Effect of Sorbinil on myo-Inositol Metabolism in Cultured Neuroblastoma Cells Exposed to Increased Glucose Levels

Neuroblastoma cells were used to determine the effect of sorbinil on myo-inositol metabolism in cells exposed to elevated levels of glucose in culture. Exposing cells to elevated levels of glucose led to an increase in levels of intracellular sorbitol. The increase in sorbitol levels was dependent on the extracellular glucose concentration. In contrast, the myo-inositol content of cells was decreased in the presence of increasing concentrations of extracellular glucose. Increasing the concentration of glucose in the culture medium caused a decrease in myo-inositol uptake and in the incorporation of extracellular myo-inositol into phospholipid. The effect of elevated glucose levels on myo-inositol metabolism and sorbitol accumulation was blocked by addition of 0.4 mM sorbinil. The ability of sorbinil to block the decrease in myo-inositol metabolism and sorbitol accumulation caused by 30 mM extracellular glucose was dependent on its concentration. Maximal effects were obtained with 0.4 mM sorbinil. However, there was some variation in the degree of effectiveness among batches of sorbinil. These results at the cellular level suggest that the intracellular accumulation of sorbitol is responsible for the alteration of myo-inositol metabolism observed in neuroblastoma cells exposed to elevated glucose concentrations.     

Characterization of the myo-inositol transport system in Trypanosoma cruzi.

myo-inositol is a growth factor for mammalian cells as well as for the pathogenic protozoa Trypanosoma cruzi. Most of the cell surface molecules in this organism rely on myo-inositol as the biosynthetic precursor for phosphoinositides and glycosylated phosphatidylinositols. The aim of this work was to investigate the process of myo-inositol translocation across the parasite cell membrane. myo-Inositol uptake was concentration-dependent in the concentration range 0.1-10 microM with maximal transport obtained at 8 microM. Using sodium-free buffers, where Na+ was replaced by choline or K+, myo-inositol uptake was inhibited by 50%. Furosemide, an inhibitor of the ouabain-insensitive Na+-ATPase, inhibited the Na+-dependent and Na+-independent myo-inositol uptake by 68 and 33%, respectively. In contrast, ouabain, an (Na++/K+) ATPase inhibitor, did not affect transport. Part of the myo-inositol uptake is mediated by active transport as it was inhibited when energy metabolism inhibitors such as carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (34%), 2,4-dinitrophenol (50%), KCN (71%) and NaN3 (69%) were added to the medium, or the temperature of the medium was lowered to 4 degrees C. The addition of glucose (5-50 mM) or mannose (10 mM) did not change the myo-inositol uptake, whereas the addition of 10 mM nonlabeled myo-inositol totally inhibited this transport, indicating that the transporter is specific for myo-inositol. Phloretin (0.3 mM) and phoridzin (5 mM), but not cytochalasin B, were efficient inhibitors of myo-inositol uptake. A portion of the accumulated myo-inositol is converted to inositol phosphates and phosphoinositides. These data show that myo-inositol transport in T. cruzi epimastigotes is mediated by at least two specific transporters - one Na+-dependent and the other Na+-independent.     

Effect of Increased Glucose Levels on Na+/K+-Pump Activity in Cultured Neuroblastoma Cells

Neuroblastoma cells were used to analyze the effect of elevated glucose levels on myo-inositol metabolism and Na+/K+-pump activity. The activity of the Na+/K+ pump in neuroblastoma cells is almost totally sensitive to ouabain inhibition. Culturing neuroblastoma cells in 30 mM glucose caused a significant decrease in Na+/K+-pump activity, myo-inositol metabolism, and myo-inositol content, compared to cells grown in the presence of 30 mM fructose. Glucose supplementation also caused a large intracellular accumulation of sorbitol. The aldose reductase inhibitor sorbinil prevented the abnormalities in myo-inositol metabolism and partially restored Na+/K+-pump activity in neuroblastoma cells cultured in the presence of elevated glucose levels. These results suggest that the accumulation of sorbitol by neuroblastoma cells exposed to elevated concentrations of extracellular glucose causes a decrease in myo-inositol metabolism and these abnormalities are associated with a reduction in Na+/K+-pump activity.     

L-Fucose Is a Potent Inhibitor of myo-Inositol Transport and Metabolism in Cultured Neuroblastoma Cells

It has been proposed that abnormal myo-inositol metabolism may be a factor in the development of diabetic complications. Studies with animal models of diabetes and cultured cells have suggested that hyperglycemia by an unknown mechanism may alter myo-inositol metabolism and content. Recently, we have shown that L-fucose, a 6-deoxy sugar whose content has been reported to be increased in diabetes, is a potent inhibitor of myo-inositol transport. To examine the effect of L-fucose on myo-inositol metabolism, neuroblastoma cells were cultured in medium supplemented with L-fucose. L-Fucose is a competitive inhibitor of Na(+)-dependent, high-affinity myo-inositol transport. The Ki for inhibition of myo-inositol transport by L-fucose is about 3 mM. L-Fucose is taken up and accumulates in neuroblastoma cells. The uptake of L-fucose is inhibited by Na+ depletion, D-glucose, glucose analogues, phloridzin, and cytochalasin B. In contrast, neither myo-inositol nor L-glucose inhibits L-fucose uptake. Chronic exposure of neuroblastoma cells to 1-30 mM L-fucose causes a decrease in myo-inositol accumulation and incorporation into inositol phospholipids, intracellular free myo-inositol content, and phosphatidylinositol levels. Na+,K(+)-ATPase transport activity is decreased by about 15% by acute or chronic exposure of neuroblastoma cells to L-fucose. Similar defects occur when neuroblastoma cells are exposed chronically to 30 mM glucose. Cell myo-inositol metabolism and Na+/K(+)-pump activity are maintained when 250 microM myo-inositol is added to the L-fucose-supplemented medium. Unlike the effect of chronic exposure of neuroblastoma cells to medium containing 30 mM glucose, the resting membrane potential of neuroblastoma cells is not altered by chronic exposure of the cells to 30 mM L-fucose. The effect of L-fucose on cultured neuroblastoma cell properties occurs at concentrations of L-fucose which may exist in the diabetic milieu. These data suggest that increased concentrations of L-fucose may have a role in myo-inositol-related defects in mammalian cells.     

Non-competitive inhibition of myo-inositol transport in cultured bovine retinal capillary pericytes by glucose and reversal by Sorbinil

myo-Inositol transport by retinal capillary pericytes in culture was characterized. The major myo-inositol transport process was sodium-dependent, ouabain-sensitive, and saturable at 40 mM, indicating a carrier-mediated process. The sodium ion concentration required to produce one-half the maximal rate of myo-inositol uptake ([Na+]0.5) did not show dependence on the external myo-inositol concentration (22.3 mM sodium for 0.005 mM myo-inositol; 18.2 mM sodium for 0.05 mM myo-inositol). myo-Inositol transport was an energy-dependent, active process functioning against a myo-inositol concentration gradient. The kinetics of the sodium-dependent system fitted a 'velocity type' co-transport model where binding of sodium ion to the carrier increased the velocity (Vmax 28 to 313 pmol myo-inositol/micrograms DNA per 20 min when [Na+] varied from 9 to 150 mM) but not the affinity for myo-inositol (Km 0.92 to 0.83 mM when [Na+] varied from 9 to 150 mM). Metabolizable hexoses (D-glucose or D-galactose; greater than 5 mM) inhibited myo-inositol uptake. Dixon-plot analysis indicated that the inhibition was non-competitive with a Ki of 22.7 mM for D-glucose and 72.6 mM for D-galactose. The inhibition was significantly reversed by Sorbinil (0.1 mM), an aldose reductase inhibitor. In contrast, high concentrations of non-metabolizable hexoses (L-glucose, 3-O-methyl-D-glucose), or partially metabolizable 2-deoxy-D-glucose, did not significantly inhibit myo-inositol uptake. The inhibitory effect of D-glucose or D-galactose on myo-inositol transport appeared to be related to glucose or galactose metabolism via the polyol pathway.     

Kinetics of myo-inositol transport in corneal endothelial cells: diverse effects of sugars and implications in corneal deutergensence [corrected

Kinetics of myo-inositol (MI) uptake into primary cultures of bovine corneal endothelial cells (CEC) were studied. Confluent corneal endothelial cells accumulated 3H-MI in a time dependent and saturable process. At a narrow range of external concentrations of 3H-MI (4-50 microM), the Na(+)-dependent MI uptake followed saturation kinetics. The apparent Km value was 20 microM with a maximum velocity (Vmax) of 16 pmol/20 min/micrograms DNA. At low external 3H-MI concentrations the uptake was dependent on Na ions, but at higher levels the Na(+)-independent fraction of MI uptake significantly increased. The uptake was sensitive to removal of Ca ions and to the presence of inhibitors such as n-ethyl maleimide, phlorizin, ouabain, and amiloride (an inhibitor of Na+/H+ exchanger). The sensitivity of MI uptake toward inhibitors and ionic changes in the bathing media was reduced as external concentrations of 3H-MI increased. Citrate at 0.5 mM increased the uptake, suggesting involvement of mitochondrial oxidative metabolism in the MI uptake. Percent release of radioactivity by 2 min, after an initial 40-min incubation with 20 microM 3H-MI, was 6.6% +/- 0.8 or 35% +/- 4 when release media contained BSS alone or BSS containing 5 mM nonradioactive MI, respectively. Efflux of radioactivity from the cells also was enhanced when release media contained 40 mM glucose. Glucose and galactose as well as nonmetabolizable glucose analogues, such as 3O-methyl glucose or alpha-methyl glucose, at high concentrations (40 mM), acutely (in the incubation media) or chronically (in the growth media) inhibited MI uptake into CEC, and the extent of inhibition was inversely proportional to the external levels of 3H-MI. However, glucose at lower levels (less than or equal to 10 mM) slightly increased MI uptake. These studies indicated that the uptake of MI into corneal endothelial cells was an Na(+)-dependent active process at a narrow range of external radioactive MI concentrations. Higher levels of MI were taken up by the cells via a passive diffusion mechanism, independent of carrier protein(s). Glucose influenced the uptake of MI in a complex manner. The increased MI efflux by glucose or by MI was perhaps due to the limited capacity of CEC for accumulation or compartmentalization of this or other solutes/osmolytes, a phenomenon that may be related to the role of CEC in maintenance of corneal deutergence. High glucose-induced inhibition of Na(+)-dependent MI uptake may be in part due to glucose regulation of Na+ fluxes and cell volume.(ABSTRACT TRUNCATED AT 400 WORDS)     

Vasopressin, insulin and peroxide(s) of vanadate (pervanadate) influence Na+ transport mediated by (Na+, K+)ATPase or Na+/H+ exchanger of rat liver plasma membrane vesicles

Uptake of 22Na+ by liver plasma membrane vesicles, reflecting Na+ transport by (Na+, K+)ATPase or Na+/H+ exchange was studied. Membrane vesicles were isolated from rat liver homogenates or from freshly prepared rat hepatocytes incubated in the presence of [Arg8]vasopressin or pervanadate and insulin. The ATP dependence of (Na+, K+)ATPase-mediated transport was determined from initial velocities of vanadate-sensitive uptake of 22Na+, the Na(+)-dependence of Na+/H+ exchange from initial velocities of amiloride-sensitive uptake. By studying vanadate-sensitive Na+ transport, high-affinity binding sites for ATP with an apparent Km(ATP) of 15 +/- 1 microM were observed at low concentrations of Na+ (1 mM) and K+ (1mM). At 90 mM Na+ and 60 mM K+ the apparent Km(ATP) was 103 +/- 25 microM. Vesiculation of membranes and loading of the vesicles prepared from liver homogenates in the presence of vasopressin increased the maximal velocities of vanadate-sensitive transport by 3.8-fold and 1.9-fold in the presence of low and high concentrations of Na+ and K+, respectively. The apparent Km(ATP) was shifted to 62 +/- 7 microM and 76 +/- 10 microM by vasopressin at low and high ion concentrations, respectively, indicating that the hormone reduced the influence of Na+ and K+ on ATP binding. In vesicles isolated from hepatocytes preincubated with 10 nM vasopression the hormone effect was conserved. Initial velocities of Na+ uptake (at high ion concentrations and 1 mM ATP) were increased 1.6-1.7-fold above control, after incubation of the cells with vasopressin or by affinity labelling of the cells with a photoreactive analogue of the hormone. The velocity of amiloride-sensitive Na+ transport was enhanced by incubating hepatocytes in the presence of 10 nM insulin (1.6-fold) or 0.3 mM pervanadate generated by mixing vanadate plus H2O2 (13-fold). The apparent Km(Na+) of Na+/H+ exchange was increased by pervanadate from 5.9 mM to 17.2 mM. Vesiculation and incubation of isolated membranes in the presence of pervanadate had no effect on the velocity of amiloride-sensitive Na+ transport. The results show that hormone receptor-mediated effects on (Na+, K+)ATPase and Na+/H+ exchange are conserved during the isolation of liver plasma membrane vesicles. Stable modifications of the transport systems or their membrane environment rather than ionic or metabolic responses requiring cell integrity appear to be involved in this regulation.     

Creatine Transport in Cultured Cells of Rat and Mouse Brain

Astroglia-rich cultures derived from brains of newborn rats or mice use a transport system for the uptake of creatine. The uptake system is saturable, Na+-dependent, and highly specific for creatine and Na+. Kinetic studies on rat cells revealed a Km value for creatine of 45 microM, a Vmax of 17 nmol x h-1 x (mg of protein)-1, and a Km value of 55 mM for Na+. The carrier is competitively inhibited by guanidinopropionate (Ki = 15 microM). No such transport system was found in neuron-rich primary cultures from embryonic rat brain. It is hypothesized that creatine transport is an astroglial rather than a neuronal function.     

Carrier-dependent and carrier-independent uptake of myo-inositol in cultured retinal pigment epithelial cells: activation by heat and concentration

Transport of myo-inositol (MI) was studied in primary cultures of bovine retinal pigment epithelial (RPE) cells. At low external concentrations (0.01-1 mM), uptake appeared to follow saturation kinetics, although the reciprocal forms of the rate equations did not fit either Lineweaver-Burk or Eadie-Hofstee plots. Increasing external concentrations dramatically changed the pattern of MI entry. At two to three orders of magnitude higher than physiological concentrations, a second saturation occurred (pseudo saturation). Cells incubated with 20 microM [3H]MI for 60 min had a ratio of intracellular to extracellular radioactivity greater than or equal to 8, indicating active transport. MI transport reduction by Na+ replacement or inhibitors (phlorizin, ouabain, amiloride, KSCN, iodoacetamide, MI analogues) was greater when RPE cells were incubated with low (20-400 microM) than with high (10-20 mM) MI concentrations. Cells incubated with 20 microM MI at 53 or 65 degrees C showed increased transport (up to 560%) compared with cells at 22 degrees C. The effect on MI uptake (20 microM) of Na+ replacement also was reduced at 53 degrees C. The uptake of MI involved at least two transport systems. The major mechanism at low external MI concentrations (physiological levels) was a carrier-mediated active process. At high external MI levels, uptake occurred by a diffusion process. A lipotropic effect of MI may be responsible for this increased rate of diffusion.     

Efficacy of glucose, ouabain and an aldose reductase inhibitor on 2-[3H] myo-inositol uptake by human, rat and rabbit erythrocytes

Myo-inositol uptake by erythrocytes from humans, rabbits and rats was studied with an isotope technique. In human erythrocytes, the inhibitory effect on myo-inositol uptake was stronger with glucose than with ouabain. However, an aldose reductase inhibitor (ONO-2235, 100 microM) or insulin (200 microU/ml) failed to correct the decrease in myo-inositol uptake in packed RBC, produced by either 10 mM glucose or 2mM ouabain. Ten mM ouabain had an inhibitory effect on myo-inositol uptake in all species, but an inhibitory effect was not observed with 20 mM glucose in rabbit erythrocytes. The results suggest that myo-inositol uptake by erythrocytes may be dependent on the active transport system via sodium-ATPase and that erythrocytes may not be a suitable model to monitor the possible effect of an aldose reductase inhibitor on myo-inositol concentrations in other tissues concerned with diabetic complications.     

Active transport of myo-inositol in rat pancreatic islets.

myo-Inositol transport by isolated pancreatic islets was measured with a dual isotope technique. Uptake was saturable with a half-maximal response at approx. 75 microM. With 50 microM-inositol, uptake was linear for at least 2 h during which time the free intracellular concentration rose to double that of the incubation medium. Inositol transport is therefore active and probably energized by electrogenic co-transport of Na+ down its concentration gradient as uptake was inhibited by ouabain, Na+ removal or depolarizing K+ concentrations. Inositol transport was abolished by cytochalasin B which binds to hexose carriers, but not by carbamoylcholine or Li+ which respectively stimulate or inhibit phosphoinositide turnover. Uptake of inositol was not affected by 3-O-methylglucose or L-glucose (both 100 mM) nor by physiological concentrations of D-glucose. The results suggest that most intracellular inositol in pancreatic islets would be derived from the extracellular medium. Since the transport mechanism is distinct from that of glucose, inositol uptake would not be inhibited during periods of hyperglycaemia.     

Transport of 1,5-anhydro-D-glucitol across plasma membranes in rat hepatoma cells

The transport of 1,5-anhydro-D-glucitol (AG) across plasma membranes was investigated in rat hepatoma cells, Reuber H-35. The AG uptake by the cells showed a concentration gradient dependency: the uptake was saturated within 40 s, which was less than one-third of the saturation time for 2-deoxy-D-glucose (DG) uptake. Furthermore, the Km value of the transport system for AG was higher than 100 mM. Though AG has a pyranoid structure resembling that of glucose, AG did not compete for cellular uptake with DG, D-glucose or 3-O-methyl-D-glucose, which are taken into cells through the glucose transporters. Conversely, the DG transport was not inhibited by AG at concentrations up to 50 mM. AG transport was hardly inhibited by 10 microM cytochalasin B, which strongly inhibits glucose transporters. In contrast, the AG transport was inhibited by 100 microM phloretin much more strongly than the DG transport when cells were preincubated with the inhibitor; the inhibition constant was 28.0 microM. The AG transport was not inhibited by 100 microM phloridzin, while the DG uptake was slightly inhibited by phloridzin. On the basis of these observations we propose that the AG uptake into rat hepatoma cells is mediated by a carrier distinct from glucose transporters.     

Characteristics of Sorbitol Uptake in Rat Glial Primary Cultures

Uptake of [U-14C]sorbitol was studied in astrogliarich rat primary cultures. Initial rate of sorbitol uptake is proportional to sorbitol concentration between 20 microM and 400 mM. Sorbitol transport is not inhibited by glucose, fructose, and a variety of structurally related polyols, or by cytochalasin B, an inhibitor of glucose transport. Phloretin, phlorizin, filipin, and n-hexanol, all compounds that alter the properties of biological membranes, and the sulfhydryl reagent p-chloromercuribenzoate inhibit sorbitol uptake to various degrees. Variation in the concentrations of extracellular Na+ and K+ does not affect transfer of sorbitol across the cell membrane. It is concluded that sorbitol is taken up into glial cells by a diffusion process, not involving a carrier and probably not through the lipid bilayer, but through a proteinaceous channel-like structure.     

Regulation of MI transport in retinal pigment epithelium by sugars, amiloride, and pH gradients: potential impairment of pump-leak balance in diabetic maculopathy

Impairment of transport and metabolism of retinal pigment epithelium (RPE) has been recognized to play a role in the development of diabetic macular edema. To understand the mechanism(s) of action of high glucose levels in alteration of RPE metabolism, primary cultures of RPE cells were used as an in vitro model of diabetic retinopathy/maculopathy. RPE cells were grown with 5 mM (control) or 40 mM glucose (a monosaccharide that enters the cells), or 40 mM sucrose (a disaccharide that does not enter the cells), and the extent of Na(+)-dependent active transport of an osmolyte ([3H]-myo-inositol, MI, 10 microM) into cells was determined. While 40 mM glucose down-regulated 3H-MI transport, 40 mM sucrose stimulated it, compared to 5 mM glucose feeding. Addition of 1 mM amiloride, an inhibitor of Na+/H+ exchanger, in the incubation media, significantly inhibited MI transport. Cells treated with high sucrose or high glucose were more sensitive toward amiloride inhibition, compared to controls. Inhibition of either pump or leak pathway alone was not sufficient to completely inhibit MI transport, but simultaneous inhibition of both pathways, by amiloride and ouabain (1 mM each), strongly inhibited osmolyte accumulation. The strongest inhibition of uptake occurred when 150 mM NaCl in the incubation media was replaced by 150 mM choline-Cl, and the percent inhibition of uptake, with choline-Cl, was highest with sucrose-fed cells, compared to normal or high glucose-fed cells. Imposition of a pH gradient [pHi (6.1) less than pH0 (8.0)] across the cell membrane, a condition that stimulates Na+/H+ exchange activity, also reduced MI accumulation. Cellular water content, measured by the extent of [3H]-3-O-methyl glucose uptake, in the presence of balanced salt solution (BSS), BSS containing half the ionic strength (hypotonic solution), or BSS containing 20 mM K+, for induction of cell swelling, varied when cells were fed with various sugars. Cells fed with high glucose were less sensitive toward media tonicity compared to normal. These results suggested that in cultured RPE cells, changes in Na+/H+ exchanger activity (intracellularly or extracellularly), through its inhibition by amiloride, its activation via intracellular acidification, or perhaps by chronic feeding with high sucrose or high glucose, affected the Na(+)-dependent active accumulation of MI. A metabolic factor involved in the development of diabetic macular edema is perhaps associated with glucose-induced alterations in Na+ fluxes (e.g., changes in Na+/H+ exchanger activity), which can secondarily influence osmolyte accumulation, impairment of pump-leak balance, and/or intracellular pH.(ABSTRACT TRUNCATED AT 400 WORDS)     

Energetics of alanine, lysine, and proline transport in cytoplasmic membranes of the polyphosphate-accumulating Acinetobacter johnsonii strain 210A.

Amino acid transport in right-side-out membrane vesicles of Acinetobacter johnsonii 210A was studied. L-Alanine, L-lysine, and L-proline were actively transported when a proton motive force of -76 mV was generated by the oxidation of glucose via the membrane-bound glucose dehydrogenase. Kinetic analysis of amino acid uptake at concentrations of up to 80 microM revealed the presence of a single transport system for each of these amino acids with a Kt of less than 4 microM. The mode of energy coupling to solute uptake was analyzed by imposition of artificial ion diffusion gradients. The uptake of alanine and lysine was driven by a membrane potential and a transmembrane pH gradient. In contrast, the uptake of proline was driven by a membrane potential and a transmembrane chemical gradient of sodium ions. The mechanistic stoichiometry for the solute and the coupling ion was close to unity for all three amino acids. The Na+ dependence of the proline carrier was studied in greater detail. Membrane potential-driven uptake of proline was stimulated by Na+, with a half-maximal Na+ concentration of 26 microM. At Na+ concentrations above 250 microM, proline uptake was strongly inhibited. Generation of a sodium motive force and maintenance of a low internal Na+ concentration are most likely mediated by a sodium/proton antiporter, the presence of which was suggested by the Na(+)-dependent alkalinization of the intravesicular pH in inside-out membrane vesicles. The results show that both H+ and Na+ can function as coupling ions in amino acid transport in Acinetobacter spp.     

The Na+-ionophore monensin enhances glucose uptake in mouse thymocytes

Monensin enhanced 2-deoxyglucose uptake and 3-O-methyl glucose transport in mouse thymocytes, but had no effect on L-glucose transport. Cytochalasin B inhibited monensin induced as well as basal glucose uptake. The enhanced 2-deoxyglucose uptake was time and dose-dependent. The increase in the rate of 2-deoxyglucose uptake induced by monensin was more rapid than that of Na+ uptake. Ouabain did not inhibit monensin-enhanced 2-deoxyglucose uptake. Monensin failed to stimulate 2-deoxyglucose uptake at low concentrations of Na+ (13 mM) or K+ (17 mM), higher concentrations of either cation were required for stimulation. Monensin enhanced glucose uptake also in Ca2+-free medium. The data indicate that the stimulation of 2-deoxyglucose uptake by monensin results from activation of carrier-mediated transport.     

Na+-linked active transport of ascorbate into cultured bovine retinal pigment epithelial cells: heterologous inhibition by glucose

The transport of ascorbate into cultured bovine retinal pigment epithelial (RPE) cells is reported. Primary or subcultured RPE cells were incubated in the presence of 10-500 microM L-[carboxyl-14C]-ascorbate for various periods of time. Accumulation of ascorbate into RPE cells followed a saturable active transport with a Km of 125 microM and a Vmax of 28 pmole/micrograms DNA/min. RPE intracellular water was calculated to be 0.8 pL/cell, and the transported cellular ascorbate concentration was 7.5 +/- 0.8 mM. Replacement of 150 mM NaCl in the incubation media with choline-Cl strongly inhibited (80 +/- 8%) ascorbate uptake into cultured RPE cells. Although the depletion of cellular ATP by 2,4-dinitrophenol and the inhibition of Na+-K+-ATPase by ouabain reduced ascorbate transport into RPE significantly, active transport of ascorbate was not entirely inhibited by these metabolic inhibitors. The ascorbate analogue, D-isoascorbate, competitively inhibited ascorbate transport into cultured RPE with a Ki of 12.5 mM. Cells grown in the presence of 5 to 50 mM alpha-D-glucose in the growth media did not differ in their ability to transport ascorbate. In contrast, the presence of alpha-D-glucose or its nonmetabolizable analogues, 3-0-methyl-glucose, alpha-methyl-glucose, and 2-deoxy-glucose, but not L-glucose or beta-D-fructose, in the incubation media inhibited ascorbate transport. myo-Inositol (10 or 20 mM) also inhibited ascorbate transport into RPE cells. The active uptake of ascorbate into cultured RPE cells was primarily coupled to the movement of sodium ion down its electrochemical gradient. A bifunctional, cotransport carrier possessing an ascorbate-binding site and a sodium-binding site may be involved in the ascorbate uptake system. The inhibition of ascorbate uptake by sugars appeared to be heterologous in nature, occurring between two distinct carrier systems, both of which were dependent on the sodium ions.     

Competitive inhibition by glucose of myo-inositol incorporation into cultured porcine aortic endothelial cells

To explore the significance of hyperglycaemia as a causal factor for the appearance of diabetic angiopathies we investigated aspects of myo-inositol metabolism in porcine aortic endothelial cells. myo-Inositol was shown to be a long-living metabolite. Its uptake into the cells was mediated by a high-affinity, Na(+)-dependent uptake system inhibitable by ouabain with an apparent KM of 18.6 mumols/l, which was responsible for more than 80% of total uptake at physiological myo-inositol concentrations. Inhibition of inositol uptake by D-glucose was exclusively competitive with an apparent Ki of 24 mmol/l as shown by Lineweaver-Burk- and Dixon-plot analysis. The specificity of competitive inhibition was studied. L-Glucose which is stereochemically related to myo-inositol in the same way as the D-isomer proved to be an equally potent inhibitor. The hexoses D-galactose, D-mannose and D-fructose inhibited myo-inositol uptake to a minor extent. D-allose and 3-O-methyl-D-glucose had no inhibitory effect indicating that the OH-group of the carbon atom in 3 position is essential for the interaction with the carrier. The acyclic hexitol sorbitol also did not compete. As expected, the aldose reductase blocker sorbinil did not influence the carrier since there is no polyol pathway operating in porcine aortic endothelial cells. In accordance with the results of the uptake experiments, the incorporation of exogenous myo-inositol into membrane phosphatidylinositol was reduced at elevated extracellular glucose levels. The results raise the possibility that hyperglycaemia impairs endothelial inositol supply.     

Delta endotoxin is a potent inhibitor of the (Na,K)-ATPase

A 68-kDa protein, delta endotoxin, produced by Bacillus thuringiensis ssp. Kurstaki inhibits ion transport, (Na,K)-ATPase, and K+-p-nitrophenylphosphatase activity catalyzed by the Na+ pump. The Ki for inhibition of the K+-p-nitrophenylphosphatase activity of purified dog kidney (Na,K)-ATPase was approximately 0.37 microM. Delta endotoxin had a similar Ki for inhibition of (Na,K)-ATPase activity when assayed at low Na+ concentration (10 mM) but the inhibition was reversed when high concentrations of Na+ (100 mM NaCl) were added to the assay. Phosphorylation of the active site aspartyl residue with 32PO3-4 was also blocked by delta endotoxin. Ouabain-sensitive 86Rb+ uptake into intact human red blood cells was not inhibited by externally added toxin; however, strophanthidin-inhibitable 22Na+ uptake into inside-out vesicles from red blood cells was completely blocked by delta endotoxin (Ki = 0.73 microM). These data suggest that delta endotoxin must enter the cell before it can inhibit the Na+ pump.     

Effect of Fructose Supplementation on Sorbitol Accumulation and myolnositol Metabolism in Cultured Neuroblastoma Cells Exposed to Increased Glucose Concentrations

Aldose reductase activity is increased in neuroblastoma cells grown in media containing 30 mM fructose and/or 30 mM glucose. Neuroblastoma cells cultured in media supplemented with increased concentrations of glucose and fructose amass greater amounts of sorbitol than do cells exposed to media containing only high glucose concentrations. The increase in sorbitol content is dependent on the fructose and glucose concentration in the media. The increase in sorbitol content caused by exposing neuroblastoma cells to media containing 30 mM glucose/30 mM fructose is due to a protein synthesis sensitive mechanism and not to an alteration in the redox state. The addition of sorbinil to media containing 30 mM glucose blocks the increase in sorbitol content. In contrast, sorbinil treatment of media containing 30 mM glucose/30 mM fructose does not totally block the increase in sorbitol levels. myo-Inositol accumulation and incorporation into inositol phospholipids and intracellular myo-inositol content are decreased in cells chronically exposed to media containing 30 mM glucose or 30 mM glucose/30 mM fructose compared to cells cultured in unsupplemented media or media containing 30 mM fructose. However, maximal depletion of myo-inositol accumulation and intracellular content occurs earlier in cells exposed to media containing 30 mM glucose/30 mM fructose than in cells exposed to media supplemented with 30 mM glucose. Sorbinil treatment of media containing 30 mM glucose/30 mM fructose maintains cellular myo-inositol accumulation and incorporation into phospholipids at near normal levels. myo-Inositol content in neuroblastoma cells chronically exposed to media containing 30 mM glucose or 30 mM glucose/30 mM fructose recovers within 72 h when the cells are transferred to unsupplemented media or media containing 30 mM fructose. In contrast, the sorbitol content of cells previously exposed to media containing 30 mM glucose or 30 mM glucose/30 mM fructose then transferred into media containing 30 mM fructose remains elevated compared to the sorbitol content of cells transferred into unsupplemented media. These data suggest that fructose may be activating or increasing sorbinil-resistant aldose reductase activity as well as partially blocking sorbitol dehydrogenase activity. The presence of increased concentrations of fructose in combination with increased glucose levels may enhance alterations in cell metabolism and properties due to increased sorbitol levels.