pharmacology.

The gastrointestinal tract (GI tract), which extracts nutrients, electrolytes, minerals, and water, is prone to injury as a result of oral drug administration. Clinical assessment of the GI tract is often limited to measurements of transit time and observations of vomiting or diarrhoea, despite the existence of methods and techniques capable of assessing specific changes in GI function at the membrane, cell, and whole animal levels. Membrane studies, record the uptake of solutes, and electrolyte transport, assessing the affects of compounds on transepithelial GI transport and flux. Such methods lend themselves to permeability, immunohistochemistry, morphology, and molecular biology techniques. Isolated cells from the GI tract or cultured cell lines provide knowledge of regulation and function at a cellular level, whilst motility patterns, taken in vivo or from biopsies, provide information at a more integrated level. In anesthetised animals, ligated segments of the intestine can be infused with test compounds, providing information about absorptive and secretory processes important for the treatment of diarrhoea. Computer simulations and modelling are used to predict the disposition of a chemical and its metabolite and can, to some extent, replace animal testing, thereby reducing development costs. Indeed, software programs can be used to simulate the dissolution, absorption, distribution, metabolism, and excretion (ADME) properties of potential drugs in the human GI tract. Finally, advances in the field of imaging, combined with endoscopy, have resulted in a wireless capsule, allowing the inspection of the GI tract anatomy and pathology without surgical intervention. It is concluded that the field of safety pharmacology could rapidly, cheaply, and routinely incorporate membrane, isolated tissue, and endoscopy techniques for GI tract testing of drugs.

Neuronal involvement in the effect of an antisecretory factor-derived peptide on induced secretion in the porcine small intestine.

The antisecretory factor is a protein inhibiting enterotoxin-induced intestinal inflammation and hypersecretion. We studied the signaling pathway of three antisecretory factor-derived peptides (A1, A3 and A4) in the proximal and distal porcine small intestine. In vivo (ligated loops), only A3 significantly reduced the cholera toxin-induced fluid accumulation and only in proximal loops. A3 and A4 reduced Escherichia coli heat-labile enterotoxin-induced fluid accumulation in the proximal segment, whereas A1 and A3 reduced the Escherichia coli heat-labile enterotoxin-induced fluid accumulation in the distal segment. The secretory response to intraluminally added 5-hydroxytryptamine was not significantly inhibited by the peptides. The amount of intraluminal 5-hydroxytryptamine accumulated in cholera toxin-stimulated loops from the proximal segment was significantly reduced by A3. In vitro,the effect of A3 on secretagogue-induced increases in short-circuit current was recorded from proximal small intestine by the Ussing chamber technique. A3 decreased the tetrodotoxin sensitive effect of substance P. The in vivo results suggest that the antisecretory effect may involve inhibition of the local release of 5-hydroxytryptamine induced by cholera toxin, but not inhibition of secretory reflexes induced by 5-hydroxytryptamine. The in vitro results suggest that the effect of A3 lies beyond the surface epithelium, and involves mucosal neuronal structures.

Effects of indomethacin on Salmonella typhimurium- and cholera toxin-induced fluid accumulation in the porcine small intestine.

The effect of the cyclooxygenase and prostaglandin E2 (PGE2) synthesis inhibitor, indomethacin, on the secretory responses induced by Salmonella serotype Typhimurium (ST) and cholera toxin (CT), in the porcine small intestine was investigated. ST (10(10) colony-forming units) and CT (56 micrograms) were instilled in tied-off intestinal loops in young anaesthetized pigs receiving intravenous indomethacin in a total dose of 7.5 mg/kg, or saline. The accumulated fluid in the loops and the luminal content of endogenous secretagogues PGE2 and 5-hydroxytryptamine (5-HT) were measured. ST induced fluid accumulation in the jejunum, whereas CT induced fluid accumulation in the jejunum and ileum. Indomethacin had no effect on the secretory responses. Indomethacin had a significant effect on the luminal content of PGE2 in jejunal ST and CT loops, whereas no effect of indomethacin was observed on the luminal content of 5-HT in ST and CT loops. In ST and CT loops, an increased content of PGE2 and 5-HT compared with test loops infused with Ringer's solution was observed. These results indicate that the porcine jejunal secretory response to ST and CT does not involve prostaglandins although indomethacin has an influence on the luminal release of PGE2 but not of 5-HT.

Effects of isoprostanes and prostanoids on porcine small intestine.

The isoprostanes are prostaglandin (PG)-like compounds formed in vivo by free-radical-catalyzed peroxidation of polyunsaturated fatty acids and are synthesized independent of cyclooxygenase. It has been debated whether the biological effects of the isoprostanes are exerted on prostanoid receptors [thromboxane A2 (TP) receptors and prostanoid E (EP) receptors] or on a "unique" isoprostane receptor. We sought to define the receptors involved in the actions of isoprostanes on the porcine small intestine. Stripped intestinal sheets were mounted in Ussing chambers, and bioelectrical parameters were recorded. Serosal application of 8-iso-PGE2 (pEC(50) = 5.71), PGE2 (pEC(50) = 6.45 and pEC(50) = 5.04), and PGF2alpha (pEC(50) = 5.07) elicited concentration-dependent increases in the short-circuit current (I(SC)). No responses were seen with 8-iso-PGF2alpha. The TP receptor agonist U46619 induced transient increase in I(SC), and the tissue responded to a further challenge to PGE2. Pretreatment with U46619 did not alter responses to a subsequent addition of either PGE2 or 8-iso-PGE2. The TP receptor antagonist SQ29,548 significantly reduced responses to the TP agonist, U46619, but did not antagonize responses to 8-iso-PGE2. Homologous and heterologous desensitization between 8-iso-PGE2, PGE2, and PGF2alpha suggested the involvement of prostanoid EP and prostanoid F (FP) receptors in the response elicited to 8-iso-PGE2. The effects of 8-iso-PGE2 were not inhibited by tetrodotoxin. Pretreatment of the tissues with bumetanide significantly reduced the increase in I(SC). The results indicate that 8-iso-PGE2 induces a Cl- secretion, and the effects involve prostanoid EP and FP receptors but not TP receptors in the porcine small intestine.

Effect of age on vasoactive intestinal polypeptide-induced short-circuit current in porcine jejunum.

Vasoactive intestinal polypeptide is a transmitter at the neuroepithelial junction of the small intestine in cholera toxin-induced secretion. We investigated whether the secretory effect in vitro of vasoactive intestinal polypeptide in porcine jejunum was changed with age. Stripped tissue preparations from three age groups, neonatal (7-11 days), young (6-8 weeks) and adult (13-15 weeks) pigs, were mounted in Ussing chambers and short-circuited. Vasoactive intestinal polypeptide produced concentration dependent increases in short-circuit current in all three age groups with EC50 values (in nM) of 14.5 +/- 1.9, 16.2 +/- 2.0 and 147 +/- 0 in neonatal, young and adult pigs, respectively. The peak increases in short-circuit current in adult pigs were significantly decreased compared with the other two age groups. To evaluate the secretory capacity, theophylline was added to tissue preparations in which baseline short-circuit current again was established. Theophylline caused a significantly lesser increase in short-circuit current in adult pigs (25.4 +/- 2.0 microA.cm-2) than neonatal (57.1 +/- 3.6 microA.cm-2) and young pigs (63.1 +/- 2.9 microA.cm-2). In conclusion, vasoactive intestinal polypeptide showed a marked decrease in the secretory response with age in porcine jejunum, at least partly caused by a reduced secretory capacity of the enterocytes.

Effects of dietary substitution with raw and heat-treated cowpea (Vigna unguiculata) on intestinal transport and pancreatic enzymes in the pig.

Tropical grain legumes represent potentially important feed for farm animals. However, diarrhoea and poor growth performance have been reported, due to the various anti-nutritional factors they contain. This study addressed in particular whether dietary cowpea impaired the growth of pigs, whether the small intestinal Na+/D-glucose coabsorptive transport capacity was decreased, whether the Cl- secretory capacity was increased, and, finally, whether these parameters were affected by heat treatment of cowpea. Pigs, 4 weeks old, were fed for 3 weeks with one of three diets: (i) standard soy, (ii) 75% of soy substituted with raw cowpea, or (iii) 75% of soy substituted with heat-treated cowpea. The absorptive and secretory capacities of the jejunum and ileum were measured with the Ussing chamber technique. Weight gain, feed intake, pancreatic protein and enzyme concentrations and levels of the blood hormones glucagon and cholecystokinin were also measured. The Na+ transport capacity was measured as the increase in short-circuit current (Isc) when D-glucose was added to the luminal side in the Ussing chambers. Isc was significantly higher in the jejunum from raw cowpea-fed pigs than in the jejunum from standard soy-fed pigs, with no difference between the two cowpea-fed groups. The phosphodiesterase inhibitor theophylline was subsequently added bilaterally, and the increase in Isc indicated the cAMP-depedent Cl- secretory capacity. In the jejunum this was significantly higher in raw and heat-treated cowpea-fed pigs than in standard soy-fed pigs. In contrast, there were no differences in the ileal transport capacities. There were no differences in the pancreatic protein and trypsin concentrations or the blood hormones, but the raw cowpea-fed pigs had significantly lower pancreatic amylase than standard soy-fed pigs. Weight gain and feed intake were lowest in the cowpea-fed groups, with no significant difference between the two groups. In conclusion, the hypothesis of impaired small intestinal absorption of D-glucose and Na+ as causing malabsorption, and therefore impaired growth, during cowpea substitution in the feed may be firmly rejected. The increased Cl- secretory capacity, although moderate, may contribute to the higher incidence of post-weaning diarrhoea in cowpea-fed pigs, as observed in other studies. Additionally, the decreased food intake, feed conversion and weight gain were unaffected by heat treatment, further suggesting involvement of heat-stable anti-nutritional factors.

Comparative aspects of actions of a short-chain phospholipid on epithelial Na+ channels and tight junction conductance.

Ion transport in both the frog skin (a high-resistance epithelium) and the rabbit nasal airway epithelium (a low-resistance epithelium) are dominated by electrogenic Na+ absorption via apical membrane amiloride-sensitive Na+ channels, and short-circuit current (ISC) is essentially a measure of Na+ absorption in both epithelia. In both epithelia, mucosal application of the short-chain phospholipid didecanoyl-L-alpha-phosphatidylcholine (DDPC) dose-dependently inhibited the amiloride-sensitive ISC and caused an initial decrease in epithelial conductance (Gt) followed by an increase in Gt to steady-state values above control level. The effects were reversible. It is concluded that DDPC (a) inhibits epithelial amiloride-sensitive Na+ channels and (b) induces an increase in paracellular tight junction conductance. These effects may involve changes in non-specific lipid-protein interactions at the cell membrane level.

Interference of a short-chain phospholipid with ion transport pathways in frog skin.

The effects of mucosal application of the short-chain phospholipid didecanoyl-L-alpha-phosphatidylcholine (DDPC; with two saturated 10-carbon acyl chains) on active Na+ transport and transepithelial conductance (G) in the frog skin (Rana temporaria) were investigated. Active Na+ transport was measured as the amiloride-sensitive short-circuit current (ISC) and G was determined from transepithelial voltage-clamp pulses under short-circuit conditions. DDPC dose-dependently inhibited ISC with an ID50 of about 0.05% (w/v) and a maximal effect ( approximately 55%) at >/= 1% DDPC. G increased to steady-state values above control level. Simultaneously, equal increases in unidirectional sucrose permeabilities (PSu; measured from [14C]sucrose fluxes) were observed, and a positive correlation was demonstrated between DDPC-induced changes in PSu and G. Since amiloride did not prevent the increase in G by DDPC, these results suggest that the DDPC-induced increase in G represents an increase in the paracellular shunt conductance. The effects of mucosal DDPC were almost fully reversible within 8 h. The results indicate that DDPC inhibits amiloride-sensitive Na+ channels in the apical membrane of the frog skin epithelium and opens a paracellular tight junction pathway. Both effects may be caused by incorporation of DDPC in the apical cell membrane.