Transient increase in Ca2+ influx in Saccharomyces cerevisiae in response to glucose: effects of intracellular acidification and cAMP levels

Influx of 45Ca2+ into Saccharomyces cerevisiae was measured under experimental conditions which enabled measurements of initial rate of transport across the plasma membrane, without interference by the vacuolar Ca2+ transport system. Addition of glucose or glycerol to the cells, after pre-incubation in glucose-free medium for 5 min, caused a rapid, transient increase in 45Ca2+ influx, reaching a peak at 3-5 min after addition of substrate. Ethanol, or glycerol added with antimycin A, had no effect on 45Ca2+ influx. We have shown previously that this increase is not mediated by an effect of the substrates on intracellular ATP levels. Changes in membrane potential accounted for only a part of the glucose-stimulated 45Ca2+ influx. The roles of intracellular acidification and changes in cellular cAMP in mediating the effects of glucose on 45Ca2+ influx were examined. After a short preincubation in glucose-free medium addition of glucose caused a decrease in the intracellular pH, [pH]i, which reached a minimum value after 3 min. A transient increase in the cellular cAMP level was also observed. Addition of glycerol also caused intracellular acidification, but ethanol or glycerol added with antimycin A had no effect on [pH]i. Artificial intracellular acidification induced by exposure to isobutyric acid or to CCCP caused a transient rise in Ca2+ influx but the extent of the increase was smaller than that caused by glucose, and the time-course was different. We conclude that intracellular acidification may be responsible for part of the glucose stimulation of Ca2+ influx.(ABSTRACT TRUNCATED AT 250 WORDS)     

Magnesium uptake by pancreatic islet cells is modulated by stimulators and inhibitors of the B-cell function

28Mg2+ uptake by rat islets was measured during incubation with various stimulators or inhibitors of insulin release. D-Glucose induced a dose-dependent increase in 28Mg2+ uptake after 10 min or 120 min. The threshold concentration was around 6 mM and the maximum effect was observed with 15-20 mM glucose. After 120 min 28Mg2+ uptake was also stimulated by the metabolized sugars mannose, N-acetylglucosamine or glyceraldehyde, was unaffected by the non-metabolized or poorly metabolized L-glucose, galactose, 3-O-methylglucose, 2-deoxyglucose, fructose or mannoheptulose and was inhibited by glucosamine. The effect of glucose was markedly impaired by mannoheptulose, glucosamine, aminooxyacetate and NH4Cl, but was only partially decreased by D600 or diazoxide, which were ineffective in a glucose-free medium. Tolbutamide or KCl slightly increased 28Mg2+ uptake. Alanine, leucine alone or with glutamine, and ketoisocaproate also stimulated 28Mg2+ uptake, whereas arginine and lysine decreased it. These changes in 28Mg2+ uptake, brought about by various modifiers of the B-cell function, are thus similar but not identical to the changes in Ca2+ uptake, and are not the consequence of insulin release. The stimulatory effect of glucose requires glucose metabolism by islet cells, but is only partially due to depolarization of the B-cell membrane.     

Derepression and carrier turnover: evidence for two distinct mechanisms of hexose transport regulation in animal cells.

Hexose uptake by hamster cells was increased five to ten fold by either substituting D-fructose for glucose or by completely omitting D-glucose from the culture medium for 24 to 48 hours. Conversely, when cycloheximide was present for 24 hours in media containing glucose, up to 20-fold decreases in hexose uptake were observed. However, these decreases in uptake activity were only observed over a narrow range of cycloheximide concentrations. After extended exposure to low concentrations of cycloheximide (0.05 to 10 mug/ml), the uptake by the fed cells decreased parallel with inhibition of protein synthesis whereas at high concentrations (greater than 50 mug/ml) uptake was increased. Cells deprived of glucose and maintained in the presence of cycloheximide did not show decreases in uptake activity. In separate experiments the high uptake rates of glucose-starved cells could be decreased by addition of glucose-free medium. The reversal was complete in 6 to 8 hours. The analog of glucose, 2-deoxy-D-glucose, did not promote the time-dependent decrease suggesting that the 6-phosphoester of glucose is not an inhibitor of transport. In addition, when cycloheximide is added at the same time as glucose, there is no decrease in uptake for at least 12 hours. We propose that turnover of components of hexose uptake systems could account for part of the control of hexose transport. Moreover, the results indicate that the turnover mechanism becomes inactive during glucose starvation and must be resynthetized following refeeding of the starved cells with glucose.     

Uptake of the glucose analogue 2-deoxyglucose by germinating mitospores of Allomyces macrogynus.

Mitospores or cysts of Allomyces macrogynus do not take up the glucose analogue 2-deoxyglucose. Uptake of 2-deoxyglucose by germlings begins at 25 min into germination, the start of the rhizoid stage, and increases in rate by approximately 50-fold until 100 min into germination. The rate remains constant from 100 to 200 min, at which time germination is completed and hyphal formation begins. The presence of glucose in the germination medium blocks the uptake of 2-deoxyglucose. Of the other sugars tested, only galactose had any effect on 2-deoxyglucose uptake. Actinomycin D treatment during germination in a glucose-containing medium prevented the appearance of the uptake system, but actinomycin D was not effective after the transfer to a glucose-free medium. Cycloheximide treatment prevented the appearance of the uptake system if it was added at the time of the transfer to the glucose-free medium; it inhibited uptake only partially if the germlings were starved of glucose before its addition. It appears, therefore, that both ribonucleic acid synthesis during germination and protein synthesis after the removal of glucose are required for the uptake of 2-deoxyglucose.     

The stimulus-secretion coupling of glucose-induced insulin release. Insulin release due to glycogenolysis in glucose-deprived islets.

1. When pancreatic islets are preincubated for 20h in the presence of glucose (83.3mM) and thereafter transferred to a glucose-free medium, theophylline (1.4mM) provokes a dramatic stimulation of insulin release. This phenomenon does not occur when the islets are preincubated for either 20h at low glucose concentration (5.6mM) or only 30 min at the high glucose concentration (83.3mM). 2. The insulinotropic action of theophylline cannot be attributed to contamination of the islets with exogenous glucose and is not suppressed by mannoheptulose. 3. The secretory response to theophylline is an immediate phenomenon, but disappears after 60min of exposure to the drug. 4. The release of insulin evoked by theophylline is abolished in calcium-depleted media containing EGTA. Theophylline enhances the net uptake of 45Ca by the islets. 5. Glycogen accumulates in the islets during the preincubation period, as judged by both ultrastructural and biochemical criteria. Theophylline significantly increases the rate of glycogenolysis during the final incubation in the glucose-free medium. 6. The theophylline-induced increase in glycogenolysis coincides with a higher rate of both lactate output and oxidation of endogenous 14C-labelled substrates. 7. These data suggest that stimulation of glycolysis from endogenous stores of glycogen is sufficient to provoke insulin release even in glucose-deprived islets, as if the binding of extracellular glucose to hypothetical plasma-membrane glucoreceptors is not an essential feature of the stimulus-secretion coupling process.     

Uptake of Ca2+ driven by the membrane potential in energy-depleted yeast cells

The time-course of 45Ca2+ influx into yeast cells was measured under non-steady-state conditions obtained by preincubating the cells in a Ca2+-free medium containing glucose and buffer. Two components were distinguished: a saturable component which reached a steady-state after about 40 s of 45Ca2+ uptake and a linear increase in cellular 45Ca2+ starting after 60-90 s. Using differential extraction methods it was determined that after 20 s of uptake, 45Ca2+ was localized in the cytoplasmic pool and in bound form with no 45Ca2+ in the vacuole. After 3 min most of the cellular 45Ca2+ was concentrated in the vacuole and in bound form. The initial rate of 45Ca2+ uptake under non-steady-state conditions thus measured 45Ca2+ transport across the plasma membrane without interference by vacuolar uptake. The effect of membrane potential (delta psi) on this transport was investigated in cells depleted of ATP. A high delta psi was produced by preincubating the cells with trifluoperazine (TFP) and subsequently washing the cells free from TFP. Substantial 45Ca2+ influx was measured in the absence of metabolic energy in cells with a high delta psi. Below a threshold value of -69.5 mV the logarithms of the initial rate of 45Ca2+ influx and of the steady-state level of the first component were linear with respect to delta psi. It is suggested that 45Ca2+ influx across the plasma membrane is mediated by channels which open when delta psi is below a threshold value. The results indicated that Ca2+ influx across the plasma membrane was driven electrophoretically by delta psi.     

Metabolic pathways in Ehrlich ascites tumor cells recovering from a low bioenergetic status

Ehrlich ascites tumor cells were found to be in a low bioenergetic status, as evaluated by acridine orange uptake and ATP content, when resuspended in a glucose medium shortly after removal from the animal. Dye uptake as well as ATP content then increased for about 2 h at room temperature. This effect was only slightly inhibited by oligomycin. Cells resuspended in a glucose-free medium initially showed high dye uptake and ATP level, which were stable over time: in this case oligomycin caused a drop in both dye uptake and ATP level. The above findings, which are indicative of a marked Crabtree effect in Ehrlich ascites tumor cells, means that it is unlikely that limiting ADP and P_i play an important role in the glucose-induced inhibition of oxidative phosphorylation in this system.     

Growth and function of primary rabbit kidney proximal tubule cells in glucose-free serum-free medium.

The properties of primary rabbit kidney proximal tubule cells in glucose-free serum-free medium have been examined. Primary rabbit kidney proximal tubule cells were observed to grow at the same rate, 1.0 doublings/day, both in glucose-free and in glucose-supplemented medium. Growth in glucose-free medium was dependent upon the presence of an additional nutritional supplement, such as glutamine, pyruvate, palmitate, lactate, or beta hydroxybutyrate. Lactate, pyruvate, and glutamate are utilized for renal gluconeogenesis in vivo. The growth of the primary rabbit kidney proximal tubule cells in glucose-free medium was also dependent upon the presence of the three growth supplements insulin, transferrin, and hydrocortisone. Insulin was growth stimulatory to the primary proximal tubule cells in glucose-free medium, although insulin causes a reduction in the phosphoenolpyruvate carboxykinase (PEPCK) activity in these cells. PEPCK is a key regulatory enzyme in the gluconeogenic pathway. In order to evaluate whether or not the primary cells have gluconeogenic capacity, their glucose content was determined. The cells contained 5 pmoles D-glucose/mg protein. However, no significant glucose was detected in the medium. Presumably, the primary cells were either utilizing or storing the glucose made by the gluconeogenic pathway. Consistent with this latter possibility, cellular glycogen levels were observed to increase with time in culture. The effect of glucose on the expression of the alpha I(IV) collagen and laminin B1 chain genes was examined. Northern analysis indicated that the level of alpha I(IV) collagen mRNA was significantly elevated in glucose containing, as compared with glucose deficient, medium. In contrast, laminin B1 chain mRNA levels were not significantly affected by the glucose content of the medium.     

Acute and chronic regulation of phosphoenolpyruvate carboxykinase mRNA by insulin and glucose

Using the well differentiated rat hepatoma Fao we have studied the regulation of phosphoenolpyruvate carboxykinase (PEPCK) mRNA by insulin and glucose and compared these results to glucose production as estimated by glucose release into the medium. Fao cells possess an active gluconeogenic pathway and, when grown in glucose-free medium, release glucose for over 8 h. Addition of the cAMP analog, 8-(4-chlorophenyl-thio) cAMP (8-CTP-cAMP) or increasing the concentration of dihydroxyacetone and oxaloacetate results in an increase in glucose release which can be suppressed by insulin at concentrations between 1 and 100 nM. These effect of cAMP and insulin are associated with parallel changes in the level of mRNAPEPCK. Insulin treatment reduces mRNAPEPCK levels in these cells by 80%; this effect is transient reaching a maximum at 2-4 h. Addition of glucose to cells grown in glucose-free (G-) medium produces a decrease in mRNAPEPCK which is similar in magnitude and kinetics to that produced by insulin. Conversely, when cells grown in normal medium are placed in G- medium mRNAPEPCK levels triple over a period of 8 h, then return toward the basal value. Cells grown in G- medium or in G- medium plus 10nM insulin for 1 yr exhibit only slightly increased levels of mRNAPEPCK and respond to both 8-CTP-cAMP, and insulin, although the response to 8-CTP-cAMP is slightly blunted. These data indicate that glucose and insulin can play independent roles in regulation of PEPCK gene expression, and that these regulatory effects are usually transient.     

Mechanisms of Toxicity and Cellular Resistance to 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine and 1-Methyl-4-Phenylpyridinium in Adrenomedullary Chromaffin Cell Cultures

Bovine adrenomedullary chromaffin (BAMC) cells, cultured in a defined medium, were used to study the mechanisms of toxicity and cellular resistance to the catecholamine neuron toxicants 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium (MPP+). The viability of the cells was assessed biochemically [cellular catecholamine content and the catalytic activities of tyrosine hydroxylase (TH) and lactate dehydrogenase (LDH)] and anatomically (by electron microscopy). When cultures of BAMC cells were exposed to MPTP or MPP+ for 3 days, a marked loss of cellular catecholamines and TH activity was observed. The addition of an inhibitor of monoamine oxidase (MAO) B (Ro 19-6327), but not MAO A (clorgyline), prevented the toxicity of MPTP but not that of MPP+. In addition, the cellular toxicity of MPP+, but not MPTP, was antagonized by desmethylimipramine, an inhibitor of cellular catecholamine uptake. The toxicity of MPP+ was time dependent, with losses of TH and the release of cellular LDH occurring after 48 h in culture. Catecholamine depletion occurred somewhat sooner, being evident after 24 h of exposure to MPP+. The cellular toxicity of MPP+ was concentration dependent and significantly enhanced by inhibitors of catecholamine vesicular uptake (reserpine, tetrabenazine, or Ro 4-1284). Electron microscopic examination of cells treated with either MPP+, tetrabenazine, or their combination revealed that MPP+ damaged BAMC cells and that this damage was markedly potentiated by the inhibition of vesicular uptake by tetrabenazine. The concentration of glucose in the culture media of untreated cells slowly decreased as a function of time. The rate of glucose consumption was markedly accelerated by MPP+ treatment and the losses in cell TH and the release of LDH into the media were preceded by a 99% depletion of glucose from the media. In cultures not treated with MPP+, lactate accumulated in the media as a function of time. Addition of MPP+ to the media increased the formation of lactate, in a concentration-dependent manner. Reserpine pretreatment further enhanced the production of lactate in response to MPP+. Culturing cells in glucose-free medium greatly potentiated the effects of MPP+ on cellular TH and catecholamines. The toxicity observed after 3 days' exposure of BAMC cells to MPP+ could be prevented when the medium was replaced with fresh medium every 24 h. The effects of glucose deprivation and reserpine were observed to be additive. The ability of MPP+ to affect mitochondrial function is determined by the capacity of the storage vesicle to sequester the pyridinium, acting as a cytosolic "buffer."(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of acetoacetate on glucose metabolism in the soleus and extensor digitorum longus muscles of the rat.

1. The effect of acetoacetate on glucose metabolism was compared in the soleus, a slow-twitch red muscle, and the extensor digitorum longus, a muscle composed of 50% fast-twitch red and 50% white fibres. 2. When incubated for 2h in a medium containing 5 mM-glucose and 0.1 unit of insulin/ml, rates of glucose uptake, lactate release and glucose oxidation in the soleus were 19.6, 18.6 and 1.47 micronmol/h per g respectively. Acetoacetate (1.7 mM) diminished all three rates by 25-50%; however, it increased glucose conversion into glycogen. In addition, it caused increases in tissue glucose, glucose 6-phosphate and fructose 6-phosphate, suggesting inhibition of phosphofructokinase. The concentrations of citrate, an inhibitor of phosphofructokinase, and of malate were also increased. 3. Rates of glucose uptake and lactate release in the extensor digitorum longus were 50-80% of those in the soleus. Acetoacetate caused moderate increases in tissue glucose 6-phosphate and possibly citrate, but it did not decrease glucose uptake or lactate release. 4. The rate of glycolysis in the soleus was approximately five times that previously observed in the perfused rat hindquarter, a muscle preparation in which acetoacetate inhibits glucose oxidation, but does not alter glucose uptake or glycolysis. A similar rate of glycolysis was observed when the soleus was incubated with a glucose-free medium. Under these conditions, tissue malate and the lactate/pyruvate ratio in the medium were decreased, and acetoacetate did not decrease lactate release or increase tissue citrate or glucose 6-phosphate. An intermediate rate of glycolysis, which was not decreased by acetoacetate, was observed when the soleus was incubated with glucose, but not insulin. 5. The data suggest that acetoacetate glucose inhibits uptake and glycolysis in red muscle under conditions that resemble mild to moderate exercise. They also suggest that the accumulation of citrate in these circumstances is linked to the rate of glycolysis, possibly through the generation of cytosolic NADH and malate formation.     

Sialic acid, galactose and fucose on the surface of Ehrlich ascites tumor cells grown in glucose-free medium in the presence of uridine

1) The content and accessibility of terminal sialic acid and galactose residues as well as the incorporation of [3H]fucose into glycoconjugates were determined in 48-h cultures of Ehrlich ascites tumor cells in a glucose-free medium supplemented with uridine, a compound which can fulfil the necessary functions of glucose. 2) Sialic-acid residues accessible to sialidase cleavage were reduced from 695 +/- 80 nmol/10(9) cells (controls) to 284 +/- 22 nmol/10(9) cells (43% of controls). In situ labeling using periodate oxidation followed by sodium borotritiide reduction revealed a tritium incorporation of 47 +/- 11% that of controls (= 4.1 x 10(5) cpm/mg protein). 3) Labeling of galactose residues of 80-90% of that of controls was achieved after treatment of the cells with galactose oxidase/sodium borotritiide. A nearly six-fold enhancement of tritium incorporation into galactose of control cells was observed after sialidase/galactose oxidase treatment and sodium borotritiide reduction (1.5----8.8 x 10(5) cpm/mg protein); only a 3.6-fold increase (1.2 x 10(5)----4.3 x 10(5) cpm/mg protein) was found with glucose-free cultured cells. It is concluded that the galactose content of the cell surface is reduced to about 50% of controls. 4) The incorporation of tritium into acid-insoluble precipitate after 24 h incubation with [3H]fucose and the activity of the acid-soluble fraction were enhanced by about 85% as compared to controls. The pattern of inhibition by tunicamycin of [3H]fucose uptake and incorporation was the same in glucose-containing standard medium and in glucose-free uridine medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of sugar transport in chick embryo fibroblasts by hexose starvation. Evidence for transcriptional regulation of transport.

Incubation of chick embryo fibroblasts in glucose-free medium resulted in a dramatic increase in the rate of 2-deoxy-D-glucose transport. The greatest increase in rate occurred during the first 20 hours of incubation in glucose-free medium and was blocked by actinomycin D, dordycepin, or cycloheximide. The conditions of 2-deoxy-D-glucose concentration and time of incubation with the sugar were determined where transport rather than phosphorylation was rate-limiting in sugar uptake. These studies demonstrated that the transport of 2-deoxy-D-glucose was rate-limiting for only 1 or 2 min when the concentration of sugar in the medium was near the Km for transport, i.e. 2mM. No difference was found in the level of hexokinase activity in homogenates prepared from cells incubated glucose-free medium or standard medium when either 2-deoxy-D-[14C]glucose or D-glucose was used as substrate. A kinetic analysis of the initial rates of 2-deoxy-D-glucose transport by Lineweaver-Burk plots showed that the Vmax for sugar transport increased from 18 to 95 nmol per mg of protein per min when fibroblasts were incubated in glucose-free medium for 40 hours. The Km remained constant at 2 mM. Analysis of the initial rates of 3-omicron-methyl-D-glucose transport by Lineweaver-Burk plots further substantiated that the increase in sugar transport was due to an increase in the Vmax for transport with the Km remaining constant. The activation energy for the transport reaction calculated from an Arrhenius plot was 17.4 Cal per mol for cells cultured in the standard medium and 17.2 Cal per mol for cells cultured in the glucose-free medium. These results are consistent with the interpretation that the Vmax increase observed in hexose-starved cells is due to an increase in the number of transport sites.     

Influence of exogenous sugars and polyols on C1-influx and efflux by the ascomycete Neocosmospora vasinfecta.

Glucose and other transportable sugars and polyols inhibited Cl- influx very soon after addition to mycelium in the process of Cl- accumulation. Under the usual experimental conditions (0.1 mM KCl, glucose greater than or equal to 2 mM) the mean percentage of inhibition of Cl- influx by glucose was 54.1 +/- 8.0 (+/- standard error; N = 26). Transport of the exogenous carbohydrate was necessary for inhibition of Cl- influx. Thus, the estimated Ki for glucose inhibition of Cl- influx (28 muM) was close to the Km for glucose transport; glycerol did not inhibit Cl- influx unless it was itself transported, and the degree of inhibition exerted by various carbohydrates correlated with their uptake rates. Inhibition was not caused by the accumulated sugar itself, as high levels (ca. 60 mM) of intramycelial 3-O-methylglucose gave rise to a stimulation of Cl- influx when the exogenous sugar was removed. It is suggested that interaction of Cl- and carbohydrate transport arises from competition for a common energy-coupling mechanism in the cell membrane. Both glucose and 3-O-methylglucose elicited Cl- efflux, but the maximal Cl- efflux rates were observed only after 40 min of incubation and only in the presence of the readily metabolizable glucose. Removal of the exogenous glucose, even after maximal Cl- efflux had been established, resulted in the rapid cessation of efflux. Studies under anaerobic conditions gave further evidence that glucose uptake was necessary and that efflux was not due to temporary depletion of energy reserves. It is proposed that glucose-induced leakage of Cl- is due to reversal of the Cl- uptake system, even though the Km for efflux is much greater than that for influx.     

Hypoxia decreases ROS level in human fibroblasts

We have previously demonstrated that cells adapt to hypoxia using different metabolic reprogramming mechanisms depending on metabolism. We now investigate how the different adapting mechanisms affect reactive oxygen species (ROS) levels, and how ROS levels and cellular metabolism are linked. We show that when skin fibroblasts grew under short-term hypoxia (1% oxygen tension) ROS level markedly decreased (-50%) whatever substrate was available to the cells. Indeed, cellular ROS level linearly and directly decreased with oxygen tension. However, these relationships cannot explain the progressive ROS level decrease observed after prolonged cells hypoxia exposure. In glucose-enriched medium reduced mitochondrial mass and greater fragmentation are observed, both clear-cut indications of mitophagy suggesting that this is responsible for cellular ROS level decrease. Otherwise, in glucose-free medium exposure to prolonged hypoxia resulted in only minor mass reduction, but significantly enhanced expression of antioxidant enzymes. Interestingly, cellular ROS levels were lower in glucose-free compared to glucose-enriched medium under either normoxic or hypoxic conditions. Taken together, these findings reveal that in primary human fibroblasts hypoxia induces a decline in ROS and that different metabolism-dependent mechanisms contribute it, besides the major oxygen concentration decrease. In addition, the present data support the notion that metabolisms generating fewer ROS are associated with lower HIF-1α stabilization.     

Nitrogen Utilization in Lemna: II. Studies of Nitrate Uptake Using NO(3)

¹³N-labeled nitrate was used to trace short-term nitrate influx into Lemna gibba L. G3 in experiments where disappearance of both radioactivity and total nitrate from the incubation medium was measured continuously and simultaneously. In plants performing net nitrate uptake from an initial nitrate concentration of 40 to 60 micromolar, there was no discrepancy between net uptake and influx, irrespective of the N status of the plants, indicating that concomitant nitrate efflux was low or nil. Plants treated with tungstate to inactivate nitrate reductase were able to take up nitrate following induction of the uptake system by exposure to a low amount of nitrate. Also, in this case, net uptake was equivalent to influx. In tungstate-treated plants preloaded with nitrate, both net uptake and influx were nil. In contrast to these observations, a clear discrepancy between net uptake and influx was observed when the plants were incubated at an initial nitrate concentration of approximately 5 micromolar, where net uptake is low and eventually ceases. It is concluded that plasmalemma nitrate transport is essentially unidirectional in plants performing net uptake at a concentration of 40 to 60 micromolar, and that transport is nil when internal nitrate sinks (vacuole, metabolism) are eliminated. The efflux component becomes increasingly important when the external concentration approaches the threshold value for net nitrate uptake (the nitrate compensation point) where considerable exchange between internal and external nitrate occurs.     

Nitrogen Utilization in Lemna: II. Studies of Nitrate Uptake Using 13NO3?

¹³N-labeled nitrate was used to trace short-term nitrate influx into Lemna gibba L. G3 in experiments where disappearance of both radioactivity and total nitrate from the incubation medium was measured continuously and simultaneously. In plants performing net nitrate uptake from an initial nitrate concentration of 40 to 60 micromolar, there was no discrepancy between net uptake and influx, irrespective of the N status of the plants, indicating that concomitant nitrate efflux was low or nil. Plants treated with tungstate to inactivate nitrate reductase were able to take up nitrate following induction of the uptake system by exposure to a low amount of nitrate. Also, in this case, net uptake was equivalent to influx. In tungstate-treated plants preloaded with nitrate, both net uptake and influx were nil. In contrast to these observations, a clear discrepancy between net uptake and influx was observed when the plants were incubated at an initial nitrate concentration of approximately 5 micromolar, where net uptake is low and eventually ceases. It is concluded that plasmalemma nitrate transport is essentially unidirectional in plants performing net uptake at a concentration of 40 to 60 micromolar, and that transport is nil when internal nitrate sinks (vacuole, metabolism) are eliminated. The efflux component becomes increasingly important when the external concentration approaches the threshold value for net nitrate uptake (the nitrate compensation point) where considerable exchange between internal and external nitrate occurs.     

Ebselen Protects Ca2+ Influx Blockage But Does Not Protect Glutamate Uptake Inhibition Caused By Hg2+

The goal of this study was to investigate the isolated and combined effect of ebselen and Hg2+ on calcium influx and on glutamatergic system. We examined the in vitro effects of 2 phenyl-1,2-benzisoselenazol-3(2H)-ona), (Ebselen) on 45Ca2+ influx in synaptosomes of rat at rest and during depolarization and glutamate uptake into synaptosomes. Entry of 45Ca was measured during exposure to mercury in non-depolarizing and depolarizing solutions. Ebselen abolished the inhibition of 45Ca2+ influx on non-depolarizing conditions; however, ebselen did no modify inhibition uptake of 45Ca2+ caused by Hg2+ in high K+ depolarizing medium. Ebselen did not modify glutamate uptake inhibition caused by Hg2+ in synaptosomes. These results indicate that ebselen has an in vitro protective effect against Hg2+ induced inhibition of Ca2+ influx into synaptosomes, depending on the depolarizing conditions of the assay. The effects of Hg2+ on glutamate uptake were not modified by ebselen, suggesting that its protection is dependent on the target protein considered.     

Transport enhancement and reversal: glucose and 3-O-methyl glucose.

In chick embryo fibroblast cultures the 15- to 30-fold enhancement of D-glucose uptake observed when cells are starved of glucose for 24 hours is not duplicated for derivatives of glucose that compete effectively for uptake and have generally been considered to use the same carrier. 2-deoxy-D-glucose, D-mannose, D-galactose and D-glucosamine are derepressed progressively less sharply in that order with glucosamine uptake never more than doubled by starvation. D-glucose at a concentration of 5.5 mM in the 24-hour conditioning medium is a strong "repressor" resulting in low "transport" behavior for each of the five sugars cited. D-glucosamine is equally effective at the same concentration. A 10-fold reduction in the concentration of glucosamine (0.55 mM) allows for the escape from repression of mannose, glucose, and deoxyglucose uptake while the others remain repressed. Mannose uptake escapes as well when the glucose concentration in the "conditioning" medium is similarly reduced. Under certain conditions of starvation and cell density dramatic effects of supplemental stimulation by insulin can be achieved. Insulin withdrawal interrupts the supplemental stimulation process. Cycloheximide, actinomycin D and cordycepin block both non-insulin and insulin-induced derepression. Short exposure (15-30 minutes) of 24-hour starved cells to glucose (5.5 mM) reduces glucose sharply but does not affect 3-O-methyl glucose uptake. If the exposure is to 2-deoxyglucose (5.5 mM) further derepression of glucose uptake results.     

Potassium ion influx measurements on cultured Chinese hamster cells exposed to 60-Hertz electromagnetic fields

Potassium ion influx was measured by monitoring ⁴²KCI uptake by Chinese hamster ovary (CHO) cells grown in suspension culture and exposed in the culture medium to 60–Hz electromagnetic fields up to 2.85 V/m. In the presence of the field CHO cells exhibited two components of uptake, the same as previously observed for those grown under normal conditions; both these components of influx were decreased when compared to sham-exposed cells. Although decreases were consistently observed in exposed cells when plotted as log_c of uptake, the differences between the means of the calculated fluxes of exposed and sham-exposed cells were quite small (on the order of 4–7%). When standard deviations were calculated, there was no significant difference between these means; however, when time-paired uptake data were analyzed, the differences were found to be statistically significant. Cells exposed only to the magnetic field exhibited similar small decreases in influx rates when compared to sham-exposed cells, suggesting that the reduction in K⁺ uptake could be attributed to the magnetic field. Additionally, intracellular K⁺ levels were measured over a prolonged exposure period (96 h), and no apparent differences in intracellular K⁺ levels were observed between field-exposed and shamexposed cultures. These results indicate that high-strength electric fields have a small effect on the rate of transport of potassium ions but no effect on long-term maintenance of intracellular K⁺.