Alteration in propagating colonic contractions by dairy proteins in isolated rat large intestine.

Gastrointestinal conditions in which the transit of contents is altered may benefit from nutritional approaches to influencing health outcomes. Milk proteins modulate the transit of contents along different regions, suggesting that they have varying effects on neuromuscular function to alter gastrointestinal motility. We tested the hypothesis that bovine whey and casein milk protein hydrolysates could have direct modulatory effects on colonic motility patterns in isolated rat large intestine. Casein protein hydrolysate (CPH), whey protein concentrate (WPC), whey protein hydrolysate (WPH), and a milk protein hydrolysate (MPH; a hydrolyzed blend of 60% whey to 40% casein) were compared for their effects on spontaneous contractile waves. These contractions propagate along the length of the isolated intact large intestine (22 cm) between the proximal colon and rectum and were detected by measuring activity at 4 locations. Milk proteins were perfused through the tissue bath, and differences in contraction amplitude and frequency were quantified relative to pretreatment controls. Propagation frequency was decreased by CPH, increased by MPH, and unaffected by intact whey proteins. The reduced motility with CPH and increased motility with MPH indicate a direct action of these milk proteins on colon tissue and provide evidence for differential modulation by hydrolysate type. These findings mirror actions on lower gastrointestinal transit reported in vivo, with the exception of WPH, suggesting that other factors are required.

Gastroparesis and lipid metabolism-associated dysbiosis in Wistar-Kyoto rats.

Altered gastric accommodation and intestinal morphology suggest impaired gastrointestinal (GI) transit may occur in the Wistar-Kyoto (WKY) rat strain, as common in stress-associated functional GI disorders. Because changes in GI transit can alter microbiota composition, we investigated whether these are altered in WKY rats compared with the resilient Sprague-Dawley (SD) rats under basal conditions and characterized plasma lipid and metabolite differences. Bead transit was tracked by X-ray imaging to monitor gastric emptying (4 h), small intestine (SI) transit (9 h), and large intestine transit (12 h). Plasma extracts were analyzed by lipid and hydrophilic interaction liquid chromatography (HILIC) and liquid chromatography-mass spectrometry (LC-MS). Cecal microbial composition was determined by Illumina MiSeq 16S rRNA amplicon sequencing and analysis using the QIIME pipeline. Stomach retention of beads was 77% for WKY compared with 35% for SD rats. GI transit was decreased by 34% (9 h) and 21% (12 h) in WKY compared with SD rats. Excluding stomach retention, transiting beads moved 29% further along the SI over 4-9 h for WKY compared with SD rats. Cecal Ruminococcus, Roseburia, and unclassified Lachnospiraceae genera were less abundant in WKY rats, whereas the minor taxa Dorea, Turicibacter, and Lactobacillus were higher. Diglycerides, triglycerides, phosphatidyl-ethanolamines, and phosphatidylserine were lower in WKY rats, whereas cholesterol esters and taurocholic acids were higher. The unexpected WKY rat phenotype of delayed gastric emptying, yet rapid SI transit, was associated with altered lipid and metabolite profiles. The delayed gastric emptying of the WKY phenotype suggests this rat strain may be useful as a model for gastroparesis.NEW & NOTEWORTHY This study reveals that the stress-prone Wistar-Kyoto rat strain has a baseline physiology of gastroparesis and rapid small intestine transit, together with metabolic changes consistent with lipid metabolism-associated dysbiosis, compared with nonstress-prone rats. This suggests that the Wistar-Kyoto rat strain may be an appropriate animal model for gastroparesis.

Tracking gastrointestinal transit of solids in aged rats as pharmacological models of chronic dysmotility.

BACKGROUND: Dysmotility in the gastrointestinal (GI) tract often leads to impaired transit of luminal contents leading to symptoms of diarrhea or constipation. The aim of this research was to develop a technique using high resolution X-ray imaging to study pharmacologically induced aged rat models of chronic GI dysmotility that mimic accelerated transit (diarrhea) or constipation. The 5-hydroxytryptamine type 4 (5-HT4 ) receptor agonist prucalopride was used to accelerate transit, and the opioid agonist loperamide was used to delay transit. METHODS: Male rats (18 months) were given 0, 1, 2, or 4 mg/kg/day prucalopride or loperamide (in dimethyl sulfoxide, DMSO) for 7 days by continuous 7-day dosing. To determine the GI region-specific effect, transit of six metallic beads was tracked over 12 h using high resolution X-ray imaging. An established rating scale was used to classify GI bead location in vivo and the distance beads had propagated from the caecum was confirmed postmortem. KEY RESULTS: Loperamide (1 mg/kg) slowed stomach emptying and GI transit at 9 and 12 h. Prucalopride (4 mg/kg) did not significantly alter GI transit scores, but at a dose of 4 mg/kg beads had moved significantly more distal than the caecum in 12 h compared to controls. CONCLUSIONS & INFERENCES: We report a novel high-resolution, non-invasive, X-ray imaging technique that provides new insights into GI transit rates in live rats. The results demonstrate that loperamide slowed overall transit in aged rats, while prucalopride increased stomach emptying and accelerates colonic transit.

The probiotic Escherichia coli Nissle 1917 inhibits propagating colonic contractions in the rat isolated large intestine.

The objective of this research was to test an in vitro motility model by investigating whether a probiotic that reduces diarrhea in humans would reduce motility in the rat colon in vitro. The probiotic Escherichia coli Nissle 1917 (EcN) the active ingredient in Mutaflor® was used as an example probiotic because it is effective for treating infectious diarrheal diseases. The effect of EcN on motility was compared in two colonic preparations. In distal colon segments EcN extract decreased the tension of spontaneous contractions by 74% and frequency by 46% compared with pre-treatment controls. In the whole large intestine the number of synchronized spontaneous propagating contractions decreased by 86% when EcN extract was applied externally and 69% when applied via the lumen compared with pre-treatment. From the inhibition produced by EcN extract in the distal colon segment a myogenic action was inferred and in the whole large intestine neural involvement was implicated. Both are consistent with its anti-diarrheal effect in humans.

An in vitro rat model of colonic motility to determine the effect of ß-casomorphin-5 on propagating contractions.

Beta-casomorphin-5 (ßCM-5) is a milk-derived bioactive peptide that slows gastro-intestinal transit (GIT) in vivo and blocks the peristaltic reflex in the guinea pig colon in vitro. We wanted to establish an in vitro model system in which effects of dairy-derived substances containing opioid peptides on intestinal motility can be assessed and used to predict in vivo outcomes. Because ßCM-5 is an opioid agonist that acts on enteric neurons, we used this substance to compare two different isolated colonic tissue preparations to determine which would more closely mimic the in vivo response previously reported in the literature. We compared and characterized the effects of ßCM-5 on spontaneous contractions in isolated segments of distal colon (1 cm length) compared with propagating contractions along the isolated intact large intestine (22 cm length). In short segments of distal colon, ßCM-5 increased the tension and frequency of spontaneous contractions in a concentration-dependent manner. At 20 µM ßCM-5 tension increased by 71 ± 17% and the frequency doubled (n = 9), effects inhibited by naloxone (n = 7) and therefore mediated by opioid receptors. In contrast 20 µM ßCM-5 disrupted propagating contractions in the large intestine preparation. At 20 µM ßCM-5 reduced the proportion of contractions initiated in the proximal colon reaching the rectum by 83 ± 11% (n = 5) and this effect was also inhibited by naloxone, consistent with altered GIT reported in vivo. Our results demonstrate that the isolated whole large intestine provides an ideal preparation that mimics the reduced propagation of GIT in vivo in response to an opioid agonist, whereas short colon segments did not. The findings of the current study reveal that preserving large segments of intact large intestine, and hence intact enteric neural circuitry provides an ideal in vitro model to investigate the effect of opioid receptor modulators on intestinal transit.

Combined effects of fungal alkaloids on intestinal motility in an in vitro rat model.

Diarrhea is caused by factors that alter absorption and secretion of water and ions across the intestinal epithelium and disrupt motility. Parasitic infection, stress, poor nutrition, and exposure to plant or fungal toxins predispose livestock to noninfectious diarrhea. This is more prevalent in sheep that graze pastures infected with wild-type endophytic fungus, suggesting the involvement of fungal alkaloids. These increase smooth muscle contraction: ergovaline/ergotamine (ergot alkaloid) activates serotonin (5-HT) receptors, and lolitrem B (indole diterpene) inhibits large-conductance Ca2+-activated K+ (BK) channels. Because of their separate mechanisms of action the objective of this study was to investigate whether they act synergistically to increase smooth muscle contraction. Effects of ergotamine (1 µM) and lolitrem B (0.1 µM) on the tension and frequency of spontaneous contractions were investigated in a longitudinal preparation of isolated distal colon. The compounds were dissolved in 0.1% dimethyl sulfoxide (DMSO) and applied separately or together for 1 h. Ergotamine increased contractile tension compared to the pretreatment control (P<0.01) and produced a short-lived increase in frequency (P<0.001). Lolitrem B increased contractile tension (P<0.05) but had no effect on frequency. When applied together, the contractile tension was greater than the sum of the compounds applied separately (P<0.05). The frequency of contractions was increased (P<0.05) but was not significantly different from that for ergotamine alone. The increased contractile tension when both compounds were applied together indicates that ergotamine and lolitrem B acted synergistically to increase smooth muscle contraction, suggesting that they would alter motility in vivo.

Mutating the highly conserved second membrane-spanning region 9' leucine residue in the alpha(1) or beta(1) subunit produces subunit-specific changes in the function of human alpha(1)beta(1) gamma-aminobutyric Acid(A) receptors.

The properties of the human alpha(1)beta(1) gamma-aminobutyric acid (GABA)(A) receptors were investigated after mutation of a highly conserved leucine residue at the 9' position in the second membrane-spanning region (TM2). The role of this residue in alpha(1) and beta(1) subunits was examined by mutating the 9' leucine to phenylalanine, tyrosine, or alanine. The mutations were in either the alpha(1) subunit (alpha*beta), the beta(1) subunit (alphabeta*), or in both subunits (alpha*beta*), and the receptors were expressed in Sf9 cells. Our results show that the rate of desensitization is increased as the size and hydrophobicity of the 9' residue in the alpha(1) subunit is increased: Y, F > L > A, T. Mutation of L9' in only the beta(1) subunit (alphabeta*) to either phenylalanine or tyrosine increased the EC(50) value for GABA at least 100 times, but the EC(50) was unchanged in alphabeta* alanine mutants. In the 9' alpha(1) mutants (alpha*beta, alpha*beta*) the GABA EC(50) was minimally affected. In alpha*beta and alpha*beta*, but not alphabeta*, the peak currents evoked by millimolar concentrations of GABA were greatly reduced. The reduction in currents could only be partially accounted for by decreased expression of the receptors These findings suggest different roles for the two types of subunits in GABA activation and later desensitization of alpha(1)beta(1) receptors. In addition, an increase in the resting membrane conductance was recorded in alanine but not in phenylalanine and tyrosine mutants, indicating that the side chain size at the 9' position is a major determinant of current flow in the closed conformation.

Mutant human alpha(1)beta(1)(T262Q) GABA(A) receptors are directly activated but not modulated by pentobarbital.

Pentobarbital activates GABA(A) receptors and enhances GABA-activated currents. A threonine residue (262) in the second membrane spanning region at the 12' position in the beta(1) subunit, alpha(1)beta(1)(T12'Q), is necessary for the potentiating action of pentobarbital. We examined whether T12'Q-mutated receptors expressed in Spodoptera frugipedra (Sf 9) cells responded to direct activation by pentobarbital. In both mutant and wild type receptors, pentobarbital (100 microM to 1 mM) evoked a current response. The pentobarbital EC(50) values were similar; 119 and 158 microM for alpha(1)beta(1) and alpha(1)beta(1)(T12'Q) receptors, respectively. The results show it is possible to discriminate between agonistic and potentiating effects of pentobarbital, suggesting these actions involve separate mechanisms.

A threonine residue in the M2 region of the beta1 subunit is needed for expression of functional alpha1beta1 GABA(A) receptors.

Although there is a high degree of homology in the M2 transmembrane segments of alpha1 and beta1 subunits, subunit-specific effects were observed in alpha1beta1 GABA(A) receptors expressed in Spodoptera frugipedra (Sf9) cells when the conserved 13' threonine residue in the M2 transmembrane region was mutated to alanine. When threonine 263 (13') was mutated to alanine in the beta1 subunit, high-affinity muscimol binding and the response to GABA were abolished. This did not occur when the threonine 263 (13') was mutated to alanine in the alpha1 subunit, but the rate of desensitisation increased and the effect of bicuculline, a competitive inhibitor, was reduced. The results show differential effects of subunits on receptor function and support a role for M2 in desensitisation.

A structural determinant of desensitization and allosteric regulation by pentobarbitone of the GABAA receptor.

Functional properties of the alpha1beta1 GABAA receptor changes in a subunit-specific manner when a threonine residue in the M2 region at the 12' position was mutated to glutamine. The rate and extent of desensitization increased in all mutants but the rate of activation was faster in the beta1 mutants. A negligible plateau current and abolition of potentiation by pentobarbitone of the GABA-activated current depended on the Thr 12' Gln mutation being present in the beta1 subunit. The Hill coefficient of the peak current response to GABA was reduced to less than one also in a beta1 subunit-specific manner. It was concluded that the beta1 subunit dominated conformational changes activated by GABA.