Stimulation of the mitogen-activated protein kinase via the A(2A)- adenosine receptor in primary human endothelial cells

Abstract

Adenosine exerts a mitogenic effect on human endothelial cells via stimulation of the A(2A)-adenosine receptor. This effect can also be elicited by the β2-adrenergic receptor but is not mimicked by elevation of intracellular cAMP levels. In the present work, we report that stimulation of the A(2A)-adenosine receptor and of the β2-adrenergic receptor activates mitogen-activated protein kinase (MAP kinase) in human endothelial cells based on the following criteria: adenosine analogues and β-adrenergic agonists cause an (i) increase in tyrosine phosphorylation of the p42 isoform and to a lesser extent of the p44 isoform of MAP kinase and (ii) stimulate the phosphorylation of myelin basic protein by MAP kinase; (iii) this is accompanied by a redistribution of the enzyme to the perinuclear region. Pretreatment of the cells with cholera toxin (to down-regulate G8α) abolishes activation of MAP kinase by isoproterenol but not that induced by adenosine analogues. In addition, MAP kinase stimulation via the A(2A)- adenosine receptor is neither impaired following pretreatment of the cells with pertussis toxin (to block G(i)-dependent pathways) nor affected by GF109203X (1 μM; to inhibit typical protein kinase C isoforms) nor by a monoclonal antibody, which blocks epidermal growth factor-dependent signaling. In contrast, MAP kinase activation is blocked by PD 098059, an inhibitor of MAP kinase kinase 1 (MEK1) activation, which also blunts the A(2A)-adenosine receptor-mediated increase in [3H]thymidine incorporation. Activation of the A(2A)-adenosine receptor is associated with increased levels of GTP-bound p21(ras). Thus, our experiments define stimulation of MAP kinase as the candidate cellular target mediating the mitogenic action of the A(2A)-adenosine receptor on primary human endothelial cells; the signaling pathway operates via p21(ras) and MEK1 but is independent of G(i), G(s), and the typical protein kinase C isoforms. This implies an additional G protein which links this prototypical G8-coupled receptor to the MAP kinase cascade.