The potential of alkaline phosphatase as a treatment for sepsis-associated acute kidney injury.

Sepsis-associated acute kidney injury (AKI) is associated with a high attributable mortality and an increased risk of developing chronic kidney failure in survivors. As a successful therapy is, as yet, unavailable, a pharmacological treatment option is clearly warranted. Recently, two small phase II clinical trials demonstrated beneficial renal effects of bovine-derived alkaline phosphatase administration in critically ill patients with sepsis-associated AKI. The rationale behind the renal protective effects remains to be fully elucidated, but is likely to be related to dephosphorylation and thereby detoxification of detrimental molecules involved in the pathogenesis of sepsis-associated AKI. A potent candidate target molecule might be endotoxin (lipopolysaccharide) from the cell wall of Gram-negative bacteria, which is associated with the development of sepsis and becomes nontoxic after being dephosphorylated by alkaline phosphatase. Another target of alkaline phosphatase could be adenosine triphosphate, a proinflammatory mediator released during cellular stress, which can be converted by alkaline phosphatase into the tissue-protective and anti-inflammatory molecule adenosine. Human recombinant alkaline phosphatase, a recently developed replacement for bovine-derived alkaline phosphatase, has shown promising results in the preclinical phase. As its safety and tolerability were recently confirmed in a phase I clinical trial, the renal protective effect of human recombinant alkaline phosphatase in sepsis-associated AKI shall be investigated in a multicenter phase II clinical trial starting at the end of this year.

Functional and RNA expression profile of adenosine receptor subtypes in mouse mesenteric arteries.

Concentration-response curves (CRCs) of adenosine receptor (AR) agonists, NECA (nonspecific), CCPA (A1 specific), CGS-216870 (A2A specific), BAY 60-6583 (A2B specific), and Cl-IB-MECA (A3 specific) for mesenteric arteries (MAs) from 4 AR knockout (KO) mice (A1, A2A, A2B, and A3) and their wild type (WT) were constructed. The messenger RNA expression of MAs from KO mice and WT were also studied. Adenosine (10 to 10 M) and NECA (10 to 10 M) induced relaxation in all mice except A2B KO mice, which only showed constriction by adenosine at 10 to 10 and NECA at 10 to 10 M. The CCPA induced a significant constriction at 10 and 10 M in all mice, except A1KO. BAY 60-6583 induced relaxation (10 to 10 M) in WT and no response in A2BKO except at 10 M. The CRCs for BAY 60-6583 in A1, A2A, and A3 KO mice shifted to the left when compared with WT mice, suggesting an upregulation of A2B AR. No responses were noted to CGS-21680 in all mice. Cl-IB-MECA only induced relaxation at concentration greater than 10 M, and no differences were found between different KO mice. The CRC for Bay 60-6583 was not significantly changed in the presence of 10 M of L-NAME, 10 M of indomethacin, or both. Our data suggest that A2B AR is the predominant AR subtype and the effect may be endothelial independent, whereas A1 AR plays a significant modulatory role in mouse MAs.

CX3CL1 is neuroprotective in permanent focal cerebral ischemia in rodents.

The chemokine CX3CL1 and its receptor CX3CR1 are constitutively expressed in the nervous system. In this study, we used in vivo murine models of permanent middle cerebral artery occlusion (pMCAO) to investigate the protective potential of CX3CL1. We report that exogenous CX3CL1 reduced ischemia-induced cerebral infarct size, neurological deficits, and caspase-3 activation. CX3CL1-induced neuroprotective effects were long lasting, being observed up to 50 d after pMCAO in rats. The neuroprotective action of CX3CL1 in different models of brain injuries is mediated by its inhibitory activity on microglia and, in vitro, requires the activation of adenosine receptor 1 (A1R). We show that, in the presence of the A1R antagonist 1,3-dipropyl-8-cyclopentylxanthine and in A1R⁻/⁻ mice, the neuroprotective effect of CX3CL1 on pMCAO was abolished, indicating the critical importance of the adenosine system in CX3CL1 protection also in vivo. In apparent contrast with the above reported data but in agreement with previous findings, cx3cl1⁻/⁻ and cx3cr1(GFP/GFP) mice, respectively, deficient in CX3CL1 or CX3CR1, had less severe brain injury on pMCAO, and the administration of exogenous CX3CL1 increased brain damage in cx3cl1⁻/⁻ ischemic mice. We also report that CX3CL1 induced a different phagocytic activity in wild type and cx3cl1⁻/⁻ microglia in vitro during cotreatment with the medium conditioned by neurons damaged by oxygen-glucose deprivation. Together, these data suggest that acute administration of CX3CL1 reduces ischemic damage via an adenosine-dependent mechanism and that the absence of constitutive CX3CL1-CX3CR1 signaling changes the outcome of microglia-mediated effects during CX3CL1 administration to ischemic brain.

Adenosine activation of A(2B) receptor(s) is essential for stimulated epithelial ciliary motility and clearance.

Mucociliary clearance, vital to lung clearance, is dependent on cilia beat frequency (CBF), coordination of cilia, and the maintenance of periciliary fluid. Adenosine, the metabolic breakdown product of ATP, is an important modulator of ciliary motility. However, the contributions of specific adenosine receptors to key airway ciliary motility processes are unclear. We hypothesized that adenosine modulates ciliary motility via activation of its cell surface receptors (A(1), A(2A), A(2B), or A(3)). To test this hypothesis, mouse tracheal rings (MTRs) excised from wild-type and adenosine receptor knockout mice (A(1), A(2A), A(2B), or A(3), respectively), and bovine ciliated bronchial epithelial cells (BBECs) were stimulated with known cilia activators, isoproterenol (ISO; 10 µM) and/or procaterol (10 µM), in the presence or absence of 5'-(N-ethylcarboxamido) adenosine (NECA), a nonselective adenosine receptor agonist [100 nM (A(1), A(2A), A(3)); 10 µM (A(2B))], and CBF was measured. Cells and MTRs were also stimulated with NECA (100 nM or 10 µM) in the presence and absence of adenosine deaminase inhibitor, erythro-9- (2-hydroxy-3-nonyl) adenine hydrochloride (10 µM). Both ISO and procaterol stimulated CBF in untreated cells and/or MTRs from both wild-type and adenosine knockout mice by ~3 Hz. Likewise, CBF significantly increased ~2-3 Hz in BBECs and wild-type MTRs stimulated with NECA. MTRs from A(1), A(2A), and A(3) knockout mice stimulated with NECA also demonstrated an increase in CBF. However, NECA failed to stimulate CBF in MTRs from A(2B) knockout mice. To confirm the mechanism by which adenosine modulates CBF, protein kinase activity assays were conducted. The data revealed that NECA-stimulated CBF is mediated by the activation of cAMP-dependent PKA. Collectively, these data indicate that purinergic stimulation of CBF requires A(2B) adenosine receptor activation, likely via a PKA-dependent pathway.

CX3CL1-induced modulation at CA1 synapses reveals multiple mechanisms of EPSC modulation involving adenosine receptor subtypes.

We characterized the role of adenosine receptor (AR) subtypes in the modulation of glutamatergic neurotransmission by the chemokine fractalkine (CX3CL1) in mouse hippocampal CA1 neurons. CX(3)CL1 causes a reversible depression of excitatory postsynaptic current (EPSC), which is abolished by the A(3)R antagonist MRS1523, but not by A(1)R (DPCPX) or A(2A)R (SCH58261) antagonists. Consistently, CX3CL1-induced EPSC depression is absent in slices from A(3)R(-/-) but not A(1)R(-/-) or A(2A)R(-/-) mice. Further, A(3)R stimulation causes similar EPSC depression. In cultured neurons, CX3CL1-induced depression of AMPA current shows A(1)R-A(3)R pharmacology. We conclude that glutamatergic depression induced by released adenosine requires the stimulation of different ARs.

What knock-out animals tell us about the effects of caffeine.

Caffeine is well known for its complex pharmacological actions, in part reflecting the multiple molecular targets of caffeine. The adenosine receptors are the primary extracellular targets of caffeine. Since caffeine has similar affinity for several adenosine receptors, it has been difficult to determine which receptor subtypes mediate caffeine's effects using pharmacological tools. The development of genetic mutant mice deficient in adenosine receptors and other signaling molecules has allowed targeted inquiry into the molecular targets by which caffeine elicits its biological effects on behavior and gene expression. This review summarizes recent work using genetic knockout models to elucidate the mechanisms of caffeine action in the brain. This review focuses on insights into caffeine action from genetic knockout models on: (1) the molecular basis for caffeine's effects on psychomotor activity; (2) the involvement of adenosine receptors in caffeine-mediated arousal and cognitive effects; and (3) a novel approach using knockout animals coupled with microarray profiling to validate multiple molecular targets of caffeine in striatal gene expression.

Involvement of A(1) adenosine receptors in osmotic volume regulation of retinal glial cells in mice.

PURPOSE: Osmotic swelling of Müller glial cells has been suggested to contribute to retinal edema. We determined the role of adenosine signaling in the inhibition of Müller cell swelling in the murine retina. METHODS: The size of Müller cell somata was recorded before and during perfusion of retinal sections and isolated Müller cells with a hypoosmolar solution. Retinal tissues were freshly isolated from wild-type mice and mice deficient in A(1) adenosine receptors (A(1)AR(-/-)), or cultured as whole-mounts for three days. The potassium conductance of Müller cells was recorded in isolated cells, and retinal slices were immunostained against Kir4.1. RESULTS: Hypotonic exposure for 4 min induced a swelling of Müller cell bodies in retinal slices from A(1)AR(-/-) mice but not wild-type mice. Pharmacological inhibition of A(1) receptors or of the ecto-5'-nucleotidase induced hypoosmotic swelling of Müller cells from wild-type mice. Exogenous adenosine prevented the swelling of Müller cells from wild-type but not A(1)AR(-/-) mice. The antiinflammatory corticosteroid, triamcinolone acetonide, inhibited the swelling of Müller cells from wild-type mice; this effect was blocked by an antagonist of A(1) receptors. The potassium conductance of Müller cells and the Kir4.1 immunolabeling of retinal slices were not different between A(1)AR(-/-) and wild-type mice, both in freshly isolated tissues and retinal organ cultures. CONCLUSIONS: The data suggest that autocrine activation of A(1) receptors by extracellularly generated adenosine mediates the volume homeostasis of Müller cells in the murine retina. The swelling-inhibitory effect of triamcinolone is mediated by enhancement of endogenous adenosine signaling.

Adenosine signaling contributes to ethanol-induced fatty liver in mice.

Fatty liver is commonly associated with alcohol ingestion and abuse. While the molecular pathogenesis of these fatty changes is well understood, the biochemical and pharmacological mechanisms by which ethanol stimulates these molecular changes remain unknown. During ethanol metabolism, adenosine is generated by the enzyme ecto-5'-nucleotidase, and adenosine production and adenosine receptor activation are known to play critical roles in the development of hepatic fibrosis. We therefore investigated whether adenosine and its receptors play a role in the development of alcohol-induced fatty liver. WT mice fed ethanol on the Lieber-DeCarli diet developed hepatic steatosis, including increased hepatic triglyceride content, while mice lacking ecto-5'-nucleotidase or adenosine A1 or A2B receptors were protected from developing fatty liver. Similar protection was also seen in WT mice treated with either an adenosine A1 or A2B receptor antagonist. Steatotic livers demonstrated increased expression of genes involved in fatty acid synthesis, which was prevented by blockade of adenosine A1 receptors, and decreased expression of genes involved in fatty acid metabolism, which was prevented by blockade of adenosine A2B receptors. In vitro studies supported roles for adenosine A1 receptors in promoting fatty acid synthesis and for A2B receptors in decreasing fatty acid metabolism. These results indicate that adenosine generated by ethanol metabolism plays an important role in ethanol-induced hepatic steatosis via both A1 and A2B receptors and suggest that targeting adenosine receptors may be effective in the prevention of alcohol-induced fatty liver.

Adenosine receptors in regulation of dendritic cell differentiation and function.

Differentiation of functional dendritic cells (DCs) critically depends on the microenvironment. DCs differentiate in hypoxic tumor sites and inflamed or damaged tissue. Because local concentrations of adenosine reach high physiologically relevant levels in these conditions, we assessed the expression of adenosine receptors and the effect of their activation on differentiation of human monocytes and mouse peritoneal macrophages and hematopoietic progenitor cells (HPCs) into myeloid DCs. Stimulation of adenosine receptors skews DC differentiation toward a distinct cell population characterized by expression of both DC and monocyte/macrophage cell surface markers. Pharmacologic analysis and experiments with cells from A(2B) adenosine receptor knockout mice identified A(2B) receptor as the mediator of adenosine effects on DCs. Unlike normal myeloid DCs, adenosine-differentiated DCs have impaired allostimulatory activity and express high levels of angiogenic, pro-inflammatory, immune suppressor, and tolerogenic factors, including VEGF, IL-8, IL-6, IL-10, COX-2, TGF-beta, and IDO. They promoted tumor growth if injected into tumors implanted in mice. Using adenosine desaminase knockout animals, we showed that DCs with proangiogenic phenotype are highly abundant under conditions associated with elevated levels of extracellular adenosine in vivo. Adenosine signaling through A(2B) receptor is an important factor of aberrant DC differentiation and generation of tolerogenic, angiogenic, and proinflammatory cells.

The ectonucleotidase cd39/ENTPDase1 modulates purinergic-mediated microglial migration.

Microglia is activated by brain injury. They migrate in response to ATP and although adenosine alone has no effect on wild type microglial migration, we show that inhibition of adenosine receptors impedes ATP triggered migration. CD39 is the dominant cellular ectonucleotidase that degrades nucleotides to nucleosides, including adenosine. Importantly, ATP fails to stimulate P2 receptor mediated migration in cd39(-/-) microglia. However, the effects of ATP on migration in cd39(-/-) microglia can be restored by co-stimulation with adenosine or by addition of a soluble ectonucleotidase. We also tested the impact of cd39-deletion in a model of ischemia, in an entorhinal cortex lesion and in the facial nucleus after facial nerve lesion. The accumulation of microglia at the pathological sites was markedly decreased in cd39(-/-) animals. We conclude that the co-stimulation of purinergic and adenosine receptors is a requirement for microglial migration and that the expression of cd39 controls the ATP/adenosine balance.

Lack of effect of extracellular adenosine generation and signaling on renal erythropoietin secretion during hypoxia.

Previous studies have yielded conflicting results as to whether extracellular adenosine generation and signaling contributes to hypoxia-induced increases in renal erythropoietin (EPO) secretion. In this study, we combined pharmacological and genetic approaches to elucidate a potential contribution of extracellular adenosine to renal EPO release in mice. To stimulate EPO secretion, we used murine carbon monoxide exposure (400 and 750 parts per million CO, 4 h), ambient hypoxia (8% oxygen, 4 h), or arterial hemodilution. Because the ecto-5-nucleotidase (CD73, conversion of AMP to adenosine) is considered the pacemaker of extracellular adenosine generation, we first tested the effect of blocking extracellular adenosine generation with the specific CD73-inhibitor adenosine 5'-(alpha,beta-methylene) diphosphate (APCP) or by gene-targeted deletion of cd73. These studies showed that neither APCP-treatment nor targeted deletion of cd73 resulted in changes of stimulated EPO mRNA or serum levels, although the increases of adenosine levels in the kidney following CO exposure were attenuated in mice with APCP treatment or in cd73(-/-) mice. Moreover, pharmacological studies using specific inhibitors of individual adenosine receptors (A1 AR, DPCPX; A 2A AR, DMPX; A 2B AR, PSB 1115; A3AR, MRS 1191) showed no effect on stimulated increases of EPO mRNA or serum levels. Finally, stimulated EPO secretion was not attenuated in gene-targeted mice lacking A1A(-/-, A2A AR-/-, A2BAR(-/-), or A3AR-/-. Together, these studies combine genetic and pharmacological in vivo evidence that increases of EPO secretion during limited oxygen availability are not affected by extracellular adenosine generation or signaling.

Gs protein-coupled adenosine receptor signaling and lytic function of activated NK cells.

The effect of adenosine and its analogues on the cytotoxic activity of IL-2-activated NK cells was investigated. Adenosine is an endogenous ligand for four different adenosine receptor (AdoR) subtypes (AdoRA1, AdoRA2A, AdoRA2B, and AdoRA3). Increased concentrations of adenosine were found in ascites of MethA sarcoma or in culture medium of 3LL Lewis lung carcinoma growing under hypoxic conditions. We hypothesize that intratumor adenosine impairs the ability of lymphokine-activated killer (LAK) cells to kill tumor cells. The effect of AdoR engagement on LAK cells cytotoxic activity was analyzed using AdoR agonists and antagonists as well as LAK cells generated from AdoR knockout mice. Adenosine and its analogues efficiently inhibited the cytotoxic activity of LAK cells. CGS21680 (AdoRA2A agonist) and 5-N-ethylcarboxamide adenosine (NECA) (AdoRA2A/ADoRA2B agonist) inhibited LAK cell cytotoxicity in parallel with their ability to increase cAMP production. The inhibitory effects of stable adenosine analog 2-chloroadenosine (CADO) and AdoRA2 agonists were blocked by AdoRA2 antagonist ZM 241385. Adenosine and its analogues impair LAK cell function by interfering with both perforin-mediated and Fas ligand-mediated killing pathways. Studies with LAK cells generated from AdoRA1-/- and AdoRA3-/- mice ruled out any involvement of these AdoRs in the inhibitory effects of adenosine. LAK cells with genetically disrupted AdoRA2A were resistant to the inhibitory effects of adenosine, CADO and NECA. However, with extremely high concentrations of CADO or NECA, mild inhibition of LAK cytotoxicity was observed that was probably mediated via AdoRA2B signaling. Thus, by using pharmacological and genetic blockage of AdoRs, our results clearly indicate the prime importance of cAMP elevating AdoR2A in the inhibitory effect of adenosine on LAK cell cytotoxicity. The elevated intratumor levels of adenosine might inhibit the antitumor effects of activated NK cells.

Emotional instability but intact spatial cognition in adenosine receptor 1 knock out mice.

Several lines of evidence point to the involvement of adenosine in the regulation of important central mechanisms such as cognition, arousal, aggression and anxiety. In order to elucidate the involvement of the adenosine A1 receptor (A1AR) in spatial learning and the control of exploratory behaviour, we assessed A1AR knockout mice (A1AR-/-) and their wild-type littermates (A1AR+/+) in a place navigation task in the water maze and in a battery of forced and free exploration tests. In the water maze, A1AR-/- mice showed normal escape latencies and were indistinguishable from controls with respect to measures of spatial performance during both training and probe trial. But despite normal performance they showed increased wall hugging, most prominently after the relocation of the goal platform for reversal training. Quantitative analysis of strategy choices indicated that wall hugging was increased mainly at the expense of chaining and passive floating, whereas the frequency of trials characterised as direct swims or focal searching was normal in A1AR-/- mice. These results indicate intact spatial cognition, but mildly altered emotional reactions to the water maze environment. In line with this interpretation, A1AR-/- mice showed normal levels and patterns of activity, but a mild increase of some measures of anxiety in our battery of forced and free exploration paradigms. These results are in line with findings published using a genetically similar line, but demonstrate that the magnitude of the changes and the range of affected behavioural measures may vary considerably depending on the environmental conditions during testing.

Reduced startle habituation and prepulse inhibition in mice lacking the adenosine A2A receptor.

Adenosine and dopamine receptors interact in the CNS to modulate behaviour, including sensorimotor gating. Prepulse inhibition (PPI) has been suggested to be an operational measure of sensorimotor gating. PPI and startle habituation are disrupted in patients with schizophrenia. In experimental animals, both parameters are modulated by dopaminergic and adenosine receptor agonists and antagonists. In the present study, we measured PPI and startle habituation in mice that lack functional adenosine A(2A) receptors. Startle amplitudes, startle habituation and PPI were significantly reduced in mice homozygous null for the adenosine A(2A) receptor (A(2A)(-/-)). In addition, differential effects of amphetamine and MK-801 on startle amplitude, startle habituation and PPI were observed between A(2A)(-/-) and wildtype controls. These data support the involvement of adenosine A(2A) receptors in regulation of PPI and startle habituation.

Focal deletion of the adenosine A1 receptor in adult mice using an adeno-associated viral vector.

Adenosine is a ubiquitous neuromodulator that increases sleep, inhibits seizures, and promotes neuroprotection. Many of these effects are mediated by A1 receptors, but A1 receptors are expressed in most brain regions, and distinguishing the precise site of action of adenosine is challenging. To test the role of adenosine in different hippocampal regions, we have used the Cre-loxP system and an adeno-associated viral (AAV) vector to focally delete endogenous adenosine A1 receptors in the hippocampus. Microinjection of an AAV vector containing the gene for Cre recombinase induced intense, focal, neuron-specific recombination in reporter mice. In a separate line of mice with loxP sites flanking the major coding exon for the adenosine A1 receptor, this AAV-Cre markedly reduced A1 receptor mRNA and focally abolished the postsynaptic response to adenosine without any change in basic electrophysiologic properties. Adenosine inhibits signaling between CA3 and CA1 neurons, but it is unclear from pharmacologic studies whether this response is caused by presynaptic or postsynaptic effects. Deletion of A1 receptors from CA3 neurons abolished this response to adenosine, but deletion of A1 receptors from CA1 neurons had no effect, demonstrating a presynaptic site of action. This transduction knock-out technique holds enormous potential for dissecting the functions of different CNS pathways.

Identification of A3 receptor- and mast cell-dependent and -independent components of adenosine-mediated airway responsiveness in mice.

Adenosine-induced bronchoconstriction is a well-recognized feature of atopic asthma. Adenosine acts through four different G protein-coupled receptors to produce a myriad of physiological effects. To examine the contribution of the A(3) adenosine receptor to adenosine-induced bronchoconstriction and to assess the contribution of mast cells to this process, we quantified airway responsiveness to aerosolized adenosine in wild-type, A(3) receptor-deficient, and mast cell-deficient mice. Compared with the robust airway responses elicited by adenosine in wild-type mice, both A(3)-deficient and mast cell-deficient mice exhibited a significantly attenuated response compared with their respective wild-type controls. Histological examination of the airways 4 h after adenosine exposure revealed extensive degranulation of airway mast cells as well as infiltration of neutrophils in wild-type mice, whereas these findings were much diminished in A(3)-deficient mice and were not different from those in PBS-treated controls. These data indicate that the airway responses to aerosolized adenosine in mice occur largely through A(3) receptor activation and that mast cells contribute significantly to these responses, but that activation of additional adenosine receptors on a cell type(s) other than mast cells also contributes to adenosine-induced airway responsiveness in mice. Finally, our findings indicate that adenosine exposure can result in A(3)-dependent airway inflammation, as reflected in neutrophil recruitment, as well as alterations in airway function.

Activation of murine lung mast cells by the adenosine A3 receptor.

Adenosine has been implicated to play a role in asthma in part through its ability to influence mediator release from mast cells. Most physiological roles of adenosine are mediated through adenosine receptors; however, the mechanisms by which adenosine influences mediator release from lung mast cells are not understood. We established primary murine lung mast cell cultures and used real-time RT-PCR and immunofluorescence to demonstrate that the A(2A), A(2B), and A(3) adenosine receptors are expressed on murine lung mast cells. Studies using selective adenosine receptor agonists and antagonists suggested that activation of A(3) receptors could induce mast cell histamine release in association with increases in intracellular Ca(2+) that were mediated through G(i) and phosphoinositide 3-kinase signaling pathways. The function of A(3) receptors in vivo was tested by exposing mice to the A(3) receptor agonist, IB-MECA. Nebulized IB-MECA directly induced lung mast cell degranulation in wild-type mice while having no effect in A(3) receptor knockout mice. Furthermore, studies using adenosine deaminase knockout mice suggested that elevated endogenous adenosine induced lung mast cell degranulation by engaging A(3) receptors. These results demonstrate that the A(3) adenosine receptor plays an important role in adenosine-mediated murine lung mast cell degranulation.

Behavioral characterization of mice lacking the A3 adenosine receptor: sensitivity to hypoxic neurodegeneration.

1. The potential neuroprotective actions of the A3 adenosine receptor (A3AR) were investigated using mice with functional deletions of the A3AR (A3AR-/-) in behavioral assessments of analgesia, locomotion, tests predictive of depression and anxiety, and the effects of mild hypoxia on cognition and neuronal survival. 2. Untreated A3AR-/- mice were tested in standard behavioral paradigms, including activity in the open field, performance in the hot-plate, tail-flick, tail-suspension, and swim tests, and in the elevated plus maze. In addition, mice were exposed repeatedly to a hypoxic environment containing carbon monoxide (CO). The cognitive effects of this treatment were assessed using the contextual fear conditioning test. After testing, the density of pyramidal neurons in the CA1, 2, and 3 subfields of the hippocampus was determined using standard histological and morphometric techniques. 3. A3AR-/- mice showed increased locomotion in the open field test, elevated plus maze (number of arm entries) and light/dark box (number of transitions). However, they spent more time immobile in two different tests of antidepressant activity (Swim and tail suspension tests). A3AR-/- mice also showed evidence of decreased nociception in the hotplate, but not tail-flick tests. Further, A3AR-/- mice were more vulnerable to hippocampal pyramidal neuron damage following episodes of carbon monoxide (CO)-induced hypoxia. One week after exposure to CO a moderate loss of pyramidal neurons was observed in all hippocampal subfields of both wild-type (A3AR+/+) and A3AR-/- mice. However, the extent of neuronal death in the CA2-3 subfields was less pronounced in A3AR+/+ than A3AR-/- mice. This neuronal loss was accompanied by a decline in cognitive function as determined using contextual fear conditioning. These histological and cognitive changes were reproduced in wild-type mice by repeatedly administering the A3AR-selective antagonist MRS 1523 (5-propyl-2-ethyl-4-propyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate 1 mg/kg i.p.). 4. These results indicate that pharmacologic or genetic suppression of A3AR function enhances some aspects of motor function and suppresses pain processing at supraspinal levels, while acting as a depressant in tests predictive of antidepressant action. Consistent with previous reports of the neuroprotective actions of A3AR agonists, A3AR-/- mice show an increase in neurodegeneration in response to repeated episodes of hypoxia.