adenosine-kinase and 5-iodotubercidin

Sudden unexpected death in epilepsy (SUDEP) is a leading cause of death in patients with refractory epilepsy. Centrally-mediated respiratory dysfunction has been identified as one of the principal mechanisms responsible for SUDEP. Seizures generate a surge in adenosine release. Elevated adenosine levels suppress breathing. Insufficient metabolic clearance of a seizure-induced adenosine surge might be a precipitating factor in SUDEP. In order to deliver targeted therapies to prevent SUDEP, reliable biomarkers must be identified to enable prompt intervention. Because of the integral role of the phrenic nerve in breathing, we hypothesized that suppression of phrenic nerve activity could be utilized as predictive biomarker for imminent SUDEP. We used a rat model of kainic acid-induced seizures in combination with pharmacological suppression of metabolic adenosine clearance to trigger seizure-induced death in tracheostomized rats. Recordings of EEG, blood pressure, and phrenic nerve activity were made concomitant to the seizure. We found suppression of phrenic nerve burst frequency to 58.9% of baseline (p < 0.001, one-way ANOVA) which preceded seizure-induced death; importantly, irregularities of phrenic nerve activity were partly reversible by the adenosine receptor antagonist caffeine. Suppression of phrenic nerve activity may be a useful biomarker for imminent SUDEP. The ability to reliably detect the onset of SUDEP may be instrumental in the timely administration of potentially lifesaving interventions.

Topics: Adenosine Kinase; Animals; Kainic Acid; Male; Phrenic Nerve; Predictive Value of Tests; Rats; Rats, Wistar; Seizures; Sudden Unexpected Death in Epilepsy; Tubercidin

Over one-third of all patients with epilepsy are refractory to treatment and there is an urgent need to develop new drugs that can prevent the development and progression of epilepsy. Epileptogenesis is characterized by distinct histopathologic and biochemical changes, which include astrogliosis and increased expression of the adenosine-metabolizing enzyme adenosine kinase (ADK; EC 2.7.1.20). Increased expression of ADK contributes to epileptogenesis and is therefore a target for therapeutic intervention. We tested the prediction that the transient use of an ADK inhibitor administered during the latent phase of epileptogenesis can mitigate the development of epilepsy.. We used the intrahippocampal kainic acid (KA) mouse model of temporal lobe epilepsy, which is characterized by ipsilateral hippocampal sclerosis with granule cell dispersion and the development of recurrent hippocampal paroxysmal discharges (HPDs). KA-injected mice were treated with the ADK inhibitor 5-iodotubercidin (5-ITU, 1.6 mg/kg, b.i.d., i.p.) during the latent phase of epileptogenesis from day 3-8 after injury; the period when gradual increases in hippocampal ADK expression begin to manifest. HPDs were assessed at 6 and 9 weeks after KA administration followed by epilepsy histopathology including assessment of granule cell dispersion, astrogliosis, and ADK expression.. 5-ITU significantly reduced the percent time in seizures by at least 80% in 56% of mice at 6 weeks post-KA. This reduction in seizure activity was maintained in 40% of 5-ITU-treated mice at 9 weeks. 5-ITU also suppressed granule cell dispersion and prevented maladaptive ADK increases in these protected mice.. Our results show that the transient use of a small-molecule ADK inhibitor, given during the early stages of epileptogenesis, has antiepileptogenic disease-modifying properties, which provides the rationale for further investigation into the development of a novel class of antiepileptogenic ADK inhibitors with increased efficacy for epilepsy prevention.

Topics: Adenosine Kinase; Animals; Anticonvulsants; Brain; Enzyme Inhibitors; Epilepsy; Male; Mice; Mice, Inbred C57BL; Tubercidin

Among the dangers to astronauts engaging in deep space missions such as a Mars expedition is exposure to radiations that put them at risk for severe cognitive dysfunction. These radiation-induced cognitive impairments are accompanied by functional and structural changes including oxidative stress, neuroinflammation, and degradation of neuronal architecture. The molecular mechanisms that dictate CNS function are multifaceted and it is unclear how irradiation induces persistent alterations in the brain. Among those determinants of cognitive function are neuroepigenetic mechanisms that translate radiation responses into altered gene expression and cellular phenotype. In this study, we have demonstrated a correlation between epigenetic aberrations and adverse effects of space relevant irradiation on cognition. In cognitively impaired irradiated mice we observed increased 5-methylcytosine and 5-hydroxymethylcytosine levels in the hippocampus that coincided with increased levels of the DNA methylating enzymes DNMT3a, TET1 and TET3. By inhibiting methylation using 5-iodotubercidin, we demonstrated amelioration of the epigenetic effects of irradiation. In addition to protecting against those molecular effects of irradiation, 5-iodotubercidin restored behavioral performance to that of unirradiated animals. The findings of this study establish the possibility that neuroepigenetic mechanisms significantly contribute to the functional and structural changes that affect the irradiated brain and cognition.

Topics: 5-Methylcytosine; Adenosine Kinase; Animals; Behavior, Animal; Brain; Dioxygenases; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA Methyltransferase 3A; DNA-Binding Proteins; Epigenomics; Hippocampus; Male; Mice; Mice, Inbred C57BL; Proto-Oncogene Proteins; Tubercidin; Whole-Body Irradiation

Extracellular adenosine has tissue-protective potential in several conditions. Adenosine levels are regulated by a close interplay between nucleoside transporters and adenosine kinase. On the basis of the evidence of the role of adenosine kinase in regulating adenosine levels during hypoxia, we evaluated the effect of adenosine kinase on lung injury. Furthermore, we tested the influence of a pharmacologic approach to blocking adenosine kinase on the extent of lung injury.. Prospective experimental animal study.. University-based research laboratory.. In vitro cell lines, wild-type and adenosine kinase+/- mice.. We tested the expression of adenosine kinase during inflammatory stimulation in vitro and in a model of lipopolysaccharide inhalation in vivo. Studies using the adenosine kinase promoter were performed in vitro. Wild-type and adenosine kinase+/- mice were subjected to lipopolysaccharide inhalation. Pharmacologic inhibition of adenosine kinase was performed in vitro, and its effect on adenosine uptake was evaluated. The pharmacologic inhibition was also performed in vivo, and the effect on lung injury was assessed.. We observed the repression of adenosine kinase by proinflammatory cytokines and found a significant influence of nuclear factor kappa-light-chain-enhancer of activated B-cells on regulation of the adenosine kinase promoter. Mice with endogenous adenosine kinase repression (adenosine kinase+/-) showed reduced infiltration of leukocytes into the alveolar space, decreased total protein and myeloperoxidase levels, and lower cytokine levels in the alveolar lavage fluid. The inhibition of adenosine kinase by 5-iodotubercidin increased the extracellular adenosine levels in vitro, diminished the transmigration of neutrophils, and improved the epithelial barrier function. The inhibition of adenosine kinase in vivo showed protective properties, reducing the extent of pulmonary inflammation during lung injury.. Taken together, these data show that adenosine kinase is a valuable target for reducing the inflammatory changes associated with lung injury and should be pursued as a therapeutic option.

Topics: Acute Lung Injury; Adenosine Kinase; Animals; B-Lymphocytes; Bronchoalveolar Lavage Fluid; Cell Line; Cytokines; Lipopolysaccharides; Lung; Mice; Pneumonia; Prospective Studies; Tubercidin

Sudden unexpected death in epilepsy (SUDEP) is a significant cause of mortality in people with epilepsy. Two postulated causes for SUDEP, cardiac and respiratory depression, can both be explained by overstimulation of adenosine receptors. We hypothesized that SUDEP is a consequence of a surge in adenosine as a result of prolonged seizures combined with deficient adenosine clearance; consequently, blockade of adenosine receptors should prevent SUDEP. Here we induced impaired adenosine clearance in adult mice by pharmacologic inhibition of the adenosine-removing enzymes, adenosine kinase and deaminase. Combination of impaired adenosine clearance with kainic acid-induced seizures triggered sudden death in all animals. Most importantly, the adenosine receptor antagonist caffeine, when given after seizure onset, increased survival from 23.75 +/- 1.35 min to 54.86 +/- 6.59 min (p < 0.01). Our data indicate that SUDEP is due to overactivation of adenosine receptors and that caffeine treatment after seizure onset might be beneficial.

Topics: Adenine; Adenosine; Adenosine Deaminase Inhibitors; Adenosine Kinase; Animals; Caffeine; Cause of Death; Death, Sudden; Disease Models, Animal; Enzyme Inhibitors; Epilepsy; Kainic Acid; Mice; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; Risk Factors; Seizures; Survival Analysis; Tubercidin

Theophylline-associated seizures (TAS) often progress to prolonged or treatment-resistant convulsions. Theophylline is a nonselective adenosine receptor antagonist. Adenosine is an endogenous anticonvulsant that can terminate seizures. Fever and young age have been reported to be risk factors for TAS. To elucidate the mechanism of TAS, we investigated the effect of theophylline and adenosine receptor ligands on hyperthermia-induced seizures in juvenile rats. The treatment dose of theophylline or control saline was injected intraperitoneally 1 h before hyperthermia-induced seizures. The seizure threshold in the theophylline group was significantly lower and seizure duration was significantly longer than those in the control group. The addition of a selective adenosine A(1) receptor agonist and an adenosine kinase inhibitor completely counteracted the effects of theophylline. Moreover, a selective A(1) antagonist caused a significantly longer seizure duration compared with the control. These findings suggest that blockage of the adenosine A(1) receptor is the main cause of TAS.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Adenosine Kinase; Animals; Behavior, Animal; Body Temperature; Brain; Disease Models, Animal; Dose-Response Relationship, Drug; Electroencephalography; Enzyme Inhibitors; Hyperthermia, Induced; Injections, Intraperitoneal; Male; Rats; Rats, Inbred Lew; Seizures; Theophylline; Tubercidin

Protein flexibility plays a significant role in drug research due to its effect on accurate prediction of ligand binding mode and activity. Adenosine kinase (AK) represents a highly flexible binding site and is known to exhibit large conformational changes as a result of substrate or inhibitor binding. Here we propose a semi-open conformation for ligand binding in human AK, in addition to the known closed and open forms. The modeling study illustrates the necessity of thorough understanding of the conformational states of protein for docking and binding mode prediction. It has been shown that predicting activity in the context of correct binding mode can improve the insight into conserved interactions and mechanism of action for inhibition of AK. Integrating the knowledge about the binding modes of ligands in different conformational states of the protein, separate pharmacophore models were generated and used for virtual screening to explore potential novel hits. In addition, 2D descriptor based clustering was done to differentiate the ligands, binding to closed, semi-open and open conformations of human AK. The results indicated that binding of all AK inhibitors cannot be described by same rules, instead, they represent a rule based preference for inhibition. This inference about tubercidins binding to semi-open conformation of human AK may facilitate in finding much extensive space for AK inhibitors.

Topics: Adenosine Kinase; Binding Sites; Cluster Analysis; Drug Design; Drug Evaluation, Preclinical; Enzyme Inhibitors; Humans; Ligands; Models, Molecular; Protein Conformation; Structural Homology, Protein; Structure-Activity Relationship; Tubercidin; User-Computer Interface

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 muM) raised extracellular adenosine, as measured with adenosine biosensors, and potently inhibited field excitatory post-synaptic potentials (fEPSPs) in an adenosine A(1)R-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(1)R 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.

Topics: Adenosine; Adenosine Kinase; Animals; Animals, Newborn; Astrocytes; Electric Stimulation; Enzyme Inhibitors; Excitatory Postsynaptic Potentials; Glial Fibrillary Acidic Protein; Hippocampus; In Vitro Techniques; Rats; Rats, Sprague-Dawley; Seizures; Synapses; Theophylline; Time Factors; Tubercidin

Erectile dysfunction (ED) in diabetes is associated with autonomic neuropathy and endothelial dysfunction. Whereas the nonadrenergic-noncholinergic (NANC)/neurogenic nitric oxide pathway has received great attention in diabetes-associated ED, few studies have addressed sympathetic overactivity.. To test the hypothesis that adenosine-induced inhibition of adrenergic-mediated contractile responses in mouse corpus cavernosum is impaired in the presence of diabetes.. The db/db (obesity and type II diabetes caused by a leptin receptor mutation) mouse strain was used as a model of obesity and type II diabetes, and standard procedures were performed to evaluate functional cavernosal responses.. Increased cavernosal responses to sympathetic stimulation in db/db mice are not associated with impaired prejunctional actions of adenosine.. Electrical field stimulation (EFS)-, but not phenylephrine (PE)-, induced contractions are enhanced in cavernosal strips from db/db mice in comparison with those from lean littermates. Direct effects of adenosine, 2-chloro-adenosine, A(1) receptor agonist C-8031 (N6 cyclopentyladenosine), and sodium nitroprusside are similar between the strips from lean and db/db mice, whereas relaxant responses to acetylcholine and NANC stimulation are significantly impaired in the cavernosal strips from db/db mice. 5'-Iodotubercidin (adenosine kinase inhibitor) and dipyridamole (inhibitor of adenosine transport), as well as the A(1) agonist C-8031, significantly and similarly inhibit contractions induced by stimulation of adrenergic nerves in the cavernosal strips from lean and db/db mice.. Results from this study suggest that corpora cavernosa from obese and diabetic db/db mice display altered neural-mediated responses that would favor penile detumescence, i.e., increased contractile response to adrenergic nerve stimulation and decreased relaxant responses upon activation of NANC nerves. However, increased cavernosal responses to adrenergic nerve stimulation are not due to impaired negative modulation of sympathetic neurotransmission by adenosine in this diabetic model.

Topics: Acetylcholine; Adenosine; Adenosine Kinase; Animals; Diabetes Mellitus, Type 2; Dipyridamole; Disease Models, Animal; Electric Stimulation; Enzyme Inhibitors; Male; Mice; Mice, Mutant Strains; Mice, Obese; Muscle Contraction; Muscle, Smooth; Nitroprusside; Penis; Phenylephrine; Phosphodiesterase Inhibitors; Tubercidin; Vasoconstrictor Agents; Vasodilator Agents

Adenosine is an endogenous neuromodulator with anticonvulsive and neuroprotective activity. Adenosine levels are normally kept in the range of 20 to 200 nmol/L by low basal expression of its main metabolic enzyme, adenosine kinase (ADK). Dysfunction of the adenosinergic system has been demonstrated to contribute to epileptogenesis. To investigate whether upregulation of ADK may render the brain more susceptible to ischemic cell death, mutant mice overexpressing an Adk transgene in brain were subjected to middle cerebral artery occlusion (MCAO). One day after either 15 or 60 mins of MCAO, wild-type (WT) animals had infarct areas encompassing about 5% and 50% of their ischemic hemisphere, respectively. In marked contrast, the volume of the infarcts increased three-fold in Adk transgenic mutants after 15 mins of MCAO, and after 60 mins of MCAO all mutants died within 24 h. Pretreatment of the mutants with the ADK inhibitor 5-iodotubercidin led to lesions similar to those in WT mice. Thus, low levels of ADK are essential to maintain adenosine-mediated neuroprotection. We conclude that pathologic overexpression of ADK as in epilepsy may also render the brain more susceptible to injury from ischemia. Consequently, ADK emerges as a rational therapeutic target to enhance neuroprotection.

Topics: Adenosine; Adenosine Kinase; Animals; Brain Ischemia; Cell Death; Enzyme Inhibitors; Excitatory Postsynaptic Potentials; Functional Laterality; Hippocampus; Infarction, Middle Cerebral Artery; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neuroprotective Agents; Promoter Regions, Genetic; Pyramidal Cells; Receptor, Adenosine A1; Tubercidin; Ubiquitin

To investigate the effect of adenosine kinase inhibition on interleukin (IL)-1beta- and lipopolysaccharide (LPS)-induced cartilage damage.. Articular cartilage was obtained from the metacarpophalangeal joints of 10 young adult horses. Following a stabilization period, weighed cartilage explants were exposed to IL-1beta (10 ng/ml) or LPS (50 microg/ml) to induce cartilage degradation. To test the potential protective effects of adenosine, these explants were simultaneously exposed to adenosine (100 microM), the adenosine kinase inhibitor 5'iodotubercidin (ITU, 1 microM) or to both adenosine and ITU. After 72 h in culture, conditioned medium was collected for evaluation of glycosaminoglycan (GAG), nitric oxide (NO), prostaglandin E2 (PGE2) and matrix metalloproteinase (MMP)-3 release.. IL-1beta and LPS stimulated significant release of GAG, NO, PGE2 and MMP-3. Incubation with ITU significantly inhibited both IL-1beta- and LPS-induced GAG release, but did not alter MMP-3 production. Exposure to ITU also reduced IL-1beta-induced PGE2 release and LPS-induced NO production. Direct adenosine supplementation did not attenuate the effects of IL-1beta or LPS, and the addition of adenosine or ITU in the absence of IL-1beta or LPS did not have any detectable effect on cartilage metabolism in this model.. The adenosine kinase inhibitor ITU attenuated experimentally induced cartilage damage in an in vitro cartilage explant model. Release of adenosine from chondrocytes may play a role in the cellular response to tissue damage in arthritic conditions and modulation of these pathways in the joint may have potential for treatment of arthropathies.

Topics: Adenosine; Adenosine Kinase; Animals; Cartilage, Articular; Culture Media, Conditioned; Dinoprostone; Enzyme Inhibitors; Glycosaminoglycans; Horses; Interleukin-1; Lipopolysaccharides; Matrix Metalloproteinase 3; Nitric Oxide; Tissue Culture Techniques; Tubercidin

Adenosine kinase (ADK) is considered to be the key regulator of the brain's endogenous anticonvulsant, adenosine. In adult brain, ADK is primarily expressed in a subpopulation of astrocytes and striking upregulation of ADK in these cells has been associated with astrogliosis after kainic acid-induced status epilepticus (KASE) in the kainic acid mouse model of temporal lobe epilepsy. To investigate the causal relationship between KASE-induced astrogliosis, upregulation of ADK and seizure activity, we have developed a novel mouse model [the Adktm1(-/-)-Tg(UbiAdk) mouse] lacking the endogenous astrocytic enzyme due to a targeted disruption of the endogenous gene, but containing an Adk transgene under the control of a human ubiquitin promoter. Mutant Adktm1(-/-)-Tg(UbiAdk) mice were characterized by increased brain ADK activity and constitutive overexpression of transgenic ADK throughout the brain, with particularly high levels in hippocampal pyramidal neurons. This ADK overexpression was associated with increased baseline levels of locomotion. Most importantly, two-thirds of the mutant mice analysed exhibited spontaneous seizure activity in the hippocampus and cortex. This was the direct consequence of transgene expression, since this seizure activity could be prevented by systemic application of the ADK inhibitor 5-iodotubercidin. Intrahippocampal injection of kainate in the mutant mice resulted in astrogliosis to the same extent as that observed in wild-type mice despite the absence of endogenous astrocytic ADK. Therefore, KASE-induced upregulation of endogenous ADK in wild-type mice is a consequence of astrogliosis. However, seizures in kainic acid-injected mutants displayed increased intra-ictal spike frequency compared with wild-type mice, indicating that, once epilepsy is established, increased levels of ADK aggravate seizure severity. We therefore conclude that therapeutic strategies that augment the adenosine system after astrogliosis-induced upregulation of ADK constitute a neurochemical rationale for the prevention of seizures in epilepsy.

Topics: Adenosine Kinase; Animals; Astrocytes; Behavior, Animal; Brain; Cerebral Cortex; Disease Models, Animal; Electroencephalography; Enzyme Inhibitors; Epilepsy, Temporal Lobe; Gliosis; Hippocampus; Kainic Acid; Locomotion; Male; Mice; Mice, Transgenic; Neurons; Transgenes; Tubercidin; Up-Regulation

Endogenous adenosine in the brain is thought to prevent the development and spread of seizures via a tonic anticonvulsant effect. Brain levels of adenosine are primarily regulated by the activity of adenosine kinase. To establish a link between adenosine kinase expression and seizure activity, we analyzed the expression of adenosine kinase in the brain of control mice and in a kainic acid-induced mouse model of mesial temporal lobe epilepsy. Immunohistochemical analysis of brain sections of control mice revealed intense staining for adenosine kinase, mainly in astrocytes, which were more or less evenly distributed throughout the brain, as well as in some neurons, particularly in olfactory bulb, striatum, and brainstem. In contrast, hippocampi lesioned by a unilateral kainic acid injection displayed profound astrogliosis and therefore a significant increase in adenosine kinase immunoreactivity accompanied by a corresponding increase of enzyme activity, which paralleled chronic recurrent seizure activity in this brain region. Accordingly, seizures and interictal spikes were suppressed by the injection of a low dose of the adenosine kinase inhibitor 5-iodotubercidin. We conclude that overexpression of adenosine kinase in discrete parts of the epileptic hippocampus may contribute to the development and progression of seizure activity.

Topics: Action Potentials; Adenosine A1 Receptor Antagonists; Adenosine Kinase; Animals; Anticonvulsants; Astrocytes; Brain; Disease Models, Animal; Disease Progression; Electroencephalography; Enzyme Inhibitors; Epilepsy, Temporal Lobe; Glial Fibrillary Acidic Protein; Hippocampus; Immunohistochemistry; Kainic Acid; Mice; Neurons; Tubercidin; Xanthines

1. Adenosine kinase (AK) inhibitors can enhance adenosine levels and potentiate adenosine receptor activation. As the AK inhibitors 5' iodotubercidin (ITU) and 5-amino-5'-deoxyadenosine (NH(2)dAdo) are nucleoside analogues, we hypothesized that nucleoside transporter subtype expression can affect the potency of these inhibitors in intact cells. 3. Three nucleoside transporter subtypes that mediate adenosine permeation of rat cells have been characterized and cloned: equilibrative transporters rENT1 and rENT2 and concentrative transporter rCNT2. We stably transfected rat C6 glioma cells, which express rENT2 nucleoside transporters, with rENT1 (rENT1-C6 cells) or rCNT2 (rCNT2-C6 cells) nucleoside transporters. 3. We tested the effects of ITU and NH(2)dAdo on [(3)H]-adenosine uptake and conversion to [(3)H]-adenine nucleotides in the three cell types. NH(2)dAdo did not show any cell type selectivity. In contrast, ITU showed significant inhibition of [(3)H]-adenosine uptake and [(3)H]-adenine nucleotide formation at concentrations < or =100 nM in rENT1-C6 cells, while concentrations > or =3 microM were required for C6 or rCNT2-C6 cells. 4. Nitrobenzylthioinosine (NBMPR; 100 nM), a selective inhibitor of rENT1, abolished the effects of nanomolar concentrations of ITU in rENT1-C6 cells. 5. This study demonstrates that the effects of ITU, but not NH(2)dAdo, in whole cell assays are dependent upon nucleoside transporter subtype expression. Thus, cellular and tissue differences in expression of nucleoside transporter subtypes may affect the pharmacological actions of some AK inhibitors.

Topics: Adenine Nucleotides; Adenosine; Adenosine Kinase; Animals; Carrier Proteins; Deoxyadenosines; Dose-Response Relationship, Drug; Enzyme Inhibitors; Equilibrative Nucleoside Transport Proteins; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Gene Expression; Membrane Proteins; Membrane Transport Proteins; Nucleoside Transport Proteins; Thioinosine; Tritium; Tubercidin; Tumor Cells, Cultured

Adenosine inhibits growth of vascular smooth muscle cells. The goals of this study were to determine which adenosine receptor subtype mediates the antimitogenic effects of adenosine and to investigate the signal transduction mechanisms involved. In rat aortic vascular smooth muscle cells, platelet-derived growth factor-BB (PDGF-BB) (25 ng/mL) stimulated DNA synthesis ([(3)H]thymidine incorporation), cellular proliferation (cell number), collagen synthesis ([(3)H]proline incorporation), total protein synthesis ([(3)H]leucine incorporation), and mitogen-activated protein (MAP) kinase activity. The adenosine receptor agonists 2-chloroadenosine and 5'-N-methylcarboxamidoadenosine, but not N(6)-cyclopentyladenosine or CGS21680, inhibited the growth effects of PDGF-BB, an agonist profile consistent with an A(2B) receptor-mediated effect. The adenosine receptor antagonists KF17837 and 1,3-dipropyl-8-p-sulfophenylxanthine, but not 8-cyclopentyl-1, 3-dipropylxanthine, blocked the growth-inhibitory effects of 2-chloroadenosine and 5'-N-methylcarboxamidoadenosine, an antagonist profile consistent with an A(2) receptor-mediated effect. Antisense, but not sense or scrambled, oligonucleotides to the A(2B) receptor stimulated basal and PDGF-induced DNA synthesis, cell proliferation, and MAP kinase activity. Moreover, the growth-inhibitory effects of 2-chloroadenosine, 5'-N-methylcarboxamidoadenosine, and erythro-9-(2-hydroxy-3-nonyl) adenine plus iodotubericidin (inhibitors of adenosine deaminase and adenosine kinase, respectively) were abolished by antisense, but not scrambled or sense, oligonucleotides to the A(2B) receptor. Our findings strongly support the hypothesis that adenosine causes inhibition of vascular smooth muscle cell growth by activating A(2B) receptors coupled to inhibition of MAP kinase activity. Pharmacological or molecular biological activation of A(2B) receptors may prevent vascular remodeling associated with hypertension, atherosclerosis, and restenosis following balloon angioplasty.

Topics: 2-Chloroadenosine; Adenine; Adenosine; Adenosine Kinase; Adenosine-5'-(N-ethylcarboxamide); Animals; Anticoagulants; Antihypertensive Agents; Aorta, Abdominal; Becaplermin; Cell Division; Dinucleoside Phosphates; Enzyme Inhibitors; Hyperplasia; Male; MAP Kinase Signaling System; Muscle, Smooth, Vascular; Oligonucleotides, Antisense; Phenethylamines; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, beta-2; Thymidine; Tritium; Tubercidin; Vasodilator Agents; Xanthines

We recently suggested that, in muscular dystrophies, the excessive accumulation of adenosine as a result of an altered purine metabolism may contribute to progressive functional deterioration and muscle cell death. To verify this hypothesis, we have taken advantage of C2C12 myoblastic cells, which can be differentiated in vitro into multinucleated cells (myotubes). Exposure of both proliferating myoblasts and differentiated myotubes to adenosine or its metabolically-stable analog, 2-chloro-adenosine, resulted in apoptotic cell death and myotube disruption. Cytotoxicity by either nucleoside did not depend upon extracellular adenosine receptors, but, at least in part, by entry into cells via the membrane nitro-benzyl-thio-inosine-sensitive transporter. The adenosine kinase inhibitor, 5-iodotubercidin, prevented 2-chloro-adenosine-induced (but not adenosine-induced) effects, suggesting that an intracellular phosphorylation/activation reaction plays a key role in 2-chloro-adenosine-mediated cytotoxicity. Conversely, adenosine cytotoxicity was aggravated by the addition of homocysteine, suggesting that adenosine effects may be due to the accumulation of S-adenosyl-homocysteine, which blocks intracellular methylation-dependent reactions. Both nucleosides markedly disrupted the myotube structure via an effect on the actin cytoskeleton; however, also for myotubes, there were marked differences in the morphological alterations induced by these two nucleosides. These results show that adenosine and 2-chloro-adenosine induce apoptosis of myogenic cells via completely different metabolic pathways, and are consistent with the hypothesis that adenosine accumulation in dystrophic muscles may represent a novel pathogenetic pathway in muscle diseases.

Topics: 2-Chloroadenosine; Acetylcysteine; Actin Cytoskeleton; Adenosine; Adenosine Kinase; Animals; Apoptosis; Cell Adhesion; Cell Line; Cytoskeleton; Dose-Response Relationship, Drug; Enzyme Inhibitors; Free Radical Scavengers; Homocysteine; Intracellular Fluid; Mice; Microscopy, Electron, Scanning; Muscle, Skeletal; Purinergic P1 Receptor Antagonists; Reactive Oxygen Species; Thioinosine; Tubercidin

The effect of adenosine kinase (AKA), adenosine deaminase (ADA) and transport inhibitors on the release of dopamine (DA) induced by methamphetamine (MTH) in rat striatum was assessed using in vivo microdialysis in freely moving rats. MTH injected in a dose of 3 x 5 mg/kg i.p. at 2-hour intervals produced a massive release of DA. This excessive release of DA was inhibited by the ADA inhibitor 2'-deoxycoformycin (DCF), the AKA inhibitor 5'-iodotubercidin (IOT) and the adenosine uptake inhibitor dilazep (DIL), each of them given locally to the striatum via a microdialysis probe at a concentration of 100 microM. Perfusion with the same concentrations of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and 5'-amino-5'-deoxyadenosine (NH(2)dAD), ADA and AKA inhibitors, respectively, induced a considerably weaker effect on DA release. The non-selective antagonist of adenosine A(1)/A(2A) receptor caffeine (75 microM) significantly prevented the inhibitory effect of DCF, IOT and DIL on the MTH-induced DA release. Intrastriatal administration of DCF, IOT and DIL (5 nmol/microl before each injection of MTH) inhibited the stereotypy induced by MTH. The striatal content of DA and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), decreased by MTH administration and measured 5 days after treatment with the toxin, was reversed by all the inhibitors at the order of potency as follows: IOT>DCF>DIL. Direct agonists of adenosine A(1) and A(1)/A(2A) receptors, N(6)-cyclopentyladenosine (CPA) and 5'-N-ethylcarboxamidoadenosine (NECA), respectively, given intrastriatally (5 nmol/microl) completely abolished the MTH-induced stereotypy and the fall in the striatal content of DA, DOPAC and HVA. The above results show that augmentation of endogenous adenosine in rat striatum by inhibition of its metabolism or uptake-despite the differences in the efficacy of various inhibitors-may provide neuroprotection against a toxic action of MTH.

Topics: Adenosine Deaminase; Adenosine Kinase; Animals; Corpus Striatum; Dilazep; Dopamine; Dopamine Uptake Inhibitors; Enzyme Inhibitors; Male; Methamphetamine; Pentostatin; Purinergic P1 Receptor Agonists; Rats; Rats, Wistar; Tubercidin

Topics: Adenine; Adenosine; Adenosine Deaminase Inhibitors; Adenosine Kinase; Adenosine Triphosphate; Cells, Cultured; Coronary Vessels; Endothelium, Vascular; Enzyme Inhibitors; Humans; Nucleotides; Pentostatin; Phosphates; Ribose; Tubercidin

Topics: 5'-Nucleotidase; Adenosine Kinase; Animals; Anti-Bacterial Agents; Antineoplastic Agents; Cell Survival; Chlorocebus aethiops; COS Cells; Enzyme Inhibitors; Humans; Kinetics; Purine Nucleosides; Ribonucleosides; Tubercidin

Inhibitors of adenosine membrane transport cause vasodilation and enhance the plasma adenosine concentration. However, it is unclear why the plasma adenosine concentration rises rather than falls when membrane transport is inhibited. We tested the hypothesis that the cytosolic adenosine concentration exceeds the interstitial concentration under well-oxygenated conditions.. In isolated, isovolumically working guinea pig hearts (n=50), the release rate of adenosine and accumulation of S-adenosylhomocysteine (after 20 minutes of 200 micromol/L homocysteine), a measure of the free cytosolic adenosine concentration, were determined in the absence and presence of specific and powerful blockers of adenosine membrane transport (nitrobenzylthioinosine 1 micromol/L), adenosine deaminase (erythro-9-hydroxy-nonyl-adenine 5 micromol/L), and adenosine kinase (iodotubericidine 10 micromol/L). Data analysis with a distributed multicompartment model revealed a total cardiac adenosine production rate of 2294 pmol. min-1. g-1, of which 8% was produced in the extracellular region. Because of a high rate of intracellular metabolism, however, 70.3% of extracellularly produced adenosine was taken up into cellular regions, an effect that was effectively eliminated by membrane transport block. The resulting approximately 2.8-fold increase of the interstitial adenosine concentration evoked near-maximal coronary dilation.. We rejected the hypothesis that the cytosolic adenosine concentration exceeds the interstitial. Rather, there is significant extracellular production, and the parenchymal cell represents a sink, not a source, for adenosine under well-oxygenated conditions.

Topics: Adenine; Adenosine; Adenosine Deaminase Inhibitors; Adenosine Kinase; Animals; Biological Transport; Bradykinin; Coronary Circulation; Cytosol; Depression, Chemical; Dipyridamole; Drug Synergism; Enzyme Inhibitors; Extracellular Space; Guinea Pigs; Heart; Models, Biological; Myocardium; Osmolar Concentration; Oxygen; Oxygen Consumption; Piperazines; Thioinosine; Tubercidin

Adenosine levels increase at seizure foci as part of a postulated endogenous negative feedback mechanism that controls seizure activity through activation of A1 adenosine receptors. Agents that amplify this site- and event-specific surge of adenosine could provide antiseizure activity similar to that of adenosine receptor agonists but with fewer dose-limiting side effects. Inhibitors of adenosine kinase (AK) were examined because AK is normally the primary route of adenosine metabolism. The AK inhibitors 5'-amino-5'-deoxyadenosine, 5-iodotubercidin, and 5'-deoxy-5-iodotubercidin inhibited maximal electroshock (MES) seizures in rats. Several structural classes of novel AK inhibitors were identified and shown to exhibit similar activity, including a prototype inhibitor, 4-(N-phenylamino)-5-phenyl-7-(5'-deoxyribofuranosyl)pyrrolo[2, 3-d]pyrimidine (GP683; MES ED50 = 1.1 mg/kg). AK inhibitors also reduced epileptiform discharges induced by removal of Mg2+ in a rat neocortical preparation. Overall, inhibitors of adenosine deaminase or of adenosine transport were less effective. The antiseizure activities of GP683 in the in vivo and in vitro preparations were reversed by the adenosine receptor antagonists theophylline and 8-(p-sulfophenyl)theophylline. GP683 showed little or no hypotension or bradycardia and minimal hypothermic effect at anticonvulsant doses. This improved side effect profile contrasts markedly with the profound hypotension, bradycardia, and hypothermia and greater inhibition of motor function observed with the adenosine receptor agonist N6-cyclopentyladenosine and opens the way to clinical evaluation of AK inhibitors as a novel, adenosine-based approach to anticonvulsant therapy.

Topics: Adenosine; Adenosine Kinase; Animals; Anticonvulsants; Cattle; Cells, Cultured; Deoxyadenosines; Electroshock; Endothelium, Vascular; Enzyme Inhibitors; Male; Microcirculation; Motor Activity; Neocortex; Pyrimidines; Radioligand Assay; Rats; Rats, Inbred Strains; Recombinant Proteins; Seizures; Structure-Activity Relationship; Tubercidin

The objective of the present study was to establish the optimal combination of inhibitors of adenosine metabolism and nucleotide precursors resulting in long-term increase in endogenous adenosine concentration without adverse metabolic consequences in non-ischemic cardiomyocytes and endothelial cells. Cardiomyocytes and endothelial cells were isolated after collagenase digestion of the rat heart. Freshly isolated cardiac myocytes or cultured endothelial cells were incubated for up to 8 h with no inhibitors or substrates or with various combinations of adenosine deaminase inhibitor: 5 micron M erythro-9(2-hydroxy-3-nonyl)adenine (EHNA), adenosine kinase inhibitors: 10 micro M 5'-iodotubercidin (ITu) or 10 micro M 5'-aminoadenosine (AA) and nucleotide precursors: 100 micro M adenine, 2.5 mm ribose and 5 mm inorganic phosphate. Nucleotide, nucleoside and base concentrations were evaluated at the end of the incubation by HPLC in cardiomyocyte or endothelial cells extracts and in incubation media. Adenosine content in cardiomyocyte suspension was enhanced after 3 h incubation in the presence of ITu+EHNA as compared to EHNA alone (2.8+/-0.2 v 0.9+/-0.2 nmol/mg protein, respectively). ATP decreased from an initial value of 22.7+/-0.7 nmol/mg protein to 18.9+/-0.7 in the presence of ITu+EHNA, while ATP was maintained at 21.8+/-0.7 nmol/mg protein with EHNA. With adenine+ITu+EHNA, the changes were similar to those observed with ITu+EHNA. However, with ribose+adenine+ITu+EHNA, ATP increased to 25. 8+/-1.2 nmol/mg protein and adenosine concentration was elevated to 3.9+/-0.3 nmol/mg protein. Similar results were observed if AA was used instead of ITu to inhibit adenosine kinase. All the changes were maintained after 8 h of incubation. Adenosine content was increased in endothelial cells incubated with ITu+EHNA to 3.1+/-0.4 nmol/mg protein as compared to 1.1+/-0.2 nmol/mg protein with EHNA alone after 3 h, while ATP decreased (18.1+/-1.1 v 22.0+/-1.4 nmol/mg protein with EHNA+ITu or EHNA, respectively). In the presence of adenine+ITu+EHNA, adenosine content increased after 3 h to 6.5+/-0.9 nmol/mg protein while ATP was elevated to 26.1+/-0.8 nmol/mg protein. Additional presence of ribose was without effect. No changes in adenylate energy charge were observed in cardiomyocytes or endothelium under any conditions studied. Inhibition of adenosine kinase and adenosine deaminase caused a decrease in ATP together with increased adenosine content both in endothelial cells a

Topics: Adenine; Adenosine; Adenosine Kinase; Adenosine Triphosphate; Animals; Cells, Cultured; Endothelium, Vascular; Enzyme Inhibitors; Heart; In Vitro Techniques; Kinetics; Male; Myocardium; Purines; Rats; Rats, Sprague-Dawley; Ribose; Tubercidin; Uridine

The purpose of this study was: 1) to evaluate a dual microdialysis technique coupled with local drug administration in the regionally ischemic rabbit heart, and; 2) to assess the ischemia-induced changes in interstitial fluid (ISF) adenosine during inhibition of adenosine deaminase or adenosine kinase.. Two microdialysis probes were implanted parallel to each other and separated by 5 mm in myocardium perfused by a branch of the left coronary artery. Probes were used to sample myocardial ISF and to deliver drugs locally to the myocardium; purine metabolite concentrations in the collected dialysate were used as indices of ISF levels. Three groups of pentobarbital-anesthetized rabbits were studied. In a control group (n = 6), both probes were perfused with Krebs-Henseleit buffer. In the second and third groups, one probe was perfused with buffer, whereas the other probe was perfused with buffer containing 1 mM erythro-2-(2-hydroxy-3-nonyl)adenine (EHNA) (n = 5), an adenosine deaminase inhibitor, or 10 microM iodotubercidin (n = 9), an adenosine kinase inhibitor. All animals were exposed to 30 min of regional myocardial ischemia followed by 60 min of reperfusion.. In the control group, similar increases in dialysate purine metabolites during ischemia were observed in both probes. Locally administered EHNA increased dialysate adenosine prior to ischemia and decreased dialysate inosine and hypoxanthine. During ischemia, the increase in dialysate adenosine in the EHNA-perfused probe was markedly augmented, while the increases in inosine and hypoxanthine were attenuated. In contrast, local infusion of iodotubercidin did not alter dialysate purine metabolites before ischemia, but there was a modest augmentation of adenosine during ischemia. These data illustrate the feasibility of dual microdialysis for assessing the effect of locally administered compounds on interstitial metabolite concentration in the regionally ischemic rabbit heart. Furthermore, adenosine deaminase inhibition has a more profound adenosine augmenting effect than adenosine kinase inhibition in the rabbit heart.

Topics: Adenine; Adenosine; Adenosine Deaminase Inhibitors; Adenosine Kinase; Animals; Chromatography, High Pressure Liquid; Extracellular Space; Hypoxanthine; Inosine; Male; Microdialysis; Myocardial Ischemia; Myocardial Reperfusion; Myocardium; Rabbits; Tubercidin

Activation of glutamate receptors triggers the release of adenosine, which exerts important inhibitory actions in the brain. Evoked adenosine release is potentiated when either adenosine kinase or adenosine deaminase are inhibited. We studied the effects of concurrent inhibition of adenosine kinase and adenosine deaminase on N-methyl-D-aspartate (NMDA)-evoked formation of extracellular adenosine in slices of rat parietal cortex, to determine if combinations of inhibitors of adenosine kinase and adenosine deaminase can produce supra-additive potentiation of this adenosine formation. Combinations of low concentrations of the adenosine kinase inhibitors 5'-amino-5'-deoxyadenosine (0.2 microM) or 5'-iodotubercidin (0.01 microM) with a low concentration of the adenosine deaminase inhibitor 2'-deoxycoformycin (0.2 microM) produced additive potentiations of NMDA-evoked adenosine release from slices of rat parietal cortex. However, combinations of low concentrations of 5'-amino-5'-deoxyadenosine (0.2 microM) or 5'-iodotubercidin (0.01 microM) with a maximal concentration of 2'-deoxycoformycin (200 microM) produced supra-additive potentiation of NMDA-evoked adenosine release. These findings suggest that such combinations of adenosine kinase inhibitors with adenosine deaminase inhibitors may provide useful strategies for developing therapies to treat disorders associated with excessive NMDA receptor activation, such as seizures, ischemic damage and neurodegenerative diseases.

Topics: Adenosine; Adenosine Deaminase Inhibitors; Adenosine Kinase; Animals; Cerebral Cortex; Deoxyadenosines; Drug Synergism; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Male; N-Methylaspartate; Pentostatin; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Tubercidin

Autophagy, measured in isolated rat hepatocytes as the sequestration of electroinjected [3H]raffinose, was moderately (17%) inhibited by adenosine (0.4 mM) alone, but more strongly (85%) in the presence of the adenosine deaminase inhibitor, 2'-deoxycoformycin (50 microM), suggesting that metabolic deamination of adenosine limited its inhibitory effectiveness. The adenosine analogs, 6-methylmercaptopurine riboside and N6,N6-dimethyladenosine, inhibited autophagy by 89% and 99%, respectively, at 0.5 mM, probably reflecting the adenosine deaminase-resistance of their 6-substitutions. 5-Iodotubercidin (10 microM), an adenosine kinase inhibitor, blocked the conversion of adenosine to AMP and largely abolished the inhibitory effects of both adenosine and its analogs, indicating that AMP/nucleotide formation was required for inhibition of autophagy. Inhibition by adenosine of autophagic protein degradation, measured as the release of [14C]valine from prelabelled protein, was similarly potentiated by deoxycoformycin and prevented by iodotubercidin. Inhibition of autophagy by added AMP, ADP or ATP (0.3-1 mM) was, likewise, potentiated by deoxycoformycin and prevented by iodotubercidin, suggesting dephosphorylation to adenosine and intracellular re-phosphorylation to AMP. Suppression of autophagy by AMP may be regarded as a feedback inhibition of autophagic RNA degradation, or as an aspect of the general down-regulation of energy-requiring processes that occurs under conditions of ATP depletion, when AMP levels are high.

Topics: Adenosine; Adenosine Deaminase Inhibitors; Adenosine Kinase; Adenosine Monophosphate; Animals; Autophagy; Cells, Cultured; Enzyme Inhibitors; Liver; Male; Pentostatin; Phosphorylation; Rats; Rats, Wistar; Tubercidin

Because proliferation of cardiac fibroblasts participates in cardiac hypertrophy/remodeling associated with hypertension and myocardial infarction, it is important to elucidate factors regulating cardiac fibroblast proliferation. Adenosine, a nucleoside abundantly produced by cardiac cells, is antimitogenic vis-à-vis vascular smooth muscle cells; however, the effect of adenosine on cardiac fibroblast proliferation is unknown. The objective of this study was to characterize the effects of exogenous and endogenous (cardiac fibroblast-derived) adenosine on cardiac fibroblast proliferation.. Growth-arrested cardiac fibroblasts were stimulated with 2.5% FCS in the presence and absence of adenosine, 2-chloroadenosine (stable adenosine analogue), or modulators of adenosine levels, including (1) erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA; adenosine deaminase inhibitor); (2) dipyridamole (adenosine transport blocker); and (3) iodotubericidin (adenosine kinase inhibitor). All of these agents inhibited, in a concentration-dependent manner, FCS-induced cardiac fibroblast proliferation as assessed by DNA synthesis ([3H]thymidine incorporation) and cell counting. EHNA, dipyridamole, and iodotubericidin increased extracellular levels of adenosine by 2.3- to 5.6-fold when added separately to cardiac fibroblasts, and EHNA+iodotubericidin or EHNA+iodotubericidin+dipyridamole increased extracellular adenosine levels by >690-fold. Both KF17837 (selective A2 antagonist) and DPSPX (nonselective A2 antagonist) but not DPCPX (selective A1 antagonist) blocked the antimitogenic effects of 2-chloroadenosine, EHNA, and dipyridamole on DNA synthesis, suggesting the involvement of A2A and/or A2B but excluding the participation of A1 receptors. The lack of effect of CGS21680 (selective A2A agonist) excluded involvement of A2A receptors and suggested a major role for A2B receptors. This conclusion was confirmed by the rank order potencies of four adenosine analogues.. Cardiac fibroblasts synthesize adenosine, and exogenous and cardiac fibroblast-derived adenosine inhibits cardiac fibroblast proliferation via activation of A2B receptors. Cardiac fibroblast-derived adenosine may regulate cardiac hypertrophy and/or remodeling by modulating cardiac fibroblast proliferation.

Topics: Adenine; Adenosine; Adenosine Deaminase Inhibitors; Adenosine Kinase; Adenosine-5'-(N-ethylcarboxamide); Adrenergic alpha-2 Receptor Agonists; Adrenergic alpha-2 Receptor Antagonists; Animals; Cattle; Cell Division; Culture Media; Dipyridamole; DNA Replication; Enzyme Inhibitors; Fibroblasts; Heart; Male; Myocardium; Phenethylamines; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, alpha-2; Tubercidin; Xanthines

Iodotubercidin is an adenosine kinase inhibitor that through its ability to increase levels of endogenous adenosine can enhance adenosine's receptor-mediated effects. We investigated whether iodotubercidin can inhibit [3H]adenosine accumulation by inhibiting transport processes in addition to inhibition of intracellular trapping of labeled adenine nucleotides. Under conditions in which extensive metabolism of intracellular adenosine was present, [3H]adenosine accumulation by DDT1 MF-2 cells was almost completely inhibited by iodotubercidin and the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)-adenine or by the nucleoside transport inhibitor nitrobenzylthioinosine. By using similar conditions, [3H]adenosine accumulation was significantly greater in Na+ buffer than in buffer containing N-methyl-D-glucamine in place of Na+; however, this effect may be explained by an observed 40% inhibition of adenosine kinase activity by N-methyl-D-glucamine. By using uptake intervals of 14 sec to represent the transport component of uptake, iodotubercidin decreased the affinity for adenosine, by about 3-fold, but had no effect on maximum velocity of transport. That these effects of iodotubercidin were due to direct interactions with nucleoside transporters was supported by findings that iodotubercidin inhibited [3H]nitrobenzylthioinosine binding to nucleoside transporters with a Ki value of 4 microM and inhibited [3H]uridine and [3H]formycin B uptake with IC50 values of 7 and 15 microM, respectively. These data suggest that iodotubercidin, at pharmacologically relevant concentrations, inhibits nucleoside transport independently of its well characterized inhibition of adenosine kinase and that N-methyl-D-glucamine must be used with caution in experiments to determine the possible presence of Na+ gradient-dependent concentrative nucleoside transporters.

Topics: Adenosine Kinase; Animals; Cricetinae; Dose-Response Relationship, Drug; Kinetics; Muscle, Smooth; Nucleosides; Radioligand Assay; Tubercidin

Adenosine released by cells during ischemia typically serves as a feedback inhibitor of further organ work. However, in ischemic intestine, adenosine appears to act via stimulatory A2b receptors to increase work in the form of chloride ion (Cl-) secretion. This unusual response may contribute to luminal fluid sequestration in intestinal ischemia. In nonischemic cells feed-forward activation of Cl- secretion does not occur despite the fact that adenosine may be continuously generated during normal cell metabolism. Thus we postulated that intestinal epithelia normally control the disposition of adenosine to prevent inappropriate activation of secretion.. Model T84 intestinal epithelia were studied by means of electrophysiologic and isotopic techniques.. Dipyridamole and nitrobenzylthioinosine (inhibitors of nucleoside transport) and iodotubercidin (an inhibitor of adenosine kinase) caused adenosine to accumulate extracellularly and induced a Cl- secretory response that was prevented by adenosine receptor blockade. Uptake of exogenous adenosine was restricted to the basolateral compartment and was blocked by nucleoside transport inhibitors.. Adenosine released from nonischemic intestinal epithelial cells is scavenged by a basolaterally restricted adenosine transporter. This system maintains extracellular adenosine levels below the prosecretory threshold and thus limits adenosine-elicited activation of Cl- secretion (and hence diarrhea) under normal conditions).

Topics: Adenosine; Adenosine Kinase; Adenosine Triphosphate; Biological Transport; Buffers; Cells, Cultured; Chlorides; Chromatography, High Pressure Liquid; Cyclic AMP; Dipyridamole; Enzyme Inhibitors; Humans; Intestinal Mucosa; Membrane Proteins; Nucleosides; Phosphodiesterase Inhibitors; Phosphorylation; Tubercidin; Vasodilator Agents

Purified adenosine kinase (AK) from Syrian hamster and bovine liver was examined for the presence of adenosine (Ad)-AMP exchange activity. The enzyme from both sources, in addition to catalyzing the conventional ATP-dependent phosphorylation of adenosine, supported an Ad-AMP exchange reaction that required ADP. Under optimal conditions both these reactions were found to occur at comparable rates. Several observations strongly indicate that the Ad-AMP exchange activity is an integral part of AK and it is likely associated with its catalytic mechanism. These observations include: (i) Both AK and Ad-AMP exchange activities show a nearly complete dependence upon the presence of pentavalent ions such as phosphate, arsenate or vanadate for catalysis; (ii) Both activities show similar heat-lability and inhibition by 5-iodotubercidin (5-ITu); (iii) In a Chinese hamster cell mutant resistant to adenosine analogs that lacked AK activity, the Ad-AMP activity was also found to be absent. The presence of a phosphoryl-enzyme intermediate, or any exchange between free 32Pi and any of the reactants, however, was not detected under the reaction conditions. Some implications of these observations regarding the catalytic mechanism of AK are discussed.

Topics: Adenosine; Adenosine Kinase; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Arsenates; Cattle; CHO Cells; Cricetinae; Enzyme Inhibitors; Enzyme Stability; Kinetics; Liver; Magnesium; Mutation; Phosphates; Temperature; Toyocamycin; Tubercidin

Inhibitors of adenosine kinase, but not adenosine deaminase, produce antinociception when administered spinally. In this study, we evaluated the relative contribution of adenosine kinase and adenosine deaminase to the regulation of adenosine release into the extracellular space within the spinal cord by determining the effects of the adenosine kinase inhibitors 5'-amino-5'-deoxyadenosine and 5-iodotubercidin, and the adenosine deaminase inhibitor 2'-deoxycoformycin on adenosine release from spinal cord slices in an in vitro perfusion system. Both 5'-amino-5'-deoxyadenosine (5-50 microM) and 5-iodotubercidin (5-50 microM), but not 2'-deoxycoformycin (50 microM), augmented adenosine release. 5-Iodotubercidin was slightly more potent and effective than 5'-amino-5'-deoxyadenosine in augmenting release except at the highest concentration, where it was considerably more effective. Combinations of 2'-deoxycoformycin (50 microM) and minimally active concentrations of 5'-amino-5'-deoxyadenosine and 5-iodotubercidin (5 microM each) produced a synergistic enhancement of release. These results support a predominant involvement of adenosine kinase in regulating extracellular adenosine levels in the spinal cord, but adenosine deaminase also can play a significant role.

Topics: Adenosine; Adenosine Deaminase Inhibitors; Adenosine Kinase; Animals; Deoxyadenosines; Dose-Response Relationship, Drug; Enzyme Inhibitors; In Vitro Techniques; Male; Pentostatin; Rats; Rats, Sprague-Dawley; Spinal Cord; Tubercidin

Adenosine kinetics in the coronary circulation were investigated in anesthetized closed-chest dogs by analysis of multiple-indicator dilution experiments. During simultaneous intracoronary bolus injections of 125I-labeled albumin, [14C]sucrose, and [3H]adenosine, dilution curves were measured by automated sampling of coronary venous blood, using high-performance liquid chromatography and isotope detection techniques. Under control conditions, only 1% of the injected [3H]adenosine was detected in coronary venous samples, compared with the reference tracer, [14C]sucrose, and the peak of the adenosine dilution curve preceded those of the reference tracers by 2-4 s. Optimized model fits to the control adenosine curves required the combination of high ratios of the capillary endothelial cell membrane permeability-surface area product to flow (5.3) and endothelial cell consumption capacity to flow (23), in addition to a broad heterogeneity of flow. Dilution curves were measured under control conditions and during increased coronary flow (nitroglycerin), inhibition of the enzyme adenosine kinase (iodotubercidin), and blockade of membrane adenosine transport (dipyridamole). Reliability of the parameter estimates was confirmed using residual analysis, sensitivity function analysis, and Monte Carlo simulation techniques. Accuracy of the model was confirmed in separate experiments in which nontracer adenosine was infused into the coronary artery and the model prediction of coronary venous adenosine concentrations was compared with measured values. Using published measurements of coronary blood flow and arterial and coronary venous plasma adenosine concentrations in open-chest dogs, the estimated in vivo interstitial adenosine concentration is 100-220 nM.

Topics: Adenosine; Adenosine Kinase; Animals; Biological Transport; Capillaries; Computer Simulation; Coronary Circulation; Dipyridamole; Dogs; Endothelium, Vascular; Female; Hemodynamics; In Vitro Techniques; Indicator Dilution Techniques; Kinetics; Male; Models, Cardiovascular; Nitroglycerin; Tubercidin; Veins

Both ischemia and hypoxia increase adenosine production in the heart. This study tested whether hypoxia increases adenosine production in the coronary artery via ecto-5'-nucleotidase and the role of protein kinase C in this condition. Canine left circumflex coronary artery was rapidly removed and incubated in 10 mL Krebs-Henseleit solution for 30 minutes. The Krebs-Henseleit solution contained 5'-iodotubercidin and 2'-deoxycoformycin, which inhibit adenosine kinase and adenosine deaminase, respectively. Adenosine production was measured in intact coronary arteries under normoxic conditions (16.2 +/- 1.2 pmol/mg protein). Adenosine production was reduced by 27% after removal of endothelium. Ecto-5'-nucleotidase activity of coronary arteries with and without endothelium was 51 +/- 6 and 41 +/- 4 nmol/mg protein per minute under normoxic conditions. Hypoxia increased adenosine production to 27.0 +/- 2.3 and 20.0 +/- 0.8 pmol/mg protein with and without endothelium. Hypoxia also increased ecto-5'-nucleotidase activity of coronary arteries with and without endothelium (74 +/- 8 and 53 +/- 5 nmol/mg protein per minute; P < .05). Increases in adenosine production under hypoxic conditions were blunted by both an inhibitor of ecto-5'-nucleotidase and inhibitors of protein kinase C. Activation of ecto-5'-nucleotidase was blunted by an inhibitor of protein kinase C. These results indicate that hypoxia increased extracellular adenosine production and activated ecto-5'-nucleotidase via activation of protein kinase C in coronary arterial smooth muscle and endothelial cells. Increased adenosine production in coronary arteries during hypoxia may contribute to coronary vasodilation and cardioprotection against ischemic injury.

Topics: 5'-Nucleotidase; Adenosine; Adenosine Deaminase Inhibitors; Adenosine Kinase; Animals; Arteries; Coronary Vessels; Dogs; Enzyme Activation; Hypoxia; In Vitro Techniques; Pentostatin; Protein Kinase C; Tubercidin

Extracellular adenosine concentrations, evaluated by microdialysis in the striatum of young and aged rats, were 66.8 +/- 0.7 and 71.6 +/- 1.0 nM, respectively. The adenosine deaminase inhibitor EHNA (100 microM) increased the extracellular adenosine levels in young rats only. The adenosine kinase inhibitor iodotubercidin (10 microM) brought about the same increase in young and aged rats. In aged rats the resting adenosine outflow was reduced by the N-methyl-D-aspartate (NMDA) receptor antagonist D-(-)-2-amino-7-phophonoheptanoic acid (D-AP7) (1 mM). It is concluded that extracellular levels of adenosine in the striatum are not affected by age, irrespective of the differences in adenosine deaminase activity and that the release of excitatory amino acids is responsible for much of resting adenosine outflow in aged but not in young rats.

Topics: 2-Amino-5-phosphonovalerate; Adenine; Adenosine; Adenosine Deaminase Inhibitors; Adenosine Kinase; Aging; Amino Acids; Animals; Corpus Striatum; Enzyme Inhibitors; Extracellular Space; Male; Microdialysis; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Tubercidin

In this study the relative importance of adenosine deaminase and adenosine kinase in regulating extracellular adenosine concentration was investigated in rat hippocampal slices labelled with [3H]-adenine. The release of [3H]-purines evoked by electrical stimulation or energy depletion (oxygen and glucose deprivation) was measured and, using high-performance liquid chromatography (HPLC), the proportion of [3H]-label in the form of [3H]-adenosine, [3H]-inosine and [3H]-hypoxanthine was determined. In addition, endogenous purine release was measured by HPLC with UV detection. 10 microM 5-iodotubericidin (5-IT), an inhibitor of adenosine kinase, significantly increased endogenous adenosine release and altered the pattern of [3H]-purine release by increasing the proportion released as [3H]-adenosine, under basal conditions and after electrical stimulation or energy depletion. 5 microM erythro-9-(2-hydroxy-3-nonyl) adenosine (EHNA), an inhibitor of adenosine deaminase, also increased endogenous adenosine release and altered the pattern of [3H]-purine release evoked by energy depletion by decreasing the proportion of [3H]-label released as [3H]-hypoxanthine and [3H]-inosine, whilst approximately doubling that of [3H]-adenosine. In contrast, adenosine release was not altered by EHNA under basal conditions or electrical stimulation. It is concluded that under conditions which provide adequate oxygen and glucose, adenosine kinase plays a much greater role than adenosine deaminase in regulating the extracellular concentration of adenosine. However, adenosine deaminase becomes important in regulating extracellular adenosine concentration when adenosine formation is increased by energy depletion.

Topics: Adenine; Adenosine; Adenosine Deaminase; Adenosine Deaminase Inhibitors; Adenosine Kinase; Animals; Basal Metabolism; Electric Stimulation; Hippocampus; In Vitro Techniques; Male; Purines; Rats; Tubercidin

Two halogenated analogues of tubercidin (7-deazaadenosine) viz. 5-iodotubercidin and 5'-deoxy-5-iodotubercidin, previously were shown to be potent inhibitors of guinea-pig brain adenosine kinase activity and adenosine uptake in guinea-pig cerebral cortex slices. A further series of halogenated tubercidin analogues have been investigated; of the 9 compounds tested, 5'-deoxy-5-iodotubercidin was the most potent adenosine kinase inhibitor while 5-iodotubercidin was the most potent in inhibiting the facilitated uptake of adenosine. These compounds may be useful for elucidating the involvement of adenosine kinase in adenosine uptake, the maintenance of intracellular adenosine levels and in the neuromodulatory actions of adenosine in the CNS.

Topics: Adenosine; Adenosine Kinase; Animals; Cerebral Cortex; Enzyme Inhibitors; Guinea Pigs; In Vitro Techniques; Tubercidin

Endogenous adenosine in the extracellular space inhibits neuronal activity. The roles of adenosine kinase, S-adenosylhomocysteine-hydrolase and adenosine deaminase activities in the regulation of the adenosine levels were investigated in rat hippocampal slices. Iodotubercidin, an inhibitor of adenosine kinase, added to the perfusion fluid at 5 microM increased the release of adenosine from the slices more than 2-fold. Iodotubercidin treatment caused inhibition of population spike discharges and hyperpolarization of pyramidal cells, mimicking the effects of exogenously applied adenosine. Adenosine dialdehyde, an inhibitor of S-adenosylhomocysteine hydrolase, and erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), an inhibitor of adenosine deaminase had little or no effect on the parameters tested. The action of iodotubercidin was greater during deaminase inhibition. The A1-receptor antagonist DPCPX had actions opposite to those of adenosine and blocked the electrophysiological effects of exogenous adenosine and of iodotubercidin. Thus adenosine kinase activity is a significant factor in the regulation of adenosine levels in the hippocampus.

Topics: Adenosine; Adenosine Kinase; Animals; Hippocampus; In Vitro Techniques; Inosine; Kinetics; Membrane Potentials; Neurons; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Tubercidin; Xanthines

Addition of micromolar concentrations of the adenosine derivative 5-iodotubercidin (Itu) initiates glycogen synthesis in isolated hepatocytes by causing inactivation of phosphorylase and activation of glycogen synthase [Flückiger-Isler and Walter (1993) Biochem. J. 292, 85-91]. We report here that Itu also antagonizes the effects of saturating concentrations of glucagon and vasopressin on these enzymes. The Itu-induced activation of glycogen synthase could not be explained by the removal of phosphorylase a (a potent inhibitor of the glycogen-associated synthase phosphatase). When tested on purified enzymes, Itu did not affect the activities of the major Ser/Thr-specific protein phosphatases (PP-1, PP-2A, PP-2B and PP-2C), but it inhibited various Ser/Thr-specific protein kinases as well as the tyrosine kinase activity of the insulin receptor (IC50 between 0.4 and 28 microM at 10-15 microM ATP). Tubercidin, which did not affect glycogen synthase or phosphorylase in liver cells, was 300 times less potent as a protein kinase inhibitor. Kinetic analysis of the inhibition of casein kinase-1 and protein kinase A showed that Itu acts as a competitive inhibitor with respect to ATP, and as a mixed-type inhibitor with respect to the protein substrate. We propose that Itu inactivates phosphorylase and activates glycogen synthase by inhibiting phosphorylase kinase and various glycogen synthase kinases. Consistent with the broad specificity of Itu in vitro, this compound decreased the phosphorylation level of numerous phosphopolypeptides in intact liver cells. Our data suggest that at least some of the biological effects of Itu can be explained by an inhibition of protein kinases.

Topics: Adenosine Kinase; Animals; Glucagon; Glycogen Synthase; Liver; Male; Peptides; Phosphorylase Kinase; Phosphorylation; Protein Kinase Inhibitors; Rats; Rats, Wistar; Tubercidin; Vasopressins

Several 5-iodotubercidin analogues in the pyrazolo[3,4-d]pyrimidine ring system were synthesized as potential inhibitors of adenosine kinase by a direct Lewis acid-catalyzed glycosylation procedure using both the preformed carbohydrate and the heterocyclic base as starting materials. The 5'-hydroxyl, -chloro, -azido, -deoxy, -amino, and -fluoro derivatives were prepared and evaluated in three systems for biological activity relative to adenosine, the true substrate, and 5-iodotubercidin, a known inhibitor. First, each compound was studied kinetically for inhibition of purified human placental adenosine kinase activity. The order of potency was: iodotubercidin > hydroxyl > amino > or = deoxy > fluoro > chloro >> azido. The Ki values for the 5'-hydroxyl and 5'-amino compounds, the two most potent inhibitors, were 80 and 150 nM, respectively. The inhibition appeared to be essentially competitive in nature, although a noncompetitive component of significance for the more potent inhibitors cannot be ruled out. Second, a bioassay was conducted in which the toxicity of 6-methylmercaptopurine riboside toward human CEM lymphoblasts was reversed by varying concentrations of the compounds. The order of effectiveness of the compounds in this system, representing a functional inhibition of adenosine kinase in cultured cells, was about the same as that with the purified enzyme, except that the 5'-chloro and 5'-fluoro compounds were ineffective. Third, the 5'-hydroxyl derivative was evaluated in vivo in a rat pleurisy inflammation model and displayed biological activity at a dose of 30 mg/kg given orally. Finally, the in vitro toxicity of each compound was assessed in CEM lymphoblasts. Results indicated that the two most potent inhibitors in the pyrazolo[3,4-d]pyrimidine ring system, the 5'-hydroxyl (7) and the 5'-amino (20), were 15-fold and 75-fold, respectively, less growth inhibitory than 5-iodotubercidin.

Topics: Adenosine Kinase; Administration, Oral; Animals; Anti-Inflammatory Agents, Non-Steroidal; Carrageenan; Cells, Cultured; Drug Evaluation, Preclinical; Humans; Kinetics; Lymphocytes; Pleurisy; Pyrazoles; Pyridines; Rats; Rats, Sprague-Dawley; Tubercidin

5-Iodotubercidin is a potent inhibitor of the enzyme adenosine kinase. It has a recognized ability to enhance interstitial fluid levels of the cerebroprotective purine, adenosine, in the hypoxic brain, and an anticonvulsant action, which is thought to be a consequence of its ability to increase extracellular adenosine levels. 5-Iodotubercidin (1 mg/kg, i.p.) was therefore tested for its ability to reduce cerebral ischemic injury in a gerbil model. Unanesthetized gerbils were subjected to a 5 min period of bilateral carotid artery occlusion and then maintained in an environmental chamber at 30 degrees C for 5 hr to counteract the hypothermia-inducing action of 5-iodotubercidin. As estimated from the extent of the increases in locomotor activity and the magnitude of hippocampal CAI layer pyramidal cell loss, 5-iodotubercidin (1 mg/kg) failed to have a cerebroprotective effect against ischemic injury.

Topics: Adenosine Kinase; Animals; Body Temperature; Brain Ischemia; Gerbillinae; Hippocampus; Male; Motor Activity; Tubercidin

Previous work has shown that normoxic isolated rat hepatocytes continuously produce adenosine from AMP and that the nucleoside is not catabolized further but immediately rephosphorylated by adenosine kinase [Bontemps, Van den Berghe and Hers (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2829-2833]. We now report the effect of anoxia on adenosine production and on the AMP/adenosine substrate cycle. In cell suspensions incubated in O2/CO2, the adenosine concentration was about 0.4 microM. It increased 30-fold in cells incubated in N2/CO2 or with 5 mM KCN, and 20-fold in cells incubated with 2 mM amytal. Adenosine production, measured in hepatocytes in which adenosine kinase and adenosine deaminase were inhibited by 5-iodotubercidin and deoxycoformycin respectively, was about 18 nmol/min per g of cells in normoxia; it increased about 2-fold in anoxia, although AMP increased 8-16-fold in this condition. From studies with inhibitors of membrane 5'-nucleotidase and of S-adenosylhomocysteine hydrolase, it was deduced that adenosine is produced by the latter enzyme and by cytosolic 5'-nucleotidase in normoxia, and by cytosolic and membrane 5'-nucleotidases in anoxia. Unlike in normoxic hepatocytes, inhibition of adenosine kinase by 5-iodotubercidin neither elevated the adenosine concentration nor enhanced total purine release from adenine nucleotides in cells treated with N2/CO2 or KCN; it had only a slight effect in cells treated with amytal. This indicates that recycling of adenosine is suppressed or profoundly inhibited in anoxia. The rate of accumulation of adenosine in anoxia was several-fold lower than the rate of its rephosphorylation upon reoxygenation. It is concluded that the elevation of adenosine in anoxic hepatocytes is much more dependent on decreased recycling of adenosine by adenosine kinase than on increased production by dephosphorylation of AMP.

Topics: 5'-Nucleotidase; Adenine Nucleotides; Adenosine; Adenosine Deaminase Inhibitors; Adenosine Kinase; Amobarbital; Animals; Carbon Dioxide; Cell Membrane; Liver; Male; Nitrogen; Oxygen; Pentostatin; Phosphoric Monoester Hydrolases; Potassium Cyanide; Rats; Rats, Wistar; Tubercidin

The purpose of this study was to determine the changes in interstitial fluid (ISF) adenosine and cerebral blood flow (CBF) during inhibition of adenosine kinase or adenosine deaminase. Brain microdialysis was used to (a) measure CBF (H2 clearance), (b) sample cerebral ISF, and (c) deliver drugs locally to the brain. Microdialysis probes were implanted bilaterally in the caudate nucleus of halothane-anesthetized rats (n = 11). One probe was perfused with artificial cerebrospinal fluid (CSF) containing iodotubercidin (IODO), an adenosine kinase inhibitor, while the other probe was perfused with erythro-2-(2-hydroxy-3-nonyl)adenine (EHNA), an adenosine deaminase inhibitor. Both probes were subsequently perfused with EHNA+IODO. Finally, 8-(p-sulfophenyl)theophylline (SPT), an adenosine receptor antagonist, was added to EHNA + IODO in one probe, while the other probe continued to receive only EHNA + IODO. CBF and dialysate adenosine levels increased with either EHNA or IODO; however, the increases were greater with IODO. EHNA + IODO further increased CBF and dialysate adenosine. The hyperemia observed with EHNA + IODO was abolished by adenosine receptor blockade. These data suggest that basal adenosine levels are influenced to a greater extent by adenosine kinase than by adenosine deaminase. In addition, the increased CBF observed with inhibition of adenosine metabolism and the attenuation of this vasodilatory response with adenosine receptor blockade support a role for adenosine in CBF regulation.

Topics: Adenine; Adenosine; Adenosine Deaminase Inhibitors; Adenosine Kinase; Animals; Brain; Cerebrovascular Circulation; Dialysis Solutions; Dose-Response Relationship, Drug; Extracellular Space; Hypoxanthine; Hypoxanthines; Inosine; Male; Rats; Rats, Wistar; Tubercidin

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)

Topics: 5'-Nucleotidase; Adenine Nucleotides; Adenosine; Adenosine Deaminase; Adenosine Deaminase Inhibitors; Adenosine Kinase; Animals; Calcimycin; Cells, Cultured; Chromatography, High Pressure Liquid; Endothelium, Vascular; Firefly Luciferin; Kinetics; Luciferases; Microspheres; Phosphorylation; Swine; Tubercidin

The adenosine kinase inhibitor 5-iodotubercidin (Itu) was found to have the following effects on glycogen metabolism in hepatocytes of fasted rats. (1) Itu strongly stimulated glycogen synthesis from different substrates (glucose, lactate plus pyruvate, dihydroxyacetone, glycerol and fructose). In cells incubated with these substrates, the well-known stimulating effect of amino acids and that of Itu was more than additive. (2) In parallel with the increase in glycogen deposition, there was an increase in synthase a and a decrease in phosphorylase a concentrations after administration of Itu. Synthase a was increased by Itu and amino acids in an additive manner, whereas the observed activation of phosphorylase after addition of amino acids was antagonized by Itu. (3) In contrast with amino acids, Itu increased neither the cell volume nor the aspartate and glutamate concentrations. (4) Itu enhanced the levels of cyclic AMP. The stimulation of glycogen deposition in the presence of Itu persisted when the cyclic AMP concentration was further increased by adenosine or 2-chloroadenosine. (5) Itu decreased the concentration of ATP, but its effects on glycogen synthesis, synthase a and phosphorylase a concentrations persisted when the ATP catabolism was prevented by adenosine. (6) The effect of Itu on glycogen synthesis was not the result of inhibition of adenosine kinase, since 5'-amino-5'-deoxyadenosine, another inhibitor of this enzyme, had no effect on glycogen deposition.

Topics: 2-Chloroadenosine; Adenosine; Adenosine Kinase; Adenosine Triphosphate; Amino Acids; Animals; Cells, Cultured; Liver; Liver Glycogen; Male; Rats; Rats, Wistar; Tubercidin

We employed an isolated guinea-pig heart model perfused at constant pressure (70 cmH2O) to test the hypothesis that inhibition of adenosine metabolism increases interstitial adenosine concentrations (as measured with epicardial discs) and coronary flow. Iodotubercidin (ITU, 1 microM) and EHNA (erythro-9-[2-hydroxy-3-nonyl] adenine, 5 microM) were used to inhibit adenosine kinase and deaminase, respectively during control conditions and during metabolic stimulation with 1 microM isoproterenol. The adenosine receptor blocker 8-phenyltheophylline (8-PT) was used during control conditions to assess whether the response seen was adenosine specific. ITU plus EHNA decreased heart rate (202 +/- 10 to 136 +/- 11 beats/min) and increased coronary flow (8.2 +/- 0.3 to 12.4 +/- 0.9 ml/min/g) without a change in MVO2, developed pressure or dP/dt. ITU plus EHNA increased adenosine concentrations in epicardial fluid (0.24 +/- 0.07 microM to 1.02 +/- 0.09 microM) and venous effluent (40 +/- 3 nM to 262 +/- 32 nM) during control conditions, and adenosine release increased from 389 +/- 96 pmols/min/g to 3480 +/- 365 pmols/min/g. 8-PT infusion reversed the effects on heart rate and coronary flow and resulted in a persistent elevation of epicardial fluid adenosine concentrations. During metabolic stimulation with 1 microM isoproterenol, ITU plus EHNA significantly limited the increase in heart rate and ventricular developed pressure and dP/dt while coronary flow increased to a significantly greater extent. Myocardial oxygen consumption was similar during metabolic stimulation between the two groups (vehicle vs. ITU plus EHNA). Epicardial fluid adenosine concentration in the vehicle-treated group increased from 0.17 +/- 0.3 microM to 0.34 +/- 0.02 microM at 15 min of isoproterenol stimulation whereas it increased from 1.10 +/- 0.02 microM to 2.90 +/- 0.46 microM in the ITU plus EHNA-treated group. Inhibition of adenosine metabolism during metabolic stimulation significantly increased venous adenosine concentrations and adenosine release and reduced inosine and hypoxanthine release proportionately. The release of adenosine+inosine+hypoxanthine was unchanged. Inhibition of adenosine metabolism provides evidence supporting the hypothesis that adenosine plays a role in regulating coronary vascular resistance as well as influencing heart rate and ventricular inotropy.

Topics: Adenine; Adenosine; Adenosine Deaminase Inhibitors; Adenosine Kinase; Animals; Coronary Circulation; Extracellular Space; Guinea Pigs; In Vitro Techniques; Isoproterenol; Male; Myocardium; Perfusion; Tubercidin; Vascular Resistance

Adenosine derivatives are frequently used in chemotherapy because of their potent antitumor, antiviral and antiparasitic activity. We investigated the metabolism of some adenosine analogues in adenosine deaminase inhibited normal and adenine phosphoribosyltransferase (APRT) deficient human erythrocytes. The ATP and GTP concentrations and the formation of unusual nucleotides were measured. Some of the analogues studied (tubercidin, 9 beta-D-arabinofuranosyladenine, 2'-deoxyadenosine, 2-chloroadenosine, neplanocin A) were phosphorylated to the corresponding nucleoside triphosphates and this process was abolished by iodotubercidin--an adenosine kinase inhibitor. With the exception of 2'-deoxyadenosine, nucleotide analogue formation was accompanied by ATP depletion. ATP decrease was not observed after adenosine kinase inhibition and ATP concentration even increased in the presence of 2'-deoxyadenosine, neplanocin A and 5'-iodo-5'-deoxyadenosine. However, the latter increment was not observed in APRT deficient erythrocytes. Bredinin, S-adenosylhomocysteine, deoxycoformycin and adenosine dialdehyde did not form nucleotide derivatives or exert any effects on ATP concentration. It is concluded that adenosine analogues can either enter the nucleotide pool via phosphorylation mechanisms, or may be converted to ATP by the pathways involving the intermediate formation of adenine.

Topics: Adenine Phosphoribosyltransferase; Adenosine Kinase; Adenosine Triphosphate; Erythrocytes; Humans; Hypoxanthine; Hypoxanthines; Pentostatin; Tubercidin

Effects of glucose and adenosine on ATP metabolism were studied using isolated round spermatids from hamsters. The ATP content of the spermatids was strongly decreased after 1 h of incubation of the cells in the presence of 0.1 mM D-glucose. Glucose (1 mM) had no effect during 18 h of incubation in the presence of 12 mM sodium DL-lactate. However, 10 mM glucose caused an almost complete loss of cellular ATP in the presence of lactate. The effect of adenosine was estimated in the absence of glucose with lactate as the energy-yielding substrate. The cellular ATP content was approximately 4 and 8 nmol/10(6) cells, after 18 h of incubation in the absence and presence of 0.1 mM adenosine, respectively. This two-fold increase was prevented by inhibitors of adenosine uptake and phosphorylation and was slowly reversed after removal of the exogenous adenosine. Treatment of the cells with adenosine had no effect on the energy charge, which was higher than 0.90, and did not alter the cellular cyclic AMP content. The suggestion that the physiological ATP content of the round spermatids is probably stabilized in the region of 4 nmol/10(6) cells is discussed.

Topics: Adenine; Adenosine; Adenosine Kinase; Adenosine Triphosphate; Animals; Cricetinae; Cyclic AMP; Dipyridamole; Dose-Response Relationship, Drug; Fructose; Galactose; Glucose; Hypoxanthine; Hypoxanthines; In Vitro Techniques; Male; Mannose; Spermatids; Tubercidin

Intracellular adenosine formation and release to extracellular space was studied in WI-L2-B and SupT1-T lymphoblasts under conditions which induce or do not induce ATP catabolism. Under induced conditions, B lymphoblasts but not T lymphoblasts, release significant amounts of adenosine, which are markedly elevated by adenosine deaminase inhibitors. In T lymphoblasts, under induced conditions, only simultaneous inhibition of both adenosine deaminase activity and adenosine kinase activities resulted in small amounts of adenosine release. Under noninduced conditions, neither B nor T lymphoblasts release adenosine, even in the presence of both adenosine deaminase or adenosine kinase inhibitors. Comparison of B and T cell's enzyme activities involved in adenosine metabolism showed similar activity of AMP deaminase, but the activities of AMP-5'-nucleotidase, adenosine kinase and adenosine deaminase differ significantly. B lymphoblasts release adenosine because of their combination of enzyme activities which produce or utilize adenosine (high AMP-5'-nucleotidase and relatively low adenosine kinase and adenosine deaminase activities). Accelerated ATP degradation in B lymphoblasts proceeds not only via AMP deamination, but also via AMP dephosphorylation into adenosine but its less efficient intracellular utilization results in the release of adenosine from these cells. In contrast, T lymphoblasts release far less adenosine, because they contain relatively low AMP-5'-nucleotidase and high adenosine kinase and adenosine deaminase activities. In T lymphoblasts, AMP formed during ATP degradation is not readily dephosphorylated to adenosine but mainly deaminated to IMP by AMP deaminase. Any adenosine formed intracellularly in T lymphoblasts is likely to be efficiently salvaged back to AMP by an active adenosine kinase. In general, these results may suggest that adenosine can be produced only by selective cells (adenosine producers) whereas other cells with enzyme combination similar to SupT1-T lymphoblasts can not produce significant amounts of adenosine even in stress conditions.

Topics: Adenosine; Adenosine Kinase; Adenosine Triphosphate; Animals; B-Lymphocytes; Cell Line; Deoxyglucose; Hypoxanthine; Hypoxanthines; Inosine; Kinetics; Models, Biological; Pentostatin; Rats; T-Lymphocytes; Tubercidin

1. The activities of ecto- and cytosolic 5'-nucleotidase (EC 3.1.3.5), adenosine kinase (EC 2.7.1.20), adenosine deaminase (EC 3.5.4.4) and AMP deaminase (EC 3.5.4.6) were compared in ventricular myocardium from man, rats, rabbits, guinea pigs, pigeons and turtles. The most striking variation was in the activity of the ecto-5'-nucleotidase, which was 20 times less active in rabbit heart and 300 times less active in pigeon heart than in rat heart. The cytochemical distribution of ecto-5'-nucleotidase was also highly variable between species. 2. Adenosine formation was quantified in pigeon and rat ventricular myocardium in the presence of inhibitors of adenosine kinase and adenosine deaminase. 3. Both adenosine formation rates and the proportion of ATP catabolized to adenosine were greatest during the first 2 min of total ischaemia at 37 degrees C. Adenosine formation rates were 410 +/- 40 nmol/min per g wet wt. in pigeon hearts and 470 +/- 60 nmol/min per g wet wt. in rat hearts. Formation of adenosine accounted for 46% of ATP plus ADP broken down in pigeon hearts and 88% in rat hearts. 4. The data show that, in both pigeon and rat hearts, adenosine is the major catabolite of ATP in the early stages of normothermic myocardial ischaemia. The activity of ecto-5'-nucleotidase in pigeon ventricle (16 +/- 4 nmol/min per g wet wt.) was insufficient to account for adenosine formation, indicating the existence of an alternative catabolic pathway.

Topics: 5'-Nucleotidase; Adenine; Adenosine; Adenosine Deaminase; Adenosine Kinase; Adult; AMP Deaminase; Animals; Columbidae; Coronary Disease; Female; Heart; Histocytochemistry; Humans; Male; Middle Aged; Myocardium; Nucleotidases; Nucleotides; Phosphoric Monoester Hydrolases; Rats; Rats, Inbred Strains; Species Specificity; Tubercidin

2-Chloroadenosine is presumably a non-metabolizable analogue of adenosine; however, this compound induced an increase in the enzymatically measured nucleotide content of isolated rat hepatocytes. HPLC separation and spectral analysis of the peaks showed that this increase may be related to the formation of 2-chloro nucleotides and that the 2-chloro nucleotides appeared in the first minutes of the incubation period. These results demonstrate that 2-chloroadenosine may be metabolized by phosphorylation in rat liver cells.

Topics: 2-Chloroadenosine; Adenosine; Adenosine Diphosphate; Adenosine Kinase; Adenosine Triphosphate; Animals; Chromatography, High Pressure Liquid; Kinetics; Liver; Male; Nucleotides; Phosphorylation; Piperazines; Rats; Rats, Inbred Strains; Spectrophotometry; Tubercidin

5'-Deoxy-5-iodotubercidin was previously reported to cause potent muscle relaxation and hypothermia when injected i.p. into mice. In normotensive rats, i.v. injection reduced blood pressure and heart rate. 5-Iodotubercidin possessed the same in vivo activities whereas tubercidin was pharmacologically almost inactive. None of these compounds interacted significantly with Al adenosine receptors, as determined by their ability to displace 3H-N6-phenylisopropyladenosine or 3H-5'-N-ethylcarboxamidoadenosine bound to rat brain membranes. Furthermore these compounds were much weaker than adenosine as agonists of adenosine-stimulated adenylate cyclase in guinea-pig brain slices (A2 receptors). A previous report showed that 5'-deoxy-5-iodotubercidin and 5-iodotubercidin were very potent inhibitors of adenosine kinase from rat or guinea-pig brain and were potent inhibitors of 3H-adenosine uptake into brain slices; relative to the halogenated derivatives, tubercidin was quite weak as an inhibitor of adenosine kinase and of adenosine uptake. We therefore propose that a significant part of the in vivo activity of the two halogenated tubercidin analogues may not be due to a direct agonist action at A1 and/or A2 adenosine sites (as proposed for a number of other metabolically-stable analogues of adenosine) but may result from an inhibition of reuptake of endogenously-released adenosine; the increased extracellular levels of adenosine resulting from this action could then interact directly with membrane receptors. Consistent with this, low concentrations of 5'-deoxy-5-iodotubercidin were shown to significantly potentiate the effects of exogenous adenosine on blood pressure and heart rate in anaesthetized rats and on adenosine-stimulated cAMP generation in guinea-pig brain slices. None of these compounds interacted with central benzodiazepine receptors. The cardiovascular and behavioural effects of 5'-deoxy-5-iodotubercidin and 5-iodotubercidin were blocked by theophylline; results from the cardiovascular studies suggest there may be different adenosine receptors in heart and blood vessels.

Topics: Adenosine; Adenosine Deaminase; Adenosine Kinase; Adenosine-5'-(N-ethylcarboxamide); Animals; Blood Pressure; Brain; Cyclic AMP; Guanosine; Guinea Pigs; Heart Rate; Male; Mice; Muscle Relaxation; Phenylisopropyladenosine; Receptors, Cell Surface; Receptors, Purinergic; Tubercidin

The enzymes responsible for the phosphorylation of deoxyadenosine and nucleoside analogs are important in the pathogenesis of adenosine deaminase deficiency and in the activation of specific anticancer and antiviral drugs. We examined the role of adenosine kinase in catalyzing these reactions using an enzyme purified 4000-fold (2.1 mumol/min/mg) from human placenta. The Km values of deoxyadenosine and ATP are 135 and 4 microM, respectively. Potassium and magnesium are absolute requirements for deoxyadenosine phosphorylation, and 150 mM potassium and 5 mM MgCl2 are critical for linear kinetics. With only 0.4 mM MgCl2 in excess of ATP levels, the Km for deoxyadenosine is increased 10-fold. ADP is a competitive inhibitor with a Ki of 13 microM with variable MgATP2-, while it is a mixed inhibitor with a Ki and Ki' of 600 and 92 microM, respectively, when deoxyadenosine is variable. AMP is a mixed inhibitor with Ki and Ki' of 177 and 15 microM, respectively, with variable deoxyadenosine; it is a non-competitive inhibitor with a Ki of 17 microM and Ki' of 27 microM with variable ATP. Adenosine kinase phosphorylates adenine arabinoside with an apparent Km of 1 mM using deoxyadenosine kinase assay conditions. The Km values for 6-methylmercaptopurine riboside and 5-iodotubercidin, substrates for adenosine kinase, are estimated to be 4.5 microM and 2.6 nM, respectively. Other nucleoside analogs are potent inhibitors of deoxyadenosine phosphorylation, but their status as substrates remains unknown. These data indicate that deoxyadenosine phosphorylation by adenosine kinase is primarily regulated by its Km and the concentrations of Mg2+, ADP, and AMP. The high Km values for phosphorylation of deoxyadenosine and adenine arabinoside suggest that adenosine kinase may be less likely to phosphorylate these nucleosides in vivo than other enzymes with lower Km values. Adenosine kinase appears to be important for adenosine analog phosphorylation where the Michaelis constant is in the low micromolar range.

Topics: Adenosine Diphosphate; Adenosine Kinase; Adenosine Monophosphate; Adenosine Triphosphate; Deoxyadenosines; Female; Humans; Hydrogen-Ion Concentration; Kinetics; Methylthioinosine; Nucleosides; Nucleotides; Phosphorylation; Phosphotransferases; Placenta; Potassium Chloride; Pregnancy; Tubercidin; Vidarabine

The growth of Leishmania donovani promastigotes and mouse L cells is differentially inhibited by several inosine analogs with modifications in the imidazole ring. The protozoal and mammalian cells also demonstrate differential metabolism of these analogs. 7-Deazainosine, 7-thia-7,9-dideazainosine, and formycin B were converted to their respective ATP analogs by both cell types. 8-Azainosine was converted to a GTP analog by mouse L cells; L. donovani did not metabolize this nucleoside. 9-Deazainosine and allopurinol riboside were metabolized only to their respective IMP analogs by L cells. L. donovani metabolized 9-deazainosine and allopurinol riboside to their ATP analogs and also metabolized 9-deazainosine to its GTP analog. All nucleosides studied were resistant to cleavage by either organism. From metabolism studies in the presence of a specific enzyme inhibitor, it was deduced that allopurinol riboside, formycin B, and 9-deazainosine were phosphorylated by at least two different routes in the mouse L cells. The metabolism of formycin B was inhibited 65% by the adenosine kinase inhibitor, 5-iodotubercidin, whereas the metabolism of allopurinol riboside (14% inhibition) and 9-deazainosine (0% inhibition) was only slightly affected by this inhibitor. The metabolism of allopurinol riboside and 9-deazainosine by L. donovani was not affected by 5-iodotubercidin. In contrast to the results of L cells, the metabolism of formycin B by L. donovani was also not affected by 5-iodotubercidin. The abilities of mouse L cells and L. donovani to metabolize these inosine analogs to the corresponding nucleotide analogs of ATP or GTP may be considered to be an activating step and correlates well with the respective cytotoxic effects of these compounds.

Topics: Adenosine Kinase; Adenosine Triphosphate; Animals; Cell Line; Chromatography, High Pressure Liquid; Enzyme Inhibitors; Fibroblasts; Guanosine Triphosphate; Inosine; Inosine Triphosphate; Leishmania; Mice; Tubercidin

Rat polymorphonuclear leucocytes or neonatal-rat heart cells in culture were treated with 2'-deoxycoformycin and 5-iodotubercidin at concentrations that inhibited adenosine deaminase (EC 3.5.4.4) and adenosine kinase (EC 2.7.1.20) inside the intact cells, and the rate of adenosine accumulation was determined. The basal rate of adenosine formation was 2% (polymorphonuclear leucocytes) or 9% (heart cells) of the maximal activity of adenosine kinase also measured in intact cells. Greatly increased rates of adenosine formation were observed during adenine nucleotide catabolism. This condition also led to a decrease in adenosine kinase activity. When isolated rat hearts were perfused with 5-iodotubercidin alone at a concentration which inhibited adenosine kinase, no increase in tissue or perfusate adenosine or inosine concentration was observed. However, perfusion with hypoxic buffer or infusion of adenosine into the coronary circulation at a rate (20 nmol/min) equivalent to 40% of the activity of adenosine kinase caused large increases in effluent perfusate adenosine and inosine concentrations. These data argue unanimously against the existence of a substrate cycle controlling adenosine concentration. They suggest instead that an increase in the rate of adenosine formation is the principal cause of elevations in adenosine concentration during ATP catabolism.

Topics: Adenosine; Adenosine Kinase; Animals; Cells, Cultured; Coformycin; Heart; In Vitro Techniques; Male; Myocardium; Neutrophils; Pentostatin; Perfusion; Rats; Rats, Inbred Strains; Tubercidin

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.

Topics: 5'-Nucleotidase; Adenosine; Adenosine Deaminase Inhibitors; Adenosine Kinase; Animals; Chromatography, High Pressure Liquid; Coformycin; Neutrophils; Nucleotidases; Pentostatin; Rats; Tubercidin