Physiology of myeloma cells grown in glucose-limited chemostat cultures

A recombinant myeloma NS1-derived clone was grown in chemostat cultures in Dulbecco's MEM/Ham's F12 (1∶1) medium containing various concentrations of glucose, at a dilution rate of 0.028 h⁻¹. Serum-supplemented cultures were virtually glucose-limited at a large range of glucose feed concentrations (0.7–5 mM). True glucose-limited cultures, however, were only established at low glucose supply levels to 1.3 mM at a maximum. In cultures obtained at higher glucose concentrations methionine was shown to be the growth-limiting compound. The pattern derived for serum-free chemostat cultures was similar, except that growth yields on glucose were much lower. Glucose was shown to be the growth-limiting substrate in cultures fed with media containing less than 4.5 mM glucose. Upon supplying glucose at higher concentrations such cultures presumably run into methionine and/or tryptophan limitation.     

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.     

Glucose transport of Escherichia coli growing in glucose-limited continuous culture.

Dilute cultures of wild-type Escherichia coli K12 and of derivatives impaired in one or other Enzyme-II component of the glucose phosphotransferase system were grown in continuous culture under glucose limitation. Cells harvested from the chemostat took up [U-14C]glucose from 0.1 mM solutions at rates directly related to the rates at which those cells had grown; the activity of the phosphotransferase system in those cells, rendered permeable with optimal accounts of toluene, parallels the ability of the cells to take up glucose. The capacity of these systems was rate-limiting for growth under the negligibly low glucose concentration in the chemostat, but was adequate to account for the stimulation of respiration observed when the cells were presented suddenly with excess glucose.     

Change from homo- to heterolactic fermentation by Streptococcus lactis resulting from glucose limitation in anaerobic chemostat cultures.

Lactic streptococci, classically regarded as homolactic fermenters of glucose and lactose, became heterolactic when grown with limiting carbohydrate concentrations in a chemostat. At high dilution rates (D) with excess glucose present, about 95% of the fermented sugar was converted to l-lactate. However, as D was lowered and glucose became limiting, five of the six strains tested changed to a heterolactic fermentation such that at D = 0.1 h(-1) as little as 1% of the glucose was converted to l-lactate. The products formed after this phenotypic change in fermentation pattern were formate, acetate, and ethanol. The level of lactate dehydrogenase, which is dependent upon ketohexose diphosphate for activity, decreased as fermentation became heterolactic with Streptococcus lactis ML(3). Transfer of heterolactic cells from the chemostat to buffer containing glucose resulted in the nongrowing cells converting nearly 80% of the glucose to l-lactate, indicating that fine control of enzyme activity is an important factor in the fermentation change. These nongrowing cells metabolizing glucose had elevated (ca. twofold) intracellular fructose 1,6-diphosphate concentrations ([FDP](in)) compared with those in the glucose-limited heterolactic cells in the chemostat. [FDP](in) was monitored during the change in fermentation pattern observed in the chemostat when glucose became limiting. Cells converting 95 and 1% of the glucose to l-lactate contained 25 and 10 mM [FDP](in), respectively. It is suggested that factors involved in the change to heterolactic fermentation include both [FDP](in) and the level of lactate dehydrogenase.     

Long-Term Changes in Chemostat Cultures of Cytophaga johnsonae

Long-term studies with a gliding, heterotrophic bacterium, Cytophaga johnsonae, were conducted in a glucose-limited chemostat at a high and a low dilution rate. To test the stability of the steady state during long-term experiments the following parameters were monitored: optical density, glucose concentration, glucose uptake potential, ATP content of the cells, and plate counts on two different agar media. Biomass remained relatively constant, although the observed changes could have been possible in both directions. During all steady states, glucose uptake showed a stepwise increase and the glucose concentration showed a corresponding decrease. Glucose uptake potential and glucose concentration in the chemostat were inversely proportional. The ATP content of the cells varied up to 33% during the steady state, but did not show a general trend. After long cultivation in all chemostats, plate counts on both agars dropped to values less than 20% of the original steady-state level. These decreases were due to an inability of the cells to grow on agar plates, not to a lack of vitality of the cells in the chemostat. This study showed that even during shorter chemostat runs, e.g., 1 week, changes in important parameters with the steady state must be expected, especially in the uptake potential and the concentration of the limiting substrate.     

Effect of endogenous methylglyoxal on Chinese hamster ovary cells grown in culture

Methylglyoxal is a ketoaldehyde that reacts readily under physiological conditions with biologically relevant ligands, such as amine and sulfhydryl groups. It is produced in mammalian cells primarily as a by-product of glycolysis. The level of glucose, L-glutamine and fetal bovine serum in culture media was found to significantly affect levels of intracellular methylglyoxal in Chinese hamster ovary cells. Medium with 25 mM glucose and 5 mM L-glutamine caused an increase in free methylglyoxal levels of 90 to 100% relative to medium containing 5 mM glucose and 2 mM L-glutamine. Both of these media compositions are representative of those found in commercially available media. Pseudomonas putida glyoxalase I was expressed in Chinese hamster ovary cells to enhance methylglyoxal detoxification. The Chinese hamster ovary cell clones showed an 80 to 90% decrease in free methylglyoxal levels. The colony-forming ability of these cells was compared to wild-type Chinese hamster ovary cells under conditions found to cause elevated methylglyoxal levels. The wild-type cells showed a 10% decrease in colony-forming ability relative to the clones. This decrease was found to be statistically significant (P>0.99) by analysis of variance. The variation in colony-forming ability amongst the clones was statistically insignificant. More importantly, the clones shoed increased colony-forming ability relative to the wild-type cells under conditions of higher methylglyoxal production with fair to good statistical significance (P>0.75 to P>0.95). This result is the first quantifiable evidence that endogenously produced methylglyoxal can negatively affect cell function under conditions found in animal cell culture.Abbreviations ANOVA analysis of variance - CHO Chinese hamster ovary cells - CFA colony-forming ability - dhfr gene for dihydrofolate reductase - DHAP dihydroxyacetone phosphate - FBS fetal bovine serum - G-3-P glyceraldehyde-3-phosphate - GloI glyoxalase I - GloII glyoxalase II - GSH reduced glutathione - HPLC high-performance liquid chromatography - IMDM Iscove's modified Dulbecco's medium - MTX methotrexate - 2-MQ 2-methylquinoxaline - 5-MQ 5-methylquinoxaline - MEM minimal essential medium - P_i inorganic phosphate - PCA perchloric acid - o-PD o-phenylenediamine     

Transient Responses of Glucose-Limited Cultures of Cytophaga johnsonae to Nutrient Excess and Starvation

Cells from glucose-limited chemostat cultures of Cytophaga johnsonae were subjected to a sudden relaxation of substrate limitation by injecting the cells into fresh batch cultures. Starvation experiments were carried out by injecting glucose-limited cells into batch cultures lacking glucose. Transient responses of biomass, glucose uptake and mineralization, ATP content, and viability on different agar media were monitored during these nutrient-shift experiments. Cells reacted differently depending on growth rate and time spent in the chemostat. Fast-growing cells showed an immediate adaptation to the new growth conditions, despite some initial overshoot reactions in ATP and uptake potential. In contrast, slowly growing cells and long-term-adapted cells showed extensive transient growth responses. Glucose uptake and mineralization potentials changed considerably during the transient growth phase before reaching new levels. During the starvation experiments, all cell types displayed a fast decrease in ATP, but the responses of the substrate uptake and mineralization potentials were strongly dependent upon the previous growth rate. Both potentials decreased rapidly in cells with high growth rates. On the other hand, cells with low growth rates maintained 80% of their uptake and mineralization potentials after 8 h of starvation. Thus, slowly growing cells are much better adapted for starvation than are fast-growing cells.     

Inhibition of fatty acid translocase cluster determinant 36 (CD36), stimulated by hyperglycemia, prevents glucotoxicity in INS-1 cells

The purpose of the present study was to determine whether exposure of pancreatic islets to glucotoxic conditions changes fatty acid translocase cluster determinant 36 (CD36) and examine the role of CD36 on the induction of glucotoxicity. We measured the changes of CD36 and insulin secretion in high glucose (30 mM) exposed INS-1 cells and CD36 suppressed INS-1 cells by transfection of CD36 siRNA. The intracellular peroxide level of INS-1 cells increased in the high glucose media compared to normal glucose (5.6mM) media. The mRNA levels of insulin and PDX-1, as well as glucose stimulated insulin secretion (GSIS) were decreased in INS-1 cells exposed to high glucose media compared to normal glucose media, while CD36 and palmitate uptake were significantly elevated with exposure to high glucose media for 12h. The inhibition of CD36 reversed the decreased GSIS and intracellular peroxide level in INS-1 cells. These results suggest that high glucose may exacerbate glucotoxicity via increasing fatty acid influx by elevation of CD36 expression, and that CD36 may be a possible target molecule for preventing glucotoxicity in pancreatic beta-cells.     

Variability of Bacillus thuringiensis under various growth conditions

When a lysogenic culture of Bacillus thuringiensis subsp. galleriae 69-6 was grown under the batch conditions, 93-99% of cells in the population produced R-form colonies and ca. 1% yielded S-form colonies. The amount of spore-forming cells was 99% in R-variants and 8% in S-variants. The quantity of S-variants rose abruptly to 99% when the culture was grown under the chemostat conditions. The number of S-variants increased with the rate and the duration of growth. The process was influenced by growth-limiting factors. Temperate phage variants capable of host culture lysis on solid media (i.e. h-mutants) were not found under the conditions of batch cultivation. However, such phage particles (h-mutants) appeared under the conditions of chemostat. The titre of these phage particles reached 10(8), 10(7) and 10(4) particles per 1 ml at limitation with yeast extract, glucose and phosphorus, respectively. Under the conditions of chemostat, the particles behaved as temperate ones and their growth was not found. Irrespective of the limitation, the phage titre did not correlate with the ratio of R and S-forms in the population. When the growth was limited with phosphorus, the quantity of S-forms increased abruptly while the spontaneous induction of the phage was inhibited. The quantity of cells capable of spore formation decreased in the cultures isolated from the chemostat and grown on MPA: 69-80% of the cells in R-forms and merely 8% in S-forms.     

Paradoxical stimulation of human sperm motility by 2-deoxyadenosine

Exposure of cryostored human spermatozoa to 2-deoxyadenosine resulted in significant increases in percentage motility, the linear velocity of progression and the frequency of sperm head rotation, which were maximal at a dose of 2.5 mM. At the same dose both adenosine and caffeine significantly increased percentage motility, although neither compound influenced the quality of sperm movement as assessed by time-exposure photomicrography. 2-Deoxyadenosine was also significantly more effective than caffeine in sustaining the motility of cryostored spermatozoa as well as in enhancing the motility of fresh and washed preparations of human spermatozoa. The ability of caffeine and 2-deoxyadenosine to influence sperm motility was counteracted by the presence of calcium in the external medium although the latter was less susceptible to such inhibition and still enhanced motility in the presence of calcium levels (1.7 mM) typical of media used for in-vitro fertilization. The mechanism of action of 2-deoxyadenosine was associated with an increase of intracellular cAMP levels, which were sustained over a time course lasting from 5 to 180 min and exhibited significant dose dependency over the range 1-10 mM. The response to 2-deoxyadenosine did not involve any changes in the steady state levels of ATP and was augmented by the presence of the phosphodiesterase inhibitors, IBMX and caffeine. We conclude that 2-deoxyadenosine is a powerful stimulator of human sperm motility and that this effect involves an increase of intracellular cAMP levels via mechanisms which do not involve the classical 'R'-site receptor mediated pathway.     

Down regulation of macrophage mannose/N-acetylglucosamine receptors by elevated glucose concentrations

The binding of the 125I-induced neoglycoprotein mannosyl-bovine serum albumin (Man-albumin) to peptone-elicited murine peritoneal macrophages was examined. Binding studies demonstrated that the extent of receptor activity for Man-albumin depended upon the glucose concentration of the medium in which the cells were cultured following peritoneal lavage and prior to the binding assay. Macrophages cultured in a medium containing a high glucose concentration (25 mM or greater) prior to the binding assay, consistently showed a reduced capacity for binding Man-albumin as compared to cells cultured in the presence of low glucose (5 mM). These results were obtained in a variety of tissue culture media or when the same medium was employed with differing amounts of added glucose (5, 25 and 50 mM). Cell toxicity and/or death was not the cause of the reduced receptor activity of macrophages cultured in high glucose as determined by morphology. Trypan blue exclusion, and the ability of these cells to actively phagocytose IgG-coated sheep red blood cells to an extent identical with those cells cultured in low glucose. Saturation binding studies and Scatchard analysis of the data demonstrated that the decreased level of binding observed with cells cultured in high glucose was the result of a reduced number of receptors and not altered receptor affinity. These studies suggest that an increased glucose concentration, such as in diabetes mellitus, can downshift the expression of the mannose/N-acetylglucosamine receptor on murine peritoneal macrophages.     

Taurine regulates insulin release from pancreatic beta cell lines

Background

Pancreatic β-cells release insulin via an electrogenic response triggered by an increase in plasma glucose concentrations. The critical plasma glucose concentration has been determined to be ~3 mM, at which time both insulin and GABA are released from pancreatic β-cells. Taurine, a β-sulfonic acid, may be transported into cells to balance osmotic pressure. The taurine transporter (TauT) has been described in pancreatic tissue, but the function of taurine in insulin release has not been established. Uptake of taurine by pancreatic β-cells may alter membrane potential and have an effect on ion currents. If taurine uptake does alter β-cell current, it might have an effect on exocytosis of cytoplasmic vesicle. We wished to test the effect of taurine on regulating release of insulin from the pancreatic β-cell.

Methods

Pancreatic β-cell lines Hit-TI5 (Syrian hamster) and Rin-m (rat insulinoma) were used in these studies. Cells were grown to an 80% confluence on uncoated cover glass in RPMI media containing 10% fetal horse serum. The cells were then adapted to a serum-free, glucose free environment for 24 hours. At that time, the cells were treated with either 1 mM glucose, 1 mM taurine, 1 mM glucose + 1 mM taurine, 3 mM glucose, or 3 mM glucose + 1 mM taurine. The cells were examined by confocal microscopy for cytoplasmic levels of insulin.

Results

In both cell lines, 1 mM glucose had no effect on insulin levels and served as a control. Cells starved of glucose had a significant reduction (p<0.001) in the level of insulin, but this level was significantly higher than all other treatments. As expected, the 3 mM glucose treatment resulted in a statistically lower (p<0.001) insulin level than control cells. Interestingly, 1 mM taurine also resulted in a statistically lower level of insulin (p<0.001) compared to controls when either no glucose or 1 mM glucose was present. Cells treated with 1 mM taurine plus 3 mM glucose showed a level of insulin similar to that of 3 mM glucose alone.

Conclusions

Taurine administration can alter the electrogenic response in β-cell lines, leading to a change in calcium homeostasis and a subsequent decrease in intracellular insulin levels. The consequence of these actions could represent a method of increasing plasma insulin levels leading to a decrease in plasma glucose levels.

     

Glucose regulation of guinea-pig sperm motility.

Washed guinea-pig spermatozoa from the vas deferens re-acquired progressive motility within 1-2 min of incubation in minimal culture medium containing pyruvate and lactate. When glucose was added, either at the beginning or after 15 min of incubation, the cells showed stimulated motility (increased straight-line velocity, linearity and beat-cross frequency, P less than 0.01). Re-acquisition of progressive motility was preceded by a significant (P less than 0.005) transient increase in sperm concentration of cyclic adenosine 5'-phosphate (cAMP) with or without glucose in the medium. Papaverine caused another large significant (P less than 0.001) increase in cAMP concentration; and 5.56mM glucose with papaverine caused a further stimulation in cAMP beyond that with papaverine alone (P less than 0.005). Although 0.05 or 5.56mM glucose plus alpha-chlorohydrin stimulated sperm motility, they did not further stimulate the increase in cAMP after 30 s of incubation. Thus, there was no apparent correlation between the glucose-stimulating effect on sperm motility and the enhancement of cAMP at 30 s. However, there was a close correlation between glucose-stimulated motility and enhancement of ATP (P less than 0.05) by glucose even under incubation conditions in which glucose caused the Crabtree effect (decrease in respiration rate).     

Induction of apoptosis in nutrient-deprived cultures of hybridoma and myeloma cells

In the present study, cell death was investigated in cultures of NS/0 myelomas and SP2/0-derived D5 hybridomas through morphological examination of cells stained with acridine orange and ethidium bromide. The relative contribution of elevated levels of lactic acid and ammonia, as well as deprivation of glutamine, cystine, and glucose on the induction of necrosis or apoptosis, was investigated. In batch culture of D5 hybridoma cells, induction of apoptotic cell death correlated with the exhaustion of glutamine, while in the case of NS/0 myelomas, it coincided with exhaustion of cystine. To determine whether limiting nutrients were the actual triggering factors for apoptosis in batch culture, exponentially growing cells were resuspended in glutamine or cystine-free media. Within 30 to 40 h, viability decreased to 50% and the nonviable cell population displayed typical apoptotic morphology, with crescents of condensed chromatin around the periphery of the nucleus, or with the entire nucleus present as one or a group of featureless, brightly staining spherical beads. Similarly, D5 hybridomas and NS/0 myelomas cultivated in glucose-free medium died mainly from apoptosis. Cells were also cultivated in fresh medium supplemented with elevated concentrations of ammonia (3.0 mM) and/or lactate (35 mM, 50 mM). This resulted in decreased viabilities and necrotic death in both cell lines. From these results, we conclude that D5 hybridomas and NS/0 myelomas deprived of essential nutrients die by apoptosis, whereas incubation in the presence of elevated levels of metabolic byproducts such as ammonia and lactate will induce necrotic cell death in these cells. (c) 1994 John Wiley & Sons, Inc.     

The onset and maintenance of hyperactivated motility of spermatozoa from the mouse

Estimates were made of the proportion of freely motile mouse spermatozoa displaying hyperactivated motility by an objective photographic method employing stroboscopic illumination under dark-field conditions and examining displacements of the sperm head and bend angles of the sperm tail. In media known to support in vitro fertilisation hyperactivation gradually appeared reaching about 40% by 6 hr incubation, and it was not promoted by 2 mM caffeine or 0.1 mM Bt₂ cAMP or washing the cells free of epididymal fluid. Raising the osmolarity of the medium to 400 mOSM with electrolytes, but not nonelectrolytes, did promote hyperactivation (60% by 2 hr) suggesting that the ionic strength of the medium was important. Hyperactivation in high ionic strength media could be prevented by removing or chelating Ca²⁺, or replacing Ca²⁺ with Ba²⁺ or Mg²⁺, when nonhyperactivated motility was maintained, but Sr²⁺, like Ca²⁺, permitted hyperactivated motility. Hyperactivation in low ionic strength medium could be promoted by the ionophore A23187, suggesting that Ca²⁺ movement into the cells is important. Of a range of glycolytic substrates tested supporting nonhyperactivated motility in the presence of lactate, only glucose supported hyperactivation. Addition to glucose— or Ca²⁺ — free, high ionic strength media after 2 hr increased hyperactivation immediately (glucose) or after a lag of 2 hr (Ca²⁺) suggesting that glucose acts on a Ca²⁺ — primed system. Removal from high ionic strength medium, chelation of Ca²⁺ or inhibition of glucose metabolism did not prevent hyperactivation continuing once it had been initiated, indicating different requirements for initiation and maintenance of this form of motility.     

L-亮氨酸对克隆的胰岛β细胞株INS-1E细胞分泌胰岛素的葡萄糖依赖性研究

目的:探讨L-亮氨酸对克隆的胰岛β细胞株INS-1E细胞分泌胰岛素的刺激作用及其葡萄糖依赖性。方法:INS-1E细胞经传代培养2 d后,在Krebs-Ringer缓冲液中37℃培养箱预培养30 min,再用含有不同浓度葡萄糖和不同浓度L-亮氨酸的改良Krebs-Ringer缓冲液培养60 min,然后留取上清液进行胰岛素测定。结果:L-亮氨酸在0.1~10 mmol.L-1范围不增加16.7mmol.L-1葡萄糖刺激的INS-1E细胞的胰岛素分泌,仅20 mmol.L-1的L-亮氨酸促进葡萄糖诱导的胰岛素分泌;10 mmol.L-1L-亮氨酸在1.1、3.3、6.7 mmol.L-1葡萄糖存在的情况下促进INS-1E细胞的胰岛素分泌,而在11.1、16.7、25 mmol.L-1葡萄糖存在的情况下无促进胰岛素分泌的作用。结论:本研究显示在无刺激胰岛素分泌的葡萄糖浓度条件下,10 mmol.L-1L-亮氨酸即显示了刺激INS-1E细胞分泌胰岛素的作用,在较高葡萄糖的条件下,10 mmol.L-1L-亮氨酸的作用减弱或消失。     

Postthaw evaluation of in vitro function of epididymal spermatozoa from four species of free-ranging African bovids

An improved understanding of reproductive physiology in nondomestic bovids is necessary for the development of assisted reproductive technologies (ARTs) for use in the conservation of endangered bovids. In this study, epididymal spermatozoa were recovered from blesbok (Damaliscus dorcas phillipsi), African buffalo (Syncerus caffer), springbok (Antidorcas marsupialis), and black wildebeest (Connochaetes gnou) following organized culls in South Africa. Our objectives were 1) to characterize the quality of epididymal spermatozoa, 2) evaluate the effectiveness of a cryopreservation protocol, and 3) compare postthaw sperm longevity (motility, viability, and acrosomal integrity) and functionality in two culture media with two capacitation reagents (caffeine and heparin). Following recovery, spermatozoa were diluted in EQ extender, slow-cooled, and frozen in the presence of 5% glycerol. Thawed spermatozoa were separated on a Percoll gradient and diluted in fertilization media (SOF for fertilization [SOFfert]; 0.6% BSA, 0.0 mM glucose, 25.0 mM NaHCO(3)) or modified SOFfert (1.2% BSA, 1.5 mM glucose, 37.0 mM NaHCO(3)) and either heparin or caffeine, and incubated for 6 h. Spermatozoa from these species maintained an average of 64% initial motility after thawing. Incubation medium and capacitation reagent had species-specific effects on the motility, viability, and acrosomal integrity of spermatozoa, suggesting ART procedures need to be optimized for each species. Springbok spermatozoa were also shown to be competent for in vitro fertilization. Information from this study concerning sperm physiology in blesbok, African buffalo, springbok, and black wildebeest will be useful in the development of ART for the conservation of these and other species of bovids.     

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)