Probiotic supplementation affects the glycan composition of mucins secreted by Brunner's glands of the pig duodenum.

The effect of a dietary probiotic blend on the carbohydrate composition of mucins secreted by the Brunner's glands in the duodenum of growing-finishing pigs was investigated by means of conventional (periodic acid-Schiff, Alcian Blue pH 2.5, high iron diamine staining) and lectin (15 lectins) histochemistry. Pigs were assigned to two dietary treatments: a control basal diet without the probiotic blend (No-Pro) and a test diet that included the probiotic blend (Pro). Duodenal tissue fragments were fixed in 4% phosphate-buffered-saline-buffered paraformaldehyde, dehydrated through a graded alcohol series, and embedded in paraffin wax. The secretory cells of the Brunner's glands from No-Pro pigs primarily produced neutral glycoproteins and a small amount of acidic non-sulphated mucins. This glycan pattern was opposite that of the Brunner's glands from Pro animals. A comparison of lectin-binding profiles of the secretory cells of Brunner's glands in these two groups showed that in Pro pigs, there was (i) a decrease in N-linked glycans containing α1,2-linked fucose (Con A, UEA I); (ii) a loss of complex types of N-glycans (PHA-L, PHA-E) terminating with lactosamine (RCA120), α1,6- and α1,3-linked fucose (LTA), and α-galactose (GSA I-B4), as well as of O-glycans with terminal Galß1,3GalNAc (PNA); and (iii) an increase in O-glycans containing GalNAc HPA. No-Pro and Pro samples showed no change in the expression of α2,6 sialoglycans and terminal GlcNAc residues and no affinity for MAL II, DBA, and SBA. These results indicate that probiotic supplementation affects the glycan composition of mucins produced in the Brunner's glands of growing-finishing pigs. These changes could effectively act on the gastrointestinal function and health status of these animals because the probiotic blend induced higher growth performance and meat quality in the test probiotic group than it did in the control basal diet group (Tufarelli et al., 2017).

Brunner's gland lesions in rats induced by a vascular endothelial growth factor receptor inhibitor.

Vascular endothelial growth factor (VEGF) receptor tyrosine kinase (RTK) inhibitors are reported to cause reversible mucosal hyperplasia (adenosis) in the duodenum of rats; however, the pathogenesis is not fully elucidated. Using lenvatinib, a VEGF RTK inhibitor, we characterized the histologic time course of this duodenal change in rats. At 4 weeks, there was degeneration and necrosis of Brunner's gland epithelium accompanied by neutrophil infiltration around the affected glands. At 13 weeks, the inflammation was more extensive, and Brunner's gland epithelium was attenuated and flattened and was accompanied by reactive hyperplasia of duodenal epithelium. At 26 weeks, the changes became more severe and chronic and characterized by marked cystic dilation, which extended to the external muscular layer. These dilated glands exhibited morphological characteristics of duodenal crypt epithelium, suggestive of replacement of disappeared Brunner's glands by regenerative duodenal crypt epithelial cells. Similar changes were not present in similar time course studies in dog and monkey studies, suggesting that this is a rodent- or species-specific change. Based on the temporal progression of Brunner's gland lesion, we identify degeneration and necrosis of the Brunner's glands as the primary change leading to inflammation, cystic dilatation, and regeneration with cells that are morphologically suggestive of duodenal crypt epithelium.

Munc18/SNARE proteins' regulation of exocytosis in guinea pig duodenal Brunner's gland acini.

AIM: To examine the molecular mechanism of exocytosis in the Brunner's gland acinar cell. METHODS: We used a submucosal preparation of guinea pig duodenal Brunner's gland acini to visualize the dilation of the ductal lumen in response to cholinergic stimulus. We correlated this to electron microscopy to determine the extent of exocytosis of the mucin-filled vesicles. We then examined the behavior of SNARE and interacting Munc18 proteins by confocal microscopy. RESULTS: One and 6 micromol/L carbachol evoked a dose-dependent dilation of Brunner's gland acini lumen, which correlated to the massive exocytosis of mucin. Munc18c and its cognate SNARE proteins Syntaxin-4 and SNAP-23 were localized to the apical plasma membrane, and upon cholinergic stimulation, Munc18c was displaced into the cytosol leaving Syntaxin-4 and SNAP-23 intact. CONCLUSION: Physiologic cholinergic stimulation induces Munc18c displacement from the Brunner's gland acinar apical plasma membrane, which enables apical membrane Syntaxin-4 and SNAP-23 to form a SNARE complex with mucin-filled vesicle SNARE proteins to affect exocytosis.

Potassium currents regulating secretion from Brunner's glands in guinea pig duodenum.

This study examined the role of outward K(+) currents in the acinar cells underlying secretion from Brunner's glands in guinea pig duodenum. Intracellular recordings were made from single acinar cells in intact acini in in vitro submucosal preparations, and videomicroscopy was employed in the same preparation to correlate these measures with secretion. Mean resting membrane potential was -74 mV and was depolarized by high external K(+) (20 mM) and the K(+) channel blockers 4-aminopyridine (4-AP), quinine, and clotrimazole. The cholinergic agonist carbachol (60-2,000 nM; EC(50) = 200 nM) caused a concentration-dependent initial hyperpolarization of the membrane and an associated decrease in input resistance. This hyperpolarization was significantly decreased by 20 mM external K(+) or membrane hyperpolarization and increased by 1 mM external K(+) or membrane depolarization. It was blocked by the K(+) channel blockers tetraethylammonium (TEA), 4-AP, quinine, and clotrimazole but not iberiotoxin. When videomicroscopy was employed to measure dilation of acinar lumen in the same preparation, carbachol-evoked dilations were altered in a parallel fashion when external K(+) was altered. The dilations were also blocked by the K(+) channel blockers TEA, 4-AP, quinine, and clotrimazole but not iberiotoxin. These findings suggest that activation of outward K(+) currents is fundamental to the initiation of secretion from these glands, consistent with the model of K(+) efflux from the basolateral membrane providing the driving force for secretion. The pharmacological profile suggests that these K(+) channels belong to the intermediate conductance group.

A novel in vitro model of Brunner's gland secretion in the guinea pig duodenum.

A novel in vitro model that combined functional and morphological techniques was employed to directly examine pathways regulating Brunner's gland secretion in isolation from epithelium. In vitro submucosal preparations were dissected from guinea pig duodenum. A videomicroscopy technique was used to measure changes in luminal diameter of glandular acini as an index of activation of secretion. Carbachol elicited concentration-dependent dilations of the lumen (EC(50) = 2 microM) by activating muscarinic receptors on acinar cells. Ultrastructural and histological analyses demonstrated that dilation was accompanied by single and compound exocytosis of mucin-containing granules and the accumulation of mucoid material within the lumen. Inflammatory mediators (histamine, PGE(1), PGE(2)) and intestinal hormones (CCK, gastrin, vasoactive intestinal polypeptide, secretin) also stimulated glandular secretion, whereas activation of submucosal secretomotor neurons by 5-hydroxytryptamine did not. This study directly demonstrates that multiple hormonal, inflammatory, and neurocrine agents activate Brunner's glands, whereas many have dissimilar effects on the epithelium. This suggests that Brunner's glands are regulated by pathways that act both in parallel to and in isolation from those controlling epithelial secretion.

Effect of secretin and somatostatin on secretion of epidermal growth factor from Brunner's glands in the rat.

The effect of secretin and somatostatin on secretion of epidermal growth factor (EGF) from Brunner's glands was investigated in rats. Secretin increased volume secretion and the median output of EGF rose from 720 fmol/5 hr (total range 460-1320) in controls to 2065 fmol/5 hr (total range 1560-2730) at a dose of 50 pmol/kg/hr of secretin. Somatostatin inhibited Brunner's gland secretion, but the total output of EGF remained unchanged. Secretin-stimulated volume secretion and secretion of EGF was significantly reduced by simultaneous infusion of somatostatin. This study has shown that secretin stimulates secretion of EGF as well as volume secretion from Brunner's glands. Somatostatin prevents the effect of secretin on Brunner's glands, which suggests a role for somatostatin in control of Brunner's gland secretion.

Effects of epidermal growth factor on alkaline secretion and mucus formation in rat duodenum exposed to luminal acid.

The effect of epidermal growth factor (EGF) on alkaline secretion and mucus formation which serve as defenses against mucosal injury was investigated using a perfusion system of the proximal duodenum in rats in situ. In control rats, intravenous or intraduodenal administration of EGF (1 or 10 micrograms/kg/hr) had no effect on mucosal alkaline secretion at high (pH 2.5-3.0) or low (pH 3.0-5.5) luminal acidities. In cysteamine-treated rats (250 mg/kg weight, intramuscular injection), mucosal alkaline secretion by intravenous EGF (10 micrograms/kg/hr) increased significantly only at levels of high luminal acidity, whereas that by intraduodenal EGF (10 micrograms/kg/hr) increased greatly at both high and low luminal acidities. Analysis by a color image processor revealed that cysteamine greatly reduced the PAS-stained mucus in the duodenal mucosa and in Brunner's glands. Intraduodenal administration of EGF significantly increased the PAS-stained mucus in the duodenal mucosa, but not in Brunner's glands. These results indicate that EGF exerts the cytoprotective effect by stimulating alkaline secretion and mucus formation in the duodenal mucosa.

Butylated hydroxyanisole (BHA) does not cause forestomach hyperplasia by inhibiting the release of gastric mucus.

High doses of BHA cause hyperplasia and subsequent neoplasia in the rodent forestomach and can inhibit gastric prostaglandin (PG) synthesis in vitro. This paper examines the hypothesis that BHA induced forestomach hyperplasia occurs in response to a reduction of gastric mucus, with consequent irritation of the forestomach. This could result from inhibition of the formation of the PG's which mediate the synthesis and release of protective mucus. Groups of 10 rats received 0 or 2% BHA in the diet for 1 or 3 weeks and a positive control group was fed a diet containing indomethacin (3.5 mg/kg), a potent inhibitor of PG synthesis. After 1 week BHA caused focal erosion and ulceration of the forestomach consistent with an irritant effect, but 2 weeks later the epithelium was healed, thickened and markedly hyperplastic. Histochemical staining for mucus showed that the development of forestomach hyperplasia was associated with increased amounts of gastric and duodenal mucus and increased numbers of serotonergic-cells in the gastric and duodenal epithelium. In contrast, indomethacin caused a marked reduction in both gastric and Brunner's gland mucus. Neither BHA nor indomethacin exerted an effect on one specific type of mucus (viz: neutral, acidic or mixed) in the stomach. These results do not support the hypothesis that forestomach hyperplasia arises from an inhibition of either the synthesis or release of gastric mucus. It is possible that the increased numbers of serotonergic-cells are related to the initial ulcerative, or subsequent hyperplastic response.

Role of vasoactive intestinal polypeptide (VIP) and endogenous somatostatin on the secretion of epidermal growth factor (EGF): studies on duodenal tissue cultures.

Epidermal Growth Factor (EGF)-containing cells have been found in Brunner's glands in the same area where several regulatory peptides are released. The present study was aimed at testing the release and the regulation of EGF secretion from cultured duodenal biopsies obtained from healthy individuals by gastroscopy. The effects and the interaction of VIP and somatostatin on the hormone release were studied. Duodenal biopsies were cultured at 37 degrees C in Mc Coy's buffer, gassed with 95% O2 and 5% CO2. After 30 min, the culture medium was decanted for the measurement of the hormones by RIA. To measure the protein content, the tissue was then homogenized; EGF detected in the culture was 11.5 ng/mg protein. The addition of VIP in the medium increased EGF mean levels to 21.6 ng/mg protein (P less than 0.01). The biopsies thus obtained were cultured with anti-somatostatin antibodies to evaluate the influence of endogenous somatostatin on EGF secretion. The inclusion of anti-somatostatin antibodies increased the EGF levels to 41.2 ng/mg protein (P less than 0.01). The combined addition of anti-somatostatin antibodies and VIP in the culture caused a mean EGF increase significantly higher than the values obtained separately by VIP and somatostatin (P less than 0.01). In conclusion, we can suggest a triangular interaction model of EGF release, where the somatostatin seems to be the negative monitor of over-secreted VIP and EGF from the gut.

Cysteamine-induced inhibition of mucosal and pancreatic alkaline secretion in rat duodenum.

To determine the effect of cysteamine on the alkaline secretion by the duodenal epithelium, pancreas, and Brunner's glands in relation to the pathogenesis of duodenal ulceration, the alkaline secretion by various types of duodenal loops was comparatively studied. The results obtained were as follows: (1) Cysteamine significantly reduced both mucosal and pancreatobiliary alkaline secretion in the proximal duodenum of rats. (2) The ratio of contribution of pancreatobiliary alkaline secretion to total neutralization of acid in the proximal duodenum was 55.9% under continuous perfusion. (3) There was no significant difference between the amounts of alkali per unit volume of the proximal and distal duodenal loops. (4) The alkaline substance secreted by the proximal duodenal mucosa was confirmed to be the bicarbonate. From these findings, it has been concluded that the impairment of bicarbonate secretion by the mucosal epithelium of proximal duodenum, not by Brunner's glands, plays a causative role in cysteamine-induced duodenal ulceration.