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A2B adenosine receptor dampens hypoxia-induced vascular leak. Blood

Departments of Anesthesiology and Intensive Care , Tübingen University Hospital, Tübingen, Germany.
Blood (Impact Factor: 10.43). 03/2008; 111(4):2024-35. DOI: 10.1182/blood-2007-10-117044
Source: PubMed

ABSTRACT Extracellular adenosine has been implicated in adaptation to hypoxia and previous studies demonstrated a central role in vascular responses. Here, we examined the contribution of individual adenosine receptors (ARs: A1AR/A2AAR/A2BAR/A3AR) to vascular leak induced by hypoxia. Initial profiling studies revealed that siRNA-mediated repression of the A2BAR selectively increased endothelial leak in response to hypoxia in vitro. In parallel, vascular permeability was significantly increased in vascular organs of A2BAR(-/-)-mice subjected to ambient hypoxia (8% oxygen, 4 hours; eg, lung: 2.1 +/- 0.12-fold increase). By contrast, hypoxia-induced vascular leak was not accentuated in A1AR(-/-)-, A2AAR(-/-)-, or A3AR(-/-)-deficient mice, suggesting a degree of specificity for the A2BAR. Further studies in wild type mice revealed that the selective A2BAR antagonist PSB1115 resulted in profound increases in hypoxia-associated vascular leakage while A2BAR agonist (BAY60-6583 [2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)-. phenyl]pyridin-2-ylsulfanyl]acetamide]) treatment was associated with almost complete reversal of hypoxia-induced vascular leakage (eg, lung: 2.0 +/- 0.21-fold reduction). Studies in bone marrow chimeric A2BAR mice suggested a predominant role of vascular A2BARs in this response, while hypoxia-associated increases in tissue neutrophils were, at least in part, mediated by A2BAR expressing hematopoietic cells. Taken together, these studies provide pharmacologic and genetic evidence for vascular A2BAR signaling as central control point of hypoxia-associated vascular leak.

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    • "After blocking for 2 h at room temperature with 5% milk in Trisbuffered saline, pH 7.6, containing 0.1% Tween 20 (TBS-T), the membranes were incubated overnight at 4 °C with a rabbit anti- A 2B R antibody (1:250; sc-28996 from Santa Cruz Biotechnology, Santa Cruz, CA, USA). This particular antibody was selected as its selectivity has been previously been validated by eliminating its immunoreactivity upon neutralising A 2B R with a siRNA (Eckle et al., 2008). After four washing periods for 10 min with TBS-T containing 0.5% milk, the membranes were incubated with the alkaline phosphatase-conjugated anti-rabbit secondary antibody (1:2000; GE Healthcare) in TBS-T containing 1% milk for 90 min at room temperature. "
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    • "This in turn leads to recruitment of immune cells to the lung and loss of pulmonary barrier function, producing the inflammation and pulmonary edema that are characteristics of hyperoxic lung injury. Adenosine is a nucleoside signaling molecule that has been recognized to play an important role in the regulation of inflammation following acute lung injury (Eckle et al. 2008a,b; Schingnitz et al. 2010; Karmouty-Quintana et al. 2013). The concentration of adenosine is normally low in the extracellular compartment, but following acute lung injury, extracellular adenosine concentrations rapidly increase (Volmer et al. 2006; Eckle et al. 2007). "
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