Accelerated degradation of adenine nucleotide in erythrocytes of patients with chronic renal failure

Recently, we have shown that erythrocytes obtained from patients with chronic renal failure (CRF) exhibited an increased rate of ATP formation from adenine as a substrate. Thus, we concluded that this process was in part responsible for the increase of adenine nucleotide concentration in uremic erythrocytes. There cannot be excluded however, that a decreased rate of adenylate degradation is an additional mechanism responsible for the elevated ATP concentration. To test this hypothesis, in this paper we compared the rate of adenine nucleotide breakdown in the erythrocytes obtained from patients with CRF and from healthy subjects.Using HPLC technique, we evaluated: (1) hypoxanthine production by uremic RBC incubated in incubation medium: (a) pH 7.4 containing 1.2 mM phosphate (which mimics physiological conditions) and (b) pH 7.1 containing 2.4 mM phosphate (which mimics uremic conditions); (2) adenine nucleotide degradation (IMP, inosine, adenosine, hypoxanthine production) by uremic RBC incubated in the presence of iodoacetate (glycolysis inhibitor) and EHNA (adenosine deaminase inhibitor). The erythrocytes of healthy volunteers served as control.The obtained results indicate that adenine nucleotide catabolism measured as a hypoxanthine formation was much faster in erythrocytes of patients with CRF than in the cells of healthy subjects. This phenomenon was observed both in the erythrocytes incubated at pH 7.4 in the medium containing 1.2 mM inorganic phosphate and in the medium which mimics hyperphosphatemia (2.4 mM) and metabolic acidosis (pH 7.1). The experiments with EHNA indicated that adenine nucleotide degradation proceeded via AMP-IMP-Inosine-Hypoxanthine pathway in erythrocytes of both patients with CRF and healthy subjects. Iodoacetate caused a several fold stimulation of adenylate breakdown. Under these conditions: (a) the rate of AMP catabolites (IMP + inosine + adenosine + hypoxanthine) formation was substantially higher in the erythrocytes from patients with CRF; (b) in erythrocytes of healthy subjects degradation of AMP proceeded via IMP and via adenosine essentially at the same rate; (c) in erythrocytes of patients with CRF the rate of AMP degradation via IMP was about 2 fold greater than via adenosine.The results presented in this paper suggest that adenine nucleotide degradation is markedly accelerated in erythrocytes of patients with CRF.     

Degradation and resynthesis of adenine nucleotides in adult rat heart myocytes

The degradation and short-term resynthesis of adenine nucleotides have been examined in a preparation of isolated rat heart myocytes. These myocyte preparations are essentially free of vascular and endothelial cells, contain levels of adenine nucleotides quite comparable to those of intact heart tissue, and retain these components remarkably well for up to 2 h of aerobic incubation in the presence of 1 mM Ca2+. When the cells are rapidly and synchronously de-energized by addition of uncoupler, an inhibitor of respiration and iodoacetate, cellular ATP is degraded almost quantitatively to AMP. The AMP is then converted to either intracellular adenosine, which accumulates to high concentrations before release to the cell exterior, or to IMP. The relative contribution of these two pathways depends on the metabolic state of the cells just prior to de-energization, with IMP production favored when respiring cells are de-energized and adenosine formation predominant when glycolyzing myocytes are subjected to this treatment. Cells de-energized by anaerobiosis in the absence of glucose lose ATP and adenine nucleotides with the production of IMP and adenosine. Upon reoxygenation, these cells restore a high adenylate energy charge and about 60% of control levels of GTP. There is a net resynthesis of 5-7 nmol of adenine nucleotides.mg-1 protein with a corresponding decline in IMP. Added [14C]adenosine labels the adenine nucleotide pool, but little net resynthesis of adenine nucleotides via adenosine kinase can be detected. It therefore appears that a rapid regeneration of adenine nucleotides can occur via the enzymes of the purine nucleotide cycle in heart myocytes and is limited by the size of the IMP pool retained.     

Differential effects of glycolytic and oxidative metabolism blockers on the Na-K pump in erythrocytes of the frog, Rana temporaria

This study was undertaken to evaluate the effects of various metabolic blockers on the Na-K-pump activity and ATP content of frog erythrocytes. To eliminate K-C1 cotransport, the frog erythrocytes were incubated in nitrate media at 20 °C. Incubation of the red cells in a glucose-free medium for 2 h had no effect on cell ATP content and K⁺ influx measured as ⁸⁶Rb uptake for 60 min. The Na⁺-K⁺-pump activity was also unchanged in the frog erythrocytes incubated in a glucose-free medium containing 10 mM 2-deoxy-D-glucose or adenosine. Unexpectedly, the treatment of red cells with 1–2 mM glycolytic blocker iodoacetate produced a 2-fold increase in the ouabain-sensitive K⁺ influx. The cell ATP content declined by 9.4% after 2 h of cell incubation with iodoacetate. Incubation of the red cells for 90 min in the presence of 2 mM cyanide, 0.01 mM antimycin A or 5 mM azide resulted in a significant reduction in K⁺ influx by about 50%, 45% and 32%, respectively. The cell ATP content diminished over 60 min and 120 min of cell incubation with 2 mM cyanide by 15.6% and 31.7% of control levels, respectively. In time-course experiments, a 50% reduction in the K⁺ influx was observed when the frog erythrocytes were incubated for only 30 min in the presence of 2 mM cyanide. In contrast, 0.01–0.10 mM rotenone, a site I inhibitor, and 0.01 mM carbonyl cyanide m-chlorophenylhydrazone, an uncoupler of oxidative phosphorylation were without effect on K⁺ influx into frog erythrocytes. These results indicate that about one-half of the Na⁺ -K⁺-pump activity in frog erythrocytes is tightly functionally coupled to cytochromes via a separate “membrane-associated” ATP pool. Accepted: 12 July 1997     

Pathways of adenine nucleotide catabolism in primary rat muscle cultures

The pathways of AMP degradation and the metabolic fate of adenosine were studied in cultured myotubes under physiological conditions and during artificially induced enhanced degradation of ATP. The metabolic pathways were gauged by tracing the flow of radioactivity from ATP, prelabelled by incubation of the cultures with [14C]adenine, into the various purine derivatives. The fractional flow from AMP to inosine through adenosine was estimated by the use of the adenosine deaminase (EC 3.5.4.4) inhibitors, coformycin and 2'-deoxycoformycin. The activities of the enzymes involved with AMP and adenosine metabolism were determined in cell extracts. The results demonstrate that under physiological conditions, there is a small but significant flow of label from ATP to diffusible bases and nucleosides, most of which are effluxed to the incubation medium. This catabolic flow is mediated almost exclusively by the activity of AMP deaminase (EC 3.5.4.6), rather than by AMP 5'-nucleotidase (EC 3.1.3.5), reflecting the markedly higher Vmax/Km ratio for the deaminase. Enhancement of ATP degradation by inhibition of glycolysis or by combined inhibition of glycolysis and of electron transport resulted in a markedly greater flux of label from adenine nucleotides to nucleosides and bases, but did not alter significantly the ratio between AMP deamination and AMP dephosphorylation, which remained around 19:1. Combined inhibition of glycolysis and of electron transport resulted, in addition, in accumulation of label in IMP, reaching about 20% of total AMP degraded. In the intact myotubes at low adenosine concentration, the anabolic activity of adenosine kinase was at least 4.9-fold the catabolic activity of adenosine deaminase, in accord with the markedly higher Vmax/Km ratio of the kinase for adenosine. The results indicate the operation in the myotube cultures, under various rates of ATP degradation, of the AMP to IMP limb of the purine nucleotide cycle. On the other hand, the formation of purine bases and nucleosides, representing the majority of degraded ATP, indicates inefficient activity of the IMP to AMP limb of the cycle, as well as inefficient salvage of hypoxanthine under these conditions.     

Metabolic regulation of ATP breakdown and of adenosine production in rat brain extracts

ATP concentration is dramatically affected in ischemic injury. From previous studies on ATP mediated purine and pyrimidine salvage in CNS, we observed that when "post-mitochondrial" extracts of rat brain were incubated with ATP at 3.6 mM, a normoxic concentration, formation of IMP always preceded that of adenosine, a well known neuroactive nucleoside and a homeostatic cellular modulator. This observation prompted us to undertake a study aimed at assessing the precise pathways and kinetics of ATP breakdown, a process considered to be the major source of adenosine in rat brain. The results obtained using post-mitochondrial extracts strongly suggest that the breakdown of intracellular ATP at normoxic concentration follows almost exclusively the pathway ATP<=>ADP<=>AMP --> IMP --> inosine<=>hypoxanthine, with little, if any, intracellular adenosine production. At low ischemic concentration, intracellular ATP breakdown follows the pathway ATP<=>ADP<=>AMP --> adenosine --> inosine<=>hypoxanthine with little IMP formation. At the same time, extracellular ATP, whose concentration is known to be enhanced during ischemia, is actively broken down to adenosine through the pathway ATP --> ADP --> AMP --> adenosine, catalysed by the well characterized ecto-enzyme cascade system. Moreover, we show that during intracellular GTP catabolism, xanthosine, in addition to guanosine, is generated through the so called "ribose 1-phosphate recycling for nucleoside interconversion". These results considerably extend our knowledge on the long debated question of the extra or intracellular origin of adenosine in CNS, suggesting that at least in normoxic conditions, intracellular adenosine is of extracellular origin.     

Depletion and recovery of ATP in V79 cells with exposure to inhibitors of glycolysis and oxidative phosphorylation

Summary Cells of the cultured hamster cell line V79 were labeled with tritiated adenosine and incubated for up to 30 min in the presence of inhibitors of glycolysis and oxidative phosphorylation. These inhibitors were (a) 5 mM KCN plus 5 mM iodoacetate, (b) 5 mM KCN plus 5 mM KF, and (c) 15 mM KCN plus 15 mM KF. The fate of the tritium label was examined during incubation with inhibitors and also during subsequent incubation in growth medium in the absence of inhibitors. The tritiated ATP pool was found to decrease in cells incubated in the presence of any of the inhibitor combinations, but only in the presence of 15 mM KCN plus 15 mM KF was this pool decreased below the level of detection. After cells were incubated with KCN plus KF, a high level of ATP was recovered when the inhibitors were removed. Cells incubated with KCN plus iodoacetate retained depletion levels of ATP. Plating efficiency and trypan blue staining showed that KCN-KF treated cells retained viability, whereas KCN-iodoacetate treated cells did not. Cells were examined for ability to take up tritiated uridine before, during, and after depletion of ATP by incubation in the presence of 15 mM KCN plus 15 mM KF. These cells were found to have a variation in uridine uptake that was related directly to intracellular ATP level. Cells in which the ATP was very low exhibited little or no uridine uptake, whereas cells in which the ATP level was near normal exhibited normal uridine uptake. This work was supported in part by Grant GM24271 from the National Institutes of Health, Bethesda, Maryland.     

Transport and metabolism of adenosine in human erythrocytes: effect of transport inhibitors and regulation by phosphate

Rapid kinetic techniques were applied to determine the effect of transport inhibitors on the transport and metabolism of adenosine in human red cells. Dipyridamole inhibited the equilibrium exchange of 500 microM adenosine by deoxycoformycin-treated cells in a similar concentration dependent manner as the equilibrium exchange and zero-trans influx of uridine with 50% inhibition being observed at about 20 nM. Intracellular phosphorylation of adenosine at an extracellular concentration of 5 microM was inhibited only by dipyridamole concentrations greater than or equal to 100 nM, which inhibited transport about 95%. Lower concentrations of dipyridamole actually stimulated adenosine phosphorylation, because the reduced influx of adenosine lessened substrate inhibition of adenosine kinase. When the cells were not treated with deoxycoformycin, greater than 95% of the adenosine entering the cells at a concentration of 100 microM became deaminated. A 95-98% inhibition of adenosine transport by treatment with dipyridamole, dilazep, or nitrobenzylthioinosine inhibited its deamination practically completely, whereas adenosine phosphorylation was inhibited only 50-85%. Whether adenosine entering the cells is phosphorylated or deaminated is strictly based on the kinetic properties of the responsible enzymes, substrate inhibition of adenosine kinase, and the absolute intracellular steady state concentration of adenosine attained. The latter approaches the extracellular concentration of adenosine, since transport is not rate limiting, except when modulated by transport inhibitors. In spite of the extensive adenosine deamination in cells incubated with 100 microM adenosine, little IMP accumulated intracellularly when the medium phosphate concentration was 1 mM, but IMP formation increased progressively with increase in phosphate concentration to 80 mM. The intracellular phosphoribosylation of adenine and hypoxanthine were similarly dependent on phosphate concentration. The results indicate that adenosine is the main purine source for erythrocytes and is very efficiently taken up and converted to nucleotides under physiological conditions, whereas hypoxanthine and adenine are not significantly salvaged. Hypoxanthine resulting from nucleotide turnover in these cells is expected to be primarily released from the cells. Adenosine was also dephosphorylated in human red cells presumably by 5'-methylthioadenosine phosphorylase, but this reaction seems without physiological significance as it occurs only at high adenosine and phosphate concentrations and if deamination is inhibited.     

The conversion of ATP to IMP by muscle surface enzymes

These experiments showed that an isolated muscle bundle could be used to study, simultaneously, ion transport and the activity of surface enzymes. Frog muscles were carefully dissected and incubated for four hours in Ringer's solution containing a tris buffer and 3 mM ATP; at various times samples of the medium were chromatographed and analysed spectrophotometrically for nucleotide content. Samples of the final medium were analysed for inorganic phoshpate and for ammonia. The results demonstrated the conversion of adenosine triphosphate to inosine monophosphate, via adenosine diphosphate and adenosine monophosphate. Under the conditions of the experiment IMP was detected on chromatograms within 20 minutes of incubation; at the end of four hours the media had concentrations of 2.3 mM IMP, 4.4 mM inorganic phosphate and 2.6 mM ammonia, showing a stoichiometric relation among the products formed. There was convincing evidence that the enzymes involved (ATPase, adenylate kinase and AMP deaminase) must be situated close to or on the muscle surface. No effect of ouabain (1 μM) on the activity of the ATPase, adenylate kinase or deaminase could be found in these experiments, but the drug inhibited Na and K recovery from a Na-loaded, K-depleted state.     

Purification and characterization of developmentally regulated AMP deaminase from Dictyostelium discoideum

AMP deaminase, the enzyme that catalyzes the conversion of adenosine monophosphate (AMP) to inosine monophosphate (IMP) and ammonia, was purified from the cellular slime mold, Dictyostelium discoideum in the nutrient-deprived state. The native enzyme had an apparent molecular weight of 199,000 daltons. Its apparent Km was 1.6 mM and its Vmax was 1.0 mumol min-1 mg-1, as measured by the release of IMP From AMP. The enzyme, like other AMP deaminases, was found to be activated by ATP, and inhibited either by GTP or inorganic phosphate. It was also specific for the deamination of AMP. Deaminase activity was increased either when vegetative cells were placed in a nutrient-deprived medium (for up to 6 h) or when vegetative cells were treated with the drug hadacidin. In cells actively growing in complete media, enzyme activity was more non-specific, hydrolyzing adenosine as well as AMP. AMP deaminase in D. discoideum appears to be stage-specific and developmentally regulated, possibly serving to regulate the adenylated nucleotide pool and the interconversion to guanylated nucleotides during early morphodifferentiation.     

Metabolic fate of extracellular NAD in human skin fibroblasts

Extracellular NAD is degraded to pyridine and purine metabolites by different types of surface-located enzymes which are expressed differently on the plasmamembrane of various human cells and tissues. In a previous report, we demonstrated that NAD-glycohydrolase, nucleotide pyrophosphatase and 5'-nucleotidase are located on the outer surface of human skin fibroblasts. Nucleotide pyrophosphatase cleaves NAD to nicotinamide mononucleotide and AMP, and 5'-nucleotidase hydrolyses AMP to adenosine. Cells incubated with NAD, produce nicotinamide, nicotinamide mononucleotide, hypoxanthine and adenine. The absence of ADPribose and adenosine in the extracellular compartment could be due to further catabolism and/or uptake of these products. To clarify the fate of the purine moiety of exogenous NAD, we investigated uptake of the products of NAD hydrolysis using U-[(14)C]-adenine-NAD. ATP was found to be the main labeled intracellular product of exogenous NAD catabolism; ADP, AMP, inosine and adenosine were also detected but in small quantities. Addition of ADPribose or adenosine to the incubation medium decreased uptake of radioactive purine, which, on the contrary, was unaffected by addition of inosine. ADPribose strongly inhibited the activity of ecto-NAD-hydrolyzing enzymes, whereas adenosine did not. Radioactive uptake by purine drastically dropped in fibroblasts incubated with (14)C-NAD and dipyridamole, an inhibitor of adenosine transport. Partial inhibition of [(14)C]-NAD uptake observed in fibroblasts depleted of ATP showed that the transport system requires ATP to some extent. All these findings suggest that adenosine is the purine form taken up by cells, and this hypothesis was confirmed incubating cultured fibroblasts with (14)C-adenosine and analyzing nucleoside uptake and intracellular metabolism under different experimental conditions. Fibroblasts incubated with [(14)C]-adenosine yield the same radioactive products as with [(14)C]-NAD; the absence of inhibition of [(14)C]-adenosine uptake by ADPribose in the presence of alpha-beta methyleneADP, an inhibitor of 5' nucleotidase, demonstrates that ADPribose coming from NAD via NAD-glycohydrolase is finally catabolised to adenosine. These results confirm that adenosine is the NAD hydrolysis product incorporated by cells and further metabolized to ATP, and that adenosine transport is partially ATP dependent.     

Inhibition by adenine compounds of the induced formation of photosynthetic pigments in Rhodopseudomonas spheroides cells under dark-semiaerobic conditions

Effects of adenine, adenosine, AMP, ADP and ATP on the inducedformation of bacteriochlorophyll and carotenoids in cell suspensionsof dark-aerobically grown Rhodopseudomonas spheroides were examinedunder dark-semiaerobic conditions where no significant cellgrowth occurred. Pigment formation was strongly inhibited by3 mM adenine, adenosine, AMP or ATP, but less strongly by ADP.Inhibition by either adenosine or ATP was completely reversible.Addition of 3 mM adenosine resulted in complete inhibition ofpigment formation, while inhibition by more than 10 min ATPdid not exceed 80%. No accumulation of any precursor-like pigmentsof either bacteriochlorophyll or carotenoids was observed incells incubated in the presence of adenine compounds. Amountsof exogenously-added adenine, adenosine, or AMP decreased significantlyduring incubation, whereas the amount of exogeneously-addedATP or ADP did not appreciably decrease. Addition of 3 mM ATPor adenosine also significantly suppressed ³H-leucine incorporationinto bacterial proteins. Nucleosides other than adenosine wereineffective in inhibiting the induced formation of photosyntheticpigments, indicating that the inhibitory action is specificto adenine compounds. It was assumed that both adenosine andATP inhibit chromatophore formation rather than a particularstep(s) in the biosynthetic pathways of the photosynthetic pigment,and that ATP exerts its effect from outside the cells, whereasadenosine does so after being taken up by the cells. (Received July 24, 1972; )     

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.     

Pathways of adenine nucleotide catabolism in primary rat muscle cultures

The pathways of AMP degradation and the metabolic fate of adenosine were studied in cultured myotubes under physiological conditions and during artificially induced enhanced degradation of ATP. The metabolic pathways were gauged by tracing the flow of radioactivity from ATP, prelabelled by incubation of the cultures with [¹⁴C]adenine, into the various purine derivatives. The fractional flow from AMP to inosine through adenosine was estimated by the use of the adenosine deaminase (EC 3.5.4.4) inhibitors, coformycin and 2′-deoxycoformycin. The activities of the enzymes involved with AMP and adenosine metabolism were determined flow of label from ATP to diffusible bases and nucleosides, most of which are effluxed to the incubation medium. This catabolic flow is mediated almost exclusively by the activity of AMP deaminase (EC 3.5.4.6), rather than by AMP 5′-nucleotidase (EC 3.1.3.5), reflecting the markedly higher V_max/K_m ratio for the deaminase. Enhancement of ATP degradation by inhibition of glycolysis or by combined inhibition of glycolysis and of electron transport resulted in a markedly greater flux of label from adenine nucleotides to nucleosides and bases, but did not alter significantly the ratio between AMP deamination and AMP dephosphorylation, which remained around 19:1. Combined inhibition of glycolysis and of electron transport resulted, in addition, in accumulation of label in IMP, reaching about 20% of total AMP degraded. In the intact myotubes at low adenosine concentration, the anabolic activity of adenosine kinase was at least 4.9-fold the catabolic activity of adenosine deaminase, in accord with the markedly higher V_max/K_m ratio of the kinase for adenosine. The results indicate the operation in the myotube cultures, under various rates of ATP degradation, of the AMP to IMP limb of the purine nucleotide cycle. On the other hand, the formation of purine bases and nucleosides, representing the majority of degraded ATP, indicates inefficient activity of the IMP to AMP limb of the cycle, as well as inefficient salvage of hypoxanthine under these conditions.     

Role of adenosine deaminase, ecto-(5''-nucleotidase) and ecto-(non-specific phosphatase) in cyanide-induced adenosine monophosphate catabolism in rat polymorphonuclear leucocytes.

1. The role of adenosine deaminase (EC 3.5.4.4), ecto-(5'-nucleotidase) (EC 3.1.3.5) and ecto-(non-specific phosphatase) in the CN-induced catabolism of adenine nucleotides in intact rat polymorphonuclear leucocytes was investigated by inhibiting the enzymes in situ. 2. KCN (10mM for 90 min) induced a 20-30% fall in ATP concentration accompanied by an approximately equimolar increase in hypoxanthine, ADP, AMP and adenosine concentrations were unchanged, and IMP and inosine remained undetectable ( less than 0.05 nmol/10(7) cells). 3. Cells remained 98% intact, as judged by loss of the cytoplasmic enzyme lactate dehydrogenase (EC 1.1.1.27). 4. Pentostatin (30 microM), a specific inhibitor of adenosine deaminase, completely inhibited hypoxanthine production from exogenous adenosine (55 microM), but did not black CN-induced hypoxanthine production or cause adenosine accumulation in intact cells. This implied that IMP rather than adenosine was an intermediate in AMP breakdown in response to cyanide. 5. Antibodies raised against purified plasma-membrane 5'-nucleotidase inhibited the ecto-(5'-nucleotidase) by 95-98%. Non-specific phosphatases were blocked by 10 mM-sodium beta-glycerophosphate. 6. These two agents together blocked hypoxanthine production from exogenous AMP and IMP (200 microM) by more than 90%, but had no effect on production from endogenous substrates. 7. These data suggest that ectophosphatases do not participate in CN-induced catabolism of intracellular AMP in rat polymorphonuclear leucocytes. 8. A minor IMPase, not inhibited by antiserum, was detected in the soluble fraction of disrupted cells.     

Different relationships between cellular adenosine or 3′-phosphorylation and cellular adenine ribonucleotide catabolism may be obtained

Treatment of BALB/c-3T3 mouse fibroblasts with 3′-led to a rapid accumulation of 3′-phosphates and the kinetics of this process has been determined. Concomitant with accumulation of these compounds, the adenine ribonucleotide pool was reduced. The kinetics of the two processes suggested that they were tightly coupled. The inhibitory effect of relatively high concentrations of coformycin indicated that IMP was an intermediate in the catabolic pathway. Similar experiments with Ehrlich ascites tumor cells were performed in Ringer-Hepes solution at pH 6.5 or 7.5 and with varying concentrations of orthophosphate. The experiments were performed with cells where ATP was [³H]-. This allowed the determination of the catabolism of adenine ribonucleotides to labeled nucleosides under conditions where added adenosine was phosphorylated. The results showed that at low phosphate concentration (5.8 mM) at pH 6.5 adenosine may be phosphorylated at a rate that was completely balanced to the concomitant catabolism of adenine ribonucleotides; that is, there was apparently a tight kinetic coupling between anabolism of adenosine and catabolism of adenine ribonucleotides. With 3′-a corresponding effect was obtained although the apparent coupling between phosphorylation of 3′-and catabolism of adenine ribonucleotides was not complete. When experiments were performed at the same pH but at high concentration of phosphate (45 mM) there was in contrast no coupling between the two processes; that is, ATP was present in constant amounts while 3′-phosphates accumulated at a high rate. In experiments with adenosine under these conditions there was still some although a relatively limited degree of apparent coupling between phosphorylation of adenosine and catabolism of adenine ribonucleotides. In both lines of cells used and with both adenosine and 3′-, the main products of the catabolism of adenine ribonucleotides were inosine and hypoxanthine. With 3′-there was in addition (about 20%) formation of xanthosine, suggesting that IMP dehydrogenase had also been activated. These results lead to the suggestion that adenosine (or 3′-) may be phosphorylated in two ways. 1) Phosphorylation may depend on an adenosine kinase unrelated to catabolism of adenine ribonucleotides. 2) Phosphorylation may be tightly coupled to catabolism of adenine ribonucleotides. A nucleoside phosphotransferase may catalyze the transfer of a phosphoryl group from IMP to adenosine (or 3′-) to form AMP (or 3′-) and inosine, a process that may be tightly coupled to an AMP deaminase reaction. The IMP formed in the latter reaction may not be released but transferred to the phosphotransferase. In contrast, the AMP formed in the phosphotransferase reaction should be in equilibrium with soluble AMP. It is assumed that a physical complex may exist, possibly in a membrane bound form, between AMP deaminase and the nucleoside phosphotransferase. © 1993 Wiley-Liss, Inc.     

Distinct roles for recombinant cytosolic 5'-nucleotidase-I and -II in AMP and IMP catabolism in COS-7 and H9c2 rat myoblast cell lines

Catabolism of AMP during ATP breakdown produces adenosine, which restores energy balance. Catabolism of IMP may be a key step regulating purine nucleotide pools. Two, cloned cytosolic 5'-nucleotidases (cN-I and cN-II) have been implicated in AMP and IMP breakdown. To evaluate their roles directly, we expressed recombinant pigeon cN-I or human cN-II at similar activities in COS-7 or H9c2 cells. During rapid (more than 90% in 10 min) or slower (30-40% in 10 min) ATP catabolism, cN-I-transfected COS-7 and H9c2 cells produced significantly more adenosine than cN-II-transfected cells, which were similar to control-transfected cells. Inosine and hypoxanthine concentrations increased only during slower ATP catabolism. In COS-7 cells, 5'-nucleotidase activity was not rate-limiting for inosine and hypoxanthine production, which was therefore unaffected by cN-II- and actually reduced by cN-I- overexpression. In H9c2 cells, in which 5'-nucleotidase activity was rate-limiting, only cN-II overexpression accelerated inosine and hypoxanthine formation. Guanosine formation from GMP was also increased by cN-II. Our results imply distinct roles for cN-I and cN-II. Under the conditions tested in these cells, only cN-I plays a significant role in AMP breakdown to adenosine, whereas only cN-II breaks down IMP to inosine and GMP to guanosine.     

Incorporation of adenosine and adenine into hypoxanthine nucleotides of fresh red blood cells

Incorporation of adenosine and adenine into hypoxanthine nucleotides of fresh red blood cells was monitored using 8-14C-adenosine and 8-14C-adenine added to the incubation medium containing adenosine, pyruvate and inorganic phosphate (APP medium). Using 8-14C-adenosine it was shown that 21.7% of the isotope contained in the incubation medium penetrated red blood cells. Of that quantity about 50% becomes incorporated into nucleotides. Of the isotope 5.3% was found in hypoxanthine nucleotides (1.3% in ITP and 4.0% in IMP). During incubation of red blood cells in APP medium fortified with the 8-14C-adenine about 95% of isotope penetrated into cells and 60% of that quantity became incorporated into nucleotides. In hypoxanthine nucleotides only trace amounts of isotope were found (0.12% in IMP and 0.13% in ITP).     

Further Studies of Sodium Transport in Feline Red Cells

The transport of radioactive sodium in high sodium cat red blood cells has been studied under various experimental conditions. It was found that iodoacetate (IAA) and iodoacetamide (IAM) inhibit Na influx by 50% whereas NaF has no effect. Reversible dyes, such as methylene blue (Mb), also inhibit this influx by 60%. Both IAA and Mb effects show a lag period of about 40 min. Cell starvation abolishes the volume-dependent Na influx which is generally observed in these cells. IAA reduces significantly the volume-dependent Na influx but does not inhibit it completely. 5 mM magnesium chloride produces a twofold increase in Na influx. On the other hand, MgCl₂ has no effect on Na transport in human red cells or on potassium or sulfate transport in cat red cells. The effect of MgCl₂ is quite rapid and does not interfere with the volume-dependent Na influx. This effect is abolished in starved cells. Reincubation of previously stored cells in buffered solutions containing glucose and MgCl₂ causes more than one order of magnitude increase in Na influx. These several observations are discussed in terms of the possibility of a link between Na transport and Na-Mg-activated ATPase.     

Energy metabolism and lipid peroxidation of human erythrocytes as a function of increased oxidative stress.

To study the influence of oxidative stress on energy metabolism and lipid peroxidation in erythrocytes, cells were incubated with increasing concentrations (0.5-10 mM) of hydrogen peroxide for 1 h at 37 degrees C and the main substances of energy metabolism (ATP, AMP, GTP and IMP) and one index of lipid peroxidation (malondialdehyde) were determined by HPLC on cell extracts. Using the same incubation conditions, the activity of AMP-deaminase was also determined. Under nonhaemolysing conditions (at up to 4 mM H2O2), oxidative stress produced, starting from 1 mM H2O2, progressive ATP depletion and a net decrease in the intracellular sum of adenine nucleotides (ATP + ADP + AMP), which were not paralleled by AMP formation. Concomitantly, the IMP level increased by up to 20-fold with respect to the value determined in control erythrocytes, when cells were challenged with the highest nonhaemolysing H2O2 concentration (4 mM). Efflux of inosine, hypoxanthine, xanthine and uric acid towards the extracellular medium was observed. The metabolic imbalance of erythrocytes following oxidative stress was due to a dramatic and unexpected activation of AMP-deaminase (a twofold increase of activity with respect to controls) that was already evident at the lowest dose of H2O2 used; this enzymatic activity increased with increasing H2O2 in the medium, and reached its maximum at 4 mM H2O2-treated erythrocytes (10-fold higher activity than controls). Generation of malondialdehyde was strictly related to the dose of H2O2, being detectable at the lowest H2O2 concentration and increasing without appreciable haemolysis up to 4 mM H2O2. Besides demonstrating a close relationship between lipid peroxidation and haemolysis, these data suggest that glycolytic enzymes are moderately affected by oxygen radical action and strongly indicate, in the change of AMP-deaminase activity, a highly sensitive enzymatic site responsible for a profound modification of erythrocyte energy metabolism during oxidative stress.     

Cyclic AMP,membrane transport and cell division. I. Effects of various chemicals on cyclic amp levels and rate of transport of nucleosides,hypoxanthine and deoxyglucose in several lines of cultured cells

Nutrient transport rates and cyclic AMP levels have been implicated in the regulation of cell proliferation. In the present study, however, changes in intracellular cyclic AMP level induced in several lines of cultured cells (normal 3T3 and SV₄₀ and polyomavirus-transformed 3T3 cells; 3T6, C6 glioma, mouse L, and Novikoff rat hepatoma cells) by treatment with papaverine, prostagladine E, or isoproterenol did not correlate with the inhibition of the uridine, hypoxanthine or deoxyglucose transport rates by these chemicals. Transport inhibitions by above chemicals or Persantin or Cytochalasin B occurred in most cell lines in the absence of any measurable change in intracellular cyclic AMP concentration. Furthermore, treatment of several cell lines with 1 mM dibutyryl cyclic AMP had no immediate effect on the transport of uridine, thymidine or deoxyglucose, although the transport capacity of the cells for uridine and thymidine, but not that for deoxyglucose, decreased progressively with time of treatment. We also observed that the uridine transport system of all cell lines derived from 3T3 cells and the hypoxanthine transport system of L cells exhibited high degrees of resistance to inhibition by the various chemicals. On the other hand, deoxyglucose transport was inhibited to about the same extent by these chemicals in all the cell lines investigated.