The effect of adenosine receptor agonists on cytokine release by human mononuclear cells depends on the specific Toll-like receptor subtype used for stimulation

In the present study, we determined whether the immunomodulatory effect of adenosine receptor stimulation depends on the Toll-like Receptor (TLR) used for stimulation of cytokine release. Therefore, human mononuclear cells were stimulated by different TLR agonists in the absence and presence of A1 (CPA), A2a (CGS21680), and A3 (Cl-IB-MECA) adenosine receptor agonists. Effects of these agonists on Il-6, Il-10, IFN-gamma, TNF-alpha, and Il-1beta production were expressed as percentage inhibition/stimulation after TLR stimulation. CGS21680 inhibited TLR4-mediated TNF-alpha release and potentiated TLR3- and TLR5-mediated IL-6 release. Cl-IB-MECA inhibited TLR4-agonist-induced IFN-gamma release. Interestingly, CPA en Cl-IB-MECA tended to inhibit cytokine release only after TLR4 stimulation. In more detail, CPA potentiated TLR5-mediated IL-6 production, TLR3-mediated IFN-gamma production and TLR3-mediated Il-1beta-production compared to TLR4-mediated stimulation. Cl-IB-MECA potentiated TLR5-mediated IL-6 and Il-1beta formation as compared to TLR4-mediated stimulation. Finally, CGS21680 potentiated TLR5-mediated IL-6 production compared to TLR1-2 stimulation, and potentiated TLR3- and TLR5-mediated IL-10 production compared to TLR1-2-mediated stimulation. In conclusion, the effect of adenosine agonists on cytokine production depends on the specific TLR agonist used for stimulation. These findings suggest that well-known anti-inflammatory effects of adenosine agonists on LPS-induced inflammation cannot be extrapolated to situations in which stimulation of other TLR subtypes is involved.     

On the role of subtype selective adenosine receptor agonists during proliferation and osteogenic differentiation of human primary bone marrow stromal cells

Purines are important modulators of bone cell biology. ATP is metabolized into adenosine by human primary osteoblast cells (HPOC); due to very low activity of adenosine deaminase, the nucleoside is the end product of the ecto-nucleotidase cascade. We, therefore, investigated the expression and function of adenosine receptor subtypes (A(1) , A(2A) , A(2B) , and A(3) ) during proliferation and osteogenic differentiation of HPOC. Adenosine A(1) (CPA), A(2A) (CGS21680C), A(2B) (NECA), and A(3) (2-Cl-IB-MECA) receptor agonists concentration-dependently increased HPOC proliferation. Agonist-induced HPOC proliferation was prevented by their selective antagonists, DPCPX, SCH442416, PSB603, and MRS1191. CPA and NECA facilitated osteogenic differentiation measured by increases in alkaline phosphatase (ALP) activity. This contrasts with the effect of CGS21680C which delayed HPOC differentiation; 2-Cl-IB-MECA was devoid of effect. Blockade of the A(2B) receptor with PSB603 prevented osteogenic differentiation by NECA. In the presence of the A(1) antagonist, DPCPX, CPA reduced ALP activity at 21 and 28 days in culture. At the same time points, blockade of A(2A) receptors with SCH442416 transformed the inhibitory effect of CGS21680C into facilitation. Inhibition of adenosine uptake with dipyridamole caused a net increase in osteogenic differentiation. The presence of all subtypes of adenosine receptors on HPOC was confirmed by immunocytochemistry. Data show that adenosine is an important regulator of osteogenic cell differentiation through the activation of subtype-specific receptors. The most abundant A(2B) receptor seems to have a consistent role in cell differentiation, which may be balanced through the relative strengths of A(1) or A(2A) receptors determining whether osteoblasts are driven into proliferation or differentiation.     

Effects of stable adenosine receptor agonists on bone marrow hematopoietic cells as inferred from the cytotoxic action of 5-fluorouracil

The aim of the study was to investigate the effects of stable adenosine receptor agonists on bone marrow hematopoiesis by utilizing the model of hematopoietic damage induced by 5-fluorouracil (5-FU), a cycle-specific cytotoxic agent. Effects of a non-selective agonist NECA activating all the known adenosine receptors (A1, A2A, A2B, A3) and of the selective agonists for A1 (CPA), A2A (CGS 21680), and A3 (IB-MECA) adenosine receptors were investigated. Experiments were performed with B10CBAF1 mice under in vivo conditions. Adenosine receptor agonists were given in single injections before 5-FU administration and the effects were determined 4 days later. The numbers of femoral marrow nucleated cells and hematopoietic progenitor cells (CFC-GM and BFU-E) were taken as indices of the effects. The non-selective agonist NECA given at a dose of 200 nmol/kg induced biphasic time-dependent effects, i.e. protection and sensitization, when given 10 h and 22 h before 5-FU administration, respectively. The use of isomolar doses of selective receptor agonists indicated that the protective effects of NECA were induced by activation of A2A and A2B receptors, while the sensitizing action of NECA was mediated via A3 receptors. In addition, it was observed that A1 receptors induced protection when activated by administration of CPA 22 h before 5-FU. These findings are discussed with respect to the action of adenosine receptor agonists on the cell cycle state and on the cell cycle-independent cellular protective mechanisms.     

Extracellular purine metabolism and signaling of CD73-derived adenosine in murine Treg and Teff cells

CD73-derived adenosine acts as potent inhibitor of inflammation, and regulatory T cells (Treg) have been shown to express CD73 as a novel marker. This study explored the role of endogenously formed adenosine in modulating NF-κB activity and cytokine/chemokine release from murine Treg and effector T cells (Teff) including key enzymes/purinergic receptors of extracellular ATP catabolism. Stimulating murine splenocytes and CD4(+) T cells with anti-CD3/anti-CD28 significantly upregulated activated NF-κB in CD73(-/-) T cells (wild type: 4.36 ± 0.21; CD73(-/-): 6.58 ± 0.75; n = 4; P = 0.029). This was associated with an augmented release of proinflammatory cytokines IL-2, TNF-α, and IFN-γ. Similar changes were observed with the CD73 inhibitor APCP (50 μM) on NF-κB and IFN-γ in wild-type CD4(+) T-cells. Treatment of stimulated CD4(+) T-cells with adenosine (25 μM) potently reduced IFN-γ release which is mediated by adenosine A2a receptors (A2aR). AMP (50 μM) also reduced cytokine release which was not inhibited by APCP. In Teff, A2aR activation (CGS21680) potently inhibited the release of IL-1, IL-2, IL-3, IL-4, IL-12, IL-13, IFN-γ, TNF-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), CCL3, and CCL4. However, in Treg, CGS21680 did not alter cytokine/chemokine release. In summary, CD73-derived adenosine tonically inhibits active NF-κB in CD4(+) T-cells, thereby modulating the release of a broad spectrum of proinflammatory cytokines and chemokines. Downregulation of P2X7 and upregulation of CD73 in Treg after antigenic stimulation may be an important mechanism to maintain the ability of Treg to generate immunosuppressive adenosine.     

Protein kinase A and Ca(v)1 (L-Type) channels are common targets to facilitatory adenosine A2A and muscarinic M1 receptors on rat motoneurons

At the rat motor endplate, pre-synaptic facilitatory adenosine A2A and muscarinic M1 receptors are mutually exclusive. We investigated whether these receptors share a common intracellular signalling pathway. Suppression of McN-A-343-induced M1 facilitation of [3H]ACh release was partially recovered when CGS21680C (an A2A agonist) was combined with the cyclic AMP antagonist Rp-cAMPS. Forskolin, rolipram and 8-bromo-cyclic AMP mimicked CGS21680C blockade of M1 facilitation. Both Rp-cAMPs and nifedipine reduced augmentation of [3H]ACh release by McN-A-343 and CGS21680C. Activation of M1 and A2A receptors enhanced Ca2+ recruitment through nifedipine-sensitive channels. Nifedipine inhibition revealed by McN-A-343 was prevented by chelerythrine (a PKC inhibitor) and Rp-cAMPS, suggesting that Ca(v)1 (L-type) channels phosphorylation by PKA and PKC is required. Rp-cAMPS inhibited [3H]ACh release in the presence of phorbol 12-myristate 13-acetate, but PKC inhibition by chelerythrine had no effect on release in the presence of 8-bromo-cyclic AMP. This suggests that the involvement of PKA may be secondary to M1-induced PKC activation. In conclusion, competition of M1 and A2A receptors to facilitate ACh release from motoneurons may occur by signal convergence to a common pathway involving PKA activation and Ca2+ influx through Ca(v)1 (L-type) channels.     

Functional coupling of the Galpha(olf) variant XLGalpha(olf) with the human adenosine A2A receptor

A recently identified novel Galphaolf variant, XLGalphaolf, is shown to functionally couple to the human adenosine A2A receptor (A2AR). In Sf9 cells expressing A2AR, beta1, and gamma2, co-expression of XLGalphaolf increased NECA-induced [35S]GTPgammaS binding from approximately 130% to 300% of basal levels. Pharmacological characteristics of A2AR ligands on these cells were evaluated by using [3H]ZM241385- and [35S]GTPgammaS- binding assays. The rank order of the equilibrium binding constants (Kd or Ki) of adenosine receptor ligands were [3H]ZM241385 approximately CGS15943 < MRS1220 < < CV1808 approximately NECA < CGS21680 approximately adenosine < IBMECA < HEMADO approximately CPA approximately CCPA. The rank order of EC50 values for agonists were CV1808 approximately NECA < adenosine approximately CGS26180 < IBMECA < HEMADO approximately CPA approximately CCPA. This pharmacology is consistent with the literature for A2AR and suggests that Sf9 cells co-expressing A2AR, beta1, gamma2, and XLGalphaolf could serve as a heterologous expression system for A2AR drug screening.     

Distinct protein kinase C isoforms mediate regulation of vascular endothelial growth factor expression by A2A adenosine receptor activation and phorbol esters in pheochromocytoma PC12 cells

Vascular endothelial growth factor (VEGF) stimulates angiogenesis during development and in disease. In pheochromocytoma (PC12) cells, VEGF expression is regulated by A(2A) adenosine receptor (A(2A)AR) activation. The present work examines the underlying signaling pathway. The adenylyl cyclase-protein kinase A cascade has no role in the down-regulation of VEGF mRNA induced by the A(2A)AR agonist, 2-[4-[(2-carboxyethyl)phenyl]ethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680). Conversely, 6-h exposure of cells to either phorbol 12-myristate 13-acetate (PMA) or protein kinase C (PKC) inhibitors mimicked the CGS21680-induced down-regulation. PMA activated PKCalpha, PKCepsilon, and PKCzeta, and CGS21680 activated PKCepsilon and PKCzeta as assessed by cellular translocation. By 6 h, PMA but not CGS21680 decreased PKCalpha and PKCepsilon expression. Neither compound affected PKCzeta levels. Following prolonged PMA treatment to down-regulate susceptible PKC isoforms, CGS21680 but not PMA inhibited the cobalt chloride induction of VEGF mRNA. The proteasome inhibitor, MG-132, abolished PMA- but not CGS21680-induced down-regulation of VEGF mRNA. Phorbol 12,13-diacetate reduced VEGF mRNA levels while down-regulating PKCepsilon but not PKCalpha expression. In cells expressing a dominant negative PKCzeta construct, CGS21680 was unable to reduce VEGF mRNA. Together, the findings suggest that phorbol ester-induced down-regulation of VEGF mRNA occurs as a result of a reduction of PKCepsilon activity, whereas that mediated by the A(2A)AR occurs following deactivation of PKCzeta.     

Sensitization by chronic diazepam treatment of A2A adenosine receptor-mediated relaxation in rat pulmonary artery

The effects of a 10-day i.p. treatment of rats with diazepam on responses to subtype selective adenosine receptor agonists were studied 3 h, 2 and 8 days after termination of diazepam treatment in isolated cardiovascular tissues possessing distinct adenosine receptors. After long-lasting diazepam exposure, the relaxation elicited by the specific A2A receptor agonist CGS 21680 was enhanced in rat main pulmonary arteries (a tissue containing A2A adenosine receptors). The increased sensitivity of A2A receptors observed 3 h and 2 days after withdrawal of diazepam was completely restored by the 8th day of the wash-out period. N6-cyclopentyladenosine (CPA)-induced suppression in mechanical activity of electrically stimulated rat atrial myocardium (a tissue containing A1 adenosine receptors) was not altered following diazepam treatment. In order to reveal the possible role of inhibition of membrane adenosine transport in the effects of diazepam (a moderate inhibitor of membrane adenosine transport), the action of a 10-day treatment with dipyridamole or S-(p-nitrobenzyl)-6-thioinosine (NBTI; prototypic adenosine uptake inhibitors) was also studied. Dipyridamole or NBTI treatment, like diazepam, increased the responsiveness of rat pulmonary artery to CGS 21680, but did not influence the cardiodepressive effect of CPA in electrically driven left atrial myocardium. The CGS 21680-induced relaxations were significantly antagonized by 10 nM ZM 241385 (a selective A2A adenosine receptor antagonist) in vessels of diazepam-treated rats. The relaxation responses to verapamil were unaltered in pulmonary arteries obtained from animals chronically treated with diazepam, dipyridamole or NBTI. These results suggest that chronic diazepam treatment is able to enhance the A2A adenosine receptor-mediated vascular functions, but does not modify the responses mediated via A1 receptors of rat myocardium, where nucleoside transport inhibitory sites of membrane are of a very low density. It is possible that sensitization of A2A adenosine receptor-mediated vasorelaxation is due to a long-lasting inhibition of membrane adenosine transporter during diazepam treatment.     

Adenosine A2A receptor stimulation potentiates nitric oxide release by activated microglia

The absence of adenosine A2A receptors, or its pharmacological inhibition, has neuroprotective effects. Experimental data suggest that glial A2A receptors participate in neurodegeneration induced by A2A receptor stimulation. In this study we have investigated the effects of A2A receptor stimulation on control and activated glial cells. Mouse cortical mixed glial cultures (75% astrocytes, 25% microglia) were treated with the A2A receptor agonist CGS21680 alone or in combination with lipopolysaccharide (LPS). CGS21680 potentiated lipopolysaccharide-induced NO release and NO synthase-II expression in a time- and concentration-dependent manner. CGS21680 potentiation of lipopolysaccharide-induced NO release was suppressed by the A2A receptor antagonist ZM-241385 and did not occur on mixed glial cultures from A2A receptor-deficient mice. In mixed glial cultures treated with LPS + CGS21680, the NO synthase-II inhibitor 1400W abolished NO production, and NO synthase-II immunoreactivity was observed only in microglia. Binding experiments demonstrated the presence of A2A receptors on microglial but not on astroglial cultures. However, the presence of astrocytes was necessary for CGS21680 potentiating effect. In light of the reported neurotoxicity of microglial NO synthase-II and the neuroprotection of A2A receptor inhibition, these data suggest that attenuation of microglial NO production could contribute to the neuroprotection afforded by A2A receptor antagonists.     

Binding of the Adenosine A2 Receptor Ligand [3H]CGS 21680 to Human and Rat Brain: Evidence for Multiple Affinity Sites

A new radiolabeled adenosine receptor agonist, 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadeno sin e (CGS 21680), apparently specific for high-affinity binding sites of the A2 subtype in rat brain, was used to identify and pharmacologically characterize adenosine receptors in human brain. The binding of [3H]CGS 21680, as determined by standard radioligand binding technique in the presence of exogenously added adenosine deaminase, reached equilibrium after 40 min at 25 degrees C. In saturation studies, a single class of high-affinity binding sites with values for KD of 22 +/- 0.5 nM and Bmax of 444 +/- 63 fmol/mg of protein were observed. Similar binding characteristics were observed regardless of whether rapid filtration or centrifugation was used to separate bound versus free ligand. Of the 14 brain regions examined, [3H]CGS 21680 binding was highest in putamen, followed by globus pallidus and caudate nucleus. The level of [3H]CGS 21680 binding in these areas of basal ganglia was identical to 5'-N-[3H]ethylcarboxamidoadenosine ([3H]NECA) binding in the presence of 50 nM N6-cyclopentyladenosine (CPA). The rank order of agonist potencies as determined by a series of competition experiments was NECA greater than or equal to CGS 21680 greater than 2-chloroadenosine greater than N6-(R)-phenylisopropyladenosine greater than N6-cyclohexyladenosine greater than N6-(S)-phenylisopropyladenosine. This potency order was the same for the binding of [3H]CGS 21680 to rat, and of [3H]NECA in the presence of 50 nM CPA to rat and human, brain membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of P1 and P2 purinoceptors in the aorta of the lizard (Agama sp.)

In the isolated Agama lizard aorta, acetylcholine (ACh; 3 nM-100 microM), noradrenaline (NA; 30 nM-0.3 mM), adrenaline (Adr; 30 nM-300 microM), adenosine 5'-triphosphate (ATP; 30 nM-1 mM), alpha,beta-methylene ATP (alpha,beta-meATP; 10 nM-10 microM), beta,gamma-methylene ATP (beta,gamma-meATP; 0.1-300 microM), 2-methylthio ATP (2-meSATP; 30 nM-30 microM) and high concentrations of uridine triphosphate (UTP; 1 microM-1 mM), all produced constriction. The P2 receptor antagonists pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS; 30 microM), suramin (0.1 mM) and Reactive blue 2 (30 microM) all raised vascular tone and could not be utilized and the antagonist 2'-O-(trinitrophenyl) ATP (TNP-ATP; 0.1 microM) had no effect on responses to the ATP analogues. alpha,beta-MeATP (3 microMx3) desensitised responses to alpha,beta-meATP (10 microM) and beta,gamma-meATP (0.3 mM), but not to ATP (0.3 mM) or 2-meSATP (30 microM). On pre-constricted aorta (EC50 concentration of either ACh or Adr), adenosine (1 microM-1 mM), the A1-selective agonist N6-cyclopentyl adenosine (CPA; 1-300 microM) [but not the A2- and A3-selective agonists CGS 21680 and IB-MECA respectively (both up to 30 microM)] and sodium nitroprusside (10 nM-100 microM) produced vasodilatation. Adenosine vasodilatation was antagonised by 8-p-sulfophenyl-theophylline (8-pSPT; 30 microM) but not by N(omega)-nitro-L-arginine methyl ester (L-NAME; 0.1 mM). ATP (up to 0.3 mM), 2-meSATP (up to 10 microM) and UTP (up to 1 mM) were not vasodilators. In summary, A1 receptors mediating relaxation and excitatory P2X1 receptors were identified in the smooth muscle of the lizard aorta. However, in contrast to mammalian aorta, P2Y receptors on endothelial cells mediating vasodilatation via nitric oxide do not appear to be present.     

A1 receptor activation decreases fatigue in mammalian slow-twitch skeletal muscle in vitro.

To test the hypothesis that adenosine improves skeletal muscle cell function, we exposed curarized mouse soleus and extensor digitorum longus (EDL) to a range of concentrations of adenosine (10(-9) M to 10(-5) M). Muscles contracted in Krebs-Henseleit bicarbonate buffer (27 degrees C, 95% O2 and 5% CO2) for 500 ms at 50 Hz once every 90 s. Soleus fatigued significantly less with adenosine present at concentrations of 10(-8) M and higher than with the Krebs-Henseleit vehicle control. Adenosine significantly improved force generation or delayed fatigue of EDL only with the initial adenosine challenge. To investigate the receptor population involved, we exposed soleus to agonists specific for A1 receptors (N6-cyclopentyladenosine, CPA), or A2 receptors (CGS 21680 hydrochloride, CGS), or A3 receptors (N6-benzyl-5'-N-ethylcarboxamidoadenosine, BNECA). CPA (A1) significantly decreased fatigue compared with the Krebs-Henseleit vehicle control at concentrations of 10(-9) M and higher. Muscles exposed to the A2 and A3 agonists did not differ from a Krebs-Henseleit plus methanol control. Phenylephrine (10(-6) M), an alpha-adrenergic agonist that increases the concentration of inositol triphosphate (IP3), significantly improved developed force in soleus. Neither a permeable cAMP analog, 8-bromo-cAMP (10(-5) M), nor a beta, agonist, isoproterenol (10(-6) M), had an effect on force generation in the soleus when compared with a saline control. Thus adenosine slowed fatigue in slow-twitch skeletal muscle through A1 receptors.     

Adenosine modulation of D-[3H]aspartate release in cultured retina cells exposed to oxidative stress

In this study we evaluated the role of adenosine receptor activation on the K+-evoked D-[3H]aspartate release in cultured chick retina cells exposed to oxidant conditions. Oxidative stress, induced by ascorbate (3.5 mM)/Fe2+ (100 microM), increased by about fourfold the release of D-[3H]aspartate, evoked by KCl 35 mM in the presence and in the absence of Ca2+. The agonist of A1 adenosine receptors, N6-cyclopentyladenosine (CPA; 10 nM), inhibited the K+-evoked D-[3H]aspartate release in control in oxidized cells. The antagonist of A1 adenosine receptor, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 50 nM), potentiated the release of D-[3H]aspartate in oxidized cells, and reverted the effect observed in the presence of CPA 10 nM. However, in oxidized cells, when DPCPX was tested together with CPA 100 nM the total release of D-[3H]aspartate increased from 5.1 +/- 0.4% to 11.4 +/- 1.0%, this increase being reverted by 3,7-dimethyl-1-propargylxanthine (DMPX; 100 nM), an antagonist of A2A adenosine receptors. In cells of both experimental conditions, the K+-evoked release of D-[3H]aspartate was potentiated by the selective agonist of A2A adenosine receptors, 2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosin e (CGS 21680; 10 nM), whereas the antagonist of these receptors, DMPX (100 nM), inhibited the release of D-[3H]aspartate in oxidized cells, but not in control cells. Adenosine deaminase (ADA; 1 U/ml), which is able to remove adenosine from the synaptic space, reduced the K+-evoked D-[3H]aspartate release, from 5.1 +/- 0.4% to 3.1 +/- 0.3% in oxidized cells, and had no significant effect in control cells. The extracellular accumulation of endogenous adenosine, upon K+-depolarization, was higher in oxidized cells than in control cells, and was reduced by the inhibitors of adenosine transporter (NBTI) and of ecto-5'-nucleotidase (AOPCP). This suggests that adenosine accumulation resulted from the outflow of adenosine mediated by the transporter, and from extracellular degradation of adenine nucleotide. Our data show that both inhibitory A1 and excitatory A2A adenosine receptors are present in cultured retina cells, and that the K+-evoked D-[3H]aspartate release is modulated by the balance between inhibitory and excitatory responses. Under oxidative stress conditions, the extracellular accumulation of endogenous adenosine seems to reach levels enough to potentiate the release of D-[3H]aspartate by the tonic activation of A2A adenosine receptors.     

Adenosine enhances glial glutamate efflux via A2a adenosine receptors

The present study investigated the effect of adenosine on glial glutamate efflux. Adenosine (from 1 nM to 100 microM) enhanced the release from cultured rat glial cells in a bell-shaped dose-responsive manner for the hippocampus and in a dose-dependent manner for the superior colliculus, and a similar increase was obtained with the A2a adenosine receptor agonist, 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS21680), but not with the A1 adenosine receptor agonist, N6-cyclohexyladenosine (CHA). Adenosine and CGS21680 also enhanced glutamate efflux from Xenopus oocytes injected with the poly (A)+ mRNAs derived from cultured glial cells for the hippocampus and the superior colliculus together with and without the A2a adenosine receptor mRNA, but instead such increase was not found in oocytes expressing A2a adenosine receptors alone. The results of the present study thus suggest that adenosine enhances glutamate efflux from glial cells via A2a adenosine receptors, and this may represent a mechanism underlying the facilitatory action of adenosine on hippocampal and superior colliculus neurotransmissions.     

Adenosine A2A receptors control the extracellular levels of adenosine through modulation of nucleoside transporters activity in the rat hippocampus

Adenosine, a neuromodulator of the CNS, activates inhibitory-A1 receptors and facilitatory-A2A receptors; its synaptic levels are controlled by the activity of bi-directional equilibrative nucleoside transporters. To study the relationship between the extracellular formation/inactivation of adenosine and the activation of adenosine receptors, we investigated how A1 and A2A receptor activation modifies adenosine transport in hippocampal synaptosomes. The A2A receptor agonist, CGS 21680 (30 nm), facilitated adenosine uptake through a PKC-dependent mechanism, but A1 receptor activation had no effect. CGS 21680 (30 nm) also increased depolarization-induced release of adenosine. Both effects were prevented by A2A receptor blockade. A2A receptor-mediated enhancement of adenosine transport system is important for formatting adenosine neuromodulation according to the stimulation frequency, as: (1) A1 receptor antagonist, DPCPX (250 nm), facilitated the evoked release of [(3)H]acetylcholine under low-frequency stimulation (2 Hz) from CA3 hippocampal slices, but had no effect under high-frequency stimulation (50 Hz); (2) either nucleoside transporter or A2A receptor blockade revealed the facilitatory effect of DPCPX (250 nm) on [3H]acetylcholine evoked-release triggered by high-frequency stimulation. These results indicate that A2A receptor activation facilitates the activity of nucleoside transporters, which have a preponderant role in modulating the extracellular adenosine levels available to activate A1 receptors.     

Adenosine receptor-mediated modulation of dopamine release in the nucleus accumbens depends on glutamate neurotransmission and N-methyl-D-aspartate receptor stimulation

Adenosine, by acting on adenosine A(1) and A(2A) receptors, exerts opposite modulatory roles on striatal extracellular levels of glutamate and dopamine, with activation of A(1) inhibiting and activation of A(2A) receptors stimulating glutamate and dopamine release. Adenosine-mediated modulation of striatal dopaminergic neurotransmission could be secondary to changes in glutamate neurotransmission, in view of evidence for a preferential colocalization of A(1) and A(2A) receptors in glutamatergic nerve terminals. By using in vivo microdialysis techniques, local perfusion of NMDA (3, 10 microm), the selective A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680; 3, 10 microm), the selective A(1) receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT; 300, 1000 microm), or the non-selective A(1)-A(2A) receptor antagonist in vitro caffeine (300, 1000 microm) elicited significant increases in extracellular levels of dopamine in the shell of the nucleus accumbens (NAc). Significant glutamate release was also observed with local perfusion of CGS 21680, CPT and caffeine, but not NMDA. Co-perfusion with the competitive NMDA receptor antagonist dl-2-amino-5-phosphonovaleric acid (APV; 100 microm) counteracted dopamine release induced by NMDA, CGS 21680, CPT and caffeine. Co-perfusion with the selective A(2A) receptor antagonist MSX-3 (1 microm) counteracted dopamine and glutamate release induced by CGS 21680, CPT and caffeine and did not modify dopamine release induced by NMDA. These results indicate that modulation of dopamine release in the shell of the NAc by A(1) and A(2A) receptors is mostly secondary to their opposite modulatory role on glutamatergic neurotransmission and depends on stimulation of NMDA receptors. Furthermore, these results underscore the role of A(1) vs. A(2A) receptor antagonism in the central effects of caffeine.     

Modulation of A2A adenosine receptor(s) by K+(ATP) channels in bovine brain striatal membranes

The modulation of adenosine receptor with K+(ATP) channel blocker, glibenclamide, was investigated using the radiolabeled A2A-receptor selective agonist [3H]CGS 21680. Radioligand binding studies in bovine brain striatal membranes (BBM) indicated that unlabeled CGS 21680 displaced the bound [3H]CGS 21680 in a concentration-dependent manner with a maximum displacement being approximately 65% at 10(-4) M. In the presence of 10(-5) M glibenclamide, unlabeled CGS 21680 increased the displacement of bound [3H]CGS 21860 by approximately 28% at 10(-4) M. [3H]CGS 21680 bound to BBM in a saturable manner to a single binding site (Kd = 10.6+/-1.71 nM; Bmax = 221.4+/-6.43 fmol/mg of protein). In contrast, [3H]CGS 21680 showed saturable binding to two sites in the presence of 10(-5) M glibenclamide; (Kd = 1.3+/-0.22 nM; Bmax = 74.3+/-2.14 fmol/mg protein; and Kd = 8.9+/-0.64 nM; Bmax = 243.2+/-5.71 fmol/mg protein), indicating modulation of adenosine A2A receptors by glibenclamide. These studies suggest that the K+(ATP) channel blocker, glibenclamide, modulated the adenosine A2A receptor in such a manner that [3H]CGS 21680 alone recognizes a single affinity adenosine receptor, but that the interactions between K+(ATP) channels and adenosine receptors.     

Adenosine A2A receptor facilitates calcium-dependent protein secretion through the activation of protein kinase A and phosphatidylinositol-3 kinase in PC12 cells

Adenosine modulates a variety of cellular functions including calcium-dependent exocytosis. Activation of adenosine A(2A) receptor (A(2A)-R) facilitates neurotransmitter release in some cell types, although the underlying mechanisms are not fully understood. In this study, we found that treatment of PC12 cells with the A(2A)-R agonist CGS21680 promotes calcium-evoked secretion of the fusion protein between neuropeptide Y and modified yellow fluorescence protein (NPY-Venus). CGS21680 treatment of PC12 cells transiently increased the phosphorylation of p38 and JNK MAP kinases and Akt, as well as that of ATF2 and CREB, reaching maximal levels at around 10-15 min of CGS21680 treatment. Importantly, pretreatment of PC12 cells with the PI3K inhibitor LY294002, together with the protein kinase A (PKA) inhibitor KT5720, significantly inhibited CGS21680 enhancement of calcium-dependent NPY-Venus release. Moreover, expression of a dominant-negative form of Akt and the PKA inhibitory polypeptide protein kinase inhibitor (PKI) co-operatively inhibited the facilitating effect of CGS21680 on secretion of NPY-Venus. These data suggest that the PI3K-Akt and PKA pathways play a critical role in A(2A)-R-mediated facilitation of calcium-dependent secretion. We also found that CGS21680 treatment promoted recruitment of the NPY-Venus-containing vesicles to the proximity of the plasma membrane at around 10-15 min of CGS21680 treatment, which may in part account for the facilitated secretion by A(2A)-R activation.     

Adenosine A1 and A2A receptor effects on G-protein cycling in beta-adrenergic stimulated ventricular membranes

In the heart beta1-adrenergic (beta1R) and adenosine A1 (A1R) and A2A (A2AR) receptors modulate contractile and metabolic function. The interaction between these receptors was investigated at the level of G-protein cycling by determining the effect of receptor agonists on the binding of GTP to G-proteins and displacement of G alpha-subunit-bound GDP by GTP. Crude membranes from rat heart or brain were stimulated by agonists for beta1R (isoproterenol; ISO), A1R (chlorocyclopentyladenosine, CCPA) and A2AR (CGS-21680; CGS). GTP binding to membranes was increased by ISO (17%), CCPA (6%) and CGS (12%). Binding values observed with incubation using ISO and CCPA together were significantly less than values obtained by the incubation of individual agents alone. With ISO, GTP binding to G alpha(s) subunits as determined by immunoprecipitation was increased 79% in heart and 87% in brain. These increases were attenuated by CCPA, an effect that was inhibited by CGS. GDP release by membranes was increased 6.9% and 4.6% by ISO and CCPA, respectively. After co-incubation of these agonists, release was increased less than determined by the addition of the individual agent responses. CGS inhibited the reduced release caused by of CCPA. Adenylyl cyclase activity stimulated by ISO was attenuated 33% by CCPA, an effect inhibited by CGS. Together, these results indicate that A1R exert an antiadrenergic action at the level of beta1R stimulated G(s)-protein cycling and that A2AR reduce this action.