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Extracellular Nucleotides and Adenosine Independently Activate AMP-Activated Protein Kinase in Endothelial Cells

Involvement of P2 Receptors and Adenosine Transporters

From the Department of Medicine (C.G.d.S., R.J., A.S., E.K.), Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Mass; and the Department of Metabolism Regulation, Institute of Biochemistry (R.J.), Warsaw University, Poland.

Correspondence to Elzbieta Kaczmarek, Beth Israel Deaconess Medical Center, Department of Medicine, Harvard Medical School, 99 Brookline Ave, Boston, MA 02215. E-mail ekaczmar{at}bidmc.harvard.edu

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 5'-nucleotidase; hence, nucleotides can be metabolized to adenosine. However, inhibition of 5'-nucleotidase had no effect on ATP/ADP/UTP-induced phospho- rylation 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 Ca²⁺ and by Ca²⁺/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 Ca²⁺ 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.

Key Words: endothelial cells • AMP-activated protein kinase • P2 receptors • adenosine • CaMKK

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