The mechanism of adenosine formation in cells. Cloning of cytosolic 5'-nucleotidase-I.

Adenosine increases blood flow and decreases excitatory nerve firing. In the heart, it reduces rate and force of contraction and preconditions the heart against injury by prolonged ischemia. Based on indirect kinetic arguments, an AMP-selective cytosolic 5'-nucleotidase designated cN-I has been implicated in adenosine formation during ATP breakdown. The molecular identity of cN-I is unknown, although an IMP/GMP-selective cytosolic 5'-nucleotidase (cN-II) and an ecto-5'-nucleotidase (e-N) have been cloned. We utilized the high abundance of cN-I in pigeon heart to purify a 40-kDa subunit for partial protein sequencing and subsequent cDNA cloning. We obtained a full-length clone encoding a novel 40-kDa peptide, unrelated to cN-II or e-N, that was most abundant in heart, brain, and breast muscle. Immunolocalization in heart showed a striated cytoplasmic location, suggesting association with contractile elements. Transient expression in COS-7 cells, generated a 5'-nucleotidase that catalyzed adenosine formation from AMP, which was increased during ATP catabolism. In conclusion, the cloning and expression of cN-I provides definitive evidence of its ability to produce adenosine during ATP breakdown.     

Ecto- and cytosolic 5'-nucleotidases in normal and AMP deaminase-deficient human skeletal muscle

In skeletal muscle, adenosine monophosphate (AMP) is mainly deaminated by AMP deaminase. However, the C34T mutation in the AMPD1 gene severely reduces AMP deaminase activity. Alternatively, intracellular AMP is dephosphorylated to adenosine via cytosolic AMP 5'-nucleotidase (cN-I). In individuals with a homozygous C34T mutation, cN-I might be a more important pathway for AMP removal. We determined activities of AMP deaminase, cN-I, total cytosolic 5'-nucleotidase (total cN), ecto-5'-nucleotidase (ectoN) and whole homogenate 5'-nucleotidase activity in skeletal muscle biopsies from patients with different AMPD1 genotypes [homozygotes for C34T mutation (TT); heterozygotes for C34T mutation (CT); and homozygotes for wild type (CC): diseased controls CC; and normal controls CC]. AMP deaminase activity showed genotype-dependent differences. Total cN activity in normal controls accounted for 57+/-22% of whole homogenate 5'-nucleotidase activity and was not significantly different from the other groups. A weak inverse correlation was found between AMP deaminase and cN-I activities (r2=0.18, p<0.01). There were no significant differences between different groups in the activities of cN-I, whole homogenate 5'-nucleotidase and ectoN, or in cN-I expression on Western blots. No correlation for age, fibre type distribution and AMPD1 genotype was found for whole homogenate nucleotidase, total cN and cN-I using multiple linear regression analysis. There was no gender-specific difference in the activities of whole homogenate nucleotidase, total cN and cN-I. The results indicate no changes in the relative expression or catalytic behaviour of cN-I in AMP deaminase-deficient human skeletal muscle, but suggest that increased turnover of AMP by cN-I in working skeletal muscle is due to higher substrate availability of AMP.     

Cloning of a mouse cytosolic 5'-nucleotidase-I identifies a new gene related to human autoimmune infertility-related protein.

Adenosine production catalysed by cytosolic 5'-nucleotidase (cN-I) regulates diverse physiological processes. We report here a mouse cN-I (mcN-I) cloned from heart and testis. The open reading frame contains several potential translation initiation sites, which yield similarly active 5'-nucleotidases. Using overexpression in COS-7 cells we showed that mcN-I, like the previously cloned pigeon cN-I, is activated by ADP and catalyses adenosine formation during ATP breakdown. The N- and C-termini of mcN-I and pcN-I are divergent. Deletion of the 12 C-terminal amino acids or the first 19 N-terminal amino acids of pcN-I does not diminish activity, although deletion of the first 31 N-terminal amino acids reduces activity by 70%. Overall mcN-I is only 66% identical to pcN-I or the recently cloned human cN-I (hcN-I), while hcN-I and pcN-I are 85% identical. We report here a partial hcN-I sequence that is only 70% identical with the published hcN-I amino acid sequence but is 87% identical with mcN-I. Both hcN-I sequences have perfect matches to distinct human genome sequences. Our data imply the existence of at least two genes for cN-I, cN-I(A), previously cloned from pigeon and human, and cN-I(B) that we report here from mouse and partially from human.     

Human cytosolic 5'-nucleotidase I: characterization and role in nucleoside analog resistance

Nucleoside analogs are important in the treatment of hematologic malignancies, solid tumors, and viral infections. Their metabolism to the triphosphate form is central to their chemotherapeutic efficacy. Although the nucleoside kinases responsible for the phosphorylation of these compounds have been well described, the nucleotidases that may mediate drug resistance through dephosphorylation remain obscure. We have cloned and characterized a novel human cytosolic 5'-nucleotidase (cN-I) that potentially may have an important role in nucleoside analog metabolism. It is expressed at a high level in skeletal and heart muscle, at an intermediate level in pancreas and brain, and at a low level in kidney, testis, and uterus. The recombinant cN-I showed high affinity toward dCMP and lower affinity toward AMP and IMP. ADP was necessary for maximal catalytic activity. Expression of cN-I in Jurkat and HEK 293 cells conferred resistance to 2-chloro-2'-deoxyadenosine, with a 49-fold increase in the IC(50) in HEK 293 and a greater than 400-fold increase in the IC(50) in Jurkat cells. Expression of cN-I also conferred a 22-fold increase in the IC(50) to 2',3'-difluorodeoxycytidine in HEK 293 cells and an 82-fold increase in the IC(50) to 2',3'-dideoxycytidine in Jurkat cells. These data indicate that cN-I may play an important role in the regulation of physiological pyrimidine nucleotide pools and may also alter the therapeutic efficacy of certain nucleoside analogs.     

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 mechanism of adenosine production in rat polymorphonuclear leucocytes.

Adenosine production in intact rat polymorphonuclear leucocytes was studied during 2-deoxyglucose-induced ATP catabolism. A cell-free system containing the cytosolic 5'-nucleotidase (EC 3.1.3.5) as the only phosphohydrolase was also studied. The rate of adenosine formation in both intact cells and the cell-free system showed a similar dependence on energy charge (([ATP] + 1/2 [ADP]/([ATP] + [ADP] + [AMP])), being maximal only at values close to 0.8. Sufficient cytosolic 5'-nucleotidase was present in intact cells to explain the observed rate of adenosine formation. We conclude that the cytosolic 5'-nucleotidase is responsible for adenosine production in rat polymorphonuclear leucocytes. This mechanism provides a direct biochemical link between the energy status of a cell and the rate of adenosine formation.     

A nucleoside transporter is functionally linked to ectonucleotidases in rat liver canalicular membrane.

Prevention of nucleoside loss in bile is physiologically desirable because hepatocytes are the main source of nucleosides for animal cells which lack de novo nucleoside biosynthesis. We have demonstrated a Na+ gradient-energized, concentrative nucleoside transport system in canalicular membrane vesicles (CMV) from rat liver by studying [3H]adenosine uptake using a rapid filtration technique. The Na(+)-dependent nucleoside transporter accepts purine, analogues of purine nucleosides and uridine; exhibits high affinity for adenosine (apparent Km, 14 microM); is not inhibited by nitrobenzylthioinosine or dipyridamole, and is present in CMV but not in rat liver sinusoidal membrane vesicles. Adenosine transport in right side-out CMV was substantially greater than with inside-out CMV. CMV also contain abundant ecto-ATPase and ecto-AMPase (5'-nucleotidase). These ectoenzymes were shown to degrade nucleotides into nucleosides which were conserved by the Na(+)-dependent nucleoside transport system.     

Adenosine Formation and Release by Embryonic Chick Neurons and Glia in Cell Culture

Adenosine formation and release were studied in 48-h-old cultured ciliary ganglia and confluent peripheral and CNS glial cultures from embryonic chicks. Metabolic poisoning induced by 30 mM 2-deoxyglucose and 2 micrograms/ml oligomycin reduced ATP concentration by 90%. An increase in adenosine accounted for 15-40% of the fall in ATP. Dilazep (3 X 10(-6) M), a nucleoside transport inhibitor, decreased both incorporation of adenosine (an index of nucleoside transport) and release of adenosine by 80-90%. Dilazep trapped the newly formed adenosine intracellularly. A concentration of alpha, beta-methylene ADP that inhibited ecto-5'-nucleotidase by 80-90% did not alter the concentration of adenosine or AMP in neurons, glia, or medium. The results demonstrate that adenosine is formed intracellularly and exported out of the cell via the nucleoside transporter. The participation of ecto-5'-nucleotidase was excluded.     

Enzymes of adenosine metabolism in the brain: diurnal rhythm and the effect of sleep deprivation

Adenosine plays a role in promoting sleep, an effect that is thought to be mediated in the basal forebrain. Adenosine levels vary in this region with prolonged wakefulness in a unique way. The basis for this is unknown. We examined, in rats, the activity of the major metabolic enzymes for adenosine - adenosine deaminase, adenosine kinase, ecto- and cytosolic 5'-nucleotidase - in sleep/wake regulatory regions as well as cerebral cortex, and how the activity varies across the day and with sleep deprivation. There were robust spatial differences for the activity of adenosine deaminase, adenosine kinase, and cytosolic and ecto-5'-nucleotidase. However, the basal forebrain was not different from other sleep/wake regulatory regions apart from the tuberomammillary nucleus. All adenosine metabolic enzymes exhibited diurnal variations in their activity, albeit not in all brain regions. Activity of adenosine deaminase increased during the active period in the ventrolateral pre-optic area but decreased significantly in the basal forebrain. Enzymatic activity of adenosine kinase and cytosolic-5'-nucleotidase was higher during the active period in all brain regions tested. However, the activity of ecto-5'-nucleotidase was augmented during the active period only in the cerebral cortex. This diurnal variation may play a role in the regulation of adenosine in relationship to sleep and wakefulness across the day. In contrast, we found no changes specifically with sleep deprivation in the activity of any enzyme in any brain region. Thus, changes in adenosine with sleep deprivation are not a consequence of alterations in adenosine enzyme activity.     

Induction and repression of enzymes involved in exogenous purine compound utilization of Bacillus cereus

5'-Nucleotidase, adenosine phosphorylase, adenosine deaminase and purine nucleoside phosphorylase, four enzymes involved in the utilization of exogenous compounds in Bacillus cereus, were measured in extracts of this organism grown in different conditions. It was found that adenosine deaminase is inducible by addition of adenine derivatives to the growth medium, and purine, nucleoside phosphorylase by metabolizable purine and pyrimidine ribonucleosides. Adenosine deaminase is repressed by inosine, while both enzymes are repressed by glucose. Evidence is presented that during growth of B. cereus in the presence of AMP, the concerted action of 5'-nucleotidase and adenosine phosphorylase, two constitutive enzymes, leads to formation of adenine, and thereby to induction of adenosine deaminase. The ionsine formed would then cause induction of the purine nucleoside phosphorylase and repression of the deaminase. Taken together with our previous findings showing that purine nucleoside phosphorylase of B. cereus acts as a translocase of the ribose moiety of inosine inside the cell (Mura, U., Sgarrella, F. and Ipata, P.L. (1978) J. Biol Chem. 253, 7905-7909), our results provide a clear picture of the molecular events leading to the utilization of the sugar moiety of exogenous AMP, adenosine and inosine as an energy source.     

Adenosine production inside rat polymorphonuclear leucocytes.

Adenosine synthesis was studied during 2-deoxyglucose-induced ATP catabolism in intact rat polymorphonuclear leucocytes. When both adenosine kinase (EC 2.7.1.20) and adenosine deaminase (EC 3.5.4.4) were selectively inhibited, adenosine accumulated. Adenosine formation took place inside the intact cells by a metabolic pathway independent of the ecto-5'-nucleotidase (EC 3.1.3.5). Distinct metabolic pathways are proposed for adenosine production from intracellular or extracellular nucleotides.     

Uptake of Adenosine by Cultured Cerebral Vascular Smooth Muscle Cells

Abstract: Adenosine uptake by cerebral smooth muscle cells is a carrier-mediated process. The K_m value for adenosine uptake is 10.0 μ M and the V _max is 0.95 nmol/ min-mg cell protein. This uptake system is inhibited by the adenosine analog 2-chloroadenosine at low adenosine concentrations. These results prove the existence of a nucleoside transport system associated with cerebral smooth muscle.     

Adenosine formation and release from neonatal-rat heart cells in culture.

The incorporation of [3H]adenosine (10 microM) into neonatal-rat heart cell nucleotides was inhibited in a concentration-dependent manner, such that 50% inhibition was obtained with 0.75 microM-dipyridamole, 0.26 microM-hexobendine or 0.22 microM-dilazep. Adenosine formation was accelerated 2.5-fold to 2.1 +/- 0.3 nmol/10(7) cells in 10 min when cells were incubated with a combination of 30 mM-2-deoxyglucose and 2 micrograms of oligomycin/ml. Of the newly formed adenosine, 6 +/- 2% was in the cells. Dipyridamole, hexobendine or dilazep (10 microM) increased the amount of adenosine in the cells and decreased that in the medium such that 45-50% of the newly formed adenosine was in the cells. Antibodies which inhibited ecto-5'-nucleotidase by 98.7 +/- 0.3% did not alter the rate of adenosine formation or its distribution between cells and medium. We conclude that adenosine was formed in the cytoplasm during catabolism of cellular ATP and was released via the dipyridamole-sensitive symmetric nucleoside transporter.     

Adenosine production and its interaction with protection of ischemic and reperfusion injury of the myocardium

Adenosine exerts cardioprotective effects on the ischemic myocardium. A flexibly mounted microdialysis probe was used to measure the concentration of interstitial adenosine and to assess the activity of ecto-5'-nucleotidase (a key enzyme responsible for adenosine production) in in vivo rat hearts. The level of adenosine during perfusion of adenosine 5'-adenosine monophosphate (AMP) was given as an index of the activity of ecto-5'-nucleotidase in the tissue. Endogenous norepinephrine (NE) activates both alpha(1)-adrenoceptors and protein kinase C (PKC), which, in turn, activates ecto-5'-nucleotidase via phosphorylation thereby enhancing the production of interstitial adenosine. Histamine-release NE activates PKC, which increased ecto-5'-nucleotidase activity and augmented release of adenosine. Opening of cardiac ATP sensitive K(+) (K(ATP)) channels may cause hydroxyl radical (.OH) generation through NE release. Lysophosphatidylcholine (LPC), an endogenous amphiphiphilic lipid metabolite, also increases the concentration of interstitial adenosine in rat hearts, through the PKC-mediated activation of endogenous ecto-5'-nucleotidase. Nitric oxide (NO) facilitates the production of interstitial adenosine, via guanosine 3',5'-cyclic monophosphate (cGMP)-mediated activation of ecto-5'-nucleotidase as another pathway. These mechanisms play an important role in high sensitivity of the cardiac adenosine system. Adenosine plays an important role as a modulator of ischemic reperfusion injury, and that the production and mechanism of action of adenosine are linked with NE release.     

Intracellular adenosine formation and its carrier-mediated release in cultured embryonic chick heart cells

Adenosine formation and release was examined in 48 hr old primary cultures of chick ventricular myocytes. Dilazep greater than hexobendine greater than dipyridamole inhibit incorporation of adenosine into chick embryonic heart cellular nucleotides in a concentration dependent manner. A combination of 30 mM 2-deoxyglucose and 2 micrograms of oligomycin/ml reduces the ATP content of the cells by 71% in 10 min. This change is accompanied by an increase in total adenosine concentration of 3.4 nmoles/10(7) cells in 10 min. Although the ATP concentration is not altered during hypoxia (95%N2/5%CO2), adenosine concentration increases by 0.52 nmoles/10(7) cells in 30 min. When nucleoside incorporation is inhibited by 85-90% by dipyridamole, dilazep or hexobendine, efflux of adenosine decreases by 70-90%, and 60-90% of the newly formed adenosine is trapped inside the cells compared to 10% in the absence of the transport inhibitors. alpha, beta -Methylene ADP inhibits the ecto 5'-nucleotidase activity by 91 +/- 6% but does not inhibit adenosine formation or alter its distribution between cells and medium, thus ruling out the involvement of this enzyme in adenosine formation. We conclude that adenosine is formed intracellularly during 2-deoxyglucose and oligomycin-induced ATP degradation and during hypoxia and that the nucleoside is released via the symmetric nucleoside transporter.     

Inhibition of hippocampal synaptic activity by ATP, hypoxia or oxygen-glucose deprivation does not require CD73

Adenosine, through activation of its A(1) receptors, has neuroprotective effects during hypoxia and ischemia. Recently, using transgenic mice with neuronal expression of human equilibrative nucleoside transporter 1 (hENT1), we reported that nucleoside transporter-mediated release of adenosine from neurons was not a key mechanism facilitating the actions of adenosine at A(1) receptors during hypoxia/ischemia. The present study was performed to test the importance of CD73 (ecto-5'-nucleotidase) for basal and hypoxic/ischemic adenosine production. Hippocampal slice electrophysiology was performed with CD73(+/+) and CD73(-/-) mice. Adenosine and ATP had similar inhibitory effects in both genotypes, with IC(50) values of approximately 25 μM. In contrast, ATP was a less potent inhibitor (IC(50) = 100 μM) in slices from mice expressing hENT1 in neurons. The inhibitory effects of ATP in CD73(+/+) and CD73(-/-) slices were blocked by the adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and were enhanced by the nucleoside transport inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBTI), consistent with effects that are mediated by adenosine after metabolism of ATP. AMP showed a similar inhibitory effect to ATP and adenosine, indicating that the response to ATP was not mediated by P2 receptors. In comparing CD73(-/-) and CD73(+/+) slices, hypoxia and oxygen-glucose deprivation produced similar depression of synaptic transmission in both genotypes. An inhibitor of tissue non-specific alkaline phosphatase (TNAP) was found to attenuate the inhibitory effects of AMP and ATP, increase basal synaptic activity and reduce responses to oxygen-glucose deprivation selectively in slices from CD73(-/-) mice. These results do not support an important role for CD73 in the formation of adenosine in the CA1 area of the hippocampus during basal, hypoxic or ischemic conditions, but instead point to TNAP as a potential source of extracellular adenosine when CD73 is absent.     

Stimulus- and cell-type-specific release of purines in cultured rat forebrain astrocytes and neurons

Adenosine is formed during conditions that deplete ATP, such as ischemia. Adenosine deaminase converts adenosine into inosine, and both adenosine and inosine can be beneficial for postischemic recovery. This study investigated adenosine and inosine release from astrocytes and neurons during chemical hypoxia or oxygen-glucose deprivation. In both cell types, 2-deoxyglucose was the most effective stimulus for depleting cellular ATP and for evoking inosine release; in contrast, oxygen-glucose deprivation evoked the greatest adenosine release. alpha,beta-Methylene ADP, an inhibitor of ecto-5'nucleotidase, significantly reduced adenosine release from astrocytes but not neurons. Dipyridamole, an inhibitor of equilibrative nucleoside transporters, inhibited both adenosine and inosine release from neurons. Erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase, reduced neuronal inosine release evoked by oxygen-glucose deprivation but not by 2-deoxyglucose treatment. These data indicate that (1). astrocytes release adenine nucleotides that are hydrolyzed extracellularly to adenosine, whereas neurons release adenosine per se, (2). inosine is formed intracellularly and released via nucleoside transporters, and (3). inosine is formed by an adenosine deaminase-dependent pathway during oxygen-glucose deprivation but not during 2-deoxyglucose treatment. In summary, the metabolic pathways for adenosine formation and release were cell-type dependent whereas the pathways for inosine formation were stimulus dependent.