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 20

of AMPK, while we excluded participation of P2Y6, P2Y11, P2X7, and other P2X
receptors [28].
Additionally, we confirmed that Ca 2+ released from the endoplasmic reticulum
plays an important role in ATP/UTP-induced phosphorylation of AMPK, as a chela-
tor of intracellular free calcium ions, BAPTA, almost completely attenuated AMPK
phosphorylation. Moreover, we identified CaMKK as the kinase responsible for
nucleotide-induced AMPK activation in ECs, whereas we excluded contributions
from LKB1, PI3K, protein kinase C (PKC) and CaMK II [28].
As mentioned earlier in this chapter, ECs express CD39 (NTPDase I), an
NTPDase that hydrolyzes nucleoside tri- and diphosphates to mono-phosphates,
as well as 5 -nucleotidase (CD73), which can further hydrolyze AMP to adeno-

sine [63, 129]. Therefore, we investigated whether phosphorylation of AMPK
induced by nucleotides was related to generation of extracellular adenosine.
Inhibition of 5

-methylene ADP (AOPCP) had no effect on
ATP/UTP/ADP-induced AMPK phosphorylation, indicating that adenosine genera-
tion is not involved in AMPK activation induced by extracellular nucleotides.
Extracellular adenosine can exert cellular responses by two mechanisms, either
through activation of cell surface P1 receptors or via uptake by nucleoside trans-
porters whereupon intracellular conversion to adenine nucleotides can affect cellular
functions. We demonstrated that adenosine but not its metabolites, inosine or hypox-
anthine, can activate AMPK. Furthermore, we established that the effect of adeno-
sine was not mediated by P1 receptors, but by adenosine uptake facilitated by nucle-
oside transporters. Finally, we clarified that the intracellular conversion of adenosine
to AMP is important for AMPK activation. We hypothesize that intracellular AMP
generated from adenosine by adenosine kinase, allosterically activates AMPK so
that it is more sensitive to further activation by upstream kinases. While search-
ing for the upstream kinases, we excluded CaMKK, as well as PI3K, and showed
that adenosine-induced phosphorylation of AMPK was dependent on activation of
LKB1. Therefore, we conclude that the pathway of adenosine-induced activation of
AMPK differs from the one identified for extracellular nucleotides [28].
We expected that AMPK activation induced by adenosine, which is converted
to AMP within the cell, would be sensitive to changes in the AMP:ATP ratio.
We observed an increase in intracellular AMP levels in response to extracellular
adenosine, however, this increase was accompanied by a corresponding increase
in ATP levels. Therefore, the ratio of AMP to ATP did not change significantly in
response to extracellular adenosine, as compared to other studies showing 6 to 25-
fold increases in the AMP:ATP ratio in adenosine treated cells [26, Hardie, 1998
#734]. We assume that the actual level of AMP is more important for activation
of AMPK than the AMP:ATP ratio [122]. Therefore, we conclude that adenosine-
induced activation of AMPK is linked to LKB1 activation and is enhanced by
increases in the intracellular AMP level.
In summary, our findings demonstrate two distinct, but converging pathways for
AMPK activation in HUVECs. One pathway induced by extracellular nucleotides is
linked to activation of P2Y1, P2Y2 and possibly P2Y4 receptors, and is dependent
on [Ca 2+ ] i and CaMKK. The second pathway induced by extracellular adenosine is

-nucleotidase with α , β