5-N-ethyl Carboxamidoadenosine Stimulates Adenosine-2b Receptor-Mediated Mitogen-Activated Protein Kinase Pathway to Improve Brain Mitochondrial Function in Amyloid Beta-Induced Cognitive Deficit Mice.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder with loss in memory as one of the cardinal features. 5-N-ethyl carboxamidoadenosine (NECA), an agonist of adenosine-2b receptor, exerts neuroprotective activity against several experimental conditions. Further, NECA activates mitogen-activated protein kinase (MAPK) and also attenuates mitochondrial toxicity in mammalian tissues other than brain. Moreover, there is no report on the role of A2b/MAPK-mediated signaling pathway in Aß-induced mitochondrial toxicity in the brain of the experimental animals. Therefore, the present study evaluated the neuroprotective activity of NECA with or without MAPK inhibitor against Aß-induced cognitive deficit and mitochondrial toxicity in the experimental rodents. Further, the effect of NECA with or without MAPK inhibitor was evaluated on Aß-induced mitochondrial toxicity in the memory-sensitive mice brain regions. Intracerebroventricular (ICV) injection of Aß 1-42 was injected to healthy male mice through Hamilton syringe via polyethylene tube to induce AD-like behavioral manifestations. NECA attenuated Aß-induced cognitive impairments in the rodents. In addition, NECA ameliorated Aß-induced Aß accumulation and cholinergic dysfunction in the selected memory-sensitive mouse HIP, PFC, and AMY. Further, NECA significantly attenuated Aß-induced mitochondrial toxicity in terms of decrease in the mitochondrial function, integrity, and bioenergetics in the brain regions of these animals. However, MAPKI diminished the therapeutic effects of NECA on behavioral, biochemical, and molecular observations in AD-like animals. Therefore, it can be speculated that NECA exhibits neuroprotective activity perhaps through MAPK activation in AD-like rodents. Moreover, A2b-mediated MAPK activation could be a promising target in the management of AD.

Reno-protective effect of NECA in diabetic nephropathy: implication of IL-18 and ICAM-1.

Diabetic nephropathy (DN) remains the most common cause of end-stage renal disease. Although, adenosine acts as a local modulator with a cytoprotective function, extracellular adenosine usually disappears quickly due to a rapid uptake into adjacent cells. Therefore, we investigated the effect of 5'-(N-ethylcarboxamido)-adenosine (NECA), a stable, nonselective adenosine receptor agonist, on diabetes-induced increases in inflammatory cytokines and adhesion molecules. The enhancement of adenosine receptor action by NECA was examined in the renal tissues of rats with streptozotocin-induced diabetes. Daily i.p. injections of NECA at 0.3 mg/kg/day were given to rats, over a two-week period, six weeks after the induction of diabetes. Morphological changes were assessed in kidney sections. Oxidative stress was examined by measuring tissue malondialdehyde. Gene expression of interleukin (IL)-18, tumor necrosis factor (TNF)-α and intercellular adhesion molecule (ICAM)-1 was measured by real-time PCR. Activation of cellular, proapoptotic pathways was demonstrated by measuring the activation of c-Jun NH(2)-terminal kinases (JNK)-mitogen-activated-protein kinase (MAPK). We found that diabetes-induced malondialdehyde formation activated the production of IL-18, TNF-α and ICAM-1, which, in turn, activated pro-apoptotic pathways in diabetic rats. Treatment with NECA protected diabetic rats by exerting hypoglycemic and antioxidant effects as well as reducing gene expression of proinflammatory cytokines. These effects were associated with deactivation of JNK-MAPK. In addition, diabetic rats treated with NECA showed mild glomerular effects and vacuolation of tubular epithelium. We can conclude that activation of adenosine receptors is a potential therapeutic target in DN. NECA acts via multiple mechanisms including: reducing diabetes-induced oxidative stress, inhibiting gene expression of IL-18, TNF-α and ICAM-1, and blocking activation of the JNK-MAPK pathway.

Adenosine signaling promotes regeneration of pancreatic ß cells in vivo.

Diabetes can be controlled with insulin injections, but a curative approach that restores the number of insulin-producing ß cells is still needed. Using a zebrafish model of diabetes, we screened ~7,000 small molecules to identify enhancers of ß cell regeneration. The compounds we identified converge on the adenosine signaling pathway and include exogenous agonists and compounds that inhibit degradation of endogenously produced adenosine. The most potent enhancer of ß cell regeneration was the adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA), which, acting through the adenosine receptor A2aa, increased ß cell proliferation and accelerated restoration of normoglycemia in zebrafish. Despite markedly stimulating ß cell proliferation during regeneration, NECA had only a modest effect during development. The proliferative and glucose-lowering effect of NECA was confirmed in diabetic mice, suggesting an evolutionarily conserved role for adenosine in ß cell regeneration. With this whole-organism screen, we identified components of the adenosine pathway that could be therapeutically targeted for the treatment of diabetes.

Intranasal administration of NECA can induce both anti-inflammatory and pro-inflammatory effects in BALB/c mice: evidence for A 2A receptor sub-type mediation of NECA-induced anti-inflammatory effects.

The role of adenosine in allergic inflammation is unclear. This study investigated the effects of the non-selective adenosine receptor agonist, 5-N-ethylcarboxamidoadenosine (NECA), on immunized only and immunized and airway challenged mice. The adenosine receptor sub-type(s) mediating the NECA effects and the A(2A) receptor mRNA expression were also investigated. In mice that were only immunized, intranasal NECA (1 mM) administration caused a significant increase in bronchoalveolar lavage total cell count (TCC), neutrophils and eosinophils (>1.5-, >6 and >60-fold, respectively). Two and four intranasal ovalbumin (OVA) challenges induced a significant (P < 0.05) increase in TCC (>2.1- and >4-fold, respectively) and eosinophils (>350- and >1700-fold, respectively). Real-time PCR analysis showed that the A(2A) receptor sub-type mRNA was significantly increased (P < 0.05) in the lung tissue of immunized mice following both two and four OVA challenges. NECA (0.3 mM) treatment caused a significant reduction in the increase induced by the two and four OVA challenges in the TCC by 46.1% and 56.6%, respectively, eosinophils by 70.1% and 75.6%, respectively, and in the A(2A) receptor sub-type mRNA by 43.2% and 41.0%, respectively. Treatment with the A(2A) receptor antagonist, 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine), SCH-58261, completely reversed both the NECA-mediated reduction in TCC and eosinophilia. Moreover, OVA challenge of immunized mice, over 2 consecutive days, resulted in a significant (P < 0.05) increase in TCC (4.5-fold) and eosinophils (>2000-fold) that was detected 72 h later. NECA (0.3 mM) treatment, at 24 and 48 h post OVA challenge, significantly reduced the increase in both TCC and eosinophils by 45.0% and 74.8%, respectively. Our data show that in immunized, but not OVA-challenged mice, high dose of NECA (1 mM) induces an inflammatory airway response. In contrast, in models of inflammation, NECA, at mainly 0.3 mM, induces a significant anti-inflammatory effect when administered prior to the induction of airway inflammation or therapeutically following its establishment. The data also indicate that the anti-inflammatory action of NECA seems to be mediated via the A(2A) receptor sub-type and hence the use of selective A(2A) receptor agonists as potential therapeutic agents in the treatment of inflammatory diseases such as asthma should be investigated further.

[Advances in the study of A2B adenosine receptor antagonists].

A2B adenosine receptor is involved in the control of mast cell degranulation, interleukin-8 synthesis and cell growth. A2B adenosine receptor antagonists may serve as novel drugs for asthma, Alzheimer' s disease, cystic fibrosis and type-II diabetes. Therefore, seeking for the highly selective A2B adenosine receptor antagonists has been one of great interest. The molecular basis, structure-activity relationship of selective A2B adenosine receptor antagonists and their interactions with A2B adenosine receptor were reviewed.

In vivo adenosine A(2B) receptor desensitization in guinea-pig airway smooth muscle: implications for asthma.

This study was aimed at characterizing the role of adenosine receptor subtypes in the contractility modulation of guinea-pig airway smooth muscle in normal and pathological settings. In vitro and in vivo experiments were performed by testing selective agonists and antagonists on isolated tracheal smooth muscle preparations and pulmonary inflation pressure, respectively, under normal conditions or following ovalbumin-induced allergic sensitization. In normal and sensitized animals, the adenosine A(2A)/A(2B) receptor agonist, NECA, evoked relaxing responses of isolated tracheal preparations precontracted with histamine, and such an effect was reversed by the adenosine A(2B) antagonist, MRS 1706, in the presence or in the absence of epithelium. The expression of mRNA coding for adenosine A(2B) receptors was demonstrated in tracheal specimens. In vitro desensitization with 100 microM NECA markedly reduced the relaxing effect of the agonist. In vivo NECA or adenosine administration to normal animals inhibited histamine-mediated bronchoconstriction, while these inhibitory effects no longer occurred in sensitized guinea-pigs. Adenosine plasma levels were significantly higher in sensitized than normal animals. In conclusion, our data demonstrate that: (i) adenosine A(2B) receptors are responsible for the relaxing effects of adenosine on guinea-pig airways; (ii) these receptors can undergo rapid adaptive changes that may affect airway smooth muscle responsiveness to adenosine; (iii) ovalbumin-induced sensitization promotes a reversible inactivation of adenosine A(2B) receptors which can be ascribed to homologous desensitization. These findings can be relevant to better understand adenosine functions in airways as well as mechanisms of action of asthma therapies targeting the adenosine system.

Adenosine receptor activation ameliorates type 1 diabetes.

Growing evidence indicates that adenosine receptors could be promising therapeutic targets in autoimmune diseases. Here we studied the role of adenosine receptors in controlling the course of type 1 diabetes. Diabetes in CD-1 mice was induced by multiple-low-dose-streptozotocin (MLDS) treatment and in nonobese diabetic (NOD) mice by cyclophosphamide injection. The nonselective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) prevented diabetes development in both MLDS-challenged mice and in cyclophosphamide-treated NOD mice. The effect of NECA was reversed by the selective A2B receptor antagonist N-(4-cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetamide (MRS 1754). The selective A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA) and A3 receptor agonist N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) were less efficacious in ameliorating the course of diabetes. NECA inhibited diabetes in A2A receptor KO mice and the selective A2A receptor agonist 2-p-(2-carboxyethyl)phenethyl-amino-5'-N-ethyl-carboxamidoadenosine (CGS21680) had no effect in normal mice, indicating a lack of role of A2A receptors. NECA failed to prevent cytokine-induced beta-cell death in vitro, but NECA strongly suppressed expression of the proinflammatory cytokines TNF-alpha, MIP-1alpha, IL-12, and IFN-gamma in pancreata, endotoxin, or anti-CD3-stimulated splenic cells, and T helper 1 lymphocytes, indicating that the beneficial effect of NECA was due to immunomodulation. These results demonstrate that adenosine receptor ligands are potential candidates for the treatment of type 1 diabetes.

NECA at reperfusion limits infarction and inhibits formation of the mitochondrial permeability transition pore by activating p70S6 kinase.

The A1/A2 adenosine agonist 5'-(N-ethylcarboxamido) adenosine (NECA) limits infarction when administered at reperfusion. The present study investigated whether p70S6 kinase is involved in this anti-infarct effect. Adult rat ventricular myocytes were isolated and incubated in tetramethylrhodamine ethyl ester (TMRE, 100 nM), which causes cells to fluoresce in proportion to their mitochondrial membrane potential. A reduction in TMRE fluorescence serves as an indicator of collapse of the mitochondrial transmembrane potential. Cells were subjected to H2O2 (200 microM), which like ischemia induces loss of mitochondrial membrane potential. Fluorescence was measured every 3 min and to facilitate quantification membrane potential was arbitrarily considered as collapsed when fluorescence reached less than 60% of the starting value. Adding NECA (1 mM) to the cells prolonged the time to fluorescence loss (48.0+/-3.2 min in the NECA group versus 29.5+/-2.2 min in untreated cells, P<0.001) and the mTOR/p70S6 kinase inhibitor rapamycin (5 nM) abolished this protection (31.3+/-3.4 min). Since cyclosporine A offered similar protection, mitochondrial permeability transition pore formation is a likely cause of the H2O2-induced loss of potential. The direct GSK-3beta inhibitor SB216763 (3 microM) also prolonged the time to fluorescence loss (49.2+/-2.1 min, P<0.001 versus control), and its protection could not be blocked by rapamycin (42.2+/-2.3 min, P<0.001 versus control). NECA treatment (100 nM) of intact isolated rabbit hearts at reperfusion after 30 min of regional ischemia decreased infarct size from 33.0+/-3.8% of the risk zone in control hearts to 11.8+/-2.0% (P<0.001), and rapamycin blocked this NECA-induced protection (38.3+/-3.7%). A comparable protective effect was seen for SB216763 (1 microM) with infarct size reduction to 13.5+/-2.3% (P<0.001). NECA treatment (200 nM) of intact rabbit hearts at reperfusion also resulted in phosphorylation of p70S6 kinase more than that seen in untreated hearts. This NECA-induced phosphorylation was blocked by rapamycin. These experiments reveal a critical role for p70S6 kinase in the signaling pathway of NECA's cardioprotection at reperfusion.

Influence of the combined treatment of LY 300164 (an AMPA/kainate receptor antagonist) with adenosine receptor agonists on the electroconvulsive threshold in mice.

7-Acetyl-5-(4-aminophenyl)-8,9-dihydro-8-methyl-7H-1,3-dioxolo-4,5H-2,3-benzodiazepine hydrochloride (LY 300164; a selective noncompetitive AMPA/kainate antagonist; 2 mg/kg) and N(6)-2-(4-aminophenyl)ethyl-adenosine (APNEA; a nonselective adenosine A(1)/A(3) receptor agonist; 2 and 3 mg/kg) significantly raised the threshold for electroconvulsions in mice. In contrast, 5'-N-ethylcarboxamidoadenosine (NECA; a nonselective adenosine A(1)/A(2) receptor agonist; up to 1 mg/kg) did not affect the electroconvulsive threshold. The combined treatment of LY 300164 with NECA or APNEA was superior to single-drug medication as regards their protective action in this seizure model. Moreover, the combinations of LY 300164 with either NECA or APNEA were devoid of motor impairment, although they produced a significant long-term memory deficit. Measurement of the plasma levels of LY 300164 alone and in combination with APNEA or NECA did not suggest pharmacokinetic phenomena as an explanation for the interaction between these drugs. APNEA did not influence the plasma concentration of LY 300164. Moreover, NECA even significantly decreased the plasma levels of the AMPA/kainate antagonist. The present study clearly indicates a strong positive interaction in terms of anticonvulsant activity between LY 300164 and the drugs acting via adenosine receptors.

Withdrawal and bidirectional cross-withdrawal responses in rats treated with adenosine agonists and morphine.

The aim of this study was to investigate whether the A1/A2 receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA), and the selective A1 agonist, N6-cyclopentyladenosine (CPA), induced physical dependence by quantifying specific antagonist-precipitated withdrawal syndromes in conscious rats. In addition, the presence of bidirectional cross-withdrawal was also investigated. The agonists were administered s.c. to groups of rats at 12 h intervals. Antagonists were administered s.c., 12 hours after the last dose, followed by observation and measurement of faecal output for 20 min. NECA (4 x 0.03 mg kg(-1), s.c) and CPA (4 x 0.03, 0.1 and 0.3 mg kg(-1), s.c.) induced physical dependence, as shown by the expression of a significant withdrawal syndrome when challenged with the adenosine A1/A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX, 0.1 mg kg(-1), s.c.) and the A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (CPDPX, 0.1 mg kg(-1), s.c.) respectively. The syndromes consisted of teeth chattering and shaking behaviours shown to occur in morphine-dependent animals withdrawn with naloxone viz, paw, body and 'wet-dog' shakes, but with the additional behaviours of head shaking and yawning. In further contrast to the opiate withdrawal syndrome, no diarrhoea occurred in the groups of animals treated with adenosine agonists and withdrawn with their respective antagonists. Bidirectional cross-withdrawal syndromes were also revealed when naloxone (3 mg kg(-1), s.c.) was administered to adenosine agonist pre-treated rats and adenosine antagonists were given to morphine pre-treated rats. This study provides further information illustrating that close links exist between the adenosine and opiate systems.

Adenosine agonists CGS 21680 and NECA inhibit the initiation of cocaine self-administration.

Administration of the adenosine antagonist caffeine will facilitate the reinstatement of cocaine self-administration responding. This suggests that adenosine receptors may play a role in the motivational systems that regulate cocaine-seeking behaviors. If so then adenosine agonists may act to block cocaine self-administration. To test this hypothesis, the effects of the nonselective adenosine agonist NECA and of the A2A selective agonist, CGS 21680 on the self-administration of cocaine were determined. In these experiments, rats were allowed to obtain intravenous cocaine infusions (0.6 mg/kg/infusion) delivered under a Fixed Ratio 5 schedule. Treatment with either NECA or CGS 21680 in comparison to vehicle administration reduced the number of infusions received per session. This, primarily, was due to a marked increase in the latency for delivery of the first cocaine infusion. Responding after drug-induced delays tended to be at control levels. Adenosine agonists are known to have sedative effects and these actions might play a role in NECA and CGS 21680-induced increases in latencies for cocaine delivery. These results indicate that the administration of adenosine agonists may inhibit cocaine-seeking behaviors. The degree to which these actions are on motivational systems as opposed to involving less specific effects remains to be fully elucidated.

New generic approach to the treatment of organophosphate poisoning: adenosine receptor mediated inhibition of ACh-release.

Current treatment of acute organophosphate (OP) poisoning includes a combined administration of a cholinesterase reactivator (oxime), a muscarinic receptor antagonist (atropine) and an anticonvulsant (diazepam). This treatment is not adequate since it does not prevent neuronal brain damage and incapacitation. Here, as in a recent review it is stated that other therapeutic approaches may improve protection. Former studies on the "direct effects" of oximes led to the conclusion that drug-induced inhibition of acetylcholine (ACh)-release shortly (1 min) after the acute OP-intoxication, could prevent and counteract convulsions and improve survival. In general, the accumulation of ACh in the synaptic cleft is considered to be responsible for the symptoms that ultimately lead to death. Therefore, prevention or suppression of this excessive accumulation of ACh could be a generic approach to antagonize OP-poisoning. Preliminary evidence for this concept has been put forward. Evaluation of drugs that would be able to prevent and counteract ACh accumulation, led to the conclusion that adenosine receptor agonists could be promising candidates. Pilot experiments demonstrated that intramuscular administration of the adenosine receptor agonists NECA (5'-N-ethylcarboxamido-adenosine) or CPA (N6-cyclopentyl adenosine) 1 min following a subcutaneous soman poisoning (1.5-2LD50) in rats, resulted in (1) prevention or postponement of chewing, salivation, convulsive activity, and respiratory distress (cholinergic symptoms), (2) improvement of survival rate (24 h), (3) a low level of extracellular brain ACh, as opposed to high levels of extracellular brain ACh in untreated animals. It is concluded that (1) adenosine agonists protect acutely soman-poisoned rats without the need of additional treatment with atropine, oxime or diazepam, (2) prevention of ACh accumulation in this way may be a new generic approach in the treatment of OP-poisoning.