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.

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).

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.

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.

[The effect of cysteamine on the duodenal defense mechanism in the vagotomized rats--with special reference to the parietal cell vagotomy with CO2-laser].

The parietal cell vagotomy with CO2-Laser defocused beam has been developed and its efficacy was confirmed in cysteamine induced duodenal ulcer in rat. Rats were classified into the truncal, parietal cell and Laser vagotomy groups and control. After cysteamine was administered, the change of the Brunner's gland were examined histologically for each group. Duodenal ulcer was seen and the depletion of the Brunner's glands was observed in all control rats. Duodenal ulcer formation was prevented in all vagotomized rats regardless of the type of vagotomy and the preservation of synthetic activity of the Brunner's gland was noted. However, multiple gastric ulcer was seen in truncal vagotomy group. Laser vagotomy was done very easily and safely. It consumed much less time and prevented duodenal ulcer formation. It prevented the cysteamine induced duodenal ulcer formation in rats by preservation of the Brunner's gland activity.

Epidermal growth factor inhibits cysteamine-induced duodenal ulcers.

The effect of the duodenal ulcerogen cysteamine on secretion of epidermal growth factor from Brunner's gland pouches was studied in the rat. Total output of immunoreactive epidermal growth factor was reduced to approximately 55%, compared with controls, 5 h after administration of cysteamine (300 mg/kg, s.c.). Furthermore, measurements on tissue extracts of the pouches revealed that 5 h after cysteamine treatment, Brunner's glands were depleted of epidermal growth factor. The effect on ulcer development of intraduodenally applied exogenous epidermal growth factor (1 micrograms/kg . h) also was studied. Luminal epidermal growth factor significantly inhibited the formation of cysteamine-induced duodenal ulcer, compared with controls receiving saline. The effect was not due to inhibition of gastric acid secretion or stimulation of duodenal bicarbonate secretion since the dose of epidermal growth factor used, when tested on chronic fistula rats, had no effect on acid secretion and did not influence bicarbonate secretion from Brunner's gland pouches. These results demonstrate that epidermal growth factor has a cytoprotective effect on the duodenal mucosa, and it is suggested that inhibition of synthesis and secretion of endogenous epidermal growth factor may be a pathogenetic factor in cysteamine-induced duodenal ulcer.

Secretory process in Brunner's glands during recovery from stimulation with a single dose of pilocarpine.

The secretory pathway and kinetics of the secretory process were studied in Brunner's glands of mice after stimulation of secretion with a parasympatho-mimetic drug. Adult male mice were injected with pilocarpine, while unstimulated controls received saline. The animals were subsequently administered an intravenous injection of 3H-threonine, and tissue was prepared for electron microscope autoradiography at intervals ranging from 5 minutes to 2 hours after injection of the radioactive precursor. Stimulation with pilocarpine resulted in discharge of secretory granules, which was reflected in a significantly lower percentage of the cell volume occupied by granules. In both control and stimulated mice, at 5 minutes after injection of 3H-threonine, the highest percentage of silver grains was found over the rough endoplasmic reticulum. The proportion of silver grains over the rough endoplasmic reticulum declined at later intervals, and a peak of labeling was observed over the Golgi apparatus at 1 hour. Labeling of the secretory granules increased in the 1- and 2-hour samples from both control and stimulated mice, although the relative concentration of radioactivity in both Golgi-associated and apical secretory granules was greater in stimulated than control glands at 1 hour. The results suggest that the secretory protein produced by Brunner's glands was synthesized by the rough endoplasmic reticulum and transported to the Golgi apparatus where secretory granules were formed in both stimulated and control glands. Depletion of secretory granules by prior stimulation resulted in no change in the kinetics of arrival of radioactivity in the cell organelles involved in the secretory process. However, the drainage of the radioactive label from the rough endoplasmic reticulum was significantly slower in the stimulated glands than in the controls. The existence of two subcompartments within the rough endoplasmic reticulum is among the possible explanations for the latter observation.

Effect of 3,4-toluenediamine on output from in situ rat Brunner's glands pouches.

Duodenal and gastric glandular mucosal damage have been observed 24 hr following single subcutaneous doses of 3,4-TDA in fed, unrestrained rats. 3,4-TDA significantly reduced secretion from in situ Brunner's glands pouches in pentobarbital anesthetized rats. The reduction in volume output with a definitely duodenal ulcerogenic dose of this compound was more than twice that observed with a minimally ulcerogenic dose, suggesting a correlation between the duodenal ulcerogenic and duodenal anti-secretory activities of this compound. The animal model described in this communication should facilitate experimentation to establish the inhibitory effect of compounds on the output of protective fluids from the proximal duodenum.

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 vasoactive intestinal polypeptide and somatostatin on secretion of epidermal growth factor and bicarbonate from Brunner's glands.

The effect of VIP and somatostatin on secretion of epidermal growth factor and bicarbonate from Brunner's glands was investigated in the rat. Vasoactive intestinal polypeptide infused in doses of 10 and 100 ng/kg/h significantly increased epidermal growth factor and bicarbonate output, but the concentrations did not change. Somatostatin infused at doses of 1, 10, 100 and 1000 ng/kg/h against a background of VIP 100 ng/kg/h inhibited in dose-dependent fashion the stimulated epidermal growth factor and bicarbonate outputs from rat Brunner's gland pouches. Also basal secretion was inhibited by somatostatin. Infusion of antisomatostatin serum stimulated Brunner's gland secretion. By immunohistochemical studies of rat duodena, it was found that epidermal growth factor, is almost exclusively present in the secretory cells of Brunner's glands. It is concluded that VIP stimulates secretion of epidermal growth factor and bicarbonate from Brunner's glands, an effect which is inhibited by somatostatin. A possible role for somatostatin in the control of Brunner's gland secretion is suggested.

Adrenergic effects on secretion of epidermal growth factor from Brunner's glands.

The influence of the sympathetic nervous system and adrenergic agonists on flow rate and secretion of epidermal growth factor (EGF) from Brunner's glands has been investigated in the rat. Chemical sympathectomy by administration of 6-hydroxydopamine increased volume secretion and output of EGF from Brunner's glands but depleted the glands of EGF. Infusion of noradrenaline, an alpha-adrenergic agonist, inhibited basal and vasoactive intestinal polypeptide (VIP) stimulated flow rate and output of EGF from Brunner's glands and increased the amount of EGF in the tissue. Vasoactive intestinal polypeptide also increased the amount of EGF in Brunner's gland tissue and this was unchanged after simultaneous infusion of VIP and noradrenaline as well as VIP and isoproterenol, a beta-adrenergic agonist. Isoproterenol had no effect on basal and VIP stimulated secretion of EGF from Brunner's glands. The presence of PAS-positive mucus in Brunner's glands was unchanged during infusion of noradrenaline whereas VIP induced a depletion of Brunner's gland mucus which in turn was prevented by simultaneous infusion of noradrenaline. This study indicates that the sympathetic nervous system influence the volume secretion, output of EGF and mucus content in Brunner's glands probably by activation of alpha-adrenergic pathways.

Effect of secretin and glucagon on Brunner's gland secretion in the rat.

Brunner's gland secretion in response to infusion of secretin and glucagon was studied in the rat. Secretin was infused in doses of 15, 150 and 1500 ng/kg/h. All dose significantly increased bicarbonate and protein output and depleted Brunner's glands of PAS-positive mucin. Bicarbonate secretion was related to plasma secretin concentration, and a marked stimulatory effect of secretin was found in very low, probably physiological, plasma concentrations. Maximal bicarbonate output was obtained at a plasma concentration of secretin about 20 pmol/l. Glucagon was infused at a rate of 1.0 micrograms/kg/h and did not influence secretion rate or cell morphology. Also large doses of 5.0 and 50.0 micrograms/kg/h had no effect on Brunner's gland secretion. It is concluded that secretin in very low plasma concentrations stimulates secretion of bicarbonate, protein and mucus from Brunner's glands in the rat, while glucagon has no effect, and it is suggested that secretin may be involved in the physiological regulation of Brunner's gland secretion.

Effects of 5-fluorouracil on exocrine glands. III. Fine structure of Brunner's glands of rats.

Effects of a pyrimidine analogue, 5-fluorouracil (Fur), have been studied by electron microscopy and by electron microscopic cytochemical techniques. Previous studies have demonstrated that rats show serious gastrointestinal disturbances 5 days after 3 daily injections of FUR (50 mg/kg). The present investigation demonstrates that Brunner's glands under the same conditions suffer certain cytological changes involving the Golgi apparatus, where a notable reduction in the number of Golgi stacks is observed. The vacuolar components in the Golgi complex appear empty. Cytochemical localizations of uridine diphosphatase and thiamine pyrophosphatase activities, however, are normal. The reaction products are localized in the distal two or three lamellae of the Golgi stack and within the secretory granules nearby. In addition reaction products are present along the apical plasma membrane on the luminal side, suggesting a possible movement of these membranes from the Golgi stack, via secretory granules, to the apical plasma membrane.

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.

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.

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.