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by calcium chelator or apyrase, an enzyme that hydrolyzes ATP and ADP to
AMP [125]. Studies with P2X1- and P2X4-knockout mice, also suggest that these
receptors are active in the endothelium [56, 57, 124].
There are also reports that P2X7 receptors are expressed in ECs [22, 91]. We
confirmed P2X7 receptor expression in HUVECs at the mRNA [119] and protein
levels (data not shown). Moreover, we localized these receptors not only to the
plasma membrane but also to the nuclear envelope, as analyzed by immunocyto-
chemistry and Western blot analysis using various cell fractions (data not shown).
P2X7 receptor expression in the nuclear membrane was earlier shown in neurons,
smooth muscle cells and epithelial cells of visceral organs of the guinea pig [6, 77].
The function of P2X7 receptors in the nuclear membrane could be to open ion chan-
nels that increase the nuclear Ca
2+
concentration, thereby affecting intra-nuclear
processes, such as the regulation of gene expression. Therefore, the discrepancy
between detection of P2X7 receptors in ECs and the lack of a calcium response to
BzATP (an agonist of this receptor) needs to be resolved.
Multiple functions for P2Y1, P2Y2 and possibly P2Y4 receptors in the vascula-
ture are well documented. These receptors have been implicated in the regulation
of angiogenesis [99], vascular tone [53], cell migration [8, 62], and mitogenic
responses [46, 117].
Although we were not able to demonstrate functional expression of P2Y6 recep-
tors in HUVECs (
i.e.
, no changes in [Ca
2+
]
i
were seen in response to UDP), there are
data showing that this receptor is functional in ECs originating from other vascular
beds. The endothelium-dependent relaxation of the aorta mediated by UDP was not
apparent in P2Y6-knockout mice [10]. Accordingly, the contractile effect of UDP
on the aorta, observed when eNOS was blocked, was also abolished in P2Y6-null
mice.
Our unpublished data demonstrate that populations of HUVECs (passage
1-4) stimulated with extracellular nucleotides respond with increases in [Ca
2+
]
i
.
Remarkably, nucleotide-induced changes in [Ca
2+
]
i
at the level of the single cell
were heterogeneous. Single cells stimulated sequentially with ADP, UTP and ATP
respond either to all three nucleotides, or only to one or two of these ligands. The
intensity of these responses, as well as the number and size of the calcium spikes
were also different between individual cells. Interestingly, with increased passage
number (from 1 to 4), HUVEC responses to ADP diminish, suggesting that either
the expression of the P2Y1 receptor, its functionality or both are decreased with
time in culture. This observation could be explained by the in vivo data showing a
decrease in P2Y1 expression in aging (19-month) rat aortic and carotid arteries, as
compared to younger (2-month) animals [78]. In contrast to HUVECs, single cells of
the EC line, EAhy926, respond to selected extracellular nucleotides (ATP, UTP and
ADP) in exactly the same way as the whole cell population. Immunocytochemical
staining of HUVECs with available antibodies to P2 receptors indicate that different
cells express varying levels of the same type of P2 receptor. We hypothesize that ECs
may exhibit heterogeneity in the context of P2 receptor subtype expression and/or
functionality. The physiological importance of this heterogeneity remains to be elu-
cidated. Our hypothesis regarding P2 receptor heterogeneity in ECs is supported by
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