External ATP-induced passive permeability change and cell lysis of cultured transformed cells: action in serum-containing growth media

External ATP causes a marked increase in the passive permeability to phosphorylated metabolites in several types of transformed cells in alkaline medium containing low concentrations of Ca2+, but not in untransformed cells. Such increased membrane permeability with external ATP was also observed in B16 melanoma cells at pH 7.4-7.5 in both Tris-buffered saline and a growth medium containing 10% calf serum and divalent ions at normal concentrations, although a higher concentration of ATP was required. The permeability change in the growth medium was significantly enhanced by calmodulin-interacting drugs, such as trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) and chlorpromazine (CPZ). As expected, prolonged exposure of the cells to ATP in the serum-containing medium led to cell lysis. This ATP-dependent cell lysis was observed only in several transformed cell lines, and not in untransformed mouse fibroblasts. These results indicate that the effect of ATP on the membrane permeability in transformed cells is elicited under the physiological conditions and this would be useful in some limited way for cancer chemotherapy management.     

Growth inhibition of transformed mouse fibroblasts by adenine nucleotides occurs via generation of extracellular adenosine

The growth of transformed mouse fibroblasts (3T6 cells) in medium containing 5% fetal bovine serum was inhibited after treatment with concentrations greater than 50 microM ATP, ADP, or AMP. Adenosine, the common catabolite of the nucleotides, had no effect on cell growth at concentrations below 1 mM. However, the following results indicate that the toxicity of ATP, ADP, and AMP is mediated by serum- and cell-associated hydrolysis of the nucleotides to adenosine. 1) ADP and AMP, but not ATP, were toxic to 3T6 cells grown in serum-free medium or medium in which phosphohydrolase activity of serum was inactivated. Under these conditions, the cells exhibited cell-associated ADPase and 5'-nucleotidase activity, but little ecto-ATPase activity. 2) Inhibition of adenosine transport in 3T6 cells by dipyridamole or S-(p-nitrobenzyl)-6-thioinosine prevented the toxicity of ATP in serum-containing medium and of ADP and AMP in serum-free medium. 3) A 16-24-h exposure to 125 microM AMP or ATP was needed to inhibit cell growth under conditions where serum- and cell-associated hydrolysis of the nucleotides generated adenosine in the medium continuously over the same time period. In contrast, 125 microM adenosine was completely degraded to inosine and hypoxanthine within 8-10 h. Furthermore, multiple doses of adenosine added to the cells at regular intervals over a 16-h period were significantly more toxic than an equivalent amount of adenosine added in one dose. Treatment of 3T6 cells with AMP elevated intracellular ATP and ADP levels and reduced intracellular UTP levels, effects which were inhibited by extracellular uridine. Uridine also prevented growth inhibition by ATP, ADP, and AMP. These and other results indicate that serum- and cell-associated hydrolysis of adenine nucleotides to adenosine suppresses growth by adenosine-dependent pyrimidine starvation.     

Biphasic Effect of ATP on In Vitro Mineralization of Dental Pulp Cells

Dental pulp cells release adenosine triphosphate (ATP) in response to intrapulpal pressure and the amount released depends on the magnitude of the pressure. ATP regulates the differentiation of stem cells into adipocytes and osteoblasts. However, it is unknown whether extracellular ATP influences the stemness and osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHEDs). Therefore, this study investigated the effects of extracellular ATP at a low (0.1 μM) and high (10 μM) concentration on the stemness and osteogenic differentiation of SHEDs. Cells were cultured in either growth medium or osteogenic medium with or without 0.1–10 μM ATP. In growth medium, both concentrations of ATP increased the mRNA expression of pluripotent and osteogenic markers. In contrast, in osteogenic medium, 0.1 μM ATP enhanced in vitro mineralization, whereas 10 μM ATP inhibited this process. In addition, 10 μM ATP stimulated the mRNA expression and activity of ectonucleotide pyrophosphatase/phosphodiesterase (ENPP), an enzyme that regulates the phosphate/pyrophosphate ratio. Thus, depending on the growth condition and its concentration, ATP stimulated stemness and in vitro mineralization or inhibited mineralization. In growth medium, both ATP concentrations stimulated pluripotent and osteogenic marker gene expression. However, in osteogenic medium, a biphasic effect was found on in vitro mineralization; the low concentration stimulated, whereas the high concentration inhibited, mineralization. We propose that ATP released due to mechanical stress modulates the stemness and differentiation of SHEDs. J. Cell. Biochem. 119: 488–498, 2018. © 2017 Wiley Periodicals, Inc.     

An incubation medium for the elevation of adenosine triphosphate and 2,3-diphosphoglycerate in fresh and long-preserved human erythrocytes.

The levels of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate in freshly drawn human erythrocytes can be tripled by a 2 h incubation at 37 degrees C in a medium containing 21 mM glucose, 1.8 mM adenine, 5 mM pyruvate, 10 mM inosine, and 96 mM phosphate. Similar incubation conditions will restore the levels of ATP and 2,3-diphosphoglycerate in erythrocytes from blood levels preserved for 12 and 15 weeks, respectively, to those of fresh cells. Omission of pyruvate from the incubation medium further increases the level of ATP slightly, but there is little elevation of 2,3-diphosphoglycerate. Under these conditions labelled pyruvate and lactate production from [14-C]glucose or [14-C]inosine is not diminished, but labelled fructose 1,6-diphosphate, rather than 2,3-diphosphoglycerate, accumulates. In addition, omission of pyruvate from the incubation medium, with a concomitant decrease in accumulation of 2,3-diphosphoglycerate, diminishes the concentration of inorganic phosphate required for optimal ATP elevation. A 5 h incubation in the glucose-adenine-pyruvate-inosine-phosphate medium elevates the levels of ATP and 2,3-diphosphoglycerate in erythrocytes from blood preserved in the cold for 15 weeks to twice that of fresh cells, indicating that the cells retain their metabolic potential even after prolonged storage at 2 degrees C. The medium may provide a method of rejuvenating 10-12 week cold-preserved erythrocytes for transfusion purposes, by a 1 h incubation at 37 degrees C.     

Effects of UV-C radiation on extension growth, H+-extrusion and transmembrane electric potential in maize coleoptile segments

The effect of 253.7 nm ultraviolet radiation on elongation growth, medium acidification and changes in electric potential difference between vacuole and external medium in cells of maize ( Zea mays L.) coleoptile segments was investigated. It was found that irradiation with 390, 1170, 3900 and 5 850 J m⁻² UV-C (ultraviolet radiation 253.7 nm) inhibited elongation growth, whereas at 195 J m⁻² stimulation of growth was observed. The administration of IAA (10⁻⁵ M ) to the incubation medium of coleoptile segments partially abolished the inhibitory effect of UV-C. The pH of the incubation medium, measured simultaneously with growth, showed that the exposure of the segments to UV-C caused inhibition of H⁺-extrusion (or stimulation of H⁺ uptake). The presence of IAA (10⁻⁵ M ) in the incubation medium promoted (except after 5850 J m⁻² irradiation) H⁺-extrusion to a level comparable with that produced by IAA in non-irradiated segments. In UV-C irradiated segments the potential difference underwent significant alterations. Irradiation of coleoptile segments with 390 J m⁻² caused a transient depolarization, which was fully reversible within 30 min, while at higher doses depolarization was irreversible. The hyperpolarization of the membrane potential (MP) in cells of maize coleoptile induced by IAA was completely nullified by subsequent irradiation with UV-C. It is suggested that UV-C inhibited IAA-induced growth by a mechanism independent of cell wall acidification.     

Extracellular ATP increases cytoplasmic free Ca2+ concentration in clonal insulin-producing RINm5F cells. A mechanism involving direct interaction with both release and refilling of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool.

Effects of extracellularly applied ATP (added as disodium salt) on stimulus-secretion coupling were investigated in clonal insulin-producing RINm5F cells. Cytoplasmic free Ca2+ concentration [( Ca2+]i), electrical activity, membrane potential, formation of InsP3 and insulin release were measured. Addition of ATP in a Ca2(+)-containing medium promoted a rapid rise in [Ca2+]i, which was followed by a slow decline towards the basal level. In a Ca2(+)-free medium, the ATP-induced increase in [Ca2+]i was smaller, but still enough to elicit insulin secretion. Upon normalization of the extracellular Ca2+ concentration, the response to ATP recovered instantaneously. The presence of glucose in the incubation medium was a prerequisite to obtain a pronounced effect of ATP in the absence of extracellular Ca2+. However, glucose did not enhance the response to ATP in a Ca2(+)-containing medium. The effect of ATP was dose-dependent, with a clearly detectable increase in [Ca2+]i at 1 microM and a maximal response being obtained at 200 microM-ATP. The response to ATP was unaffected by activating adenylate cyclase by forskolin, but was abolished by 10 nM of the phorbol ester phorbol 12-myristate 13-acetate. The effects of ATP on [Ca2+]i could not be accounted for by a generalized increase in plasma-membrane permeability, as evident from the failure of the nucleotide to increase the fluorescence of the nuclear stain ethidium bromide. After stimulation with ATP there was an increase in membrane potential, in both the absence and the presence of extracellular Ca2+. Blockage of the voltage-activated Ca2+ channals with D-600, in a Ca2(+)-containing medium, decreased the effect of ATP on [Ca2+]i slightly. Patch-clamp measurements using the cell-attached patch configuration revealed that the RINm5F cells produce spontaneous action potentials, the frequency of which increased markedly on addition of ATP. Whole-cell recordings demonstrated that the increase in spike frequency was not associated with the development of an inward current, but was rather accountable for by a decrease in the activity of the ATP-regulated K+ channels. Addition of 200 microM-ATP stimulated phospholipase C activity, as evident from the formation of InsP3, both in the absence and in the presence of extracellular Ca2+. Thus in the absence of extracellular Ca2+ the stimulatory effect of ATP on insulin release can be explained by InsP3-induced mobilization of intracellularly bound Ca2+. Hence, in the RINm5F cells extracellular ATP acts in a manner similar to other Ca2(+)-mobilizing agents.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purinergic agonist ATP is a comitogen in thyroid FRTL-5 cells

Several growth factors may stimulate proliferation of thyroid cells. This effect has, in part, been dependent on calcium entry. In the present study using FRTL-5 cells, we show that in addition to its effect on calcium fluxes, ATP acts as a comitogen in these cells. In medium containing 5% serum, but no TSH, ATP stimulated the incorporation of ³H-thymidine in a dose- and time-dependent manner in the cells. At least a 24-h incubation with ATP was necessary to observe the enhanced (30–50%) incorporation of ³H-thymidine and an increased (30%) cell number. The effect of ATP was dependent on insulin in the incubation medium. Furthermore, ATP enhanced the TSH-mediated incorporation of ³H-thymidine. The effect of ATP was apparently mediated via a G-protein dependent mechanism, as no stimulation of thymidine incorporation was observed in cells treated with pertussis toxin. The effect of ATP was not dependent on the activation of protein kinase C (PKC), as ATP was effective in cells with downregulated PKC. ATP rapidly phosphorylated mitogen activated protein (MAP) kinase in FRTL-5 cells. In addition, ATP stimulated the expression of a 62 kDa c-fos dependent protein in a dose- and time-dependent manner. Our results thus suggest that extracellular ATP, in the presence of insulin, may be a cofactor in the regulation of thyroid cell proliferation, probably by phosphorylating MAP kinase and stimulating the expression of c-fos. © 1996 Wiley-Liss, Inc.     

Inhibition of growth by erythritol catabolism in Brucella abortus.

The growth of Brucella abortus (US-19) in a complex tryptose-yeast extract medium containing D-glucose is inhibited by 10 mM erythritol. The enzymes of the erythritol pathway, except for D-erythrulose 1-phosphate dehydrogenase (D-glycero-2-tetrulose 1-phosphate:nicotinamide adenine dinucleotide (NAD+) 4-oxidoreductase) were detected in the soluble and membrane fractions of cell extracts. Glucose catabolism by cell extracts was inhibited by erythritol, whereas, phosphorylated intermediates of the hexose monophosphate pathway were converted to pyruvic acid with oxygen consumption. Erythritol kinase (EC 2.7.1.27; adenosine 5'-triphosphate (ATP): erythritol 1-phosphotransferase) was found to be eightfold higher in activity than the hexokinase in cell extracts. In vivo, ATP is apparently consumed with the accumulation of D-erythrulose 1-phosphate (D-glycero-2-tetrulose 1-phosphate) and no substrate level phosphorylation. ATP levels dropped 10-fold in 30 min after addition of erythritol to log phase cells in tryptose-yeast extract medium with D-glucose as the carbon source. These data suggest bacteriostasis in the presence of erythritol results from the ATP drain caused by erythritol kinase.     

Uridine, but not cytidine can sustain growth of Ehrlich ascites tumor cells in glucose-deprived medium with altered proliferation kinetics

Cell cycle kinetics and energy metabolism of Ehrlich ascites tumor cells grown in glucose-deprived medium supplemented with uridine, were investigated in order to extend our knowledge about the significance of the metabolic conversion of glucose for cell cycle progression of these cells. Viability (dye exclusion test) of uridine supplemented cells was not affected, although growth was reduced to 50 to 60% of the controls. Uridine did not significantly impair growth of controls in standard medium up to 20 mM. Studies on cell cycle progression using flow cytometry (BrdU-H33258 technique) revealed an accumulation of cells in G2M phase, which can be explained by a delay in the passage of G2M-compartment of about 12 +/- 2 h. After a 24 h culture period, 60% of the cell populations were found in G2M (30% in control cultures). This fraction increased to about 70% in the following passage. Protein and DNA synthesis corresponded to the proliferation rate. Oxygen uptake was increased by about 50%, glutamine consumption by 30%, lactate production was reduced to below 10% of the controls. The ATP/ADP concentration ratio was found in a physiological range of 4 to 6. It was calculated that cells grown in standard medium produced 60% of ATP via oxidative pathways and 40% via glycolysis; however, in glucose-free, uridine-supplemented medium the values are more than 90% and less than 10%. No significant differences in total ATP production were observed. Growth of the cells in glucose-deprived medium could not be sustained by cytidine. All our data substantiate the present concept that glycolytic ATP-production is not essential for maintenance of viability and growth of these cells.     

Alterations in the survival of X-irradiated cells by 2,4-dinitrophenol depending on ATP deprivation.

The dose-survival curve of cultured melanoma cells was changed by post-irradiation treatment with 2,4-dinitrophenol (DNP). The parameters of the curves were Do = 147 R and n = 5 . 6 for untreated cells and Do = 143 R, n = 7 . 9 and Do = 142 R, n = 2 . 0 for the cells treated with 10(-5) M DNP and 5 x 10(-5) M DNP in phosphate-buffered saline, respectively. The content of ATP in the cell decreased to 5% of the control level after treatment with either concentration of DNP. The recovery of ATP content was rapid and complete after 2 hours' incubation in culture medium after the removal of 10(-5) M DNP, but was retarded and incomplete after 4 hours with 5 x 10(-5) M DNP. Thus prolonged ATP deprivation with a high concentration of DNP results in an inhibition of recovery and a reduction in the n-value.     

An immediate and steep increase in ATP concentration in response to reduced turgor pressure in Escherichia coli B

Osmotic treatment with sodium chloride of Escherichia coli B growing in the logarithmic phase induced an immediate increase in ATP concentration in response to increased concentrations of added solute in its growth medium in the first 10 min of the addition. After that, ATP concentration decreased gradually. Sodium arsenate and potassium fluoride almost abolished the ATP increase. The time course of the increase was quite different from that of cells treated with inhibitors of protein synthesis. The osmotic treatment did not decrease the viability of cells. In addition, there was no degradation of RNA by 5 min after sodium chloride addition, and, further, the lag time of ATP increase was extended by an inhibitor of nucleotide synthesis. These results indicated that a major fraction of the stress-increased ATP resulted from de novo synthesis, and that it was mainly dependent upon the reaction of substrate-level phosphorylation, which is regulated by turgor pressure.     

The involvement of cellular ATP in receptor-mediated internalization of epidermal growth factor and hormone-induced internalization of beta-adrenergic receptors

Beta-Adrenergic receptors and epidermal growth factor receptors are both expressed on the cell surface of human astrocytoma cells. Incubation with a catecholamine or epidermal growth factor results in rapid internalization of the respective receptor. The internalized receptors co-migrate in light fractions on sucrose gradients. Astrocytoma cells maintain a constant ATP concentration by either glycolytic or mitochondrial ATP production. When cells are incubated in a medium depleted of substrates for glycolysis and gluconeogenesis, addition of inhibitors of mitochondrial ATP synthesis causes a rapid reduction in cellular ATP content. An immediate return to control ATP levels occurs upon addition of an appropriate nutrient, such as glucose. Decreasing the cellular ATP content to less than 10% of control markedly inhibits internalization of beta-adrenergic receptors and epidermal growth factor. The inhibition of endocytosis is reversed as soon as the intracellular ATP content is restored. Previous work by others (Clarke, B.L., and Weigel, P.H. (1985) J. Biol. Chem. 260, 128-133) suggested that ATP is not required for internalization (per se) of asialoglycoprotein in hepatocytes but was required for recycling of the asialoglycoprotein receptor. In contrast, our results indicate that in astrocytoma cells the process of internalization of epidermal growth factor and beta-adrenergic receptors, per se, is highly ATP dependent.     

Transcriptomic and physiological analyses of the dinoflagellate Karenia mikimotoi reveal non‐alkaline phosphatase‐based molecular machinery of ATP utilisation

The ability to utilize dissolved organic phosphorus (DOP) is important for phytoplankton to survive the scarcity of dissolved inorganic phosphorus (DIP), and alkaline phosphatase (AP) has been the major research focus as a facilitating mechanism. Here, we employed a unique molecular ecological approach and conducted a broader search for underpinning molecular mechanisms of adenosine triphosphate (ATP) utilisation. Cultures of the dinoflagellate Karenia mikimotoi were set up in L1 medium (+P), DIP‐depleted L1 medium (–P) and ATP‐replacing‐DIP medium (ATP). Differential gene expression was profiled for ATP and +P cultures using suppression subtractive hybridisation (SSH) followed by 454 pyrosequencing, and RT‐qPCR methods. We found that ATP supported a similar growth rate and cell yield as L1 medium and observed DIP release from ATP into the medium, suggesting that K. mikimotoi cells were expressing extracellular hydrolases to hydrolyse ATP. However, our SSH, qPCR and enzymatic activity assays indicated that 5′‐nucleotidase (5NT), rather than AP, was responsible for ATP hydrolysis. Further gene expression analyses uncovered that intercellular purine metabolism was significantly changed following the utilisation of ATP. Our findings reveal a multi‐faceted machinery regulating ATP utilisation and P metabolism in K. mikimotoi, and underscore AP activity is not the exclusive indicator of DOP utilisation.     

Maintenance of growth transformation with Epstein-Barr virus is mediated by secretion of autocrine growth factors in two serum-free B-cell lines.

The characteristics of two tamarin (Saguinus oedipus) B-cell lines (sfBIT and sfBT) growth-transformed by Epstein-Barr virus (EBV) that proliferate continuously in serum-free medium are described. sfBIT was established by selecting cells for growth in RPMI 1640 supplemented with insulin, transferrin, and selenium (J. E. Shaw, R. G. Petit, and K. Leung, J. Virol. 61:4033-4037, 1987). sfBT, a subline of sfBIT cells reported here for the first time, required transferrin as the only protein supplement for continuous growth in RPMI 1640. Growth of sfBT cells was linear with human transferrin at 10(-2) to 10 micrograms/ml. Transferrin at 5 micrograms/ml yielded a culture density of 5 X 10(5) to 1 X 10(6) cells per ml, a cell doubling time of 2 to 3 days, and a culture viability greater than 95%. sfBIT and sfBT cells released transforming virus during continuous growth in serum-free culture medium without EBV-inducing agents. The spent medium of both serum-free lines supported cell growth at low culture density (1 x 10(4) to 5 X 10(4) cells per ml), but growth was arrested at low culture density with fresh serum-free medium. A procedure to measure growth-promoting activity (GPA) was established, and it revealed that the GPA of spent medium was greater than that of fresh medium for both serum-free cell lines. When fresh and spent media were dialyzed (molecular weight cutoff, 3,500) and subsequently concentrated by lyophilization, only the GPA of spent medium increased. We conclude that maintenance of growth transformation of tamarin cells latently infected with EBV is mediated by growth factors that are entirely autocrine in origin.     

Fermentation of Glucose, Fructose, and Xylose by Clostridium thermoaceticum: Effect of Metals on Growth Yield, Enzymes, and the Synthesis of Acetate from CO2

Clostridium thermoaceticum ferments xylose, fructose, and glucose with acetate as the only product. In fermentations with mixtures of the sugars, xylose is first fermented, then fructose, and last, glucose. Fructose inhibits the fermentation of glucose, and this inhibition appears to be due to a repression of the synthesis of an enzyme needed for glucose utilization. Addition of metals to the culture medium increases the cell yield drastically from about 7 to 18 g per liter, and Y(glucose) values between 40 and 50 are obtained. According to the postulated pathways of the fermentation of glucose and synthesis of acetate from CO(2) by C. thermoaceticum, 3 mol of ATP are available as energy for growth. Thus a Y(adenosine 5'-triphosphate) of 13 to 16 is obtained. Because the normal Y(ATP) value is 10.5, this could mean that an additional source of ATP is available by an unknown mechanism. The addition of metals also increases the nicotinamide adenine dinucleotide phosphate-dependent formate dehydrogenase activity, the overall reaction ((14)CO(2) --> acetate), and the incorporation of the methyl group of 5-methyltetrahydrofolate into acetate. These reactions are catalyzed very efficiently by cells harvested in early growth, whereas cells obtained at the end of a fermentation have very low formate dehydrogenase activity and capacity to incorporate CO(2) into acetate. The following enzymes involved in the synthesis of acetate from CO(2) and in the metabolism of pyruvate are present in extracts of C. thermoaceticum: 10-formyltetrahydrofolate synthetase, 5,10-methenyltetrahydrofolate cyclohydrolase, 5,10-methylenetetrahydrofolate dehydrogenase, 5,10-methylenetetrahydrofolate reductase, phosphate acetyltransferase, and acetate kinase. These enzymes are not or are very little affected by the addition of metals to the growth medium.The amount of corrinoids in cells from early growth is low, whereas it is high in cells harvested late in growth. The opposite is found for the activity of delta-aminolevulinate dehydratase, which is high at the beginning of growth and low at the end.     

Sensitization of cultured Chinese hamster cells to 42 degrees C hyperthermia by pentalenolactone, an inhibitor of glycolytic ATP synthesis

The antibiotic pentalenolactone, a specific inhibitor of glyceraldehydephosphate dehydrogenase, was used to investigate the effect of glycolytic adenosine triphosphate (ATP) synthesis on the survival response of aerobic and hypoxic Chinese hamster cells treated with 42 degrees C hyperthermia. Data obtained with aerobic cells, incubated in balanced salt solutions supplemented with different substrates for ATP production, showed that 50 microM pentalenolactone blocked ATP synthesis via glycolysis but not by oxidative phosphorylation. The glycolytic inhibition was reversed upon transfer of the cells to antibiotic-free medium, and minimal cytotoxicity (less than 20 per cent) was observed. Hypoxic cultures were obtained by incubating dense cell suspensions (2 X 10(6)/ml) to produce metabolic oxygen depletion. Concomitant with the development of hypoxia, pentalenolactone-treated cells became ATP-depleted; cellular ATP levels were reduced by about 70-fold as compared to hypoxic cells in the antibiotic-free medium. The ATP-depleted cells were more sensitive to killing by hyperthermia. Comparison of the 42 degrees C survival curves for control and the antibiotic-treated hypoxic cells yielded a dose-modifying factor of 4 (5 per cent survival level). The results indicate that inhibition of glycolytic ATP synthesis, for example by pentalenolactone, can selectively sensitize hypoxic cells to the lethal effects of mild hyperthermia.     

Hexose transport derepressed and refractory to purine regulation in NAD(H)-depleted Nil cells

Nil hamster fibroblasts depleted of NAD(H) by growth in medium devoid of nicotinamide (NAm^?MEM) exhibit up to 2-3-fold higher rates of glucose transport. Derepression of glucose transport is observed only when Nil cells have become severely depleted of both intracellular NAD(H) and ATP, despite the continued presence of 5.5 mM D-glucose in the growth medium. Neither the initial rate of transport, approximated from 3-O-methylglucose uptake, nor accumulation of D-glucose itself is repressed upon restoring nicotinamide to the medium. Exposure of the cells to NAD⁺ (10^?5 M), however, leads to a sharp curtailment of transport within 2 to 3 hours. The purines, hypoxanthine and guanine, that sharply reduce glucose transport capacity of normal cells, have no significant effect upon transport activity of NAD(H)-depleted cells.     

ATP potently modulates anion channel-mediated excitatory amino acid release from cultured astrocytes

Volume-dependent ATP release andsubsequent activation of purinergic P2Y receptors have been implicatedas an autocrine mechanism triggering activation of volume-regulatedanion channels (VRACs) in hepatoma cells. In the brain ATP is releasedby both neurons and astrocytes and participates in intercellularcommunication. We explored whether ATP triggers or modulates therelease of excitatory amino acid (EAAs) via VRACs in astrocytes inprimary culture. Under basal conditions exogenous ATP (10 µM)activated a small EAA release in 70-80% of the cultures tested.In both moderately (5% reduction of medium osmolarity) andsubstantially (35% reduction of medium osmolarity) swollen astrocytes,exogenous ATP greatly potentiated EAA release. The effects of ATP weremimicked by P2Y agonists and eliminated by P2Y antagonists or the ATPscavenger apyrase. In contrast, the same pharmacological maneuvers didnot inhibit volume-dependent EAA release in the absence of exogenous ATP, ruling out a requirement of autocrine ATP release for VRAC activation. The ATP effect in nonswollen and moderately swollen cellswas eliminated by a 5-10% increase in medium osmolarity or byanion channel blockers but was insensitive to tetanus toxin pretreatment, further supporting VRAC involvement. Our data suggest that in astrocytes ATP does not trigger EAA release itself but actssynergistically with cell swelling. Moderate cell swelling and ATP mayserve as two cooperative signals in bidirectional neuron-astrocytecommunication in vivo.

     

Direct and Continuous Detection of ATP Secretion from Primary Monolayer Cultures of Bovine Adrenal Chromaffin Cells

A method was developed for direct and continuous detection of secretion of ATP from primary monolayer cultures of bovine adrenal chromaffin cells. ATP, which is costored with catecholamines within adrenal chromaffin cells, was released into the incubation medium, where it reacted with firefly luciferin-luciferase producing light detected by a photomultiplier located directly below the culture well. Acetylcholine, nicotine, the Ca2+ ionophore A23187, BaCl2, and KCl induced release of ATP. Induction of release of ATP by acetylcholine was dose dependent, with a threshold at 10(-7) M and a maximum at 10(-4) M. The dose-response curve for nicotine was bell shaped, with a threshold at 10(-7) M, a maximum at 10(-5) M, and diminished release at higher concentrations, an observation indicative of desensitization. Investigation of the initial rates of ATP secretion revealed that 10(-4) M nicotine actually induced release of ATP at a faster rate than 10(-5) M nicotine. However, the rate of ATP release evoked by 10(-4) M nicotine began to decline by 6 s, a result indicating the onset of receptor desensitization, whereas release induced by 10(-5) M nicotine continued unabated. Induction of release of ATP by acetylcholine or nicotine was biphasic, with a rapid, initial phase of release followed by a plateau at 0.5-1.5 min and a second phase of release beginning at 1.5-2 min, reaching a maximum by 2-3 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between intracellular ATP and the sodium pump activity in dog renal tubules

To examine the potential effect of the cellular ATP concentration and of the phosphate potential on the function of the sodium pump in intact renal cells, the ATP content of dog cortical tubules was first modified by a 30-min preincubation with one of the following effectors: 5 or 10 mM fructose, 2.5 mM adenosine 5'-monophosphate (AMP), or 2.5 mM adenosine in the presence of substrates (10 mM glutamine + 1 mM glutamate with either 10 mM lactate (low ATP) or 10 mM pyruvate (high ATP)). The tubules were then incubated in Krebs-Henseleit saline using two different phosphate concentrations and the same substrate mixture. The ATP content in tubular cells was modified by these treatments, ranging from 2.2 to 5.7 mM. The oxygen uptake by the tubules was measured before and after application of a small amount of nystatin (0.05 mM, 6 mumol/g wet wt.), added to impose an identical and submaximal increment of work to the Na(+)-K+ ATPase in tubules, irrespective of their ATP condition. This manoeuvre was followed by the addition of 1 mM ouabain to inhibit the sodium pump and quantify the respiration related to the activity of the Na+ pump. No significant effect of the ATP content on the respiratory cost of the Na(+)-K+ ATPase activity was noted when the [ATP] was above the normal concentration of approximately 3.0 mM before or after introduction of nystatin. In a second group of experiments, tubules were treated with 0.1 mM digitonin (13 mumol/g wet wt.) and resuspended in intracellular-like and sodium-free medium. The respiration was measured before and after the addition of increasing Mg-ATP concentrations (0-12 mM). A fixed quantity of Na+ (20 mM) was then introduced before ouabain was applied. The oxygen uptake was measured in these three conditions. We observed a fixed increment of ouabain-sensitive respiration upon stimulation of the pump activity by sodium at ATP concentrations ranging from 2 to 7 mM. The same observation applied when the free energy released from ATP hydrolysis ranged from -50 to -56 kJ.mol-1 and when the [ATP]/[ADP].[Pi] ratio ranged from 1.5 to 7.5 mM-1. These results suggest that the Na+:ATP stoichiometry of the Na(+)-K+ ATPase is not modified by [ATP] in dog cortical tubules when the ATP content is at or above the physiological value. Furthermore, the stoichiometry of the pump does not appear to change when the phosphate potential and (or) the free energy released from ATP hydrolysis are altered.