Histochemical analysis of molluscan stomach and intestinal alkaline phosphatase: A sialoglycoprotein

Summary Though sialoprotein nature of alkaline phosphatase of certain mammalian organs has been suggested by biochemical investigations, no histochemical techniques have yet been applied to elucidate this concept. With this view, the alkaline phosphatase of stomach and intestine of a mollusc—Semperula maculata—was analysed histochemically to elucidate its sialoglycoprotein nature. The localisation of alkaline phosphatase and sialic acid was investigated by employing well known and standard histochemical techniques.Alkaline phosphatase was localised selectively in the brush border of the mucosa of stomach and intestine, it was Mg⁺⁺ nonsensitive but showed a structure-linked sensitivity to phenylalanine. The sialomucins were selectively localised in the brush border, whereas the goblet cells contained both the sialomucins and sulfomucins, and the connective tissue of lamina propria contained sulfomucins. The localisation of alkaline phosphatase and sialomucins in the brush border uniquely coincided with each other. The alkaline phosphatase activity in the brush border was completely lost after neuraminidase treatment at 37.5° C for 16 h. Such effect of neuraminidase on alkaline phosphatase activity was pH dependent and controlled by velocity of reaction. Heat-inactivated neuraminidase showed no effect on alkaline phosphatase activity.These histochemical results have been interpreted as suggesting a sialoglycoprotein nature of alkaline phosphatase in the brush border, and sialic acid somehow seems to be essential for enzyme activity. These results, thus, indicate necessity of visualising some of the sialo-glycoproteins as macromolecules with catalytic activity.     

Acute dieldrin toxicity: effect on the uptake of glucose and leucine and on brush border enzymes in monkey intestine

Administration of a single oral dose of dieldrin (20 mg/kg body wt.) to rhesus monkeys considerably elevated the uptake of glucose and the activities of brush border sucrase, lactase, maltase and alkaline phosphatase in intestine compared to control animals. Leucine uptake and leucine amino peptidase activity was significantly depressed in pesticide-treated animals. Kinetic studies with brush border sucrase revealed that augmentation of enzyme activity in pesticide-fed animals was due to an increase in the disaccharidase content.     

Subcellular localization of enterokinase (Enteropeptidase EC 3.4.21.9) in rat small intestine

The subcellular localization of enterokinase is controversial. In this study, enterokinase was extracted from a soluble fraction and a brush border fraction of rat small intestine by differential centrifugation. The soluble fraction contained 41% of the initial enterokinase activity while the brush border fraction contained only 4.6% of the initial activity. In contrast, alkaline phosphatase monitored as a brush border marker, yielded 26.3 in the brush border fraction and only 6% in the soluble fraction. Further separation of the soluble fraction on a Sepharose 4B column revealed three peaks of enterokinase activity. One small peak (3%) of a bound enzyme (M_r, 2·10^?6) and two larger peaks of free enzyme (M_r, 3·10⁵ and 9·10⁵). In contrast, alkaline phosphatase major fraction was in a high molecular weight peak of bound enzyme. When the brush border fraction was chromatographed only a single peak of bound enterokinase and alkaline phosphatase were found. In the lower part of the small intestine, no brush border-bound enterokinase was found, while the peak of alkaline phosphatase was the same as in the upper intestine. These data suggest that enterokinase activity in the rat intestine is mainly in a free form localized in the mucin and soluble fraction and to a negligible extent in the brush border.     

Influence of duodenal secretions and its components on release and activities of human brush-border enzymes

The in vitro effects of human duodenal secretions and various combinations of its components on activity and release of enzymes from the human brush border were examined. Sucrase retained activity for 90 min in duodenal secretions, and maltase was almost as stable; lactase lost activity rapidly and alkaline phosphatase was of intermediate stability. Inactivation of lactase could only be partly (50%) attributed to luminal proteases, bile salts and phospholipids played no role. Rate of release of an enzyme from the brush border bore no relationship to its rate of inactivation. When individual proteases were studied, elastase was the most potent for releasing disaccharidases from the brush border; trypsin was ineffective alone but augmented the effect of elastase. Sucrase and maltase were activated by proteolytic release, but activation was abolished by simultaneous exposure of brush borders to bile salts. Lactase was released and rapidly inactivated by proteinases, while alkaline phosphatase appeared to be inactivated without significant release. These results show that there are significant interactions between luminal factors which have been inapparent when studying them in isolation. Loss of functionally useful enzyme does not follow release of sucrase or maltase from the brush border into the lumen but does follow release of lactase. Study of the susceptibility of lactase to inactivation by luminal factors in the various forms of lactose intolerance is warranted.     

Histochemical distribution of digestive enzymes in intestine of goldline, Sarpa salpa L. 1758

Histochemical localization of non‐specific esterase, alkaline and acid phosphatase in the intestine of free‐living goldline (Sarpa salpa L. 1758) was investigated. Fish were caught in the vicinity of the town of Zadar (Adriatic Sea, Croatia), and samples of three parts of the intestine proper (anterior, middle and posterior) as well as the rectum were used for presentation of non‐specific esterases, alkaline phosphatase and acid phosphatase. Non‐specific esterase activity was found in the cytoplasm and brush border of enterocytes in all investigated intestinal segments and the rectum. The activity was stronger in the middle and posterior part of the intestine but weaker in the anterior segment of the intestine as well as in the rectum. Intestinal alkaline phosphatase was detected in the brush border and supranuclear cytoplasm of enterocytes of all investigated intestinal segments. Enzymatic activity gradually decreased in a posterior direction. Acid phosphatase activity was observed as a fine granular reaction product in the supranuclear region of enterocytes and was almost equal in all investigated intestinal segments as well as in the rectum. The possible role of enzymes in intracellular digestion and transport is discussed.     

Chemiluminescent assay of enzymes using proenhancers and pro-anti-enhancers

Enhanced chemiluminescent assays for hydrolase enzymes have been developed using proehancer and pro-anti-enhancer substrates. Alkaline phosphatase is measured using disodium para-iodophenyl phosphate (proenhancer) which is converted to para-iodophenol and this in turn enhances the light emission from the horseradish peroxidase catalysed chemiluminescent oxidation of luminol by peroxide. An alternative strategy uses para-nitrophenyl phosphate which is converted by alkaline phosphatase to para-nitrophenol which inhibits the enhanced chemiluminescent reaction. The detection limit for the enzyme using the proenhancer and pro-anti-enhancer assays was 100 attomoles and 1 picornole, respectively. The proenhancer strategy was effective in assays for beta-D-galactosidase, beta-D -glucosidase and aryl sulfatase. A limited comparison of the proenhancer and a conventional colorimetric assay for an alkaline phosphatase label in an enzyme immunoassay for alpha-fetoprotein showed good agreement.     

Enzymes of the human intestinal brush border membrane. Identification after gel electrophoretic separation

The position of a number of human intestine brush border membrane enzyme activities in polyacrylamide gels after electrophoresis has been determined. These activities are, in order from the origin, maltase/glucoamylase, lactase/phlorizin hydrolase, maltase/sucrase/isomaltase, enteropeptidase, trehalase and γ-glutamyltransferase. Leucylnaphthylamide hydrolyzing activity was inactivated by sodium dodecylsulfate and its position was not determined. The positions of the activities have been correlated with the positions of protein bands previously determined. One such band situated between enteropeptidase and alkaline phosphatase has not been identified.     

Enzymes of the human intestinal brush border membrane. Identification after gel electrophoretic separation.

The postition of a number of human intestine brush border membrane enzyme activities in polyacrylamide gels after electrophoresis has been determined. These activities are, in order from the origin, maltase/glucoamylase, lactase/phlorizin hydrolase, maltase/sucrase/isomaltase, enteropeptidase, trehalase and gamma-glutamyl-transferase. Leucylnaphthylamide hydrolyzing activity was inactivated by sodium dodecylsulfate and its position was not determined. The positions of the activities have been correlated with the positions of protein bands previously determined. One such band situated between enteropeptidase and alkaline phosphatase has not been identified.     

Characterization of brush border membrane-bound alkaline phosphatase activity in different segments of the porcine small intestine

This study was conducted to characterize enterocyte apical membrane-bound alkaline phosphatase activity in different segments of the porcine small intestine. Duodenal, jejunal, and distal ileal segments were isolated from three 26-kg pigs and enterocyte brush border membrane, enriched between 19- and 24-fold in sucrase specific activity, was prepared by Mg(2+) precipitation and differential centrifugation. With P-nitrophenyl phosphate as substrate, the optimum pH for porcine brush border membrane-bound alkaline phosphatase activity was defined to be 10.5 for all three segments. At the optimal pH, the kinetics of membrane-bound alkaline phosphatase were determined for the three intestinal segments. The affinity of this enzyme (K(m), mM) in the jejunum (0.64 +/- 0.07) was four times greater than that in the duodenum (2.75 +/- 0.59) and the distal ileum (2.71 +/- 1.14). These results indicate that different isomers of membrane-bound alkaline phosphatase might have been expressed in different segments of porcine small intestine. The maximal specific activity (V(max), micromol/mg protein . min) of this enzyme was highest in the duodenal (7.74 +/- 0.95), intermediate in the jejunal (4.31 +/- 0.18), and lowest in the distal ileal (3.53 +/- 0.84) brush border membrane. Therefore, the maximal specific activity of brush border membrane-bound alkaline phosphatase along the intestinal longitudinal axis in growing pigs decreases from the duodenum toward the distal ileum.     

Organ culture of suckling rat intestine: Comparative study of various hormones on brush border enzymes

Summary Jejunal mucosa of 6 d-old rats were cultured for 24 and 48 h in the presence of thyroxine, insulin, pentagastrin, glucagon, epidermal growth factor (EGF) or dibutyryl-A-3:5-MP cyclic with or without dexamethasone (DX). The enzymes were assayed on the purified brush borders. The various agents added alone to the basic culture medium had no effect with the exception of DX on the levels of enzyme activities. Dexamethasone alone induced sucrase, stimulated maltase, and protected other brush border enzyme activities (aminopeptidase, lactase, and alkaline phosphatase). When added to DX-supplemented medium, only the following factors modified the levels of enzymatic activities observed with DX alone. Insulin (10⁻⁶ M) increased maltase, alkaline phosphatase, and lactase activity to a greater extent than DX at 24 h culture, the effect being maintained at 48 h on alkaline phosphatase only. At 48 h culture, both EGF (10⁻⁸ M) and dbcAMP (10⁻³ M) decreased DX-induced sucrase activity. The latter agent also depressed DX-stimulated aminopeptidase activity. This work was supported by the Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, and a grant 79.7.1243 from the Délégation Générale a la Recherche Scientifique et Technique. P. M. S. is a recipient of a grant from Fondation de la Recherche Médicale (France).     

Isolation and characterization of Brush border fragments from mosquito mesenterons

Brush border fragments were isolated from homogenates of mesenterons from the mosquito, Culex tarsalis, by a combination of Ca²⁺ precipitation and differential centrifugation. These preparations were routinely enriched seven- to eightfold for the brush border marker enzyme, leucine aminopeptidase. Alkaline phosphatase, a putative brush border marker for both vertebrate and invertebrate brush borders, was found to be unsuitable for Cx. tarsalis. Isoelectric focusing electrophoresis coupled with histochemical enzyme detection was used to enumerate isozymic species of nonspecific esterases [3], leucine aminopeptidase [1], and alkaline phosphatase [1] in isolated brush border fragments. Leucine aminopeptidase activity was solubilized by papain digestion, suggesting an extrinsic active site for this membrane-bound enzyme. The predominant nonspecific esterase isozyme remained membrane-bound. Conventional staining (ie, Coomassie Blue and silver) of proteins separated by isoelectric focusing, sodium dodecylsulfate, and two-dimensional electrophoresis indicated a simple pattern for brush border fragments, with two proteins predominating among the 11–14 routinely detected.     

Effect of antiulcerogenic drug, ranitidine, on intestinal absorption and digestive functions in mice

Oral administration of antiulcerogenic drug ranitidine significantly inhibits glucose and amino acid uptake in small intestinal segments. It also inhibits activities of brush border membrane disaccharidases and alkaline phosphatase but increases the activity of leucine aminopeptidase. Kinetic analysis reveals noncompetitive and mixed type of inhibition for disaccharidases and alkaline phosphatase, respectively. In vitro addition of the drug to membrane preparation shows similar type of results as seen in vivo with the inhibition constant (ki) for sucrase, lactase, maltase and alkaline phosphatase as 12.5, 5, 11.5 and 19.5 mM, respectively.     

Age related effects of organochlorine insecticide lindane on intestinal brush border membrane in rats.

The effect of oral administration of lindane (gamma-HCH) has been studied on the intestine in 10-day, 20-day and 100-day old rats. In 10 day-old suckling pups exposed to lindane, there was a significant decrease in the activities of sucrase (29%), lactase (20%) and that of alkaline phosphatase (24%) compared to control. Sialic acid content of the brush borders was significantly decreased (29%) in 10-day old as well as in 20- and 100-day old rats (20 and 25% respectively), while fucose content of the membranes was significantly enhanced in all the age groups upon pesticide treatment. Among the brush border lipids, cholesterol content was significantly increased in all the age groups studied, the maximum increase of 35% being observed in 10-day-old rats. Membrane phospholipids were also increased in 20- and 100-day old animals (22% each) on lindane exposure. The present studies indicated that brush border membranes of suckling rat intestine were more susceptible to pesticide induced changes compared to older animals.     

Histochemical distribution of potassium-dependent p-nitrophenylphosphatase in the calf intestine

Summary Potassium-dependentp-nitrophenylphosphatase was demonstrated, using the lead citrate method of Mayaharaet al. (1980), in frozen sections of calf intestine fixed in formalin-calcium. The calcium chloride included in the fixative was shown to improve the localization of the reaction markedly. The phosphatase activity observed in the basolateral cell borders of the surface epithelium in the small intestine and colon was reduced by 10mm oubain and by substitution of sodium ions for potassium ions, confirming that the reaction was representative for the second step in the Na⁺/K⁺-ATPase complex. The intensity of the basolateral enzyme reaction was in the order: colon > duodenum, proximal jejunum > ileum > middle and distal jejunum. The crypts reacted weakly. A reaction in the brush border of the proximal jejunum and duodenum and a granular reaction in the supranuclear cytoplasm of the epithelial cells was not influenced by oubain. The staining pattern for the potassium-dependent phosphatase differed from that of alkaline phosphatase and Mg²⁺-dependent ATPase, which gave a reaction that was restricted to the brush border.     

Imaging of chemiluminescent signals with cooled CCD camera systems

We investigated imaging of chemiluminescent signals from 1,2-dioxetanes with cooled CCD cameras. Non-radioactive detection methods for biomolecules utilizing these chemiluminescent substrates for alkaline phosphatase have been developed. Applications which have been successfully adapted to this technology include Southern and Northern blotting, immunoblotting, ELISA methods and DNA sequencing. Dephosphorylation of the dioxetane CSPD by alkaline phosphatase generates an unstable anion that decomposes resulting in light production. The wavelength of the emitted light is approximately 460nm. We have utilized Photometrics Star and MXC 200L cooled CCD cameras for direct imaging of chemiluminescent signals. Benefits of utilizing a CCD detector include rapid data digitization and more accurate quantitation of chemiluminescent signals compred to film-based densitometry owing to the significantly greater dynamic range. Chemiluminescent images from dot blots of biotinylated DNA, Southern blots and DNA sequencing gel blots were obtained. In a chemiluminescent microtitre plate assay, serial dilutions of alkaline phosphatase spanning four orders of magnitude can be detected. Our results indicate that the digitization of chemiluminescent signal data with cooled CCD cameras is an excellent alternative to ³²P detection methods utilizing storage phosphor screen imaging systems.     

Expression of sodium-glucose co-transporter and brush border disaccharidases in Giardia lamblia infected rat intestine

The absorption of D-glucose and brush border membrane disaccharidases in the intestine of rat during infection by Giardia lamblia has been studied. The level of mRNA encoding Na+/glucose co-transporter (SGLT1) and brush border sucrase and lactase activities were also analyzed. At the peak of infection, i.e, day 7, 11 and 15 post-infection, there was a marked decrease in the signal of 4.5 kb and 2.8 kb mRNAs encoding SGTL1 compared to the controls. A similar decrease in sucrase and lactase mRNA's (6.5 kb and 6.8 kb respectively) was also observed under these conditions. This corresponds to observed decrease in the rate of Na(+)-dependent D-glucose uptake and low activities of brush border sucrase and lactase under these conditions. There was no change in Na(+)-independent D-glucose uptake in giardia infected rat intestine. These findings suggest that the down regulation of the expression of SGLT1 and brush border sucrase and lactase activities may be responsible for the observed malabsorption in G. lamblia infection.     

Histophysiology of digestion and observations on the structure of the alimentary canal in the ectosymbiont Chaetogaster limnaei limnaei Baer, 1827 (Annelida: Oligochaeta)

The naidid oligochaete Chaetogaster limnaei limnaei has an alimentary canal consisting of a mouth, pharynx with a dorsal pharyngeal pad, esophagus, stomach, anterior and posterior intestine, and anus. The diet is omnivorous but limited by particle size. Unattached food organisms are sucked into the pharynx while sessile organisms are plucked from the substratum. Granules of acid mucosubstances that stain purple with neutral red are secreted into the stomach lumen after food enters, rapidly increasing the acidity from pH 3 to 1.5. Acid induced lysis of the organisms initiates autolysis before the food is passed into the alkaline, pH 7 to 8, anterior intestine. Ciliated intestinal cells showed arylamidase, acid phosphatase and C-esterase active granules indicating primary lysosomes with secondary lysosomes being recognized in electron micrographs suggesting intracellular digestion. Arylamidase and alkaline phosphatase activity appears in the intestinal margins during the alkaline phase of digestion. Scattered, pyramidal cells found only in the anterior intestine contain yellow refractile spheres. The spheres stain alcian blue pH 2.5 and bromophenol blue positive and exhibit a strong acid phosphatase activity all the time with A-esterase active granules surrounding them. Glycogen and lipids are stored mainly in the chlorogague cells. Many of the yellow refractile granules in the stomach and intestinal cells are bacteria.     

Ultrastructure of Enteronephridia and General Description of the Alimentary Canal in Trochonerilla mobilis and Nerillidium troglochaetoides (Polychaeta,Nerillidae)

The alimentary canals of Trochonerilla mobilis and Nerillidium troglochaetoides consist of a ventral pharyngeal organ, oesophagus, stomach, intestine, and rectum. Prominent salivary glands lying lateral to the oesophagus discharge their secretions into the buccal cavity. Ciliated canals, the enteronephridia, embedded in the intestinal epithelium, open into the stomach near its border to the intestine. The ventral pharynx comprises a muscle bulb connected to a tonguelike organ by an investing muscle. The whole alimentary canal is ciliated except for the intestine of T. mobilis. The stomach is built up of absorptive cells and posteriorly also of secretory cells, whereas the intestine consists of only one cell type which is considered to be mainly absorptive. A typical microvillar brush border is present only in the intestine of T. mobilis; elsewhere the density of microvilli is low or the cells have irregular apical processes. In N. troglochaetoides the intestine has a ventral ciliary gutter laterally bordered by cells with highly specialized microvilli. The enteronephridia — 3 in N. troglochaetoides and 13 in T. mobilis — are unicellular tubes up to 130 μm long with a microvillar brush border and other cytological features typical for nephridial ducts. These structures are not known in any other polychaete taxon.     

Brush border membrane and amino acid transport

Fundamental differences in midgut structure, physiology, brush border proteins, and transporters among Leptinotarsa decemlineata, lepidopteran caterpillars, other insect taxa, and vertebrates are reviewed. The effects of dietary protein concentration on Manduca sexta midgut amino acid transport and brush border membrane proteins are reported. M. sexta fed diet with reduced protein had elevated levels of leucine aminopeptidase in the brush border membrane. No changes in amino acid transport or alkaline phosphatase activity due to dietary differences were detected. Changes in brush border proteins could affect the toxicity and pathogenicity of microbial agents. © 1996 Wiley-Liss, Inc.     

Organ culture of suckling rat intestine: comparative study of various hormones on brush border enzymes

Jejunal mucosa of 6 d-old rats were cultured for 24 and 48 h in the presence of thyroxine, insulin, pentagastrin, glucagon, epidermal growth factor (EGF) or dibutyryl-A-3:5-MP cyclic with or without dexamethasone (DX). The enzymes were assayed on the purified brush borders. The various agents added alone to the basic culture medium had no effect with the exception of DX on the levels of enzyme activities. Dexamethasone alone induced sucrase, stimulated maltase, and protected other brush border enzyme activities (aminopeptidase, lactase, and alkaline phosphatase). When added to DX-supplemented medium, only the following factors modified the levels of enzymatic activities observed with DX alone. Insulin (10(-6) M) increased maltase, alkaline phosphatase, and lactase activity to a greater extent than DX at 24 h culture, the effect being maintained at 48 h on alkaline phosphatase only. At 48 h culture, both EGF (10(-8) M) and dbcAMP (10(-3) M) decreased DX-induced sucrase activity. The latter agent also depressed DX-stimulated aminopeptidase activity.