A role for the low-affinity A2B adenosine receptor in regulating superoxide generation by murine neutrophils.

The formation of adenosine dampens inflammation by inhibiting most cells of the immune system. Among its actions on neutrophils, adenosine suppresses superoxide generation and regulates chemotactic activity. To date, most evidence implicates the G(s) protein-coupled A(2A) adenosine receptor (AR) as the primary AR subtype responsible for mediating the actions of adenosine on neutrophils by stimulating cAMP production. Given that the A(2B)AR is now known to be expressed in neutrophils and that it is a G(s) protein-coupled receptor, we examined in this study whether it signals to suppress neutrophil activities by using 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulfanyl]acetamide (BAY 60-6583), a new agonist for the human A(2B)AR that was confirmed in preliminary studies to be a potent and highly selective agonist for the murine A(2B)AR. We found that treating mouse neutrophils with low concentrations (10(-9) and 10(-8) M) of BAY 60-6583 inhibited formylated-methionine-leucine-phenylalanine (fMLP)-stimulated superoxide production by either naive neutrophils, tumor necrosis factor-α-primed neutrophils, or neutrophils isolated from mice treated systemically with lipopolysaccharide. This inhibitory action of BAY 60-6583 was confirmed to involve the A(2B)AR in experiments using neutrophils obtained from A(2B)AR gene knockout mice. It is noteworthy that BAY 60-6583 increased fMLP-stimulated superoxide production at higher concentrations (>1 µM), which was attributed to an AR-independent effect. In a standard Boyden chamber migration assay, BAY 60-6583 alone did not stimulate neutrophil chemotaxis or influence chemotaxis in response to fMLP. These results indicate that the A(2B)AR signals to suppress oxidase activity by murine neutrophils, supporting the idea that this low-affinity receptor for adenosine participates along with the A(2A)AR in regulating the proinflammatory actions of neutrophils.

Adenosine suppresses lipopolysaccharide-induced tumor necrosis factor-alpha production by murine macrophages through a protein kinase A- and exchange protein activated by cAMP-independent signaling pathway.

Adenosine is generated during tissue hypoxia and stress, which reduces inflammation by suppressing the activity of most immune cells. Among its various actions, adenosine suppresses the production of proinflammatory cytokines including tumor necrosis factor (TNF)-alpha, through the cAMP-elevating A(2A) adenosine receptor (AR) subtype. In this study, we examined the signaling mechanisms by which A(2A)AR activation inhibits TNF-alpha production in thioglycollate-elicited mouse peritoneal macrophages. Pretreating murine macrophages with the nonselective AR agonist adenosine-5'-N-ethylcarboxamide (NECA), the A(2A)AR agonist 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680), or the cAMP-elevating agent forskolin reduced TNF-alpha production in response to lipopolysaccharide (LPS) by greater than 60%. All of these agents increased cAMP production in macrophages and activated protein kinase A (PKA). However, we were surprised to find that treating macrophages with three different PKA inhibitors or small interfering RNA-mediated knockdown of the exchange protein activated by cAMP (Epac-1) failed to block the suppressive actions of NECA or forskolin on LPS-induced TNF-alpha release. Instead, okadaic acid was effective at low concentrations that selectively inhibit protein serine/threonine phosphatases. Subsequent studies showed that NECA and forskolin decreased LPS-induced steady-state TNF-alpha mRNA levels; this effect was due to a decreased rate of transcription based on assays examining the rate of generation of primary TNF-alpha transcripts. Treatment with NECA or forskolin did not interfere with LPS-induced translocation or DNA binding of the RelA/p65 subunit of nuclear factor-kappaB or phosphorylation of inhibitor of nuclear factor-kappaB-alpha, extracellular signal-regulated kinase 1/2, c-Jun NH(2)-terminal kinase, or p38 kinase. Our results suggest that AR activation inhibits LPS-induced TNF-alpha production by murine macrophages at the level of gene transcription through a unique cAMP-dependent, but PKA- and Epac-independent, signaling pathway involving protein phosphatase activity.

Characterization of the A2B adenosine receptor from mouse, rabbit, and dog.

We have cloned and pharmacologically characterized the A(2B) adenosine receptor (AR) from the dog, rabbit, and mouse. The full coding regions of the dog and mouse A(2B)AR were obtained by reverse transcriptase-polymerase chain reaction, and the rabbit A(2B)AR cDNA was obtained by screening a rabbit brain cDNA library. It is noteworthy that an additional clone was isolated by library screening that was identical in sequence to the full-length rabbit A(2B)AR, with the exception of a 27-base pair deletion in the region encoding amino acids 103 to 111 (A(2B)AR(103-111)). This 9 amino acid deletion is located in the second intracellular loop at the only known splice junction of the A(2B)AR and seems to result from the use of an additional 5' donor site found in the rabbit and dog but not in the human, rat, or mouse sequences. [(3)H]3-Isobutyl-8-pyrrolidinoxanthine and 8-[4-[((4-cyano-[2,6-(3)H]-phenyl)carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)xanthine ([(3)H]MRS 1754) bound with high affinity to membranes prepared from human embryonic kidney (HEK) 293 cells expressing mouse, rabbit, and dog A(2B)ARs. Competition binding studies performed with a panel of agonist (adenosine and 2-amino-3,5-dicyano-4-phenylpyridine analogs) and antagonist ligands identified similar potency orders for the A(2B)AR orthologs, although most xanthine antagonists displayed lower binding affinity for the dog A(2B)AR compared with A(2B)ARs from rabbit and mouse. No specific binding could be detected with membranes prepared from HEK 293 cells expressing the rabbit A(2B)AR(103-111) variant. Furthermore, the variant failed to stimulate adenylyl cyclase or calcium mobilization. We conclude that significant differences in antagonist pharmacology of the A(2B)AR exist between species and that some species express nonfunctional variants of the A(2B)AR due to "leaky" splicing.

Adenosine inhibits tumor necrosis factor-alpha release from mouse peritoneal macrophages via A2A and A2B but not the A3 adenosine receptor.

Adenosine is elaborated in injured tissues where it suppresses inflammatory responses of essentially all immune cells, including production of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha). Most of the anti-inflammatory actions of adenosine have been attributed to signaling through the A(2A) adenosine receptor (A(2A)AR). Previously, however, it has been shown that the A(3)AR agonist N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (IB-MECA) potently inhibited TNF-alpha release from macrophages obtained from A(2A)AR "knockout" (A(2A)KO) mice, suggesting that the A(3)AR may also regulate cytokine expression. Here, we confirmed that the A(2A)AR is the primary AR subtype that suppresses TNF-alpha release from thioglycollate-elicited mouse peritoneal macrophages induced by both Toll-like receptor-dependent (TLR) and TLR-independent stimuli, but we determined that the A(2B)AR rather than the A(3)AR mediates the non-A(2A)AR actions of adenosine since 1) the ability of IB-MECA to inhibit TNF-alpha release was not altered in macrophages isolated from A(3)KO mice, and 2) the A(2B)AR antagonist 1,3-dipropyl-8-[4-[((4-cyanophenyl)carbamoylmethyl)oxy]phenyl]xanthine (MRS 1754) blocked the ability of the nonselective AR agonist adenosine-5'-N-ethylcarboxamide (NECA) to inhibit TNF-alpha release from macrophages isolated from A(2A)KO mice. Although A(2B)ARs seem capable of inhibiting TNF-alpha release, the A(2A)AR plays a dominant suppressive role since MRS 1754 did not block the ability of NECA to inhibit TNF-alpha release from macrophages isolated from wild-type (WT) mice. Furthermore, the potency and efficacy of adenosine to inhibit TNF-alpha release from WT macrophages were not influenced by blocking A(2B)ARs with MRS 1754. The data indicate that adenosine suppresses TNF-alpha release from macrophages primarily via A(2A)ARs, although the A(2B)AR seems to play an underlying inhibitory role that may contribute to the anti-inflammatory actions of adenosine under select circumstances.

Cl-IB-MECA [2-chloro-N6-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide] reduces ischemia/reperfusion injury in mice by activating the A3 adenosine receptor.

We used pharmacological agents and genetic methods to determine whether the potent A(3) adenosine receptor (AR) agonist 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (Cl-IB-MECA) protects against myocardial ischemia/reperfusion injury in mice via the A(3)AR or via interactions with other AR subtypes. Pretreating wild-type (WT) mice with Cl-IB-MECA reduced myocardial infarct size induced by 30 min of coronary occlusion and 24 h of reperfusion at doses (30 and 100 mug/kg) that concomitantly reduced blood pressure and stimulated systemic histamine release. The A(3)AR-selective antagonist MRS 1523 [3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine-carboxylate], but not the A(2A)AR antagonist ZM 241385 [4-{2-7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl}phenol], blocked the reduction in infarct size provided by Cl-IB-MECA, suggesting a mechanism involving the A(3)AR. To further examine the selectivity of Cl-IB-MECA, we assessed its cardioprotective effectiveness in A(3)AR gene "knock-out" (A(3)KO) mice. Cl-IB-MECA did not reduce myocardial infarct size in A(3)KO mice in vivo and did not protect isolated perfused hearts obtained from A(3)KO mice from injury induced by global ischemia and reperfusion. Additional studies using WT mice treated with compound 48/80 [condensation product of p-methoxyphenethyl methylamine with formaldehyde] to deplete mast cell contents excluded the possibility that Cl-IB-MECA was cardioprotective by releasing mediators from mast cells. These data demonstrate that Cl-IB-MECA protects against myocardial ischemia/reperfusion injury in mice principally by activating the A(3)AR.

Comparison of three different A1 adenosine receptor antagonists on infarct size and multiple cycle ischemic preconditioning in anesthetized dogs.

A(1) adenosine receptor (AR) antagonists are effective diuretic agents that may be useful for treating fluid retention disorders including congestive heart failure. However, antagonism of A(1)ARs is potentially a concern when using these agents in patients with ischemic heart disease. To address this concern, the present study was designed to compare the actions of the A(1)AR antagonists CPX (1,3-dipropyl-8-cyclopentylxanthine), BG 9719 (1,3-dipropyl-8-[2-(5,6-epoxynorbornyl)]xanthine), and BG 9928 (1,3-dipropyl-8-[1-(4-propionate)-bicyclo-[2,2,2]octyl]xanthine) on acute myocardial ischemia/reperfusion injury and ischemic preconditioning (IPC) in an in vivo dog model of infarction. Barbital-anesthetized dogs were subjected to 60 min of left anterior descending coronary artery occlusion followed by 3 h of reperfusion, after which infarct size was assessed by staining with triphenyltetrazolium chloride. IPC was elicited by four 5-min occlusion/5-min reperfusion cycles produced 10 min before the 60-min occlusion. Multiple-cycle IPC produced a robust reduction ( approximately 65%) in infarct size; this effect of IPC on infarct size was not abrogated in dogs pretreated with any of the three AR antagonists. Surprisingly, in the absence of IPC, pretreatment with CPX or BG 9928 before occlusion or immediately before reperfusion resulted in significant reductions ( approximately 40-50%) in myocardial infarct size. However, treatment with an equivalent dose of BG 9719 had no similar effect. We conclude that the A(1)AR antagonists BG 9719, BG 9928, and CPX do not exacerbate cardiac injury and do not interfere with IPC induced by multiple ischemia/reperfusion cycles. We discuss the possibility that the cardioprotective actions of CPX and BG 9928 may be related to antagonism of A(2B)ARs.