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development of vascular disorders [28, 67, 68]. On the other hand, other investiga-
tors have shown endothelial nucleotide-inactivating ectoenzymes NTPDase1/CD39
and ecto-5
-nucleotidase/CD73 can be upregulated during acute hypoxia and inflam-
mation. This increases the intravascular adenosine concentrations and dampens
excessive inflammatory responses by affecting endothelial barrier function, adhe-
sion and transmigration of lymphoid cells, and expression of other molecules
involved in the adhesion cascade [22, 46, 85].
Specific purine-converting activities were quantified by thin-layer chromatog-
raphy (TLC) using cultured endothelial cells as enzyme sources and saturating
concentrations of
3
H-labeled and non-labelled nucleotides as appropriate substrates,
as described previously [89]. In these studies, ATPase/NTPDase was evaluated as
the rate of 500
M[
3
H]ATP dephosphorylation into
3
H-metabolites, that were
quantified as pooled ADP, AMP and nucleoside fractions (Fig. 5.5a). Likewise,
ADPase/NTPDase (Fig. 5.5b) and ecto-5
-nucleotidase (Fig. 5.5c) activities of the
cells were determined by their ability to hydrolyse 500
μ
M[
3
H]ADP and 300
M
[
3
H]AMP, respectively. Backward adenylate kinase (Fig. 5.5d) and NDP kinase
(Fig. 5.5e) activities were also determined by extent of ATP-induced phosphoryla-
tion of 500
μ
μ
M[
3
H]AMP or [
3
H]ADP into high-energy
3
H-phosphoryls. Incubation
times in these assays varied between 20 and 40 min, and were chosen so that the
amount of metabolites did not exceed 10-15% of initially introduced substrate.
Consistent with our previous findings on human umbilical vein endothelial
cells (HUVEC) [89], current studies using various cultured bovine endothelial
cells also demonstrated the co-existence of extensive network of purine-converting
endothelial ectoenzymes that regulate nucleotide levels via two counteracting,
ATP-consuming and ATP-regenerating, pathways (Fig. 5.5f). Though, further com-
parative analysis of major exchange activities revealed substantial differences
among the studied endothelial cells. Specifically, cultured VVEC hydrolyze various
3
H-labelled nucleotides with relative ATPase/ADPase/AMPase ratios of
μ
4.4/9.3/1
and in addition, were able to sequentially phosphorylate exogenous AMP via ADP
to ATP in the presence of
∼
-phosphate-donating ATP. Compared to VVEC, cul-
tured MPAEC and especially MVEC (all isolated from chronically hypoxic calves)
were characterized by substantially higher ATP- and ADP-inactivating capability,
and at the same time, displayed relatively low activities of ecto-5
-nucleotidase
as well as nucleotide-phosphorylating enzymes adenylate kinase and NDP kinase
(Fig. 5.5a-e).
The lower rates of [
3
H]ADP (Fig. 5.6b) hydrolysis in VVEC compared to
MPAEC and MVEC is in agreement with previous reports demonstrating that
decreases of ATP diphosphohydrolase activity/CD39 can be observed during
oxidative stress-induced endothelial cell activation [1, 48, 67]. No significant
hypoxia-dependent shifts were determined in studies with other purinergic enzymes,
ecto-ATPase (Fig. 5.6a), adenylate kinase (Fig. 5.6d) and NDP kinase (Fig. 5.6e).
Along with evidence for selectively diminished ADP- and AMP-inactivating capac-
ity of the hypoxic VVEC, the unchanged activities of other ectoenzymes may
additionally serve as suitable “internal controls” for comparable assay conditions
among the endothelial cell types. Furthermore, it could be even more relevant to
γ
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