Depletion of Phospholipid Arachidonoyl-Containing Molecular Species in a Human Schwann Cell Line Grown in Elevated Glucose and Their Restoration by an Aldose Reductase Inhibitor

Abstract: In experimental diabetic neuropathy, defective arachidonic acid metabolism characterized by a decrease in the proportion of glycerophospholipid arachidonoyl-containing molecular species (ACMS) occurs and has been implicated in the pathogenesis of the disorder. In this study, we evaluated the suitability of a tumor-derived human Schwann cell line (NF1T) as a model to investigate the mechanism underlying the loss of ACMS. NF1T cells grown in 30 versus 5.5 m M glucose undergo a marked reduction in ACMS in phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol, in a manner resembling that of diabetic nerve. The depletion of ACMS can be reversed on transferring the cells from 30 m M glucose to medium containing physiological levels of glucose. Cells maintained in 5.5 m M glucose plus 25 m M mannitol or sorbitol did not exhibit decreased ACMS levels, indicating that osmotic effects were not responsible for ACMS depletion. However, growth in 25 m M fructose elicited a reduction of ACMS similar to that produced by 30 m M glucose. Excessive glucose flux through the polyol pathway has been implicated in the neural and vascular abnormalities associated with diabetes. Therefore, we examined the effects of polyol pathway inhibitors, including two aldose reductase inhibitors, zopolrestat and sorbinil, and a sorbitol dehydrogenase inhibitor (SDI), CP166,572, on ACMS levels in NF1T cells cultured in elevated glucose concentrations. At 200 µ M , zopolrestat fully and sorbinil partially corrected ACMS depletion. The SDI at concentrations up to 100 µ M failed to affect diminished ACMS levels. Neither zopolrestat nor the SDI restored ACMS levels reduced in the presence of elevated fructose concentrations. These findings suggest that enhanced flux through the polyol pathway and, in particular, elevated aldose reductase activity may play a significant role in the reduction of ACMS levels in the cells brought about by elevated glucose levels.     

Slow Orthograde Axonal Transport of Radiolabelled Protein in Sciatic Motoneurones of Rats with Short-Term Experimental Diabetes: Effects of Treatment with an Aldose Reductase Inhibitor or myo-Inositol

Abstract: This study examined the effect of streptozotocin diabetes of 5 weeks duration on the profile of slow orthogradely transported radiolabelled protein in rat sciatic motoneurones. The diabetic rats showed a retardation of the tail of the slow-component profile. This selective retardation was unaffected by treatment with an aldose reductase inhibitor, although this treatment reduced the accumulation of sorbitol and prevented the depletion of myo -inositol in the sciatic nerves of the treated diabetic rats. Other groups, treated with myo -inositol, had normal or elevated sciatic nerve myo -inositol levels in the presence of accumulated sorbitol. The axonal transport profiles from both control and diabetic myo-inositol-treated groups gave normal tail velocities but an altered shape such that retardation of the tail of the profile may have been present in both. The study concludes that rats with 5 weeks streptozotocin diabetes show retardation of the velocity of the most slowly transported proteins in sciatic motoneurones, and that this defect is not linked to the polyol pathway.     

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.     

Effects of ascorbic acid, aminoguanidine, sorbinil and zopolrestat on sorbitol and betaine contents in cultured rat renal cells

Aldose reductase inhibitors (ARI) have been developed to reduce the conversion of high glucose levels to sorbitol, an important renal osmolyte that may rise to damaging levels in many tissues during diabetic hyperglycemia. Ascorbic acid (AA) and aminoguanidine (AMG) have also been reported to reduce sorbitol levels in diabetes: AMG in rat kidney, and AA in guinea pig lens and human erythrocytes. We tested the effects of AMG, AA, and Pfizer’s sorbinil and zopolrestat for 48 h on primary rat renal cell cultures, established from renal inner medullas of male Wistar rats 8–12 weeks old. Osmolyte contents in scraped cells were analysed by HPLC: 100 µM sorbinil and 20 µM zopolrestat decreased sorbitol levels (P<0.05 and P<0.001, respectively), and increased the content of another osmolyte, betaine (P<0.01 and <0.01, respectively). The quantity of ATP in cells was unchanged, suggesting no short-term problems. In contrast, 10 mM AMG and 10 mM AA had no effect on sorbitol contents (in contrast to some previous studies). We then tested aldose reductase (AR) activity in crude homogenates of rat lens and renal inner medulla, with glyceraldehyde substrate. For both tissues, 5 µM zopolrestat inhibited AR activity by 92–94% (P<0.002); 10 mM AA by 16–20% (P<0.02); and 10 mM aminoguanidine by 22–24% (P<0.03). We conclude that AMG and AA are not readily usable as inhibitors of renal AR.     

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.     

Reduced Na+/K+ ATPase transport activity,resting membrane potential,and bradykinin-stimulated phosphatidylinositol synthesis by polyol accumulation in cultured neuroblastoma cells

In these studies we examined the effect of polyol accumulation on neural cellmyo-inositol metabolism and properties. Neuroblastoma cells were cultured for two weeks in media containing 30 mM glucose, fructose, galactose or mannose with or without 0.4 mM sorbinil or 250 Mmyo-inositol. Chronic exposure of neuroblastoma cells to media containing 30 mM glucose, galactose, or mannose caused a decrease inmyo- inositol content and myo-[2-³H]inositol accumulation and incorporation into phosphoinositides compared to cells cultured in unsupplemented medium or medium containing 30 mM fructose as an osmotic control. These monosaccharides each caused an increase in intracellular polyol levels with galactitol > sorbitol = mannitol accumulation. Chronic exposure of neuroblastoma cells to media containing 30 mM glucose, galactose, or mannose caused a significant decrease in Na⁺/K⁺ ATPase transport activity, resting membrane potential, and bradykinin-stimulated³²P incorporation into phosphatidylinositol compared to cells cultured in medium containing 30 mM fructose. In contrast, basal incorporation of³²P into phosphatidylinositol or basal and bradykinin-stimulated³²P incorporation into phosphatidylinositol 4,5-bisphosphate were not effected. Each of these cellular functions as well asmyo-inositol metabolism and content and polyol levels remained near control values when 0.4 mM sorbinil, an aldose reductase inhibitor, was added to the glucose, galactose, or mannose supplemented media.myo-Inositol metabolism and content and bradykinin-stimulated phosphatidylinositol synthesis were also maintained when media containing 30 mM glucose, galactose, or mannose was supplemented with 250 Mmyo-inositol. The results suggest that polyol accumulation induces defects in neural cellmyo-inositol metabolism and certain cell functions which could, if they occurred in vivo, contribute to the pathological defects observed in diabetic neuropathy.     

Osmotic regulation of aldose reductase protein synthesis in renal medullary cells

Renal medullary cells are normally exposed to high extracellular NaCl as part of the urinary concentrating mechanism. They react to this stress by accumulating sorbitol and other organic osmolytes. PAP-HT25, a line of epithelial cells derived from rabbit renal inner medulla, expresses this response. In hypertonic medium, these cells accumulate large amounts of sorbitol. There is a large increase in the amount of aldose reductase, which catalyzes production of sorbitol from glucose. The purpose of the present study was to investigate whether the aldose reductase protein increases because of faster synthesis or slower degradation. We measured the rate of synthesis and degradation of aldose reductase protein by pulse-chase with [35S]methionine, followed by immunoprecipitation with specific antiserum and autoradiography. The protein synthesis rate was 6 times greater in cells grown in hypertonic (500 mosmol/kg) medium, than in those grown in normal (300 mosmol/kg) medium. When control cells were switched to hypertonic medium, the synthesis rate increased 15-fold by 24 h, then decreased to 11-fold after 48 h. In contrast, synthesis rate continued to increase past 24 h when accumulation of sorbitol was prevented by inhibiting aldose reductase activity with Tolrestat. Thus, there is a feedback mechanism by which cellular sorbitol accumulation inhibits aldose reductase protein synthesis. Degradation of aldose reductase protein was slow (only about 25% in 3 days) and was not affected by osmolality. Thus, the osmoregulatory increase in aldose reductase protein is due to an increase in its synthesis rate and not to any change in its degradation.     

Purified rat lens aldose reductase. Polyol production in vitro and its inhibition by aldose reductase inhibitors.

The production of polyols in vitro by highly purified aldose reductase (EC 1.1.1.21) was monitored by g.l.c. In the presence of NADPH aldose reductase reduced glucose, galactose and xylose to the respective polyols sorbitol, galactitol and xylitol. The rates of formation of these polyols closely mirrored the Km values for the substrates obtained from kinetic measurements that monitored the rate of disappearance of NADPH. No polyol production occurred in the absence of purified aldose of purified aldose reductase, and analysis by g.l.c. revealed only the presence of unchanged monosaccharides. Addition of the aldose reductase inhibitor sorbinil to purified rat lens aldose reductase incubated with xylose in the presence of NADPH resulted in decreased xylitol production. However, aldose reductase inhibitors produced no effect in altering the rate of Nitro Blue Tetrazolium formation from either glucose or xylose, indicating that the observed inhibition in vitro does not result from a free-radical-scavenger effect.     

Inositol Uptake and Metabolism in Molluscan Neuronal Tissue

Abstract: The uptake of myo -[³H]inositol into neurones from Lymnaea stagnalis has been demonstrated to be a sodium-dependent process, saturable with a K _m of approximately 50 μ M and shown to be linear with time for at least 120 min. The rate of transport of myo -inositol into the cell appears to influence directly its incorporation into neuronal lipids. Using anion-exchange high-performance liquid chromatography, we have demonstrated a high rate of breakdown of phosphatidylinositol 4,5–bisphosphate in Lymnaea nerve under basal conditions. Stimulation with carbamylcholine enhanced production of inositol 1–phosphate, inositol bisphosphate, inositol 1,4,5–trisphosphate, and inositol 1,3,4–trisphosphate. Formation of inositol tetrakisphosphate was not detected. Electrical stimulation also caused an increased formation of inositol phosphates. These results provide evidence for an active myo -inositol transport system in molluscan neurones and suggest that the hydrolysis of inositol lipids may play a role as an intracellular signalling system in this tissue.     

l-DOPA Inhibits Complex IV of the Electron Transport Chain in Catecholamine-Rich Human Neuroblastoma NB69 Cells

Abstract: l -3,4-Dihydroxyphenylalanine ( l -DOPA) is toxic for human neuroblastoma cells NB69 and its toxicity is related to several mechanisms including quinone formation and enhanced production of free radicals related to the metabolism of dopamine via monoamine oxidase type B. We studied the effect of l -DOPA on activities of enzyme complexes in the electron transport chain (ETC) in homogenate preparations from the human neuroblastoma cell line NB69. As a preliminary step we compared the activity of ETC in cellular homogenates with that of purified mitochondria from NB69 cells and rat brain. Specific activities for complex I, complex II–III, and complex IV in NB69 cells were, respectively, 65, 96, and 32% of those in brain mitochondria. Complex I activity was inhibited in a dose-dependent way by 1-methyl-4-phenylpyridinium ion with an EC₅₀ of ∼150 µ M . Treatment with 0.25 m M l -DOPA for 5 days reduces complex IV activity to 74% of control values but does not change either complex I or citrate synthase. Ascorbic acid (1 m M ), which protects NB69 cells from l -DOPA-induced neurotoxicity, increases complex IV activity to 133% of the control and does not change other ETC complexes. Ascorbic acid also reverses l -DOPA-induced reduction of complex IV activity in NB69 cells. This observation might indicate that the protection observed with ascorbic acid is related to complex IV activation. In vitro incubation with l -DOPA (0.125–4 m M ) for 2 min produced a dose-dependent reduction of complex IV without change in complex I and II–III activities.     

Inhibition of ascorbic acid transport in human neutrophils by glucose.

Because of the structural similarity between glucose and ascorbic acid, we investigated the effect of glucose on uptake and accumulation of ascorbic acid in isolated normal human neutrophils. Ascorbic acid accumulation was determined using high-performance liquid chromatography with coulometric electrochemical detection, in conjunction with liquid scintillation spectrometry. Ascorbic acid accumulation in neutrophils is mediated by a high and a low affinity transport activity. In neutrophils from different volunteers, glucose inhibited uptake and accumulation of ascorbic acid by both transport activities 3-9-fold. The mechanism of inhibition was different for each transport activity: inhibition of the high affinity transport activity was noncompetitive, while inhibition of the low affinity activity was competitive. Glucose-induced inhibition of both ascorbic acid transport activities occurred in neutrophils of all donors tested and was fully reversible. Although the mechanism of ascorbic acid accumulation appeared to be different than that for glucose transport, other monosaccharides and glucose transport inhibitors also inhibited ascorbic acid accumulation. These are the first data to suggest that ascorbic acid accumulation in neutrophils can be regulated by compounds of similar structure.     

Inhibition of human brain aldose reductase and hexonate dehydrogenase by alrestatin and sorbinil

Abstract: Human brain aldose reductase and hexonate dehydrogenase are inhibited by alrestatin (AY 22,284) and sorbinil (CP 45,634). Inhibition by alrestatin is noncompetitive for both enzymes, and slightly stronger for hexonate dehydrogenase ( K _I values 52-250 μ M ) than for aldose reductase ( K _I values 170-320 μ M ). Sorbinil inhibits hexonate dehydrogenase far more potently than aldose reductase, K _I values being 5 μ M for hexonate dehydrogenase and 150 μ M for aldose reductase. The inhibition of hexonate dehydrogenase by sorbinil is noncompetitive with respect to both aldehyde and NADPH substrates, and is thus kinetically similar to the inhibition by alrestatin. However, sorbinil inhibition of aldose reductase is uncompetitive with respect to glyceraldehyde and noncompetitive with NADPH as the varied substrate. Inhibition of human brain aldose reductase by these two inhibitors is much less potent than that reported for the enzyme from other sources.     

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.     

Hyperglycemia Promotes Schwann Cell De-differentiation and De-myelination via Sorbitol Accumulation and Igf1 Protein Down-regulation

Diabetes mellitus (DM) is frequently accompanied by complications, such as peripheral nerve neuropathy. Schwann cells play a pivotal role in regulating peripheral nerve function and conduction velocity; however, changes in Schwann cell differentiation status in DM are not fully understood. Here, we report that Schwann cells de-differentiate into immature cells under hyperglycemic conditions as a result of sorbitol accumulation and decreased Igf1 expression in those cells. We found that de-differentiated Schwann cells could be re-differentiated in vitro into mature cells by treatment with an aldose reductase inhibitor, to reduce sorbitol levels, or with vitamin D₃, to elevate Igf1 expression. In vivo DM models exhibited significantly reduced nerve function and conduction, Schwann cell de-differentiation, peripheral nerve de-myelination, and all conditions were significantly rescued by aldose reductase inhibitor or vitamin D₃ administration. These findings reveal mechanisms underlying pathological changes in Schwann cells seen in DM and suggest ways to treat neurological conditions associated with this condition.     

Molecular cloning of cDNA coding for kidney aldose reductase. Regulation of specific mRNA accumulation by NaCl-mediated osmotic stress

Cells generally respond to long-term hyperosmotic stress by accumulating nonperturbing organic osmolytes. Unlike bacteria, in which molecular mechanisms involved in the increased accumulation of osmolytes have been identified, those in multicellular organisms are virtually unknown. In mammals, during antidiuresis, cells of the renal inner medulla are exposed to high and variable extracellular NaCl. Under these conditions, the cells contain a high level of sorbitol and other osmolytes which help balance the high extracellular osmolality. PAP-HT25 is a continuous line of cells derived from rabbit renal inner medulla. When medium osmolality is increased by raising the NaCl concentration, these cells accumulate sorbitol. The sorbitol is synthesized from glucose in a reaction catalyzed by aldose reductase. When the medium is made hyperosmotic, aldose reductase activity increases because of a larger increase in the amount of enzyme. This increase is produced by the accelerated rate of synthesis of aldose reductase protein. The purpose of the present studies was to examine the mechanism of this increase in aldose reductase protein by measuring the relative abundance of aldose reductase mRNA. A cDNA clone coding for rabbit kidney aldose reductase was isolated. Antisense RNA probes transcribed from this clone hybridized specifically with a 1.5-1.6 kilobase mRNA in Northern blots. Cells grown chronically in hyperosmotic medium had a relative abundance of this specific mRNA which was six times that of cells grown in isoosmotic medium. When cells grown in isoosmotic medium were switched to hyperosmotic medium, the level of aldose reductase mRNA peaked (18-fold) at 18-24 h. The induction of aldose reductase mRNA by osmotic stress was reversible. Our finding of increased abundance of a specific mRNA in direct response to hyperosmotic stress represents the first report of such an effect in animals.     

Benzoxazinone-thiosemicarbazones as antidiabetic leads via aldose reductase inhibition: Synthesis,biological screening and molecular docking study

Aldose reductase is an important enzyme in the polyol pathway, where glucose is converted to fructose, and sorbitol is released. Aldose reductase activity increases in diabetes as the glucose levels increase, resulting in increased sorbitol production. Sorbitol, being less cell permeable tends to accumulate in tissues such as eye lenses, peripheral nerves and glomerulus that are not insulin sensitive. This excessive build-up of sorbitol is responsible for diabetes associated complications such as retinopathy and neuropathy. In continuation of our interest to design and discover potent inhibitors of aldo-keto reductases (AKRs; aldehyde reductase ALR1 or AKR1A, and aldose reductase ALR2 or AKR1B), herein we designed and investigated a series of new benzoxazinone-thiosemicarbazones (3a-r) as ALR2 and ALR1 inhibitors. Most compounds exhibited excellent inhibitory activities with IC₅₀ values in lower micro-molar range. Compounds 3b and 3l were found to be most active ALR2 inhibitors with IC₅₀ values of 0.52 ± 0.04 and 0.19 ± 0.03 μM, respectively, both compounds were more effective inhibitors as compared to the standard ALR2 inhibitor (sorbinil, with IC₅₀ value of 3.14 ± 0.02 μM).     

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.     

Effects of ascorbic acid on the uptake of serotonin in differentiated neuroblastoma cells

Ascorbic acid causes concentration-dependent and time-dependent effects on [³H]-serotonin (³H-5HT) uptake into differentiated neuroblastoma N-2a cells. Preincubation of cells with ascorbic acid inhibits both passive diffusion and active transport of ³H-5HT (0.1 μM). The kinetic characteristics of the active uptake process change with ascorbic acid treatment, resulting in an increase in the Km from 0.27 μM to 3.0 μM and in the Vmax from 453 to 2369 fmol/min/10⁶ cells. This inhibitory effect of ascorbic acid appears to be due to its reducing properties.     

Effect of glucose on Na, K-ATPase activity in cultured bovine aortic endothelial cells.

The effect of high concentrations of glucose on Na, K-ATPase activity and the polyol pathway was studied using cultured bovine aortic endothelial cells. Na, K-ATPase activity was expressed as ouabain-sensitive K+ uptake. A significant decrease in Na, K-ATPase activity with an intracellular accumulation of sorbitol was found in confluent endothelial cells incubated with 400 mg/dl glucose for 96 h. However, there was no significant change in the Na, K-ATPase activity or sorbitol content of the cells incubated with 100 mg/dl glucose plus 300 mg/dl mannitol. The decrease in Na, K-ATPase induced by the high glucose concentration was restored by the simultaneous addition of 10(-4) M ponalrestat (ICI 128,436; Statil), an aldose reductase inhibitor. The addition of this agent also significantly reduced the increase in sorbitol induced by high glucose levels. These results suggest that the decrease in Na, K-ATPase activity induced in cultured aortic endothelial cells by high concentrations of glucose may be caused in part by the accumulation of sorbitol.