Acute effects of alcohol on sleep are mediated by components of homeostatic sleep regulatory system: An Editorial Highlight for 'Lesions of the basal forebrain cholinergic neurons attenuates sleepiness and adenosine after alcohol consumption' on page 710.

Alcohol causes adenosine buildup, which inhibits wake-active neurons via adenosine A1 receptors thus disinhibiting sleep active neurons and also stimulates sleep-active neurons via A2A receptors, causing sleep. This editorial highlights the study entitled, "Lesions of the basal forebrain cholinergic neurons attenuates sleepiness and adenosine after alcohol consumption" by Sharma and colleagues. They report that the wake-promoting basal forebrain (BF) cholinergic neurons play a crucial role in mediating acute alcohol-induced sleep via adenosinergic signaling.

Adenosine 2A receptors modulate reward behaviours for methamphetamine.

Addiction to methamphetamine (METH) is a global health problem for which there are no approved pharmacotherapies. The adenosine 2A (A2 A ) receptor presents a potential therapeutic target for METH abuse due to its modulatory effects on striatal dopamine and glutamate transmission. Notably, A2 A receptor signalling has been implicated in the rewarding effects of alcohol, cocaine and opiates; yet, the role of this receptor in METH consumption and seeking is essentially unknown. Therefore, the current study used A2 A knockout (KO) mice to assess the role of A2 A in behaviours relevant to METH addiction. METH conditioned place preference was absent in A2 A KO mice compared with wild-type (WT) littermates. Repeated METH treatment produced locomotor sensitization in both genotypes; however, sensitization was attenuated in A2 A KO mice in a dose-related manner. METH intravenous self-administration was intact in A2 A KO mice over a range of doses and schedules of reinforcement. However, the motivation to self-administer was reduced in A2 A KO mice. Regression analysis further supported the observation that the motivation to self-administer METH was reduced in A2 A KO mice even when self-administration was similar to WT mice. Sucrose self-administration was also reduced in A2 A KO mice but only at higher schedules of reinforcement. Collectively, these data suggest that A2 A signalling is critically required to integrate rewarding and motivational properties of both METH and natural rewards.

A2 Adenosine Receptor-mediated Cardioprotection Against Reperfusion Injury in Rat Hearts Is Associated With Autophagy Downregulation.

This study was undertaken to determine and confer the cardioprotective effects of the adenosine A2 receptor (A2AR) and its impact on myocardial autophagy in the setting of reperfusion. We established a rat ischemia model by subjecting rats to 30 minutes of ischemia (I) and 120 minutes of reperfusion (R). The A2AR agonists CGS21680 (A2aAR specific) and BAY60-6583 (A2bAR specific) were administered separately and in combination 5 minutes before reperfusion (postconditioning). No visible improvements in the rats' hemodynamic changes in response to either CGS or BAY were observed compared with untreated control groups (I/R). BAY significantly reduced infarct sizes, whereas CGS did not. Electron microscopy, enzyme-linked immunosorbent assay and TUNEL apoptosis staining results demonstrated that CGS and BAY play cardioprotective roles by maintaining mitochondria structural stability, decreasing serum cardiac troponin I (cTnI) concentrations and decreasing the number of apoptotic cells. CGS21680 and BAY60-6583 slightly increased the expression (vs. I/R group) of Bcl-2 and significantly attenuated the expression of Beclin-1, LC3B, and LAMP-2, as analyzed by Western blot, compared with the I/R alone group. Notably, BAY60-6583 exerts a predominant effect on mitochondria structural stabilization, apoptotic inhibition, and attenuation of LC3B/LAMP-2 expression. No synergistic effects were observed for the 2 agonists. Our data suggest that A2AR-mediated cardioprotection is associated with Beclin-1-induced autophagy downregulation in the setting of reperfusion. A2bAR activation exerts stronger cardioprotective effects against I/R injury compared with A2aAR.

Adenosine and dopamine receptors coregulate photoreceptor coupling via gap junction phosphorylation in mouse retina.

Gap junctions in retinal photoreceptors suppress voltage noise and facilitate input of rod signals into the cone pathway during mesopic vision. These synapses are highly plastic and regulated by light and circadian clocks. Recent studies have revealed an important role for connexin36 (Cx36) phosphorylation by protein kinase A (PKA) in regulating cell-cell coupling. Dopamine is a light-adaptive signal in the retina, causing uncoupling of photoreceptors via D4 receptors (D4R), which inhibit adenylyl cyclase (AC) and reduce PKA activity. We hypothesized that adenosine, with its extracellular levels increasing in darkness, may serve as a dark signal to coregulate photoreceptor coupling through modulation of gap junction phosphorylation. Both D4R and A2a receptor (A2aR) mRNAs were present in photoreceptors, inner nuclear layer neurons, and ganglion cells in C57BL/6 mouse retina, and showed cyclic expression with partially overlapping rhythms. Pharmacologically activating A2aR or inhibiting D4R in light-adapted daytime retina increased photoreceptor coupling. Cx36 among photoreceptor terminals, representing predominantly rod-cone gap junctions but possibly including some rod-rod and cone-cone gap junctions, was phosphorylated in a PKA-dependent manner by the same treatments. Conversely, inhibiting A2aR or activating D4R in daytime dark-adapted retina decreased Cx36 phosphorylation with similar PKA dependence. A2a-deficient mouse retina showed defective regulation of photoreceptor gap junction phosphorylation, fairly regular dopamine release, and moderately downregulated expression of D4R and AC type 1 mRNA. We conclude that adenosine and dopamine coregulate photoreceptor coupling through opposite action on the PKA pathway and Cx36 phosphorylation. In addition, loss of the A2aR hampered D4R gene expression and function.

The adenosine A2A receptor agonist CGS 21680 decreases ethanol self-administration in both non-dependent and dependent animals.

There is emerging evidence that the adenosinergic system might be involved in drug addiction and alcohol dependence. We have already demonstrated the involvement of A2A receptors (A2AR) in ethanol-related behaviours in mice. Here, we investigated whether the A2AR agonist CGS 21680 can reduce ethanol operant self-administration in both non-dependent and ethanol-dependent Wistar rats. To rule out a potential involvement of the A1R in the effects of CGS 21680, we also tested its effectiveness to reduce ethanol operant self-administration in both heterozygous and homozygous A1R knockout mice. Our results demonstrated that CGS 21680 (0.065, 0.095 and 0.125 mg/kg, i.p.) had a bimodal effect on 10% ethanol operant self-administration in non-dependent rats. The intermediate dose was also effective in reducing 2% sucrose self-administration. Interestingly, the intermediate dose reduced 10% ethanol self-administration in dependent animals more effectively (75% decrease) when compared with non-dependent animals (57% decrease). These results suggest that the A2AR are involved in CGS 21680 effects since the reduction of ethanol self-administration was not dependent upon the presence of A1R in mice. In conclusion, our findings demonstrated the effectiveness of the A2AR agonist CGS 21680 in a preclinical model of alcohol addiction and suggested that the adenosinergic pathway is a promising target to treat alcohol addiction.

Adenosine receptor signaling modulates permeability of the blood-brain barrier.

The blood-brain barrier (BBB) is comprised of specialized endothelial cells that form the capillary microvasculature of the CNS and is essential for brain function. It also poses the greatest impediment in the treatment of many CNS diseases because it commonly blocks entry of therapeutic compounds. Here we report that adenosine receptor (AR) signaling modulates BBB permeability in vivo. A(1) and A(2A) AR activation facilitated the entry of intravenously administered macromolecules, including large dextrans and antibodies to ß-amyloid, into murine brains. Additionally, treatment with an FDA-approved selective A(2A) agonist, Lexiscan, also increased BBB permeability in murine models. These changes in BBB permeability are dose-dependent and temporally discrete. Transgenic mice lacking A(1) or A(2A) ARs showed diminished dextran entry into the brain after AR agonism. Following treatment with a broad-spectrum AR agonist, intravenously administered anti-ß-amyloid antibody was observed to enter the CNS and bind ß-amyloid plaques in a transgenic mouse model of Alzheimer's disease (AD). Selective AR activation resulted in cellular changes in vitro including decreased transendothelial electrical resistance, increased actinomyosin stress fiber formation, and alterations in tight junction molecules. These results suggest that AR signaling can be used to modulate BBB permeability in vivo to facilitate the entry of potentially therapeutic compounds into the CNS. AR signaling at brain endothelial cells represents a novel endogenous mechanism of modulating BBB permeability. We anticipate these results will aid in drug design, drug delivery and treatment options for neurological diseases such as AD, Parkinson's disease, multiple sclerosis and cancers of the CNS.

Dysfunction of neurotransmitter modulation system on adrenergic nerves of caudal artery in type 2 diabetic Goto-Kakizaki rats.

The Goto-Kakizaki (GK) rat is a non-obese and spontaneous model of mild Type 2 diabetes mellitus. In the present study, we compared the regulatory mechanisms of endogenous norepinephrine (NE) release from sympathetic nerves of caudal arteries of 12-week-old GK rats and age-matched normal Wistar rats. Electrical stimulation (ES) evoked significant NE release from caudal arteries of Wistar and GK rats. The amounts of NE released by ES were almost equal in Wistar and GK rats, although the NE content in caudal artery of GK rats was significantly lower than that of Wistar rats. We examined the effects of an α2-adrenoceptor agonist, clonidine (CLO), and an α2-adrenoceptor antagonist, yohimbine (YOH), on the release of endogenous NE evoked by ES. CLO significantly reduced NE release from caudal arteries of Wistar but not GK rats. On the other hand, YOH significantly increased NE release from both rats. Furthermore, we examined the effects of an A1-adenosine receptor agonist, 2-chloroadenosine (2CA), and an A1-adenosine receptor antagonist, 8-sulfophenyltheophylline (8SPT), on the release of endogenous NE evoked by ES. 2CA significantly reduced NE release from caudal arteries of Wistar but not GK rats. On the other hand, 8SPT did not affect NE release from both rats. These results suggest that the dysfunction of negative feedback regulation of NE release via presynaptic receptors on sympathetic nerves in GK rats may be involved in the autonomic nervous system dysfunction associated with diabetic autonomic neuropathy.

Pollen metabolome analysis reveals adenosine as a major regulator of dendritic cell-primed T(H) cell responses.

BACKGROUND: Water-soluble components from pollen modulate dendritic cell (DC) functions, such as IL-12 secretion and 3'-5'-cyclic adenosine monophosphate (cAMP) signaling and migration, possibly contributing to the establishment of a T(H)2-dominated immune response against pollen. Because these effects could not solely be attributed to the previously identified pollen-associated lipid mediators, the pollen metabolome was analyzed for candidate immunomodulatory substances. OBJECTIVE: We sought to perform an analysis of the effect of pollen-associated adenosine on DC function and T(H) cell differentiation. METHODS: Fractions of aqueous pollen extracts (APEs) were generated by means of ultrafiltration and were subjected simultaneously to biological tests and metabolome analysis (ultra-high-resolution mass spectrometry) and ultraperformance liquid chromatography. Effects of pollen-derived adenosine on monocyte-derived DC cAMP signaling, cytokine response, and capacity to differentiate T(H) cells were studied. RESULTS: The less than 3-kd fraction of APEs comprised thousands of substances, including adenosine in micromolar concentrations. Pollen-derived adenosine mediated A2 receptor-dependent induction of cAMP and inhibition of IL-12p70 in DCs. APEs digested with adenosine deaminase failed to mediate IL-12 inhibition. DCs of nonatopic donors exposed to APEs showed an adenosine-dependent reduced capacity to differentiate T(H)1 cells and an enhanced capacity to induce regulatory T cells and IL-10. DCs of atopic donors failed to induce IL-10 but instead induced IL-5 and IL-13. CONCLUSION: This study identifies adenosine out of thousands of metabolites as a potent immunoregulatory substance in pollen. It acts on the level of the DC, with differential effects in atopic and nonatopic donors.

Adenosine A2 receptors are involved in the activation of ATP-sensitive K+ currents during metabolic inhibition in guinea pig ventricular myocytes.

It has been hypothesized that an interaction among adenosine A(1) receptors, protein kinase C (PKC) activation, and ATP-sensitive potassium channels (K(ATP)) mediates ischemic preconditioning in experiments on different animal species. The purpose of this study was to determine if activation of K(ATP) is functionally coupled to A(1) receptors and (or) PKC activation during metabolic inhibition (MI) in guinea pig ventricular myocytes. Perforated-patch using nystatin and conventional whole-cell recording methods were used to observe the effects of adenosine and adenosine-receptor antagonists on the activation of K(ATP) currents during MI induced by application of 2,4-dinitrophenol (DNP) and 2-deoxyglucose (2DG) without glucose, in the presence or absence of a PKC activator, phorbol 12-myristate 13-acetate (PMA). Adenosine accelerated the time course activation of K(ATP) currents during MI under the intact intracellular condition or dialyzed condition with l mmol/L ATP in the pipette solution. The accelerated effect of adenosine activation of K(ATP) under MI was not reversed by a nonselective Al adenosine receptor antagonist, 8-(p-sulfophenyl)theophylline (SPT), or a specific Al adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). However, the adenosine A(2) receptor antagonist alloxazine reversed the time course activation of the K(ATP) current under MI. An adenylate cyclase activator, forskolin, did not further abbreviate the time course activation of K(ATP) with or without adenosine. Application of a PKC blocker, chelerythrine, reversed the time course activation of K(ATP) by adenosine under MI. In addition, pretreatment with a PKC activator, PMA, had similar effects to adenosine, while adenosine did not further shorten the time required for activation of K(ATP) currents during MI with PMA pretreatment. There is no direct evidence of activation of K(ATP) currents by adenosine A(1) receptor during metabolic inhibition under our experimental condition. However, adenosine A(2) receptor activation is involved in the K(ATP) channel activation in the guinea pig ventricular myocytes, of which effect is not mediated through the increase in intracellular cAMP. Adenosine seems to interact with PKC activation to open K(ATP) during MI, but a possible link between the adenosine A(2) receptor and PKC activation in this process needs further elucidation.

Methylxanthines and sleep.

Caffeine is widely used to promote wakefulness and counteract fatigue induced by restriction of sleep, but also to counteract the effects of caffeine abstinence. Adenosine is a physiological molecule, which in the central nervous system acts predominantly as an inhibitory neuromodulator. Adenosine is also a sleep-promoting molecule. Caffeine binds to adenosine receptors, and the antagonism of the adenosinergic system is believed to be the mechanism through which caffeine counteracts sleep in humans as well as in other species. The sensitivity for caffeine varies markedly among individuals. Recently, genetic variations in genes related to adenosine metabolism have provided at least a partial explanation for this variability. The main effects of caffeine on sleep are decreased sleep latency, shortened total sleep time, decrease in power in the delta range, and sleep fragmentation. Caffeine may also decrease the accumulation of sleep propensity during waking, thus inducing long-term harmful effects on sleep quality.

Modulation of Blood-Brain Barrier Permeability by Activating Adenosine A2 Receptors in Oncological Treatment.

The blood-brain barrier (BBB) plays an important protective role in the central nervous system and maintains its homeostasis. It regulates transport into brain tissue and protects neurons against the toxic effects of substances circulating in the blood. However, in the case of neurological diseases or primary brain tumors, i.e., gliomas, the higher permeability of the blood-derived substances in the brain tissue is necessary. Currently applied methods of treatment for the primary brain neoplasms include surgical removal of the tumor, radiation therapy, and chemotherapy. Despite the abovementioned treatment methods, the prognosis of primary brain tumors remains bad. Moreover, chemotherapy options seem to be limited due to low drug penetration into the cancerous tissue. Modulation of the blood-brain barrier permeability may contribute to an increase in the concentration of the drug in the CNS and thus increase the effectiveness of therapy. Interestingly, endothelial cells in cerebral vessels are characterized by the presence of adenosine 2A receptors (A2AR). It has been shown that substances affecting these receptors regulate the permeability of the BBB. The mechanism of increasing the BBB permeability by A2AR agonists is the actin-cytoskeletal reorganization and acting on the tight junctions. In this case, the A2AR seems to be a promising therapy target. This article aims to assess the possibility of increasing the BBB permeability through A2AR agonists to increase the effectiveness of chemotherapy and to improve the results of cancer therapy.

Adenosine A(1) and A(2A) receptors in mouse prefrontal cortex modulate acetylcholine release and behavioral arousal.

During prolonged intervals of wakefulness, brain adenosine levels rise within the basal forebrain and cortex. The view that adenosine promotes sleep is supported by the corollary that N-methylated xanthines such as caffeine increase brain and behavioral arousal by blocking adenosine receptors. The four subtypes of adenosine receptors are distributed heterogeneously throughout the brain, yet the neurotransmitter systems and brain regions through which adenosine receptor blockade causes arousal are incompletely understood. This study tested the hypothesis that adenosine A(1) and A(2A) receptors in the prefrontal cortex contribute to the regulation of behavioral and cortical arousal. Dependent measures included acetylcholine (ACh) release in the prefrontal cortex, cortical electroencephalographic (EEG) power, and time to waking after anesthesia. Sleep and wakefulness were also quantified after microinjecting an adenosine A(1) receptor antagonist into the prefrontal cortex. The results showed that adenosine A(1) and A(2A) receptors in the prefrontal cortex modulate cortical ACh release, behavioral arousal, EEG delta power, and sleep. Additional dual microdialysis studies revealed that ACh release in the pontine reticular formation is significantly altered by dialysis delivery of adenosine receptor agonists and antagonists to the prefrontal cortex. These data, and early brain transection studies demonstrating that the forebrain is not needed for sleep cycle generation, suggest that the prefrontal cortex modulates EEG and behavioral arousal via descending input to the pontine brainstem. The results provide novel evidence that adenosine A(1) receptors within the prefrontal cortex comprise part of a descending system that inhibits wakefulness.

Adenosine protected against pulmonary edema through transporter- and receptor A2-mediated endothelial barrier enhancement.

We have previously demonstrated that adenosine plus homocysteine enhanced endothelial basal barrier function and protected against agonist-induced barrier dysfunction in vitro through attenuation of RhoA activation by inhibition of isoprenylcysteine-O-carboxyl methyltransferase. In the current study, we tested the effect of elevated adenosine on pulmonary endothelial barrier function in vitro and in vivo. We noted that adenosine alone dose dependently enhanced endothelial barrier function. While adenosine receptor A(1) or A(3) antagonists were ineffective, an adenosine transporter inhibitor, NBTI, or a combination of DPMX and MRS1754, antagonists for adenosine receptors A(2A) and A(2B), respectively, partially attenuated the barrier-enhancing effect of adenosine. Similarly, inhibition of both A(2A) and A(2B) receptors with siRNA also blunted the effect of adenosine on barrier function. Interestingly, inhibition of both transporters and A(2A)/A(2B) receptors completely abolished adenosine-induced endothelial barrier enhancement. The adenosine receptor A(2A) and A(2B) agonist, NECA, also significantly enhanced endothelial barrier function. These data suggest that both adenosine transporters and A(2A) and A(2B) receptors are necessary for exerting maximal effect of adenosine on barrier enhancement. We also found that adenosine enhanced Rac1 GTPase activity and overexpression of dominant negative Rac1 attenuated adenosine-induced increases in focal adhesion complexes. We further demonstrated that elevation of cellular adenosine by inhibition of adenosine deaminase with Pentostatin significantly enhanced endothelial basal barrier function, an effect that was also associated with enhanced Rac1 GTPase activity and with increased focal adhesion complexes and adherens junctions. Finally, using a non-inflammatory acute lung injury (ALI) model induced by alpha-naphthylthiourea, we found that administration of Pentostatin, which elevated lung adenosine level by 10-fold, not only attenuated the development of edema before ALI but also partially reversed edema after ALI. The data suggest that adenosine deaminase inhibition may be useful in treatment of pulmonary edema in settings of ALI.

Adenosine receptor interactions alter cardiac contractility in rat heart.

1. The effect of the adenosine A(2) receptor (AdoA(2)R) agonist N(6)-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA) on adenosine A(1) receptor (AdoA(1)R)-mediated negative inotropic responses was investigated in rat heart. 2. Hearts from male Wistar rats (250-350 g) were perfused with Krebs'-Henseleit solution at constant flow in non-recirculating Langendorff mode. Hearts were paced at 5 Hz (5 ms duration, supramaximal voltage) via ventricular electrodes. After 30 min equilibration, (R)-N(6)-phenylisopropyl adenosine (R-PIA) concentration-response curves were constructed in the absence or presence of DPMA. 3. In paced hearts, R-PIA induced concentration-dependent decreases in triple product (heart rate x peak systolic developed pressure x dP / dt(max)), which were significantly attenuated by 1 nmol / L DPMA with a shift in pEC(50) from 8.0 +/- 0.5 (n = 9) in control hearts to 6.63 +/- 1.03 (n = 5) in treated tissues (P < 0.05). The AdoA(2A)R antagonist 8-(3-chlorostyryl)caffeine (1 micromol / L) and the adenylyl cyclase inhibitor cis-N-(2-phenylcyclopentyl)-azacyclotridec-1-en-2-amine hydrochloride (MDL12330A; 100 nmol / L) reversed the effects of DPMA on AdoA(1)R-mediated negative inotropic actions, whereas the AdoA(2B)R antagonist alloxazine (3 micromol / L) had no effect on DPMA activity. 4. The results of the present study show that stimulation of the AdoA(2)R attenuates AdoA(1)R-dependent reductions in inotropic state. The receptor involved appears to be the AdoA(2A)R and its action involves stimulation of adenylyl cyclase activity.

Adenosine A2A receptors enable the synaptic effects of cannabinoid CB1 receptors in the rodent striatum.

Adenosine A(2A), cannabinoid CB(1) and metabotropic glutamate 5 (mGlu(5)) receptors are all highly expressed in the striatum. The aim of the present work was to investigate whether, and by which mechanisms, the above receptors interact in the regulation of striatal synaptic transmission. By extracellular field potentials (FPs) recordings in corticostriatal slices, we demonstrated that the ability of the selective type 1 cannabinoid receptor (CB(1)R) agonist WIN55,212-2 to depress synaptic transmission was prevented by the pharmacological blockade or the genetic inactivation of A(2A)Rs. Such a permissive effect of A(2A)Rs towards CB(1)Rs does not seem to occur pre-synaptically as the ability of WIN55,212-2 to increase the R2/R1 ratio under a protocol of paired-pulse stimulation was not modified by ZM241385. Furthermore, the effects of WIN55,212-2 were reduced in slices from mice lacking post-synaptic striatal A(2A)Rs. The selective mGlu(5)R agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) potentiated the synaptic effects of WIN55,212-2, and such a potentiation was abolished by A(2A)R blockade. Unlike the synaptic effects, the ability of WIN55,212-2 to prevent NMDA-induced toxicity was not influenced by ZM241385. Altogether, these results show that the state of activation of A(2A)Rs regulates the synaptic effects of CB(1)Rs and that A(2A)Rs may control CB(1) effects also indirectly, namely through mGlu(5)Rs.

Activation of A(2) adenosine receptors dilates cortical efferent arterioles in mouse.

Adenosine can induce vasodilatation and vasoconstriction of the renal afferent arteriole of the mouse. We determined here its direct effect on efferent arterioles of mouse kidneys. Using isolated-perfused cortical efferent arterioles, we measured changes in luminal diameter in response to adenosine. Extraluminal application of adenosine and cyclohexyladenosine had no effect on the luminal diameter. When the vessels were constricted by the thromboxane mimetic U46619, application of adenosine and 5'-N-ethylcarboxamido-adenosine dilated the efferent arterioles in a dose-dependent manner. We also found that the adenosine-induced vasodilatation was inhibited by the A(2)-specific receptor blocker 3,7-dimethyl-1-propargylxanthine. In the presence of this inhibitor, adenosine failed to alter the basal vessel diameter of quiescent efferent arterioles. Using primer-specific polymerase chain reaction we found that the adenosine A(1), A(2a), A(2b), and A(3) receptors were expressed in microdissected mouse efferent arterioles. We conclude that adenosine dilates the efferent arteriole using the A(2) receptor subtype at concentrations compatible with activation of the A(2b) receptor.

New evidence for purinergic signaling in the olfactory bulb: A2A and P2Y1 receptors mediate intracellular calcium release in astrocytes.

Purinergic receptors play a key role in neuron-glia and glia-neuron interactions. In the present study, we have recorded cytosolic Ca(2+) responses using confocal imaging in astrocytes of acute olfactory bulb slices from mice (postnatal days 3-8). By application of agonists and antagonists, we identified two types of receptors, P2Y(1) and A(2A), that mediated Ca(2+) responses attributable to Ca(2+) release from intracellular stores in the astrocytes. Both receptor types were activated by application of ATP and ADP; however, when enzymatic ATP degradation was suppressed by the alkaline phosphatase inhibitor levamisole, ATP only activated MRS2179-sensitive P2Y(1) but not ZM241385-sensitive A(2A) receptors. The dose-response curve for A(2A) receptors activated by adenosine revealed an EC(50) of 0.3 microM, one order of magnitude smaller than the EC(50) of 5 microM determined for P2Y(1) receptors activated by ADP. Electrical stimulation of the olfactory nerve in the presence of glutamate receptor blockers to suppress excitation of postsynaptic neurons evoked Ca(2+) responses in most of the astrocytes, which were inhibited by blocking both P2Y(1) and A(2A) receptors. Our results indicate that olfactory nerve terminals release not only glutamate, but also ATP, which activates P2Y(1) receptors and, after degradation of ATP to adenosine, A(2A) receptors in astrocytes.

A1 adenosine receptor activation promotes angiogenesis and release of VEGF from monocytes.

Adenosine is a proangiogenic purine nucleoside released from ischemic and hypoxic tissues. Of the 4 adenosine receptor (AR) subtypes (A1, A2A, A2B, and A3), the A2 and A3 have been previously linked to the modulation of angiogenesis. We used the chicken chorioallantoic membrane (CAM) model to determine whether A1 AR activation affects angiogenesis. We cloned and pharmacologically characterized chicken AR subtypes to evaluate the selectivity of various agonists and antagonists. Application of the A1 AR-selective agonist N6-cyclopentyladenosine (CPA; 100 nmol/L) to the CAM resulted in a 40% increase in blood vessel number (P<0.01), which was blocked by the A1 AR-selective antagonist C8-(N-methylisopropyl)-amino-N6-(5'-endohydroxy)-endonorbornan-2-yl-9-methyladenine (WRC-0571; 1 micromol/L). Selective A2A AR agonists did not stimulate angiogenesis in the CAM. In an ex vivo rat aortic ring model of angiogenesis that includes cocultured endothelial cells, fibroblasts, and smooth muscle cells, 50 nmol/L CPA did not directly stimulate capillary formation; however, medium from human mononuclear cells pretreated with CPA, but not vehicle, increased capillary formation by 48% (P<0.05). This effect was blocked by WRC-0571 (1.5 micromol/L) or anti-VEGF antibody (1 microg/mL). CPA (5 nmol/L) stimulated a 1.7-fold increase in VEGF release from the mononuclear cells. This is the first study to show that A1 AR activation induces angiogenesis. Stimulation of A2 ARs on endothelial cells results in proliferation and tube formation, and A2 and A3 ARs on inflammatory cells modulate release of angiogenic factors. We conclude that adenosine promotes a coordinated angiogenic response through its interactions with multiple receptors on multiple cell types.

Adenosine receptor antagonists improve short-term object-recognition ability of spontaneously hypertensive rats: a rodent model of attention-deficit hyperactivity disorder.

The strain of spontaneously hypertensive rats (SHR) is considered a genetic model for the study of attention-deficit hyperactivity disorder (ADHD), as it displays hyperactivity, impulsivity and poorly sustained attention. Recently, we have shown the involvement of adenosinergic neuromodulation in the SHR's short-term and long-term memory impairments. In this study, we investigated the performance of male and female SHR in a modified version of the object-recognition task (using objects with different structural complexity) and compared them with Wistar rats, a widely used outbred rat strain for the investigation of learning processes. The suitability of the SHR strain to represent an animal model of ADHD, as far as mnemonic deficits are concerned, was pharmacologically validated by the administration of methylphenidate, the first-choice drug for the treatment of ADHD patients. The role of adenosine A1 and A2A receptors in object discrimination was investigated by the administration of caffeine (nonselective antagonist) or selective adenosine receptor antagonists. Wistar rats discriminated all the objects used (cube vs. pyramid; cube vs. T-shaped object), whereas SHR only discriminated the most structurally distinct pairs of objects (cube vs. pyramid). Pretraining administration of methylphenidate [2 mg/kg, intraperitoneal (i.p.)], caffeine (1-10 mg/kg, i.p.), the selective adenosine receptor antagonists DPCPX (8-cyclopenthyl-1,3-dipropylxanthine; A1 antagonist, 5 mg/kg, i.p.) and ZM241385 (A2A antagonist, 1.0 mg/kg, i.p.), or the association of ineffective doses of DPCPX (3 mg/kg) and ZM241385 (0.5 mg/kg), improved the performance of SHR in the object-recognition task. These findings show that the discriminative learning impairments of SHR can be attenuated by the blockade of either A1 or A2A adenosine receptors, suggesting that adenosinergic antagonists might represent potentially interesting drugs for the treatment of ADHD.

A2A receptors in inflammation and injury: lessons learned from transgenic animals.

Adenosine regulates the function of the innate and adaptive immune systems through targeting virtually every cell type that is involved in orchestrating an immune/inflammatory response. Of the four adenosine receptors (A(1), A(2A), A(2B), A(3)), A(2A) receptors have taken center stage as the primary anti-inflammatory effectors of extracellular adenosine. This broad, anti-inflammatory effect of A(2A) receptor activation is a result of the predominant expression of A(2A) receptors on monocytes/macrophages, dendritic cells, mast cells, neutrophils, endothelial cells, eosinophils, epithelial cells, as well as lymphocytes, NK cells, and NKT cells. A(2A) receptor activation inhibits early and late events occurring during an immune response, which include antigen presentation, costimulation, immune cell trafficking, immune cell proliferation, proinflammatory cytokine production, and cytotoxicity. In addition to limiting inflammation, A(2A) receptors participate in tissue remodeling and reparation. Consistent with their multifaceted, immunoregulatory action on immune cells, A(2A) receptors have been shown to impact the course of a wide spectrum of ischemic, autoimmune, infectious, and allergic diseases. Here, we review the regulatory roles of A(2A) receptors in immune/inflammatory diseases of various organs, including heart, lung, gut, liver, kidney, joints, and brain, as well as the role of A(2A) receptors in regulating multiple organ failure and sepsis.