Extracellular Purines in Endothelial Cell Barrier Regulation - Extracellular ATP and Adenosine as Regulators of Endothelial Cell Function - page 42

demonstrated Ca 2+ -dependent activation of endothelial nitric oxide (NO) synthase
(eNOS) via direct interaction with Ca 2+ /calmodulin and/or via phosphorylation by
Ca 2+ /calmodulin-dependent protein kinase II (CaMKII)) [38, 87]. NO production
leads to activation of guanylyl cyclase (GA), cGMP synthesis and stimulation of
cGMP-dependent protein kinase (PKG) [53, 68]. While this pathway provides a
negative feedback control of Ca 2+ influx through down-regulation of endoplasmic
reticulum (ER) IP 3 -sensitive channels and plasma membrane Ca 2+ -influx channels
[25, 113], it can also increase Ca 2+ uptake by ER via activation of ER Ca 2+ ATPa s e s
[25]. Therefore, eNOS/GA/PKG cascade can down-regulate Ca 2+ -mediated signal-
ing leading to endothelial barrier dysfunction. In HUVEC, stimulatory phosphory-
lation of eNOS at Ser-1177 was shown to be adenosine-independent, but mediated
by several nucleotides (ATP, UTP and ADP). Inhibitory analysis demonstrated an
involvement of P2Y1, P2Y2, and possibly P2Y4 purinoceptors in the activation of
eNOS via [Ca 2+ ] i elevation and DAG-dependent PKC

[24]. Another protein tar-
get of activated PKG is vasodilator-stimulated phosphoprotein (VASP), a protein
regulating actin polymerization and assembly [57]. Phosphorylated by PKG/PKA,
VASP has been shown to localize to endothelial cell-cell junctions: in association
with TJs and AJs [20, 79]. Although an entire role of the VASP phosphorylation
in endothelial contraction/relaxation remains unclear, its modification correlates
with enhancement of endothelial barrier function in purinoceptor agonist-stimulated
cells [20, 54].
The P2Y2 receptor was also shown to activate G

δ

α 12 protein signaling. This acti-

vation requires an interaction of the receptor with α v β 3-integrin and can be inhibited

either by v-integrin antisense oligonucleotides or by point mutation in an integrin-

binding sequence of the P2Y2 receptor [58]. Activation of G

α

12 protein positively
modulate Rho-guanine nucleotide exchange factor (p115Rho-GEF) via its interac-
tion with G

α

[47].
This, in turn, promotes RhoA/ROK activation and phosphorylation of MLC 20 and
MLC phosphatase.
Elevation of cytosolic Ca 2+ may be, apparently, attributed to any P1 or P2Y
purinoceptor-mediated pathway in EC. Indeed, beside IP 3 -mediated Ca 2+

α 12 subunit [56] as well as via phosphorylation by activated PKC α

release

i, Ca 2+ influx from extracellular space may be stim- stimulated by G α q/11 and G α

s via cAMP-activated Ca 2+ -channel, While [Ca 2+ ] i is essential for
activation of eNOS and stimulation of endothelium-derived release of vasorelaxant,
NO [29, 43], elevation of cytosolic Ca 2+ is certainly a negative factor for endothelial
integrity. Nevertheless, as was shown in purine-activated EC, Ca 2+ influx is rather
transient and does not interfere with barrier-enhancing effects [54, 67].

ulated by G α

3.2.2 Adenosine-Activated Signaling and Endothelial
Monolayer Integrity

Extracellular adenosine or ATP may have a positive, as well as a negative effect on
the integrity of endothelium, depending on a prevalence of particular P1 and P2Y
purinoceptors. Although extracellular ATP itself should have a positive effect on