Cell Surface ATP Synthase: A Potential Target for Anti-Angiogenic Therapy - Extracellular ATP and Adenosine as Regulators of Endothelial Cell Function - page 147

by apyrase prevents the shear stress-dependent influx of Ca 2+ in HPAEC, indicating
that Ca 2+ transport into these cells depends on the presence of extracellular ATP
[51]. Yamamato et al. propose that cell surface F 1 F 0 ATP synthase is the source for
shear stress-induced ATP production, since incubation of HPAEC with the ATP syn-
thase inhibitors angiostatin, oligomycin, piceatannol, or antibody directed against
the

subunit of ATP synthase prevents the rise in extracellular ATP levels and
Ca 2+ influx without affecting intracellular ATP levels (Fig. 9.7b) [49, 51]. Addition
of exogenous extracellular ATP overcomes the inhibitory effect of ATP synthase
inhibitors on Ca 2+ flow into the endothelial cells (Fig. 9.7c).
Yamamoto et al. went on to demonstrate that ATP induces Ca 2+ influx through
P2X 4 —a purinoreceptor with greater sensitivity to ATP than ADP. siRNA knock-
down of this ATP-gated cation channel inhibits shear flow-induced Ca 2+ responses
in HPAEC. The G protein-coupled P2Y purinoreceptors do not appear to be
involved, as the shear stress-induced calcium rise is due to influx of external Ca 2+
rather than Ca 2+ release from internal stores. P2Y receptor activation would have
stimulated inositol 1,4,5-trisphosphate (IP 3 ) production, leading to release of Ca 2+
from intracellular stores. Thus, ATP synthase production of extracellular ATP plays
a role in flow-induced pulmonary artery endothelial cell signaling by activating Ca 2+
influx through the P2X 4 cation channel. Differences do exist in the level of shear
stress-induced ATP production by endothelial cells from distinct sources, indicating
that involvement of ATP synthase in endothelial response to shear stress may vary in
different vascular beds. For example, although flow-stimulated Ca 2+ influx occurs
in HUVEC, this response requires the addition of exogenous ATP [1, 48]. The
authors attribute this incongruent finding to the relatively lower shear-stress induced
endogenous ATP production by HUVEC relative to HPAEC [51]. Alternatively, they
propose a difference in P2X receptor ATP affinity in HUVEC versus HPAEC due to
potential variations in P2X subunit multimerization.
Regardless of the potentially disparate roles of ATP in different vascular beds,
a recent study in mice deficient in the P2X 4 receptor ( P2rx4 − / − ) indicates that
purine signaling through this ligand-gated cation channel does have in vivo rele-
vance to vascular regulation. These knockout mice display abnormal flow-induced
calcium responses and impaired vasodilation in response to increased blood flow.
The authors attribute this decreased vasodilation to the lack of nitric oxide release by
P2rx4 − / − mice in response to shear stress, in contrast to the dose-dependent increase
in NO production by endothelial cells from wild-type mice with normal vasodila-
tory response. The P2X 4 receptor also appears to be involved in vascular remodeling
as demonstrated by the response of P2rx4 − / − mice to a chronic decrease in blood
flow via external carotid artery ligation. While the common carotid artery diameter
decreases in wild-type mice, it does not change in the knockout mice [50]. Further
work is required to elucidate how shear stress stimulates ATP production by ATP
synthase, however current evidence does suggest that flow-dependent increase in
ATP is important in acute and chronic vascular changes. It should be noted that other
groups have demonstrated flow-induced release of ATP from intracellular stores,
and the relative contributions of this released ATP versus ATP generated by ATP
synthase remains unclear [7, 20].

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