Nucleotides and Novel Signaling Pathways in Endothelial Cells: Possible Roles in Angiogenesis, Endothelial Dysfunction and Diabetes Mellitus - Extracellular ATP and Adenosine as Regulators of Endothelial Cell Function - page 19

Fig. 2.1 A scheme of the P2
receptor-initiated effects
(activation of FAK and PI3K,
and interaction with integrin)
leading to EC migration and
cytoskeletal changes

VEGF receptor-2 (VEGFR-2) [99, 100, 107] (discussed in Chapter 4 of this
topic). Whether described here nucleotide-mediated effects observed in HUVECs
are VEGFR-2-dependent or not, remain to be determined.

2.3.2 Extracellular Nucleotides and Adenosine Activate AMPK
in HUVECs

AMPK is a heterotrimeric Ser/Thr kinase consisting of a catalytic α subunit and

regulatory subunits. AMPK is activated in response to various stimuli that

induce an increase in the intracellular AMP:ATP ratio caused by intracellular ATP
depletion. This decrease in intracellular ATP levels can originate from pathological
cellular stresses, such as heat shock, hypoxia or ischemia, and from physiological
exercise-induced skeletal muscle contraction [54]. AMPK can also be phosphory-
lated and activated by the mechanism independent of changes in the intracellular
AMP:ATP ratio [32]. AMPK is activated up to 5-fold by allosteric modification by
AMP, and 50-100-fold by phosphorylation of Thr-172 on the

β and γ

α

subunit by upstream
kinases, including the tumor suppressor kinase, LKB1 and calcium/calmodulin-
dependent protein kinase kinase (CaMKK) [58, 113]. AMPK has been localized to
skeletal muscle, brain, liver, pancreas, as well as ECs [30]. However, mechanisms
of AMPK activation, and the function of this kinase in the endothelium still are not
fully characterized. We aimed to determine whether extracellular nucleotides are
involved in the activation of AMPK.
Studies from our laboratory indicate that the extracellular nucleotides, ATP,
UTP and ADP, as well as adenosine, induce phosphorylation of AMPK and
its downstream target, acetyl-CoA carboxylase (ACC), in time- and nucleotide
concentration-dependent manners. Further, by pharmacological analysis, we
showed that P2Y1, P2Y2 and possibly P2Y4 receptors are involved in the activation