Effect of A2A adenosine receptor stimulation and antagonism on synaptic depression induced by in vitro ischaemia in rat hippocampal slices.
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1. In the present study we investigated the role of A2A adenosine receptors in hippocampal synaptic transmission under in vitro ischaemia-like conditions. 2. The effects of adenosine, of the selective A2A receptor agonist, CGS 21680 (2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoade nos ine ), and of selective A2A receptor antagonists, ZM 241385 (4-(2-[7-amino-2-(2-furyl)- inverted question mark1,2,4 inverted question mark-triazolo inverted question mark2,3-a inverted question mark inverted question mark1,3, 5 inverted question marktriazin-5-ylamino]ethyl)phenol) and SCH 58261 (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2, 4-triazolo[1,5-c]pyrimidine), have been evaluated on the depression of field e.p.s.ps induced by an in vitro ischaemic episode. 3. The application of 2 min of in vitro ischaemia brought about a rapid and reversible depression of field e.p.s.ps, which was completely prevented in the presence of the A1 receptor antagonist DPCPX (1, 3-dipropyl-8-cyclopentylxanthine) (100 nM). On the other hand both A2A receptor antagonists, ZM 241385 and SCH 58261, by themselves did not modify the field e.p.s.ps depression induced by in vitro ischaemia. 4. A prolonged application of either adenosine (100 micronM) or CGS 21680 (30, 100 nM) before the in vitro ischaemic episode, significantly reduced the synaptic depression. These effects were antagonized in the presence of ZM 241385 (100 nM). 5. SCH 58261 (1 and 50 nM) did not antagonize the effect of 30 nM CGS 21680 on the ischaemia-induced depression. 6. These results indicate that in the CA1 area of the hippocampus the stimulation of A2A adenosine receptors attenuates the A1-mediated depression of synaptic transmission induced by in vitro ischaemia. (+info)
Adenosine A(2A) receptor mRNA regulation by nerve growth factor is TrkA-, Src-, and Ras-dependent via extracellular regulated kinase and stress-activated protein kinase/c-Jun NH(2)-terminal kinase.
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We have shown previously that nerve growth factor (NGF) down-regulates adenosine A(2A) receptor (A(2A)AR) mRNA in PC12 cells. To define cellular mechanisms that modulate A(2A)AR expression, A(2A)AR mRNA and protein levels were examined in three PC12 sublines: i) PC12nnr5 cells, which lack the high affinity NGF receptor TrkA, ii) srcDN2 cells, which overexpress kinase-defective Src, and iii) 17.26 cells, which overexpress a dominant-inhibitory Ras. In the absence of functional TrkA, Src, or Ras, NGF-induced down-regulation of A(2A)AR mRNA and protein was significantly impaired. However, regulation of A(2A)AR expression was reconstituted in PC12nnr5 cells stably transfected with TrkA. Whereas NGF stimulated the mitogen-activated protein kinases p38, extracellular regulated kinase 1 and 2 (ERK1/ERK2), and stress-activated protein kinase/c-Jun NH(2)-terminal kinase (SAPK/JNK) in PC12 cells, these kinases were activated only partially or not at all in srcDN2 and 17.26 cells. Inhibiting ERK1/ERK2 with PD98059 or inhibiting SAPK/JNK by transfecting cells with a dominant-negative SAPKbeta/JNK3 mutant partially blocked NGF-induced down-regulation of A(2A)AR expression in PC12 cells. In contrast, inhibiting p38 with SB203580 had no effect on the regulation of A(2A)AR mRNA and protein levels. Treating SAPKbeta/JNK3 mutant-transfected PC12 cells with PD98059 completely abolished the NGF-induced decrease in A(2A)AR mRNA and protein levels. These results reveal a role for ERK1/ERK2 and SAPK/JNK in regulating A(2A)AR expression. (+info)
Afferent arteriolar adenosine A2a receptors are coupled to KATP in in vitro perfused hydronephrotic rat kidney.
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Adenosine is known to exert dual actions on the afferent arteriole, eliciting vasoconstriction, by activating A1 receptors, and vasodilation at higher concentrations, by activating lower-affinity A2 receptors. We could demonstrate both of these known adenosine responses in the in vitro perfused hydronephrotic rat kidney. Thus, 1.0 microM adenosine elicited a transient vasoconstriction blocked by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and 10-30 microM adenosine reversed KCl-induced vasoconstriction. However, when we examined the effects of adenosine on pressure-induced afferent arteriolar vasoconstriction, we observed a third action. In this setting, a high-affinity adenosine vasodilatory response was observed at concentrations of 10-300 nM. This response was blocked by both 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) and glibenclamide and was mimicked by 2-phenylaminoadenosine (CV-1808) (IC50 of 100 nM), implicating adenosine A2a receptors coupled to ATP-sensitive K channels (KATP). Like adenosine, 5'-N-ethylcarboxamidoadenosine (NECA) elicited both glibenclamide-sensitive and glibenclamide-insensitive vasodilatory responses. The order of potency for the glibenclamide-sensitive component was NECA > adenosine = CV-1808. Our findings suggest that, in addition to the previously described adenosine A1 and low-affinity A2b receptors, the renal microvasculature is also capable of expressing high-affinity adenosine A2a receptors. This renal adenosine receptor elicits afferent arteriolar vasodilation at submicromolar adenosine levels by activating KATP. (+info)
Cross talk between A(1) and A(2A) adenosine receptors in the hippocampus and cortex of young adult and old rats.
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Adenosine modulates synaptic transmission by acting on inhibitory A(1) and facilitatory A(2A) receptors, the densities of which are modified in aged animals. We investigated how A(2A) receptor activation influences A(1) receptor function and whether this interaction is modified in aged rats. In hippocampal and cortical nerve terminals from young adult (6 wk), but not old rats (24 mo), the A(2A) receptor agonist, 2-[4-(2-carboxyethyl) phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680; 30 nM) decreased the binding affinity of a selective A(1) receptor agonist, cyclopentyladenosine (CPA), an effect prevented by the A(2A) antagonist, (4-(2-[7-amino-2-(2-furyl (1,2,4)-triazolo(2,3-a (1,3,5)triazin-5-yl-aminoethyl)phenol (ZM 241385, 20 nM). This effect of CGS 21680 required intact nerve terminals and was also observed in the absence of Ca(2+). This A(2A)-induced "desensitization" of A(1) receptors was prevented by the protein kinase C inhibitor, chelerythrine (6 microM), and was not detected in the presence of the protein kinase C activator, phorbol-12,13-didecanoate (250 nM), which itself caused a reduction in binding affinity for CPA. The protein kinase A inhibitor, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (10 microM), and the protein kinase A activator, 8-Br-cAMP (1 mM), had no effects on the A(2A)-induced A(1) receptor desensitization. This A(2A)-induced A(1) receptor desensitization had a functional correlation because CGS 21680 (10 nM) attenuated by 40% the inhibition caused by CPA (10 nM) on CA1 area population spike amplitude in hippocampal slices. This A(2A)/A(1) interaction may explain the attenuation by adenosine deaminase (2 U/ml), which removes tonic A(1) inhibition, of the facilitatory effect of CGS 21680 on synaptic transmission. The requirement of tonic A(1) receptor activation for CGS 21680 to induce facilitation of synaptic transmission was reinforced by the observation that the A(1) receptor antagonist, 1, 3-dipropyl-8-cyclopentylxanthine (20 nM) prevented CGS 21680 (10 nM) facilitation of population spike amplitude. The present results show the ability of A(2A) receptors to control A(1) receptor function in a manner mediated by protein kinase C, but not protein kinase A, in young adult but not in aged rats. (+info)
Ligand-activation of the adenosine A2a receptors inhibits IL-12 production by human monocytes.
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Adenosine (ADO) exerts potent anti-inflammatory and immunosuppressive effects. In this paper we address the possibility that these effects are partly mediated by inhibition of the secretion of IL-12, a proinflammatory cytokine and a major inducer of Th1 responses. We demonstrate that 5'-N-ethylcarboxamidoadenosine (NECA), a nonspecific ADO analogue, and 2-p-(2-carbonyl-ethyl)phenylethylamino-5'-N-ethylcarboxamidoadenos ine (CGS-21680), a specific A2a receptor agonist, dose-dependently inhibited, in whole blood ex vivo and monocyte cultures, the production of human IL-12 induced by LPS and Stapholococcus aureus Cowan strain 1. However, the A1 receptor agonist 2-Chloro-N6-cyclopentyladenosine and the A3 receptor agonists N6-Benzyl-NECA and 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-be ta-d -ribofuranuronamide expressed only weak inhibitory effects. On the other hand, NECA and CGS-21680 dose-dependently potentiated the production of IL-10. The differential effect of these drugs on monocyte IL-12 and IL-10 production implies that these effects are mediated by A2a receptor signaling rather than by intracellular toxicity of ADO analogue's metabolites. Moreover, CGS-21680 inhibited IL-12 production independently of endogenous IL-10 induction, because anti-IL-10 Abs failed to prevent its effect. The selective A2a antagonist 8-(3-Chlorostyryl) caffeine prevented the inhibitory effect of CGS-21680 on IL-12 production. The phosphodiesterase inhibitor Ro 20-1724 dose-dependently potentiated the inhibitory effect of CGS-21680 and, furthermore, Rp-cAMPS, a protein kinase A inhibitor, reversed the inhibitory effect of CGS-21680, implicating a cAMP/protein kinase A pathway in its action. Thus, ligand activation of A2a receptors simultaneously inhibits IL-12 and stimulates IL-10 production by human monocytes. Through this mechanism, ADO released in excess during inflammatory and ischemic conditions, or tissue injury, may contribute to selective suppression of Th1 responses and cellular immunity. (+info)
Adenosine receptor occupancy suppresses chemoattractant-induced phospholipase D activity by diminishing membrane recruitment of small GTPases.
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Adenosine (Ado) is an important autocrine modulator of neutrophil functions. In this study, we determined the effects of endogenous Ado on fMet-Leu-Phe (fMLP)-induced phospholipase D (PLD) activity in neutrophils. The removal of extracellular Ado by Ado deaminase (ADA) or the blockade of its action by the A2a receptor antagonists 8-(3-chlorostyryl) caffeine (CSC) or CGS15943 markedly increased fMLP-induced PLD activation. The concentration-dependent stimulatory effects of CSC and CGS15943 were abolished by a pretreatment of neutrophil suspensionswith ADA. In contrast, the selective A2a receptor agonist CGS21680 suppressed fMLP-induced PLD activation. Furthermore, inhibition by CGS21680 of fMLP-induced PLD activity was reversed by CSC or CGS15943. The removal of Ado by ADA or the blockade of its action by CSC or CGS15943, markedly increased the membrane recruitment of cytosolic protein kinase Calpha (PKCalpha), RhoA, and ADP-ribosylation factor (ARF) in response to fMLP. As shown for PLD activity, the stimulatory effect of Ado receptor antagonists on PLD cofactors translocation was abolished by a pretreatment of the cells with ADA. Moreover, the membrane translocation of both PKCalpha, RhoA, and ARF in response to fMLP was attenuated by CGS21680 and this effect of the A2a receptor agonist was antagonized by CSC or CGS15943. These data demonstrate that Ado released by neutrophils in the extracellular milieu inhibits PLD activation by blocking membrane association of ARF, RhoA, and PKCalpha through Ado A2a receptor occupancy. (Blood. 2000;95:519-527) (+info)
Localization of adenosine A2a receptor in retinal development and oxygen-induced retinopathy.
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PURPOSE: To investigate the association of adenosine A2a receptors (A2aR) with retinal vasculogenesis and angiogenesis that occurs in the canine model of oxygen-induced retinopathy (OIR). METHODS: One-day-old dogs were exposed to 100/o oxygen for 4 days and killed in oxygen (5 days old) and at 3, 10, 17, and 23 days after exposure to hyperoxia. Room air control animals were killed at 1, 5, 8, 15, 22, and 28 days of age. Immunolocalization of A2aR was performed on frozen sections, and reaction product density was quantified using microdensitometry. Cell types were identified in serial sections using antibodies against von Willebrand factor (endothelial cells) and GFAP (astrocytes), and enzyme histochemistry for menadione-dependent a-glycerophosphate dehydrogenase (M-a-GPDH) (to label angioblasts and developing blood vessels). RESULTS: A2aR immunoreactivity was associated with forming blood vessels and angioblasts in the nerve fiber layer (NFL) of peripheral retina. As development progressed, vascular labeling decreased, whereas labeling of neuronal elements increased. In OIR, A2aR immunoreactivity in the NFL was reduced after exposure to hyperoxia and significantly elevated in the inner retina throughout vascularized retina and in advance of forming vasculature in all oxygen-treated animals returned to room air. A2aR immunoreactivity was also prominent in fronds of intravitreal neovascularization. CONCLUSIONS: A2aR immunoreactivity was associated with developing retinal vessels. As development progressed, vascular-associated A2aR labeling decreased and, concomitantly, labeling of neuronal elements increased. A2aR immunoreactivity was significantly elevated at the edge of forming vasculature in all animals returned to room air after hyperoxia and in intravitreal neovas cular formations. These results provide additional evidence for the importance of A2aR and its ligand adenosine in retinal vascular development and in the vasoproliferative stage of canine OIR. (+info)
Regulation of the phosphorylation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa in vivo by dopamine D1, dopamine D2, and adenosine A2A receptors.
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Dopamine D(1), dopamine D(2), and adenosine A(2A) receptors are highly expressed in striatal medium-sized spiny neurons. We have examined, in vivo, the influence of these receptors on the state of phosphorylation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32). DARPP-32 is a potent endogenous inhibitor of protein phosphatase-1, which plays an obligatory role in dopaminergic transmission. A dose-dependent increase in the state of phosphorylation of DARPP-32 occurred in mouse striatum after systemic administration of the D(2) receptor antagonist eticlopride (0.1-2.0 mg/kg). This effect was abolished in mice in which the gene coding for the adenosine A(2A) receptor was disrupted by homologous recombination. A reduction was also observed in mice that had been pretreated with the selective A(2A) receptor antagonist SCH 58261 (10 mg/kg). The eticlopride-induced increase in DARPP-32 phosphorylation was also decreased by pretreatment with the D(1) receptor antagonist SCH 23390 (0.125 and 0.25 mg/kg) and completely reversed by combined pretreatment with SCH 23390 (0.25 mg/kg) plus SCH 58261 (10 mg/kg). SCH 23390, but not SCH 58261, abolished the increase in DARPP-32 caused by cocaine (15 mg/kg). The results indicate that, in vivo, the state of phosphorylation of DARPP-32 and, by implication, the activity of protein phosphatase-1 are regulated by tonic activation of D(1), D(2), and A(2A) receptors. The results also underscore the fact that the adenosine system plays a role in the generation of responses to dopamine D(2) antagonists in vivo. (+info)