A simple assay for adenylate cyclase in intact cells by affinity elution chromatography.

A method using the principle of affinity elution chromatography is described for the assay of adenylate cyclase in intact human platelets. By incubating platelet-rich plasma in the presence of radioactively labelled adenine, the ATP pool of the cells was prelabelled. Formation of labelled cyclic AMP from ATP was determined by extracting the platelets with HC1O4. After removal of the latter as KC1O4, the extract containing cyclic AMP and other adenine nucleotides was adsorbed in a NN-diethyl-N-2-hydroxypropylamino (QAE)-cellulose column. The column was washed, and subsequently cyclic AMP was specifically eluted with a cyclic AMP-dependent protein kinase and the radioactivity of the eluate was determined.     

A separation method for the assay of adenylylcyclase, intracellular cyclic AMP, and cyclic-AMP phosphodiesterase using tritium-labeled substrates.

A method for the separation of cyclic AMP from adenosine and polyvalent adenine nucleotides is described. The method consists of the sequential elution of adenosine and cyclic AMP from a single column of acidic aluminum oxide (alumina) with dilute hydrochloric acid and ammonium acetate. Adenosine, adenine, xanthine, and hypoxanthine are rapidly eluted with the application of 0.005 N hydrochloric acid while cyclic AMP remains adsorbed to the alumina. A subsequent application of 0.1 M ammonium acetate elutes more than 90% of the cyclic AMP. Under these conditions, polyvalent nucleotides (AMP, ADP, and ATP) remain adsorbed to the alumina. The method permits the measurement of adenylylcyclase activity using [3H]ATP as the labeled substrate. The same technique can be used to measure the accumulation of cyclic AMP in intact cells after labeling the ATP pool with [3H]adenine. With slight modification, the technique can be used to measure the activity of cyclic-AMP phosphodiesterase using [3H]cyclic AMP as the substrate. The proposed technique provides rapid, highly reproducible assays using inexpensive, disposable columns.     

A simple and sensitive method for monitoring drug-induced cell injury in cultured cells

A simple, sensitive method has been developed for evaluating cell injury noninvasively in monolayer cells in culture. The cell ATP pool was radiolabeled by incubating the cells with [14C]adenine. The uptake and incorporation of [14C]adenine was shown to proportional to the number of cells. As determined by HPLC, about 65-70% of the incorporated 14C label was in the ATP pool, 15-20% was in the ADP pool, and the rest was in the 5'-AMP pool. When prelabeled cells were exposed to toxic drugs (acetaminophen, calcium ionophore A-23187, or daunomycin) there was a marked decrease in cell ATP with a concomitant increase in leakage of labeled nucleotides, mainly 5'-AMP and 5'IMP. We have shown that leakage of 14C label into the medium from the prelabeled cells may be employed for quantitation of cell injury. This new measure of toxicity was shown to correlate very well with LDH leakage from the cells, which is a well accepted measure of cell injury. The leakage of 5'-[14C]AMP also correlated very well with the reduction of cell ATP in cardiac myocytes. This method has been used for monitoring drug-induced toxicity in liver cells, cardiac myocytes, and LB cells.     

Mechanism of action of ATP on intestinal epithelial cells. Cyclic AMP-mediated stimulation of active ion transport

ATP, ADP and AMP but not adenosine increased cyclic AMP in dispersed enterocytes prepared from guinea pig small intestine. This action of ATP was augmented by IBMX and was reproduced by App(NH)p or App(CH2)p. ATP also increased the formation of cyclic [14C]AMP in enterocytes that had been preincubated with [14C]adenine. Gpp(NH)p and NaF each caused persistent activation of adenylate cyclase in plasma membranes from enterocytes and ATP caused significant augmentation of this persistent activation. In addition to increasing cellular cyclic AMP and augmenting Gpp(NH)p and NaF-stimulated persistent activation of adenylate cyclase, ATP increased the Isc across mounted strips of small intestine and inhibited net absorption of fluid and electrolytes in segments of everted small intestine. These results indicate that intestinal epithelial cells possess a receptor that interacts with ATP and other adenine nucleotides and that receptor occupation by ATP causes activation of adenylate cyclase, increased cyclic AMP and changes in active ion transport across intestinal mucosa.     

Mechanism of action of ATP on intestinal epithelial cells: Cyclic AMP-mediated stimulation of active ion transport

ATP, ADP and AMP but not adenosine increased cyclic AMP in dispersed enterocytes prepared from guinea pig small intestine. This action of ATP was augmented by IBMX and was reproduced by App(NH)p or App(CH₂)p. ATP also increased the formation of cyclic [¹⁴C]AMP in enterocytes that had been preincubated with [¹⁴C]adenine. Gpp(NH)p and NaF each caused persistent activation of adenylate cyclase in plasma membranes from enterocytes and ATP caused significant augmentation of this persistent activation. In addition to increasing cellular cyclic AMP and agumenting Gpp(NH)p and NaF-stimulated persistent activation of adenylate cyclase, ATP increased the I_sc across mounted strips of small intestine and inhibited net absorption of fluid and electrolytes in segments of everted small intestine. These results indicate that intestinal epithelial cells possess a receptor that interacts with ATP and other adenine nucleotides and that receptor occupation by ATP causes activation of adenylate cyclase, increased cyclic AMP and changes in active ion transport across intestinal mucosa.     

Fluorometric Determination of Adenosine Nucleotide Derivatives as Measures of the Microfouling, Detrital, and Sedimentary Microbial Biomass and Physiological Status

Adenosine, adenine, cyclic adenosine monophosphate (AMP), AMP, nicotinamide adenine dinucleotide, adenosine diphosphate, and adenosine triphosphate (ATP) were recovered quantitatively from aqueous portions of lipid extracts of microfouling, detrital, and sedimentary microbial communities. These could be detected quantitatively in the picomolar range by forming their 1-N⁶-etheno derivatives and analyzing by high-pressure liquid chromatography with fluorescence detection. Lipid extraction and subsequent analysis allowed the simultaneous measurement of the microbial community structure, total microbial biomass with the quantitative recovery of the adenine-containing cellular components, which were protected from enzymatic destruction. This extraction and fluorescent derivatization method showed equivalency with the luciferin-luciferase method for bacterial ATP measurements. Quick-freezing samples in the field with dry ice-acetone preserved the ATP and energy charge (a ratio of adenosine nucleotides) for analysis at remote laboratories. The metabolic lability of ATP in estuarine detrital and microfouling communities, as well as bacterial monocultures of constant biomass, showed ATP to be a precarious measure of biomass under some conditions. Combinations of adenosine and adenine nucleotides gave better correlations with microbial biomass measured as extractable lipid phosphate in the detrital and microfouling microbial communities than did ATP alone. Stresses such as anoxia or filtration are reflected in the rapid accumulation of intracellular adenosine and the excretion of adenosine and AMP into the surrounding milieu. Increases in AMP and adenosine may prove to be more sensitive indicators of metabolic status than the energy charge.     

Adenosine and adenine nucleotides stimulation of skin (epidermal) adenylate cyclase.

Adenosine, AMP, ADP and ATP activated adenylate cyclase in pig skin (epidermis) slices resulting in the accumulation of cyclic AMP. This effect was highly potentiated by the addition of the cyclic AMP-phosphodiesterase inhibitor, papaverine. But another inhibitor, theophylline, strongly blocked the activation of adenylate cyclase by adenosine and adenine nucleotides. Theophylline apparently competed with adenosine for the cell surface receptor. Like theophylline, the addition of adenine alone caused no accumulation of cyclic AMP, but it significantly inhibited the stimulatory effect of adenosine. Guanosine, or guanine, cytidine, uridine, or thymidine nucleotides had no effect on the accumulation of cyclic AMP. Among other adenine nucleotides we tested, adenosine 5'-monophosphoramidate, but not adenosine 5'-monosulfate significantly increased cyclic AMP especially with the addition of papaverine. Neither 2'- nor 3'-adenylic acid were effective. Our data indicate that pig epidermis has four specific and independent adenylate cyclase systems for adenosine (and adenine nucleotides), histamine, epinephrine and prostaglandin E.     

Adenosine and adenine nucleotides stimulation of skin (epidermal) adenylate cyclase

Adenosie, AMP, ADP and ATP activated adenylate cyclase in pig skin (epidermis) slices resulting in the accumulation of cyclic AMP. This effect was highly potentiated by the addition of the cyclic AMP-phophodiesterase inhibitor, papaverine. But another inhibitor, theophylline, strongly blocked the activation of adenylate cyclase by adenosine and adenine nucleotides. Theophylline apparently competed with adenosine for the cell suface receptor. Like theophylline, the addition of adenine alone caused no accumulation of cyclic AMP, but it significantly inhibited the stimulatory effect of adenosine. Guanosine, or guanine, cytidine, uridine, or thymidine nucleotides has no effect on the accumulation of cyclic AMP. Among other adenine nucleotides was tested, adenosine 5′-monophosphoramidate, but not adenosine 5′-monosulfate, significantly increased cyclic AMP especially with the addition of papaverine. Neither 2′- nor 3′-adenylic acid were effective. Our data indicate that pig epidermis has four specific and independent adenylate cyclase systems for adenosine (and adenine nucleotides), histamine, epinephrine and prostaglandin E.     

Involvement of inositol 1,4,5-trisphosphate and calcium in the action of adenine nucleotides on aortic endothelial cells

ADP and ATP, in the 1-100 microM range of concentrations, increased the formation of inositol phosphates in bovine aortic endothelial cells. The accumulation of inositol trisphosphate in response to adenine nucleotides was rapid (maximum at 15 s) and transient. This material was identified as the biologically active isomer inositol 1,4,5-trisphosphate on the basis of its retention time by high-performance liquid chromatography on an anion-exchange resin. AMP and adenosine have no effect on inositol phosphates. The action of ATP and ADP was mimicked with an equal potency and activity by their phosphorothioate analogs, ATP gamma S and ADP beta S, and with a lower potency by adenosine 5'-(beta,gamma-imido)triphosphate, whereas adenosine 5'-(alpha,beta-methylene)triphosphate, was inactive. In the same range of concentrations, ADP and ATP induced an efflux of 45Ca2+ from prelabeled bovine aortic endothelial cells and increased the fluorescence emission by cells loaded with quin-2. Here, too, AMP and adenosine were completely inactive. The outflow of 45Ca2+ induced by ADP was partially maintained in a calcium-free medium. These data suggest that in aortic endothelial cells, P2-purinergic receptors, of the P2Y subtype, are coupled to the hydrolysis of phosphatidylinositol bisphosphate by a phospholipase C. It is likely that the release of prostacyclin and endothelium-derived relaxing factor in response to ADP and ATP is a consequence of this initial event.     

Relationship of adenosine-3', 5'-monophosphate to other adenine nucleotides in human platelets

Labeled cyclic AMP and other adenine nucleotides in human platelets were distinctly separated by means of thin-layer chromatography. In lysates of human platelets, ATP was decomposed following the major route: ATP→ADP→AMP→IMP→inosine→hypoxanthine. In contrast, cyclic AMP synthesis occurred rapidly following the breakdown of ATP and leveled off after 30–60 min of incubation. Cyclic AMP synthesis in platelet lysates was 1.02 ± 0.39 nanomoles/hr/mg protein. The level of cyclic AMP formed was related to the 5′-AMP level, although the former did not exceed 5 % of the latter.     

Adenosine Receptor Activation and the Regulation of Tyrosine Hydroxylase Activity in PC 12 and PC 18 Cells

We compared the response of rat PC12 cells and a derivative PC18 cell line to the effects of adenosine receptor agonists, antagonists, and adenine nucleotide metabolizing enzymes. We found that theophylline (an adenosine receptor antagonist), adenosine deaminase, and AMP deaminase all decreased basal cyclic AMP content and tyrosine hydroxylase activity in the PC12 cells, but not in PC18 cells. Both cell lines responded to the addition of 2-chloroadenosine and 5'-N-ethylcarboxamidoadenosine, adenosine receptor agonists, by exhibiting an increase in tyrosine hydroxylase activity and cyclic AMP content. The latter finding indicates that both cell lines contained an adenosine receptor linked to adenylate cyclase. We found that the addition of dipyridamole, an inhibitor of adenosine uptake, produced an elevation of cyclic AMP and tyrosine hydroxylase activity in both cell lines. Deoxycoformycin, an inhibitor of adenosine deaminase, failed to alter the levels of cyclic AMP or tyrosine hydroxylase activity. This suggests that uptake was the primary inactivating mechanism of adenosine action in these cells. We conclude that both cell types generated adenine nucleotides which activate the adenosine receptor in an autocrine or paracrine fashion. We found that PC12 cells released ATP in a calcium-dependent process in response to activation of the nicotinic receptor. We also measured the rates of degradation of exogenous ATP, ADP, and AMP by PC12 cells. We found that the rates of metabolism of the former two were at least an order of magnitude greater than that of AMP. Any released ATP would be rapidly metabolized to AMP and then more slowly degraded to adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of adenine nucleotides by thin-layer chromatography combined with the dansylation reaction

A new method was developed for the qualitative and quantitative determining of adenine nucleotides, based on thin-layer chromatography and dansilation. Chromatography mixtures 1,4-dioxan-water-ammonia (40:20:8) or 1,4-dioxan-water-propanol 2-ammonia (40:20:10:8) were used for separation of dansilated derivatives of ATP. ADP, AMP. The intensity of luminescence and the areas of spots served as measures of nucleotide quantities and were estimated using a microdensitometer. The method was applied to estimate the adenine nucleotide pool in E. coli K-12 cells.     

Effects of adenosine 5'-monophosphate and adenosine 3',5'-cyclic monophosphate on l cells.

The adenine nucleotides, 5'-AMP and 3',5'-cyclic AMP block L cells in the S-phase of the cell cycle. The intracellular level of cyclic AMP is reduced after incubation of cells with 5'-AMP, and rates of uridine transport are increased after incubation with either 5'-AMP or cyclic AMP. On the contrary, cyclic AMP levels are increased and uridine transport decreased in cells treated with an inhibitor of the cyclic AMP phosphodiesterase. This inhibitor partially reverses the growth-inhibitory effect of cyclic AMP, indicating that a breakdown product is the effective inhibitor of growth. The inhibition of cell growth induced by the adenine nucleotides is prevented by uridine, suggesting that the block in S is due to a lack of availability of pyrimidines.     

Role of AMP on the activation of glycogen synthase and phosphorylase by adenosine, fructose, and glutamine in rat hepatocytes

The mechanism for glycogen synthesis stimulation produced by adenosine, fructose, and glutamine has been investigated. We have analyzed the relationship between adenine nucleotides and glycogen metabolism rate-limiting enzymes upon hepatocyte incubation with these three compounds. In isolated hepatocytes, inhibition of AMP deaminase with erythro-9-(2-hydroxyl-3nonyl)adenine further increases the accumulation of AMP and the activation of glycogen synthase and phosphorylase by fructose. This ketose does not increase cyclic AMP or the activity of cyclic AMP-dependent protein kinase. Adenosine raises AMP and ATP concentration. This nucleotide also activates glycogen synthase and phosphorylase by covalent modification. The correlation coefficient between AMP and glycogen synthase activity is 0.974. Nitrobenzylthioinosine, a transport inhibitor of adenosine, blocks (by 50%) the effect of the nucleoside on AMP formation and glycogen synthase but not on phosphorylase. 2-Chloroadenosine and N6-phenylisopropyladenosine, nonmetabolizable analogues of adenosine, activate phosphorylase (6-fold) without increasing the concentration of adenine nucleotides or the activity of glycogen synthase. Cyclic AMP is not increased by adenosine in hepatocytes from starved rats but is in cells from fed animals. [Ethylenebis (oxyethylenenitrilo)]tetraacetic acid (EGTA) blocks by 60% the activation of phosphorylase by adenosine but not that of glycogen synthase. Glutamine also increases AMP concentration and glycogen synthase and phosphorylase activities, and these effects are blocked by 6-mercaptopurine, a purine synthesis inhibitor. Neither adenosine nor glutamine increases glucose 6-phosphate. It is proposed that the observed efficient glycogen synthesis from fructose, adenosine, and glutamine is due to the generation of AMP that activates glycogen synthase probably through increases in synthase phosphatase activity. It is also concluded that the activation of phosphorylase by the above-mentioned compounds can be triggered by metabolic changes.     

Metabolism and excretion of exogenous adenosine 3':5'-monophosphate and guanosine 3':5'-monophosphate. Studies in the isolated perfused rat kidney and in the intact rat.

Isolated rat kidneys were perfused with a recirculating medium containing exogenous adenosine 3':5'-monophosphate (cyclic AMP) or guanosine 3':5'-monophosphate (cyclic GMP) at an initial concentration of 0.1 mM. Both cyclic nucleotides were rapidly removed from the perfusate. Urinary excretion accounted for about 20% and 40% of the respective cyclic AMP and cyclic GMP lost from the perfusate. The metabolism of the cyclic nucleotides was studied by 14C-labeled cyclic nucleotides in the perfusate. During 60 min, 30% of added cyclic [14C]AMP was metabolized to renal [14C]adenine nucleotides (ATP, ADP, and AMP) and 30% to perfusate [14C]uric acid. Similarly, 20% of cyclic[14C]GMP was metabolized to renal [14C]guanine nucleotides (GTP, GDP, and GMP) and 30% to perfusate [14C]uric acid. Urine contained principally unchanged 14C-labeled cyclic nucleotide. Addition of 0.1 mM cyclic AMP to the perfusate elevated the renal ATP and ADP contents 2-fold. Addition of 0.1 mM of either cyclic AMP or cyclic GMP to the perfusate also elevated the renal production of uric acid 2- to 3-fold. The production and distribution of metabolites of exogenous cyclic nucleotides were also studied in the intact rat. Within 60 min after injection, 3.3 mumol of either 14C-labeled cyclic AMP or cyclic GMP was cleared from the plasma. Kidney cortex and liver were the principal tissues for 14C accumulation. Urinary excretion accounted for about 20 and 45% of the cyclic [14C]AMP and cyclic [14C]GMP lost from the plasma, respectively. The 14C found in the kidney and liver was present almost entirely as the respective purine mono-, di-, and trinucleotides. The other principal metabolite was [14C]allantoin, found in the urine and, to a lesser extent, the liver. The urine contained mostly unchanged 14C-labeled cyclic nucleotide. Unlike the findings with the perfused kidney, [14C]uric acid was not a significant metabolite of the 14C-labeled cyclic nucleotides in these in vivo experiments.     

Stimulation of glycogenolysis by adenine nucleotides in the perfused rat liver.

Infusion of adenine nucleotides and adenosine into perfused rat livers resulted in stimulation of hepatic glycogenolysis, transient increases in the effluent perfusate [3-hydroxybutyrate]/[acetoacetate] ratio, and increased portal vein pressure. In livers perfused with buffer containing 50 microM-Ca2+, transient efflux of Ca2+ was seen on stimulation of the liver with adenine nucleotides or adenosine. ADP was the most potent of the nucleotides, stimulating glucose output at concentrations as low as 0.15 microM, with half-maximal stimulation at approx. 1 microM, and ATP was slightly less potent, half-maximal stimulation requiring 4 microM-ATP. AMP and adenosine were much less effective, doses giving half-maximal stimulation being 40 and 20 microM respectively. Non-hydrolysed ATP analogues were much less effective than ATP in promoting changes in hepatic metabolism. ITP, GTP and GDP caused similar changes in hepatic metabolism to ATP, but were 10-20 times less potent than ATP. In livers perfused at low (7 microM) Ca2+, infusion of phenylephrine before ATP desensitized hepatic responses to ATP. Repeated infusions of ATP in such low-Ca2+-perfused livers caused homologous desensitization of ATP responses, and also desensitized subsequent Ca2+-dependent responses to phenylephrine. A short infusion of Ca2+ (1.25 mM) after phenylephrine infusion restored subsequent responses to ATP, indicating that, during perfusion with buffer containing 7 microM-Ca2+, ATP and phenylephrine deplete the same pool of intracellular Ca2+, which can be rapidly replenished in the presence of extracellular Ca2+. Measurement of cyclic AMP in freeze-clamped liver tissue demonstrated that adenosine (150 microM) significantly increased hepatic cyclic AMP, whereas ATP (15 microM) was without effect. It is concluded that ATP and ADP stimulate hepatic glycogenolysis via P2-purinergic receptors, through a Ca2+-dependent mechanism similar to that in alpha-adrenergic stimulation of hepatic tissue. However, adenosine stimulates glycogenolysis via P1-purinoreceptors and/or uptake into the cell, at least partially through a mechanism involving increase in cyclic AMP. Further, the hepatic response to adenine nucleotides may be significant in regulating hepatic glucose output in physiological and pathophysiological states.     

Effect of ascorbic acid on ACTH-induced cyclic AMP formation and steroidogenesis in isolated adrenal cells of vitamin E-deficient rats.

Isolated adrenal cells from Vitamin E-deficient and control rats were prepared by a trypsin digestion method. Cyclic adenosine 3',5'-monophosphate (cyclic AMP) formation was studied in response to adrenocorticotropin (ACTH) in the presence and absence of ascorbate by measuring the conversion of prelabeled adenosine 5'-triphosphate [14C]ATP to cyclic [14C]AMP. Ascorbate (0.5 mM) inhibited ACTH-induced cyclic [14C]AMP formation in adrenal cells isolated from Vitamin E-deficient rats but had no effect in the control cells. The inhibitory effect of ascorbate on ACTH-induced cyclic AMP formation in Vitamin E-deficient rats decreased as the concentration of ACTH increased. In Vitamin E-deficient rats ascorbate inhibited ACTH-induced cyclic [14C]AMP formation after 30 min of incubation. There was no further significant accumulation of cyclic [14C]AMP at 60 min or 120 min although in the absence of ascorbate cyclic [14C]AMP continued to be formed. The in vitro addition of alpha-tocopherol reduced the inhibition of ACTH-induced cyclic [14C]AMP formation by ascorbate in Vitamin E-deficient rats. These studies suggest that alpha-tocopherol and ascorbate may affect ACTH-induced cyclic AMP formation through interaction with the membrane-bound enzyme adenylate cyclase.     

Fluorometric determination of adenine nucleotides and adenosine by ion-paired, reverse-phase, high-performance liquid chromatography

A sensitive and specific assay for measurement of adenine nucleotides and adenosine by paired-ion high-performance liquid chromatography is described. The 1,N6-ethenoderivatives of ATP (epsilon-ATP), ADP (epsilon-ADP), AMP (epsilon-AMP), and adenosine (epsilon-Ado), formed by reaction with chloroacetaldehyde at 37 degrees C, were separated under isocratic conditions in 20 min. These compounds are strongly fluorescent at an emission wavelength of 280 nm, rendering a lowest detection limit of 2-5 pmol per injection. The detector responded linearly over the measured ranges (5-100 pmol for epsilon-Ado and 5-4000 pmol for nucleotides). Specificity was confirmed enzymatically. alpha, beta-Methyleneadenosine 5'-diphosphate could be used as an internal standard for measurement of the nucleotides. Significant amounts of NADH appeared as a separate peak in hypoxic tissue. Recoveries from snap-frozen kidney were 88, 92, 76, and 63% for AMP, ADP, ATP, and adenosine, with SD for recovery of 1.0, 10.5, 8.3, and 5.6%, respectively. This method was successfully used to measure adenine nucleotides and adenosine in oxygenated and hypoxic perfused rat kidneys.     

Simultaneous analysis of adenosine 3',5'-cyclic monophosphate accumulation and adenosine 5'-triphosphate metabolism in cultured cells preincubated with [2-3H]adenine.

A simple and sensitive method based on metabolic labeling was developed for the simultaneous analysis of cyclic AMP accumulation and ATP metabolism in small numbers of cultured cells. Cells are preincubated overnight with [2-3H]adenine to label the ATP pool to a high specific activity. After cell stimulation the metabolites are extracted in a small volume of aqueous acetic acid and chloroform and separated without further manipulation by one-dimensional thin-layer chromatography and the radioactivity incorporated is determined by liquid scintillation counting. With ATP labeled to about 6 Ci/mmol, the lower limit of cyclic AMP detection is 2 fmol, a sensitivity that is comparable to the radioimmunoassay of acetylated cyclic AMP. In primary neurons and a neural cell line, for example, levels of ATP and its metabolites change when large amounts of cyclic AMP are generated, each with its unique pattern. ATP is also depleted when metabolic energy is consumed concomitantly with stimulation of cyclic AMP production by agonists, probably as a result of an increase in ion pump activity following cation influx. As ATP is utilized for cyclic AMP production and simultaneously for many other processes, an assessment of its metabolism in parallel with that of cyclic AMP is critical. We suggest that the method described here is particularly advantageous over other methods for this purpose.     

Catabolism of adenine nucleotides in suspension-cultured plant cells

Profiles of the catabolism of adenine nucleotides in cultured plant cells were investigated. Adenine nucleotides, prelabelled by incubation of suspension-cultured Catharantus roseus cells with [8-14C]adenosine, were catabolized rapidly and most of the radioactivity appeared in 14CO2. Allantoin and allantoic acid, intermediates of the oxidative catabolic pathway of purines, were temporarily labelled. When the cells, prelabelled with [8-14C]adenosine, were incubated with high concentrations of adenosine, the rate of catabolism of adenine nucleotides increased. The results suggest that the relative rate of catabolism of adenine nucleotides is strongly dependent on the concentration of adenine nucleotides in the cells. Studies using allopurinol, coformycin and tiazofurin, inhibitors of enzymes involved in purine metabolism, suggest that participation of AMP deaminase and xanthine oxidoreductase in the catabolism of adenine nucleotides in plant cells. AMP deaminase was found in extracts from C. roseus cells and its activity increased significantly in the presence of ATP. In contrast, no adenosine deaminase or adenine deaminase activity was detected. Qualitative differences in the catabolic activity of AMP were observed between suspension-cultured cells from different species of plants.