Chronic Exposure to Adenosine Receptor Agonists and Antagonists Reciprocally Regulates the A1 Adenosine Receptor-Adenylyl Cyclase System in Cerebellar Granule Cells

Abstract: Chronic treatment with the adenosine receptor antagonist caffeine evokes an up-regulation of A₁ adenosine receptors and increased coupling of the receptor to G proteins in rat brain membranes. However, chronic agonist exposure has not been explored. Primary cultures of cerebellar granule cells were exposed chronically to A₁ adenosine receptor agonists and antagonists. Exposure to the A₁ adenosine receptor agonist N ⁶-cyclopentyladenosine resulted in (1) a time- and concentration-dependent reduction in the density of receptors labeled by 1,3-[³H]dipropyl-8-cyclopentylxanthine, (2) an enhanced ability of guanyl nucleotides to decrease the fraction of A₁ adenosine receptor sites displaying high affinity for 2-chloroadenosine, and (3) a functional uncoupling of receptors from adenylyl cyclase (EC 4.6.1.1). The adenosine antagonists caffeine and 8- p -sulfophenyltheophylline produced alterations in A₁ adenosine receptor homeostasis that were antipodal to those associated with agonist treatment. Antagonist exposure (1) increased the density of A₁ adenosine receptors in cerebellar granule cell membranes, (2) blunted the effect of guanyl nucleotides on receptor coupling to G proteins, and (3) increased the functional coupling of receptors to adenylyl cyclase inhibition. Forskolin treatment of cerebellar granule cells did not affect receptor density, suggesting that cyclic AMP is not involved in the regulation of A₁ adenosine receptor expression.     

Desensitisation of the Adenosine A1 Receptor by the A2A Receptor in the Rat Striatum

Abstract: The influence of the adenosine A_2A receptor on the A₁ receptor was examined in rat striatal nerve terminals, a model for other cells in which these receptors are coexpressed. Incubation of striatal synaptosomes with the A_2A receptor agonist 2- p -(2-carboxyethyl)phenethylamino-5'- N -ethylcarboxamidoadenosine (CGS 21680) caused the appearance of a low-affinity binding site for the A₁ receptor agonist 2-chloro- N ⁶-cyclopentyladenosine (CCPA). This effect was blocked by the A_2A receptor antagonist ZM241385 and by the protein kinase C inhibitor chelerythrine, but not by the protein kinase A inhibitor N -(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004). The effect was not seen with striatal membranes or with hypotonically lysed synaptosomes. These results demonstrate a protein kinase C-mediated heterologous desensitisation of the A₁ receptor by the A_2A receptor.     

Activation of Adenosine A2 Receptors Stimulates Phosphoinositide Metabolism in Rat Peripheral Nerve

Abstract: The effects of adenosine analogues on phosphoinositide metabolism in rat sciatic nerve were examined. Sciatic nerve segments were prelabeled with [³H]-cytidine and incubated in the presence of LiCl and varying concentrations of adenosine analogues. The formation of [³H]cytidine monophosphate phosphatidic acid ([³H]-CMP-PA) was determined as an index of phosphoinositide breakdown. Liponucleotide accumulation was elevated significantly in the presence of 5'- N -ethylcarboxamidoadenosine (NECA), a nonselective analogue, and two different A₂-selective analogues, N ⁶-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl]adenosine and 2- p -(2-carboxyethyl)phenethylamino-NECA (CGS 21680), but not by N ⁶-cyclopentyladenosine, an A₁-selective analogue. The stimulatory action of CGS 21680 was blocked by the A₂-selective adenosine receptor antagonists 3,7-dimethyl-1-propargylxanthine (DMPX) and 1,3-dipropyl-7-methylxanthine. Inositol phosphate formation was also stimulated to a comparable degree by CGS 21680 and this response was antagonized by DMPX. Carbamylcholine, which was previously shown to stimulate phosphoinositide breakdown, also enhanced the accumulation of CMP-PA. When adenosine analogues and carbamylcholine were simultaneously present, their effects were additive. Taken together, these data suggest that an adenosine receptor, possibly of the A₂ subtype, is coupled to enhanced phosphoinositide hydrolysis in peripheral nerve. However, adenosine-receptor activation does not appear to modulate phosphoinositide hydrolysis stimulated via muscarinic receptors.     

Adenosine A2b Receptors Mediate an Increase in Interleukin (IL)-6 mRNA and IL-6 Protein Synthesis in Human Astroglioma Cells

Abstract: The cytokine interleukin (IL)-6 has recently been demonstrated to play a role in the pathology of Alzheimer's disease (AD). The mechanisms leading to increased IL-6 levels in brains of AD patients are still unknown. Because in experimental animals ischemia increases both the level of cytokines and the extracellular concentrations of adenosine in the brain, we hypothesized that these two phenomena may be functionally connected and that adenosine might increase IL-6 gene expression in the brain. Here we show that the mixed A₁ and A₂ agonist 5'-( N -ethylcarboxamido)adenosine (NECA) induces an increase in IL-6 mRNA levels and protein synthesis in the human astrocytoma cell line U373 MG. The A₁-specific agonists R -phenylisopropyladenosine and cyclopentyladenosine are much less potent, and the A_2a-specific agonist CGS-21680 shows only marginal effects. Increased levels of mRNA are already found within 30 min after NECA treatment. The A_2a-selective antagonists 8-(3-chlorostyryl)caffeine and KF17837 [( E )-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine], which have also some antagonistic properties at A_2b receptors, and the nonspecific adenosine antagonist 8-phenyltheophylline were equipotent at inhibiting the NECA-induced increase in IL-6 protein synthesis, whereas the specific A₁ antagonist 8-cyclopentyl-1,3-dipropylxanthine is much less potent. The results indicate that adenosine A_2b receptors participate in the regulation of the IL-6 gene in astrocytoma cells.     

Effect of glutamate intake during gestation on adenosine A(1) receptor/adenylyl cyclase pathway in both maternal and fetal rat brain

Pregnant Wistar rats were orally treated with 1 g/L l -glutamate during the entire gestational period and the status of adenosine A₁ receptor (A₁R)/adenylyl cyclase transduction pathway from maternal and fetal brain was analyzed. Glutamate consumption, estimated from the loss of water from the drinking bottles, was 110 ± 4.6 mg/kg/day. In mother brains glutamate intake did not significantly alter the B _max value, although the K _d value was significantly decreased. However in fetus brain, a significant decrease in B _max was observed, without an alteration of K _d value. Similar results were observed by western blot assays using specific A₁R antibody, suggesting a down-regulation of A₁R in fetal brain. Concerning α subunits of inhibitory G proteins (Gi), αGi₃ protein was slightly but significantly decreased in maternal brain without alterations of either Gi₁ or Gi₂. In contrast, αGi₁ and αGi₂ isoforms were increased in fetal brain. On the other hand, basal, forskolin, and forskolin plus GTPγS-stimulated adenylyl cyclase activity was significantly decreased in both maternal and fetal brain, and this was more prominent in fetal than in maternal brain. Finally, A₁R functionality was significantly decreased in mother brain whereas no significant differences were detected in fetus brain. These results suggest that glutamate administered to pregnant rats modulates A₁R signaling pathways in both tissues, showing an A₁R down-regulation in fetal brain, and desensitization in maternal brain.     

Adenosine Receptor Agonists Inhibit K+-Evoked Ca2+ Uptake by Rat Brain Cortical Synaptosomes

Abstract: The uptake of Ca²⁺ by a K⁺-depolarized rat brain cerebral cortical crude synaptosomal preparation (P₂ fraction) was investigated. The characteristics of the Ca²⁺ uptake system are similar to those observed by other investigators. The preparation is also a suitable model with which to study the effects of adenosine on Ca²⁺ uptake and neurotransmitter release, as it is generally accepted that K⁺-evoked Ca²⁺ uptake is intimately related to depolarization-induced release of neurotransmitters. We have demonstrated that an extracellular receptor is involved in mediating the adenosine-evoked inhibition of K⁺-evoked Ca²⁺ uptake. The pharmacological properties of the receptor suggest that it may be similar in some respects to the A₂-receptor associated with adenylate cyclase. The adenosine uptake inhibitor, dipyridamole, potentiated the action of adenosine, suggesting that re-uptake is important in controlling the extracellular adenosine concentration and thus in the regulation of the adenosine receptor. The adenosine receptor antagonist theophylline inhibited the effects of adenosine. Calmodulin inhibited K⁺- evoked uptake of Ca²⁺ by the synaptosomal fraction.     

Modulation of [3H]Acetylcholine Release from Cultured Amacrine-Like Neurons by Adenosine A1 Receptors

Abstract: We have investigated the effect of endogenous adenosine on the release of [³H]acetylcholine ([³H]ACh) in cultured chick amacrine-like neurons. The release of [³H]ACh evoked by 50 m M KCl was mostly Ca²⁺ dependent, and it was increased in the presence of adenosine deaminase and in the presence of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an adenosine A₁ receptor antagonist. The effect of adenosine on [³H]ACh release was sensitive to pertussis toxin (PTX) and was due to a selective inhibition of N-type Ca²⁺ channels. Ligand binding studies using [³H]DPCPX confirmed the presence of adenosine A₁ receptors in the preparation. Using specific inhibitors of the plasma membrane adenosine carriers and of the ectonucleotidases, we found that the extracellular accumulation of adenosine in response to KCl depolarization was due to the release of endogenous adenosine per se and to the extracellular conversion of released nucleotides into adenosine. Activation of adenosine A₁ receptors was without effect on the intracellular levels of cyclic AMP under depolarizing conditions, but it inhibited the accumulation of inositol phosphates. Our results indicate that in cultured amacrine-like neurons, the Ca²⁺-dependent release of [³H]ACh evoked by KCl is under tonic inhibition by adenosine, which activates A₁ receptors. The effect of adenosine on the [³H]ACh release may be due to a direct inhibition of N-type Ca²⁺ channels and/or secondary to the inhibition of phospholipase C and involves the activation of PTX-sensitive G proteins.     

[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.     

Adenosine A1 Receptor Inhibition of Glutamate Exocytosis and Protein Kinase C-Mediated Decoupling

Abstract: The adenosine modulation of glutamate exoeytosis from guinea pig cerebrocortical synaptosomes is investigated. Endogenously leaked adenosine is sufficient to cause a partial tonic inhibition of 4-aminopyridine-evoked glutamate release, which can be relieved by adenosine deaminase. The adenosine A₁ receptor is equally effective in mediating inhibition of glutamate exocytosis evoked by 4-aminopyridine (where K⁺-channel activation would inhibit release) and by elevated KC1 (where K⁺-channel activation would have no effect), arguing for a central role of Ca²⁺-channel modulation. In support of this, the plateau phase of depolarization-evoked free Ca²⁺ elevation is decreased by adenosine with both depolarization protocols. No effect of adenosine agonists is seen on membrane potential in polarized or KC1- or 4-aminopyridine-stimulated synaptosomes. The interaction of protein kinase C with the A₁ receptormediated inhibition is examined. Activation of protein kinase C by 4β-phorbol dibutyrate has been shown previously by this laboratory to modulate glutamate release via K⁺-channel inhibition, and is shown here to have an additional action of decoupling the adenosine inhibition of glutamate exocytosis.     

Adenosine Deaminase Interacts with A1 Adenosine Receptors in Pig Brain Cortical Membranes

Abstract: Adenosine deaminase is an enzyme of purine metabolism that has largely been considered to be cytosolic. A few years ago, adenosine deaminase was reported to appear on the surface of cells. Recently, it has been demonstrated that adenosine deaminase interacts with a type II membrane protein known as either CD26 or dipeptidylpeptidase IV. In this study, by immunoprecipitation and affinity chromatography it is shown that adenosine deaminase and A₁ adenosine receptors interact in pig brain cortical membranes. This is the first report in brain demonstrating an interaction between a degradative ectoenzyme and the receptor whose ligand is the enzyme substrate. By means of this interaction adenosine deaminase leads to the appearance of the high-affinity site of the receptor, which corresponds to the receptor-G protein complex. Thus, it seems that adenosine deaminase is necessary for coupling A₁ adenosine receptors to heterotrimeric G proteins.     

Modulation of adenosine A1 and A2A receptors in C6 glioma cells during hypoxia: involvement of endogenous adenosine

During hypoxia, extracellular adenosine levels are increased to prevent cell damage, playing a neuroprotective role mainly through adenosine A₁ receptors. The aim of the present study was to analyze the effect of hypoxia in both adenosine A₁ and A_2A receptors endogenously expressed in C6 glioma cells. Two hours of hypoxia (5% O₂) caused a significant decrease in adenosine A₁ receptors. The same effect was observed at 6 h and 24 h of hypoxia. However, adenosine A_2A receptors were significantly increased at the same times. These effects were not due to hypoxia-induced alterations in cells number or viability. Changes in receptor density were not associated with variations in the rate of gene expression. Furthermore, hypoxia did not alter HIF-1α expression in C6 cells. However, HIF-3α, CREB and CREM were decreased. Adenosine A₁ and A_2A receptor density in normoxic C6 cells treated with adenosine for 2, 6 and 24 h was similar to that observed in cells after oxygen deprivation. When C6 cells were subjected to hypoxia in the presence of adenosine deaminase, the density of receptors was not significantly modulated. Moreover, DPCPX, an A₁ receptor antagonist, blocked the effects of hypoxia on these receptors, while ZM241385, an A_2A receptor antagonist, was unable to prevent these changes. These results suggest that moderate hypoxia modulates adenosine receptors and cAMP response elements in glial cells, through a mechanism in which endogenous adenosine and tonic A₁ receptor activation is involved.     

Bidirectional Regulation of GABAA Receptor α1 and α6 Subunit Expression by a Cyclic AMP-Mediated Signalling Mechanism in Cerebellar Granule Cells in Primary Culture

Abstract: Forskolin treatment of cerebellar granule cells in culture resulted in bidirectional regulation of the expression of GABA_A receptor α1 and α6 subunits. Thus, forskolin applied at 2 days in vitro (DIV) increased expression of the α1 subunit but decreased the expression of the α6 subunit. Values with respect to control cultures, both assayed at 9 DIV by immunoblotting, were 310 ± 48% for α1 and 25 ± 16% for the α6 subunit. Similar effects were evoked following chronic treatment with both dibutyryl cyclic AMP and 3-isobutyl-1-methylxanthine. Dideoxyforskolin had no effect on the level of expression of either the α1 or the α6 GABA_A receptor subunits. The changes in subunit expression were accompanied by a 1.7-fold increase in number of total specific [³H]Ro 15-4513 binding sites expressed by intact cerebellar granule cells. This increase in total binding sites was accommodated by a 2.7-fold increase in number of diazepam-sensitive Ro 15-4513 binding sites in accordance with the observed increase in α1 subunit expression. The number of diazepam-insensitive subtype of binding sites were not significantly changed. These results suggest that GABA_A receptor subtype expression can be differentially regulated by intracellular cyclic AMP concentration.     

Dual Signalling by the Adenosine A2a Receptor Involves Activation of Both N- and P-Type Calcium Channels by Different G Proteins and Protein Kinases in the Same Striatal Nerve Terminals

Abstract: Many G_s-linked receptors have been reported to use multiple signalling pathways in transfected cells but few in their normal cell environment. We show that the adenosine A_2a receptor uses two signalling pathways to increase the release of acetylcholine from striatal nerve terminals. One pathway involves activation of G_s, adenylyl cyclase, protein kinase A, and P-type calcium channels; the other is mediated by a cholera toxin-insensitive G protein, protein kinase C, and N-type calcium channels. The effects of these two pathways are not additive, the second pathway being inhibited by the first; but they are equally sensitive to the A_2a receptor antagonist KF17837. This demonstrates that the A_2a receptor activates two signalling systems in striatal cholinergic neurons.     

Adenosine A1 Receptors Are Associated with Cerebellar Granule Cells

The cerebellum of mouse appears to have only the adenosine A1 receptor, which decreases adenylate cyclase activity, and not the A2 receptor, which increases adenylate cyclase activity. The adenosine analog N6-(L-phenylisopropyl)adenosine (PIA), stimulates the A1 receptor in a membrane preparation and decreases basal adenylate cyclase activity by 40%. The EC50 for PIA is approximately 50 nM. To associate the A1 receptor with a cerebellar cell type, three different neurological mutant mouse strains were studied: staggerer (Purkinje and granule cell defect), nervous (Purkinje cell defect), and weaver (granule cell defect). PIA was unable to effect a maximal decrease in adenylate cyclase activity of membranes prepared from cerebella of the staggerer and weaver mice in comparison with the respective littermate control mice. In contrast, membranes from nervous mice and their littermates showed similar PIA dose-response curves. Moreover, the diminished PIA response observed in the weaver cerebellum, when compared with the control littermate, was not detected in the striatum. This suggests no overall brain defect in the adenosine A1 receptors coupled to adenylate cyclase of the weaver mouse. We conclude that a loss of granule cells coincides with an attenuated response to PIA, implying that the A1 receptors are associated with the granule cells of the cerebellum.     

Rat-1 Fibroblasts Engineered with GAD65 and GAD67 cDNAs in Retroviral Vectors Produce and Release GABA

Abstract: The effects of the adenosine A₁ agonist N ₆-cyclohexyladenosine (CHA) on MPTP-induced dopamine (DA) depletion in the striatum of C57BL/6 mice were studied. Twenty hours after a single injection of MPTP (30 mg/kg, s.c.), the toxin caused 62% depletion of striatal DA. CHA (0.2–3 mg/kg, s.c.), when given together with MPTP, prevented the toxin-induced DA depletion in a dose-dependent manner. This protective action was apparently mediated by the A¹ receptors, because this effect was selectively antagonized by pretreating the animals with the A₁ antagonist 8-cyclopentyl-1,3-dipropylxanthine (25 mg/kg, i.p.) but not with the A₂ antagonist 1,3-dipropyl-7-methylxanthine (25 mg/kg, i.p.). When CHA (3 mg/kg) was injected 5 h after MPTP administration, at which point striatal DA levels were already reduced significantly, a rapid and complete recovery of the striatal DA levels occurred. These neurochemical data suggest that the A₁ agonist CHA is potentially useful as a neuroprotective agent against MPTP-induced toxicity.     

Inhibition of Voltage-Sensitive Calcium Channels by the A2A Adenosine Receptor in PC12 cells

Abstract: The role of the A_2A adenosine receptor in regulating voltage-sensitive calcium channels (VSCCs) was investigated in PC12 cells. Ca²⁺ influx induced by membrane depolarization with 70 m M K⁺ could be inhibited with CGS21680, an A_2A receptor-specific agonist. Both L- and N-type VSCCs were inhibited by CGS21680 treatment. Effects of adenosine receptor agonists and antagonists indicate that the typical A_2A receptor mediates inhibition of VSCCs. Cholera toxin (CTX) treatment for 24 h completely eliminated the CGS21680 potency. Similar inhibitory effects on VSCCs were obtained by membrane-permeable activators of protein kinase A (PKA). These effects were blocked by Rp -adenosine-3',5'-cyclic monophosphothioate, a PKA inhibitor. The data suggest that activation of the A_2A receptor leads to inhibition of VSCCs via a CTX-sensitive G protein and PKA. ATP pretreatment caused a reduction in subsequent rise in cytosolic free Ca²⁺ concentration induced by 70 m M K⁺, presumably by inactivation of VSCCs. Simultaneous treatment with ATP and CGS21680 produced significantly greater inhibition of VSCCs than treatment with CGS21680 or ATP alone. Furthermore, the CGS21680-induced inhibition of VSCCs was not affected by the presence of reactive blue 2. CGS21680 still significantly inhibited ATP-evoked Ca²⁺ influx without VSCC activity after cobalt or 70 m M K⁺ pretreatment. These data suggest that the A_2A receptor-sensitive VSCCs differ from those activated by ATP treatment. Although A_2A receptors induce inhibition of VSCCs as well as ATP-induced Ca²⁺ influx, the two inhibitory effects are clearly distinct from each other.     

Inhibition of Striatal GABA Release by the Adenosine A2a Receptor Is Not Mediated by Increases in Cyclic AMP

Abstract: The adenosine A_2a receptor inhibition of potassium (15 m M )-evoked GABA release from striatal nerve terminals has been examined. High extracellular calcium concentrations (4 m M ) reduced the effect of the A_2a receptor agonist CGS-21680 (1 n M ). CGS-21680 inhibited GABA release in the presence of the L-type calcium channel blocker nifedipine, which itself inhibited evoked GABA release (by 16 ± 4%). ω-Conotoxin inhibited the evoked release by 45 ± 4% and prevented the action of CGS-21680. Forskolin and 8-bromo cyclic AMP both stimulated evoked GABA release at low concentrations, but at higher concentrations they abolished the inhibition by CGS-21680 without affecting the evoked release. The nonselective protein kinase inhibitor H-7 inhibited both the evoked release and the inhibition by CGS-21680, whereas the selective protein kinase A and G inhibitor HA-1004 had no effect on either evoked release or the action of CGS-21680. Pretreatment with pertussis toxin did not affect the A_2a receptor-mediated inhibition. Therefore, the effect of A_2a receptor stimulation was not mediated by protein kinases A or G but was inhibited by elevated cyclic AMP levels and mimicked by inhibitors of the N-type calcium channel and protein kinase C.