Extracellular Purines in Endothelial Cell Barrier Regulation - Extracellular ATP and Adenosine as Regulators of Endothelial Cell Function - page 46

3.2.4 Down-Regulation (Desensitization)
of Purinoceptor-Mediated Signaling

P1 and P2Y receptor-mediated signaling can be down-regulated at different levels
of the signaling cascades. Activated PKA has inhibitory effect on some adenylyl
cyclase isoforms (AC5 and AC6) via its direct phosphorylation. PKA phosphoryla-
tion of AC5 directly inhibits its activity, whereas phosphorylation of AC6 at Ser674
disrupts G

α

s-binding [18, 49].
Moreover, PKA phosphorylation enhances activities of cAMP-specific phospho-
diesterases (PDEs), particularly, PDE4 isoforms [26, 114], diminishing intracellular
levels of cAMP more efficiently.
Activated GPCRs are phosphorylated by specific GPCR kinases (GRKs) docked
to the receptors by membrane-bound free G

dimers [72]. This phosphorylation
serves as a signal for receptor internalization. Phosphorylated receptors are able to
form complexes with adaptor proteins of clathrin-coated pits (such as

βγ

-arrestin)
and are internalized by clathrin-dependent endocytosis followed by either recycling
back to the cell surface or lysosomal degradation [46]. Interestingly,

β

-arrestin asso-
ciated with purinoceptors may also serve as a scaffold for a signaling complex
Raf-1/MEK1/2/ERK1/2 on the surface of endosomes [46]. In this case, activated
ERK1/2 is not translocated to the cell nucleus but switched to phosphorylation of
cytoplasmic protein substrates. Ubiquitous GRK2 is one of cytoplasmic ERK1/2
substrates. ERK1/2-dependent phosphorylation stimulates GRK2 activity and hence
positively regulates purinoceptor internalization and desensitization [98]. ERK2
can also phosphorylate PDE4 with either positive or negative effect on activities
of different PDE4 isoforms providing a fine modulation of intracellular cAMP
levels [48].
A variety in presentation and expression levels of purinoceptors in EC of different
origin determines a complexity of agonist-mediated cell response due to a possible
cross-talk among several pathways with opposed effects on endothelial functions.
Because of that, a usage of selective agonists/antagonists, either natural or synthetic,
has been considered as a very attractive way for pharmacological manipulation of
endothelial barrier function. Extensive research in this field already has provided
several highly-specific P1 and P2Y receptor agonists and antagonists.

β

3.3 Barrier-Protective Potency of Receptor-Specific Purinergic
Agonists

Selective activation of endothelial purinoreceptor(s) responsible for barrier pro-
tection might form a basis for the treatment of various disorders. The therapeutic
potential of purinoreceptors is rapidly expanding field in pharmacology and some
selective agonists became recently available. One recent study demonstrates that the
human bronchial epithelia express P2Y6 receptors on both apical and basolateral
membranes and that the cAMP/PKA pathway regulates apical but not basolateral