Circulating or locally released nucleotides are rapidly metabolized by surface ectoenzymes (1). Ecto-5′nucleotidase (CD73) is a membrane-bound glycoprotein that functions to hydrolyze extracellular nucleotides into bioactive nucleoside intermediates (2). Surfacebound CD73 converts AMP to adenosine, which, when released, can activate seven transmembrane-spanning adenosine receptors (3, 4) or can be internalized through dipyridamole-sensitive carriers (5). These pathways have been shown to activate such diverse endpoints as adenine nucleotide recycling during cellular hypoxia (6), stimulation of epithelial electrogenic chloride secretion (responsible for mucosal hydration) (7), regulation of lymphocyte-epithelial adhesion (8), and promotion of endothelial and epithelial barrier function (4). Rather little is known about the regulation of CD73, and whether regulated expression provides a physiologic role. A number of studies have suggested that CD73 contributes to the protective aspects of adenine nucleotide metabolism during hypoxia and ischemia (9). For example, brief periods of ischemia preceding sustained ischemia, termed ischemic preconditioning, appear to result in large part from adenosine metabolism via increased CD73 activity (9). Increased CD73 activity in ischemic preconditioning has been attributed to adenosine receptor activation, protein kinase C activation, and α 1-adrenoreceptor activation (10). Few studies have addressed whether the CD73 gene is transcriptionally regulated. The cloned CD73 gene promoter contains a cAMP response element (CRE) (11), i.e., consensus DNA

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At sites of ongoing inflammation, polymorphonuclear leukocytes (PMN, neutrophils) migrate across vascular endothelia, and such transmigration has the potential to disturb barrier properties and can result in intravascular fluid loss and edema. It was recently appreciated that endogenous pathways exist to dampen barrier disruption during such episodes and may provide an important anti-inflammatory link. For example, during transmigration, PMN-derived adenosine activates endothelial adenosine receptors and induces a cAMP-dependent resealing of endothelial barrier function. In our study reported here, we sought to understand the link between cyclic nucleotide elevation and increased endothelial barrier function. Initial studies revealed that adenosine-induced barrier function is tightly linked to activation of protein kinase A (PKA). Because PKA selectively phosphorylates serine and threonine residues, we screened zonula occludens-1 (ZO-1) immunoprecipitates for the existence of such phosphorylated proteins as targets for barrier regulation. This analysis revealed a dominantly phosphorylated band at 50 kDa. Microsequencing identified this protein as vasodilator-stimulated phosphoprotein (VASP), an actin binding protein with multiple serine/threonine phosphorylation sites. Immunofluorescent microscopy revealed that VASP localizes to endothelial junctional complexes and colocalizes with ZO-1, occludin, and junctional adhesion molecule-1 (JAM-1). To address the role of phospho-VASP in regulation of barrier function, we generated a phosphospecific VASP antibody targeting the Ser157 residue phosphorylation site, the site preferred by PKA. Immunolocalization studies with this antibody revealed that upon PKA activation, phospho-VASP appears at cell-cell junctions. Transient transfection of truncated VASP fragments revealed a parallel increase in barrier function. Taken together, these studies reveal a central role for phospho-VASP in the coordination of PKA-regulated barrier function, such as occurs during episodes of inflammation. E ndothelial cells that line blood vessels are the primary determinants of vascular permeability [1], and structural and functional integrity of the endothelium is crucial in determining overall vascular permeability. Endothelial injury, such as occurs during leukocyte-endothelial interactions, may result in increased paracellular permeability, decreased barrier function, and subsequent intravascular loss of fluid and local tissue edema [2,3]. During transendothelial migration (TEM), polymorphonuclear leukocyte (PMN)-derived adenosine activates endothelial adenosine receptors and induces a cAMP-dependent sealing of endothelial monolayers [4,5]. Elevated levels of cAMP could enhance barrier function by activating protein kinase A (PKA) and subsequent phosphorylation of key complex-associated proteins [6,7]. At present, the intracellular signals that link cAMP elevation and vascular barrier function have not been identified.Regulation of junctional permeability is coupled to actin-based systems, a...