Stimulation of the Mitogen-activated Protein Kinase via the A2A-Adenosine Receptor in Primary Human Endothelial Cells

Abstract

When released into or formed in the extracellular space, adenosine acts as an autacoid via interaction with four types of G protein 1 -coupled receptors, termed A 1 -, A 2A -, A 2B -, and A 3 -adenosine receptor. These receptors are encoded by distinct genes and can be differentiated based on their affinities for adenosine analogues and methylxanthine antagonists (1, 2). In addition, the receptors can be classified based on their mechanism of signal transduction; A 1 -and A 3 -adenosine receptors interact with pertussis toxin-sensitive G proteins of the G i and G o family (3, 4, 5), whereas A 2A -and A 2B -adenosine receptors stimulate adenylyl cyclase via G s (6, 7).Adenosine is ubiquitously released by hypoxic tissues in large amounts; the nucleoside has therefore been proposed as one of the angiogenic factors that link the altered metabolism in oxygen-deprived cells to the formation of new capillaries (8).Earlier observations suggested that adenosine acts as an endothelial mitogen in vivo (9, 10). The mitogenic effect of adenosine has been verified in cultured endothelial cells derived from several vascular beds (11-13). In human endothelial cells, the proliferative response is mediated by the A 2A -adenosine receptor, an effect mimicked by stimulation of the endothelial ␤ 2 -adrenergic receptor (14). However, the mechanism by which adenosine analogues promote endothelial cell growth is not clear; there is, in particular, the apparent paradox that persistent stimulation of the signaling cascade composed of G s , adenylyl cyclase, and protein kinase A, which is downstream of A 2A -adenosine receptor, inhibits endothelial cell proliferation (14 -16). In the present work, we have therefore searched for additional effector mechanisms. We report that, in human endothelial cells, the A 2A -adenosine receptor stimulates the mitogen-activated protein kinase; this activation is independent of G s , G i , and typical protein kinase C isoforms but is associated with activation of p21 ras . EXPERIMENTAL PROCEDURESMaterials-Cell culture media were from Life Technologies, Inc., and cell culture dishes were from Greiner (Krems, Austria). [␥-32 P]ATP and [ 32 P]orthophosphate were from DuPont NEN. Cholera toxin, CPA, (Ϫ)isoproterenol, 8-Br-cAMP, collagenase (type IV), TPA, protein ASepharose, rabbit anti-rat IgG, forskolin, and epidermal growth factor (EGF) were obtained from Sigma, pertussis toxin was from Peninsula Laboratories (St. Helens, UK); bFGF, guanine nucleotides, and adenosine deaminase were from Boehringer Mannheim (FRG); XAC was from Research Biochemicals (Natick, MA), GF109203X was from Calbiochem, molecular weight standards (covering the range from 14 to 97 kDa) and reagents for SDS-polyacrylamide gel electrophoresis were from Bio-Rad. PD 098059, an inhibitor of MAP kinase kinase 1 (17), was from New England BioLabs (Beverly, MA). NECA and CGS 21680 were

Regulation of Endothelial CD73 by Adenosine: Paracrine Pathway for Enhanced Endothelial Barrier Function

“…These receptors are classified according to use of pertussis toxin-sensitive pathways (A 1 and A 3 ) or adenylate cyclase activation pathways (A 2A and A 2B ) (5). Endothelial cells of many origins express constitutive adenosine receptors (5), primarily of the A 2A and A 2B subtypes (27)(28)(29)(30). The present studies define a transcriptional pathway mediated by adenosine receptor activation, including but not limited to CD73.…”

Both Gs and Gi Proteins Are Critically Involved in Isoproterenol-induced Cardiomyocyte Hypertrophy

“…The activation of adenylyl cyclase induces an increase in cAMP levels, which in turn activates PKA (14,19,50,51). In addition, a previous report indicated that down-regulation of G s␣ abolishes ISO-induced ERK activation in human endothelial cells (25), suggesting that G s␣ is required for activation of ERKs by ␤-AR. Our previous (7) and present studies also showed that ISO activates ERKs through cAMP/PKA in cardiac myocytes, supporting that G s protein plays an essential role in the activation of ERKs.…”

“…On the contrary, in some cell types such as PC12 cells, Swiss-3T3 cells, and S49 mouse lymphoma cells, cAMP activates ERKs and potentiates the effects of growth factors on differentiation and gene expression (20 -24). In human endothelial cells, down-regulation of the ␣ subunit of G s (G s␣ ) abolishes ␤-AR-mediated ERK activation by ISO (25), suggesting that G s␣ -dependent cAMP elevation and PKA activation are responsible for the activation of ERKs. We and others have also demonstrated that ␤-AR agonists including ISO significantly activate ERKs and increase protein synthesis through cAMP/PKA in cardiac myocytes (6,7).…”

Signal pathway involved in the development of hypoxic preconditioning in rat hepatocytes

“…8 The apparent discrepancy between these findings and a role of adenosine A 2A receptors can be explained considering the possible coupling of (MEK1) or pertussis toxin-inhibitable G i proteins. [26][27][28] It is noteworthy that stimulation of adenosine A 2A -receptor protects liver sinusoidal endothelial cells against reperfusion injury 9 and reduces TNF-␣ release by lipopolysaccharide-stimulated Kupffer cells. 29 However, in both these cell types a cyclic-AMP dependent pathway appears to be coupled with the stimulation of adenosine A 2A -receptors.…”