Laura M. Kreckler

Cl-IB-MECA [2-Chloro-N6-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide] Reduces Ischemia/Reperfusion Injury in Mice by Activating the A Adenosine Receptor

We used pharmacological agents and genetic methods to determine whether the potent A3 adenosine receptor (AR) agonist 2-chloro-N6-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide (Cl-IB-MECA) protects against myocardial ischemia/reperfusion injury in mice via the A3AR 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 μg/kg) that concomitantly reduced blood pressure and stimulated systemic histamine release. The A3AR-selective antagonist MRS 1523 [3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine-carboxylate], but not the A2AAR 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 A3AR. To further examine the selectivity of Cl-IB-MECA, we assessed its cardioprotective effectiveness in A3AR gene “knock-out” (A3KO) mice. Cl-IB-MECA did not reduce myocardial infarct size in A3KO mice in vivo and did not protect isolated perfused hearts obtained from A3KO 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 A3AR.

Adenosine Suppresses Lipopolysaccharide-Induced Tumor Necrosis Factor-α 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)-α, through the cAMP-elevating A2A adenosine receptor (AR) subtype. In this study, we examined the signaling mechanisms by which A2AAR activation inhibits TNF-α production in thioglycollate-elicited mouse peritoneal macrophages. Pretreating murine macrophages with the nonselective AR agonist adenosine-5′-N-ethylcarboxamide (NECA), the A2AAR agonist 2-[p-(2-carboxyethyl)phenethylamino]-5′-N-ethylcarboxamidoadenosine (CGS 21680), or the cAMP-elevating agent forskolin reduced TNF-α 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-α 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-α mRNA levels; this effect was due to a decreased rate of transcription based on assays examining the rate of generation of primary TNF-α transcripts. Treatment with NECA or forskolin did not interfere with LPS-induced translocation or DNA binding of the RelA/p65 subunit of nuclear factor-κB or phosphorylation of inhibitor of nuclear factor-κB-α, extracellular signal-regulated kinase 1/2, c-Jun NH2-terminal kinase, or p38 kinase. Our results suggest that AR activation inhibits LPS-induced TNF-α 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.

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 Gs protein-coupled A2A adenosine receptor (AR) as the primary AR subtype responsible for mediating the actions of adenosine on neutrophils by stimulating cAMP production. Given that the A2BAR is now known to be expressed in neutrophils and that it is a Gs 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 A2BAR that was confirmed in preliminary studies to be a potent and highly selective agonist for the murine A2BAR. 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 A2BAR in experiments using neutrophils obtained from A2BAR 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 A2BAR signals to suppress oxidase activity by murine neutrophils, supporting the idea that this low-affinity receptor for adenosine participates along with the A2AAR in regulating the proinflammatory actions of neutrophils.

Characterization of the A2B Adenosine Receptor from Mouse, Rabbit, and Dog

We have cloned and pharmacologically characterized the A2B adenosine receptor (AR) from the dog, rabbit, and mouse. The full coding regions of the dog and mouse A2BAR were obtained by reverse transcriptase-polymerase chain reaction, and the rabbit A2BAR 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 A2BAR, with the exception of a 27-base pair deletion in the region encoding amino acids 103 to 111 (A2BAR103-111). This 9 amino acid deletion is located in the second intracellular loop at the only known splice junction of the A2BAR 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. [3H]3-Isobutyl-8-pyrrolidinoxanthine and 8-[4-[((4-cyano-[2,6-3H]-phenyl)carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)xanthine ([3H]MRS 1754) bound with high affinity to membranes prepared from human embryonic kidney (HEK) 293 cells expressing mouse, rabbit, and dog A2BARs. 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 A2BAR orthologs, although most xanthine antagonists displayed lower binding affinity for the dog A2BAR compared with A2BARs from rabbit and mouse. No specific binding could be detected with membranes prepared from HEK 293 cells expressing the rabbit A2BAR103-111 variant. Furthermore, the variant failed to stimulate adenylyl cyclase or calcium mobilization. We conclude that significant differences in antagonist pharmacology of the A2BAR exist between species and that some species express nonfunctional variants of the A2BAR due to “leaky” splicing.