Effect of pH on sodium-stimulated D-glucose uptake in rat small intestine.
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Biomedical subjects
Publications and source records attributed to A Mahmood.
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The effect of isatin (indole-2,3-dione) on D-glucose uptake has been studied in rat intestine. Isatin at 6 mM concentration significantly inhibited both the sugar uptake and transmural (mucosal to serosal side) transport in the intestine. The suppression of glucose uptake by isatin was irreversible. Similar to the action of various SH-group-reacting agents, isatin inhibited the sugar uptake, presumably by binding to membrane sulfhydryl groups through a covalent linkage. Isatin-induced reduction in glucose uptake was unaffected by pH (between 5.5 and 8.4) and by DTT addition to incubation medium. Inhibition of sugar uptake by isatin and harmaline was additive in nature; this suggested that these compounds interact at different sites on the microvillus membrane surface.
The purpose of this work was to assess the toxic lectin ricin (RCAII) as a probe for the study of intestinal permeability in the developing small bowel. Jejunal explants from suckling and adult rabbits were exposed to varying dosages of RCAII for 30 min at 25 degrees C and then cultured in toxin-free medium. The RCAII dose required to inhibit protein synthesis during 6 h of culture increased from 0.1 microgram/ml in 4-day-old rabbits to 25 micrograms/ml in weanling rabbits. RCAII cytotoxicity was almost completely blocked by 0.1 M lactulose in all age groups. The kinetics of 125I-RCAII binding to purified microvillus membranes were determined by incubating a fixed concentration of membrane protein (30 micrograms) with increasing concentrations of labeled lectin (2-18 micrograms/ml). Binding attained saturation with adult but not with suckling animal membranes. The latter yielded a curvilinear relationship in Scatchard plots, suggesting either several classes of binding sites or negative cooperativity. RCAII binding was confined to the delipidated fraction of the membranes and decreased by 42% from 6 days old to adult age. The extreme sensitivity of colostral epithelium to RCAII is probably related to the high level of endocytosis exhibited by the immature membrane of suckling rabbits. The development of increasing resistance to the toxin, and associated decrease in binding, might be related to disappearance of saccharide sites in productive surface receptors occurring in the developmental course of intestinal glycosylation.
Cortisone, thyroxine, epidermal growth factor, or insulin were administered to 8-day-old rats for 4 days. In comparison to saline-injected controls, cortisone treatment: 1) lowered the sialic acid and raised the fucose content of the intestinal microvillus membranes, 2) increased [3H]fucose incorporation into these membranes, and 3) decreased the membrane binding of 125I-wheat germ agglutinin, while increasing the binding of 125I-ulex europeus agglutinin I and 125I-peanut agglutinin. Thyroxine treatment had similar effects on fucose content and 125I-ulex europeus agglutinin I binding, but did not alter [3H] fucose incorporation or sialic acid content. At the doses used, epidermal growth factor and insulin had no significant effects. The effect of cortisone treatment on sialic acid and fucose was commensurate with a 5- to 6-day acceleration of postnatal intestinal maturation. The changes in lectin binding, however, suggested qualitative differences between developmental and cortisone-induced membrane glycosylation. In addition, this study demonstrates significant quantitative and qualitative differences in the response of intestinal glycosylation to pharmacologic doses of the four hormones.
We present evidence of a change from sialylation to fucosylation of intestinal microvillus membrane oligosaccharides during postnatal development in the rat. The initial high sialic acid to fucose molar ratio in native and delipidated membranes was completely reversed after weaning. The specific binding of 125I-labeled wheat germ agglutinin to neuraminidase-sensitive sites in the native and delipidated membranes decreased markedly from early suckling to weaning ages. The binding of 125I-labeled Ulex europeus agglutinin I showed an age-related pattern opposite to that of wheat germ agglutinin. The changes in membrane reactivities to these lectins were entirely consistent with the existence of a developmentally-controlled shift from terminal sialyl to fucosyl substitutions among various glycoconjugate classes. This could play a key role on the functional transformation experienced by the intestinal epithelium of suckling rats.
The transport of manganese from extrinsically labeled human milk, bovine milk and infant formula was studied by the everted intestinal sac method. Tissue/mucosal flux data indicated that transport of manganese into the intestinal tissue was significantly greater with bovine milk and formula than from human milk. Similarly, the total flux of manganese from the mucosal to serosal surface was less when human milk was used. Smaller molecular weight manganese binding ligands isolated from the milk samples enhanced the mucosal to tissue movement of manganese as contrasted to the higher molecular weight manganese binding ligands. Most significantly the data suggest that the transport and uptake of manganese is less in the presence of human milk and its isolated manganese fractions than it is in bovine milk or infant formula.
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[U-14C]oxalic acid and 45Ca uptake was measured in control and vitamin B6-deficient rats. Calcium and oxalate uptake rates were significantly increased from the intestine of vitamin B6-deficient rats as compared to pair-fed controls. Oxalate uptake in pair-fed control rats follows a passive diffusion. In pyridoxine-deficient rats, the oxalate uptake increases nonlinearly as the oxalate concentration in the incubation medium increased, indicating a two-component system--a saturable sodium-independent uptake and a linear nonsaturable passive-diffusion component. The brush border membrane composition reveals that membrane sialic acid, cholesterol, and protein contents were markedly reduced. These aberrations in the chemical composition of brush border membrane may be responsible for the enhanced oxalic acid uptake in vitamin B6-deficient rats.
The effect of dietary thiamin deficiency has been studied on intestinal functions and chemical composition of brush border membranes in rats. Intestinal uptake of glucose, glycine, alanine, and leucine was significantly stimulated in thiamin deficiency compared to pair-fed control group. Studies with glucose and glycine revealed that stimulation of the absorption process occurs only in the presence of Na+ but not in its absence. Km measured in the presence of 140 mM Na+ for glucose and glycine uptakes was reduced by 56 and 41%, respectively, but Vmax remained unaltered in vitamin deficiency. There was no change in these parameters in Na+-free medium (Km = 31.3 and 23.3 mM; Vmax = 17.2 to 19.7 and 13.5 to 16.4 mumol/10 min/g wet tissue, respectively) under these conditions. The activities of brush border sucrase, lactase, maltase, alkaline phosphatase, and leucine aminopeptidase were reduced by 42 to 66% in thiamin deficiency, compared to pair-fed controls. Kinetic studies with sucrase and alkaline phosphatase evinced that a decrease in Vmax (61 and 64%, respectively) with no change in Km (33.8 and 4.3 mM, respectively) was responsible for observed impairment in the enzyme activities in thiamin deficiency. Microvillus membrane proteins expressed on dry membrane basis were reduced by 20% in thiamin-deficient intestine. There was no difference in membrane sialic acid, cholesterol, phospholipids, and triglycerides fractions under these conditions. It is suggested that thinning of the microvillus membrane may be implicated in observed aberrations of intestinal functions in thiamin-deprived animals.
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The intestinal microvillus membrane of suckling rats has a large number of unsubstituted and sialyl-substituted sites for 125I-labeled peanut agglutinin in glycopeptides, indicating that the membrane surface is rich in beta, D-Gal(1 leads to 3)D-GalNAc residues. The membrane loses all the unsubstituted and about half of the sialyl-substituted PNA-reactive sites during weaning. Simultaneously, there occur increases in the bindings of 125I-labeled soybean lectin and 125I-Ricinus communis toxin to unsubstituted sites in glycopeptides. This indicates appearance of new terminal nonreducing D-GalNAc and D-Gal in glycopeptides in the mature membranes. The developmental loss of PNA reactive sites from the microvillus membrane in rat intestine is probably related to D-GalNAc substitution in O-glycans of mucin-type glycoproteins, and to steric hindrance arising in the course of glycosylation.
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Radiolabelled U-14C oxalic acid uptake was measured in the intestine of scorbutic and ascorbic acid (AA) supplemented guinea pigs. The feeding of vitamin C deficient diet to the animals for 26 days resulted in a significant fall in the ascorbic acid levels in the various tissues studied. Supplementation of vitamin C (10, 25 or 50 mg per 200 g body weight) increased ascorbic acid levels of spleen, adrenals, liver and leucocytes. The intestinal uptake of oxalate follows a passive diffusion mechanism in normally fed guinea pigs. The oxalate uptake rate was significantly increased (p less than 0.001) in the vitamin C administered group. Vitamin C depletion significantly decreased the oxalate uptake rate as compared to control animals. The changes observed in the uptake rate appear to be related with the chemical aberrations produced in the brush border membranes.
The effect of a single oral dose of pp'DDT (100 mg/kg body wt.) has been studied on the intestinal uptake of certain nutrients and on brush border enzymes in rats. Intestinal uptake of leucine, and phenylalanine was considerably increased but there was no change in the absorption of glucose and alanine in DDT fed rats, compared to controls. The activities of brush border sucrase, alkaline phosphatase and Na+, K+-ATPase were significantly depressed in pesticide treated animals, but leucine aminopeptidase levels remained unaffected under these conditions. Analysis of the chemical composition of the microvillus membranes revealed a considerable enhancement in total lipids, phospholipids and triglyceride contents of the membranes in DDT exposed rats, but membrane protein, sialic acid and cholesterol fractions did not record any change. 1-14C-acetate incorporation into various lipid classes was studied to explain the observed increase in membrane lipids in DDT exposed animals.
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It is well established that Giardia infection causes malabsorption. However, the precise mechanism of such a malabsorption is not known. To investigate this, transport studies, using the tissue accumulation technique, were carried out in mice infected with G. lamblia obtained from human stools. There was a significant fall in the transport of D-glucose, L-alanine and glycine in the infected animals compared with the controls. Kinetics of the D-glucose and glycine transport system were examined by measuring the tissue uptake in the presence of different concentrations of the substrate. For glucose, the affinity constant (Km) for the transport site was the same (4 . 37mM) in normal and infected animals but the maximal transport rate (V max) was considerably reduced in infected animals (158 . 7 mu moles/hr/g tissue) compared with (357 . 1 microgram moles/hr/g tissue) in controls. Results with glycine were similar; the Km was similar in control and infected animals (5 . 7 mM) whereas the V max was reduced in infected animals (27 . 02 microgram moles/hr/g tissue) compared with controls (45 . 5 micrograms moles/hr/g tissue). Analysis of the intestinal enzymes showed a significant decrease in the levels of brush border sucrase, lactase and alkaline phosphatase in infected animals; the cellular enzymes, LDH, GOT and GPT remained unaffected. The observed aberrations in the transport functions and brush border enzymes suggest that G. lamblia causes malabsorption by damaging the epithelial membrane of the enterocyte.