Adenosine Kinase Activity Research Articles

Genetic Disruption of Adenosine Kinase in Mouse Pancreatic β-Cells Protects Against High-Fat Diet-Induced Glucose Intolerance.

Islet β-cells adapt to insulin resistance through increased insulin secretion and expansion. Type 2 diabetes typically occurs when prolonged insulin resistance exceeds the adaptive capacity of β-cells. Our prior screening efforts led to the discovery that adenosine kinase (ADK) inhibitors stimulate β-cell replication. Here, we evaluated whether ADK disruption in mouse β-cells affects β-cell mass and/or protects against high-fat diet (HFD)-induced glucose dysregulation. Mice targeted at the Adk locus were bred to Rip-Cre and Ins1-Cre/ERT1Lphi mice to enable constitutive (βADKO) and conditional (iβADKO) disruption of ADK expression in β-cells, respectively. Weight gain, glucose tolerance, insulin sensitivity, and glucose-stimulated insulin secretion (GSIS) were longitudinally monitored in normal chow (NC)-fed and HFD-fed mice. In addition, β-cell mass and replication were measured by immunofluorescence-based islet morphometry. NC-fed adult βADKO and iβADKO mice displayed glucose tolerance, insulin tolerance and β-cell mass comparable to control animals. By contrast, HFD-fed βADKO and iβADKO animals had improved glucose tolerance and increased in vivo GSIS. Improved glucose handling was associated with increased β-cell replication and mass. We conclude that ADK expression negatively regulates the adaptive β-cell response to HFD challenge. Therefore, modulation of ADK activity is a potential strategy for enhancing the adaptive β-cell response.

Role of the purinergic signaling in epilepsy.

Adenine nucleotides and adenosine are signaling molecules that activate purinergic receptors P1 and P2. Activation of A1 adenosine receptors has an anticonvulsant action, whereas activation of A2A receptors might initiate seizures. Therefore, a significant limitation to the use of A1 receptor agonists as drugs in the CNS might be their peripheral side effects. The anti-epileptic activity of adenosine is related to its increased concentration outside the cell. This increase might result from the inhibition of the equilibrative nucleoside transporters (ENTs). Moreover, the implantation of implants or stem cells into the brain might cause slow and persistent increases in adenosine concentrations in the extracellular spaces of the brain. The role of adenosine in seizure inhibition has been confirmed by results demonstrating that in patients with epilepsy, the adenosine kinase (ADK) present in astrocytes is the only purine-metabolizing enzyme that exhibits increased expression. Increased ADK activity causes intensified phosphorylation of adenosine to 5'-AMP, which therefore lowers the adenosine level in the extracellular spaces. These changes might initiate astrogliosis and epileptogenesis, which are the manifestations of epilepsy. Seizures might induce inflammatory processes and vice versa. Activation of P2X7 receptors causes intensified release of pro-inflammatory cytokines (IL-1β and TNF-α) and activates metabolic pathways that induce inflammatory processes in the CNS. Therefore, antagonists of P2X7 and the interleukin 1β receptor might be efficient drugs for recurring seizures and prolonged status epilepticus. Inhibitors of ADK would simultaneously inhibit the seizures, prevent the astrogliosis and epileptogenesis processes and prevent the formation of new epileptogenic foci. Therefore, these drugs might become beneficial seizure-suppressing drugs.

South (S)- and North (N)-Methanocarba-7-Deazaadenosine Analogues as Inhibitors of Human Adenosine Kinase.

Adenosine kinase (AdK) inhibitors raise endogenous adenosine levels, particularly in disease states, and have potential for treatment of seizures, neurodegeneration, and inflammation. On the basis of the South (S) ribose conformation and molecular dynamics (MD) analysis of nucleoside inhibitors bound in AdK X-ray crystallographic structures, (S)- and North (N)-methanocarba (bicyclo[3.1.0]hexane) derivatives of known inhibitors were prepared and compared as human (h) AdK inhibitors. 5'-Hydroxy (34, MRS4202 (S); 55, MRS4380 (N)) and 5'-deoxy 38a (MRS4203 (S)) analogues, containing 7- and N(6)-NH phenyl groups in 7-deazaadenine, robustly inhibited AdK activity (IC50 ∼ 100 nM), while the 5'-hydroxy derivative 30 lacking the phenyl substituents was weak. Docking in the hAdK X-ray structure and MD simulation suggested a mode of binding similar to 5'-deoxy-5-iodotubercidin and other known inhibitors. Thus, a structure-based design approach for further potency enhancement is possible. The potent AdK inhibitors in this study are ready to be further tested in animal models of epilepsy.

Adenosine A2A receptors in neuronal outgrowth: a target for nerve regeneration?

Adenosine A2A receptors in neuronal outgrowth: a target for nerve regeneration?

The roles of purinergic signaling in psychiatric disorders.

Ecto-purines and ecto-pyrimidines are present in the extracellular space of the central nervous system (CNS). Together with P1 and P2 receptors and nucleotides metabolizing ecto-enzymes, they make signaling system involved in neurotransmission, the modulation of sensory signals, including pain stimuli conduction, and the induction of apoptosis and necrosis of the cells. Purines and pyrimidines have a dual effect: positive (neuroprotective) of nucleosides, and negative (pro-inflammatory and pro-apoptotic) of nucleotides. Adenosine-5'-triphosphate (ATP) in the CNS triggers the pro-inflammatory reactions, predominantly by activation of the P2X7 receptor, which results in production and release of pro-inflammatory cytokines. In contrast to ATP, adenosine acts generally as an anti-inflammatory agent and plays an important role in neuroprotection. Currently, it is believed that the initiation of CNS diseases, including mental disorders, is caused by any imbalance between the concentration of ATP and adenosine in the extracellular space. Genetic tests provide also the evidence for the participation of purinergic signaling in psychiatric disorders. It is believed that any action leading to the effective increase of adenosine concentration: activation of nucleotide metabolizing ecto-enzymes (mainly NTPDases - nucleoside triphosphate diphosphohydrolases), inhibition of adenosine deaminase and/or adenosine kinase activity as well as therapies using P1 receptor agonists (adenosine or its analogues) might be beneficial in therapy of psychiatric disorders.

Reciprocal regulation of insulin and plasma 5'-AMP in glucose homeostasis in mice.

A previous investigation has demonstrated that plasma 5'-AMP (pAMP) exacerbates and causes hyperglycemia in diabetic mice. However, the crosstalk between pAMP and insulin signaling to regulate glucose homeostasis has not been investigated in depth. In this study, we showed that the blood glucose level was more dependent on the ratio of insulin to pAMP than on the absolute level of these two factors. Administration of 5'-AMP significantly attenuated the insulin-stimulated insulin receptor (IR) autophosphorylation in the liver and muscle tissues, resulting in the inhibition of downstream AKT phosphorylation. A docking analysis indicated that adenosine was a potential inhibitor of IR tyrosine kinase. Moreover, the 5'-AMP treatment elevated the ATP level in the pancreas and in the isolated islets, stimulating insulin secretion and increasing the plasma level of insulin. The insulin administration decreased the 5'-AMP-induced hyper-adenosine level by the up-regulation of adenosine kinase activities. Our results indicate that blood glucose homeostasis is reciprocally regulated by pAMP and insulin.

A Complex Containing SNF1-Related Kinase (SnRK1) and Adenosine Kinase in Arabidopsis

SNF1-related kinase (SnRK1) in plants belongs to a conserved family that includes sucrose non-fermenting 1 kinase (SNF1) in yeast and AMP-activated protein kinase (AMPK) in animals. These kinases play important roles in the regulation of cellular energy homeostasis and in response to stresses that deplete ATP, they inhibit energy consuming anabolic pathways and promote catabolism. Energy stress is sensed by increased AMP:ATP ratios and in plants, 5′-AMP inhibits inactivation of phosphorylated SnRK1 by phosphatase. In previous studies, we showed that geminivirus pathogenicity proteins interact with both SnRK1 and adenosine kinase (ADK), which phosphorylates adenosine to generate 5′-AMP. This suggested a relationship between SnRK1 and ADK, which we investigate in the studies described here. We demonstrate that SnRK1 and ADK physically associate in the cytoplasm, and that SnRK1 stimulates ADK in vitro by an unknown, non-enzymatic mechanism. Further, altering SnRK1 or ADK activity in transgenic plants altered the activity of the other kinase, providing evidence for in vivo linkage but also revealing that in vivo regulation of these activities is complex. This study establishes the existence of SnRK1-ADK complexes that may play important roles in energy homeostasis and cellular responses to biotic and abiotic stress.

Ecto-5'-Nucleotidase (CD73)-Mediated Formation of Adenosine Is Critical for the Striatal Adenosine A2A Receptor Functions

Adenosine is a neuromodulator acting through inhibitory A1 receptors (A1Rs) and facilitatory A2ARs, which have similar affinities for adenosine. It has been shown that the activity of intracellular adenosine kinase preferentially controls the activation of A1Rs, but the source of the adenosine activating A2ARs is unknown. We now show that ecto-5'-nucleotidase (CD73), the major enzyme able to convert extracellular AMP into adenosine, colocalizes with A2ARs in the basal ganglia. In addition to astrocytes, striatal CD73 is prominently localized to postsynaptic sites. Notably, CD73 coimmunoprecipitated with A2ARs and proximity ligation assays confirmed the close proximity of CD73 and A2ARs in the striatum. Accordingly, the cAMP formation in synaptosomes as well as the hypolocomotion induced by a novel A2AR prodrug that requires CD73 metabolization to activate A2ARs were observed in wild-type mice, but not in CD73 knock-out (KO) mice or A2AR KO mice. Moreover, CD73 KO mice displayed increased working memory performance and a blunted amphetamine-induced sensitization, mimicking the phenotype of global or forebrain-A2AR KO mice, as well as upon pharmacological A2AR blockade. These results show that CD73-mediated formation of extracellular adenosine is responsible for the activation of striatal A2AR function. This study points to CD73 as a new target that can fine-tune A2AR activity, and a novel therapeutic target to manipulate A2AR-mediated control of striatal function and neurodegeneration.

How systemic inflammation modulates adenosine metabolism and adenosine receptor expression in humans in vivo

Adenosine modulates inflammation and prevents associated organ injury by activation of its receptors. During sepsis, the extracellular adenosine concentration increases rapidly, but the underlying mechanism in humans is unknown. We aimed to determine the changes in adenosine metabolism and signaling both in vivo during experimental human endotoxemia and in vitro. We studied subjects participating in three different randomized double-blind placebo-controlled trials. In order to prevent confounding by the different pharmacological interventions in these trials, analyses were performed on data of placebo-treated subjects only. Intensive care research unit at the Radboud University Nijmegen Medical Center. In total, we used material of 24 healthy male subjects. Subjects received 2 ng/kg Escherichia coli endotoxin (lipopolysaccharide) intravenously. Following experimental endotoxemia, endogenous adenosine concentrations increased. Expression of 5'ectonucleotidase messenger RNA was upregulated (p = .01), whereas adenosine deaminase messenger RNA was downregulated (p = .02). Furthermore, both adenosine deaminase and adenosine kinase activity was significantly diminished (both p ≤ .0001). A2a and A2b receptor messenger RNA expression was elevated (p = .02 and p = .04, respectively), whereas messenger RNA expression of A1 and A3 receptors was reduced (both, p = .03). In vitro, lipopolysaccharide dose-dependently attenuated the activity of both adenosine deaminase and adenosine kinase (both p ≤ .0001). Adenosine metabolism and signaling undergo adaptive changes during human experimental endotoxemia promoting higher levels of adenosine thereby facilitating its inflammatory signaling.

Abstract 3878: A highly sensitive assay for the discovery of inhibitors of the histone methyltransferase DOT1L to treat MLL-rearranged leukemia

Abstract MLL-rearranged leukemia is an aggressive form of cancer characterized by specific chromosomal rearrangements of the mixed lineage leukemia (MLL) gene. In normal cells, MLL, a histone methyltransferase, methylates the lysine 4 residue on histone 3 (H3K4) and thereby regulates the expression of genes which control hematopoietic development. In leukemic cells, chromosomal rearrangements fuse the N-terminus of MLL with one of more than 50 partner proteins, giving rise to oncogenic fusion proteins that lack H3K4 methyltransferase activity. These fusion proteins inappropriately recruit DOT1L, which methylates H3K79, producing an abnormal methylation pattern and activation of genes normally suppressed during differentiation. Elevated expression of HOXA9 in particular leads to a block in normal hematopoetic differentiation and the onset of leukemia. A solid body of evidence has shown DOT1L to be crucial for the development and maintenance of MLL-rearranged leukemias; consequently, it is a promising target for therapeutic intervention. We have focused on developing an assay suitable for high-throughput screening (HTS) to detect compounds that inhibit DOT1L enzymatic activity. DOT1L catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to H3K79 and generates S-adenosylhomocysteine (SAH) as a byproduct. The enzyme displays a very low rate of methylation of histone octamers or nucleosomes and no activity towards individual histones or histone-derived peptides. Thus DOT1L HTS requires a highly sensitive assay that is able to accommodate protein-protein and protein-DNA complexes as substrates. An assay using radiolabeled SAM has sufficient sensitivity, but is relatively laborious and not well-suited for HTS. We tested several previously reported non-radioactive assay formats, but none enabled robust detection of DOT1L activity. Therefore, we developed a coupled enzyme assay in which SAH is converted to homocysteine and adenosine using SAH hydrolase. In a second enzyme-coupled step, adenosine kinase generates AMP and ADP from the SAH-derived adenosine and added ATP. Finally, ADP is detected using the luciferase-based Promega ADP-Glo assay. The luminescent signal provides a highly sensitive measure of ADP at a concentration as low as 10 nM. The readout is directly proportional to SAH production; thus DOT1L inhibitors are identified by a reduction in this signal. We report development of the assay, including careful optimization of the activity of adenosine kinase to enhance sensitivity, and assay validation demonstrating DOT1L inhibition by the non-selective SAM-competitive inhibitor sinefungin, and pilot HTS of a 2,000-compound library. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3878. doi:1538-7445.AM2012-3878

Adenosine dysfunction in epilepsy

Extracellular levels of the brain's endogenous anticonvulsant and neuroprotectant adenosine largely depend on an astrocyte-based adenosine cycle, comprised of ATP release, rapid degradation of ATP into adenosine, and metabolic reuptake of adenosine through equilibrative nucleoside transporters and phosphorylation by adenosine kinase (ADK). Changes in ADK expression and activity therefore rapidly translate into changes of extracellular adenosine, which exerts its potent anticonvulsive and neuroprotective effects by activation of pre- and postsynaptic adenosine A(1) receptors. Increases in ADK increase neuronal excitability, whereas decreases in ADK render the brain resistant to seizures and injury. Importantly, ADK was found to be overexpressed and associated with astrogliosis and spontaneous seizures in rodent models of epilepsy, as well as in human specimen resected from patients with hippocampal sclerosis and temporal lobe epilepsy. Several lines of evidence indicate that overexpression of astroglial ADK and adenosine deficiency are pathological hallmarks of the epileptic brain. Consequently, adenosine augmentation therapies constitute a powerful approach for seizure prevention, which is effective in models of epilepsy that are resistant to conventional antiepileptic drugs. The adenosine kinase hypothesis of epileptogenesis suggests that adenosine dysfunction in epilepsy undergoes a biphasic response: an acute surge of adenosine that can be triggered by any type of injury might contribute to the development of astrogliosis via adenosine receptor-dependent and -independent mechanisms. Astrogliosis in turn is associated with overexpression of ADK, which was shown to be sufficient to trigger spontaneous recurrent electrographic seizures. Thus, ADK emerges as a promising target for the prediction and prevention of epilepsy.

Overexpression of ADK in human astrocytic tumors and peritumoral tissue is related to tumor‐associated epilepsy

Adenosine kinase (ADK), a largely astrocyte-based metabolic enzyme, regulates adenosine homeostasis in the brain. Overexpression of ADK decreases extracellular adenosine and consequently leads to seizures. We hypothesized that dysfunction in the metabolism of tumor astrocytes is related to changes in ADK expression and that those changes might be associated with the development of epilepsy in patients with tumors. We compared ADK expression and cellular distribution in surgically removed tumor tissue (n = 45) and peritumoral cortex (n = 20) of patients with glial and glioneuronal tumors to normal control tissue obtained at autopsy (n = 11). In addition, we compared ADK expression in tumor patients with and without epilepsy. To investigate ADK expression, we used immunohistochemistry and Western blot analysis. ADK activity measurement was performed in surgical specimens of astrocytomas World Health Organization (WHO) grade III (n = 3), peritumoral cortex (n = 3), and nonepileptic cortex (n = 3). Immunohistochemistry predominantly showed cytoplasmic labeling in tumors and peritumoral tissue containing infiltrating tumor cells. ADK immunoreactivity was significantly stronger in tumor and peritumoral tissue compared to normal white matter and normal cortex, especially in astrocytoma WHO grade III, as confirmed by Western blot analysis and ADK activity measurements. Importantly, we found a significantly higher expression of ADK in the peritumoral infiltrated tissue of patients with epilepsy than in patients without epilepsy. These results suggest a dysregulation of ADK in astrocytic brain tumors. Moreover, the upregulation of ADK observed in peritumoral infiltrated tissue of glioma patients with epilepsy supports the role of this enzyme in tumor-associated epilepsy.

Molecular Characterization of Chinese Hamster Cells Mutants Affected in Adenosine Kinase and Showing Novel Genetic and Biochemical Characteristics

BackgroundTwo isoforms of the enzyme adenosine kinase (AdK), which differ at their N-terminal ends, are found in mammalian cells. However, there is no information available regarding the unique functional aspects or regulation of these isoforms.ResultsWe show that the two AdK isoforms differ only in their first exons and the promoter regions; hence they arise via differential splicing of their first exons with the other exons common to both isoforms. The expression of these isoforms also varied greatly in different rat tissues and cell lines with some tissues expressing both isoforms and others expressing only one of the isoforms. To gain insights into cellular functions of these isoforms, mutants resistant to toxic adenosine analogs formycin A and tubercidin were selected from Chinese hamster (CH) cell lines expressing either one or both isoforms. The AdK activity in most of these mutants was reduced to <5% of wild-type cells and they also showed large differences in the expression of the two isoforms. Thus, the genetic alterations in these mutants likely affected both regulatory and structural regions of AdK. We have characterized the molecular alterations in a number of these mutants. One of these mutants lacking AdK activity was affected in the conserved NxxE motif thereby providing evidence that this motif involved in the binding of Mg2+ and phosphate ions is essential for AdK function. Another mutant, FomR-4, exhibiting increased resistance to only C-adenosine analogs and whose resistance was expressed dominantly in cell-hybrids contained a single mutation leading to Ser191Phe alteration in AdK. We demonstrate that this mutation in AdK is sufficient to confer the novel genetic and biochemical characteristics of this mutant. The unusual genetic and biochemical characteristics of the FomR-4 mutant suggest that AdK in this mutant might be complexed with the enzyme AMP-kinase. Several other AdK mutants were altered in surface residues that likely affect its binding to the adenosine analogs and its interaction with other cellular proteins.ConclusionsThese AdK mutants provide important insights as well as novel tools for understanding the cellular functions of the two isoforms and their regulation in mammalian cells.

Adenosine kinase determines the degree of brain injury after ischemic stroke in mice.

Adenosine kinase (ADK) is the major negative metabolic regulator of the endogenous neuroprotectant and homeostatic bioenergetic network regulator adenosine. We used three independent experimental approaches to determine the role of ADK as a molecular target for predicting the brain's susceptibility to ischemic stroke. First, when subjected to a middle cerebral artery occlusion model of focal cerebral ischemia, transgenic fb-Adk-def mice, which have increased ADK expression in striatum (164%) and reduced ADK expression in cortical forebrain (65%), demonstrate increased striatal infarct volume (126%) but almost complete protection of cortex (27%) compared with wild-type (WT) controls, indicating that cerebral injury levels directly correlate to levels of ADK in the CNS. Second, we demonstrate abrogation of lipopolysaccharide (LPS)-induced ischemic preconditioning in transgenic mice with brain-wide ADK overexpression (Adk-tg), indicating that ADK activity negatively regulates LPS-induced tolerance to stroke. Third, using adeno-associated virus-based vectors that carry Adk-sense or -antisense constructs to overexpress or knockdown ADK in vivo, we demonstrate increased (126%) or decreased (51%) infarct volume, respectively, 4 weeks after injection into the striatum of WT mice. Together, our data define ADK as a possible therapeutic target for modulating the degree of stroke-induced brain injury.

The Interaction between geminivirus pathogenicity proteins and adenosine kinase leads to increased expression of primary cytokinin-responsive genes

Pathogenicity proteins (AL2/C2) of begomo- and curtoviruses suppress silencing through inhibition of the methyl cycle, as a consequence of inhibiting adenosine kinase (ADK). ADK phosphorylates cytokinin nucleosides, helping maintain a pool of bioactive cytokinins through interconversion of free-bases, nucleosides and nucleotides. We provide evidence that inhibiting ADK affects expression of primary cytokinin-responsive genes. Specifically, we demonstrate increased activity of a primary cytokinin-responsive promoter in adk mutant Arabidopsis plants, and in response to silencing ADK expression or inhibiting ADK activity in transient assays. Similar changes in expression are observed in geminivirus infected tissue and when AL2/C2 are over-expressed. Increased cytokinin-responsive promoter activity may therefore be a consequence of an ADK/AL2/C2 interaction. Application of exogenous cytokinin increases susceptibility to geminivirus infection, characterized by a reduced mean latent period and enhanced viral replication. Thus, ADK appears to be a high value target of geminiviruses that includes increasing expression of primary cytokinin-responsive genes.

Effect of insulin and glucose on adenosine metabolizing enzymes in human B lymphocytes.

In diabetes several aspects of immunity are altered, including the immunomodulatory action of adenosine. Our study was undertaken to investigate the effect of different glucose and insulin concentrations on activities of adenosine metabolizing enzymes in human B lymphocytes line SKW 6.4. The activity of adenosine deaminase in the cytosolic fraction was very low and was not affected by different glucose concentration, but in the membrane fraction of cells cultured with 25 mM glucose it was decreased by about 35% comparing to the activity in cells maintained in 5 mM glucose, irrespective of insulin concentration. The activities of 5'-nucleotidase (5'-NT) and ecto-5'-NT in SKW 6.4 cells depended on insulin concentration, but not on glucose. Cells cultured with 10(-8) M insulin displayed an about 60% lower activity of cytosolic 5'-NT comparing to cells maintained at 10(-11) M insulin. The activity of ecto-5'-NT was decreased by about 70% in cells cultured with 10(-8) M insulin comparing to cells grown in 10(-11) M insulin. Neither insulin nor glucose had an effect on adenosine kinase (AK) activity in SKW 6.4 cells or in human B cells isolated from peripheral blood. The extracellular level of adenosine and inosine during accelerated catabolism of cellular ATP depended on glucose, but not on insulin concentration. Concluding, our study demonstrates that glucose and insulin differentially affect the activities of adenosine metabolizing enzymes in human B lymphocytes, but changes in those activities do not correlate with the adenosine level in cell media during accelerated ATP catabolism, implying that nucleoside transport is the primary factor determining the extracellular level of adenosine.

Mycobacterial adenosine kinase is not a typical adenosine kinase

Adenosine kinase (AK) is only found in eukaryotes. Recently, a Mycobacterium tuberculosis (MTub) protein exhibiting greater sequence similarity to ribokinases (RK) was identified as AK. We have expressed AKs from MTub, human and Chinese hamster (CH) cells in Escherichia coli and also AK from human and MTub in AK-deficient CH cells. While both E. coli and CH cells expressing mammalian AKs efficiently metabolized various adenosine analogs, those expressing MTub-AK were completely inactive. The AK activity of the MTub protein was very low (50-fold lower than E. coli RK) and it was not stimulated by phosphate or inhibited by several AK inhibitors. These results raise questions over MTub protein’s true function and whether it functions as AK in cells.

An Adenosine Kinase Exists inXanthomonas campestrisPathovar campestris and Is Involved in Extracellular Polysaccharide Production, Cell Motility, and Virulence

Adenosine kinase (ADK) is a purine salvage enzyme and a typical housekeeping enzyme in eukaryotes which catalyzes the phosphorylation of adenosine to form AMP. Since prokaryotes synthesize purines de novo and no endogenous ADK activity is detectable in Escherichia coli, ADK has long been considered to be rare in bacteria. To date, only two prokaryotes, both of which are gram-positive bacteria, have been reported to contain ADK. Here we report that the gram-negative bacterium Xanthomonas campestris pathovar campestris, the causal agent of black rot of crucifers, possesses a gene (designated adk(Xcc)) encoding an ADK (named ADK(Xcc)), and we demonstrate genetically that the ADK(Xcc) is involved in extracellular polysaccharide (EPS) production, cell motility, and pathogenicity of X. campestris pv. campestris. adk(Xcc) was overexpressed as a His(6)-tagged protein in E. coli, and the purified His(6)-tagged protein exhibited ADK activity. Mutation of adk(Xcc) did not affect bacterial growth in rich and minimal media but led to an accumulation of intracellular adenosine and diminutions of intracellular ADK activity and ATP level, as well as EPS. The adk(Xcc) mutant displayed significant reductions in bacterial growth and virulence in the host plant.

Erythrocytic Adenosine Monophosphate as an Alternative Purine Source in Plasmodium falciparum

Plasmodium falciparum is a purine auxotroph, salvaging purines from erythrocytes for synthesis of RNA and DNA. Hypoxanthine is the key precursor for purine metabolism in Plasmodium. Inhibition of hypoxanthine-forming reactions in both erythrocytes and parasites is lethal to cultured P. falciparum. We observed that high concentrations of adenosine can rescue cultured parasites from purine nucleoside phosphorylase and adenosine deaminase blockade but not when erythrocyte adenosine kinase is also inhibited. P. falciparum lacks adenosine kinase but can salvage AMP synthesized in the erythrocyte cytoplasm to provide purines when both human and Plasmodium purine nucleoside phosphorylases and adenosine deaminases are inhibited. Transport studies in Xenopus laevis oocytes expressing the P. falciparum nucleoside transporter PfNT1 established that this transporter does not transport AMP. These metabolic patterns establish the existence of a novel nucleoside monophosphate transport pathway in P. falciparum.

Astrocytic adenosine kinase regulates basal synaptic adenosine levels and seizure activity but not activity-dependent adenosine release in the hippocampus

Adenosine is an endogenous inhibitor of excitatory synaptic transmission with potent anticonvulsant properties in the mammalian brain. Given adenosine's important role in modulating synaptic transmission, several mechanisms exist to regulate its extracellular availability. One of these is the intracellular enzyme adenosine kinase (ADK), which phosphorylates adenosine to AMP. We have investigated the role that ADK plays in regulating the presence and effects of extracellular adenosine in area CA1 of rat hippocampal slices. Inhibition of ADK activity with 5′-iodotubercidin (IODO; 5 μM) raised extracellular adenosine, as measured with adenosine biosensors, and potently inhibited field excitatory post-synaptic potentials (fEPSPs) in an adenosine A 1R-dependent manner. In nominally Mg 2+-free aCSF, which facilitated the induction of electrically-evoked epileptiform activity, adenosine biosensor recordings revealed that seizures were accompanied by the transient release of adenosine. Under these conditions, IODO also inhibited the fEPSP and greatly suppressed epileptiform activity evoked by brief, high-frequency stimulation. During spontaneous seizures evoked by the A 1R antagonist CPT, adenosine release was unaffected by IODO. This suggests that ADK activity does not limit activity-dependent adenosine release. On the basis of strong ADK immunoreactivity in GFAP-positive cells, astrocytes are likely to play a key role in regulating basal adenosine levels. It is this action of ADK on the basal adenosine tone that is permissive to seizure activity, and, by extension, other forms of activity-dependent neuronal activity such as synaptic plasticity.