Inhibition Of Sugar Transport Research Articles

Biochemical Effects of Fluoride on Oral Bacteria

Fluoride inhibition of carbohydrate metabolism by the acidogenic plaque microflora is well-established, although it has not always been appreciated that oral bacteria vary considerably in their susceptibility to fluoride. Early studies demonstrated that the F-induced reduction in acid production was due, in part, to the inhibition of the glycolytic enzyme, enolase, which converts 2-P-glycerate to P-enolpyruvate. The decreased output of PEP in the presence of F, in turn, results in the inhibition of sugar transport via the PEP phosphotransferase system (PTS). Bacterial accumulation of fluoride involves the transport of HF, a process requiring a transmembrane pH difference or pH gradient, which is generated only by metabolically active cells. The uptake of HF into the more alkaline cytoplasm results in the dissociation of HF to H+ and F- and, if allowed to continue, the accumulation of protons acidifies the cytoplasm, causing a reduction in both the proton gradient and enzyme activity. Current information indicates that in addition to enolase, F- also inhibits the membrane-bound, proton-pumping H+/ATPase, which is involved in the generation of proton gradients through the efflux of protons from the cell at the expense of ATP. Thus, fluoride has the dual action of dissipating proton gradients and preventing their generation through its action on H+/ATPase. The collapse of transmembrane proton gradient, in turn, reduces the ability of cells to transport solutes via mechanisms involving proton motive force. In spite of these known effects on the bacterial cell, there is no general agreement that the anti-microbial effects of F contribute to the anti-caries effect of fluoride.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochalasin B‐induced redistribution of cytokeratin filaments in PtK1 cells

Indirect immunofluorescence demonstrated a dramatic reorganization of cytokeratin filaments produced by cytochalasin B (CB) treatment of PtK1 cells. Much of the normal cytokeratin network became arranged into a latticework consisting of bundles of cytokeratin filaments that radiated from, and interconnected, distinct foci. Electron microscopy showed foci to be dense granular regions through which bundles of cytokeratin filaments looped. Composition of the foci included actin, myosin, and alpha-actinin, as shown by labeling with rhodamine phalloidin or specific antisera. Simultaneous treatment with CB and colchicine was not required for lattice formation, but did produce more extensive development than did CB alone. In cells treated only with CB, the microtubule network remained intact, even in regions of extensive lattice formation. These results contrast sharply with those of Knapp et al (J. Cell Biol. 97:1788 [1983b]), who found lattice formation dependent upon simultaneous CB and colchicine treatment. Time-course and dose-response studies of CB treatment showed lattice formation to follow disruption of stress fibers and the concentration of actin into distinct patches that marked the location of lattice foci. Overall results suggest a structural association between microfilaments and cytokeratin filaments that produces the lattice pattern upon CB-induced disruption of stress fibers. Lattice formation was not limited to a specific cell-cycle stage, since G1, G2, and M cells displayed the lattice. Treatment of cells with dihydro-CB and experiments with enucleated cells showed that lattice formation was dependent upon neither the inhibition of sugar transport nor the nuclear extrusion effects of CB.

The influence of uncouplers on proton-sugar symport in Saccharomyces fragilis

Uncouplers of oxidative phosphorylation inhibit proton-sugar symport in Saccharomyces fragilis. However, they do not induce efflux of accumulated sugar. It is shown that the effect cannot be explained by uncoupler-induced alterations in the transmembrane potential or transmembrane pH difference. It is also indicated that a decrease in intracellular pH is not involved in inhibition of sugar transport. It is argued that inhibition of transport by uncouplers is most likely caused by a direct interaction with the translocator.

The nature of salicylate inhibition of sugar transport in yeast

A study was made in yeast of the cellular site associated with inhibition of sugar transport by salicylate. Time-course and stereospecificity studies were done on the uptake of salicylic acid (sal), meta-hydroxybenzoic acid (m-OH), D-glucose (gluc) and 3-0-methyl-D-glucose (3-OMG) at 20 and 30°C. Sal added simultaneously with 3-OMG had little effect on initial uptake rates of the latter. Inhibition of 3-OMG transport occurred only after appreciable uptake of sal. Rates of glucose utilization in cells preincubated with sal increased with time despite increased drug in the medium as sal decreased in the cells. Benzoic acid also inhibited 3-OMG transport. m-OH or para-hydroxybenzoic acid entered cells but neither substance inhibited 3-OMG transport nor affected the inhibition by sal of 3-OMG transport. Benzyl and salicyl alcohols had no effect on 3-OMG transport. The results indicate a stereospecific binding site or sites for sal located in the cell interior and associated with inhibition of sugar transport. The known inhibition of sugar transport by sal in susceptible mammalian cells, including human cells, may also occur through binding of sal to intracellular, stereospecific sites.

The Nature of Salicylate Inhibition of Sugar Transport in Yeast

The Nature of Salicylate Inhibition of Sugar Transport in Yeast. Scharff, T.G., Badr, M.Z. and Doyle, RJ. (1982). Fundam. Appl. Toxicol. 2:168-172. A study was made in yeast of the cellular site associated with inhibition of sugar transport by salicylate. Time-course and stereospecifcity studies were done on the uptake of salicylic acid (sal), meta-hydroxybenzoic acid (m-OH), D-glucose (gluc) and 3-0-methyl-D-glucose (3-OMG) at 20 and 30°C. Sal added simultaneously with 3-OMG had little effect on initial uptake rates of the latter. Inhibition of 3-OMG transport occurred only after appreciable uptake of sal. Rates of glucose utilization in cells preincubated with sal increased with time despite increased drug in the medium as sal decreased in the cells. Benzoic acid also inhibited 3-OMG transport. m-OH or para-hydroxybenzoic acid entered cells but neither substance inhibited 3-OMG transport nor affected the inhibition by sal of 3-OMG transport. Benzyl and salicyl alcohols had no effect on 3-OMG transport. The results indicate a stereospecific binding site or sites for sal located in the cell interior and associated with inhibition of sugar transport. The known inhibition of sugar transport by sal in susceptible mammalian cells, including human cells, may also occur through binding of sal to intracellular, stereospecific sites.

The relationship between high-affinity binding of cytochalasin B to 3T3 cells and inhibition of sugar transport and cell motility.

Transport and motility inhibitors have been used to classify different types of high-affinity cytochalasin B (CB) binding sites in 3T3 cells. The potency of phloretin and phlorizin as inhibitors of sugar uptake paralleled their effectiveness in displacing high-affinity bound CB from the cells, indicating that the two compounds compete with CB for binding to sites associated with sugar transport proteins. On the other hand, cytochalasins D and E, which did not inhibit sugar uptake, inhibited binding of CB to a portion of the high-affinity sites, most probably those associated with actin-containing cytoskeletal-contractile structures. A small amount of high-affinity CB binding remained in the presence of both phloretin and cytochalasin E, indicating that the cells have a third class of sites which is not related to either sugar transport or cell motility, When isolated membranes were examined, it was found that a fraction of each class of high-affinity CB binding sites were associated with the fraction. In contrast, only sites sensitive to cytochalasin D were recovered in a soluble extract of the cells.

Saturable and nonsaturable hexose uptake in cultured human skin fibroblasts.

The saturable transport of 2-deoxy-D-glucose across the cell membrane of cultured human skin fibroblasts was measured in sparse and confluent cultures. The contribution of nonsaturable sugar uptake to total sugar uptake was monitored by determining L-glucose uptake. The uptake of 2-deoxy-D-glucose was studied as a function of time and substrate concentration. Greater than 70% of transported 2-deoxy-D-glucose was phosphorylated after incubation for 2 min or less at all substrate concentrations employed (0.1 to 3.0 mM), and phosphorylation paralleled sugar uptake at these time intervals. Experiments with cytochalasin B demonstrated that an inhibition of transport was always paralleled by an equal inhibition of sugar phosphorylation.The kinetic constants for the uptake and phosphorylation of 2-deoxy-D-glucose and the inhibition of transport by competing sugars and cytochalasin B were calculated from Line-weaver-Burk plots. The Km and Vmax for saturable sugar uptake were calculated for sparse and confluent cultures after subtracting the contribution of nonsaturable sugar uptake. The resulting Km values for sugar uptake in the sparse and confluent cultures were 1.21 ± 0.04 and 0.88 ± 0.2 mM respectively. The corresponding Vmax values were 15.5 ± 1 nmol/mg protein∙min−1 for the sparse cultures and 10.1 ± 1 nmol/mg protein∙min−1 for the confluent cultures. In both sparse and confluent cultures, the Ki values for the competitive inhibition of sugar transport by D-glucose and 3-O-methyl-D-glucose were 0.8 and 2.7 mM respectively; the Ki value for the noncompetitive inhibition of sugar transport by cytochalasin B was 0.5 μM. The Km values for sugar phosphorylation by cell-free homogenates of sparse and confluent cultures were 0.57 ± 0.1 and 0.6 ± 0.1 mM respectively, while their respective Vmax values were 160 ± 53 and 139 ± 43 nmol/mg protein∙min−1.The data are in agreement with the concept that in cultured human skin fibroblasts sugar transport is the rate-limiting step in 2-deoxy-D-glucose metabolism and that phosphorylation is distinct from transport.

Effects of photoactivated porphyrins at the cell surface of leukemia L1210 cells

When murine leukemia L1210 cells are exposed to certain porphyrins, in the presence of light, a rapid loss of cell viability occurs. We have examined structure-activity relationships, using a series of porphyrins, and have studied early effects of these agents, to elucidate their mode of action. In the system employed here, only water-soluble porphyrins were cytotoxic. The first step in cytotoxicity involved binding of porphyrin to the cell surface. Porphyrins unable to bind were inactive. An important determinant of drug binding was the partition coefficient of a porphyrin between octanol and water. In the presence of light, presumably via a singlet oxygen intermediate, a variety of effects on the cell surface and cell membrane were then produced. These included inhibition of nucleoside and amino acid transport, perturbation of permeability barriers to actinomycin D uptake, enhanced binding of the fluorescent probe 8-anilino-1-naphthalenesulfonate, inhibition of activity of 5′-nucleotidase, an ectoenzyme, and altered cell-surface properties, measured with a two-phase aqueous polymer system. In the L1210 cell line, the most potent compound tested was deuteroporphyrin IX which produced the effects mentioned above at a 5 × 10−6 level; this drug level also prevented subsequent cell division. A tenfold higher drug level caused inhibition of intracellular nucleoside kinase activity, along with inhibition of sugar transport and of the fluorogenic interaction between 8-anilino-1-naphthalenesulfonic acid and cell companents. We conclude that the initial site of photoactivated porphyrin toxicity is at or near the cell surface.

The inhibition of sugar transport and oxidation in fat cell ghosts by colchicine

Colchicine inhibits glucose oxidation and the uptake of 2-deoxy-D-glucose in fat cell ghosts but has no effect on glucose oxidation by fat cell homogenates. This inhibition is rapid, reversible, and temperature-independent. Insulin-stimulated glucose oxidation and 2-deoxy-D-glucose transport are also inhibited by colchicine to an extent comparable to the basal processes.

Isoosmotic transport of fluid across the hamster small intestine in the presence of phlorizin-induced inhibition of sugar transport.

Experiments were performed to investigate whether the fluid transported across the small intestine is isoosmotic with the mucosal solution when the active transport of glucose is partially inhibited. Everted hamster mid small intestine was incubated in one of the following four mucosal solutions: (1) Isotonic control, Krebs-Ringer bicarbonate solution containing 10 mM glucose (KRBSG), (2) Isotonic with phlorizin, KRBSG + 5X10-5 M phlorizin, (3) Hypertonic control, KRBSG + 50 mM mannitol, (4) Hypertonic with phlorizin, KRBSG + 50 MM mannitol + 5x10-5 M phlorizin. The serosal surface of the intestine was not bathed. Results indicate that the transported fluid was always isoosmotic with any of the mucosal solutions used. When the mucosal solution was made hypertonic with mannitol, the concentration of glucose and electrolytes in the absorbate increased, and as a result, the absorbate became hypertonic and isoosmotic with the mucosal solution. The presence of phlorizin either in the isotonic or in the hypertonic mucosal solution decreased the glucose concentration of the absorbate, but the transported fluid became isoosmotic with the mucosal solution due to a higher concentration of Na, K, and their associated anions. Phlorizin caused a decrease in the transmural potential difference. In spite of this, the presence of this glucoside in the mucosal solution increased the transport of sodium in relation to glucose transport. It is suggested that, at the concentrations used, phlorizin inhibits sodium movement through the electrogenic pathway, but increases the transport of this ion through the non-electrogenic route. This increase in neutral sodium transport seems to compensate for the low concentration of glucose in the absorbate, so that the absorbate becomes isoosmotic with the mucosal solution whether the latter is isotonic or hypertonic. It is suggested further that isoosmotic transport of fluid is an inherent property of the small intestine and that there may be an osmoregulatory mechanism in the gut which controls this process.

The effect of phospholipase D on Escherichia coli membrane vesicles: A re-examination

1. 1 The action of commercial cabbage phospholipase D (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) on Escherichia coli membrane vesicles prepared according to Kaback (1971) Methods in Enzymology (Jakoby, W. B., ed.), Vol. 22, pp 99–120, Academic Press, New York) was re-investigated. 2. 2 With experimental conditions originally described by Milner and Kaback ((1970) Proc. Natl. Acad. Sci. U.S. 65, 683–690), the hydrolytic activity of phospholipase D is absent due to a lack of Ca 2+ in the incubation medium. 3. 3 Even though the hydrolytic activity of the enzyme is absent, the phospholipase D preparation inhibits uptake of α-methyl- d-glucoside by membrane vesicles. 4. 4 Under incubation condition where the hydrolytic activity of the phospholipase D is present, the majority of phospholipid hydrolysed in membrane vesicle is phosphatidylethanolamine. 5. 5 The origin findings of Milner and Kaback which indicated phospholipase D specifically attacks phosphatidylglycerol in E. coli membrane vesicles could not be reproduced. Inhibition of sugar transport in membrane vesicle by phospholipase D is not due to hydrolysis of phospholipids but probably is caused by some other component in the enzyme preparation.

The Inhibition of Sugar Transport in Chick Embryo Fibroblasts by Cytochalasin B: EVIDENCE FOR A MEMBRANE-SPECIFIC EFFECT

Abstract The mold metabolites, cytochalasin A and B, have been shown to be potent inhibitors of 2-deoxy-d-glucose uptake in cultured cells (Kletzien, R. F., Perdue, J. F., and Springer, A. (1972) J. Biol. Chem. 247, 2964–2966). Cytochalasin B also inhibits the uptake of d-galactose and d-glucosamine in chick embryo fibroblasts at concentrations similar to those used to inhibit the uptake of 2-deoxy-d-glucose. A concentration of cytochalasin B which inhibited 50% of sugar uptake did not affect the ability of the cells to concentrate 2-deoxy-d-glucose as the phosphorylated sugar nor did the drug inhibit the phosphorylation of 2-deoxy-d-glucose by cellular homogenates. Therefore, the inhibition of sugar uptake by cytochalasin B was due to an inhibition of transport across the plasma membrane. This inhibition was analyzed by three methods and was of the linear competitive type with an apparent dissociation constant (Ki) for the inhibitor-carrier complex of 1.45 x 10-7 m.

Inhibition of sugar transport by plasma from cystic-fibrosis patients

<h2>Summary</h2> Plasma from cystic fibrosis (C.F.) M patients inhibited uptake of a glucose analogue, arbutin, by rat jejunal tissue in vitro. Some inhibitory activity was seen in control plasma, but much greater inhibition occurred in plasma from C.F. children with almost complete segregation from age-matched controls. Plasma from parents of the C.F. children had an intermediate range of inhibitory activity overlapping both C.F. and control groups. The mean inhibition level in the C.F. children (homozygotes) was approximately twice that of the parents (heterozygotes). Manifestation of the abnormality in a 2/1 ratio is in keeping with the proposed autosomal recessive nature of the disease. This observation suggests a close relationship between the transport-inhibition activity and the gene defect in C.F.

Regulation of sugar transport in muscle: Effect of increased external potassium in vitro

1. (1) Membrane transport of 3-O- methyl- d[ 14C]glucose and intracellular levels of Na + and K + were measured in “intact” rat hemidiaphragms, in vitro. 2. (2) Incubation in media with high K + content led to decreased Na + and increased K + levels in the muscle cells, corresponding to stimulation of the Na + pump. 3. (3) In parallel with the decrease in internal Na + and increase in internal K + levels, sugar transport both into and out of the cells was decreased. This effect was greater in the presence of a submaximal concentration of insulin but was also observed in its absence. 4. (4) “High-potassium” media were prepared by isosmotic substitution of K + for Na + or by addition of KCl to normal medium. The resulting decrease in external Na + or increase in osmolarity, respectively, both stimulated sugar transport and partially counteracted the inhibitory effect of high K +. 5. (5) The inhibition of sugar transport by high K + is the converse to the previously described stimulation of sugar transport following inhibition of the Na + pump. The results thus support our earlier suggestion that the internal concentrations of Na + and/or K + may regulate sugar transport in muscle.

Inhibition of sugar transport in erythrocytes by fluorodinitrobenzene.

Inhibition of sugar transport in erythrocytes by fluorodinitrobenzene.

The Attachment of Phloretin and Analogues to Human Erythrocytes in Connection with Inhibition of Sugar Transport

The Attachment of Phloretin and Analogues to Human Erythrocytes in Connection with Inhibition of Sugar Transport