Adenosine A1 and A2A receptors are involved on guanosine protective effects against oxidative burst and mitochondrial dysfunction induced by 6-OHDA in striatal slices.

6-Hydroxydopamine (6-OHDA) is the most used toxin in experimental Parkinson's disease (PD) models. 6-OHDA shows high affinity for the dopamine transporter and once inside the neuron, it accumulates and undergoes non-enzymatic auto-oxidation, promoting reactive oxygen species (ROS) formation and selective damage of catecholaminergic neurons. In this way, our group has established a 6-OHDA in vitro protocol with rat striatal slices as a rapid and effective model for screening of new drugs with protective effects against PD. We have shown that co-incubation with guanosine (GUO, 100 µM) prevented the 6-OHDA-induced damage in striatal slices. As the exact GUO mechanism of action remains unknown, the aim of this study was to investigate if adenosine A1 (A1R) and/or A2A receptors (A2AR) are involved on GUO protective effects on striatal slices. Pre-incubation with DPCPX, an A1R antagonist prevented guanosine effects on 6-OHDA-induced ROS formation and mitochondrial membrane potential depolarization, while CCPA, an A1R agonist, did not alter GUO effects. Regarding A2AR, the antagonist SCH58261 had similar protective effect as GUO in ROS formation and mitochondrial membrane potential. Additionally, SCH58261 did not affect GUO protective effects. The A2AR agonist CGS21680, although, completely blocked GUO effects. Finally, the A1R antagonist DPCPX, and the A2AR agonist CGS21680 also abolished the preventive guanosine effect on 6-OHDA-induced ATP levels decrease. These results reinforce previous evidence for a putative interaction of GUO with A1R-A2AR heteromer as its molecular target and clearly indicate a dependence on adenosine receptors modulation to GUO protective effect.

Electroacupuncture Pretreatment Alleviates Cerebral Ischemia-Reperfusion Injury by Increasing GSK-3ß Phosphorylation Level via Adenosine A1 Receptor.

OBJECTIVE: To observe the effect of adenosine A1 receptor in the hippocampus of mice on GSK-3ß phosphorylation level and elucidate the underlying mechanisms of electroacupuncture pretreatment by activating Α1 receptor mediating cerebral ischemia-reperfusion injury. METHOD: The model of middle cerebral artery occlusion (MCAO) was established and grouped into electroacupuncture pretreatment group (EA group), MCAO group, and sham-operated group (Sham group). The neurobehavioral manifestation, the volume of cerebral infarction, and its related protein changes in mice in each group were observed. Then, adenosine Α1 receptor antagonist and agonist were injected intraperitoneally to observe the effects of A1 receptor on the phosphorylation level of GSK-3ß phosphorylation level and elucidate the underlying mechanisms of electroacupuncture pretreatment by activating Α1 receptor mediating cerebral ischemia-reperfusion injury. RESULTS: (1) Compared with the MCAO group (24 hours after reperfusion), the infarct size in the EA group decreased significantly, and the Garcia neurological score and phosphorylation level of GSK-3ß phosphorylation level and elucidate the underlying mechanisms of electroacupuncture pretreatment by activating Α1 receptor mediating cerebral ischemia-reperfusion injury. ß phosphorylation level and elucidate the underlying mechanisms of electroacupuncture pretreatment by activating Α1 receptor mediating cerebral ischemia-reperfusion injury. ß phosphorylation level and elucidate the underlying mechanisms of electroacupuncture pretreatment by activating Α1 receptor mediating cerebral ischemia-reperfusion injury. CONCLUSIONS: Electroacupuncture pretreatment can increase GSK-3ß phosphorylation level via activating A1 receptor, to protect neurons in ischemia-reperfusion injury.ß phosphorylation level and elucidate the underlying mechanisms of electroacupuncture pretreatment by activating Α1 receptor mediating cerebral ischemia-reperfusion injury.

Role of the adenosine system and glucose restriction in the acute anticonvulsant effect of caprylic acid in the 6 Hz psychomotor seizure test in mice.

Although several studies have reported the acute anticonvulsant activity of caprylic acid in animal seizure models, little is known about the mechanism underlying this effect. Recently, the role of adenosine in the efficacy of the ketogenic diet has been postulated. Therefore, the present study aimed to evaluate the possible involvement of the adenosine system (in non-fasted mice) as well as the role of glucose restriction (in fasted and non-fasted mice) in the acute anticonvulsant activity of caprylic acid in the 6 Hz psychomotor seizure threshold test. We showed that the anticonvulsant effect of caprylic acid (30 mmol/kg, p.o.) was reversed by a selective adenosine A1 receptor antagonist (DPCPX, 1mg/kg, i.p.) and a selective adenosine A2A receptor antagonist (KW-6002, 1 mg/kg, p.o.) but not by glibenclamide (1 pg/mouse, i.c.v.) - the ATP-sensitive potassium (KATP) channel blocker. Co-administration of an ineffective dose of caprylic acid (20 mmol/kg) with an ineffective dose of adenosine transporter inhibitor (dipyridamole, 50 mg/kg, i.p.) significantly raised the threshold for the 6 Hz-induced seizures. A high dose of glucose (2 g/kg) significantly only diminished the anticonvulsant effect of caprylic acid (30 mmol/kg) in non-fasted mice, and this was accompanied by an increase in blood glucose level and no changes in ketone body level as compared to the caprylic acid-treated group. In both fasted and non-fasted mice treated with glucose and caprylic acid, a significant decrease in trunk blood pH occurred as compared to the control group. No alternations in motor coordination or muscular strength were noted with any drug treatment, apart from the caprylic acid and glibenclamide combination, where a significant decrease in the muscle strength was observed. The present study provides a new insight into the role of the adenosine system and low glucose usage in the mechanisms underlying the anticonvulsant effects of caprylic acid in the 6 Hz seizure test.

Chronic treatment with ticagrelor limits myocardial infarct size: an adenosine and cyclooxygenase-2-dependent effect.

OBJECTIVE: In a phase III clinical trial (PLATelet inhibition and patient Outcomes, PLATO), ticagrelor provided better clinical outcomes than clopidogrel in patients with acute coronary syndromes. In addition to P2Y12-receptor antagonism, ticagrelor prevents cell uptake of adenosine and has proven able to augment adenosine effects. Adenosine protects the heart against ischemia-reperfusion injury. We compared the effects of clopidogrel and ticagrelor on myocardial infarct size (IS). APPROACH AND RESULTS: Rats received oral ticagrelor (0, 75, 150, or 300 mg/kg/d) or clopidogrel (30 or 90 mg/kg/d) for 7 days and underwent 30-minute coronary artery ligation and 24-hour reperfusion. Area at risk was assessed by blue dye and IS by 2,3,5-triphenyl-tetrazolium-chloride. Cyclooxygenase-2 (COX2) enzyme activity was assessed by ELISA and expression by real-time polymerase chain reaction. Mechanism responsible was explored using adenosine-receptor antagonist (CGS15943, an A2A/A1 antagonist) or cyclooxygenase inhibition by either aspirin (5, 10, or 25 mg/kg) or specific cyclooxygenase-1 (SC560) or COX2 (SC5815) inhibitors. Ticagrelor, dose-dependently, reduced IS, whereas clopidogrel had no effect. Adenosine-receptor antagonism blocked the ticagrelor effect and COX2 inhibition by SC5815, or high-dose aspirin attenuated the IS-limiting effect of ticagrelor, whereas cyclooxygenase-1 inhibition or low-dose aspirin had no effect. Ticagrelor, but not clopidogrel, upregulated COX2 expression and activity. Also this effect was blocked by adenosine-receptor antagonism. Ticagrelor, but not clopidogrel, increased Akt and endothelial nitric oxide synthase phosphorylation. CONCLUSIONS: Ticagrelor, but not clopidogrel, reduces myocardial IS. The protective effect of ticagrelor was dependent on adenosine-receptor activation with downstream upregulation of endothelial nitric oxide synthase and COX2 activity.

Role of adenosine and the orexinergic perifornical hypothalamus in sleep-promoting effects of ethanol.

STUDY OBJECTIVES: Strong clinical and preclinical evidence suggests that acute ethanol promotes sleep. However, very little is known about how and where ethanol acts to promote sleep. We hypothesized that ethanol may induce sleep by increasing extracellular levels of adenosine and inhibiting orexin neurons in the perifornical hypothalamus. DESIGN: Experiments 1 and 2: Within-Subject Design; Experiment 3: Between-Subject Design. SETTING: N/A. PATIENTS OR PARTICIPANTS: N/A. INTERVENTIONS: N/A. MEASUREMENTS AND RESULTS: Using adult male Sprague-Dawley rats as our animal model, we performed three experiments to test our hypothesis. Our first experiment examined the effect of A1 receptor blockade in the orexinergic perifornical hypothalamus on sleep- promoting effects of ethanol. Bilateral microinjection of the selective A1 receptor antagonist 1,3-dipropyl-8-phenylxanthine (500 µM; 250 nL/side) into orexinergic perifornical hypothalamus significantly reduced nonrapid eye movement sleep with a concomitant increase in wakefulness, suggesting that blockade of adenosine A1 receptor attenuates ethanol-induced sleep promotion. Our second experiment examined adenosine release in the orexinergic perifornical hypothalamus during local ethanol infusion. Local infusion of pharmacologically relevant doses of ethanol significantly and dose-dependently increased adenosine release. Our final experiment used c-Fos immunohistochemistry to examine the effects of ethanol on the activation of orexin neurons. Acute ethanol exposure significantly reduced the number of orexin neurons containing c-Fos, suggesting an inhibition of orexin neurons after ethanol intake. CONCLUSIONS: Based on our results, we believe that ethanol promotes sleep by increasing adenosine in the orexinergic perifornical hypothalamus, resulting in A1 receptor-mediated inhibition of orexin neurons.

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 A2A receptor antagonist). Further, 5'-N-ethylcarboxamidoadenosine (0.005 mg/kg, i.p., non-selective A1 /A2 receptor agonist), or N(6) -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 A2A 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 A2A receptors in mice. Thus, curcumin may prove to be an effective adjunct in treatment of convulsions.

Activation of central adenosine A(2A) receptors lowers the seizure threshold of hyperthermia-induced seizure in childhood rats.

Adenosine is a potent neuromodulator in the central nervous system (CNS). The functional deterioration of adenosine A(1) receptors in the CNS was reported to cause a failure of termination of seizures and to a lower seizure threshold of hyperthermia-induced seizures (HS) in childhood rats, which may contribute to adenosine-related convulsive disorders such as theophylline-associated seizures in childhood patients. In contrast to the inhibitory effect of adenosine A(1) receptors, the function of adenosine A(2A) receptors remains controversial. To clarify the function of adenosine A(2A) receptors in childhood convulsive disorders associated to hyperthermia, we investigated the in vivo interaction between adenosine A(2A) receptors and their ligands in HS in childhood rats. Adenosine selective A(2A) receptor ligands were injected intraperitoneally before HS. We measured brain temperature at the onset of seizures and the mortality rate after HS. We found that brain temperature at seizure onset was significantly higher in the A(2A) receptor antagonist group compared with that in the control group (p<0.05), and there was no significant difference in mortality among the groups. In contrast, brain temperature at seizure onset was significantly lower in the A(2A) receptor agonist group compared with that in the control group (p<0.05), and mortality was significantly higher in the A(2A) agonist group compared with that in the control group (p<0.001). The activation of the adenosine A(2A) receptor might enhance seizures associated to hyperthermia in the childhood human brain, and be involved in the pathogenesis of sudden unexpected death in epilepsy (SUDEP) in childhood patients with convulsive disorders.

A role for adenosine A(1) receptor blockade in the ability of caffeine to promote MDMA "Ecstasy"-induced striatal dopamine release.

Co-administration of caffeine profoundly enhances the acute toxicity of 3,4 methylenedioxymethamphetamine (MDMA) in rats. The aim of this study was to determine the ability of caffeine to impact upon MDMA-induced dopamine release in superfused brain tissue slices as a contributing factor to this drug interaction. MDMA (100 and 300µM) induced a dose-dependent increase in dopamine release in striatal and hypothalamic tissue slices preloaded with [(3)H] dopamine (1µM). Caffeine (100µM) also induced dopamine release in the striatum and hypothalamus, albeit to a much lesser extent than MDMA. When striatal tissue slices were superfused with MDMA (30µM) in combination with caffeine (30µM), caffeine enhanced MDMA-induced dopamine release, provoking a greater response than that obtained following either caffeine or MDMA applications alone. The synergistic effects in the striatum were not observed in hypothalamic slices. As adenosine A(1) receptors are, one of the main pharmacological targets of caffeine, which are known to play an important role in the regulation of dopamine release, their role in the modulation of MDMA-induced dopamine release was investigated. 1µM 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a specific A(1) antagonist, like caffeine, enhanced MDMA-induced dopamine release from striatal slices while 1µM 2,chloro-N(6)-cyclopentyladenosine (CCPA), a selective adenosine A(1) receptor agonist, attenuated this. Treatment with either SCH 58261, a selective A(2A) receptor antagonist, or rolipram, a selective PDE-4 inhibitor, failed to reproduce a caffeine-like effect on MDMA-induced dopamine release. These results suggest that caffeine regulates MDMA-induced dopamine release in striatal tissue slices, via inhibition of adenosine A(1) receptors.