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receptors (coupled directly to Gq protein) stimulates phospholipase C, followed
by an increase in inositol 1,4,5 trisphosphate and release of Ca
2+
from intracel-
lular stores [120]. P2Y11 receptors additionally mediate a stimulation of adenylate
cyclase to generate cAMP. In contrast, activation of the P2Y12, P2Y13, and P2Y14
receptors (coupled to Gi protein) causes an inhibition of adenylate cyclase activity
and a decrease in intracellular cAMP levels [12].
It is clear now, that extracellular nucleotides, acting via P2 receptors and adeno-
sine acting via P1/adenosine receptors (A1, A2a, A2b, A3), activate multiple
signaling pathways in various cells, including ECs.
2.2 P2 Receptors in ECs and Calcium Responses to Extracellular
Nucleotides
Human umbilical vein endothelial cells (HUVECs) express multiple P2X and P2Y
receptor subtypes, including P2X1, P2X2, P2X4, P2X6, P2X7, P2Y1, P2Y2, P2Y4,
P2Y6, and P2Y11 receptors, as determined by PCR and/or quantitative PCR and
partially confirmed by Western blot analysis when reliable antibodies were avail-
able ([55, 94, 121] and our unpublished data). P2Y1, P2Y2 and P2X4 receptors are
highly expressed in ECs, compared to other P2 receptor subtypes.
Early cellular responses to extracellular nucleotides include elevation of [Ca
2+
]
i
due to calcium influx from the extracellular space or release from intracellular stores
[24]. Calcium ions function as second messengers, regulating numerous cellular
processes. Brief spikes and waves in the intracellular free calcium ion concentration
([Ca
2+
]
i
) regulate the expression of selected genes [37, 71]. We analyzed calcium
responses by confocal microscopy in HUVECs loaded with fluo-3, a fluorescent
Ca
2+
indicator. ATP, ADP, UTP and their slowly hydrolysable analogs (ATP
γ
S and
S) mediated significant increases in [Ca
2+
]
i
. However, UDP (P2Y6 recep-
tor ligand), BzATP (P2X7 and P2Y11 receptor ligand) and
ADP
β
α
-
β
-meATP (a ligand
for P2X receptors) in the concentration range studied (1-200
M) did not induce
increases in [Ca
2+
]
i
, suggesting that only P2Y1, P2Y2 and possibly P2Y4 receptors
are functionally active in HUVECs [62]. Moreover, the nucleotide-induced increase
in [Ca
2+
]
i
was inhibited by BAPTA (an intracellular Ca
2+
chelator) and thapsigargin
(which depletes Ca
2+
from intracellular stores by inhibiting endoplasmic reticulum
Ca
2+
ATPase) but not by EGTA (a chelator of extracellular Ca
2+
) [62]. This indi-
cates that ATP, ADP and UTP-induced changes in [Ca
2+
]
i
depend on Ca
2+
release
from intracellular stores and therefore P2Y rather than P2X receptors are respon-
sible for nucleotide-mediated increases in [Ca
2+
]
i
, and most likely for Ca
2+
-related
signaling events in HUVEC. This is confirmed by other studies suggesting that P2Y,
and not P2X, receptors are functionally active in the endothelium [115].
In contrast to these results, the roles of some P2X receptor subtypes have
been reported in ECs. This includes P2X4 receptors, whose involvement in the
EC response to shear stress is recognized. In human pulmonary artery ECs, ATP
released in response to shear stress mediates Ca
2+
μ
influx, which was inhibited
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