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Regulation of vessel contraction/relaxation by extracellular nucleotides has been
well known for many years and is associated with NO generation [19] in response to
a conformational activation of eNOS by Ca
2+
/CaM [64, 75]. However, until recently
the impact of extracellular nucleotides on eNOS phosphorylation at Ser-1177 had
not been fully elucidated. We showed that ADP, ATP, and UTP induce Ser-1177-
eNOS phosphorylation, indicating that P2Y1, P2Y2 and possibly P2Y4 receptors
are the main receptors involved in purinergic activation of eNOS in ECs [29]. We
excluded P2Y6 and P2Y11 receptors, as their respective ligands, UDP and BzATP,
did not induce eNOS phosphorylation.
Our results also demonstrate that nucleotide-induced eNOS phosphorylation is
Ca
2+
-dependent, as chelation of intracellular Ca
2+
with BAPTA completely inhib-
ited Ser-1177 phosphorylation. However, activation of Ca
2+
-dependent kinases
upstream of eNOS activation, including CaMKII and CaMKK [102] is not involved
in nucleotide-induced eNOS phosphorylation on Ser-1177, even though extracellu-
lar nucleotides can activate CaMKK in ECs [28].
We also excluded AMPK, a reported upstream activator of eNOS [116]
that is activated by extracellular nucleotides in ECs [28]. Inhibition of AMPK
with a pharmacological inhibitor, Compound C, or by expression of dominant-
negative AMPK
α
2 did not decrease eNOS phosphorylation induced by extracellular
nucleotides [29].
Likewise, pharmacological inhibitors of PI3K/Akt, ERK, p38, PKA, PKG, and
PKC
did not affect nucleotide-induced eNOS phosphorylation. In contrast, phar-
macological inhibitors of PKC
α
expression with siRNA
decreased nucleotide-induced eNOS activation in ECs. Additionally, we confirmed
that nucleotide-induced eNOS phosphorylation correlates with increases in cGMP
levels, likely caused by NO generation. Accordingly, we propose that extracellu-
lar nucleotides induce eNOS activation, through Ca
2+
-dependent conformational
changes and PKC
δ
or suppression of PKC
δ
-dependent phosphorylation of eNOS (Fig. 2.3).
In contrast to our results, Brown et al. showed that eNOS is activated by P2Y
receptors via the PKC
δ
isoform [14]. Results from Ian Bird's group suggest roles
for Ca
2+
and ERK1/2 in pregnancy-related eNOS activation in uterine arteries [35].
In other studies, P2X1 [56] and P2X4 [124] receptor activation has been implicated
in NO-dependent vasodilation.
α
Fig. 2.3
A scheme of the proposed signaling pathway of the extracellular nucleotide-induced
phosphorylation of eNOS in EC
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