Early activation of the p42/p44 ^MAPK pathway mediates adenosine‐induced nitric oxide production in human endothelial cells: a novel calcium‐insensitive mechanism

Abstract

ABSTRACT Adenosine is released from the myocardium, endothelial cells, and skeletal muscle in ischemia and is an important regulator of coronary blood flow. We have already shown that acute (2 min) activation of A 2a purinoceptors stimulates NO production in human fetal umbilical vein endothelial cells (1) and now report a key role for p42/p44 mitogen‐activated protein kinases (p42/p44 MAPK ) in the regulation of the L‐arginine‐nitric oxide (NO) signaling pathway. Expression of mRNA for the A 2a ‐, A 2b ‐, and A 3 ‐adenosine receptor subtypes was abundant whereas A 1 ‐adenosine receptor mRNA levels were negligible. Activation of A 2a purinoceptors by adenosine (10 fM) or the A 2a receptor agonist CGS21680 (100 nM) resulted in an increase in L‐arginine transport and NO release that was not mediated by changes in intracellular Ca 2 + , pH, or cAMP. Stimulation of endothelial cells with adenosine was associated with a membrane hyperpolarization and phosphorylation of p42/p44 MAPK . L‐NAME abolished the adenosine‐induced hyperpolarization and stimulation of L‐arginine transport whereas sodium nitroprusside activated an outward potassium current. Genistein (10 fM) and PD98059 (10 fM), an inhibitor of MAPK kinase 1/2 (MEK1/2), inhibited adenosine‐stimulated L‐arginine transport, NO production, and phosphorylation of p42/p44 MAPK . We found no evidence for activation of eNOS via the serine/threonine kinase Akt/ PKB (protein kinase B) in adenosine‐stimulated cells. Our results provide the first evidence that adenosine stimulates the endothelial cell L‐arginine‐NO pathway in aCa 2 + ‐insensitive manner involving p42/p44 MAPK , with release of NO leading to a membrane hyperpolarization and activation of L‐arginine transport.—Wyatt, A. W., Steinert, J. R., Wheeler‐Jones, C. P. D., Morgan, A. J., Sugden, D., Pearson, J. D., Sobrevia, L., Mann, G. E. Early activation of the p42/p44 MAPK pathway mediates adenosine‐induced nitric oxide production in human endothelial cells: a novel calcium‐insensitive mechanism. FASEB J . 16, 1584–1594 (2002)