Role Of Extracellular Adenosine Research Articles

CD73 Downregulation Decreases In Vitro and In Vivo Glioblastoma Growth

Glioblastoma is the worst and most common primary brain tumor. Here, we demonstrated the role of CD73, an enzyme responsible for adenosine (ADO) production, in glioblastoma progression. ADO increased glioma cell viability via A1 receptor sensitization. CD73 downregulation decreased glioma cell migration and invasion by reducing metalloproteinase-2 and vimentin expression and reduced cell proliferation by 40%, which was related to necrosis and sub-G1 phase blockage of cell cycle. Those effects also involved the stimulation of Akt/NF-kB pathways. Additionally, CD73 knockdown or enzyme inhibition potentiated temozolomide cytotoxic effect on glioma cells by decreasing the IC50 value and sensitizing cells to a non-cytotoxic drug concentration. CD73 inhibition also decreased in vivo rat glioblastoma progression. Delivery of siRNA-CD73 or APCP reduced tumor size by 45 and 40%, respectively, when compared with control. This effect was followed by a parallel 95% reduction of ADO levels in cerebrospinal fluid, indicating the role of extracellular ADO in in vivo glioma growth. Treatment did not induce systemic damage or mortality. Altogether, we conclude that CD73 is an interesting target for glioblastoma treatment and its inhibition may provide new opportunities to improve the treatment of brain tumors. Graphical Abstract ᅟ.

A CD38/CD203a/CD73 ectoenzymatic pathway independent of CD39 drives a novel adenosinergic loop in human T lymphocytes

The tumor microenvironment is characterized by of high levels of extracellular nucleotides that are metabolized through the dynamic and sequential action of cell surface enzymes (ectoenzymes). These ectoenzymes operate according to their spatial arrangement, as part of (1) continuous (molecules on the same cell) or (2) discontinuous (molecules on different cells) pathways, the latter being facilitated by restricted cellular microenvironment. The outcome of this catabolic activity is an increase in the local concentration of adenosine, a nucleoside involved in the control of inflammation and immune responses. The aim of the work presented here was to demonstrate that a previously unexplored enzymatic pathway may be an alternate route to produce extracellular adenosine. Our data show that this new axis is driven by the nucleotide-metabolizing ectoenzymes CD38 (an NAD+ nucleosidase), the ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1, also known as CD203a or PC-1) and the 5′ ectonucleotidase (5′-NT) CD73, while bypassing the canonical catabolic pathway mediated by the nucleoside tri- and diphosphohydrolase (NTPDase) CD39. To determine the relative contributions of these cell surface enzymes to the production of adenosine, we exploited a human T-cell model allowing for the modular expression of the individual components of this alternative pathway upon activation and transfection. The biochemical analysis of the products of these ectoenzymes by high-performance liquid chromatography (HPLC) fully substantiated our working hypothesis. This newly characterized pathway may facilitate the emergence of an adaptive immune response in selected cellular contexts. Considering the role for extracellular adenosine in the regulation of inflammation and immunogenicity, this pathway could constitute a novel strategy of tumor evasion, implying that these enzymes may represent ideal targets for antibody-mediated therapy.

CD73-generated adenosine facilitatesToxoplasma gondiidifferentiation to long-lived tissue cysts in the central nervous system

Toxoplasma gondii is an obligate intracellular protozoan pathogen that traffics to the central nervous system (CNS) following invasion of its host. In the CNS, T. gondii undergoes transformation from a rapidly dividing tachyzoite to a long-lived, slow-dividing bradyzoite contained within cysts. The role of extracellular adenosine in T. gondii pathogenesis has not been previously investigated. T. gondii uses host purines such as adenosine for its energy needs, as it is unable to make its own. Here, we show that CD73(-/-) mice, which lack the ability to generate extracellular adenosine, are protected from T. gondii chronic infection, with significantly fewer cysts and reduced susceptibility to reactivation of infection in the CNS independent of host effector function. Parasite dissemination to the brain was unimpaired in CD73(-/-) hosts, suggesting that the reduced cyst number is due to impaired parasite differentiation in the CNS. Confirming this, T. gondii tachyzoites formed fewer cysts following alkaline pH stress in astrocytes isolated from CD73(-/-) mice compared with wild type, and in fibroblasts treated with a CD73 inhibitor. Cyst formation was rescued in CD73(-/-) astrocytes supplemented with adenosine, but not with adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine. Furthermore, mice lacking adenosine receptors had no defect in cyst formation. Based on these findings, we conclude that CD73 expression promotes Toxoplasma bradyzoite differentiation and cyst formation by a mechanism dependent on the generation of adenosine, but independent of adenosine receptor signaling. Overall, these findings suggest that modulators of extracellular adenosine may be used to develop therapies aimed at defending against human toxoplasmosis.

CD73-generated adenosine limits immunopathology during acute Toxoplasma gondii infection (164.17)

Abstract As an obligate intracellular pathogen, the apicomplexan parasite Toxoplasma gondii elicits robust innate and adaptive immune responses that must be limited to prevent immunopathology. The breakdown of extracellular ATP to adenosine by the cell-surface enzyme CD73 plays an important role in the resolution of inflammation. The role of extracellular adenosine in the immune response to T. gondii infection has not been previously elucidated. In comparison to wildtype C57BL/6 mice, CD73-knockout mice infected with T. gondii by interperitoneal injection were highly susceptible to immune-mediated pathology, with significantly increased morbidity and mortality associated with marked infiltration of neutrophils and T cells into the peritoneal cavity. Peritoneal exudate cells from infected CD73KO mice generated higher levels of the inflammatory mediators nitric oxide, TNF-alpha, and IL1-beta, without enhanced parasite killing or clearance. The increased morbidity and mortality seen in CD73KO mice could be rescued by treating mice with the broad spectrum adenosine receptor agonist 5'-N-ethylcarboxamido adenosine. In addition, mice deficient in the adenosine receptor A2A were more susceptible to immunopathology during intraperitoneal infection with T. gondii compared to wildtype mice. Thus extracellular adenosine is a key molecule that regulates the immune response to an intracellular pathogen and promotes host survival.

Preserving postischemic reperfusion in the kidney: a role for extracellular adenosine

Several adenosine receptor subtypes on endothelial, epithelial, mesangial, and inflammatory cells have been implicated in ischemic acute kidney injury, a life-threatening condition that frequently complicates the care of hospitalized patients. In this issue of the JCI, Grenz and coworkers provide novel insight into how preservation of postischemic renal perfusion by endothelial cell adenosine A2B receptors is antagonized by adenosine reuptake into proximal tubule cells by equilibrative nucleotide transporter 1, which can be inhibited by dipyridamole. The work suggests that adenosine A2B receptor agonists and inhibition of equilibrative nucleoside transporters by dipyridamole may have therapeutic potential in ischemic acute kidney injury, a condition for which there are currently no specific therapeutic interventions.

The Role of Extracellular Adenosine in Chemical Neurotransmission in the Hippocampus and Basal Ganglia: Pharmacological and Clinical Aspects

Now there is general agreement that the purine nucleoside adenosine is an important neuromodulator in the central nervous system, playing a crucial role in neuronal excitability and synaptic/non-synaptic transmission in the hippocampus and basal ganglia. Adenosine is derived from the breakdown of extra- or intracellular ATP and is released upon a variety of physiological and pathological stimuli from neuronal and non-neuronal sources, i.e. from glial cells and exerts effects diffusing far away from release sites. The resultant elevation of adenosine levels in the extracellular space reaches micromolar level, and leads to the activation A1, A2A, A2B and A3 receptors, localized to pre- and postsynaptic as well as extrasynaptic sites. Activation of presynaptic A1 receptors inhibits the release of the majority of transmitters including glutamate, acetylcholine, noradrenaline, 5-HT and dopamine, whilst the stimulation of A2A receptors facilitates the release of glutamate and acetylcholine and inhibits the release of GABA. These actions underlie modulation of neuronal excitability, synaptic plasticity and coordination of neural networks and provide intriguing target sites for pharmacological intervention in ischemia and Parkinson’s disease. However, despite that adenosine is also released during ischemia, A1 adenosine receptors do not participate in the modulation of excitotoxic glutamate release, which is nonsynaptic and is due to the reverse operation of transporters. Instead, extrasynaptic A1 receptors might be responsible for the neuroprotection afforded by A1 receptor activation.

CD73 and extracellular adenosine confer susceptibility to Toxoplasma gondii infection (38.7)

Abstract After tissue injury or infection, high levels of ATP inside cells is liberated and can serve as a danger signal that activates immune cells and promotes inflammation. The subsequent breakdown of extracellular ATP to adenosine by the cell-surface enzyme CD73 plays an important role in the resolution of the inflammatory response. Interestingly, the protozoan pathogen Toxoplasma gondii requires host adenosine for its purine needs as it cannot synthesize purines de novo. The role of extracellular adenosine in the immune response to T. gondii infection has not been previously elucidated. To investigate the role of CD73-generated adenosine in the immune response to infection with Toxoplasma gondii, wild-type C57BL/6 and CD73-/- mice were inoculated with T. gondii cysts by intragastric gavage. CD73-/- mice were less susceptible to T. gondii infection, with reduced intestinal inflammation and tissue damage during the acute stages of infection, and reduced parasite burden in the brain during chronic infection. In addition, cysts isolated from infected CD73-knockout mice were attenuated in their ability to cause pathology and mortality when inoculated into naïve wildtype or CD73-/- mice. In vitro, peritoneal macrophages and bone-marrow derived dendritic cells lacking CD73 were less permissive to the intracellular replication of T. gondii tachyzoites. These findings are the first to demonstrate that lack of CD73-generated adenosine impairs T. gondii pathogenesis.

The role of extracellular adenosine in the basal ganglia for sleep-wake regulation

The role of extracellular adenosine in the basal ganglia for sleep-wake regulation

Role of Extracellular Adenosine in Acute Lung Injury

Acute lung injury (ALI) is a lung disease characterized by pulmonary edema and severe hypoxia. The past decade hosted a search for endogenous mechanisms controlling lung inflammation and pulmonary edema during ALI. As such, recent evidence indicates extracellular adenosine in orchestrating the resolution of pulmonary edema and inflammation during ALI.

Extracellular nucleotide metabolism in Leishmania: influence of adenosine in the establishment of infection

Leishmaniasis is a parasitic disease with a variety of clinical forms, which are related to the Leishmania species involved. In the murine model, Leishmania amazonensis causes chronic non-healing lesions in Leishmania braziliensis- or Leishmania major-resistant mouse strains. In this study, we investigated the involvement of the pathway of extracellular nucleotide hydrolysis, with special focus on the role of extracellular adenosine, in the establishment of Leishmania infection. Our results show that the more virulent parasite—L. amazonensis—hydrolyzes higher amounts of ATP, ADP and AMP than the two other species, probably due to the higher expression of membrane NTPDase. Corroborating the idea that increased production of adenosine is important to lesion development and establishment of tissue parasitism, we observed that increased 5′-nucleotidase activity in L. braziliensis or addition of adenosine at the moment of infection with this parasite resulted in an increase in lesion size and parasitism as well as a delay in lesion healing. Furthermore, inhibition of adenosine receptor A2B led to decreased lesion size and parasitism. Thus, our results suggest that the conversion of ATP, a molecule with pro-inflammatory activity, into adenosine, which possesses immunomodulatory properties, may contribute to the establishment of infection by Leishmania.

Adenosine A2A Receptors Are Essential for Long-Term Potentiation of NMDA-EPSCs at Hippocampal Mossy Fiber Synapses

The physiological conditions under which adenosine A2A receptors modulate synaptic transmission are presently unclear. We show that A2A receptors are localized postsynaptically at synapses between mossy fibers and CA3 pyramidal cells and are essential for a form of long-term potentiation (LTP) of NMDA-EPSCs induced by short bursts of mossy fiber stimulation. This LTP spares AMPA-EPSCs and is likely induced and expressed postsynaptically. It depends on a postsynaptic Ca2+ rise, on G protein activation, and on Src kinase. In addition to A2A receptors, LTP of NMDA-EPSCs requires the activation of NMDA and mGluR5 receptors as potential sources of Ca2+ increase. LTP of NMDA-EPSCs displays a lower threshold for induction as compared with the conventional presynaptic mossy fiber LTP; however, the two forms of LTP can combine with stronger induction protocols. Thus, postsynaptic A2A receptors may potentially affect information processing in CA3 neuronal networks and memory performance.

A novel mechanism of vasoregulation: ADP‐induced relaxation of the porcine isolated coronary artery is mediated via adenosine release

In this study, we have investigated the mechanism of ADP-induced relaxation of porcine coronary artery (PCA) rings. The P2Y receptor agonists ADP and ADPbetaS produced concentration-dependent relaxation of endothelium-denuded PCA smooth muscle with pD2 values of 5.3 and 4.9, respectively. RT-polymerase chain reaction (RT-PCR) and immunoblotting demonstrated mRNA and protein expression of P2Y1 and A2A adenosine receptors in the PCA. The nonselective P2 antagonist PPADS or the P2Y1-selective antagonist MRS2179 failed to alter ADP- or ADPbetaS-induced relaxations. Relaxations to ADP were, however, blocked by the A2A adenosine receptor-selective antagonists ZM241385 and SCH58261 (apparent pK(B) values of 9.2 and 8.9, respectively). We excluded roles for direct occupancy of A2A adenosine receptors by ADP or ADPbetaS as well as metabolism to adenosine as mechanisms for ADP-evoked relaxations. However, ADP responses were significantly enhanced in the presence of the ENT1 nucleoside transporter inhibitors dipyridamole and NBTI and were significantly inhibited by adenosine deaminase, indicating a role for extracellular adenosine. Suprafusion of [3H]-adenine-labeled PCA segments showed that ADP induced the release of a number of purines, including adenosine. These data suggest that ADP mediates relaxation of the PCA via a novel mechanism that involves adenine nucleotide-evoked adenosine release and the subsequent activation of A2A receptors.

Extracellular Adenosine in the Human Brain During Sleep and Sleep Deprivation: An in Vivo Microdialysis Study

To examine the pattern of extracellular adenosine in the human brain during sleep deprivation, sleep, and normal wake. Following recovery from implantation of clinical depth electrodes, epilepsy patients remained awake for 40 continuous hours, followed by a recovery sleep episode. Neurology ward at UCLA Medical Center. Seven male epilepsy patients undergoing depth electrode localization of pharmacologically refractory seizures. All subjects were implanted with depth electrodes, a subset of which were customized to contain microdialysis probes. Microdialysis samples were collected during normal sleep, sleep deprivation, and recovery sleep from human amygdalae (n = 8), hippocampus (n = 1), and cortex (n = 1). In none of the probes did we observe an increase in extracellular adenosine during the sleep deprivation. There was a significant, though very small, diurnal oscillation (2.5%) in 5 of the 8 amygdalae. There was no effect of epileptogenicity on the pattern of extracellular adenosine. Our observations, along with those in animal studies, indicate that the role of extracellular adenosine in regulating sleep pressure is not a global brain phenomenon but is likely limited to specific basal forebrain areas. Thus, if energy homeostasis is a function of sleep, an increased rate of adenosine release into the extracellular milieu of the amygdala, cortex, or hippocampus is unlikely to be a marker of such a process.

Extracellular Adenosine in the Human Brain During Sleep and Sleep Deprivation: An in Vivo Microdialysis Study

To examine the pattern of extracellular adenosine in the human brain during sleep deprivation, sleep, and normal wake. Following recovery from implantation of clinical depth electrodes, epilepsy patients remained awake for 40 continuous hours, followed by a recovery sleep episode. Neurology ward at UCLA Medical Center. Seven male epilepsy patients undergoing depth electrode localization of pharmacologically refractory seizures. All subjects were implanted with depth electrodes, a subset of which were customized to contain microdialysis probes. Microdialysis samples were collected during normal sleep, sleep deprivation, and recovery sleep from human amygdalae (n=8), hippocampus (n=1), and cortex (n=1). In none of the probes did we observe an increase in extracellular adenosine during the sleep deprivation. There was a significant, though very small, diurnal oscillation (2.5%) in 5 of the 8 amygdalae. There was no effect of epileptogenicity on the pattern of extracellular adenosine. Our observations, along with those in animal studies, indicate that the role of extracellular adenosine in regulating sleep pressure is not a global brain phenomenon but is likely limited to specific basal forebrain areas. Thus, if energy homeostasis is a function of sleep, an increased rate of adenosine release into the extracellular milieu of the amygdala, cortex, or hippocampus is unlikely to be a marker of such a process. Zeitzer JM; Morales-Villagran A; Maidment NT et al. Extracellular adenosine in the human brain during sleep and sleep deprivation: an in vivo microdialysis study.

The Role of Extracellular Adenosine in Regulating Mossy Fiber Synaptic Plasticity

Hippocampal mossy fiber synapses show unique molecular features and dynamic range of plasticity. A recent paper proposed that the defining features of mossy fiber synaptic plasticity are caused by a local buildup of extracellular adenosine (Moore et al., 2003). In this study, we reassessed the role of ambient adenosine in regulating mossy fiber synaptic plasticity in mouse and rat hippocampal slices. Synaptic transmission was highly sensitive to activation of presynaptic adenosine A1 receptors (A1Rs), which reduced transmitter release by >75%. However, most of A1Rs were not activated by ambient adenosine. Field potentials increased only by 20-30% when A1Rs were fully blocked with the A1R antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (1 microM). Moreover, blocking A1Rs hardly altered paired-pulse facilitation, frequency facilitation, or posttetanic potentiation. Frequency facilitation was similar in A1R-/- mice and when measured with NMDA receptor-mediated EPSCs in CA3 pyramidal cells in the presence of DPCPX. Additional experiments suggested that the results obtained by Moore et al. (2003) can partially be explained by their usage of a submerged recording chamber and elevated divalent cation concentrations. In conclusion, a reduction of the basal release probability by ambient adenosine does not underlie presynaptic forms of plasticity at mossy fiber synapses.

A Role for Extracellular Adenosine in Time-Dependent Reversal of Long-Term Potentiation by Low-Frequency Stimulation at Hippocampal CA1 Synapses

The involvement of adenosine on the development of time-dependent reversal of long-term potentiation (LTP) by low-frequency stimulation (LFS) was investigated at Schaffer collateral-CA1 synapses of rat hippocampal slices. A train of LFS (2 Hz, 10 min, 1200 pulses) had no long-term effects on synaptic transmission but produced lasting depression of previously potentiated responses. This reversal of LTP (depotentiation) was observed when the stimulus was delivered </=3 min after induction of LTP. However, application at 10 min after induction had no detectable effect on potentiation. This time-dependent reversal of LTP by LFS appeared to be mediated by extracellular adenosine, because it was mimicked by bath-applied adenosine and was specifically inhibited by the selective A(1) adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (100 nM). The effect of adenosine could be mimicked by 5-HT(1A) receptor agonist buspirone, but the LFS-induced depotentiation could not be antagonized by 5-HT(1A) receptor antagonist NAN-190. The source of extracellular adenosine in response to LFS appeared to be attributable to the efflux of cAMP. In addition, this LFS-induced depotentiation was blocked by bath application of adenylyl cyclase activator forskolin or injection of a cAMP analog Sp-adenosine cAMP (10 mM) into postsynaptic neurons. Moreover, the selective protein phosphatase 1 and 2A inhibitors okadaic acid and calyculin A prevented the LFS-induced depotentiation. These results thus suggest that increasing extracellular adenosine appears to underlie the LFS-induced depotentiation via acting on the A(1) receptor subtype to interrupt the cAMP-dependent biochemical processes leading to the LTP expression.

Release of Oxygen Metabolites From Chemoattractant-Stimulated Neutrophils Is Inhibited by Resting Platelets: Role of Extracellular Adenosine and Actin Polymerization

The effect of human platelets on chemoattractant-induced generation of oxygen metabolites in neutrophils was investigated, using luminol-enhanced chemiluminescence (CL). Resting platelets inhibited the extracellular, but not the intracellular, production of oxygen radicals in formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)-stimulated neutrophils. Maximal effect was obtained at the physiological neutrophil/platelet ratio of 1/50. Similar results were acquired by adding supernatants of platelets, indicating a role for a soluble factor. Removal of extracellular adenosine by adenosine deaminase (ADA), or blocking of adenosine-receptors by theophylline, antagonized the inhibitory effects of platelets (or the equivalent supernatant) on the neutrophil respiratory burst. In contrast, accumulation of adenosine by apyrase enhanced the inhibition. Exogenous adenosine mimicked the effects of platelets on the fMet-Leu-Phe-induced respiratory burst. To further assess the role of platelet-derived adenosine, the platelets were fixed with paraformaldehyde. We found that fixed platelets, as well as their supernatant, inhibited the fMet-Leu-Phe-induced CL-response to the same extent as viable cells. These effects were also reversed by ADA and theophylline, respectively. A prior removal of adenosine in the platelet suspension by ADA, followed by treatment with erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) to inactivate ADA, did not reverse the inhibitory action of platelets on the fMet-Leu-Phe-induced CL-response in neutrophils. However, if adenosine receptors of neutrophil at the same time were blocked with theophyline, the inhibition was significantly reduced. Platelets markedly increased the generation of adenosine in a neutrophil suspension. The effect was antagonized by S-(4-Nitrobenzyl)-6-thioguanosine (NBTG), but unaffected by alpha, beta-methyl-eneadenosine5'diphosphate (AMP-CP), indicating that the platelet-dependent accumulation of adenosine is due to an increased release of endogenous adenosine from neutrophils and not to a degradation of extracellular AMP. In correlation, NBTG, but not AMP-CP, reversed the platelet-mediated inhibition of the fMet-Leu-Phe-induced CL-response in neutrophils. Consequently, these data suggest that a platelet-derived factor increases the release of endogenously formed adenosine from neutrophils, terminating the production of oxygen radicals. The inhibition of oxidase activity was also associated with a platelet-induced polymerization of actin in the margin of the neutrophils. Treatment of neutrophils with cytochalasin B reversed the effects of platelets, both on F-actin content and CL-response. In summary, resting platelets limit the release of oxygen radicals from chemoattractant-stimulated neutrophils, thus preventing excessive damage to host tissues in the vascular space. This effect is suggested to be associated with an increase generation of neutrophil-derived adenosine enhancing an autoregulatory inhibitory pathway, and a peripheral accumulation of actin filaments forming a barrier for extracellular release of reactive oxygen radicals.

Calcium infusion induces myocardial ischaemia in patients with coronary artery disease by a mechanism possibly adenosine mediated

In the first part of the study five healthy volunteers were submitted to i.v. infusion of 0.2 mM.kg-1 b.w. of calcium gluconate over 20 min. Total calcium (atomic absorption method), ionized calcium (ion-selective electrode method) and adenosine (HPLC technique) were measured at the following times: 0 (basal), 5, 10, 15, 20 (end of infusion), 25, 30, 35, and 50 min. The increase in total and ionized calcium serum levels was associated with a significant increase in adenosine plasma levels (from 207 +/- 41 to 362 +/- 63 nM.l-1, P < 0.001). Since the increase in adenosine plasma levels, obtained either with adenosine or dipyridamole (an adenosine reuptake inhibitor), has been used to test the coronary reserve in coronary artery disease (CAD) patients, in the second part of the study we compared the effects of calcium infusion with dipyridamole in 15 subjects. Pharmacological stress tests were evaluated by monitoring two-dimensional echocardiography and ECG. Ten patients had positive results with both the dipyridamole stress test and the calcium infusion. Our results show that calcium infusion induces an increase in adenosine plasma levels that can elicit a dipyridamole-like coronary steal, thus suggesting the central role of extracellular adenosine in myocardial ischaemia.

Role of Extracellular Adenosine in Ethanol‐Induced Desensitization of Cyclic AMP Production

The decrease in receptor-stimulated cyclic AMP production after chronic ethanol exposure was suggested previously to be secondary to an ethanol-induced increase in extracellular adenosine. The present study was undertaken to ascertain whether a similar mechanism was responsible for the ethanol-induced desensitization of cyclic AMP production in PC12 pheochromocytoma cells. The acute addition of ethanol in vitro significantly increased both basal cyclic AMP content and extracellular levels of adenosine. A 4-day exposure to ethanol decreased basal as well as 2-chloroadenosine- and forskolin-stimulated cyclic AMP contents. No change in cyclic AMP content was observed after a 2-day exposure of PC12 cells to ethanol. Inclusion of adenosine deaminase during the chronic ethanol treatment significantly decreased extracellular levels of adenosine, yet the percentage decrease in 2-chloroadenosine- and forskolin-stimulated cyclic AMP levels after chronic ethanol exposure was not changed by the inclusion of the adenosine deaminase. Similar results were obtained when the chronic treatment was carried out with serum-free defined media. The ethanol-induced desensitization could not be mimicked by chronic exposure of PC12 cells to adenosine analogues. A 24-h exposure of PC12 cells to 2-chloroadenosine resulted in a decrease in the subsequent ability of this adenosine analogue to stimulate cyclic AMP content, but basal and forskolin-stimulated cyclic AMP levels were increased. Similar results were obtained after a 4-day exposure of PC12 cells to 2-chloroadenosine or 5'-N-ethylcarboxamido-adenosine. The present results indicate that the ethanol-induced decrease in receptor-stimulated cyclic AMP content in PC12 cells is not due to an increase in extracellular adenosine.