Biology Reference
In-Depth Information
PLC Beta. While the initial peak was largely independent of extracellular Ca
2+
,the
subsequent sustained phase (beyond 2 min) was entirely dependent on extracellular
Ca
2+
. Moreover, P-UAEC but not NP-UAEC were capable of rapid (within seconds)
[Ca
2+
]i oscillations superimposed upon the down slope of the initial [Ca
2+
]i peak.
In addition to these observations on the [Ca
2+
]i response itself, a causal relation-
ship between [Ca
2+
]i and NO was also implied by a strong correlation between the
dose-dependency for [Ca
2+
]i responses and NO
∗
production in NP- and P-UAEC
[6]. A previous review describes these initial studies in more detail [3] and we will
not repeat that here. Instead we will now move on to focus on the more recent stud-
ies which have gone on to examine the molecular basis of the [Ca
2+
]i responses
in single cells as well as in groups of cells over a longer period of time (30 min
of stimulation). We will also relate this to recent data from direct imaging of NO
and [Ca
2+
]i simultaneously in freshly isolated endothelium. Such additional stud-
ies have allowed us to identify the molecular signaling components underlying
these events and how pregnancy has altered the Ca
2+
signaling pathway to func-
tion in a different way to achieve dramatically enhanced eNOS activation during
pregnancy.
11.2 Investigating Differences in P2Y Receptors, G Protein
Alpha Subunits and Phospholipase C B3 Expression
in P-UAEC Vs. NP-UAEC
Early studies had suggested a P2Y class receptor was most likely responsible for
the [Ca
2+
]i response [6]. More recent studies have since confirmed that multi-
ple subtypes of P2Y and P2X receptors are present in UAEC [11], but further
that the expression levels of those receptor classes coupled to PLC in particular
are not significantly different in P-UAEC when compared to NP-UAEC. In spite
of the wide range of P2X and P2Y receptor subtypes in UAEC, comparison of
[Ca
2+
]i responses to a wide range of selective purinergic agonists with differential
affinities for members of the P2X and P2Y receptor families have now clearly estab-
lished that the primary receptor mediating the stimulation of [Ca
2+
]i itself is indeed
P2Y2 [11].
At a post receptor level, additional western blot analysis of all G protein alpha
subtypes known to couple to the PLC beta signaling pathway further confirmed
that Gq, the isoform most likely to mediate this P2Y2 receptor response, is present
in equal amounts in both NP- and P-UAEC [11]. Alpha subunit isoforms for Gi,
Go, Gs and Gz were also identified, again in amounts that were not different in
NP- vs. P-UAEC. At the level of phospholipase C itself, microarray analysis has
confirmed that PLC beta 3 isoform is highly abundant and again expressed at sim-
ilar levels in NP- and P-UAEC [9]. Thus enhanced [Ca
2+
]i signaling in P-UAEC
is not due to simple changes in P2X or P2Y receptor expression, and the same
P2Y2 receptor mediates the effect in each case. We must conclude, therefore, that
Search WWH ::
Custom Search