Extracellular Purines in Endothelial Cell Barrier Regulation - Extracellular ATP and Adenosine as Regulators of Endothelial Cell Function - page 43

endothelial integrity (via specific P2Y receptors coupled to heterotrimeric G α s and

G q/11 proteins), a quick degradation of ATP by cell-surface ectonucleotidases to

ADP and adenosine [80, 95] may be followed by activation of ADP-specific P2Y
and adenosine-specific P1 receptors. Therefore, the resulting effect may reflect an
interference of opposite signaling cascades. The response mediated by A1 and A3
receptors can definitively be a cause of the integrity loss. First, signaling pathways
activated by these purinoceptors shift MLC 20 to its phosphorylated form. Elevation
of [Ca 2+ ] i activate Ca 2+ /calmodulin-dependent phosphorylation of MLC 20 along
with RhoA/ROK-dependent inhibitory phosphorylation of regulatory myosin phos-
phatase target subunit (MYPT1) of MLC phosphatase, PP1, which, in turn, causes
stress fiber formation with following polarization of EC. Second, an activation of
Src-family kinases can increase clathrin- and caveolin-dependent endocytosis of
adhesive proteins via phosphorylation of caveolin-1 and dynamin-2 [63], stimu-
late disassembly of AJs via tyrosine phosphorylation of p120-catenin and SHP-2
[3, 50, 99] and, therefore, may enhance a loss of cell-cell adhesion and formation of
intercellular gaps.
On the other hand, signaling cascades activated by A2 receptors can decrease
endothelial permeability by shifting MLC 20 to its dephosphorylated form via
PKA-dependent inhibition of RhoA/ROK pathway. As was recently demonstrated,
PKA phosphorylation of RhoGDI at Ser-174 prevents RhoA activation by throm-
bin [74].
Activation of ERK1/2 is a common event in purinoceptor-mediated signaling.
As has been shown in model experiments (Chinese hamster ovary cells stably
transfected with human P1 receptors), all four adenosine receptors may activate
ERK1/2 phosphorylation when stimulated by the non-P1 receptor agonist, NECA,
although intensities of the activation were not equal. The most potent ERK1/2
activator was the A2B receptor, whereas the highest level of ERK1/2 phosphory-
lation was obtained in A1 and A3 receptor-transfected cells [88, 89]. However,
the activation of ERK1/2 is, likely, cell/tissue-specific, and was not detected in
some types of cells expressed endogenous P1 receptors upon agonist stimula-
tion [60]. Although ERK1/2 may be responsible for Ca 2+ -independent NO release
by endothelium [110], its functions in EC may have a dual role, because of
an involvement in barrier-protective and barrier-disruptive mechanisms. In cul-
tured EC stimulated by extracellular ATP, ERK1/2 activity has been shown to
be dispensable as an enhancement of endothelial barrier function under normal
conditions [54], but it was important for ATP-mediated survival of hyperoxia-
challenged EC [1].

α

3.2.3 Purinoceptor-Mediated Signaling as a Regulator
of Endothelial Integrity at Various Pathological States

A number of agents which negatively affect endothelial integrity have been shown
to activate mechanisms interfering with purinoceptor-mediated signaling. Activation
of the RhoA/ROK signaling pathway followed by actin stress fiber formation and a