Cell biology. Purinergic chemotaxis.
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Adenosine↗
Biomedical subjects
Joel Linden
Publications and source records attributed to Joel Linden.
Subcellular distributions of adenosine A1 and A2A receptors in the rat dorsomedial nucleus of the solitary tract at the level of the area postrema.
Adenosine A1 and A2A receptors mediate distinct cardiovascular components of defense reactions that are ascribed, in part, to opposing actions within the nucleus tractus solitarius. To assess the cellular sites of relevance to these actions, we examined the light and electron microscopic immunolabeling of adenosine A1 and A2A receptors in the rat dorsomedial nucleus of the solitary tract at the level of the area postrema (dmNTS-AP), a region crucial for cardiovascular regulation involving vagal baroreceptor afferents. Immunoreactivity for each receptor was independently localized to distinct segments of plasma membranes and endomembranes in somatodendritic, axonal, and glial profiles. The dendritic labeling for each receptor also was detected within and near asymmetric, excitatory-type synapses. Of all peroxidase labeled profiles exclusive of somata, approximately 58% were A1- and 39% were A2A-labeled dendrites. Dendrites and astrocytic glia were the profiles that most often expressed both subtypes of adenosine receptors. The axonal labeling for A2A receptors was seen mainly in unmyelinated axons, whereas the A1 receptors were prominently localized within axon terminals. These terminals often formed single or multisynaptic excitatory-type junctions or single symmetric synapses on dendrites, a few of which expressed A1 and A2A receptors. These results provide the first ultrastructural evidence that A1 and A2A receptors have distributions conductive to their dual involvement in modulating the output of single neurons and glial function in the dmNTS-AP, where the predominate presynaptic effects of adenosine are mediated through A1 receptors.
Adenosine↗
Potent and orally bioavailable 8-bicyclo[2.2.2]octylxanthines as adenosine A1 receptor antagonists.
In the search for a selective adenosine A1 receptor antagonist with greater aqueous solubility than the compounds currently in clinical trials as diuretics, a series of 1,4-substituted 8-cyclohexyl and 8-bicyclo[2.2.2]octylxanthines were investigated. The binding affinities of a variety of cyclohexyl and bicyclo[2.2.2]octylxanthines for the rat and human adenosine A1, A2A, A2B, and A3 receptors are presented. Bicyclo[2.2.2]octylxanthine 16 exhibited good pharmaceutical properties and in vivo activity in a rat diuresis model (ED50=0.3 mg/kg po). Optimization of the bridgehead substituent led to propionic acid 29 (BG9928), which retained high potency (hA1, Ki=7 nM) and selectivity for the adenosine A1 receptor (915-fold versus adenosine A2A receptor; 12-fold versus adenosine A2B receptor) with improved oral efficacy in the rat diuresis model (ED50=0.01 mg/kg) as well as high oral bioavailability in rat, dog, and cynomolgus monkey.
Adenosine A1 Receptor Antagonists↗
Adenosine A2A receptor activation reduces hepatic ischemia reperfusion injury by inhibiting CD1d-dependent NKT cell activation.
Ischemia reperfusion injury results from tissue damage during ischemia and ongoing inflammation and injury during reperfusion. Liver reperfusion injury is reduced by lymphocyte depletion or activation of adenosine A2A receptors (A2ARs) with the selective agonist 4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]- prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester (ATL146e). We show that NKT cells are stimulated to produce interferon (IFN)-gamma by 2 h after the initiation of reperfusion, and the use of antibodies to deplete NK1.1-positive cells (NK and NKT) or to block CD1d-mediated glycolipid presentation to NKT cells replicates, but is not additive to, the protection afforded by ATL146e, as assessed by serum alanine aminotransferase elevation, histological necrosis, neutrophil accumulation, and serum IFN-gamma elevation. Reduced reperfusion injury observed in RAG-1 knockout (KO) mice is restored to the wild-type (WT) level by adoptive transfer of NKT cells purified from WT or A2AR KO mice but not IFN-gamma KO mice. Additionally, animals with transferred A2AR-/- NKT cells are not protected from hepatic reperfusion injury by ATL146e. In vitro, ATL146e potently inhibits both anti-CD3 and alpha-galactosylceramide-triggered production of IFN-gamma by NKT cells. These findings suggest that hepatic reperfusion injury is initiated by the CD1d-dependent activation of NKT cells, and the activation of these cells is inhibited by A2AR activation.
Animals↗
Adenosine A2A receptors in bone marrow-derived cells but not in forebrain neurons are important contributors to 3-nitropropionic acid-induced striatal damage as revealed by cell-type-selective inactivation.
Endogenous adenosine acting at the adenosine A2A receptor (A2AR) can modify brain injury in a variety of neurological disorder models. However, both A2AR activation and inactivation have been shown to be neuroprotective in different situations, raising the intriguing possibility that A2ARs in distinct cellular elements may have different and even opposing effects. In this study, we developed three novel transgenic models to dissect out cell-type-specific actions of A2ARs on striatal damage by the mitochondrial toxin 3-nitropropionic acid (3-NP). Whereas global inactivation of A2ARs exacerbated 3-NP-induced neurological deficit behaviors and striatal damage, selective inactivation of A2ARs in forebrain neurons (using the Cre/loxP strategy) did not affect neurological deficit or striatal damage after the acute systemic treatment of 3-NP and intrastriatal injection of malonate. However, selective inactivation of A2ARs in bone marrow-derived cells (BMDCs) by transplanting bone marrow cells from global A2AR knock-out (KO) mice into wild-type C57BL/6 mice produced a similar phenotype of global A2AR KO mice, i.e., exacerbation of 3-NP-induced striatal damage. Thus, cell-type-selective inactivation of A2ARs reveals that A2ARs in BMDCs but not in forebrain neurons are an important contributor to striatal damage induced by mitochondrial dysfunction.
Adenosine A2 Receptor Antagonists↗
Myocardial infarct-sparing effect of adenosine A2A receptor activation is due to its action on CD4+ T lymphocytes.
BACKGROUND: We previously used adenosine A2A receptor (A2AR) knockout (KO) mice and bone marrow transplantation to show that the infarct-sparing effect of A2AR activation at reperfusion is primarily due to effects on bone marrow-derived cells. In this study we show that CD4+ but not CD8+ T lymphocytes contribute to myocardial ischemia/reperfusion injury. METHOD AND RESULTS: After a 45-minute occlusion of the left anterior descending coronary artery and reperfusion, T cells accumulate in the infarct zone within 2 minutes. Addition of 10 microg/kg of the A2AR agonist ATL146e 5 minutes before reperfusion produces a significant reduction in T-cell accumulation and a significant reduction in infarct size (percentage of risk area) measured at 24 hours. In Rag1 KO mice lacking mature lymphocytes, infarct size is significantly smaller than in C57BL/6 mice. Infarct size in Rag1 KO mice is increased to the level of B6 mice by adoptive transfer of 50 million CD4+ T lymphocytes derived from C57BL/6 or A2AR KO but not interferon-gamma KO mice. ATL146e completely blocked the increase in infarct size in Rag1 KO mice reconstituted with B6 but not A2AR KO CD4+ T cells. The number of neutrophils in the reperfused heart at 24 hours after infarction correlated well with the number of lymphocytes and infarct size. CONCLUSIONS: These results strongly suggest that the infarct-sparing effect of A2AR activation is primarily due to inhibition of CD4+ T-cell accumulation and activation in the reperfused heart.
Animals↗
Influence of differential stability of G protein βγ dimers containing the γ11 subunit on functional activity at the M1 muscarinic receptor, A1 adenosine receptor, and phospholipase C-β.
Ggamma11 is an unusual guanine nucleotide-binding regulatory protein (G protein) subunit. To study the effect of different Gbeta-binding partners on gamma11 function, four recombinant betagamma dimers, beta1gamma2, beta4gamma2, beta1gamma11, and beta4gamma11, were characterized in a receptor reconstitution assay with the G(q)-linked M1 muscarinic and the G(i1)-linked A1 adenosine receptors. The beta4gamma11 dimer was up to 30-fold less efficient than beta4gamma2 at promoting agonist-dependent binding of [35S]GTPgammaS to either alpha(q) or alpha(i1). Using a competition assay to measure relative affinities of purified betagamma dimers for alpha, the beta4gamma11 dimer had a 15-fold lower affinity for G(i1) alpha than beta4gamma2. Chromatographic characterization of the beta4gamma11 dimer revealed that the betagamma is stable in a heterotrimeric complex with G(i1) alpha; however, upon activation of alpha with MgCl2 and GTPgammaS under nondenaturing conditions, the beta4 and gamma11 subunits dissociate. Activation of purified G(i1) alpha:beta4gamma11 with Mg+2/GTPgammaS following reconstitution into lipid vesicles and incubation with phospholipase C (PLC)-beta resulted in stimulation of PLC-beta activity; however, when this activation preceded reconstitution into vesicles, PLC-beta activity was markedly diminished. In a membrane coupling assay designed to measure the ability of G protein to promote a high-affinity agonist-binding conformation of the A1 adenosine receptor, beta4gamma11 was as effective as beta4gamma2 when coexpressed with G(i1) alpha and receptor. However, G(i1) alpha:beta4gamma11-induced high-affinity binding was up to 20-fold more sensitive to GTPgammaS than G(i1) alpha:beta4gamma2-induced high-affinity binding. These results suggest that the stability of the beta4gamma11 dimer can modulate G protein activity at the receptor and effector.
Animals↗
Cutting edge: Critical role for A2A adenosine receptors in the T cell-mediated regulation of colitis.
A(2A) adenosine receptors (A(2A)AR) inhibit inflammation, although the mechanisms through which adenosine exerts its effects remain unclear. Although the transfer of regulatory Th cells blocks colitis induced by pathogenic CD45RB(high) Th cells, we show that CD45RB(low) or CD25+ Th cells from A(2A)AR-deficient mice do not prevent disease. Moreover, CD45RB(high) Th cells from A(2A)AR-deficient mice were not suppressed by control CD45RB(low) Th cells. A(2A)AR agonists suppressed the production of proinflammatory cytokines by CD45RB(high) and CD45RB(low) T cells in association with a loss of mRNA stability. In contrast, anti-inflammatory cytokines, including IL-10 and TGF-beta, were minimally affected. Oral administration of the A(2A)AR agonist ATL313 attenuated disease in mice receiving CD45RB(high) Th cells. These data suggest that A(2A)AR play a novel role in the control of T cell-mediated colitis by suppressing the expression of proinflammatory cytokines while sparing anti-inflammatory activity mediated by IL-10 and TGF-beta.
Adenosine A2 Receptor Agonists↗
A selective adenosine A2A receptor agonist, ATL-146e, prevents concanavalin A-induced acute liver injury in mice.
BACKGROUND AND AIMS: Concanavalin A (Con A) activates T lymphocytes and induces CD4+ T cell-mediated hepatic injury in mice. Pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and interleukin-6 (IL-6), are critical mediators in this experimental model. Activation of adenosine A2A receptors reduces the production of various pro-inflammatory cytokines and suppresses T cell activation. A selective adenosine A2A receptor agonist (ATL-146e) has been shown to be a potent inhibitor of inflammation by increasing intracellular cyclic AMP (cAMP) in leukocytes. The aim of the present study was to determine whether ATL-146e could ameliorate Con A-induced hepatic injury, reduction of pro-inflammatory cytokine production. METHODS: Balb/c mice were injected with 25mg/kg Con A with or without a single injection of ATL-146e (0.5-50 microg/kg), 5 min prior to Con A administration. Liver enzymes, histology, and serum levels of tumor necrosis factor-alpha, interferon-gamma, and interleukin-6 were examined. We also assessed the effects of ATL-146e on pro-inflammatory cytokine production with CD4+ T cell. RESULTS: Pretreatment with ATL-146e significantly reduced serum levels of liver enzymes (P<0.001). The serum pro-inflammatory cytokines were all increased after Con A administration and reduced to near normal levels by ATL-146e. ATL-146e also inhibited CD4+ T cell pro-inflammatory cytokine production. CONCLUSION: A selective adenosine A2A receptor agonist, ATL-146e, can prevent concanavalin A-induced hepatic injury that is presumably mediated by its anti-inflammatory properties.
Adenosine A2 Receptor Agonists↗
Adenosine metabolism and cancer. Focus on "Adenosine downregulates DPPIV on HT-29 colon cancer cells by stimulating protein tyrosine phosphatases and reducing ERK1/2 activity via a novel pathway".
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Adenosine↗
The allosteric enhancer PD81,723 increases chimaeric A1/A2A adenosine receptor coupling with Gs.
PD81,723 {(2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluromethyl)-phenyl]methanone} is a selective allosteric enhancer of the G(i)-coupled A1 AR (adenosine receptor) that is without effect on G(s)-coupled A2A ARs. PD81,723 elicits a decrease in the dissociation kinetics of A1 AR agonist radioligands and an increase in functional agonist potency. In the present study, we sought to determine whether enhancer sensitivity is dependent on coupling domains or G-protein specificity of the A1 AR. Using six chimaeric A1/A2A ARs, we show that the allosteric effect of PD81,723 is maintained in a chimaera in which the predominant G-protein-coupling domain of the A1 receptor, the 3ICL (third intracellular loop), is replaced with A2A sequence. These chimaeric receptors are dually coupled with G(s) and G(i), and PD81,723 increases the potency of N6-cyclopentyladenosine to augment cAMP accumulation with or without pretreatment of cells with pertussis toxin. Thus PD81,723 has similar functional effects on chimaeric receptors with A1 transmembrane sequences that couple with G(i) or G(s). This is the first demonstration that an allosteric regulator can function in the context of a switch in G-protein-coupling specificity. There is no enhancement by PD81,723 of G(i)-coupled A2A chimaeric receptors with A1 sequence replacing A2A sequence in the 3ICL. The results suggest that the recognition site for PD81,723 is on the A1 receptor and that the enhancer acts to directly stabilize the receptor to a conformational state capable of coupling with G(i) or G(s).
Adenosine↗
Renal ischemia-reperfusion injury and adenosine 2A receptor-mediated tissue protection: the role of CD4+ T cells and IFN-gamma.
A(2A) adenosine receptor (A(2A)R)-expressing bone marrow (BM)-derived cells contribute to the renal protective effect of A(2A) agonists in renal ischemia-reperfusion injury (IRI). We performed IRI in mice lacking T and B cells to determine whether A(2A)R expressed in CD4+ cells mediate protection from IRI. Rag-1 knockout (KO) mice were protected in comparison to wild-type (WT) mice when subjected to IRI. ATL146e, a selective A(2A) agonist, did not confer additional protection. IFN-gamma is an important early signal in IRI and is thought to contribute to reperfusion injury. Because IFN-gamma is produced by kidney cells and T cells we performed IRI in BM chimeras in which the BM of WT mice was reconstituted with BM from IFN-gamma KO mice (IFN-gamma KO-->WT chimera). We observed marked reduction in IRI in comparison to WT-->WT chimeras providing additional indirect support for the role of T cells. To confirm the role of CD4+ A(2A)R in mediating protection from IRI, Rag-1 KO mice were subjected to ischemia-reperfusion. The protection observed in Rag-1 KO mice was reversed in Rag-1 KO mice that were adoptively transferred WT CD4+ cells (WT CD4+-->Rag-1 KO) or A(2A) KO CD4+ cells (A(2A) KO CD4+-->Rag-1 KO). ATL146e reduced injury in WT CD4+-->Rag-1 KO mice but not in A(2A) KO CD4+-->Rag-1 KO mice. Rag-1 KO mice reconstituted with CD4+ cells derived from IFN-gamma KO mice (IFN-gamma CD4+-->Rag-1 KO) were protected from IRI; ATL146e conferred no additional protection. These studies demonstrate that CD4+ IFN-gamma contributes to IRI and that A(2A) agonists mediate protection from IRI through action on CD4+ cells.
Adenosine A2 Receptor Agonists↗
N6-ethyl-2-alkynyl NECAs, selective human A3 adenosine receptor agonists.
A series of N6-ethyl-2-alkynyl NECA (5'-N-ethylcarboxamidoadenosine) analogs were synthesized and their binding affinity with the four human adenosine receptors was evaluated. One of the compounds ZR1121 shows high affinity with hA3 receptor and its selectivity over hA1 receptor is 1-2 log orders greater than IB-MECA or Cl-IB-MECA, the currently employed selective A3 agonists.
Adenosine A3 Receptor Agonists↗
Norbornyllactone-substituted xanthines as adenosine A(1) receptor antagonists.
During the search for second-generation adenosine A(1) receptor antagonist alternatives to the clinical candidate 8-(3-oxa-tricyclo[3.2.1.0(2,4)]oct-6-yl)-1,3-dipropyl-3,7-dihydro-purine-2,6-dione (BG9719), we developed a series of novel xanthines substituted with norbornyl-lactones that possessed high binding affinities for adenosine A(1) receptors and in vivo activity.
Adenosine A1 Receptor Antagonists↗
Attenuation of gastric mucosal inflammation induced by aspirin through activation of A2A adenosine receptor in rats.
AIM: To determine whether a specific adenosine A(2A) receptor agonist (ATL-146e) can ameliorate aspirin-induced gastric mucosal lesions in rats, and reduce neutrophil accumulation and production of pro-inflammatory cytokines. METHODS: Gastric lesions were produced by oral gavage of aspirin (200 mg/kg) and HCl (0.15 mol/L, 8.0 mL/kg). 4-{3-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester (ATL-146e, 2.5-5 mug/kg, IP) was injected 30 min before the administration of aspirin. Tissue myeloperoxidase (MPO) concentration in gastric mucosa was measured as an index of neutrophil infiltration. Gastric mucosal concentrations of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) were determined by ELISA. Also, we examined the effect of ATL-146e on tissue prostaglandin E2 (PGE2) production and gastric secretion. RESULTS: Intragastric administration of aspirin induced multiple hemorrhagic erosions in rat gastric mucosa. The total length of gastric erosions (ulcer index) in control rats was 29.8+/-7.75 mm and was reduced to 3.8+/-1.42 mm after pretreatment with 5.0 g/kg ATL-146e (P<0.01). The gastric contents of MPO and pro-inflammatory cytokines were all increased after the administration of aspirin and reduced to nearly normal levels by ATL-146e. Gastric mucosal PGE2 concentration was not affected by intraperitoneal injection of ATL-146e. CONCLUSION: The specific adenosine A(2A) receptor agonist, ATL-146e, has potent anti-ulcer effects presumably mediated by its anti-inflammatory properties.
Animals↗
Comparison of systemic and retrograde delivery of adenosine A2A agonist for attenuation of spinal cord injury after thoracic aortic cross-clamping.
BACKGROUND: Paraplegia remains a devastating complication of thoracic aortic surgery, which has been attenuated by retrograde adenosine and systemic adenosine A2A receptor activation. We hypothesized that despite retrograde spinal perfusion of an adenosine A2A agonist (ATL-146e), systemic therapy produces superior spinal cord protection with reduced inflammation. METHODS: Forty pigs underwent 30-minute thoracic aortic cross-clamping. Pigs received: no therapy (control); retrograde saline (retrograde control); retrograde ATL-146e; systemic ATL-146e; systemic ATL-146e with retrograde saline; or systemic and retrograde ATL-146e. Retrograde therapies were given during ischemia. Systemic ATL-146e (0.06 microg.kg(-1).min(-1)) was given intravenously for 3 hours at reperfusion. At 24 hours, motor function was assessed using the Tarlov scale. Tissue was analyzed for neuronal viability, microtubule-associated protein-2 expression, and neutrophil sequestration (myeloperoxidase activity). RESULTS: Four pigs received retrograde barium showing both radiographic and histologic spinal cord perfusion. Tarlov scores at 24 hours were significantly improved versus both control groups in all ATL groups except the combined ATL-146e group (all p < 0.05). Neuronal viability by hematoxylin and eosin stain was significantly preserved in systemic ATL groups compared with both control groups (all p < 0.05). Microtubule-associated protein-2 expression was significantly preserved compared with both control groups in all systemic ATL groups. Systemic ATL significantly lowered myeloperoxidase activity versus both control groups (p < 0.01). CONCLUSIONS: Both retrograde and systemic ATL-146e therapies attenuate ischemic spinal cord injury, but combining the two routes was less effective. Given comparable results between the two routes and the simplicity of systemic delivery, peripheral venous ATL-146e at reperfusion should be preferred for spinal cord protection in thoracic aortic surgery.
Adenosine A2 Receptor Agonists↗
Functional and cytoarchitectural spinal cord protection by ATL-146e after ischemia/reperfusion is mediated by adenosine receptor agonism.
BACKGROUND: ATL-146e protects the spinal cord from ischemia/reperfusion injury, presumably via adenosine A(2A) receptor activation, but this relationship remains unproven. We hypothesized that spinal cord functional and cytoarchitectural preservation from ATL-146e would be lost with simultaneous administration of the specific adenosine A(2A) antagonist ZM241385 (ZM), thus proving that adenosine A(2A) receptor activation is responsible for the protective effects of this compound. METHODS: New Zealand White rabbits underwent 45 minutes of infrarenal aortic cross-clamping. Groups (n = 10) included sham, ischemia, ischemia plus ATL-146e (ATL-146E), ischemia plus ZM, or ischemia with both compounds (agonist-antagonist). Tarlov scores were recorded every 12 hours. After 48 hours, the spinal cord was fixed for histology and microtubule-associated protein 2 immunohistochemistry. RESULTS: Tarlov scores at 48 hours were significantly better in the sham and ATL-146E groups (5.0 and 3.9, respectively) compared with the other three groups (all < or =1.3; P < .001). On hematoxylin and eosin, neuronal viability was higher in the sham, ATL-146E, and agonist-antagonist groups compared with the control and ZM groups (P < .05). Microtubule-associated protein 2 expression was preserved in the sham and ATL-146E groups but was lost in the ATL + ZM, ZM241385, and control groups. CONCLUSIONS: ATL-146e preserves the spinal cord in terms of both cytoarchitecture and function after reperfusion of the ischemic spinal cord, but this preservation is not completely blocked by competitive adenosine A(2A) receptor antagonism. Although ATL-146e does seem to partially function through activation of the adenosine A(2A) receptor, the neuroprotective mechanism may not be limited to this particular receptor.
Adenosine A2 Receptor Agonists↗
New insights into the regulation of inflammation by adenosine.
Adenosine, long known as a regulator of cardiovascular function, has recently been identified as a significant paracrine inhibitor of inflammation that acts primarily by activation of A2A adenosine receptors (A2AARs) on lymphoid or myeloid cells. In this issue of the JCI, Yang et al. describe a proinflammatory phenotype resulting from deletion of the gene encoding the A2B adenosine receptor (A2BAR) in the mouse, suggesting that activation of the A2BAR can also have antiinflammatory effects (see the related article beginning on page 1913). Nevertheless, the role of the A2BAR remains enigmatic since its activation can either stimulate or inhibit the release of proinflammatory cytokines in different cells and tissues.
Adenosine↗