Transgenic over expression of ectonucleotide triphosphate diphosphohydrolase-1 protects against murine myocardial ischemic injury

Modulation of purinergic signaling is critical to myocardial homeostasis. Ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD-1; CD39) which converts the proinflammatory molecules ATP or ADP to AMP is a key regulator of purinergic modulation. However, the salutary effects of transgenic over expression of ENTPD-1 on myocardial response to ischemic injury have not been tested to date. Therefore we hypothesized that ENTPD-1 over expression affords myocardial protection from ischemia–reperfusion injury via specific cell signaling pathways. ENTPD-1 transgenic mice, which over express human ENTPDase-1, and wild-type (WT) littermates were subjected to either ex vivo or in vivo ischemia–reperfusion injury. Infarct size, inflammatory cell infiltrate and intracellular signaling molecule activation were evaluated. Infarct size was significantly reduced in ENTPD-1 versus WT hearts in both ex vivo and in vivo studies. Following ischemia–reperfusion injury, ENTPD-1 cardiac tissues demonstrated an increase in the phosphorylation of the cellular signaling molecule extracellular signal-regulated kinases 1/2 (ERK 1/2) and glycogen synthase kinase-3β (GSK-3β). Resistance to myocardial injury was abrogated by treatment with a non-selective adenosine receptor antagonist, 8-SPT or the more selective A_2B adenosine receptor antagonist, MRS 1754, but not the A₁ selective antagonists, DPCPX. Additionally, treatment with the ERK 1/2 inhibitor PD98059 or the mitochondrial permeability transition pore opener, atractyloside, abrogated the cardiac protection provided by ENTPDase-1 expression. These results suggest that transgenic ENTPDase-1 expression preferentially conveys myocardial protection from ischemic injury via adenosine A_2B receptor engagement and associated phosphorylation of the cellular protective signaling molecules, Akt, ERK 1/2 and GSK-3β that prevents detrimental opening of the mitochondrial permeability transition pore.     

Potential use of DPCPX as probe for in vivo localization of brain A1 adenosine receptors

The suitability of (³H)DPCPX (8-cyclopentyl-1,3-dipropylxanthine), a xanthine derivative, as an vivo probe for labelling adenosine A₁ receptors was studied in rats. [³H]DPCPX (nM) penetrated largely into the brain (0.8% of the injected dose per gram of brain tissue 5 min after injection). Brain concentrations stayed at a plateau level from 5 to 15 min after the injection. The distribution in the different brain regions was heterogeneous with the highest amount of [³H]DPCPX in cerebellum and hippocampus and the lowest concentrations in hypothalamus and brain stem. Displacement (45–70% of total radioactivity) was obtained by the injection of 250 nM of cold DPCPX or cyclopentylxanthine, an analog of DPCPX. The ex vivo autoradiographic distribution of [³H]DPCPX was similar to the in vitro autoradiographic distribution of tritiated A₁ adenosine receptor ligand as [³H]CHA. These results suggest the potential use of DPCPX for further in vivo investigation of A₁ adenosine receptors with techniques such as positron emission tomography.     

Depression of synaptic transmission and evoked NMDA Ca2+ influx in hippocampal neurons by adenosine and its blockade by LTP or ischemia

Neuronal Ca²⁺ influx in response to repetitive synaptic activation was determined in rat hippocampal slices by measuring the evoked decreases of the extracellular Ca²⁺ concentration with ion-sensitive electrodes in the synaptic layer (ΔCaSpad) and in the CA₁ pyramidal cell layer (ΔCaSpyr). The generation of NMDA receptor-mediated Ca²⁺ fluxes (NMDA Ca²⁺ fluxes) was uncovered by measuring the proportion of ΔCa blocked by the NMDA receptor antagonist 2-amino-5-phosphonovalerate (APV); 1 μmol adenosine increased the critical input frequency required to generate synaptic MNDA Ca²⁺ influx. This effect is apparently exerted by limiting the postsynaptic membrane depolarization and is no longer seen, if the NMDA Ca²⁺ channels have lost their Mg-dependent voltage sensitivity. It has been recently reported by Ben-Ari and co-workers [(1992) Trends Neurosci 15:333–339] that such a loss of voltage sensitivity, leading to an “upregulation” and persistent generation of NMDA Ca²⁺ currents, Can be elicited by an activation of the protein kinase C (PKC) and may be responsible for the initiation of synaptic long-term potentiation (LTP) and for ischemia-induced nerve cell damage. Consistent with this hypothesis, we found that the depressive effect of 1 μmol adenosine on the generation of NMDA Ca²⁺ influx or synaptic transmission can be no longer elicited in hippocampal slices after LTP and after preceding transient brain ischemia in vivo. These findings suggest that the mosaic of different adenosine actions includes some which are related to PKC activation. Accordingly, we observed a synaptic modulation by adenosine which was characterized by a reduced Mg-sensitivity and blocked by phorbol ester treatment. © 1993 Wiley-Liss, Inc.     

Blockade of adenosine A(1) receptors prevents methylphenidate-induced impairment of object recognition task in adult mice

Methylphenidate (MPH) is the preferred treatment used for attention-deficit/hyperactivity disorder (ADHD). Recently, misuse for MPH due to its apparent cognitive enhancer properties has been reported. Adenosine is a neuromodulator known to exert influence on the dopaminergic neurotransmission, which is the main pharmacological target of MPH. We have reported that an overdosage of MPH up-regulates adenosine A₁ receptors in the frontal cortex, but this receptor was not involved in its anxiolytic effects. In this study, the role of adenosine A₁ receptor was investigated on MPH-induced effects on aversive and recognition memory in adult mice. Adult mice received acute and chronic (15 days) administration of methylphenidate (5 mg/kg, i.p.), or an acute overdosage (50 mg/kg, i.p) in order to model misuse. Memory was assessed in the inhibitory avoidance and object recognition task. Acute administration 5 mg/kg improved whereas 50 mg/kg disrupted recognition memory and decreased performance in the inhibitory avoidance task. Chronic administration did not cause any effect on memory, but decreased adenosine A₁ receptors immunocontent in the frontal cortex. The selective adenosine A₁ receptor antagonist, (DPCPX 1 mg/kg, i.p.), prevented methylphenidate-triggered recognition memory impairment. Our findings showed that recognition memory rather than aversive memory was differently affected by acute administration at both doses. Memory recognition was fully impaired by the overdosage, suggesting that misuse can be harmful for cognitive functions. The adenosinergic system via A₁ receptors may play a role in the methylphenidate actions probably by interfering with dopamine-enhancing properties of this drug.     

Adenosine and memory storage:

Rationale: Caffeine is a non-selective A₁/A₂ adenosine receptor antagonist which is known to improve cognitive performance in humans. This effect of caffeine has been attributed to its antagonism of adenosine receptors. Objective: The present study was devised to identify the role of A₁ and A_2A adenosine receptors in the facilitation of memory consolidation in mice performing a passive avoidance task. Methods: Adult albino Swiss male mice were used. The mice were trained in a step-through inhibitory avoidance task in which they were punished by a foot-shock (0.4 mA, 5 Hz, for 3 s) delivered through the grid floor. Caffeine (0.1, 0.3, 1.0 and 3.0 mg/kg), SCH 58261 (0.1, 0.3, 1.0 and 3.0 mg/kg) and DPCPX (0.1, 0.3, 1.0 and 3.0 mg/kg) were injected IP immediately or 180 min after training. The retention test was performed 24 h after training. Results: Caffeine and the selective A_2A adenosine receptor antagonist SCH 58261 facilitated retention when administered immediately after training, but not when administered 180 min later. The dose response was a bell-shaped curve. Conversely, post-training administration of the selective A₁ adenosine receptor antagonist DPCPX did not affect retention. Caffeine and SCH 58261 had no effect in mice not given the foot-shock on the training trial, a finding indicating that the drug’s effect on retention was specific. Conclusions: These results suggest that A_2A but not A₁ adenosine receptors are involved in memory retention and consolidation. Received: 14 February 1999 / Final version: 23 April 1999     

The effects of selective A1 and A2a adenosine receptor antagonists on cerebral ischemic injury in the gerbil

Cerebral ischemia of 5 min duration was induced in unanesthetized Mongolian gerbils by bilateral occlusion of the carotid arteries. The extent of ischemic injury was assessed behaviorally by measuring the increases in locomotor activity following ischemia and by a histopathological assessment of the extent of CA1 hippocampal pyramidal cell injury and loss 5 days after ischemia. The A_2a adenosine receptor selective antagonists 8-(3-chlorostyryl) caffeine (CSC; 0.1 mg/kg i.p.) and 4-amino-l-phenyl[1,2,4]-triazolo[4,3-a] quinoxaline (CP 66,713; 0.1 mg/kg i.p.) reduced the extent of ischemia-induced injury. An A1 selective receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1.0 mg/kg i.p.), enhanced ischemia-evoked injury. These results suggest that adenosine A_2a receptor antagonists may be useful for the prevention of cerebral injuries resulting from stroke or cardiac arrest.     

Effect of Curcumin Against Pentylenetetrazol‐Induced Seizure Threshold in Mice: Possible Involvement of Adenosine A1 Receptors

Curcumin, obtained from Curcuma longa, has been in use for manifold human disorders. The present study explores the effect of curcumin against pentylenetetrazol (PTZ) seizure threshold in mice. The possible involvement of adenosine receptor(s) mechanism was also investigated. Minimal dose of PTZ (i.v., mg/kg) needed to induce different phases of convulsions were recorded as an index of seizure threshold. Curcumin (20–120 mg/kg, p.o.) produced an increase in seizure threshold for convulsions induced by PTZ i.v. infusion. The anticonvulsant effect of curcumin (80 mg/kg) was prevented by 8‐phenyltheophylline (0.5 mg/kg, i.p., non‐selective adenosine receptor antagonist) and 8‐cyclopentyl‐1,3‐dipropylxanthine (5 mg/kg, i.p., adenosine A₁ receptor antagonist) but not by 8‐(3‐cholorostryl)caffeine (4 mg/kg, i.p., adenosine A_2A receptor antagonist). Further, 5′‐N‐ethylcarboxamidoadenosine (0.005 mg/kg, i.p., non‐selective A₁/A₂ receptor agonist), or N⁶‐cyclohexyladenosine (0.2 mg/kg, i.p., adenosine A₁ receptor agonist), was able to potentiate the anticonvulsant action of curcumin. In contrast, 5′‐(N‐cyclopropyl) carboxamidoadenosine (0.1 mg/kg, i.p., adenosine A_2A receptor agonist) failed to potentiate the effect of curcumin. This study demonstrated the anticonvulsant effect of curcumin against PTZ i.v. seizure threshold via a direct or indirect activation of adenosine A₁ but not A_2A receptors in mice. Thus, curcumin may prove to be an effective adjunct in treatment of convulsions. Copyright © 2013 John Wiley & Sons, Ltd.     

Fructose-1,6-bisphosphate reduces inflammatory pain-like behaviour in mice: role of adenosine acting on A1 receptors

Background and purpose:

D-Fructose-1,6-bisphosphate (FBP) is an intermediate in the glycolytic pathway, exerting pharmacological actions on inflammation by inhibiting cytokine production or interfering with adenosine production. Here, the possible antinociceptive effect of FBP and its mechanism of action in the carrageenin paw inflammation model in mice were addressed, focusing on the two mechanisms described above.

Experimental approach:

Mechanical hyperalgesia (decrease in the nociceptive threshold) was evaluated by the electronic pressure-metre test; cytokine levels were measured by elisa and adenosine was determined by high performance liquid chromatography.

Key results:

Pretreatment of mice with FBP reduced hyperalgesia induced by intraplantar injection of carrageenin (up to 54%), tumour necrosis factor α (40%), interleukin-1 β (46%), CXCL1 (33%), prostaglandin E₂ (41%) or dopamine (55%). However, FBP treatment did not alter carrageenin-induced cytokine (tumour necrosis factor α and interleukin-1 β) or chemokine (CXCL1) production. On the other hand, the antinociceptive effect of FBP was prevented by systemic and intraplantar treatment with an adenosine A₁ receptor antagonist (8-cyclopentyl-1,3-dipropylxanthine), suggesting that the FBP effect is mediated by peripheral adenosine acting on A₁ receptors. Giving FBP to mice increased adenosine levels in plasma, and adenosine treatment of paw inflammation presented a similar antinociceptive mechanism to that of FBP.

Conclusions and implications:

In addition to anti-inflammatory action, FBP also presents an antinociceptive effect upon inflammatory hyperalgesia. Its mechanism of action seems dependent on adenosine production but not on modulation of hyperalgesic cytokine/chemokine production. In turn, adenosine acts peripherally on its A₁ receptor inhibiting hyperalgesia. FBP may have possible therapeutic applications in reducing inflammatory pain.     

腺苷A1受体拮抗剂对L—NAME预处理第二窗心肌保护作用的影响

目的探讨药物L—NAME预处理后第二窗心肌保护作用是否与腺苷A1受体激活有关。方法将30只SD大鼠随机分成3组,每组10只,即缺血对照组、L—NAME预处理组及拮抗剂组,每组均于给药后24h建立离体大鼠心脏Langendorff灌注模型,全心缺血60min,再灌注120min,于S15、R1、R60、R1204个时间点观察HR、LVDP、CF的变化,检测冠脉流出液中cTnI的变化,电镜观察缺血后心肌的超微结构改变。结果全组心率缺血前无差别,拮抗剂组缺血后心率较L—NAME组慢,两者差异有统计学意义（P〈0．05）,与缺血对照组无差别;拮抗剂组的LVDP较L—NAME预处理组低,两者差异有统计学意义（P〈0．05）。与缺血对照组无差别;拮抗剂组的CF较L—NAME预处理组少,两者差异有统计学意义（P〈0．05）,与缺血对照组无差别：拮抗剂组的cTnI较L-NAME预处理组显著增高（P〈0．05）,与缺血对照组无差别;电镜下拈抗剂组及缺血对照组心肌超微结构的损伤程度较L—NAME预处理组重。结论腺苷A1受体拮抗剂DPCPX阻断了L—NAME预处理第二窗的心肌保护作用,腺苷A1受体可能是L-NAME预处理引起心肌保护作用的启动因子。     

Adenosine antagonists have differential effects on induction of long-term potentiation in hippocampal slices

How adenosine leakage and tetanic release might affect long-term potentiation (LTP) was investigated by applying adenosine antagonists 8(p-sulfophenyl)theophylline (8SPT) or 8-cyclopentyl-3, 7-dihydro-1, 3-dipropyl-1H-purine-2, 6-dione (DPCPX) to slices, while recording CA1 field EPSPs and population spikes. In the first series of experiments, we applied weak double tetani (at 100 Hz, for 1 s) that were subliminal for evoking LTP in initial control runs. In the presence of 8SPT—at concentrations (10–50 μM) which block both A₁ and A₂ receptors—the same tetani consistently evoked LTP of population spikes but not of excitatory postsynaptic potentials (EPSPs), whereas DPCPX (50 nM), which blocks only A₁ receptors, facilitated LTP of both EPSPs and population spikes. These results are consistent with previous evidence that tetanic adenosine release on the one hand depresses LTP via A₁ receptors but on the other facilitates LTP via A₂ receptors. In a second set of experiments, 8SPT (50–100 μM) did not prevent the induction of LTP of both EPSPs and population spikes by stronger tetanic stimulation. Therefore A₂ receptor activation is not essential for the induction of LTP when stronger tetani are applied. Overall, the main effect of endogenous adenosine release is to oppose LTP induction. © 1995 Wiley-Liss, Inc.