Adenosine Salvage Research Articles

Nucleoside Transport as a Potential Target for Chemotherapy in Malaria

Malaria constitutes an enormous drain on the health and economies of many countries and causes more than a million deaths annually. Moreover, resistance to existing antimalarial drugs is a growing problem, rendering the search for new targets urgent. Protozoan parasites of the genus Plasmodium that cause malaria lack the ability to synthesise the purine ring de novo and so are reliant upon salvage of purines, including hypoxanthine, inosine and adenosine, from the host. The transport systems responsible for uptake of these precursors are therefore promising targets for novel antimalarial drugs. In humans, purine uptake into many cell types is mediated by members of the Equilibrative Nucleoside Transporter (ENT) family, in particular hENT1 and hENT2. Genome sequencing has revealed that P. falciparum and P. vivax, the species responsible for the majority of malaria cases, each also possesses four members of this family, and in P. falciparum transcripts of each are expressed in the erythrocytic stages of the parasite responsible for clinical disease. One of the proteins, PfENT1, is known to be present in the parasite plasma membrane, and the kinetic properties of the heterologously expressed transporter are consistent with its representing the major purine uptake system in the trophozoite. Importantly, its inhibitor specificity and permeant selectivity differ from those of the host. In this review we discuss the possibility of exploiting these differences to develop novel antimalarial drugs that either selectively inhibit purine uptake into the pararasite or are selectively delivered by the transporter to the parasite cytoplasm.

Role of adenosine salvage in wound-induced adenylate biosynthesis in potato tuber slices

Levels of ATP and other nucleotides increased in wounded potato tuber slices, maintained on moist paper for 24 h after preparation. The relative expression intensity of genes encoding adenosine kinase ( AK) and adenine phosphoribosyltransferase ( APRT) in wounded slices was greater than the intensity of genes of the de novo pathway, glycineamide ribonucleotide formyltransferase ( GART) and 5-aminoimidazole ribonucleotide synthetase ( AIRS). In vitro activities of adenosine kinase (ATP:adenosine 5′-phosphotransferase; EC 2.7.1.20) and adenine phosphoribosyltransferase (AMP:pyrophosphate phospho- d-ribosyltransferase; EC 2.4.2.7) increased during wounding. Adenosine nucleosidase (adenosine ribohydrolase; EC 3.2.2.7) activity was negligible in freshly prepared slices, but its activity is dramatically enhanced in wounded slices. In situ adenosine salvage activity, estimated from the incorporation of radioactivity from exogenously supplied [8- 14C]adenosine into nucleotides and RNA, increased more than five times in the wounded slices. These results strongly suggest that greater expression of the genes encoding enzymes of adenosine salvage during wounding is closely related to the increased supply of adenine nucleotides in the wounded slices.

Changes of purine and pyrimidine nucleotide biosynthesis during shoot initiation from epicotyl explants of white spruce (Picea glauca)

Nucleotide metabolism was investigated during white spruce organogenesis by following the metabolic fate of 14C-labeled adenine, adenosine and inosine, as purine precursors, and orotic acid, uridine, and uracil, as pyrimidine intermediates. Key enzymes of purine and pyrimidine metabolism were also assayed during the organogenic process. White spruce epicotyl explants cultured on shoot-forming (SF) medium had a better ability to utilize adenine and adenosine for nucleotide and nucleic acid synthesis, compared to tissue cultured on non-shoot forming (NSF) medium. High levels of salvage products were observed in SF tissue after 10 days in culture, when shoot formation was initiated along the epicotyl axis of the explants. Such a differential utilization of purine precursors was mainly due to the higher specific activity of the two adenine and adenosine salvage enzymes, adenine phosphoribosyltransferase (APRT) and adenosine kinase (AK), measured in SF tissue. Similar catabolism of inosine was observed in both SF and NSF conditions during the 30 days of culture. For pyrimidines, the higher activities of the de novo, salvage, and degradation pathways observed in SF tissue, compared to NSF tissue throughout the course of the experiment, clearly denote a faster turnover of pyrimidine nucleotides in the former. Taken together, these results suggest that a better utilization of purine bases and nucleosides for nucleotide and nucleic acid synthesis, as well as a more rapid turnover of pyrimidine nucleotides, represent a physiological switch, which occurs during the initiation and continuation of the organogenic process in white spruce.

Adenine and adenosine salvage pathways in erythrocytes and the role of S‐adenosylhomocysteine hydrolase

This article is devoted to the study of redundancy and yield of salvage pathways in human erythrocytes. These cells are not able to synthesize ATP de novo. However, the salvage (recycling) of certain nucleosides or bases to give nucleotide triphosphates is operative. As the salvage pathways use enzymes consuming ATP as well as enzymes producing ATP, it is not easy to see whether a net synthesis of ATP is possible. As for pathways using adenosine, a straightforward assumption is that these pathways start with adenosine kinase. However, a pathway bypassing this enzyme and using S-adenosylhomocysteine hydrolase instead was reported. So far, this route has not been analysed in detail. Using the concept of elementary flux modes, we investigate theoretically which salvage pathways exist in erythrocytes, which enzymes belong to each of these and what relative fluxes these enzymes carry. Here, we compute the net overall stoichiometry of ATP build-up from the recycled substrates and show that the network has considerable redundancy. For example, four different pathways of adenine salvage and 12 different pathways of adenosine salvage are obtained. They give different ATP/glucose yields, the highest being 3:10 for adenine salvage and 2:3 for adenosine salvage provided that adenosine is not used as an energy source. Implications for enzyme deficiencies are discussed.

Adenosine Kinase Inhibition and Suppression of RNA Silencing by Geminivirus AL2 and L2 Proteins

Most plant viruses are initiators and targets of RNA silencing and encode proteins that suppress this adaptive host defense. The DNA-containing geminiviruses are no exception, and the AL2 protein (also known as AC2, C2, and transcriptional activator protein) encoded by members of the genus Begomovirus has been shown to act as a silencing suppressor. Here, a three-component, Agrobacterium-mediated transient assay is used to further examine the silencing suppression activity of AL2 from Tomato golden mosaic virus (TGMV, a begomovirus) and to determine if the related L2 protein of Beet curly top virus (BCTV, genus Curtovirus) also has suppression activity. We show that TGMV AL2, AL2(1-100) (lacking the transcriptional activation domain), and BCTV L2 can all suppress RNA silencing directed against a green fluorescent protein (GFP) reporter gene when silencing is induced by a construct expressing an inverted repeat GFP RNA (dsGFP). We previously found that these viral proteins interact with and inactivate adenosine kinase (ADK), a cellular enzyme important for adenosine salvage and methyl cycle maintenance. Using the GFP-dsGFP system, we demonstrate here that codelivery of a construct expressing an inverted repeat ADK RNA (dsADK), or addition of an ADK inhibitor (the adenosine analogue A-134974), suppresses GFP-directed silencing in a manner similar to the geminivirus proteins. In addition, AL2/L2 suppression phenotypes and nucleic acid binding properties are shown to be different from those of the RNA virus suppressors HC-Pro and p19. These findings provide strong evidence that ADK activity is required to support RNA silencing, and indicate that the geminivirus proteins suppress silencing by a novel mechanism that involves ADK inhibition. Further, since AL2(1-100) is as effective a suppressor as the full-length AL2 protein, activation and silencing suppression appear to be independent activities.

Purine Salvage Pathways in the Apicomplexan Parasite Toxoplasma gondii

We have exploited a variety of molecular genetic, biochemical, and genomic techniques to investigate the roles of purine salvage enzymes in the protozoan parasite Toxoplasma gondii. The ability to generate defined genetic knockouts and target transgenes to specific loci demonstrates that T. gondii uses two (and only two) pathways for purine salvage, defined by the enzymes hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) and adenosine kinase (AK). Both HXGPRT and AK are single-copy genes, and either one can be deleted, indicating that either one of these pathways is sufficient to meet parasite purine requirements. Fitness defects suggest both pathways are important for the parasite, however, and that the salvage of adenosine is more important than salvage of hypoxanthine and other purine nucleobases. HXGPRT and AK cannot be deleted simultaneously unless one of these enzymes is provided in trans, indicating that alternative routes of functionally significant purine salvage are lacking. Despite previous reports to the contrary, we found no evidence of adenine phosphoribosyltransferase (APRT) activity when parasites were propagated in APRT-deficient host cells, and no APRT ortholog is evident in the T. gondii genome. Expression of Leishmania donovani APRT in transgenic T. gondii parasites yielded low levels of activity but did not permit genetic deletion of both HXGPRT and AK. A detailed comparative genomic study of the purine salvage pathway in various apicomplexan species highlights important differences among these parasites.

Comparison of adenosine metabolism in leaves of several mangrove plants and a poplar species

A possible increased demand for ATP in salt- tolerant mangrove plants was studied by the comparison of metabolic fates of [8- 14C] adenosine in leaf disks of several mangrove plants and of poplar. In mangrove trees, Rhizophora stylosa, Bruguiera gymnorrhiza, Kandelia candel and Sonneratia alba, 56–92% of [8- 14C]adenosine taken up by leaf disks was converted during 3 h incubation to salvage products, i.e., nucleotides and RNA. Synthesis of nucleotides including ATP was stimulated by salt stress induced by 250 mM NaCl. In leaf disks of Avicennia marina, a mangrove shrub that produces glycinebetaine as compatible solutes, 46% of radioactivity entered salvage products when [8- 14C] adenosine was continuously supplied to the leaf disks. Hydrolysis of adenosine to adenine was extremely active in this mangrove shrub. This is probably due to the high activity of adenosine nucleosidase (EC 3.2.2.7). In leaf disks of another mangrove shrub, Lumnitzera racemosa, only limited amounts of [8- 14C]adenosine were metabolised (< ca. 30% taken up by leaf disks), but synthesis of ATP and ADP was stimulated by salt stress. In Pemphis acidula leaf disks, adenosine salvage activity was low and more than 30% of adenosine was hydrolysed to adenine. In leaf disks of poplar, a non-salt-resistant plant, ca. 40% of [8- 14C] adenosine was converted to salvage products during 3 h of incubation, but the rate was slightly reduced by treatment with 250 mM NaCl. The present results suggest that large mangrove trees generally have efficient adenosine salvage ability, which is stimulated by salt. Lesser salvage activity is found in small size mangrove shrubs, although salt generally still enhances salvage activity.

5′-Adenosine monophosphate and adenosine metabolism, and adenosine responses in mouse, rat and guinea pig heart

We examined myocardial 5′-adenosine monophosphate (5′-AMP) catabolism, adenosine salvage and adenosine responses in perfused guinea pig, rat and mouse heart. M V o 2 increased from 71±8 μl O 2/min per g in guinea pig to 138±17 and 221±15 μl O 2/min per g in rat and mouse. V o 2/beat was 0.42±0.03, 0.50±0.03 and 0.55±0.04 μl O 2/g in guinea pig, rat and mouse, respectively. Resting and peak coronary flows were highest in mouse vs. rat and guinea pig, and peak ventricular pressures and Ca 2+ sensitivity declined as heart mass increased. Net myocardial 5′-AMP dephosphorylation increased significantly as mass declined (3.8±0.5, 9.0±1.4 and 11.0±1.6 nmol/min per g in guinea pig, rat and mouse, respectively). Despite increased 5′-AMP catabolism, coronary venous [adenosine] was similar in guinea pig, rat and mouse (45±8, 69±10 and 57±14 nM, respectively). Comparable venous [adenosine] was achieved by increased salvage vs. deamination: 64%, 41% and 39% of adenosine formed was rephosphorylated while 23%, 46%, and 50% was deaminated in mouse, rat and guinea pig, respectively. Moreover, only 35–45% of inosine and its catabolites derive from 5′-AMP (vs. IMP) dephosphorylation in all species. Although post-ischemic purine loss was low in mouse (due to these adaptations), functional tolerance to ischemia decreased with heart mass. Cardiovascular sensitivity to adenosine also differed between species, with A 1 receptor sensitivity being greatest in mouse while A 2 sensitivity was greatest in guinea pig. In summary: (i) cardiac 5′-AMP dephosphorylation, V o 2, contractility and Ca 2+ sensitivity all increase as heart mass falls; (ii) adaptations in adenosine salvage vs. deamination limit purine loss and yield similar adenosine levels across species; (iii) ischemic tolerance declines with heart mass; and (iv) cardiovascular sensitivity to adenosine varies, with increasing A 2 sensitivity relative to A 1 sensitivity in larger hearts.

Distinct regional distribution of human equilibrative nucleoside transporter proteins 1 and 2 (hENT1 and hENT2) in the central nervous system

Nucleoside transport processes play an important role in human cells in salvage of nucleosides used in the biosynthesis of nucleic acids and in regulating endogenous adenosine concentrations in the human central nervous system (CNS). By altering the levels of adenosine available to interact with cell–surface receptors, nucleoside transporters have profound effects on the ability of adenosine to modulate neurotransmission, vascular tone and other physiological events. Although the human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) are believed to play a crucial role in modulating brain function, their distribution within the major divisions of the human CNS is not known. In this work, antibodies specific for hENT1 and hENT2 were produced against fragments of the transporter proteins and used for immunoblot analysis of enriched membrane fractions prepared from several regions of the human brain. While hENT1 was most prevalent in the frontal and parietal lobes of the cerebral cortex, thalamus, midbrain and basal ganglia, hENT2 was concentrated in the cerebellum and brainstem regions, particularly the pons. The apparent reciprocal distribution of hENT1 and hENT2 in human brain suggests that these nucleoside transporter proteins are produced in distinct regions of the CNS where they function in nucleoside salvage and/or regulation of exogenous adenosine. Within the brain regions that were investigated, the pattern of hENT1 distribution correlated well with adenosine A 1 receptor abundance. The regional co-localization of hENT1 and A 1 receptor protein suggests an important role of hENT1-mediated transport process in the control of neuromodulatory actions mediated by adenosine A 1 receptors in human brain.

Purine metabolism during white spruce somatic embryo development: salvage of adenine, adenosine, and inosine

Contribution of the adenine, adenosine and inosine salvage to the purine nucleotide and nucleic acid biosynthesis during white spruce ( Picea glauca) somatic embryo maturation was estimated by in situ assays using [8- 14C]adenine, [8- 14C]adenosine and [8- 14C]inosine. The salvage of adenine and adenosine was high during the initial stages of embryo maturation, characterized by rapid cell proliferation, but it declined upon further embryo development. Inosine salvage activity was always much lower than that observed for adenine and adenosine. Consistent with these results, activities of adenine phosphoribosyltransferase (APRT) and adenosine kinase (AK) measured in the embryo extracts in vitro were much higher than the activity of inosine kinase (IK) during all stages of embryo development. Utilization of adenosine and inosine for nucleotide and nucleic acid synthesis was found to be regulated by the enzymes AK and IK, as the pattern of their activities was very similar to the activity of adenosine and inosine salvage, estimated with exogenously supplied precursors. However, little correlation between salvage of adenine and activity of APRT was found throughout somatic embryo maturation. As no adenosine nucleosidase activity was found in white spruce embryos, adenosine, but not adenine, seems to be the major end product of adenylate catabolism and becomes the predominant substrate for purine salvage in vivo. Thus, adenosine salvage appeared to have the most important role in white spruce embryos. Studies on the metabolic fate of [8- 14C]adenine and [8- 14C]adenosine suggest that turnover of adenine nucleotides is rapid, as some of them are utilized for nucleic acid synthesis. In contrast, most of [8- 14C]inosine taken up by the embryos seems to be directly catabolized by the conventional purine catabolic pathway via ureides in all stages of embryo maturation.

Adenosine kinase of Arabidopsis. Kinetic properties and gene expression.

To assess the functional significance of adenosine salvage in plants, the cDNAs and genes encoding two isoforms of adenosine kinase (ADK) were isolated from Arabidopsis. The ADK1- and ADK2-coding sequences are very similar, sharing 92% and 89% amino acid and nucleotide identity, respectively. Each cDNA was overexpressed in Escherichia coli, and the catalytic activity of each isoform was determined. Both ADKs had similar catalytic properties with a K(m) and V(max)/K(m) for adenosine of 0.3 to 0.5 microM and 5.4 to 22 L min(-1) mg(-1) protein, respectively. The K(m) and V(max)/K(m) for the cytokinin riboside N(6)(isopentenyl) adenosine are 3 to 5 microM and 0.021 to 0.14 L min(-1) mg(-1) protein, respectively, suggesting that adenosine is the preferred substrate for both ADK isoforms. In Arabidopsis plants, both ADK genes are expressed constitutively, with the highest steady-state mRNA levels being found in stem and root. ADK1 transcript levels were generally higher than those of ADK2. ADK enzyme activity reflected relative ADK protein levels seen in immunoblots for leaves, flowers, and stems but only poorly so for roots, siliques, and dry seeds. The catalytic properties, tissue accumulation, and expression levels of these ADKs suggest that they play a key metabolic role in the salvage synthesis of adenylates and methyl recycling in Arabidopsis. They may also contribute to cytokinin interconversion.

Ribose-enhanced Synthesis of UTP, CTP, and GTP from Parent Nucleosides in Cardiac Myocytes

The participation of ribose and its metabolites in some nucleoside salvage reactions is well established. Isolated adult rat cardiac myocytes were used as a model system to determine whether ribose acts as a general stimulant of salvage reactions in cardiac muscle, or whether only certain classes of nucleosides are affected by ribose. Myocytes were incubated with [3H]-adenosine, [3H]-cytidine, [3H]-guanosine, [3H]-thymidine, or [3H]-uridine for 30 or 60 min in the presence or absence of 5 mM ribose. The cells were extracted and the extracts assayed for [3H]-nucleoside and [3H]-nucleotide products. Salvage synthesis of cytosine, guanine and uracil nucleotides from the parent nucleosides was stimulated by ribose. Guanosine and uridine salvage appeared saturated at 50 microM external nucleoside (the dose response of cytidine salvage was not examined). Adenosine salvage was unaffected by ribose addition; the response to increasing external adenosine concentration was non-Michaelis-Menten, showing a peak of activity at 25 microM external nucleoside. Thymidine salvage was also unaffected by ribose, and was saturated at 50 microM external thymidine. These data suggest that adenosine and thymidine are metabolized to their respective nucleotide monophosphates by kinase activity. Cytidine, guanosine, and uridine salvage are stimulated by ribose, and must therefore be metabolized in part by nucleoside phosphorylase and phosphoribosyltransferase activity.

Hypoxia-induced inhibition of adenosine kinase potentiates cardiac adenosine release.

To elucidate the physiological role of the AMP-adenosine metabolic cycle and to investigate the relation between AMP and adenosine formation, the O2 supply of isolated guinea pig hearts was varied (95% to 10% O2). The net adenosine formation rate (AMP-->adenosine) and coronary venous effluent adenosine release rate were measured; free cytosolic AMP was determined by 31P-nuclear magnetic resonance. Switching from 95% to 40% O2 increased free AMP and adenosine formation 4-fold, whereas free cytosolic adenosine and venous adenosine release rose 15- to 20-fold. In the AMP range from 200 to 3000 nmol/L, there was a linear correlation between free AMP and adenosine formation (R2 = .71); however, adenosine release increased several-fold more than formation. At 95% O2, only 6% of the adenosine formed was released; however, this fraction increased to 22% at 40% O2, demonstrating reduced adenosine salvage. Selective blockade of adenosine deaminase and adenosine kinase indicated that flux through adenosine kinase decreased from 85% to 35% of adenosine formation in hypoxia. Mathematical model analysis indicated that this apparent decrease in enzyme activity was not due to saturation but to the inhibition of adenosine kinase activity to 6% of the basal levels. The data show (1) that adenosine formation is proportional to the AMP substrate concentration and (2) that hypoxia decreases adenosine kinase activity, thereby shunting myocardial adenosine from the salvage pathway to venous release. In conclusion, because of the normal high turnover of the AMP-adenosine metabolic cycle, hypoxia-induced inhibition of adenosine kinase causes the amplification of small changes in free AMP into a major rise in adenosine. This mechanism plays an important role in the high sensitivity of the cardiac adenosine system to impaired oxygenation.

Rapid turnover of the AMP-adenosine metabolic cycle in the guinea pig heart.

The intracellular flux rate through adenosine kinase (adenosine-->AMP) in the well-oxygenated heart was investigated, and the relation of the AMP-adenosine metabolic cycle (AMP<-->adenosine) to transmethylation (S-adenosylhomocysteine [SAH]-->adenosine) and coronary flow was determined. Adenosine kinase was blocked in isolated guinea pig hearts by infusion of iodotubercidin in the presence of the adenosine deaminase blocker erythro-9-(2-hydroxy-3-nonyl)adenine (5 mumol/L). Iodotubercidin (1 nmol/L to 4 mumol/L) caused graded increases in venous effluent concentrations of adenosine, from 8 +/- 3 to 145 +/- 32 nmol/L (mean +/- SEM, n = 3), and in coronary flow, which increased to maximal levels. Flow increases were completely abolished by adenosine deaminase (5 to 10 U/mL). Interstitial adenosine concentrations, estimated using a mathematical model, increased from 22 nmol/L during control conditions to 420 nmol/L during maximal vasodilation. The possibility that iodotubercidin caused increased venous adenosine by interfering with myocardial energy metabolism was ruled out in separate 31P nuclear magnetic resonance experiments. To estimate total normoxic myocardial production of adenosine (AMP-->adenosine<--SAH), the time course of coronary venous adenosine release was measured during maximal inhibition of adenosine kinase with 30 mumol/L iodotubercidin. Adenosine release increased more than 15-fold over baseline, reaching a new steady-state value of 3.4 +/- 0.3 nmol.min-1 x g-1 (n = 5) after 4 minutes. In parallel experiments, the relative roles of AMP hydrolysis and transmethylation (SAH hydrolysis) were determined, using adenosine dialdehyde (10 mumol/L) to block SAH hydrolase. In these experiments, adenosine release increased to similar levels of 3.4 +/- 0.5 nmol.min-1 x g-1 (n = 6) during inhibition of adenosine deaminase and adenosine kinase. It is concluded that (1) maximal increases in coronary flow are elicited by increases in interstitial adenosine concentration to approximately 400 nmol/L, (2) more than 90% of the adenosine produced in the heart is normally rephosphorylated to AMP without escaping into the venous effluent, (3) AMP hydrolysis is the dominant pathway for cardiac adenosine production under normoxic conditions, and (4) the high rate of adenosine salvage is due to rapid turnover of a metabolic cycle between AMP and adenosine. Rapid cycling may serve to amplify the relative importance of AMP hydrolysis over transmethylation in controlling cytosolic adenosine concentrations.

Inhibition of adenosine deaminase and nucleoside transport: Utility in a model of homograft cardiac valve preimplantation processing

Human cardiac valves are increasingly used in the reconstruction of ventricular outflow tracts and offer performance advantages over porcine and mechanical prostheses; the durability of these replacements has been associated with leaflet interstitial cell viability and a presumed sustained function after implantation. Preimplantation tissue preparation entails sequential steps that are potentially cytotoxic and may therefore affect functional cell survival at thaw. We defined the metabolic consequences of each interval using semilunar cusps from 118 porcine valves to model a homograft preparation with 40 minutes of fixed cadaveric (harvest) ischemia. Fifty-eight valves served as controls and were first processed according to standard cryopreservation protocol; nucleosides were extracted at the end of each step to differentiate independent contributions to high-energy phosphate depletion. Sixty simultaneously harvested leaflets were administered the nucleoside transport inhibitor p-nitrobenzy-thionosine (NBMPR) and the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) at procurement, to attempt adenosine salvage and restitution of processing-incurred adenine nucleotide losses. High-performance liquid chromatography was used to compare adenosine triphosphate, diphosphate, and monophosphate and diffusible nucleopurines of the control and EHNA/NBMPR-treated groups. Control results indicate that disruption of the adenosine triphosphate-diphosphate cycle occurs independently with antibiotic disinfection and cryopreservation. However, throughout all preparation steps, adenine nucleotides were maintained at harvest (baseline) concentrations in the EHNA/NBMPR valves. This suggests that salvage therapy may protect a significant number of cells from net high-energy phosphate catabolism. If, with further study, the durability of transplanted valves is concluded to benefit from retained leaflet interstitial cell viability, such enhancement of metabolic tolerance to the obligatory processing may facilitate functional recovery.

Formation and salvage of adenosine by macrovascular endothelial cells.

Contribution of extracellular adenine nucleotide degradation to adenosine formation and internal salvage of adenosine via adenosine kinase were quantified in macrovascular porcine endothelial cells. Microcarrier beads covered with endothelial cells were kept in a perfusion column at a flow rate of 2 ml/min. Total adenine nucleotide (AN) release was quantified with a sensitive firefly luciferin-luciferase assay after enzymatic rephosphorylation of AMP and ADP to ATP. Adenosine (ADO) was measured by radioimmunoassay or high-pressure liquid chromatography (HPLC) techniques. Basal AN and ADO release under steady-state conditions were 2.2 and 13.8 pmol.min-1 x ml column volume (CV)-1, respectively. Inhibition of adenosine deaminase with erythro-9-(2-hydroxy-3-nonyl)adenine (5 x 10(-6) M) enhanced ADO release by 3.3 pmol.min-1 x ml CV-1, and AN release remained unchanged (2.8 pmol.min-1 x ml CV-1). Inhibition of adenosine kinase by 5-iodotubercidine (10(-5) M) greatly enhanced ADO release by 97.7 pmol.min-1 x ml CV-1, while AN release was unaffected. Inhibition of ecto-5'-nucleotidase by alpha,beta-methylene-ADP (5 x 10(-5) M) enhanced AN release from 2.6 to 8.2 pmol.min-1 x ml CV-1 and reduced ADO release by an equivalent extent. Stimulation of endothelial cells with Ca ionophore A23187 dose dependently augmented AN and ADO release to 2,013.2 and 92.5 pmol.min-1 x ml CV-1, respectively. Thrombin (1 U/ml) enhanced AN release from 5.0 to 8.7 pmol.min-1 x ml CV-1, whereas several other endothelium-dependent and -independent vasodilators including acetylcholine, bradykinin, isoproterenol, and norepinephrine were proven to have no significant effect.(ABSTRACT TRUNCATED AT 250 WORDS)

148 PATHWAYS OF ATP CATABOLISM IN PRIMARY RAT CARDIOMYOCYTE CULTURES

The pathways of ATP catabolism were investigated in cultured beating cardiomyocytes. The activity of the enzymes involved in AMP degradation was assayed in cell extracts, whereas fluxes of label from ATP to the various purine derivatives were measured in intact cells. Under physiological conditions, in accord with a 7 fold higher activity of AMP deaminase than that of AMP 5′-Nucleotidase, cells degraded AMP through deamination to IMP. Mild ATP degradation, induced by inhibition of glycolysis by iodoacetate, caused no alterations in the degradation pathways (more than 85% through deamination to IMP). However, fast ATP degradation (83% of adenine nucleotides/10 min), induced by simultaneous inhibition of glycolysis and electron transport (by antimycin A), caused increased dephosphorylation of AMP to adenosine (50% of total AMP-degradation). IMP was rapidly degraded to inosine, hypoxanthine, xanthine and uric acid, which were effluxed from the cells. The cardiomyocyte extracts were found to contain a significant activity of PNP. Despite the presence of HGPRT, salvage of hypoxanthine to IMP, both at physiological as well as at conditions associated with ATP degradation, was slow. The salvage of adenosine appeared to be efficient at physiological conditions, but not at fast rates of ATP degradation.

149 ENZYMES OF PURINE NUCLEOTIDE METABOLISM IN MATURE RAT BRAlN NEURONS IN CULTURE

Several enzymes of purine nucleotide metabolism were assayed in mature (10-14 days old) rat brain neurons in culture: No activity could be detected for xanthine oxidase and uricase. The cultures exhibited de novo nucleotide synthesis. The results indicate that purine nucleotides can be synthesized in the mature rat brain cultured cells by de novo and by salvage from hypoxanthine, adenine and adenosine. From the Vmax values of the enzymes and from the known Km values, it appears that the degradation of adenine nucleotides through deamination to IMP has a slightly better capacity than that through dephosphorylation to adenosine and that the capacity for adenosine salvage is better than that for its deamination. The absence of xanthine oxidase activity is in favor of hypoxanthine salvage to IMP, but the presence of guanase competes with the salvage of guanine.

Pathways of adenine nucleotide catabolism in primary rat cardiomyocyte cultures

The pathways of adenine nucleotide catabolism were investigated in cultured beating cardiomyocytes. The activity of the enzymes involved in AMP degradation was assayed in cell extracts. Fluxes of label from ATP to the various purine derivatives were measured in intact cells. Under physiological conditions, cells degraded AMP through deamination to IMP. IMP was rapidly degraded to inosine, hypoxanthine, xanthine and uric acid, which were effluxed from the cells. This is in accord with the fact that the activity of AMP deaminase (EC 3.5.4.6) was 7-fold that of AMP 5'-Nucleotidase (EC 3.1.3.5). Mild ATP-degradation, induced by inhibition of glycolysis by iodoacetate, caused no alterations in the degradation pathways (more than 85% through deamination to IMP). However, fast ATP-degradation (83% of adenine nucleotides/10 min), induced by simultaneous inhibition of glycolysis and electron transport (by antimycin A), caused increased dephosphorylation of AMP to adenosine (50% of total AMP-degradation). The cardiomyocyte extracts were found to contain a significant activity of purine nucleoside phosphorylase (EC 2.4.2.1). Despite the presence of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8), salvage of hypoxanthine to IMP, both at physiological as well as at conditions associated with ATP degradation, was slow. The salvage of adenosine appeared to be efficient at physiological conditions, but not at fast rates of ATP degradation.

Salvage mechanisms for regeneration of adenosine triphosphate in rat cardiac myocytes

Since mitochondrial oxidative phosphorylation does not produce sufficient adenosine triphosphate for rapid restoration of contractile function in myocardium reoxygenated after ischaemia alternative non-oxidative routes by which purine nucleosides or bases may be used to increase the cytoplasmic adenine nucleotide pool were studied. Comparative rates of uptake and salvage of adenosine, adenine, and hypoxanthine were determined using myocytes isolated from adult rat heart. For each precursor reactions limiting the overall rate at which substrate in the extracellular fluid is incorporated into the intracellular adenosine triphosphate pool were identified. Adenosine was salvaged at twice the rate seen with adenine in the presence of ribose, whereas the rate of salvage of hypoxanthine, in the presence of ribose, was only 5% of that for adenosine. Adenine may be an advantageous substrate since high concentrations of adenine are not inhibitory to salvage and do not influence cardiac haemodynamics. Salvage of hypoxanthine appeared to be limited by the rate of adenylosuccinate synthetase, which was present at less than 1% of the adenylosuccinase rate in rat, rabbit, and beef heart. In addition, since salvage of bases is dependent on the availability of phosphoribosylpyrophosphate rates of synthesis of phosphoribosylpyrophosphate were measured in whole myocytes from ribose and in cytoplasmic extracts from ribose and from ribose-5-phosphate. Metabolic sites were identified at which interventions designed to accelerate salvage rates might best be directed.