SUMMARYExtracellular nucleotides bind to type-2 purinergic/pyrimidinergic (P2) receptors that mediate various responses, such as cell activation, proliferation and apoptosis, implicated in inflammatory processes. The role of P2 receptors and their associated signal transduction pathways in endothelial cell responses has not been fully investigated. Here, it is shown that stimulation of human umbilical vein endothelial cells (HUVEC) with extracellular ATP or UTP increased intracellular free calcium ion concentrations ([Ca 2+ ] i ), induced phosphorylation of focal adhesion kinase (FAK), p130 cas and paxillin, and caused cytoskeletal rearrangements with consequent cell migration. Furthermore, UTP increased migration of HUVEC in a phosphatidylinositol 3-kinase (PI3-K)-dependent manner. BAPTA or thapsigargin inhibited the extracellular nucleotide-induced increase in [Ca 2+ ] i , a response crucial for both FAK phosphorylation and cell migration. Furthermore, long-term exposure of HUVEC to ATP and UTP, agonists of the G protein-coupled P2Y2 and P2Y4 receptor subtypes, caused upregulation of α v integrin expression, a cell adhesion molecule known to directly interact with P2Y2 receptors. Our results suggest that extracellular nucleotides modulate signaling pathways in HUVEC influencing cell functions, such as cytoskeletal changes, cellular adhesion and motility, typically associated with integrin-activation and the action of growth factors. We propose that P2Y2 and possibly P2Y4 receptors mediate those responses that are important in vascular inflammation, atherosclerosis and angiogenesis.

Abstract-AMP-activated protein kinase (AMPK) plays a key role in the regulation of energy homeostasis and is activated in response to cellular stress, including hypoxia/ischemia and hyperglycemia. The stress events are accompanied by rapid release of extracellular nucleotides from damaged tissues or activated endothelial cells (EC) and platelets. We demonstrate that extracellular nucleotides (ATP, ADP, and UTP, but not UDP) and adenosine independently induce phosphorylation and activation of AMPK in human umbilical vein EC (HUVEC) by the mechanism that is not linked to changes in AMP:ATP ratio. HUVEC express NTPDases, as well as 5Ј-nucleotidase; hence, nucleotides can be metabolized to adenosine. However, inhibition of 5Ј-nucleotidase had no effect on ATP/ADP/UTP-induced phosphorylation of AMPK, indicating that AMPK activation occurred as a direct response to nucleotides. Nucleotide-evoked phosphorylation of AMPK in HUVEC was mediated by P2Y1, P2Y2, and/or P2Y4 receptors, whereas P2Y6, P2Y11, and P2X receptors were not involved. The nucleotide-induced phosphorylation of AMPK was affected by changes in the concentration of intracellular Ca 2ϩ and by Ca 2ϩ /calmodulin-dependent kinase kinase (CaMKK), although most likely it was not dependent on LKB1 kinase. Adenosine-induced phosphorylation of AMPK was not mediated by P1 receptors but required adenosine uptake by equilibrative nucleoside transporters followed by its (intracellular) metabolism to AMP. Moreover, adenosine effect was Ca 2ϩ and CaMKK independent, although probably associated with upstream LKB1. We hypothesize that P2 receptors and adenosine transporters could be novel targets for the pharmacological regulation of AMPK activity and its downstream effects on EC function. is an evolutionarily conserved enzyme that acts as an ultrasensitive energy charge sensor and regulates cell energy metabolism. 1 AMPK is a heterotrimeric Ser/Thr kinase consisting of a catalytic ␣ subunit and regulatory ␤ and ␥ subunits. AMPK is activated in response to ATP depletion associated with an increase in the intracellular AMP:ATP ratio. This ATP consumption can originate from pathological cellular stress, such as heat shock, hypoxia, or ischemia, and from physiological exerciseinduced skeletal muscle contraction. 2 AMPK can also be phosphorylated and activated by the mechanism independent of changes in AMP:ATP levels. 3 AMPK is activated allosterically by AMP (up to 5-fold) and by phosphorylation of Thr172 on the ␣ subunit (50-to 100-fold), which is catalyzed by upstream kinases, including LKB1 and CaMKK. 4,5 Activated AMPK turns on catabolic pathways that generate ATP and turns off pathways that consume ATP by phosphorylation of multiple targets. These include glycogen synthase, acetylcoenzyme A carboxylase (ACC), and hydroxymethylglutarylcoenzyme A (HMG-CoA) reductase. Because AMPK is central in controlling the metabolism of glucose and fatty acids, its role in obesity and type 2 diabetes is of major importance. AMPK is expressed in skeletal muscle, brain, liver, and pancre...

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