Tina C. Wan

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.

Activation of the A3 Adenosine Receptor Suppresses Superoxide Production and Chemotaxis of Mouse Bone Marrow Neutrophils

Adenosine is formed in injured/ischemic tissues, where it suppresses the actions of essentially all cells of the immune system. Most of the anti-inflammatory actions of adenosine have been attributed to signaling through the Gs protein-coupled A2A adenosine receptor (AR). Here, we report that the A3AR is highly expressed in murine neutrophils isolated from bone marrow. Selective activation of the A3AR with (2S,3S,4R,5R)-3-amino-5-[6-(2,5-dichlorobenzylamino)purin-9-yl]-4-hydroxytetrahydrofuran-2-carboxylic acid methylamide (CP-532,903) potently inhibited mouse bone marrow neutrophil superoxide generation and chemotaxis induced by various activating agents. The selectivity of CP-532,903 was confirmed in assays using neutrophils obtained from A2AAR and A3AR gene “knockout” mice. In a model of thioglycollate-induced inflammation, treating mice with CP-532,903 inhibited recruitment of leukocytes into the peritoneum by specifically activating the A3AR. Collectively, our findings support the theory that the A3AR contributes to the anti-inflammatory actions of adenosine on neutrophils and provide a potential mechanistic explanation for the efficacy of A3AR agonists in animal models of inflammation (i.e., inhibition of neutrophil-mediated tissue injury).

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.

Evidence that the acute phase of ischemic preconditioning does not require signaling by the A2B adenosine receptor

Ischemic preconditioning (IPC) is a protective phenomenon in which brief ischemia renders the myocardium resistant to subsequent ischemic insults. Here, we used A(2B)AR gene knock-out (A(2B)KO)/β-galactosidase reporter gene knock-in mice and the A(2B)AR antagonist ATL-801 to investigate the potential involvement of the A(2B)AR in IPC, focusing on the acute phase of protection. Cardioprotection provided by acute IPC elicited by two 3-min occlusion/3-min reperfusion cycles was readily apparent in an isolated, Langendorff-perfused mouse heart model in studies using hearts from A(2B)KO mice. IPC equivalently improved the recovery of contractile function following 20 min of global ischemia and 45 min of reperfusion in both WT and A(2B)KO hearts by ~30-40%, and equivalently decreased the release of cardiac troponin I during the reperfusion period (from 5969 ± 925 to 1595 ± 674 ng/g and 4376 ± 739 to 2278 ± 462 ng/g using WT and A(2B)KO hearts, respectively). Similarly, the infarct size-reducing capacity of acute IPC in an in vivo model of infarction was fully manifested in experiments using A(2B)KO mice, as well as in experiments using rats pretreated with ATL-801. We did observe, however, a marked reduction in infarct size in rats following administration of the selective A(2B)AR agonist BAY 60-6583 (~25% reduction at a dose of 1.0mg/kg). While supportive of its concept as a cardioprotective receptor, these experiments indicate that the mechanism of the early phase of IPC is not dependent on signaling by the A(2B)AR. We present the idea that the A(2B)AR may contribute to the later stages of IPC dependent on the induction of stress-responsive genes.

Adenosine A1 receptors heterodimerize with β1- and β2-adrenergic receptors creating novel receptor complexes with altered G protein coupling and signaling

G protein coupled receptors play crucial roles in mediating cellular responses to external stimuli, and increasing evidence suggests that they function as multiple units comprising homo/heterodimers and hetero-oligomers. Adenosine and β-adrenergic receptors are co-expressed in numerous tissues and mediate important cellular responses to the autocoid adenosine and sympathetic stimulation, respectively. The present study was undertaken to examine whether adenosine A1ARs heterodimerize with β1- and/or β2-adrenergic receptors (β1R and β2R), and whether such interactions lead to functional consequences. Co-immunoprecipitation and co-localization studies with differentially epitope-tagged A1, β1, and β2 receptors transiently co-expressed in HEK-293 cells indicate that A1AR forms constitutive heterodimers with both β1R and β2R. This heterodimerization significantly influenced orthosteric ligand binding affinity of both β1R and β2R without altering ligand binding properties of A1AR. Receptor-mediated ERK1/2 phosphorylation significantly increased in cells expressing A1AR/β1R and A1AR/β2R heteromers. β-Receptor-mediated cAMP production was not altered in A1AR/β1R expressing cells, but was significantly reduced in the A1AR/β2R cells. The inhibitory effect of the A1AR on cAMP production was abrogated in both A1AR/β1R and A1AR/β2R expressing cells in response to the A1AR agonist CCPA. Co-immunoprecipitation studies conducted with human heart tissue lysates indicate that endogenous A1AR, β1R, and β2R also form heterodimers. Taken together, our data suggest that heterodimerization between A1 and β receptors leads to altered receptor pharmacology, functional coupling, and intracellular signaling pathways. Unique and differential receptor cross-talk between these two important receptor families may offer the opportunity to fine-tune crucial signaling responses and development of more specific therapeutic interventions.

EFFECT OF HUMAN 15-LIPOXYGENASE-1 METABOLITES ON VASCULAR FUNCTION IN MOUSE MESENTERIC ARTERIES AND HEARTS

Lipoxygenases regulate vascular function by metabolizing arachidonic acid (AA) to dilator eicosanoids. Previously, we showed that endothelium-targeted adenoviral vector-mediated gene transfer of the human 15-lipoxygenase-1 (h15-LO-1) enhances arterial relaxation through the production of vasodilatory hydroxyepoxyeicosatrienoic acid (HEETA) and trihydroxyeicosatrienoic acid (THETA) metabolites. To further define this function, a transgenic (Tg) mouse line that overexpresses h15-LO-1 was studied. Western blot, immunohistochemistry and RT-PCR results confirmed expression of 15-LO-1 transgene in tissues, especially high quantity in coronary arterial wall, of Tg mice. Reverse-phase HPLC analysis of [(14)C]-AA metabolites in heart tissues revealed enhanced 15-HETE synthesis in Tg vs. WT mice. Among the 15-LO-1 metabolites, 15-HETE, erythro-13-H-14,15-EETA, and 11(R),12(S),15(S)-THETA relaxed the mouse mesenteric arteries to the greatest extent. The presence of h15-LO-1 increased acetylcholine- and AA-mediated relaxation in mesenteric arteries of Tg mice compared to WT mice. 15-LO-1 was most abundant in the heart; therefore, we used the Langendorff heart model to test the hypothesis that elevated 15-LO-1 levels would increase coronary flow following a short ischemia episode. Both peak flow and excess flow of reperfused hearts were significantly elevated in hearts from Tg compared to WT mice being 2.03 and 3.22 times greater, respectively. These results indicate that h15-LO-1-derived metabolites are highly vasoactive and may play a critical role in regulating coronary blood flow.

IL13 Promotes in vivo Neonatal Cardiomyocyte Cell Cycle Activity and Heart Regeneration

There is great interest in identifying signaling mechanisms by which cardiomyocytes (CMs) can enter the cell cycle and promote endogenous cardiac repair. We have previously demonstrated that IL-13 stimulated cell cycle activity of neonatal CMs in vitro. However, the signaling events that occur downstream of IL-13 in CMs and the role of IL-13 in CM proliferation and regeneration in vivo have not been explored. Here, we tested the role of IL-13 in promoting neonatal CM cell cycle activity and heart regeneration in vivo and investigated the signaling pathway(s) downstream of IL-13 specifically in CMs. Compared with control, CMs from neonatal IL-13 knockout (IL-13-/-) mice showed decreased proliferative markers and coincident upregulation of the hypertrophic marker brain natriuretic peptide ( Nppb) and increased CM nuclear size. After apical resection in anesthetized newborn mice, heart regeneration was significantly impaired in IL-13-/- mice compared with wild-type mice. Administration of recombinant IL-13 reversed these phenotypes by increasing CM proliferation markers and decreasing Nppb expression. RNA sequencing on primary neonatal CMs treated with IL-13 revealed activation of gene networks regulated by ERK1/2 and Akt. Western blot confirmed strong phosphorylation of ERK1/2 and Akt in both neonatal and adult cultured CMs in response to IL-13. Our data demonstrated a role for endogenous IL-13 in neonatal CM cell cycle and heart regeneration. ERK1/2 and Akt signaling are important pathways known to promote CM proliferation and protect against apoptosis, respectively; thus, targeting IL-13 transmembrane receptor signaling or administering recombinant IL-13 may be therapeutic approaches for activating proregenerative and survival pathways in the heart. NEW & NOTEWORTHY Here, we demonstrate, for the first time, that IL-13 is involved in neonatal cardiomyocyte cell cycle activity and heart regeneration in vivo. Prior work has shown that IL-13 promotes cardiomyocyte cell cycle activity in vitro; however, the signaling pathways were unknown. We used RNA sequencing to identify the signaling pathways activated downstream of IL-13 in cardiomyocytes and found that ERK1/2 and Akt signaling was activated in response to IL-13.

Cardiovascular Biology of the A3 Adenosine Receptor

The goal of this chapter is to review current theories regarding the potential involvement of the A3 receptor in mediating the actions of adenosine in the cardiovascular system. The Gi protein-coupled A3 adenosine receptor is the last adenosine receptor subtype to be discovered and remains poorly characterized in terms of its molecular biology and biological functions. Recent evidence suggests that the A3 receptor may mediate the actions of adenosine to regulate vascular tone and angiogenesis, either directly or indirectly by stimulating the release of mediators from mast cells. Substantial evidence has accumulated to suggest that the A3 receptor is responsible for some of the beneficial effects of adenosine in reducing injury caused by ischemia and reperfusion. Readers are reminded that the A3 receptor has proven to be the most difficult adenosine receptor subtype to research due to its unique pharmacological properties and unusual species differences in terms of its pharmacology, tissue expression, and biological function. The theories described in this chapter remain controversial and require additional verification as new tools become available to study this fascinating member of the adenosine receptor family.