Facilitation of noradrenaline release by adenosine A(2A) receptors in the epididymal portion and adenosine A(2B) receptors in the prostatic portion of the rat vas deferens

The adenosine-receptor modulation of noradrenaline release was compared in prostatic and epididymal portions of rat vas deferens. In both portions, tritium overflow elicited by electrical stimulation (100 pulses/8 Hz) was reduced by the adenosine A(1) receptor agonist, N(6)-cyclopentyladenosine, and enhanced by the nonselective receptor agonist, 5'-N-ethylcarboxamidoadenosine, in the presence of the adenosine A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 20 and 100 nM). The adenosine A(2A) receptor agonist, 2-p-(2-carboxyethyl)phenethyl-amino-5'-N-ethylcarboxamidoadenosine, increased tritium overflow, but only in the epididymal portion. The enhancement caused by NECA was prevented by the adenosine A(2A) receptor antagonist, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385; 20 nM), in the epididymal and by the adenosine A(2B) receptor antagonist, alloxazine (1 microM), in the prostatic portion. Inhibition of adenosine uptake enhanced tritium overflow in both portions, an effect blocked by ZM 241385 in the epididymal and by alloxazine in the prostatic portion. The results indicate that adenosine exerts an adenosine A(1) receptor-mediated inhibition, in both portions, and facilitation mediated by adenosine A(2A) receptors in the epididymal and by A(2B) receptors in the prostatic portion.     

Region-specific neuroprotective effect of ZM 241385 towards glutamate uptake inhibition in cultured neurons

Active uptake by neurons and glial cells is the main mechanism for maintaining extracellular glutamate at low, non-toxic concentrations. Adenosine A_2A receptors regulate extracellular glutamate levels by acting on both the release and the uptake of glutamate. The aim of this study was to evaluate whether the inhibition of the effects of glutamate uptake blockers by adenosine A_2A receptor antagonists resulted in neuroprotection. In cortical and striatal neuronal cultures, the application of l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC, a transportable competitive inhibitor of glutamate uptake), induced a dose-dependent increase in lactate dehydrogenase (LDH) levels, an index of cytotoxicity. Such an effect of PDC was significantly reduced by pre-treatment with the adenosine A_2A receptor antagonist ZM 241385 (50 nM) in striatal, but not cortical, cultures. The protective effects of ZM 241385 were specifically due to a counteraction of PDC effects, since ZM 241385 was totally ineffective in preventing the cytotoxicity induced by direct application of glutamate to cultures. These results indicate that adenosine A_2A receptor antagonists prevent the toxic effects induced by a transportable competitive inhibitor of glutamate uptake, that such an effect specifically occurs in the striatum and that it does not depend on a direct blockade of glutamate-induced toxicity.     

Tonic adenosine A1 and A2A receptor activation is required for the excitatory action of VIP on synaptic transmission in the CA1 area of the hippocampus

Adenosine can regulate synaptic transmission through modulation of the action of other neurotransmitters. The influence of adenosine on VIP enhancement of synaptic transmission in hippocampal slices was investigated. Facilitation of fEPSP slope by 1 nM VIP (23.3+/-1.3%) was turned into an inhibition (-12.1+/-3.4%) when extracellular endogenous adenosine was removed using adenosine deaminase (ADA, 1U/ml). Blockade of adenosine A(1) receptors with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10 nM) or of A(2A) receptors with ZM241385 (20 nM) attenuated the effect of VIP. When both DPCPX and ZM241385 were present the effect of VIP was abolished. In the presence of ADA, selective A(1) receptor activation with N(6)-cyclopentyladenosine (CPA, 15 nM) or A(2A) receptor-activation with CGS21680 (10 nM) partially readmitted the excitatory effect of VIP on fEPSPs. In contrast, facilitation of PS amplitude by 1 nM VIP (19.1+/-1.2%) was attenuated in the presence of ADA or DPCPX but was not changed by ZM241385. CPA, in the presence of ADA, fully restored the effect of VIP on PS amplitude. In conclusion, VIP facilitation of synaptic transmission to hippocampal pyramidal cell dendrites is dependent on both A(1) and A(2A) receptor activation by endogenous adenosine. VIP effects on PS amplitude are only dependent on A(1) adenosine receptor activation. This differential sensitivity to adenosine modulation might be due to the different VIP circuits contributing to VIP effects on pyramidal cell dendrites and pyramidal cell bodies.     

ZM 241385, an adenosine A(2A) receptor antagonist, inhibits hippocampal A(1) receptor responses

4-(2-[7-amino-2-(2-furyl?1,2,4?-triazolo?2,3a?-?1,3, 5?triazin-5-yl-amino]ethyl)phenol (ZM 241385) has been used as an antagonist of adenosine A(2A) receptors, exhibiting high selectivity over adenosine A(1) receptors. We now report that ZM 241385 (10-50 nM) attenuated the inhibitory action of N(6)-cyclopentyladenosine (10 nM) and R(-)-N(6)-phenylisopropyladenosine (R-PIA, 20 nM), two selective adenosine A(1) receptor agonists, on hippocampal population spike amplitude. This effect is unlikely to be a direct antagonism of adenosine A(1) receptor since the K(i) of ZM 241385 to displace [3H]PIA (2 nM) binding, from hippocampal membranes ranged from 0.8 to 1.9 microM. These results question the usefulness of ZM 241385 to define adenosine A(2A) receptors actions in functional studies.     

Adenosine A2A receptor stimulation decreases GAT-1-mediated GABA uptake in the globus pallidus of the rat

We examined modulation of [(3)H]GABA uptake in slices of the rat globus pallidus because stimulation of adenosine A(2A) receptors increases extracellular GABA in this structure. Pharmacological analysis showed that GAT-1 is the main transporter present in these slices. Both adenosine and the A(2A) agonist CGS 21680 reduced GABA uptake. Antagonist ZM 241385 prevented these effects. Agents that increase protein kinase A activity like forskolin and 8-bromo-cAMP also inhibited GABA uptake. The inhibition of uptake produced by these substances and by CGS 21680 was prevented by the protein kinase A blocker H-89. The protein phosphatase blocker okadaic acid reduced uptake; this effect and the response to CGS 21680 were not additive. The effective concentrations of adenosine (EC(50)=15.2microM) are within the range measured in the interstitial fluid under some physiological conditions. Thus, inhibition of uptake may be important in increasing interstitial GABA during endogenous adenosine release.     

The role of adenosine A(2) receptors in the regulation of TNF-alpha production and PGE(2) release in mouse peritoneal macrophages

The adenosine A(2) receptors are known to mediate most of the anti-inflammatory activities of adenosine. In lipopolysaccharides (LPS)-stimulated macrophages adenosine strongly inhibits TNF-alpha release, but may also enhance PGE(2) generation. The aims of this study were to determine the relative contributions of the A(2A) and A(2B) receptor subclasses in these two effects and to determine whether the enhanced release of PGE(2) contributes to the inhibition of TNF-alpha release. In LPS-stimulated mouse macrophages, adenosine potently inhibited TNF-alpha production and also potentiated PGE(2) release, though less potently (IC(50)=250 nM vs EC(50) approximately 8 microM, respectively). The non-selective adenosine receptor agonist NECA, and the selective A(2A) receptor agonist CGS21680 also inhibited TNF-alpha production even more potently (IC(50)=4.8 and 2.3 nM, respectively). NECA, but not CGS21680, also enhanced PGE(2) production. The selective A(2A) receptor antagonist ZM241385 (30 nM), but not the selective A(2B) receptor antagonist MRS1754 (30 nM), blocked the inhibitory effect of NECA and CGS21680 on TNF-alpha release. On the other hand, MRS1754, but not ZM241385, abolished the PGE(2) potentiating effect of NECA. Pre-treatment with indomethacin (1 microM) abolished adenosine-induced PGE(2) release enhancement but did not prevent the inhibition of TNF-alpha release. These results show that in this system, the inhibition of TNF-alpha release by adenosine is mediated by the A(2A) receptors whereas the enhancement of PGE(2) release appears to be mediated by the A(2B) receptors. The results also show that while exogenous PGE(2) is a potent inhibitor of TNF-alpha release, the enhanced PGE(2) release induced by adenosine does not appear to contribute to the inhibition of TNF-alpha release.     

Functional interaction between pre-synaptic α6β2-containing nicotinic and adenosine A2A receptors in the control of dopamine release in the rat striatum

Background and Purpose

Pre-synaptic nicotinic ACh receptors (nAChRs) and adenosine A_2A receptors (A_2ARs) are involved in the control of dopamine release and are putative therapeutic targets in Parkinson''s disease and addiction. Since A_2ARs have been reported to interact with nAChRs, here we aimed at mapping the possible functional interaction between A_2ARs and nAChRs in rat striatal dopaminergic terminals.

Experimental Approach

We pharmacologically characterized the release of dopamine and defined the localization of nAChR subunits in rat striatal nerve terminals in vitro and carried out locomotor behavioural sensitization in rats in vivo.

Key Results

In striatal nerve terminals, the selective A_2AR agonist {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 inhibited, while the A_2AR antagonist ZM241385 potentiated the nicotine-stimulated [³H]dopamine ([³H]DA) release. Upon blockade of the α6 subunit-containing nAChRs, the remaining nicotine-stimulated [³H]DA release was no longer modulated by A_2AR ligands. In the locomotor sensitization experiments, nicotine enhanced the locomotor activity on day 7 of repeated nicotine injection, an effect that no longer persisted after 1 week of drug withdrawal. Notably, ZM241385-injected rats developed locomotor sensitization to nicotine already on day 2, which remained persistent upon nicotine withdrawal.

Conclusions and Implications

These results provide the first evidence for a functional interaction between nicotinic and adenosine A_2AR in striatal dopaminergic terminals, with likely therapeutic consequences for smoking, Parkinson''s disease and other dopaminergic disorders.     

Adenosine A2A receptor antagonism increases striatal glutamate outflow in dopamine-denervated rats

The objective of the work was to study, by in vivo microdialysis, the effect of the adenosine 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) on glutamate outflow in the striata of unilateral 6-hydroxydopamine-infused rats. Two vertical microdialysis probes were implanted bilaterally in both the denervated striatum and in the intact striatum. Glutamate concentrations in the dialysate were determined by high-performance liquid chromatography (HPLC). Infusion of the adenosine A(2A) receptor antagonist SCH 58261 (50 nM), through the microdialysis fiber, significantly increased glutamate outflow from the denervated striatum while it decreased glutamate outflow from the intact striatum. The opposite effects of SCH 58261 on glutamate outflow in the intact and 6-hydroxydopamine-lesioned striatum might be attributed to blockade of striatal adenosine A(2A) receptors located on either striatal indirect output pathways or glutamatergic terminals. These results may be relevant to our understanding of the mechanism of action of adenosine A(2A) receptor antagonists in Parkinson's disease.     

Functional expression of adenosine A2A and A3 receptors in the mouse dendritic cell line XS-106

There is increasing evidence to suggest that adenosine receptors can modulate the function of cells involved in the immune system. For example, human dendritic cells derived from blood monocytes have recently been described to express functional adenosine A1, A2A and A3 receptors. Therefore, in the present study, we have investigated whether the recently established murine dendritic cell line XS-106 expresses functional adenosine receptors. The selective adenosine A3 receptor agonist 1-[2-chloro-6[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-beta-D-ribofuranuronamide (2-Cl-IB-MECA) inhibited forskolin-mediated [3H]cyclic AMP accumulation and stimulated concentration-dependent increases in p42/p44 mitogen-activated protein kinase (MAPK) phosphorylation. The selective adenosine A2A receptor agonist 4-[2-[[-6-amino-9-(N-ethyl-beta-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzene-propanoic acid (CGS 21680) stimulated a robust increase in [3H]cyclic AMP accumulation and p42/p44 MAPK phosphorylation. In contrast, the selective adenosine A1 receptor agonist CPA (N6-cyclopentyladenosine) did not inhibit forskolin-mediated [3H]cyclic AMP accumulation or stimulate increases in p42/p44 MAPK phosphorylation. These observations suggest that XS-106 cells express functional adenosine A2A and A3 receptors. The non-selective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) inhibited lipopolysaccharide-induced tumour necrosis factor-alpha (TNF-alpha) release from XS-106 cells in a concentration-dependent fashion. Furthermore, treatment with Cl-IB-MECA (1 microM) or CGS 21680 (1 microM) alone produced a partial inhibition of lipopolysaccharide-induced TNF-alpha release (when compared to NECA), whereas a combination of both agonists resulted in the inhibition of TNF-alpha release comparable to that observed with NECA alone. Treatment of cells with the adenosine A2A receptor selective antagonists 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5ylamino]ethyl)phenol (ZM 241385; 100 nM) and 5-amino-2-(2-furyl)-7-phenylethyl-pyrazolo[4,3-e]-1,2,4-triazolo[1,5c]pyrimidine (SCH 58261; 100 nM) and the adenosine A3 receptor selective antagonist N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-benzeneacetamide (MRS 1220; 100 nM) partially blocked the inhibitory effects of NECA on lipopolysaccharide-induced TNF-alpha release. Combined addition of MRS 1220 and SCH 58261 completely blocked the inhibitory effects of NECA on lipopolysaccharide-induced TNF-alpha release. In conclusion, we have shown that the mouse dendritic cell line XS-106 expresses functional adenosine A2A and A3 receptors, which are capable of modulating TNF-alpha release.     

Solubilisation and immunoprecipitation of rat striatal adenosine A(2A) receptors.

In the present study, we have sought to solubilise adenosine A(2A) receptors from rat striatal membranes using a variety of different detergents. Of the detergents tested, 1% CHAPS (3-[(3-deoxycholic acid (cholamidopropyl) dimethylammonio]-1-propanesulfonate) yielded optimal conditions for solubilisation (in the presence of 3 mg/ml protein, 44% of receptor was solubilised, 50% of total protein was solubilised). An antipeptide antibody was raised against a 15 amino-acid sequence within the predicted third intracellular loop region of the human and rat adenosine A(2A) receptor. The antibody was coupled to protein A immobilised on sepharose CL-4B and used to immunoprecipitate adenosine A(2A) receptors from solubilised rat striatal preparations. Radioligand-binding studies were performed using the selective adenosine A(2) antagonist [(3)H]ZM 241385 (4-(2-[7-amino-2-(2-fury1)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol). Using [(3)H]ZM 241385, the pharmacology of immunoprecipitated adenosine A(2A) receptors was composed to striatal membrane bound adenosine A(2A) receptors and detergent solubilised adenosine A(2A) receptors. [(3)H]ZM 241385 labelled a single saturable binding site with high affinity in all three preparations (membrane bound K(d) 2.7 nM+/-1.0; solubilised K(d) 1.9 nM+/-0.3; immunoprecipitated K(d) 2.2 nM+/-0.7). Additionally, all three assays confirmed a rank order of potency for displacers consistent with adenosine A(2A) receptor pharmacology: ZM 241385>KW 6002 ((E)-8-[2-(3,4-dimethoxyphenyl)ethynyl]-1-3-diethyl-3,7-dihydro-7-methyl-1-purine 2,6 dione)>CGS 21680, (2-(4-(2 carboxyethyl)phenylethylamino)-5'-N-ethylcarboxamidoadenosine)>DPCPX (8-cyclopentyl-1,3-dipropylxanthine). We conclude that we have solubilised and immunoprecipitated adenosine A(2A) receptors from rat striatum and that their pharmacology is consistent with native striatal adenosine A(2A) receptors.     

Hypotensive effects of intravenously administered uridine and cytidine in conscious rats: involvement of adenosine receptors

In the present study, we investigated the cardiovascular effects of intravenously injected uridine or cytidine, and the role of adenosine receptors in mediating these effects, in conscious normotensive rats. Intravenous (i.v.) administration of uridine (124, 250, 500 mg/kg) dose-dependently decreased arterial pressure and heart rate. Cytidine (124, 250, 500 mg/kg; i.v.) produced slight dose-related hypotension without changing heart rate. Plasma uridine and cytidine concentrations increased time- and dose-dependently while plasma adenosine levels did not change after injection of the respective nucleosides. Pretreatment with intravenous caffeine (20 mg/kg), 8-phenyltheophylline (8-PT) (1 mg/kg), nonselective adenosine receptor antagonists, or 8-p-sulfophenyltheophylline (8-SPT) (20 mg/kg), a nonselective adenosine receptor antagonist which does not cross the blood-brain barrier, abolished the cardiovascular effects of uridine (250 mg/kg; i.v.) or cytidine (250 mg/kg; i.v.). Intracerebroventricular (i.c.v.) caffeine (200 microg) or 8-SPT (50 microg) pretreatment did not change the magnitude of the cardiovascular responses induced by nucleosides. Intravenous 8-cyclopenthyl-1,3-dipropylxanthine (DPCPX) (5 mg/kg), a selective adenosine A(1) receptor antagonist, greatly attenuated the cardiovascular responses to uridine and cytidine. Pretreatment with 3,7,-dimethyl-1-propargylxanthine (DMPX) (2 mg/kg), an adenosine A(1)/A(2) receptor antagonist, attenuated hypotension induced by uridine and blocked the arterial pressure decrease in response to cytidine. Uridine-induced bradycardia was blocked by DMPX. 4-(2-[7-amino-2-(2-furyl[1,2,4]-triazolo[2,3-a[1,3,5]triazin-5-yl-aminoethyl)phenol (ZM241385) (1 mg/kg; i.v.), a selective adenosine A(2A) receptor antagonist, pretreatment produced an only very small blockade in the first minute of the hypotensive effects of uridine without affecting the bradycardia. ZM241385 pretreatment completely blocked cytidine's hypotensive effect. In Langendorff-perfused rat heart preparation, uridine (10(-3) M), but not cytidine, decreased the heart rate. Our results show that intravenously injected uridine or cytidine is able to decrease arterial pressure by activating peripheral adenosine receptors. The data also implicates that the mainly adenosine A(1) receptor activation is involved in the uridine-induced cardiovascular effects, while both adenosine A(1) and A(2A) receptor activations mediate the cytidine's effects.     

Adenosine agonists can both inhibit and enhance in vivo striatal dopamine release

The effects on striatal dopamine (DA) release and metabolism by local administration of adenosine analogues with different affinities for the A1 and A2 adenosine receptors, were investigated using the technique of microdialysis. 2-Chloroadenosine (2-CADO), decreased levels of both dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) in striatal dialysates at concentrations of 1-10 microM; a similar effect was also seen after administration of N6-(2-phenylisopropyl)adenosine (R(-)-PIA) and 5'-N-ethylcarboxamido-adenosine (NECA) at a concentration of 10 microM. The inhibitory action of 2-CADO on DA release and metabolism was blocked by the selective A1 antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT), suggesting mediation by A1 receptors. 2-CADO at a concentration of 100 microM increased levels of both DA and DOPAC in striatal perfusates, an effect which was not inhibited by CPT. This stimulatory action on extracellular DA concentration in striatum was also seen with 10 microM 5'-(N-cyclopropyl)-carboxamidoadenosine (CPCA) a relatively selective A2 adenosine agonist. These results suggest that adenosine agonists enhance or depress striatal DA release depending on the nature and concentration of the drug.     

The role of adenosine A1 and A2A receptors of entorhinal cortex on piriform cortex kindled seizures in rats.

In this research the role of adenosine A1 and A2A receptors of the entorhinal cortex on piriform cortex kindled seizures was investigated. In piriform cortex kindled rats, N6-cyclohexyladenosine (CHA), a selective A1 receptor agonist, 1,3-dimethyl-8 cyclopenthylxanthine (CPT), a selective A1 receptor antagonist, CGS21680 hydrochloride (CGS), a selective A2A receptor agonist and ZM241385 (ZM), a selective A2A receptor antagonist were injected into the entorhinal cortex bilaterally. Five minutes later, animals were stimulated and seizure parameters were recorded. CHA (10 and 100microM) decreased the afterdischarge duration (ADD), stage 5 seizure duration (S5D), and seizure duration (SD), and increased the latency to stage 4 of the seizure (S4L) significantly. Bilateral microinjection of CPT (100microM) increased ADD, S(5)D, and SD, and reduced S(4)L significantly. Pretreatment of animals with CPT (50microM) before CHA (100microM), reduced the effect of CHA on seizure parameters. On the other hand, CGS (1mM) increased only ADD. Bilateral microinjection of ZM had no effect on seizure parameters. However, pretreatment of animals with ZM (200microM) before CGS (1mM), eliminated the excitatory effect of CGS on seizure parameters. These results suggest that activation of A1 receptors of the entorhinal cortex has an anticonvulsant, but activation of A2A receptors of this region has a proconvulsive effect on piriform cortex kindled seizures.     

Antagonism of coronary artery relaxation by adenosine A2A-receptor antagonist ZM241385

We have tested the existence of functional A2A adenosine receptor in porcine coronary artery using, for the first time, the new A2A antagonist ZM241385. Nonselective agonist NECA and A2A-selective agonist CGS21680 produced concentration-dependent relaxation of prostaglandin F2alpha (PGF2alpha)-precontracted endothelium intact (E+) and denuded (E-) rings. Relaxation was significantly greater in E+ rings than in E-rings. A2A adenosine receptor-selective antagonist, ZM241385 (10(-6) M), significantly attenuated the relaxation responses. The antagonism of ZM241385 was compared with that of SCH58261 (10(-6)M), another A2A adenosine receptor-selective antagonist, which also significantly attenuated the relaxation response to both agonists. However, ZM241385 produced a significantly greater shift of the relaxation-response curves to the right compared with SCH58261 both in E+ and E- rings. The data show for the first time that ZM241385 is a potent A2A-receptor antagonist in porcine coronary artery and a useful tool to study A2A-receptor function.     

Allosteric modulation of A(2A) adenosine receptors by amiloride analogues and sodium ions

Allosteric regulation of rat A(2A) adenosine receptors by amiloride, amiloride analogues, and sodium ions was studied by investigating their ability to influence the dissociation of [(3)H]4-2-[7-amino-2-(2-furyl)-1,2,4-triazolo[1,5-a][1,3, 5]triazin-5-yl-amino]ethylphenol ([(3)H]ZM241385) from receptors in rat striatal membranes. Both amiloride and its analogues accelerated the dissociation, the analogues being more potent than amiloride itself. In contrast, sodium ions decreased the rate of [(3)H]ZM241385 dissociation in a concentration-dependent manner, and this rate was not influenced by guanosine triphosphate, N-ethylmaleimide, suramin, or the selective A(2A) adenosine receptor antagonist, 5-amino-2-(2-furyl)-7(2-phenylethyl)pyrazolo[4,3-e]-1,2, 4-triazolo[1,5-c]pyrimidine (SCH58261). The effect of competition between the amiloride analogue 5-(N,N-hexamethylene)amiloride (HMA) and sodium ions on [(3)H]ZM241385 dissociation was also explored. The addition of sodium ions resulted in a concentration-dependent rightward shift of the HMA response curve. The slopes of the HMA concentration-response curves in the presence and absence of sodium ions were not significantly different, which suggests that sodium ions and amiloride analogues act at a common allosteric site on the A(2A) adenosine receptor. There was a lack of correlation between the displacement of ligand binding and the allosteric potencies of the amiloride analogues.     

Modulation of A2A adenosine receptors and associated Galphas proteins by ZM 241385 treatment of porcine coronary artery

Functional regulation and expression of the adenosine A2A receptor and associated G-protein were investigated in porcine coronary artery exposed to an A2A receptor antagonist, ZM 241385 (4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol). The arteries were incubated for 3 days in culture medium in the absence (control) and presence (treated) of 10 microM ZM 241385. Changes in isometric tension by adenosine receptor agonists were evaluated in endothelium-free tissues. ZM 241385-treatment produced a statistically significant rightward displacement of CGS-21680, NECA, and CAD concentration-response curves compared with the respective controls (P < 0.05). The EC50, expressed in nM, values in treated and control tissues were: 617.3 +/- 23 versus 24.9 +/- 1.5 for CGS-21680 (2-(p-(2-carboxyethyl)phenethylamino)-5'N-ethylcarboxamidoadenosine), 27.4 +/- 6.3 versus 3.06 +/- 0.8 for NECA (5'-N-ethylcarboxamidoadenosine), and 5786.2 +/- 160 versus 89.2 +/- 24.1 for CAD (chloroadenosine). However, the relaxing effect of forskolin remained unchanged in treated and control tissues. The concentration-response curves for NECA, CAD, and CGS-21680 were also displaced to the right when cAMP levels were measured in treated and control smooth muscle cells while no differences were observed with forskolin. Quantitative Western blot analysis demonstrated that the density of A2A receptors increased in ZM 241385-treated artery. We also showed a significant decrease in Galphas protein levels after ZM 241385 treatment compared with control. Taken together, these data indicate that prolonged blockade of A2A receptors in the coronary artery leads to desensitization of the functional effects of adenosine agonists by a mechanism that involves decreases in cAMP production. This was associated with an up-regulation of A2A receptors and a decrease in Galphas protein expression.     

Adenosine receptors co-operate with NMDA preconditioning to protect cerebellar granule cells against glutamate neurotoxicity

N-Methyl-D-aspartate (NMDA) preconditioning is evoked by subtoxic concentrations of NMDA (50 microM), which has been shown previously to lead to transient resistance to subsequent lethal dose of glutamate or NMDA in cultured neurons. The purpose of this study was to investigate the participation of adenosine A1 and A2A receptors on NMDA preconditioning against glutamate-induced cellular damage in cerebellar granule cells. NMDA preconditioning prevented the stimulatory effect induced by glutamate on AMP hydrolysis, but not on ADP hydrolysis. The neuroprotection evoked by NMDA preconditioning against glutamate-induced cellular damage was prevented by the presence of adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dimethylxanthine (CPT, 100 nM), but not by the adenosine A2A receptors antagonist, (4-(2[7-amino-2-(2-furyl {1,2,4}-triazolo{2,3-a{1,3,5}triazian-5-yl-aminoethyl)phenol (ZM 241385, 50 nM). Interestingly, a long-term treatment with CPT or ZM 241385 alone protected cells against glutamate-induced neurotoxicity. Moreover, the functionality of adenosine A1 receptor was not affected by NMDA preconditioning, but this treatment promoted adenosine A2A receptor desensitization, measured by cAMP accumulation. Taken together, the results described herein suggest that the neuroprotection evoked by NMDA preconditioning against cellular damage elicited by glutamate occurs through mechanisms involving adenosine A2A receptors desensitization co-operating with adenosine A1 receptors activation in cerebellar granule cells.     

Antagonistic interaction between adenosine A2A and dopamine D2 receptors modulates the social recognition memory in reserpine-treated rats

Increasing evidence suggests that antagonistic interactions between specific subtypes of adenosine and dopamine receptors in the basal ganglia are involved in the control of motor activity. However, there are few studies investigating this interaction in other brain regions and its role in additional functions. In the present study, we evaluated whether reserpine-treated rats (1.0 mg/kg, i.p.) exhibit altered social recognition memory abilities. The effects of acute administration of the dopamine receptor agonists 7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3 benzazepine (SKF 38393, dopamine D(1) receptor agonist) and quinpirole (dopamine D(2) receptor agonist), together with the adenosine receptor antagonists caffeine (non-selective), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, adenosine A(1) receptor antagonist) and 4-(2-[7-amino-2-{2-furyl}{1,2,4}triazolo-{2,3-a}{1,3,5}triazin-5-yl-amino]ethyl)phenol (ZM241385, adenosine A(2A) receptor antagonist), were also investigated. Twenty-four hours after treatment, reserpine-treated rats exhibited a significant disruption in the ability to recognize a juvenile rat after a short period of time. These animals did not show any motor deficit. The social recognition disruption induced by reserpine was reversed by acute treatment with quinpirole (0.05-0.1 mg/kg, i.p.), caffeine (10.0-30.0 mg/kg, i.p.) or ZM241385 (0.5-1.0 mg/kg, i.p.), but not with SKF 38393 (0.5-3.0 mg/kg, i.p.) or DPCPX (0.5-3.0 mg/kg, i.p.). Moreover, a synergistic response was observed following the co-administration of 'non-effective' doses of ZM241385 (0.1 mg/kg, i.p.) and quinpirole (0.01 mg/kg, i.p.). These results reinforce and extend the notion of antagonistic interactions between adenosine and dopamine receptors, and demonstrate, for the first time, that the blockade of adenosine A(2A) receptors and the activation of dopamine D(2) receptors can reverse the social recognition deficits induced by reserpine in rats.     

Adenosine A2A receptors inhibit the N-methyl-D-aspartate component of excitatory synaptic currents in rat striatal neurons

The effects of the adenosine A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethyl-amino-5'-N-ethylcarboxamidoadenosine (CGS 21680) on currents mediated by excitatory amino acid receptors were examined in rat striatal brain slices. In a Mg(2+)-free superfusion medium, CGS 21680 decreased the amplitude of excitatory postsynaptic currents (EPSCs) in about 70% of striatal neurons. The inhibitory effect of CGS 21680 disappeared both in the presence of the adenosine A(2A) receptor antagonist 8-(3-chlorostyryl) caffeine and the NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (AP-5). NMDA-induced currents were also depressed by CGS 21680 in a subset of striatal cells, whereas alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-induced currents were not affected. The results suggest that adenosine A(2A) receptor agonists inhibit the NMDA component of the EPSC.     

The in vitro pharmacology of ZM 241385, a potent, non-xanthine A2a selective adenosine receptor antagonist.

1. This paper describes the in vitro pharmacology of ZM 241385 (4-(2-[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3-a][1,3,5]triazin- 5-yl amino]ethyl) phenol), a novel non-xanthine adenosine receptor antagonist with selectivity for the A2a receptor subtype. 2. ZM 241385 had high affinity for A2a receptors. In rat phaeochromocytoma cell membranes, ZM 241385 displaced binding of tritiated 5'-N-ethylcarboxamidoadenosine (NECA) with a pIC50 of 9.52, (95% confidence limits, c.l., 9.02-10.02). In guinea-pig isolated Langendorff hearts, ZM 241385 antagonized vasodilatation of the coronary bed produced by 2-chloroadenosine (2-CADO) and 2-[p-(2-carboxyethyl) phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680) with pA2 values of 8.57 (c.l., 8.45-8.68) and 9.02 (c.l., 8.79-9.24) respectively. 3. ZM 241385 had low potency at A2b receptors and antagonized the relaxant effects of adenosine in the guinea-pig aorta with a pA2 of 7.06, (c.l., 6.92-7.19). 4. ZM 241385 had a low affinity at A1 receptors. In rat cerebral cortex membranes it displaced tritiated R-phenylisopropyladenosine (R-PIA) with a pIC50 of 5.69 (c.l., 5.57-5.81). ZM 241385 antagonized the bradycardic action of 2-CADO in guinea-pig atria with a pA2 of 5.95 (c.l., 5.72-6.18). 5. ZM 241385 had low affinity for A3 receptors. At cloned rat A3 receptors expressed in chinese hamster ovary cells, it displaced iodinated aminobenzyl-5'-N-methylcarboxamido adenosine (AB-MECA) with a pIC50 of 3.82 (c.l., 3.67-4.06). 6. ZM 241385 had no significant additional pharmacological effects on the isolated tissues used in these studies at concentrations three orders of magnitude greater than those which block A2a receptors.(ABSTRACT TRUNCATED AT 250 WORDS)