Extracellular Purines in Endothelial Cell Barrier Regulation - Extracellular ATP and Adenosine as Regulators of Endothelial Cell Function - page 38

3.1 Introduction

The vascular endothelium is a semi-selective diffusion barrier between the plasma
and interstitial fluid and is critical for normal vessel wall homeostasis. Endothelial
permeability is known to be regulated by the balance between centripetal and cen-
trifugal intracellular forces, provided by the contractile machinery and the elements
opposing contraction, respectively. The latter include tethering complexes, respon-
sible for cell-cell and cell-substrate contacts, and systems granting cell rigidity and
preventing cell collapse, such as actin filaments, microtubules and intermediate fil-
aments [28]. Some naturally occurring substances such as sphingosine-1-phosphate
[27] and the second messenger cAMP [39] are known to enhance the endothelial
cells (EC) barrier. Recently, much attention has been given to the therapeutic poten-
tial of purinergic agonists and antagonists for the treatment of cardiovascular and
pulmonary diseases [4, 14, 75, 97]. Purines (ATP, ADP and adenosine) function
as intercellular signaling molecules, which are released to extracellular compart-
ments from different sources in the body and subsequently reach the target organs
[15]. Accumulating experimental data suggest that ATP [54, 55] and other purines
are promising physiologically relevant barrier-protective agents as they are readily
present in the EC microenvironment in vivo, and they decrease transendothelial per-
meability in vitro. ATP can be released into the bloodstream from platelets [6] and
red blood cells [7, 16] and its concentrations may temporarily exceed 100 Min

blood [19]. Furthermore, the endothelium is a source of ATP locally within the vas-
cular bed, and ATP is released constitutively across the apical membrane of EC
under basal conditions [91]. Enhanced release of ATP is observed from ECs in
response to various stimuli, including hypotonic challenge [91], calcium agonists
[91], shear stress [71], thrombin [71], ATP itself [11], and LPS [12]. Extracellular
ATP can either signal directly [34, 107] or is rapidly degraded into adenosine
leading to additional vascular nucleoside signaling [31-33, 59]. Purine action is
mediated by cell surface P1 and P2Y purinergic receptors (purinoceptors) [76, 102].
Both P1 and P2Y purinoceptors belong to the superfamily of G-protein-coupled
receptors (GPCR). GPCRs consist of seven transmembrane domains, three extracel-
lular and three intracellular loops, extracellular N- and intracellular C-termini. The
receptors are coupled to heterotrimeric G-proteins serving for them as guanidine
exchange factors (GEFs). Heterotrimeric G-proteins, immediate targets for acti-
vated purinoceptors, exist as

μ

αβγ trimers. G βγ is a non-dissociated dimer, whereas

G -subunit is dissociated from the complex after GTP binding. There are 39 differ-

ent G-protein subunits identified in mammalian cells: 21

α

α -subunits, 6 β -subunits

and 12 -subunits. Such number of the subunits means a remarkable variety of

the heterotrimers, although, likely, not all of them may be formed in vivo due to
tissue-specific expression of some subunits and other factors [69].
Purinoceptors are activated upon extracellular adenosine (P1) and
ATP/ADP/UTP/UDP-glucose (P2Y) stimulation. To date, twelve G-protein-
coupled purinoceptors were identified in mammalian cells: four P1 receptors (A1,
A2A, A2B, A3) and eight P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11,
P2Y12, P2Y13, P2Y14) [90, 103] (Table 3.1). In EC of different origin, expressions

γ