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endothelial cadherin (VE-cadherin) involved in Ca
2+
-dependent homotypic contacts
with adjacent cells. Cytoplasmic domain of VE-cadherin is linked to the cortical
actin ring via
-catenins which stabilizes AJ, as well as determines a mecha-
nism for dynamic reorganization of cell-cell contacts. Actin-mediated disassem-
bly/stabilization of AJ is determined mainly by phosphorylation/dephosphorylation
equilibrium of actin-associated 20-kDa myosin light chain protein (MLC
20
).
Phosphorylation status of MLC
20
plays an important role in cytoskeleton orga-
nization in EC and, therefore, is crucial for endothelium integrity [40, 101].
Phosphorylation of MLC
20
by Ca
2+
/calmodulin-dependent MLC kinase leads to an
actin-myosin contraction and centripetal force-driven AJ results in a loss of endothe-
lial integrity and hyperpermeability. In contrast, cell signaling pathways leading to
deposphorylation of MLC
20
by MLC phosphatase, protein phosphatase 1 (PP1),
results in the formation of a thick cortical actin ring, cell relaxation and spreading.
Agonist-mediated activation of purinoceptors on the surface of EC may enhance
as well as decrease a barrier function of the endothelium (Fig. 3.1). Stimulation of
A2A, A2B, or P2Y11 receptors coupled to G
β
/
α
α
s proteins [21, 22, 51, 73] leads to
direct interaction of dissociated G
s with plasma membrane adenylyl cyclase and
activation of the cAMP synthesis [111]. The cAMP is a classical activator of protein
kinase A (PKA), although it may be involved in modulation of other pathways. As it
was recently shown for 5
-N-ethylcarboxamidoadenosine (NECA)-stimulated A2B
receptors in human umbilical vein EC (HUVEC), generation of cAMP may lead
to alternative, PKA-independent Epac1/Rap1/B-Raf pathway resulting in ERK1/2
activation [35]. The cAMP-dependent activation of PKA has indispensible conse-
quences as a potent positive regulator of endothelial monolayer integrity. Indeed,
activated PKA shifts EC to relaxed status by prevention of MLC
20
phosphoryla-
tion and, therefore, formation of stress fibers. Purinoceptors A1, A3, and P2Y14
are involved in Gi protein-mediated signaling [35, 51]. Activation of these receptors
leads to an inhibition of adenylyl cyclase via its direct association with free G
α
α
i
[70]. Gi protein-derived free G
βγ
dimers interact with PI3-kinase (PI3-K) or phos-
pholipase C
) and initiate respective signaling pathways [44, 78, 90]. PI3-K
activates PKB/Akt (via phosphatidyl-3,4,5-triphosphate (PIP
3
) generation and acti-
vation of PDK1) [42] and ERK1/2 (via Ras/Raf-1/MEK1/2) [44]. PLC activation
(and, therefore, generation of inositol 1,4,5-triphosphate (IP
3
) and diacylglycerol
(DAG))mayfollowbyCa
2+
influx due to stimulation of plasma membrane and
endoplasmic reticulum Ca
2+
-channels [78]. Elevation of [Ca
2+
]
i
and DAG levels
routinely follows by membrane translocation and activation of conventional (
β
(PLC
β
α
β
,
I,
β
isoform has been shown
to activate the RhoA/Rho kinase (ROK) pathway by direct phosphorylation of the
upstream effectors, RhoGDI and RhoGEF [52], which leads to phosphorylation of
MLC
20,
as well as to regulation of TJ disassembly via phosphorylation of p120
and
II,
γ
) and novel (
δ
,
,
η
,
θ
) PKC isoforms [66]. PKC
α
i proteins has also been
shown to promote an upregulation of p38 MAPK [44] and may possibly acti-
vate JNK by ROK-dependent phosphorylation [62]. The p38 MAPK can initiate
stress fiber formation via phosphorylation of actin-capping protein hsp27 and its
further dissociation from actin filaments [45, 86]. Another important event related
β
-catenin [82]. Adenosine-mediated activation of G
α
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