Adenosine 2A receptor modulates inflammation and phenotype in experimental abdominal aortic aneurysms.

Activation of the adenosine 2A receptor (A2AR) reduces inflammation in models of acute injury but contribution in development of chronic abdominal aortic aneurysms (AAAs) is unknown. Elastase perfusion to induce AAA formation in A2AR-knockout (A2ARKO) and C57BL6/J wild-type (WT) mice resulted in nearly 100% larger aneurysms in A2ARKO compared to WT at d 14 (P<0.05), with evidence of greater elastin fragmentation, more immune cell infiltration, and increased matrix metallatoproteinase (MMP) 9 expression (P<0.05). Separately, exogenous A2AR antagonism in elastase-perfused WT mice also resulted in larger aneurysms (P<0.05), while A2AR agonism limited aortic dilatation (P<0.05). Activated Thy-1.2(+) T lymphocytes from WT mice treated in vitro with A2AR antagonist increased cytokine production, and treatment with A2AR agonist decreased cytokine production (P<0.05 for all). Primary activated CD4(+) T lymphocytes from A2ARKO mice exhibited greater chemotaxis (P<0.05). A2AR antagonist increased chemotaxis of activated CD4(+) cells from WT mice in vitro, and A2AR agonist reduced this effect (P<0.05). A2AR activation attenuates AAA formation partly by inhibiting immune cell recruitment and reducing elastin fragmentation. These findings support augmenting A2AR signaling as a putative target for limiting aneurysm formation.

The adenosine A2A receptor - myocardial protectant and coronary target in endotoxemia.

BACKGROUND: Cardiac injury and dysfunction are contributors to disease progression and mortality in sepsis. This study evaluated the cardiovascular role of intrinsic A2A adenosine receptor (A2AAR) activity during lipopolysaccharide (LPS)-induced inflammation. METHODS: We assessed the impact of 24 h of LPS challenge (20 mg/kg, IP) on cardiac injury, coronary function and inflammatory mediator levels in Wild-Type (WT) mice and mice lacking functional A2AARs (A2AAR KO). RESULTS: Cardiac injury was evident in LPS-treated WTs, with ~7-fold elevation in serum cardiac troponin I (cTnI), and significant ventricular and coronary dysfunction. Absence of A2AARs increased LPS-provoked cTnI release at 24 h by 3-fold without additional demise of contraction function. Importantly, A2AAR deletion per se emulated detrimental effects of LPS on coronary function, and LPS was without effect in coronary vessels lacking A2AARs. Effects of A2AAR KO were independent of major shifts in circulating C-reactive protein (CRP) and haptoglobin. Cytokine responses were largely insensitive to A2AAR deletion; substantial LPS-induced elevations (up to 100-fold) in IFN-γ and IL-10 were unaltered in A2AAR KO mice, as were levels of IL-4 and TNF-α. However, late elevations in IL-2 and IL-5 were differentially modulated by A2AAR KO (IL-2 reduced, IL-5 increased). Data demonstrate that in the context of LPS-triggered cardiac and coronary injury, A2AAR activity protects myocardial viability without modifying contractile dysfunction, and selectively modulates cytokine (IL-2, IL-5) release. A2AARs also appear to be targeted by LPS in the coronary vasculature. CONCLUSIONS: These experimental data suggest that preservation of A2AAR functionality might provide therapeutic benefit in human sepsis.

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.

Adenosine A1 receptors and microglial cells mediate CX3CL1-induced protection of hippocampal neurons against Glu-induced death.

Fractalkine/CX3CL1 is a neuron-associated chemokine, which modulates microglia-induced neurotoxicity activating the specific and unique receptor CX3CR1. CX3CL1/CX3CR1 interaction modulates the release of cytokines from microglia, reducing the level of tumor necrosis factor-alpha, interleukin-1-beta, and nitric oxide and induces the production of neurotrophic substances, both in vivo and in vitro. We have recently shown that blocking adenosine A(1) receptors (A(1)R) with the specific antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) abolishes CX3CL1-mediated rescue of neuronal excitotoxic death and that CX3CL1 induces the release of adenosine from microglia. In this study, we show that the presence of extracellular adenosine is mandatory for the neurotrophic effect of CX3CL1 as reducing adenosine levels in hippocampal cultures, by adenosine deaminase treatment, strongly impairs CX3CL1-mediated neuroprotection. Furthermore, we confirm the predominant role of microglia in mediating the neuronal effects of CX3CL1, because the selective depletion of microglia from hippocampal cultures treated with clodronate-filled liposomes causes the complete loss of effect of CX3CL1. We also show that hippocampal neurons obtained from A(1)R(-/-) mice are not protected by CX3CL1 whereas A(2A)R(-/-) neurons are. The requirement of functional A(1)R for neuroprotection is not unique for CX3CL1 as A(1)R(-/-) hippocampal neurons are not rescued from Glu-induced cell death by other neurotrophins such as brain-derived neurotrophic factor and erythropoietin, which are fully active on wt neurons.

Inactivation of neuronal forebrain A receptors protects dopaminergic neurons in a mouse model of Parkinson's disease.

Adenosine A(2A) receptors antagonists produce neuroprotective effects in animal models of Parkinson's disease (PD). As neuroinflammation is involved in PD pathogenesis, both neuronal and glial A(2A) receptors might participate to neuroprotection. We employed complementary pharmacologic and genetic approaches to A(2A) receptor inactivation, in a multiple MPTP mouse model of PD, to investigate the cellular basis of neuroprotection by A(2A) antagonism. MPTP.HCl (20 mg/kg daily for 4 days) was administered in mice treated with the A(2A) antagonist SCH58261, or in conditional knockout mice lacking A(2A) receptors on forebrain neurons (fbnA(2A)KO mice). MPTP-induced partial loss of dopamine neurons in substantia nigra pars compacta (SNc) and striatum (Str), associated with increased astroglial and microglial immunoreactivity in these areas. Astroglia was similarly activated 1, 3, and 7 days after MPTP administration, whereas maximal microglial reactivity was detected on day 1, returning to baseline 7 days after MPTP administration. SCH58261 attenuated dopamine cell loss and gliosis in SNc and Str. Selective depletion of A(2A) receptors in fbnA(2A)KO mice completely prevented MPTP-induced dopamine neuron degeneration and gliosis in SNc, and partially counteracted gliosis in Str. Results provide evidence of a primary role played by neuronal A(2A) receptors in neuroprotective effects of A(2A) antagonists in a multiple MPTP injections model of PD. With the symptomatic antiparkinsonian potential of several A(2A) receptor antagonists being pursued in clinical trials, this study adds to the rationale for broader clinical benefit and use of these drugs early in the treatment of PD.

Mice heterozygous for both A1 and A(2A) adenosine receptor genes show similarities to mice given long-term caffeine.

Caffeine is believed to exert its stimulant effects by blocking A(2A) and A(1) adenosine receptors (A(2A)R and A(1)R). Although a genetic knockout of A(2A)R eliminates effects of caffeine, the phenotype of the knockout animal does not resemble that of caffeine treatment. In this study we explored the possibility that a mere reduction of the number of A(1)Rs and A(2A)Rs, achieved by deleting one of the two copies of the A(1)R and A(2A)R genes, would mimic some aspects of long-term caffeine ingestion. The A(1)R and A(2A)R double heterozygous (A(1)R-A(2A)R dHz) mice indeed had approximately one-half the number of A(1)R and A(2A)R, and there were little compensatory changes in A(2B) or A(3) adenosine receptor (A(2B)R or A(3)R) expression. The ability of a stable adenosine analog to activate receptors was shifted to the right by caffeine and in A(1)R-A(2A)R dHz tissue. Caffeine (0.3 g/l in drinking water for 7-10 days) and A(1)R-A(2A)R dHz genotype increased locomotor activity (LA) and decreased heart rate without significantly influencing body temperature. The acute stimulatory effect of a single injection of caffeine was reduced in A(1)R-A(2A)R dHz mice and in mice treated long term with oral caffeine. Thus at least some aspects of long-term caffeine use can be mimicked by genetic manipulation of the A(1)R and A(2A)R.

[Establishment and evaluation of brain adenosine A2A receptors inactivation model of mice].

To establish a model of inactivation adenosine A2A receptors in brain tissues of mice, we transplanted bone marrow cells (BMCs) from wild type (WT) C57BL/6 mice into A2A receptor knockout (A2A KO) C57BL/6 mice which were previously fractionated total body irradiation of 6.2 Gyx2. Six weeks later, we identified and evaluated the model. The results showed that the sexual chromagene pattern on white blood cells of recipient mice changed from female pattern to male pattern and there were 95.9% of A2AR+ cells in peripheral white blood cells of recipient mice, whereas there was no significant difference of A2AR mRNA level in brains between these recipient mice and A2AR KO mice. Furthermore, there was no significant difference of breathing frequency, brain water content and level of glutamate between the model mice and WT mice. These results indicated that we established successfully a mouse model of inactivation adenosine A2A receptors in brain tissues. This may provide a new and efficient strategy to study the effect of adenosine A2A receptors in disease and injuries of central nervous system.

[(3)H]Adenosine uptake in brainstem membranes of CD-1 mice lacking the adenosine A(2a) receptor.

Previous studies in our laboratory have demonstrated a decrease in [(3)H]nitrobenzylthioinosine binding sites in the brainstem of adenosine A(2a) receptor knockout mice, particularly in the brain nuclei involved in central control of cardiovascular function [Brain Research 877 (2000) 160]. The present study aimed to correlate this decrease, shown using autoradiography, with a functional change using a previously described method of [(3)H]adenosine uptake in a membrane preparation from the brainstem of wildtype CD - 1 and homozygous mutant mice lacking the adenosine A(2a) receptor. A statistically significant decrease was shown in the mean V(MAX) value obtained from homozygous mutant preparations (4.7 +/- 1.3 fmol/mg protein/20 s, P < 0.05, n = 4) compared to that obtained from wildtype controls (51.6 +/- 4.2 fmol/mg protein/20 s, n = 4). Competition studies using nucleoside uptake inhibitors showed a statistically significant increase in the log IC(50) values for dipyridamole (Wildtype: -4.3 +/- 0.2, Homozygous mutant: -8.3 +/- 0.4, n=5, P < 0.05) and dilazep (Wildtype: -3.9 +/- 0.8, Homozygous mutant: -8.3 +/- 0.8, n=5, P < 0.05) in the preparations using homozygous mutant tissue. The present study, in conjunction with the results of previous studies [Brain Research 877 (2000) 160], indicates that components of purinergic neurotransmission system have apparently adjusted in compensation for the lack of the A(2a) receptor.