adenosine-kinase and 1-3-dipropyl-8-cyclopentylxanthine

Adenosine kinase (AK) inhibitor is a potential candidate for controlling pain, but some AK inhibitors have problems of adverse effects such as motor impairment. ABT-702, a non-nucleoside AK inhibitor, shows analgesic effect in animal models of pain. Here, we investigated the effects of ABT-702 on synaptic transmission via nociceptive and motor reflex pathways in the isolated spinal cord of neonatal rats. The release of adenosine from the spinal cord was measured by HPLC. ABT-702 inhibited slow ventral root potentials (sVRPs) in the nociceptive pathway more potently than monosynaptic reflex potentials (MSRs) in the motor reflex pathway. The inhibitory effects of ABT-702 were mimicked by exogenously applied adenosine, blocked by 8CPT (8-cyclopentyl-1,3-dipropylxanthine), an adenosine A1 receptor antagonist, and augmented by EHNA (erythro-9-(2-hydroxy-3-nonyl) adenine), an adenosine deaminase (ADA) inhibitor. Equilibrative nucleoside transporter (ENT) inhibitors reversed the effects of ABT-702, but not those of adenosine. ABT-702 released adenosine from the spinal cord, an effect that was also reversed by ENT inhibitors. The ABT-702-facilitated release of adenosine by way of ENTs inhibits nociceptive pathways more potently than motor reflex pathways in the spinal cord via activation of A1 receptors. This feature is expected to lead to good analgesic effects, but, caution may be required for the use of AK inhibitors in the case of ADA dysfunction or a combination with ENT inhibitors.

Topics: Adenine; Adenosine; Adenosine A1 Receptor Antagonists; Adenosine Deaminase; Adenosine Kinase; Analgesics; Animals; Animals, Newborn; Enzyme Inhibitors; Membrane Potentials; Morpholines; Motor Neurons; Neural Pathways; Nociceptive Pain; Pyrimidines; Reflex; Spinal Cord; Tissue Culture Techniques; Xanthines

Adenosine is an important inhibitory neuromodulator that regulates neuronal excitability. Several studies have shown that nitric oxide induces release of adenosine. Here we investigated the mechanism of this release. We studied the effects of nitric oxide on evoked field excitatory postsynaptic potentials (fEPSPs) recorded in the CA1 area of rat hippocampal slices. The nitric oxide donor 1,1-diethyl-2-hydroxy-2-nitroso-hydrazine sodium (DEA/NO; 100 microm) depressed the fEPSP by 77.6 +/- 4.1%. This effect was abolished by the adenosine A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 400 nm), indicating that the nitric oxide effect was mediated by adenosine accumulation. The DEA/NO effect was unaltered by the 5'-ectonucleotidase inhibitor alpha,beta-methylene-adenosine 5'-diphosphate (AMP-CP; 100 microm), indicating that extracellular adenosine did not derive from ATP or cAMP release. The guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazole[4,3-a]quinoxaline-1-one (ODQ; 5 microm) did not affect nitric oxide depression of the fEPSPs, indicating that nitric oxide-mediated adenosine release was not mediated through a cGMP signaling cascade. This conclusion was confirmed by the observation that 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP; 1 mm) reversibly depressed the fEPSP by 24.9 +/- 4.5%, but this effect was not blocked by adenosine antagonists. Adenosine kinase inhibitor 5-iodotubercidin (ITU; 7 microm) occluded the nitric oxide effects by 74%, suggesting that inhibition of adenosine kinase activity contributes to adenosine release. In conclusion, exogenous nitric oxide evokes adenosine release by a cGMP-independent pathway. Intracellular cGMP elevation partially inhibits the fEPSP but not through adenosine release. Although a direct block of adenosine kinase by nitric oxide can not be excluded, the depression of adenosine kinase activity may be due to inhibition by its own substrate adenosine.

Topics: Adenosine; Adenosine Kinase; Animals; Cyclic GMP; Enzyme Inhibitors; Excitatory Postsynaptic Potentials; Hippocampus; Hydrazines; Male; Nitric Oxide; Organ Culture Techniques; Oxadiazoles; Quinoxalines; Rats; Rats, Sprague-Dawley; Synaptic Transmission; Thionucleotides; Xanthines

Volatile anaesthetics depress excitatory signal transmission by potentiating the inhibitory action of GABAA receptors and there is strong evidence that this is related with anaesthesia. Using primary hippocampal cultures we analyzed the possibility that the volatile anaesthetics enflurane and sevoflurane depress excitatory signal transmission by activation of adenosine A1 receptors.. Primary rat hippocampal cultures on 4 cm poly-L-lysine coated glass coverslips were loaded with the Ca2+-indicator fluo-3 and incorporated in a gastight, temperature-controlled perfusion chamber. The intracellular Ca2+-concentration was monitored with a confocal laser-scanning microscope (BioRad) using the 488 nm laser line of a krypton-argon laser for excitation and the Lasersharp Acquisition software for analysis.. Continuous perfusion in Mg2+-free medium generated spontaneous synchronized calcium oscillations, which were dose dependently depressed by the volatile anaesthetics enflurane and sevoflurane (0.25-1 minimum alveolar concentration). Addition of 100 nmol of 2-chloro-N6-cyclopentyladenosine, a specific adenosine A1 receptor antagonist, partly reversed the anaesthetic-induced inhibition of the oscillation amplitude of the oscillating cells. The effect of the anaesthetics was mimicked by the addition of S-(p-nitrobenzyl)-6-thioguanosine, an adenosine transport inhibitor and by the addition of 5-amino-5-deoxyadenosine, an inhibitor of adenosine kinase.. The volatile anaesthetics sevoflurane and enflurane activate adenosine A1 receptors in primary rat hippocampal cultures. This effect is mediated by liberation of adenosine most likely by an interaction of the volatile anaesthetics with adenosine transport or key enzymes in adenosine metabolism.

Topics: Adenosine; Adenosine A1 Receptor Antagonists; Adenosine Kinase; Anesthetics, Inhalation; Animals; Calcium Signaling; Cells, Cultured; Deoxyadenosines; Dose-Response Relationship, Drug; Enflurane; Guanosine; Hippocampus; Magnesium; Methyl Ethers; Neurons; Purinergic P1 Receptor Antagonists; Rats; Receptor, Adenosine A1; Receptors, N-Methyl-D-Aspartate; Sevoflurane; Signal Transduction; Thionucleosides; Xanthines

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

Adenosine is an inhibitor of neuronal activity in the brain. The local release of adenosine from grafted cells was evaluated as an ex vivo gene therapy approach to suppress synchronous discharges and epileptic seizures. Fibroblasts were engineered to release adenosine by inactivating the adenosine-metabolizing enzymes adenosine kinase and adenosine deaminase. After encapsulation into semipermeable polymers, the cells were grafted into the brain ventricles of electrically kindled rats, a model of partial epilepsy. Grafted rats provided a nearly complete protection from behavioral seizures and a near-complete suppression of afterdischarges in electroencephalogram recordings, whereas the full tonic-clonic convulsions in control rats remained unaltered. Thus, the local release of adenosine resulting in adenosine concentrations <25 nM at the site of action is sufficient to suppress seizure activity and, therefore, provides a potential therapeutic principle for the treatment of drug-resistant partial epilepsies.

Topics: Adenosine; Adenosine Deaminase; Adenosine Kinase; Aggression; Animals; Brain; Cell Line; Cell Transplantation; Cricetinae; Epilepsies, Partial; Exploratory Behavior; Fibroblasts; Genetic Therapy; Kindling, Neurologic; Male; Mice; Mice, Knockout; Motor Activity; Phenytoin; Rats; Rats, Inbred Strains; Seizures; Social Behavior; Xanthines

1. Intracellular current clamp recordings were made from CA1 pyramidal neurones in rat hippocampal slices. Experiments were performed in the presence of ionotropic glutamate receptor antagonists and gamma-aminobutyric acid (GABA) receptor antagonists to block all fast excitatory and inhibitory synaptic transmission. A single stimulus, delivered extracellularly in the stratum oriens, caused a reduction in spike frequency adaptation in response to a depolarizing current step delivered 2 s after the stimulus. A 2- to 10-fold increase in stimulus intensity evoked a slow excitatory postsynaptic potential (EPSP) which was associated with a small increase in input resistance. The peak amplitude of the EPSP occurred approximately 2.5 s after the stimulus and its magnitude (up to 30 mV) and duration (10-50 s) increased with increasing stimulus intensity. 2. The slow EPSP was unaffected by the metabotropic glutamate receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine ((+)-MCPG; 1000 microM) but was greatly enhanced by the acetylcholinesterase inhibitor physostigmine (1-5 microM). Both the slow EPSP and the stimulus-evoked reduction in spike frequency adaptation were inhibited by the muscarinic acetylcholine receptor (mAChR) antagonist atropine (1-5 microM). These results are consistent with these effects being mediated by mAChRs. 3. Both the mAChR-mediated EPSP (EPSPm) and the associated reduction in spike frequency adaptation were reversibly depressed (up to 97%) by either adenosine (100 microM) or its non-hydrolysable analogue 2-chloroadenosine (CADO; 0.1-5.0 microM). These effects were often accompanied by postsynaptic hyperpolarization (up to 8 mV) and a reduction in input resistance (up to 11%). The selective adenosine A1 receptor agonists 2-chloro-N6-cyclopentyladenosine (CCPA; 0.1-0.4 microM) and R(-)N6-(2-phenylisopropyl)-adenosine (R-PIA; 1 microM) both depressed the EPSPm. In contrast, the adenosine A2A receptor agonist 2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680; 0.5-1.0 microM) did not significantly affect the EPSPm. 4. The selective adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 0.2 microM) fully reversed the depressant effects of both adenosine (100 microM) and CADO (1 microM) on the EPSPm and the stimulus-evoked reductions in spike frequency adaptation. 5. DPCPX (0.2 microM) alone caused a small but variable mean increase in the EPSPm of 22 +/- 19% and enabled activation of an EPS

Topics: Adenosine; Adenosine Kinase; Animals; Atropine; Cyclic AMP; Dose-Response Relationship, Drug; Electrophysiology; Excitatory Amino Acid Antagonists; Female; GABA Antagonists; Glutamic Acid; Hippocampus; Membrane Potentials; Parasympatholytics; Parasympathomimetics; Physostigmine; Quinoxalines; Rats; Rats, Wistar; Receptors, Muscarinic; Receptors, Purinergic P1; Synaptic Membranes; Synaptic Transmission; Xanthines

The acute antiinflammatory effects of methotrexate are mediated, at least in part, by increased extracellular adenosine concentrations at inflamed sites. This observation suggests that other agents that increase extracellular adenosine concentrations might also reduce inflammation. Since adenosine can be rapidly taken up by cells, phosphorylated by adenosine kinase, and maintained intracellularly as adenine nucleotides, we investigated whether a potent inhibitor of adenosine kinase, GP-1-515, could increase exudate adenosine concentration and thereby diminish inflammation in the murine air pouch model of inflammation.. We studied the effect of various oral doses of GP-1-515 on carrageenan-induced inflammation in air pouches induced on BALB/c mice. Adenosine concentration in pouch exudates was determined by high performance liquid chromatography, and intensity of inflammation was determined by leukocyte counts in the exudate fluid.. There was a greater concentration of adenosine in the pouch exudates of animals treated with GP-1-515 than of those treated with saline (P < 0.002). GP-1-515 inhibited, in a dose-dependent manner (P < 0.01), leukocyte accumulation in the murine air pouch in response to carrageenan. Inhibition of inflammation by GP-1-515 in this model depended upon increased adenosine concentration in the inflamed pouch since injection of adenosine deaminase into the air pouch with the carrageenan completely reversed the antiinflammatory effects of GP-1-515 at all doses of GP-1-515 tested. Moreover, as previously demonstrated, the antiinflammatory effects of adenosine were mediated via occupancy of adenosine A2 receptors, since the specific adenosine A2 receptor antagonist 3,7-dimethyl-1-propargylxanthine, but not the A1 receptor antagonist 8-cyclopentyl-dipropylxanthine, completely reversed the antiinflammatory effects of GP-1-515. GP-1-515 also decreased tumor necrosis factor alpha levels in the air pouch exudates by 51%, most likely as a result of the direct action of adenosine on macrophages.. These results indicate that the antiinflammatory actions of GP-1-515 are mediated by adenosine. The development of agents that promote adenosine release at sites of inflammation is a novel strategy for the treatment of inflammatory diseases such as rheumatoid arthritis.

Topics: Adenosine; Adenosine Deaminase; Adenosine Kinase; Animals; Anti-Inflammatory Agents; Anti-Inflammatory Agents, Non-Steroidal; Female; Leukocytes; Mice; Mice, Inbred BALB C; Purinergic P1 Receptor Antagonists; Ribonucleosides; Theobromine; Tumor Necrosis Factor-alpha; Xanthines

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