Purine uptake and release in rat C6 glioma cells: nucleoside transport and purine metabolism under ATP-depleting conditions

Adenosine, through activation of membrane-bound receptors, has been reported to have neuroprotective properties during strokes or seizures. The role of astrocytes in regulating brain interstitial adenosine levels has not been clearly defined. We have determined the nucleoside transporters present in rat C6 glioma cells. RT-PCR analysis, (3)H-nucleoside uptake experiments, and [(3)H]nitrobenzylthioinosine ([(3)H]NBMPR) binding assays indicated that the primary functional nucleoside transporter in C6 cells was rENT2, an equilibrative nucleoside transporter (ENT) that is relatively insensitive to inhibition by NBMPR. [(3)H]Formycin B, a poorly metabolized nucleoside analogue, was used to investigate nucleoside release processes, and rENT2 transporters mediated [(3)H]formycin B release from these cells. Adenosine release was investigated by first loading cells with [(3)H]adenine to label adenine nucleotide pools. Tritium release was initiated by inhibiting glycolytic and oxidative ATP generation and thus depleting ATP levels. Our results indicate that during ATP-depleting conditions, AMP catabolism progressed via the reactions AMP --> IMP --> inosine --> hypoxanthine, which accounted for >90% of the evoked tritium release. It was surprising that adenosine was not released during ATP-depleting conditions unless AMP deaminase and adenosine deaminase were inhibited. Inosine release was enhanced by inhibition of purine nucleoside phosphorylase; ENT2 transporters mediated the release of adenosine or inosine. However, inhibition of AMP deaminase/adenosine deaminase or purine nucleoside phosphorylase during ATP depletion produced release of adenosine or inosine, respectively, via the rENT2 transporter. This indicates that C6 glioma cells possess primarily rENT2 nucleoside transporters that function in adenosine uptake but that intracellular metabolism prevents the release of adenosine from these cells even during ATP-depleting conditions.     

Mononucleotide Metabolism in the Rat Brain After Transient Ischemia

Nucleotide metabolism was studied in rats during and following the induction of 10 min of forebrain ischemia (four-vessel occlusion model). Purine and pyrimidine nucleotides, nucleotides, and bases in forebrain extracts were quantitated by HPLC with an ultraviolet detector. Ischemia resulted in a severe reduction in the concentration of nucleoside triphosphates (ATP, GTP, UTP, and CTP) and an increase in the concentration of AMP, IMP, adenosine, inosine, hypoxanthine, and guanosine. During the recovery period, both the phosphocreatine level and adenylate energy charge were rapidly and completely restored to the normal range. ATP was only 78% of the control value at 180 min after ischemic reperfusion. Levels of nucleosides and bases were elevated during ischemia but decreased to values close to those of control animals following recirculation. Both the decrease in the adenine nucleotide pool and the incomplete ATP recovery were caused by insufficient reutilization of hypoxanthine via the purine salvage system. The content of cyclic AMP, which transiently accumulated during the early recirculation period, returned to the control level, paralleling the decrease of adenosine concentration, which suggested that adenylate cyclase activity during reperfusion is modulated by adenosine A2 receptors. The recovery of CTP was slow but greater than that of ATP, GTP, and UTP. The GTP/GDP ratio was higher than that of the control animals following recirculation.     

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.     

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.     

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.     

The pathway of adenylate catabolism in Azotobacter vinelandii. Evidence for adenosine monophosphate nucleosidase as the regulatory enzyme.

Cell-free, dialyzed extracts from Azotobacter vinelandii rapidly dephosphorylate [U-14C]ATP to labeled ADP and AMP, which is then degraded to hypoxanthine, the end product of AMP catabolism under the experimental conditions which were used. The intermediates of the pathway from ATP to hypoxanthine have been identified by thin layer chromatography and quantitated by the 14-C content. The concentrations of intermediates present during the production of hypoxanthine are consistent with AMP nucleosidase being responsible for AMP degradation in these extracts. This result was confirmed in experiments which utilized rabbit antibody prepared against purified AMP nucleosidase. The antibody inhibited AMP nucleosidase activity in cell-free extracts but did not inhibit adenine demanase or adenosine deaminase from the same extracts. In the presence of antibody prepared against purified AMP nucleosidase, the dialyzed extracts showed a marked reduction in the production of hypoxanthine from ATP. Other enzymes which could be responsible theoretically for the conversion of AMP to hypoxanthine were not detected by standard assay procedures. These results are consistent with AMP degradation proceeding by way of AMP nucleosidase to yield adenine and ribose 5-phosphate. The adenine is then converted to hypoxanthine by adenine deaminase. Both of these enzymes were present in sufficient quantities to account for the observed rates of hypoxanthine formation. The rate of hypoxanthine formation decreases during the time course of the [U-14-C]ATP degradation experiments, even though the concentration of AMP remains high. This decrease in the rate of hypoxanthine formation as a function of time is attributed to the decreasing ATP and increasing P0-4 concentrations, since ATP is an activator of AMP nucleosidase and P0-4 is an inhibitor. These observations suggest that the in vivo activity of AMP nucleosidase could also be regulated by changes in the relative ratios of ATP:AMP:P0-4.     

PURINE NUCLEOTIDE METABOLISM IN THE CAT BRAIN AFTER ONE HOUR OF COMPLETE ISCHEMIA

—Complete cerebral ischemia was produced in normothermic anaesthetized cats by clamping the innominate and the left subclavian arteries combined with lowering the blood pressure. After 1 h of ischemia, ATP was no longer present in detectable amounts. Total adenine nucleotides were reduced to 34 per cent of the normal level. The breakdown of guanine nucleotides was less marked, with small amounts of GTP still being present at the end of the ischemic period. In animals with signs of functional recovery after 3–7 h of recirculation, ATP was resynthesized to 62 per cent of the control level. Total adenine nucleotides increased to 68 per cent and the adenylate energy change—[ATP + 1/2 ADP]/[AMP + ADP + ATP]—was re-established to within 7 per cent of the pre-ischemic value. Radiochromatography of nucleotides following intravenous injection of [¹⁴C]formate indicated a marked enhancement of postischemic purine de novo synthesis. Purine nucleosides and free bases which accumulated during ischemia, were partially re-utilized by salvage pathways: adenosine was rephosphorylated to AMP by adenosine kinase (EC 2.7.1.20); inosine and hypoxanthine were re-used via IMP in a reaction mediated by hypoxanthine phosphoribosyltransferase (EC 2.4.2.8).     

Adenine nucleotide metabolism in Azotobacter vinelandii. Two metabolic pathways of AMP degradation

AMP-degrading pathways in Azotobacter vinelandii cells were investigated. AMP nucleosidase (EC 3.2.2.4) was rapidly synthesized and reached a maximum at 24 h, while the activity of 5-nucleotidase (EC 3.1.3.5) specific for AMP, which was negligible during the logarithmic phase of the growth, first appeared in 24 h-cultures, and reached a maximum after complete exhaustion of sucrose from the growth medium (70 h).Cell-free extracts of A. vinelandii of 48 h-cultures hydrolyzed AMP to ribose 5-phosphate and adenine in the presence of ATP, and adenine was deaminated to hypoxanthine. When ATP was excluded, AMP was dephosphorylated to adenosine, which was further metabolized to inosine, and finally to hypoxanthine. Hypoxanthine thus formed was reutilized for the salvage synthesis of IMP under the conditions where 5-phosphoribosyl 1-pyrophosphate was able to be supplied. These results suggest that the levels of ATP can determine the rate of AMP degradation by the AMP nucleosidase- and 5-nucleotidase-pathways. The role of ATP in the AMP degradation was discussed in relation to the regulatory properties of AMP nucleosidase, inosine nucleosidase (EC 3.2.2.2) and adenosine deaminase (EC 3.5.4.4).     

Determination of concentrations of adenosine and other purines in human term placenta by reversed-phase high-performance liquid chromatography with photodiode-array detection: evidence for pathways of purine metabolism in the placenta

A robust analytical method, using reversed-phase high-performance liquid chromatography with gradient elution and photodiode-array detection, was used to measure six purines and β-NAD⁺ in acid-soluble extracts of samples taken from six different regions of human term placenta. Resolution of the analyte peaks in chromatographic profiles of the extracts, and the use of optimized integration, allowed simultaneous quantitation of all seven analytes from a single chromatogram. Peak purity was confirmed via on-line analysis of peak spectra, utilizing the purity parameter treatment of spectral data. Major placental purines were adenosine, inosine, hypoxanthine and adenine. Except for adenine, concentrations of the purines varied by two-fold or more between different regions of each placenta, but concentration ratios, i.e., adenosine/inosine and inosine/hypoxanthine, were similar. The findings indicate that the pathway of ATP breakdown to hypoxanthine in ischemic human term placenta is via adenosine, and that regional differences in placental concentrations of adenosine and its metabolites may result from regional differences in degree of ischemia.     

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.     

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.     

The relationship between constitutive ATP release and its extracellular metabolism in isolated rat kidney glomeruli.

ATP and adenosine are important extracellular regulators of glomerular functions. In this study, ATP release from glomeruli suspension and its extracellular metabolism were investigated. Basal extraglomerular ATP concentration (1nM) increased several fold during inhibition of ecto-ATPase activity, reflecting the basal ATP release rate. Mechanical perturbation increased the amounts of ATP released from glomeruli. ATP added to glomeruli was almost completely degraded within 20 minutes. In that time, AMP was the main product of extracellular ATP metabolism. Significant accumulation of AMP was observed after 5 min (194 +/-16 microM) and 20 min (271 +/-11 microM), whereas at the same time concentration of adenosine was only 10 muM. A competitive inhibitor of ecto-5-nucleotidase alpha-beta-methylene-ADP (AOPCP), decreased extraglomerular ATP and adenosine concentration by 80% and 50%, respectively. Similarly, AMP (100 microM) also markedly reduced extraglomerular ATP accumulation, whereas IMP, its deamination product, was not effective. P1, P5-diadenosine pentaphosphate (Ap5A) - an inhibitor of ecto-adenylate kinase prevented significantly the disappearance of ATP from extraglomerular media caused by AMP. These findings demonstrate that the decrease in extracellular ATP concentration observed after addition of AOPCP or AMP is caused by the presence of ecto-adenylate kinase activity in the glomeruli. The enzyme catalyses reversible reaction 2ADP<->ATP+AMP, and a rise in the AMP concentration can lead to fall in ATP level. The present study provides evidence the extraglomerular accumulation of ATP reflects both release of ATP from glomeruli cells and its metabolism by ecto-enzymes. Our data suggest that AMP, produced from ATP in the Bowman's capsular space, might plays a dual role as a substrate for ecto-adenylate kinase and ecto-nucleotidase reactions being responsible for the regulation of intracapsular ATP and adenosine concentration. We conclude that AMP degrading and converting ecto-enzymes effectively determine the balance between ATP and adenosine concentration and thus the activation of P2 and/or adenosine receptors.     

Effect of glucose on AMP-catabolite release during fatty acid perfusion in normal and ischemic rat hearts

Isolated rat hearts were perfused retrogradely with a modified, oxygenated Tyrode solution containing 0.5 mM palmitate (complexed to albumin in a molar ratio of 6:1) with or without 11mM glucose. Fatty acid perfusion induced a decrease in contractile behaviour which was partly counteracted by glucose. The energy charge $\text{{([}\text{ATP}\text{] +}\text{1}\text{2}\text{[}\text{ADP}\text{])/([}\text{ATP}\text{] + [}\text{ADP}\text{] + [}\text{AMP}\text{]}}$ of the tissue was not altered although a significant drop was observed in creatine phosphate/ATP ratio in the absence of glucose. The release of AMP-catabolites, adenosine, inosine and hypoxanthine, occurring during fatty acid perfusion was reduced by glucose. In the absence of glucose fatty acids still induce lactate release indicating an enhanced glycogenolysis. In ischemic hearts the fatty acid-induced decrease in mechanical performance was significantly more severe when glucose was absent, while the glucose protection could also be observed in the energy charge of the ischemic tissue and the release of AMP-catabolites in the coronary effluent. The results suggest that loss of adenosine, inosine and hypoxanthine might contribute to the detrimental actions of a high fatty acid/albumin ratio upon the myocardium and confirms the protective action of glucose.     

Utilization of exogenous purine compounds in Bacillus cereus. Translocation of the ribose moiety of inosine.

Intact cells of Bacillus cereus catalyze the breakdown of exogenous AMP to hypoxanthine and ribose 1-phosphate through the successive action of 5'-nucleotidase, adenosine deaminase, and inosine phosphorylase. Inosine hydrolase was not detectable, even in crude extracts. Inosine phosphorylase causes a "translocation" of the ribose moiety (as ribose 1-phosphate) inside the cell, while hypoxanthine remains external. Even though the equilibrium of the phosphorolytic reaction favors nucleoside synthesis, exogenous inosine (as well as adenosine and AMP) is almost quantitatively transformed into external hypoxanthine, since ribose 1-phosphate is readily metabolized inside the cell. Most likely, the translocated ribose 1-phosphate enters the sugar phosphate shunt, via its prior conversion into ribose 5-phosphate, thus supplying the energy required for the subsequent uptake of hypoxanthine in B. cereus.     

Characterization of purine nucleotide metabolism in primary rat cardiomyocyte cultures

Primary rat cardiomyocyte cultures were utilized as a model for the study of purine nucleotide metabolism in the heart muscle, especially in connection with the mechanisms operating for the conservation of adenine nucleotides. The cultures exhibited capacity to produce purine nucleotides from nonpurine molecules (de novo synthesis), as well as from preformed purines (salvage synthesis). The conversion of adenosine to AMP, catalyzed by adenosine kinase, appears to be the most important physiological salvage pathway of adenine nucleotide synthesis in the cardiomyocytes. The study of the metabolic fate of IMP formed from [14C]formate or [14C]hypoxanthine and that of AMP formed from [14C]adenine or [14C]adenosine revealed that in the cardiomyocyte the main flow in the nucleotide interconversion pathways is from IMP to AMP, whereas the flux from AMP to IMP appeared to be markedly slower. Following synthesis from labeled precursors by either de novo or salvage pathways, most of the radioactivity in purine nucleotides accumulated in adenine nucleotides, and only a small proportion of it resided in IMP. The results suggest that the main pathway of AMP degradation in the cardiomyocyte proceeds through adenosine rather than through IMP. About 90% of the total radioactivity in purines effluxed from the cells during de novo synthesis from [14C]formate or following prelabeling of adenine nucleotides with [14C]adenine were found to reside in hypoxanthine. The activities in cell extracts of AMP 5'-nucleotidase and IMP 5'-nucleotidase, which catalyze nucleotide degradation, and of AMP deaminase, a key enzyme in the purine nucleotide cycle, were low. The nucleotidase activity resembles, and that of the AMP deaminase contrasts the respective enzyme activities in extracts of cultured skeletal-muscle myotubes. The results indicate that in the cardiomyocyte, in contrast to the myotube, the main mechanism operating for conservation of nucleotides is prompt phosphorylation of AMP, rather than operation of the purine nucleotide cycle. The primary cardiomyocyte cultures are a plausible model for the study of purine nucleotide metabolism in the heart muscle.     

Metabolic fate of AMP, IMP, GMP and XMP in the cytosol of rat brain: an experimental and theoretical analysis

A systematic study of the metabolic fate of AMP, IMP, GMP and XMP (NMP) in the presence of cytosol from rat brain is here presented; the kinetics of both disappearance of NMP, and appearance of their degradation products was followed by HPLC. In the absence of ATP, AMP was preferentially degraded to adenosine with concomitant appearance of inosine and hypoxanthine. In the presence of ATP, AMP was preferentially degraded via IMP. The nucleosides generated in the course of the reactions are further degraded, almost exclusively, via nucleoside phosphorylase using as cofactor the P(i) generated in the reaction mixture. In order to quantify the effect of each one of the enzymes involved in the degradation of NMP, two complementary approaches were followed: (i) the V:(max) and K:(m) values of the enzymes acting in the intermediate steps of the reactions were determined; (ii) these data were introduced into differential equations describing the concentration of the nucleotides and their degradation products as a function of the time of incubation. Factors affecting kinetic parameters of the equation velocity as a function of ATP concentration were introduced when required. The differential equations were solved with the help of Mathematica 3.0. The theoretical method can be used to simulate situations not feasible to be carried out, such as to measure the influence of nM-microM concentrations of ATP on the metabolism of AMP.     

Adenine nucleotides and adenosine metabolism in pig kidney proximal tubule membranes

Exogenous adenosine triphosphate (ATP) added to brush-border membrane vesicles was rapidly degraded mainly to inosine according to the high ecto-nucleotidase activities in these vesicles. In the absence of phosphate, inosine was slowly transformed into hypoxanthine, and xanthine oxidase and dehydrogenase activities were not detected. The presence of ecto-adenosine deaminase and ecto-adenosine monophosphate (AMP) nucleotidase was shown. The ecto-adenosine deaminase was inhibited by deoxycoformycin and was also detected in rat renal brush-border membrane vesicles. Using orthovanadate, levamisole, and α, β-methylene adenosine diphosphate as possible inhibitors, alkaline phosphatase was shown to be the main agent responsible for ecto-AMP nucleotidase activity. In pig renal basolateral membrane vesicles and in whole cell extracts from pig renal cortex, ecto-AMP nucleotidase was the limiting factor in ATP degradation. Comparing the ATP catabolism in the whole cell cortical extract with the catabolism in the same sample precleared of membranes, it was shown that ectonucleotidase activity is mainly bound to the membranous components. It is also shown that the whole cell extract of pig renal cortex has hypoxanthine phosphoribosyl transferase activity, and it seems probable that the rapid and specific formation of luminal inosine and its transport into the cell in competition with adenosine may start the purine salvage pathway through the synthesis of IMP from hypoxanthine. © Wiley-Liss, Inc.     

Genetic and physiological characterization of the purine salvage pathway in the archaebacterium Methanobacterium thermoautotrophicum Marburg.

The enzymes involved in the purine interconversion pathway of wild-type and purine analog-resistant strains of Methanobacterium thermoautotrophicum Marburg were assayed by radiometric and spectrophotometric methods. Wild-type cells incorporated labeled adenine, guanine, and hypoxanthine, whereas mutant strains varied in their ability to incorporate these bases. Adenine, guanine, hypoxanthine, and xanthine were activated by phosphoribosyltransferase activities present in wild-type cell extracts. Some mutant strains simultaneously lost the ability to convert both guanine and hypoxanthine to the respective nucleotide, suggesting that the same enzyme activates both bases. Adenosine, guanosine, and inosine phosphorylase activities were detected for the conversion of base to nucleoside. Adenine deaminase activity was detected at low levels. Guanine deaminase activity was not detected. Nucleoside kinase activities for the conversion of adenosine, guanosine, and inosine to the respective nucleotides were detected by a new assay. The nucleotide-interconverting enzymes AMP deaminase, succinyl-AMP synthetase, succinyl-AMP lyase, IMP dehydrogenase, and GMP synthetase were present in extracts; GMP reductase was not detected. The results indicate that this autotrophic methanogen has a complex system for the utilization of exogenous purines.     

Uptake of AMP, ADP, and ATP in Escherichia coli W

The uptake activity ratio for AMP, ADP, and ATP in mutant (T-1) cells of Escherichia coli W, deficient in de novo purine biosynthesis at a point between IMP and 5-aminoimidazole-4-carboxiamide-1-β-D-ribofuranoside (AICAR), was 1:0.43:0.19. This ratio was approximately equal to the 5'-nucleotidase activity ratio in E. coli W cells. The order of inhibitory effect on [2-3H]ADP uptake by T-1 cells was adenine > adenosine > AMP > ATP. About 2-fold more radioactive purine bases than purine nucleosides were detected in the cytoplasm after 5 min in an experiment with [8-1?C]AMP and T-1 cells. Uptake of [2-3H]adenosine in T-1 cells was inhibited by inosine, but not in mutant (Ad-3) cells of E. coli W, which lacked adenosine deaminase and adenylosuccinate lyase. These experiments suggest that AMP, ADP, and ATP are converted mainly to adenine and hypoxanthine via adenosine and inosine before uptake into the cytoplasm by E. coli W cells.     

Cellular Level of Purine Compounds in Ischemic Gerbil Brain by High Performance Liquid Chromatography

Abstract: The cellular level of ATP and related compounds in ischemic gerbil brain was investigated by high performance liquid chromatography (HPLC). Brain samples were obtained in situ following ligation of the common carotid artery. AMP and ADP peaks in the brain extracts in the ischemic group became much larger whereas the ATP peak decreased dramatically. The most striking finding was an extensive increase of adenosine: 50–100 fold. The levels of inosine and hypoxanthine also increased greatly in typical symptomatic gerbil.