Effect of tannic acid on brush border disaccharidases in mammalian intestine.

Tannic acid is a glucoside (penta-m-digallolyl-glucose), which exhibits a wide variety of physiological functions. Around neutral pH, 0.4 mM tannic acid produced 84% inhibition of rat brush border sucrase activity, but 35-40% enzyme inhibition was observed in the rabbit intestine at 0.08 mM concentration. In the mice, 74-77% enzyme inhibition was observed at 0.05 mM concentration of tannic acid. The observed inhibition was reversible in rat intestine. Tannic acid (0.2 mM) also inhibited lactase (18% in adult and 71% in suckling animals), maltase (76%) and trehalase (88%) activities in rat intestine. pH versus activity curves showed that 0.2 mM tannic acid inhibited enzyme activity in rat by 91% at pH 5.5 which was reduced to 14% at pH 8.5 compared to the respective controls. In the rabbit 18-60% enzyme inhibition was noticed below pH 7.0, however at pH 8.5, it was of the order of 38%. Kinetic analysis revealed that tannic acid is a competitive inhibitor of rat brush border sucrase at pH 6.8. Effect of tannic acid together with various -SH group reacting reagents revealed that the enzyme inhibition is additive in nature, suggesting the distinct nature of binding sites on the enzyme for these compounds. The results suggest that tannic acid is a potent inhibitor of intestinal brush border disaccharidases, and could modulate the intestinal functions.

Susceptibility of lactase to luminal proteases in developing rat intestine.

BACKGROUND: Postnatal development of rat intestine is associated with a decline in brush-border lactase activity. This phenomenon is similar to the adulthood hypolactasia in humans. However, the mechanism underlying this process is not understood. METHODS: The effect of luminal proteases from adult rat intestine on the intestinal lactase activity in animals aged 7, 14, 21 and 30 days was studied in in vitro experiments. Lactase levels were estimated using enzyme assays and Western blot analysis. RESULTS: Incubation of purified brush borders with increasing concentrations of luminal proteases reduced the lactase activity in intestine of 7-day-old rats, but not in that of adult animals. Western blot analysis revealed low signal of the 220-kDa lactase protein in 7-day-old animals, but not that of older weaned animals. CONCLUSIONS: Our findings suggest that luminal proteases may be responsible for the maturational decline in intestinal lactase activity.

Tissue alterations in the Guinea pig lateral prostate following antiandrogen flutamide therapy

The flutamide antiandrogenic effects on the Guinea pig male prostate morphology in puberal, post-puberal and adult ages were evaluated in the present study. Daily-treated group animals received flutamide subcutaneous injection at a dose of 10 mg/Kg body weight for 10 days. The control group animals received a pharmacological vehicle under the same conditions. The lateral prostate was removed, fixed and processed for light and transmission electron microscopy. The results revealed an increase of the acinus diameter in the treated puberal animals and straitness in the stromal compartment around the acini. The epithelial cells exhibited cubic phenotype. In the post-puberal and adult animals, a decrease of the acinus diameter was observed, as well as an increase of the smooth muscle layer and presence of the folds at epithelium. The ultrastructural evaluation of the secretory cells in the treated group demonstrated endomembrane enlargement, mainly in the rough endoplasmic reticulum and Golgi apparatus. In addition, a decrease of the microvilli and alterations in the distribution patterns and density of the stromal fibrillar components were observed. In conclusion, the flutamide treatment exerts tissue effects on the lateral prostate, promoting stroma/epithelium alterations.

Apoptogenic effect of the lipophilic o-naphthoquinone CG 10-248 on rat hepatocytes: light and electron microscopy studies

CG 10-248 (3,4-dihydro-2,2-dimethyl-9-chloro-2H-naphtho[1,2b]pyran-5,6-dione; CG-NQ), a beta-lapachone analogue, modified the ultrastructure of rat hepatocytes, as demonstrated by light and electron microscopy. After 4 h incubation with 100 microM CG-NQ, the following effects were observed: (a) nuclear chromatin condensation; (b) chromatin fragmentation; (c) displacement of mitochondria, concentrated around the nucleus; (d) disruption or expansion of mitochondrial outer or inner membranes, respectively; (e) displacement and alteration of endoplasmic reticulum (rough and smooth); (f) decrease of microvilli; (g) blebbing of plasma membrane and production of apoptotic bodies formed by folding of plasma membrane fragments around mitochondria or peroxysomes; and (h) production of hydrogen peroxide. Expression of such effects varied according to hepatocyte samples and taken together strongly support an apoptotic action of CG-NQ dependent on reactive oxygen species.

Effect of Salmonella typhimurium toxin on the expression of rabbit intestinal functions.

BACKGROUND & OBJECTIVES: Infection by Salmonella Typhimurium is one of the leading causes of intestinal dysfunction, however the underlying mechanism of this effect is largely unknown. Hence the effect of enterotoxin secreted by Salmonella Typhimurium-(S-LT) was studied on D-glucose absorption and brush border enzymes in rabbit ileum. mRNA levels encoding these proteins were also analysed. METHODS: Adult male New Zealand white rabbits were used. The polymyxine B extract of enterotoxin obtained from Salmonella Typhimurium was tested for the presence of enterotoxicity by rabbit ileal loop test. D-glucose uptake by ileal tissue was measured by the tissue accumulation method. Intestinal brush border membranes were isolated and the effect of S-LT on various brush border enzymes studied. RESULTS: S-LT significantly inhibited (P < 0.01) the absorption of Na+ dependent D-glucose uptake but had no effect on Na+ independent sugar uptake in rabbit ileum. The activities of brush border sucrase (72% P < 0.001) and lactase (47% P < 0.01) and alkaline phosphatase (43% P < 0.01) were also significantly reduced in infected animals as compared to the controls. Northern blot analysis revealed that mRNA levels encoding Na+ glucose co-transporter (SGLT1), brush border lactase and sucrase activities were unaffected in Salmonella infected rabbit ileal loops. INTERPRETATION & CONCLUSION: The findings suggest that the intestinal dysfunctions observed in Salmonella infection are unrelated to mRNA expression encoding Na+ glucose co-transporter and brush border enzyme proteins in rabbit ileum.

Effects of endosulfan on intestinal functions in protein-malnourished rats.

In rats fed 18% protein diet, administration of endosulfan (2mg/kg body weight daily for 7 days) significantly decreased the brush border sialic acid and increased the hexoses contents. The intestinal uptake of glucose was increased while that of glycine and calcium was reduced. Brush border enzymes and lipids were not affected. However, in protein malnourished rats (fed 8% protein) exposed to endosulfan, brush border sucrase and peptidase activities were enhanced, while alkaline phosphatase activity was decreased compared to untreated malnourished animals. Membrane sialic acid content was low while fucose and cholesterol levels were augmented in endosulfan fed malnourished animals. The uptake of glucose and glycine was elevated under these conditions. These results Suggest that the nutritional status of the animals has an important bearing on thc susceptibility of intestinal tissue to endosulfan toxicity in rats.

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.

Effect of insecticidal crystal proteins of Bacillus thuringiensis var. israelensis on the enzymes of rat intestinal brush border membrane vesicles.

In vivo treatment of intestinal brush border membrane vesicles with solubilized insecticidal crystal proteins (ICP) from the two strains of B. thuringiensis var. israelensis (VCRC B17 and VCRC MB24) caused no adverse effect on gamma glutamyl transpeptidase, Na+K+ATPase, sucrase and lactase enzymes. But, exposure of membrane vesicles to solubilized ICP's in vitro, lead to significant reduction in the activity of Na+K+ATPase, sucrase and lactase enzymes.

Inhibition of brush border sucrase by indoline 2,3-dione (isatin) in rat small intestine.

Isatin (15-25 mM) inhibited rat brush border sucrase by 40% in presence of Na+ and the inhibition was enhanced to over 60% in sodium free medium. Sucrase inhibition by isatin was dependent on pH. Kinetic analysis revealed a pure capacity type (Vmax-effect) inhibition of sucrase activity by isatin in presence of sodium. But it changed to affinity type (K-effect) in sodium free medium. The value of Ki was around 20-25 mM under these conditions. Enzyme inhibition by isatin was alleviated by increasing Na+ or sucrose concentrations. Other monovalent cations like K+, Li+ and Cs+ were also effective in restoring the enzyme activity to control levels. The effectiveness of the metal ions in alleviating the enzyme inhibition was in the order of Na+ > Cs+ > K+ > Li+.

Modulation of monkey small intestinal brush border membrane D-glucose transport by nonesterified fatty acids.

Brush border membranes isolated from monkey intestinal mucosa was found to contain considerable amount of nonesterified fatty acids. Incubation of brush border membranes with fatty acid free albumin selectively removed the free fatty acids more than 80% without altering the level of phospholipids or cholesterol. The sodium dependent D-glucose transport was stimulated by the albumin treatment. Kinetic study showed that albumin treatment did not alter the apparent affinity (Km) of the transporter for glucose whereas the maximal velocity (Vmax) was increased significantly. The sodium dependent D-glucose transport was inhibited by the exogenously added unsaturated fatty acids. Saturated fatty acids and methyl esters of unsaturated fatty acids showed no inhibition. Based on these results, it may be concluded that free fatty acids inhibit the sodium dependent intestinal D-glucose transport either by directly interacting with the transport protein or by abolishing the sodium gradient.

Studies on the role of iron binding ligands and the intestinal brush border receptors in iron absorption.

With a view to identifying ligands that could be used as promoters of iron absorption, the affinity of a number of iron chelating agents and the efficiency with which they can donate iron to the brush border receptors has been studied. A number of organic and inorganic compounds were found to chelate iron and keep it soluble at pH 7.5 of the intestinal lumen. This ligand-bound iron was taken up by the intestinal brush border receptors with varying degree of efficiency; ascorbic acid being the most effective and EDTA and citrate the least effective in donating the chelated iron to the receptors. Several polyphosphate compounds, used as food additives, chelated iron and kept it in solution but showed moderate potency for donating iron to the receptors.

Kinetic characteristics of soluble and brush border alkaline phosphatase and sucrase activities in developing rat intestine: effect of hormones.

Suckling rat intestine contains 35 and 65% of the cytosolic and membrane-bound alkaline phosphatase (AP) activities. The corresponding values for sucrase were 20 and 80% respectively. The amount of the soluble enzymes was reduced to 7-11% in adult rat intestine. Administration of cortisone, thyroxine or insulin to suckling animals induced adult type distribution of the enzymes. There were apparent differences in kinetic characteristics of soluble and brush border enzymes, but the kinetic properties of the normally developed and hormone-induced AP and sucrase were essentially similar. This suggested identical nature of these enzymes under these conditions. A biphasic Arrhenius plot was obtained for AP in weaned and hormone injected pups with a break point around 18 degrees C, while the soluble enzyme yielded a monophasic curve (Ea = 8-11 kcal/mole). Arrhenius plot for sucrase was monophasic in the suckling, hormone-injected and adult rat intestine (Ea = 8.3-15.1 kcal/mole). Membrane-bound enzymes were generally labile, while soluble enzyme activities were stable to heat treatment (sucrase at 50 degrees C and AP at 60 degrees C) in various experimental groups.