Adenosine A2A and A2B receptor expression in neuroendocrine tumours: potential targets for therapy

The clinical management of neuroendocrine tumours is complex. Such tumours are highly vascular suggesting tumour-related angiogenesis. Adenosine, released during cellular stress, damage and hypoxia, is a major regulator of angiogenesis. Herein, we describe the expression and function of adenosine receptors (A(1), A(2A), A(2B) and A(3)) in neuroendocrine tumours. Expression of adenosine receptors was investigated in archival human neuroendocrine tumour sections and in two human tumour cell lines, BON-1 (pancreatic) and KRJ-I (intestinal). Their function, with respect to growth and chromogranin A secretion was carried out in vitro. Immunocytochemical data showed that A(2A) and A(2B) receptors were strongly expressed in 15/15 and 13/18 archival tumour sections. Staining for A(1) (4/18) and A(3) (6/18) receptors was either very weak or absent. In vitro data showed that adenosine stimulated a three- to fourfold increase in cAMP levels in BON-1 and KRJ-1 cells. The non-selective adenosine receptor agonist (adenosine-5'N-ethylcarboxamide, NECA) and the A(2A)R agonist (CGS21680) stimulated cell proliferation by up to 20-40% which was attenuated by A(2B) (PSB603 and MRS1754) and A(2A) (SCH442416) receptor selective antagonists but not by the A(1) receptor antagonist (PSB36). Adenosine and NECA stimulated a twofold increase in chromogranin A secretion in BON-1 cells. Our data suggest that neuroendocrine tumours predominantly express A(2A) and A(2B) adenosine receptors; their activation leads to increased proliferation and secretion of chromogranin A. Targeting adenosine signal pathways, specifically inhibition of A(2) receptors, may thus be a useful addition to the therapeutic management of neuroendocrine tumours.     

Adenosine inhibits the release of interleukin-1beta in activated human peripheral mononuclear cells

The effects of adenosine and subtype-specific activators of adenosine receptors (A1, A2A, A2B and A3) were studied on the release of interleukin-1beta (IL-1beta) from peripheral mononuclear cells, monocytes and lymphocytes. In the cells activated by the protein kinase C specific phorbol ester (phorbol 12-myristate 13-acetate) and Ca(2+) ionophore (A23187) both adenosine and the subtype-specific receptor agonists, CPA (A1), CGS 21680 (A2A) and IB-MECA (A3) induced a concentration-dependent inhibition of IL-1beta release. The rank order of potency in the inhibition of IL-1beta release was CPA=CGS 21680>IB-MECA>adenosine>NECA (in the presence of A1, A2A and A3 receptor inhibitors). The inhibitory actions of CPA, CGS 21680 or IB-MECA were significantly reduced in the presence of DPCPX, ZM 243185 or MRS 1191 as subtype-specific antagonists on A1, A2A and A3 adenosine receptors, respectively. It can be concluded that adenosine inhibits the release of IL-1beta from the activated human peripheral mononuclear cells. In this process A1, A2A and A3 receptors are involved.     

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)     

Expression of A1 adenosine receptors in the developing avian retina: in vivo modulation by A2A receptors and endogenous adenosine

Little is known about the mechanisms that regulate the expression of adenosine receptors during CNS development. We demonstrate here that retinas from chick embryos injected in ovo with selective adenosine receptor ligands show changes in A1 receptor expression after 48 h. Exposure to A1 agonist N⁶‐cyclohexyladenosine (CHA) or antagonist 8‐Cyclopentyl‐1, 3‐dipropylxanthine (DPCPX) reduced or increased, respectively, A1 receptor protein and [³H]DPCPX binding, but together, CHA+DPCPX had no effect. Interestingly, treatment with A_2A agonist 3‐[4‐[2‐[[6‐amino‐9‐[(2R,3R,4S,5S)‐5‐(ethylcarbamoyl)‐3,4‐dihydroxy‐oxolan‐2‐yl]purin‐2‐yl]amino] ethyl]phenyl] propanoic acid (CGS21680) increased A1 receptor protein and [³H]DPCPX binding, and reduced A_2A receptors. The A_2A antagonists 7‐(2‐phenylethyl)‐5‐amino‐2‐(2‐furyl)‐pyrazolo‐[4,3‐e]‐1,2,4‐trizolo[1,5‐c] pyrimidine (SCH58261) and 4‐(2‐[7‐amino‐2‐[2‐furyl][1,2,4]triazolo[2,3‐a][1,3,5]triazo‐5‐yl‐amino]ethyl)phenol (ZM241385) had opposite effects on A1 receptor expression. Exposure to CGS21680 + CHA did not change A1 receptor levels, whereas CHA + ZM241385 or CGS21680 + DPCPX had no synergic effect. The blockade of adenosine transporter with S‐(4‐nitrobenzyl)‐6‐thioinosine (NBMPR) also reduced [³H]DPCPX binding, an effect blocked by DPCPX, but not enhanced by ZM241385. [³H]DPCPX binding kinetics showed that treatment with CHA reduced and CGS21680 increased the Bmax, but did not affect Kd values. CHA, DPCPX, CGS21680, and ZM241385 had no effect on A1 receptor mRNA. These data demonstrated an in vivo regulation of A1 receptor expression by endogenous adenosine or long‐term treatment with A1 and A_2A receptors modulators.     

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.     

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.     

Different roles of adenosine A1, A2A and A3 receptors in controlling kainate-induced toxicity in cortical cultured neurons

Adenosine is a neuromodulator that can control brain damage through activation of A(1), A(2A) and A(3) receptors, which are located in both neurons and other brain cells. We took advantage of cultured neurons to investigate the role of neuronal adenosine receptors in the control of neurotoxicity caused by kainate and cyclothiazide. Both A(1), A(2A) and A(3) receptors were immunocytochemically identified in cortical neurons. Activation of A(1) receptors with 100 nM CPA did not modify the extent of neuronal death whereas the A(1) receptor antagonist, DPCPX (50 nM), attenuated neurotoxicity by 28 +/- 5%, and effect similar to that resulting from the removal of endogenous adenosine with 2U/ml of adenosine deaminase (27 +/- 3% attenuation of neurotoxicity). In the presence of adenosine deaminase, DPCPX had no further effect and CPA now exacerbated neurotoxicity by 42 +/- 4%. Activation of A(2A) receptor with 30 nM CGS21680 attenuated neurotoxicity by 40 +/- 8%, an effect prevented by the A(2A) receptor antagonists, SCH58261 (50 nM) or ZM241385 (50 nM), which by themselves were devoid of effect. Finally, neither A(3) receptor activation with Cl-IB-MECA (100-500 nM) nor blockade with MRS1191 (5 microM) modified neurotoxicity. These results show that A(1) receptor activation enhances and A(2A) receptor activation attenuates neurotoxicity in cultured cortical neurons, indicating that these two neuronal adenosine receptors directly control neurodegeneration. Interestingly, the control by adenosine of neurotoxicity in cultured neurons is similar to that observed in vivo in newborn animals and is the opposite of what is observed in adult brain preparations where A(1) receptor activation and A(2A) receptor blockade are neuroprotective.     

The Adenosine Receptors Present on the Plasma Membrane of Chromaffin Cells Are of the A2b Subtype

The adenosine receptors in the plasma membrane of chromaffin cells from bovine adrenal medulla were characterized. The presence of A1 receptors was discounted owing to the absence of R-[3H]phenylisopropyladenosine (R-PIA) and [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX) binding. The binding of the specific A2a ligand CGS-21680 was low. In contrast, the binding of 5'-(N-[3H]-ethylcarboxamidoadenosine ([3H]NECA) was relatively high (1.7 pmol/mg of protein at a ligand concentration up to 90 nM). This binding did not correspond to non-adenosine receptor NECA binding sites because the specific [3H]-NECA binding was similar when unlabeled adenosine, NECA, or R-PIA was used to measure the nonspecific binding. The rank order of potency of different ligands for the displacement of specific [3H]NECA binding was DPCPX greater than NECA greater than chloroadenosine greater than R-PIA greater than theophylline = CGS-21680. These results indicate that the receptors present on the plasma membrane of chromaffin cells are exclusively of the A2b subtype.     

Interaction of Adenosine and Guanine Derivatives in the Rat Hippocampus: Effects on Cyclic AMP Levels and on the Binding of Adenosine Analogues and GMP

Guanine nucleotides (GN) have been implicated in many intracellular mechanisms. Extracellular actions, probably as glutamate receptor antagonists, have also been recently attributed to these compounds. GN may have a neuroprotective role by inhibiting excitotoxic events evoked by glutamate. Effects of extracellular GN on adenosine-evoked cellular responses have also been reported. However, the exact mechanism of such interaction is not known. In the present study, we showed that GN potentiated adenosine-induced cAMP accumulation in slices of hippocampus from young rats. However, neither GMP nor the metabotropic glutamate receptor agonist, 1S,3R-ACPD, inhibited the binding of the adenosine receptor agonist [³H]NECA (when binding to adenosine A2 receptors), or the binding of the adenosine A2a receptor agonist [³H]CGS 21680 in hippocampal membrane preparations. GppNHp, probably by interacting with G-proteins, decreased [³H]CGS 21680 binding. [³H]GMP binding was assayed in order to evaluate the GN sites which are not G-proteins. [³H]GMP binding was inhibited by GMP and GppNHp, but not by 1S,3R-ACPD. The interaction of endogenous adenosine with the GMP-binding sites was determined by incubating membranes in the presence or absence of adenosine deaminase (ADA). NECA, CADO, CGS 21680 and CPA (only at the highest concentration used) increased GMP binding in the presence of ADA. However, in the absence of ADA, the control levels of GMP binding were as high as in the presence of added ADA plus adenosine agonists, indicating that endogenous adenosine modulates the binding of GMP. If this site has a neuroprotective role, adenosine may be increasing its neuromodulator and proposed protective action.     

A2B adenosine receptor blockade inhibits growth of prostate cancer cells

The role of the A_2B adenosine receptor (AR) in prostate cell death and growth was studied. The A_2B AR gene expression quantified by real-time quantitative RT-PCR and Western blot analysis was the highest among four AR subtypes (A₁, A_2A, A_2B, and A₃) in all three commonly used prostate cancer cell lines, PC-3, DU145, and LNCaP. We explored the function of the A_2B AR using PC-3 cells as a model. The A_2B AR was visualized in PC-3 cells by laser confocal microscopy. The nonselective A_2B AR agonist NECA and the selective A_2B AR agonist BAY60-6583, but not the A_2A AR agonist CGS21680, concentration-dependently induced adenosine 3′,5′-cyclic monophosphate (cyclic AMP) accumulation. NECA diminished lactate dehydrogenase (LDH) release, TNF-α-induced increase of caspase-3 activity, and cycloheximide (CHX)-induced morphological changes typical of apoptosis in PC-3 cells, which were blocked by a selective A_2B AR antagonist PSB603. NECA-induced proliferation of PC-3 cells was diminished by siRNA specific for the A_2B AR. The selective A_2B AR antagonist PSB603 was shown to inhibit cell growth in all three cell lines. Thus, A_2B AR blockade inhibits growth of prostate cancer cells, suggesting selective A_2B AR antagonists as potential novel therapeutics.     

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.     

Effects of adenosinergic drugs on hypoxia-induced electrophysiological changes in rat hippocampal slices.

The effects of adenosinergic antagonists caffeine and DPCPX, and of the adenosinergic agonists L-PIA, CPA and CGS 21680 were investigated on fully and partially reversible hypoxia-induced electrophysiological changes in rat hippocampal slices. The influence of a high potassium solution and of the N-methyl-D-aspartate antagonist dizocilpine (MK 801) was also tested. The latency to obtain a 50% decrease in the amplitude of the CA1 population spike (CA1 PS) during a short- (5-10 min) lasting hypoxic period was significantly increased (P less than 0.01) by slice perfusion with caffeine (50 microM), DPCPX (0.2 microM), and by increasing (from 3 to 4 mM) the potassium concentration in the medium bathing the hippocampal slices. The latency was significantly decreased (P less than 0.01) by slice perfusion with L-PIA (0.2 microM) and CPA (0.05 microM). It was not significantly modified by CGS 21680 (5 microM). The incidence of reappearance of the CA1 PS during reoxygenation after long- (45 min) lasting hypoxia was significantly increased (P less than 0.05) by slice perfusion with MK 801 (50 microM), while it was not significantly affected by slice perfusion with caffeine (50 microM) or DPCPX (0.2 microM) or L-PIA (0.2 microM) or CPA (0.05 microM) or CGS 21680 (5 microM). The results indicate a prevalent involvement of the A1 adenosine receptors in the early mechanisms underlying hypoxia-induced reversible changes. Adenosine seems to have a limited role in the late mechanisms occurring after a long-lasting hypoxic period.     

The A2a/A2b receptor antagonist ZM-241385 blocks the cardioprotective effect of adenosine agonist pretreatment in in vivo rat myocardium

There is increasing evidence for interactions among adenosine receptor subtypes in the brain and heart. The purpose of this study was to determine whether the adenosine A(2a) receptor modulates the infarct size-reducing effect of preischemic administration of adenosine receptor agonists in intact rat myocardium. Adult male rats were submitted to in vivo regional myocardial ischemia (25 min) and 2 h reperfusion. Vehicle-treated rats were compared with rats pretreated with the A(1) agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA, 10 mug/kg), the nonselective agonist 5'-N-ethylcarboxamidoadenosine (NECA, 10 mug/kg), or the A(2a) agonist 2-[4-(2-carboxyethyl)phenethylamino]-5'-N-methylcarboxamidoadenosine (CGS-21680, 20 mug/kg). Additional CCPA- and NECA-treated rats were pretreated with the A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 100 mug/kg), the A(2a)/A(2b) antagonist 4-(-2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a} {1,3,5}triazin-5-yl-amino]ethyl)phenol (ZM-241385, 1.5 mg/kg) or the A(3) antagonist 3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine carboxylate (MRS-1523, 2 mg/kg). CCPA and NECA reduced myocardial infarct size by 50% and 35%, respectively, versus vehicle, but CGS-21680 had no effect. DPCPX blunted the bradycardia associated with CCPA and NECA, whereas ZM-241385 attenuated their hypotensive effects. Both DPCPX and ZM-241385 blocked the protective effects of CCPA and NECA. The A(3) antagonist did not alter the hemodynamic effects of CCPA or NECA, nor did it alter adenosine agonist cardioprotection. None of the antagonists alone altered myocardial infarct size. These findings suggest that although preischemic administration of an A(2a) receptor agonist does not induce cardioprotection, antagonism of the A(2a) and/or the A(2b) receptor blocks the cardioprotection associated with adenosine agonist pretreatment.     

Adenosine A2A Receptor Deficiency Up-Regulates Cystatin F Expression in White Matter Lesions Induced by Chronic Cerebral Hypoperfusion

In previous studies, we have shown that the inactivation of the adenosine A2A receptor exacerbates chronic cerebral hypoperfusion-induced white matter lesions (WMLs) by enhancing neuroinflammatory responses. However, the molecular mechanism underlying the effect of the adenosine A2A receptor remains unknown. Recent studies have demonstrated that cystatin F, a potent endogenous cysteine protease inhibitor, is selectively expressed in immune cells in association with inflammatory demyelination in central nervous system diseases. To understand the expression of cystatin F and its potential role in the effect of A2A receptor on WMLs induced through chronic cerebral hypoperfusion, we investigated cystatin F expression in the WMLs of A2A receptor gene knockout mice, the littermate wild-type mice and wild-type mice treated daily with the A2A receptor agonist {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 or both {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 and A2A receptor antagonist {"type":"entrez-protein","attrs":{"text":"SCH58261","term_id":"1052882304","term_text":"SCH58261"}}SCH58261 after chronic cerebral hypoperfusion. The results of quantitative-PCR and western blot analysis revealed that cystatin F mRNA and protein expression were significantly up-regulated in the WMLs after chronic cerebral hypoperfusion. In addition, cystatin F expression in the corpus callosum was significantly increased in A2A receptor gene knockout mice and markedly decreased in mice treated with {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 on both the mRNA and protein levels. Additionally, {"type":"entrez-protein","attrs":{"text":"SCH58261","term_id":"1052882304","term_text":"SCH58261"}}SCH58261 counteracted the attenuation of cystatin F expression produced by {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 after chronic cerebral hypoperfusion. Moreover, double immunofluorescence staining revealed that cystatin F was co-localized with the activated microglia marker CD11b. In conclusion, the cystatin F expression in the activated microglia is closely associated with the effect of the A2A receptors, which may be related to the neuroinflammatory responses occurring during the pathological process.     

Study of Adenosine A2 Receptors in Membrane Preparations from Optic Tectum of Chicks

Binding properties of the subtypes of adenosine A2 receptors in membrane preparations and the effects of adenosine receptor ligands on cAMP accumulation in slices from the optic tectum of neonatal chicks have been investigated. [³H]2-[4-(2-p-carboxyethyl)phenylamino]-5'-N-ethylcarboxaminoadenosine (CGS 21680), a selective ligand for adenosine A2a receptors, did not bind to optic tectal membranes, as observed with rat striatal membranes. CGS 21680 also did not induce cyclic AMP accumulation in optic tectum slices. However, 5'-N-ethylcarboxamidoadenosine (NECA), 2-chloro-adenosine or adenosine induced a 2.5- to 3-fold increase on cyclic AMP accumulation in this preparation. [³H]NECA binds to fresh non-washed-membranes obtained from optic tectum of chicks, displaying one population of binding sites, which can be displaced by NECA, 8-phenyltheophylline, 2-chloro-adenosine, but is not affected by CGS 21680. The estimated K_D value was 400.90 ± 80.50 nM and the B_max was estimated to be 2.51 ± 0.54 pmol/mg protein. Guanine nucleotides, which modulate G-proteins activity intracellularly, are also involved in the inhibition of glutamate responses by acting extracellularly. Moreover, we have previously reported that guanine nucleotides potentiate, while glutamate inhibits, adenosine-induced cyclic AMP accumulation in slices from optic tectum of chicks. However, the guanine nucleotides, GMP or GppNHp and the metabotropic glutamate receptors agonist, 1S,3R-ACPD did not alter the [³H]NECA binding observed in fresh non-washed-membranes. Therefore, the adenosine A2 receptor found in the optic tectum must be the adenosine A2b receptor which is available only in fresh membrane preparations, and its not modulated by guanine nucleotides or glutamate analogs.     

Characterization of adenosine receptors in Mullus surmuletus

The intent of the present study was to investigate adenosine receptor sites in brain membranes of the saltwater teleost fish, Mullus surmuletus, using the A₁ receptor selective agonist, [³H]CHA, and A_2a receptor selective agonist [³H]CGS 21680. The A₁ selective agonist, [³H]CHA, bound saturably, reversibly and with high affinity to a single-class of binding sites (K_d 1.47 nM; B_max 100–190 fmol/mg protein, dependent on fish length). The A_2a selective agonist, [³H]CGS 21680, also bound saturably, reversibly and with relative high affinity to a single-class of binding sites (K_d 44.2 nM; B_max 150–300 fmol/mg protein dependent on fish length). In equilibrium competition experiments, adenosine analogous, NECA, CGS 21680, CHA, CPA, S-PIA, R-PIA, CPCA, DPMA, and xanthine antagonists, DPCPX, XAC, and THEO all displaced [³H]CHA and [³H]CGS 21680 specifically bound to brain membranes from Mullus surmuletus. Specific binding of both [³H]CHA and [³H]CGS 21680 was inhibited by GDPβS. For [³H]CHA the IC₅₀ value was 2.5 ± 0.1 μM, while for [³H]CGS 21680 the IC₅₀ value was 7.7 ± 0.3 μM. Our results indicate that the high affinity binding sites for [³H]CHA have some pharmacological characteristics of mammalian A₁ adenosine receptors, while the binding sites for [³H]CGS 21680 appear to be virtually identical to the binding sites for [³H]CHA.     

Caffeine inhibition of rat carotid body chemoreceptors is mediated by A2A and A2B adenosine receptors

Caffeine, an unspecific antagonist of adenosine receptors, is commonly used to treat the apnea of prematurity. We have defined the effects of caffeine on the carotid body (CB) chemoreceptors, the main peripheral controllers of breathing, and identified the adenosine receptors involved. Caffeine inhibited basal (IC50, 210 microm) and low intensity (PO2 approximately 66 mm Hg/30 mm K+) stimulation-induced release of catecholamines from chemoreceptor cells in intact preparations of rat CB in vitro. Opposite to caffeine, 5'-(N-ethylcarboxamido)adenosine (NECA; an A2 agonist) augmented basal and low-intensity hypoxia-induced release. 2-p-(2-Carboxyethyl)phenethyl-amino-5'-N-ethylcaboxamido-adenosine hydrochloride (CGS21680), 2-hexynyl-NECA (HE-NECA) and SCH58621 (A2A receptors agents) neither affected catecholamine release nor altered the caffeine effects. The 8-cycle-1,3-dipropylxanthine (DPCPX; an A1/A2B antagonist) and 8-(4-{[(4-cyanophenyl)carbamoylmethyl]-oxy}phenyl)-1,3-di(n-propyl)xanthine (MRS1754; an A2B antagonist) mimicking of caffeine indicated that caffeine effects are mediated by A2B receptors. Immunocytochemical A2B receptors were located in tyrosine hydroxylase positive chemoreceptor cells. Caffeine reduced by 52% the chemosensory discharges elicited by hypoxia in the carotid sinus nerve. Inhibition had two components with pharmacological analysis indicating that A2A and A2B receptors mediate, respectively, the low (17 x 10(-9) m) and high (160 x 10(-6) m) IC50 effects. It is concluded that endogenous adenosine, via presynaptic A2B and postsynaptic A2A receptors, can exert excitatory effects on the overall output of the rat CB chemoreceptors.     

Excitatory Transmitter Amino Acid Release from the Ischemic Rat Cerebral Cortex: Effects of Adenosine Receptor Agonists and Antagonists

The effects of selective adenosine receptor agonists [N6-cyclopentyladenosine (CPA) and N-ethylcarboxamidoadenosine (NECA)] and antagonists [8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and 9-chloro-2-(2-furanyl)-5,6-dihydro-1,2,4-triazolo[1,5-c]quinazoline-5-im ine (CGS-15943A)] on aspartate and glutamate release from the ischemic rat cerebral cortex were studied with the cortical cup technique. Cerebral ischemia (for 20 min) was elicited by four-vessel occlusion. Excitatory amino acid releases were compared from control ischemic rats and drug-treated rats. Basal levels of aspartate and glutamate release were not greatly affected by pretreatment with the adenosine receptor agonists or antagonists. However, CPA (10(-10) M) and NECA (10(-9) M) significantly inhibited the ischemia-evoked release of aspartate and glutamate into cortical superfusates. The ability to block ischemia-evoked release of excitatory amino acids was not evident at higher concentrations of CPA (10(-6) M) or NECA (10(-5) M). The selective A1 receptor antagonist DPCPX also had no effect on release when administered at a low dosage (0.01 mg/kg, i.p.) but blocked the ischemia-evoked release of aspartate and glutamate at a higher dosage (0.1 mg/kg). Evoked release was inhibited by the selective A2 receptor antagonist CGS-15943A (0.1 mg/kg, i.p.). Thus, adenosine and its analogs may suppress ischemia-evoked release of excitatory neurotransmitter amino acids via high-affinity A1 receptors, whereas coactivation of lower-affinity A2 receptors may block (or reverse) the A1-mediated response.     

A novel fused 1,2,4-triazine aryl derivative as antioxidant and nonselective antagonist of adenosine A(2A) receptors in ethanol-activated liver stellate cells

It has been detected that hepatic adenosine A(2A) receptors play an active role in the pathogenesis of hepatic fibrosis and suggest a novel therapeutic target in the treatment and prevention of hepatic cirrhosis. In this paper we examined if our new triazine derivative (IMT) can inhibit ethanol-induced activation of HSCs measured as increased α-SMA, collagen synthesis and enhanced oxidative stress in rat liver stellate cells. We also investigated its influence on cytokines (TGF-β, TNF-α) synthesis, MMP-2 and TIMP-1 production and ethanol-induced intracellular signal transduction. Moreover, with using of known adenosine A(2A) receptor agonist (CGS 21680), and antagonist (SCH 58261) we examined if this triazine derivative acts on adenosine receptors. We detected a strong antagonistic action of new triazine derivative (IMT) on ethanol-induced rat liver stellate cells activation, observed as a significant decrease in α-SMA, collagen synthesis, reactive oxygen species production, TGF-β, TNF-α, MMP-2 and TIMP-1 production as well as JNK, p38MAPK, NFκB, IκB, Smad3 phosphorylation. Moreover, IMT strongly inhibited activation of stellate cells by known selective agonist of adenosine A(2A) receptor (CGS 21680). When known A(2A) receptor antagonist (SCH 58261) was used together with IMT this effect was not spectacular. Additionally, only slight enhancement of inhibition was observed when cells were pretreated both IMT with SCH 58261, hence we suppose that IMT acts as nonselective antagonist of A(2A) receptors, and, besides its antioxidant activity, also by this way inhibited ethanol-induced stellate cell activation.     

[3H]SCH 58261, a Selective Adenosine A2A Receptor Antagonist, Is a Useful Ligand in Autoradiographic Studies

Abstract: We have characterized the new potent and selective nonxanthine adenosine A_2A receptor antagonist SCH 58261 as a new radioligand for receptor autoradiography. In autoradiographic studies using agonist radioligands for A_2A receptors ([³H]CGS 21680) or A₁ receptors ( N ⁶-[³H]cyclohexyladenosine), it was found that SCH 58261 is close to 800-fold selective for rat brain A_2A versus A₁ receptors ( K _i values of 1.2 n M versus 0.8 µ M ). Moreover, receptor autoradiography showed that [³H]SCH 58261, in concentrations below 2 n M , binds only to the dopamine-rich regions of the rat brain, with a K _D value of 1.4 (0.8–1.8) n M . The maximal number of binding sites was 310 fmol/mg of protein in the striatum. Below concentrations of 3 n M , the nonspecific binding was <15%. Three adenosine analogues displaced all specific binding of [³H]SCH 58261 with the following estimated K _i values (n M ): 2-hex-1-ynyl-5'- N -ethylcarboxamidoadenosine, 3.9 (1.8–8.4); CGS 21680, 130 (42–405); N ⁶-cyclohexyladenosine, 9,985 (3,169–31,462). The binding of low concentrations of SCH 58261 was not influenced by either GTP (100 µ M ) or Mg²⁺ (10 m M ). The present results show that in its tritium-labeled form, SCH 58261 appears to be a good radioligand for autoradiographic studies, because it does not suffer from some of the problems encountered with the currently used agonist radioligand [³H]CGS 21680.