Expression of duodenase-like protein in epitheliocytes of Brunner's glands in human duodenal mucosa.

A duodenase, a protease structurally related to human cathepsin G, was found earlier in bovine duodenal mucosa. It was demonstrated that under the influence of duodenase an enteropeptidase zymogen is activated in vitro showing the possible participation of duodenase in the cascade of activation of digestive enzymes. To identify a duodenase functional analog in human duodenum, an immunofluorescence study of duodenal mucosa was conducted by confocal microscopy using antibodies to human cathepsin G and to bovine duodenase. The previously unknown place of synthesis and secretion of cathepsin G - Paneth cells located at the bottom of Lieberkuhn crypts - was revealed. Binding of cathepsin G-specific antibodies in a rough endoplasmic reticulum zone and in the cryptal duct was observed. Duodenase-specific immunofluorescence but not that of cathepsin G was found in the epitheliocytes and secretory ducts of Brunner's glands, which are characteristic sites of duodenase biosynthesis in cattle. Binding of CD14-specific antibodies in the Brunner's glands, where the antibodies co-localized with the antibodies to duodenase, was also demonstrated. These data indicate the presence of a protein immunologically similar to duodenase in the human duodenal mucosa. Our study demonstrated the absence of its co-localization with cathepsin G in Brunner's glands.

Glucose-6-phosphate dehydrogenase in small intestine of rabbit: biochemical properties and subcellular localization.

Biochemical properties and cellular and subcellular distribution patterns of glucose-6-phosphate dehydrogenase (G6PD) were investigated in small intestine of rabbits. The specific activity of G6PD in fresh homogenates of small intestine was 19 +/- 9 IU/g protein. This value did not change significantly after dialysis. The kinetic and electrophoretic properties of the partially purified enzyme were similar to those found in other rabbit tissues. Enzyme histochemical analysis of G6PD activity using the tetrazolium salt method showed high activity in epithelial cells of villi and crypts of Lieberkuhn. The activity in acinar cells of Brunner's glands was lower than that in epithelium, whereas cells of the muscularis externa showed a very low activity. Immunohistochemical analysis showed that the amounts of G6PD protein were lower in the epithelium than in Brunner's glands and muscularis externa. The differences between distribution patterns of activity and protein of G6PD may reflect the presence of inactive enzyme molecules in Brunner's glands and muscularis externa or posttranslational activation of G6PD in epithelium. Electron microscopic immunocytochemical analysis performed with gold-labelled antibodies showed the presence of G6PD protein throughout the cytoplasm and at smooth endoplasmic reticulum in enterocytes. In Paneth cells and cells of Brunner's glands, G6PD was found in the cytoplasm, at rough endoplasmic reticulum and Golgi complex. Immunolabelling was not found in mitochondria or nuclei. Our findings show that G6PD is heterogeneously distributed in cells of the small intestine and that the enzyme is associated with rough and smooth endoplasmic reticulum to support synthetic functions in these compartments by NADPH production.

Neural pathways regulating Brunner's gland secretion in guinea pig duodenum in vitro.

This study examined the neural pathways innervating Brunner's glands using a novel in vitro model of acinar secretion from Brunner's glands in submucosal preparations from the guinea pig duodenum. Neural pathways were activated by focal electrical stimulation and excitatory agonists, and videomicroscopy was used to monitor dilation of acinar lumen. Electrical stimulation of perivascular nerves evoked large dilations that were blocked by TTX (1 microM) or the muscarinic receptor antagonist 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride (1 microM). The nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium iodide (100 microM) had no effect, and the nerve-evoked responses were not inhibited by hexamethonium (200 microM). Dilations were abolished in preparations from chronically vagotomized animals. Activation of submucosal ganglia significantly dilated submucosal arterioles but not Brunner's glands. Effects of electrical stimulation of perivascular and submucosal nerves were not altered by guanethidine. Capsaicin and substance P also dilated arterioles but had no effect on Brunner's glands. Cholinergic (choline acetyltransferase-immunoreactive) nerve fibers were found in Brunner's glands. These findings demonstrate that Brunner's glands are innervated by cholinergic vagal fibers but not by capsaicin-sensitive or intrinsic enteric nerves.

Dynamic regulation of mucus gel thickness in rat duodenum.

We examined the dynamic regulation of mucus gel thickness (MGT) in vivo in rat duodenum in response to luminal acid, cyclooxygenase (COX) inhibition, and exogenous PGE(2). An in vivo microscopic technique was used to measure MGT with fluorescent microspheres in urethan-anesthetized rats. Duodenal mucosa was topically superfused with pH 7.0 or pH 2.2 solutions with or without PGE(2) and indomethacin treatments. Glycoprotein concentration of duodenal loop perfusates was measured with periodic acid/Schiff (PAS) or Alcian blue (AB) staining. MGT and perfusate glycoprotein concentration were stable during a 35-min perfusion with pH 7.0 solution. Acid exposure increased MGT and PAS- and AB-positive perfusate glycoprotein concentrations. Indomethacin pretreatment increased both PAS- and AB-positive perfusate glycoprotein at baseline; subsequent acid superfusion decreased perfusate glycoproteins and gel thickness. PGE(2) (1 mg/kg iv) simultaneously increased MGT and PAS-positive perfusate glycoprotein concentrations followed by a transient increase in AB-positive glycoprotein concentration, suggesting contributions from goblet cells and Brunner's glands. Parallel changes in MGT and perfusate glycoprotein concentration in response to luminal acid and PGE(2) suggest that rapid MGT variations reflect alterations in the balance between mucus secretion and exudation, which in turn are regulated by a COX-related pathway. Luminal acid and PGE(2) augment mucus secretion from goblet cells and Brunner's glands.

[Duodenase--a potential activator of cascade of digestive proteases]. / Duodenaza--potentsial'nyi aktivator kaskada pishchevaritel'nykh proteinaz.

The substrate specificity of duodenase from bovine duodenum mucosa to synthetic and natural polypeptides was studied. Amino acid residues preferential for duodenase in the P1 and P2 positions of the substrate were determined. It was shown that the enzyme is synthesized in epithelial secretory cells of duodenal (Brunner's) glands and enters, as part of the secreta, into the lumen of the duodenum. The possible role of duodenase as an activator of proenteropeptidase is discussed.

Immunocytochemical demonstration that human duodenal Brunner's glands may participate in intestinal defence.

The immunocytochemical demonstration of IgA and IgM in some secretory units of human Brunner's glands, associated with the presence of secretory component in all secretory cells, indicates the possibility that these glands assist the function of the intestinal crypts in transporting immunoglobulins into the gut lumen. In addition, the presence of muramidase (lysozyme) in the cells of the secretory units suggests that Brunner's glands continuously secrete bactericidal enzyme, thus reinforcing the function of the Paneth cells as contributors to nonspecific defence (innate immunity) in the intestinal tract.

[The immunochemical detection of the site of duodenase synthesis and secretion: its functional role in the proteolytic carrier system]. / Immunokhimicheskoe vyiavlenie mesta sinteza i sekretsii duodenazy: ee funktsional'naia rol' v proteoliticheskom konveire.

A new protease (duodenase) was discovered, isolated from the bovine duodenum, and purified. The protease manifested two types of specific features: (1) dual tripsin- and chymotripsin-like specifics in respect to exoproteolysis of peptides and their derivatives, and (2) conformational specifics in respect to endoproteolysis of peptides. The duodenase seems to be synthetized and secreted in the duodenal (Brunner) glands.

Pancreatic secretory trypsin inhibitor in human Brunner's glands.

Brunner's glands (duodenal glands) in humans are located mainly in the two proximal thirds of the duodenum. They are known to produce and secrete mucin. In recent years, human Brunner's glands have also been shown to express immunoreactivity toward epidermal growth factor-urogastrone (EGF-uro) and lysozyme. These proteins are considered to have a protective function within the gastrointestinal canal. Human pancreatic secretory trypsin inhibitor (PSTI) was recently identified in Brunner's glands. This present study was done by an immunohistochemical method, using monospecific polyclonal antibodies against human PSTI and human lysozyme, respectively. McManus/Alcian blue mucin staining was used to clarify the distribution of mucin. We found immunoreactive PSTI (irPSTI) in seven out of ten specimens. Lysozyme and mucin were present in all ten. While virtually all cells were stained for lysozyme and mucin, irPSTI was restricted to separate lobules and to cells in the ducts.

[The trypsin activity in the chyme and parietal layer of the mucus in experimental exclusion of the Brunner's gland section of the duodenum in rats]. / Aktivnost' tripsina v khimuse i pristenochnom sloe slizi pri éksperimental'nom vykliuchenii brunnerovskogo otdela dvenadtsatiperstnoi kishki u krys.

The duodenal glands secretion into intestine was excluded by surgical ablation or ligation of Brunner's glands region. The trypsin activity in chyme and adherent mucous layer was reduced following these manipulations. Possible reasons for changes established are discussed.

Effect of ayurvedic medicines on beta-glucuronidase activity of Brunner's glands during recovery from cysteamine induced duodenal ulcers in rats.

Biochemical and histochemical studies revealed decreased beta-glucuronidase activity in the Brunner's glands of duodenal ulcerated rats. The enzyme activity showed gradual increase during recovery. Rats treated with a mixture of Ayurvedic medicines (Glycyrrhiza glabra, Terminalia chebula, Piper longum and Shanka Bhasma) recovered faster with concomitant increase in beta-glucuronidase activity in the Brunner's glands. It can be concluded that Ayurvedic medicines used do not act as antacid but improve the secretory status of Brunner's glands involved in the protection against duodenal ulcer.

Tridimensional architecture of the Golgi apparatus and its components in mucous cells of Brunner's glands of the mouse.

The three-dimensional structure of the Golgi apparatus and its components has been analyzed in thin and thick sections of mucous cells of mouse Brunner's glands by using low- and high-voltage electron microscopes and a stereoscopic approach. In thick sections of glands impregnated with osmium or treated to detect nicotinamide adenine dinucleotide phosphatase (NADPase) or thiamine pyrophosphatase (TPPase) activity, the Golgi apparatus appeared, at low magnification, as a continuous network located in the supranuclear region. At higher magnifications and in thin sections of tissue postfixed with reduced osmium and stained with lead citrate or treated to demonstrate phosphatase activity, the following components were observed: on the cis-face of the Golgi stacks, an osmiophilic tubular network referred to as the cis-element; a cis-saccular-compartment composed of a distended porous saccule slightly reactive for NADPase and three or four underlying NADPase-positive, flattened, poorly fenestrated saccules; a trans-saccular-compartment consisting of four to six TPPase-positive saccules or sacculo-tubular elements, prosecretory granules, and "peeling off" trans-tubular networks. The saccules of the cis-compartment were often perforated by large pores in register. The cavities thus formed in the stacks were called wells and were pan-shaped with a mouth directed toward the cis-face of the stacks and a bottom closed by TPPase-positive saccules. The wells always contained 80-nm vesicles. The saccules of the trans-compartment were involved in the formation of secretory granules according to the following proposed sequence of transformation. The secretion product appeared initially as a granular material evenly distributed throughout a slightly distended, poorly fenestrated saccule. These saccules appeared to transform into fenestrated elements with irregular pores and with parts of them taking on the appearance of a tubular network; they were thus referred to as sacculotubular elements. The secretory material initially distributed throughout these elements accumulated in nodular dilatations randomly distributed along the tubular portions of the elements. The dilatations, considered as prosecretory granules, increased in size as they drained the secretory material from the rest of the sacculotubular elements. Such prosecretory granules, large and irregular in shape, "peeled off" from the stacks of saccules with residual saccular or tubular structures still attached to them, some of the latter forming trans-tubular networks. The prosecretory granules detached from such membranous residues, condensed, and finally transformed into spherical secretion granules.

Topography of cholinergic perikarya and nerve fibres as well as cholinergic vesicles in the rat duodenum.

Using the acetylthiocholine staining method, it was possible to visualize acetylcholinesterase (AChE)-stained neuronal cell bodies and nerve endings as well as AChE-positive vesicles in the rat duodenum. AChE-reactive perikarya were seen with certainty only in the myenteric plexus. They were 40 micron in diameter and were mostly localized in groups within the ganglia (3-6 neurons per ganglion). Some thick, AChE-reactive nerve processes, running over a long distance in interconnecting nerve fibre strands, had their origin from AChE-containing myenteric plexus perikarya. AChE-stained nerve fibres were detected in the myenteric and submucosal plexus as well as in the longitudinal and circular smooth muscle cell layer. AChE-positive nerve fibres were in close contacts with blood vessels, probably arterioles, Brunner's gland cells and epithelial cells. A conspicuously high density of AChE-positive nerve fibres was noted in the longitudinal smooth muscle layer, while AChE-stained nerve fibres were visualized only sporadically in the circular smooth muscle layer. Some Brunner's gland cells and epithelial cells contained AChE-reactive vesicles, which were constantly localized on the basal cell portion. The present findings might indicate that acetylcholine possesses important physiological roles as neurotransmitter and/or neuromodulator in the rat duodenum.

Myelin figures in the basal-granulated cells of human Brunner's glands.

Peculiar myelin figures were abundantly found in some basal-granulated cells including S, D1 and I cells in human Brunner's glands. Intense acid phosphatase activity was found in the periphery of the myelin figures, indicating that they were secondary lysosomes or residual bodies. The acid phosphatase activity was also found in some secretory granules. There were some secretory granules which were partly membranous in content, suggesting the initial stage of their degradation into myelin figures. There were also features indicating the fusion of secretory granules with the myelin figures. All these findings suggest that the myelin figures are the products of lysosomal degradation of secretory granules. The rate of occurrence of basal-granulated cells containing myelin figures in Brunner's glands tended to be higher in subjects with duodenal ulcer than in cases of gastric cancer or ulcer.

Carbonic anhydrase in the monkey stomach and intestine.

The distribution of carbonic anhydrase in the stomach and intestine of the cynomolgus monkey (Macaca fascicularis) was studied by the histochemical method of Hansson. In the gastric surface epithelium high enzyme activity was found in the cytoplasm and at the lateral cell borders. The parietal cells in the gastric glands also showed high enzyme activity, while the chief cells were less active. The mucous cells in the pyloric glands and in the Brunner's glands demonstrated a staining pattern similar to that of gastric surface cells. The mucosa of the duodenum and the jejunum was less intensely stained than the gastric mucosa. The enzyme activity was located at the lateral cell borders of the enterocytes, with weak or no cytoplasmic activity. Goblet cells and Paneth cells were unstained. In the ileum a small number of epithelial cells displayed high enzyme activity; their identity is not clear at present. In the cecum and colon large amounts of cytoplasmic carbonic anhydrase were found in the surface epithelium and in the upper part of the glands. Capillaries showing clear enzyme staining were found in the mucosa of all tissues; they were often located close to the surface epithelium and the glands. In the stomach, cecum and colon the distribution of the enzyme in the monkey appears very similar to that reported for other mammalian species, but in the small intestine clear differences exist. The functional role of carbonic anhydrase at the various sites in the gastrointestinal tract is only partly understood.

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.