Activities Of Brush Border Membrane Enzymes Research Articles

Effect of uranyl nitrate on enzymes of carbohydrate metabolism and brush border membrane in different kidney tissues

Uranium, the heaviest of the naturally occurring elements is widely present as environmental contaminant from natural deposits, industrial emissions and most importantly from modern weapons. Histopathological examinations revealed that uranyl nitrate (UN) exposure caused severe damage to pars recta of renal proximal tubule. However, biochemical events involved in cellular response to renal injury are not completely elucidated. We hypothesized that UN exposure would severely damage kidney tissues and alter their metabolic functions. Rats were treated with a single nephrotoxic dose of UN (0.5mg/kg body weight) i.p. After 5d, effect of UN was studied on the activities of various enzymes of carbohydrate metabolism, brush border membrane (BBM) and oxidative stress in different kidney tissues. Activity of lactate dehydrogenase increased whereas activities of isocitrate, succinate and malate dehydrogenases, glucose-6-phosphatase and fructose-1,6-bisphosphatase significantly decreased by UN exposure. Activity of glucose-6-phosphate dehydrogenase decreased whereas that of NADP-malic enzyme increased. The activities of BBM enzymes were significantly lowered and after dissociation from BBM excreted in urine. Lipid peroxidation and the activities of superoxide dismutase and glutathione peroxidase increased whereas catalase activity decreased by UN. UN treatment caused specific alterations in the activities of metabolic and membrane enzymes and perturbed antioxidant defenses.

The Influence of Age on Intestinal Dipeptidyl Peptidase IV (DPP IV/CD26), Disaccharidases, and Alkaline Phosphatase Enzyme Activity in C57BL/6 Mice

The objective of this study was to determine and describe the age-related changes in intestinal brush border membrane enzyme activities that occur in C57Bl/6 mice. Specifically, jejunal, duodenal, and ileal dipeptidyl peptidase IV/CD26, disaccharidase (lactase, sucrase, and maltase), and alkaline phosphatase activities were determined. A significant correlation between analyzed intestinal brush border membrane enzyme activities and animal age was found. Our study revealed that intestinal dipeptidyl peptidase IV/CD26, lactase, sucrase, maltase, and alkaline phosphatase activities decline significantly with age (p < .05). Nevertheless, the horizontal (duodenum to ileum) enzyme activity patterns are not affected by age.

Effect of cisplatin on brush border membrane enzymes and anti-oxidant system of rat intestine

Cisplatin (CP) is a widely used antineoplastic agent which exhibits gastrointestinal toxicity. The present work was done to study the effect of administration of CP on brush border membrane (BBM) enzymes and anti-oxidant system of rat intestine. Male Wistar rats were given a single intraperitoneal dose of CP (6 mg/kg body weight) and then sacrificed 1, 3, 5 and 7 days after this treatment. Control animals were given saline only. The administration of CP led to significant decline in the specific activities of BBM enzymes both in the mucosal homogenates and isolated membrane vesicles. Kinetic studies showed that the V max of the enzymes was decreased in BBM vesicles from CP treated rats while the K m remained unchanged. The activities of catalase, Cu–Zn superoxide dismutase, glucose 6-phosphate dehydrogenase and glutathione reductase decreased while the activities of glutathione S-transferase and thioredoxin reductase increased in CP treated animals compared to the control group. Lipid peroxidation and total sulfhydryl groups were also altered upon CP treatment indicating the generation of oxidative stress. The maximum changes in all the parameters studied above were 3 days after administration of CP and then recovery took place on days 5 and 7. Thus, the administration of CP leads to significant alterations in the activities of BBM enzymes and the anti-oxidant status of rat intestine.

Changes in Growth and Function of Chick Small Intestine Epithelium Due to Early Thermal Conditioning

The effect of exposure to heat at 3 d of age on small intestine functionality and development was assayed by measuring villus size, proliferating enterocytes, and brush-border membrane (BBM) enzyme expression and activity. Results showed that thermal conditioning caused an immediate effect characterized by lowered triiodothyronine (T3) level, reduced feed intake, and depressed enterocyte proliferation and BBM enzyme activity. A second series of effects, observed 48 h posttreatment, was characterized by elevated T3, increased feed intake, increased enterocyte proliferation, and higher expression and activity of BBM enzymes. The association between ambient temperature, feed intake, growth rate, and plasma T3 levels was reflected in the structure and function of the intestinal tract. The results suggest that thermal conditioning at an early age influences T3 concentrations, which in turn alter the intestinal capacity to proliferate, grow, and digest nutrients. However, these experiments were not able to discriminate between effects due to feed intake and those due to thermal conditioning. The treatments modulated changes in the intestinal tract following thermal treatment.

Expression of the H Type 1 Blood Group Antigen during Enterocytic Differentiation of Caco-2 Cells

We made a comparative study of the structures of the oligosaccharides on the glycoproteins from Caco-2 human colonic adenocarcinoma cells, before and after differentiation. Enterocytic differentiated Caco-2 cells highly express H type 1 blood group antigen on the cell surface as well as activities of brush border membrane hydrolases, such as dipeptidyl peptidase IV and alkaline phosphatase. A strong correlation was observed between the amounts of H type 1 blood group antigen and the degrees of differentiation. Structural analysis with use of lectin affinity high performance liquid chromatography revealed that typical mucin-type sugar chains of the glycoproteins from undifferentiated cells have H type 2 group, linear polylactosamines, and core 1 structure. On the other hand, differentiated cells newly contain H type 1 and Le(b) groups and core 2 structure. Mucins with H type 1 group make contact with brush border membrane enzymes on differentiated cells. Furthermore benzyl 2-acetamide-2-deoxy-alpha-D-galactopyranoside inhibited both expression of H type 1 group on the cell surface and enhancement of brush border membrane enzyme activities even in the presence of a differentiating inducer. These results suggest that the mucin-type sugar chains with H type 1 group have important functions regarding differentiation of Caco-2 cells.

Toxicological and biochemical effects of different beverages on human intestinal cells

The objective of the present study was to examine and compare the effects of frequently consumed beverages on the human intestinal cell line, Caco-2, in terms of toxicity, growth, and differentiation. For this purpose, Caco-2 cells were incubated for 24 h in the presence of: a mineral water, fresh orange juice, packaged orange juice, a cola drink, an energy drink, black, camomille, and green teas, and drip coffee. Toxicity was evaluated firstly by measuring the lactate dehydrogenase leakage from the cells and secondly by performing the MTT assay. Cell differentiation was determined by measuring two brush border membrane enzyme activities. Growth rates of Caco-2 cells were evaluated by cell counting. The results indicated that there were no significant differences between the beverages in toxicity and cell proliferation. Cells exposed to fresh orange juice exhibited higher tetrazolium reduction rates in the MTT assay (121.3% of control). These cells also showed higher succinate cytochrome c reductase activities than the other samples, implying that the contents of fresh orange juice, such as ascorbic acid, stimulated mitochondrial metabolism. The alkaline phosphatase and aminopeptidase N activities of the Caco-2 cells lay between 61.50 and 110.00%, indicating a partial influence of some beverages on Caco-2 differentiation.

Cinnamic acid inhibits proliferation and modulates brush border membrane enzyme activities in Caco-2 cells

The objective of the present study was to examine whether cinnamic acid exerts antitumor activity against colon cancer cells in vitro. For this purpose we investigated the effect of cinnamic acid on cell proliferation and on the differentiation markers alkaline phosphatase, sucrase and aminopeptidase N in human colon adenocarcinoma cells (Caco-2). Cinnamic acid (2.5–8.0 mM) prolonged the doubling time and inhibited the DNA synthesis of growing cells. The antiproliferative effect occurred rapidly after 2 h of treatment with 8.0 mM cinnamic acid and reached nearly maximal values after 8 h of treatment. Sucrase and aminopeptidase N activities were stimulated under cinnamic acid treatment (4.0–8.0 mM), while alkaline phosphatase activity was inhibited in postconfluent cells (8.0 mM). Similar effects on enzyme activities were seen in non-proliferating cells. Cinnamic acid did not alter the adhesion to collagen matrix or cell viability. Intracellular cAMP levels were decreased significantly after 1 h of treatment with 8.0 mM cinnamic acid, suggesting that cinnamic acid induces its effects on enzyme activities partly by modulating the cAMP signaling pathway.

Importance of milk and diet on the ontogeny and adaptation of the intestine.

Importance of milk and diet on the ontogeny and adaptation of the intestine.

The Effect of Epidermal Growth Factor on Mucosal Function after Ileal Resection

Since epidermal growth factor (EGF) enhances gut mucosal regeneration, the present study was undertaken to evaluate the effect of EGF on brush-border membrane enzyme activity and glutamine uptake in the intestinal remnant following extensive small bowel resection. Twenty-four adult male New Zealand White rabbits were divided into three groups: Group 1 ( n = 12) served as controls. Groups 2 and 3 ( n = 6 each) underwent a 50-60% mid-jejunoileal resection with anastomosis of the remaining intestine, leaving 90 cm between the pylorus and the ileocecal valve. Group 3 rabbits had a subcutaneous osmotic pump implanted to deliver EGF for 7 days at 0.3 μg/kg/hr. Rabbits from Groups 2 and 3 were sacrificed 3 weeks postoperation. Mucosa from the proximal and distal segments of the remaining intestine was analyzed for wet/dry weight, maltase and aminooligopeptidase activity, and glutamine uptake. There was a twofold increase in mucosal dry weight/cm of intestine in rabbits without EGF at 3 weeks (Group 2) and a fourfold increase in those given EGF (Group 3). The maltase enzyme capacity (UEnzyme/rabbit) increased from 37 ± 10 in controls (Group 1) to 167 ± 30 without EGF and 207 ± 30 with EGF. The aminooligopeptidase enzyme capacity (UEnzyme/ rabbit) increased from 55 ± 10 to 147 ± 20 and 226 ± 30 in Groups 1, 2, and 3, respectively. Glutamine uptake capacity (μ M glutamine/min) also increased significantly, from 63 ± 19 in Group 1 to 88 ± 6 without EGF and 162 ± 18 with EGF ( P < 0.001). We conclude that EGF greatly increases mucosal mass and brush-border membrane enzyme activity in the residual small intestine following extensive bowel resection which significantly increases the absorptive and digestive capacity.

Duodenal brush-border mucosal glucose transport and enzyme activities in aging man and effect of bacterial contamination of the small intestine.

Duodenal biopsies were collected from 38 subjects (24 female and 14 male) ranging in age from 55 to 91 years. Evidence of bacterial contamination of the small bowel (BCSB) was sought at the same time by bacterial culture of duodenal aspirates and by hydrogen and [14C]glycocholic acid breath tests; subjects were considered to be positive for BCSB if any one of the three tests was abnormal. Biopsies were analyzed for six brush-border membrane enzyme activities: maltase, sucrase, lactase, alkaline phosphatase, leucine aminopeptidase, and alpha-glucosidase. Analysis of covariance with age as the covariate indicated no significant effect of age on the specific activities of these enzymes. Mucosal Na(+)-dependent glucose transport was quantified in brush-border membrane vesicles prepared from the biopsies. In all groups, glucose transport at 20-30 sec was greater (ranging from mean values of 2.45 to 3.66 times) than at 45 min, consistent with Na(+)-coupled glucose transport, and no significant effect of age was observed. BCSB had no significant effect on specific activities of any of the duodenal mucosal hydrolases but was associated with reduced (P = 0.05) brush-border glucose transport. None of the variables studied was significantly affected by the gender of subjects. In conclusion, these biochemical data do not support the contention that reduced capacity for carbohydrate absorption in the elderly is explained by reductions in duodenal brush-border mucosal disaccharidase activities or glucose transport.

Effects of Small Peptides as Intraluminal Substrates on Transport Carriers for Amino Acids and Peptides.

The effects of small peptides on brush-border membrane enzyme activities and on transport carriers for amino acids and dipeptides were investigated by measurement of these enzyme activities and the transmural potential difference in the small intestines of guinea pigs fed elemental diets containing either small peptides (SP) or amino acids (AA) as the nitrogen source. Brush-border membrane aminopeptidase activities were significantly higher in the group fed the diet containing SP. In addition, the transmural potential differences induced by L-leucine and glycyl-L-leucine (Gly-Leu) were significantly higher in the SP group. The maximum potential differences for L-leucine and Gly-Leu were significantly higher in the SP group, whereas the half saturation values did not differ between the two groups. The same effect of a SP diet was seen in rats, when the uptake of L-[U-14C]leucylglycine (L-[14C]Leu-Gly) into intestinal brush-border membrane vesicles was measured. Some 70% of L-[14C]Leu-Gly was not hydrolyzed by aminopeptidase, but was found as a dipeptide in the brush-border membrane vesicles. These results indicate that small peptides as intraluminal substrates increase brush-border membrane aminopeptidase activities and the activity of carriers for amino acids and dipeptides.

Regulation of glycolytic metabolism during long-term primary culture of renal proximal tubule cells

Adequate oxygenation was a major factor regulating the induction of glycolytic metabolism in primary cultures of rabbit renal proximal tubule cells during short-term (less than 1 day) and long-term (1-7 day) culture. As measured by cellular lactate content, glucose consumption, and lactate dehydrogenase activity, less glycolytic metabolism was induced in cultured cells that were constantly aerated than in cells that were held stationary. When oxidative metabolism is supported by providing 2-10 mM heptanoate (HEP) as a metabolic substrate glycolytic metabolism further decreased during short-term, but not long-term culture. Cellular proliferation did not play a major role in regulating the induction of glycolytic metabolism, since glycolytic metabolism increased before cell growth had occurred, did not decline once logarithmic cell growth had ceased, and was stimulated less by cell growth than by inadequate oxygenation. Fructose-1,6-bisphosphatase and alkaline phosphatase, representative markers of gluconeogenic and brush-border membrane enzyme activities, respectively, declined during culture regardless of culture conditions or the presence of HEP. Therefore, glycolytic metabolism can be effectively minimized by constantly aerating cultured proximal tubule cells and can be further reduced by the addition of HEP during short-term culture.

Caco‐2 cells cultured in serum‐free medium as a model for the study of enterocytic differentiation in vitro

Caco-2 cells, which express spontaneous enterocytic differentiation at confluency, is one of the most relevant in vitro models for the study of differentiation and regulation of intestinal functions. However, these cells are normally cultured in the presence of 15-20% serum which renders extremely complex the identification of the factors involved in the regulation of both proliferation and differentiation. This study has been devoted to the establishment of chemically defined culture conditions which can sustain growth and differentiation of Caco-2 cells. The replacement of serum by ITS (insulin, transferrin, and selenium) allowed for normal structural and functional differentiation of cells as revealed by the establishment of cell polarity and the expression of brush-border membrane enzyme markers (sucrase, maltase, lactase, alkaline phosphatase, gamma-glutamyltransferase, aminopeptidase N, and dipeptidyl-dipeptidase IV), although the levels of sucrase activity were lower in ITS-supplemented medium. Coating petridishes with either type IV collagen or basement membrane proteins (Matrigel) did not improve the differentiation of cells, brush-border membrane enzyme activities being, in fact, lower when the cells were grown on these substrata. When triiodothyronine (T3, 5 x 10(-8) M) was added to the ITS-supplemented medium, disaccharidase and alkaline phosphatase activities were significantly increased while gamma-glutamyltransferase activity was diminished by T3 and stimulated by epidermal growth factor (1.6 x 10(-6) M). On the other hand, hydrocortisone (HC, 10(-6) M) did not modify disaccharidase and peptidase activities. These data clearly show that Caco-2 cells can be maintained in serum-free medium and that this system allows the study of the factors involved in the regulation of the differentiation of enterocyte in vitro.

Binding of soybean agglutinin to small intestinal brush border membranes and brush border membrane enzyme activities in Atlantic salmon ( Salmo salar)

To test for possible toxicity of soybean lectin for salmon, the location of soybean agglutinin binding sites in the small intestinal brush border membranes (BBM) was determined quantitatively by an enzyme-linked lectin sorbent assay (ELLSA). The assay revealed substantial binding in both the proximal ( B max 1764±170 n M per mg BBM protein; K d 2.16±0.23·10 −5) and distal ( B max 2096±79 n M per mg BBM protein; K d 1.58±0.11·10 −5) small intestine. The higher maximum binding and the lower dissociation constant for the distal small intestine might indicate a greater sensitivity to the toxic effect of soybean lectin in this region. Enzyme activities for alkaline phosphatase (EC 3.1.3.1) and sucrase isomaltase (EC 3.2.1.48) in brush border membrane preparations were very low in both the proximal and distal small intestine. A high enzyme activity was found for aminopeptidase (EC 3.4.11.2). Activities of sucrase isomaltase and aminopeptidase were lower in the distal than in the proximal small intestine.

Intestinal brush border membrane enzyme activities of rats fed chemically defined diets containing oligopeptides or amino acids as the nitrogen source.

Intestinal brush border membrane enzyme activities of rats fed chemically defined diets containing oligopeptides or amino acids as the nitrogen source.

Brush border enzymes as markers for ascending pyelonephritis: active immunization with pili

Activities of renal brush border membrane (BBM) enzymes, alkaline phosphatase, maltase and leucine aminopeptidase, were determined in control, pyelonephritic and immunized-infected rats. The activities of all enzymes decreased significantly (P < 0.05) in obstructed kidney while activities of alkaline phosphatase and leucine aminopeptidase increased significantly (P < 0.05) in unobstructed kidney in early stages of infection. The affinity constant (Km) of all enzymes remained unaltered in control and experimental groups. Significant differences (P < 0.05) in the activities of BBM enzymes of infected and immunized-infected animals suggested a protective role of active immunization with pili.

Common characteristics for Na+-dependent sugar transport in Caco-2 cells and human fetal colon.

The recent demonstration that the human colon adenocarcinoma cell line Caco-2 was susceptible to spontaneous enterocytic differentiation led us to consider the question as to whether Caco-2 cells would exhibit sodium-coupled transport of sugars. This problem was investigated using isotopic tracer flux measurements of the nonmetabolizable sugar analog alpha-methylglucoside (AMG). AMG accumulation in confluent monolayers was inhibited to the same extent by sodium replacement, 200 microM phlorizin, 1 mM phloretin, and 25 mM D-glucose, but was not inhibited further in the presence of both phlorizin and phloretin. Kinetic studies were compatible with the presence of both a simple diffusive process and a single, Na+-dependent, phlorizin- and phloretin-sensitive AMG transport system. These results also ruled out any interaction between AMG and a Na+-independent, phloretin-sensitive, facilitated diffusion pathway. The brush-border membrane localization of the Na+-dependent system was inferred from the observations that its functional differentiation was synchronous with the development of brush-border membrane enzyme activities and that phlorizin and phloretin addition 1 hr after initiating sugar transport produced immediate inhibition of AMG uptake as compared to ouabain. Finally, it was shown that brush-border membrane vesicles isolated from the human fetal colonic mucosa do possess a Na+-dependent transport pathway(s) for D-glucose which was inhibited by AMG and both phlorizin and phloretin. Caco-2 cells thus appear as a valuable cell culture model to study the mechanisms involved in the differentiation and regulation of intestinal transport functions.

Comparative study of some intestinal brush border membrane enzymes during perinatal development of the mouse

1. 1. Intestinal brush border membrane (bbm) fractions have been isolated from fetal and neonatal mice. 2. 2. The existence of discordant developmental patterns of intestinal enzymatic activity derived from total homogenate and bbm fraction was confirmed. 3. 3. It originates chiefly from two phenomena: (a) variations in the state of purity of brush border fractions, and (b) loss of brush border membrane enzyme activities in supernatant that increases with age. 4. 4. The phenomenon of solubility for glucoamylase and alkaline phosphatase is already present two days before birth.

Nephrotoxicity of cefotiam (CGP 14221/E) in rats and rabbits.

Cefotiam (CGP 14221/E; SCE 963), a semisynthetic cephalosporin, was administered as a single dose by i.v. injection to rats l(up to 1.8 g/kg body-weight) and rabbits (up to 1.7 g/kg body-weight). Cephaloridine served as positive control (1.0 and 0.75 g/kg in rats; 0.3 and 0.28 g/kg in rabbits). The animals were sacrificed 24 h after injection and the kidneys preserved for routine histology and enzyme histochemistry (alkaline phosphatase, aminopeptidase, succinate dehydrogenase, esterase). Serum samples (Na+, K+, Cl-, urea, creatinine, LDH, alkaline phosphatase) and 24-h urine (Na+, K+, Cl-, urea, creatinine, protein, LDH, aminopeptidase) were analysed before and 24 h after injection. Minimal, irregularly scattered, degenerative changes in the proximal tubules which were not dose-dependent in degree were observed in rat kidneys following cefotiam injection. A slight dose-dependent degeneration in up to 50% of proximal tubular cells with loss of brush-border membrane enzyme activity was observed in rabbit kidneys. In both animal species the ability to concentrate urine was retained and urea and creatinine serum levels hardly affected. Following cefotiam injection a dose-dependent 4-fold excretion of urinary protein but not of LDH was observed in rabbits only. By contrast, cephaloridine caused extensive degeneration and necrosis in up to 90% of proximal tubular cells in both rats and rabbits, which was accompanied with formation of enzymically active hyaline casts, loss of urine-concentrating capacity of the kidney, elevated serum levels of urea and creatinine and an increased urinary excretion of LDH (60-fold in rats, 20-fold in rabbits) and protein (3-fold in rats, 10-fold in rabbits). Histochemistry and electron microscopy of rabbit kidneys suggested a loss of microvilli from proximal tubule cells by endocytosis and thus degeneration following injection of large doses of cefotiam, whereas cell disruption and necrosis prevailed after cephaloridine. The action of cefotiam on the proximal tubule cells is, therefore, not only quantitatively but possibly also qualitatively different from that of cephaloridine. Semiquantitative evaluation of tubular injuries in alkaline phosphatase-stained kidney sections and measurements of LDH and protein content in 24-h urine samples were advantageous in determining the quantity and the quality of nephrotoxic effects.

Organ culture of adult mouse intestine

The proteins, the DNA content and the brush border membrane enzyme activities of adult mouse intestinal explants have been measured during a 24-hr organ culture. These activities were not modified in comparison with the controls at the beginning of the culture. The activity of the enzymes secreted in the medium during the 24-hr culture was equal to the activity present in the explants at the start of the culture. These results show that several metabolic functions of the intestine are fully preserved in organ culture.